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Broad-spectrum crop protection technologies, such as abamectin and bifenthrin, are globally relied upon to curb the existential threats from economic crop pests such as the generalist herbivore Tetranychus urticae Koch (TSSM). However, the rising cost of discovering and registering new acaricides, particularly for specialty crops, along with the increasing risk of pesticide resistance development, underscores the urgent need to preserve the efficacy of currently registered acaricides. This study examined the overall genetic mechanism underlying adaptation to abamectin and bifenthrin in T. urticae populations from commercial hop fields in the Pacific Northwestern region of the USA. A transcriptomic study was conducted using four populations (susceptible, abamectin-resistant, and two bifenthrin-resistant populations). Differential gene expression analysis revealed a notable disparity, with significantly more downregulated genes than upregulated genes in both resistant populations. Gene ontology enrichment analysis revealed a striking consistency among all three resistant populations, with downregulated genes predominately associated with chitin metabolism. In contrast, upregulated genes in the resistant populations were linked to biological processes, such as peptidase activity and oxidoreductase activity. Proteolytic activity by peptidase enzymes in abamectin- and bifenthrin-resistant TSSM populations may suggest their involvement in acaricide metabolism. These findings provide valuable insights into the molecular mechanisms underlying acaricide resistance in the TSSM. This knowledge can be utilized to develop innovative pesticides and molecular diagnostic tools for effectively monitoring and managing resistant TSSM populations. more...

What limits the size of nature's most extreme structures? For weapons like beetle horns, one possibility is a tradeoff associated with mechanical levers: as the output arm of the lever system-the beetle horn-gets longer, it also gets weaker. This "paradox of the weakening combatant" could offset reproductive advantages of additional increases in weapon size. However, in contemporary populations of most heavily weaponed species, males with the longest weapons also tend to be the strongest, presumably because selection drove the evolution of compensatory changes to these lever systems that ameliorated the force reductions of increased weapon size. Therefore, we test for biomechanical limits by reconstructing the stages of weapon evolution, exploring whether initial increases in weapon length first led to reductions in weapon force generation that were later ameliorated through the evolution of mechanisms of mechanical compensation. We describe phylogeographic relationships among populations of a rhinoceros beetle and show that the "pitchfork" shaped head horn likely increased in length independently in the northern and southern radiations of beetles. Both increases in horn length were associated with dramatic reductions to horn lifting strength-compelling evidence for the paradox of the weakening combatant-and these initial reductions to horn strength were later ameliorated in some populations through reductions to horn length or through increases in head height (the input arm for the horn lever system). Our results reveal an exciting geographic mosaic of weapon size, weapon force, and mechanical compensation, shedding light on larger questions pertaining to the evolution of extreme structures., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.) more...

The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism., (© 2023. The Author(s).) more...

The elaborate ornaments and weapons of sexual selection, such as the vast array of horns observed in scarab beetles, are some of the most striking outcomes of evolution. How these novel traits have arisen, develop, and respond to condition is governed by a complex suite of interactions that require coordination between the environment, whole-animal signals, cell-cell signals, and within-cell signals. Endocrine factors, developmental patterning genes, and sex-specific gene expression have been shown to regulate beetle horn size, shape, and location, yet no overarching mechanism of horn shape has been described. Recent advances in microscopy and computational analyses combined with a functional genetic approach have revealed that patterning genes combined with intricate epithelial folding and movement are responsible for the final shape of a beetle head horn., (Copyright © 2021. Published by Elsevier Ltd.) more...

The two-spotted spider mite, Tetranychus urticae, is a polyphagous pest feeding on over 1100 plant species, including numerous highly valued economic crops. The control of T. urticae largely depends on the use of acaricides, which leads to pervasive development of acaricide resistance. Cytochrome P450-mediated metabolic detoxification is one of the major mechanisms of acaricide resistance in T. urticae. NADPH-cytochrome P450 reductase (CPR) plays as a crucial co-factor protein that donates electron(s) to microsomal cytochrome P450s to complete their catalytic cycle. This study seeks to understand the involvement of CPR/P450 in acaricide resistance in T. urticae. The full-length cDNA sequence of T. urticae's CPR (TuCPR) was cloned and characterized. TuCPR was ubiquitously transcribed in different life stages of T. urticae and the highest transcription was observed in the nymph and adult stages. TuCPR was constitutively over-expressed in six acaricide resistant populations compared to a susceptible one. TuCPR transcriptional expression was also induced by multiple acaricides in a time-dependent manner. Down-regulation of TuCPR via RNA interference (RNAi) in T. urticae led to reduced enzymatic activities of TuCPR and cytochrome P450s, as well as a reduction of resistance to multiple acaricides, abamectin, bifenthrin, and fenpyroximate. The outcome of this study highlights CPR as a potential novel target for eco-friendly control of T. urticae and other related plant-feeding pests., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.) more...

