Kemily Nunes da Silva Moya ¹, Sofia Angiole Cavalcante ¹, Mesaqueuri Mota Nonato¹, Kelly Regina Pereira da Silva ¹, Claudia Maria Ríos-Velasquez ¹, Juliana de Saldanha da Gama Fischer Carvalho ², Giuseppe Palmisano ³, Priscila Ferreira de Aquino ¹

The anticipation of larval hatching in Aedes aegypti eggs exposed to the fungus Purpureocillium lilacinum CFAM0094 represents an intriguing biological phenomenon with relevant implications for vector control. Although the virulence of other entomopathogenic fungi is well documented, the molecular mechanisms underlying early host responses remain poorly understood. Proteomic analysis, through its ability to map global changes in protein expression, offers a powerful approach to investigate such interactions with functional insight. Accordingly, this study aimed to apply shotgun proteomics to identify molecular mechanisms potentially involved in the induction of premature larval hatching in Ae. aegypti eggs exposed to P. lilacinum. The fungal strain P. lilacinum CFAM0094 was reactivated from the Amazon Fungal Collection (CFAM) of the Leônidas & Maria Deane Institute (ILMD/Fiocruz Amazônia). Ae. aegypti eggs were obtained from adult mosquitoes maintained at the Insectary of the Laboratory of Ecology of Transmissible Diseases in the Amazon (LEDTA/ILMD). For proteomic analysis, ovicidal bioassays were conducted using 600 eggs per replicate, aged 3 to 5 days, exposed to 1x10⁷ conidia.mL⁻¹ of the fungal strain for five days. Micotized eggs were homogenized using glass beads (5 mm) and subjected to protein extraction with phenol–ammonium acetate 0.1 M, followed by SDS-PAGE and in-gel tryptic digestion. Peptides were analyzed by nanoLC-MS/MS on an Orbitrap Exploris 120 mass spectrometer. Bioinformatic analysis was performed using PatternLab V software. Functional annotation included Gene Ontology (GO) enrichment, KEGG pathway analysis, and protein-protein interaction mapping. A total of 1,008 proteins were identified in the fungal-exposed egg group, of which 362 were exclusive to this condition. Additionally, 458 fungal proteins were detected, including infection-related enzymes such as subtilisin-like proteases, catalase-peroxidase, and carbohydrate-binding domain proteins. In total, 130 host proteins showed differential abundance (49 upregulated and 81 downregulated). Upregulated proteins were associated with amino acid and fatty acid catabolism and peptidase activity, including 3-hydroxybutyrate dehydrogenase, uricase, and serine protease inhibitors, suggesting metabolic reprogramming and stress response. Downregulated proteins included paramyosin, ribosomal subunits, translation initiation factor eIF3 subunit L, and other structural components, indicating suppression of protein synthesis and loss of cellular integrity. GO and KEGG analyses reinforced this pattern: the upregulated group was enriched in catabolic pathways, while the downregulated group was primarily involved in translation processes and redox metabolism. The protein–protein interaction network of the downregulated proteins, revealed functionally organized connections centered around the ribosome, indicating a coordinated translational shutdown in response to infection. Altogether, these findings suggest that P. lilacinum triggers an intense metabolic response in Ae. aegypti eggs, marked by the activation of catabolic pathways and the coordinated suppression of protein biosynthesis. This highlights the fungus\'s entomopathogenic potential and suggests that the observed premature hatching may be linked to stress-induced developmental modulation. Therefore, this study contributes to advancing innovative and sustainable strategies for the control of arbovirus vectors. 

Agradecimentos: The authors thanks FAPEAM (POSGRAD Program and Public Call No. 010/2021 – Priority Areas in CT&I), CAPES (Funding Code 001), and PROEP/ILMD-FIOCRUZ AMAZÔNIA – LDMAIS for financial support