Trypsin-like serine peptidase profiles in the egg, larval, and pupal stages of Aedes albopictus

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.

Trypsin inhibitor fromLeucaena leucocephalaseeds delays and disrupts the development ofAedes aegypti,a multiple-disease vector

“…It is well known that mature eggs from Aedes mosquitos have the same serine proteolytic profile as the first larval instar. This is due to the fact that mature embryos are very similar to first instars, although their metabolism is lower than that of larvae . LTI is a water‐soluble protein with low molecular mass (around 20 kDa), and once in solution, this protein may enter into the eggshell through its micropyle pore, which has a diameter of 3.55 µm, and the influx of trypsin inhibitor may cause blockage of trypsin‐like enzymes or other proteases, which are essential for mature embryo nutrition at the first moments before hatching as a larva, possibly leading to death.…”

The Developmental Transcriptome ofAe. albopictus,a Major Worldwide Human Disease Vector

“…Following the larval stage, the mosquito then enters the pupal stage, the final aquatic stage in the mosquito's life cycle. Clusters 11, 12 and 15 include genes enriched for structural constituent of cuticle, oxidoreductase, peptidase, and serine-type peptidase activity, which are likely involved in immunity and the hydrolysis of nutrients (Saboia-Vahia et al 2013). Steroid biosynthetic and metabolic processes are also prevalent which suggests hormones like ecdysteroids, which are crucial for metamorphosis, are preparing the pupa to molt into an adult mosquito (Margam et al 2006).…”

Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions

“…The substrates were prepared in buffer containing 100 mM Tris-HCl pH 7.8 and 20 mM CaCl 2 . 40 L of each substrate (4 mM BAPNA or 3.5 mM SAPNA) plus 150 L buffer (pH 7.5, which has been shown to be the ideal pH for determining proteolytic activity in earlier studies (Cuervo et al, 2008;Saboia-Vahia et al, 2013;Xavier et al, 2005) and a closely related pH, 7.8) and 10 g ES alone or previously incubated for 10 min with 10 mM PMSF, TLCK or TPCK seeded per well in 96-well polysorb plates. The resulting mixtures were incubated for 1 h at 37 • C and optical density (OD) was measured at 405 nm using an MJ ELISA Multiskan reader.…”

“…According to the proteolytic enzymes described in insects so far, these molecules include a large complex of proteases whose most dominant family consists of serine proteases which have been reported as being the insects' main class of digestive enzymes (Casu et al, 1994;Johnston et al, 1995;Kerlin and Hughes, 1992;Sandeman et al, 1990;Young et al, 1996). This family plays several roles involving physiological mechanisms, such as hydrolysis of nutrients from food, blood clotting, the immune response, signal transduction, cell-cell recognition, hormonal activation, stress responses, inflammation and cell development (O'Connell et al, 2006).…”