Insect Molecular Biology最新文献

Many species are threatened by climate change and must rapidly respond to survive in changing environments. Epigenetic modifications, such as DNA methylation, can facilitate plastic responses by regulating gene expression in response to environmental cues. Understanding epigenetic responses is therefore essential for predicting species' ability to rapidly adapt in the context of global environmental change. Here, we investigated the functional significance of different methylation-associated cellular processes on temperature-dependent life history in seed beetles, Callosobruchus maculatus Fabricius 1775 (Coleoptera: Bruchidae). We assessed changes under thermal stress in (1) DNA methyltransferase (Dnmt1 and Dnmt2) expression levels, (2) genome-wide methylation and (3) reproductive performance, with (2) and (3) following treatment with 3-aminobenzamide (3AB) and zebularine (Zeb) over two generations. These drugs are well-documented to alter DNA methylation across the tree of life. We found that Dnmt1 and Dnmt2 were expressed throughout the body in males and females, but were highly expressed in females compared with males and exhibited temperature dependence. However, whole-genome methylation did not significantly vary with temperature, and only marginally or inconclusively with drug treatment. Both 3AB and Zeb led to profound temperature-dependent shifts in female reproductive life history trade-off allocation, often increasing fitness compared with control beetles. Mismatch between magnitude of treatment effects on DNA methylation versus life history effects suggest potential of 3AB and Zeb to alter reproductive trade-offs via changes in DNA repair and recycling processes, rather than or in addition to (subtle) changes in DNA methylation. Together, our results suggest that epigenetic mechanisms relating to Dnmt expression, DNA repair and recycling pathways, and possibly DNA methylation, are strongly implicated in modulating insect life history trade-offs in response to temperature change.

In this study, we identified and assembled a strain of American nodavirus (ANV) in the Phlebotomus papatasi-derived PP9ad cell line. This strain most closely resembles Flock House virus and ANV identified in the Drosophila melanogaster S2/S2R cell line. Through small RNA sequencing and analysis, we demonstrate that ANV replication in PP9ad cells is primarily targeted by the exogenous small interfering RNA (exo-siRNA) pathway, with minimal engagement from the PIWI-interacting RNA (piRNA) pathway. In mosquitoes such as Aedes and Culex, the PIWI pathway is expanded and specialised, which actively limits virus replication. This is unlike in Drosophila spp., where the piRNA pathway does not restrict viral replication. In Lutzomyia sandflies (family Psychodidae), close relatives of Phlebotomus species and Drosophila, there appears to be an absence of virus-derived piRNAs. To investigate whether this absence is due to a lack of PIWI pathway proteins, we analysed the piRNA and siRNA diversity and repertoire in PP9ad cells. Previous assemblies of P. papatasi genome (Ppap_1.0) have revealed a patchy repertoire of the siRNA and piRNA pathways. Our analysis of the updated P. papatasi genome (Ppap_2.1) has shown no PIWI protein expansion in sandflies. We found that both siRNA and piRNA pathways are transcriptionally active in PP9ad cells, with genomic mapping of small RNAs generating typical piRNA signatures. Our results suggest that the piRNA pathway may not respond to virus replication in these cells, but an antiviral response is mounted via the exo-siRNA pathway.

Arylalkylamine N-acetyltransferase (aaNAT) is a crucial enzyme that catalyses the transfer of acetyl groups from acetyl coenzyme A to arylalkylamines and arylamines. Evolutionary studies have identified a distinct class of aaNATs specific to mosquitoes, yet their functions remain elusive. This study focuses on Ae-aaNAT7, a mosquito-unique gene in Aedes aegypti (Diptera:Culicidae), to explore its functionality. Temporal and spatial expression analysis of Ae-aaNAT7 mRNA revealed high expression during embryonic development and in first-instar larvae, with notable expression in the limbs of adult mosquitoes based on tissue expression profiling. By further employing CRISPR/Cas9 technology for loss-of-function studies, our investigation revealed a reduction in the area of white spotting in the limbs of Ae-aaNAT7 mutant adult mosquitoes. Further investigation revealed a significant decrease in the fecundity and hatchability of the mutants. Dissection of the ovaries from Ae-aaNAT7 heterozygous mutants showed a noticeable reduction in the oocyte area compared with wild type. Dissection of the exochorion of the eggs from Ae-aaNAT7 homozygous mutants consistently revealed a striking absence of mature embryos. In addition, RNA interference experiments targeting Ae-aaNAT7 in males resulted in a reduction in fecundity, but no effect on hatchability was observed. These collective insights underscore the substantial impact of Ae-aaNAT7 on reproduction and its pivotal contribution to adult limb pigmentation in Ae. aegypti. These revelations offer insights pivotal for the strategic design of future insecticide targets.

