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.

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.

Sericin Composition in the Silk of Antheraea yamamai.

The silks produced by caterpillars consist of fibroin proteins that form two core filaments, and sericin proteins that seal filaments into a fiber and conglutinate fibers in the cocoon. Sericin genes are well-known in Bombyx mori (Bombycidae) but have received little attention in other insects. This paper shows that Antheraea yamamai (Saturniidae) contains five sericin genes very different from the three sericin genes of B. mori. In spite of differences, all known sericins are characterized by short exons 1 and 2 (out of 3-12 exons), expression in the middle silk gland section, presence of repeats with high contents of Ser and charged amino acid residues, and secretion as a sticky silk component soluble in hot water. The B. mori sericins represent tentative phylogenetic lineages (I) BmSer1 and orthologs in Saturniidae, (II) BmSer2, and (III) BmSer3 and related sericins of Saturniidae and of the pyralid Galleria mellonella. The lineage (IV) seems to be limited to Saturniidae. Concerted evolution of the sericin genes was apparently associated with gene amplifications as well as gene loses. Differences in the silk fiber morphology indicate that the cocktail of sericins linking the filaments and coating the fiber is modified during spinning. Silks are composite biomaterials of conserved function in spite of great diversity of their composition.

Mutation in the Drosophila melanogaster adenosine receptor gene selectively decreases the mosaic hyperplastic epithelial outgrowth rates in wts or dco heterozygous flies.

Adenosine (Ado) is a ubiquitous metabolite that plays a prominent role as a paracrine homeostatic signal of metabolic imbalance within tissues. It quickly responds to various stress stimuli by adjusting energy metabolism and influencing cell growth and survival. Ado is also released by dead or dying cells and is present at significant concentrations in solid tumors. Ado signaling is mediated by Ado receptors (AdoR) and proteins modulating its concentration, including nucleoside transporters and Ado deaminases. We examined the impact of genetic manipulations of three Drosophila genes involved in Ado signaling on the incidence of somatic mosaic clones formed by the loss of heterozygosity (LOH) of tumor suppressor and marker genes. We show here that genetic manipulations with the AdoR, equilibrative nucleoside transporter 2 (Ent2), and Ado deaminase growth factor-A (Adgf-A) cause dramatic changes in the frequency of hyperplastic outgrowth clones formed by LOH of the warts (wts) tumor suppressor, while they have almost no effect on control yellow (y) clones. In addition, the effect of AdoR is dose-sensitive and its overexpression leads to the increase in wts hyperplastic epithelial outgrowth rates. Consistently, the frequency of mosaic hyperplastic outgrowth clones generated by the LOH of another tumor suppressor, discs overgrown (dco), belonging to the wts signaling pathway is also dependent on AdoR. Our results provide interesting insight into the maintenance of tissue homeostasis at a cellular level.

The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly.

Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1.

A comprehensive gene expression analysis of the unique three-layered cocoon of the cecropia moth, Hyalophora cecropia.

The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein components of these cocoons in relation to the morphology of the silk gland and its affiliation to the different layers of the cocoon. The study used transcriptomic and proteomic analyses to identify 91 proteins associated with the silk cocoons, 63 of which have a signal peptide indicating their secretory nature. We checked the specificity of their expression in different parts of the SG and the presence of the corresponding protein products in each cocoon layer. Differences were observed among less abundant proteins with unclear functions. The representation of proteins in the inner envelope and intermediate space was similar, except for a higher proportion of probable contaminating proteins, mostly originating from the gut. On the other hand, the outer envelope contains a number of putative enzymes with unclear function. However, the protein most specific to the outer layer has sequence homology to putative serine/threonine kinase-like proteins and some adhesive proteins, and its closest homolog in Bombyx mori was found in the scaffold silk. This research provides valuable insights into the silk production of the cecropia moth, highlighting both similarities and differences to other moth species.

Dominant suppressor genes of p53-induced apoptosis in Drosophila melanogaster.

