Evaluation of adult emergence and larval root injury for Cry3Bb1‐resistant populations of the western corn rootworm

The transgenic maize (Zea mays L.) event MON 88017 produces the Bacillus thuringiensis Berliner (Bt) toxin Cry3Bb1 to provide protection from western corn rootworm (Diabrotica virgifera virgifera LeConte) larval feeding. In response to reports of reduced performance of Cry3Bb1‐expressing maize at two locations in Illinois, we conducted a two‐year experiment at these sites to characterize suspected resistance, as well as to evaluate root injury and adult emergence. Single‐plant bioassays were performed on larvae from each population that was suspected to be resistant. Results indicate that these populations had reduced mortality on Cry3Bb1‐expressing maize relative to susceptible control populations. No evidence of cross‐resistance between Cry3Bb1 and Cry34/35Ab1 was documented for the Cry3Bb1‐resistant populations. Field studies were conducted that included treatments with commercially available rootworm Bt hybrids and their corresponding non‐Bt near‐isolines. When compared with their near‐isolines, larval root injury and adult emergence were typically reduced for hybrids expressing Cry34/35Ab1 either alone or in a pyramid. In many instances, larval root injury and adult emergence were not significantly different for hybrids expressing mCry3A or Cry3Bb1 alone when compared with their non‐Bt near‐isolines. These findings suggest that Cry34/35Ab1‐expressing Bt maize may represent a valuable option for maize growers where Cry3Bb1 resistance is either confirmed or suspected. Consistent trends in adult size (head capsule width and dry mass) for individuals recovered from emergence cages were not detected during either year of this experiment. Because of the global importance of transgenic crops for managing insect pests, these results suggest that improved decision‐making for insect resistance management is needed to ensure the durability of Bt maize.     

Effects of adult emergence timing on susceptibility and fitness of Cry3Bb1‐resistant western corn rootworms

Field‐evolved resistance by the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte to the Cry3Bb1 trait expressed in maize, has been documented in areas of Nebraska USA. Currently, only limited information is available on life‐history traits of Cry3Bb1‐resistant field populations. Therefore, the Gassmann on‐plant bioassay was used to investigate the potential variability among four Cry3Bb1‐resistant WCR field collections made in 2011–2012 by focusing on the key parameters: larval survival, developmental stage and weight with specific emphasis on the impact of adult emergence timing on these parameters in subsequent progeny. Key results: In three of four collections, the susceptibility of larval progeny from adults that emerged early or late within a generation from Cry3Bb1 plants was similar. Each of the three collections exhibited complete resistance; that is, survival on Cry3Bb1 plants was greater or equal to survival on non‐Bt isoline plants. Bioassays from an additional field collection from one site 2 years (2013) after the original collection (2011) (both from Cry3Bb1 maize) indicated that resistance to Cry3Bb1 was maintained over time at the site despite Bt trait rotation in 2012. In general, comparative WCR life‐history parameter data from Cry3Bb1 and isoline maize indicate that fitness of field collections exhibiting complete resistance was similar on each hybrid. The mean proportion of larvae in third instar and mean weight of larvae recovered in bioassays from progeny of early‐ and late‐emerged adults was not significantly affected by emergence period. This suggests that delays in development and associated mean adult emergence commonly observed in populations that are susceptible to Cry3Bb1 may become smaller as populations become resistant to Cry3Bb1. Results from this article will inform Cry3Bb1 resistance mitigation efforts and contribute to the development of sustainable WCR management programmes.     

Quantifying rates of random mating in western corn rootworm emerging from Cry3Bb1‐expressing and refuge maize in field cages

