Search

Insect biomass is declining globally, likely driven by climate change and pesticide use, yet systematic studies on the effects of various chemicals remain limited. In this work, we used a chemical library of 1024 molecules-covering insecticides, herbicides, fungicides, and plant growth inhibitors-to assess the impact of sublethal pesticide doses on insects. In Drosophila melanogaster , 57% of chemicals affected larval behavior, and a higher proportion compromised long-term survivability. Exposure to sublethal doses also induced widespread changes in the phosphoproteome and changes in development and reproduction. The negative effects of agrochemicals were amplified when the temperature was increased. We observed similar behavioral changes across multiple insect species, including mosquitoes and butterflies. These findings suggest that widespread sublethal pesticide exposure can alter insect behavior and physiology, threatening long-term population survival.

Data from prior research indicate the prepupal stage of the monarch butterfly life cycle is more sensitive to clothianidin exposure than the larval stage. A set of experiments was conducted to determine if the dietary clothianidin exposures that cause prepupal mortality are environmentally relevant. Monarch larvae were raised from egg to pupae on clothianidin-contaminated swamp milkweed plants (Asclepias incarnata). Larval growth as well as larval and prepupal survival were monitored throughout the experiments, in which the exposures ranged from 1.4 to 2793.1 ng/g leaf. Exposures of 5.4 to 46.9 ng/g leaf resulted primarily in prepupal mortality, whereas higher exposures of 1042.4 to 2793.1 ng/g leaf resulted exclusively in larval mortality, indicating the prepupal stage is more sensitive to clothianidin exposure than the larval stage. A median lethal concentration and a 10% lethal concentration of 37 and 6 ng/g leaf, respectively, were estimated for prepupal mortality. Both effect concentrations are within the range of clothianidin concentrations reported in leaves collected from wild milkweed plants, indicating prepupal mortality is an environmentally relevant effect. Environ Toxicol Chem 2024;43:2039-2044. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Pieris rapae (Lepidoptera: Pieridae) poses a significant threat to Brassicaceae crops, leading to substantial losses annually. Repeated insecticide applications are widely used to protect crops and increase the resistance of P. rapae. Exploring the biochemical and molecular basis of insecticide tolerance in P. rapae is crucial for achieving effective insect suppuration and implementing resistance control strategies. In our research, emamectin benzoate (EBZ) resistance was developed in P. rapae strain through selective pressure over 15 generations. Moreover, the biochemical mechanisms underlying resistance to EBZ and its potential cross-resistance to other insecticides were studied. Additionally, the expression levels of cytochrome P450 (CYP450) and glutathione-s-transferase (GST) genes in P. rapae were quantitatively assessed upon exposure to EBZ using real-time PCR. Our data exhibited that the LC 50 value of susceptible strain (Sus) and EBZ resistance strain (EBZ-R) were 0.009 and 8.09 mg/L, with a resistance ratio (RR) reaching 898.8-fold. The EBZ-R stain displayed notably low cross-resistance to lambda-cyhalothrin, spinetoram, and cypermethrin. However, it demonstrated a moderate level of cross-resistance to deltamethrin. Conversely, no cross-resistance was noted to chlorantraniliprole and indoxacarb. Notably, enzyme inhibitors of detoxification enzymes revealed that piperonyl butoxide (PBO) and diethyl maleate (DEM) enhanced the EBZ toxicity to the resistant strain, indicating the potential involvement of CYP450 and GST in avermectin resistance. A remarkable enhancement in CYP450 and GST activity was observed in the EBZ-R stain. CYP450 and GST genes are upregulated in the EBZ-R stain compared to the Sus strain, which serves as a basis for comprehending the mechanism behind P. rapae resistance to EBZ. The molecular docking analysis demonstrated that EBZ has a high binding affinity with CYP6AE120 and PrGSTS1 with docking energy values of -20.19 and -22.57 kcal/mol, respectively. Our findings offer valuable insights into crafting efficient strategies to monitor and manage resistance in P. rapae populations in Egypt., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Glucosinolates are token stimuli in host selection of many crucifer specialist insects, but the underlying molecular basis for host selection in these insects remains enigmatic. Using a combination of behavioral, electrophysiological, and molecular methods, we investigate glucosinolate receptors in the cabbage butterfly Pieris rapae. Sinigrin, as a potent feeding stimulant, elicited activity in larval maxillary lateral sensilla styloconica, as well as in adult medial tarsal sensilla. Two P. rapae gustatory receptor genes PrapGr28 and PrapGr15 were identified with high expression in female tarsi, and the subsequent functional analyses showed that Xenopus oocytes only expressing PrapGr28 had specific responses to sinigrin; when ectopically expressed in Drosophila sugar sensing neurons, PrapGr28 conferred sinigrin sensitivity to these neurons. RNA interference experiments further showed that knockdown of PrapGr28 reduced the sensitivity of adult medial tarsal sensilla to sinigrin. Taken together, we conclude that PrapGr28 is a gustatory receptor tuned to sinigrin in P. rapae, which paves the way for revealing the molecular basis of the relationships between crucifer plants and their specialist insects., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist.

Insecticide exposure has been identified as a contributing stressor to the decline in the North American monarch butterfly Danaus plexippus L. (Lepidoptera: Nymphalidae) population. Monarch toxicity data are currently limited and available data focuses on lethal endpoints. This study examined the 72-h toxicity of two pyrethroid insecticides, bifenthrin and β-cyfluthrin, and their effects on growth and diet consumption. The toxicity of bifenthrin to caterpillars was lower than β-cyfluthrin after 72 h. Survival was the most sensitive endpoint for bifenthrin, but diet consumption and caterpillar growth were significantly reduced at sublethal levels of β-cyfluthrin. Using AgDRIFT spray drift assessment, the aerial application of bifenthrin or β-cyfluthrin is predicted to pose the greatest risk to fifth-instar caterpillars, with lethal insecticide deposition up to 28 m for bifenthrin and up to 23 m for β-cyfluthrin from treated edges of fields. Low boom ground applications are predicted to reduce distances of lethal insecticide exposure to 2 m from the treated field edge for bifenthrin and β-cyfluthrin. Growth and survival of fifth-instar monarch caterpillars developing within the margins of a treated field may be significantly impacted following foliar applications of bifenthrin or β-cyfluthrin. These findings provide evidence that pyrethroid insecticides commonly used for soybean pest control are a potential risk to monarch caterpillars in agricultural landscapes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

Field cultivation of Genetically Modified (GM) Bt-plants has a potential environmental risk toward non-target Lepidoptera (NTLs) larvae through the consumption of Bt-maize pollen. The Bt-maize Cry protein targeting Lepidoptera species detrimental to the crop is also expressed in pollen which is dispersed by wind and can thus reach habitats of NTLs. To better assess the current ecological risk of Bt-maize at landscape scales, we developed a spatially-explicit exposure-hazard model considering (i) the dynamics of pollen dispersal obtained by convolving GM plants emission with a dispersal kernel and (ii) a toxicokinetic-toxicodynamic (TKTD) model accounting for the impact of toxin ingestion on individual lethal effects. We simulated the model using real landscape observations in Catalonia (Spain): GM-maize locations, flowering dates, rainfall time series and larvae emergence date of the European peacock butterfly Aglais io. While in average, the additional mortality appears to be negligible, we show significant additional mortality at sub-population level, with for instance a mortality higher than 40% within the 10m for the 10% most Bt-sensitive individuals. Also, using Pareto optimality we capture the best trade-off between isolation distance and additional mortality: up to 50 m are required to significantly buffer Bt-pollen impact on NTLs survival at the individual level. Our study clears up the narrow line between diverging conclusions: those claiming no risk by only looking at the average regional effect of Bt on NTLs survival and those pointing out a significant threaten when considering the variability of individuals mortality., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neonicotinoid pesticides harm nontarget insects, but their sublethal effects on butterflies are understudied. We exposed larvae of 3 butterfly species (Pieris rapae, Colias philodice, and Danaus plexippus) to low levels of the neonicotinoid imidacloprid in their host plants and followed individuals to adulthood. Imidacloprid altered adult body size, especially in female monarchs, but its effects varied across maternal families, highlighting the importance of considering genetic variation in ecotoxicological testing. Environ Toxicol Chem 2020;39:2228-2236. © 2020 SETAC., (© 2020 SETAC.)