One of a major function of programmed cell death (apoptosis) is the removal of cells which suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes which, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS-promoters at both ends which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS-promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, seven genes were identified which suppressed the p53-induced apoptosis. In four mutants, the suppression effect resulted from single genes activated by one UAS-promoter (Pka-R2, Rga, crol, Spt5). In the other three (Orct2, Polr2M, stg), deleting either UAS-promoter eliminated the suppression effect. In qPCR experiments we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eucaryotic genomes there are co-expressed gene clusters. Three of the DEP insertion mutants are included and two are in close vicinity of separate co-expressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.

Functional conservation and structural diversification of silk sericins in two moth species.

Sericins are hydrophilic structural proteins produced by caterpillars in the middle section of silk glands and layered over fibroin proteins secreted in the posterior section. In the process of spinning, fibroins form strong solid filaments, while sericins seal the pair of filaments into a single fiber and glue the fiber into a cocoon. Galleria mellonella and the previously examined Bombyx mori harbor three sericin genes that encode proteins containing long repetitive regions. Galleria sericin genes are similar to each other and the protein repeats are built from short and extremely serine-rich motifs, while Bombyx sericin genes are diversified and encode proteins with long and complex repeats. Developmental changes in sericin properties are controlled at the level of gene expression and splicing. In Galleria , MG-1 sericin is produced throughout larval life until the wandering stage, while the production of MG-2 and MG-3 reaches a peak during cocoon spinning.

Selective elimination/RNAi silencing of FMRF-related peptides and their receptors decreases the locomotor activity in Drosophila melanogaster.

Five neuropeptide genes are classified in the FMRF-related (FaRP) group: the Fmrf, dromyosuppressin (Dms), drosulfakinin (Dsk), neuropeptide F (npf) and short neuropeptide F (sNPF) genes coding for 8, 1, 2, 1 and 4 peptides, respectively. In order to compare their effects on the locomotor activity of Drosophila adults, we made RNAi knockdown of the peptides and their specific receptor genes. In addition, we constructed Gal4 drivers with three distinct parts of the Fmrf gene's 5' regulatory sequence (RS8-Gal4, RS11-Gal4, RS17-Gal4), and used them to ablate FMRF-positive neurons inducing apoptosis by expressing the reaper (rpr) gene. We examined the locomotor activity of flies by measuring the mean velocity of movement (MVM) following repeated air-puffs. Locomotor activity was decreased by RNAi knockdown induced in the CNS by the elav-Gal4 driver. According to the MVM curve profiles, RNAi knockdown most effectively decreased the velocity when the DmsR-1 and DmsR-2 genes were silenced together (DmsR-1-RNAi/elav-Gal4; DmsR-2-RNAi/+). Similar effect was observed in Dsk-RNAi/ elav-Gal4; DskR-2-RNAi/+, while moderate effects were found in three other combinations (Fmrf-RNAi/elav-Gal4; FR-RNAi/+, Dms-RNAi/ elav-Gal4;DmsR-2-RNAi/+, CCKLR-17D1-RNAi/elav-Gal4; CCKLR-17D3-RNAi/+), and weak effect in DmsR-2-RNAi/elav-Gal4; DmsR-1-RNAi/+. Male and female flies were not different in this respect. In the cell ablation experiment, the MVM profiles of the female flies were different from the controls when the UAS-rpr transgene was driven by RS8-Gal4 or RS17-Gal4. The RS11-Gal4 and Fmrf-Gal4 drivers were ineffective. In the males only the RS17-Gal4 showed a weak effect. RNAi silencing of the FaRP and FaRP-receptor genes effectively decreased the startle-induced locomotor activity of flies. Ablation of FMRF-positive neurons by the RS8-Gal4 and/or RS17-Gal4 drivers also decreased the flies' activity.

Identification of silk components in the bombycoid moth Andraca theae (Endromidae) reveals three fibroin subunits resembling those of Bombycidae and Sphingidae.