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is the most significant pest of field maize, Zea mays L. (Poaceae), in the USA. Maize plants expressing Bt toxins targeting the corn rootworm complex have been widely adopted and are the primary insecticidal control measure for this pest in North America. Insect resistance management tactics using various refuge structures have been adopted to ensure Bt products will retain durability. An assumption of the refuge strategy is that males and females emerging from Bt and refuge plantings mate randomly; this has not been tested in the field. We conducted cage studies using field populations of WCR in Indiana, USA, to generate empirical field data on mating rates between beetles emerging from Cry3Bb1‐expressing Bt and refuge maize plants. Two refuge configurations were tested; all refuge plants were labeled using the stable isotope ¹⁵N. This mark persists in adult beetles after eclosion, allowing for collection and analysis of isotopic ratios of all beetles. Additional data collected included adult emergence rates, timing and sex ratios for each of the treatments, and head capsule size and dry weights of beetles collected. Treatment had a significant effect on dry weight; mean dry weight decreased in Bt‐only treatments. Fisher's exact test of proportions of mating pairs of refuge and Bt insects indicated that mating was not random in 20% strip refuges and 5% seed blend treatments. We found high percentages of beetles that fed on Bt‐expressing plants as larvae, suggesting that mating between resistant beetles may not be rare even if random mating did occur.     

Baseline susceptibility of western corn rootworm (Coleoptera: Chrysomelidae) to clothianidin

Abstract:  Western corn rootworm, Diabrotica virgifera virgifera LeConte, neonate susceptibility to clothianidin, a contact and systemic neonicotinoid insecticide, was determined from both laboratory and field-collected populations. Neonates were exposed to filter paper treated with increasing clothianidin concentrations and mortality was evaluated after 24 h. Additionally, two populations were exposed to an artificial diet which was surface treated with clothianidin. Although larvae were five- to six-fold more sensitive to treated diet, results with treated filter paper were more reliable in terms of control mortality and required much less manipulation of rootworm larvae. Therefore, initial baseline comparisons were conducted using the filter paper assays. The variation among populations exposed to treated filter paper was generally low, 4.4-fold among laboratory populations tested; however, there was a 14.5-fold difference in susceptibility among all populations tested. In general, clothianidin was very toxic to rootworm neonates, with LC₅₀ values ranging from 1.5 to 21.9 ng/cm². These results indicate the practicability and sensitivity of the paper filter disc assay to establish baseline susceptibility levels, which is an essential first step in resistance management. A baseline response provides a reference for tracking shifts in susceptibility following commercialization of a control agent so that early changes in susceptibility can be detected.     

Mortality impact of MON863 transgenic maize roots on western corn rootworm larvae in the field

Mortality of western corn rootworm (Diabrotica virgifera virgifera LeConte) due to feeding on MON863 transgenic maize (Zea mays L.) expressing the Cry3Bb1 protein was evaluated at three Missouri sites in both 2003 and 2004 and at one site each in South Dakota, Nebraska and Iowa in 2004. To do this, survivorship relative to survivorship on isoline maize (i.e. the same genetic background, but without Cry3Bb1) was evaluated. Comparisons were made using low (1650–2500 eggs/m) and high (3300–3500 eggs/m) western corn rootworm egg densities. Significantly fewer beetles were recovered from MON863 than from isoline maize. Emergence from MON863 as a percentage of viable eggs ranged from 0.02% to 0.10%, whereas percentage emergence from isoline maize ranged from 1.09% to 7.14%. Survivorship on MON863 relative to survivorship on isoline averaged 1.51% when averaged across all environments and both years, so mortality because of the Cry3Bb1 protein averaged 98.49%. The average time delay to 50% cumulative beetle emergence from MON863 was 18.3 days later than from isoline maize. Females comprised 56% and 71% of total beetles recovered from MON863 in 2003 and 2004, respectively. Results are discussed in relation to insect resistance management (IRM) of western corn rootworm.     

Characterizing laboratory colonies of western corn rootworm (Coleoptera: Chrysomelidae) selected for survival on maize containing event DAS-59122-7

