EU Funded Project FP6-2004-SSP-4-022623   INRA_08D01.09_30MAR07_v01.00




          D01.09 - Literature survey on
         WCR ecology finalised (WP1 Task 2.1)


                  Diabr-Act -
             Harmonise the strategies for fighting
              Diabrotica virgifera virgifera


                   Specific Support Action
                  (FP6-2004-SSP-4- 022623)




Contract Start Date : 01-06-2006
Duration : 24 months
Project Applicant : ARVALIS – Institut du Végétal




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        Document Classification
               D01.09 - Literature survey on WCR ecology finalised (WP1 Task
Title
               2.1)
               D01.09
Deliverable
               1
Reporting Period:
Contractual Date of Delivery Project Month 11
               30 03 2007
Actual Date of Delivery

                        Guillemaud P08 INRA
Authors
                        WP01 Basic Ecology and Integrated crop management
Work package
                        PU Public
Dissemination
                        REPORT
Nature
                        V01.00
Version
                        INRA08_D01.09_30MAR07_v01.00
Doc ID Code
                        Report literature survey WCR ecology
Keywords

Document History
Name                      Remark           Version               Date
Thomas Guillemaud                Final version       01.00                30 03 2007

Document Abstract
A literature survey has been conducted on the ecology of the western corn rootworm, Diabrotica
virgifera virgifera. This survey details a large part of the literature available on this subject as well
as unpublished results of current research.


 The information contained in this report is subject to change without notice and should not be construed as a commitment by any members of the
Diabr-Act Consortium. The Diabr-Act Consortium assumes no responsibility for the use or inability to use any procedure or protocol which might be
described in this report. The information is provided without any warranty of any kind and the Diabr-Act Consortium expressly disclaims all implied
          warranties, including but not limited to the implied warranties of merchantability and fitness for a particular use.




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Table of Contents

  Abbreviations ................................................................................................................................... 5
  Short description .............................................................................................................................. 6
CONTRIBUTORS:........................................................................................................................................................... 6
INTRODUCTION ............................................................................................................................................................ 6
WESTERN CORN ROOTWORM COURTSHIP AND MATING BEHAVIOR/PHYSIOLOGY &
OVIPOSITION TIMING................................................................................................................................................. 8
  I Courtship and Mating .................................................................................................................... 8
  II Oviposition: timing and amount of viable eggs ......................................................................... 12
  III References Cited ....................................................................................................................... 13
DIURNAL RHYTHMS OF WESTERN CORN ROOTWORM ACTIVITY........................................................... 16
  I Conditions that influence expression of diurnal rhythms. ........................................................... 16
  II Periodicity of general WCR activity. ......................................................................................... 16
  III Periodicity of adult emergence. ................................................................................................ 16
  IV Periodicity of female pheromone calling, male response to pheromone, and mating. ............. 17
  V Periodicity of WCR flight, interfield movement, and abundance in corn, soybean and other
  crops. .............................................................................................................................................. 17
  VI Periodicity of oviposition..........................................................................................................18
  VII A caution about diurnal periodicities. ..................................................................................... 18
  VIII References Cited .................................................................................................................... 18
NUTRITIONAL ECOLOGY ........................................................................................................................................ 20
  I Adults...........................................................................................................................................20
    I-1 Feeding hosts, role of wild plants.........................................................................................20
    I-2 Chemical and physical cues: biotic/abiotic .......................................................................... 21
  II Larvae.........................................................................................................................................22
    II-1 Host selection......................................................................................................................22
    II-2 Chemical and physical cues ................................................................................................ 23
  III Case of the variant - Oviposition site selection and behavior...................................................24
  IV References Cited ....................................................................................................................... 25
WESTERN CORN ROOTWORM POPULATION DYNAMICS............................................................................. 28
  I Egg Diapause ............................................................................................................................... 28
  II Larval and adult dynamics ......................................................................................................... 29
  III Non-trophic (direct or indirect) interactions with other arthropods..........................................32
  IV References Cited ....................................................................................................................... 32
MICRO- SHORT AND LONG RANGE WESTERN CORN ROOTWORM MOVEMENT ................................. 40
  I Micro-range ................................................................................................................................. 40
  II Short range WCR movement ..................................................................................................... 40
  III Interfield movement..................................................................................................................42
  IV Long distance movement .......................................................................................................... 44
  V References Cited ........................................................................................................................ 45
SPATIAL DISTRIBUTION AT VARIOUS SCALES ................................................................................................ 49
  I Within plants or within roots ....................................................................................................... 49

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  II Among fields (including crop phenology, rotation history, tillage, landscape features) ........... 49
  III Dynamics of spread in North America & Europe....................................................................50
    III- 1 Dynamics of spread in North America ............................................................................. 50
    III- 2 Dynamics of spread in Europe..........................................................................................52
    III-3 Dynamics of spread of the rotation-resistant variant in the U.S........................................53
  IV Introduction routes of invading populations in Europe ............................................................ 53
  V References Cited ........................................................................................................................ 55
GENETICS: TOOLS AND APPLICATIONS............................................................................................................. 61
  I Variable genetic markers + population genetics description:......................................................61
    I-1 Microsatellites ...................................................................................................................... 61
    I-2 AFLP .................................................................................................................................... 62
    I-3 Cytoplasmic markers............................................................................................................62
    I-4 Applications..........................................................................................................................63
  II Genomic resources ..................................................................................................................... 66
  III Laboratory colonies selected for insecticide resistance, rotation resistance, and non-diapause.
  ........................................................................................................................................................ 67
  IV References Cited ....................................................................................................................... 69
RECENT ADAPTIVE CHARACTERS: RESISTANCE TO PESTICIDES AND CROP ROTATION................ 74
  I Corn rootworm biology and management. .................................................................................. 74
  II Rootworm Adaptation: Cyclodiene Resistance..........................................................................74
  III Rootworm Adaptation: Organophosphate Resistance. ............................................................ 77
  IV Rootworm Adaptation: Adaptation to Crop Rotation...............................................................80
  V References Cited ........................................................................................................................ 83
AN ABBREVIATED REVIEW OF THE LITERATURE FOR THE VARIANT WESTERN CORN
ROOTWORM................................................................................................................................................................. 86
  I State of the art..............................................................................................................................86
  II References Cited.........................................................................................................................90




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       Abbreviations
WCR: Western Corn Rootworm
NCR: Northern Corn Rootworm
MCR: Mexican Corn Rootworm
SCR: Southern Corn Rootworm




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Short description
This Deliverable D01.09 intends to present a literature survey that has been conducted on the
ecology of the western corn rootworm, Diabrotica virgifera virgifera. This survey details a large
part of the literature available on this subject as well as unpublished results of current research.


Contributors:
  •  Stefan Vidal, Institute of Plant Pathology and Plant Protection, Georg-August University,
    Germany
  •  Nora Levay, Université de Godollo, Hungary
  •  Joachim Möser, Institute of Plant Pathology and Plant Protection, Georg-August University,
    Germany
  •  Ivan Hiltpold Université de Neuchatel, Switzerland
  •  Bruce Hibbard, USDA-ARS, University of Missouri, Columbia, USA
  •  Lance Meinke, Department of Entomology, University of Nebraska Lincoln, USA
  •  Michael Gray, Department of Crop Sciences, University of Illinois, USA
  •  Thomas Sappington, USDA-ARS, Ames, USA
  •  Nicholas Miller, USDA-ARS, Ames, USA
  •  Joseph Spencer, Section of Ecological Entomology, Illinois Natural History Survey,
    Champaign, USA
  •  Blair Siegfried, Department of Entomology, University of Nebraska Lincoln, USA
  •  Rosana Giordano, Dpt of Plant and Soil Science, University of Vermont, USA
  •  Lorenzo Furlan, University of Padova, Italy
  •  Thomas Guillemaud, INRA, UMR 1112, France
  •  David Onstad, University of Illinois, USA
  •  Judit Pap, Université de Godollo, Hungary
  •  Ferenth Toth, Université de Godollo, Hungary
  •  Joseph Kiss, Université de Godollo, Hungary


Introduction
The western corn rootworm (WCR), Diabrotica virgifera virgifera is currently the major pest of
corn in North America, and is about to get the same status in Europe where it is present for more
than 15 years. Since its first observation in 1992 in former Yugoslavia, WCR has reached various
western and south eastern European countries, from the UK to Bulgaria, and from Poland to Serbia.
This past and recent situations in North America and Europe have led the scientific community of
entomologists and agronomists to produce large efforts to understand the basic biology and ecology
of this species. Since the beginning of the XXth century, this chrysomelid has been the subject of
about a thousand scientific papers, most of them dealing with ecology, sensu lato, i.e. the biology of
the animal in relation with its environment. Recently, the production has grown at an increasing
speed: approximately a thousand authors wrote about 800 papers referenced in the cab-abstract web
database since 1973, most of them being published after the eighties. A priori, this rich literature
deals primarily with basic biology, nutritional relationships, plant-insect interactions, population
dynamics and response to management practices. The present report aims at providing a detailed a
posteriori description of the scientific production concerning WCR ecology by surveying the past
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       and recent work on WCR ecology. It is important to note that the present report does not
refer to scientific productions dealing with management practices (biological control, insecticide
treatments, plant breeding, etc.) as these topics are already covered and reviewed in various sub-
packages of the Diabr’act project. This state of the art report, written by a panel of the most
recognized experts in the field including both American and European scientists, consists of three
main sections on the subjects of behavior, population dynamics and genetics and adaptations. More
specifically, sexual behavior (courtship, mating, and oviposition), diurnal activity and nutritional
ecology are part of a large behavioral section, population growth; dispersal and geographical
distribution constitute the population dynamics section; and finally, the description of genetic tools
and application, and of recent adaptive characters provide the structure of the last section. The two
first main subjects refer to a literature that has been partially reviewed in previous publications (e.g.
Chiang, 1973; Levine and Oloumi-Sadeghi, 1991; Vidal, Kuhlman & Edwards, 1995), however
they provide a new, actualized and recent state of the art of the fields. The last large section dealing
with genetics and adaptation provides a completely new survey on a recent scientific field. The
review also surveys ongoing work that is not yet published but that deserves to be reported.

Vidal, S., Kuhlmann, U., and C. R. Edwards, eds. 1995. Western Corn Rootworm: Ecology and
Management. CABI Publishing, Wallingford, Oxfordshire, UK. Pp. 121-144.
Chiang, H.C. 1973. Bionomics of the northern and western corn rootworms. Annual Review of
Entomology 18: 47-72.
Levine, E. and H. Oloumi-Sadeghi. 1991. Management of diabroticite rootworms in corn. Annual
Review of Entomology 36: 229-255.




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Western Corn Rootworm Courtship and Mating Behavior/Physiology
& Oviposition timing
I Courtship and Mating
The western corn rootworm (Diabrotica virgifera virgifera LeConte, WCR) is a protandrous
species; first emergence of WCR males precedes that of females by ca. 5 days (chiefly because the
post-hatching development of males is faster than females) (Branson 1987). The WCR mating
system is a scramble competition polygyny (Thornhill and Alcock 1983); males are always more
abundant than is necessary to inseminate all the available females. Despite protandry, Quiring and
Timmins (1990), note that 97.8% of the male emergence period still overlaps with that of females.
Once the males emerge, ca. 5-7 days of post-emergence development are required for 80% of the
male population to reach sexual maturity (characterized by responsiveness to female sex
pheromone) (Guss 1976). WCR females are sexually mature upon adult emergence (Hammack
1995), many mate within just hours of emergence (Ball 1957, Hill 1975, Lew and Ball 1979).
The first indication that a sex pheromone was involved in WCR reproduction came from the
unpublished thesis of Cates (1968). Ball and Chaudhury (1973) were first to publish evidence of a
sex pheromone extractable from females that elicited behavioral responses in males. They were
also able to demonstrate elevated male WCR trap catch on female-extract baited sticky traps in the
field.
The major active component of the female-produced WCR sex pheromone is 8R-methyl-2R-
decenyl-propanoate (Guss et al. 1982). The WCR sex pheromone was the first to be identified and
synthesized for a species of Chrysomelidae (Guss et al. 1982); however, the first evidence for sex
pheromone use by a Chrysomelid beetle came from another diabroticite, the banded cucumber
beetle, Diabrotica baleata LeConte (Cuthbert and Reid 1964). The same molecule is also the active
component of the sex pheromone of the Mexican corn rootworm, Diabrotica virgifera zeae (MCR);
the two closely-related species (Krysan et al. 1980) have almost identical response profiles to the
pheromone (Guss et al. 1984). 8R-methyl-2R-decenyl-propanoate is also the sex phermone of the
northern corn rootworm, Diabrotica barberi (Smith and Lawrence) (NCR) (Guss et al. 1982,
Dobson and Teal 1987); the lack of pheromone species-specificity among rootworms means that
sex pheromone is not a reproductive isolating mechanism (Dobson and Teal 1987). The shared sex
pheromone is one reason why pairings between NCR males and WCR females are observed in the
field (Ball 1957); however, post-mating barriers and other factors prevent any significant
hybridization between these species in the field (Krysan and Guss 1978). Another stereoisomer,
8R-methyl-2S-decenyl-propanoate (with no biological activity in WCR) can inhibit NCR responses
to the 8R,2R isomer (Guss et al. 1985, Dobson and Teal 1987). The 8R,2S isomer is not present in
the NCR pheromone but apparently is the sex pheromone of Diabrotica longicornis; this
circumstance may reproductively isolate NCR from D. longicornis (Guss et al. 1985).
In flight tunnel studies (Dobson and Teal 1987), WCR responses (i.e., directed flight toward a
pheromone lure) could be elicited at release rates of 1.18 ng/hr; Guss et al. (1984) reported the
threshold dose was ca. 10 ng. Guss et al. (1984), also present a WCR male dose response to
synthetic 8R-Methyl-2R-Decenyl-Propanoate that increased steadily with increasing lure-loading
rate from 0.25 ug to the maximum of 1000 ug; Guss et al. (1982), had earlier reported a similar
increasing response to traps baited with between 0.001 ug to 100 ug/trap. Male WCR in the field
exhibit bimodal morning and evening peaks of activity in response to pheromone sources (Bartelt
and Chiang 1977, Dobson and Teal 1986), though in the laboratory the greatest response was
observed in the morning (Dobson and Teal, 1987). The NCR displays a late-night peak of
responsiveness to the same compound.

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       WCR antennae are sexually dimorphic. The flagellar segments of the male antennae are
longer and bear 25% more sensilla than the female flagellum (Staetz et al. 1976). There is also a
sexual dimorphism in expression of an acetylcholinesterase (Est-a, a possible odorant metabolizing
enyme); it is only found in the 5 most distal segments of male antennae (Newman et al. 1993). In
addition, the structure and distribution of sensilla on the male antennnae are also hypothesized to be
a sex-specific specializations for detection of the WCR sex pheromone (Staetz et al. 1976, Newman
et al. 1993).
Lew and Ball (1978) identified secretory-type epithelial cells with associated cuticular pore
openings at tip of the adult female WCR abdomen as the likely sites of pheromone release. When a
female is ready to broadcast her readiness to accept a mate, she assumes a ‘calling’ posture where
the body axis is oriented parallel to the substrate (Hammack 1995). During calling, the tip of the
abdomen may be everted exposing the dorsal and ventral intersegmental membranes between the 7th
and 8th abdominal segments; in come cases, the everted tip of may be pulse slightly during calling
(Hammack 1995). Many newly-emerged females (54%) begin calling on the day of adult
emergence (Hammack 1995). The likelihood of calling behavior was greatest during the day after
emergence (96.4%); by the 3rd day, all females have called (Hammack 1995). During laboratory
observations, Hammack (1995) reported that the greatest proportion of females called between 0730
and 1230 when 70% of females assumed the calling posture. However, Hammack (1995) also
noted that there was considerable variability between individuals in calling patterns and that only
ca. 80% of females were attractive to mate-seeking males. In field studies using sticky traps baited
with virgin females, Bartelt and Chiang (1977) could not identify any pattern of female calling
based on trap capture data.
In the hours after adult emergence, newly-emerged females are observed to crawl upward on the
corn plants near to where they emerged or to fly short distances to nearby plants where they fed,
preened or remained motionless (Quiring and Timmins 1987). This observation is consistent with
the calling posture described by Hammack (1995), who reported that females assuming the calling
posture at were significantly more likely to be sexually receptive (74.4%) to males than those who
did not call (35.5%) (Hammack 1995). A current calling posture was not necessary for a female to
be approached by a male; 70% of females that mated during observations were not calling at the
time they were approached by mate-seeking males (Hammack 1995). Females that were not mated
in the Hammack (1995) study continued to call for several days; however, given a normal field-
abundance of males, an extended period of female virginity is unlikely; most WCR females are
likely mated within a few hours after emergence. That most mating females are still teneral (i.e.,
their elytra are still pale-colored because they have not been completely sceritized) a process which
takes 12-24 hours (Cates 1968) is evidence supporting rapid mating. Quiring and Timmins (1990)
reported that 96.6% of females in mating pairs were teneral (97.2% of males were not).
Mate-seeking WCR males respond strongly to pheromone and move to rapidly locate and mate with
nearby ‘calling’ females. Males respond to intial perception of pheromone by becoming agitated,
they raise their antennae, and move them back-and-forth in an excited fashion (Lew and Ball 1979).
Under conditions favorable for flight, males responding to pheromone detection will orient their
bodies into the direction of an odor plume, open their wings and take flight (Lew and Ball 1979).
Dobson and Teal (1987) characterized a slow hovering flight with the body held in a vertical
orientation as evidence of a positive response to pheromone. There are no published field studies
indicating the maximum distance at which males are capable of responding to a calling female, nor
any studies addressing the impact of changing density of calling females on that distance.
Unpublished field observations of caged calling females suggest that a calling female at ear height
will be discovered by a mate-seeking male within ca. 6.6 minutes. Most males arriving at calling
females likely came from nearby; none of 100s of fluorescent-powder marked males released 10
rows (7.6 m) away from the caged virgins in this study were ever recovered at a virgin cage (JLS,
unpublished data).

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      During laboratory observations, excited males near to calling females (e.g., males placed
in arenas with calling females) would hold their antennae extended forward and approach a
potential mate from the rear, touching her abdomen with their antennae (Lew and Ball 1979).
Pheromone alone is not adequate to evoke a full mating response; Dobson and Teal (1987) used the
observation that WCR males did not attempt copulation with lures to suggest they require additional
visual or other stimulation to evoke that response.
Once in contact with a female, the male then may lunge onto the female and grasp her elytra with
his first two pairs of legs while resting the hind legs on the substrate. An unreceptive female could
attempt to discourage or dislodge the mounted male by turning the tip of her abdomen downward or
attempting to dislodge him by kicking and attempting to walk away (Lew and Ball 1979).
If not immediately rejected, the mounted male will continue physical courtship activity for 10-60
min before copulation (Lew and Ball 1979). During this pre-copulatory period, the male may
repeatedly tap or stroke the female head and antennae with his own antennae. In the spotted
cucumber beetle (Diabrotica undecimpunctata howardi Barber, SCB) the stroking behavior is
known to promote relaxation of the female vaginal duct which allows the male’s needle-like
intromittent organ, the adeagus, to access the bursa copulatrix and begin delivering the liquid
spermatophore components (Tallamy et al. 2002a). A faster stroking rate in SCB males is
associated with a greater likelihood of being accepted as a mate (Tallamy et al. 2002b). In WCR,
Lew and Ball (1979) report that antennal tapping quieted the female allowing the male to insert his
adeagus. Among less-receptive females, the male tapping may become more rapid and be followed
by aggressive thrusting and pumping of the aedeagus, into the opening of the female reproductive
tract. Once in copulo, the male lowers or bends his abdominal segments downward and his
antennae cease moving and are folded backward over the male elytra. In SCB, posterior folding of
the antennae over the elytra is a sign that a spermatophore is being successfully transferred
(Tallamy et al. 2000). Periodically during the period of WCR copulation, the male will stroke the
sides of the female body with the mesothorasic legs; Lew and Ball (1979) suggest this is also to
calm the female.
During copulation, the male anchors his metathorasic legs under the tip of the female abdomen to
maintain his genital hold and to resist female attempts to kick him off. Hairless, planar ovoid
patches found only on the basitarsi of the prothorasic and mesothorasic legs of male WCR and NCR
may aid males in gripping the female elytra and help to maintaining their mating posture during
copulation (Hammack and French 2007). Periodically he engages in vigorous side-to-side rocking
motions, punctuated by bouts of less intense forward and backward thrusting. While in copulo, the
female is free to walk about and can often be observed feeding and grooming; the male does not
have any opportunity to feed during copulation. The entire mating processes lasts 3-4 hours (Ball
1957, Lew and Ball 1979, Sherwood and Levine 1993). At its conclusion, the male may remain
atop the female in a mate-guarding posture until disturbed or dislodged by the female. The mating
sequence is similar in the lab or field (Lew and Ball 1979).
While in copulo, the male deposits a large spermatophore in the female’s bursa copulatrix.
Spermatophores are produced from proteinaceaous secretions of the male accessory glands (Gillot
2003). The WCR spermatophore consists of a two parts: a milky, gelatinous portion with a layered
structure that is deposited first in the anterior lobe of the bursa, it is followed by a pale-red/brown
spherical posterior portion that is deposited in the posterior lobe of the bursa. Copulation disruption
studies reveal that the milky, gelatinous portion of the spermatophore is depositied during the first
hour of mating, and that the spherical portion is deposited between 1-1.5 hr (Lew and Ball 1980).
Sperm are stored in a single darkly-pigmented, sickle-shaped spermathecae that is ducted to the
bursa copulatrix (WCR internal anatomy is similar to that of SCB, see Mendoza and Peters 1968).
Within 2.0 hours after initiation of mating, 35% of females have some sperm stored in their
spermatheca; 100% have some stored sperm by 4.0 hours after mating initiation (Lew and Ball


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       1980). Sperm can be found in the spermatophore, which remains in the bursa, for up to
3 days after mating.
The spermatophore is slowly degraded in the female reproductive tract and no trace remains after
ca. 7 days (Lew and Ball 1980). Stored sperm are viable for at least 40 days (Lew and Ball 1980)
and likely much longer; Branson and Johnson (1973) reported some females produced viable eggs
or more than 60 days after mating, while Hill (1975) had females laying viable eggs 76 days after
mating.
Quiring and Timmins (1990) measured mating frequency and the spermatophore mass for 10 large
(>10.5 mg; mean =11.71 ±0.47 mg) and 10 small (<7.00 mg; mean = 6.03 ±0.20 mg) WCR males.
Larger males were found to initiate their first mating at an earlier age (2.0 vs. 3.2 d) and to mate
more frequently (15.8 ±1.2 times vs. 9.7 ±2.1 times) than smaller males; regardless of male size,
over 90% of all mating activity occurred during the first 40 days after adult emergence (Quiring and
Timmins 1990). Peak mating activity occurred during the 2nd and 3rd weeks which corresponded
with the peak of female emergence (Quiring and Timmins 1990). Branson et al. (1977) reported
that males mated 8.2 times in 41.6 days; one male mated 14 times. Some large and small males in
the Quiring and Timmins (1990) study were able to mate twice per day; no males mated three times
in a day. In some earlier studies, males have been reported to mate with up to four females in day
(Hill 1975). Surprizingly, mean (±SEM) spermatophore size was not significantly different
between large (0.57 ±0.02 mg) and small males (0.54 ±0.03 mg). When males mated twice in a
day, the mean spermatophore size was not different from the mean spermatophore size when only
one was produced during a day (0.50 ±0.04 mg).
Deposition of the spermatophore involves a significant transfer of resources from male to female.
Depending on the size of the male, the mass of material transferred to a female equals 5-9% of the
male‘s body mass. Because spermatophore size did not vary between male size, small males
transferred a greater proportion of their body mass with each spermatophore. In the Quiring and
Timmins (1990) study, some twice-mating males could transfer up to 24% of their initial body mass
to the female.
For the majority of WCR females, a single mating likely provides all the sperm necessary to
fertilize eggs over a lifetime (Branson and Johnson 1973, Hill 1975); a single mating is adequate to
maintain an elevated rate of egg development that supports ovipositon for 4-5 weeks after mating
(Sherwood and Levine 1993). It is speculated that WCR females may remate late in life if their
sperm supply is exhausted (Branson et al. 1977). Branson et al. (1977) used evidence from matings
with sterilized males combined with later mating opportunties with untreated males to suggest that
females will not remate as long as they are actively laying eggs. Though egg-laying rates of
variously mated females were not presented in their study, the results of Branson et al. (1977) imply
that presence of viable sperm are not needed to stimulate egg laying. Rarely, females with two
spematophores are found (Branson et al. 1977, J.L. Spencer, unpublished dissection data); this
suggests that even after an intial mating, females can remain attractive/receptive to mate-seeking
males for a short while.
As is common among many other insects, the act of mating and/or the substances transferred in the
WCR spermatophore are believed to reduce female mating receptivity and enhance fecundity
(Gillot 2003). Hammack (1995) reported that once previously calling females were mated, calling
behavior was suppressed; only 1 of 30 mated females was observed to assume the postural display
typical of calling females. In a study where matings were prematurely terminated, Sherwood and
Levine (1993) found that females from matings interrupted before sperm had been transferred had
an increased likelihood of laying eggs compared to unmated females, but laid significantly fewer
eggs than females from completed matings or interrupted matings where some sperm were
transferred. A significant (i.e., at 10-16 d post mating) increase in egg development (characteristic
of all mated females, regardless of mating duration) quickly waned in females mated for just 1 h
(Sherwood and Levine 1993). Females mated for 1 h, laid 72.1% of all the eggs they would ever lay

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      between 6 and10 d after mating; females from matings where sperm were transferred
deposited eggs at a steady pace over the first 27 d post-mating (Sherwood and Levine 1993). In this
study, no eggs were laid within 5 days of mating (these females would have been 10 days post-
emergence when the first eggs were laid (Sherwood and Levine 1993). Other studies report pre-
ovipositional periods (timing is based on post-emergence days) ranging from 12.2 days (Hill 1975)
to 14.3 days (Branson and Johnson 1973) to a 13-18 day estimate for the time until females are
gravid and 21 days as age at oviposition (Short and Hill 1972). Based on the reduced egg-laying in
the incompletely mated females, the missing or diminished volume of semen or spermatophore
components may play a role in sustaining full fertility. The fecundity enhancing effects of a mating
can be realized by an interspecific NCR x WCR cross. Egg production following interspecific
matings is increased, yet there is very poor fertility (Krysan and Guss 1978, Hintz and George
1979). The same is true for crosses between WCR and Mexican corn rootworm (Diabrotica
virgifera zeae, MCR) (Krysan et al. 1980). Despite compatibility at several levels, there is evidence
that post-mating barriers to hybridization exist between WCR and its close relatives NCR and
MCR.

II Oviposition: timing and amount of viable eggs
The following description of mating, behavior, maturation, and oviposition is based on the model
by Onstad et al. (2001a). It is possible that research published since 2000 could lead to
improvements in the model.
For females, the preoviposition period lasts 13 days including the first day as a teneral adult
(Branson and Johnson 1973, Hill 1975). Under normal conditions in which the female mates during
the first 13 days, eggs are oviposited for 60 days during the first oviposition period (Branson and
Johnson 1973, Hill 1975, Fisher et al. 1991). The second nonovipositing period continues until a
second mating occurs. Thus, after the first oviposition period, the female becomes receptive to
mating again. Upon remating, the female enters the second oviposition stage. Under laboratory
conditions, females oviposited (and likely remated) during at least 100 days (Branson and Johnson
1973, Elliott et al. 1990b), but Elliott et al. (1990a) observed very little oviposition after 8 weeks by
females fed corn nutrients from the field. In the model, Onstad et al. (2001a) allow the second
oviposition stage to last up to 40 days.
     Because Onstad et al. (2001a) are interested in possible remating, they model the
development and survival of adults independently. Four factors determine adult survival in the
model: first date of 0o C in the fall, toxicity of transgenic crop, nutritional status, and age. The
daily survival rate based on nutrition depends on the crop. For corn, the daily survival rate for
females is calculated from data of Elliott et al. (1990a). Quiring and Timmins (1990) observed a
daily survival rate of 0.95 for males feeding on old corn tissues. Therefore, Onstad et al. (2001a)
use the same function for males and females.
     For females, age, t, becomes an important factor after 6 weeks (Elliott et al. 1990a,b). Thus,
females in the teneral period, preoviposition period, and the first 29 days of the first oviposition
period have Sage(t) = 1. Based on data from 3 sources (Elliott et al. 1990a, Regime D; Elliott et al.
1990b, 23o and 26.5o C; Fisher et al. 1991), Onstad et al. (2001a) calculated a daily survival rate
Sage(t) = 0.98 for females after the first 42 days under optimal nutrition. With this value, no more
than 53% of the females will be alive after the first oviposition period.
Although Hill (1975) did not observe any nonvirgin females mating a second time, Branson et al.
(1977) observed 5 out of 30 previously mated females mating after egg laying stopped. Onstad et al.
(2001a) assume that mated female beetles do not remate once they begin to oviposit, but a second
mating is possible after the first oviposition period ends (Branson et al. 1977). After the completion
of the oviposition period following the first mating (age 73 days), Onstad et al. (2001a) allow a
proportion (< 0.20) of the females to mate again. Because males live for a maximum of 44 days and

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      females oviposit over a maximum of 60 days, any remating by a female must occur with
a younger male, because all males in a female's emergence cohort are dead.
    The maximum fecundity for once-mated females held under realistic, but close to optimal,
nutritional conditions averages 440 viable eggs (Boetel and Fuller 1997). Elliott et al. (1990a) and
Fisher et al. (1991) observed fecundities of 441 total eggs and 266 viable eggs, respectively.
Onstad et al. (2001a) created a function that distributes the 440 over the entire oviposition period.
Although in reality eggs are deposited temporally in batches or clutches by single females, they
modeled oviposition at the population level on a daily basis. They used the function 6(t-13) x e-
0.115(t-13) to determine the daily oviposition rate per female in age t, where t=14 to 73. This
function mimics the curves observed by Branson and Johnson (1973) and Elliott et al. (1990a,b)
when survival of females is accounted for. This function has a peak of about 19 eggs/female on day
22, and over 97% of the eggs oviposited by a single cohort of females are produced by 55 days of
age. They also used 6t x e-0.115t to mimic the shape of the second oviposition period, which
means the oviposition after day 74 observed by Branson and Johnson (1973). However, for the
second period, Onstad et al. (2001a) used 25% of the calculated value to produce lower oviposition
by older females.
    Over an 8-week oviposition period, egg viability declines from approximately 80% to about
30% (Fisher et al. 1991). Therefore, Onstad et al. (2001a) used 1-0.01t to calculate the proportion
of viable eggs produced by each cohort of females of age t during the first oviposition period, and a
constant 0.3 for the second oviposition period.
    Some of the best descriptions of the oviposition period for an entire population were
produced by Hein and Tollefson (1985) and Hein et al. (1988).

