Evolution of horn length and lifting strength in the Japanese rhinoceros beetle Trypoxylus dichotomus.

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.

Cell Cycle Progression Determines Wing Morph in the Polyphenic Insect Nilaparvata lugens.

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.

The activin signaling transcription factor Smox is an essential regulator of appendage size during regeneration after autotomy in the crayfish.

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 ß (TGF-ß) 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-ß 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.

Rhinoceros beetle horn development reveals deep parallels with dung beetles.

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.

Host quality induces phenotypic plasticity in a wing polyphenic insect.

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.

Endocrine regulation of a dispersal polymorphism in winged insects: a short review.

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.

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae.

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.

Ecological Trade-offs between Migration and Reproduction Are Mediated by the Nutrition-Sensitive Insulin-Signaling Pathway.

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.

JNK signaling mediates wing form polymorphism in brown planthoppers (Nilaparvata lugens).

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.

FOXO links wing form polyphenism and wound healing in the brown planthopper, Nilaparvata lugens.

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.

Transcriptome-based identification of ABC transporters in the western tarnished plant bug Lygus hesperus.

ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate diverse physiological functions by coupling ATP hydrolysis with substrate transport across lipid membranes. In insects, these proteins play roles in metabolism, development, eye pigmentation, and xenobiotic clearance. While ABC transporters have been extensively studied in vertebrates, less is known concerning this superfamily in insects, particularly hemipteran pests. We used RNA-Seq transcriptome sequencing to identify 65 putative ABC transporter sequences (including 36 full-length sequences) from the eight ABC subfamilies in the western tarnished plant bug (Lygus hesperus), a polyphagous agricultural pest. Phylogenetic analyses revealed clear orthologous relationships with ABC transporters linked to insecticide/xenobiotic clearance and indicated lineage specific expansion of the L. hesperus ABCG and ABCH subfamilies. The transcriptional profile of 13 LhABCs representative of the ABCA, ABCB, ABCC, ABCG, and ABCH subfamilies was examined across L. hesperus development and within sex-specific adult tissues. All of the transcripts were amplified from both reproductively immature and mature adults and all but LhABCA8 were expressed to some degree in eggs. Expression of LhABCA8 was spatially localized to the testis and temporally timed with male reproductive development, suggesting a potential role in sexual maturation and/or spermatozoa protection. Elevated expression of LhABCC5 in Malpighian tubules suggests a possible role in xenobiotic clearance. Our results provide the first transcriptome-wide analysis of ABC transporters in an agriculturally important hemipteran pest and, because ABC transporters are known to be important mediators of insecticidal resistance, will provide the basis for future biochemical and toxicological studies on the role of this protein family in insecticide resistance in Lygus species.

Identification of an alternative knockdown resistance (kdr)-like mutation, M918L, and a novel mutation, V1010A, in the Thrips tabaci voltage-gated sodium channel gene.

BACKGROUND: Knockdown resistance (kdr) has been identified as a main mechanism against pyrethroid insecticides in many arthropod pests including in the onion thrips, Thrips tabaci. To characterize and identify pyrethroid-resistance in onion thrips in Washington state, we conducted insecticide bioassays and sequenced a region of the voltage gated sodium channel gene from several different T. tabaci populations. RESULTS: Field collected Thrips tabaci were found to have large variations in resistance to the pyrethroid insecticide lambda-cyhalothrin. We identified two single nucleotide substitutions in our analysis of a partial sequence of the T. tabaci voltage-gated sodium channel gene. One mutation resulted in the non-synonymous substitution of methionine with leucine (M918L), which is well known to be responsible for super knockdown resistance in some pest species. Another non-synonymous substitution, a valine (GTT) to alanine (GCT) replacement at amino acid 1010 (V1010A) was identified in our study and was associated with lambda-cyhalothrin resistance. CONCLUSION: We have characterized a known kdr mutation and identified a novel mutation in the voltage-gated sodium channel gene of Thrips tabaci associated with resistance to lambda-cyhalothrin. This gene region and these mutations are expected to be useful in the development of a diagnostic test to detect kdr resistance in many onion thrips populations.

Soldier morphogenesis in the damp-wood termite is regulated by the insulin signaling pathway.