Event DAS‐59122‐7 is a novel transgenic trait designed to protect the roots and yield potential of maize from the insect pest corn rootworm Diabrotica spp. (Col.: Chrysomelidae). The increased pest status of corn rootworm, exceptional efficacy of this trait, and anticipated increases in farm efficiency and grower and environmental safety will drive adoption of this trait. Strong grower acceptance of this trait highlights the importance of science‐based and practical resistance management strategies. A non‐diapause trait was introgressed into two laboratory colonies of Diabrotica virgifera virgifera collected from geographically distinct locations: Rochelle, IL and York, NE. Both colonies were divided and each reared on maize containing event DAS‐59122‐7 or its near isoline. Selected and unselected colonies were evaluated for phenotypic change in larval development, injury potential and survival to adulthood during 10 and 11 generations. The F1 generation of both selected colonies displayed increased larval development, survivorship and measurable, but economically insignificant increases in injury potential on DAS‐59122‐7 maize. Survival rates of 0.4 and 1.3% in F1 generations of both selected colonies corroborate field estimates of survival on DAS‐59122‐7 maize. Over later generations, total phenotypic variation declined gradually and irregularly. Despite the absence of random mating, the tolerance trait could not be fixed in either population after 10 or 11 generations of selection. An allele conferring major resistance to DAS‐59122‐7 was not identified in either selected colony. The assessment also concluded that major resistance gene(s) are rare in populations of D. v. virgifera in the United States, and that a minor trait(s) conferring a low level of survival on DAS‐59122‐7 maize was present. The tolerance trait identified in this study was considered minor with respect to its impact on DAS‐59122‐7 maize efficacy, and the role this trait may play in total effective refuge for major resistance genes with recessive inheritance is the basis of future work.     

Assessment of fitness costs in Cry3Bb1‐resistant and susceptible western corn rootworm (Coleoptera: Chrysomelidae) laboratory colonies

Abstract Maize production in the United States is dominated by plants genetically modified with transgenes from Bacillus thuringiensis (Bt). Cry3Bb delta endotoxins expressed by Bt maize specifically target corn rootworms (genus Diabrotica) and have proven highly efficacious. However, development of resistance to Bt maize, especially among western corn rootworm (Diabrotica virgifera virgifera) populations, poses a significant threat to the future viability of this pest control biotechnology. The structured refuge insect resistance management (IRM) strategy implemented in the United States for Bt maize adopts a conservative approach to managing resistance by assuming no fitness costs of Bt resistance, even though these trade‐offs strongly influence the dynamics of Bt resistance within numerous agricultural pest species. To investigate the effects of Bt resistance on fitness components of western corn rootworm, we compared survivorship, fecundity and viability of five Bt‐resistant laboratory lines reared on MON863 (YieldGard Rootworm), a Bt maize product that expresses Cry3Bb1 delta endotoxin, and on its non‐transgenic isoline. Analysis of performance on the isoline maize demonstrated no fitness costs associated with Bt resistance. In fact, resistant lines emerged approximately 2–3 days earlier than control lines when reared on both MON863 and the isoline, indicating that selection for Bt resistance resulted in a general increase in the rate of larval development. In addition, resistant lines reared on Bt maize displayed higher fecundity than those reared on the isoline, which may have significant management implications. These data will be valuable for formulating improved IRM strategies for a principal agricultural pest of maize.     

Heterorhabditis sp. not attracted to synthetic (E)‐β‐caryophyllene,a volatile emitted by roots upon feeding by corn rootworm

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a destructive pest of maize, Zea mays L. in North America and Europe. Larvae and pupae can be controlled with entomopathogenic nematodes (EPN) of the genus Heterorhabditis. When maize is attacked by WCR, the roots of some maize varieties emit (E)‐β‐caryophyllene that attract EPN to the pest larvae. To use synthetic (E)‐β‐caryophyllene in bioassays for the genetic selection of EPN strains with enhanced chemotactic response to a volatile emitted from the damaged root when attacked by larvae of the WCR, different laboratory bioassays were tested. Three sand assays and one agar assay used synthetic (E)‐β‐caryophyllene as an attractant for H. megidis. In none of the assays, attraction of the nematodes to (E)‐β‐caryophyllene was recorded. Possible reasons why (E)‐β‐caryophyllene was not attracting EPN in the bioassays are discussed.     

Estimating western corn rootworm (Coleoptera: Chrysomelidae) larval susceptibility to event DAS‐59122‐7 maize

