Protein digestion in cereal aphids (Sitobion avenae) as a target for plant defence by endogenous proteinase inhibitors

https://doi.org/10.1016/j.jinsphys.2011.03.024Get rights and content

Abstract

Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo. Protein could partially substitute for free amino acids in diet, although it could not support complete development. Recombinant wheat proteinase inhibitors (PIs) fed in diet were antimetabolic to cereal aphids, even when normal levels of free amino acids were present. PIs inhibited proteolysis by aphid gut extracts in vitro, and digestion of protein fed to aphids in vivo. Wheat subtilisin/chymotrypsin inhibitor, which was found to inhibit serine and cysteine proteinases, was more effective in both inhibitory and antimetabolic activity than wheat cystatin, which inhibited cysteine proteases only. Digestion of ingested protein is unlikely to contribute significantly to nutritional requirements when aphids are feeding on phloem, and the antimetabolic activity of dietary proteinase inhibitors is suggested to result from effects on proteinases involved in degradation of endogenous proteins.

Highlights

► Cereal aphids can hydrolyse ingested proteins and utilise their amino acids. ► Cysteine proteases with chymotrypsin-like specificity are present in gut extracts. ► Recombinant wheat protease inhibitors can block activity of aphid gut proteases. ► Recombinant wheat protease inhibitors are antimetabolic to cereal aphids. ► Effects of protease inhibitors are indirect, and not dependent on protein digestion.

Introduction

Aphids (order Hemiptera; superfamily Aphidoidae) are pests of cultivated and wild plants. They are responsible for major crop losses in world agriculture, not only by direct effects on plant growth, but also by acting as vectors for transmission of viruses. Aphids have a specialised feeding habit, extracting phloem and/or xylem sap from vascular tissue by means of a stylet, a piercing tubular structure formed by fusion of the normal insect mouth parts. The aphid diet is determined by the composition of the sap which is ingested, and thus is normally sugar-rich, but deficient in nitrogen. Phloem sap contains a limited selection of free amino acids, mainly aspartate, glutamate, asparagine, glutamine and serine, as the main nitrogen source (Douglas, 2006). The symbiotic association between aphids and bacteria like Buchnera aphidicola enables the insect to use a diet which is nutritionally inadequate, by exploiting the capacity of the bacteria to synthesise amino acids lacking in the diet (Gunduz and Douglas, 2009).

Besides free amino acids, phloem sap also contains a variety of proteins, including small peptides, enzymes, redox regulators, proteinase inhibitors, and lectins (Kehr, 2006), as a potential nitrogen source for nutrition. Protein concentrations in phloem sap vary from 0.3 to as high as 60 mg ml−1 depending upon plant species. Although the view that aphids and other sap-feeding hemipterans do not carry out proteolysis of ingested phloem sap proteins has been widely stated, evidence from both biochemistry and molecular biology suggests that this is not the case, and that the capacity for digestive proteolysis is present. Assays of gut extracts from the sap-feeding hemipteran rice brown planthopper (Nilaparvata lugens) showed that proteolytic activity was present, with contributions from both serine and cysteine proteinases; cDNA species encoding both types of enzyme were isolated from gut tissue (Foissac et al., 2002). The hemipteran plant bug, Lygus hesperus, has been shown to be able to digest ingested protein by using tracking experiments following green fluorescent protein and casein through the digestive system (Habibi et al., 2002), confirming earlier results in which the hemipteran silverleaf whitefly (Bemesia argentifolii) was fed 35S labelled cotton leaf proteins, which were digested to free amino acids excreted via honeydew or used in de novo protein synthesis (Salvucci et al., 1998).

Aphids appear to use cysteine proteinases as the major type of enzyme for protein digestion in the gut. The major proteolytic activity in the gut of pea aphid (Acyrthosiphon pisum) was characterised as cathepsin L-like (Cristofoletti et al., 2003), and a gut-specific cathepsin-L like cysteine proteinase from the cotton aphid, Aphis gossypii, was cloned and characterised (Deraison et al., 2004). The determination of a genome sequence for pea aphid has shown that this insect contains a large family of genes coding for cathepsin-B like cysteine proteinases, many of which were expressed at relatively high levels in gut (Rispe et al., 2007).

