General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor

Abstract

The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a “shotgun” approach, based on a general up regulation of a large set of endoproteinases.

Introduction

Since their discovery, plant proteinase inhibitors have been considered potential defense compounds against insects and pathogens (Ryan, 1990). One of the problems of using proteinase inhibitor proteins for crop protection, however, is that some of the pests do not show the expected adverse effects caused by the presence of the inhibitors in their diets (Purcell et al., 1992). Broadway and Duffey (1986), in a seminal work, showed that an increase in gut proteinase activities is one of the strategies of insects to cope with the presence of proteinase inhibitors in their diets. A closer look into the adaptation of insects to the inhibitors was made later on by Maarten A Jongsma's group using Spodoptera exigua and the potato proteinase inhibitor (PPI) II (Jongsma et al., 1995). They observed that larvae of S. exigua are able to adapt to the proteinase inhibitors by inducing gut proteinase activity that is insensitive to the inhibitors. Since then, inhibitor adaptation has attracted the interest of several researchers, who aim at understanding the adaptation mechanism, to ultimately design better strategies to utilize proteinase inhibitors in crop protection (Broadway and Villani, 1995; Bolter and Jongsma, 1995; Brito et al., 2001).

Spodoptera frugiperda, better known as fall armyworm, is a generalist insect that feeds on important crops. Although S. frugiperda mainly derives its proteinase activity from midgut serine proteinases (Ferreira et al., 1994a, Ferreira et al., 1994b), the insect larvae show a remarkable capacity to adapt to the presence of soybean proteinase inhibitors (SPI) in their diets (Paulillo et al., 2000).

A persistent question concerning the mechanism of adaptation is if changes in endoproteinases in the guts of insects are a matter of de novo synthesis, or a general up regulation of the entire arsenal of the insect's proteinases, or a selective increase in just the inhibitor-insensitive proteinases. The present work examines the gene expression responses of endoproteinases of S. frugiperda when challenged by SPI. We identified 13 different chymotrypsins and 9 different trypsins. Of those, we were able to follow the gene expression of 8 chymotrypsins and 3 trypsins during an adaptation period of 48 h. The proteinase activity elicited by the presence of the SPI in the diet and the set of genes found to be up regulated point to a general up regulation of a large set of endoproteinases. Altogether, the data indicate that S. frugiperda caterpillars make use of a proteinase “shotgun” approach to cope with the presence of serine proteinase inhibitors in their diet.