Transgenic insect -resistant maize has been assessed thoroughly in the field but not in storage. The effects of several transgenic Bacillus thuringiensis (Bt) maize hybrids on the interactions between stored -grain insects (Indianmeal moth, Plodia interpunctella, or maize weevil, Sitophilus zeamais) and the storage fungus Aspergillus flavus were evaluated in this study under laboratory conditions. Grain of Bt and non -Bt maize hybrids, with or without the conidia of A. flavus, were infested with P. interpunctella larvae or S. zeamais adults separately and stored at 32 degrees C and 80-85 % relative humidity. In Bt maize hybrids with lepidopteran resistance genes expressing Cry1Ab, Cry1Ab x Cry1F, and Cry1Ab x Cry1F x Vip3Aa proteins, the mortality of P. interpunctella was 100 % at the end of the 28 -day storage period. In Bt hybrids with coleopteran resistance genes expressing mCry3A and mCry3A x Cry34A/35Ab1 proteins, mortality of S. zeamais was 100 %. In the non -Bt grain, aflatoxin levels at the end of the storage period increased by almost two -fold in the presence of either P. interpunctella or S. zeamais (p <= 0.01). Insect infestation did not enhance A. flavus or aflatoxin contamination on transgenic hybrids because of deterred insect activity. In the non -Bt hybrid, A. flavus caused increased mortality, reduced survivorship, and lower growth indices of both insects, limiting their feeding in the A. flavus coated conventional grain. As a result, insect damage and grain weight loss were significantly higher in the absence of A. flavus. Insect infestation only enhanced aflatoxin contamination in the absence of transgenic insect resistance genes. This study demonstrated that Bt proteins were effective against the stored -grain insects P. interpunctella and S. zeamais and suggests that the use of transgenic hybrids can mitigate the risk of insect infestation, and A. flavus and aflatoxin contamination in stored grain.

Field experiments were conducted to evaluate the roles of two corn borers, Asian corn borer (ACB), Ostrinia furnacalis and yellow peach moth (YPM), Conogethes punctiferalis, in Fusarium verticillioides infection using green fluorescent protein (GFP) as a marker. Effects of insect injury, manual injury, and insecticide application on fumonisin production also were assessed. In this study, third instars of ACB and YPM significantly increased GFP-tagged F. verticillioides infection compared with the control, regardless of the fungal inoculation method. Besides acquiring F. verticillioides spores from leaf surfaces and transmitting them to ears, larvae of the ACB and YPM also injure maize ears, which allows F. verticillioides from leaves or silk to infect ears more easily. This suggests that ACB and YPM larvae are vectors of F. verticillioides, which can increase the occurrence of ear rot. Manual injuries significantly increased GFP-tagged F. verticillioides infection of ears, while effective insect control significantly reduced F. verticillioides infection of ears. Insecticide control of borers also significantly reduced fumonisin content in kernels. Larval infestations significantly increased fumonisins in kernels to levels higher than or very close to the European Union threshold (4,000 mu g kg(-1)). Significant and high correlations among corn borer attack, F. verticillioides severity, and kernel fumonisin levels were discovered, confirming the important role of ACB and YPM activity in F. verticillioides infection and kernel fumonisin production.

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.

The North American monarch butterfly (Danaus plexippus) is a candidate species for listing under the Endangered Species Act. Multiple factors are associated with the decline in the eastern population, including the loss of breeding and foraging habitat and pesticide use. Establishing habitat in agricultural landscapes of the North Central region of the United States is critical to increasing reproduction during the summer. We integrated spatially explicit modeling with empirical movement ecology and pesticide toxicology studies to simulate population outcomes for different habitat establishment scenarios. Because of their mobility, we conclude that breeding monarchs in the North Central states should be resilient to pesticide use and habitat fragmentation. Consequently, we predict that adult monarch recruitment can be enhanced even if new habitat is established near pesticide-treated crop fields. Our research has improved the understanding of monarch population dynamics at the landscape scale by examining the interactions among monarch movement ecology, habitat fragmentation, and pesticide use.

