Crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) are widely planted and enable management of key insect pests while reducing the use of conventional insecticides. However, the evolution of Bt resistance could diminish these benefits. Fitness costs of Bt resistance occur in the absence of Bt toxin when individuals with resistance alleles show a reduction in fitness relative to susceptible individuals, and they can delay the evolution of resistance. Ecological factors including host-plant variety can affect the magnitude of fitness costs, and consequently, the degree to which fitness costs delay resistance. In this study, we measured fitness costs of resistance to Bt toxin Cry1F in the European corn borer Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) using Cry1F-resistant and Cry1F-susceptible strains sharing a similar genetic background. Fitness costs were tested on three lines of maize, Zea mays L., by measuring larval survival and development in two greenhouse experiments with plants in either the vegetative or reproductive stage. Both experiments showed that maize line significantly affected larval survival and developmental rate. However, larval survival, mass, and developmental rate did not differ between the Cry1F-resistant and susceptible strains, indicating a lack of fitness costs of resistance to Cry1F for the larval fitness components measured in this experiment. Future experiments should test for fitness costs of Cry1F resistance affecting survival to adulthood and adult life-history parameters.
Plant cell walls are the largest reservoir of organic carbon on earth. To breach and utilize this carbohydrate-rich protective barrier, microbes secrete plant cell wall degrading enzymes (PCWDEs) targeting pectin, cellulose and hemicelluloses. There is a growing body of evidence that genomes of some herbivorous insects also encode PCWDEs, raising questions about their evolutionary origins and functions. Among herbivorous beetles, pectin-degrading polygalacturonases (PGs) are found in the diverse superfamilies Chrysomeloidea (leaf beetles, long-horn beetles) and Curculionoidea (weevils). Here our aim was to test whether these arose from a common ancestor of beetles or via horizontal gene transfer (HGT), and whether PGs kept their ancestral function in degrading pectin or evolved novel functions. Transcriptome data derived from 10 beetle species were screened for PG-encoding sequences and used for phylogenetic comparisons with their bacterial, fungal and plant counterparts. These analyses revealed a large family of PG-encoding genes of Chrysomeloidea and Curculionoidea sharing a common ancestor, most similar to PG genes of ascomycete fungi. In addition, 50 PGs from beetle digestive systems were heterologously expressed and functionally characterized, showing a set of lineage-specific consecutively pectin-degrading enzymes, as well as conserved but enzymatically inactive PG proteins. The evidence indicates that a PG gene was horizontally transferred ?200 million years ago from an ascomycete fungus to a common ancestor of Chrysomeloidea and Curculionoidea. This has been followed by independent duplications in these two lineages, as well as independent replacement in two sublineages of Chrysomeloidea by two other subsequent HGTs. This origin, leading to subsequent functional diversification of the PG gene family within its new hosts, was a key event promoting the evolution of herbivory in these beetles.
A variety of management methods to control the soybean aphid (Aphis glycines Matsumura) have been investigated since its invasion into North America in 2000, among them plant resistance has emerged as a viable option for reducing aphid damage to soybeans and preventing outbreaks. Plant resistance methods often use natural soybean plant defenses that impose stress on aphids by reducing fitness and altering behavior. Research efforts have heavily focused on identification and development of aphid resistant soybean varieties, leaving much unknown about soybean aphid response to stressful host plant defenses. In this study, we aimed to 1) evaluate lifetime fitness consequences and phenotypic variation in response to host plant-induced stress and 2) investigate whether trade-offs involving fitness costs and/or cross-virulence to multiple antibiotic soybean varieties exists. We compared aphid survival and reproduction during and after a short period of exposure to soybeans with the Rag2 resistance gene and measured aphid clonal variation in response to Rag2 soybeans. In addition, we measured the performance of Rag2 virulent and avirulent aphids on five soybean varieties with various forms of antibiotic resistance. Our results indicate that plant defenses impose high levels of stress and have long-term fitness consequences, even after aphids are removed from resistant plants. We identified one aphid clone that was able to colonize Rag2 among the seven clones tested, suggesting that virulent genotypes may be prevalent in natural populations. Finally, although we did not find evidence of cross-virulence to multiple antibiotic soybean varieties, our results suggest independent mechanisms of aphid virulence to Rag1 and Rag2 that may involve fitness costs.
Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (?-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ?-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1?) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, ?-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.
Cellulose is an important nutritional resource for a number of insect herbivores. Digestion of cellulose and other polysaccharides in plant-based diets requires several types of enzymes including a number of glycoside hydrolase (GH) families. In a previous study, we showed that a single GH45 gene is present in the midgut tissue of the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). However, the presence of multiple enzymes was also suggested by the lack of a significant biological response when the expression of the gene was silenced by RNA interference. In order to clarify the repertoire of cellulose-degrading enzymes and related GH family proteins in D. v. virgifera, we performed next-generation sequencing and assembled transcriptomes from the tissue of three different developmental stages (eggs, neonates, and third instar larvae). Results of this study revealed the presence of seventy-eight genes that potentially encode GH enzymes belonging to eight families (GH45, GH48, GH28, GH16, GH31, GH27, GH5, and GH1). The numbers of GH45 and GH28 genes identified in D. v. virgifera are among the largest in insects where these genes have been identified. Three GH family genes (GH45, GH48, and GH28) are found almost exclusively in two coleopteran superfamilies (Chrysomeloidea and Curculionoidea) among insects, indicating the possibility of their acquisitions by horizontal gene transfer rather than simple vertical transmission from ancestral lineages of insects. Acquisition of GH genes by horizontal gene transfers and subsequent lineage-specific GH gene expansion appear to have played important roles for phytophagous beetles in specializing on particular groups of host plants and in the case of D. v. virgifera, its close association with maize.
Transgenic corn hybrids that express toxins from Bacillus thuringiensis has suppressed European corn borer populations and reduced the pest status of this insect throughout much of the U.S. Corn Belt. A major assumption of the high-dose/refuge strategy proposed for insect resistance management and Bt corn is that the frequency of resistance alleles is low so that resistant pests surviving exposure to Bt corn will be rare.
Transgenic expression of Bacillus thuringiensis (Bt) crystalline (Cry) toxins by crop plants result in reduced insect feeding damage, but sustainability is threatened by the development of resistance traits in target insect populations. We investigated Bt toxin resistance trait in a laboratory colony of the European corn borer, Ostrinia nubilalis, selected for increased survival when exposed to Cry1Ab and correlated survival on Cry1Ab toxin with a constitutive ?146.2 ± 17.3-fold reduction in midgut aminopeptidase N1 (apn1) transcript levels. A 7.1 ± 1.9-fold reduction apn3 transcript level was also correlated with Cry1Ab resistance. Quantitative trait locus (QTL) mapping identified a single major genome region controlling Cry1Ab resistance on linkage group 24 (LG24), and a minor QTL on LG27. Both QTL were independent of apn1 and apn3 loci on LG02. Positional mapping identified genetic markers that may assist in the identification of causal gene(s) within QTL intervals. This study indicates that genetic factor(s) may act in trans to reduce both apn1 and apn3 expression in Cry1Ab resistant O. nubilalis larvae, and suggest that gene regulatory pathways can influence Bt resistance traits. These findings show that gene interactions (epistasis) may influence Bt resistance in target insect populations.