The silk produced by Lepidoptera caterpillars is a mixture of proteins secreted by the transformed labial glands, the silk glands (SG). The silk fiber consists of insoluble filamentous proteins that form a silk core and are produced in the posterior part of the SG and soluble coat proteins consisting of sericins and various other polypeptides secreted in the middle part of the SG. We constructed a silk gland specific transcriptome of Andraca theae and created a protein database required for peptide mass fingerprinting. We identified major silk components by proteomic analysis of cocoon silk and by searching for homologies with known silk protein sequences from other species. We identified 30 proteins including a heavy chain fibroin, a light chain fibroin and fibrohexamerin (P25) that form the silk core, as well as members of several structural families that form the silk coating. To uncover the evolutionary relationships among silk proteins, we included orthologs of silk genes from several recent genome projects and performed phylogenetic analyses. Our results confirm the recent molecular classification that the family Endromidae appears to be slightly more distant from the family Bombycidae. Our study provides important information on the evolution of silk proteins in the Bombycoidea, which is needed for proper annotation of the proteins and future functional studies.

Characterization of the Drosophila adenosine receptor: the effect of adenosine analogs on cAMP signaling in Drosophila cells and their utility for in vivo experiments.

Adenosine receptors (AR) belonging to the G protein-coupled receptor family influence a wide range of physiological processes. Recent elucidation of the structure of human A2AR revealed the conserved amino acids necessary for contact with the Ado moiety. However, the selectivity of Ado analogs for AR subtypes is still not well understood. We have shown previously that the Drosophila adenosine receptor (DmAdoR) evokes an increase in cAMP and calcium concentration in heterologous cells. In this study, we have characterized the second-messenger stimulation by endogenous DmAdoR in a Drosophila neuroblast cell line and examined a number of Ado analogs for their ability to interact with DmAdoR. We show that Ado can stimulate cAMP but not calcium levels in Drosophila cells. We found one full and four partial DmAdoR agonists, as well as four antagonists. The employment of the full agonist, 2-chloroadenosine, in flies mimicked in vivo the phenotype of DmAdoR over-expression, whereas the antagonist, SCH58261, rescued the flies from the lethality caused by DmAdoR over-expression. Differences in pharmacological effect of the tested analogs between DmAdoR and human A2AR can be partially explained by the dissimilarity of specific key amino acid residues disclosed by the alignment of these receptors.

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.

Characterization of silk genes in Ephestia kuehniella and Galleria mellonella revealed duplication of sericin genes and highly divergent sequences encoding fibroin heavy chains.

Silk is a secretory product of numerous arthropods with remarkable mechanical properties. In this work, we present the complete sequences of the putative major silk proteins of E. kuehniella and compare them with those of G. mellonella, which belongs to the same moth family Pyralidae. To identify the silk genes of both species, we combined proteomic analysis of cocoon silk with a homology search in transcriptomes and genomic sequences to complement the information on both species. We analyzed structure of the candidate genes obtained, their expression specificity and their evolutionary relationships. We demonstrate that the silks of E. kuehniella and G. mellonella differ in their hydrophobicity and that the silk of E. kuehniella is highly hygroscopic. In our experiments, we show that the number of genes encoding sericins is higher in G. mellonella than in E. kuehniella. By analyzing the synteny of the chromosomal segment encoding sericin genes in both moth species, we found that the region encoding sericins is duplicated in G. mellonella. Finally, we present the complete primary structures of nine fibH genes and proteins from both families of the suborder Pyraloidea and discuss their specific and conserved features. This study provides a foundation for future research on the evolution of silk proteins and lays the groundwork for future detailed functional studies.

The Exact Timing of Microinjection of Parthenogenetic Silkworm Embryos Is Crucial for Their Successful Transgenesis.

