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The diverse colors and patterns found in Lepidoptera are important for success of these species. Similar to the wings of adult butterflies, lepidopteran larvae exhibit diverse color variations to adapt to their habitats. Compared with butterfly wings, however, less attention has been paid to larval body colorations and patterns. In the present study, we focus on the yellow-y gene, which participates in the melanin synthesis pathway. We conducted CRISPR/Cas9-mediated targeted mutagenesis of yellow-y in the tobacco cutworm Spodoptera litura. We analyzed the role of S. litura yellow-y in pigmentation by morphological observation and discovered that yellow-y is necessary for normal black pigmentation in S. litura. We also showed species- and tissue-specific requirements of yellow-y in pigmentation in comparison with those of Bombyx mori yellow-y mutants. Furthermore, we found that almost none of the yellow-y mutant embryos hatched unaided. We provide evidence that S. litura yellow-y has a novel important function in egg hatching, in addition to pigmentation. The present study will enable a greater understanding of the functions and diversification of the yellow-y gene in insects., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The functions of the Ionotropic Receptor (IR) family have been well studied in Drosophila melanogaster, but only limited information is available in Lepidoptera. Here, we conducted a large-scale genome-wide analysis of the IR gene repertoire in 13 moths and 16 butterflies. Combining a homology-based approach and manual efforts, totally 996 IR candidates are identified including 31 pseudogenes and 825 full-length sequences, representing the most current comprehensive annotation in lepidopteran species. The phylogeny, expression and sequence characteristics classify Lepidoptera IRs into three sub-families: antennal IRs (A-IRs), divergent IRs (D-IRs) and Lepidoptera-specific IRs (LS-IRs), which is distinct from the case of Drosophila IRs. In comparison to LS-IRs and D-IRs, A-IRs members share a higher degree of protein identity and are distinguished into 16 orthologous groups in the phylogeny, showing conservation of gene structure. Analysis of selective forces on 27 orthologous groups reveals that these lepidopteran IRs have evolved under strong purifying selection (dN/dS≪1). Most notably, lineage-specific gene duplications that contribute primarily to gene number variations across Lepidoptera not only exist in D-IRs, but are present in the two other sub-families including members of IR41a, 76b, 87a, 100a and 100b. Expression profiling analysis reveals that over 80% (21/26) of Helicoverpa armigera A-IRs are expressed more highly in antennae of adults or larvae than other tissues, consistent with its proposed function in olfaction. However, some are also detected in taste organs like proboscises and legs. These results suggest that some A-IRs in H. armigera likely bear a dual function with their involvement in olfaction and gustation. Results from mating experiments show that two HarmIRs (IR1.2 and IR75d) expression is significantly up-regulated in antennae of mated female moths. However, no expression difference is observed between unmated female and male adults, suggesting an association with female host-searching behaviors. Our current study has greatly extended the IR gene repertoire resource in Lepidoptera, and more importantly, identifies potential IR candidates for olfactory, gustatory and oviposition behaviors in the cotton bollworm., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Plant volatiles are vital cues in the location of hosts for feeding and oviposition for Lepidoptera moths. The noctuid Helicoverpa assulta is a typical polyphagous moth, regarded as a good model for studying the olfactory reception of plant volatiles. In this study, four full-length genes encoding odorant receptors HassOR24, HassOR40, HassOR41, and HassOR55 expressed in antenna in H. assulta were functionally characterized. The highly expressed HassOR40 was narrowly tuned to a few structurally-related plant volatiles: geranyl acetate, geraniol and nerolidol. By systematically analyzing responses of single neuron in both trichoid sensilla and basiconic sensilla using single sensillum recording, the specific neuron B in one type of short trichoid sensilla was found to be mainly activated by the same chemicals as HassOR40 with high sensitivity, and with no significant difference between male and female neurons. Thus, a clear "receptor-neuron" relationship in H. assulta was demonstrated here, suggesting that HassOR40/HassOrco are expressed in neuron B of short trichoid sensilla. The active tobacco volatile nerolidol, recognized by this receptor-neuron line, elicits significant behavioral attraction of both sexes in H. assulta adults. The results indicate that we identified a receptor-neuron route for the peripheral coding of a behaviorally relevant host volatile in H. assulta., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The silks of silkworm and waxworm contain abundant antimicrobial proteins, including protease inhibitors and seroins. Protease inhibitors have antifungal activities, whereas seroins have antiviral and antibacterial activities. In order to obtain insights into the structure, evolution, and expression of seroins, we performed an extensive survey based on the available genome, transcriptome, and expressed sequence tags datasets. Sixty-four seroins were identified in 32 lepidopteran species. The phylogenetic and structural analyses revealed that seroins can be classified into