Diamide insecticides, such as chlorantraniliprole, cyantraniliprole, and tetrachlorantraniliprole, are a new class of insecticides that selectively target insects by affecting calcium homeostasis. While this class of insecticides are effective on a wide range of insect pests, the toxicities of diamide insecticides vary among species and life stages. In this study, we addressed the mechanism underlying the different responses of Plutella xylostella and Pieris rapae to diamide insecticides. The susceptibility to insecticides of P. xylostella and P. rapae larvae was assessed 2 and 4 days after exposure to chlorantraniliprole, cyantraniliprole, and tetrachlorantraniliprole. P. xylostella larvae treated with distilled water (Group A), chlorantraniliprole (Group B), cyantraniliprole (Group C), and tetrachlorantraniliprole (Group D) and P. rapae larvae treated with distilled water (Group E), chlorantraniliprole (Group F), cyantraniliprole (Group G) and tetrachlorantraniliprole (Group H) were subjected to metabolomics analysis. The differential metabolites in the B vs. F, C vs. G, and D vs. H groups were analyzed, followed by pathway enrichment analysis. Chlorantraniliprole, cyantraniliprole, and tetrachlorantraniliprole all showed high toxicities for P. xylostella and P. rapae larvae. P. rapae larvae were more sensitive to the diamide insecticides than P. xylostella larvae. There were 65 overlapped differential metabolites between P. xylostella and P. rapae larvae treated with these three diamide insecticides. Pathway analysis showed that the differential metabolites were closely related with fatty acid biosynthesis and metabolism-related pathways. The differential regulation of fatty acid biosynthesis and metabolism may contribute to the different response to diamide insecticides in P. xylostella and P. rapae., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The long-term decline of monarch butterflies has been attributed to loss of their milkweed (Asclepias sp.) host-plants after the introduction of herbicide-tolerant crops. However, recent studies report pesticide residues on milkweed leaves that could act as a contributing factor when ingested as part of their larval diet. In this study, we exposed monarch larvae to six pesticides (insecticide: clothianidin; herbicides: atrazine, S-metolachlor; fungicides: azoxystrobin, pyraclostrobin, trifloxystrobin) on their primary host-plant, A. syriaca. Each was tested at mean and maximum levels reported from published analyses of milkweeds bordering cropland and thus represent field-relevant concentrations. Monarch lethal and sub-lethal responses were tracked over their complete development, from early instar larvae to adult death. Overall, we found no impact of any pesticide on immature development time and relatively weak effects on larval herbivory or survival to adulthood. Comparatively stronger effects were detected for adult performance; namely, a 12.5% reduction in wing length in response to the fungicides azoxystrobin and trifloxystrobin. These data collectively suggest that monarch responses to host-plant pesticides are largely sublethal and more pronounced in the adult stage, despite exposure only as larvae. This outcome has important implications for risk assessment and the migratory success of monarchs in North America.

The monarch butterfly, Danaus plexippus (Lepidoptera: Nymphalidae), is threatened by substantial loss of habitat, extreme weather events linked to global climate change, and nontarget impacts of broad-spectrum insecticides. To investigate the impact of chronic ingestion of pyrethroids on monarchs, wild-type Florida D. plexippus were reared on milkweed (Asclepias curassavica) that was exposed to ultra-low volume applications of DeltaGard by a truck-mounted fogger, at distances of 25 and 50 m. We observed significant negative impacts on monarchs reared on milkweed at 25 m from the DeltaGard spray route, including significant decreases in survival, and significantly longer development times, compared with untreated controls. Larvae reared on host plants closest to the truck spray route were 3 times more likely to experience a mortality event than the control cohort in trial 1 and 6 times in trial 2. Survival of monarch caterpillars reared on milkweed sprayed at 50 m was not significantly different from controls. For monarchs that survived to adulthood, we did not observe statistically significant differences among cohorts for variables measured. These data demonstrate that ultra-low volume treatments of pyrethroids can result in significant mortality in monarchs, but that the effects diminish with distance from the spray route., (Copyright © 2020 by The American Mosquito Control Association, Inc.)

Natural resource managers are concerned about the impacts of aerial ultra-low volume spray (ULV) of insecticides for mosquito control (i.e., mosquito adulticides) and seek science-driven management recommendations that reduce risk but allow vector control for nearby human populations. Managers at the National Key Deer Refuge (Florida Keys, FL) are concerned for ULV effects upon conservation efforts for imperiled butterflies (Florida leafwing [Anaea troglodyta floridalis] and Bartram's hairstreak [Strymon acis bartrami] butterflies). No-spray zones were designated for protection of those butterflies, but their effectiveness for mitigation is unclear. To address this uncertainty, cholinesterase activity (ChE) and mortality were monitored for caged butterflies gulf fritillary [Agraulis vanilla] and great southern white [Ascia monuste]) deployed on the Refuge during three aerial ULV applications of the insecticide naled. Residue samplers also were deployed to estimate butterfly exposure. Spray efficacy against mosquitoes was assessed by deploying caged mosquitoes at the same locations as the butterflies. Average naled residue levels on filter paper samplers in the target area (1882-2898 µg/m 2 ) was significantly greater than in the no-spray zone (9-1562 µg/m 2 ). Differences between the no-spray zone and target area for butterfly mortality and ChE were inconsistent. Average mortality was significantly lower, and average ChE was significantly higher in the no-spray zone for larvae of one species but not for larvae of the other species. Mosquito mortality did not differ significantly between the two areas. Data from the present study reflect the inconsistent effectiveness of no-spray zones on the Refuge using standard methods employed at the time by the vector control agency in the Florida Keys and possibly by other vector control agencies in similar coastal environments. Furthermore, these findings helped to guide the design and to improve the conservation value of future no-spray zone delineations while allowing for treatment in areas where mosquito control is necessary for vector-borne disease reduction.

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., (© 2020 SETAC.)

