Juvenile hormone biosynthetic genes are critical for regulating reproductive diapause in the cabbage beetle.

In insects, the juvenile hormone (JH) biosynthetic pathway regulates the in vivo JH titer. Thus, its downregulation potentially contributes to the lowering of JH titers typically observed in insects undergoing reproductive diapause, a developmental arrest at the adult stage. However, no systematic evidence has yet been presented to demonstrate the physiological and genetic roles of JH biosynthetic genes in reproductive diapause. In this work, we performed RNA interference (RNAi)-based reverse genetic analyses by targeting JH biosynthetic genes, followed by analysis of the reproductive diapause traits in Colaphellus bowringi, an economically important cabbage beetle. We identified a total of 22 genes encoding homologues of enzymes involved in the mevalonate pathway and the JH branch of JH biosynthesis in C. bowringi. Among these, 18 genes showed significant downregulation of their expression in the long day-induced diapausing females, compared to the short day-induced reproductive females. RNAi knockdown of almost any one of the 18 genes in reproductive females reduced the expression of the JH-responsive gene, Krüppel homolog1 (Kr-h1), indicating a lowered circulating JH. Most importantly, depleting transcripts of 3-hydroxy-3-methylglutaryl-CoA reductase 2 (HMGR2), farnesyl-pyrophosphate synthase 1 (FPPS1) and juvenile hormone acid methyltransferase 1 (JHAMT1) induced diapause-associated traits, including immature and inactive ovaries, large accumulations of lipids and adult burrowing behavior. Meanwhile, genes related to ovarian development, lipid accumulation and stress response showed expression patterns like those of diapausing females. RNAi-mediated diapause phenotypes could be reversed to reproductive phenotypes by application of methoprene, a JH receptor agonist. These results suggest that photoperiodic reproductive diapause in C. bowringi is triggered by transcriptional suppression of JH biosynthetic genes, with HMGR2, FPPS1 and JHAMT1 playing a critical role in this process. This work provides sufficient evidence to reveal the physiological roles of JH biosynthetic genes in reproductive diapause.

Innovation of heterochromatin functions drives rapid evolution of essential ZAD-ZNF genes in Drosophila.

Contrary to dogma, evolutionarily young and dynamic genes can encode essential functions. We find that evolutionarily dynamic ZAD-ZNF genes, which encode the most abundant class of insect transcription factors, are more likely to encode essential functions in Drosophila melanogaster than ancient, conserved ZAD-ZNF genes. We focus on the Nicknack ZAD-ZNF gene, which is evolutionarily young, poorly retained in Drosophila species, and evolves under strong positive selection. Yet we find that it is necessary for larval development in D. melanogaster. We show that Nicknack encodes a heterochromatin-localizing protein like its paralog Oddjob, also an evolutionarily dynamic yet essential ZAD-ZNF gene. We find that the divergent D. simulans Nicknack protein can still localize to D. melanogaster heterochromatin and rescue viability of female but not male Nicknack-null D. melanogaster. Our findings suggest that innovation for rapidly changing heterochromatin functions might generally explain the essentiality of many evolutionarily dynamic ZAD-ZNF genes in insects.

Genomic architecture of a genetically assimilated seasonal color pattern.

Developmental plasticity allows genomes to encode multiple distinct phenotypes that can be differentially manifested in response to environmental cues. Alternative plastic phenotypes can be selected through a process called genetic assimilation, although the mechanisms are still poorly understood. We assimilated a seasonal wing color phenotype in a naturally plastic population of butterflies (Junonia coenia) and characterized three responsible genes. Endocrine assays and chromatin accessibility and conformation analyses showed that the transition of wing coloration from an environmentally determined trait to a predominantly genetic trait occurred through selection for regulatory alleles of downstream wing-patterning genes. This mode of genetic evolution is likely favored by selection because it allows tissue- and trait-specific tuning of reaction norms without affecting core cue detection or transduction mechanisms.

A chimeric gene paternally instructs female sex determination in the haplodiploid wasp Nasonia.

Various primary signals direct insect sex determination. In hymenopteran insects, the presence of a paternal genome is needed to initiate female development. When absent, uniparental haploid males develop. We molecularly and functionally identified the instructor sex-determination gene, wasp overruler of masculinization (wom), of the haplodiploid wasp Nasonia vitripennis This gene contains a P53-like domain coding region and arose by gene duplication and genomic rearrangements. Maternal silencing of wom results in male development of haploid embryos. Upon fertilization, early zygotic transcription from the paternal wom allele is initiated, followed by a timely zygotic expression of transformer (tra), leading to female development. Wom is an instructor gene with a parent-of-origin effect in sex determination.