five subfamilies: seroin 1, seroin 2, seroin 3, seroin 2 + 1, and seroin 3 + 3. It is interesting that seroin 2 + 1 contains two tandem seroin domains, seroin 2 and seroin 1, whereas seroin 3 + 3 has two tandem seroin 3 domains. Each seroin domain contains a proline-rich N-terminal motif and a conserved C-terminal motif. The transcriptome and EST data indicated that seroin 1 and seroin 2 genes were expressed in the silk gland but seroin 3 genes were not. Semi-quantitative RT-PCR and western blot analyses suggested that seroin 1 and seroin 2 were constantly accumulated in the silk gland of silkworm during the fifth instar, and then secreted into cocoon silk during spinning. Immunofluorescence analyses indicated that seroin 1 was secreted into the fibroin and sericin layers, whereas seroin 2 protein was only secreted into the sericin layer. However, the antimicrobial activity of seroin 2 was more effective than that of seroin 1. The presence of seroin 1 in the fibroin layer suggested that this protein not only acts as an antimicrobial protein, but might also play a role in the assembly and secretion of fibroins. Seroin 3, which was first identified here, might be related to pheromone synthesis or recognition, as it was highly expressed in male antennae and in the pheromone gland., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Genome editing using transcription activator-like effector nuclease (TALEN) has been applied for various model organisms but not yet for agricultural pest insects. In this study, TALEN-mediated mutagenesis of the gene encoding odorant receptor co-receptor (Orco) of an important agricultural pest Ostrinia furnacalis (OfurOrco) was carried out. Of the two pairs of TALEN constructs designed, one generated somatic and germline mutations at rates of 70.8% and 20.8%, respectively. Physiological and behavioral analyses using a gas chromatograph-electroantennographic detector system and a wind tunnel, respectively, revealed that antennal responses to sex pheromone components were decreased to trace levels, and behavioral responses were abolished in OfurOrco mutants. This study demonstrated that TALEN-mediated mutagenesis is applicable to pest insects, and these results will open the way for a better understanding of chemosensory systems in wild insects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with the allylic diphosphate primer dimethylallyl diphosphate (DMAPP; C5) to generate the C15 prenyl chain (FPP) used for protein prenylation as well as sterol and terpene biosynthesis. Here, we designed and prepared a series of pyridinium bisphosphonate (PyrBP) compounds, with the aim of selectively inhibiting FPPS of the lepidopteran insect order. FPPSs of Drosophila melanogaster and the spruce budworm, Choristoneura fumiferana, were inhibited by several PyrBPs, and as hypothesized, larger bisphosphonates were more selective for the lepidopteran protein and completely inactive towards dipteran and vertebrate FPPSs. Cell growth of a D. melanogaster cell line was adversely affected by exposure to PyrPBs that were strongly inhibitory to insect FPPS, although their effect was less pronounced than that observed upon exposure to the electron transport disrupter, chlorfenapyr. To assess the impact of PyrBPs on lepidopteran insect growth and development, we performed feeding and topical studies, using the tobacco hornworm, Manduca sexta, as our insect model. The free acid form of a PyrBP and a known bisphosphonate inhibitor of vertebrate FPPS, alendronate, had little to no effect on larval M. sexta; however, the topical application of more lipophilic ester PyrBPs caused decreased growth, incomplete larval molting, cuticle darkening at the site of application, and for those insects that survived, the formation of larval-pupal hybrids. To gain a better understanding of the structural differences that produce selective lepidopteran FPPS inhibition, homology models of C. fumiferana and D. melanogaster FPPS (CfFPPS2, and DmFPPS) were prepared. Docking of substrates and PyrBPs demonstrates that differences at the -3 and -4 positions relative to the first aspartate rich motif (FARM) are important factors in the ability of the lepidopteran enzyme to produce homologous isoprenoid structure and to be selectively inhibited by larger PyrBPs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Hexamerins are large hemolymph-proteins that accumulate during the late larval stages of insects. Hexamerins have emerged from hemocyanin, but have lost the ability to bind oxygen. Hexamerins are mainly considered as storage proteins for non-feeding stages, but may also have other functions, e.g. in cuticle formation, transport and immune response. The genome of the hornworm Manduca sexta harbors six hexamerin genes. Two of them code for arylphorins (Msex2.01690, Msex2.15504) and two genes correspond to a methionine-rich hexamerin (Msex2.10735) and a moderately methionine-rich hexamerin (Msex2.01694), respectively. Two other genes do not correspond to any known hexamerin and distantly resemble the arylphorins (Msex2.01691, Msex2.01693). Five of the six hexamerin genes are clustered within ∼45 kb on scaffold 00023, which shows conserved synteny in various lepidopteran genomes. The methionine-rich hexamerin gene is located at a distinct site. M. sexta and other Lepidoptera have lost the riboflavin-binding hexamerin. With the exception of Msex2.01691, which displays low mRNA levels throughout the life cycle, all hexamerins are most highly expressed during pre-wandering phase of the 5th larval instar of M. sexta, supporting their role as storage proteins. Notably, Msex2.01691 