New genes often acquire male-specific functions but rarely become essential in Drosophila.

Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, our phylogenomic analyses reveal that young Drosophila genes are frequently restricted to the nonessential male reproductive system. We performed large-scale CRISPR/Cas9 mutagenesis of "conserved, essential" and "young, RNAi-lethal" genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, we detected widespread signatures of positive selection on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function.

Low affinity binding sites in an activating CRM mediate negative autoregulation of the Drosophila Hox gene Ultrabithorax.

Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that decreases Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.

Transcriptional profiling and physiological roles of Aedes aegypti spermathecal-related genes.

BACKGROUND: Successful mating of female mosquitoes typically occurs once, with the male sperm being stored in the female spermatheca for every subsequent oviposition event. The female spermatheca is responsible for the maintenance, nourishment, and protection of the male sperm against damage during storage. Aedes aegypti is a major vector of arboviruses, including Yellow Fever, Dengue, Chikungunya, and Zika. Vector control is difficult due to this mosquito high reproductive capacity. RESULTS: Following comparative RNA-seq analyses of spermathecae obtained from virgin and inseminated females, eight transcripts were selected based on their putative roles in sperm maintenance and survival, including energy metabolism, chitin components, transcriptional regulation, hormonal signaling, enzymatic activity, antimicrobial activity, and ionic homeostasis. In situ RNA hybridization confirmed tissue-specific expression of the eight transcripts. Following RNA interference (RNAi), observed outcomes varied between targeted transcripts, affecting mosquito survival, egg morphology, fecundity, and sperm motility within the spermathecae. CONCLUSIONS: This study identified spermatheca-specific transcripts associated with sperm storage in Ae. aegypti. Using RNAi we characterized the role of eight spermathecal transcripts on various aspects of female fecundity and offspring survival. RNAi-induced knockdown of transcript AeSigP-66,427, coding for a Na+/Ca2+ protein exchanger, specifically interfered with egg production and reduced sperm motility. Our results bring new insights into the molecular basis of sperm storage and identify potential targets for Ae. aegypti control.

Intracellular dynamics of polydnavirus innexin homologues.

Polydnaviruses associated with ichneumonid parasitoid wasps (Ichnoviruses) encode large numbers of genes, often in multigene families. The Ichnovirus Vinnexin gene family, which is expressed in parasitized lepidopteran larvae, encodes homologues of Innexins, the structural components of insect gap junctions. Here, we have examined intracellular behaviours of the Campoletis sonorensis Ichnovirus (CsIV) Vinnexins, alone and in combination with a host Innexin orthologue, Innexin2 (Inx2). QRT-PCR verified that transcription of CsIV vinnexins occurs contemporaneously with inx2, implying co-occurrence of Vinnexin and Inx2 proteins. Confocal microscopy demonstrated that epitope-tagged VinnexinG (VnxG) and VinnexinQ2 (VnxQ2) exhibit similar subcellular localization as Spodoptera frugiperda Inx2 (Sf-Inx2). Surface biotinylation assays verified that all three proteins localize to the cell surface, and cytochalasin B and nocodazole that they rely on actin and microtubule cytoskeletal networks for localization. Immunomicroscopy following co-transfection of constructs indicates extensive co-localization of Vinnexins with each other and Sf-Inx2, and live-cell imaging of mCherry-labelled Inx2 supports that Vinnexins may affect Sf-Inx2 distribution in a Vinnexin-specific fashion. Our findings support that the Vinnexins may disrupt host cell physiology in a protein-specific manner through altering gap junctional intercellular channel communication, as well as indirectly by affecting multicellular junction characteristics.

Steroid signaling in mature follicles is important for Drosophila ovulation.