The evolution of resistance to the Bacillus thuringiensis ( Bt ) toxins by insect pests is a major threat to Bt technology. However, the rate of resistance can be slowed with appropriate integrated insect resistance management (IRM) strategies. Surveys were conducted to identify alternate host species for Maruca vitrata (commonly called the legume pod borer or Maruca ) that could serve as refuges for Pod-Borer Resistant (PBR) cowpea in three West African countries (Ghana, Nigeria, and Burkina Faso). Survey sites included 25 in northern Ghana, 44 in northern Nigeria, and 52 in north-central and southwestern Burkina Faso. Alternate hosts of Maruca identified from plant species belonging to the Fabaceae family that showed signs of Maruca damage on cowpea tissues were collected and dissected. Larvae that were found during these dissections were reared to adult moths in the laboratory then identified to species. The alternate host plants including species of Crotolaria , Sesbania , Tephrosia , and Vigna were the most frequently encountered among sites and locations. Flowering and podding of these plants overlapped with flowering and podding of the nearby (~200 m) cowpea crop. Abundance of these wild hosts and overlapping flowering patterns with the cowpea crop in most locations have the potential to sustain ample numbers of Bt susceptible Maruca that will mate with possible resistant Maruca and deter resistance development. Further quantitative studies, however, are required from each location to determine if actual Maruca production from alternate hosts is sufficient for a PBR IRM strategy. If verified, this approach would be compatible with the high dose/refuge IRM strategy that includes alternate hosts and non- Bt crops as refuges.

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.

Conservation of North America's eastern monarch butterfly (Danaus plexippus) population would require establishment of milkweed (Asclepias spp.) and nectar plants in the agricultural landscapes of the north central United States. A variety of seed-treatment and foliar insecticides are used to manage early- and late-season pests in these landscapes. Thus, there is a need to assess risks of these insecticides to monarch butterfly life stages to inform habitat conservation practices. Chronic and acute dietary toxicity studies were undertaken with larvae and adults, and acute topical bioassays were conducted with eggs, pupae, and adults using 6 representative insecticides: beta-cyfluthrin (pyrethroid), chlorantraniliprole (anthranilic diamide), chlorpyrifos (organophosphate), imidacloprid, clothianidin, and thiamethoxam (neonicotinoids). Chronic dietary median lethal concentration values for monarch larvae ranged from 1.6 × 10-3 (chlorantraniliprole) to 5.3 (chlorpyrifos) μg/g milkweed leaf, with the neonicotinoids producing high rates of arrested pupal ecdysis. Chlorantraniliprole and beta-cyfluthrin were generally the most toxic insecticides to all life stages, and thiamethoxam and chlorpyrifos were generally the least toxic. The toxicity results were compared to insecticide exposure estimates derived from a spray drift model and/or milkweed residue data reported in the literature. Aerial applications of foliar insecticides are expected to cause high downwind mortality in larvae and eggs, with lower mortality predicted for adults and pupae. Neonicotinoid seed treatments are expected to cause little to no downslope mortality and/or sublethal effects in larvae and adults. Given the vagile behavior of nonmigratory monarchs, considering these results within a landscape-scale context suggests that adult recruitment will not be negatively impacted if new habitat is established in close proximity of maize and soybean fields in the agricultural landscapes of the north central United States. Environ Toxicol Chem 2021;40:1761-1777. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Establishment and maintenance of milkweed plants (Asclepias spp.) in agricultural landscapes of the north central United States are needed to reverse the decline of North America's eastern monarch butterfly (Danaus plexippus) population. Because of a lack of toxicity data, it is unclear how insecticide use may reduce monarch productivity when milkweed habitat is placed near maize and soybean fields. To assess the potential effects of foliar insecticides, acute cuticular and dietary toxicity of 5 representative active ingredients were determined: beta-cyfluthrin (pyrethroid), chlorantraniliprole (anthranilic diamide), chlorpyrifos (organophosphate), and imidacloprid and thiamethoxam (neonicotinoids). Cuticular median lethal dose values for first instars ranged from 9.2 × 10-3 to 79 μg/g larvae for beta-cyfluthrin and chlorpyrifos, respectively. Dietary median lethal concentration values for second instars ranged from 8.3 × 10-3 to 8.4 μg/g milkweed leaf for chlorantraniliprole and chlorpyrifos, respectively. To estimate larval mortality rates downwind from treated fields, modeled insecticide exposures to larvae and milkweed leaves were compared to dose-response curves obtained from bioassays with first-, second-, third-, and fifth-instar larvae. For aerial applications to manage soybean aphids, mortality rates at 60 m downwind were highest for beta-cyfluthrin and chlorantraniliprole following cuticular and dietary exposure, respectively, and lowest for thiamethoxam. To estimate landscape-scale risks, field-scale mortality rates must be considered in the context of spatial and temporal patterns of insecticide use. Environ Toxicol Chem 2020;39:923-941. © 2020 SETAC.

