Most of the information on the proteolytic enzymes of mosquitoes is comprised from data collected at the adult stages and is often directed towards the understanding of blood digestion [38–40]. The aim of this study was to investigate the expression of active peptidases from the preimaginal stages of Ae. albopictus. To this end, we characterized the proteolytic profile from whole extracts of eggs, larvae and pupae using SDS-substrate gel. The peptidase activities were dependent on the time, pH and temperature of incubation. The proteolytic profile from all of the analyzed stages was composed primarily of trypsin-like serine peptidases, and each stage exhibited a specific and complex profile of these enzymes. Additionally, 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.
Although the genome of Ae. albopictus has not yet been completely sequenced, the expression of multiples genes coding for trypsin-like serine peptidases has been reported in the genome of different mosquitoes species, such as Anopheles gambiae, Ae. aegypti and Culex quinquefasciatus, accounting for 345, 380 and 403 putative genes, respectively [41–44]. Compared to other diptera species, such as Drosophila melanogaster, which has 260 genes encoding trypsin-like serine peptidases, the mosquito genome exhibits a larger genetic repertoire of these genes, likely due to gene expansion events [45, 46]. The maintenance of a variety of trypsin-like coding genes may be related to their key role in numerous physiological processes of the insect, such as digestion, immunity, reproduction, development, signal transduction and wound healing [47–52]. However, the characterization of active trypsin-like peptidases is scarce, and little is known about the biochemical nature of these enzymes in the preimaginal stages.
In the present study, we observed that Ae. albopictus larval instars displayed a clear proteolytic pattern that was detected after 2 hours of reaction. In contrast, it has been reported that Ae. aegypti exhibits a zymographic profile after only 1 hour of reaction . These results suggest that the larval peptidases in these species present (i) distinct kinetic activities or (ii) differential quantitative expression. Such differences could also be related to species-specific nutritional characteristics of the larvae. In this regard, the larvae of Ae. albopictus feed more slowly and eat smaller amounts of nutrients at one time compared to the larvae of Ae. aegypti. This slower intake of food could lead to slower or reduced activation/expression of peptidases involved in nutrient digestion. Post-feeding induction of peptidase expression has been observed, particularly in adults of Ae. aegypti[53–55]. Here, zymographic analyses revealed that the expression of peptidases increases at each larval instar. This result was corroborated by the quantitative in-solution assays using the fluorogenic substrate Z-Phe-Arg-AMC. Quantitative differential expression in larval instars has also been observed by other authors, specifically in the intestine, using alternative methods [21, 39, 56]. In the present study, the proteolytic classes of the enzymes were characterized using different inhibitors, revealing that peptidases from the four larval instars were inhibited by PMSF, an inhibitor of trypsin and chymotrypsin, and 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]. Although no other peptidase classes were detected under the conditions used here, we cannot rule out the possibility that other classes of peptidases could be detected under different experimental conditions.
The four larval instars of Ae. albopictus showed proteolytic activity in a broad range of pH values, with optimal activity between pH 7.5 and 10. Accordingly, the larvae of Ae. aegypti, An. stephensi and Cx. quinquefasciatus displayed high enzymatic activities at an alkaline pH [22, 23, 58]. These strong proteolytic activities may be associated with larval nutrition because these stages are highly detritivorous and need to eat large amounts of food to obtain their basic nutrients. Indeed, the main peptidases expressed by larvae are thought to be involved in the processing of nutrients and have optimal activity at an alkaline pH [13, 58]. In addition, we demonstrated that larval instars exhibit a complex profile of active peptidases composed by at least eight bands of trypsin-like serine peptidases. These results are in agreement with a recent transcriptome study showing that 12 serine peptidases-like genes were preferential expressed in the larvae of Ae. aegypti[39, 59]. The broad spectrum of enzymatic activities detected in the larvae of Ae. albopictus could be related to their survival in aquatic environments that are lacking nutrients [13, 58].