Unexpected mutual regulation underlies paralogue functional diversification and promotes epithelial tissue maturation in Tribolium.

Insect Hox3/zen genes represent an evolutionary hotspot for changes in function and copy number. Single orthologues are required either for early specification or late morphogenesis of the extraembryonic tissues, which protect the embryo. The tandemly duplicated zen paralogues of the beetle Tribolium castaneum present a unique opportunity to investigate both functions in a single species. We dissect the paralogues' expression dynamics (transcript and protein) and transcriptional targets (RNA-seq after RNAi) throughout embryogenesis. We identify an unexpected role of Tc-Zen2 in repression of Tc-zen1, generating a negative feedback loop that promotes developmental progression. Tc-Zen2 regulation is dynamic, including within co-expressed multigene loci. We also show that extraembryonic development is the major event within the transcriptional landscape of late embryogenesis and provide a global molecular characterization of the extraembryonic serosal tissue. Altogether, we propose that paralogue mutual regulation arose through multiple instances of zen subfunctionalization, leading to their complementary extant roles.

Glycosylhydrolase genes control respiratory tubes sizes and airway stability.

Tight barriers are crucial for animals. Insect respiratory cells establish barriers through their extracellular matrices. These chitinous-matrices must be soft and flexible to provide ventilation, but also tight enough to allow oxygen flow and protection against dehydration, infections, and environmental stresses. However, genes that control soft, flexible chitin-matrices are poorly known. We investigated the genes of the chitinolytic glycosylhydrolase-family 18 in the tracheal system of Drosophila melanogaster. Our findings show that five chitinases and three chitinase-like genes organize the tracheal chitin-cuticles. Most of the chitinases degrade chitin from airway lumina to enable oxygen delivery. They further improve chitin-cuticles to enhance tube stability and integrity against stresses. Unexpectedly, some chitinases also support chitin assembly to expand the tube lumen properly. Moreover, Chitinase2 plays a decisive role in the chitin-cuticle formation that establishes taenidial folds to support tube stability. Chitinase2 is apically enriched on the surface of tracheal cells, where it controls the chitin-matrix architecture independently of other known cuticular proteins or chitinases. We suppose that the principle mechanisms of chitin-cuticle assembly and degradation require a set of critical glycosylhydrolases for flexible and not-flexible cuticles. The same glycosylhydrolases support thick laminar cuticle formation and are evolutionarily conserved among arthropods.

Intracellular dynamics of polydnavirus innexin homologues.

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

Immediate early gene kakusei potentially plays a role in the daily foraging of honey bees.

kakusei is a non-coding RNA that is overexpressed in foraging bee brain. This study describes a possible role of the IEG kakusei during the daily foraging of honey bees. kakusei was found to be transiently upregulated within two hours during rewarded foraging. Interestingly, during unrewarded foraging the gene was also found to be up-regulated, but immediately lowered when food was not rewarded. Moreover, the kakusei overexpression was diminished within a very short time when the time schedule of feeding was changed. This indicates the potential role of kakusei on the motivation of learned reward foraging. These results provide evidence for a dynamic role of kakusei during for aging of bees, and eventually its possible involvement in learning and memory. Thus the kakusei gene could be used as search tool in finding distinct molecular pathways that mediate diverse behavioral components of foraging.

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

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

Deletion mutant of sPLA2 using CRISPR/Cas9 exhibits immunosuppression, developmental retardation, and failure of oocyte development in legume pod borer, Maruca vitrata.

Phospholipase A2 (PLA2) catalyzes release of free fatty acids linked to phospholipids at sn-2 position. Some of these released free fatty acids are used to synthesize eicosanoids that mediate various physiological processes in insects. Although a large number of PLA2s form a superfamily consisting of at least 16 groups, few PLA2s have been identified and characterized in insects. Furthermore, physiological functions of insect PLA2s remain unclear. Clustered regularly interspaced short parlindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been a useful research tool to validate gene function. This study identified and characterized a secretory PLA2 (sPLA2) from legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), and validated its physiological functions using CRISPR/Cas9. An open reading frame of M. vitrata sPLA2 (Mv-sPLA2) encoding 192 amino acids contained signal peptide, calcium-binding domain, and catalytic site. Phylogenetic analysis indicated that Mv-sPLA2 was related to other Group III sPLA2s. Mv-sPLA2 was expressed in both larval and adult stages. It was inducible by immune challenge. RNA interference (RNAi) of Mv-sPLA2 significantly suppressed cellular immunity and impaired larval development. Furthermore, RNAi treatment in female adults prevented oocyte development. These physiological alterations were also observed in a mutant line of M. vitrata with Mv-sPLA2 deleted by using CRISPR/Cas9. Mv-sPLA2 was not detected in the mutant line from western blot analysis. Addition of an eicosanoid, PGE2, significantly rescued oocyte development of females of the mutant line. These results suggest that Mv-sPLA2 plays crucial role in immune, developmental, and reproductive processes of M. vitrata.

