摘要: Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. In this study, we identified a testis-specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2H2 type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin-protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587-Gal4 > CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, we found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92E(F06346) mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, our results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila.

摘要: Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Despite important progress in understanding various aspects of silkworm biology, the hormone signaling pathway in the silkworm remains poorly understood. Genome-wide screening using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9)-based libraries has recently emerged as a novel method for analyzing genome function, enabling further research into essential genes, drug targets, and virus-host interaction. Previously, we constructed a genome-wide CRISPR/Cas9-based library of the silkworm (Bombyx mori) and successfully revealed the genes involved in biotic or abiotic stress factor responses. In this study, we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action. Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus. Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity, interfere with intracellular nutrition and energy metabolism, and eventually cause cell apoptosis. The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes, which had increased tolerance to 20E. Our findings provide a panoramic overview of signaling in response to 20E in the silkworm, underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

摘要: Sap-sucking insects often transmit plant viruses but also carry insect viruses, which infect insects but not plants. The impact of such insect viruses on insect host biology and ecology is largely unknown. Here, we identified a novel insect-specific virus carried by brown citrus aphid (Aphis citricidus), which we tentatively named Aphis citricidus picornavirus (AcPV). Phylogenetic analysis discovered a monophyletic cluster with AcPV and other unassigned viruses, suggesting that these viruses represent a new family in order Picornavirales. Systemic infection with AcPV triggered aphid antiviral immunity mediated by RNA interference, resulting in asymptomatic tolerance. Importantly, we found that AcPV was transmitted horizontally by secretion of the salivary gland into the feeding sites of plants. AcPV influenced aphid stylet behavior during feeding and increased the time required for intercellular penetration, thus promoting its transmission among aphids with plants as an intermediate site. The gene expression results suggested that this mechanism was linked with transcription of salivary protein genes and plant defense hormone signaling. Together, our results show that the horizontal transmission of AcPV in brown citrus aphids evolved in a manner similar to that of the circulative transmission of plant viruses by insect vectors, thus providing a new ecological perspective on the activity of insect-specific viruses found in aphids and improving the understanding of insect virus ecology.

摘要: After a millennium of domestication, numerous silkworm mutants have emerged that exhibit transparent epidermis, which is caused by abnormally low levels of uric acid. We identified the Bombyx mori gene Bmcap (BMSK0003832) as the homolog of cappuccino, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1) that has been extensively characterized in human, mouse, and insect species, by analyzing the amino acid sequences of putative purine metabolism genes. Using the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 system, we disrupted Bmcap, resulting in decreased uric acid levels in the silkworm epidermis and a translucent skin phenotype. In the Bmcap mutant, the purine metabolism, nitrogen metabolism, pyrimidine metabolism, and membrane system were altered compared to the wild type. Biogenesis of lysosome-related organelle complex genes play a role in the pigmentation and biogenesis of lysosome-related organelles (LROs) in platelets, melanocytes, and megakaryocytes. LROs exhibit unique morphologies and functions in various tissues and cells. Investigation of the Bmcap mutant will enhance our understanding of the uric acid metabolic pathway in silkworms, and this mutant offers a valuable silkworm model for LRO studies.

摘要: RNA interference (RNAi) is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells. However, silencing efficacy varies greatly among different insect species. Recently, we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection. The disappearance of double-stranded RNA (dsRNA) could be a potential factor that restricts RNAi efficiency. Here, we found that dsRNA can be degraded in midgut fluids, and a dsRNase of A. lucorum (AldsRNase) was identified and characterized. Sequence alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects' dsRNases. The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase. AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle, with peaks at the 4th instar ecdysis in the whole body. The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA. When comparing the substrate specificity of AldsRNase, 3 specific substrates (dsRNA, small interfering RNA, and dsDNA) were all degraded, and the most efficient degradation is dsRNA. Subsequently, immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells. Through cloning and functional study of AldsRNase, the enzyme activity and substrate specificity of the recombinant protein, as well as the subcellular localization of nuclease, the reason for the disappearance of dsRNA was explained, which was useful in improving RNAi efficiency in A. lucorum and related species.

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摘要: Monochamus alternatus is the primary carrier of pine wood nematodes, which pose a serious threat to Pinus spp. in many countries. Newly emerging M. alternatus adults feed on heathy host pines, while matured adults transfer to stressed host pines for mating and oviposition. Several odorant-binding proteins (OBPs) of M. alternatus have been proved to aid in the complex process of host location. To clarify the corresponding relations between OBPs and pine volatiles, more OBPs need to be studied. In this research, MaltOBP19 showed a specific expression in the antennae and mouthparts of M. alternatus, and it was marked in 4 types of antenna sensilla by immunolocalization. Fluorescence binding assays demonstrated the high binding affinity of MaltOBP19 with camphene and myrcene in vitro. In Y-tube olfactory experiments, M. alternatus adults were attracted by camphene and RNAi of OBP19 via microinjection significantly decreased their attraction index. Myrcene induced phobotaxis, but RNAi had no significant effect on this behavior. Further, we found that ingesting dsOBP19 produced by a bacteria-expressed system with a newly constructed vector could lead to the knockdown of MaltOBP19. These results suggest that MaltOBP19 may play a role in the process of host conversion via the recognition of camphene, which has been identified to be strongly released in stressed host pines. In addition, it is proved that knockdown of OBP can be achieved by oral administration of bacteria-expressed double-stranded RNA in M. alternatus adults, providing a new perspective in the control of M. alternatus.