The use of parthenogenetic silkworm (Bombyx mori) strains, which eliminate the problem of recombination, is a useful tool for maintaining transgenic clonal lines. The generation of genetically identical individuals is becoming an important tool in genetic engineering, allowing replication of an existing advantageous trait combination without the mixing that occurs during sexual reproduction. Thus, an animal with a particular genetic modification, such as the ability to produce transgenic proteins, can reproduce more rapidly than by natural mating. One obstacle to the widespread use of parthenogenesis in silkworm genetic engineering is the relatively low efficiency of downstream transgenesis techniques. In this work, we seek to optimize the use of transgenesis in conjunction with the production of parthenogenetic individuals. We found that a very important parameter for the introduction of foreign genes into a parthenogenetic strain is the precise timing of embryo microinjection. Our modification of the original method increased the efficiency of transgene injection as well as the survival rate of injected embryos. We also provide a detailed description of the methodological procedure including a graphical overview of the entire protocol.

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.

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.

Equilibrative nucleoside transporter 2 regulates associative learning and synaptic function in Drosophila.

Nucleoside transporters are evolutionarily conserved proteins that are essential for normal cellular function. In the present study, we examined the role of equilibrative nucleoside transporter 2 (ent2) in Drosophila. Null mutants of ent2 are lethal during late larval/early pupal stages, indicating that ent2 is essential for normal development. Hypomorphic mutant alleles of ent2, however, are viable and exhibit reduced associative learning. We additionally used RNA interference to knock down ent2 expression in specific regions of the CNS and show that ent2 is required in the alpha/beta lobes of the mushroom bodies and the antennal lobes. To determine whether the observed behavioral defects are attributable to defects in synaptic transmission, we examined transmitter release at the larval neuromuscular junction (NMJ). Excitatory junction potentials were significantly elevated in ent2 mutants, whereas paired-pulse plasticity was reduced. We also observed an increase in stimulus dependent calcium influx in the presynaptic terminal. The defects observed in calcium influx and transmitter release probability at the NMJ were rescued by introducing an adenosine receptor mutant allele (AdoR(1)) into the ent2 mutant background. The results of the present study provide the first evidence of a role for ent2 function in Drosophila and suggest that the observed defects in associative learning and synaptic function may be attributable to changes in adenosine receptor activation.

Characterization of two closely related α-amylase paralogs in the bark beetle, Ips typographus (L.).

Ips typographus (L.), the eight-spined spruce bark beetle, causes severe damage throughout Eurasian spruce forests and suitable nuclear markers are needed in order to study its population structure on a genetic level. Two closely related genes encoding α-amylase in I. typographus were characterized and named AmyA and AmyB. Both α-amylase paralogs consisted of six exons and five introns. AmyA encodes a polypeptide of 483 amino acids, whereas AmyB has two alternative transcripts encoding polypeptides of 483 and 370 amino acids. The expression levels of both genes were high during larval stage and adulthood. The AmyB transcripts were absent in the pupal stage. A modification of the allozyme staining method allowed us to detect two clusters of bands on the electrophoretic gel that may correspond to the two α-amylase genes. There was a correlation between the lack of AmyB expression in pupa and the absence of the fast migrating isozyme cluster at this stage, suggesting that the faster migrating isoforms are products of the AmyB gene, whereas the slowly migrating bands are derived from the AmyA.

The Filippi's Glands of Giant Silk Moths: To Be or Not to Be?

The Filippi's glands (FGs), formerly "Lyonet's glands", are paired accessory organs associated with the silk glands. They are unique to Lepidoptera caterpillars and their exact role is not clear. The FGs are thought to be involved in the construction of a silk cocoon in bombycoid moths. FGs can differ in size and shape, therefore, in this study we attempt to find a correlation between FG morphology and phylogenetic position within the Bombycoidea. We use light and electron microscopy to examine the presence and morphology of FGs in a range of wild (giant) silk moths and several related species. Our results confirm that the majority of studied silk moth species have complex type of FGs that continuously increase in size during larval development. We identified several species of giant silk moths and two hawk moth species that completely lack FGs throughout their larval development. Finally, in several hawk moth species in which FGs are well developed during the first larval stage, these glands do not grow and remain small during later larval growth. Our results suggest that FGs are not critical for spinning and that loss of FGs occurred several times during the evolution of saturniids and sphingids. Comparison of FGs in different moths is an important first step in the elucidation of their physiological significance.