Thrips tabaci is a key pest of onions, especially in the Pacific Northwestern USA. Management of T. tabaci is dominated by the application of various insecticides. However, T. tabaci is known to develop insecticide resistance which possibly leads to control failures, crop loss, and environmental concern. Here, we evaluated resistance status of T. tabaci populations from conventional and organic commercial onion fields to three widely used insecticides: oxamyl, methomyl, and abamectin with on-field concentration-mortality bioassays. The biochemistry and molecular mechanisms underlying resistance to these insecticides were also investigated by using enzymatic assays and detecting resistance-associated mutations. Field-evolved resistance to oxamyl, methomyl and abamectin were detected in most of the T. tabaci populations collected from conventional onion farms. At the labeled field rate, all the tested insecticides, particularly methomyl and oxamyl, had significantly reduced efficacy. Enzymatic assays of insecticide target and detoxification enzymes indicated that T. tabaci populations in Western USA onions harbor multiple mechanisms of resistance including enhanced activities of detoxification enzymes and target site insensitivity. Our results provide new information in understanding the dynamics of T. tabaci adaptation to multiple insecticides, which will help to design sustainable insecticide resistance management strategies for T. tabaci. Furthermore, this study provides the foundation for future research in identifying the biochemical and molecular markers associated with insecticide resistance in T. tabaci., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.) more...

Wing polyphenism is a phenomenon in which one genotype can produce two or more distinct wing phenotypes adapted to the particular environment. What remains unknown is how wing pad development is controlled downstream of endocrine signals such as insulin and JNK pathways. We show that genes important in cellular proliferation, cytokinesis, and cell cycle progression are necessary for growth and development of long wings. Wing pad cellular development of the long-winged morph was characterized by a highly structured epithelial layer with microvilli-like structures. Cells of adult short wing pads are largely in the G2/M phase of the cell cycle, whereas those of long wings are largely in G1. Our study is the first to report the comparative developmental and cellular morphology and structure of the wing morphs and to undertake a comprehensive evaluation of the cell cycle genes necessary for wing development of this unique, adaptive life history strategy., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.) more...

Multiple acaricide resistance in Tetranychus urticae continues to threaten crop production globally, justifying the need to adequately study resistance for sustainable pest management. Most studies on acaricide resistance have focused on the acute contact toxicity of acaricides with little or no information on the behavioral responses elicited after acaricide exposure. Furthermore, the impact of physiological resistance on these behavioral responses remains unknown in most pest species, including T. urticae. We tested the effect of acaricide resistance on contact toxicity, irritancy and repellency of mitochondrial electron transport inhibitor of complex I (MET-I) and mite growth inhibitor (MGI) acaricides on multiple T. urticae strains. We also tested whether acaricides with similar physiological target site/mode of action also elicit similar behavioral effects on T. urticae strains. MET-I acaricides (fenazaquin, fenpyroximate, and pyrabiden) and MGIs (clofentezine, hexythiazox and etoxazole) elicited a dose-dependent irritant and repellent effect on T. urticae. Selection of strains for physiological resistance to these acaricides affected the behavioral response of T. urticae, especially in MET-I resistant strains, that showed reduced irritancy and repellency to MET-I acaricides. Behavioral response also affected the oviposition of T. urticae, where strains generally showed preferential oviposition away from the acaricides. The outcome of this study highlights negative consequences of acaricide resistance that can potentially affect T. urticae management. more...