Eusocial insects exhibit various morphological castes associated with the division of labor within a colony. Termite soldiers possess defensive traits including mandibles that are greatly exaggerated and enlarged, as compared to termite reproductives and workers. The enlarged mandibles of soldiers are known to result from dynamic morphogenesis during soldier differentiation that can be induced by juvenile hormone and its analogs. However, the detailed developmental mechanisms still remain unresolved. Because the insulin/insulin-like growth factor signaling (IIS) pathway has been shown to regulate the relative sizes of organs (i.e., allometry) in other insects, we examined the expression profiles of major IIS factors in the damp-wood termite Hodotermopsis sjostedti, during soldier differentiation. The relative expression patterns of orthologs for termite InR (HsjInR), PKB/Akt (HsjPKB/Akt), and FOXO (HsjFOXO) suggest that HsjInR and HsjPKB/Akt were up-regulated in the period of elongation of mandibles during soldier development. In situ hybridization showed that HsjInR was strongly expressed in the mandibular epithelial tissues, and RNA interference (RNAi) for HsjInR disrupted soldier-specific morphogenesis including mandibular elongation. These results suggest that signaling through the IIS pathway is required for soldier-specific morphogenesis. In addition, up-regulation of the IIS pathway in other body tissues occurred at earlier stages of development, indicating that there is tissue-specific IIS regulation. Because the IIS pathway is generally thought to act upstream of JH in insects, our results suggest the damp-wood termite may have evolved a novel feedback loop between JH and IIS that enables social interactions, rather than nutrition, to regulate caste determination.

Cloning and characterization of an mRNA encoding an insulin receptor from the horned scarab beetle Onthophagus nigriventris (Coleoptera: Scarabaeidae).

The insulin signaling pathway is the primary signaling pathway coupling growth with nutritional condition in all animals. Sensitivity to circulating levels of insulin has been shown to regulate the growth of specific traits in a dose-dependent manner in response to environmental conditions in a diversity of insect species. Alternative phenotypes in insects manifest in a variety of morphologies such as the sexually dimorphic and male dimorphic horned beetles. Large males of the sexually dimorphic dung beetle Onthophagus nigriventris develop a thoracic horn up to twice the length of the body whereas small males and females never develop this horn. The regulation of this dimorphism is known to be nutrition dependent for males. We focused on the insulin signaling pathway as a potential regulator of this dimorphism. We sequenced a full-length gene transcript encoding the O. nigriventris insulin receptor (OnInR), which is the receptor for circulating insulin and insulin-like peptides in animals. We show that the predicted OnInR protein is similar in overall amino acid identity to other insulin receptors (InRs) and is most closely related phylogenetically to insect InRs. Expression of the OnInR transcript was found during development of imaginal tissues in both males and females. However, expression of OnInR in the region where a horn would grow of small males and female was significantly higher than in the horn tissues of large males at the end of growth. This variation in OnInR expression between sexes and morphs indicates a role for the InR in polymorphic horn development.

A mechanism of extreme growth and reliable signaling in sexually selected ornaments and weapons.

Many male animals wield ornaments or weapons of exaggerated proportions. We propose that increased cellular sensitivity to signaling through the insulin/insulin-like growth factor (IGF) pathway may be responsible for the extreme growth of these structures. We document how rhinoceros beetle horns, a sexually selected weapon, are more sensitive to nutrition and more responsive to perturbation of the insulin/IGF pathway than other body structures. We then illustrate how enhanced sensitivity to insulin/IGF signaling in a growing ornament or weapon would cause heightened condition sensitivity and increased variability in expression among individuals--critical properties of reliable signals of male quality. The possibility that reliable signaling arises as a by-product of the growth mechanism may explain why trait exaggeration has evolved so many different times in the context of sexual selection.

Flight behavior of the rhinoceros beetle Trypoxylus dichotomus during electrical nerve stimulation.

Neuronal stimulation is an intricate part of understanding insect flight behavior and control insect itself. In this study, we investigated the effects of electrical pulses applied to the brain and basalar muscle of the rhinoceros beetle (Trypoxylus dichotomus). To understand specific neuronal stimulation mechanisms, responses and flight behavior of the beetle, four electrodes were implanted into the two optic lobes, the brain's central complex and the ventral nerve cord in the posterior pronotum. We demonstrated flight initiation, turning and cessation by stimulating the brain. The change undergone by the wing flapping in response to the electrical signal was analyzed from a sequence of images captured by a high-speed camera. Here, we provide evidence to distinguish the important differences between neuronal and muscular flight stimulations in beetles. We found that in the neural potential stimulation, both the hind wing and the elytron were suppressed. Interestingly, the beetle stopped flying whenever a stimulus potential was applied between the pronotum and one side of the optic lobe, or between the ventral nerve cord in the posterior pronotum and the central complex. In-depth experimentation demonstrated the effective of neural stimulation over muscle stimulation for flight control. During electrical stimulation of the optic lobes, the beetle performed unstable flight, resulting in alternating left and right turns. By applying the electrical signal into both the optic lobes and the central complex of the brain, we could precisely control the direction of the beetle flight. This work provides an insight into insect flight behavior for future development of insect-micro air vehicle.