The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species. In addition, similar expression of the putative cadherin- and APN-like protein receptors were observed at 260 and 135 kDa, respectively. A high degree of similarity (98% amino acid sequence identity) of cDNA sequences for both putative receptor sequences was observed. The Cry1F ligand blot revealed that O. furnacalis and O. nubilalis BBMV exhibited slightly different binding patterns, with strong binding to putative proteins at 150 and 140 kDa, respectively. Both proteins appeared to also bind Cry1Ab, although the signal intensity was much reduced with Cry1Ab. O. furnacalis showed an additional but weaker band at 210 kDa relative to the 150 kDa band. Diatraea saccharalis (Fabricius), which was used as an outgroup species, exhibited different binding patterns than either Ostrinia species, with both Cry1Ab and Cry1F toxins binding to a 210 kDa protein. These results support the previous experiments indicating that O. nubilalis and O. furnacalis share similar patterns of susceptibility to Cry toxins.
RNA interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that the efficiency of RNAi varies between different species, the mode of RNAi delivery, and the genes being targeted. There is also variation in the duration of transcript suppression. At present, we have a limited capacity to predict the ideal experimental strategy for RNAi of a particular gene/insect because of our incomplete understanding of whether and how the RNAi signal is amplified and spread among insect cells. Consequently, development of the optimal RNAi protocols is a highly empirical process. This limitation can be relieved by systematic analysis of the molecular physiological basis of RNAi mechanisms in insects. An enhanced conceptual understanding of RNAi function in insects will facilitate the application of RNAi for dissection of gene function, and to fast-track the application of RNAi to both control pests and develop effective methods to protect beneficial insects and non-insect arthropods, particularly the honey bee (Apis mellifera) and cultured Pacific white shrimp (Litopenaeus vannamei) from viral and parasitic diseases.
: The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is a significant corn pest throughout the United States corn belt. Rootworm larvae feed on corn roots causing yield losses and control expenditures that are estimated to exceed US$1 billion annually. Traditional management practices to control rootworms such as chemical insecticides or crop rotation have suffered reduced effectiveness due to the development of physiological and behavioral resistance. Transgenic maize expressing insecticidal proteins are very successful in protecting against rootworm damage and preserving corn yield potential. However, the high rate of grower adoption and early reliance on hybrids expressing a single mode of action and low-dose traits threatens the durability of commercialized transgenic rootworm technology for rootworm control. A summary of current transgenic approaches for rootworm control and the corresponding insect resistance management practices is included. An overview of potential new modes of action based on insecticidal proteins, and especially RNAi targeting mRNA coding for essential insect proteins is provided.
Chemical analysis shows that honey bees (Apis mellifera) and hive products contain many pesticides derived from various sources. The most abundant pesticides are acaricides applied by beekeepers to control Varroa destructor. Beekeepers also apply antimicrobial drugs to control bacterial and microsporidial diseases. Fungicides may enter the hive when applied to nearby flowering crops. Acaricides, antimicrobial drugs and fungicides are not highly toxic to bees alone, but in combination there is potential for heightened toxicity due to interactive effects.
The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (>12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn. Genetic linkage maps with segregating AFLP markers show that the Cry1F resistance trait is controlled by a single quantitative trait locus (QTL) on linkage group 12. The map position of single nucleotide polymorphism (SNP) markers indicated that midgut Bt toxin-receptor genes, alkaline phosphatase, aminopeptidase N, and cadherin, are not linked with the Cry1F QTL. Evidence suggests that genes within this genome interval may give rise to a novel Bt toxin resistance trait for Lepidoptera that appears independent of known receptor-based mechanisms of resistance.
Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, but insect adaptation can reduce their efficacy. The genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. Previous results suggested that the modified toxins would be effective only if resistance was linked with mutations in genes encoding toxin-binding cadherin proteins. Here we report evidence from five major crop pests refuting this hypothesis. Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of Plutella xylostella and Ostrinia nubilalis in which resistance was not linked with cadherin mutations. Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains.