The fruits of Melia toosendan Sieb. et Zucc. (Meliaceae) are a source of bioactive limonoids that can be used as effective pesticides. In this study, two novel limonoids, 6-acetylsendanal and 6-ketocinamodiol, were isolated together with fourteen known compounds, namely four protolimonoids, six trichilin-class limonoids, and four C-seco limonoids. The structures of the new compounds were determined by extensive spectroscopic analyses (HR-ESI-MS, UV, IR, 1D and 2D NMR). The bioassay results revealed that eleven of the extracted limonoids exhibited interesting antifeedant activities against the larvae of Pieris rapae with AFC 50 values in the range of 0.11-1.79 mm. Particularly, mesendanin H, with an AFC 50 value of 0.11 mm, exhibited a higher activity than the positive control toosendanin. Information on new bioactive limonoids may provide further insight into M. toosendan as a source of bioactive components., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

While the diversity of sex pheromone communication systems across insects is well documented, the mechanisms that lead to such diversity are not well understood. Sex pheromones constitute a species-specific system of sexual communication that reinforces interspecific reproductive isolation. When odor blends evolve, the efficacy of male-female communication becomes compromised, unless preference for novel blends also evolves. We explore odor learning as a possible mechanism leading to changes in sex pheromone preferences in the butterfly Bicyclus anynana. Our experiments reveal mating patterns suggesting that mating bias for new blends can develop following a short learning experience, and that this maternal experience impacts the mating outcome of offspring without further exposure. We propose that odor learning can be a key factor in the evolution of sex pheromone blend recognition and in chemosensory speciation.

In the Southern High Plains of the United States, beef cattle feed yards and row crop agriculture are predominant sources of agrochemical usage. Beef cattle feed yards use large quantities of veterinary pharmaceuticals to promote cattle growth and health, along with insecticides to control insect pests, whereas row crop-based agriculture relies on herbicides, fungicides, and insecticides to increase yields. Previous studies have documented the occurrence of agrochemicals beyond feed yard and row crop agriculture boundaries in uncultivated, marginal areas, raising concern that migratory pollinators and pollinators indigenous to the Southern High Plains frequenting these remaining habitat corridors may become exposed to toxic agrochemicals. Larvae of the painted lady butterfly (Vanessa cardui) were used to investigate the potential toxicity of agrochemicals used on feed yards and in row crop agriculture among pollinators. Moxidectin, an antiparasiticide used on beef cattle feed yards, was determined to be extremely toxic to V. cardui larvae, with a lethal dose at which 50% of larvae died of 2.1 ± 0.1 ng/g. Pyraclostrobin, clothianidin, and permethrin all delayed V. cardui development. However, moxidectin was the only chemical that produced significant toxic effects at environmentally relevant concentrations. These results indicate that agrochemicals originating from feed yards have the potential to adversely impact the development of pollinator larvae occurring in the Southern High Plains. Environ Toxicol Chem 2019;38:2629-2636. © 2019 SETAC., (© 2019 SETAC.)

Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness 1 . Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits 2-4 , and new genome editing technology is facilitating functional validation of these mutations in whole organisms 1,5 . We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins 6-11 . Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na + /K + -ATPase) 7-11 , the physiological target of cardiac glycosides 12 . Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization 13,14 . We then performed CRISPR-Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage 11,15 . We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides 16 , culminating in triple mutant 'monarch flies' that were as insensitive to cardiac glycosides as monarch butterflies. 'Monarch flies' retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators 17-19 . The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis 13,14,20-22 . Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities 2,6-11,15,17-19 .

Color patterns often function as camouflage to protect insects from predators. In most swallowtail butterflies, younger larvae mimic bird droppings but change their pattern to mimic their host plants during their final molt. This pattern change is determined during the early fourth instar by juvenile hormone (JH-sensitive period), but it remains unclear how the prepatterning process is controlled. Using Papilio xuthus larvae, we performed transcriptome comparisons to identify three camouflage pattern-associated homeobox genes [ clawless , abdominal-A , and Abdominal-B ( Abd-B )] that are up-regulated during the JH-sensitive period in a region-specific manner. Electroporation-mediated knockdown of each gene at the third instar caused loss or change of original fifth instar patterns, but not the fourth instar mimetic pattern, and knockdown of Abd-B after the JH-sensitive period had no effect on fifth instar patterns. These results indicate the role of these genes during the JH-sensitive period and in the control of the prepatterning gene network.

Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na + /K + ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na + /K + ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed's diverse community of specialist cardenolide-sequestering insect herbivores.

Coordinated social behaviour is fundamental for ant ecological success. However, even distantly-related organisms, such as plants, have evolved the ability to manipulate ant collective performances to their own advantage. In the parasitic system encompassing Maculinea butterflies, Myrmica ants, and Origanum vulgare plants, the ant-plant interaction elicits the release of a volatile terpenoid compound (carvacrol) which is used by the gravid butterfly to locate the ideal oviposition site. Here we show that this ant-plant association is maintained by the effect of O. vulgare terpenoids on ant behaviour and that food plants might gain protection by Myrmica ants by chemically manipulating workers to forage in their surroundings. The variation in the locomotor ability of three ant species (Formica cinerea, Tetramorium caespitum, and Myrmica scabrinodis) was studied after treatment with the two major O. vulgare terpenoid volatile compounds (i.e., carvacrol and thymol). The brain levels of three biogenic amines (dopamine, tyramine and serotonin) were analysed in ants exposed to the O. vulgare terpenoids by HPLC-ESI-MS/MS. Carvacrol and thymol increased the locomotor activity of all ant species tested, but if blended reduced the movement propensity of Myrmica scabrinodis. Dopamine and tyramine production was positively correlated with the worker locomotor activity. In Myrmica ants, both brain biogenic ammines were negatively correlated with the aggressive behaviour. Blends of O. vulgare volatiles affected the locomotor ability while increased the aggressiveness of Myrmica workers by altering the aminergic regulation in the ant brains. This behavioural manipulation, might enhance partner fidelity and plant protection. Our findings provide new insights supporting a direct role of plant volatiles in driving behavioural changes in social insects through biogenic amine modulation., Competing Interests: The authors have declared that no competing interests exist.

Heavy metal pollution is a major problem in urban and industrial environments, and has a myriad of negative effects on animals. Quantifying the amount of population-level variation that exists for heavy metal tolerance and how plastic responses to heavy metals play out across generations are essential for understanding how animals respond to pollution. As an initial step toward studying transgenerational effects and population-level variation in concert, we brought cabbage white butterflies (Pieris rapae) from two populations-collected from St. Paul, MN, and Davis, CA-into common conditions and fed them a diet dosed with nickel. To measure transgenerational effects, we reared a second generation in a fully factorial design, within each population, to achieve all combinations of parent and offspring exposure to nickel or control diets. Across both generations, we quantified survival and other fitness-related traits, including development time, body size, and egg size and number. We found both population differences and complex transgenerational effects, including a positive effect of nickel on survival and development time in one of the populations. Overall, nickel exposure was stressful in one population, mainly after two generations of exposure, and had neutral or slightly positive effects on the other. We found no evidence for costs of mismatch between parental and offspring environments. While the reasons for the differences observed between the two populations are unclear, the variation in nickel tolerance observed in this species suggests that some organisms may be less affected by low levels of heavy metal pollution in urban and industrial areas than expected.