III References Cited
Ball, H.J. 1957. On the biology and egg-laying habits of the western corn rootworm. Journal of
Economic Entomology, 50:126-128.
Ball, H.J. and M.F.B. Chaudhury. 1973. A sex attractant of the western corn rootworm. Journal of
Economic Entomology, 66:1051-1053.
Bartelt, R.J. and H.C. Chiang. 1977. Field studies involving the sex-attractant pheromones of the
western and northern corn rootworm beetles. Environmental Entomology, 6:853-861.
Boetel, M. A., and B. W. Fuller. 1997. Seasonal emergence-time effects on adult longevity,
fecundity, and egg viability of northern and western corn rootworm (Coleoptera: Chrysomelidae).
Environ. Entomol. 26:1208-1212.
Branson, T.F. 1987. The contribution of prehatch and posthatch development to protandry in the
chrysomelid, Diabrotica virgifera virgifera. Entomologia Experimentalis et Applicata, 43:205-208.
Branson, T.F., P.L. Guss, and J.J. Jackson. 1977. Mating frequency of the western corn rootworm.
Annals of the Entomological Society of America, 70: 506-508.
Branson, T.F and R.D. Johnson. 1973. Adult western corn rootworms: oviposition, fecundity, and
longevity in the laboratory. Journal of Economic Entomology, 66:417-418.
Cates, M.D. 1968. Behavioral and physiological aspects of mating and oviposition by the adult
western corn rootworm, Diabrotica virgifera virgifera LeConte. Ph.D. Thesis, University of
Nebraska, Lincoln, Nebraska. 91 pp.
Cuthbert, Jr., F.P. and W.J. Reid, Jr. 1964. Studies of sex attractant of banded cucumber beetle.
Journal of Economic Entomology, 57:247-250.
Dobson, I.D. and P.E.A. Teal. 1986. Field studies of the temporal response patterns of male
Diabrotica virgifera virgifera LeConte and D. barberi Smith and Lawrence to 8R-methyl-2R-decyl
propanoate. Physiological Entomology, 11:405-410.
Dobson, I.D. and P.E.A. Teal. 1987. Analysis of long-range reproductive behavior of male
Diabrotica virgifera virgifera LeConte and D. barberi Smith and Lawrence to stereoisomers of 8-

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      methyl-2-decyl propanoate under laboratory conditions. Journal of Chemical Ecology,
13:1331-1341.
Elliott, N. C., R. D. Gustin, and S. L. Hanson. 1990a. Influence of adult diet on the reproductive
biology and survival of the western corn rootworm, Diabrotica virgifera virgifera . Entomol. Exp.
Appl. 56:15-21.
Elliott, N. C., D. R. Lance, and S. L. Hanson. 1990b. Quantitative description of the influence of
fluctuating temperatures on the reproductive biology and survival of the western corn rootworm,
Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). Can. Ent. 122:59-68.
Fisher, J. R., G. R. Sutter, and T. F. Branson. 1991. Influence of corn planting date on the survival
and on some reproductive parameters of Diabrotica virgifera virgifera (Coleoptera:
Chrysomelidae). Environ. Entomol. 20:185-189.
Gillott, C. 2003. Male accessory gland secretions: modulators of female reproductive physiology
and behavior. Annual Review of Entomology, 48:163-184.
Guss, P.L. 1976. The sex pheromone of the western corn rootworm (Diabrotica virgifera).
Environmental Entomology, 5:219-223.
Guss, P.L., P.E. Sonnet, R.L. Carney, T.F. Branson, and J.H. Tumlinson. 1984. Response of
Diabrotica virgifera virgifera, D. v. zeae, and D. porracea to stereoisomers of 8-methyl-2-decyl
propanoate. Journal of Chemical Ecology, 10:1123-1131.
Guss, P.L., P.E. Sonnet, R.L. Carney, J.H. Tumlinson, and P.J. Wilkin. 1985. Response of
northern corn rootworm, Diabrotica barberi Smith and Lawrence, to stereoisomers of 8-methyl-2-
decyl propanoate. Journal of Chemical Ecology, 11:21-26.
Guss, P.L., J.H. Tumlinson, P.E. Sonnet, and A.T. Proveaux. 1982. Identification of a female-
produced sex pheromone of the western corn rootworm. Journal of Chemical Ecology, 8:545-556.
Hammack, L. 1995. Calling behavior in female western corn rootworm beetles (Coleoptera:
Chrysomelidae). Annals of the Entomological Society of America, 88:562-569.
Hammack, L. and B.W. French. 2007. Sexual dimorphism of basitarsi in pest species of
Diabrotica and Cerotoma (Coleoptera: Chrysomelidae). Annals of the Entomological Society of
America, 100:59-63.
Hein, G. L., and J. J. Tollefson. 1985. Seasonal oviposition of northern and western corn rootworms
(Coleoptera: Chrysomelidae) in continuous cornfields. J. Econ. Entomol. 78:1238-1241.
Hein, G. L., J. J. Tollefson, and R. E. Foster. 1988. Adult northern and western corn rootworm
(Coleoptera: Chrysomelidae) population dynamics and oviposition. J. Kansas Entomol. Soc.
61:214-223.
Hill, R.E. 1975. Mating, oviposition patterns, fecundity and longevity of the western corn
rootworm. Journal of Economic Entomology, 68:311-315.
Hintz, A.M. and B.W. George. 1979. Successful laboratory hybridization of Diabrotica virgifera
(western corn rootworm) and Diabrotica longicornis (northern corn rootworm) (Coleoptera:
Chrysomelidae). Journal of the Kansas Entomological Society, 52:324-330.
Krysan, J.L. and P.L. Guss. 1978. Barriers to hybridization between Diabrotica virgifera and D.
longicornis barberi (Coleoptera: Chrysomelidae). Annals of the Entomological Society of America,
71:931-934.
Krysan, J.L., R.F. Smith, T.F. Branson, and P.L. Guss. 1980. A new subspecies of Diabrotica
virgifera (Coleoptera: Chrysomelidae): description, distribution, and sexual compatibility. Annals
of the Entomological Society of America, 73:123-130.
Lew, A.C. and H.J. Ball. 1978. The structure of the apparent pheromone-secreting cells if female
Diabrotica virgifera. Annals of the Entomological Society of America, 71:685-688.
Lew, A.C. and H.J. Ball. 1979. The mating behavior of the western corn rootworm Diabrotica
virgifera virgifera (Coleoptera: Chrysomelidae). Annals of the Entomological Society of America,
72: 391-393.


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      Lew, A.C. and H.J. Ball. 1980. Effect of copulation time on spermatozoan transfer of
Diabrotica virgifera (Coleoptera: Chrysomelidae). Annals of the Entomological Society of
America, 73:360-361.
Mendoza, C.E. and D.C. Peters. 1968. Morphology and histology of the reproductive systems of
adult southern corn rootworms. Annals of the Entomological Society of America, 61:1279-1284.
Newman, S.M., I.C. McDonald, Jr., and B. Triebold. 1993. Antennal sexual dimorphism in
Diabrotica virgifera virgifera (Le Conte) (Coleoptera: Chrysomelidae): male specific structures,
ultrastructure of a unique sensillum, and sites of esterase activity. International Journal of Insect
Morphology and Embryology. 22: 535-547.
Onstad, D. W., C. A. Guse, J. L. Spencer, E. Levine, and M. Gray. 2001a. Modeling the adaptation
to transgenic corn by western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol.
94:529-540.
Quiring, D.T. and P.R. Timmins. 1990. Influence of reproductive ecology on feasibility of mass
trapping Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Journal of Applied Ecology,
27:965-982.
Sherwood, D.R. and E. Levine. 1993. Copulation and its duration affects female weight,
oviposition, hatching patterns, and ovarian development in the western corn rootworm (Coleoptera:
Chrysomelidae). Journal of Economic Entomology, 86: 1664-1671.
Short, D.E. and R.F. Hill. 1972. Adult emergence, ovarian development, and oivposition sequence
of the western corn rootworm in Nebraska. Journal of Economic Entomology, 65:685-689.
Staetz, C.A., H.J. Ball, and S.D. Carlson. 1976. Antennal morphology of Diabrotica virgifera adults
(Coleoptera: Chrysomelidae). Annals of the Entomological Society of America, 69:695-698.
Tallamy, D.W., M.B. Darlington, J.D. Pesek, and B.E. Powell. 2002a. Copulatory courtship
signals male genetic quality in cucumber beetles. Proceedings of the Royal Society of London,
Series B, 270:77-82.
Tallamy, D.W., P.M. Gorski, and J.K. Burzon. 2000. Fate of male-derived cucurbitacins in spotted
cucumber beetle females. Journal of Chemical Ecology, 26:413-427.
Tallamy, D.W., B.E. Powell, and J.A. McClafferty 2002b. Male traits under cryptic female choice
in the spotted cucumber beetle (Coleoptera: Chrysomelidae). Behavioral Ecology, 13:511-518.
Thornhill, R. and J. Alcock. 1983. The Evolution of Insect Mating Systems. Harvard University
Press, Cambridge, Massachusetts.




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Diurnal Rhythms of Western Corn Rootworm Activity
I Conditions that influence expression of diurnal rhythms.
Many WCR activities are limited by extremes in a number of abiotic environmental factors. Among
these are temperature, windspeed, insolation (including day:night cycles) and precipitation.
Conditions that are too cold (<15°C) or warm (>ca. 32°C), too windy (>2.0 m/s), rainy, or too dark
will prevent WCR adults from expressing some behavior (Isard et al. 1999). For activities like adult
emergence or intrafield flight, expression of which is likely gated at some level by an innate
circadian oscillator, an out-of-range environmental condition(s) can prevent their expression.
Dobson and Teal (1986) describe such a condition in their study of WCR and NCR responses to
pheromone-baited traps. Evening and nighttime temperatures near thresholds for beetle activity
(15°C) suppressed normal expression of activity for both species; an expected NCR period of
activity was shifted to the next morning where it overlapped with the peak of WCR activity.

II Periodicity of general WCR activity.
Witkowski et al. (1975) measured diurnal peaks of WCR flight activity in Iowa cornfields. The
patterns they gleaned from sticky traps indicated that WCR flight activity was bimodal, with peaks
of greatest activity occurring during the hours just before sunset and the hours just after sunrise.
Temperatures between 22.2°C – 27.0°C were characteristic of the best periods for flight activity.
VanWoerkom et al. (1980) measured adult male and female patterns of locomotor activity, however
they worked in the laboratory using an actograph (an activity recording device that measures the
movement of insects held within the device). In this study, WCR adults at constant temperature
expressed a high level of activity between 1700-0800 hours, with reduced activity from 0900-1700
hours; a pattern almost identical to that of Witkowski et al (1975). VanWoerkom et al. (1980) also
noted that maximum male activity occurred at a slightly cooler temperature (25-27°C) than
maximum female activity (27-29°C); overall males were more active than females.
Naranjo (1990) used a vertical flight mill (Wales et al. 1985) design to measured diurnal trivial
flight activity patterns among tethered males and females adults in the laboratory. Though the
activity peaks and troughs in this well-replicated study are not as distinct as others, the same
bimodal morning and evening pattern of peak flight activity is evident.

III Periodicity of adult emergence.
Quiring and Timmins (1990) present patterns of daily male and female adult emergence in Ontario
cornfield that overlap almost perfectly. Peak daily emergence for both male and female WCR
occurred at ca. 0800 hours with a secondary emergence peak following at ca. 2000 hours. The
emergence minimum for both sexes occurred in the middle of the day between 1200 noon and 1600
hours. A coincident increase, decrease, and increase in the number of mating pairs observed
between the peaks of adult female WCR emergence, provides strong circumstantial evidence for the
rapid exploitation of emerging females by mate -seeking males. During extensive collections of
WCR mating pairs in Urbana, Illinois cornfields, during 2005 and 2006, nearly identical collection
patterns were observed. In the Urbana study, mating pairs were also most abundant around 0800
hours (unpublished JLS field data).




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   IV Periodicity of female pheromone calling, male response to
pheromone, and mating.
  The major active component of the female-produced WCR sex pheromone is 8R-methyl-2R-
  decenyl-propanoate (Guss et al. 1982). The pattern of pheromone emission during calling, was
  maximal among WCR females between 0730 and 1230 hours when 70% of females assumed the
  calling posture (Hammack 1995). 8R-methyl-2R-decenyl-propanoate is also the active
  component of the sex pheromone of the Mexican corn rootworm, Diabrotica virgifera zeae
  (MCR) (Guss et al. 1984) and the northern corn rootworm, Diabrotica barberi (Smith and
  Lawrence) (NCR) (Guss et al. 1982). The two closely-related WCR and MCR (Krysan et al.
  1980) have almost identical response profiles to the pheromone (Guss et al. 1984). Male WCR in
  the field exhibit bimodal morning and evening peaks of activity in response to pheromone
  sources (Bartelt and Chiang 1977, Dobson and Teal 1986), though in the laboratory the greatest
  response was observed in the morning (Dobson and Teal, 1987). The NCR displays a late-night
  peak of responsiveness to the same compound (Dobson and Teal 1986).

V Periodicity of WCR flight, interfield movement, and abundance in
corn, soybean and other crops.
A diagnostic characteristic of areas where rotation-resistant WCR populations are present is high
WCR abundance in crops outside of cornfields (Levine et al. 2002, Spencer et al. 2005, Schroeder
et al. 2005). While unusual WCR abundance in a rotated crop is symptomatic of rotation resistance,
the condition is a consequence of frequent interfield movement by WCR adults. Thus, factors that
influence local WCR flight may affect daily measures of WCR abundance. Interfield western corn
rootworm flights typically occur during morning and in the evening before sunset (Witkowski et al.
1975, Coats et al. 1986, Naranjo 1990, Grant and Seevers 1990, Isard et al. 2000), and can involve
substantial numbers of individuals. Extrapolation of captures from eight malaise traps positioned at
the borders of a 1.64-ha soybean field in east central Illinois suggests that 0.25 million western corn
rootworm flew across the borders of the field on a single day (Isard et al. 2000). Flight activity is
influenced by atmospheric conditions and can be limited by strong winds, low air temperatures,
darkness, and precipitation (VanWoerkom et al. 1983, Grant and Seevers 1990, Isard et al. 1999).
In areas where rotation-resistant WCR are present, there is a strong daily periodicity in WCR
abundance in soybean fields (and fields of other crops outside of cornfields) (Spencer et al. 1999)
that is associated with periodicity in the WCR flight activity along field margins (Isard et al. 2000).
In these areas, the daily peak populations of WCR in soybean fields are found in the morning and
evening (around the times of peak interfield flight), with significantly lower WCR abundance
during midday (Spencer et al. 1999). These interfield movement patterns are confirmed by
identification of corn and soybean tissues in gut contents. The proportion of WCR adults collected
in soybean fields with both corn and soybean tissues in their gut contents (indicative of recent
movement into soybeans after feeding in a cornfield) rises and falls as WCR move in and out of
soybean fields during the course of one day (Spencer et al. 1999, Spencer et al. 2005)
Flight periodicity extends beyond local interfield (trivial) flight to include the dispersal flights of
recently mated females ascending from their natal cornfields. Sampled from scaffolding towers at
high elevation (10 m) with aerial insect nets, these dispersing females are smaller, younger, and
carry less developed oocytes than females collected within crop canopies or flying above the
soybean canopy (Spencer et al. 2005). Like females nearer the ground, they are most likely to in fly
in the early to mid-morning or near dusk (Isard et al. 2004). While conditions like high winds or
low temperatures may limit flight, within a range of conditions conducive to flight, the level of
activity was not strictly dependent on the values of particular factors. Rather, WCR ascent and


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      peaks of high elevation flight corresponded to predictable changes in the atmospheric
conditions above the fields (Isard et al. 2004).

VI Periodicity of oviposition.
Perhaps the generally hidden location (within soil cracks and other soil openings) of egg-laying
females explains why there are no published accounts of diurnal periodicity in WCR oviposition in
the field. Reduced ovipositional fidelity among rotation-resistant WCR populations (Spencer et al.
2005) has broadened the area where egg-laying females could possibly be found; this will further
complicate any attempts to measure field ovipositional periodicity in the affected areas of he U.S.A.
Corn Belt. Ball (1971) published results from a laboratory study of diurnal WCR egg-laying
patterns. Peak egg laying occurred between 0800-1200 hours. Ball (1971) hypothesized that the
combination of general activity patterns (which he suggests peaks between 1600-2000 hours) with
soil moisture, shown by Cates (1968) to influence the number of eggs laid by females, may explain
the timing of egg laying.

VII A caution about diurnal periodicities.
It is important to know whether a measured quantity is subject to diurnal periodicity. Quantities
whose measurements are subject predictable diurnal variation, force researchers to either limit
measurement periods to just a portion of the day or broaden the interval to encompass the entire
day, and thus factor out within day variation. A diurnal rhythm of sensitivity to the insecticide
Diazinon, reported by Ball (1969), is an excellent example. In that 1967-1968 study, the daily
topical LC50 (ug/g) varied by 60-33% between the period of greatest sensitivity during the day and
the period of minimum sensitivity during the night and early morning; these periods roughly
describe intervals when WCR activity is at its greatest and least, respectively, as reported by Cates
(1968).

VIII References Cited
Ball, H.J. 1969. Diurnal rhythm of sensitivity to diazinon in adult western corn rootworm. Journal
of Economic Entomology, 62:1097-1098.
Ball, H.J. 1971. Laboratory observations on the daily oviposition cycle in the western corn
rootworm. Journal of Economic Entomology, 64:1319-1320.
Bartelt, R.J. and H.C. Chiang. 1977. Field studies involving the sex-attractant pheromones of the
western and northern corn rootworm beetles. Environmental Entomology, 6:853-861.
Cates, M.D. 1968. Behavioral and physiological aspects of mating and oviposition by the adult
western corn rootworm, Diabrotica virgifera virgifera LeConte. Ph.D. Thesis, University of
Nebraska, Lincoln, Nebraska. 91 pp.
Coats, S.A., J.J. Tollefson, and J.A. Mutchmor. 1986. Study of migratory flight in western corn
rootworm (Coleoptera: Chrysomelidae). Environmental Entomology, 15:620-625.
Dobson, I.D. and P.E.A. Teal. 1986. Field studies of the temporal response patterns of male
Diabrotica virgifera virgifera LeConte and D. barberi Smith and Lawrence to 8R-methyl-2R-decyl
propanoate. Physiological Entomology, 11:405-410.
Dobson, I.D. and P.E.A. Teal. 1987. Analysis of long-range reproductive behavior of male
Diabrotica virgifera virgifera LeConte and D. barberi Smith and Lawrence to stereoisomers of 8-
methyl-2-decyl propanoate under laboratory conditions. Journal of Chemical Ecology, 13:1331-
1341.
Grant, R.H. and K.P. Seevers. 1990. The vertical movement of adult western corn rootworms
(Diabrotica virgifera virgifera) relative to the transport of momentum and heat. Agricultural and
Forest Meteorology, 49:191-203.

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       Guss, P.L., P.E. Sonnet, R.L. Carney, T.F. Branson, and J.H. Tumlinson. 1984.
Response of Diabrotica virgifera virgifera, D. v. zeae, and D. porracea to stereoisomers of 8-
methyl-2-decyl propanoate. Journal of Chemical Ecology, 10:1123-1131.
Guss, P.L., J.H. Tumlinson, P.E. Sonnet, and A.T. Proveaux. 1982. Identification of a female-
produced sex pheromone of the western corn rootworm. Journal of Chemical Ecology, 8:545-556.
Hammack, L. 1995. Calling behavior in female western corn rootworm beetles (Coleoptera:
Chrysomelidae). Annals of the Entomological Society of America, 88:562-569.
Isard, S.A., M.A. Nasser, J.L. Spencer, and E. Levine. 1999. The influence of weather on western
corn rootworm flight activity at the borders of a soybean field in east central Illinois. Aerobiologia,
15:95-104.
Isard, S.A., Spencer, J.L., Nasser, M.A., and E. Levine. 2000. Aerial movement of western corn
rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae): Diel periodicity of flight
activity in soybean fields. Environmental Entomology, 29:226-234.
Isard, S.A., J.L. Spencer, T.R. Mabry, and E. Levine. 2004. Influence of atmospheric conditions
on high elevation flight of western corn rootworm (Coleoptera: Chrysomelidae). Environmental
Entomology, 33:650-656.
Levine, E., J.L. Spencer, S.A. Isard, D.W. Onstad, and M.E. Gray. 2002. Adaptation of the
western corn rootworm to crop rotation: evolution of a new strain in response to a management
practice. American Entomologist, 48:94-107.
Naranjo, S.E. 1990. Comparative flight behavior of Diabrotica virgifera virgifera and D. barberi
in the laboratory. Entomologia Experimentalis et Applicata, 55:79-90.
Schroeder J.B. S.T. Ratcliffe, and M.E. Gray. 2005. Effect of four cropping systems on variant
western corn rootworm (Coleoptera: Chrysomelidae) adult and egg densities and subsequent larval
injury in rotated maize. Journal of Economic Entomology, 98:1587-1593.
Spencer, J.L., S.A. Isard, and E. Levine. 1999. Western corn rootworm injury in first-year corn:
what’s new, pp. 13-27. In 1999 Illinois Crop Protection Technology Conference, University of
Illinois at Urbana-Champaign.
Spencer, J.L., T.R. Mabry, E. Levine, and S.A. Isard. 2005. Movement, Dispersal, and Behavior of
Western Corn Rootworm Adults in Rotated Corn and Soybean Fields. In Western Corn Rootworm:
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Wallingford, Oxfordshire, UK. Pp. 121-144.
Quiring, D.T. and P.R. Timmins. 1990. Influence of reproductive ecology on feasibility of mass
trapping Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Journal of Applied Ecology,
27:965-982.
VanWoerkom, G.J., F.T. Turpin, and J.R. Barret, Jr. 1980. Influence of constant and changing
temperatures on locomotor activity of adult western corn rootworms (Diabrotica virgifera) in the
laboratory. Environmental Entomology, 9:32-34.
VanWoerkom, G.J., F.T. Turpin, and J.R. Barret, Jr. 1983. Wind effect on western corn rootworm
(Coleoptera: Chrysomelidae) flight behavior. Environmental Entomology, 12:196-200.
Wales, P.J., C.S. Barfield, and N.C. Leppla. 1985. Simultaneous monitoring of flight and
oviposition of individual velvetbean caterpillar moths. Physiological Entomology, 10:467-472.
Witkowski, J.F., J.C. Owens, and J.J. Tollefson. 1975. Diel activity and vertical flight distribution
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352.




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Nutritional ecology
I Adults
I-1 Feeding hosts, role of wild plants
I-1-1 State of the art
Adult Diabrotica virgifera virgifera nutritional ecology has implications on several import aspects
of WCR biology like invasion potential, fecundity and population buildup. Because most economic
damage is done by larval feeding and not adults, adult nutritional ecology has not been studied as
thoroughly as larval ecology. Significant yield loss is generally not likely except in seed production
(Kiss, pers. communication). Threshold levels for the economic impact of adults on seed production
are not well established.
The adult beetles feed on all aboveground parts of maize plants, especially maize pollen and silk
(Chiang 1973; Ludwig and Hill 1975, Moeser and Vidal 2005). Ball (1957) hypothesized that the
nutritional ecology of adult WCR was based on the availability of maize tissue changing in time.
Results from Moeser and Vidal (2005) support this idea: Beetles started feeding on the leaves, then
on pollen and silk, finally on kernel and pollen from alternate host plants like weeds or crops. After
the depletion of their primary food source (maize pollen and silk), beetles started to feed on other
maize tissue or weed pollen. The feeding on weed pollen depended on their availability in the field.
Later during the growing season, flowering weeds outside the field became very attractive and were
used to a large extent by female D. v. virgifera. Males used a wider range of alternate pollen
resources, but use each pollen source less intensively (Moeser and Vidal 2005). In maize fields in
the USA, Ludwig and Hill (1975) similarly found that more males than females had corn plus weed
pollen in their gut.
The knowledge of the impact of alternate pollen sources and different mixtures of food on
population buildup or maintenance of viable populations in the absence of maize and also on
longevity and fecundity of the females is scattered at best: Pavuk and Stinner (1994) concluded
from their studies that weeds in maize fields had no significant effect on WCR populations,
although higher numbers of beetles were encountered in mixed weeds plots. As Siegfried and
Mullin (1990) pointed out that the longevity of females was significantly reduced when fed
exclusively on alternative food like squash blossoms or sunflower inflorescences compared to
females maintained on corn ears, although the former diet keeps them alive and leads to the
production of viable eggs. The impact of some alternate pollen resources seems not clear at the
moment, because lab and field data are contradictory. Mullin et al. (1991) found a decreased
longevity when adults fed on floral parts of sunflower compared to corn. They even isolated and
identified antifeedants from sunflower and Solidago canadensis pollen, suggesting that Asteraceae
do not serve well as food sources for D. v. virgifera. Moeser and Vidal (2005) found Asteraceae
(sunflower) pollen to be one of the major pollen resources used by field caught adults in
southeastern Europe. The data suggesting decreased longevity derived from no-choice tests with
beetles feeding exclusively on sunflower for their whole lifespan (Mullin et al. 1991). Under natural
conditions a mixing of different dietary components would most likely take place, therefore the no-
choice experiments may have little explanatory value.
For many herbivorous insects, pollen is the most nourishing food and necessary for egg production
(Wheeler 1996). For D. v. virgifera maize pollen and green silk (Elliot et al., 1990) served best for
egg production but only maize tissues were tested in this study, so weed pollen could contribute as
well. If the more intense use of alternative food resources by females leads to a higher fecundity, a
higher population density or faster population buildup remains yet to be studied.

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      The influence of adult feeding behavior on oviposition (and adaptation to crop rotation),
migration, invasion success, and chances of keeping a viable population alive in the absence of
maize is largely not understood. O´Neal et al. (2002) found that corn phenology influenced feeding
choices between corn and soybean. They hypothesized that corn phenology may have contributed
to the selection for the soybean biotype, where changes in adult feeding behavior led to changes in
oviposition behavior as well.
The only study from Europe regarding adult nutritional ecology is describing conditions in
Southeastern Europe (Moeser and Vidal 2005). Although the weeds which were present in the
studied fields in southern Hungary are widespread in all European maize production areas, weed
composition may change and therefore feeding ecology may change too. This leads to the question
if landscape diversity (= many food sources vs. very limited number of food sources) has an impact
on adult nutritional ecology and all the related fields.
With the commercialization of transgenic corn, which is resistant against corn rootworms not only
larval feeding on maize is affected. Possible sublethal effects of pollen and silk consumption on
adult longevity or fecundity need to be studied as well.

I-2 Chemical and physical cues: biotic/abiotic
There has been a great deal of work on certain aspects of adult WCR – cucurbit interactions. The
chemical ecology of adult WCR in response to plant kairomones has been reviewed by Metcalf and
Metcalf (1992). Aspects focusing primarily on WCR-cucurbit interactions have been recently
reviewed by Tallamy et al. (2005) and the specifics of pharmacophagy in these interactions on a
more general scale have been reviewed by Gillespie et al. (2004). Because of the availability of
these reviews, these sections will not be included here.
Although there has been a great deal of work on WCR phagostimulation in relation to the
interaction of WCR and cucurbit plants as described in the above reviews, relatively little has been
done in relation to its main host, maize. Amino acids present in pollen lead to an increase in
feeding of WCR on maize and squash pollen in comparison to sunflower and goldenrod in a no-
choice experiment (Hollister and Mullin 1999). They attributed these findings to the presence of a
combination of specific amino acids. Lin and Mullin (1999) used a bioassay-driven fractionation to
characterize phagostimulants in sunflower pollen. Lipids, including triglycerides, free fatty acids,
phosphatidylethanolamines, phosphatidic acids, and phosphatidylcholines were highly
phagostimulatory.
The female-produced sex pheromone of the western corn rootworm was identified as 8-methyl-2-
decanol propropanoate (Guss et al. 1982). D. v. virgifera, D. v. zeae, and D. barberi, all respond
preferentially to the 2R, 8R configuration (Guss et al. 1984). Wilkin et al. (1986) performed
electroantennogram (EAG) bioassays of gas-liquid chromatographic (GLC) fractions of volatiles
collected from virgin females from WCR, SCR, and Diabrotica longicornis. Only one active
fraction was found from each species, corresponding to structurally known sex pheromones for
WCR and SCR. However, in wind tunnel bioassays, Dobson and Teal (1987) reported that WCR
and NCR will land on a pheromone source but will not attempt to copulate with it. They assumed
that other components were needed to induce copulation behavior. Hammack (1985) noted that
calling was observed in the laboratory on the day that teneral females emerged from the soil.
Frequency of the behavior peaked 1 day later. Females called throughout the day but were most
active during the first half of photophase on a photoperiod of 14:10 (L:D) h. Virgin females mated
significantly faster and in greater numbers if they had recently engaged in calling activity. Most
females ceased calling within 24 h after insemination. Attempts have been made to evaluate the sex
pheromone for monitoring (Lance 1988, Meinke et al. 1989), but, in part, because it targets the male
the sex pheromone is not being used in WCR IPM.