Susceptibility of Diabrotica virgifera virgifera (LeConte) larvae to DAS‐59122‐7 maize was evaluated using a laboratory technique that measures rootworm survival to adulthood on maize seedlings. This method produces direct measures of larval susceptibility using realistic exposure to the same range of insecticidal protein concentrations found in field‐grown DAS‐59122‐7 maize roots. First, second and third instars were reared to adulthood on DAS‐59122‐7 maize seedlings or a non‐transgenic, near‐isoline maize. Data on survival, adult gender ratio, adult weight and median emergence were collected. Overall, larval susceptibility to DAS‐59122‐7 maize was lower than earlier predictions ( Storer et al. 2006 ). Neonate survival on DAS‐59122‐7 maize was approximately 33% of isoline survival after 17 days, and the same 33% recovered and developed to adulthood when the isoline maize was substituted. Survival rate on DAS‐59122‐7 maize increased with instar. The mean survivorship was 0.5%, 26% and 65% when exposure to DAS‐59122‐7 maize began at the first, second and third instars, respectively. Exposure to DAS‐59122‐7 maize led to sub‐lethal effects on adult gender ratio, weight and median emergence. These effects decreased when exposure to DAS‐59122‐7 maize began at later instars. The killing effect of DAS‐59122‐7 maize on rootworm larvae appeared to result from the combined chronic effects and absence of a suitable host as perceived by the larvae. The relevance of these data and the methodology of estimating rootworm susceptibility to plant‐incorporated protectants are discussed in the context of the US Environmental Protection Agency’s functional definition of ‘high dose’ and use of refuge for resistance management ( EPA 1998a ). Based on these results it is evident that DAS‐59122‐7 maize does not meet the functional definitions of high dose as described by EPA (1998a,b) and ILSI (1999) , and the utility of refuge, refuge size and refuge placement for delaying rootworm resistance should be further investigated.     

Selection for Cry3Bb1 resistance in a genetically diverse population of nondiapausing western corn rootworm (Coleoptera: Chrysomelidae)

Five short-diapause laboratory lines of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), were selected for resistance to MON863, a variety of corn genetically modified with the Bacillus thuringiensis Berliner (Bt) transgene that expresses the Cry3Bb1 delta-endotoxin. Three of the selected lines were developed by incremental increase in the duration of exposure to MON863 over 11 generations (moderate selected lines). Two selected lines were developed from a control group by constant exposure to MON863 for at least 14 d posthatch over seven generations (intense selected lines). At the end of the experiment, survivorship, as measured by adult emergence, was approximately 4 times higher in each of the selected lines reared on MON863 compared with control lines. Estimates of realized heritabilities (h2) were 0.16 and 0.15 for the moderate and intense selected lines, respectively, and are consistent with h2 estimates reported previously from a variety of pest insects. These lines provide data necessary for evaluating the potential for Bt resistance within diabroticite beetles and will be useful for developing improved insect resistance management strategies.     

Volatile semiochemical impact on trapping and distribution in maize of northern and western corn rootworm beetles (Coleoptera: Chrysomelidae)

Abstract 1 Field studies evaluated plant attractants and analogues as tools to move corn rootworm beetles (Diabrotica spp.) into areas to be treated with toxic baits for population suppression via mass removal/annihilation of reproductive adults. 2 When dispensed from sticky traps in maize, 2‐phenyl‐1‐ethylamine and 2‐phenyl‐1‐ethanol captured more northern corn rootworm, Diabrotica barberi, than did 4‐methoxyphenethanol. Only 2‐phenyl‐1‐ethanol attracted the western corn rootworm, Diabrotica virgifera virgifera, but not until maize matured beyond milk stage. 3 Attraction of D. barberi to the amine, alone or blended with 2‐phenyl‐1‐ethanol, occurred before and after maize flowered but not during intervening silk or blister stages. Attraction recurred during early milk stage at or before 50% emergence of adult female D. barberi or D. v. virgifera, respectively, and before populations declined for the season. 4 Synergistic interaction of 2‐phenyl‐1‐ethylamine with 2‐phenyl‐1‐ethanol in attracting D. barberi females did not occur until maize matured to late milk stage. 5 The amine‐alcohol blend (0.44 point sources m^?2) doubled the density of D. barberi but not D. v. virgifera when applied to small plots within mostly milk‐stage or younger maize. Traps without bait within attractant‐treated plots captured more female, but not male, D. barberi than did traps in untreated control plots, hinting that females accounted for most of the observed increase in beetle density. 6 The results suggest that attractants can be used despite phenological limitations to concentrate preovipositional females within field areas and thus to complement a variety of corn rootworm control strategies.     