The diamondback moth (Plutella xylostella) and small white cabbage butterfly (Pieris rapae) are the two main serious pests of cruciferous crops (Brassicaceae) that have developed resistance to chemical control methods. In order to avoid such resistance and also the adverse effects of chemical pesticides on the environment, alternative methods have usually been suggested, including the use of plant enzyme inhibitors. Here, the inhibitory effects of proteinaceous inhibitors extracted from wheat, canola, sesame, bean and triticale were evaluated against the digestive α-amylases, larval growth, development and nutritional indecs of the diamondback moth and small white cabbage butterfly. Our results indicated that triticale and wheat extracts inhibited α-amylolytic activity in an alkaline pH, which is in accordance with the moth and butterfly gut α-amylase optimum pH. Dose-dependent inhibition of two crucifer pests by triticale and wheat was observed using spectrophotometry and gel electrophoresis. Implementation of specificity studies showed that wheat and triticale-proteinaceous extract were inactive against Chinese and purple cabbage amylase. Triticale and wheat were resistant against insects' gut proteases. Results of the feeding bioassay indicated that triticale-proteinaceous extract could cause a significant reduction in survival and larval body mass. The results of the nutritional indecs also showed larvae of both species that fed on a Triticale proteinaceous inhibitor-treated diet had the lowest values for the efficiency of conversion of ingested food and relative growth rate. Our observations suggested that triticale shows promise for use in the management of crucifer pests.

The small white butterfly, Pieris rapae (Lepidoptera: Pieridae), is an important pest on Brassicaceae plants, causing heavy crop loss each year. Cytochrome P450 monooxygenase (CYP) is a superfamily of enzymes involved in the detoxification of various xenobiotic compounds, including insecticides. However, little is known about the role of CYP genes in P. rapae. In this study, we identified 63 CYP genes in P. rapae, and analyzed their phylogenetic relationships, exon-intron structures and genomic locations. Moreover, our insecticide-response transcription profiling showed that LD 5 doses of lambda-cyhalothrin, chlorantraniliprole, and abamectin significantly increased expression of five (CYP4M59, CYP6AE119, CYP6AE120, CYP6AE121, and CYP6BD18), three (CYP4AU1, CYP6AE120, and CYP6AW1), and five (CYP4L40, CYP4AU1, CYP6AE119, CYP6AW1, and CYP6BD19) CYP genes, respectively; and LD 20 doses of the three pesticides significantly upregulated six (CYP4M59, CYP6AE119, CYP6AE120, CYP6AE121, CYP4AU1, and CYP6BD18), six (CYP4G168, CYP4L40, CYP4AU1, CYP6AE120, CYP6AW1, and CYP6BD19), and five (CYP4L40, CYP4AU1, CYP6AB108, CYP6AE119, and CYP6BD19) genes, respectively. When we used LD 50 doses of the three insecticides, we reported significantly elevated expression levels of five (CYP4M59, CYP6AE119, CYP6AE120, CYP6BD17, and CYP6BD18), eight (CYP4G168, CYP4L40, CYP4AU1, CYP6AE120, CYP6AE121, CYP6AW1, CYP6BD18, and CYP6BD19), and six (CYP4L40, CYP4S34, CYP6AB108, CYP6AE119, CYP6AE120, and CYP6BD19) genes, respectively. Our expression analysis also revealed that five (CYP4G168, CYP4G169, CYP4S34, CYP6AW1, and CYP6CT3) and three (CYP4L40, CYP6AN33, and CYP6BD17) CYP genes were mainly expressed in the midgut and fat body, respectively, and one CYP gene (CYP6AE119) in the Malpighian tubules. This is the first large-scale report into the characterization of CYP genes in P. rapae., (© 2018 Wiley Periodicals, Inc.)

Certain butterflies utilize plant-acquired alkaloids for their own chemical defense and/or for producing male sex pheromone; a trait known as pharmacophagy. Males of the danaine butterfly, Parantica sita, have been reported to ingest pyrrolizidine alkaloids (PAs) as adults to produce two PA-derived sex pheromone components, viz. danaidone (major) and 7R-hydroxydanaidal. We found, however, that not all PAs that can be precursors for the pheromone serve for mating success of males. Here we show that although the sex pheromone is regarded as a requisite for successful mating, uptake of specific PA(s) (lycopsamine-type PAs) is also imperative for the males to achieve copulation. The increase in the levels of two biogenic amines, octopamine and/or serotonin, in the brain and thoracic ganglia of males fed with specific PA(s) suggested that these alkaloids most likely enhance male mating activity. The results can present new evidence for the evolutionary provenance of pharmacophagous acquisition of PAs in PA-adapted insects.

Premise of the Study: Pachypodium (Apocynaceae) is a genus of iconic stem-succulent and poisonous plants endemic to Madagascar and southern Africa. We tested hypotheses about the mode of action and macroevolution of toxicity in this group. We further hypothesized that while monarch butterflies are highly resistant to cardenolide toxins (a type of cardiac glycoside) from American Asclepias, they may be negatively affected by Pachypodium defenses, which evolved independently., Methods: We grew 16 of 21 known Pachypodium spp. and quantified putative cardenolides by HPLC and also by inhibition of animal Na + /K + -ATPase (the physiological target of cardiac glycosides) using an in vitro assay. Pachypodium extracts were tested against monarch caterpillars in a feeding bioassay. We also tested four Asclepias spp. and five Pachypodium spp. extracts, contrasting inhibition of the cardenolide-sensitive porcine Na + /K + -ATPase to the monarch's resistant form., Key Results: We found evidence for low cardenolides by HPLC, but substantial toxicity when extracts were assayed on Na + /K + -ATPases. Toxicity showed phylogenetic signal, and taller species showed greater toxicity (this was marginal after phylogenetic correction). Application of Pachypodium extracts to milkweed leaves reduced monarch growth, and this was predicted by inhibition of the sensitive Na + /K + -ATPase in phylogenetic analyses. Asclepias extracts were 100-fold less potent against the monarch compared to the porcine Na + /K + -ATPase, but this difference was absent for Pachypodium extracts., Conclusions: Pachypodium contains potent toxicity capable of inhibiting sensitive and cardenolide-adapted Na + /K + -ATPases. Given the monarch's sensitivity to Pachypodium, we suggest that these plants contain novel cardiac glycosides or other compounds that facilitate toxicity by binding to Na + /K + -ATPases., (© 2018 Botanical Society of America.)

