摘要: American foulbrood (AFB) disease, caused by the bacterium Paenibacillus larvae, is a devastating disease affecting honeybee (Apis mellifera L.) populations worldwide. Commonly treated with antibiotics, which have negative impacts on both honeybees and the environment, there is an urgent need for alternatives in AFB control. This study aimed to investigate the effects of gallic acid (GA) on honeybee larvae challenged with P. larvae spores and explore its modulation of larval microbiota. Our results demonstrated that in the presence of P. larvae spores, coadministration of 125 mu g/mL GA significantly increased the survival rate and body weight of honeybee larvae. Molecular docking analyses revealed that GA competitively binds to spore germination proteins YndE and GerM, with affinities comparable to L-tyrosine and stronger than uric acid, respectively, suggesting interference with P. larvae spore germination. 16S rRNA gene amplicon sequencing revealed that GA treatment augmented bacterial diversity and enriched lactic acid bacteria (LAB) in honeybee larvae. Whole-genome sequencing of 2 LAB strains, Apilactobacillus kunkeei GL-2 and Enterococcus faecium GL-6, isolated from GA-treated larvae, unveiled their potential to produce antimicrobial secondary metabolites and bacteriocins, which may contribute to their competitive advantages against P. larvae. Notably, the E. faecium GL-6 strain possessed genes encoding gallate decarboxylase, enabling GA utilization, and 2 putative bacteriocinogenic genetic clusters for enterolysin A and enterocin L50 a/b. These findings suggest that GA and the GL-6 strain hold potential as preventive measures against AFB disease in honeybees through modulation of gut microbiota and competitive inhibition of P. larvae.

摘要: Respiration is a vital process essential for organism survival, with most terrestrial insects relying on a sophisticated tubular tracheal network. In the current study, a gene with repetitive sequence was identified within the silkworm genome. Designated as BmMuc91C, it contains a dozen repeated motifs PSSSYGAPX and GGYSSGGX in its sequence. BmMuc91C exhibits specific expression in the tracheal system of silkworm larvae, with significantly higher expression levels during the molting stage. Overexpression of BmMuc91C in individual silkworms resulted in a marked increase in tracheal diameter, particularly during the molting stage. Immunofluorescence staining using a BmMuc91C antibody revealed a noticeable thickening of the apical extracellular matrix in the trachea. Tensile testing confirmed a considerable enhancement in tracheal elasticity. Additionally, a BmMuc91C mutation strain of silkworms was generated using the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 system. Although no significant differences were observed in the growth, development, and molting of BmMuc91C mutant silkworms, mechanical tests demonstrated a decrease in tracheal elasticity. Transcriptomic techniques revealed that a significant number of cuticular and chitin-binding proteins were among the differentially expressed genes between mutant and wild-type silkworms. Furthermore, the recombined BmMuc91C protein was successfully expressed using the Escherichia coli system. Cross-linking experiments with horseradish peroxidase demonstrated the formation of macromolecular complexes of BmMuc91C, which exhibited spontaneous luminescent properties under ultraviolet light. This research sheds light on the role of elastic proteins in insect tracheae and provides valuable insights for the development of elastic biomaterials.

摘要: Diapause is an adaptive strategy employed by insects to endure adverse environmental conditions and is characterized by reduced metabolic activity, primarily due to a decreased respiratory rate. AMP-activated protein kinase (AMPK) serves as an intracellular energy regulator, modulating energy metabolism in response to metabolic fluctuations. However, its role in pupal diapause of the cotton bollworm, Helicoverpa armigera, remains unclear. In this study, we found that AMPK and its active form, P-AMPK, are highly expressed in diapause-destined pupae. Furthermore, activation of AMPK delayed the development of nondiapause-destined pupae, suggesting a critical role for AMPK in the regulation of pupal diapause in H. armigera. Manipulating AMPK activity in H. armigera epidermal (HaEpi) cells and pupae significantly influenced the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), which our laboratory previously reported as a key inducer of pupal diapause through the reduction of mitochondrial activity in H. armigera. Histone deacetylase 4 (HDAC4), a shuttle protein phosphorylated by AMPK which translocates between the cytoplasm and the nucleus, was found to exhibit significantly higher expression in diapause-destined pupal brains compared to their nondiapause counterparts. AMPK in both HaEpi cells and pupae positively regulated the protein levels of P-HDAC4 by binding to the HDAC4 promoter. Additionally, HDAC4 was shown to enhance HIF-1 alpha expression in diapause-destined individuals. HDAC4 binds to and deacetylates heat shock protein 70 (HSP70), and reduced acetylation of HSP70 was found to significantly elevate HIF-1 alpha protein levels. The AMPK-HIF-1 alpha signaling pathway appears to play a pivotal role in reducing mitochondrial activity and facilitating diapause induction in H. armigera pupae.