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       Although observed by many field entomologists previously, Prystupa et al. (1988) was
first to formally demonstrate that WCR are attracted to maize silk. Abou-Fakhr et al. (1996)
demonstrated that the senescing portions (the brown portion that protrudes from the tip of the ear)
of maize silks elicited strong EAG responses from WCR adults, while the green portions (under the
husk or very young, green silk beyond the husk) did not elicit a significant EAG response. Hibbard
et al. (1997) went on to isolate and identify the primary EAG-active components from brown maize
silk as tridecan-2-one, (E,E)-3,5-octadien-2-one, (E,Z)-2,6-nonadienal, and (E)-2-nonenal.
Hammack (1996) demonstrated that (E)-6,10-dimethyl-5,9-undecadien-2-one (geranylacetone) was
highly attractive to D. barberi and also attractive to WCR. In a reevaluation of the most EAG-
active fractions from maize silk, Hibbard et al. (1997) found 6,10-dimethyl-5,9-undecadien-2-one
as one of the smaller peaks present. The phenyl propanoids 2-phenethanol and benzyl alchohol
were also present, but were not found in the fractions with the most EAG-activity.
Lance (1992) documented clearly that odors influenced WCR oviposition in choice tests.
Factors triggering enhanced oviposition included homogenates of WCR ovaries, homogenates of
male abdomens, excised maize roots, several bacterial cultures, and pure carbon dioxide. Since the
homogenates were deactivated by sorbate, a bacteriostatic agent, most treatments were likely active
due to enhanced levels of carbon dioxide.

II Larvae
II-1 Host selection
II-1-1 State of the art
In studies to determine the larval host range of WCR, Branson and Ortman (1967, 1970) observed
larval survival for at least ten days on 18 of 44 grass species evaluated in experiments in Petri
dishes without soil. All larvae that survived 10 d developed to the 2nd instar. No WCR larvae
developed on any of the 27 broadleaf species evaluated. Their studies provided a foundation for
further work, particularly regarding the “grasses only” larval host hypothesis of this species.
Branson and Krysan (1981) suggested that “D. virgifera became a specialist on corn in the tropics
or subtropics and ‘followed’ the diffusion of corn into the temperate United States” based primarily
on the fact that corn was by far the best larval host identified. Krysan and Smith (1987) stated that
“it is reasonable to conclude that the presence of D. v. virgifera in the U.S. does not predate the
presence of corn.” Neither Branson and Krysan (1981) nor Krysan and Smith (1987) provided any
evidence that maize was grown in western Kansas in the 1860s, the time and location that WCR
was first collected (LeConte 1868), but their view is the accepted dogma today. However, the “best
host” is not necessarily the original host. Another virgifera group species, Diabrotica longicornis,
does not utilize maize when feral, but larvae develop on maize at a rate equal or faster than larvae of
its sibling species, Diabrotica barberi, the northern corn rootworm (NCR), which specializes on
maize (Golden and Meinke 1991). WCR and NCR adult emergence has not been observed outside
corn fields, but this was almost certainly occurring prior to corn monoculture. According to the
literature, corn was not present in the region of western Kansas where WCR was first identified
(Weatherwax 1954; Goodman 1987). What was supporting WCR larvae at the time? In
greenhouse studies of the dominant prairie grasses present at the time, WCR adult production from
one of the wheatgrass species was not significantly different than adult production from corn
(Oyediran et al. 2004a). Of 60 grass species evaluated for WCR growth and development, larvae
survived for at least 10 days on 57 species and grew to the 2nd instar on 50 of the 60 grass species
evaluated (Clark and Hibbard 2004, Oyediran et al. 2004a, Wilson and Hibbard 2004).
In an elegant set of experiments, Moeser and Vidal (2004a, b) measured the growth of WCR larvae
and the amount of food consumed to calculated food conversion indexes for a series of alternate
hosts and maize varieties respectively. In addition, they evaluated the carbon/nitrogen ratio and the

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      phytosterol content of the alternate hosts and maize varieties, respectively. For alternate
hosts, the plant species with higher nitrogen content were less suitable for WCR development.
Phytosterol content influenced the amount of food consumed, but not the weight gain of the insects.
For maize, nitrogen content was positively correlated with converting root biomass into insect
biomass and phytosterol content influenced larval weight gain and the amount of infested food. The
change in weight of the second instar larvae evaluated ranged from strongly positive to strongly
negative values for both alternate hosts and maize varieties. Unfortunately, in both studies any
differences in weight gain of the insects and food conversion indices are confounded with any
differences in the speed that a plant species or maize variety senesces, since cut roots were the only
food source for six days. Olmer and Hibbard (2006) evaluated survival and weight gain of neonate
and second instar larvae on senescing maize and Setaria faberi. When infested five days after
spraying with glyphosate, no neonate WCR larvae survived. When infested on the day of
glyphosate spray, a few neonate larvae were recovered after five days, but weight gain for both
neonate and second instar larvae was negative when infested on the day of glyphosate spray.
Glyphosate sprayed maize remained suitable for rootworm development longer than glyphosate
sprayed S. faberi.

II-2 Chemical and physical cues
WCR larvae utilize a complicated set of behaviors for host location and host acceptance. WCR
larvae orient toward carbon dioxide (Strnad et al. 1986, Hibbard and Bjostad 1988, Bernklau and
Bjostad 1998a,b), which is released by respiring roots of corn and other species (Massimino et al.
1980). The most recent refereed literature states that carbon dioxide is the only volatile chemical
used by neonate WCR larvae in host location (Bernklau and Bjostad 1998b). WCR larvae were
attracted to a range of CO2 concentrations from 2 to 100 mmol/mol, and can detect gradients as low
as 12% (Bernklau and Bjostad 1998a). In choice tests in soil bioassays, significantly more neonate
WCR larvae were attracted to a synthetic CO2 with a higher concentration of CO2 (11.2 mmol/mol)
than to growing corn with a CO2 concentration of 1.36 mmol/mol (Bernklau et al. 2004). Bernklau
et al. (2004) was able to disrupt the host-location ability of neonate WCR larvae in the laboratory
and in the field by placing CO2-generating materials into the soil, thereby preventing larvae from
locating the roots of growing corn.
Reports previous to Bernklau and Bjostad (1998a, b), used second instar larvae in bioassays to
isolate and identify corn semiochemicals in addition to CO2 (Hibbard and Bjostad 1990). This
work indicated that 6-methoxy-2-benzoxazolinone (MBOA) (Bjostad and Hibbard 1992) and long-
chain free fatty acids (Hibbard et al. 1994) were involved in orientation of second instar WCR
larvae. Bernklau and Bjostad (1998a, b) did not evaluate second instar larvae. Although, MBOA
showed some promise in increasing the efficacy of insecticides in the laboratory, it did not
consistently increase the efficacy of insecticides in the field (Hibbard et al. 1995).
The liquid extracted from germinating corn contains compounds that stimulate feeding by neonate
WCR larvae. In feeding bioassays, neonate WCR larvae fed vigorously on paper disks treated with
liquid pressed from corn roots, or with a solvent extract of corn roots, but not on disks treated with
distilled water (Bernklau 2003; Bernklau and Bjostad 2005). In subsequent studies, the efficacy of
thiamethoxam insecticide was significantly increased when feeding stimulants (corn extract) were
added (Bernklau and Bjostad 2005). The amount of insecticide required for 50% mortality of WCR
larvae (within 30 minutes) was 1 ppm when corn extract was added. With the water control, the
highest mortality rate obtained (within 30 minutes) was only 40% and this required 10 fold the
concentration of insecticide tested with feeding stimulants. Larval mortality after 24 hours was
significantly higher for corn extract-treated disks with 0.01, 0.1, 1 or 10 ppm thiamethoxam than for
the same concentrations of insecticide with just water. The amount of insecticide required for 50%
larval mortality after 24 hours was reduced from 1 ppm without corn extract, to 0.01 ppm when

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      corn extract was present. This experiment demonstrated a 100-fold increase in the
efficacy of thiamethoxam with the addition of larval feeding stimulants (Bernklau 2003, Bernklau
and Bjostad 2005). Further unpublished work indicated that the efficacy of thiamethoxam on clay
granules in soil could be increased with the addition of feeding stimulants (Bernklau, personal
communication). Tefluthrin, nor other insecticides commonly used on clay granules were not
evaluated.
Strnad and Dunn (1990) documented that the behavior of neonate WCR larvae was dramatically
different after removal from a 5 min exposure to the roots of hosts than after removal from a 5 min
exposure to the roots of nonhosts. If a neonate WCR larva had been exposed to host roots, it
exhibited a localized searching behavior. In the localized behavior, WCR traveled relatively slow,
with increased turns and path crossings and less overall area covered. If a neonate WCR larva was
exposed to nonhosts, they exhibited a ranging behavior with increased rate of travel, fewer turns
and path crossings, and a greater overall area covered.

III Case of the variant - Oviposition site selection and behavior
In Europe, the case of rotation resistant variant known in US Corn Belt is often interpreted in a
confused way that WCR population can develop in crops other than maize (soybean in this case). It
is well known from the literature that WCR larvae may feed on root system of various grassy weed
species. Field tests in Europe confirmed that WCR larvae can survive and develop to adult stage on
roost system of Setaria species though in much less percentage compared to maize (Breitenbach et
al. 2005). From adult gut analysis and field observations in Europe it is also known that WCR
adults may feed with pollen of grassy and broad leaf weed species (Moeser and Hibbard 2005), of
sunflower (Hatvani and Horváth 2002) and with floral parts and fruit of pumpkin.
Selection of host plants and feeding behavior of WCR adults on crops other than maize is crucial
from two different aspects.
- One is the survival of the population in non-corn plant stands or in non-corn habitats and therefore
the contribution of non-corn plants to maintenance of WCR populations.
- The other aspect is the stimulus and attractiveness of non-corn plants to WCR adults (especially to
females) for leaving corn fields, feeding elsewhere and contribution to potential egg laying outside
of corn fields.
Feeding of adult WCR in non-corn plants does not necessarily results in egg laying in the same
habitat where they fed. On the other hand, feeding of WCR adults and potential egg laying in
outside of corn fields is important in crops or plant stands (crop and weeds, set aside areas) that will
be rotated to corn is subsequent year.
Besides feeding behavior of either WCR larvae or adults additional factors should be considered
that certainly impact the hazard of egg laying. These factors in non-corn plant stands where WCR
adults feed are as follows:
-    microclimatic conditions (air humidity) of the plant stand;
-    soil conditions and suitability for egg laying;
-    availability of alternative food sources, specific plant stages (volunteer winter wheat, weeds
with pollen source after an early harvested spring crop like green pea) for WCR adults around
mating and egg laying period;
-    irrigation practices (if any) and coincidence of various suitable factors for egg laying;
A kind of continuous observation of rotation systems and follow up of reported cases seems
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       IV References Cited
Abou-Fakhr, E. M., B. E. Hibbard, D. K. Jewett, and L. B. Bjostad. 1996. Electroantennogram
responses of western corn rootworm adults (Coleoptera: Chrysomelidae) in relation to maize silk
morphology and phenology. Environ. Entomol. 25: 430-435.
Ball, H. J. 1957. On the biology and egg-laying habits of the western corn rootworm. J. of Econ.
Entomol. 50: 126-128.
Bernklau, E. J. 2003. Behavioral effects of carbon dioxide on western corn rootworm and
subterranian termites with implications for pest management. Ph.D., Colorado State University,
Fort Collins, CO.
Bernklau, E. J., and L. B. Bjostad. 1998a. Behavioral responses of first instar western corn
rootworm (Coleoptera: Chrysomelidae) to CO2 in a glass bead bioassay. J. Econ. Entomol. 91:
444-456.
Bernklau, E. J., and L. B. Bjostad. 1998b. Re-investigation of host location by western corn
rootworm larvae (Coleoptera: Chrysomelidae): CO2 is the only volatile attractant. J. Econ.
Entomol. 91: 1331-1340.
Bernklau, E. J., and L. B. Bjostad. 2005. Insecticide enhancement with feeding stimulants in corn
for western corn rootworm larvae (Coleoptera: Chrysomelidae). J. Econ. Entomol. 98: 1150-1156.
Bernklau, E. J., E. A. Fromm, and L. B. Bjostad. 2004. Disruption of host location of western corn
rootworm larvae (Coleoptera: Chrysomelidae) with carbon dioxide. J. Econ. Entomol. 97: 330-339.
Bjostad, L. B. and B. E. Hibbard. 1992. 6-Methoxy-2-benzoxazolinone: A semiochemical for host
location by western corn rootworm larvae. J. Chem. Ecol. 18: 931-944.
Branson, T. F. and E. E. Ortman. 1967a. Host range of the western corn rootworm. J. Econ.
Entomol. 60: 201-203.
Branson, T. F. and E. E. Ortman. 1967b. Host range of larvae of the western corn rootworm:
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Branson, T. F. and J. L. Krysan. 1981. Feeding and oviposition behavior and life cycle strategies of
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Breitenbach, S.; Heimbach, U. and K.H. Lauer (2005): Field tests on the host range of the larvae of
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Chiang, H. C. 1973. Bionomics of the northern and western corn rootworm. Annu. Rev. Entomol.
18: 47-72.
Clark, T. L., and B. E. Hibbard. 2004. A comparison of non-maize hosts to support western corn
rootworm (Coleoptera:Chrysomelidae) larval biology. Environ. Entomol. 33: 681-689.
Dobson, I. D and P. E. A. Teal. 1987. Analysis of long-range reproductive behavior of male
Diabrotica virgifera virgifera LeConte and D. barberi Smith and Lawrence to stereoisomers of 8-
methyl-2-decyl propanoate under laboratory conditions. J. Chem. Ecol. 13: 1331-1341.
Elliot, N. C., R. D. Gustin, and S. L. Hanson. 1990. Influence of adult diet on the reproductive
biology and survival of the western corn rootworm, Diabrotica virgifera virgifera. Entomol. Exp.
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Gillespie, J.J., Kjer, K.M., Riley, E.G. and Tallamy, D.W. 2004. The evolution of cucurbitacin
pharmacophagy in rootworms: insight from Luperini paraphyly, pp. 37-57. In P. H. Jolivet, J. A.
Santiago-Blay, and M. Schmitt, (eds), New Developments in the Biology of Chrysomelidae.
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Golden, K. L., and L. J. Meinke. 1991. Immature development, fecundity, longevity, and egg
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Goodman, M. M. 1987. The history and evolution of maize. Critical Revs. Plant Sci. 7: 197-220.

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       Guss, P. L., J. H. Tumlinson, P. E. Sonnet, and A. T. Proveaux. 1982. Identification of
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(in Hungarian). Növényvédelem 38, 513-517.
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volatile semiochemicals from corn seedlings. J. Chem. Ecol. 14: 1523-1539.
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western corn rootworm larvae. J. Chem. Ecol. 16: 3425--3439.
Hibbard, B.E., E.J. Bernklau, and L.B. Bjostad. 1994. Long-chain fatty acids: Semiochemicals for
host location by western corn rootworm larvae. J. Chem. Ecol. 20: 3335-3344.
Hibbard, B. E., P. F.B., S. D. Pilcher, M. E. Schroeder, D. K. Jewett, and L. B. Bjostad. 1995.
Germinating corn extracts and 6-methoxy-2-benzoxazolinone: Western corn rootworm (Coleoptera:
Chrysomelidae) larval attractants evaluated with soil insecticides. J. Econ. Entomol. 88: 716-724.
Hibbard, B. E., Randolph, T. L., Bernklau, E. J., Abou-Fakhr, E. M. and Bjostad, L. B. 1997.
Electroantennogram-active components of maize silk for adults of the western corn rootworm
(Coleoptera: Chrysomelidae). Environ. Entomol. 26: 285-295.
Hibbard, B.E., M.L. Higdon, D.P. Duran, Y.M. Schweikert, and M.R. Ellersieck. 2004. Role of
egg density on establishment and plant-to-plant movement by western corn rootworm larvae
(Coleoptera: Chrysomelidae). J. Econ. Entomol. 97: 871-882.
Hollister, B. and C. A. Mullin. 1999. Isolation and identification of primary metabolite feeding
stimulants for adult Western Corn Rootworm, Diabrotica virgifera virgifera LeConte, from host
pollen. J. Chem. Ecol. 25: 1263-1280.
Krysan, J. L. and R. F. Smith. 1987. Systematics of the virgifera species group of Diabrotica
(Coleoptera: Chrysomelidae: Galerucinae). Entomography 5: 375-484.
Lance, D. R. 1988. Potential of 8-methyl-2-decyl propanoate and plant-derived volatiles for
attracting corn rootworm beetles (Coleoptera: Chrysomelidae) to toxic bait. J. Econ. Entomol. 81:
1359-1362.
Lance, D. R. 1992. Odors influence choice of oviposition sites by Diabrotica virgifera virgifera
(Coleoptera: Chrysomelidae). J. Chem. Ecol. 18: 1227-1237.
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59.
Lin, S. and C. A. Mullin. 1999. Lipid, polyamide, and flavonol phagostimulants for adult westen
corn rootworm from sunflower (Helianthus annuus L.) pollen. J. Agric. and Food Chem. 47: 1223-
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Rootworm in Northeast Nebraska. Environ. Entomol. 4: 435-438.
Massimino, D., M. Andre, C. Richaud, A. Daguenet, J. Massimino, and J. Vivoli. 1980. Evolution
horaire au cours d'une journee normale de la photosynthese, de la transpiration, de la respiration
foliare et racinaire et de la nutrition N.P.K. chez Zea mays. Physiol. Plant 48: 512-518.
Metcalf, R.L. and Metcalf, E.R. 1992. Plant Kairomones in Insect Ecology and Control. Routledge,
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      Meinke, L. J. Z. Mayo, and T. J. Weissling. 1989. Pheromone delivery system: western
corn rootworm (Coleoptera: Chrysomelidae) pheromone encapsulation in a starch borate matrix. J.
Econ. Entomol. 82: 1830-1835.
Moeser, J., and S. Vidal. 2004a. Do alternative host plants enhance the invasion of the maize pest
Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae, Galerucinae) in Europe? Environ.
Entomol. 33: 1169-1177.
Moeser, J., and S. Vidal. 2004b. Response of larvae of invasive maize pest Diabrotica virgifera
virgifera (Coleoptera: Chrysomelidae) to carbon/nitrogen ratio and phytosterol content of European
maize varieties. J. Econ. Entomol. 97: 1335-1341.
Moeser, J., and S. Vidal. 2005. Nutritional resources used by the invasive maize pest Diabrotica
virgifera virgifera in its new South-east-European distribution range. Entomol Exp. Appl. 114: 55-
63.
Mullin, C. A., A. A. Alfatafta, J. L. Harman, A. A. Serino and S. L. Everett, 1991. Corn rootworm
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bioregulators. Symposium series 449. American Chemical Society, Washington, DC. pp. 278-292.
Olmer, K.J. and B.E. Hibbard. The nutritive value of senescing maize and Setaria roots for western
corn rootworm development. IWGO (International Working Group on Ostrinia and other maize
pests) 22nd Conference, Vienna, Austria, 5-8 November 2006.
O´Neal, M E., C. D. DiFonzo, and D. A. Landis. 2002. Western Corn Rootworm (Coleoptera:
Chrysomelidae) feeding on corn and soybean leaves affected by corn phenology. Environ. Entomol.
31: 285-292.
Oyediran, I. O., B. E. Hibbard, and T. L. Clark. 2004. Prairie grasses as hosts of the western corn
rootworm (Coleoptera: Chrysomelidae). Environ. Entomol. 33: 740-747.
Pavuk, D. M. and B. R. Stinner. 1994. Influence of weeds within Zea mays crop plantings on
populations of adult Diabrotica barberi and Diabrotica virgifera virgifera. Agriculture Ecosystems
and Environment 50: 165-175.
Prystupa, B., C. R. Ellis, and P. E. A. Teal. 1988. Attraction of adult Diabrotica (Coleoptera:
Chrysomelidae) to corn silks and analysis of the host-finding response. J. Chem. Ecol. 14: 635-
651.
Siegfried, B. D. and C. A. Mullin. 1990. Effects of alternative host plants on longevity, oviposition,
and emergence of western and northern corn rootworms (Coleoptera: Chrysomelidae). Environ.
Entomol. 19: 474-480.
Strnad, S. P., and P. E. Dunn. 1990. Host search behavior of neonate western corn rootworm
(Diabrotica virgifera virgifera). J. Insect Physiol. 36: 201-205.
Strnad, S. P., M. K. Bergman, and W. C. Fulton. 1986. First-instar western corn rootworm
(Coleoptera: Chrysomelidae) response to carbon dioxide. Environ. Entomol. 15: 839-842.
Tallamy, D. W., B. E. Hibbard, T. L. Clark, and J. J. Gillespie. 2005. Western Corn Rootworm,
Cucurbits, and Cucurbitacins, pp. 67-93. In Western Corn Rootworm: Ecology and Management.
Stefan Vidal, Ulrich Kuhlmann, and Richard Edwards (eds.), CABI publishers, Wallingford, United
Kingdom.
Weatherwax, P. 1954. Indian corn in old America, 253 pp., Macmillan, New York.
Wilkin, P. J., J. L. Krysan, and P. L. Guss. 1986. Electroantennogram responses of corn
rootworms and other closely related Diabrotica (Coleoptera: Chrysomelidae) to sex pheromones
and synthetic attractants. J. Kans. Entomol. Soc. 59: 571-579.
Wilson, T. A., and B. E. Hibbard. 2004. Host suitability of nonmaize agroecosystem grasses for the
western corn rootworm (Coleoptera: Chrysomelidae). Environ. Entomol. 25: 1167-1172.
Wheeler, D. 1996. The role of nourishment in oogenesis. Annu. Rev. Entomol. 41: 407-431.




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Western corn rootworm population dynamics
I Egg Diapause
The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is univoltine,
overwintering as an egg in the soil (Ball 1957, Chiang 1973). As the overwintering stage, the egg is
exposed to harsh conditions for an extended period, resulting in overall high mortality (Gustin
1981, 1986, Godfrey et al. 1995, Levine and Oloumi-Sadeghi 1991, Toepfer and Kuhlmann 2006).
Eggs surviving the winter determine the maximum level of damage to be inflicted in an infested
field, and the dynamics of egg dormancy is a key to understanding the species ecology as a whole
(Schaafsma et al. 1991, Woodson and Gustin 1993, Ellsbury et al. 1998, Ellsbury and Lee 2004).
Winter dormancy consists of two phases, an initial obligate diapause followed by facultative
quiescence governed by environmental conditions (Krysan 1978). In temperate regions, eggs are
oviposited in the soil during July to September. Embryogenesis presumably begins at oviposition
or soon after, but development halts within about 11-13 days at 20oC as the egg enters diapause
(Krysan 1972, Branson 1976b). Krysan et al. (1977) reported 100% of eggs examined had embryos
in the diapause form by 18 d post-oviposition at 25 oC. Continuous chilling of the egg before
reaching diapause is "detrimental" (B. W.George unpub. data, cited in Chiang 1973), and a 2-3
week period at 25 oC is routinely employed when rearing eggs through to hatch to allow
prediapause development before chilling (Jackson 1986). At the point of entering diapause, the
embryo is immersed in the yolk as an undifferentiated germinal disc about 90 µm in length, and it
remains at this stage until diapause is terminated (Krysan 1972). Reported mean duration of
diapause (also referred to as "diapause intensity") varies widely among studies and latitude, ranging
from 78-163 d, and is quite variable among individuals within a population as well (Branson 1976b,
Krysan 1982). Termination of diapause does not require chilling or other known environmental
cues, but is an event apparently governed by time (Krysan et al. 1977, Krysan 1978). Under natural
conditions in temperate regions, termination occurs during midwinter when soil temperatures are
still below 11 oC, the thermal threshold for development (Wilde 1971, Schaafsma et al. 1991,
Levine et al. 1992). Thus, postdiapause eggs remain dormant in a facultative state of chill-
quiescence until the soil temperature warms above 11 oC (Krysan 1978, Gustin 1981, Krysan et al.
1984).
Postdiapause development then commences until hatch, which begins by 14-20 d and peaks by 18-
43 d post-chill at constant 20-25 oC in the laboratory, again depending on the study and on the
latitude of the subject population (Wilde 1971, Branson 1976a,b, 1987, Krysan et al. 1984, Fisher
1989, Schaafsma et al. 1991, Levine et al. 1992). Fluctuating laboratory rearing temperatures
tracking outdoor soil temperatures in South Dakota increased post-chill time to hatch by 45-50 days
over that (19-24 d) at a constant 25 oC (Fisher 1989). Musick and Fairchild (1971) found egg hatch
in northwest Missouri was usually completed within 35 days. Both linear and nonlinear models
have been developed to predict postdiapause development time to egg hatch based on accumulation
of heat units (Fisher 1989, Elliott et al. 1990, Schaafsma et al. 1991, 1993, Levine et al. 1992). The
optimum temperature for postdiapause egg development is about 28 oC (Schaafsma et al. 1991).
Numerous factors like microclimate, tillage, and soil characteristics affect soil temperature at a
given depth, and thus affect predictability of egg hatch (Pruess et al. 1968, Bergman and Turpin
1986, Elliott et al. 1990, Schaafsma et al. 1993, Godfrey et al. 1995).
In addition to warm enough temperatures, postdiapause eggs require uptake of water to complete
development (Krysan 1978). In the absence of water, postdiapause development stalls at various
stages, with embryos ranging mostly from 300-600 µm in length. The eggs remain in this
facultative state of dry-quiescence until water contacts the egg and is absorbed (Krysan 1978).

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       Water is not absorbed during diapause itself, indicating a change in egg permeability
after diapause is terminated (Krysan 1978). A decrease in specific gravity of eggs is associated
with water uptake and embryo development (Palmer et al. 1976, 1977).
Evidence supports a tropical or subtropical origin of WCR, with subsequent colonization of
temperate North America (Branson and Krysan 1981, Krysan 1982). WCR/Mexican corn
rootworm (MCR), Diabrotica virgifera zeae Krysan and Smith, egg diapause in Mexico occurs
during the dry season, when maize is not available, followed by a dry-quiescence (Krysan et al.
1977, Branson et al. 1978). Onset of the rainy season (summer) at planting time allows
continuation of postdiapause development and egg hatch in synchrony with host development
(Krysan et al. 1977, Branson and Krysan 1981, Cocke et al. 1994). Thus, it is thought that egg
diapause in WCR/MCR evolved as a strategy to survive dry conditions, and that diapause
preadapted the WCR to survive the cold season in temperate regions (Krysan et al. 1977, Krysan
1982, Ellsbury et al. 1998).
There is a geographic cline in diapause duration, which decreases from south to north (Wilde et al.
1972, Krysan and Branson 1977, Krysan et al. 1977, Krysan 1982, Schaafsma et al. 1991, Levine et
al. 1992). Diapause duration is under genetic control, with artificial selection for early hatching
(i.e., shorter diapause duration; Krysan 1978) resulting in a "non-diapause" colony of WCR
(Branson 1976b), still widely used in research because of its short generation time (Kim et al.
2007). Diapause duration in MCR averaged 253 d compared to 73 d for WCR from South Dakota
under the same conditions (Krysan and Branson 1977). Reciprocal crosses between these two
populations and with the non-diapause selected colony produced offspring with intermediate
diapause durations, but with evidence of a maternal effect (Krysan and Branson 1977). A long
diapause duration is thought to be necessary in the tropics to maintain dormancy until the true rainy
season begins, rather than relying on dry-quiescence which might break prematurely with an
unseasonal winter shower (Krysan et al. 1977). In the north, diapause need only be long enough to
ensure dormancy until winter, after which cold-quiescence is an efficient enforcer of dormancy until
spring (Krysan et al. 1977, Krysan 1978, 1982, Ellsbury et al. 1998).