Background: Investigating the impact of pesticides on non-target organisms is essential for sustainable integrated pest management programs. We therefore assessed the toxicity of ten insecticides to the brassica caterpillar Ascia monuste and its ant predator Solenopsis saevissima and examined the effect that the insecticide synergists had on toxicity to the predator. We also assessed the residual period of control and impact of the insecticides during the brassica growing cycle., Results: All insecticides except flubendiamide exhibited mortality above the threshold required by Brazilian legislation (80%). Chlorantraniliprole, cyantraniliprole, indoxacarb and spinosad exhibited lower toxicity to the ant predator than they did to the brassica caterpillar. The results obtained for synergized insecticides suggest that selectivity to the predator was due the involvement of cytochrome P450-dependent monooxygenases. Chlorfenapyr and cyantraniliprole exhibited the highest residual periods of control to the brassica caterpillar, whereas malathion had the greatest impact on the predator., Conclusion: Most of the insecticides efficiently controlled the brassica caterpillar, but not all exhibited selectivity to the predator. Therefore, due to the distinctive responses of organisms with respect to residual periods of control and the impact of the insecticides, spraying frequency must be strongly considered in integrated pest management programs. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Insect glutathione S-transferases (GSTs) play essential roles in the detoxification of insecticides and other xenobiotic compounds. The cabbage white butterfly, Pieris rapae, is an economically important agricultural pest. In this study, 17 cDNA sequences encoding putative GSTs were identified in P. rapae. All cDNAs include a complete open reading frame and were designated PrGSTd1-PrGSTz2. Based on phylogenetic analysis, PrGSTs were divided into six classes (delta, epsilon, omega, sigma, theta and zeta). The exon-intron organizations of these PrGSTs were also analysed. Recombinant proteins of eight PrGSTs (PrGSTD1, PrGSTD2, PrGSTE1, PrGSTE2, PrGSTO1, PrGSTS1, PrGSTT1 and PrGSTZ1) were heterologously expressed in Escherichia coli, and all of these proteins displayed glutathione-conjugating activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Expression patterns in various larval tissues, at different life stages, and following exposure to sublethal doses of abamectin, chlorantraniliprole or lambda-cyhalothrin were determined by reverse transcription-quantitative PCR. The results showed that PrGSTe3, PrGSTs1, PrGSTs2, and PrGSTs4 were mainly transcribed in the fat body, while PrGSTe2 was expressed predominantly in the Malpighian tubules. Four genes (PrGSTe2, PrGSTo4, PrGSTs4 and PrGSTt1) were mainly expressed in fourth-instar larvae, while others were ubiquitously expressed in egg, larval, pupa and/or adult stages. Abamectin treatment significantly upregulated ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTt1). Chlorantraniliprole and lambda-cyhalothrin treatment significantly upregulated nine genes (PrGSTd1, PrGSTd2, PrGSTe1, PrGSTe2, PrGSTe3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTz1) and ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo2, PrGSTs1, PrGSTs2, PrGSTs3 and PrGSTz2), respectively. These GSTs are potentially involved in the detoxification of insecticides., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Serotonin (5-hydroxytryptamine, 5-HT) plays a key role in modulating diverse physiological processes and behaviors in both protostomes and deuterostomes. These functions are mediated through the binding of serotonin to its receptors, which are recognized as potential insecticide targets. We investigated the sequence, pharmacology and tissue distribution of three 5-HT receptors (Piera5-HT 1A , Piera5-HT 1B , Piera5-HT 7 ) from the small white butterfly Pieris rapae, an important pest of cultivated cabbages and other mustard family crops. Activation of Piera5-HT 1A or Piera5-HT 1B by 5-HT inhibited the production of cAMP in a dose-dependent manner. Stimulation of Piera5-HT 7 with 5-HT increased cAMP level significantly. Surprisingly, with the exception of 5-methoxytryptamine, agonists including α-methylserotonin, 8-Hydroxy-DPAT and 5-carboxamidotryptamine activated these receptors poorly. The results are consistent with previous findings in Manduca sexta. All three receptors were blocked by methiothepin, but ketanserin and yohimbine were not effective. The selective mammalian 5-HT receptor antagonists SB 216641 and SB 269970 displayed potent inhibition effects on Piera5-HT 1B and Piera5-HT 7 respectively. The results we achieved here indicate that the pharmacological properties of Lepidoptera 5-HT receptors are quite different from those in other insects and vertebrates and may contribute to development of new selective pesticides. This study offers important information on three 5-HT receptors from P. rapae that will facilitate further analysis of the functions of 5-HT receptors in insects., (Copyright © 2017. Published by Elsevier Ltd.)

Six new highly oxygenated grayanane diterpenoids, neopierisoids G-L, 1-6, together with 10 known related compounds, 7-16, were identified from the flowers of the poisonous plant Pieris japonica. The structures were elucidated on the basis of comprehensive NMR spectroscopy and mass analysis. The relative configurations of 1-6 were elucidated by analysis of ROESY spectra and comparison of NMR data with the analogues. The absolute configurations of 1-6 were established by the X-ray diffraction analysis of 1 and comparison of the CD spectra of 1-6. Compared with the skeleton of the normal grayanane diterpenoids, compounds 1-6 shared an unusual seco A ring moiety. The antifeedant activities of compounds 1-16 against Pieris brassicae were evaluated by using a dual-choice bioassay, and compounds 1-10 with a normal grayanane skeleton showed potent antifeedant activity against P. brassicae. The structure-activity relationships of antifeedant activities of 1-16 against P. brassicae are discussed.

Phytophagous insects have to deal with toxic defense compounds from their host plants. Although it is known that insects have evolved genes and mechanisms to detoxify plant allochemicals, how specialist and generalist precisely respond to specific secondary metabolites at the molecular level is less understood. Here we studied the larval performance and transcriptome of the generalist moth Heliothis virescens and the specialist butterfly Pieris brassicae feeding on Arabidopsis thaliana genotypes with different glucosinolate (GS) levels. H. virescens larvae gained significantly more weight on the GS-deficient mutant quadGS compared to wild-type (Col-0) plants. On the contrary, P. brassicae was unaffected by the presence of GS and performed equally well on both genotypes. Strikingly, there was a considerable differential gene expression in H. virescens larvae feeding on Col-0 compared to quadGS. In contrast, compared to H. virescens, P. brassicae displayed a much-reduced transcriptional activation when fed on both plant genotypes. Transcripts coding for putative detoxification enzymes were significantly upregulated in H. virescens, along with digestive enzymes and transposable elements. These data provide an unprecedented view on transcriptional changes that are specifically activated by GS and illustrate differential molecular responses that are linked to adaptation to diet in lepidopteran herbivores., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The common grass yellow butterfly, Eurema mandarina (formerly Eurema hecabe mandarina) (Lepidoptera, Pieridae), recently has been separated taxonomically from a subtropical population of Eurema hecabe in Japan. This species is widely distributed in the temperate region of Japan, and feeds mainly on various ligneous plants within the Fabaceae. We attempted to identify an oviposition stimulant for E. mandarina from its primary hosts, Albizia julibrissin and Lespedeza cuneata. In both hosts, crude extract and an aqueous fraction elicited oviposition responses from gravid females. A polar subfraction of the aqueous fraction also stimulated high oviposition-stimulatory activity, comparable to the original aqueous fraction, suggesting that E. mandarina females use water-soluble compounds for host recognition. Subsequent activity-directed fractionation by ion exchange chromatography indicated that one of the key substances was contained in the neutral/amphoteric fraction. Chemical analyses revealed that the active fractions of both hosts contained D-(+)-pinitol as the major component. We examined female responses to authentic D-pinitol and found that it induced oviposition responses at concentrations greater than 0.1 %. Since this cyclitol is omnipresent in Fabaceae, we conclude that D-pinitol plays a role in mediating oviposition of E. mandarina on fabaceous plants.

