Search

Varroa mites (Varroa destructor) are parasitic mites that, combined with other factors, are contributing to high levels of honey bee (Apis mellifera) colony losses. A Varroa-active dsRNA was recently developed to control Varroa mites within honey bee brood cells. This dsRNA has 372 base pairs that are homologous to a sequence region within the Varroa mite calmodulin gene (cam). The Varroa-active dsRNA also shares a 21-base pair match with monarch butterfly (Danaus plexippus) calmodulin mRNA, raising the possibility of non-target effects if there is environmental exposure. We chronically exposed the entire monarch larval stage to common (Asclepias syriaca) and tropical (Asclepias curassavica) milkweed leaves treated with concentrations of Varroa-active dsRNA that are one- and ten-fold higher than those used to treat honey bee hives. This corresponded to concentrations of 0.025-0.041 and 0.211-0.282 mg/g leaf, respectively. Potassium arsenate and a previously designed monarch-active dsRNA with a 100% base pair match to the monarch v-ATPase A mRNA (leaf concentration was 0.020-0.034 mg/g) were used as positive controls. The Varroa mite and monarch-active dsRNA's did not cause significant differences in larval mortality, larval or pupal development, pupal weights, or adult eclosion rates when compared to negative controls. Irrespective of control or dsRNA treatment, larvae that consumed approximately 7500 to 10,500-mg milkweed leaf within 10 to 12 days had the highest pupal weights. The lack of mortality and sublethal effects following dietary exposure to dsRNA with 21-base pair and 100% base pair match to mRNAs that correspond to regulatory genes suggest monarch mRNA may be refractory to silencing by dsRNA or monarch dsRNase may degrade dsRNA to a concentration that is insufficient to silence mRNA signaling.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.

The legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is an insect pest species of crops grown by subsistence farmers in tropical regions of Africa. We present the de novo assembly of 3729 contigs from 454- and Sanger-derived sequencing reads for midgut, salivary, and whole adult tissues of this non-model species. Functional annotation predicted that 1320 M. vitrata protein coding genes are present, of which 631 have orthologs within the Bombyx mori gene model. A homology-based analysis assigned M. vitrata genes into a group of paralogs, but these were subsequently partitioned into putative orthologs following phylogenetic analyses. Following sequence quality filtering, a total of 1542 putative single nucleotide polymorphisms (SNPs) were predicted within M. vitrata contig assemblies. Seventy one of 1078 designed molecular genetic markers were used to screen M. vitrata samples from five collection sites in West Africa. Population substructure may be present with significant implications in the insect resistance management recommendations pertaining to the release of biological control agents or transgenic cowpea that express Bacillus thuringiensis crystal toxins. Mutation data derived from transcriptome sequencing is an expeditious and economical source for genetic markers that allow evaluation of ecological differentiation.

We report the assembly of the 14,054 bp near complete sequencing of the mitochondrial genome of the legume pod borer (LPB), Maruca vitrata (Lepidoptera: Crambidae), which we subsequently used to estimate divergence and relationships within the lepidopteran lineage. The arrangement and orientation of the 13 protein-coding, 2 rRNA, and 19 tRNA genes sequenced was typical of insect mitochondrial DNA sequences described to date. The sequence contained a high A+T content of 80.1% and a bias for the use of codons with A or T nucleotides in the 3rd position. Transcript mapping with midgut and salivary gland ESTs for mitochondrial genome annotation showed that translation from protein-coding genes initiates and terminates at standard mitochondrial codons, except for the coxI gene, which may start from an arginine CGA codon. The genomic copy of coxII terminates at a T nucleotide, and a proposed polyadenylation mechanism for completion of the TAA stop codon was confirmed by comparisons to EST data. EST contig data further showed that mature M. vitrata mitochondrial transcripts are monocistronic, except for bicistronic transcripts for overlapping genes nd4/nd4L and nd6/cytb, and a tricistronic transcript for atp8/atp6/coxIII. This processing of polycistronic mitochondrial transcripts adheres to the tRNA punctuated cleavage mechanism, whereby mature transcripts are cleaved only at intervening tRNA gene sequences. In contrast, the tricistronic atp8/atp6/coxIII in Drosophila is present as separate atp8/atp6 and coxIII transcripts despite the lack of an intervening tRNA. Our results indicate that mitochondrial processing mechanisms vary between arthropod species, and that it is crucial to use transcriptional information to obtain full annotation of mitochondrial genomes.