If proteolysis in the aphid gut makes a significant contribution to nutrition, then these insects may be subject to the antimetabolic effects of plant protein proteinase inhibitors (PIs). PIs have an established role in defence of plants against herbivorous insect pests which feed by chewing and ingesting plant tissue; the wounding response leads to synthesis of PIs in Solanaceae and other plant species. While feeding of aphids on some plant hosts (tomato) does not elicit synthesis of PIs via the wounding response (Stout et al., 1998), in other cases (barley) PIs are produced in response to aphid feeding (Casaretto and Corcuera, 1998).

Results from bioassays based on artificial diets have shown that PIs can produce antimetabolic effects when fed to aphids. In agreement with reports of cathepsin L-like activity in aphid gut, oryzacystatin I (OC-I), a cysteine proteinase inhibitor from rice (Oryzacystatin-OC-I) caused growth reductions of up to 40% and reduced fecundity in pea aphid (A. pisum), cotton aphid (A. gossypii) and peach potato aphid (Myzus persicae) when fed at levels up to 0.25 mg/ml (Rahbé et al., 2003a). Expression of OC-I in leaves and phloem sap of transgenic oil seed rape plants and eggplants also caused limited antimetabolic effects (growth impairment and reduced fecundity) in aphids developing on the transgenic plants (Rahbé et al., 2003a, Ribeiro et al., 2006). Serine PIs also show insecticidal effects towards aphids. For example, a systematic study of isoforms of Bowman-Birk type PIs from pea seeds showed varying antimetabolic effects, including significant mortality, to pea aphid (A. pisum). The effects were associated with inhibitory activity towards chymotrypsin (Rahbé et al., 2003b). This result was consistent with previous assays of various PIs towards pea aphids (Rahbé and Febvay, 1993, Rahbé et al., 1995) and with the effects of synthetic peptides containing chymotrypsin inhibitory sites. However, chymotrypsin-like activity was not detected in A. pisum gut using two chromogenic substrates (Rahbé et al., 2003b), and a direct effect of the inhibitor on gut proteolysis was not considered likely. More relevantly to the present study, effects of 5 plant serine PIs were determined on 3 species of cereal aphids, Diuraphis noxia, Schizaphis graminum and Rhopalosiphum padi. Potato inhibitors PI-I and PI-II (which inhibit chymotrypsin) showed significant antimetabolic effects, increasing mortality and reducing production of nymphs, suggesting that potato PIs were potential candidates for the control of cereal aphid species (Tran et al., 1997).

This paper presents evidence to suggest that the cereal aphid, S. avenae, can hydrolyse ingested proteins to supplement its nutrition. Inhibitory effects of two endogenous PIs from wheat, a plant host of the aphid, on digestive proteolysis by the aphid have been determined in vitro and in vivo. These PIs are shown to cause antimetabolic effects (both on survival and growth) when fed to S. avenae, and could potentially contribute to defence of wheat against aphid herbivores.

Section snippets

Insect culture and diets

Parthenogenetic females of cereal aphid, Sitobion avenae (F.), were maintained on oat (Avena sativa L. cv. Coastblack) seedlings maintained at 18 °C in a long-day regime of 16 h of light and 8 h of dark. For artificial diet feeding trials, 2-day-old parthenogenetic apterous aphids were transferred to sterile diet of formulation ‘A’ (Prosser and Douglas, 1992) containing 150 mM amino acids and 500 mM sucrose. Feeding chambers containing 10 aphids were maintained under the same environmental

Proteolytic activity in S. avenae gut extracts

Extracts of total soluble proteins from whole cereal aphids and dissected guts had similar levels of proteolytic activity towards a protein substrate (labelled casein), when compared on a per insect basis, suggesting that guts contained the majority of the proteolytic activity in the insect (result not shown). In agreement with this conclusion, no proteolytic activity above background could be detected in aphid haemolymph. All subsequent results were obtained with protein extracts prepared from

Discussion

The data presented in this paper establishes that cereal aphids are able to digest ingested protein, and can use it to partially compensate for a lack of free amino acids in their diet. However, dietary protein is not an adequate substitute for free amino acids, suggesting that these insects do not have sufficient protein digestive capacity to supply their nutritional requirements. A recent analysis of sieve tube exudate from wheat by aphid stylectomy has shown that mean total amino acid

Acknowledgement

The authors thank BBSRC for funding to support this research (Crop Science Initiative; Grant Number BB/E006280/1).

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