Effect of Cry3Bb1-expressing transgenic corn on plant-to-plant movement by western corn rootworm larvae (Coleoptera: Chrysomelidae)

Dispersal of larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in specific combinations of transgenic corn expressing the Cry3Bb1 protein and nontransgenic, isoline corn was evaluated in a 2-yr field study. In total, 1,500 viable western corn rootworm eggs were infested in each subplot. Each year, plant damage and larval recovery were evaluated among four pedigree combinations (straight transgenic; straight nontransgenic corn; nontransgenic corn with a transgenic central, infested plant; and transgenic corn with a nontransgenic central, infested plant) on six sample dates between egg hatch and pupation. For each subplot, the infested plant, three successive plants down the row (P1, P2, and P3), the closest plant in the adjacent row of the plot, and a control plant were sampled. The number of western corn rootworm larvae recovered from transgenic rootworm-resistant plants adjacent to infested nontransgenic plants was low and not statistically significant in either 2001 or 2002. In 2001, significantly fewer larvae were recovered from transgenic rootworm-resistant plants than from nontransgenic plants when both were adjacent to infested, nontransgenic plants. In 2002, significantly more neonate western corn rootworm larvae were recovered from nontransgenic plants adjacent to infested, transgenic rootworm-resistant plants than nontransgenic plants adjacent to infested, nontransgenic plants on the second sample date. Together, these data imply that both neonate and later instar western corn rootworm larvae prefer nontransgenic roots to transgenic rootworm-resistant roots when a choice is possible. However, when damage to the infested, nontransgenic plant was high, western corn rootworm larvae apparently moved to neighboring transgenic rootworm-resistant plants and caused statistically significant, although only marginally economic, damage on the last sample date in 2001. Implications of these data toward resistance management plan are discussed.     

Polymorphic microsatellite loci from the western corn rootworm (Insecta: Coleoptera: Chrysomelidae) and cross‐amplification with other Diabrotica spp

Corn rootworms (Diabrotica spp.) make up the major insect pest complex of corn in the US and Europe, and there is a need for molecular markers for genetics studies. We used an enrichment strategy to develop microsatellite markers from the western corn rootworm (Diabrotica virgifera virgifera). Of 54 loci isolated, 25 were polymorphic, and of these, 17 were surveyed for variability in 59 wild individuals. In addition, the potential for cross‐amplification of these microsatellites was surveyed for Mexican, northern, and southern corn rootworms. Nine microsatellite loci showed Mendelian inheritance and are likely to be useful in population genetics studies.     

Validation of a novel resistance monitoring technique for corn rootworm (Coleoptera: Chrysomelidae) and event DAS-59122-7 maize

Rootworm control tactics have recently expanded to include transgenic maize, which express insecticidal proteins from Bacillus thuringiensis (Bt) to reduce larval injury and protect yield potential. Exceptional root protection, increased grower efficiency and improved safety have led to rapid adoption of this technology in the USA. As a result, there is a recognized need for resistance management programmes aimed at delaying rootworm resistance. An essential component of resistance management programmes is the development and implementation of effective resistance monitoring techniques. Five test populations of Diabrotica virgifera virgifera (LeConte) were used to evaluate the sensitivity of two techniques used to describe population susceptibility to the Bt proteins expressed in event DAS‐59122‐7 maize: a diet bioassay employing purified proteins applied to artificial diet and a novel technique using sub‐lethal measures of larval development on seedling maize. Test populations included Rochelle‐US, an unselected susceptible colony, three populations composed of 5%, 25% or 50% Rochelle‐S mixed with Rochelle‐US, and the Rochelle‐S selected colony. Rochelle‐S was derived from the same founding population as Rochelle‐US, but selected for survival on DAS‐59122‐7 maize. Selections identified a minor trait conferring increased tolerance, and greenhouse plant efficacy evaluations confirmed that after 10 generations of selection with no random mating, Rochelle‐S caused significantly more root injury to DAS‐59122‐7 than Rochelle‐US. Rochelle‐S present at 5% of the test population resulted in measurable but not significant increase in injury to DAS‐59122‐7 maize. The diet bioassay was relatively insensitive to the susceptibility differences between the Rochelle‐US and Rochelle‐S populations. Neither LC₅₀ nor EC₅₀ estimates produced statistically significant differentiation between test populations with 0%, 5%, 25% or 50% Rochelle‐S. The sub‐lethal assay clearly identified differences between Rochelle‐S and Rochelle‐US and an increased rate of larval development was measurable when the test population contained only 5% of Rochelle‐S.     

