Insect Molecular Biology最新文献

The western flower thrips, Frankliniella occidentalis, poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye-colour genes, white (Fo-w) and cinnabar (Fo-cn), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo-w or Fo-cn specific guide RNAs. Homozygous Fo-w and Fo-cn knockout lines were established by crossing mutant females and males. The Fo-w knockout line revealed an age-dependent modification of eye-colour phenotype. Specifically, while young larvae exhibit orange-coloured eyes, the colour transitions to bright red as they age. Unexpectedly, loss of Fo-w function also altered body colour, with Fo-w mutants having a lighter coloured body than wild type, suggesting a dual role for Fo-w in thrips. In contrast, individuals from the Fo-cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene function and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.

Bumblebees are crucial pollinators, providing essential ecosystem services and global food production. The success of pollination services relies on the interaction between sensory organs and the environment. The antenna functions as a versatile multi-sensory organ, pivotal in mediating chemosensory/olfactory information, and governs adaptive responses to environmental changes. Despite an increasing number of RNA-sequencing studies on insect antenna, comprehensive antennal transcriptome studies at the different life stages were not elucidated systematically. Here, we quantified the expression profile and dynamics of coding/microRNA genes of larval head and antennal tissues from early- and late-stage pupa to the adult of Bombus terrestris as suitable model organism among pollinators. We further performed Pearson correlation analyses on the gene expression profiles of the antennal transcriptome from larval head tissue to adult stages, exploring both positive and negative expression trends. The positively correlated coding genes were primarily enriched in sensory perception of chemical stimuli, ion transport, transmembrane transport processes and olfactory receptor activity. Negatively correlated genes were mainly enriched in organic substance biosynthesis and regulatory mechanisms underlying larval body patterning and the formation of juvenile antennal structures. As post-transcriptional regulators, miR-1000-5p, miR-13b-3p, miR-263-5p and miR-252-5p showed positive correlations, whereas miR-315-5p, miR-92b-3p, miR-137-3p, miR-11-3p and miR-10-3p exhibited negative correlations in antennal tissue. Notably, based on the inverse expression relationship, positively and negatively correlated microRNA (miRNA)–mRNA target pairs revealed that differentially expressed miRNAs predictively targeted genes involved in antennal development, shaping antennal structures and regulating antenna-specific functions. Our data serve as a foundation for understanding stage-specific antennal transcriptomes and large-scale comparative analysis of transcriptomes in different insects.

DNA methylation in insects is generally low in abundance, and its role is not well understood. It is often localised in protein coding regions and associated with the expression of ‘housekeeping’ genes. Few studies have explored DNA methylation dynamics during lifecycle stage transitions in holometabolous (metamorphosing) insects. Using targeted mass spectrometry, we have found a significant difference in global DNA methylation levels between larvae, pupae and adults of Helicoverpa armigera (Lepidoptera: Noctuidae) Hübner, a polyphagous pest of agricultural importance. Whole-genome bisulfite sequencing confirmed these observations and pointed to non-CG context being the primary explanation for the difference observed between pupa and adult. Non-CG methylation was enriched in genes specific to various signalling pathways (Hippo signalling, Hedgehog signalling and mitogen-activated protein kinase (MAPK) signalling) and ATP-dependent chromatin remodelling. Understanding the function of this epigenetic mark could be a target in future studies focusing on integrated pest management.

