Control of coleopteran insect pests through RNA interference.

Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. Toward this end, we demonstrate that ingestion of double-stranded (ds)RNAs supplied in an artificial diet triggers RNA interference in several coleopteran species, most notably the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte. This may result in larval stunting and mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage in a growth chamber assay, suggesting that the RNAi pathway can be exploited to control insect pests via in planta expression of a dsRNA.

Impact of MON863 transgenic roots is equivalent on western corn rootworm larvae for a wide range of maize phenologies.

The effects of maize, Zea mays L., phenology on establishment, damage, and adult emergence of the western corn rootworm, Diabrotica virgifera virgifera LeConte, on MON863 transgenic maize expressing the Cry3Bb1 protein and its isoline was evaluated in field trials in 2002 and 2003. As expected, plant damage, western corn rootworm larval recovery, and adult emergence were significantly lower on MON863 than isoline maize. The average weight of larvae and adults recovered from MON863 and isoline maize was generally not significantly different. If western corn rootworm larvae were able to establish on transgenic rootworm-resistant plants, larval growth was relatively normal. Plant damage, the number of western corn rootworm larvae recovered, and adult emergence from MON863 did not significantly differ between egg hatch times from widely varying phenologies in either year of the study. Although the extractable level of Cry3Bb1 decreased significantly from vegetative (V)4 to V9 maize in previous studies, in the current study, the amount of Cry3Bb1 did not vary from V3 to R3 in a way that affected neonate survival by western corn rootworm larvae in the field.

Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) larval feeding behavior on transgenic maize (MON 863) and its isoline.

Diabrotica species (Coleoptera: Chrysomelidae) larval behavior studies have posed a challenge to researchers because of the subterranean life cycle of this pest. To fully understand how the western corn rootworm, Diabrotica virgifera virgifera LeConte, injures the maize, Zea mays L., root system, its behavior must be studied. For example, larvae that can detect an area of the root that has a lower amount of toxin, whether from an insecticide or a transgenic maize plant, have an increased chance of survival. This study assessed D. v. virgifera larval feeding behavior on rootworm-susceptible maize and maize containing a biotechnology-derived trait (MON 863) with resistance to D. v. virgifera first instar feeding. Maize plants were grown in a medium that allowed for direct observation and measurements during feeding of larval stadia. Neonates were placed on maize seedlings, and data were taken at 3, 6, 9, and 12 d postinfestation on resistant and susceptible maize. On rootworm-susceptible maize, neonate larvae aggregated at the root tips and began actively feeding, and then they moved to older root tissue. Conversely, some larvae that ingested Cry 3Bb1 from the resistant maize exhibited no movement. Other larvae on the resistant maize moved continuously, sampling root hairs or root tissue but not actively feeding. The continuously moving larvae had visibly empty guts, suggesting possible nonpreference for the resistant root. This study contributes to our understanding of D. v. virgifera larval behavior and provides insight into questions surrounding the potential evolution of behavioral and biochemical resistance to Cry3Bb1.

Effect of Bacillus thuringiensis cry3Bb1 protein on the feeding behavior and longevity of adult western corn rootworms (Coleoptera: Chrysomelidae).

The first transgenic corn hybrids expressing the Bacillus thuringiensis (Bt) Cry3Bb1 protein to control corn rootworm (Diabrotica spp.) larvae were registered for commercial use in 2003. This study was conducted to investigate the effect of Cry3Bb1 protein in combination with a cucurbitacin bait on adult feeding and longevity of both organophosphate-resistant and -susceptible western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). In choice and no-choice tests, possible repellency to the Bt protein was quantified by comparing beetle consumption of cellulose disks treated with three concentrations of Bt in combination with a feeding stimulant (Invite EC) to disks treated with stimulant alone. A lethal-time assay also was conducted to examine survival of beetles exposed to Bt protein in their diet. Results from these assays indicate that adult rootworms are not significantly deterred by the presence of Cry3Bb1 on the treated discs and that ingestion of toxin does not adversely affect adult longevity.

Quantitative trait loci for maternal performance for offspring survival in mice.

