Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

Abstract

We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.Keywords: Oxysarcodexia thornax -Sarcophagidae -chymotrypsin-like serine proteasesThe genus Oxysarcodexia belongs to the Sarcophagidae family (flesh flies) whose major biological feature is the ovo-larvipary (Pape 1996). Females may deposit first instar larvae directly onto the vertebrate host at predisposing sites (causing secondary myiasis), carcasses, decomposing organic matter, and dung (Panu et al. 2000). Genus Oxysarcodexia is widely distributed in the Neotropical regions (Lopes 1946), and in Brazil, O. thornax (Walker) is found in both urban and rural areas of several states. This species is abundant in the state of Rio de Janeiro, occurring preferentially in the summer season, with the population peak between January and February (Oliveira et al. 2002).In biological systems, proteases may carry out (i) a regulatory function by activation or inactivation of specific proteins via selective proteolysis, and (ii) a nonspecific proteolytic function involved in general protein hydrolysis. Such proteolytic mechanisms are highly regulated and are involved in a variety of physiologic processes, such as programmed cell death, stress responses (heat shock and anoxia), skeletal muscle atrophy, cellcell recognition, signal transduction and learning, morphogenesis, and photoreceptor light adaptation (Mykles 1998). Different classes of proteases such as cysteine proteases, metalloproteases and serino proteases have been described both in adults and larvae of several species of the Diptera (Han et al. 1997, Rosenfeld & Vanderberg 1998, Cho et al. 1999, Noriega et al. 2002, Vierstraete et al. 2003, Okuda et al. 2005, Fazito do Vale et al. 2007, Pires et al. 2007, Rodrigues et al. 2007). Despite the existence of information about morphology, taxonomy and ecology of this genus, as far as we know, no data has been published on the biochemical traits of O. thornax. In the present study, the proteolytic profile of O. thornax first instar larvae was investigated using zymographic analysis. O. thornax adults were collected as previously described (Oliveira et al. 2002) and females were kept in a 50 ml plastic vial for 24 h at room temperature. Following death of the females, abundant live first instar larvae (L1) were released and immediately collected. Larvae were ...

Proteolytic profiling and comparative analyses of active trypsin-like serine peptidases in preimaginal stages of Culex quinquefasciatus

“…aegypti and Lu. longipalpis were shown to exhibit serine peptidase activity over a wide range of alkaline pH [32,33,44]. …”

“…The resulting extracts were centrifuged at 14000 ×  g for 30 min at 4°C to remove insoluble material and protein concentration was determined using the Pierce Protein assay, following the manufacturers protocol. Afterwards, samples were resolved as previously described [32]. Briefly, 10 μg of protein from each sample was mixed with SDS-PAGE sample buffer (125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 0.002% bromophenol blue) and loaded in 12% or 10% polyacrylamide gels co-polymerized with 0.1% porcine gelatin for separation at 4°C with a constant voltage of 110 V. Peptidase activity was detected as previously reported [33] with few modifications.…”

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

“…albopictus and Culex quinquefasciatus larvae [21-23]. These enzymes have also been associated with the digestion of nutrients by larvae of other diptera species, such as Dermatobia hominis, Lutzomya longipalpis , Musca domestica , and Oxysarcodexia thornax [24-27]. In addition, trypsins secreted in the lumen of the gut have been implicated in the process of pathogen establishment in vector mosquitoes.…”

CARACTERIZAÇÃO BIOQUÍMICA DO PRODUTO DE EXCREÇÃO/SECREÇÃO DE LARVAS DE Cochliomyia hominivorax (DIPTERA: CALLIPHORIDAE)

“…Por exemplo, em O. ovis, a atividade enzimática máxima dos produtos E/S de L3 foi observada em pH 8,0 (20) . O pH ótimo observado para as proteases oriundas de produtos E/S de L. sericata, L. cuprina e Oxysarcodexia thornax esteve entre 7,0 e 9,5 (9,21,22) . Ainda em L. cuprina, Guerrini et al (23) relataram que o maior crescimento e sobrevivência das larvas foi conseguido com pH 8,0-9,0 em ovelhas infestadas artificialmente.…”

“…Em O. ovis (3) e em L. cuprina (35) , a clivagem de IgG foi atribuída a uma protease digestiva identificada como serina protease. Estudos realizados por Cuervo et al (22) identificaram também que as principais proteases expressas por O. thornax pertencem à classe de serina proteases. Pires et al (27) detectaram atividades de cisteína e serina proteinases em larvas de segundo e terceiro estádios de D. hominis e mostraram uma maior atividade de serina proteinase quando comparada à atividade de cisteína proteinase para esta espécie.…”