The natural biodiversity that is found in tropical areas offers countless biotechnological opportunities; especially if we take in account that many biomolecules from several microorganisms have supported for many years, different industrial applications in areas such as pharmacology, agro-industry, bioprocess, environmental technology, and bioconversion. In order to find new lignocellulolytic enzymes and evaluate bamboo fibers as substrate, Schizophyllum commune a fungus with broad distribution was isolated and grown during 15 days in liquid culture medium containing 1% lignocellulosic fibers from bamboo, banana stem, and sugarcane bagasse. The enzymatic activity of xylanase, mannanase, polygalacturonase, CMCase, FPase, and avicelase were evaluated. Sugarcane bagasse and banana stem showed to induce higher hollocellulase activity when compared with bamboo as the main carbon source. The physical mechanism that the fungus uses to degrade bamboo was observed not only in fibers naturally infected but also in healthy fibers that were treated and untreated with enzyme solution. SEM analysis showed the structural disruption and invasion of the vascular bundles, parenchyma cells, and parenchymatous tissues as a consequence of the presence of this fungus and the catalytic action of its enzymes into the plant tissue.
The cross-resistance spectrum and biochemical mechanism of resistance to the Bacillus thuringiensis Cry1Ab toxin was studied in a field-derived strain of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) that was further selected in the laboratory for high levels (>1000-fold) of resistance to Cry1Ab. The resistant strain exhibited high levels of cross-resistance to Cry1Ac and Cry1Aa but only low levels of cross-resistance (<4-fold) to Cry1F. In addition, there was no significant difference between the levels of resistance to full-length and trypsin-activated Cry1Ab protein. No differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in the resistant strain. Significantly reduced binding of radiolabeled Cry1Aa was observed in the resistant strain whereas binding of Cry1Ab and Cry1Ac was practically the same in both resistant and susceptible strains. The interpretation of the overall data seems to suggest the involvement of an alteration in the binding of Cry1A toxins to a common receptor, which is more clearly revealed by the binding assays using radiolabeled Cry1Aa.
Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study, we identified 10 aminopeptidase-like genes, which have previously been identified as putative Bt toxin receptors in other insects and examined their expression in relation to Cry1Ab toxicity and resistance. Expression analysis for the 10 aminopeptidase-like genes revealed that most of these genes were expressed predominantly in the larval midgut, but there was no difference in the expression of these genes in Cry1Ab resistant and susceptible strains. This suggested that altered expression of these genes was unlikely to be responsible for resistance in these ECB strains. However, we found that there were changes in two amino acid residues of the aminopeptidase-P like gene (OnAPP) involving Glu(305) to Lys(305) and Arg(307) to Leu(307) in the two Cry1Ab-resistant strains as compared with three Cry1Ab-susceptible strains. The mature OnAPP contains 682 amino acid residues and has a putative signal peptide at the N-terminus, a predicted glycosylphosphatidyl-inositol (GPI)-anchor signal at the C-terminal, three predicted N-glycosylation sites at residues N178, N278 and N417, and an O-glycosylation site at residue T653. We used a feeding based-RNA interference assay to examine the role of the OnAPP gene in Cry1Ab toxicity and resistance. Bioassays of Cry1Ab in larvae fed diet containing OnAPP dsRNA resulted in a 38% reduction in the transcript level of OnAPP and a 25% reduction in the susceptibility to Cry1Ab as compared with larvae fed GFP dsRNA or water. These results strongly suggest that the OnAPP gene could be involved in binding the Cry1Ab toxin in the ECB larval midgut and that mutations in this gene may be associated with Bt resistance in these two ECB strains.
To test the efficacy of RNA interference (RNAi) as a method for target-site screening in Diabrotica virgifera virgifera LeConte (Coleptera: Chrysomelidae) larvae, genes were identified and tested for which clear RNAi phenotypes had been identified in the Coleopteran model, Tribolium castaneum. Here the cloning of the D. v. vergifera orthologs of laccase 2 (DvvLac2) and chitin synthase 2 (DvvCHS2) is reported. Injection of DvvLac2-specific double-stranded RNA resulted in prevention of post-molt cuticular tanning, while injection of DvvCHS2-specific dsRNA reduced chitin levels in midguts. Silencing of both DvvLac2 and DvvCHS2 was confirmed by RT-PCR and quantitative RT-PCR. As in T. castaneum, RNAi-mediated gene silencing is systemic in Diabrotica. The results indicate that RNAi-induced silencing of D. v. vergifera genes provides a powerful tool for identifying potential insecticide targets.