Flying insects have the highest known mass-specific demand for oxygen, which makes it likely that reduced availability of oxygen might limit sustained flight, either instead of or in addition to the limitation due to metabolite resources. The Glanville fritillary butterfly (Melitaea cinxia) occurs as a large metapopulation in which adult butterflies frequently disperse between small local populations. Here, we examine how the interaction between oxygen availability and fuel use affects flight performance in the Glanville fritillary. Individuals were flown under either normoxic (21 kPa O2) or hypoxic (10 kPa O2) conditions and their flight metabolism was measured. To determine resource use, levels of circulating glucose, trehalose and whole-body triglyceride were recorded after flight. Flight performance was significantly reduced in hypoxic conditions. When flown under normoxic conditions, we observed a positive correlation among individuals between post-flight circulating trehalose levels and flight metabolic rate, suggesting that low levels of circulating trehalose constrains flight metabolism. To test this hypothesis experimentally, we measured the flight metabolic rate of individuals injected with a trehalase inhibitor. In support of the hypothesis, experimental butterflies showed significantly reduced flight metabolic rate, but not resting metabolic rate, in comparison to control individuals. By contrast, under hypoxia there was no relationship between trehalose and flight metabolic rate. Additionally, in this case, flight metabolic rate was reduced in spite of circulating trehalose levels that were high enough to support high flight metabolic rate under normoxic conditions. These results demonstrate a significant interaction between oxygen and energy availability for the control of flight performance., (© 2016. Published by The Company of Biologists Ltd.)

Plants damaged by herbivores emit volatile organic compounds (VOCs) that are used by parasitoids for host location. In nature, however, plants are exposed to multiple abiotic and biotic stresses of varying intensities, which may affect tritrophic interactions. Here, we studied the effects of ozone exposure and feeding by Pieris brassicae larvae on the VOCs emitted by Brassica nigra and the effects on oriented flight of the parasitoid Cotesia glomerata. We also investigated the oriented flight of C. glomerata in a wind-tunnel with elevated ozone levels. Herbivore-feeding induced the emission of several VOCs, while ozone alone had no significant effect. However, exposure to 120 ppb ozone, followed by 24 hr of herbivore-feeding, induced higher emissions of all VOCs as compared to herbivore-feeding alone. In accordance, herbivore-damaged plants elicited more oriented flights than undamaged plants, whereas plants exposed to 120 ppb ozone and 24 hr of herbivore-feeding elicited more oriented flights than plants subjected to herbivore-feeding alone. Ozone enrichment of the wind-tunnel air appeared to negatively affect orientation of parasitoids at 70 ppb, but not at 120 ppb. These results suggest that the combination of ozone and P. brassicae-feeding modulates VOC emissions, which significantly influence foraging efficiency of C. glomerata.

Nutritional enhancement of crops using genetic engineering can potentially affect herbivorous pests. Recently, oilseed crops have been genetically engineered to produce the long-chain omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at levels similar to that found in fish oil; to provide a more sustainable source of these compounds than is currently available from wild fish capture. We examined some of the growth and development impacts of adding EPA and DHA to an artificial diet of Pieris rapae, a common pest of Brassicaceae plants. We replaced 1% canola oil with EPA: DHA (11:7 ratio) in larval diets, and examined morphological traits and growth of larvae and ensuing adults across 5 dietary treatments. Diets containing increasing amounts of EPA and DHA did not affect developmental phenology, larval or pupal weight, food consumption, nor larval mortality. However, the addition of EPA and DHA in larval diets resulted in progressively heavier adults (F 4, 108 = 6.78; p = 0.011), with smaller wings (p < 0.05) and a higher frequency of wing deformities (R = 0.988; p = 0.001). We conclude that the presence of EPA and DHA in diets of larval P. rapae may alter adult mass and wing morphology; therefore, further research on the environmental impacts of EPA and DHA production on terrestrial biota is advisable.

Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Here, we report that Pteromalus puparum venom impairs the antimicrobial activity of its host Pieris rapae. Inhibition zone results showed that bead injection induced the antimicrobial activity of the host hemolymph but that venom inhibited it. The cDNAs encoding cecropin and lysozyme were screened. Relative quantitative PCR results indicated that all of the microorganisms and bead injections up-regulated the transcript levels of the two genes but that venom down-regulated them. At 8 h post bead challenge, there was a peak in the transcript level of the cecropin gene, whereas the peak of lysozyme gene occurred at 24 h. The transcripts levels of the two genes were higher in the granulocytes and fat body than in other tissues. RNA interference decreased the transcript levels of the two genes and the antimicrobial activity of the pupal hemolymph. Venom injections similarly silenced the expression of the two genes during the first 8 h post-treatment in time- and dose-dependent manners, after which the silence effects abated. Additionally, recombinant cecropin and lysozyme had no significant effect on the emergence rate of pupae that were parasitized by P. puparum females. These findings suggest one mechanism of impairing host antimicrobial activity by parasitoid venom.

Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to cardenolide toxins based on amino acid substitutions of their cellular sodium-potassium pump (Na(+)/K(+)-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated cardenolides, we show that resistant Na(+)/K(+)-ATPases are not necessary to cope with dietary cardenolides. By contrast, sequestration of cardenolides in the body (as a defence against predators) is associated with the three levels of Na(+)/K(+)-ATPase resistance. To estimate the potential physiological burden of cardenolide sequestration without Na(+)/K(+)-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na(+)/K(+)-ATPase of sequestering and non-sequestering species. Haemolymph cardenolides dramatically impair non-adapted Na(+)/K(+)-ATPase, but had systematically reduced effects on Na(+)/K(+)-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na(+)/K(+)-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects., (© 2015 The Author(s).)

As it pertains to insect herbivores, the preference-performance hypothesis posits that females will choose oviposition sites that maximize their offspring's fitness. However, both genetic and environmental cues contribute to oviposition preference, and occasionally "oviposition mistakes" occur, where insects oviposit on hosts unsuitable for larval development. Pieris virginiensis is a pierine butterfly native to North America that regularly oviposits on an invasive plant, Alliaria petiolata, but the caterpillars are unable to survive. Alliaria petiolata has high concentrations of the glucosinolate sinigrin in its tissues, as well as a hydroxynitrile glucoside, alliarinoside. We investigated sinigrin as a possible cause of mistake oviposition, and sinigrin and alliarinoside as possible causes of larval mortality. We found that sinigrin applied to leaves of Cardamine diphylla, a major host of P. virginiensis that does not produce sinigrin, had no effect on oviposition rates. We tested the effect of sinigrin on larval performance using two host plants, one lacking sinigrin (C. diphylla) and one with sinigrin naturally present (Brassica juncea). We found no effect of sinigrin application on survival of caterpillars fed C. diphylla, but sinigrin delayed pupation and decreased pupal weight. On B. juncea, sinigrin decreased survival, consumption, and caterpillar growth. We also tested the response of P. virginiensis caterpillars to alliarinoside, a compound unique to A. petiolata, which was applied to B. oleracea. We found a significant reduction in survival, leaf consumption, and caterpillar size when alliarinoside was consumed. The 'novel weapon' alliarinoside likely is largely responsible for larval failure on the novel host A. petiolata. Sinigrin most likely contributes to the larval mortality observed, however, we did not observe any effect of sinigrin on oviposition by P. virginiensis females. Further research needs to be done on non-glucosinolate contact cues, and volatile signals that may induce P. virginiensis oviposition.