In addition, the zymographic patterns of larvae from Ae. albopictus and Ae. aegypti share a band of proteolytic activity with the same intensity at approximately 28 kDa. Additionally, several bands between 36 and 72 kDa observed in the enzymatic profile of Ae. albopictus match those of Ae. aegypti, although with different intensities. These data could indicate that (i) the expression of genes coding for some active trypsin-like serine peptidases isoforms is conserved among Aedes species and (ii) that other isoforms are species-specific. In fact, this conservation is expected given the importance of these enzymes for the life cycle of these mosquitoes [21, 56, 59]. On the other hand, bands of proteolytic activities with different intensities within the same stage of development, among distinct stages and between the two species may indicate that some genes coding for trypsin are differentially expressed or that some isoforms with specific catalytic features are differentially regulated during the life cycle of the insect. Complex mechanisms regulating the expression of trypsin in insects have been previously described [39, 53–56, 60].
We next sought to investigate the stability of the proteolytic profile in larvae obtained from the eggs of females reared in a colony for long time periods compared with larvae hatched from eggs collected in the natural environment. Our results demonstrate that the enzymatic pattern did not change, suggesting that the qualitative expression of peptidases is stable under the experimental conditions used here. In other words, such stability suggests that the genes coding for these enzymes could be under strong selective pressure so that the proteolytic profile is maintained in both natural and colony conditions. These results indicate that inbreeding during prolonged maintenance of the colony does not alter the expression of the genes coding for the trypsin-like serine peptidases detected in zymographic assays of the larvae. In addition, such observations suggest that trypsin-like serine peptidases of Ae. albopictus could be constitutively expressed during all developmental stages.
The proteolytic profile of Ae. albopictus eggs was similar to that exhibited by the larval instars, specifically the L1 instar, in the number and intensity of bands. Three bands of enzymatic activity between 17 and 28 kDa were observed in both the eggs and larvae, although they displayed different intensities. The similarity of the active peptidase profile observed between the eggs and L1 may be explained by the fact that the eggs were assayed at the final stage of maturation when the embryo is very similar to the young L1. In addition, the proteolytic activities from the eggs likely had lower intensity because before they hatched, the larvae were lethargic, presenting low metabolism inside the eggs.
The pupal stage exhibited a complex and stage-specific proteolytic profile, composed of six bands of activity. Compared to the larval profile, which showed activities in a wide pH range (3.5 – 10), the pupal stage presented activities exclusively between pH 7.5 and 10. Peptidases from the larval extracts could be observed after 2 hours of incubation, whereas peptidases from the pupal stage were detected only after 24 hours of reaction. These observations indicate a reduction in the expression of active digestive peptidases after the last larval molt . However, although pupae do not feed, serine peptidase activities could be related to the proteolysis of the remaining larval tissue during metamorphosis. In support of this idea, in Sarcophaga peregrina, a 26 kDa trypsin protein was isolated from the corpus luteum, an organ that develops temporarily in the pupae and serves to disintegrate the gut of immature stages and reshape it to form the adult insect's gut [50, 61–63].
The effect of temperature on trypsin-like serine peptidase activities from larvae was also tested. When compared to the control condition (37°C), the proteolytic activities of all larval instars were susceptible to low temperatures (4°C and 10°C) and highly stimulated at high temperatures (50°C). Although enzymatic activity decayed at 60°C, a clear profile could still be observed at this temperature. The strong decrease of proteolytic activity could be due to the thermal denaturation of the enzymes. Similar results were described for trypsin and chymotrypsin from the larvae of Tenebrio molitor[64, 65] and for serine peptidases from Oestrus ovis and Ae. aegypti. Because temperature is one of the most important environmental conditions for sustaining life on earth, the adaptation of an organism to extreme environments requires the optimization of its enzymatic repertoire. In this sense, the investigation of thermostable proteins, which are highly conserved in phylogenetically related groups of organisms, could help to identify changes in amino acid sequences that could be associated with thermal adaptation . Because Ae. aegypti and Ae. albopictus are related species that originated from different environments, a detailed study comparing the sequence and structure of the trypsin isoforms could provide important information on the molecular basis of the thermal stability of these enzymes.