Macrophages play a nutritive role in post-metamorphic maturation in Drosophila.

In the body of multicellular organisms, macrophages play an indispensable role in maintaining tissue homeostasis by removing old, apoptotic and damaged cells. In addition, macrophages allow significant remodeling of body plans during embryonic morphogenesis, regeneration and metamorphosis. Although the huge amount of organic matter that must be removed during these processes represents a potential source of nutrients, their further use by the organism has not yet been addressed. Here, we document that, during metamorphosis, Drosophila larval adipose tissue is infiltrated by macrophages, which remove dying adipocytes by efferocytosis and engulf leaking RNA-protein granules and lipids. Consequently, the infiltrating macrophages transiently adopt the adipocyte-like metabolic profile to convert remnants of dying adipocytes to lipoproteins and storage peptides that nutritionally support post-metamorphic development. This process is fundamental for the full maturation of ovaries and the achievement of early fecundity of individuals. Whether macrophages play an analogous role in other situations of apoptotic cell removal remains to be elucidated.

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 Trail-Following Communication in Stylotermes faveolus and S. halumicus (Blattodea, Isoptera, Stylotermitidae).

Stylotermitidae appear peculiar among all termites, feeding in trunks of living trees in South Asia only. The difficulty to collect them limits the ability to study them, and they thus still belong to critically unknown groups in respect to their biology. We used a combination of microscopic observations, chemical analysis and behavioural tests, to determine the source and chemical nature of the trail-following pheromone of Stylotermes faveolus from India and S. halumicus from Taiwan. The sternal gland located at the 5th abdominal segment was the exclusive source of the trail-following pheromone in both S. faveolus and S. halumicus, and it is made up of class I, II and III secretory cells. Using gas chromatography coupled mass spectrometry, (3Z)-dodec-3-en-1-ol (DOE) was identified as the trail-following pheromone which elicits strong behavioural responses in workers at a threshold around 10- 4 ng/cm and 0.1 ng/gland. Our results confirm the switch from complex aldehyde trail-following pheromones occurring in the basal groups to simpler linear alcohols in the ancestor of Kalotermitidae and Neoisoptera.

Concurrent Infection of the Human Brain with Multiple Borrelia Species.

Lyme disease (LD) spirochetes are well known to be able to disseminate into the tissues of infected hosts, including humans. The diverse strategies used by spirochetes to avoid the host immune system and persist in the host include active immune suppression, induction of immune tolerance, phase and antigenic variation, intracellular seclusion, changing of morphological and physiological state in varying environments, formation of biofilms and persistent forms, and, importantly, incursion into immune-privileged sites such as the brain. Invasion of immune-privileged sites allows the spirochetes to not only escape from the host immune system but can also reduce the efficacy of antibiotic therapy. Here we present a case of the detection of spirochetal DNA in multiple loci in a LD patient's post-mortem brain. The presence of co-infection with Borrelia burgdorferi sensu stricto and Borrelia garinii in this LD patient's brain was confirmed by PCR. Even though both spirochete species were simultaneously present in human brain tissue, the brain regions where the two species were detected were different and non-overlapping. The presence of atypical spirochete morphology was noted by immunohistochemistry of the brain samples. Atypical morphology was also found in the tissues of experimentally infected mice, which were used as a control.

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.

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.

Extended longevity of termite kings and queens is accompanied by extranuclear localization of telomerase in somatic organs and caste-specific expression of its isoforms.

Kings and queens of termites are endowed with an extraordinary longevity coupled with lifelong fecundity. We recently reported that termite kings and queens display a dramatically increased enzymatic activity and abundance of telomerase in their somatic organs when compared to short-lived workers and soldiers. We hypothesized that this telomerase activation may represent a noncanonical pro-longevity function, independent of its canonical role in telomere maintenance. Here, we explore this avenue and investigate whether the presumed noncanonical role of telomerase may be due to alternative splicing of the catalytic telomerase subunit TERT and whether the subcellular localization of TERT isoforms differs among organs and castes in the termite Prorhinotermes simplex. We empirically confirm the expression of four in silico predicted splice variants (psTERT1-A, psTERT1-B, psTERT2-A, psTERT2-B), defined by N-terminal splicing implicating differential localizations, and C-terminal splicing giving rise to full-length and truncated isoforms. We show that the transcript proportions of the psTERT are caste- and tissue-specific and that the extranuclear full-length isoform TERT1-A is relatively enriched in the soma of neotenic kings and queens compared to their gonads and to the soma of workers. We also show that extranuclear TERT protein quantities are significantly higher in the soma of kings and queens compared to workers, namely due to the cytosolic TERT. Independently, we confirm by microscopy the extranuclear TERT localization in somatic organs. We conclude that the presumed pleiotropic action of telomerase combining the canonical nuclear role in telomere maintenance with extranuclear functions is driven by complex TERT splicing.