Monarch butterflies (Danaus plexippus) frequently consume milkweed in and near agroecosystems and consequently may be exposed to pesticides like neonicotinoids. We conducted a dose response study to determine lethal and sublethal doses of clothianidin using a 36-h exposure scenario. We then quantified clothianidin levels found in milkweed leaves adjacent to maize fields. Toxicity assays revealed LC10, LC50, and LC90 values of 7.72, 15.63, and 30.70 ppb, respectively. Sublethal effects (larval size) were observed at 1 ppb. Contaminated milkweed plants had an average of 1.14±0.10 ppb clothianidin, with a maximum of 4 ppb in a single plant. This research suggests that clothianidin could function as a stressor to monarch populations.

Integrated Pest Management (IPM) is a decision making process used to manage pests that relies on many tactics, including cultural and biological control, which are practices that conserve beneficial insects and mites, and when needed, the use of conventional insecticides. However, systemic, soil-applied neonicotinoid insecticides are translocated to pollen and nectar of flowers, often for months, and may reduce survival of flower-feeding beneficial insects. Imidacloprid seed-treated crops (0.05 mg AI (active ingredient) /canola seed and 1.2 mg AI/corn seed) translocate less than 10 ppb to pollen and nectar. However, higher rates of soil-applied imidacloprid are used in nurseries and urban landscapes, such as 300 mg AI/10 L (3 gallon) pot and 69 g AI applied to the soil under a 61 (24 in) cm diam. tree. Translocation of imidacloprid from soil (300 mg AI) to flowers of Asclepias curassavica resulted in 6,030 ppb in 1X and 10,400 ppb in 2X treatments, which are similar to imidacloprid residues found in another plant species we studied. A second imidacloprid soil application 7 months later resulted in 21,000 ppb in 1X and 45,000 ppb in 2X treatments. Consequently, greenhouse/nursery use of imidacloprid applied to flowering plants can result in 793 to 1,368 times higher concentration compared to an imidacloprid seed treatment (7.6 ppb pollen in seed- treated canola), where most research has focused. These higher imidacloprid levels caused significant mortality in both 1X and 2X treatments in 3 lady beetle species, Coleomegilla maculata, Harmonia axyridis, and Hippodamia convergens, but not a fourth species, Coccinella septempunctata. Adult survival were not reduced for monarch, Danaus plexippus and painted lady, Vanessa cardui, butterflies, but larval survival was significantly reduced. The use of the neonicotinoid imidacloprid at greenhouse/nursery rates reduced survival of beneficial insects feeding on pollen and nectar and is incompatible with the principles of IPM.

Three Florida native larval butterflies (Junonia coenia, Anartia jatrophae, Eumaeus atala) were used in the present study to determine the acute toxicity, hazard, and risk of a 24h ingestion of leaves contaminated with the adult mosquito control insecticides permethrin, naled, and dichlorvos to late 4th and early 5th in-star caterpillars. Based on 24-h LD50s for ingestion, naled was more acutely toxic than permethrin and dichlorvos to caterpillars. Hazard quotients using the ratio of the highest doses and the 90th percentile doses from field measurements in host plant foliage following actual mosquito control applications to the toxicological benchmarks from laboratory toxicity tests indicate potential high acute hazard for naled compared to permethrin and dichlorvos. Based on probabilistic ecological risk methods, naled exposure doses in the environment also presented a higher acute risk to caterpillars than permethrin and dichlorvos. The acute toxicity laboratory results and ecological risk assessment are based only on dietary ingestion and single chemical doses. It does not include other typical exposure scenarios that may occur in the environment. It is thus plausible to state that the ecological risk assessment presented here underestimates the potential risks in the field to caterpillars. However, one assumption that is scientifically feasible and certainly real from the results - if the environmental exposure doses of mosquito control operations are similar or higher to those presented here in leaves from the field, after applications, there will likely be significant mortalities and other adverse effects on caterpillar populations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A comprehensive probabilistic terrestrial ecological risk assessment (ERA) was conducted to characterize the potential risk of mosquito control insecticide (i.e., naled, it's metabolite dichlorvos, and permethrin) usage to adult butterflies in south Florida by comparing the probability distributions of environmental exposure concentrations following actual mosquito control applications at labeled rates from ten field monitoring studies with the probability distributions of butterfly species response (effects) data from our laboratory acute toxicity studies. The overlap of these distributions was used as a measure of risk to butterflies. The long-term viability (survival) of adult butterflies, following topical (thorax/wings) exposures was the environmental value we wanted to protect. Laboratory acute toxicity studies (24-h LD50) included topical exposures (thorax and wings) to five adult butterfly species and preparation of species sensitivity distributions (SSDs). The ERA indicated that the assessment endpoint of protection, of at least 90% of the species, 90% of the time (or the 10th percentile from the acute SSDs) from acute naled and permethrin exposures, is most likely not occurring when considering topical exposures to adults. Although the surface areas for adulticide exposures are greater for the wings, exposures to the thorax provide the highest potential for risk (i.e., SSD 10th percentile is lowest) for adult butterflies. Dichlorvos appeared to present no risk. The results of this ERA can be applied to other areas of the world, where these insecticides are used and where butterflies may be exposed. Since there are other sources (e.g., agriculture) of pesticides in the environment, where butterfly exposures will occur, the ERA may under-estimate the potential risks under real-world conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The conditional expression of alternative life strategies is a widespread feature of animal life and a pivotal adaptation to life in seasonal environments. To optimally match suites of traits to seasonally changing ecological opportunities, animals living in seasonal environments need mechanisms linking information on environmental quality to resource allocation decisions. The butterfly Bicyclus anynana expresses alternative adult life histories in the alternating wet and dry seasons of its habitat as endpoints of divergent developmental pathways triggered by seasonal variation in preadult temperature. Pupal ecdysteroid hormone titers are correlated with the seasonal environment, but whether they play a functional role in coordinating the coupling of adult traits in the alternative life histories is unknown. Here, we show that manipulating pupal ecdysteroid levels is sufficient to mimic in direction and magnitude the shifts in adult reproductive resource allocation normally induced by seasonal temperature. Crucially, this allocation shift is accompanied by changes in ecologically relevant traits, including timing of reproduction, life span, and starvation resistance. Together, our results support a functional role for ecdysteroids during development in mediating strategic reproductive investment decisions in response to predictive indicators of environmental quality. This study provides a physiological mechanism for adaptive developmental plasticity, allowing organisms to cope with variable environments.

In herbivorous insects, food selection depends on sensitivity to specific chemical stimuli from host-plants as well as to secondary metabolites (bitter) and to sugars (phagostimulatory). Bitter compounds are noxious, unpalatable or both and evoke an aversive feeding response. Instead, sugars and sugar alcohols play a critical role in determining and enhancing the palatability of foods. We assumed that peripheral taste sensitivity may be related to the width of the host selection. Our model consists of two closely phylogenetically related Papilionid species exhibiting a difference in host plant choice: Papilio hospiton and Papilio machaon. The spike activity of the lateral and medial maxillary styloconic taste sensilla was recorded following stimulation with several carbohydrates, nicotine and NaCl, with the aim of characterizing their gustatory receptor neurons and of comparing their response patterns in the light of their different acceptability in feeding behaviour. The results show that: a) each sensillum houses phagostimulant and phagodeterrent cells; b) the spike activity of the gustatory neurons in response to different taste stimuli is higher in P. hospiton than in P. machaon; c) sugar solutions inhibit the spike activity of the deterrent and salt cells, and the suppression is higher in P. machaon than in P. hospiton. In conclusion, we propose that the different balance between the phagostimulant and phagodeterrent inputs from GRNs of maxillary sensilla may contribute in determining the difference in food choice and host range.

