Insect Molecular Biology最新文献

E75, a member of the nuclear receptor gene family, regulates diverse biological processes, including ovarian development and fat body remodelling. The brown planthopper (Nilaparvata lugens Stål) (Hemiptera:Delphacidae), a major rice pest, poses a significant threat to rice production. However, the role of Nilaparvata lugens E75 (NlE75) in development, particularly ovarian development and its underlying mechanisms, remains largely unknown. In this study, we found that downregulation of NlE75 expression significantly reduced the eclosion rate, especially in the third-, fourth- and fifth-instar nymphs. Moreover, decreased NlE75 levels affected fat body morphology as well as ovarian and testicular development, leading to delayed ovarian maturation and reduced egg production. NlE75 knockdown drastically reduced the total number of eggs laid. We also found that NlE75 may participate in the transcriptional regulation of Vitellogenin (Vg), Vitellogenin-like1 (Vg-like1) and Vitellogenin-like2 (Vg-like2) and that downregulation of NlE75 significantly reduced Vg protein expression. In summary, we found that NlE75 is required for lipid droplet formation in the fat body, as well as for ovary and vas deferens development. Furthermore, downregulation of NlE75 altered fat body morphology and reduced lipid content, suggesting decreased energy reserves. Importantly, NlE75 was found to be involved in the transcriptional regulation of cell cycle-related genes, influencing DNA replication and the expression of genes controlling cell cycle progression. Flow cytometry analysis further revealed significant disruption of ovarian cell cycles following NlE75 downregulation, suggesting that E75 may function in cell cycle progression to coordinate development and reproduction in N. lugens. The involvement of NlE75 in regulating cell cycle-related genes and cell cycle progression in N. lugens represents a novel finding. This study provides a valuable reference for related research in other species, offering new insights into the mechanisms of NlE75 function and its interaction with both JH and ecdysone signalling.

Sexual signalling by pheromones is essential for mate finding and mate choice in moths and plays an important role in reproductive isolation. Acetates (i.e., acetate esters) produced by females of Heliothis (Chloridea) subflexa Fabricius, 1777 (Lepidoptera: Noctuidae) attract conspecific males but repel Heliothis virescens Fabricius, 1777 (Lepidoptera: Noctuidae) males. A QTL (quantitative trait locus) harbouring carboxylesterases and lipases was previously shown to affect acetates, and CRISPR/Cas9-induced knockouts increased acetate amounts by blocking hydrolysis of the esters as expected. A second, unlinked QTL, containing a cluster of three different carboxylesterases (CXEs), unexpectedly yielded decreased acetate amounts. In one of these genes, esterase CXE24, we found a naturally occurring transposable element insertion in exon 8. A CRISPR/Cas9-induced frameshift at the same position yielded the same results. The paradox was resolved by a CRISPR/Cas9-induced frameshift in exon 2 of CXE24 which increased acetate amounts. The frameshift in exon 2 produced a truncated protein lacking the substrate binding site and the catalytic triad, while the frameshift in exon 8 removed only the third residue of the catalytic triad. In silico modelling showed that the exon-8-truncated protein could not hydrolyse the esters by itself, which likely explains the decreased acetate amounts. To place our findings in an evolutionary context, we explored variation in the esterase cluster in 16 species of Lepidoptera with completely sequenced genomes. Geographic and temporal variation in acetates has been observed in H. subflexa, and variation in the frequency of the transposable element could be a possible explanation.

Colour pattern formation is a key model for studying evolutionary and developmental mechanisms. In the fruit fly Drosophila guttifera, which exhibits distinctive polka-dot wing pigmentation, we investigated the roles of two putative melanin synthesis genes, yellow and tan, using CRISPR/Cas9-mediated genome editing. We established multiple mutant strains with lesions in either gene and found that both genes were essential for normal pigmentation intensity in wing spots, though the patterns themselves persisted. Double mutants showed further reduction in pigmentation, indicating additive effects but not complete loss of patterning. Ectopic expression of wingless failed to induce normal pigmentation in yellow or tan mutants, demonstrating that both genes act downstream of wingless and are required for its pigmentation-inducing function. Furthermore, mosaic phenotypes in G0 individuals revealed quasi-cell-autonomous functions of tan, suggesting that pigmentation in D. guttifera wings depends on local availability of precursors rather than solely on transport via wing veins. This study establishes D. guttifera as a genetically tractable system for functional analyses and contributes to understanding the molecular basis of insect colour pattern formation.