Identification of Lygus hesperus by DNA barcoding reveals insignificant levels of genetic structure among distant and habitat diverse populations.

BACKGROUND: The western tarnished plant bug Lygus hesperus is an economically important pest that belongs to a complex of morphologically similar species that makes identification problematic. The present study provides evidence for the use of DNA barcodes from populations of L. hesperus from the western United States of America for accurate identification. METHODOLOGY/PRINCIPAL FINDINGS: This study reports DNA barcodes for 134 individuals of the western tarnished plant bug from alfalfa and strawberry agricultural fields in the western United States of America. Sequence divergence estimates of <3% reveal that morphologically variable individuals presumed to be L. hesperus were accurately identified. Paired estimates of F(st) and subsequent estimates of gene flow show that geographically distinct populations of L. hesperus are genetically similar. Therefore, our results support and reinforce the relatively recent (<100 years) migration of the western tarnished plant bug into agricultural habitats across the western United States. CONCLUSIONS/SIGNIFICANCE: This study reveals that despite wide host plant usage and phenotypically plastic morphological traits, the commonly recognized western tarnished plant bug belongs to a single species, Lygus hesperus. In addition, no significant genetic structure was found for the geographically diverse populations of western tarnished plant bug used in this study.

Juvenile hormone regulates extreme mandible growth in male stag beetles.

The morphological diversity of insects is one of the most striking phenomena in biology. Evolutionary modifications to the relative sizes of body parts, including the evolution of traits with exaggerated proportions, are responsible for a vast range of body forms. Remarkable examples of an insect trait with exaggerated proportions are the mandibular weapons of stag beetles. Male stag beetles possess extremely enlarged mandibles which they use in combat with rival males over females. As with other sexually selected traits, stag beetle mandibles vary widely in size among males, and this variable growth results from differential larval nutrition. However, the mechanisms responsible for coupling nutrition with growth of stag beetle mandibles (or indeed any insect structure) remain largely unknown. Here, we demonstrate that during the development of male stag beetles (Cyclommatus metallifer), juvenile hormone (JH) titers are correlated with the extreme growth of an exaggerated weapon of sexual selection. We then investigate the putative role of JH in the development of the nutritionally-dependent, phenotypically plastic mandibles, by increasing hemolymph titers of JH with application of the JH analog fenoxycarb during larval and prepupal developmental periods. Increased JH signaling during the early prepupal period increased the proportional size of body parts, and this was especially pronounced in male mandibles, enhancing the exaggerated size of this trait. The direction of this response is consistent with the measured JH titers during this same period. Combined, our results support a role for JH in the nutrition-dependent regulation of extreme mandible growth in this species. In addition, they illuminate mechanisms underlying the evolution of trait proportion, the most salient feature of the evolutionary diversification of the insects.

Misdiagnosis of spider bites: bacterial associates, mechanical pathogen transfer, and hemolytic potential of venom from the hobo spider, Tegenaria agrestis (Araneae: Agelenidae).

The European spider Tegenaria agrestis (Walckenaer) (hobo spider) has been implicated as a spider of medical importance in the Pacific Northwest since its introduction in the late 1980s. Studies have indicated that the hobo spider causes necrotic tissue lesions through hemolytic venom or through the transfer of pathogenic bacteria introduced by its bite. Bacterial infections are often diagnosed as spider bites, in particular the pathogenic bacteria methicillin-resistant Staphylococcus aureus (MRSA). This study examines three aspects of the potential medical importance of hobo spiders in part of its introduced range, Washington State. First, the bacterial diversity of the spider was surveyed using a polymerase chain reaction-based assay to determine whether the spider carries any pathogenic bacteria. Second, an experiment was conducted to determine the ability of the spiders to transfer MRSA. Third, the venom was evaluated to assess the hemolytic activity. We found 10 genera of ubiquitous bacteria on the exterior surface of the spiders. In addition, none of the spiders exposed to MRSA transferred this pathogen. Finally, the hemolytic venom assay corroborates previous studies that found hobo spider venom was not deleterious to vertebrate red blood cells.