Comprehensive analysis of silk proteins and gland compartments in Limnephilus lunatus, a case-making trichopteran.

Caddisfly larvae produce silk containing heavy and light fibroins, similar to the silk of Lepidoptera, for the construction of underwater structures. We analyzed the silk of Limnephilus lunatus belonging to the case-forming suborder Integripalpia. We analyzed the transcriptome, mapped the transcripts to a reference genome and identified over 80 proteins using proteomic methods, and checked the specificity of their expression. For comparison, we also analyzed the transcriptome and silk proteome of Limnephilus flavicornis. Our results show that fibroins and adhesives are produced together in the middle and posterior parts of the silk glands, while the anterior part produces enzymes and an unknown protein AT24. The number of silk proteins of L. lunatus far exceeds that of the web-spinning Plectrocnemia conspersa, a previously described species from the suborder Annulipalpia. Our results support the idea of increasing the structural complexity of silk in rigid case builders compared to trap web builders.

Genome sequence and silkomics of the spindle ermine moth, Yponomeuta cagnagella, representing the early diverging lineage of the ditrysian Lepidoptera.

Many lepidopteran species produce silk, cocoons, feeding tubes, or nests for protection from predators and parasites for caterpillars and pupae. Yet, the number of lepidopteran species whose silk composition has been studied in detail is very small, because the genes encoding the major structural silk proteins tend to be large and repetitive, making their assembly and sequence analysis difficult. Here we have analyzed the silk of Yponomeuta cagnagella, which represents one of the early diverging lineages of the ditrysian Lepidoptera thus improving the coverage of the order. To obtain a comprehensive list of the Y. cagnagella silk genes, we sequenced and assembled a draft genome using Oxford Nanopore and Illumina technologies. We used a silk-gland transcriptome and a silk proteome to identify major silk components and verified the tissue specificity of expression of individual genes. A detailed annotation of the major genes and their putative products, including their complete sequences and exon-intron structures is provided. The morphology of silk glands and fibers are also shown. This study fills an important gap in our growing understanding of the structure, evolution, and function of silk genes and provides genomic resources for future studies of the chemical ecology of Yponomeuta species.

Mutation in Drosophila concentrative nucleoside transporter 1 alters spermatid maturation and mating behavior.

Concentrative nucleoside transporters (Cnts) are unidirectional carriers that mediate the energy-costly influx of nucleosides driven by the transmembrane sodium gradient. Cnts are transmembrane proteins that share a common structural organization and are found in all phyla. Although there have been studies on Cnts from a biochemical perspective, no deep research has examined their role at the organismal level. Here, we investigated the role of the Drosophila melanogaster cnt1 gene, which is specifically expressed in the testes. We used the CRISPR/Cas9 system to generate a mutation in the cnt1 gene. The cnt1 mutants exhibited defects in the duration of copulation and spermatid maturation, which significantly impaired male fertility. The most striking effect of the cnt1 mutation in spermatid maturation was an abnormal structure of the sperm tail, in which the formation of major and minor mitochondrial derivatives was disrupted. Our results demonstrate the importance of cnt1 in male fertility and suggest that the observed defects in mating behavior and spermatogenesis are due to alterations in nucleoside transport and associated metabolic pathways.

Using the multi-omics approach to reveal the silk composition in Plectrocnemia conspersa.

Similar to Lepidoptera, the larvae of Trichoptera are also capable of producing silk. Plectrocnemia conspersa, a predatory species belonging to the suborder Annulipalpia, builds massive silken retreats with preycapturing nets. In this study, we describe the silk glands of P. conspersa and use the multi-omics methods to obtain a complete picture of the fiber composition. A combination of silk gland-specific transcriptome and proteomic analyses of the spun-out fibers yielded 27 significant candidates whose full-length sequences and gene structures were retrieved from the publicly available genome database. About one-third of the candidates were completely novel proteins for which there are no described homologs, including a group of five pseudofibroins, proteins with a composition similar to fibroin heavy chain. The rest were homologs of lepidopteran silk proteins, although some had a larger number of paralogs. On the other hand, P. conspersa fibers lacked some proteins that are regular components in moth silk. In summary, the multi-omics approach provides an opportunity to compare the overall composition of silk with other insect species. A sufficient number of such studies will make it possible to distinguish between the basic components of all silks and the proteins that represent the adaptation of the fibers for specific purposes or environments.

The Role of Filippi's Glands in the Silk Moths Cocoon Construction.