II Larval and adult dynamics
Larvae occupy plant roots and the soil around roots; the pre-pupal stage forms a discrete earthen
cell for pupation (Chiang 1973, Krysan 1999). Initial larval eclosion from eggs is variable over
years but usually occurs during late May to early June in the U.S. Corn Belt and in Central and
Eastern European countries. From 1985-2006, initial eclosion has occurred between May 22 and
June 16 at Ithaca, Nebraska, U.S.A. (Meinke, unpub.). Eclosion duration has been reported over a
mean of 29 days for males and 32 days for females (Musick and Fairchild 1971, Branson 1976a,
Palmer et al. 1977, Krysan et al. 1984, Levine et al. 1992). Larval development progresses through
3 instars (George and Hintz 1966, Hammack et al. 2003). Males complete larval/pupal
development faster than females within a temperature range of 18-30oC (i.e., 0.9 to 2.8 days
depending on temperature) but both sexes have a lower threshold of development near 9oC (Jackson
and Elliott 1988). Rate of development of immature stages is temperature dependent (Kuhlman et
al. 1970, Fisher 1986, Jackson and Elliott 1988). Average female neonate larva to adult emergence
times ranged from 45.0 - 20.7 days at 18 - 30oC (Jackson and Elliott 1988). The greatest mean head
capsule width and survival of adults occurs between 21-30oC; temperatures greater than 30oC are
detrimental to immature development (Jackson and Elliott 1988).
In the U.S. Corn Belt, WCR adult emergence may begin in late June to early July with peak
emergence often occurring during July (Quiring and Timmins 1990, Darnell et al. 2000, Nowatzki
2001). This general pattern is consistent in Central and Eastern European countries (i.e., Croatia,
Serbia, Hungary) and U.S. geographical areas inside and outside of the areas where crop rotation
resistant (Pierce and Gray 2006) or organophosphate resistant populations occur. In Croatia, initial

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       adult emergence was recorded from 17 June-2 July during 1998-2000 (Bazok 2001) and
50% emergence occurred during the first - third weeks of July (Bazok 2001, Bayar et al. 2003).
The duration of WCR emergence can be highly variable among fields and years. In Iowa U.S.A.,
duration of emergence from 78 continuous corn fields over a six year period averaged 33.4 days for
males and 51.3 days for females (Nowatzki 2001). Various models have been developed to predict
western corn rootworm adult emergence patterns that have been based on calendar date, air, or soil
degree-day accumulations and specific development thresholds (Ruppel et al. 1978, Bergman and
Turpin 1986, Hein et al. 1988, Fisher et al. 1990, Davis et al. 1996, Nowatzki et al. 2002).
Adult males begin emerging before females, and peak (50%) cumulative male emergence also
occurs earlier than peak female cumulative emergence (Darnell et al. 2000, Nowatzki et al. 2002,
Bayar et al. 2003). This is a function of differences between sexes in both postdiapause embryonic
development and posthatch immature development (Branson 1987). Sex ratio of total emerged
adults can be highly variable among fields and years (eg. Sutter et al. 1991, Darnell et al. 2000). A
1:1 male:female sex ratio has been observed especially when larval densities and associated root
injury are low (Weiss et al. 1985, Meinke unpub.). However, male:female sex ratios from field
emergence cages are often skewed toward one sex or the other in continuous corn (Darnell et al.
2000, Sutter et al. 1991).
Various factors can affect adult emergence timing, total emergence, size, and the sex ratio of total
emerged adults. Delayed planting of corn delays initial adult emergence and reduces total
emergence (Musick et al. 1980, Bergman and Turpin 1984, Meinke 1995). The effects of late
planting in relation to western corn rootworm egg hatch patterns presumably reduces availability of
corn roots and larval colonization sites resulting in mortality of earliest eclosing larvae (Bergman
and Turpin 1984, Boetel et al. 2003). The presence of grassy weeds (i.e., yellow foxtail, Setaria
pumila ) that can serve as alternate western corn rootworm larval hosts in cornfields can also delay
adult emergence and reduce mean beetle size when compared to weed-free corn (Ellsbury et al.
2005). This is consistent with published data that indicate yellow foxtail is a sub-optimal larval
host (Branson and Ortman 1967, 1970; Clark and Hibbard 2004). Adult emergence may be delayed
in conservation tillage systems, but total cumulative emergence is often comparable across tillage
systems (Gray and Tollefson 1988a). Cooler soil temperatures in the conservation tillage systems
than tilled systems especially during the egg-larval periods may contribute to adult emergence
delays observed in reduced tillage fields (Gray and Tollefson 1988a). Reduced row spacing in a
cornfield (i.e., 38 cm versus 76 cm row spacing) can lead to greater total adult emergence per m2
(Nowatzki et al. 2002).
As density of larvae on corn roots and associated root injury increases (especially very high
densities), total adult emergence and mean adult size decreases (Branson and Sutter 1985, Weiss et
al. 1985, Elliott et al. 1989). High larval densities also lead to prolonged mean development time
from neonate larva to adult (Weiss et al. 1985, Elliott et al. 1989, Sutter et al. 1991). Onstad et al.
(2006) recently developed equations from published data that clarify density-dependent effects on
survival from egg to adult.
Total adult emergence, and in some cases emergence patterns and sex ratios of emerged adults can
be altered in cornfields where soil insecticide applications have been used for rootworm control
(Sutter et al. 1991, Gray et al. 1992, Boetel et al. 2003). While different effects on adult parameters
have been reported for various insecticides, it is unclear whether effects on western corn rootworm
population dynamics are directly caused by sublethal exposure to the insecticide, or indirectly as a
result of environment/insecticide/rootworm density interactions. Under low to moderate larval
densities, WCR larvae reared on transgenic corn plants expressing the Cry3Bb1 protein develop
slower, initial adult emergence is delayed, 50% adult emergence is delayed, and the sex ratio of
emerged beetles becomes skewed more toward females when compared to a non-transgenic isoline
(Monsanto unpub., Meinke, unpub., Becker 2006). The mechanism is unclear, but it appears to be a


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       complex interaction of behavioral and physiological responses of larvae to the Cry3Bb1
protein (Clark et al. 2006, Becker 2006).
WCR population densities vary within and among fields over time (Darnell et al. 2000, Park and
Tollefson 2005) . Adults can often be found in and around cornfields until early fall or first frost
(Short and Hill 1972, Krnjajić 1995, Bazok 2001, Komaromi et al. 2001, Komaromi and Kiss 2004,
Ivezic et al. 2006) and peak densities are often correlated with peak emergence patterns, namely late
July to early August (Darnell et al. 1999, Komaromi et al. 2000, Bazok 2001, Komaromi and Kiss
2004). Because of all of the abiotic and biotic factors that may potentially interact to cause WCR
mortality (Toepfer and Kuhlmann 2005) or affect fitness, it is difficult to predict changes in density
within and among years. The number of beetles emerging from within a continuous cornfield is
often a primary contributor to the total population density in a field but emigration and immigration
rates can significantly affect the population density during a specific period (Meinke 1995, Darnell
et al. 2000). Total seasonal adult densities are often greater in continuous corn than first year corn
(Godfrey and Turpin 1983, Kiss et al. 2005), but peak densities may be later in first year corn
(Godfrey and Turpin 1983) or crops rotated with corn (e.g., variant in Illinois, U.S.A., Pierce and
Gray 2006). Contrasts in plant phenology can greatly affect adult densities within and among fields
during a season (Darnell et al. 1999, 2000; Pierce and Gray 2006). The mobility and reproductive
potential of adult females enables the western corn rootworm to recolonize areas very quickly
where densities have been eliminated by crop rotation (Godfrey and Turpin 1983) or greatly
reduced by adult management (Pruess et al. 1974). The western corn rootworm has clearly adapted
to use of corn as a superior larval host (Branson and Krysan 1981, Clark and Hibbard 2004, Wilson
and Hibbard 2004), so a high proportion of hectares planted as continuous corn greatly favors
survival and the build-up of population densities in a region over time (Hill and Mayo 1980).
Various types of models have been developed to address questions about WCR population
dynamics. Elliott and Hein (1991) created and analyzed a single-season deterministic model of
WCR population dynamics and corn crop phenology. They emphasized the influence of weather,
particularly temperature, on population processes. They tested the simulated population dynamics
against independent field data collected by the authors. Their simulations matched the field data
well, but they stated that better adult immigration functions may be needed for the model to work
well under many other situations. Also, the results were sensitive to some temperature-dependent
functions and to age-specific oviposition. This model may be the best to use for predicting accurate
single-season population dynamics.
Onstad et al. (2001) and subsequent extensions of the model (Onstad et al. 2003, Crowder and
Onstad 2005, Crowder et al. 2005) are deterministic models of WCR population dynamics and
genetics that simulate multiple generations. Onstad (2006) added a larval submodel. Onstad et al.
(2001) tested the population dynamics of the model against field data of Hein and Tollefson (1985)
and Godfrey and Turpin (1983). Onstad et al. (2001) determined that the carrying capacity (the
asymptotic densities of WCR) of a typical cornfield with continuously planted corn, is 15 million
eggs per ha in autumn and 0.7 million emerging adults. Onstad et al. (2003) simulated a higher
carrying capacity of 2 million adults per ha.
  Storer (2003) created a stochastic, spatially-explicit, simulation model of western corn rootworm
  population dynamics and genetics. All processes surrounding crop phenology, insect phenology,
  insect survival, and insect dispersal are probabilistic in the model. He tested the model’s
  population dynamics against field data of Hein et al. (1988). From his modeling effort, Storer
  (2003) concluded that local dispersal is poorly understood, and that the model is highly sensitive
  to dispersal parameters. Spatial heterogeneity in the agroecosystem, such as non-agricultural
  land and barriers to dispersal, could be very important.




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       Hemerik et al. (2004) used a deterministic, temperature-based simulation model of the
entire life cycle to predict the population success and spread of WCR. They predicted that WCR
would not invade the Netherlands until 2018 because of limitations due to suboptimal temperatures.
  Caprio et al. (2006) used a stochastic, individual-based, simulation model of population
  dynamics and genetics to study the evolution of resistance to organophosphate insecticide
  targeted at adults in Nebraska, U.S.A.. The model also simulated multi-field spatial dynamics.
  The simulated population dynamics were qualitatively tested against field data of Short and Hill
  (1972).

III Non-trophic (direct or indirect) interactions with other arthropods
The arthropods that potentially could interact with the western corn rootworm in European
cornfields can be divided into two main groups; those primarily found at the soil surface or in the
soil, and those found on/in corn plant tissues above the soil line. This would include pest and non-
pest arthropods. Examples of pest arthropods found in each environment that can occur at
moderate-high densities are listed below.
Pest species attacking seeds and roots mainly in early season: Wireworms in the genus Agriotes
are often present (i.e., Agriotes brevis, Agriotes sordidus, Agriotes ustulatus, Agriotes litigiosus,
Agriotes lineatus, Agriotes obscurus, Agriotes sputator, Agriotes rufipalpis, Agriotes proximus.),
and in some Eastern European countries Tanymecus dilaticollis may be an important pest. Several
cutworm species (i.e., Agrotis ipsilon, Agrotis segetum ) can be present on a sporatic basis.
Pest species attacking leaves, ear, stalk of developed plants: The key pest is Ostrinia nubilalis
(Lepidoptera, Crambidae) which is widespread in Europe. Several other stalk borers, Sesamia
cretica and Sesamia nonagrioides can be important pests in southern European regions. Other
Lepidoptera (Helicoverpa armigera, Helicoverpa zeae, Heliothis sp, Spodoptera spp.), several
species of Thysanoptera, Tetranychus urticae (limited outbreaks can be recorded in dry summers in
the border of the corn fields), and aphids (genera Rhopalosiphum and Sitobion) can be occasional
pests in field corn.
To date, very little data is available on non-trophic (direct or indirect) interactions of the WCR with
other arthropods in cornfields. An extensive field survey was carried out in Hungary in 2004
(Veres and Tóth unpublished). A total of 53 corn fields were sampled by shaking the silk of the
corn ears. Arthropods falling from the corn silk were recorded (57 morphotaxa, 7288 individuals,
308 D. v. virgifera adults). Number of WCR adults correlated negatively with most other arthropod
groups, but most values were non-significant. A significant negative correlation was found in
relation with predatory and phytophagous mites (Acari; r=-0.27 and -0.20 respectively, df=52) and
predatory thrips (Aeolothrips spp.; r=-0.23, df=52). Significant positive correlation was found only
with cereal leaf beetles (Oulema spp.; r=+0.21, df=52). A possible explanation of this phenomenon
is that feeding (silk clipping) of WCR adults may destroy the silk as a microhabitat. This
disturbance seems to be unfavourable to certain small-sized arthropod taxa.
While WCR adults may have an impact on microhabitats on the corn plants, western corn rootworm
larval damage can change the structure and characteristics of the whole habitat (the cornfield) by
increasing the number of lodged or underdeveloped plants. White and Andow (2006) found that
habitat modification of WCR larval damage can dramatically reduce parasitism rate of the European
corn borer (Ostrinia nubilalis). Explanation of this indirect impact is that Macrocentrus grandii, a
specialist parasitoid of O. nubilalis, prefers high and dense corn stands.

IV References Cited
Ball, H. J. 1957. On the biology and egg-laying habits of the western corn rootworm. J. Econ.
Entomol. 50: 126-128.


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Wilde, G., H. C. Chiang, E. T. Hibbs, and D. E. Lawson. 1972. Variation in egg hatch among
western and northern corn rootworms from six midwestern states. J. Kans. Entomol. Soc. 45: 259-
263.
Wilson, T. A., and B. E. Hibbard. 2004. Host suitability of nonmaize agroecosystem grasses for
the western corn rootworm (Coleoptera: Chrysomelidae). Environ. Entomol. 33: 1102-1108.
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      White, J. A. and Andow, D. A. 2006 Habitat modification contributes to associational
resistance between herbivores. Oecologia 148 (3): 482-490.
Woodson, W. D., and R. D. Gustin. 1993. Low temperature effects on hatch of western corn
rootworm eggs (Coleoptera: Chrysomelidae). J. Kans. Entomol. Soc. 66: 104–107.




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Micro- Short and Long Range Western Corn Rootworm Movement
I Micro-range
Upon hatch from different locations within the soil profile, physical and chemical factors interact to
affect first instar larval movement and subsequent establishment on corn roots. Because the first
instar is small and soft-bodied, movement throughout the soil is likely to primarily accomplished by
using the interconnected air-filled pores in the soil with relatively little actual burrowing (Gustin
and Schumacker 1989). Larval movement is limited by increasing soil bulk density (Strnad and
Bergman 1987a, Ellsbury et al. 1994) and pore sizes smaller than larval head capsule width (Gustin
and Schumacker 1989). Pore size distribution is directly related to the texture, structure, and bulk
density of a soil. First instars move farther in fine textured soils than coarse soils (MacDonald and
Ellis 1990). The interaction of soil texture and soil moisture affects movement. Within a soil
texture, very wet or very dry soil conditions can restrict movement of first instars (MacDonald and
Ellis 1990). Very wet conditions can reduce available air-filled pores as many spaces become
saturated with water. Establishment success on maize plants is independent of egg density (Hibbard
et al. 2004), but might be affected by the number of infestation points around a plant (Wilson et al.
2006). In artificial infestations with varying infestation depths, infestation rates, and distances from
a maize row, Chaddha (1990) documented that there was no significant difference in adult
emergence between infestation depths of 7.5 cm or 15 cm. However, as distances from the corn
row increased, plant damage decreased. Adult emergence was also affected by distances from the
corn row, but to a greater extent from the lower infestation rate, perhaps because of density-
dependent mortality from the higher infestation rate at the closer infestation distance.
Larval migration is not complete when the neonate reaches a host plant. Strnad and Bergman
(1987b) demonstrated that as larvae grow, they re-distribute, moving to younger root whorls that
emerge from the stalk. Hibbard et al. (2003) document not only move within the plant, but also at
least three plants down a corn row and across 46 cm row. Although egg density appeared not to be
an important factor in percent larval establishment (Hibbard et al. 2004), it was an important factor
in plant damage and, secondarily, subsequent larval movement. In general, damage was highest in
the plots infested with the most viable eggs and decreased as distance from the infested plant
decreased. Post-establishment movement generally occurred about the time that significant damage
began to appear, rather than at the time of establishment. These data imply that plant-to-plant
movement was motivated by a search for food and was density-dependent only because damage
was density-dependent. If crowding caused larval movement rather than a lack of food, movement
would be expected to have occurred earlier in Hibbard et al. (2004). Larval movement from egg
hatch to adult emergence had been reported to be as high as 100 cm (Suttle et al. 1967, Short and
Luedtke 1970). Although Branson (1986) questioned these data for procedural reasons, Hibbard et
al. (2004) document movement as far as 61 cm and it is not inconceivable that movement up to 100
cm from egg hatch to adult emergence could take place. Compaction of interrow soil from wheel
traffic can help prevent larval movement into corn from soybean areas planted to corn the previous
year in a strip intercropping system (Ellsbury et al. 1999).

II Short range WCR movement
The dichotomy between the biology of rotation-resistant WCR (WCR from populations where egg
laying occurs in corn and rotated crops) and rotation-susceptible or ‘wildtype’ WCR (populations
were egg laying occurs almost exclusively in cornfields) is chiefly due to differences in the degree
of interfield movement. Broad adoption of annual crop rotation in the eastern Corn Belt is believed
to have selected for existing WCR phenotypes with reduced ovipositional fidelity to cornfields

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       (Levine et al. 2002, Spencer et al. 2005). Over a period of ca. 20 years, the reproductive
benefits accruing to females who deposited at least some eggs in soybean and other rotated crop
fields, created a WCR population where little ovipositional fidelity to cornfields remained. In a
landscape where soybean is annually rotated to corn with almost 95-98% certainty, WCR females
that deposit eggs outside of cornfields will produce more surviving offspring that females with
perfect fidelity to cornfields. In the case of rotation resistance, selection did not broaden
ovipositional host specificity to include new plants, but rather the effect was to relax a very strict
ovipositional fidelity to cornfields.
Intrafield movement and general activity within cornfields. A variety of methods have been used to
study intrafield (and interfield) WCR movement, including mass marking techniques (Lance and
Elliott, 1990; Naranjo, 1990a; Oloumi-Sadeghi and Levine, 1990; Spencer et al., 1999, Toepfer et
al. 2006) and more recently Rubidium labeling of WCR adults (Nowatzki et al. 2003) and
transgenic tissue detection (Spencer et al. 2003). Tethered flight mills have also been employed to
gauge the capacity of WCR adults to engage in short and sustained flights (Coats et al. 1986,
Naranjo 1990a).
WCR male movement in response to sex pheromone emitted by calling females is one of the first
types of intrafield movement engaged in by WCR each summer. Detection of ingested transgenic
corn tissue as described in Spencer et al. (2003) has been used to study movement of mate-seeking
WCR males between fields YieldGard®Rootworm transgenic corn and adjacent non-transgenic
corn refuges. Early during the 2006 growing season, before corn pollination began, the average
mate-seeking WCR male moved at 17.56 ±1.0 m/d through corn, with moving ≥48 m in 24 h
(movement rate determinations were based on detection of ingested transgenic corn tissue in WCR
collected at measured distances from known sources of transgenic corn). Male movement rates
declined slightly to 15.04 ±1.3 m/d as female emergence peaked during corn pollination; average
male movement rate for the first month of the growing season averaged 16.17 ±0.8 m/d. Calculated
movement rates for males collected while in copulo (20.17±1.2 m/d) were significantly greater than
rates for unpaired, mate-seeking males (16.17±0.8 m/d). In a preliminary version of this study in
2005, mating and non-mating male movement rates were not different (JL Spencer unpublished
field data)
Female WCR also engage in intrafield movement, it may be divided into pre-mating and post-
mating/pre-dispersal movement. In the first hours after adult emergence, short distances are moved
by unmated, teneral females (i.e., they are pale colored because their exoskeleton is not yet fully
sclerotized, a process that is completed ca.12-24 h after emergence) (Quiring and Timmins 1990).
Many are observed to crawl upward on the corn plants near to where they emerged or to fly short
distances to nearby plants where they feed, preen or remain motionless (Quiring and Timmins
1990). During this period they are also very likely to initiate calling behavior by exposing a pair of
abdominal pheromone-secreting glands and broadcasting their readiness to mate (Hammack 1995).
Most calling females are rapidly mated (Ball 1957, Hill 1975, Lew and Ball 1979).
During measurement of movement rates for each individual in field-collected WCR mating pairs,
evidence was obtained indicating that some non-teneral (i.e., older than 24 h post-emergence)
females from mating pairs had moved as far as 48 m (the greatest distance from source areas at
which moving WCR were sampled) before they were eventually mated (Spencer, unpublished field
data). These data suggest that if not mated, apparently sedentary calling females can become quite
mobile. In fact, mean movement rates of these ‘mature’ mating females (23.36 ±1.6 m/d) were not
different from the movement rates of the males they were coupled with (20.17 ±1.2 m/d); no
measurable pre-mating movement by teneral females from a mating pairs was observed.
Once mated, female WCR remain in their natal cornfield and feed for a few days to a week before
ascending from the cornfield and dispersing presumably to a different cornfield. The ascent and
dispersal flight of recently-mated females coincides with morning and evening periods of peak
WCR fight activity (Isard et al. 2004). Dispersing females ascend steeply. Identification of

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        transgenic corn tissue (using transgenic tissue detection methods of Spencer et al.
(2003)) in the gut contents of females netted from the tops of 10 m scaffolding towers indicates that
most of the females (these populations of high-flying WCR are 85% female (Isard et al. 2004))
originated from the field below or next to the collection tower. Coats et al. (1986) observed that
15% of female tethered in flight mills engaged in sustained flights (>30 minutes in duration) during
their lifespan. Using a vertical flight mill, Naranjo (1990b) estimated that 24% of females engaged
in sustained flights (>20 minutes in duration). Dissection of female WCR collected at variety of
heights and locations near a cornfield (Spencer et al. 2005) suggest that the proportion of dispersing
females in the population is perhaps 3-4x greater than the flight-mill based estimates. In the
Spencer et al. (2005) samples, 84% of females collected at high elevation (10 m) contained a
spermatophore, 43.7% of females contained a large spermatophore (indicative of recent mating).
The spermatophore, which is deposited in the female by the male during mating is slowly degraded
during 5-7 days after mating. The presence of a large spermatophore or collapsing remnants of one
in a female’s reproductive tract is an indication that she was recently mated. Other characteristics
of these females (low mass and poorly developed oocyctes) also suggest they are young and
recently mated.
The high proportion of females in the dispersing population and the approximate timing of the
dispersal parallels historical patterns of WCR dispersal from the period before evolution of rotation-
resistant WCR. When continuous cornfields were the only major source areas for WCR emergence
(as is still true in parts of the U.S. Corn Belt and areas of Europe where WCR were most recently
established), the timing of post-mating dispersal was observable by monitoring for the arrival of
WCR adults in rotated cornfields. The WCR populations in these fields climb ca. one week after
WCR emergence in nearby continuous cornfields and are female biased (Godfrey and Turpin 1983).

III Interfield movement
Once a dispersing female has settled in a cornfield, she will continue to feed and provision batches
of eggs. Over the bulk of the U.S. Corn Belt, this means that WCR females will engage in more
intrafield movement, perhaps punctuated by a succession of periodic interfield flights into
phenologically less-mature cornfields (Naranjo 1994). These interfield flights are likely stimulated
by detection of any of a number of attractive volatile stimuli that emanate from pollinating corn and
developing corn silks or in response to unfavorable conditions in the current field (i.e., advancing
crop maturity) (Roscoe and Mayo 1974, Witkowski and Owens 1979, Prystupa et al. 1988, Naranjo
1994, Hammack 1996, Darnell et al. 2000) or opportunities to exploit weedy sources of pollen late
in the growing season (Campbell and Meinke 2006). The presence of volunteer corn plants in
soybean fields or other rotated crops is highly attractive, many WCR will orient to these plants,
sometimes resulting in significant egg laying in their vicinity (Shaw et al. 1978).
Where rotation-resistant WCR are present, post-dispersal movement of females includes intrafield
movement as well as frequent interfield movement. The magnitude of interfield movement from
corn into soybean and other rotated crops is evident in the prodigious WCR abundances measurable
outside of cornfields throughout the growing season, even while corn is pollinating. Beginning ca.
1 week after the first WCR detected in cornfields, they can be observed moving around and feeding
in nearby soybean fields (Levine et al. 2002). WCR are typically more abundant in soybean fields
than cornfields for most of growing season. It is important to note that, like wildtype populations,
rotation-resistant WCR also engage in interfield movement to exploit phenologically less-mature
cornfields.
Crop phenology has been hypothesized to play a key role in evolution of rotation resistance. Pierce
(2003) tested whether phenology differences alone could bring egg-laying WCR into soybean fields
under field conditions and found that differences in corn phenology can influence egg laying by
rotation-resistant WCR in corn; however, a wide phenology difference was apparently not sufficient

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       to drive females into soybean nor to stimulate egg laying by WCR from an area where
rotation-resistant WCR were not known to be present. O’Neal et al. (2002, 2004) hold an
alternative view, having concluded that advancing corn phenology is the force that moves rotation-
resistant WCR out of corn and into soybean. While WCR abundance in soybean does increase over
the course of the growing season (O’Neal et al. 1999), a phenology-based mechanism for rotation
resistance does not account for the large numbers of WCR adults found in soybean while corn is
still an attractive host (Rondon and Gray, 2003)
Most (ca. 60%) of the rotation-resistant WCR in soybean fields or collected while moving between
cornfields and other rotated crops are female (Levine et al. 2002; O’Neal et al. 1999; Rondon and
Gray 2003). This proportion is reminiscent of the proportion of females that was previously typical
of first-year corn (Godfrey and Turpin 1983) prior to the spread of WCR rotation resistance. After
rotation-resistant enter a soybean field, many feed on soybean tissue (despite a lack of nutritive
value to WCR (Mabry and Spencer 2003)). During the growing season, ca. 56-86%% of WCR
females in soybean contained identifiable soybean tissue in their gut contents (JLS 1996–2001
dissection data). A similar proportion of the much less abundant WCR population from soybean
fields in rotation-susceptible regions also contain ingested soybean tissue, an indication that
soybean herbivory is not a unique characteristic of rotation-resistant beetles.
Although readily eaten, soybean tissue does not support WCR egg development (Mabry and
Spencer 2003). In the laboratory, few field-collected WCR that eat only soybean tissue live ≥1
week. However, WCR adults that eat mixed diets that alternate between corn and soybean tissue,
are as vigorous as WCR maintained on a continual diet of corn plant tissues (Mabry and Spencer.
2003). Soybean herbivory also significantly affects movement and egg-laying behavior. Mabry et
al. (2004) found that when WCR were switched from corn to soybean or soybean to corn diets,
WCR feeding on soybean were significantly more active than WCR feeding on corn tissues. The
same insects also laid significantly more eggs on days when soybean tissue was their only available
diet (Mabry et al. 2004). Using a behavioral assay, Knolhoff et al. (2006) found that female WCR
from rotation-resistant populations were faster to escape an arena than rotation-susceptible
populations; greater general activity levels in rotation-resistant populations may explain the
abundance of moving WCR. A predisposition toward greater activity when feeding on soybean
combined with behavioral effects of soybean herbivory may provide the proximate mechanism
behind the back-and-forth movement of rotation-resistant WCR from soybean fields back into
adjacent cornfields (Mabry et al. 2004, Spencer et al. 2005).
Imminent egg laying cannot be the only force leading to interfield movement. Gravid females or
those capable of maturing some eggs account for only 20% of females that make interfield flights
into soybean fields from cornfields (Mabry and Spencer 2003). The season-long presence of many
females without mature eggs in soybean fields suggests that egg laying is not the reason most
females leave corn for soybean. In addition, because soybean is such a poor quality food for WCR
(Mabry and Spencer 2003), the 80% of females that enter soybean fields without sufficient reserves
to mature eggs clearly must return to a cornfield to feed before they can lay eggs (Mabry et al.
2004).
Interfield movement rates for WCR moving from corn into three types of rotated crops have been
measured using transgenic tissue detection (Spencer et al. 2003) of the Cry 3Bb1 protein as an
ingestible marker. Differences in WCR movement rates between corn and soybean fields, or other
rotated crops like wheat, suggest a possible explanation for crop-to-crop variability in WCR egg
laying. Spencer et al. (2003-2005 field data) found that movement from corn into soybean was
slower that movement from corn into wheat. Corn in rotation with wheat escaped yield-reducing
root injury from WCR larvae, while corn after soybean or wheat double-cropped with soybean
suffered injury likely to reduce yields. The generally hotter and drier conditions prevailing in wheat
and wheat stubble (compared to the soybean canopy) may promote more rapid movement (ca. 6.8-
7.2 m/d from corn into wheat vs. 4.8-5.1 m/d into soybean) and consequently the few gravid

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       females that accumulate there have less time to lay eggs. Using similar arrays of the
same crops, Schroeder et al. (2005) also found that corn rotated with wheat offered the greatest
potential preventing economic injury in rotated corn.
Transgenic tissue detection (Spencer et al. 2003) is also being used to study movement of WCR
females returning to corn after being in nearby soybean fields. In this study, ingestion of Roundup
Ready® soybean tissue in WCR females captured at the edges of cornfields is used as a marker to
detect evidence of interfield movement from soybean fields into non-Roundup Ready® cornfields.
Part of this study involves collection of WCR from commercial non-Roundup Ready corn and an
adjacent Roundup Ready soybean fields from across the entire state of Illinois. Analyses of these
data reveal that in areas where rotation resistance is known to cause economic injury in rotated
corn, 7% of females in corn at the edge of soybeans fields test positive for ingested Roundup
Ready® soybean tissue—an indication that they recently moved into that cornfield after feeding in
soybean. In contrast, just 0.7% of females from the first row of the cornfield test positive for recent
soybean herbivory in areas without a rotation-resistance problem. The significant difference in
proportion of soybean ‘positive’ WCR females suggests that the level of soybean herbivory among
WCR at a corn-soybean field interface (measured by detecting soybean tissue in WCR gut contents
with transgenic-tissue detection) may be useful to help rapidly assess the local threat of rotation
resistance.

IV Long distance movement
During the post-mating, pre-ovipositional dispersal period, favorable atmospheric conditions (e.g.,
instability due to heating of air near the ground and the passage of summertime convective storms)
and light winds promote flight and favor ascent from cornfields (Witkowski et al. 1975,
VanWoerkom et al. 1983). A diel periodicity in flight tendency is reflected in peaks of flight during
early-late morning and early evening; Isard et al. (1999; 2000) review many of the factors that
influence flight patterns. During the passage of summertime storms, some airborne WCR adults
may be drawn into storms and carried 10’s of miles before being washed out of the storm in rain
(Grant and Seevers 1989). When WCR-bearing storms pass over Lake Michigan, evidence of storm
transport can be found in the piles of WCR beetles that wash-up along the waterline (Grant and
Seevers 1989).
Using WCR tethered to automated flight mills, Coats et al. (1986) and Naranjo (1990b) measured
the flight capability of WCR adults and found that 15% and 24% of females, respectively, engaged
in sustained flights of greater than 20-30 minutes (the longest flights in the Naranjo (1990b) and
Coats et al. (1986) studies were 150 minutes and 4 hours, respectively). In both studies, sustained
flight was a phenomenon of younger, mated females; the incidence declined steeply as females aged
no sustained female flights occurred after age 6 and 9 days in the Naranjo (1990b) and Coats et al.
(1986) studies, respectively. In a follow-up study, Coats et al. (1987) investigated the physiology of
these sustained or ‘migratory’ flights; juvenile hormone levels were found to be important
moderators of WCR. Converted to distance traveled, the sustained fliers in the Coats et al. (1986)
could have traveled up to 24 Km in a single flight or 39.4 km in a series of flights during one day.
Such a capacity to engage in sustained flight in the field would endow an individual with the
potential to cover a great distance in a few days. Though extremely long sustained flights were
uncommon, the capabilities for long duration flight by some exceptional young females are
illustrative of the capacity for rapid geographic expansion in WCR populations. The history of
WCR range expansion in the U.S. during the mid-late 20th century (Metcalf 1983) testifies to the
long distance mobility of the WCR. The hypothesis that insecticide resistance may have played a
role in accelerating the range expansion is intriguing (Metcalf 1983).