Members of the phylum Arthropoda, comprising over 80% of total animal species, have evolved regenerative abilities, but little is known about the molecular mechanisms mediating this process. Transforming growth factor β (TGF-β) signaling mediates a diverse set of essential processes in animals and is a good candidate pathway for regulation of regeneration in arthropods. In this study we investigated the role of activin signaling, a TGF-β superfamily pathway, in limb regeneration in the crayfish. We identified and cloned a downstream transcription factor in the activin pathway, Smox, and characterized its function with regard to other elements of the activin signaling pathway. Gene knockdown of Smox by RNAi induced regeneration of complete but smaller pereopods after autotomy. This indicates that activin signaling via Smox functions in regulation of pereopod growth and size. The expression levels of both Smox and the activin receptor babo were closely correlated with molting. The expression level of Smox increased when babo was knocked down by RNAi, indicating that Smox and babo transcription are linked. Our study suggests that the Babo-Smox system in activin signaling is conserved in decapods, and supports an evolutionary conservation of this aspect of molecular signaling during regeneration between protostomes and deuterostomes., (© 2018 Wiley Periodicals, Inc.) more...

Among the most dramatic examples of sexual selection are the weapons used in battles between rival males over access to females. As with ornaments of female choice, the most "exaggerated" sexually selected weapons vary from male to male more widely than other body parts (hypervariability), and their growth tends to be more sensitive to nutritional state or physiological condition compared with growth of other body parts ("heightened" conditional expression). Here, we use RNAseq analysis to build on recent work exploring these mechanisms in the exaggerated weapons of beetles, by examining patterns of differential gene expression in exaggerated (head and thorax horns) and non-exaggerated (wings, genitalia) traits in the Asian rhinoceros beetle, Trypoxylus dichotomus. Our results suggest that sexually dimorphic expression of weaponry involves large-scale changes in gene expression, relative to other traits, while nutrition-driven changes in gene expression in these same weapons are less pronounced. However, although fewer genes overall were differentially expressed in high- vs. low-nutrition individuals, the number of differentially expressed genes varied predictably according to a trait's degree of condition dependence (head horn > thorax horn > wings > genitalia). Finally, we observed a high degree of similarity in direction of effects (vectors) for subsets of differentially expressed genes across both sexually dimorphic and nutritionally responsive growth. Our results are consistent with a common set of mechanisms governing sexual size dimorphism and condition dependence., (© 2018 John Wiley & Sons Ltd.) more...

Beetle horns are attractive models for studying the evolution of novel traits, as they display diverse shapes, sizes, and numbers among closely related species within the family Scarabaeidae. Horns radiated prolifically and independently in two distant subfamilies of scarabs, the dung beetles (Scarabaeinae), and the rhinoceros beetles (Dynastinae). However, current knowledge of the mechanisms underlying horn diversification remains limited to a single genus of dung beetles, Onthophagus. Here we unveil 11 horn formation genes in a rhinoceros beetle, Trypoxylus dichotomus. These 11 genes are mostly categorized as larval head- and appendage-patterning genes that also are involved in Onthophagus horn formation, suggesting the same suite of genes was recruited in each lineage during horn evolution. Although our RNAi analyses reveal interesting differences in the functions of a few of these genes, the overwhelming conclusion is that both head and thoracic horns develop similarly in Trypoxylus and Onthophagus, originating in the same developmental regions and deploying similar portions of appendage patterning networks during their growth. Our findings highlight deep parallels in the development of rhinoceros and dung beetle horns, suggesting either that both horn types arose in the common ancestor of all scarabs, a surprising reconstruction of horn evolution that would mean the majority of scarab species (~35,000) actively repress horn growth, or that parallel origins of these extravagant structures resulted from repeated co-option of the same underlying developmental processes., Competing Interests: The authors have declared that no competing interests exist. more...

Food quality is a critical environmental condition that impacts an animal's growth and development. Many insects facing this challenge have evolved a phenotypically plastic, adaptive response. For example, many species of insect exhibit facultative wing growth, which reflects a physiological and evolutionary trade-off between dispersal and reproduction, triggered by environmental conditions. What the environmental cues are and how they are transduced to produce these alternative forms, and their associated ecological shift from dispersal to reproduction, remains an important unsolved problem in evolutionary ecology. In this study, we investigated the role that host quality has on the induction of wing development in a wing polyphenic insect exhibiting strong tradeoffs in investment between dispersal and reproduction, the brown planthopper, a serious rice pest in Asia. As rice plants grow, the short-winged brown planthopper dominates the population, but a shift occurs as the plants mature and senesce in the field such that long-winged brown planthoppers emerge and migrate. It remains unknown how changes in the rice plant induce development of the long-winged morph, despite recent discoveries on the role of the insulin and JNK signaling pathways in wing development. We found that by mimicking the glucose concentration of senescing rice plants, we significantly increased the proportion of long-winged female planthoppers. The effects of glucose on wing morph are additive with previously described effects of density. Our results show that host quality both directly regulates phenotypic plasticity and interacts with other factors such as density to produce the appropriate phenotype for specific environmental conditions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.) more...