is most highly expressed in the brain, suggesting a divergent function. Phylogenetic analyses showed that hexamerin evolution basically follows insect systematics. Lepidoptera display an unparalleled diversity of hexamerins, which exceeds that of other hexapod orders. In contrast to previous analyses, the lepidopteran hexamerins were found monophyletic. Five distinct types of hexamerins have been identified in this order, which differ in terms of amino acid composition and evolutionary history: i. the arylphorins, which are rich in aromatic amino acids (∼20% phenylalanine and tyrosine), ii. the distantly related arylphorin-like hexamerins, iii. the methionine-rich hexamerins, iv. the moderately methionine rich hexamerins, and v. the riboflavin-binding hexamerins., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chorion proteins of Lepidoptera have a tripartite structure, which consists of a central domain and two, more variable, flanking arms. The central domain is highly conserved and it is used for the classification of chorion proteins into two major classes, A and B. Annotated and unreviewed Lepidopteran chorion protein sequences are available in various databases. A database, named LepChorionDB, was constructed by searching 5 different protein databases using class A and B central domain-specific profile Hidden Markov Models (pHMMs), developed in this work. A total of 413 Lepidopteran chorion proteins from 9 moths and 1 butterfly species were retrieved. These data were enriched and organised in order to populate LepChorionDB, the first relational database, available on the web, containing Lepidopteran chorion proteins grouped in A and B classes. LepChorionDB may provide insights in future functional and evolutionary studies of Lepidopteran chorion proteins and thus, it will be a useful tool for the Lepidopteran scientific community and Lepidopteran genome annotators, since it also provides access to the two pHMMs developed in this work, which may be used to discriminate A and B class chorion proteins. LepChorionDB is freely available at http://bioinformatics.biol.uoa.gr/LepChorionDB., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Silk secreted by the larvae of Hydropsyche angustipennis (Trichoptera) contains serpins HaSerp2A and HaSerp2B that are homologous to serpin 2 known from several lepidopterans and some other insects. The gene HaSerp2A is 2684 bp downstream from the HaSerp2B gene. The genes possess identical exon/intron segmentation (9 exons) and their sequences are nearly identical: only 8 out of 1203 nt differ in the coding region, 4 out of 567 nt in the introns and 2 out of 52 nt in 3' UTR. Both genes are highly expressed in the silk glands whereas expression in larval carcass devoid of the silk glands is hard to detect. Translation products of the genes consist of 401 amino acids, are 98.8% identical, and are secreted as 45 kDa proteins into silk. Homologous genes in similar tandem arrangement occur on chromosome 15 of Bombyx mori (Lepidoptera). The upstream gene BmSerp2B is modified in several exons and does not seem to produce functional mRNA. The gene BmSerp2A contains two copies of exon 9, of which only the second one is used. One kind of mRNA does and the other does not include exon 1, which encodes a signal peptide. The mRNA yielding secreted BmSerp2A is expressed in the posterior, and that encoding the cytoplasmic BmSerp2A in the middle silk gland region; both kinds are strongly expressed in the anterior region. The data indicate that (1) A duplication of serpin 2 gene occurred either before Trichoptera and Lepidoptera diverged as separate orders or independently in early phylogeny of either order; (2) In the caddisfly H. angustipennis, both genes are expressed specifically in the silk glands and generate proteins deposited in the silk; (3) Only one gene seems to be functional in B. mori and is expressed in a cytoplasmic and in a secreted forms in diverse organs, including the silk glands., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Helicoverpa zea is one of the most costly insect pests of food and fiber crops throughout the Americas. Pyrethroid insecticides are widely applied for its control as they are effective and relatively inexpensive; however, resistance to pyrethroids threatens agricultural systems sustainability because alternative insecticides are often more expensive or less effective. Although pyrethroid resistance has been identified in this pest since 1990, the mechanisms of resistance have not yet been elucidated at the molecular level. Pyrethroids exert their toxicity by prolonging the open state of the voltage-gated sodium channel. Here we report the cDNA sequence of the H. zea sodium channel alpha-subunit homologous to the para gene from Drosophila melanogaster. In field-collected males which were resistant to cypermethrin as determined by the adult vial test, we identify known resistance-conferring mutations L1029H and V421M, along with two novel mutations at the V421 residue, V421A and V421G. An additional mutation, I951V, may be the first example of a pyrethroid resistance mutation caused by RNA editing. Identification of the sodium channel cDNA sequence will allow for testing hypotheses on target-site resistance for insecticides acting on this channel through modeling and expression studies. Understanding the mechanisms responsible for resistance will greatly improve our ability to identify and predict resistance, as well as preserve susceptibility to pyrethroid insecticides., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth (Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.