Abstract Asian corn borer, Ostrinia furnacalis (Guenée), is an important insect pest of maize throughout most of Asia. The rind of a maize stalk is a key barrier against corn borer larvae boring into the plant. There is a need to better understand the relationship between stalk strength and O. furnacalis larval injury, particularly for elite maize genotypes. To determine whether stalk strength is involved in maize resistance to O. furnacalis larval injury, 39 maize lines were evaluated in 2012 and 2013. Rind penetration strength (RPS) was measured at tassel (VT) and milk (R3) stages as a possible stalk resistance trait for O. furnacalis. RPS of primary ear internode at VT and R3 accounted for 37 and 38% of the variance in O. furnacalis injury (measured as number of holes) for simulated (artificially infested) first and second generation O. furnacalis, respectively. Relationships between stalk RPS values and tunnel length were weak. Results suggest that harder stalks have enhanced resistance to stalk boring but not to pith feeding or tunneling of O. furnacalis larvae. The RPS measures could provide classical maize breeders an important tool for evaluating stalk strength and corn borer resistance in maize. The assessments should focus on the internodes primary ear or above/below primary ear during both VT stage for first generation and R3 stage for second generation O. furnacalis resistance.

Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis (Bt) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non-Bt GE Crops (Washington DC, USA, 4–5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms.

Abstract Survival probability is fundamental for understanding population dynamics. Methods for estimating survival probability from field data typically require marking individuals, but marking methods are not possible for arthropod species that molt their exoskeleton between life stages. We developed a novel Bayesian state‐space model to estimate arthropod larval survival probability from stage‐structured count data. We performed simulation studies to evaluate estimation bias due to detection probability, individual variation in stage duration, and study design (sampling frequency and sample size). Estimation of cumulative survival probability from oviposition to pupation was robust to potential sources of bias. Our simulations also provide guidance for designing field studies with minimal bias. We applied the model to the monarch butterfly (Danaus plexippus), a declining species in North America for which conservation programs are being implemented. We estimated cumulative survival from egg to pupation from monarch counts conducted at 18 field sites in three landcover types in Iowa, USA, and Ontario, Canada: road right‐of‐ways, natural habitats (gardens and restored meadows), and agricultural field borders. Mean predicted survival probability across all landcover types was 0.014 (95% CI: 0.004–0.024), four times lower than previously published estimates using an ad hoc estimator. Estimated survival probability ranged from 0.002 (95% CI: 7.0E−7 to 0.034) to 0.058 (95% CI: 0.013–0.113) at individual sites. Among landcover types, agricultural field borders in Ontario had the highest estimated survival probability (0.025 with 95% CI: 0.008–0.043) and natural areas had the lowest estimated survival probability (0.008 with 95% CI: 0.009–0.024). Monarch production was estimated as adults produced per milkweed stem by multiplying survival probabilities by eggs per milkweed at these sites. Monarch production ranged from 1.0 (standard deviation [SD] = 0.68) adult in Ontario natural areas in 2016 to 29.0 (SD = 10.42) adults in Ontario agricultural borders in 2015 per 6809 milkweed stems. Survival estimates are critical to monarch population modeling and habitat restoration efforts. Our model is a significant advance in estimating survival probability for monarch butterflies and can be readily adapted to other arthropod species with stage‐structured life histories.

Monarch butterfly overwintering numbers have declined over the past 20 years. Restoring habitat that includes milkweeds, the only host plants for larval monarch butterflies, is necessary to increase monarch numbers within the breeding range. The value of different milkweed species for restoration will depend, in part, on the extent to which they are utilized by ovipositing females. The number of eggs laid on different species over a season will be a function of plant size and phenology as well as female preference. We examined seasonal egg deposition and females' oviposition choices by comparing the number of eggs laid by free-flying wild monarchs on each of nine native milkweed species occurring in Iowa (Asclepias syriaca, Asclepias tuberosa, Asclepias incarnata, Asclepias verticillata, Asclepias exaltata, Asclepias hirtella, Asclepias speciosa, Asclepias sullivantii, and Cynanchum laeve). One plot, consisting of clusters of each of the nine species, was established at each of 14 sites across the state of Iowa. Eggs were counted weekly in June, July and August 2015–2017. The highest egg totals were recorded on A. incarnata and A. syriaca in all years. Fewer eggs were counted on A. exaltata, A. hirtella, A. tuberosa, A. verticillata, and C. laeve. Our results show that monarchs prefer some milkweed species over others, but that they can use all nine native milkweed species for oviposition.