The study of fitness costs associated with resistance to toxins from Bacillus thuringiensis Berliner (Bt) in Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) is important for understanding resistance evolution and for evaluating resistance management practices that prevent or mitigate resistance to transgenic corn, Zea mays L. Resistant individuals identified from a field collection in Kandiyohi, MN, were used to generate a Cry1Ab-resistant strain. We used susceptible and resistant strains with similar genetic background to establish crosses and estimate dominance of fitness costs by measuring fitness components and population parameters determined by fertility life tables. Spermatophore volume and mating frequency also were compared to identify potential effects of resistance on fertility. Inheritance of fitness costs in O. nubilalis varied from recessive to incompletely recessive among the parameters evaluated. Selection for resistance to Cry1Ab significantly reduced the fitness of O. nubilalis. Resistant insects exhibited reduced pupal weight and increased developmental time compared with susceptible and F1 larvae derived from reciprocal crosses of resistant and susceptible parents. In addition, it was observed that resistant insects exhibited a higher proportion of unsuccessful matings and lower fertility than the susceptible strain. Despite the differences observed in resistant insects, our results did not indicate strong evidence of fitness costs in the F1 progeny.
DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction.
Atrazine is a widely used herbicide that has been reported to induce the activity of certain detoxification enzymes and to affect insecticide toxicity in organisms experiencing simultaneous exposure to both atrazine and insecticides. In this study, the effects of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) exposure on protein expression in male and female Drosophila melanogaster adults in both microsomal and cytosolic cell fractions was investigated by 2-dimensional gel electrophoresis. Differentially expressed proteins (vs. controls) were identified using matrix assisted laser desorption-time (MALDI-TOF) of flight mass spectrometry (MS). We identified a total of 28 proteins associated with energy production including glycolysis and mitochondrial respiration as differentially expressed and nine proteins associated with detoxification and response to oxidative stress. Most of these proteins were expressed in one sex or the other but not in both. Surprisingly, the only proteins associated with detoxification were identified as glutathione transferases. No cytochrome P450s were identified which have previously been shown to be responsive to atrazine exposure in D. melanogaster and proposed to be associated with insecticide/atrazine interactions. Results of this investigation support the role of atrazine in affecting mitochondrial electron transport and oxidative stress. However, the role of atrazine in pesticide interactions remains uncertain.
A laboratory study was conducted to determine the toxicity of indoxacarb and chlorantraniliprole to Eastern subterranean termites, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) resulting from topical applications and exposure to treated soil. Soils with varying organic matter (0.57-3.64%) and chemical characteristics were used in termiticide bioassays. Lethal dose resulting from topical application indicated that chlorantraniliprole was two- to 11-fold more toxic than indoxacarb. Lethal concentration assays yielded opposite results where concentrations of indoxacarb in soil that caused either 50 or 90% mortality of R. flavipes workers at 48 and 144 h were two- to six-fold lower than chlorantraniliprole. The bioavailability of indoxacarb and chlorantraniliprole was negatively correlated with soil organic matter. Our results suggest that indoxacarb is more bioavailable to termites in soil than chlorantraniliprole based on calculated bioavailability ratios. However, how these laboratory results correlate to actual field application data and termite efficacy is unknown, and more research is needed. These compounds seem to have excellent activity on termites and have potential to provide new modes of action and new chemistry as liquid termiticides.