Male hilarine flies (Diptera: Empididae: Empidinae) present prospective mates with silk-wrapped gifts. The silk is produced by specialised cells located in the foreleg basitarsus of the fly. In this report, we describe 2.3 kbp of the silk gene from a hilarine fly (Hilara spp.) that was identified from highly expressed mRNA extracted from the prothoracic basitarsus of males. Using specific primers, we found that the silk gene is expressed in the basitarsi and not in any other part of the male fly. The silk gene from the basitarsi cDNA library matched an approximately 220 kDa protein from the silk-producing basitarsus. Although the predicted silk protein sequence was unlike any other protein sequence in available databases, the architecture and composition of the predicted protein had features in common with previously described silks. The convergent evolution of these features in the Hilarini silk and other silks emphasises their importance in the functional requirements of silk proteins.

The non-steroidal ecdysone agonist, RH-5992, exhibits ecdysteroid activities in vivo as well as in vitro more effectively than 20-hydroxyecdysone (20E). Using the IAL-PID2 cells derived from imaginal wing discs of last larval instar of Plodia interpunctella, we investigated the action of RH-5992 in the control of cell growth. Its effects on the proliferative activity of IAL-PID2 cells, the induction level in G2/M arrest and on the expression rate of Plodia B cyclin (PcycB), ecdysone B1-isoform (PIEcR-B1) and Ultraspiracle-2 isoform (PIUSP-2) were examined. From these cellular and molecular assays, our results brought evidence that RH-5992, like 20E, induced an inhibition on cell proliferation by blocking IAL-PID2 cells in G2/M phase. Moreover, this G2/M arrest was preceded by a decrease in the expression level of PcycB and a high induction of PIEcR-B1, PIUSP-2 mRNAs. Dose-response experiments revealed that RH-5992 was even more potent than 20E. On these parameters, we therefore suggest that the differential observed in the expression level of USP and EcR by RH-5992 and 20E could contribute to the difference observed for the biological potency of these two compounds.

Evolution of resistance by pests could cut short the success of transgenic plants producing toxins from Bacillus thuringiensis, such as Bt cotton. The most common mechanism of insect resistance to B. thuringiensis is reduced binding of toxins to target sites in the brush border membrane of the larval midgut. We compared toxin binding in resistant and susceptible strains of Pectinophora gossypiella, a major pest of cotton worldwide. Using Cry1Ab and Cry1Ac labeled with (125)I and brush border membrane vesicles (BBMV), competition experiments were performed with unlabeled Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Ja, Cry2Aa, and Cry9Ca. In the susceptible strain, Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ja bound to a common binding site that was not shared by the other toxins tested. Reciprocal competition experiments with Cry1Ab, Cry1Ac, and Cry1Ja showed that these toxins do not bind to any additional binding sites. In the resistant strain, binding of (125)I-Cry1Ac was not significantly affected; however, (125)I-Cry1Ab did not bind to the BBMV. This result, along with previous data from this strain, shows that the resistance fits the "mode 1" pattern of resistance described previously in Plutella xylostella, Plodia interpunctella, and Heliothis virescens.

The insect midgut is generally lined with a unique protective chitin/protein structure, the peritrophic membrane (PM). We demonstrated that in Trichoplusia ni larvae, the majority of PM proteins were assembled with chitin as a consequence of their chitin binding properties. These proteins could be dissociated from the PM in vitro by Calcofluor, a well-known chemical with chitin binding properties. The chitin binding characteristics of PM proteins were confirmed by their high affinity binding in vitro to regenerated chitin. In vivo assays demonstrated that Calcofluor could inhibit PM formation in five lepidopteran insects tested. The inhibition of T. ni PM formation by Calcofluor, was accompanied by increased larval susceptibility to baculovirus infection. Continuous inhibition of PM formation by Calcofluor resulted in retarded larval development and mortality. The destructive effect of Calcofluor on PM formation was demonstrated to be transient and reversible depending on the presence of Calcofluor within the midgut. In addition, degradation of the insect intestinal mucin was observed concurrently with the inhibition of PM formation by Calcofluor. Our studies revealed a potential novel approach to develop strategies for insect control by utilizing chitin binding molecules to specifically target PM formation in a broad range of insect pest species.