Changes in food availability and crowding are two critical environmental conditions that impact an animal's trajectory toward either reproduction or migration. Many insects facing this challenge have evolved wing polymorpisms that allow them to respond to changing conditions. When conditions favor reproduction, wing polymorphic species produce adults that either have no wings or short, non-functional wings; however, when conditions favor migration, adults with functional wings and robust flight muscles develop. Here we review three recently reported signaling pathways regulating wing polyphenism in wing polymorphic crickets, aphids, and brown planthoppers: juvenile horomone/ecdysone signaling, insulin signaling, and Jun-N-terminal Kinase (JNK) signaling. Understanding how these pathways respond to nutrition, stress and crowding with the appropriate adaptive phenotype is an important step in understanding how life-history trade-offs evolve., (Copyright © 2017 Elsevier Inc. All rights reserved.) more...

Mite growth inhibitors (MGIs), such as etoxazole and hexythiazox, are valuable IPM tools for Tetranychus urticae control in hops due to their unique mode of action and selectivity. Hence, it is necessary to standardize bioassay methods to evaluate the efficacy of MGIs, monitor resistance, and identify mechanisms underlying MGI resistance in the field. Here, we developed a three-tiered approach for evaluating ovicidal toxicity of MGIs to T. urticae, which simulated different MGI exposure scenarios in the field. The most effective bioassay method was direct exposure of T. urticae eggs to MGIs. With this method, four field-collected T. urticae populations showed low-to-moderate resistance to MGIs. Cross-resistance among MGIs and from MGIs to bifenazate and bifenthrin was detected. Besides target site insensitivity, enhanced cytochrome P450 and esterase activities also contribute to the MGI resistance in hop yard-collected T. urticae populations. Low-to-moderate MGI resistance in T. urticae populations may be mediated by multiple mechanisms. Positive selection pressure on the I1017F mutation is moderate in field-collected T. urticae populations. Further studies are required to identify metabolic detoxification genes that confer resistance to MGIs for precise resistance monitoring. more...

One of the defining features of the evolutionary success of insects is the morphological diversification of their appendages, especially mouthparts. Although most insects share a common mouthpart ground plan, there is remarkable diversity in the relative size and shapes of these appendages among different insect lineages. One of the most prominent examples of mouthpart modification can be found in the enlargement of mandibles in stag beetles (Coleoptera, Insecta). In order to understand the proximate mechanisms of mouthpart modification, we investigated the function of appendage-patterning genes in mandibular enlargement during extreme growth of the sexually dimorphic mandibles of the stag beetle Cyclommatus metallifer. Based on knowledge from Drosophila and Tribolium studies, we focused on seven appendage patterning genes (Distal-less (Dll), aristaless (al), dachshund (dac), homothorax (hth), Epidermal growth factor receptor (Egfr), escargot (esg), and Keren (Krn). In order to characterize the developmental function of these genes, we performed functional analyses by using RNA interference (RNAi). Importantly, we found that RNAi knockdown of dac resulted in a significant mandible size reduction in males but not in female mandibles. In addition to reducing the size of mandibles, dac knockdown also resulted in a loss of the serrate teeth structures on the mandibles of males and females. We found that al and hth play a significant role during morphogenesis of the large male-specific inner mandibular tooth. On the other hand, knockdown of the distal selector gene Dll did not affect mandible development, supporting the hypothesis that mandibles likely do not contain the distal-most region of the ancestral appendage and therefore co-option of Dll expression is unlikely to be involved in mandible enlargement in stag beetles. In addition to mandible development, we explored possible roles of these genes in controlling the divergent antennal morphology of Coleoptera., (Copyright © 2016 Elsevier Inc. All rights reserved.) more...

Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest., Competing Interests: The authors have declared that no competing interest exists. more...

Juvenile hormone (JH) is a key insect growth regulator frequently involved in modulating phenotypically plastic traits such as caste determination in eusocial species, wing polymorphisms in aphids, and mandible size in stag beetles. The jaw morphology of stag beetles is sexually-dimorphic and condition-dependent; males have larger jaws than females and those developing under optimum conditions are larger in overall body size and have disproportionately larger jaws than males raised under poor conditions. We have previously shown that large males have higher JH titers than small males during development, and ectopic application of fenoxycarb (JH analog) to small males can induce mandibular growth similar to that of larger males. What remains unknown is whether JH regulates condition-dependent trait growth in other insects with extreme sexually selected structures. In this study, we tested the hypothesis that JH mediates the condition-dependent expression of the elaborate horns of the Asian rhinoceros beetle, Trypoxylus dichotomus. The sexually dimorphic head horn of this beetle is sensitive to nutritional state during larval development. Like stag beetles, male rhinoceros beetles receiving copious food produce disproportionately large horns for their body size compared with males under restricted diets. We show that JH titers are correlated with body size during the late feeding and early prepupal periods, but this correlation disappears by the late prepupal period, the period of maximum horn growth. While ectopic application of fenoxycarb during the third larval instar significantly delayed pupation, it had no effect on adult horn size relative to body size. Fenoxycarb application to late prepupae also had at most a marginal effect on relative horn size. We discuss our results in context of other endocrine signals of condition-dependent trait exaggeration and suggest that different beetle lineages may have co-opted different physiological signaling mechanisms to achieve heightened nutrient-sensitive weapon growth., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.) more...

Wing polyphenism is considered to be an adaptive trade-off between migration (long winged forms) and reproduction (short winged forms), determined by various environmental conditions. The c-Jun NH2-terminal kinase (JNK) is crucial for the regulation of the activity of a number of transcription factors, and is activated under stress and environmental fluctuations where it functions in maintaining cell viability and proliferation. We used RNA interference and a pharmacological inhibitor of JNK to test the role of JNK signaling in regulating the wing dimorphism of the brown planthopper, Nilaparvata lugens. Silencing NlJNK increased the proportion of short winged female adults, reminiscent of the effect of silencing inhibitory components of the insulin-signaling pathway, such as NlAkt. However, silencing of the JNK-activated transcription factors NlJun and NlFos did not change the wing form ratio significantly, indicating that NlJNK may not act through NlJun and NlFos in mediating this process. In summary, JNK signaling may play a role in determining wing polymorphism in N. lugens females., (Copyright © 2016 Elsevier Ltd. All rights reserved.) more...

Crowding and changes in food availability are two critical environmental conditions that impact an animal's trajectory toward either migration or reproduction. Many insects facing this challenge have evolved wing polyphenisms. When conditions favor reproduction, wing polyphenic species produce adults that either have no wings or short, non-functional wings. Facultative wing growth reflects a physiological and evolutionary trade-off between migration and reproduction, triggered by environmental conditions. How environmental cues are transduced to produce these alternative forms, and their associated ecological shift from migration to reproduction, remains an important unsolved problem in evolutionary ecology. The brown planthopper, a wing polymorphic insect exhibiting strong trade-offs in investment between migration and reproduction, is one of the most serious rice pests in Asia. In this study, we investigated the function of four genes in the insulin-signaling pathway known to couple nutrition with growth, PI3 Kinase (PI3K), PDK1, Akt (Protein Kinase B), and the forkhead gene FOXO. Using a combination of RNA interference and pharmacological inhibitor treatment, we show that all four genes contribute to tissue level regulation of wing polymorphic development in this insect. As predicted, silencing of the NlPI3K, NlAkt and NlPDK1 through dsRNA and with the pharmacological inhibitor Perifosine resulted in short-winged brown planthoppers, whereas knockdown of NlFOXO resulted in long-winged planthoppers. Morphometric analyses confirm that phenotypes from our manipulations mimic what would be found in nature, i.e., major parameters such as bristle number, wing area and body weight are not significantly different from non-experimental animals. Taken together, these data implicate the insulin-signaling pathway in the transduction of environmental factors into condition-dependent patterns of wing growth in insects. more...