DNA methylation has been proposed as an epigenetic driver of phenotypic plasticity in social insects, yet experimental evidence remains limited. Even less is known about the role of epigenetic mechanisms underlying behavioural and social polymorphism. We quantified CpG methylation for the socially polymorphic harvester ant Pogonomyrmex californicus across larvae, pupae, workers and queens using Oxford Nanopore Technologies (ONT) sequencing. These results were compared against the current gold standard whole-genome bisulfite sequencing (WGBS). Methylation sites were highly correlated between WGBS and ONT, validating the use of ONT for high-throughput epigenomic profiling. Genome-wide methylation was low (~3%), consistent with findings in other (Hymenoptera: Formicidae), and highly clustered within gene bodies, especially exons, while introns, intergenic DNA, promoters and transposable elements were hypo-methylated. Gene body methylation (GBM) correlated positively with gene expression in queens, corroborating previous reports for other insects, suggesting a conserved regulatory role for DNA methylation in insects. A comparison between developmental stages revealed significant stage-specific differences in GBM frequencies. Workers and queens, although from different populations, shared a substantial core of methylated loci enriched for olfactory-receptor activity and biosynthetic pathways, processes that are central to caste-specific behaviour and physiology. These shared methylation signatures, coupled with stage-dependent variability, highlight DNA methylation as a possible factor in developmental stages and caste differentiation. In the future, it is essential to disentangle the effects of caste and variation between populations. Our study establishes P. californicus as a powerful model for dissecting how epigenetic modifications interface with gene expression to generate developmental and complex social organization, which is largely unexplored.

Insect Taiman (Tai) is a basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) transcription factor that plays a role in juvenile hormone (JH) signalling during the larval stage in various insect species, as well as in 20-hydroxyecdysone (20E) signalling during the oogenesis of Drosophila melanogaster. It forms a heterodimer with another bHLH-PAS transcription factor Methoprene-tolerant (Met) mediating JH signalling and acts as a steroid receptor coactivator of the functional 20E receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP). However, whether Tai plays the dual functions in other insects deserves further exploration. In this study, we determined the role of Tai in Henosepilachna vigintioctomaculata, a Coleopteran potato pest mainly distributed in northern China. HvTai mRNA levels were higher just before and right after the ecdysis within each larval instar and at early and late stages of prepupae than those in the intermediate days. RNA interference (RNAi)-aided knockdown of Tai at the second, penultimate and final larval instar stages caused 100% mortalities 9, 11 and 10 days post dsRNA administration. All Tai RNAi larvae failed to complete moulting; they were wrapped in the old larval exuviae, gradually darkened and shrivelled and ultimately died. The Tai-depleted larvae did not complete gut clearing; the larval guts contained food residues. Moreover, knockdown of Tai disrupted both JH and 20E pathway genes. Our findings demonstrated that Tai was necessary for metamorphosis in H. vigintioctomaculata. The findings also imply that H. vigintioctomaculata is an ideal model for further exploration of how Tai contributes specifically to hormone signalling.

Krüppel homolog1 (Kr-h1), a transcription factor with a DNA-binding motif of eight C₂H₂ zinc fingers, exerts species-specific functions by mediating juvenile hormone (JH) signal in certain physiological processes during female reproduction in insects. Nevertheless, the regulatory role of Kr-h1 on vitellogenesis deserves further exploration. Here, we found that Kr-h1 was actively expressed from 0- to 12-day-old females and among detected tissues including fat body and ovary in two potato-defoliating beetles, Henosepilachna vigintioctopunctata and H. vigintioctomaculata. RNA interference (RNAi) for Kr-h1 significantly lengthened the preoviposition period, dramatically impaired ovarian development, and decreased oviposition. Weaker yolk accumulation occurred in the Kr-h1 RNAi oocytes, in contrast to a larger quantity of yolk granules in controls. Correspondingly, Vg transcript levels in the fat bodies and ovaries and of VgR amounts in the ovarian tissues were substantially reduced in the Kr-h1 knockdown samples. In H. vigintioctomaculata, RNAi for the other two JH signal genes, HvmHairy and HvmGroucho, was also performed. Silencing either or both genes affected neither female reproduction nor the mRNA levels of Vg and VgR. Therefore, our findings suggest Kr-h1, rather than Hairy/Groucho, regulates vitellogenesis in the two Henosepilachna species. Moreover, targeting Kr-h1 could potentially be a new way to control potato pests.