Although agricultural intensification is thought to pose a significant threat to species, little is known about its role in driving biodiversity loss at regional scales. I assessed the effects of a major component of agricultural intensification, agricultural chemical use, and land-cover and climatic variables on butterfly diversity across 81 provinces in Turkey, where agriculture is practiced extensively but with varying degrees of intensity. I determined butterfly species presence in each province from data on known butterfly distributions and calculated agricultural chemical use as the proportion of agricultural households that use chemical fertilizers and pesticides. I used constrained correspondence analyses and regression-based multimodel inference to determine the effect of environmental variables on species composition and richness, respectively. The variation in butterfly species composition across the provinces was largely explained (78%) by the combination of agricultural chemical use, particularly pesticides, and climatic and land-cover variables. Although overall butterfly richness was primarily explained by climatic and land-cover variables, such as the area of natural vegetation cover, threatened butterfly richness and the relative number of threatened butterfly species decreased substantially as the proportion of agricultural households using pesticides increased. These findings suggest that widespread use of agricultural chemicals, or other components of agricultural intensification that may be collinear with pesticide use, pose an imminent threat to the biodiversity of Turkey. Accordingly, policies that mitigate agricultural intensification and promote low-input farming practices are crucial for protecting threatened species from extinction in rapidly industrializing nations such as Turkey. Efectos del Uso Extensivo de Agroquímicos sobre la Diversidad de Mariposas en Provincias Turcas., (© 2013 Society for Conservation Biology.)

For almost a century, the iridescence of tropical Morpho butterfly scales has been known to originate from 3D vertical ridge structures of stacked periodic layers of cuticle separated by air gaps. Here we describe a biological pattern of surface functionality that we have found in these photonic structures. This pattern is a gradient of surface polarity of the ridge structures that runs from their polar tops to their less-polar bottoms. This finding shows a biological pattern design that could stimulate numerous technological applications ranging from photonic security tags to self-cleaning surfaces, gas separators, protective clothing, sensors, and many others. As an important first step, this biomaterial property and our knowledge of its basis has allowed us to unveil a general mechanism of selective vapor response observed in the photonic Morpho nanostructures. This mechanism of selective vapor response brings a multivariable perspective for sensing, where selectivity is achieved within a single chemically graded nanostructured sensing unit, rather than from an array of separate sensors.

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na(+)/K(+) -ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so-called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide-containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na(+)/K(+) -ATPase gene and by enzymatic assays with extracted Na(+)/K(+) -ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom-up selection by plant compounds and top-down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. Intracellular calcium level has a definite role in innate and adaptive immune signaling. However, very few information are accessible about calcium transients of invertebrate immunocytes, especially of insect hemocytes, the effector cells of insect immunity. In this study, we show that the RyR-stimulating agent flubendiamide inhibit hemocyte spreading and phagocytosis in the cabbage white butterfly, Pieris rapae. Furthermore, we cloned a cDNA encoding a ryanodine receptor (PrRyR) from the hemocytes of P. rapae. It encodes 5107 amino acids with a predicted molecular weight of 578.2 kDa. PrRyR shares a common feature with known RyRs: a well-conserved COOH-terminal domain with two consensus calcium-binding EF-hands and six transmembrane domains, and a large hydrophilic NH2-terminal domain. In the larval stage, PrRyR was highly expressed in epidermis tissue and also expressed in hemocytes at a moderate level. In the adult stage, PrRyR was expressed at high levels in thoraces and legs, while low levels in abdomens and antennae. Quantitative real-time PCR analysis showed that its expression did not display any significant change in response to bacterial challenge. Western blot analysis and immunohistochemistry assay displayed that PrRyR was detected and presented on hemocytes. We also showed that flubendiamide, a RyR-activating insecticide, induced Ca(2+) release and thereby confirmed functional expression of the PrRyR in the hemocytes of P. rapae., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Two new chlorinated multiacylated 3,4-seco-grayanane diterpenoids, neopierisoids A and B (1 and 2), were isolated from flowers of the poisonous plant Pieris japonica and were identified from spectroscopic analysis and X-ray diffraction data. Both compounds showed obvious antifeedant activity against Pieris brassicae with an EC50 of 10.07 μg/cm(2) for 1 and 5.33 μg/cm(2) for 2, indications of toxic properties. Chlorinated 3,4-seco-grayanane diterpenoids in P. japonica may play a defensive role against herbivores.

Herbicides are the most commonly applied pesticides in agroecosystems, and therefore pose potentially significant ecotoxicological risks to plants and insects. Glyphosate is the most common herbicide worldwide, and glyphosate-resistant weeds are quickly becoming serious challenges in some agroecosystems. Because of this resistance epidemic and the recent development of crops with resistance to dicamba or 2,4-D, herbicide-use patterns are likely to change. Presently, dicamba and 2,4-D cause most herbicide-drift damage to nontarget plants despite limited agricultural usage, but the effects of these synthetic auxin herbicides on insects have been poorly explored. To understand the influence of dicamba on insects, we applied several sublethal, drift-level rates of dicamba to soybean, Glycine max L., and Carduus thistle, and measured growth and survival of Helicoverpa zea (Boddie) and Vanessa cardui (L.) larvae, respectively. For thistle, we measured percent nitrogen content before and after dicamba application. We also performed direct toxicity bioassays on the two caterpillar species with several rates of dicamba. Dicamba was not directly toxic to larvae of either species, and H. zea showed no negative effects when feeding on soybeans dosed with dicamba. We did, however, detect significant negative, indirect effects of higher rates of dicamba on V. cardui larval and pupal mass, total nitrogen of thistles post application, and thistle biomass in the presence of V. cardui larvae. Notably, thistle biomass was not related to dicamba dose in absence of larvae. Our results indicate that dicamba can indirectly influence the performance of some caterpillar species, possibly by altering plant nutritional content.

The search for general patterns in the production and allocation of plant defense traits will be facilitated by characterizing multivariate suites of defense, as well as by studying additional plant taxa, particularly those with available genomic resources. Here, we investigated patterns of genetic variation in phytochemical defenses (phenylpropanoid glycosides, PPGs) in Mimulus guttatus (yellow monkeyflower). We grew plants derived from several natural populations, consisting of multiple full-sibling families within each population, in a common greenhouse environment. We found substantial variation in the constitutive multivariate PPG phenotype and in constitutive levels of individual phytochemicals within plants (among leaves of different ages), within populations (among full-sibling families), and among populations. Populations consisting of annual plants generally, but not always, had lower concentrations of phytochemicals than did populations of perennial plants. Populations differed in their plastic response to artificial herbivory, both in the overall multivariate PPG phenotype and in the individual phytochemicals. The relationship between phytochemistry and another defense trait, trichomes, differed among populations. Finally, we demonstrated that one of the PPGs, verbascoside, acts as a feeding stimulant rather than a feeding deterrent for a specialist herbivore of M. guttatus, the buckeye caterpillar (Junonia coenia Nymphalidae). Given its available genetic resources, numerous, easily accessible natural populations, and patterns of genetic variation highlighted in this research, M. guttatus provides an ideal model system in which to test ecological and evolutionary theories of plant-herbivore interactions.