Filippi's glands (FGs), formerly also called Lyonet's glands, are accessory secretory structures of the labial (silk) glands of lepidopteran caterpillars, which were implicated to play an important role in the maturation of the silk material and the construction of the cocoon. In our previous study, we have identified several species of giant silk moths that completely lack the FGs. Interestingly, the absence of FGs in these species correlates with the construction of a loose cocoon architecture. We investigated the functions of FGs by their surgical extirpation in the last instar larvae of the silkworm, Bombyx mori. We found that the absence of FGs altered the structure of the resulting cocoon, in which the different layers of silk were separated. In further experiments, we found no effects of the absence of FGs on larval cocoon formation behavior or on changes in cocoon mass or lipid content. Differential proteomic analysis revealed no significant contribution of structural proteins from FGs to silk cocoon material, but we identified several low abundance proteins that may play a role in posttranslational modifications of some silk proteins. Proteomic analysis also revealed a difference in phosphorylation of the N-terminal sequence of fibroin-heavy chain molecule. Thus, FGs appear to affect silk stickiness during spinning by regulating posttranslational modifications. This could also explain the link that exists between the absence of these glands and the formation of loose cocoons in some giant silk moth species.

Comparison of Silks from Pseudoips prasinana and Bombyx mori Shows Molecular Convergence in Fibroin Heavy Chains but Large Differences in Other Silk Components.

Many lepidopteran larvae produce silk feeding shelters and cocoons to protect themselves and the developing pupa. As caterpillars evolved, the quality of the silk, shape of the cocoon, and techniques in forming and leaving the cocoon underwent a number of changes. The silk of Pseudoips prasinana has previously been studied using X-ray analysis and classified in the same category as that of Bombyx mori, suggesting that silks of both species have similar properties despite their considerable phylogenetic distance. In the present study, we examined P. prasinana silk using 'omics' technology, including silk gland RNA sequencing (RNA-seq) and a mass spectrometry-based proteomic analysis of cocoon proteins. We found that although the central repetitive amino acid sequences encoding crystalline domains of fibroin heavy chain molecules are almost identical in both species, the resulting fibers exhibit quite different mechanical properties. Our results suggest that these differences are most probably due to the higher content of fibrohexamerin and fibrohexamerin-like molecules in P. prasinana silk. Furthermore, we show that whilst P. prasinana cocoons are predominantly made of silk similar to that of other Lepidoptera, they also contain a second, minor silk type, which is present only at the escape valve.

Adenosine Receptor and Its Downstream Targets, Mod(mdg4) and Hsp70, Work as a Signaling Pathway Modulating Cytotoxic Damage in Drosophila.

Adenosine (Ado) is an important signaling molecule involved in stress responses. Studies in mammalian models have shown that Ado regulates signaling mechanisms involved in "danger-sensing" and tissue-protection. Yet, little is known about the role of Ado signaling in Drosophila. In the present study, we observed lower extracellular Ado concentration and suppressed expression of Ado transporters in flies expressing mutant huntingtin protein (mHTT). We altered Ado signaling using genetic tools and found that the overexpression of Ado metabolic enzymes, as well as the suppression of Ado receptor (AdoR) and transporters (ENTs), were able to minimize mHTT-induced mortality. We also identified the downstream targets of the AdoR pathway, the modifier of mdg4 (Mod(mdg4)) and heat-shock protein 70 (Hsp70), which modulated the formation of mHTT aggregates. Finally, we showed that a decrease in Ado signaling affects other Drosophila stress reactions, including paraquat and heat-shock treatments. Our study provides important insights into how Ado regulates stress responses in Drosophila.

Silk of the common clothes moth, Tineola bisselliella, a cosmopolitan pest belonging to the basal ditrysian moth line.

Many lepidopteran larvae produce silk secretions to build feeding tubes and cocoons that play important protective roles in their lives. Recent research on the silk of bombycoid and pyralid moths has shown that it contains several highly abundant silk components with remarkable mechanical properties. It was also found to contain a number of other proteins of which the functions have yet to be identified. To gain an overview of the silk composition in more primitive lepidopteran species and to identify the core silk components common to most species, we analyzed the cocoon proteins of Tineola bisselliella, which belongs to the basal ditrysian moth line. Using de novo transcriptome sequencing combined with mass spectrometry (MS)-based proteomics, we detected more than 100 secretory proteins in the silk cocoons. Fibroin, sericins, and protease inhibitors were found to be the most abundant proteins, along with several novel candidate silk components. We also verified the tissue and developmental stage specificity of the silk protein expression and characterized the morphology of both the silk glands and silk in T. bisselliella. Our study provides a detailed analysis of silk in the primitive moth, expands the known set of silk-specific genes in Lepidoptera, and helps to elucidate their evolutionary relationships.