Zinc excretion is crucial for zinc homeostasis. However, the mechanism of zinc excretion has not been well characterized. Zinc homeostasis in Drosophila seems well conserved to mammals. In this study, we screened all members of the zinc transporters ZnT (SLC30) and Zip (SLC39) for their potential roles in Drosophila hindgut, an insect organ that belongs to the excretory system. The results indicated that Catecholamines up (Catsup, CG10449), a ZIP member localized to the Golgi, is responsible for zinc homeostasis in the hindgut of Drosophila hindgut-specific knockdown of Catsup leads to a developmental arrest in the larval stage, which could be rescued well by human ZIP7. Further study suggested that Catsup RNAi in the hindgut reduced zinc levels in the excretory system (containing the Malpighian tubule and hindgut) but exhibited systemic zinc overload. Besides, more calculi were observed in the Malpighian tubules of Catsup RNAi flies. The developmental arrest and calculi in the Malpighian tubules of hindgut-specific Catsup RNAi flies could be rescued by dietary zinc restriction but hypersensitivity to zinc. These results will help us understand the fundamental process of zinc excretion in higher eukaryotes.

Agricultural insect pests (AIPs) are widely successful in adapting to natural and anthropogenic stressors, repeatedly overcoming population bottlenecks and acquiring resistance to intensive management practices. Although they have been largely overlooked in evolutionary studies, AIPs are ideal systems for understanding rapid adaptation under novel environmental conditions. Researchers have identified several genomic mechanisms that likely contribute to adaptive stress responses, including positive selection on de novo mutations, polygenic selection on standing allelic variation and phenotypic plasticity (e.g., hormesis). However, new theory suggests that stress itself may induce epigenetic modifications, which may confer heritable physiological changes (i.e., stress-resistant phenotypes). In this perspective, we discuss how environmental stress from agricultural management generates the epigenetic and genetic modifications that are associated with rapid adaptation in AIPs. We summarise existing evidence for stress-induced evolutionary processes in the context of insecticide resistance. Ultimately, we propose that studying AIPs offers new opportunities and resources for advancing our knowledge of stress-induced evolution.

The silkworm (Bombyx mori) is an important model lepidopteran insect and can be used to identify pesticide resistance-related genes of great significance for biological control of pests. Uridine diphosphate glucosyltransferases (UGTs), found in all organisms, are the main secondary enzymes involved in the metabolism of heterologous substances. However, it remains uncertain if silkworm resistance to fenpropathrin involves UGT. This study observes significant variations in BmUGT expression among B. mori strains with variable fenpropathrin resistance post-feeding, indicating BmUGT's role in fenpropathrin detoxification. Knockdown of BmUGT with RNA interference and overexpression of BmUGT significantly decreased and increased BmN cell activity, respectively, indicating that BmUGT plays an important role in the resistance of silkworms to fenpropathrin. In addition, fenpropathrin residues were significantly reduced after incubation for 12 h with different concentrations of a recombinant BmUGT fusion protein. Finally, we verified the conservation of UGT to detoxify fenpropathrin in Spodoptera exigua: Its resistance to fenpropathrin decreased significantly after knocking down SeUGT. In a word, UGT plays an important role in silkworm resistance to fenpropathrin by directly degrading the compound, a function seen across other insects. The results of this study are of great significance for breeding silkworm varieties with high resistance and for biological control of pests.

Exportin 1 (XPO1) is the major karyopherin-β nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including cell-cycle progression, transcription and translation. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is a plant-infecting rhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue-specific expression patterns with higher expression in the ovaries compared with the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared with controls, but no effect was observed for males. PmXPO1 knockdown experiments revealed a role for PmXPO1 in ovary function and egg production. Oviposition and egg hatch on plants were dramatically reduced in females treated with dsRNA PmXPO1. These results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection.