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Naranjo, S.E. 1994. Flight orientation of Diabrotica virgifera virgifera and D. barberi
(Coleoptera: Chrysomelidae) at habitat interfaces. Annals of the Entomological Society of
America, 87: 383-394.
Nowatzki, T.M., B. Niimi, K.J. Warren, S. Putnam, L.J. Meinke D.C. Gosselin, F.E. Harvey, T.E
Hunt, and B.D. Siegfried. 2003. In-field labeling of western corn rootworm adults (Coleoptera:
Chrysomelidae) with rubidium. Journal of Economic Entomology, 96:1750-1759.
O’Neal, M.E., M.E. Gray, and C.A. Smyth. 1999. Population characteristics of a western corn
rootworm (Coleoptera: Chrysomelidae) strain in east-central Illinois corn and soybean fields
Journal of Economic Entomology, 92: 1301-1310.
O’Neal, M.E., C.D. DiFonzo, and D.A. Landis. 2002. Western corn rootworm (Coleoptera:
Chrysomelidae) feeding on corn and soybean leaves affected by corn phenology. Environmental
Entomology, 31:285-292.




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       O’Neal, M.E., D.A. Landis, J.R. Miller, and C. D. DiFonzo. 2004. Corn phenology
influences Diabrotica virgifera virgifera emigration and visitation to soybean in laboratory assays.
Environmental Entomology, 33:35-44.
Oloumi-Sadeghi, H. and Levine, E. (1990) A simple, effective, and low-cost method for mass
marking adult western corn rootworms (Coleoptera: Chrysomelidae). Journal of Entomological
Science, 25:170-175.
Pierce, C.M.F., and M.E. Gray. 2006. Western corn rootworm, Diabrotica virgifera virgifera
LeConte (Coleoptera: Chrysomelidae), oviposition: a variant’s response to maize phenology.
Environmental Entomology, 35:423-434.
Pierce, C.M.F. 2003. Population dynamics of a western corn rootworm, Diabrotica virgifera
virgifera LeConte (Coleoptera: Chrysomelidae), variant in commercial corn and soybean fields in
east central Illinois. Ph. D. dissertation, University of Illinois, Urbana, IL.
Prystupa, B, C.R. Ellis, and P.E.A. Teal. 1988. Attration of adult Diabrotica (Coleoptera:
Chrysomelidae) to corn silks and analysis of the host-finding response. Journal of Chemical
Ecology, 14:653-651.
Quiring, D.T. and P.R. Timmins. 1990. Influence of reproductive ecology on feasibility of mass
trapping Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Journal of Applied Ecology,
27:965-982.
Rondon, S.I., and M.E. Gray. 2003. Captures of western corn rootworm (Coleoptera:
Chrysomelidae) adults with Pherocon AM and vial traps in four crops in east central Illinois.
Journal of Economic Entomology, 96: 737-747.
Schroeder, J.B. 2005. Variant western corn rootworm, Diabrotica virgifera virgifera LeConte,
population responses to cropping diversity and atmospheric conditions. M. S. thesis, University of
Illinois, Urbana IL.
Shaw, J.T., J.H. Paullus, and W.H. Luckmann. 1978. Corn rootworm oviposition in soybeans.
Journal of Economic Entomology, 71:189-191.
Short, D. E., and R. J. Luedtke. 1970. Larval migration of the western corn rootworm. J. Econ.
Entomol. 63: 325-326.
Spencer J.L., S.A. Isard, and E. Levine. 1999. Free flight of western corn rootworm (Coleoptera:
Chrysomelidae) to corn and soybean plants in a walk-in wind tunnel. Journal of Economic
Entomology, 92: 146-155.
Spencer, J.L., T.R. Mabry, E. Levine, and S.A. Isard. 2005. Movement, Dispersal, and Behavior of
Western Corn Rootworm Adults in Rotated Corn and Soybean Fields. In Western Corn Rootworm:
Ecology and Management. S. Vidal, U. Kuhlmann, and C. R. Edwards, eds. CABI Publishing,
Wallingford, Oxfordshire, UK. Pp. 121-144.
Spencer, J.L.; T.R. Mabry, and T.T. Vaughn. 2003. Use of transgenic plants to measure insect
herbivore movement. Journal of Economic Entomology, 96:1738-1749.
Strnad, S. P., and M. K. Bergman. 1987a. Movement of first-Instar western corn rootworms
(Coleoptera: Chrysomelidae) in soil. Environ. Entomol. 16: 975-978.
Suttle, P. J., G. J. Musick, and M. L. Fairchild. 1967. Study of larval migration of the western corn
rootworm. J. Econ. Entomol. 60: 1226-1228.
Toepfer, S., N. Levay, and J. Kiss. 2006. Adult movements of newly introduced alien Diabrotica
virgifera virgifera (Coleoptera: Chrysomelidae) from non-host habitats. Bulletin of Entomological
Research, 96:327-335.
Van Woerkom, G.J., F.T. Turpin, and J.R. Barret, Jr. 1983. Wind effect on western corn rootworm
(Coleoptera: Chrysomelidae) flight behavior. Environmental Entomology, 12:196-200.
Wilson, T.A., T.L. Clark, and B.E. Hibbard. 2006. Number of point sources of western corn
rootworm (Coleoptera: Chrysomelidae) eggs in artificial infestations affects larval establishment
and plant damage. J. Kans. Entomol. Soc. 79: 119-128.


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      Witkowski, J.F. and J.C. Owens. 1979. Corn rootworm behavior in response to trap
corn. Iowa State Journal of Research, 53:317-324.
Witkowski, J.F., J.C. Owens, and J.J. Tollefson. 1975. Diel activity and vertical flight distribution
of adult western corn rootworms in Iowa cornfields. Journal of Economic Entomology, 68: 351-
352.




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Spatial distribution at various scales
I Within plants or within roots
Early studies (Apple and Patel 1963, Sechriest 1969) documented that more rootworm larvae are in
corn roots than in soil early in the season. Among the best studies on rootworm distribution within
the root system is that by Strnad and Bergman (1987). They used a lactophenol and acid fuchsin
stain (Goodey 1937) to fix and visualize rootworm larvae in corn roots. Neonate WCR larvae were
found predominantly on nodes 3, 4, and 5 in one year and nodes 1, 2, and 3 the second year. Third
instar larvae were found predominantly on nodes 6, 7, and 8 the first year and nodes 6 and 7 the
second year. Significantly more first and second instars were oriented toward the root tip than the
root base. Orientation within corn roots was not as clear cut with third instar larvae. First instars
burrowed into root branches as small as 0.5 mm. Most first instar larvae were in roots 2 mm or less.
In addition, significantly more first instar larvae than a random 33% were found in the distal third of
the root than in the middle or proximal thirds. Later instar larvae tended to leave the initial roots
they were feeding on and move toward the newer nodes of roots developing at the base of the stalk.

II Among fields (including crop phenology, rotation history, tillage, landscape
features)
Inter-field movement of such mobile insects as the WCR has an important influence on the
population dynamics within single crop fields (Naranjo 1991). Therefore, characterization of such
movement and the ecological factors that affect it are critical for understanding the ecology of this
pest.
Changes in corn phenology can trigger inter-field flights (Chiang 1973), which can result in rapid
changes in population densities (Darnell et al. 2000). The flowering stage of corn is the most
attractive stage for WCR adults (Naranjo 1991; Lance et al. 1989; Darnell et al. 1999), so
differences in planting date can generate differential attractiveness of fields later in the season.
Naranjo (1991) studied the movements of adults between early- and late planted corn fields (22
days apart). Using a simulation analysis he found that the net emigration from an early planted
cornfield to a late planted field was 61.9% on a season-long basis, and that the majority of
emigrating beetles were reproductively mature females. Darnell et al. (2000) pointed out that
differences in phenological stages among cornfields result in an aggregated distribution of adults,
which should be considered when developing adult sampling programs.
Campbell and Meinke (2006) examined habitat use by adult Diabrotica species at prairie-corn
interfaces, and found that (especially for WCR) contrasts in plant phenology seem to be key factors
in habitat choice between corn and noncorn habitats. WCR adults preferred a primary habitat (corn)
but migrated towards secondary habitats if the relative attractiveness of the former decreased. This
result is similar to the findings of O’Neal et al. (2002), who showed in laboratory assays that the
decrease of preferred corn tissues (fresh leaves and silks) increased acceptance of alternative food
sources. Toepfer et al. (2006) found in their mark-release-recapture experiment that adult WCR
beetles can make significant use of alternative host plants during a migratory flight, and be arrested
for some time in attractive noncorn habitats. In contrast, when no alternative food sources were
provided in a noncorn habitat type, the beetles’ flight orientation in some cases was nonrandom,
being slightly oriented toward surrounding cornfields within a radius of 1500m (significant only for
males).
Directional movement of adults can be influenced either by environmental factors such as wind, or
by long-distance recognition and attraction by other crops or habitats (Spencer et al., 1999).
Naranjo (1994) described random movement of adults within corn growing areas, which is partially

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       in agreement with the findings of Toepfer et al. (2006). In 47% of cases in their
mark-release-recapture experiment, random flight of adults was observed. Nevertheless, in 33%
and 20% of cases, unidirectional and bidirectional oriented flight was observed, respectively.
Wind has a strong effect on flight initiation and orientation of WCR. Van Woerkom et al. (1983)
showed that WCR adults can move upwind when wind speed is less than 0.5 ms-1 and can move
crosswind if wind speed is less than 2 ms-1. At higher wind speeds adults do not initiate flight,
however, storms can transport large numbers of beetles long distances (Onstad et al., 1999).
Topefer et al. (2006) found a significant correlation (R=0.55) between beetle movement and mean
wind direction during the daytime, but adult flight orientation could not be explained adequately by
mean wind direction.
Isard et al. (2000) found that peaks in WCR flight activity occurred between 0700-1100h in the
morning and 1700-1900h in the afternoon, with no captures in malaise traps between sunset and
sunrise. This is partially in agreement with Coats et al. (1986) who observed peak daily flight
activity on flight mills between 1800-2400h. Witkowski et al. (1975) reported that the peak of
flight occurs 2-3 hours after sunrise and before sunset. This periodicity was further characterised by
a temperature range of 22.2 to 27.0 oC.
The height of WCR flight in the Witkowski et al. (1975) study did not differ between sexes below
1.8 m. However, between 1.8 m and 3 m there were significantly more females captured than
males. Spencer et al. (2005) sampled the migrating population at 10 m height and found that the
majority of migrants were young, newly mated females without mature eggs but with sperm in their
spermatheca, and with residues of corn tissue in their gut. Coats et al. (1986) showed that females
older than 9 days did not initiate sustained flight at all on flight mills.
Beckler et al. (2004) studied adult WCR distribution and abundance in relation to landscape
attributes and found that total abundance depended on availability of continuous vs. first-year corn
patches. They suggest that the natal continuous-corn fields reach their carrying capacity relatively
fast, thus, WCR adults disperse away from their natal field in search of new habitats. Large
first-year corn patches nearby the natal fields are more attractive than small distant patches.

III Dynamics of spread in North America & Europe

III- 1 Dynamics of spread in North America
The WCR is a native of North America, and almost certainly originated in the tropics (Smith 1966,
Branson and Krysan 1981). It is univoltine with an egg diapause, originally an adaptation to
survive the dry season when its primary host, corn, is unavailable (Krysan et al. 1977, Branson et al.
1978). Colonization of temperate North America may have accompanied the spread of cultivated
corn out of Mexico beginning about 1300 years ago (Krysan et al. 1977), with diapause having pre-
adapted the WCR to survive cold winters in dormancy (Krysan et al. 1977, Branson and Krysan
1981, Krysan 1982, Ellsbury et al. 1998). It is possible that WCR can develop on other species of
grass (Branson and Ortman 1970, Clark and Hibbard 2004, Moeser and Vidal 2004, Oyediran et al.
2004, 2005, Chege et al. 2005), but utilization of alternate hosts with corn nearby is probably
uncommon (Campbell and Meinke 2006).
Historical knowledge of WCR distribution began with its description by LeConte in 1868 from
specimens he collected in what is now Wallace County in far western Kansas (Smith and Lawrence
1967). Smith and Lawrence (1967) suggest it may have been collected by Say in 1820 in central or
southeastern Colorado, but the specimens have been lost. For many years it was simply one more
of hundreds of obscure New World diabroticite species until it started damaging sweet corn in north
central Colorado in 1909 (Gillette 1912). Before it became a serious pest of corn in the 1940s,
populations were low (Branson and Krysan 1981), and reports of its presence were scattered and
infrequent. In addition to Colorado and western Kansas, pre-1940 reports include New Mexico,

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       Arizona, and Sonora, Mexico (Bryson et al. 1953), southwestern Nebraska (Tate and
Bare 1946), and apparently south-central and far western South Dakota (Kantack 1965). Judging
from these accounts, WCR may have been resident in low numbers in northwestern Nebraska,
eastern Wyoming, and southeastern Montana as well.
Around 1940, WCR was routinely damaging corn in the southern tier of counties in southwestern
Nebraska, and it became a problem in Norton County cornfields in northwestern Kansas in 1945
(Bryson et al. 1953, Burkhardt and Bryson 1955). This marked the beginning of a dramatic
eastward range expansion, with WCR reaching New Jersey, Maryland, and Delaware along the
Atlantic Coast by the mid-1980s (Krysan 1986, Sutter 1999). This species is now established from
Montana (Smith and Lawrence 1967, Krysan 1978) and North Dakota (Glogoza and Boetel 2005)
in the northwest, to Quebec (Meloche et al. 2005) and New England (Boucher 2006) in the
northeast, and to northern Georgia (Hudson and All 1996) and Alabama (Flanders 2006) in the
southeast. The MCR was first reported as a pest of corn in Texas in 1977 (Sutter and Stewart
1995), and is now established in Oklahoma and most corn-growing regions of Texas, south through
eastern Mexico to Costa Rica (Giordano et al. 1997, Stewart 1999).
Except in areas where rotation resistance has spread (Levine et al. 2002), continuous corn
production is necessary for WCR populations to build because of this species' life history
characteristics of fidelity to corn for oviposition, univoltinism, the egg as the overwintering stage,
and relatively poor survival of larvae on non-corn hosts (Krysan et al. 1977, Branson and Krysan
1981, Levine and Oloumi-Sadeghi 1991, Onstad et al. 1999, Boriani et al. 2006). The initiation of
the range expansion was due to the increased adoption of planting corn following corn after World
War II, which in turn was made possible by the availability of soil insecticides and irrigation in
western Nebraska and Kansas (Smith 1966, Chiang and Flaskerd 1969, Luckmann et al. 1974,
Ruppel 1975, Hill and Mayo 1980, Metcalf 1986, Kuhar et al. 1997, Sutter 1999, Isard et al. 2001,
2004). Grower preference for planting continuous corn created conditions suitable for rapid
population growth, and thus increased the number of individuals dispersing from an infested area.
Furthermore, the high percentage planting of continuous corn also increased survival potential of
progeny from founding females that dispersed ahead of the front.
Metcalf (1983, 1986) noted that the initial expansion through Nebraska and Kansas (Ball 1957,
Burkhardt and Bryson 1955, Hill and Mayo 1980) was relatively slow (~20-50 km/yr) compared to
that of the subsequent expansion across the Midwest, reaching west-central Wisconsin by 1964
(~190 km/yr) and northwest Indiana by 1968 (~110 km/yr). He pointed out that the accelerated
period of range expansion coincided with development of resistance to cyclodiene insecticides, first
detected in southern Nebraska in 1959 (Ball and Weekman 1962). Cyclodiene resistance spread
throughout the WCR range, so that populations along the expanding species boundary were nearly
uniformly resistant by 1964 (Hamilton 1965, Siegfried and Mullin 1989). Metcalf (1983, 1986)
suggested that the resistant insects were behaviorally different so that they dispersed farther per year
than susceptible beetles, but this proposal is unsupported and unnecessary. Rate of expansion
across the U.S. actually varied widely depending on location, year, and direction. Based on the map
of WCR range expansion drawn by Metcalf (1983), Onstad et al. (1999) calculated a variety of rates
of spread depending on the direction and time period. The rate generally declined from 1963 to
1979. The eastward flow was calculated to be 138, 115, and 38 km/yr, respectively, for the periods
1963-64, 1964-68, and 1968-74. From 1968-74, the eastward rate was 38 km/yr and the
northeastward rate was much greater at 77 km/yr. From 1963-79 the eastward flow of WCR had a
rate of 63 km/yr on average, whereas the northeastward flow from 1963-73 had a rate of 92 km/yr.
Other rates reported or estimated from the literature include: from western Nebraska and Kansas to
Maryland and Delaware, averaged ~64-80 km/yr over 25-30 years (Sutter 1999); western Kansas to
northeastern Illinois, averaged ~60 km/yr over 15 years (Smith 1966); southeastern to east-central
South Dakota, ~80-120 km/yr (Kantack 1965); east-central South Dakota to southeastern North
Dakota, ~20 km/yr over 6 years (Kantack 1965, McBride 1972); within Minnesota ranged from

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       ~20-200 km/yr (Chiang and Flaskerd 1969); within Virginia ranged from ~40-180 km/yr
(Youngman and Day 1993); western Ontario to eastern Ontario from 1976-1990, averaged ~ 45
km/yr (Foott and Timmins 1977, Meloche and Hermans 2004); eastern Ontario into western Quebec
in 2000, ~45 km/yr (Meloche et al. 2001). Note that the range of rates within the state of Minnesota
alone (20-200 km/yr) (Chiang and Flaskerd 1969) encompasses the range of rates before (20 km/yr)
and after (190 km/yr) development of insecticide resistance as estimated by Metcalf (1983, 1986).
One of the main factors in North America affecting rate of spread of WCR was prevailing direction
of wind and storm fronts (Ruppel 1975, Grant and Seevers 1989, Onstad et al. 1999, Isard et al.
2001, 2004) resulting in differential rates of spread depending on direction. Mated, preoviposition
WCR females are prone to engage in long-distance flight activity (Coats et al. 1986, Naranjo 1990,
1991, Isard et al. 2004), and it is presumed that wind influences the direction and distance of such
flights (Grant and Seevers 1989, Onstad et al. 1999, 2003, Isard et al. 1999, 2001, 2004).
Dispersing females can sometimes travel long distances, as evidenced by founder populations of
WCR that developed out ahead of the main front of infestation (Chiang and Flaskerd 1969, Chiang
1973, Ruppel 1975), and ahead of the advancing front of rotation resistance (Onstad et al. 1999).
Ruppel (1975) observed the invasion of WCR into Michigan from 1971 to 1974. The rates of
spread calculated by Onstad et al. (1999) varied from 66 km/yr to 125 km/yr depending on the
direction (northward, eastward, or northeastward) and counties chosen for estimation. Onstad et al.
(1999) calculated a typical rate of 96 km/yr for this time period in Michigan. In Ohio, Clement et
al. (1979) observed the southeastward to eastward invasion of western corn rootworm during 1974
to 1978. Onstad et al. (1999) calculated a mean rate of spread of 44 km/yr. The lower rate of
spread in Ohio (Clement et al. 1979) compared to that for Michigan (Ruppel 1975) may have been
due to the prevalence of northeastward flowing storms depositing WCR in northeastern counties in
the lower part of Michigan. This phenomenon would not be apparent in Ohio because the invasion
began in northwestern counties (Onstad et al. 1999).
Another major factor affecting rate of WCR spread was the percentage of hectarage in continuous
corn production ahead of the front (Chiang and Flaskerd 1969, Youngman and Day 1993). For
example, invasion of western, central, and north-central Virginia during 1985-1988, where only
39% of the corn hectarage was rotated, was much more rapid than in eastern and southeastern parts
of the state from 1989-1992 where 92% of corn hectarage was rotated (Youngman and Day 1993).
Eastward expansion of rotation-resistance recently stalled in Michigan, Indiana, and Ohio, and
models suggest this is due to increased landscape diversity ahead of the front (Onstad et al. 2003).
However, this trait is not selectively neutral, and the mechanism for landscape diversity effects on
rate of spread is unknown and likely more complicated than its effects on simple spread of wild-
type populations into virgin territory (Onstad et al. 2001a).

III- 2 Dynamics of spread in Europe
Range expansion and population development of WCR in Europe has been well documented over
the past decade through intensive regional cooperation (Kiss et al 2005). The rate of spread has
differed greatly from year to year, averaging 40 km/yr, but ranging from 1-5 to 80 km/yr. This
variation depends on several factors, but one of the main ones is the topographical nature of the
region. In particular, mountains are barriers that often block or slow spread. High mountains can
be penetrated by WCR adults via road and railway passes along narrow valleys, as happened
towards Ukraine from Hungary through the Verecke Pass in the Carpathian Mountains (Yakobtsuk
et al, 2006). WCR adults were collected in color and pheromone traps at elevations of 800-900 m,
where small corn plots, or even home gardens with a few corn plants, offered suitable food and
survival sites for WCR under less than favorable highland conditions (Pai and Kiss, unpublished).
The observed spread of WCR in Central Europe also is a function of population density in the
source region from which adults spread. Continuous corn production allowing WCR populations to

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      build before being rotated, due to late detection of severe plant lodging, maintains a
source of dispersing WCR. Thus, late detection and subsequent population build up contributes to
an increased rate of spread. An example is a case of late detection in Lombardy in North Italy.
However, applying careful population estimation for rotation decisions and relevant management
practices, clearly results in lower WCR population densities and therefore decreased rate of spread
(Boriani et al 2006). A recent Commission Decision and Recommendation present a broad spectrum
of management options (annual or triannual rotation, seed treatment, soil insecticide application,
etc.) that will be implemented in coming years and is expected to impact WCR spread in Europe.
For details see COMMISSION DECISION of 11 August 2006 (2006/564/EC) and COMMISSION
RECOMMENDATION of 11 August (2006 2006/565/EC).

III-3 Dynamics of spread of the rotation-resistant variant in the U.S.
Spread of the rotation-resistant variant of the WCR in the U.S. is well documented (reviewed in
Levine et al. 2002), with the origin pinpointed to a 3-km2 area near Piper City in Ford County, IL
beginning in 1986 (Levine and Oloumi-Sadeghi 1996). According to the model of Onstad et al.
(1999), the rotation-resistant variant of WCR spread 10-30 km per year from 1986 to 1997
depending on the directions of the prevailing storms and winds. The maximum modeled rate is 33
km per year, which is much lower than the observations before 1979. Of course, the early invasion
of eastern US did not involve a major fitness cost associated with oviposition outside of cornfields.
Model results supported the hypothesis that the population of western corn rootworm infesting
soybean originated in Ford County, Illinois. The predictions of the simple model fit an independent
set of observations well on 3 of 4 fronts or directions up to 1997.
Onstad et al. (2003) improved the first model and used it to study hypotheses that attempt to explain
the spread of resistance through 2001. Their primary hypothesis is that increased landscape
diversity slows the rate of regional spread of the rotation-resistant western corn rootworm over
several years. The rate of spread of the western corn rootworm variant was significantly slower
from 1998-2001 than from 1986-1997. The rate of spread from 1986-1997 was approximately 27-
33 km/yr to the east and 8.5 km/yr to the west. From 1998-2001, the rate of spread slowed to less
than 16 km/yr to the east and 7.75 km/yr to the west.

IV Introduction routes of invading populations in Europe
  The description of the introduction routes of WCR allows determining the pathway and the
  number of independent introductions into Europe. It is crucial because, in the case of multiple
  introductions, the genetic variability and the probability that adaptive alleles (e.g. insecticide
  resistance alleles) are present in Europe may be larger than expected for a single invasion event.
  Multiple introductions also increase the probability of eventual introduction of the rotation-
  resistant variant and possibly of the northern corn rootworm into Europe. Given the timing and
  locations of first detections of WCR in Europe (Kiss et al. 2005), it was assumed that the isolated
  outbreak populations were “leap-frogging” out of the expanding Eastern Europe population.
  Studies of the introduction routes of WCR into Europe have relied on an Approximate Bayesian
  Computation (ABC) framework (Beaumont et al. 2002). The details of the ABC approach can be
  found in the supplementary online information that accompanies Miller et al. (2005). The ABC
  approach allows to combine genetic (microsatellite) and historical (years when European
  outbreaks of WCR were first detected) data. It can be used to make quantitatitve comparisons of
  different putative introduction scenarios and to estimate some of the demographic parameters
  associated with the introduction process.
  The initial analysis of introduction routes (Miller et al. 2005) made use of the microsatellites
  employed by Kim and Sappington (2005) plus an additional locus, identified from EST data by

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      USDA-ARS (Kim et al. 2007a). The analysis was confined to five Western European
  outbreaks: Roissy (south of Paris, detected in 2002), Alsace (eastern France, detected in 2003),
  Val d’Oise (north of Paris, detected in 2004), Piedmont (north-western Italy, detected in 2000)
  and Friuli (north-eastern Italy, detected in 2003). It was assumed that the extensive population in
  Central and south-eastern Europe (CSE Europe) was: a) a direct introduction from North
  America and b) a single introduction (samples from CSE Europe studied to this point were
  genetically homogenous). It was also assumed that the homogenous population that extends from
  the Corn Belt to the east coast of North America (Kim and Sappington 2005) represented the
  original source population.
  The results of this study were startling (Figure 1). There had been three direct introductions into
  Europe from North America: CSE Europe, Piedmont and Roissy. There had also been two intra-
  European introductions from Roissy into Alsace and from CSE Europe into Friuli.




  Figure 1: Most likely scenarios of invasion into Europe by the western corn rootworm, deduced
  from the Approximate Bayesian Computations. For each European outbreak, the arrow indicates
   its most likely origin and the posterior weights of the introduction scenarios are in parentheses.


  Subsequent to the initial study, a significant amount of work has been carried out on WCR’s
  introduction into Europe that is, as yet, unpublished. An important question is whether the
  assumption that the Corn Belt represents the original source population is valid. The
  determination of the source populations of the European outbreaks is crucial because it
  determines the adaptive characters that may be present or will be present in European western
  corn rootworms. The distribution of insecticide resistance and adaptation to crop rotation is
  known to be heterogeneous in the USA. Thus, depending on the origin of the European invading
  populations, the detrimental effects of the invasions may greatly vary. Moreover, if introductions
  from North America to Europe are a chronic event, the spread of insecticide resistance and
  rotation resistance throughout North America may be a real danger for European agriculture.
  The question of the origin of the European outbreaks has been addressed by calculating average
  assignment likelihoods [the probability of an individual’s genotype arising in a population with
  given allele frequencies (Rannala & Mountain, 1997)] for the three direct introductions towards
  the samples of Kim and Sappington (2005) plus a sample from Arizona. The results of this
  analysis indicated that the source population is unlikely to be Arizona or Texas. The most likely
  source population differed for the three introductions (Kansas for CSE Europe, Delaware for
  Piedmont and New York for Roissy). However, in each case the difference between the most
  likely source and the rest of the Corn Belt - East Coast region was not significant. Thus it may be
  concluded that the source for the European introductions lies somewhere in the homogenous
  Corn Belt - East Coast population, but we cannot be more precise at present.


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      A second important question, given the finding of multiple transatlantic introductions, is
  whether there has been more than one introduction into CSE Europe. This has been investigated
  by testing for heterogeneity of microsatellite allele frequencies between samples from within
  CSE Europe. Globally, there appears to have been a single introduction as allele frequencies
  were generally homogenous within CSE Europe. There was some weak but significant
  heterogeneity between samples from Serbia and those near (at the time of sampling) the
  expansion front in Hungary and Austria. This heterogeneity appears to be due to an isolation-by-
  distance effect rather than multiple introductions.
  The demographic parameters of the introduction process are also of interest. In particular,
  knowledge of the minimum number of founders needed for a successful introduction and the
  typical time delay between introduction and detection would be helpful to European plant
  protection agencies. The models of introduction used to analyse the introduction routes into
  France and Italy were rather complex. They included parameters for the population size in North
  America, the number of founders arriving in Europe, the duration of the post-introduction
  bottleneck, the stable size of the European populations, the time delay between introduction and
  detection and several mutational parameters. Unfortunately, the microsatellite data did not
  contain information concerning many of these parameters. In particular, there was no
  information on the growth, post-introduction, of the European populations, which were best
  modelled as having a constant size equal to the initial number of founders. As a consequence,
  there was no information on the delay between introduction and detection. Nevertheless,
  estimates of the number of founders could be obtained and were reasonably precise. The
  estimates from CSE Europe, Piedmont and Roissy (with 5% and 95% quantiles in parentheses)
  were 37 (26 and 56), 31 (22 and 49) and 28 (19 and 53) respectively. Because of the lack of
  information on population growth these values are means of effective population size of the
  European outbreaks over the lifespan of these outbreaks. In other words they are overestimates
  of the true number of effective founders. Estimates of the minimum number of founders were
  obtained assuming a bottleneck’s length of one generation. The estimates from CSE Europe,
  Piedmont and Roissy were 3 (2 and 6), 5 (3 and 8) and 6 (4 and 16) respectively. Because a
  bottleneck’s length of only one generation is highly improbable in the case of WCR these
  numbers are underestimates of the true number of effective founders. This analysis does at least
  suggest that several individuals, as oppose to one or two fertilised females, are needed for a
  successful introduction.