Background: Genes in the sex determination pathway are important regulators of sexually dimorphic animal traits, including the elaborate and exaggerated male ornaments and weapons of sexual selection. In this study, we identified and functionally analyzed members of the sex determination gene family in the golden metallic stag beetle Cyclommatus metallifer, which exhibits extreme differences in mandible size between males and females., Results: We constructed a C. metallifer transcriptomic database from larval and prepupal developmental stages and tissues of both males and females. Using Roche 454 pyrosequencing, we generated a de novo assembled database from a total of 1,223,516 raw reads, which resulted in 14,565 isotigs (putative transcript isoforms) contained in 10,794 isogroups (putative identified genes). We queried this database for C. metallifer conserved sex determination genes and identified 14 candidate sex determination pathway genes. We then characterized the roles of several of these genes in development of extreme sexual dimorphic traits in this species. We performed molecular expression analyses with RT-PCR and functional analyses using RNAi on three C. metallifer candidate genes--Sex-lethal (CmSxl), transformer-2 (Cmtra2), and intersex (Cmix). No differences in expression pattern were found between the sexes for any of these three genes. In the RNAi gene-knockdown experiments, we found that only the Cmix had any effect on sexually dimorphic morphology, and these mimicked the effects of Cmdsx knockdown in females. Knockdown of CmSxl had no measurable effects on stag beetle phenotype, while knockdown of Cmtra2 resulted in complete lethality at the prepupal period. These results indicate that the roles of CmSxl and Cmtra2 in the sex determination cascade are likely to have diverged in stag beetles when compared to Drosophila. Our results also suggest that Cmix has a conserved role in this pathway. In addition to those three genes, we also performed a more complete functional analysis of the C. metallifer dsx gene (Cmdsx) to identify the isoforms that regulate dimorphism more fully using exon-specific RNAi. We identified a total of 16 alternative splice variants of the Cmdsx gene that code for up to 14 separate exons. Despite the variation in RNA splice products of the Cmdsx gene, only four protein isoforms are predicted. The results of our exon-specific RNAi indicated that the essential CmDsx isoform for postembryonic male differentiation is CmDsxB, whereas postembryonic female specific differentiation is mainly regulated by CmDsxD., Conclusions: Taken together, our results highlight the importance of studying the function of highly conserved sex determination pathways in numerous insect species, especially those with dramatic and exaggerated sexual dimorphism, because conservation in protein structure does not always translate into conservation in downstream function. more...

Polyphenisms such as wing dimorphisms and caste determination are important in allowing animals to adapt to changing environments. The brown planthopper Nilaparvata lugens, one of the most serious insect agricultural pests, includes two wing forms, the long wing form (macropterous) and the short wing form (brachypterous). Long wings are specialized for migration, while short wings are found in individuals specialized for reproduction. While studying wing form polyphenism in the brown planthopper, we excised single wing pads from 4th instar nymphs in order to preserve transcriptional records to correlate with adult wing form. Surprisingly, we found that excision of one wing pad from a pair of the forewings changed the wing morph of the other wing after development to the adult, resulting in the short wing morph. Further experiments showed that not only excision or slicing of the wing pad, but also needle punctures in the abdomen all caused a significant increase in the proportion of nymphs developing into short winged adults. Thus wounding appears to cause a shift to short wing development. We then tested the transcriptional expression in N. lugens of the transcription factor FOXO, which has been shown to help mediate both wing polyphenism in brown planthoppers and wound healing in mice, after excision of the wing pad. Both NlFOXO and its downstream target Nl4EBP increased significantly after wing pad excision. These results indicate that FOXO mediates both wing development and wound healing in N. lugens, which results in an interesting linkage of these two physiological processes., (Copyright © 2015 Elsevier Ltd. All rights reserved.) more...