Abstract Over the past two decades, the population of monarch butterflies east of the Rocky Mountains has experienced a significant decline in overwintering numbers. Habitat restoration that includes planting milkweeds is essential to boost monarch numbers within the breeding range. Milkweeds are the only host plants for larval monarch butterflies, but female oviposition preference for different milkweed species, especially those with overlapping ranges, is not well documented. We examined the relative inclination to lay eggs on nine milkweed species native to Iowa (no choice), and oviposition preference (choice) among the four most commonly occurring Iowa species (Asclepias incarnata, Asclepias syriaca, Asclepias tuberosa, and Asclepias verticillata). In both experiments, eggs were counted daily for four days. The milkweeds tested were Asclepias exaltata (poke milkweed), Asclepias hirtella (tall green milkweed), A. incarnata (swamp milkweed), Asclepias speciosa (showy milkweed), Asclepias sullivantii (prairie milkweed), A. syriaca (common milkweed), A. tuberosa (butterfly milkweed), A. verticillata (whorled milkweed), and Cynanchum laeve (honeyvine milkweed). When females were given only a single species on which to lay eggs, there were significant differences among milkweed species in the average number of eggs laid; A. incarnata had the highest average egg count. When females were given a choice among A. incarnata, A. syriaca, A. tuberosa, and A. verticillata, there were also differences among milkweed species in the number of eggs laid; again, A. incarnata had the highest average number of eggs laid. Additionally, females laid more total eggs when four plants of different milkweed species were available than when there were four plants of a single milkweed species. Our results show that monarch butterflies will lay eggs on all nine milkweeds, but that there are clear preferences for some milkweed species over others.

Subsidies and fines are compared to voluntary and mandatory refuge insurance (insurance for pest damage on Bt corn refuge) as mechanisms for securing grower compliance with EPA refuge mandates. A conceptual model partially ranks mechanisms. Tradeoffs between mechanisms using grower welfare, payments to growers, and monitoring frequency are quantified empirically. Grower welfare is lowest with mandatory insurance because growers pay all costs, and is highest with direct refuge subsidies because public funds or companies subsidize all costs. Assuming typical premium loads and ignoring distribution considerations, we develop monitoring budgets for fines and subsidies, above which voluntary or mandatory insurance is better.

Genetically engineered crops offer farmers a new option for controlling pests. The high efficacy of these pesticidal crops, combined with the potential for widespread adoption, has raised concerns that pest resistance may prematurely diminish their value. In response to these concerns, the Environmental Protection Agency requires resistance management plans. Current resistance management plans rely on a high-dose refuge strategy. This analysis extends the current framework for evaluating high-dose refuge strategies to include a measure of agricultural productivity and conventional pesticide use. The economic tradeoff relative to agricultural productivity, conventional pesticide use, and pest resistance is assessed when Bt corn is planted to control the European corn borer.

Microsatellite markers are difficult to apply within lepidopteran studies due to the lack of locus-specific PCR amplification and the high proportion of null alleles, such that erroneous estimations of population genetic parameters often result. Herein single nucleotide polymorphism (SNP) markers are developed from Ostrinia nubilalis (Lepidoptera: Crambidae) using next-generation expressed sequence tag (EST) data. A total of 2742 SNPs were predicted within a reference assembly of 7414 EST contigs, and a subset of 763 were incorporated into 24 multiplex PCR reactions. To validate this pipeline, 5 European and North American sample sites were genotyped at 178 SNP loci, which indicated 84 (47.2%) were in Hardy-Weinberg equilibrium. Locus-by-locus FST, analysis of molecular variance (AMOVA), and STRUCTURE analyses indicate significant genetic differentiation may exist between European and North American O. nubilalis. The observed genetic diversity was significantly lower among European sites, which may be the result from genetic drift, natural selection, a genetic bottleneck, or ascertainment bias due to North American origin of EST sequence data. SNPs are an abundant mutation data molecular genetic marker development in non-model species with shared ancestral SNPs showing application within closely related species. These markers offer advantages over microsatellite markers for genetic and genomic analyses of Lepidoptera, but the source of mutation data may affect the estimation of population parameters and likely need be considered in the interpretation of empirical data.

PCR-based O. nubilalis population and pedigree analysis indicated female specificity of a (GAAAAT)n microsatellite, and male specificity of a CAYCARCGTCACTAA repeat unit marker. These loci were respectively named Ostrinia nubilalis W-chromosome 1 (ONW1) and O. nubilalis Z-chromosome 1 (ONZ1). Intact repeats of three, four, or five GAAAAT units are present among ONW1 alleles, and biallelic variation exists at the ONZ1 locus. Screening of 493 male at ONZ1 and 448 heterogametic females at ONZ1 and ONW1 loci from eleven North American sample sites was used to construct genotypic data. Analysis of molecular variance (AMOVA) and F-statistics indicated no female haplotype or male ONZ1 allele frequency differentiation between voltinism ecotypes. Four subpopulations from northern latitudes, Minnesota and South Dakota, showed the absence of a single female haplotype, a significant deviation of ONZ1 data from Hardy-Weinberg expectation, and low-level geographic divergence from other subpopulations. Low ONZ1 and ONW1 allele diversity could be attributed either to large repeat unit sizes, low repeat number, reduced effective population (Ne) size of sex chromosomes, or the result of recent O. nubilalis introduction and population expansion, but likely could not be due to inbreeding. These sequences have been deposited in GenBank AF442958, and AY102618 to AY102620.