Functional Analysis of Adipokinetic Hormone Signaling in Bombyx mori.

Insect adipokinetic hormones (AKHs) are short peptides produced in the corpora cardiaca and are responsible for mobilizing energy stores from the fat body to the hemolymph. Three related peptides, AKH1, AKH2, and AKH/corazonin-related peptide (ACP) as well as three AKH receptors have been reported in Bombyx mori. AKH1 and AKH2 are specific for the AKHR1 receptor, whereas ACP interacts with the other two AKHRs. To assess the effect of the two silkworm AKHs and ACP in the regulation of energy homeostasis we examined the expression pattern of the three peptides and their receptors as well as their effect on the level of carbohydrates and lipids in the hemolymph. Our results support the hypothesis that only AKH1 and AKH2 peptides together with the AKHR1 receptor are involved in the maintenance of energy homeostasis. Because Bombyx AKHR1 (BmAKHR1) seems to be a true AKHR we generated its mutation. The BmAKHR1 mutant larvae display significantly lower carbohydrate and lipid levels in the hemolymph and reduced sensitivity to starvation. Our study clarifies the role of BmAKHR1 in energy homeostasis.

Identification of Regulatory Host Genes Involved in Sigma Virus Replication Using RNAi Knockdown in Drosophila.

The Drosophila melanogaster sigma virus, a member of the Rhabdoviridae family, specifically propagates itself in D. melanogaster. It contains six genes in the order of 3'-N-P-X-M-G-L-5'. The sigma virus is the only arthropod-specific virus of the Rhabdoviridae family. Sigma-virus-infected Drosophila may suffer from irreversible paralysis when exposed to a high CO2 concentration, but generally, no other symptoms are reported. A recent study reported that host gene expression in immune pathways was not changed in sigma-virus-infected Drosophila, which does not necessarily suggest that they are not involved in virus-host interactions. The present study aimed to identify host genes associated with sigma virus replication. Immune pathways JAK-STAT and IMD were selected for detailed study. The results showed that the genome copy number of the sigma virus increased after knocking down the immune pathway genes domeless and PGRP-LC in Drosophila S2 cells. The knocking down of domeless and PGRP-LC significantly up-regulated the expression of the L gene compared to the other viral genes. We propose that the immune pathways respond to sigma virus infection by altering L expression, hence suppressing viral replication. This effect was further tested in vivo, when D. melanogaster individuals injected with dsdome and dsPGRP-LC showed not only an increase in sigma virus copy number, but also a reduced survival rate when treated with CO2. Our study proved that host immunity influences viral replication, even in persistent infection. Knocking down the key components of the immune process deactivates immune controls, thus facilitating viral expression and replication. We propose that the immunity system of D. melanogaster regulates the replication of the sigma virus by affecting the L gene expression. Studies have shown minimal host-virus interaction in persistent infection. However, our study demonstrated that the immunity continued to affect viral replication even in persistent infection because knocking down the key components of the immune process disabled the relevant immune controls and facilitated viral expression and replication.

Drosophila imaginal disc growth factor 2 is a trophic factor involved in energy balance, detoxification, and innate immunity.

Drosophila imaginal disc growth factor 2 (IDGF2) is a member of chitinase-like protein family (CLPs) able to induce the proliferation of imaginal disc cells in vitro. In this study we characterized physiological concentrations and expression of IDGF2 in vivo as well as its impact on the viability and transcriptional profile of Drosophila cells in vitro. We show that IDGF2 is independent of insulin and protects cells from death caused by serum deprivation, toxicity of xenobiotics or high concentrations of extracellular adenosine (Ado) and deoxyadenosine (dAdo). Transcriptional profiling suggested that such cytoprotection is connected with the induction of genes involved in energy metabolism, detoxification and innate immunity. We also show that IDGF2 is an abundant haemolymph component, which is further induced by injury in larval stages. The highest IDGF2 accumulation was found at garland and pericardial nephrocytes supporting its role in organismal defence and detoxification. Our findings provide evidence that IDGF2 is an important trophic factor promoting cellular and organismal survival.