V References Cited
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      Yakobtsuk, V. I., A. J. Sikura, B. Pai, and J. Kiss. 2006. If the western corn rootworm
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Genetics: Tools and applications
I Variable genetic markers + population genetics description:

I-1 Microsatellites
Microsatellites are short tandem repeats of DNA sequences, which are highly polymorphic, and
evenly distributed and abundant in genomes, occupying ~50,000-100,000 loci in eukaryotes (Litt
and Luty 1989). Microsatellites can be scored relatively easily using PCR amplification followed
by genotyping with an automated sequencer. For these reasons and because of their co-dominant
mode of inheritance, microsatellite loci are generally the markers of choice for numerous
applications in ecological genetics (Parker et al., 1998; Goldstein and Schlötterer, 1999; Zhang and
Hewitt 2003, Lowe 2004), and are often employed in linkage mapping and quantitative trait loci
studies (Primrose 2003). They are especially valuable for inferring levels of genetic variation and
patterns of population structure among closely related or recently diverged populations (Roderick
1996, Haig 1998, Donnelly and Townson 2000, Kim and Sappington 2005b, 2006). These markers
can be used to estimate rate and patterns of gene flow, and to directly identify immigrants in a
population and their likely region of origin using population assignment analyses (Waser and
Strobeck 1998, Davies et al. 1999, Paetku et al. 2004, Nardi et al. 2005, Miller et al. 2005, Kim et
al. 2006).
The introduction and continuing range expansion of WCR in Europe, concern that WCR may
develop resistance to transgenic Bt-corn, and ongoing spread of the rotation-resistant variety in
North America have combined to put a premium on obtaining estimates of gene flow and
understanding population genetic structure of this insect (Sappington et al. 2006). Several
laboratories in the U.S. and Europe were interested in developing microsatellite markers for WCR
so that population genetics studies could be performed. One disadvantage of microsatellites as
markers is that they are expensive and time-consuming to develop. They are usually species-
specific, so they must be developed anew for each species (Zane et al. 2002), although certain loci
sometimes can be amplified across closely related species. The Diabrotica Genetics Consortium
was organized in 2004 in part to coordinate efforts in marker development (Sappington et al. 2006).
Kim and Sappington (2005a) tested 17 microsatellites from 54 that were initially isolated using the
biotin-enrichment methods of Kijas et al. (1994) and Ronald et al. (2000), and found that nine of
these will likely be useful in population genetics studies. The others showed signs of harboring null
alleles, which are caused by a mutation in the flanking region of the microsatellite locus that
prevents primer binding and PCR amplification (Callen et al. 1993, Behura 2006). An individual
with a null allele is often falsely genotyped as a homozygote at that locus, when in fact it may be a
heterozygote with the real allele being artifactually invisible. Null alleles thus bias the results in
population genetics analyses, and microsatellites that are known to have one should be avoided
(Pemberton et al. 1995, Liewlaksaneeyanawin et al. 2002, Dakin and Avise 2004). About 59% of
WCR microsatellites have null alleles (Kim et al. 2007a). U. Stolz, E. Waits, and M. Bagley of the
USEPA have developed a number of microsatellites from a combined MCR and NCR genomic
library (unpublished data), and these are expected to be published soon. It is anticipated that the
markers that amplify MCR loci also will amplify in WCR, as was the reciprocal case for previously
developed WCR markers, all of which amplified MCR loci (Kim and Sappington 2005a).
Microsatellites designed from sequences mined from a WCR EST database were tested against
conventionally derived microsatellites, and performed comparably in population genetics
applications (Kim et al. 2007a). Thus, EST data mining represents a less-expensive alternative for


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      future marker development, and one such marker has already seen service in population
studies of WCR (Miller et al. 2005).
Because coordination of marker development occurred early for WCR, the opportunity was siezed
upon to develop a standard core set of microsatellite markers to be recommended for use in future
population genetics studies (Sappington et al. 2006). Laboratories in the U.S. (USDA-ARS, Iowa,
and USEPA, Ohio) and France (INRA, Sophia-Antipolis) evaluated 22 potential markers for six
desireable characteristics: high polymorphism, readability and repeatability on different sequencing
systems, lack of null alleles, selective neutrality, cross-species amplification, and no linkage
between loci. Six markers performed well under these strict criteria (Kim et al. 2007b). Use of
these markers in future studies will facilitate comparisons of data generated by different
laboratories, and will allow direct sharing of genotype data, which can save significant time and
resources.

I-2 AFLP
A second class of molecular marker that is being applied to studies of WCR is Amplified Fragment
Length Polymorphism (AFLP) (Vos et al. 1995). Briefly, the AFLP technique involves digesting
genomic DNA with a pair of restriction enzymes. Synthetic oligonucleotide adapters are then
ligated to the sticky ends of the digested DNA. Because the sequences of the adapters are known,
they can then be used as targets for PCR primers. There is considerable scope for “tuning” AFLP
protocols to suit organisms with different genome sizes and base compositions. In particular, the
choice of restriction enzymes or the number of “selective” bases at the 3’ end of the PCR primers
(extending beyond the adapters) can be varied to increase or decrease the number of fragments that
are amplified during a single PCR reaction. Typically, one of the PCR primers is radioactively or
fluorescently labeled to allow visualization and sizing of the amplified fragments by traditional gel
electrophoresis or using automated DNA sequencer technology.
The term Amplified Fragment Length Polymorphism is, in a sense, a slight misnomer. The
observed polymorphisms are not generally in the length of the amplified fragments but in their
presence or absence. This is due to sequence polymorphisms at the recognition sites for the
restriction enzymes. If a recognition site is missing, a much larger fragment (primed from the next
recognition site) is produced. In general, these larger fragments will fall outside the size range being
visualized and effectively are lost. Consequently, unlike microsatellites, AFLP markers are
inherited as dominant markers, in that both homozygotes for the positive allele and heterozygotes
for the positive and negative alleles produce a single fragment, and therefore cannot be
distinguished.
The key advantage of AFLP is that it can provide hundreds of polymorphic markers quickly and
cheaply. This makes it an attractive system for use in linkage mapping, genome scanning and other
applications that demand large numbers of markers. The dominant nature of AFLPs may be a
disadvantage in population studies, especially if no co-dominant markers are available to verify that
populations conform to Hardy-Weinberg expectations.
The use of AFLP to study WCR is in its infancy. To date, no studies have been published.
Nevertheless, some unpublished results are available. An AFLP protocol designed for Colorado
potato beetle (Hawthorne 2001) has been used successfully to study WCR in both David
Hawthorne’s laboratory at the University of Maryland (pers comm), and at INRA (Miller et al.
2007). This protocol uses two six-cutter restriction enzymes, EcoRI and PstI with two or three
selective nucleotides.

I-3 Cytoplasmic markers
Mitochondrial DNA and more generally DNA from cytoplasmic organelles present desirable
properties (Hartl & Clark, 1997, p361). Cytoplasmic organelles are inherited maternally so that

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       ancestry relationships are more easily inferred from cytoplasmic than from nuclear
genetic material: mitochondrial or cytoplasmic bacteria gene lineages correspond to female
individual lineages while nuclear gene lineages do not simply correspond to individual lineages.
Moreover cytoplasmic organelles (very often) do not recombine (Wallis, 1999) so that each of them
can be considered as a single locus. Phylogenetic or phylogeographic relationships are thus directly
inferred from similarity of cytoplasmic DNA sequences between tatxa or between individuals.
Additional properties make mitochondrial DNA a good candidate for population genetics analysis:
-it evolves rapidly and thus shows a large amount of variability,
-it is a short molecule thus it is easy to study
-it is present in several copies in each cell so that it is easy to extract and conserve
Several cytoplasmic molecular markers have been used to elucidate the phylogenetic relationship
and population genetic diversity of the western corn rootworm, Diabrotica virgifera, and the
Mexican corn rootworm, Diabrotica virgifera zeae, as well as the diversity of the endosymbiotic
bacteria Wolbachia that occurs in some populations of these two subspecies.
The mitochondrial cytochrome oxidase I (COI) gene and the second internal transcribed spacer
region (ITS-2) have been used to infer the phylogenetic relationship of thirteen Diabrotica species
belonging to the fucata and virgifera species group (Simon et al. 1994; Clark et al. 2001b). The
mitochondrial genes ATPase 6 and 8, and ND5 were evaluated as to their level of genetic diversity
in D. virgifera populations and were found to be lower than those obtained for COI (Giordano R.
unpublished).
Wolbachia is an intracellular bacterium that is widespread in arthropods and can cause several sex
altering phenotypes (Stevens et al. 2001). The most common phenotype is cytoplasmic
incompatibility. Wolbachia infected females can successfully mate with both infected and
uninfected males and produce infected offspring, while uninfected females are at a disadvantage
and can only mate with likewise uninfected males. As a result, Wolbachia spreads or drives into
uninfected populations of insects, carrying with it the concomitant host cellular cytoplasmic
components such as mitochondria. Thus, the spread of a single strain of Wolbachia in an existing
naïve population, or the spread of an invasive insect species from a few founder individuals,
infected with a single strain of Wolbachia, can be tracked by measuring mitochondrial haplotype
diversity.
Wolbachia bacteria in western and Mexican corn rootworm populations have been identified using
the following genes: The 16S rRNA gene was amplified using primers 21F and 994R and 99F and
1492R (O’Neill et al. 1992; Giordano et al. 1997). The ftsZ gene, whose product contributes to the
septation of bacterial cells was amplified using primers ftsZf1 and ftsZr1(Werren et al. 1995;
Giordano et al. 1997). The wsp gene encoding the major surface protein of Wolbachia has been
amplified using primers 81F and 691R (Braig et al. 1998; Zhou et al. 1998; Giordano R.
unpublished).

I-4 Applications
I-4-1- Microsatellites
Several published and unpublished studies have used microsatellites to study WCR populations. All
these studies use some of the six loci proposed for the standard core set. However, none have used
the core set in its entirety, which has only recently been formulated.
Kim and Sappington (2005) used seven loci to investigate the degree of geographical population
genetic structuring in the United States. They examined samples at a broad spatial scale ranging
from Texas to New York. They found very little significant genetic differentiation between samples
ranging from the Corn Belt to the east coast, although their sample from Illinois was significantly
different from those from Pennsylvania and Delaware. A principal component analysis also
indicated that the Illinois sample was slightly different from the rest of the Corn Belt.

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       Microsatellites also were used by Miller et al. (2006) to study WCR population structure
at a finer geographical scale. The main objective of this study was to test whether the rotation
tolerant variant form of WCR is a genetically distinct population. Samples of adult WCR were
taken from paired maize and soybean fields in the region of Illinois where the variant was present.
An additional sample was obtained from western Illinois, where the variant was absent at the time.
To increase the number of wild type samples, the Iowa and Ohio data of Kim and Sappington
(2005) also were included in the analysis. In keeping with the results of Kim and Sappington
(2005), the study found that there was no significant geographical population structure.
Furthermore, there was no evidence for a genetically distinct variant population.
A study carried out at INRA (Miller et al. 2007) compared samples of pupae from rotation tolerant
populations in Illinois and wild type populations in Iowa. A weak but statistically significant
genetic differentiation was detected between the two states. This result differs from those of Kim
and Sappington (2005) and Miller et al. (2006) in terms of statistical significance. However, it
should be noted that Kim and Sappington (2005) did find Illinois to be slightly (albeit non-
significantly) different from the rest of the Corn Belt. The slight discrepancy between the published
studies and this one may indicate that there is a low level of general genetic differentiation between
variant and wild type WCR. The Miller et al. (2007) study sampled pupae, ensuring that the
oviposition site was known. The earlier studies used adults which, in Illinois, may have been
admixed samples of variants and wild type.
There are several ongoing studies of WCR, using microsatellites that are, as yet, unpublished.
Researchers at US-EPA in Cincinnati are engaged in several projects. One of these is a study of
geographical population structure, similar to that of Kim and Sappington (2005) but with a larger
number of samples. In a report to the Diabrotica genetics consortium they stated “Preliminary
analysis shows that there is a low yet significant amount of population differentiation across the
USA, with New Mexico, Texas and southern Kansas sites showing the greatest difference from the
rest of the North American collections.” They also reported that they have “estimates of population
sizes based on temporal comparisons.” They also plan to use microsatellites in a project to map
QTL involved in tolerance to Bt-transgenic maize.
Microsatellites also have been used to study European WCR populations. The details of this work
are described in the section “Introduction routes of invading populations in US & Europe”.
Microsatellites were used to show that there have been multiple introductions of WCR into Europe
from North America (Miller et al. 2005), and to estimate some of the demographic parameters
associated with these introductions (Ciosi, unpublished).
I-4-2- AFLP
Miller et al. (2007) used AFLP for a genome scan to seek markers associated with the rotation
tolerant variant. Although this project did not find any markers that were strongly linked to the
variant, it did demonstrate that AFLP is a valid and feasible technique for studying natural
populations of WCR. Further details of this study are given in the section “Recent Adaptive
Characters”. Researchers at the University of Maryland and US-EPA in Cincinnati are using AFLP
to map genes for resistance to insecticides and transgenic Bt maize respectively. The status of these
projects are unknown. There is also a project at Purdue University to use AFLP to seek markers
associated with variant WCR. Again, the status of this project is unknown.
I-4-3 Cytoplasmic markers:
-Phylogenetic studies
Most published work using cytoplasmic markers in WCR is related to phylogenetic studies and
molecular species determination. PCR-RFLP on ND4 (Szalanski & Powers 1996), 12S-N4,
CB2HC1 (Szalanski et al. 1999) and COI (Clark et al. 2001a) were used to find diagnostic markers
of Diabrotica species with various degrees of success. ND4 locus allowed distinguishing among D.
undecipunctata, D. barberi and WCR (Szalanski et al. 1996) and the COI gene studied by Clark et

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       al. (2001a) provided a key to distinguish among 12 species of Diabrotica. However
Szalanski et al. (1999) could not find any diagnostic maker to distinguish MCR from WCR nor any
differentiation between WCR subpopulations. DNA sequences obtained from COI (Szalanski et al.
2000; Clark et al. 2001b; Gillespie et al. 2003) and COII (Szalanski, 2000) proved useful to infer
phylogenetic relationships between Diabrotica (Szalanski et al. 2000; Clark et al. 2001b) and
Luperini (Gillespie et al. 2003) species.
-Wolbachia and mitochondrial DNA diversity in populations of D. v. virgifera and D. v. zeae.
Though similar in many respects (Lance et al. 1992, Spurgeon et al. 2004), there are some
ecological and physiological differences between D. v. virgifera and D. v. zeae the western and
Mexican corn rootworm (Woodson and Chandler 2000). Based on differences in elytral maculation
patterns, the absence of pre-mating barriers to reproduction, unidirectional incompatibility and
geographic distribution, Krysan et al. (1980) designated the two populations of D. v. virgifera and
D. v. zeae as subspecies. This subspecies characterization is useful because the latter superficially
resembles D. barberi and some populations of D. longicornis, while populations of D. longicornis
from New Mexico, Arizona and Mexico have black elytral markings similar to D. v. virgifera.
Both D. v. virgifera and D. v. zeae are thought to have originated in Central America and invasively
spread in the U.S. in relatively recent times (Smith 1966, Branson and Krysan 1981, Krysan and
Smith 1987). First collected in western Kansas in 1865 by Le Conte (Krysan and Smith 1987), D. v.
virgifera began to expand its range concomitantly with the spread and increase of continuous maize
production in North America in the mid 20th century (Chiang 1973; Metcalf 1983), and reached the
east coast by about 1990. In the Americas D. v. virgifera and D. v. zeae are now distributed from
Arizona and the Dakotas to New York and Virginia and into southern Mexico (Krysan and Smith
1987). Early genetic studies of D. v. virgifera and D. v. zea populations showed little genetic
diversity within and between the two subspecies (Krysan et al. 1989, Szalanski et al. 1999). This,
in spite of the fact that some local populations show unique phenotypic characteristics such as
pesticide resistance (Ball and Weekman 1963; Meinke et al. 1998), crop-rotation-adaptation
(Stewart et al. 1995; Levine and Oloumi-Sadeghi 1996), and presence or absence of infection with
Wolbachia (Giordano et al. 1997).
Giordano et al. (1997) tested whether the unidirectional incompatibility first determined by Krysan
et al. 1980, between D. v. virgifera and D. v zeae was due to the presence of Wolbachia bacteria
present in populations of D. v. virgifera. They found that full compatibility was restored when
tetracycline treated and thus uninfected D. v. virgifera males mated with D. v. zeae females. A
study of D. v. virgifera and D. v. zeae populations throughout the U.S. showed that a single strain of
Wolbachia (wDiavir) is present in D. v. virgifera beetles from northern Texas to NY, while
populations in southeastern Arizona and populations of D. v. zeae in Oklahoma and Texas are not
infected. Moreover, populations of D. v. virgifera in the northern Mexican state of Durango, where
a hybrid zone occurs between D. v. virgifera and D. v. zeae (Krysan and Smith 1987), were also
found to be uninfected with Wolbachia.
Using historical information with regards to the recent spread of D. v. virgifera in the corn belt and
the pattern of Wolbachia infection distribution, Giordano et al. (1997) proposed that the infected
population of D. v. virgifera in the northern U.S. had likely undergone at least a cytoplasmic
bottleneck and that as a result the genetic diversity of this very large population would be reduced
with respect to its probable uninfected parental population in southeastern Arizona or Mexico. To
test this hypothesis we conducted a study of mitochondrial haplotype distribution of populations of
D. v. virgifera and D. v. zea in the U.S. and the three Mexican States of Durango, Jalisco and
Oaxaca, using the mitochondrial genes, cytochrome oxidase I (COI) (1247 bp) and ATPase 6 and 8
(949 bp). In the regions we sampled we have detected a total of 33 COI and 10 ATPase haplotypes.
Both genes indicate a similar pattern of geographic mitochondrial variation. This data indicates that
the highest degree of variation is found in populations of D. v. virgifera and D. v. zea in
Southeastern Arizona and Mexico while the lowest levels are to be found in the Corn Belt.

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       Given the historical record and likely region of origin of D. v. virgifera it is not
surprising to find higher genetic variation in Mexico, but it is surprising to find comparable levels
of variation in southeastern Arizona as in Mexico. This distribution of mitochondrial variation
supports our hypothesis that the Wolbachia infected D. v. virgifera populations in the Corn Belt of
the US harbor less variation than its purported uninfected parental population to the south, and that
this is the consequence of a bottleneck which occurred prior to the spread of the infected progeny of
possibly a single infected female. Moreover, this preliminary analysis of COI haplotypes recovered
from the D. v. virgifera and D. v. zeae populations sampled indicates that geography is a better
predictor of genetic relationship than the subspecies designation. It is also not surprising that the
Wolbachia infection profile of these beetle populations is also more complex in Mexico. In North
America we have detected uninfected and singly infected D. v. virgifera populations. In Mexico we
have found unifected D. v. virgifera in the northern state of Durango and uninfected D.v. zeae in
Jalisco. While individuals of D. v. zeae sampled from Oaxaca were found to be uninfected, or
infected with one or two new strains of Wolbachia, wDiazea1 and wDiazea2. Up to this point, our
data lead us to conclude that these Wolbachia infections have only recently been introduced in this
population.
Genetic information of cytoplasmic maternally inherited components, mitochondria and Wolbachia,
coupled with nuclear markers, obtained from populations on a broad spatial scale, can be used to
more accurately detect population differentiation and bottlenecks in populations of D. v. virgifera
and D. v. zeae.

II Genomic resources
The genomes of living organisms contain many elements, including genes coding for proteins. The
portion of genes expressed as mature mRNA, collectively known as the transcriptome, represents
only a small percentage of the complete genome but contains much of the information of interest
(Joneneel 2000). A snapshot of the transcriptome of a particular tissue or cell type can be obtained
by producing a cDNA library and sequencing a sufficiently large number of individual clones to
ensure that most of the information present in the library has been extracted. Because there can be
many genes from which more than one EST is derived, clustering of overlapping sequences through
computation comparison of overlapping ends and based on the degree of sequence identity or
similarity is employed to identify contiguous sequences or “contigs.” Unique sequences or
“unigenes” are represented by sequences identified in only one clone. At present, EST databases
developed from adult heads (S. Ratcliffe, University of Illinois; personal communication) and from
the larval guts (Siegfried et al. 2005) represent the only genomic resources currently available for
western corn rootworms.
The first and currently most extensive EST database was developed from adult head tissues. A
normalized cDNA library was constructed using the RNA extracted from the heads of adult western
corn rootworms, and random sequencing of individual clones from the cDNA library generated the
EST sequences. A total of 16,172 high quality sequences were obtained. After assembly, a unique
set of 6,397 clones were identified. Gene Ontology (The Gene Ontology Consortium 2001;
http://www.geneontology.org) was used as the primary annotation for the assembled EST set to
assign a molecular function and biological process associated with each unique sequence. The 6,397
unique sequences were used to BLAST (Basic Local Alignment Search Tool) against known
sequences from Drosophila melanogaster. From the BLAST results, 3,197 sequences have at least
one hit (E < 10-6; the lower the E value, the more significant the score) and 2,013 sequences have
been associated with at least one Gene Ontology term. There are 1,413 unique GO terms that are
represented by the data set. The EST database can be searched for annotation and similarity
comparison by contacting Dr. Susan Ratcliffe, University of Illinois, Department of Entomology.



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      The second EST database was derived from larval gut tissue. The cDNA library from
mRNA extracted from dissected D. v. virgifera larval midguts contained 2.53 x 1010 primary clones.
Sequencing of 2,880 clones was conducted of which 1,528 usable sequences were assembled into
190 contiguous sequences (contigs) and 501 unique sequences. The average length of readable
sequences was 635 nucleotides. Each unique sequence was searched against the non-redundant
GenBank database using the BLAST algorithm. Of the 691 unique sequences, 27% (187) did not
return any significant (E≤10-5) BLASTX match. Of the remaining 504 sequences, 71% had best
matches to insect sequences; specifically 42% to Drosophila melanogaster and 29% to other
insects. Those sequences returning a significant BLASTX match were ascribed a putative
biological process and molecular function (The Gene Ontology Consortium 2001;
http://www.geneontology.org) based on the single “best hit” match. Molecular functions
correspond to activities that can be performed by individual gene products, while biological process
are accomplished by one or more ordered assemblies of molecular functions. Strikingly, 80% of the
sequences predicted proteins with either catalytic activities (61.8%) or binding functions (18.8%).
Correspondingly, 74% of sequences were predicted to encode proteins involved in either
metabolism (64.5%) or transport (9.1%).
Recently, a cDNA microarray chip was constructed using from a combination of unique cDNA’s
from adult head and larval midgut tissues representing approximately 6600 genes (G. Robinson,
University of Illinois; personal communication). This array has been used to compare hybridization
of cDNA samples from previously identified as organophosphate susceptible and resistant D. v.
virgifera strains to identify key genes that are differentially expressed and which may be associated
with resistance. Results from comparison revealed an esterase gene among the genes most
significantly different in expression between the two strains. Differential expression was verified
by quantitative RT-PCR. Enhanced activity of esterase isozymes has been previously shown to be
associated with resistance (Zhou et al. 2002), although specific esterase genes have not previously
been identified. These results illustrate the utility of the array for comparing gene expression
among different populations of interest.
A preliminary estimate of the size of the D. v. virgifera genome indicates that it is rather large (~2.5
Gbp). Rapid improvements in the efficiency of genome sequencing and reduced costs may make
such an effort more attractive in the future. EST databases will continue to be important resource
for identifying genes of interest. However, the EST databases must be expanded to include other
tissues (e.g., larval fat body) and additional sequencing of existing libraries should be conducted to
ensure more complete transcriptome coverage.

-Internet-based glossaries of Genomic and Bioinformatic terms:
NCBI: http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/glossary2.html
City University of New York - College of Staten Island:
http://www.library.csi.cuny.edu/~davis/Bio_326/bioinfo_glossary.html

III Laboratory colonies selected for insecticide resistance, rotation resistance, and
non-diapause.
Though not a trivial undertaking, WCR can be reared continuously in the laboratory (Jackson
1986), and a number of colonies are maintained in the U.S. and Europe. The USDA-ARS North
Central Agricultural Research Laboratory (NCARL) (formerly the Northern Grain Insects Research
Laboratory) in Brookings, South Dakota maintains many colonies, including the original line
selected for non-diapause in the early 1970s (Branson 1976). WCR undergoes an egg diapause
(Krysan 1978). A chill period of 4 months is needed to synchronize hatch (Krysan et al. 1984,
Jackson 1986), resulting in long generation times which are often inconvenient for research
activities. Branson (1976) selected a laboratory population for short diapause-duration, creating a

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       "non-diapause" colony after nine generations. Six generations per year are possible with
this line (Branson et al. 1981), making it attractive for use by researchers. However, by 2003 this
line had been in culture without outcrossing for about 190 generations (Kim and Sappington 2007),
so loss or change of genetic make-up of the colony has been a concern. Larvae from artificial
infestations of non-diapause eggs have compared favorably with wild-type populations in field tests
(Hibbard et al. 1999), except when soil temperatures are low at time of infestation (Branson et al.
1981). Kim et al. (2007c) used microsatellite markers (Kim and Sappington 2005a,b, Miller et al.
2005) to compare genetic variability in the non-diapause line at NCARL with that of other NCARL
diapause colonies and wild populations. The non-diapause colony has lost about 15-39% of its
genetic diversity relative to wild populations, depending on the measure, but neutral genetic
variation in the diapause colonies is similar to that of wild populations. If researchers desire a non-
diapause colony for selection experiments, it is recommended that they introgress the non-diapause
trait into a wild-type background to increase the amount of genetic variation available for selection,
rather than working with the non-diapause colony directly (Kim et al. 2007c).
Insecticide resistant strains of western corn rootworm are currently maintained by NCARL. These
strains have been characterized for resistance both to the cyclodiene insecticide, aldrin and the
organophosphate insecticide, methyl-parathion. Resistance characterization is based on dose-
response assays for aldrin, diagnostic methyl-parathion bioassays and on biochemical
characterization of non-specific esterase activity that is associated with methyl-parathion resistance.
The initial identification of organophosphate resistance among field populations was based on
survival at a diagnostic concentration of methyl-parathion, derived from the LC99 from what was
considered to be a susceptible population (Zhou et al. 2002). This technique identified several
populations that exhibit >90% survival at the diagnostic concentration. However, because the assay
was not completely discriminating among resistant genotypes, it is not possible to determine the
relative frequency of resistance among field collections. Because the resistance is at least partially
conferred by increased hydrolytic metabolism (Miota et al. 1998), biochemical assays that visualize
esterase isoenzymes from individual beetles on native polyacrylamide electrophoresis gels indicate
that there is high frequency of resistance alleles. As with the diagnostic insecticide bioassays, this
technique does not distinguish heterozygous individuals from resistant homozygotes (Parimi et al.
2003), and it is not possible to estimate allele frequencies using this technique.
Results of aldrin bioassays from both field and laboratory populations (Parimi et al. 2006) indicate
the presence of high levels of resistance. Aldrin resistance has remained consistently high among
field populations over the four decades since aldrin resistance was first reported and with drastically
reduced selective pressures since the chemical class was banned in 1972. However, there is
apparently considerable variation in resistance levels and a general decline in resistance among
Nebraska populations where resistance was first identified. If resistance to cyclodienes were
associated with a selective disadvantage for resistant phenotypes in the absence of the insecticide,
the frequency of resistance alleles in natural populations will decline over time following the
cessation of insecticide usage (McKenzie, 1996). The only population to exhibit complete
susceptibility to aldrin was the non-diapause laboratory strain that has been in culture since 1968.
This strain was derived from a field collection made in an area where resistance was reported to
have been present at the time of collection (Metcalf 1986). Because up to six generations of the
non-diapause strain can be reared in the laboratory in a single year (Branson et al. 1981), slight
fitness disadvantages may have been manifested in the loss of resistance over a shorter period of
time relative to univoltine field populations. It should also be noted that the non-diapause
population has likely undergone a rather restrictive genetic bottle-neck during selection for the non-
diapause trait (Kim et al. 2007c). Therefore, the genes conferring resistance could have been lost
during selection for the non-diapause trait and are unrelated to possible fitness disadvantages.
WCR larvae have been successfully reared wholly and partially on transgenic Bt-corn for more than
eight generations at the USDA-ARS, Plant Genetics Research laboratory in Columbia, Missouri (B.

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      Hibbard, unpublished data). Four colonies were initiated from reciprocal crosses
between wild-type beetles collected in Kansas and beetles from the NCARL non-diapause colony
(Branson 1976). Combined F1 progeny produced 241,000 eggs (called generation 0) from which
four colonies were initiated. Each of the colonies is fed optimally as adults, but differs in larval
diet: non-Bt (isoline) only; exposed to Bt corn as neonates but reared on isoline; Bt corn only from
second instar to pupation; and reared solely on Bt corn. These are referred to as Colonies 0, 1, 2,
and 3, and each colony has been under selection for at least eight generations.
After three generations, larvae from each colony were evaluated for survival and weight gain on
both isoline and Bt corn (B. Hibbard, unpublished data). In greenhouse trials, more WCR larvae
were recovered from isoline corn than from Bt corn for each colony, except Colony 3, the colony
reared totally on Bt corn. The number of Colony 3 larvae recovered from Bt corn was not
significantly different than for any of the colonies recovered from isoline corn. In addition, for each
colony except Colony 3, the average dry weight of larvae recovered from isoline corn was
significantly greater than those recovered from Bt corn. Not only were more Colony 3 larvae
recovered from Bt corn, but they weighed more and had a greater head-capsule width than larvae of
other colonies emerging from Bt corn. Interestingly, the average dry weight of Colony 2 larvae
recovered from Bt corn was also significantly greater than the average dry weight of Colony 0 or 1
larvae recovered from Bt corn. In subsequent evaluations of the progeny of generation 6, the level
of resistance of the colony fully reared on Bt corn was between 12 and 38 fold in the field, as
indicated by relative survival on Bt corn vs. isoline corn.
Preliminary analyses were conducted (Kim and Sappington, unpublished data) to characterize
genetic diversity at six microsatellite loci within Missouri Colonies 0 and 3 after three generations
of selection, using methods similar to those of Kim et al. (2007c). Number of alleles and
heterozygosity decreased slightly within both colonies after three generations, but more so in
Colony 3. Pairwise comparisons indicated significant genetic differentiation between the starting
Colony and Colony 3 after three generations of selection. Despite the loss of genetic diversity in
the Missouri colonies compared to wild populations, Colony 3 responded rapidly to selection on Bt
corn as described above (B. Hibbard, unpublished data).