Abstract Cowpea [Vigna unguiculata (L) Walp.] is an important staple legume in the diet of many households in sub-Saharan Africa. Its production, however, is negatively impacted by many insect pests including bean pod borer, Maruca vitrata F., which can cause 20–80% yield loss. Several genetically engineered cowpea events that contain a cry1Ab gene from Bacillus thuringiensis (Bt) for resistance against M. vitrata were evaluated in Nigeria, Burkina Faso, and Ghana (West Africa), where cowpea is commonly grown. As part of the regulatory safety package, these efficacy data were developed and evaluated by in-country scientists. The Bt-cowpea lines were planted in confined field trials under Insect-proof netting and artificially infested with up to 500 M. vitrata larvae per plant during bud formation and flowering periods. Bt-cowpea lines provided nearly complete pod and seed protection and in most cases resulted in significantly increased seed yield over non-Bt control lines. An integrated pest management strategy that includes use of Bt-cowpea augmented with minimal insecticide treatment for protection against other insects is recommended to control pod borer to enhance cowpea production. The insect resistance management plan is based on the high-dose refuge strategy where non-Bt-cowpea and natural refuges are expected to provide M. vitrata susceptible to Cry1Ab protein. In addition, there will be a limited release of this product until a two-toxin cowpea pyramid is released. Other than South African genetically engineered crops, Bt-cowpea is the first genetically engineered food crop developed by the public sector and approved for release in sub-Saharan Africa.

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.

The population of monarch butterflies east of the Rocky Mountains has noticeably declined over the past two decades. The decline is due, in part, to loss of breeding and forage habitat in the Southern and Midwestern USA. To support a resilient overwintering population of six hectares of occupied forest canopy, approximately 1.6–1.8 billion additional ramets of milkweed are needed in the summer breeding range. Milkweed establishment that facilitates natural behavior of monarchs is necessary for effective conservation restoration. This study explored the effect of milkweed ramet density on larval search behavior, milkweed utilization, and survival without predation, parasitism, or competition. Under our experimental greenhouse conditions, monarch larvae abandoned their natal ramet, and subsequent ramets, prior to the pre-pupal wandering stage and before all available leaf biomass on a ramet was consumed. This is consistent with previous field observations. Larvae consumed biomass from three or four milkweed ramets that totaled the approximate biomass of single 10–35 cm ramet. Movement behavior suggests that isolated ramets may not support development through pupation, even though an isolated ramet could provide enough biomass. Our results suggest milkweed patches containing at least two to four ramets of closely-spaced common milkweed would provide sufficient biomass for development and increase the likelihood that larvae moving in random directions would encounter non-natal ramets to support development. Larval movement behavior and biomass requirements are critical aspects of monarch larval biology that should be considered in habitat restoration and maintenance plans, monitoring survey designs and protocols, and population modeling.

Most of Venezuela's agriculture occurs in the dry tropical forest life zone; this region covers 38% of the country and accounts for about 85% of the national honey production. When harvesting honey many Venezuelan beekeepers remove Africanized bees from honey supers by smoking the supers with large amounts of smoke, then by brushing or shaking out any remaining bees. Beekeepers in Venezuela, like many beekeepers around the world, seldom agree on beekeeping methods. Many Venezuelan beekeepers are trying hybrids from different strains in an attempt to find less defensive and better honey-producing bees. Hybrids produced from the Carniolan subspecies are popular among several of the beekeepers. Most crops in Venezuela prior to Africanization were pollinated by native bees, primarily stingless bees. Honey in Venezuela is used primarily for medicinal purposes and for the baking and candy industries. Most of the honey produced by the commercial beekeepers is sold at wholesale prices to national supermarkets.

This chapter examines the problems queen producers might encounter with Africanized honey bee. Producers in areas of Africanization will have to consider whether or not they will be able to produce acceptable queens at reasonable costs. The chapter considers methods to evaluate mating control. A Mating Control Index is provided as a possible aid for bee breeders. Mating nuclei stocked with Africanized bees produce fewer mated queens and abscond more often than those stocked with European bees. Cordovan workers result from cordovan queens mating with cordovan drones; wild-type workers result from cordovan queens mating with wild-type drones. The mating control experiments suggest that some queen producers should be able to control 90-95% of the matings without substantially changing their procedures. Such levels of mating control should be acceptable even in areas of Africanized bees. Natural matings of queens can be influenced by either manipulating populations of drones, or by using methods that isolate queens with desirable drones.

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.

The Iowa Monarch Conservation Consortium was established in March 2015. Adding milkweeds to agricultural landscapes is one of many Consortium goals. In order to further monarch butterfly conservation efforts, scientists need more information about milkweed phenology and persistence on the landscape, and how monarchs are using these plants because milkweeds are now absent from most agricultural fields.