Although ecdysteroid signaling regulates multiple steps in oogenesis, it is not known whether it regulates Drosophila ovulation, a process involving a matrix metalloproteinase-dependent follicle rupture. In this study, we demonstrated that ecdysteroid signaling is operating in mature follicle cells to control ovulation. Moreover, knocking down shade (shd), encoding the monooxygenase that converts ecdysone (E) to the more active 20-hydroxyecdysone (20E), specifically in mature follicle cells, blocked follicle rupture, which was rescued by ectopic expression of shd or exogenous 20E. In addition, disruption of the Ecdysone receptor (EcR) in mature follicle cells mimicked shd-knockdown defects, which were reversed by ectopic expression of EcR.B2 but not by EcR.A or EcR.B1 isoforms. Furthermore, we showed that ecdysteroid signaling is essential for the proper activation of matrix metalloproteinase 2 (Mmp2) for follicle rupture. Our data strongly suggest that 20E produced in follicle cells before ovulation activates EcR.B2 to prime mature follicles to be responsive to neuronal ovulatory stimuli, thus providing mechanistic insights into steroid signaling in Drosophila ovulation.

Isolation of AccGalectin1 from Apis cerana cerana and its functions in development and adverse stress response.

The galectins are a family of lectins that play important roles in development, immunity, and the regulation of cellular responses. Much research has focused on the functions of galectins in mammals, though less in insects. Here, we identified the AccGalectin1 gene in Apis cerana cerana for the first time and explored its functions. The open reading frame of AccGalectin1 is 1449 base pairs and encodes a 482-amino-acid protein. AccGalectin1 expression was high during the transition between developmental stages and was high in the head, thorax, and epidermis compared with its levels in other tissues. In addition, the expression of AccGalectin1 was induced by several adverse stresses, including both abiotic and biotic stresses. A disk diffusion assay of recombinant AccGalectin1 protein revealed possible roles in protecting cells from oxidative stress. Furthermore, the expression levels of multiple oxidative genes (AccCAT, AccTpx1, AccTrx2, etc) were increased after AccGalectin1 was knocked down in Apis cerana cerana using RNA interference. We also observed that the malondialdehyde content in the AccGalectin1-silenced bees was higher than that in the control bees, while the antioxidant enzymatic activities of superoxide dismutase and peroxidase were lower. Considering these results, we suggest that AccGalectin1 may be indispensable for protecting honeybees from biotic and abiotic damage by participating in the oxidative resistance response and the immune response. These results may provide insight into the precise functions of galectins in mammals and other insects.

Modular cis-regulatory logic of yellow gene expression in silkmoth larvae.

Colour patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (ie yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remain poorly understood. To elucidate this, molecular studies involving dipterans have largely analysed the cis-regulatory regions of pigmentation genes and have revealed cis-regulatory modularity. Here, we used well-developed transgenic techniques in Bombyx mori and demonstrated that cis-regulatory modularity controls tissue-specific expression of the yellow gene. We first identified which body parts are regulated by the yellow gene via black pigmentation. We then isolated three discrete regulatory elements driving tissue-specific gene expression in three regions of B. mori larvae. Finally, we found that there is no apparent sequence conservation of cis-regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans-regulatory landscapes of the yellow gene differ significantly between the two taxa. The results of this study confirm that lepidopteran species use cis-regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms.

Silkworm storage protein Bm30K-19G1 has a certain antifungal effects on Beauveria bassiana.

Storage proteins in the 30 K family are ubiquitous in the hemolymph of insects and play important roles in adult metamorphosis, development, egg formation, carrier transport and even host immunity. Some studies have shown that the 30 K proteins can inhibit apoptosis and have certain antifungal effects. The silkworm protein Bm30K-19G1 is a low molecular weight apolipoprotein that is abundant in hemolymph of fifth instar larvae. Our previous transcriptome sequencing, real-time PCR analysis and proteomic studies showed that the expression level of the 30 K protein gene was significantly up-regulated in the silkworm infected with Beauveria bassiana. In this study, the ORF sequence of Bm30K-19G1 was amplified by PCR. The sequence is 1311 bp in length and encodes a 436 amino acid peptide. Bm30K-19G1 was expressed in all tested tissues of fifth instar larvae, with highest expression in fat body and the lowest expression in the midgut. Bm30K-19G1 protein was successfully expressed in the prokaryotic expression system using pET-28a(+) as vector and E. coli Arctic Express (DE3) as the expression bacterium strain. The expressed recombinant Bm30K-19G1 protein has an inhibitory effect on the conidial germination and hyphal growth of B. bassiana. Bm30K-19G1 also inhibited the growth and reproduction of B. bassiana in vivo; the median lethal time of infected silkworms was postponed by 6.4 h and the time for death of all infected larvae was postponed by 10 h. The results indicated that the silkworm storage protein 30K-19G1 is an antifungal protein against B. bassiana and help to elucidate the molecular mechanism of silkworm resistance against B. bassiana.

Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin.

When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to hexanoic acid but not to Cry3Aa toxin.

The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila.

Alternative pre-mRNA splicing (AS) is a critical regulatory mechanism that operates extensively in the nervous system to produce diverse protein isoforms. Fruitless AS isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, we show that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ∼1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.

Electrical interactions between photoreceptors in the compound eye of Periplaneta americana.

The compound eye of Periplaneta americana contains two spectral classes of photoreceptors: narrow-band UV-sensitive and broad-band green-sensitive. In intracellular recordings, stimulation of green-sensitive photoreceptors with flashes of relatively bright UV/violet light produced anomalous delayed depolarization after the end of the normal light response, whereas stimulation of UV-sensitive photoreceptors with green light elicited biphasic responses characterized by initial transient hyperpolarization followed by prolonged delayed depolarization. To explore the basis for these findings, we used RNA interference to selectively suppress expression of the genes encoding green opsin (GO1), UV opsin (UVO) or both. The hyperpolarizing component in UV-sensitive photoreceptors was eliminated and the delayed depolarization was reduced after GO1 knockdown, suggesting that the hyperpolarization represents fast inhibitory interactions between green- and UV-sensitive photoreceptors. Green-sensitive photoreceptor responses of GO1 knockdowns to flashes of UV/violet were almost exclusively biphasic, whereas residual responses to green had normal kinetics. Knockdown of UVO reduced the responses of UV-sensitive photoreceptors but had minor effects on delayed depolarization in green-sensitive photoreceptors. Angular sensitivity analysis indicated that delayed depolarization of green-sensitive photoreceptors by violet light originates from excitation of (an)other photoreceptor(s) in the same ommatidium. The angle at which the maximal delayed depolarization was observed in green-sensitive photoreceptors stimulated with violet light did not match the angle of the maximal transient depolarization. In contrast, no significant mismatch was observed for delayed depolarization elicited by green light. These results suggest that the cellular sources of the normal transient and additional delayed depolarization by violet light are separate and distinct.

Genetic architecture of natural variation in Drosophila melanogaster aggressive behavior.

Aggression is an evolutionarily conserved complex behavior essential for survival and the organization of social hierarchies. With the exception of genetic variants associated with bioamine signaling, which have been implicated in aggression in many species, the genetic basis of natural variation in aggression is largely unknown. Drosophila melanogaster is a favorable model system for exploring the genetic basis of natural variation in aggression. Here, we performed genome-wide association analyses using the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and replicate advanced intercross populations derived from the most and least aggressive DGRP lines. We identified genes that have been previously implicated in aggressive behavior as well as many novel loci, including gustatory receptor 63a (Gr63a), which encodes a subunit of the receptor for CO2, and genes associated with development and function of the nervous system. Although genes from the two association analyses were largely nonoverlapping, they mapped onto a genetic interaction network inferred from an analysis of pairwise epistasis in the DGRP. We used mutations and RNAi knock-down alleles to functionally validate 79% of the candidate genes and 75% of the candidate epistatic interactions tested. Epistasis for aggressive behavior causes cryptic genetic variation in the DGRP that is revealed by changing allele frequencies in the outbred populations derived from extreme DGRP lines. This phenomenon may pertain to other fitness traits and species, with implications for evolution, applied breeding, and human genetics.

Comparative Transcriptome Analyses Uncover Key Candidate Genes Mediating Flight Capacity in Bactrocera dorsalis (Hendel) and Bactrocera correcta (Bezzi) (Diptera: Tephritidae).