摘要: Bombyx mori ELAV-like-1 (BmEL-1) and B. mori ELAV-like-2 (BmEL-2) are 2 members of the ELAV-like family of RNA-binding proteins. Mutations in Bmel-1 and Bmel-2 resulted in 5.8% and 28.5% decreases in larval weight on the 3rd day of the 5th instar larva (L5D3), respectively. Triglycerides (TG) are the most important energy resource and are the main component of neutral fat (NF) in animals. To investigate the role of Bmelav-like genes in the synthesis and decomposition of TG, transcriptomic, and metabolic analyses were performed on the whole bodies on the 1st day of the 2nd instar larvae (L2D1) and on fat bodies on L5D3 of Bmel-1- and Bmel-2- mutants, respectively. As compared with the control silkworm, differentially expressed genes generated in both mutants were mainly enriched in lysine degradation, fatty acid (FA) metabolism, and unsaturated FAs biosynthesis. The diglyceride and phosphatide contents were significantly lower in Bmel-1- and Bmel-2- fat bodies than those of the control group. Consistently, the NF content of both mutants' fat bodies were reduced by 50% and 60%, respectively. BmEL-2 positively regulates BmAGPAT gamma (B. mori 1-acyl-sn-glycerol-3-phosphate acyltransferase gamma, LOC101741736) and BmFaF2 (B. mori fatty acid synthetase-associated factor 2, LOC101739090) expression by binding to the specific regions of their 3 ' untranslated regions in BmN cells. This study suggests that BmEL-2 may be an important regulator of BmAGPAT gamma and BmFAF2 expression and thereby participates in TG metabolism in the silkworm fat body.

摘要: The gut is not only used by insects as an organ for the digestion of food and absorption of nutrients but also as an important barrier against the invasion and proliferation of pathogenic microorganisms. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an insect-specific virus, predominantly colonizes the midgut epithelial cells of the silkworm, thereby jeopardizing its normal growth. However, there is limited knowledge of the cellular immune responses to viral infection and whether the infection is promoted or inhibited by different types of cells in the silkworm midgut. In this study, we used single-nucleus RNA sequencing to identify representative enteroendocrine cells, enterocytes, and muscle cell types in the silkworm midgut. In addition, by analyzing the transcriptional profiles of various subpopulations in the infected and uninfected groups, we found that BmCPV infection suppresses the response of the antiviral pathways and induces the expression of BmHSP70, which plays a role in promoting BmCPV replication. However, certain immune genes in the midgut of the silkworm, such as BmLebocin3, were induced upon viral infection, and downregulation of BmLEB3 using RNA interference promoted BmCPV replication in the midgut of B. mori. These results suggest that viral immune evasion and active host resistance coexist in BmCPV-infected silkworms. We reveal the richness of cellular diversity in the midgut of B. mori larvae by single-nucleus RNA sequencing analysis and provide new insights into the complex interactions between the host and the virus at the single-cell level.

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摘要: In insects, the juvenile hormone (JH) and 20-hydroxyecdysone (20E) pathways jointly regulate fecundity, but only methyl farnesoate (MF) and ponasterone A exist in mites. Comparative transcriptomic analysis in Panonychus citri showed that E75B was significantly downregulated when exposed to lufenuron. Knockdown of E75B significantly affects the expression of vitellogenin (Vg), Fushi tarazu factor 1 (Ftz-f1) and juvenile hormone acid O-methyltransferase (JHAMT), reducing fecundity in mites. The knockdown of Ftz-f1 produced a more significant effect than the knockdown of E75B, indicating that the ponasterone A pathway positively regulates fecundity in P. citri. After the knockdown of JHAMT, the expression levels of both Vg and Ftz-f1 and fecundity were significantly increased, along with the inhibition of Kr-h1, suggesting that JHAMT was negatively correlated with fecundity in the regulatory network. Knockdown of Kr-h1 inhibited the expression of Vg and Ftz-f1 and fecundity, and whether the drop in fecundity is caused by Kr-h1 or Ftz-f1 is unclear. Subsequent feeding with MF induced Kr-h1 and Vg expression, whereas no significant effects were observed for JHAMT and Ftz-f1. Therefore, the MF pathway stimulates fecundity independently. RNA interference (RNAi) showed that JHAMT and Ftz-f1 inhibited each other, resulting in opposite effects of MF and ponasterone A pathways on steady-state fecundity when either factor changed. Meanwhile, JHAMT knockdown led to increased fecundity, indicating that the stimulating effect of the ponasterone A pathway was greater than the inhibiting effect of the MF pathway, and demonstrating the dominant role of the ponasterone A pathway. Therefore, the interaction between JHAMT and Ftz-f1 may be closely associated with the maintenance of MF-ponasterone A regulatory network homeostasis and is involved in the reduction of fecundity in P. citri induced by exposure to lufenuron.