Field screening maize germplasm for resistance and tolerance to western corn rootworms (Col.: Chrysomelidae)†

Abstract: In the hopes of lessening the current reliance on soil insecticides, developing a viable alternative for transgenic maize hybrids, and providing sustainable options for Europe, researchers recently have been developing novel maize lines that exhibit resistance and/or tolerance to corn rootworm larvae. Here we report the results of a 2‐year field experiment in a northern growing region assessing the resistance and tolerance of 10 experimental synthetic maize populations selected for varying levels of damage from western corn rootworm larvae, Diabrotica virgifera virgifera LeConte (Col.: Chrysomelidae) and four maize hybrids. Maize non‐preference, antibiosis and tolerance to rootworms was evaluated using previously established methods, including: the Iowa 1–6 root damage rating scale, root fresh weight, compensatory root growth ratings and adult rootworm emergence. Among the experimental synthetic maize populations, BS29‐11‐01 was the most susceptible, and had a mean root damage rating that was greater than the highly susceptible maize hybrid B37 × H84. This line also had the lowest mean root fresh weight and one of the lowest mean compensatory root growth ratings. In contrast, CRW8‐3 appeared to be tolerant to western corn rootworms, and had the lowest mean root damage rating, which was comparable with that of the non‐transgenic hybrid DeKalb^® 46‐26.     

Baseline susceptibility of western corn rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin

Susceptibility to Cry3Bb1 toxin from Bacillus thuringiensis (Bt) was determined for western corn rootworm, Diabrotica virgifera virgifera LeConte, neonates from both laboratory and field populations collected from across the Corn Belt. Rootworm larvae were exposed to artificial diet treated with increasing Cry3Bb1 concentrations, and mortality and growth inhibition were evaluated after 4-7 d. The range of variation in Bt susceptibility indicated by growth inhibition was similar to that indicated by mortality. Although interpopulation variation in susceptibility was observed, the magnitude of the differences was comparable with the variability observed between generations of the same population. In general, the toxin was not highly toxic to larvae and estimated LC50 and EC50 values were several times higher than those reported for lepidopteran-specific Cry toxins by using similar bioassay techniques. These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among rootworm populations and provide a baseline for estimating potential shifts in susceptibility that might result from selection and exposure to Cry3Bb1-expressing corn hybrids.     

Field measures of western corn rootworm (Coleoptera: Chrysomelidae) mortality caused by Cry34/35Ab1 proteins expressed in maize event 59122 and implications for trait durability

Maize, Zea mays L., has been transformed to express the Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis strain PS149B1. These two proteins act together as a binary insecticidal protein that is effective against corn rootworm (Coleoptera: Chrysomelidae) species. The design of the resistance management plan to preserve the long-term durability of this trait largely depends on the level of rootworm mortality induced by Cry34/35Ab1 corn rootworm-protected maize (frequently referred to as "dose" in this context). Here, we report on studies that showed Cry34/35Ab1-expressing maize event 59122 caused 99.1 to 99.98% mortality of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae, after adjusting adult emergence numbers for density-dependent mortality. In two of three studies, there was a short delay in time to 50% adult emergence from 59122 maize plots compared with control plots, although emergence was completed at approximately the same time from both types of maize. These data support an expectation that alleles conferring resistance to the Cry34/35Ab1 proteins in western corn rootworm will be functionally nearly completely to completely recessive on 59122 maize and that there is unlikely to be assortative mating of Cry34/35Ab1-resistant and susceptible rootworms. When incorporated into simulation models of rootworm adaptation to transgenic maize, these findings suggest that a 20% refuge is likely to be highly effective at prolonging the durability of 59122 maize.     