Microsatellite loci are standard genetic markers for population genetic analysis, whereas single nucleotide polymorphisms (SNPs) are more recent tools that require assessment of neutrality and appropriate use in population genetics. Twelve SNP markers were used to describe the genetic structure of Diabrotica virgifera virgifera (LeConte; Coleoptera: Chrysomelidae) in the United States of America and revealed a high mean observed heterozygosity (0.40 +/- 0.059) and low global F(ST) (0.029). Pairwise F(ST) estimates ranged from 0.007 to 0.045, and all but 2 populations showed significant levels of genetic differentiation (P < or = 0.008). Population parameters and conclusions based on SNP markers were analogous to that obtained by use of microsatellite markers from the identical population samples. SNP-based F(ST) estimates were 3-fold higher than corresponding estimates from microsatellites, wherein lower microsatellite F(ST) estimates likely resulted from an overestimate of migration rates between subpopulations due to convergence of allele size (homoplasy). No significant difference was observed in the proportion of SNP or microsatellite markers loci that were nonneutral within populations. SNP markers provided estimates of population genetic parameters consistent with those from microsatellite data, and their low back mutation rates may result in reduced propensity for error in estimation of population parameters.
The high dose plus refuge is one of the major components of the resistance management plan mandated for transgenic corn expressing Cry toxins from Bacillus thuringiensis Berliner (Bt) that targets the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae). This strategy was based on assumptions such as functional recessive inheritance, which has not been previously tested for O. nubilalis. The authors used a field-derived resistant strain of O. nubilalis to define the nature of resistance to Cry1Ab toxin by examining the inheritance and on-plant survival of susceptible and resistant insects and their F(1) progeny.
Transgenic corn expressing Cry1Ab (a Bacillus thuringiensis toxin) is highly effective in the control of Ostrinia nubilalis. For its toxic action, Cry1Ab has to bind to specific insect midgut proteins. To date, in three Lepidoptera species resistance to a Cry1A toxin has been conferred by mutations in cadherin, a protein of the Lepidoptera midgut membrane. The implication of cadherin in the resistance of an Ostrinia nubilalis colony (Europe-R) selected with Bacillus thuringiensis Cry1Ab protoxin was investigated. Several major mutations in the cadherin (cdh) gene were found, which introduced premature termination codons and/or large deletions (ranging from 1383 to 1701bp). The contribution of these major mutations to the resistance was analyzed in resistant individuals that survived exposure to a high concentration of Cry1Ab protoxin. The results indicated that the presence of major mutations was drastically reduced in individuals that survived exposure. Previous inheritance experiments with the Europe-R strain indicated the involvement of more than one genetic locus and reduced amounts of the cadherin receptor. The results of the present work support a polygenic inheritance of resistance in the Europe-R strain, in which mutations in the cdh gene would contribute to resistance by means of an additive effect.
The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is native to Asia and was recently (2000) detected in North America. Since then, it has become a significant threat to U.S. soybean production. Although neonicotinoid insecticide seed treatments, imidacloprid and thiamethoxam, have been suggested as a method of control, the season-long efficacy is still uncertain. Therefore, the use of imidacloprid and thiamethoxam seed treatments to control soybean aphid in Nebraska were examined. Soybean aphid populations were monitored weekly in an irrigated field study planted during the later half of the typical Nebraska planting window during 2005 and 2006. Imidacloprid and thiamethoxam were quantified by leaves through time, and leaf specific bioassays were conducted. In 2005, aphid populations were very low; however, in 2006 aphid numbers were significantly higher than 2005, reaching approximately 1,200 aphids per plant in the untreated plots. Aphid injury significantly reduced yield and individual seed size in 2006. Imidacloprid significantly reduced aphid densities in 2006 but not below the economic threshold. In 2006, thiamethoxam held aphid densities below the economic threshold. Leaf specific bioassays and leaf specific imidacloprid and thiamethoxam quantification indicated that thiamethoxam was present in the plant at higher concentrations and for a longer period.