摘要: Entomopathogenic fungi have been widely used as the main mycoinsecticide for controlling agricultural and forest pests. The effector molecules of these mycopathogens have evolved to adapt to their hosts. The role of fungal effectors in evading the host immune system in insects remains mainly unclear. We characterized the widely distributed fungal effector necrosis-inducing-like secreted protein 1 (NLS1) in the entomopathogenic fungus Metarhizium robertsii. Our findings revealed the presence of M. robertsii NLS1 (MrNLS1) in host hemocytes during the early stage of hemocoel infection. MrNLS1 knock down (Delta MrNLS1) reduced fungal pathogenicity during infection and altered the expression of host immune genes. The molecular docking results and the yeast 2-hybrid assay confirmed that MrNLS1 interacts with the host defense protein Hdd11. The phylogenetic analysis indicated that Hdd11 is conserved across a broad range of Lepidoptera species. Knock down of hdd11 in Helicoverpa armigera, Bombyx mori, and Galleria mellonella markedly suppressed their immune responses against M. robertsii. However, no significant difference was observed in the mean lethal time between hdd11-knockdown Lepidoptera species infected with Delta MrNLS1 and those infected with wild-type M. robertsii. Therefore, in Lepidoptera insects, Hdd11 is essential for fungal defense. In conclusion, M. robertsii infects Lepidoptera insects by targeting host Hdd11 through its protein MrNLS1, thereby suppressing the host immune response. Our findings clarify the molecular mechanisms underlying fungal infection pathogenesis.

摘要: Oviposition preferences of plant-feeding predators remain a complex topic, as such omnivores choose oviposition sites by assessing both plant characteristics and the quality and quantity of nearby animal food sources. Orius predators are omnivores that oviposit endophytically, thus plant characteristics play an important role in their oviposition choices. In this study, we assessed the oviposition and foraging preferences of O. laevigatus and O. majusculus on vegetative and flowering chrysanthemum plants, and assessed the survival of their offspring on differently aged tissues. Our results show a preference of O. laevigatus for young and tender chrysanthemum tissues, where the survival of the nymphs was longer on a plant diet. In contrast, O. majusculus selected older plant parts when laying its eggs, and nymphs did not survive long on any of the plant tissues offered. The foraging activity of Orius females for animal prey (Ephestia kuehniella eggs) did not reveal any specific pattern for either of the two predators. Furthermore, we tested the plasticity of the within-plant oviposition preferences of O. laevigatus, by offering sentinel prey (E. kuehniella eggs) on distinct plant parts. We found that more eggs were laid in older plant tissue when animal prey was offered lower on the plant. Overall, our findings show that oviposition choices of Orius predators are based on a dynamic interplay between plant characteristics, presence of animal and/or floral food sources among other factors, and that differences may well occur between closely related species based on the importance of plant resources in their diet.

摘要: The clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9)-mediated gene editing technology has revolutionized the study of fundamental biological questions in various insects. Diverse approaches have been developed to deliver the single-guide RNA (sgRNA) and Cas9 to the nucleus of insect embryos or oocytes to achieve gene editing, including the predominant embryonic injection methods and alternative protocols through parental ovary delivery. However, a systematic comparative study of these approaches is limited, especially within a given insect. Here, we focused on revealing the detailed differences in CRISPR/Cas9-mediated gene editing between the embryo and ovary delivery methods in the beetle Tribolium castaneum, using the cardinal and tyrosine hydroxylase (TH) as reporter genes. We demonstrated that both genes could be efficiently edited by delivering Cas9/sgRNA ribonucleoproteins to the embryos by microinjection, leading to the mutant phenotypes and indels in the target gene sites. Next, the Cas9/sgRNA complex, coupled with a nanocarrier called Branched Amphiphilic Peptide Capsules (BAPC), were delivered to the ovaries of parental females to examine the efficacy of BAPC-mediated gene editing. Although we observed that a small number of beetles' progeny targeting the cardinal exhibited the expected white-eye phenotype, unexpectedly, no target DNA indels were found following subsequent sequencing analysis. In addition, we adopted a novel approach termed direct parental CRISPR (DIPA-CRISPR). However, we still failed to find gene-editing events in the cardinal or TH gene-targeted insects. Our results indicate that the conventional embryonic injection of CRISPR is an effective method to initiate genome editing in T. castaneum. However, it is inefficient by the parental ovary delivery approach.