Abstract
BackgroundAedes albopictus, a ubiquitous mosquito, is one of the main vectors of dengue and yellow fever, representing an important threat to public health worldwide. Peptidases play key roles in processes such as digestion, oogenesis, and metamorphosis of insects. However, most of the information on the proteolytic enzymes of mosquitoes is derived from insects in the adult stages and is often directed towards the understanding of blood digestion. The aim of this study was to investigate the expression of active peptidases from the preimaginal stages of Ae. albopictus.MethodsAe. albopictus eggs, larvae, and pupae were analyzed using zymography with susbtrate-SDS-PAGE. The pH, temperature and peptidase inhibitor sensitivity was evaluated. In addition, the proteolytic activities of larval instars were assayed using the fluorogenic substrate Z-Phe-Arg-AMC.ResultsThe proteolytic profile of the larval stage was composed of 8 bands ranging from 17 to 130 kDa. These enzymes displayed activity in a broad range of pH values, from 5.5 to 10.0. The enzymatic profile of the eggs was similar to that of the larvae, although the proteolytic bands of the eggs showed lower intensities. The pupal stage showed a complex proteolytic pattern, with at least 6 bands with apparent molecular masses ranging from 30 to 150 kDa and optimal activity at pH 7.5. Peptidases from larval instars were active from 10°C to 60°C, with optimal activity at temperatures between 37°C and 50°C. The proteolytic profile of both the larval and pupal stages was inhibited by phenyl-methyl sulfonyl-fluoride (PMSF) and Nα-Tosyl L-lysine chloromethyl ketone hydrochloride (TLCK), indicating that the main peptidases expressed during these developmental stages are trypsin-like serine peptidases.ConclusionThe preimaginal stages of Ae. albopictus exhibited a complex profile of trypsin-like serine peptidase activities. A comparative analysis of the active peptidase profiles revealed differential expression of trypsin-like isoforms among the preimaginal stages, suggesting that some of these enzymes are stage specific. Additionally, a comparison of the peptidase expression between larvae from eggs collected in the natural environment and larvae obtained from the eggs of female mosquitoes maintained in colonies for a long period of time demonstrated that the proteolytic profile is invariable under such conditions.
Highlights
Aedes albopictus, a ubiquitous mosquito, is one of the main vectors of dengue and yellow fever, representing an important threat to public health worldwide
A comparison of the peptidase expression between the larvae from eggs collected from a natural environment and larvae obtained from eggs of female mosquitoes maintained in colonies for a long period of time showed that the proteolytic profile is invariable under these conditions
The proteolytic classes of the enzymes were characterized using different inhibitors, revealing that peptidases from the four larval instars were inhibited by phenyl-methyl sulfonyl-fluoride (PMSF), an inhibitor of trypsin and chymotrypsin, and Tosyl L-lysine chloromethyl ketone hydrochloride (TLCK), an inhibitor of trypsin. These results indicate that the enzymes were predominantly trypsin-like serine peptidases in larval instars of Ae. albopictus, which is in agreement with previous descriptions that suggest the occurrence of trypsin in other species from diptera [13,21,24,25,57]
Summary
A ubiquitous mosquito, is one of the main vectors of dengue and yellow fever, representing an important threat to public health worldwide. During the last three decades, the mosquito Aedes (Stegomyia) albopictus has spread from Southeast Asia to Africa, the Middle East, Europe and America This species has demonstrated a strong ecological plasticity that allows for rapid adaptation to diversified habitats, including urban environments [1,2,3,4,5,6]. Within the serine peptidase family, trypsin and chymotrypsin are the most abundant digestive enzymes in the midgut of several insect species [17]. These enzymes are characterized as having a His-Asp-Ser catalytic triad and the same basic tridimensional structure, consisting of two six-stranded β-barrels that contain the active site, the substrate recognition region and the zymogen activation domain [18]. Despite the high similarity of serine peptidases among mosquito species, each enzyme has a unique set of accessory catalytic residues that are thought to be important for determining substrate specificity [19,20]
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