Genes regulating wing patterning in Drosophila melanogaster show reduced expression under exposure of Daminozide, the fruit ripening retardant.

In our previous study we demonstrated that the fruit ripening retardant Daminozide or Alar causes change in life history traits, distortion of adult wing structure, DNA damage in brain cells and mutagenic effects in fruit fly Drosophila melanogaster. As a continuation of the previous study the present work is designed to explore the metabolic modification of Daminozide following ingestion, the effects of Daminozide on the expression of genes which are pivotal for wing development and molecular interactions of Daminozide with those proteins involved in wing patterning. We demonstrated through reporter gene construct assay using X-gal staining method and transgenic Drosophila melanogaster stocks that the vestigial, wingless and decapentaplegic genes in wing imaginal disc from 3rd instar larvae exhibited reduced expression when exposed to Daminozide in compare to control larvae. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of those genes confirmed that exposure to Daminozide reduces the transcription level of those genes. In silico approach with molecular docking study revealed Daminozide may bind and interfere with the optimal functioning of expressed wing signaling proteins.

Identification of an Apis cerana cerana MAP kinase phosphatase 3 gene (AccMKP3) in response to environmental stress.

MAP kinase phosphatase 3 (MKP3), a member of the dual-specificity protein phosphatase (DUSP) superfamily, has been widely studied for its role in development, cancer, and environmental stress in many organisms. However, the functions of MKP3 in various insects have not been well studied, including honeybees. In this study, we isolated an MKP3 gene from Apis cerana cerana and explored the role of this gene in the resistance to oxidation. We found that AccMKP3 is highly conserved in different species and shares the closest evolutionary relationship with AmMKP3. We determined the expression patterns of AccMKP3 under various stresses. qRT-PCR results showed that AccMKP3 was highly expressed during the pupal stages and in adult muscles. We further found that AccMKP3 was induced in all the stress treatments. Moreover, we discovered that the enzymatic activities of peroxidase, superoxide dismutase, and catalase increased and that the expression levels of several antioxidant genes were affected after AccMKP3 was knocked down. Collectively, these results suggest that AccMKP3 may be associated with antioxidant processes involved in response to various environmental stresses.

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

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

Identification of an inositol-3-phosphate synthase 1-B gene (AccIPS1-B) from Apis cerana cerana and its role in abiotic stress.

Inositol phosphate synthase (IPS) is a rate-limiting enzyme in myo-inositol biosynthesis, which can regulate stress responses in plants and animals. However, there are few studies on the function of IPS in insects, especially in Apis cerana cerana. In this study, the inositol-3-phosphate synthase 1-B gene (AccIPS1-B) was isolated from Apis cerana cerana, and its connection to antioxidant defence was investigated. The open reading frame of AccIPS1-B was 1542 bp, encoding a 513 amino acid polypeptide. Quantitative real-time PCR analysis revealed that the expression level of AccIPS1-B was highest in pupae of Apis cerana cerana, and it was expressed at higher levels in the thorax than in other tissues tested. Moreover, the expression of AccIPS1-B was significantly upregulated by abiotic stresses. The recombinant AccIPS1-B also displayed significant tolerance to cumene hydroperoxide and HgCl2. In addition, knockdown of AccIPS1-B significantly suppressed the expression of most of the antioxidant genes and decreased the antioxidant enzymatic activities of SOD, POD, and GST. Taken together, these findings indicate that AccIPS1-B may be involved in the response to antioxidant defence and development in Apis cerana cerana.

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

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

Convergent eusocial evolution is based on a shared reproductive groundplan plus lineage-specific plastic genes.

Eusociality has convergently evolved multiple times, but the genomic basis of caste-based division of labor and degree to which independent origins of eusociality have utilized common genes remain largely unknown. Here we characterize caste-specific transcriptomic profiles across development and adult body segments from pharaoh ants (Monomorium pharaonis) and honey bees (Apis mellifera), representing two independent origins of eusociality. We identify a substantial shared core of genes upregulated in the abdomens of queen ants and honey bees that also tends to be upregulated in mated female flies, suggesting that these genes are part of a conserved insect reproductive groundplan. Outside of this shared groundplan, few genes are differentially expressed in common. Instead, the majority of the thousands of caste-associated genes are plastically expressed, rapidly evolving, and relatively evolutionarily young. These results emphasize that the recruitment of both highly conserved and lineage-specific genes underlie the convergent evolution of novel traits such as eusociality.

Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of "Qiufeng", a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and ßFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

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

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

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

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