DNA methylase 1 (Dnmt1) is an important regulatory factor associated with biochemical signals required for insect development. It responds to changes in the environment and triggers phenotypic plasticity. Meanwhile, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)—a destructive invasive pest—can rapidly invade and adapt to different habitats; however, the role of Dnmt1 in this organism has not been elucidated. Accordingly, this study investigates the mechanism(s) underlying the rapid adaptation of Tuta absoluta to temperature stress. Potential regulatory genes were screened via RNAi (RNA interference), and the DNA methylase in Tuta absoluta was cloned by RACE (Rapid amplification of cDNA ends). TaDnmt1 was identified as a potential regulatory gene via bioinformatics; its expression was evaluated in response to temperature stress and during different development stages using real-time polymerase chain reaction. Results revealed that TaDnmt1 participates in hot/cold tolerance, temperature preference and larval development. The full-length cDNA sequence of TaDnmt1 is 3765 bp and encodes a 1254 kDa protein with typical Dnmt1 node-conserved structural features and six conserved DNA-binding active motifs. Moreover, TaDnmt1 expression is significantly altered by temperature stress treatments and within different development stages. Hence, TaDnmt1 likely contributes to temperature responses and organismal development. Furthermore, after treating with double-stranded RNA and exposing Tuta absoluta to 35°C heat shock or −12°C cold shock for 1 h, the survival rate significantly decreases; the preferred temperature is 2°C lower than that of the control group. In addition, the epidermal segments become enlarged and irregularly folded while the surface dries up. This results in a significant increase in larval mortality (57%) and a decrease in pupation (49.3%) and eclosion (50.9%) rates. Hence, TaDnmt1 contributes to temperature stress responses and temperature perception, as well as organismal growth and development, via DNA methylation regulation. These findings suggest that the rapid geographic expansion of T absoluta has been closely associated with TaDnmt1-mediated temperature tolerance. This study advances the research on ‘thermos Dnmt’ and provides a potential target for RNAi-driven regulation of Tuta absoluta.

Ribosomal protein L13 (RPL13) is highly conserved in evolution. At present, the properties and functions of RPL13 have not been characterised in insects. In this study, Bombyx mori RPL13 (BmRPL13) was first found to be specifically recruited to the sites of ultraviolet (UV)-induced DNA damage and contributed to UV damage repair. Escherichia coli expressing BmRPL13 showed better resistance to UV radiation. After knocking down the expression of BmRPL13 in BmN cells, the repair speed of UV-damaged DNA slowed down. The further results showed that BmRPL13 interacted with B. mori nucleopolyhedrovirus (BmNPV) ORF65 (Bm65) protein to locate at the UV-induced DNA damage sites of BmNPV and helped repair UV-damaged viral DNA.

Akirin is a nuclear protein that controls development in vertebrates and invertebrates. The function of Akirin has not been assessed in any Coleopteran insects. We found that high levels of akirin transcripts in Henosepilachna vigintioctopunctata, a serious Coleopteran potato defoliator (hereafter Hvakirin), were present at prepupal, pupal and adult stages, especially in larval foregut and fat body. RNA interference (RNAi) targeting Hvakirin impaired larval development. The Hvakirin RNAi larvae arrested development at the final larval instar stage. They remained as stunted larvae, gradually blackened and finally died. Moreover, the remodelling of gut and fat body was inhibited in the Hvakirin depleted larvae. Two layers of cuticles, old and newly formed, were noted in the dsegfp-injected animals. In contrast, only a layer of cuticle was found in the dsakirin-injected beetles, indicating the arrest of larval development. Furthermore, the expression of three transforming growth factor-β cascade genes (Hvsmox, Hvmyo and Hvbabo), a 20-hydroxyecdysone (20E) receptor gene (HvEcR) and six 20E response genes (HvHR3, HvHR4, HvE75, HvBrC, HvE93 and Hvftz-f1) was significantly repressed, consistent with decreased 20E signalling. Conversely, the transcription of a juvenile hormone (JH) biosynthesis gene (Hvjhamt), a JH receptor gene (HvMet) and two JH response genes (HvKr-h1 and HvHairy) was greatly enhanced. Our findings suggest a critical role of Akirin in larval development in H. vigintioctopunctata.

Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3′ untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.

The prominent role of the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway in animals is to silence transposable elements and maintain genome stability, ensuring proper gametogenesis in gonads. GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) is an evolutionarily conserved protein located on the outer mitochondrial membrane of germ cells and plays vital roles in the piRNA pathway and spermatogenesis in mammals. In the model insect Drosophila melanogaster, GASZ is essential for piRNA biogenesis and oogenesis, whereas its biological functions in non-drosophilid insects are still unknown. Here, we describe a comprehensive investigation of GASZ functions in the silkworm, Bombyx mori, a lepidopteran model insect, by using a binary transgenic CRISPR/Cas9 system. The BmGASZ mutation did not affect growth and development, but led to sterility in both males and females. Eupyrene sperm bundles of mutant males exhibited developmental defects, while the apyrene sperm bundles were normal, which were further confirmed through double copulation experiments with sex-lethal mutants, which males possess functional eupyrene sperm and abnormal apyrene sperm. In female mutant moths, ovarioles were severely degenerated and the eggs in ovarioles were deformed compared with that of wild type (WT). Further RNA-seq and RT-qPCR analysis revealed that amounts of piRNAs and transposon expression were dysregulated in gonads of mutants. In summary, this study has demonstrated vital roles of BmGASZ in gametogenesis through regulating the piRNA pathway in B. mori.

Zheng, H., Chen, C., Liu, C., Song, Q. and Zhou, S. (2020) Rhythmic change of adipokinetic hormones diurnally regulates locust vitellogenesis and egg development. Insect Molecular Biology, 29, 283–292. Available from: https://doi.org/10.1111/imb.12633

In the article by Zheng et al. (2020), an incorrect grant number was given in the Acknowledgments.

The correct text should be:

This work was supported by the National Natural Science Foundation of China (NSFC) (U1804232 and 31630070) and the 111 project of China (D16014).

We apologize for this error.

Cordyceps cicadae (Hypocreales: Cordycipitaceae) is a renowned entomopathogenic fungus used as herbal medicine in China. However, wild C. cicadae resources have been threatened by heavy harvesting. We hypothesised that Bombyx mori L. (Lepidoptera: Bombycidae) could be a new alternative to cultivate C. cicadae due to the low cost of rearing. Bacterial communities are crucial for the formation of Cordyceps and for promoting the production of metabolites. To better understand the bacterial community structure associated with Cordyceps, three Claviciptaceae fungi were used to explore the pathogenicity of the silkworms. Here, fifth-instar silkworms were infected with C. cicadae, Cordyceps cateniannulata (Hypocreales: Cordycipitaceae) and Beauveria bassiana (Hypocreales: Cordycipitaceae). Subsequently, we applied high-throughput sequencing to explore the composition of bacterial communities in silkworms. Our results showed that all three fungi were highly pathogenic to silkworms, which suggests that silkworms have the potential to cultivate Cordyceps. After fungal infection, the diversity of bacterial communities in silkworms decreased significantly, and the abundance of Staphylococcus increased in mummified larvae, which may play a role in the death process when the host suffers infection by entomopathogenic fungi. Furthermore, there were high similarities in the bacterial community composition and function in the C. cicadae and C. cateniannulata infected samples, and the phylogenetic analysis suggested that these similarities may be related to the fungal phylogenetic relationship. Our findings reveal that infection with different entomopathogenic fungi affects the composition and function of bacterial communities in silkworms and that the bacterial species associated with Cordyceps are primarily host dependent, while fungal infection affects bacterial abundance.