REPAT (response to pathogen) is an immune-associated gene family that plays important roles in insect immune response to pathogens. Although nine REPAT genes have been identified in Spodoptera frugiperda (Lepidoptera: Noctuidae) currently, their functions and mechanisms in the immune response to pathogens still remain unclear. Therefore, SfREPAT38, a pathogen response gene (REPAT) of S. frugiperda, was characterised and its function was analysed. The results showed that SfREPAT38 contains a signal peptide and a transcription activator MBF2 (multi-protein bridging factor 2) domain. Quantitative real-time polymerase chain reaction analysis showed that SfREPAT38 was highly expressed in the sixth-instar larvae (L6) and was the highest in expression in the midgut of L6. We found that the expression of SfREPAT38 could be activated by challenge with four microbial pathogens (Bacillus thuringiensis, Metarhizium anisopliae, Spodoptera exigua nuclearpolyhedrosis and Escherichia coli), except 12 h after E. coli infection. Furthermore, the SfREPAT38 expression levels significantly decreased at 24, 48 and 72 h after SfREPAT38 dsRNA injection or feeding. Feeding with SfREPAT38 dsRNA significantly decreased the weight gain of S. frugiperda, and continuous feeding led to the death of S. frugiperda larvae from the fourth day. Moreover, SfREPAT38 dsRNA injection resulted in a significant decrease of weight gain on the fifth day. Silencing SfREPAT38 gene down-regulated the expression levels of immune genes belonging to the Toll pathway, including SPZ, Myd88, DIF, Cactus, Pell and Toll18W. After treatment with SfREPAT38 dsRNA, S. frugiperda became extremely sensitive to the B. thuringiensis infection, and the survival rate dramatically increased, with 100% mortality by the eighth day. The weight of S. frugiperda larvae was also significantly lower than that of the control groups from the second day onwards. In addition, the genes involved in the Toll signalling pathway and a few antibacterial peptide related genes were down-regulated after treatment. These results showed that SfREPAT38 is involved in the immune response of S. frugiperda larvae through mediating Toll signalling pathway.

The silkworm, Bombyx mori, is a complete metamorphosed economic insect, and the silk gland is a significant organ for silk protein synthesis and secretion. The silk gland completely degenerates during pupation, but the regulatory mechanism of programmed cell death (PCD) has not yet been understood. In the present study, we investigated the non-genetic pathway of 20E-induced PCD in the posterior silk gland (PSG) based on intracellular Ca²⁺ levels. Silk gland morphology and silk gland index indicated rapid degeneration of silk gland during metamorphosis from mature silkworm (MS) to pupal day 1 (P1), and Ca²⁺ levels within the PSG were found to peak during the pre-pupal day 1 (PP1) stage. Moreover, the results of autophagy and apoptosis levels within the PSG showed that autophagy was significantly increased in MS-PP1 periods, and significantly decreased in PP2 and P1 periods. Apoptosis was almost absent in MS-PP1 periods and significantly increased in PP2 and P1 periods. Additionally, western blotting results showed that autophagy preceded apoptosis, and the autophagy-promoting ATG5 was cleaved by calpain to the autophagy-inhibiting and apoptosis-promoting NtATG5 since PP1 period, while decreased autophagy was accompanied by increased apoptosis. Collectively, these findings suggest that Ca²⁺ is a key factor in the shift from autophagy to apoptosis.

The fluctuation in temperature poses a significant challenge for poikilothermic organisms, notably insects, particularly in the context of changing climatic conditions. In insects, temperature adaptation has been driven by polygenes. In addition to genes that directly affect traits (core genes), other genes (peripheral genes) may also play a role in insect temperature adaptation. This study focuses on two peripheral genes, the GRIP and coiled-coil domain containing 2 (GCC2) and karyopherin subunit beta 1 (KPNB1). These genes are differentially expressed at different temperatures in the cosmopolitan pest, Plutella xylostella. GCC2 and KPNB1 in P. xylostella were cloned, and their relative expression patterns were identified. Reduced capacity for thermal adaptation (development, reproduction and response to temperature extremes) in the GCC2-deficient and KPNB1-deficient P. xylostella strains, which were constructed by CRISPR/Cas9 technique. Deletion of the PxGCC2 or PxKPNB1 genes in P. xylostella also had a differential effect on gene expression for many traits including stress resistance, resistance to pesticides, involved in immunity, trehalose metabolism, fatty acid metabolism and so forth. The ability of the moth to adapt to temperature via different pathways is likely to be key to its ability to remain an important pest species under predicted climate change conditions.