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To elucidate the cascade effect between the V-type proton ATPase catalytic subunit A (V-ATPase A), a key regulator of cellular energy metabolism, and the autophagy-related gene HcAtg8 in the fall webworm Hyphantria cunea, the HcAtg8 gene was cloned by PCR. The bioinformatic characteristics and phylogenetic relationships were analyzed. The HcAtg8 protein was expressed in vitro using a prokaryotic expression system and detected via Western blot. Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of HcAtg8 in different developmental stages and tissues (foregut, midgut, and hindgut). Additionally, after silencing HcV-ATPase A, histopathological changes in midgut tissues related to autophagy and the expression of HcAtg8 were examined. The results showed that the cloned coding region of HcAtg8 was 354 bp in length, encoding 117 amino acids. Phylogenetic analysis indicated that HcAtg8 clustered closely with BmAtg8 from Bombyx mori, suggesting a close evolutionary relationship. A recombinant expression vector pMAL-c2X-HcAtg8 was constructed, and a 56.4 kD HcAtg8 protein was obtained by prokaryotic expression in vitro, with the highest expression level observed after 8 h of induction. HcAtg8 was expressed across all developmental stages and gut tissues, with the highest expression observed in pupae. Among gut tissues, expression was highest in the midgut. After silencing HcV-ATPase A, the number of autophagosomes in the treated group was 11.0, significantly higher than that in the control group (3.5), representing a 3.1-fold increase. The number of lipid droplets in the treated group was 73.8, significantly higher than that in the control group (12.5), representing a 5.9-fold increase. Silencing HcV-ATPase A disrupted the acidic environment of lysosomes, leading to the accumulation of autophagosomes and related metabolites. qRT-PCR results showed that the expression level of HcAtg8 was 6.1-fold higher than that of the control at 24 h, peaked at 375.2-fold at 48 h, and subsequently decreased to 2.5-fold at 72 h, consistent with the histological observations. These results indicate that silencing HcV-ATPase A leads to the upregulation of HcAtg8 expression and the accumulation of autophagosomes and lipid droplets, thereby disrupting midgut cellular homeostasis, and resulting in cellular dysfunction or death.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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