Flight capacity is important for invasive pests during entry, establishment and spreading. Both Bactroceradorsalis Hendel and Bactroceracorrecta Bezzi are invasive fruit flies but their flight capacities differ. Here, a tethered flight mill test demonstrated that B. dorsalis exhibits a greater flight capacity than B. correcta. RNA-Seq was used to determine the transcriptomic differences associated with the flight capacity of two Bactrocera species. Transcriptome data showed that 6392 unigenes were differentially expressed between the two species in the larval stage, whereas in the adult stage, 4104 differentially expressed genes (DEGs) were identified in females, and 3445 DEGs were observed in males. The flight capacity appeared to be correlated with changes in the transcriptional levels of genes involved in wing formation, flight muscle structure, energy metabolism, and hormonal control. Using RNA interference (RNAi) to verify the function of one DEG, the epidermal growth factor receptor (EGFR), we confirmed the role of this gene in regulating wing development, and thereby flight capacity, in both species. This work reveals the flight mechanism of fruit flies and provides insight into fundamental transcriptomics for further studies on the flight performance of insects.

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx.

The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing.

Plant-mediated RNAi silences midgut-expressed genes in congeneric lepidopteran insects in nature.

BACKGROUND: Plant-mediated RNAi (PMRi) silencing of insect genes has enormous potential for crop protection, but whether it works robustly under field conditions, particularly with lepidopteran pests, remains controversial. Wild tobacco Nicotiana attenuata and cultivated tobacco (N. tabacum) (Solanaceae) is attacked by two closely related specialist herbivores Manduca sexta and M. quinquemaculata (Lepidoptera, Sphingidae). When M. sexta larvae attack transgenic N. attenuata plants expressing double-stranded RNA(dsRNA) targeting M. sexta's midgut-expressed genes, the nicotine-ingestion induced cytochrome P450 monooxygenase (invert repeat (ir)CYP6B46-plants) and the lyciumoside-IV-ingestion induced ß-glucosidase1 (irBG1-plants), these larval genes which are important for the larvae's response to ingested host toxins, are strongly silenced. RESULTS: Here we show that the PMRi procedure also silences the homologous genes in native M. quinquemaculata larvae feeding on irCYP6B46 and irBG1-transgenic N. attenuata plants in nature. The PMRi lines shared 98 and 96% sequence similarity with M. quinquemaculata homologous coding sequences, and CYP6B46 and BG1 transcripts were reduced by ca. 90 and 80%, without reducing the transcripts of the larvae's most similar, potential off-target genes. CONCLUSIONS: We conclude that the PMRi procedure can robustly and specifically silence genes in native congeneric insects that share sufficient sequence similarity and with the careful selection of targets, might protect crops from attack by congeneric-groups of insect pests.

Comparative transcriptomics of convergent evolution: different genes but conserved pathways underlie caste phenotypes across lineages of eusocial insects.

An area of great interest in evolutionary genomics is whether convergently evolved traits are the result of convergent molecular mechanisms. The presence of queen and worker castes in insect societies is a spectacular example of convergent evolution and phenotypic plasticity. Multiple insect lineages have evolved environmentally induced alternative castes. Given multiple origins of eusociality in Hymenoptera (bees, ants, and wasps), it has been proposed that insect castes evolved from common genetic "toolkits" consisting of deeply conserved genes. Here, we combine data from previously published studies on fire ants and honey bees with new data for Polistes metricus paper wasps to assess the toolkit idea by presenting the first comparative transcriptome-wide analysis of caste determination among three major hymenopteran social lineages. Overall, we found few shared caste differentially expressed transcripts across the three social lineages. However, there is substantially more overlap at the levels of pathways and biological functions. Thus, there are shared elements but not on the level of specific genes. Instead, the toolkit appears to be relatively "loose," that is, different lineages show convergent molecular evolution involving similar metabolic pathways and molecular functions but not the exact same genes. Additionally, our paper wasp data do not support a complementary hypothesis that "novel" taxonomically restricted genes are related to caste differences.

Gene expression and localization analysis of Bombyx mori bidensovirus and its putative receptor in B. mori midgut.

