The midgut of Aedes albopictus females expresses active trypsin-like serine peptidases

Abstract

BackgroundAedes 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.MethodsIn the present work, we further analyzed the peptidase expression in the midgut of sugar-fed females using 1DE-substrate gel zymography, two-dimensional electrophoresis (2DE), mass spectrometry (MS), and protein identification based on similarity.ResultsThe combination of zymography, in solution assays using fluorescent substrates and 2DE-MS/MS allowed us to identify the active serine peptidase “fingerprint” in the midgut of A. albopictus females. Zymographic analysis revealed a proteolytic profile composed of at least 13 bands ranging from ~25 to 250 kDa, which were identified as trypsin-like serine peptidases by using specific inhibitors of this class of enzymes. Concomitant use of the fluorogenic substrate Z-Phe-Arg-AMC and trypsin-like serine protease inhibitors corroborated the zymographic findings. Our proteomic approach allowed the identification of two different trypsin-like serine peptidases and one chymotrypsin in protein spots of the alkaline region in 2DE map of the A. albopictus female midgut. Identification of these protein coding genes was achieved by similarity to the A. aegypti genome sequences using Mascot and OMSSA search engines.ConclusionThese results allowed us to detect, identify and characterize the expression of active trypsin-like serine peptidases in the midgut of sugar-fed A. albopictus females. In addition, proteomic analysis allowed us to confidently assign the expression of two trypsin genes and one chymotrypsin gene to the midgut of this mosquito. These results contribute to the gene annotation in this species of unknown genome and represent a small but important step toward the protein-level functional and localization assignment of trypsin-like serine peptidase genes in the Aedes genus.

In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus

“…This assay corroborated the results observed by zymography and allowed a quantitative analysis of proteolytic activities at the distinct pH. In agreement with the results obtained previously for other Diptera, trypsin-like serine peptidases of C. quinquefasciatus are highly active at alkaline pH [ 34 , 52 , 53 , 55 ]. PMSF, a specific inhibitor of serine peptidases, revealed that the profile of active peptidases expressed in the midgut of females fed a sugar diet is due to serine peptidases (Fig.…”

“…Despite such factors that could impede the regular migration of the peptidases, we cannot rule out the possibility that some peptidases could be extensively processed at the post-translational level, increasing their apparent molecular mass in the gel and allowing their association with other proteins in the midgut extract [ 49 – 51 ]. Despite these possibilities, zymographic analysis is a highly reproducible method for the study of the proteolytic profiles in different Culicidae species, suggesting that such high molecular mass enzymes are common findings and that they are not experimental artifacts [ 34 , 52 , 53 ]. Similarly, other authors have observed such results in other insects [ 54 ].…”

Expression of active trypsin-like serine peptidases in the midgut of sugar-feeding female Anopheles aquasalis

“…The higher molecular mass bands could be due to the sample preparation procedure, in which the proteins are not completely denatured and reduced, enabling protein aggregation and/or oligomerization that slows the electrophoretic migration; therefore, the apparent molecular mass of peptidases may increase in the zymographic gel. Despite this possibility, the zymographic profiles observed here are highly reproducible and agree with previous reports of high molecular mass peptidase activities detected by zymographic methods [ 26 , 52 – 56 ].…”

“…aquasalis females that fed on sugar. In addition, our results show a drastic difference in the trypsin activity profile among Culicidae members fed on sugar [ 52 ]. These findings could suggest the existence of distinct mechanisms of trypsin regulation in such mosquito species.…”

French invasive Asian tiger mosquito populations harbor reduced bacterial microbiota and genetic diversity compared to Vietnamese autochthonous relatives

“…Although, this observation may also be explained by the relatively homogeneity of the habitats of Aedes spp. mosquitoes (low oxygen pressure, a pH comprised between 8 and 10) (Dillon and Dillon, 2004 ; del Pilar Corena et al, 2005 ; Saboia-Vahia et al, 2014 ), and the establishment of habitat-specific bacterial associations. However, some factors such as the type of nutrients ingested and temperature (not regulated in ectotherms) can strongly vary between French and Vietnamese populations.…”