Mesaqueuri Mota Nonato¹, Kemily Nunes da Silva Moya¹, Sofia Angiole Cavalcante¹, Kelly Regina Pereira da Silva¹, Claudia Maria Ríos-Velasquez¹, Juliana de Saldanha da Gama Fischer Carvalho², Giuseppe Palmisano³, Priscila Ferreira de Aquino¹

Aedes aegypti has high vector competence, transmitting arboviruses that cause dengue and Zika. Vector control has been the main strategy of health authorities to mitigate this problem. Among the control alternatives, fungus-based products have been studied due to their high specificity and low environmental impact. However, there are still gaps in the literature on the molecular level action of these microorganisms in Ae. aegypti. In this context, proteomics emerges as an approach that can identify and characterize proteins associated with the infection process and the host\'s immune response. This study aimed to identify the biochemical changes in Ae. aegypti eggs treated with Penicillium citrinum, analyzing the possible impact associated with post-hatching larval viability. For this purpose, Ae. aegypti eggs were obtained from the insectarium of the Laboratory Infectious Diseases Ecology in the Amazon (LIDEA/ILMD), while the P. citrinum strain was reactivated and acquired from the Amazon Fungus Collection of Fiocruz Amazônia. Thus, 600 eggs were exposed to the P. citrinum inoculum (1x10⁸ conidia/mL) for 10 days. Additionally, a control group was prepared with eggs free of fungal treatment. After this period, the group treated with fungal propagules and the control were macerated, the proteins precipitated with phenol and then solubilized in urea/thiourea (7M/2M). The samples were subjected to one-dimensional electrophoresis (SDS-PAGE) and in-gel tryptic digestion. The resulting peptides were desalted and analyzed using the nanoLC-MS/MS system coupled to the Orbitrap Exploris 120 mass spectrometer. The data obtained were analyzed using PatternLab for Proteomics V software, using the Peptide Spectrum Match (PSM) approach, and RStudio. As a result, 1,141 proteins with at least two unique peptides were obtained, of which 1,136 proteins are from Ae. aegypti and 5 belong to P. citrinum. In addition, the Venn diagram indicated that the two groups have 784 proteins in common, with 5 proteins exclusive to the fungus-treated eggs and 13 proteins present only in the control group. The CDF Score analysis indicated that an uncharacterized protein of fungal origin was the most abundant in the group exposed to P. Citrinum; this protein is possibly associated with serine-type endopeptidase activity and extracellular proteolysis processes. Therefore, the hypothesis is that this biomolecule may be acting in the degradation of host structures. It was also possible to observe that the samples treated with fungal propagules presented a prevalence of structural proteins, in addition to others associated with post-transcriptional regulation and metabolic processes. Among the upregulated proteins, one stands out for its association with lipid recognition and activation of immune responses, whereas the downregulated ones are related to glycolytic metabolism and carbohydrate degradation. Thus, through proteomic analysis, it was possible to identify proteins that may favor energy imbalance and regular embryo development. Therefore, the identification of proteins expressed under these conditions can provide information that may result in the development of more assertive biocontrol strategies for Aedes aegypti.

Agradecimentos: FAPEAM (POSGRAD Program and Notice No. 010/2021 - Priority Areas of Science, Technology, and Innovation), CAPES (Funding Code 001), and PROEP/ILMD-FIOCRUZ AMAZÔNIA – LDMAIS.