Background: The mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus. Methods: The proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 μM of the fluorogenic substrate Z-Phe-Arg-AMC. Results: Comparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C. Conclusion: The combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the repertoire of proteolytic enzymes in preimaginal stages of the insect. Finally, differences in the trypsin-like serine peptidase profile of preimaginal stages were observed, suggesting that such enzymes exert specific functions during the different stages of the life cycle of the insect.

Anopheles aquasalis is a dipteran of the family Culicidae that is widely distributed in the coastal regions of South and Central America. This species acts as a vector of Plasmodium vivax, an important etiological agent of malaria, which represents a serious public health problem. In mosquitoes, trypsin-like serine proteases are important in blood meal digestion, immune responses and reproductive functions. The study of peptidases expressed in the mosquito midgut is essential to understanding the mechanisms of parasite-host interaction and the physiological process of nutrient digestion. Our study aimed to identify and characterize the proteolytic activities in the midgut of sugar-fed An. aquasalis females using zymographic analyses (substrate-SDS-PAGE), in-solution assays and mass spectrometry. Here, we used a zymographic analysis to further biochemically characterize the proteolytic profile of the midgut of sugar-feeding An. aquasalis females. The trypsin peptidases migrated between ~17 and ~76 kDa and displayed higher proteolytic activities between pH 7.5 and 10 and at temperatures between 37 °C and 50 °C. Four putative trypsin-like serine peptidases were identified using mass spectrometry and data mining. The molecular masses of these peptidases were similar to those observed using zymography, which suggested that these peptidases could be responsible for some of the observed proteolytic bands. Taken together, our results contribute to the gene annotation of the unknown genome of this species, to the tissue location of these peptidases, and to the functional prediction of these crucial enzymes, which all impact further studies of this species.

Aedes albopictus is widely distributed across tropical and sub-tropical regions and is associated with the transmission of several arboviruses. Although this species is increasingly relevant to public health due its ability to successfully colonize both urban and rural habitats, favoring the dispersion of viral infections, little is known about its biochemical traits, with all assumptions made based on studies of A. aegypti. In previous studies we characterized the peptidase profile of pre-imaginal stages of A. albopictus and we reported the first proteomic analysis of the midgut from sugar-fed females of this insect species.

Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins. We used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features. Zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far. We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.

Visceral leishmaniasis (VL) is a neglected disease caused by Leishmania parasites. Although significant morbidity and mortality in tropical and subtropical regions of the world are associated with VL, the low investment for developing new treatment measures is chronic. Moreover, resistance and treatment failure are increasing for the main medications, but the emergence of resistance phenotypes is poorly understood at the protein level. Here, we analyzed the development of resistance to miltefosine upon experimental selection in a L. infantum strain. Time to miltefosine resistance emergence was ~six months and label-free quantitative mass-spectrometry-based proteomics analyses revealed that this process involves a remodeling of components of the membrane and mitochondrion, with significant increase in oxidative phosphorylation complexes, particularly on complex IV and ATP synthase, accompanied by increased energy metabolism mainly dependent on β-oxidation of fatty acids. Proteins canonically involved in ROS detoxification did not contribute to the resistant process whereas sterol biosynthesis enzymes could have a role in this development. Furthermore, changes in the abundance of proteins known to be involved in miltefosine resistance such as ABC transporters and phospholipid transport ATPase were detected. Together, our data show a more complete picture of the elements that make up the miltefosine resistance phenotype in L. infantum.

Aedes albopictus is a vector for several fatal arboviruses in tropical and sub-tropical regions of the world. The midgut of the mosquito is the first barrier that pathogens must overcome to establish infection and represents one of the main immunologically active sites of the insect. Nevertheless, little is known about the proteins involved in the defense against pathogens, and even in the processing of food, and the detoxification of metabolites. The identification of proteins exclusively expressed in the midgut is the first step in understanding the complex physiology of this tissue and can provide insight into the mechanisms of pathogen-vector interaction. However, identification of the locally expressed proteins presents a challenge because the Ae. albopictus genome has not been sequenced.