Toxic and behavioural effects of free fatty acids on western corn rootworm (Coleoptera: Chrysomelidae) larvae

Feeding behaviour, feeding intensity and staying behaviour of neonate western corn rootworm (Diabrotica virgifera virgifera LeConte) larvae were evaluated in response to synthetic feeding stimulant blends. All of the treatments contained a 3‐sugar blend (glucose : fructose : sucrose, 30 : 4 : 4 mg/ml) and one of twelve free fatty acids. Each free fatty acid was tested in this blend at three different concentrations. The addition of the 12 : 0, 16 : 0, 16 : 1, 18 : 0, 18 : 1, 18 : 2 and 18 : 3 free fatty acids to the sugar blend significantly (P < 0.05) increased the percentage of larvae feeding, but did not increase food consumption per larva. Most of the free fatty acids elicited staying behaviour. At the lowest dose (0.1 mg/ml), all of the free fatty acids except the 18 : 0 and the 20 : 0 elicited staying by significantly more larvae than the sugar blend, and at the highest dose (1.0 mg/ml), eight free fatty acids (8 : 0, 10 : 0, 12 : 0, 14 : 0, 16 : 1, 18 : 1, 18 : 2 and 18 : 3) caused more larvae to stay compared to the sugar blend. Larvae were visibly impaired after exposure to some of the free fatty acids. At the highest dose, the 8 : 0, 10 : 0, 12 : 0, 14 : 0, 16 : 1, 18 : 1 and 18 : 2 free fatty acids were toxic to the larvae. At least 60% of larvae were impaired after exposure to the 12 : 0, 16 : 1 and 18 : 2 free fatty acids and the 8 : 0 and 10 : 0 free fatty acids caused 100% impairment or death. Synthetic blends were compared with liquid from crushed maize roots and with a methanol extract of maize roots. Feeding intensity and staying behaviour on the root liquid and the root extract were significantly greater than on any of the synthetic blends, suggesting the presence of additional compounds in maize roots that serve as feeding cues for western corn rootworm larvae.     

Susceptibility of northern corn rootworm Diabrotica barberi (Coleoptera: Chrysomelidae) to mCry3A and eCry3.1Ab Bacillus thuringiensis proteins

The susceptibility of the northern corn rootworm Diabrotica barberi (Smith & Lawrence) to mCry3A and eCry3.1Ab proteins derived from Bacillus thuringiensis (Bt) was determined using a diet bioassay. Northern corn rootworm neonates were exposed to different concentrations of mCry3A and eCry3.1Ab, incorporated into artificial diet. Larval mortality was evaluated after 7 d. The mCry3A and eCry3.1Ab proteins were found to be toxic to the northern corn rootworm larvae. The LC₅₀ and LC₉₉ values for mCry3A were 5.13 and 2482.31 μg/mL, respectively. For eCry3.1Ab, the LC₅₀ and LC₉₉ values were 0.49 and 213.01 μg/mL. Based on the estimated lethal concentrations, eCry3.1Ab protein was more efficacious to northern corn rootworm larvae than mCry3A. These lethal concentration values will be used as diagnostic doses for routine annual monitoring for change in susceptibility of field collected northern corn rootworm to mCry3A, and eCry3.1Ab toxins.     

Sensitivity of the cereal leaf beetle Oulema melanopus (Coleoptera: Chrysomelidae) to Bt maize-expressed Cry3Bb1 and Cry1Ab

The sensitivity of the cereal leaf beetle, Oulema melanopus (Coleoptera: Chrysomelidae), to maize-expressed Bacillus thuringiensis (Bt) proteins was investigated in the present study. Neonate larvae of O. melanopus were caged on leaves of Cry3Bb1-expressing (MON88017) or Cry1Ab-expressing (MON810) Bt maize, the corresponding near-isolines, or two non-related, conventional maize varieties. Larval survival was reduced on Cry3Bb1-expressing, but not on Cry1Ab-expressing maize compared with conventional varieties. Differences among conventional varieties were also present. The amount of eaten leaf material, developmental time to prepupal stage, and prepupal weight did not differ between Bt maize varieties and their corresponding near-isolines. In an additional feeding study with newly emerged adults, survival and beetle weight did not differ when leaves of Cry3Bb1-expressing maize or the near-isoline were offered as food over 3 weeks. ELISA measurements revealed that larvae feeding on Bt maize contained rather high Cry3Bb1 or Cry1Ab levels, which were in the same order of magnitude as the leaves. In contrast, concentrations in feces were one order, and concentrations in prepupae and adults two orders of magnitude lower.