Defensive glands in Stylotermitidae (Blattodea, Isoptera).

The large abundance of termites is partially achieved by their defensive abilities. Stylotermitidae represented by a single extant genus, Stylotermes, is a member of a termite group Neoisoptera that encompasses 83% of termite species and 94% of termite genera and is characterized by the presence of the frontal gland. Within Neoisoptera, Stylotermitidae represents a species-poor sister lineage of all other groups. We studied the structure of the frontal, labral and labial glands in soldiers and workers of Stylotermes faveolus, and the composition of the frontal gland secretion in S. faveolus and Stylotermes halumicus. We show that the frontal gland is a small active secretory organ in soldiers and workers. It produces a cocktail of monoterpenes in soldiers, and some of these monoterpenes and unidentified proteins in workers. The labral and labial glands are developed similarly to other termite species and contribute to defensive activities (labral in both castes, labial in soldiers) or to the production of digestive enzymes (labial in workers). Our results support the importance of the frontal gland in the evolution of Neoisoptera. Toxic, irritating and detectable monoterpenes play defensive and pheromonal functions and are likely critical novelties contributing to the ecological success of these termites.

The rate of DNA synthesis in ovaries, fat body cells, and pericardial cells of the bumblebee (Bombus terrestris) depends on the stage of ovarian maturation.

Bumblebees are important pollinators of plants worldwide and they are kept for commercial pollination. By studying the process of oogenesis, we can understand their ontogenetic developmental strategy and reproduction. We describe the anatomy of the ovary of the bumblebee Bombus terrestris using 3D reconstruction by confocal microscopy. We found that an oocyte is accompanied by 63 endopolyploidy nurse cells. The number of nurse cells nuclei decreased during oogenesis and the cells are finally absorbed by the oocyte. We monitored the rate of DNA synthesis in vivo during 12 h in ovaries, fat body, and pericardial cells in B. terrestris queens and workers of different ages. The DNA replication activity was detected on the basis of visualization of incorporated 5-ethynyl-2'-deoxyuridine. DNA synthesis detected in differentiated nurse cells indicated endoreplication of nuclei. The dynamics of mitotic activity varied among different ages and statuses of queens. In 3- to 8-day-old virgin queens, intense mitotic activity was observed in all tissue types investigated. This might be related to the initial phase of oogenesis and the development of the hepato-nephrotic system. In 15- to 20-day-old mated pre-diapause queens, DNA synthesis was exclusively observed in the ovaries, particularly in the germarium and the anterior part of the vitellarium. In 1-year-old queens, replication occurred only in the peritoneal sheath of ovaries and in several cells of the fat body. The similar DNA synthesis patterns in the ovaries of mated pre-diapause queens, ovipositing workers, and non-egg-laying workers show that mitotic activity is related not only to age but also to the stage of ovarian maturation and is relatively independent of caste affiliation.

3D reconstruction of larval and adult brain neuropils of two giant silk moth species: Hyalophora cecropia and Antheraea pernyi.

We present a detailed analysis of the brain anatomy of two saturniid species, the cecropia silk moth, Hyalophora cecropia, and the Chinese oak silk moth, Antheraea pernyi, including 3D reconstructions of the major brain neuropils in the larva and in male and female adults. The 3D reconstructions, prepared from high-resolution optical sections, showed that the corresponding neuropils of these saturniid species are virtually identical. Similarities between the two species include a pronounced sexual dimorphism in the adults in the form of a male-specific assembly of markedly enlarged glomeruli forming the so-called macroglomerular complex. From the reports published to date, it can be concluded that the neuropil architecture of saturniids resembles that of other nocturnal moths, including the sibling family Sphingidae. In addition, compared with previous anatomical data on diurnal lepidopteran species, significant differences were observed in the two saturniid species, which include the thickness of the Y-tract of the mushroom body, the size of the main neuropils of the optic lobes, and the sexual dimorphisms of the antennal lobes.

No bioaccumulation of Cry protein in the aphidophagous predator Harmonia axyridis.

The uptake of insecticidal Cry1Ab from genetically engineered (GE) maize, via herbivore Rhopalosiphum padi, to a predator Harmonia axyridis and its potential intergenerational transfer were investigated. Cry1Ab concentration was found to be 400-fold lower in R. padi compared to GE maize, and more than two-fold lower in H. axyridis. For 62% of H. axyridis samples, Cry1Ab was under the limit of detection (LOD), for another 13% were under the limit of quantification (LOQ). The concentration of Cry1Ab was similar between H. axyridis exposed short-term and long-term with the exception of adults after long-term. There was no correlation between Cry1Ab in females and eggs and neonates. The performance of H. axyridis was comparable between Cry1Ab and control. Histological investigation did not show any pathological changes in the digestive and reproductive systems. The detected route of exposure is unlikely to be important for functional biological control by H. axyridis in agroecosystem.

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.

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 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.

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.

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.

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.