Lepidoptera represents more than 160,000 insect species which include some of the most devastating pests of crops, forests, and stored products. However, the genomic information on lepidopteran insects is very limited. Only a few studies have focused on developing expressed sequence tag (EST) libraries from the guts of lepidopteran larvae. Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt) toxins, and for discovering new targets for novel toxins for use in pest management. This study analyzed the ESTs generated from the larval gut of the European corn borer (ECB, Ostrinia nubilalis), one of the most destructive pests of corn in North America and the western world. Our goals were to establish an ECB larval gut-specific EST database as a genomic resource for future research and to explore candidate genes potentially involved in insect-Bt interactions and Bt resistance in ECB.
The biochemical mechanism of resistance to the Bacillus thuringiensis Cry1F toxin was studied in a laboratory-selected strain of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) showing more than 3000-fold resistance to Cry1F and limited cross resistance to other Cry toxins. Analyses of Cry1F binding to brush border membrane vesicles of midgut epithelia from susceptible and resistant larvae using ligand immunoblotting and Surface Plasmon Resonance (SPR) suggested that reduced binding of Cry1F to insect receptors was not associated with resistance. Additionally, no differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in the resistant strain. Considering these results along with previous evidence of relatively narrow spectrum of cross resistance and monogenic inheritance, the resistance mechanism in this Cry1F selected strain of O. nubilalis appears to be specific and may be distinct from previously identified resistance mechanisms reported in other Lepidoptera.
Acaricides are used to treat honey bee (Apis mellifera L.) colonies to control the varroa mite (Varroa destructor Anderson & Trueman), a worldwide threat to honey bee health. Although acaricides control a serious honey bee parasite and mitigate bee loss, they may cause harm to bees as well. We topically applied five acaricides, each with a different mode of action, to young adult queen and worker bees to generate dose-response curves and LD50. Twenty-four hours after treatment, queens were found to be three-times more tolerant of tau-fluvalinate and six-times more tolerant of thymol than workers when adjusted for body weight differences between workers (108 mg) and queens (180 mg). Queens survived the highest administered doses of fenpyroximate (1620 microg/g) and coumaphos (2700 microg/g) indicating that queens are at least 11-fold more tolerant of coumaphos and at least 54-fold more tolerant of fenpyroximate than workers. However, queens treated with as little as 54 microg/g of fenpyroximate exhibited reduced survival over 6 wk after treatment. Amitraz was the only acaricide tested for which queens were not more tolerant than workers. The striking difference in acaricide tolerance of queen and worker honey bees suggests physiological differences in how the two castes are affected by xenobiotics.
A novel insect ?-1,4-endoglucanase (DvvENGaseI) gene belonging to the glycoside hydrolase family (GHF) 45 was identified from the western corn rootworm, Diabrotica virgifera virgifera. The cDNA of the DvvENGaseI consisted of a 720 bp open reading frame encoding a 239 amino-acid protein. Analysis of the amino acid sequence revealed that DvvENGaseI exhibits 60% protein sequence identity when compared with an endoglucanase belonging to GHF45 from another beetle, Leptinotarsa decemlineata. Western blot analyses using a polyclonal antiserum developed from a partial peptide sequence revealed that DvvENGaseI expression coincided with body regions corresponding to the fore-, mid- and hindgut, although regions corresponding to the midgut and hindgut were the primary sites for DvvENGaseI expression. Functional analysis of the DvvENGaseI by RNA interference (RNAi) indicated that nearly complete knock-down of gene expression could be obtained by injection of dsRNA based on qRT-PCR and western blot analysis. However, suppression only resulted in slight developmental delays suggesting that this gene may be part of a larger system of cellulose degrading enzymes.
Neonicotinoid insecticides are generally efficacious against many turfgrass pests, including several important phloem-feeding insects. However, inconsistencies in control of western chinch bugs, Blissus occiduus, have been documented in field efficacy studies. This research investigated the efficacy of three neonicotinoid insecticides (clothianidin, imidacloprid and thiamethoxam) against B. occiduus in buffalograss under field conditions and detected statistically significant differences in B. occiduus numbers among treatments. A subsequent study documented the relative quantity and degradation rate of these insecticides in buffalograss systemic leaf tissues, using HPLC.