IV References Cited
Ball, H. J. and Weekman, G. T. 1963 Differencial resistance of corn rootworms to insecticides in
Nebraska and adjoning states. Journal of Economic Entomology 56: 553-555.
Behura, S. K. 2006. Molecular marker systems in insects: current trends and future avenues. Mol.
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Recent adaptive characters: resistance to pesticides and crop rotation
I Corn rootworm biology and management.
The western corn rootworm, Diabrotica virgifera virgifera LeConte, is arguably the single most
important pest of field corn, Zea mays L. (Levine and Oloumi-Sadeghi 1991, Sappington et al.
2006), throughout most of the U.S. Corn Belt both in terms of crop losses and the use of synthetic
insecticides. Managing corn rootworm populations to minimize risk of economic loss is extremely
difficult, in part because of its nearly unlimited capacity to evolve resistance both to chemical
insecticides (Metcalf 1986, Meinke et al. 1998, Parimi et al. 2005, Siegfried 2005) and cultural
control practices such as crop rotation (Levine et al. 2002). Recent management practices have
relied extensively on neurotoxic and nonspecific synthetic insecticides that are directed against both
larvae and adults. Corn rootworm management strategies that include prescriptive insecticide
applications have not been widely adopted (Gray and Steffey 1995) placing increased pressure on
the limited number of options available to growers.
Novel control techniques are being developed and marketed for corn rootworm management. The
two most recent and significant developments involve transgenic corn hybrids expressing
insecticidal genes from Bacillus thuringiensis (Bt) and seed treatments employing neonicotinoid
insecticides. Both technologies have the potential to drastically reduce the environmental and
human health risks associated with conventional rootworm management practices (e.g., soil
insecticides). However, the remarkable history of western corn rootworm adaptation to the selective
pressures imposed by recent pest management practices (reviewed below) necessitates the proactive
implementation of management strategies designed to sustain these novel management alternatives.
Moreover, because of the invasive nature of this pest, proactive intervention for the purposes of
mitigating invasions or minimizing the spread of resistance outbreaks are likely to be important
aspects of future management decisions.

II Rootworm Adaptation: Cyclodiene Resistance.
Cyclodiene insecticides were commonly used as soil treatments for the control of both western and
northern corn rootworms from the late 1940s to early 1960s. Benzene hexachloride (Muma et al.,
1949), aldrin, chlordane (Ball and Hill, 1953) and heptachlor (Ball and Roselle, 1954) were the
recommended active ingredients for control of root feeding larvae during this period. Control
failures with these compounds were first noted in Nebraska in 1959 (Roselle et al., 1959), and
further evaluations in 1960 (Roselle et al., 1960) and 1961 (Roselle et al., 1961) revealed the
magnitude and rapid development of the resistance. During 1961, western corn rootworm adults
were collected from different fields in Nebraska and susceptibility to aldrin and heptachlor was
determined by topical application (Ball and Weekman, 1962; 1963). Differences in susceptibility
among field populations provided the first direct evidence of resistance evolution.
The development of cyclodiene resistance coincided with a rapid eastward range expansion. By
1980 the distribution of D. v. virgifera covered most of the U.S. Corn Belt, including areas where
cyclodienes were not widely used as soil insecticides (Metcalf 1986). Resistance has persisted in
populations for many years after the use of these compounds was discontinued (Siegfried and
Mullin 1989) even in areas where the insecticides were not commonly used as soil insecticides.
Parimi et al. (2006) reported the presence of high levels of resistance in both the laboratory-reared
and field-collected adult western corn rootworms based on topical bioassays with the cyclodiene
insecticide, aldrin. Aldrin resistance apparently has remained consistently high among field
populations over the four decades since resistance was first reported. These high resistance levels
have persisted in spite of reduced selective pressures since the chemical class was banned in 1972.

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       However, considerable variation in resistance levels among populations was detected; a
general decline in resistance among Nebraska populations and consistently higher levels of
resistance in more eastern populations were noted. The general trend for higher resistance levels
among populations where selection pressures are believed to have been lowest is puzzling. The use
of broadcast applications of cyclodiene insecticides was generally confined to the western Corn Belt
where resistance was first identified, and the higher resistance levels in eastern North America seem
counter to the geographic gradient in selection intensity.
The only population examined to exhibit complete susceptibility to aldrin was a non-diapause
laboratory strain that was established from field collections in 1968. This strain was derived from a
field collection made in an area where resistance was reported to have been present at the time of
collection (Metcalf 1986). Because up to 4 generations of the non-diapause strain can be reared in
the laboratory in a single year, slight fitness disadvantages may have been manifested in the loss of
resistance over a shorter period of time relative to field populations. It should also be noted that the
non-diapause population has likely undergone a rather restrictive genetic bottle-neck during
selection for the non-diapause trait. Therefore, in selecting for a non-diapause trait, the genes
conferring resistance could may have been lost and the susceptibility of this strain unrelated to
possible fitness disadvantages.
The mechanisms of cyclodiene resitsance in western corn rootworms remains uncertain.
Comparisons of detoxification enzyme activities and in vivo and in vitro aldrin metabolism among
resistant western corn rootworms and the closely related but susceptible northern corn rootworm
revealed no consistent differences (Siegfried and Mullin 1990). Recent research has suggested that
target site insensitivity associated with GABA (γ-aminobutyric acid) receptor-ionophore is a likely
resistance mechanism. As a receptor for the major inhibitory neurotransmitter in insects, the
GABA receptor is an important target for a number of insecticides including the cyclodienes
(ffrench-Constant et al., 2000 and Ramond-Delpech et al. 2005). Since a GABA-receptor subunit-
encoding a resistance-associated mutation (Rdl) was first isolated from a dieldrin resistant strain of
Drosophila melanogaster (ffrench-Constant et al. 1991), Rdl-like mutations have been found in
several other insect orders (ffrench-Constant et al. 2000). Although cyclodiene resistance is
historically very widespread and in the past accounted for over 60% of reported cases of resistance
(Georghiou 1969), cyclodienes themselves have been largely withdrawn from use, and therefore, in
relative terms, the overall number of cyclodiene resistant species has been declining. Importantly,
cyclodiene resistance still remains a model of target site-mediated resistance and has been used in a
number of instances to glean information regarding the genetic architecture of resistance (reviewed
by ffrench-Constant 2000).
In most cases studied, resistance appears to involve insensitivity of the GABA receptor, caused by a
conserved point mutation which results in an amino acid substitution of an alanine either to serine
or glycine within the 2nd transmembrane domain (M2) (Hosie et al 1997). Significant progress has
been made recently in two specific areas related to cyclodiene resistance in western corn
rootworms: 1) identification of a Rdl mutation associated with cyclodiene resitance and 2)
identification of significant variation in susceptibility to the cyclodiene insecticide aldrin among
western corn rootworm populations. Because of the rapid range expansion and persistence of
cyclodiene resistance in D. v. virgifera, detectable variation in susceptibility, and availability of
molecular markers, cyclodiene resistance in this species represents a potentially important model
for understanding the evolution and movement of target site-mediated resistance genes.




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      As discussed previously, we have recently reported the presence of high levels of
resistance in both the laboratory-reared and field-collected adult western corn rootworms to the
cyclodiene insecticide, aldrin (Parimi et al. 2006). However, there was considerable variation in
resistance levels and a general decline in resistance among Nebraska populations. To confirm
differences in susceptibility of field populations, a diagnostic bioassay was developed for the
cyclodiene, aldrin based on the LC99 for the susceptible non-diapause lab colony. The calculated
LC99 (16.5 µg/vial) was used as a diagnostic concentration to compare susceptibility of field
collected adults. The assay was repeated at least ten times on four different population (Saunders
County, NE; Story County, IA Champaign County, IL; and Center County, PA) collected in 2006.
Results of diagnostic aldrin bioassays indicated a wide range of susceptibility with a pattern of
eastward increases in resistance among field populations (Fig.1). It is clear from these results that
cyclodiene resistance is still widely persistent, although considerable variation in the resistance
level suggests the frequency of resistance conferring alleles is also variable.
               100
       Mortality (%)




                  77.1
               80
                       59.6
               60
               40
                            12.3
                            12.3
               20
                                5.6
               0
                  NE   IA     IL   PA
                      Populations
Figure 1. Susceptibility of D. v. virgifera adults collected from NE, IA, IL and PA to a diagnostic
   concentration of aldrin corresponding the LC99 of the susceptible non-diapause strain.

To determine the possible involvement of an Rdl mutation associated with cyclodiene resistance in
western corn rootworms, a combination of degenerate PCR and RACE (Rapid Amplification of
cDNA Ends) PCR with gene-specific primers resulted in a complete cDNA for a GABA receptor
gene of 1397 bp including the poly A tail (referred to as DvvA). DvvA has an open reading frame
(ORF) of 1266 nucleotides encoding a putative protein of 422 amino acids (Fig. 2), including three
transmembrane domains (M1-M3), which are general features of chemically-gated ion channels
(Schofield et al. 1987). The ORF is followed by a 129 bp non-transcribed sequence, and a
polyadenylyation signal (AATAAA) adjacent to the poly(dA) at the 3` terminus. A BLASTX
search of GeneBank indicated that the conceptual translation of DvvA closely aligned with the
isoform A of a GABA receptor from Tribolium castaneum with 86% identity (Fig. 2). Identities of
GABA receptor genes from other insect species are shown in Table 1. Importantly, the conserved
point mutation associated with cyclodiene resistance in at least ten different insect species
(Thompson et al 1993) was observed in our deduced AA sequence (A to S, indicated by asterisk,
Fig. 2) from aldrin resistant individuals based on survival at the diagnostic aldrin concentration..




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Table 1. BLASTx search of GABA receptor genes and percent identities of deduce AA sequence.
            Sequence Identity                  Sequence Identity
   Species                    Species
            Protein     %                 Protein     %
   Tribolium                   Heliothis
   castaneum     393/452     86    virescens        345/414     83
   Apis                     Drosophila
   mellifera     356/408     87    melanogaster       304/326     93
   Plutella                   Lucilia
   xylostella     242/299     80    cuprina         353/456     77
   Anopheles                   Musca
   gambiae      342/472     72    domestica        350/481     72

To determine to the potential for the Rdl mutation to serve as a molecular diagnostic, allele specific
primers were designed with two combinations of primers, F1+R, and F+R1 that should discriminate
among different genotypes. Primers F and R were designed to end in either a T or A residue,
respectively, which selectively amplified the resistance allele only (Figure 3). Three genotypes,
R/R, R/S and S/S, were readily identified by allele-specific PCR reactions among individuals of D.
v. virgifera collected from Mead, Nebraska in 2006 (Figure 3). The genotypes were also found to be
correlated with the resistant level of individual rootworm adults (data not shown).
             1                        50
T.castaneum     (1) MGHSRVVWPAVLLALALPWASAG-SPGAGGSYLGDVNISAILDSFSVSYD
                                       Figure 2. Comparison of the deduced
D.v.v.(A)      (1) MGRSRMVWPAVLLTLVAFGGAFAGSPGAGGSYVGGVNVSAILDSFSVSYD
             51                       100
                                       amino acid sequence of the GABA
T.castaneum     (50) KRVRPNYGGPPVEVGVTMYVLSISSLSEVKMDFTLDFYFRQFWTDPRLAF
D.v.v.(A)      (51) KRVRPNYGGPPVDVGVTMYVLSISSLSEVQMDFTLDFYFRQFWTDPRLAF
                                       receptor gene from resistant strain of D. v.
             101                      150
T.castaneum    (100) RKRPGVETLSVGSEFIKNIWVPDTFFVNEKQSYFHIATTSNEFIRIHHSG
                                       virgifera with an isoform A from
D.v.v.(A)     (101) RKRPSVEILSVGSEFIKNIWVPDTFFVNEKHSSFHMATTSNEFIRIHHSG
             151                      200
                                       Tribolium castaneum. Completely
T.castaneum    (150) SITRSIRLTITASCPMNLQYFPMDRQLCHIEIESFGYTMRDIRYKWNEGP
D.v.v.(A)     (151) SITRSIRLTITASCPMNLQYFPMDRQVCHIEIESFGYTMRDIRYKWNEGP
                                       conserved regions are underlined, *
             201                      250
T.castaneum    (200) NSVGVSNEVSLPQFKVLGHRQRAMEISLTTGNYSRLACEIQFVRSMGYYL
                                       indicates the position where the A to S/G
D.v.v.(A)     (201) NSVGVSNEVSLPQFKVLGHRQRAMEISLTTGNYSRLACEIQFVRSMGYYL
                                    YYL
             251                      300
                                       substitution has been observed in
T.castaneum    (250) IQIYIPSGLIVIISWVSFWLNRNATPARVALGVTTVLTMTTLMSSTNAAL
D.v.v.(A)     (251) IQIYIPSGLIVIISWVSFWLNRNATPARVSLGVTTVLTMTTLMSSTNAAL
                                       cyclodiene-resistant species (ffrench-
                 M1          *  M2
             3 01                      350
                                       Constant et al. 2000). The transmembrane
T.castaneum    (300) PKISYVKSIDVYLGTCFVMVFASLLEYATVGYMAKRIQMRKNRFLAIQKI
D.v.v.(A)     (301) PKISYVKSIDVYLGTCFVMVFASLLEYATVGYMAKRIQMQKNRFLAIQKI
                                       domain (M1-M3) is highlighted in blue.
                       M3
             3 51                      400
T.castaneum    (350) AEQKKLNVDGGPDSDHAPKQTVSRPIGHHLFQEVRFKVHDPKAHSKGGTL
D.v.v.(A)     (351) AEQKKLNVDGG-----PDDHAPKQTVR--------FTVHDPKAHSKGGTL
             4 01                      450
T.castaneum    (401) ESTVNGGRGG-GGGGGP-DEEAAAPIPQHIIHPNKDINKLYGITPSDIDK
D.v.v.(A)     (389) KSTVNGGRGGDRGGAG-PDKEAGAPIPQHIIHHHYY*INCIVSRI*VTVF
             451                 491
                                        Ladder   S/S    R/S    R/R
T.castaneum    (449) YSRIVFPVCFVCFNLMYWIIYLHISDVVADDLVLLEEDK
          (439) LLI*KLDSIEPRKYLNKVKKVAQKKKKKK----------
D.v.v.(A)
                                       1 kb plus F1+R F+R1 F1+R F+R1 F1+R F+R1
Figure 3. Allele-specific PCR. S/S, R/R and R/S
indicate the genotype of homozygous susceptible,
resistant and heterozygous resistant individual
respectively. F1+R and F+R1 are the pair of primers.




III Rootworm Adaptation: Organophosphate Resistance.
Organophosphate and carbamate insecticides were introduced following the failure of cyclodienes
and successfully replaced these compounds as the predominant rootworm insecticides throughout
the U.S. Corn Belt. Both organophosphates and carbamates are still used as soil insecticides and as

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      foliar insecticides in adult management programs. Both soil insecticides and adult
rootworm management have been adopted as primary management tools where irrigated,
continuous corn is planted over large acreages throughout the Platte River valley of central
Nebraska. However, in some areas of Nebraska, aerially applied Penncap-M® (methyl parathion)
was used almost exclusively (Meinke 1995) over relatively large areas and in consecutive years.
Control failures of aerially applied methyl-parathion were first reported in the early 1990s, and
resistance to organophosphate and carbamate active ingredients was documented in rootworm
adults from a number of Nebraska populations (Meinke et al. 1998). The distribution of resistant
rootworms was initially restricted to areas of the state where adult management had been practiced
in excess of 10 years, while areas relying on soil insecticides and crop rotation apparently remained
susceptible. It has also been determined that rootworm larvae are resistant to a number of
organophosphate and carbamate active ingredients, and that the same metabolic mechanisms are
present across life stages (Miota et al. 1998, Wright et al. 2000). However, the spectrum of
resistance is relatively narrow and does not appear to affect efficacy of other more commonly used
active ingredients.
Slight insensitivity of acetylcholinesterases to inhibition by methyl-paraoxon has been identified in
resistant Phelps County beetles (Miota et al. 1998), and elevated cytochrome P450-based
metabolism of carbaryl (Scharf et al. 1999a) has been noted in populations from both York and
Phelps Counties. However, synergism studies using the esterase inhibitor DEF indicate a common
involvement of esterases in both methyl parathion (Miota et al. 1998) and carbaryl resistance
(Scharf et al. 1999b). Insecticide metabolism studies employing [14C] ethyl parathion (Miota et al.
1998) and [14C] carbaryl (Scharf et al. 1999a) showed increased formation of hydrolysis products
and disappearance of parent insecticides. Relative to susceptible rootworm larvae, elevated esterase
activity has also been noted in populations resistant to multiple acetylcholinesterase inhibiting
insecticides (Wright et al. 2000). Together, these findings emphasize the relative importance of
hydrolytic metabolism as a cross-resistance conferring mechanism which occurs across life stages
and over relatively large geographic areas.
In 1996, a diagnostic bioassay was developed for quickly assessing the resistance status of field–
collected rootworm populations. Based on the dose response curves of representative resistant and
susceptible populations (Fig. 1), a diagnostic concentration corresponding to the LC99 of a standard
susceptible colony was determined. This concentration was used to assess the resistance by
identifying the proportion of a given population that exceeds 1% survival at the LC99 for a
susceptible population. Based on sampling results over a 4-year period using the diagnostic
concentration of methyl-parathion, resistance exhibited significant expansion both in distribution
and in intensity (Figure 2).
Initial sampling of rootworm susceptibility in 1996 indicated the presence of two distinct resistant
areas based on the presence of susceptible populations that separate these two regions. However,
by 1998 significantly increased levels of tolerance were observed in the areas of York and Hamilton
counties, and areas previously identified as being susceptible had become highly resistant (Adams
County; Figure 3). Although resistance appears to be growing both in intensity and in geographic
range, there are still populations of rootworms that remain susceptible to methyl parathion in close
proximity to resistant populations. Furthermore, in areas where aerially applied methy-parathion no
longer provides effective control of adult rootworms, growers are adopting other management
practices such as crop rotation and use of soil insecticides.




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                                                                          0 - 25%                 RESISTANT
                                                                          26 - 50%
                                                                          51 - 75%

                                                                                                                      Figure 4. Distribution of methyl-parathion
                                                                          76 - 100%             SUSCEPTIBLE


                                                                                                                      resistant western corn rootworms based on
   Custer
                   Valley    Greeley
                                       Platte        Colfax  Dodge
                                                              Custer          Valley    Greeley




                                                                                                                      percent survival at diagnostic concentration after
                                                                                                Platte        Colfax   Dodge



                                 Nance
                                                                                         Nance




                                                                                                                      4 hr exposure; N=50-100.
                                                Butler
                   Sherman   Howard               Polk
                                                                           Sherman   Howard
                                                                                                        Butler
                                                                                                    Polk
                                 Merrick
                                                                                         Merrick

                                                      Saunders
Dawson
                                                                                                               Saunders
                                                Seward         Dawson
                         Hall            York                                   Buffalo  Hall
              Buffalo
                                                      Lancaster                                    York        Seward
                                                                                                               Lancaster

                               Hamilton

                                                                                        Hamilton

            Phelps                                                  Gosper
                    Kearney
     Gosper                                                              Phelps




                           In representative resistant populations, esterase
                                                                           Kearney
                          Adams

                                      Fillmore                                           Adams
                               Clay               Saline
                                                                                        Clay     Fillmore
Furnas




                           activity toward α- and β-naphtholic esters was
          Harlan       Franklin                                                                                Saline
                                                                     Harlan
                                                            Furnas
                                                                           Franklin




                           ca. 5 to 6-fold elevated in relation to susceptible
                                                                                 Webster
                         Webster                                                            Nuckolls
                               Nuckolls                                                             Thayer
                                       Thayer                                                                       Gage
                                                                                                        Jefferson
                                               Jefferson    Gage




     1996           1998
                           baselines (Miota et al. 1998). Visualization of
esterase isoenzymes from mass homogenates of rootworm abdomens on native polyacrylamide gels
has consistently shown a similar trend (Fig. 5A; Zhou et al. 2002). On these native gels, 3 esterase
isoenzyme groups are typically visible, with one showing elevated activity in resistant populations
(group 2). Interestingly, when individual abdomens are homogenized and separated on native
polyacrylamide gels (i.e., 1 abdomen per lane), only the group 2 esterases of resistant individuals
are visible (Fig. 5B). Representative high, intermediate, and low activity-level populations, with
their corresponding survival at diagnostic concentrations of methyl parathion are shown in Figure 6.
During the 1998 growing season, 26 western corn rootworm populations of varying methyl
parathion susceptibility (based on survival at a diagnostic concentration of methyl parathion) were
evaluated to determine the proportion of individuals in these populations having elevated esterase
activity by native PAGE. A linear regression comparing these results is shown in Figure 6, and
demonstrates that elevated esterase activity-frequency is well correlated with survival on methyl
parathion diagnostic concentrations (r2 = 0.898) (Zhou et al. 2002).
                         le




                                                               Population #1                           Population #2                  Population #3
                       tib

                        t
                      tan
                      ep




                                                               (99 % Mortality)                         (69 % Mortality)                (8 % Mortality)
                     sis
                    sc




                                                                                                                                  Figure 5. Native PAGE of
                   Re
                   Su




                                                                                                                                  adult rootworm homogenates
                                   } Group 1
                                                                                                                                  stained for general esterase
                                   } Group 2                                                                                           activity.
                                   } Group 3
                                                                                                                        RESISTANT
                                                            SUSCEPTIBLE

          A. 20 beetles/homogenate                                                           B. Individual homogenates



                         1 00

                             80
                                                                                                                        Figure 6. Correlation of % mortality at
                             60
                                                                                                                        diagnostic concentration with percentage of
% Bioassay
Mortality                                               r2 = 0.898
                                                                                                                        rootworms displaying elevated Group 2
                             40                       y = 101.34 - 0.93x
                                                                                                                        esterases.
                                                    n = 26 populations
                             20

                               0
                                                                                                                     100
                                   0                   20              40               60                  80
                                             % Elevated “Group 2” Esterases
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Importantly, the development and spread of methyl-parathion resistance provides an important tool
in validating models of resistance evolution. This approach was recently used to validate a
stochastic model of the evolution of resistance to adulticidal sprays of methyl-parathion in western
corn rootworm populations in Nebraska (Caprio et al. 2006). When resistance was examined as a
genetic phenomenon, the rate of increase of the resistance allele depended almost entirely on
genetic factors (LC50 values), the characteristics of the pesticide (residual activity), and the variance
associated with emergence of adults. When resistance was measured as failure of methyl-parathion
to reduce populations below threshold levels (0.5 gravid females per plant), parameters that
contributed to population growth rate (mortality and fecundity) were also important. These data
suggest two important phases in resistance evolution in corn rootworms: a genetic phase associated
with negative growth rates and rapid changes in resistance allele frequencies and a rebound phase
associated with positive growth rates and near fixation of the resistance allele.
The precise molecular nature of resistance to organophophate insecticides remains elusive. Specific
cytochrome P450 gene fragments have been identified that appear to be over-expressed in resistant
populations (Scharf et al. 2001) but their involvement is as yet unconfirmed. With respected to
enhanced hydrolytic metabolism, there is a clear indication that over-expressed carbolyesterases are
associated with resistance and enhanced hydrolytic metabolism of insecticide substrates has been
documented in resistant strains (Miota et al. 1998, Scharf et al. 1999a) but specific esterase genes
have yet to be identified. Recently, a cDNA microarray chip was constructed using from a
combination of unique cDNA’s from adult head and larval midgut tissues representing
approximately 6600 genes (G. Robinson, University of Illinois; personal communication). This
array has been used to compare hybridization of cDNA samples from previously identified
organophosphate susceptible and resistant D. v. virgifera strains to identify key genes genes that are
differentially expressed and which may be associated with resistance. Results from comparison
revealed an esterase gene with high similarity to a Tribolium castaneum esterase among the genes
most significantly different in expression.

IV Rootworm Adaptation: Adaptation to Crop Rotation.
Damage to first year maize was first observed in Ford County, Illinois in 1987 (Levine and Oloumi-
Sadeghi 1996). By 1995, maize producers across a large section of east central Illinois experienced
severe root injury in their rotated maize fields. The problem has now spread thoughout most of
Illinois and into several nearby states (Figure 7). The mechanism behind this phenomenon was
unclear. Although prolonged diapause of western corn rootworm (Diabrotica virgifera virgifera
LeConte) eggs is possible, Levine et al. (1992) indicated that only 0.14% of 4,202 eggs they
examined hatched after two simulated winters in the laboratory. Because of the very low number of
western corn rootworm eggs that prolonged their diapause, it seemed very unlikely that this
mechanism was the causal factor for the widespread and severe root injury in rotated maize that
occurred in east central Illinois and northern Indiana in 1995. Other potential explanations for the
failure of crop rotation to limit root injury in rotated maize included a pyrethroid repellancy
hypothesis (Levine and Oloumi-Sadeghi 1996). Because maize seed-production fields frequently
receive multiple applications of pyrethroid insecticides to prevent excessive corn
earworm(Helicoverpa zea) injury, it was hypothesized that western corn rootworm females were
being repelled from these maize seed-production fields and laying their eggs in the soil of nearby
soybean fields. As the number of maize acres in rotated fields with significant corn rootworm larval
injury increased across eastern Illinois, this hypothesis was abandoned as the most viable
explanation. Oviposition by western corn rootworms in soybean fields has been reported previously
(Shaw et al. 1978); however, the primary reason for egg laying in soybeans was attributed to

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      volunteer maize in soybean fields. This explanation was not viable as the primary factor
responsible for the widespread damage to rotated maize fields in the mid-1990s. Weed management
is excellent in most commercial maize production fields and significant volunteer grass infestations
are not common. Researchers from Purdue University (Sammons et al. 1997), reported that a
variant of the western corn rootworm was attracted to soybean foliage. Western corn rootworm
adults collected from Indiana consumed more soybean leaf tissue than beetles obtained from Iowa
or Nebraska, states with more continuous maize production. This research was conducted within a
laboratory setting and the researchers urged caution in interpreting the results until field studies
could confirm these observations. They concluded that “This variant preferred soybean
environments over corn environments.” Spencer et al. (1999) utilized a walk-in wind tunnel
experiment to evaluate the flight of western corn rootworms and found “… no evidence for
attraction to soybeans based on postflight plant association.” The beetles they used in their
experiments were collected from problem and non-problem areas (where damage to rotated maize
occurred and did not occur). By the late 1990s, considerable confusion still existed regarding the
mechanism behind the western corn rootworm’s adaptation to crop rotation.



                       Figure 7. Expansion over time of the problem of damage to
                       first year maize.




                   Researchers have made considerable progress in determining
                   why variant western corn rootworm adults (particularly
                   females) leave maize and disperse to adjacent soybean fields.
                   O’Neal et al. (2002) conducted no-choice and choice assays in
                   laboratory experiments with western corn rootworm adults
                   collected from Illinois, Nebraska, and Michigan. They
                   observed that maize phenology affected the amount of soybean
foliage that was consumed by western corn rootworm adults. Greater levels of soybean tissue were
eaten when more mature reproductive stage maize was present in contrast with younger vegetative
stage maize. No significant differences in the amount of soybean foliage consumed by adults
collected from these three states were observed. In a follow-up investigation (O’Neal et al. 2004),
again utilizing laboratory olfactometer chamber assays, found that western corn rootworm adults
moved to chambers containing soybean foliage more readily when corn foliage in other chambers
began to senesce. O’Neal et al. (2004) argued: “Collectively, our data are not supportive of the
genetically based behavioral change model for rotation failure. No direct evidence was obtained for
any difference in behaviour of Illinois versus Nebraska D. v. virgifera.”
In contrast to the O’Neal et al. (2002, 2004) laboratory investigations, Pierce and Gray (2006)
reached a different conclusion with respect to the role of maize phenology on oviposition of the
variant western corn rootworm in soybean fields. Pierce and Gray (2006a) conducted a large scale
(32 hectares) experiment near Champaign, Illinois near the epicenter of the variant western corn
rootworm population. Eight treatments of soybean and maize that varied in planting date and
maturity groupings were established to achieve considerable variation in plant phenology across this
experimental landscape. All treatments were replicated four times. After maize developed beyond
the R2 stage (full ear size and brown silks present), densities of adult western corn rootworms
began to increase in soybean plots. Oviposition occurred across all maize and soybean treatments;

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       however, those maize plots that were planted first (mid-April), and consequently
matured earlier, had approximately 1/2 as many eggs as maize that was planted 1 month later (mid-
May). Late-planted maize (mid-May) and the four soybean treatments had roughly equal egg
densities. An experiment that served as the control for this research was established in northwestern
Illinois, an area of the state in which the variant western corn rootworm was not established at the
time of the experiment. This experiment was conducted on 6.7 hectares and included two maize and
two soybean treatments that varied considerably in their phenology due to planting date and
maturity group of the cultivars. Despite the exaggerated differences in crop phenology (maize and
soybeans) at this northwestern Illinois experiment, western corn rootworm oviposition did not occur
in soybeans. These results stand in stark contrast to the results of the Champaign experiment. The
authors of this research offered the following hypothesis: “We also hypothesize the intensive
selection pressure that resulted from decades of crop rotation in east central Illinois resulted in a
variant D.v. virgifera that responds to the phenology of maize by expanding its ovipositional range
of crops, most notably to include soybeans.” These authors believe that this response to phenology
has an underlying genetic component, as evidenced by the different ovipositional patterns that
occurred between Champaign and northwestern Illinois western corn rootworm populations when
exposed to different phonological choices of maize and soybean treatments.
Mabry and Spencer (2003) evaluated the survival and oviposition of variant western corn rootworm
adults offered soybean foliage. Based upon the results of their laboratory investigations they
hypothesized that “the presence of D. virgifera in soybean fields, and other locations outside of
cornfields, is likely to be a function of an increase in general D. virgifera activity that may be
symptomatic of a relaxed affinity to cornfields.” The results of Pierce and Gray (2006 a) suggest
that maize phenology may be associated with this “relaxed affinity” to maize. Mabry and Spencer
(2003) suggested that western corn rootworm females that feed on soybean foliage may increase
their level of stress resulting in premature oviposition within soybean fields. In essence, variant
western corn rootworm females that feed on soybean foliage, in the interiors of very large soybean
fields (hundreds of hectares), may never be able to disperse back to maize for egg-laying purposes.
Mabry et al. (2004) conducted additional behavioral studies regarding the role that corn and
soybean diets may have on the survivorship, dispersal characteristics, and oviposition of the variant
western corn rootworm. Western corn rootworm larvae were collected from Urbana (variant area)
and Monmouth (non-variant area), Illinois. These researchers determined that the variant western
corn rootworm population did not have any greater tendencies to feed on soybean leaves or survive
on them as compared with the non-variant population. Exposure to soybean foliage caused elevated
levels of nutritional stress in both populations triggering an increase in oviposition rate and beetle
activity. The authors offered the following conclusion: “… these data suggest that soybean plays no
direct role in the D. v. virgifera circumvention of crop rotation.”
Knolhoff et al. (2006) reported on the development of a behavioral assay used to characterize
variant and non-variant western corn rootworm females. Beetles used in the assays were collected
from Urbana, IL (variant area) and Iowa and Wisconsin (non-variant areas). Beetles collected from
Urbana were characterized as “more active” than beetles obtained from non-variant areas where
crop rotation remains an effective cultural management tactic. At present, the authors note that
“This assay does not yet provide a clear threshold to differentiate between rotation-resistant and
wild- type phenotypes.” but suggest that it may be possible to modify the assay to do so. Knolhoff
et al. (2006) contend that the results of their behavioral assay support the hypothesis that there is a
genetic difference between variant and non-variant western corn rootworm populations. As yet, no
gene or genes have been identified to prove this hypothesis.
Although a genetic basis for the variant behavior has not been proven, it seems highly likely.
Computer simulation studies by Onstad et al. (2001) have shown that the selection pressure
imposed by the level of crop rotation typical of east-central Illinois would favor an adaptive allele
that confers reduced specificity to maize. Further simulation studies by Onstad et al. (2003) have

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      shown that the growth of the area in which first year maize is at risk can be explained by
the dispersal (aided by storms) of a rotation-adapted (i.e. genetically-determined) population.
Consequently, there have been a number of attempts to identify genetic markers associated with the
variant phenomenon. Miller et al. (2006) analyzed the variation at eight microsatellite loci and
could not determine any genetic differentiation between adult western corn rootworms collected in
soybeans and those obtained from maize in areas categorized as variant or non-variant. Miller et al.
(2006) suggested that “genetic markers for rotation resistance probably will be found only at the
gene or genes responsible, or at tightly linked loci.” Following on from this study Miller and
Guillemaud. (unpublished) attempted to identify Amplified Fragment Length Polymorphisms
(AFLPs) associated with the variant. This latter study made use of a more targeted sampling
strategy than the former. Samples were collected as pupae from first year maize in east-central
Illinois and from continuous maize in central Iowa. Thus the Illinois samples were guaranteed to be
the offspring of females that had oviposited into soybean (i.e. variant females) whereas, (given their
geographical location) the Iowa samples could safely be assumed to be wild type. Of 253 AFLP
markers that were analyzed, only one showed any evidence of being associated with the variant -
wild type difference. However, the level of differentiation between the two types at this locus was
modest and the marker cannot be used to discriminate between them. This may be because the
marker is only loosely linked to a gene involved in the variant behavior. The failure of this study to
identify a marker strongly linked to a “variant gene” may well be a consequence of the large size of
the WCR genome.
A candidate-gene approach to the problem has met with some success although the results are still
somewhat preliminary. Garabagi et al. (unpublished) have cloned a homolog of the Drosophila for
gene. The for gene encodes a cyclic GMP-dependent protein kinase. In Drosophilla, allelic
variation at this locus results in products with different levels of activity and ultimately, two distinct
phenotypes for foraging behavior: “rover” and sitter”. Interestingly, Garabagi et al. (unpublished)
found that transcription levels of the WCR for homolog were 25% higher in lab colonies derived
from variant populations than in their wild type counterparts.