Maternal performance refers to the effect that the environment provided by mothers has on their offspring's phenotypes, such as offspring survival and growth. Variations in maternal behavior and physiology are responsible for variations in maternal performance, which in turn affects offspring survival. In our study we found females that failed to nurture their offspring and showed abnormal maternal behaviors. The genetic architecture of maternal performance for offspring survival was investigated in 241 females of an F(2) intercross of the SM/J and LG/J inbred mouse strains. Using interval-mapping methods we found two quantitative trait loci (QTL) affecting maternal performance at D2Mit17 + 6 cM and D7Mit21 + 2 cM on chromosomes 2 and 7, respectively. In a two-way genome-wide epistasis scan we found 15 epistatic interactions involving 23 QTL distributed across all chromosomes except 12, 16, and 17. These loci form several small sets of interacting QTL, suggesting a complex set of mechanisms operating to determine maternal performance for offspring survival. Taken all together and correcting for the large number of significant factors, QTL and their interactions explain almost 35% of the phenotypic variation for maternal performance for offspring survival in this cross. This study allowed the identification of many possible candidate genes, as well as the relative size of gene effects and patterns of gene action affecting maternal performance in mice. Detailed behavior observation of mothers from later generations suggests that offspring survival in the first week is related to maternal success in building nests, grooming their pups, providing milk, and/or manifesting aggressive behavior against intruders.

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.

Use of transgenic plants to measure insect herbivore movement.

Use of ingested transgenic corn tissue as a marker for measuring movement of adult Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae; western corn rootworm) was investigated. Laboratory observations of beetles feeding on corn foliage, pollen, silks, or soybean foliage provided background on feeding patterns. The interval between food consumption and its appearance in feces (gut passage time) ranged from 102.7 +/- 11 min for soybean foliage to 56.7 +/- 2.9 min for corn silks. In a laboratory assay, protein expression tests identified the presence of Cry3Bb1 protein inside 50% of adult D. virgifera for up to 16 h after they had last consumed Cry3Bb1 protein-expressing corn silks from 'YieldGard Rootworm' corn plants (Monsanto Co.). Cry3Bb1 protein could not be detected by 32 h postfeeding. The proportion of Cry3Bb1 protein-positive beetles declined linearly with increasing time since feeding on 'YieldGard Rootworm' tissue. Approximately 20% of adult D. virgifera collected near 'YieldGard Rootworm' corn plots tested positive for Cry3Bb1 protein, indicating 'YieldGard Rootworm' tissue consumption within the last 16-32 h. Based on a 16- to 32-h postfeeding detection interval for Cry3Bb1 protein and the distance between 'YieldGard Rootworm' sources and sites where Cry3Bb1-positive insects were collected, 85.3% of males and females moved < or = 4.6-9.1 m/d through R2-R3 stage corn. Among Cry3Bb1-positive adults that left corn and were captured in an adjacent soybean field, 86.4% of males and 93.1% of females moved < or = 4.6-9.1 m/d through soybean. Detection of transgenic plant tissues in mobile insect herbivores is a novel application of biotechnology to the study of insect movement.

A global approach to resistance monitoring.

Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) have been grown in many parts of the world since 1996. In the United States, the Environmental Protection Agency (EPA) has required that industry submit insect resistance management (IRM) plans for each Bt corn and cotton product commercialized. A coalition of stakeholders including the EPA, USDA, academic scientists, industry, and grower organizations have cooperated in developing specific IRM strategies. Resistance monitoring (requiring submission of annual reports to the EPA), and a remedial action plan addressing any contingency if resistance should occur, are important elements of these strategies. At a global level, Monsanto conducts baseline susceptibility studies (prior to commercialization), followed by monitoring studies on target pest populations, for all of its commercialized Bt crop products. To date, Monsanto has conducted baseline/monitoring studies in Argentina, Australia, Brazil, Canada, China, Colombia, India, Mexico, the Philippines, South Africa, Spain, and the United States. Examples of pests on which resistance monitoring has been conducted include cotton bollworm, Helicoverpa zea, European corn borer, Ostrinia nubilalis, pink bollworm, Pectinophora gossypiella, Southwestern corn borer, Diatraea grandiosella, tobacco budworm, Heliothis virescens, and western corn rootworm, Diabrotica virgifera virgifera, in the United States, cotton bollworm, Helicoverpa armigera, in China, India and Australia, and H. virescens and H. zea in Mexico. No field-selected resistance to Bt crops has been documented.

Quantitative trait loci for body size components in mice.

Do body size components, such as weights of internal organs and long bone lengths, with different functions and different developmental histories also have different genetic architectures and pleiotropic patterns? We examine murine quantitative trait loci (QTL) for necropsy weight, four long bone lengths, and four organ weights in the LG/J x SM/J intercross. Differences between trait categories were found in number of QTL, dominance, and pleiotropic patterns. Ninety-seven QTLs for individual traits were identified: 52 for long bone lengths, 30 for organ weights, and 15 for necropsy weight. Results for long bones are typically more highly significant than for organs. Organ weights were more frequently over- or underdominant than long bone lengths or necropsy weight. The single-trait QTLs map to 35 pleiotropic loci. Long bones are much more frequently affected in groups while organs tend to be affected singly or in pairs. Organs and long bones are found at the same locus in only 11 cases, 8 of which also include necropsy weight. Our results suggest mainly separate genetic modules for organ weights and long bone lengths, with a few loci that affect overall body size. Antagonistic pleiotropy, in which a locus has opposite effects on different characteristics, is uncommon.