Feeding damage caused by the western corn rootworm, Diabrotica virgifera virgifera, is destructive to corn plants in North America and Europe where control remains challenging due to evolution of resistance to chemical and transgenic toxins. A BAC library, DvvBAC1, containing 109,486 clones with 104 ± 34.5?kb inserts was created, which has an ~4.56X genome coverage based upon a 2.58?Gb (2.80?pg) flow cytometry-estimated haploid genome size. Paired end sequencing of 1037 BAC inserts produced 1.17?Mb of data (~0.05% genome coverage) and indicated ~9.4 and 16.0% of reads encode, respectively, endogenous genes and transposable elements (TEs). Sequencing genes within BAC full inserts demonstrated that TE densities are high within intergenic and intron regions and contribute to the increased gene size. Comparison of homologous genome regions cloned within different BAC clones indicated that TE movement may cause haplotype variation within the inbred strain. The data presented here indicate that the D. virgifera virgifera genome is large in size and contains a high proportion of repetitive sequence. These BAC sequencing methods that are applicable for characterization of genomes prior to sequencing may likely be valuable resources for genome annotation as well as scaffolding.
The European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) has been a major pest of corn and other crops in North America since its accidental introduction nearly a hundred years ago. Wide adoption of transgenic corn hybrids that express toxins from Bacillus thuringiensis, referred to as Bt corn, has suppressed corn borer populations and reduced the pest status of this insect in parts of the Corn Belt. Continued suppression of this pest, however, will depend on managing potential resistance to Bt corn, currently through the high-dose refuge (HDR) strategy. In this review, we describe what has been learned with regard to O. nubilalis resistance to Bt toxins either through laboratory selection experiments or isolation of resistance from field populations. We also describe the essential components of the HDR strategy as they relate to O. nubilalis biology and ecology. Additionally, recent developments in insect resistance management (IRM) specific to O. nubilalis that may affect the continued sustainability of this technology are considered.
RNA interference (RNAi) is commonly used in insect functional genomics studies and usually involves direct injection of double-stranded RNA (dsRNA). Only a few studies have involved exposure to dsRNAs through feeding. For western corn rootworm (Diabrotica virgifera virgifera) larvae, ingestion of dsRNA designed from the housekeeping gene, vacuolar ATPase (vATPase) triggers RNAi causing growth inhibition and mortality; however, the effect of dsRNA feeding on adults has not been examined. In this research, WCR adults were fed with vATPase-dsRNA-treated artificial diet containing a cucurbitacin bait, which is a proven feeding stimulant for chrysomelid beetles of the subtribe Diabroticina to which rootworms belong.
Concern about the role of pesticides in honey bee decline has highlighted the need to examine the effects of sublethal exposure on bee behaviors. The video-tracking system EthoVisionXT (Noldus Information Technologies) was used to measure the effects of sublethal exposure to tau-fluvalinate and imidacloprid on honey bee locomotion, interactions, and time spent near a food source over a 24-h observation period. Bees were either treated topically with 0.3, 1.5, and 3 µg tau-fluvalinate or exposed to 0.05, 0.5, 5.0, 50, and 500 ppb imidacloprid in a sugar agar cube. Tau-fluvalinate caused a significant reduction in distance moved at all dose levels (p < 0.05), as did 50 and 500 ppb imidacloprid (p < 0.001). Bees exposed to 50 and 500 ppb spent significantly more time near the food source than control bees (p < 0.05). Interaction time decreased as time in the food zone increased for both chemicals. This study documents that video-tracking of bee behavior can enhance current protocols for measuring the effects of pesticides on honey bees at sublethal levels. It may provide a means of identifying problematic compounds for further testing.
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