Bombyx mori bidensovirus (BmBDV), which causes fatal flacherie disease in the silkworm, replicates only in midgut columnar cells. The viral resistance expressed by some silkworm strains, which is characterized as non-susceptibility irrespective of the viral dose, is determined by a single gene, nsd-2. We previously identified nsd-2 by positional cloning and found that this gene encodes a putative amino acid transporter that might function as a receptor for BmBDV. In this study, we investigated the relationship between the part of the midgut expressing nsd-2 (resistance gene), +(nsd-2) (susceptibility gene) and BmBDV propagation. Quantitative RT-PCR (qRT-PCR) analysis using total RNA isolated from the anterior, middle, and posterior parts of the midgut showed that nsd-2 and +(nsd-2) were strongly expressed in the posterior part of the midgut. The expression levels of both genes were very low in the anterior and middle parts. The qRT-PCR analysis showed that the expression levels of BmBDV-derived transcripts were correlated with the levels of +(nsd-2) expression. However, BmBDV-derived transcripts were clearly detected in all parts of the midgut. These results suggest that the infectivity of BmBDV depends mainly on the expression level of +(nsd-2) in the midgut and that viral infection is supported even by very faint expression of +(nsd-2). By contrast, the expression levels of +(nsd-2) were exceedingly low or undetectable in the middle part of the midgut, indicating that BmBDV infection might occur via another mechanism, independent of +(nsd-2), in the middle part of the midgut.

Autonomous regulation of the insect gut by circadian genes acting downstream of juvenile hormone signaling.

In temperate regions, the shortening day length informs many insect species to prepare for winter by inducing diapause. The adult diapause of the linden bug, Pyrrhocoris apterus, involves a reproductive arrest accompanied by energy storage, reduction of metabolic needs, and preparation to withstand low temperatures. By contrast, nondiapause animals direct nutrient energy to muscle activity and reproduction. The photoperiod-dependent switch from diapause to reproduction is systemically transmitted throughout the organism by juvenile hormone (JH). Here, we show that, at the organ-autonomous level of the insect gut, the decision between reproduction and diapause relies on an interaction between JH signaling and circadian clock genes acting independently of the daily cycle. The JH receptor Methoprene-tolerant and the circadian proteins Clock and Cycle are all required in the gut to activate the Par domain protein 1 gene during reproduction and to simultaneously suppress a mammalian-type cryptochrome 2 gene that promotes the diapause program. A nonperiodic, organ-autonomous feedback between Par domain protein 1 and Cryptochrome 2 then orchestrates expression of downstream genes that mark the diapause vs. reproductive states of the gut. These results show that hormonal signaling through Methoprene-tolerant and circadian proteins controls gut-specific gene activity that is independent of circadian oscillations but differs between reproductive and diapausing animals.

Insights into insect wing origin provided by functional analysis of vestigial in the red flour beetle, Tribolium castaneum.

Despite accumulating efforts to unveil the origin of insect wings, it remains one of the principal mysteries in evolution. Currently, there are two prominent models regarding insect wing origin: one connecting the origin to the paranotal lobe and the other to the proximodorsal leg branch (exite). However, neither hypothesis has been able to surpass the other. To approach this conundrum, we focused our analysis on vestigial (vg), a critical wing gene initially identified in Drosophila. Our investigation in Tribolium (Coleoptera) has revealed that, despite the well-accepted view of vg as an essential wing gene, there are two groups of vg-dependent tissues in the "wingless" first thoracic segment (T1). We show that one of these tissues, the carinated margin, also depends on other factors essential for wing development (such as Wingless signal and apterous), and has nubbin enhancer activity. In addition, our homeotic mutant analysis shows that wing transformation in T1 originates from both the carinated margin and the other vg-dependent tissue, the pleural structures (trochantin and epimeron). Intriguingly, these two tissues may actually be homologous to the two proposed wing origins (paranotal lobes and exite bearing proximal leg segments). Therefore, our findings suggest that the vg-dependent tissues in T1 could be wing serial homologs present in a more ancestral state, thus providing compelling functional evidence for the dual origin of insect wings.

Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance.

Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/ß-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcαE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10(6) M(-1) s(-1)) indicative of a natural substrate.