Diet, obesity, and hyperglycemia in LG/J and SM/J mice.

OBJECTIVE: To examine the differential response of obesity- and diabetes-related traits to a high- or low-fat diet in LG/J and SM/J mice. We also examined food consumption in these strains. RESEARCH METHODS AND PROCEDURES: Mice were placed on a high- or low-fat diet after weaning. Animals were weighed once per week and subjected to glucose tolerance tests at 20 weeks. At sacrifice, fat pads and internal organs were removed along with serum samples. For food consumption, LG/J and SM/J mice of each sex were assigned to a high-fat or low-fat diet after reaching maturity. Mice were weighed three times per week, and food consumed was determined by subtraction. RESULTS: LG/J animals consume more total food, but SM/J animals consume more food per gram of body weight. LG/J mice grow faster to 10 weeks but slower from 10 to 20 weeks, have higher cholesterol and free fatty acid levels, and have lower basal glucose levels and better response to a glucose challenge than SM/J mice. For most traits, SM/J mice respond more strongly to a high-fat diet than LG/J mice, including body weight and growth, basal glucose levels, organ weights, fat distribution, and circulating triglycerides and cholesterol levels. DISCUSSION: Obesity-related phenotypes, as well as response to increased dietary fat, differ genetically between LG/J and SM/J and can, therefore, be mapped. This study indicates that the cross of SM/J and LG/J mice would be an excellent model system for the study of gene-by-diet interaction in obesity.

Pleiotropic effects on mandibular morphology II: differential epistasis and genetic variation in morphological integration.

The evolution of morphological modularity through the sequestration of pleiotropy to sets of functionally and developmentally related traits requires genetic variation in the relationships between traits. Genetic variation in relationships between traits can result from differential epistasis, where epistatic relationships for pairs of loci are different for different traits. This study maps relationship quantitative trait loci (QTLs), specifically QTLs that affect the relationship between individual mandibular traits and mandible length, across the genome in an F2 intercross of the LG/J and SM/J inbred mouse strains (N = 1045). We discovered 23 relationship QTLs scattered throughout the genome. All mandibular traits were involved in one or more relationship QTL. When multiple traits were affected at a relationship QTL, the traits tended to come from a developmentally restricted region of the mandible, either the muscular processes or the alveolus. About one-third of the relationship QTLs correspond to previously located trait QTLs affecting the same traits. These results comprise examples of genetic variation necessary for an evolutionary response to selection on the range of pleiotropic effects.

Pleiotropic effects on mandibular morphology I. Developmental morphological integration and differential dominance.

Pleiotropy refers to a single genetic locus that affects more than one phenotypic trait. Pleiotropic effects of genetic loci are thought to play an important role in evolution, reflecting functional and developmental relationships among phenotypes. In a previous study, we examined pleiotropic effects displayed by quantitative trait loci (QTLs) on murine mandibular morphology in relation to mandibular structure and function. In replicating most of our previous QTLs and increasing our sample size, this study strengthens and extends our earlier results. As in our previous study, we find that QTL effects tend to be restricted to developmentally or functionally related traits. In addition, we examine patterns of differential dominance for pleiotropic QTL effects. Differential dominance occurs when dominance patterns for a single locus vary among traits. We find that multivariate overdominance is a common and substantial phenomenon, and may potentially provide an explanation for the persistence of heterozygosity in natural populations.

EPISTASIS AND THE EVOLUTION OF ADDITIVE GENETIC VARIANCE IN POPULATIONS THAT PASS THROUGH A BOTTLENECK.

Traditional models of genetic drift predict a linear decrease in additive genetic variance for populations passing through a bottleneck. This perceived lack of heritable variance limits the scope of founder-effect models of speciation. We produced 55 replicate bottleneck populations maintained at two male-female pairs through four generations of inbreeding (average F = 0.39). These populations were formed from an F2 intercross of the LG/J and SM/J inbred mouse strains. Two contemporaneous control strains maintained with more than 60 mating pairs per generation were formed from this same source population. The average level of within-strain additive genetic variance for adult body weight was compared between the control and experimental lines. Additive genetic variance for adult body weight within experimental bottleneck strains was significantly higher than expected under an additive genetic model This enhancement of additive genetic variance under inbreeding is likely to be due to epistasis, which retards or reverses the loss of additive genetic variance under inbreeding for adult body weight in this population. Therefore, founder-effect speciation processes may not be constrained by a loss of heritable variance due to population bottlenecks.

Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte.

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.