Male stag beetles possess colossal mandibles, which they wield in combat to obtain access to females. As with many other sexually selected weapons, males with longer mandibles win more fights. However, variation in the functional morphology of these structures, used in male-male combat, is less well understood. In this study, mandible bite force, gape, structural strength, and potential tradeoffs are examined across a wide size range for one species of stag beetle, Cyclommatus metallifer. We found that not only does male mandible size demonstrate steep positive allometry, but the shape, relative bite force, relative gape, and safety factor of the mandibles also change with male size. Allometry in these functionally important mandibular traits suggests that larger males with larger mandibles should be better fighters, and that the mandibles can be considered an honest signal of male fighting ability. However, negative allometry in mandible structural safety factor, wing size, and flight muscle mass suggest significant costs and a possible limit on the size of the mandibles. J. Exp. Zool. 325A:3-12, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.) more...

The two-spotted spider mite, Tetranychus urticae Koch is a major pest that feeds on >1,100 plant species. Many perennial crops including hop (Humulus lupulus) are routinely plagued by T. urticae infestations. Hop is a specialty crop in Pacific Northwest states, where 99% of all U.S. hops are produced. To suppress T. urticae, growers often apply various acaricides. Unfortunately T. urticae has been documented to quickly develop resistance to these acaricides which directly cause control failures. Here, we investigated resistance ratios and distribution of multiple resistance-associated mutations in field collected T. urticae samples compared with a susceptible population. Our research revealed that a mutation in the cytochrome b gene (G126S) in 35% tested T. urticae populations and a mutation in the voltage-gated sodium channel gene (F1538I) in 66.7% populations may contribute resistance to bifenazate and bifenthrin, respectively. No mutations were detected in Glutamate-gated chloride channel subunits tested, suggesting target site insensitivity may not be important in our hop T. urticae resistance to abamectin. However, P450-mediated detoxification was observed and is a putative mechanism for abamectin resistance. Molecular mechanisms of T. urticae chemical adaptation in hopyards is imperative new information that will help growers develop effective and sustainable management strategies. more...

Background: Insects exhibit a diversity of environmentally sensitive phenotypes that allow them to be an extraordinarily successful group. For example, mandible size in male stag beetles is exquisitely sensitive to the larval nutritional environment and is a reliable signal of male condition., Results: To date, studies of how such phenotypically plastic traits develop have focused on two types of mechanistic processes. Local, tissue-specific genetic mechanisms specify the shape and approximate final size of structures, whereas whole-animal hormonal signaling mechanisms modulate trait growth in response to environmental circumstance, including the body size and nutritional state of each individual. Hormones such as juvenile hormone, ecdysteroids, and/or ligands of the insulin-signaling pathway specify whether traits grow and regulate how much growth occurs across a diversity of insect groups. What remains to be shown is how the local, tissue-specific developmental genetic pathways interact with these whole animal hormonal signaling pathways during development to yield phenotypically plastic patterns of trait growth., Conclusions: Because the Fat/Hippo signaling pathway coordinates trait growth and development through its interactions with morphogens and hormonal pathways, we propose that Fat/Hippo signaling is a missing mechanistic link coordinating environmentally sensitive trait development in insects. Developmental Dynamics 244:1039-1045, 2015. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.) more...

Many male animals have evolved exaggerated traits that they use in combat with rival males to gain access to females and secure their reproductive success. But some male animals invest in nuptial gifts that gains them access to females. Both these reproductive strategies are costly in that resources are needed to produce the weapon or nuptial gift. In closely related species where both weapons and nuptial gifts are present, little is known about the potential evolutionary trade-off faced by males that have these traits. In this study, we use dobsonflies (order Megaloptera, family Corydalidae, subfamily Corydalinae) to examine the presence and absence of enlarged male weapons versus nuptial gifts within and among species. Many dobsonfly species are sexually dimorphic, and males possess extremely enlarged mandibles that they use in battles, whereas in other species, males produce large nuptial gifts that increase female fecundity. In our study, we show that male accessory gland size strongly correlates with nuptial gift size and that when male weapons are large, nuptial gifts are small and vice versa. We mapped weapons and nuptial gifts onto a phylogeny we constructed of 57 species of dobsonflies. Our among-species comparison shows that large nuptial gift production evolved in many species of dobsonfly but is absent from those with exaggerated weapons. This pattern supports the potential explanation that the trade-off in resource allocation between weapons and nuptial gifts is important in driving the diversity of male mating strategies seen in the dobsonflies, whereas reduced male-male competition in the species producing large spermatophores could be an alternative explanation on their loss of male weapons. Our results shed new light on the evolutionary interplay of multiple sexually selected traits in animals., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.) more...