Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

Abstract

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min -1 mg protein -1 , at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min -1 mg protein -1 . The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC 50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC 50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

Mining an Ostrinia nubilalis midgut expressed sequence tag (EST) library for candidate genes and single nucleotide polymorphisms (SNPs)

“…High pH largely determines redox state of the midgut, and affects the solubility and structure of lepidopteran digestive enzymes (Johnson & Felton, 1998). Physiological conditions may have resulted in the selection of high pH optima among lepidopteran digestive enzymes that are secreted into the lumen by midgut tissues (Pereira et al ., 2005). Midgut physiology also is influenced by the pH and secondary compounds of ingested plant material (Johnson & Felton, 1998), whereby midgut enzyme structure and expression also has adapted to function in the presence of plant defensive compounds (Jongsma et al ., 1995; Duffy & Stout, 1996).…”

Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner)

“…Membranebound trypsin activity has been described in lepidopteran larvae gut cells and there are some evidences that it is the precursor of the trypsin soluble form (JORDÃO et al, 1999). In A. gemmatalis, the membrane-bound trypsin-like activity corresponded to 18% of total trypsin-like activity in the midgut (PEREIRA et al, 2005). Thus, in order to improve the knowledge about the A. gemmatalis digestive proteases, this study describes the purification of an anionic membrane-bound trypsin-like enzyme from the fifth instar larvae.…”

“…Evidences that in these insects the soluble trypsin is derived from a trypsin form that is associated with vesicle membranes led to a model for trypsin secretion in larval midgut (JORDÃO et al, 1999). Membranebound trypsin-like activity has already been reported in gut extracts from A. gemmatalis, but no further purification was attempted (OLIVEIRA et al, 2005;PEREIRA et al, 2005;. Considering the economical relevance of soybean culture, the damages caused by A. gemmatalis attack and the fundamental role of trypsin-like enzymes in these insect digestion, analysis of biochemical aspects of its digestive processes involving enzyme characterization is important to go further on the development of pest control methods acting through the digestive system, especially based on disruption of protein metabolism using proteinase inhibitors.…”

Caracterização enzimática de isoformas de cisteíno protease de Anticarsia gemmatalis (Hübner, 1818)

“…Quando essas são inibidas in vitro, torna-se aparente a atividade de cisteíno protease, desde que obedecidas as exigências reacionais dessa classe de proteases, ou seja, adição de um agente redutor para garantir que a cisteína do centro ativo esteja cataliticamente disponível e, caso a enzima não esteja purificada, a adição de um inibidor de serino protease. Pereira et al (2005) utilizaram azocaseína para medir atividade proteásica de A. gemmatalis e afirmaram que esse inseto não possui atividade de cisteíno protease em seu intestino, quando utilizaram 2-marcaptoetanol e L-cisteína no meio de reação, os quais não aumentaram a hidrólise de azocaseína, porém não utilizaram um inibidor de serino protease. Quando utilizaram o inibidor de cisteíno protease ácido iodoacético, a atividade proteásica total caiu 4%.…”