摘要: The Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae), a globally distributed storage pest, relies on odors that are emitted from stored foods to select a suitable substrate for oviposition. However, the molecular mechanism underlying the chemical communication between P. interpunctella and its host remains elusive. In this study, 130 chemosensory genes were identified from the transcriptomes of 7 P. interpunctella tissues, and the quantitative expression levels of all 56 P. interpunctella odorant receptor genes (PintORs) were validated using real-time quantitative polymerase chain reaction. The functional characteristics of 5 PintORs with female antennae-biased expression were investigated using 2-electrode voltage clamp recordings in Xenopus laevis oocytes. PintOR23 was found to be specifically tuned to acetophenone. Acetophenone could elicit a significant electrophysiological response and only attracted mated females when compared with males and virgin females. In addition, molecular docking predicted that the hydrogen bonding sites, TRP-335 and ALA-167, might play key roles in the binding of PintOR23 to acetophenone. Our study provides valuable insights into the olfactory mechanism of oviposition substrate detection and localization in P. interpunctella and points toward the possibility of developing eco-friendly odorant agents to control pests of stored products.

摘要: High fecundity is a common characteristic of insect pests which increases the difficulty of population control. Serine/threonine kinase Akt is an indispensable component of the insulin signaling pathway. Silencing of LsAkt severely hinders reproduction in Lasioderma serricorne, a stored product insect pest. However, the post-transcriptional pathway of LsAkt in L. serricorne remains unknown. This study identified 2 binding sites of miR-9c-5p and novel-mir50 in the coding sequences of LsAkt. The expression profiles of 2 microRNAs (miRNAs) and LsAkt displayed an opposite pattern during the adult stages. Luciferase reporter assay showed that novel-mir50 and miR-9c-5p could downregulate the expression of LsAkt. Overexpression of miR-9c-5p and novel-mir50 by injection of mimics inhibited the expression of LsAkt and reduced oviposition, decreased egg hatchability, and blocked ovarian development. It also decreased the expression of genes involved in ovarian development (LsVg and LsVgR) and the nutritional signaling pathway (LsTOR, LsS6K, and Ls4EBP), and reduced the phosphorylation of Akt. Conversely, injection of miR-9c-5p and novel-mir50 inhibitors induced the expressions of LsAkt, LsVg, LsVgR, LsTOR, LsS6K, and Ls4EBP, enhanced Akt phosphorylation level, and accelerated ovarian development. Injection of bovine insulin downregulated the expression of miR-9c-5p and novel-mir50 and upregulated the LsAkt expression. It also rescued the reproductive development defects associated with miR-9c-5p/novel-mir50 overexpression, forming a positive regulatory loop of insulin signaling. These results indicate that miR-9c-5p/novel-mir50 regulates the female reproduction of L. serricorne by targeting Akt in response to insulin signaling. The data also demonstrate the effects of the insulin/miRNA/Akt regulatory axis in insect reproduction.

摘要: Pheromone receptors (PRs) are key proteins in the molecular mechanism of pheromone recognition, and exploring the functional differentiation of PRs between closely related species helps to understand the evolution of moth mating systems. Pheromone components of the agricultural pest Mythimna loreyi have turned into (Z)-9-tetradecen-1-yl acetate (Z9-14:OAc), (Z)-7-dodecen-1-yl acetate (Z7-12:OAc), and (Z)-11-hexadecen-1-yl acetate, while the composition differs from that of M. separata in the genus Mythimna. To understand the molecular mechanism of pheromone recognition, we sequenced and analyzed antennal transcriptomes to identify 62 odorant receptor (OR) genes. The expression levels of all putative ORs were analyzed using differentially expressed gene analysis. Six candidate PRs were quantified and functionally characterized in the Xenopus oocytes system. MlorPR6 and MlorPR3 were determined to be the receptors of major and minor components Z9-14:OAc and Z7-12:OAc. MlorPR1 and female antennae (FA)-biased MlorPR5 both possessed the ability to detect pheromones of sympatric species, including (Z,E)-9,12-tetradecadien-1-ol, (Z)-9-tetradecen-1-ol, and (Z)-9-tetradecenal. Based on the comparison of PR functions between M. loreyi and M. separata, we analyzed the differentiation of pheromone recognition mechanisms during the evolution of the mating systems of 2 Mythimna species.