Germ cells depend on germline genes for their specification, migration and differentiation during development. Among these, the ATP-dependent RNA helicase gene vasa (vas) is one of the most conserved germline genes across animal phyla. Most organisms have a single copy of vas, but duplications are observed in some lineages. The evolutionary basis for this duplication remains unclear. Most insects, such as the fly Drosophila melanogaster, possess a single vas gene, but the pea aphid Acyrthosiphon pisum and related species have four paralogs. Previously, we identified Ap-vas1 as the germline-specific gene in aphids. Here, we examine the developmental expression of Ap-vas2, Ap-vas3 and Ap-vas4 at both mRNA and protein levels, compare their patterns with those of Ap-vas1 and analyse their developmental roles. Using antibodies specific to each Ap-Vas protein for immunostaining, we find that Ap-Vas2-4 proteins are not enriched in germ cells but exhibit distinct somatic expression patterns, suggesting roles in somatic development, such as chromosome segregation and nuage formation. We further assessed the divergent N-terminal regions of Ap-Vas proteins using chimeric constructs in Drosophila oocytes. Only the N-terminus of Ap-Vas1 partially mediated germ-plasm localisation, whereas those of Ap-Vas2-4 significantly impaired posterior localisation. These findings suggest how divergence in the N-terminal regions of Vas proteins may underlie their functional diversification in germline and somatic cell development across insects.

In insects, eye pigmentation is vital for various adaptive functions, including foraging, mating and predator avoidance. Due to its visible and often heritable variation, altered eye pigmentation in mutants provides an excellent model for studying biosynthetic pathways and identifying new genes involved in pigmentation. Eye mutants can also be valuable in science communication as they offer simplified examples to help the public understand complex genetic concepts. In this study, we used a community science-based approach to identify the inheritance pattern and mutation(s) responsible for white-eye pigmentation in honey bees. With the help of the beekeeping community, we identified a honey bee queen that produced a fraction of haploid sons (called drones) with white-eyes. As the queen was wild-type, we predicted that the mutation is most likely recessive to wild-type. Using genome-wide association and differentiation scans of wild-type and white-eyed drone brothers, we identified a single elevated region (52 kb) of chromosome 11. This region contains four non-coding RNA (ncRNA) and one protein-coding gene. We identified an eight-base pair region with two SNPs and a four-nucleotide deletion that are likely responsible for the phenotype. The mutation likely affects the expression and/or downstream effects of an uncharacterized ncRNA (LOC100578475). Our efforts highlight the value of community-based science in novel gene discovery. We hope this serves, not only as a new example for the burgeoning field of honey bee functional genomics, but also as a teaching tool for both researchers and educators.

Diapause is a crucial biological adaptation that enables many insects to endure unfavourable environmental conditions and maintain species survival. The silkworm, Bombyx mori, a classic model organism exhibiting embryonic diapause, provides an excellent system for investigating diapause mechanisms. In this study, we evaluated the effectiveness of electrical stimulation in terminating diapause and explored its underlying regulatory mechanisms during the termination process in silkworms. Specifically, electrical stimulation significantly improved the economic traits of silkworms compared to HCl treatment. Transcriptome sequencing was performed to analyse dynamic gene regulation during diapause termination following electrical stimulation in two silkworm strains: the Chinese lineage strain Qiufeng and the Japanese lineage strain Baiyu. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) revealed, for the first time, that the mechanism of diapause termination in silkworms is lineage-specific. Weighted gene co-expression network analysis (WGCNA) further indicated that this lineage-specific diapause release is primarily driven by the differential expression of genes involved in the carbohydrate biosynthesis pathway. These findings both validate the practical application of electrical stimulation in sericulture and reveal fundamental differences in diapause regulation between silkworm lineages, advancing our understanding of this crucial biological process.

Moulting and metamorphosis are fundamental physiological processes in the growth and development of holometabolous insects, primarily regulated by ecdysteroids and juvenile hormone (JH), which are synthesized and secreted by the prothoracic gland and corpora allata, respectively. However, the signalling regulatory network involved in the synthesis of these hormones and their effects is complex and interactive and involves many unidentified functional genes. In this study, we conducted a basic bioinformatics analysis of the CAD96CA gene and obtained CAD96CA mutants at the individual level in domestic silkworms using the CRISPR-Cas9 technology. We analysed the growth, development and silk protein synthesis phenotypes of the mutants and detected the synthesis and signalling effects of 20E and JH. The results revealed that knocking out the CAD96CA gene resulted in inhibited larval growth, reduced silk protein production, hindered larval-pupal transition and led to larval mortality. The synthesis of 20E and its signalling pathways, as well as the signalling pathways of JH, were all affected to varying degrees following CAD96CA knockout. Our study elucidates the role of CAD96CA in the growth and development of silkworms and provides a reference for studying metamorphosis mechanisms.