V References Cited
Ball, H. J., Hill, R.E, 1953. You can control corn rootworm. Nebraska Exp. Sta. Quart. Winter
1952-53, Nebraska Agr. Exp. Sta., Lincoln, Nebraska.
Ball, H. J., Roselle, R.E., 1954. You can control corn rootworms. Univ. Nebraska. Ext. Circ. 1567.
Ball, H. J., Weekman, G. T., 1962. Insecticide resistance in the adult western corn rootworm in
Nebraska. J. Econ. Entomol. 55, 439-41.
Ball, H.J., Weekman, G.T., 1963. Differential resistance of corn rootworms to insecticides in
Nebraska and adjoining states. J. Econ. Entomol. 56, 553-555.
Caprio, M.J., T.J. Nowatzki, B.D. Siegfried, L.J. Meinke, R.J. Wright, and L.D. Chandler. 2006.
Assessing the risk of resistance to aerial applications of methyl-Parathion in the western corn
rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 99: 483-493.
ffrench-Constant, R. H., Anthony, N., Aronstein, K., Rocheleau, T. and Stilwell, G. (2000)
Cyclodiene insecticide resistance: From molecular to population genetics. Annual Review of
Entomology 45: 449-466.
ffrench-Constant, R., Mortlock, D. P., Shaffer, C. D., Macintyre, R. J. and Roush, R. T. (1991)
Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate
GABAA receptor locus. Proc Natl Acad Sci U S A 88: 7209-7213.
Georghiou, G.P. 1969. The magnitude of the resistance problem. In Pesticide Resistance:
Strategies and Tactics for Management, ed. National Academoy of Sciences, pp. 14-43.
Washington, D.S.: Natl. Acad. Press.



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       Hosie, A.M., K. Aronstein, D.B. Sattelle, and R. ffrench-Constant. 1997. Molecular
biology if insect neuronal GABA recptors. Trends Neurosci. 20: 578-83.
Knolhoff, L.M., D.W. Onstad, J.L. Spencer, and E. Levine. 2006. Behavioral differences between
rotation-resistant and wild-type Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae).
Environ. Entomol. 35(4): 1049-1057.
Levine, E., and H. Oloumi-Sadeghi. 1996. Western corn rootworm (Coleoptera: Chrysomelidae)
larval injury to corn grown for seed production following soybeans grown for seed production. J.
Econ. Entomol. 89(4): 1010-1016.
Levine, E., H. Oloumi-Sadeghi, and C.R. Ellis. 1992. Thermal requirements, hatching patterns, and
prolonged diapause in western corn rootworm (Coleoptera: Chrysomelidae) eggs. J. Econ. Entomol.
85(6): 2425-2432.
Mabry, T.R., and J.L. Spencer. 2003. Survival and oviposition of a western corn rootworm variant
feeding on soybean. Entomologia Experimentalis et Applicata 109: 113-121.
Mabry, T.R., J.L. Spencer, E. Levine, and S.A. Isard. 2004. Western corn rootworm (Coleoptera:
Chrysomelidae) behavior is affected by alternating diets of corn and soybean. 2004. Environ.
Entomol. 33(4): 860-871.
Miller, N.J., K.S. Kim, S.T. Ratcliffe, A. Estoup, D. Bourguet, and T. Guillemaud. 2006. Absence
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Siegfried. 1998. Mechanisms of methyl and ethyl parathion resistance in the western corn rootworm
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Muma, M. H., Hill, R. E., Hixson, E., 1949. Soil treatments for corn rootworm control. J. Eco.
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O’Neal, M.E., C.D. DiFonzo, and D.A. Landis. 2002. Western corn rootworm (Coleoptera:
Chrysomelidae) feeding on corn and soybean leaves affected by corn phenology. Environ. Entomol.
31(2): 285-292.
O’Neal, M.E., D.A. Landis, J.R. Miller, and C.D. DiFonzo. 2004. Corn phenology influences
Diabrotica virgifera virgifera emigration and visitation to soybean in laboratory assays. Environ.
Entomol. 33(1): 35-44.
Onstad, D.W., D.W. Crowder, P.D. Mitchell, C.A. Guse, J.L. Spencer, E. Levine, and M.E. Gray.
2003. Economics versus alleles: balancing integrated pest management and insect resistance
management for rotation-resistant western corn rootworm (Coleoptera: Chrysomelidae). J. Econ.
Entomol. 96(6): 1872-1885.
Onstad, D.W., D.W. Crowder, S.A. Isard, E. Levine, J.L. Spencer, M.E. O’Neal, S.T. Ratcliffe,
M.E. Gray, L.W. Bledsoe, C.D. DiFonzo, J.B. Eisely, and C.R. Edwards. 2003. Does landscape
diversity slow the spread of rotation-resistant western corn rootworm (Coleoptera: Chrysomelidae)?
Environ. Entomol. 32(5): 992-1001.
Pierce, C.M.F., and M.E. Gray. 2006a. Western corn rootworm, Diabrotica virgifera virgifera
LeConte (Coleoptera: Chrysomelidae), oviposition: a variant’s response to maize phenology.
Environ. Entomol. 35(2): 423-434.
Parimi, S., L.J. Meinke, B.W. French, L.D. Chandler, and B.D. Siegfried. 2006. Persistence and
stability of methyl-parathion and aldrin resistance in western corn rootworms. Crop Protection.
25:269-274.
Roselle, R. E., Anderson, L. W., Simpson, R. G., Webb M. C., 1959. Annual report for 1959,
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channels: molecular target of neuroactive insecticides. Invertebrate Neuroscience: 119-133.
Roselle, R. E., Anderson, L. W., Simpson, R. C., and Bergman, P. W., 1960. Annual report for
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      Roselle, R. E., Anderson, L. W., Bergman, P. W., 1961. Annual report for 1961,
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Sammons, A.E., C.R. Edwards, L.W. Bledsoe, P.J. Boeve, and J.J. Stuart. 1997. Behavioral and
feeding assays reveal a western corn rootworm (Coleoptera: Chrysomelidae) variant that is attracted
to soybean. Environ. Entomol. 26(6): 1336-1342.
Scharf, M.E., L.J. Meinke, R.J. Wright, L.D. Chandler, and B.D. Siegfried. 1999a. Metabolism of
carbaryl by insecticide-resistant and -susceptible western corn rootworm populations (Coleoptera:
Chrysomelidae). Pestic. Biochem. Physiol. 63: 85-96.
Scharf, M.E., L.J. Meinke, B.D. Siegfried, R.J. Wright and L.D. Chandler. 1999b. Carbaryl
susceptibility, diagnostic concentration determination, and synergism for U.S. populations of
western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 92: 33-39.
Scharf, M.E., S. Parimi, L.J. Meinke, and B.D. Siegfried. 2001. Expression and induction of three
family 4 cytochrome P450 (CYP4) genes identified from insecticide-resistant and susceptible
western corn rootworms, Diabrotica virgifera virgifera. Insect Mol. Biol. 10: 139-146.
Spencer, J.L., S.A. Isard, and E. Levine. 1999. Free flight of western corn rootworm (Coleoptera:
Chrysomelidae) to corn and soybean plants in a walk-in wind tunnel. J. Econ. Entomol. 92(1): 146-
155.
Shaw, J.T., J.H. Paullus, and W.H. Luckmann. 1978. Corn rootworm oviposition in soybeans. J.
Econ. Entomol. 71(2): 189-191.
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rootworms (Coleoptera: Chrysomelidae). J. Econ. Entomol. 95: 1261-1266.




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An Abbreviated Review of the Literature for the Variant Western
Corn Rootworm
I State of the art
Corn rootworm injury to the root systems of first-year maize (rotated maize) is not a new
phenomenon. Historically, the reason behind injury to first-year maize has largely been attributed to
a prolonged diapause of the northern corn rootworm (Diabrotica barberi Smith and Lawrence).
This problem (prolonged diapause) remains an important issue for producers in some areas of the
U.S. Corn Belt (Iowa, Minnesota, South Dakota). In 1995, maize producers across a large section of
east central Illinois experienced severe root injury in their rotated maize fields. The mechanism
behind this phenomenon was unclear. Although prolonged diapause of western corn rootworm
(Diabrotica virgifera virgifera LeConte) eggs is possible, Levine et al. (1992) indicated that only
0.14% of 4,202 eggs they examined hatched after two simulated winters in the laboratory. This was
the first published proof of prolonged diapause in the western corn rootworm. Because of the very
low number of western corn rootworm eggs that prolonged their diapause, it seemed very unlikely
that this mechanism was the causal factor for the widespread and severe root injury in rotated maize
that occurred in east central Illinois and northern Indiana in 1995. Other potential explanations for
the failure of crop rotation to limit root injury in rotated maize included a pyrethroid repellancy
hypothesis (Levine and Oloumi-Sadeghi 1996). Because maize seed-production fields frequently
receive multiple applications of pyrethroid insecticides to prevent excessive corn
earworm(Helicoverpa zea) injury, it was hypothesized that western corn rootworm females were
being repelled from these maize seed-production fields and laying their eggs in the soil of nearby
soybean fields. As the number of maize acres in rotated fields with significant corn rootworm larval
injury increased across eastern Illinois, this hypothesis was abandoned as the most viable
explanation. Oviposition by western corn rootworms in soybean fields has been reported previously
(Shaw et al. 1978); however, the primary reason for egg laying in soybeans was attributed to
volunteer maize in soybean fields. This explanation was not viable as the primary factor responsible
for the widespread damage to rotated maize fields in the mid-1990s. Weed management is excellent
in most commercial maize production fields and significant volunteer grass infestations are not
common. Researchers from Purdue University (Sammons et al. 1997), reported that a variant of the
western corn rootworm was attracted to soybean foliage. Western corn rootworm adults collected
from Indiana consumed more soybean leaf tissue than beetles obtained from Iowa or Nebraska,
states with more continuous maize production. This research was conducted within a laboratory
setting and the researchers urged caution in interpreting the results until field studies could confirm
these observations. They concluded that “This variant preferred soybean environments over corn
environments.” Spencer et al. (1999) utilized a walk-in wind tunnel experiment to evaluate the
flight of western corn rootworms and found “… no evidence for attraction to soybeans based on
postflight plant association.” The beetles they used in their experiments were collected from
problem and non-problem areas (where damage to rotated maize occurred and did not occur). By
the late 1990s, considerable confusion still existed regarding the mechanism behind the western
corn rootworm’s adaptation to crop rotation. Gray et al. (1998) published a more complete review
of western and northern corn rootworm adaptations to crop rotation as a cultural practice. Levine et
al. (2002) provided a very complete review of this issue with an emphasis on the variant western
corn rootworm.
One of the first IPM (integrated pest management) projects for the variant western corn rootworm
was to begin characterizing field populations of the western corn rootworm collected in soybean
fields across east central Illinois. O’Neal et al. (1999) sampled twenty sites (soybean and maize

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       fields) across seven Illinois counties in eastern Illinois from late July to early September
in 1996 and 1997. They reported that numbers of western corn rootworm adults and the percentage
of females were greater in soybean fields than in maize fields. These researchers utilized Pherocon
AM (Hein and Tollefson 1985) and vial traps (Levine and Gray 1994) in both soybean and maize
fields. They postulated that “soybean fields are acceptable oviposition sites for western corn
rootworms in east-central Illinois.” In a subsequent IPM project, O’Neal et al. (2001) reported on
the potential usefulness of Pherocon AM traps as a sampling tool that producers could use in
soybean fields to predict the severity of larval damage in rotated maize the following season. This
research was conducted in producers’ (n = 17) fields across east-central Illinois. At the conclusion
of the research, O’Neal et al. (2001) reported that they could account for approximately 27% of the
variation in root injury based upon adult counts with Pherocon AM traps. Densities of 5 beetles
caught per trap per day in soybean fields were found to be sufficient to reach the economic injury
index of 3.0 (moderate level of root pruning, Hills and Peters 1971) the following season in rotated
maize if left untreated. Implementation of this IPM approach has been very modest across areas of
the U.S. Corn Belt in which the variant western corn rootworm has become established. Many
producers view the deployment of Pherocon AM traps as too labor intensive and prefer to manage
this insect pest prophylactically with soil insecticides or transgenic hybrids. Consequently, the use
of transgenic maize rootworm hybrids (particularly the “stacked” hybrids, those with multiple
events) will continue to increase significantly.
The agricultural landscape of the eastern U.S. Corn Belt primarily consists of large tracts of maize
and soybean hectares that are rotated annually. Researchers began to explore the potential use of
other crops in the rotation to deter egg-laying by the variant western corn rootworm. Rondon and
Gray (2004) determined that “the western corn rootworm oviposits in maize, soybean, oat stubble,
and alfalfa.” They concluded: “Lack of oviposition preference of the western corn rootworm variant
demonstrated in this experiment represents a reasonable explanation of why the effectiveness of the
rotation strategy to control western corn rootworm has diminished.” Rondon and Gray (2003) found
that western corn rootworm adults were common inhabitants of maize, soybean, oat stubble, and
alfalfa. In their research plots, following emergence, densities of females began to decline in maize
and increase in the other crops. Further studies regarding the potential utility of other crop rotation
sequences in deterring oviposition of the variant western corn rootworm were performed by
Schroeder et al. (2005). These authors measured adult densities (vial traps) and also took egg
samples in several crops: maize, soybean, winter wheat double-cropped with soybeans, and wheat
alone. Oviposition occurred in all four cropping systems. The authors stated that “… the use of
wheat demonstrated the most potential for preventing yield reducing levels of root injury in rotated
corn.” Oviposition by the variant western corn rootworm occurred at the lowest level in fields with
wheat stubble.
Researchers have made considerable progress in determining why variant western corn rootworm
adults (particularly females) leave maize and disperse to adjacent soybean fields. O’Neal et al.
(2002) conducted no-choice and choice assays in laboratory experiments with western corn
rootworm adults collected from Illinois, Nebraska, and Michigan. They observed that maize
phenology affected the amount of soybean foliage that was consumed by western corn rootworm
adults. Greater levels of soybean tissue were eaten when more mature reproductive stage maize was
present in contrast with younger vegetative stage maize. No significant differences in the amount of
soybean foliage consumed by adults collected from these three states were observed. In a follow-up
investigation (O’Neal et al. 2004), again utilizing laboratory olfactometer chamber assays, found
that western corn rootworm adults moved to chambers containing soybean foliage more readily
when corn foliage in other chambers began to senesce. O’Neal et al. (2004) argued: “Collectively,
our data are not supportive of the genetically based behavioral change model for rotation failure. No
direct evidence was obtained for any difference in behaviour of Illinois versus Nebraska D. v.
virgifera.”

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       In contrast to the O’Neal et al. (2002, 2004) laboratory investigations, Pierce and Gray
(2006) reached a different conclusion with respect to the role of maize phenology on oviposition of
the variant western corn rootworm in soybean fields. Pierce and Gray (2006a) conducted a large
scale (32 hectares) experiment near Champaign, Illinois near the epicenter of the variant western
corn rootworm population. Eight treatments of soybean and maize that varied in planting date and
maturity groupings were established to achieve considerable variation in plant phenology across this
experimental landscape. All treatments were replicated four times. After maize developed beyond
the R2 stage (full ear size and brown silks present), densities of adult western corn rootworms
began to increase in soybean plots. Oviposition occurred across all maize and soybean treatments;
however, those maize plots that were planted first (mid-April), and consequently matured earlier,
had approximately ½ as many eggs as maize that was planted 1 month later (mid-May). Late-
planted maize (mid-May) and the four soybean treatments had roughly equal egg densities. An
experiment that served as the control for this research was established in northwestern Illinois, an
area of the state in which the variant western corn rootworm was not established at the time of the
experiment. This experiment was conducted on 6.7 hectares and included two maize and two
soybean treatments that varied considerably in their phenology due to planting date and maturity
group of the cultivars. Despite the exaggerated differences in crop phenology (maize and soybeans)
at this northwestern Illinois experiment, western corn rootworm oviposition did not occur in
soybeans. These results stand in stark contrast to the results of the Champaign experiment. The
authors of this research offered the following hypothesis: “We also hypothesize the intensive
selection pressure that resulted from decades of crop rotation in east central Illinois resulted in a
variant D.v. virgifera that responds to the phenology of maize by expanding its ovipositional range
of crops, most notably to include soybeans.” These authors believe that this response to phenology
has an underlying genetic component, as evidenced by the different ovipositional patterns that
occurred between Champaign and northwestern Illinois western corn rootworm populations when
exposed to different phonological choices of maize and soybean treatments. In a separate series of
on-farm studies, Pierce and Gray (2006b) further reported on the ovipositional patterns of western
corn rootworms in soybean and maize fields of east central Illinois. They determined that
oviposition was approximately “90% complete by 9, 17, and 14 August in maize and by 15, 12, and
26 August in soybean fields from 1999 through 2001, respectively.”
Mabry and Spencer (2003) evaluated the survival and oviposition of variant western corn rootworm
adults offered soybean foliage. Based upon the results of their laboratory investigations they
hypothesized that “the presence of D. virgifera in soybean fields, and other locations outside of
cornfields, is likely to be a function of an increase in general D. virgifera activity that may be
symptomatic of a relaxed affinity to cornfields.” The results of Pierce and Gray (2006 a) suggest
that maize phenology may be associated with this “relaxed affinity” to maize. Mabry and Spencer
(2003) suggested that western corn rootworm females that feed on soybean foliage may increase
their level of stress resulting in premature oviposition within soybean fields. In essence, variant
western corn rootworm females that feed on soybean foliage, in the interiors of very large soybean
fields (hundreds of hectares), may never be able to disperse back to maize for egg-laying purposes.
Mabry et al. (2004) conducted additional behavioral studies regarding the role that corn and
soybean diets may have on the survivorship, dispersal characteristics, and oviposition of the variant
western corn rootworm. Western corn rootworm larvae were collected from Urbana (variant area)
and Monmouth (non-variant area), Illinois. These researchers determined that the variant western
corn rootworm population did not have any greater tendencies to feed on soybean leaves or survive
on them as compared with the non-variant population. Exposure to soybean foliage caused elevated
levels of nutritional stress in both populations triggering an increase in oviposition rate and beetle
activity. The authors offered the following conclusion: “… these data suggest that soybean plays no
direct role in the D. v. virgifera circumvention of crop rotation.” Knolhoff et al. (2006) reported on
the development of a behavioral assay used to characterize variant and non-variant western corn

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      rootworm females. Beetles used in the assays were collected from Urbana, IL (variant
area) and Iowa and Wisconsin (non-variant areas). Beetles collected from Urbana were
characterized as “more active” than beetles obtained from non-variant areas where crop rotation
remains an effective cultural management tactic. Knolhoff et al. (2006) contend that the results of
their behavioral assay support the hypothesis that there is a genetic difference between variant and
non-variant western corn rootworm populations. As yet, no gene or genes have been identified to
prove this hypothesis. Miller et al. (2006) analyzed the variation at eight microsatellite loci and
could not determine any genetic differentiation between adult western corn rootworms collected in
soybeans and those obtained from maize in areas categorized as variant or non-variant. Miller et al.
(2006) suggested that “genetic markers for rotation resistance probably will be found only at the
gene or genes responsible, or at tightly linked loci.” Perhaps those western corn rootworm females
characterized as variant phenotypes by the techniques described by Knolhoff et al. (2006) can be
used to more efficiently locate potential markers linked with the rotation-resistant trait.
Since 1995, the variant western corn rootworm continues to disperse throughout the agricultural
landscape of the U.S. Corn Belt. Reports of damage to rotated maize by western corn rootworms are
increasingly common in the northern 2/3 of Illinois and Indiana, southern Michigan, western Ohio,
and in extreme southeastern Iowa. The dispersal characteristics of the variant western corn
rootworm have been described as a “result of high-altitude daytime flight” (Isard et al. 2004).
Lower temperatures, high winds, and darkness reduced beetle flight activity. Modeling studies on
the influence of landscape diversity (Onstad et al. 2003) revealed that as cropping complexity
increased the rate at which the variant western corn rootworm dispersed across a region slowed.
These results suggest that because of a more diversified system of crops in many areas of Europe,
the variant western corn rootworm (if found to be present) may be more restricted in its ability to
disperse. Onstad et al. (2003) modeled six management strategies (combination of transgenic
hybrids and crop rotation schemes) over a 15-year period and determined that resistance to rotation
may evolve in less than 15 years. The rate at which rotation resistance developed increased as the
percentage of landscape that was rotated increased. In the simulations, each combination of
management tactics was effective in delaying resistance to crop rotation if the resistance allele was
recessive. A successful management option in delaying rotation resistance was the use of a 3-year
rotation of maize, soybean, and wheat. The wheat was characterized as unattractive regarding
oviposition and preceded maize in the rotation. In subsequent simulations, Crowder et al. (2005a)
determined that planting transgenic maize to rotated fields is an effective strategy that can
potentially prevent resistance development to crop rotation and transgenic maize where variant
western corn rootworm populations exist or become established. Crowder et al. (2005a) made the
following recommendation: “If farmers are located in an area where rotation-resistant western corn
rootworms already pose a problem or may cause problems in the near future, they should consider a
management approach of planting transgenic corn only in first-year cornfields.” Further economic
analyses concerning the use of transgenic maize hybrids in areas of the U.S. Corn Belt in which the
rotation resistant variant western corn rootworm is established clearly indicated that the planting of
a transgenic corn rootworm hybrid in first-year maize fields was the most economical management
approach (Crowder et al. 2005b).
Significant concerns have arisen among academics that many producers in the commercial maize
and soybean fields of the U.S. Corn Belt have abandoned some important IPM principles, most
notably the abandonment of scouting and economic thresholds to base their decisions regarding the
use of crop management inputs such as pesticides or transgenic maize hybrids. Modeling results of
Crowder et al. (2006) would help support the move away from some of these more traditional IPM
approaches in areas where the variant western corn rootworm has become established: “Sampling
was never effective in areas where rotation resistance is already a severe problem. Therefore,
planting transgenic corn every season may not only be the most economical but also the safest


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      strategy for risk-averse farmers to ensure maximum returns in areas with rotation-
resistant phenotypes.”
Kaster and Gray (2005) describe the management challenges that have arisen with the introduction
of western corn rootworms in Europe. Miller et al. (2005) established a strong case for the multiple
introductions (at least three) of western corn rootworms into Europe as compared with a single
entry point. As in the U.S., it will continue to be of great interest to characterize European
populations as variant or non-variant. The results of this discovery will help to refine our
management recommendations for European producers.
Global atmospheric carbon dioxide increased by 20% in the last century. By 2050, the global
concentration of carbon dioxide is predicted to increase another 48%. Schroeder et al. (2006)
determined that variant western corn rootworm egg levels increased three fold in soybean plots with
combined elevated carbon dioxide and ozone concentrations as compared with plots with ambient
levels of these gases. If projected increases of these gases by the mid-21st century are correct, we
may find variant western corn rootworms thriving under these atmospheric conditions assuming
cropping patterns remain the same. These assumptions are far from certain.

II References Cited
Crowder, D.W., D.W. Onstad, M.E. Gray, C.M.F. Pierce, A.G. Hager, S.T. Ratcliffe, and K.L.
Steffey. 2005a. Analysis of the dynamics of adaptation to transgenic corn and crop rotation by
western corn rootworm (Coleoptera: Chrysomelidae) using a daily time-step model. 2005. J. Econ.
Entomol. 98(2): 534-551.
Crowder, D.W., D.W. Onstad, M.E. Gray, P.D. Mitchell, J.L. Spencer, and R.J. Brazee. 2005b.
Economic analysis of dynamic management strategies utilizing transgenic corn for control of
western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 98(3): 961-975.
Crowder, D.W., D.W. Onstad, and M.E. Gray. 2006. Planting transgenic insecticidal corn based on
economic thresholds: consequences for integrated pest management and insect resistance
management. J. Econ. Entomol. 99(3): 899-907.
Hein, G.L., and J.J. Tollefson. 1985. Use of the Pherocon AM trap as a scouting tool for predicting
damage by corn rootworm (Coleoptera: Chrysomelidae) larvae. J. Econ. Entomol. 78: 200-203.
Hills, T.M., and D.C. Peters. 1971. A method of evaluating postplant insecticide treatments for
control of western corn rootworm larvae. J. Econ. Entomol. 64: 764-765.
Isard, S.A., J.L. Spencer, T.R. Mabry, and E. Levine. 2004. Influence of atmospheric conditions on
high-elevation flight of western corn rootworm (Coleoptera: Chrysomelidae). Environ. Entomol.
33(3): 650-656.
Gray, M.E., E. Levine, and H. Oloumi-Sadeghi. 1998. Adaptation to crop rotation: western and
northern corn rootworms respond uniquely to a cultural practice. Recent Res. Devel. Entomol. 2:
19-31.
Kaster, L.V., and M.E. Gray. 2005. European corn borers and western corn rootworms: old and new
invasive maize pests challenge farmers on European and North American continents. Maydica 50:
235-245.
Knolhoff, L.M., D.W. Onstad, J.L. Spencer, and E. Levine. 2006. Behavioral differences between
rotation-resistant and wild-type Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae).
Environ. Entomol. 35(4): 1049-1057.
Levine, E., and M.E. Gray. 1994. Use of cucurbitacin vial traps to predict corn rootworm
(Coleoptera: Chrysomelidae) larval injury in a subsequent crop of corn. J. Entomol. Sci. 29: 590-
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Levine, E., and H. Oloumi-Sadeghi. 1996. Western corn rootworm (Coleoptera: Chrysomelidae)
larval injury to corn grown for seed production following soybeans grown for seed production. J.
Econ. Entomol. 89(4): 1010-1016.

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      Levine, E., H. Oloumi-Sadeghi, and C.R. Ellis. 1992. Thermal requirements, hatching
patterns, and prolonged diapause in western corn rootworm (Coleoptera: Chrysomelidae) eggs. J.
Econ. Entomol. 85(6): 2425-2432.
Levine, E., J.L. Spencer, S.A. Isard, D.W. Onstad, and M.E. Gray. 2002. Adaptation of the western
corn rootworm to crop rotation: evolution of a new strain in response to a management practice.
Am. Entomol. 48(2): 94-107.
Mabry, T.R., and J.L. Spencer. 2003. Survival and oviposition of a western corn rootworm variant
feeding on soybean. Entomologia Experimentalis et Applicata 109: 113-121.
Mabry, T.R., J.L. Spencer, E. Levine, and S.A. Isard. 2004. Western corn rootworm (Coleoptera:
Chrysomelidae) behavior is affected by alternating diets of corn and soybean. 2004. Environ.
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