No abstract

The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real-time PCR (RT-PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN-responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a Blast search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, whereas others exhibited developmental stage-specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN whereas structural, defence and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.

1 The soybean cysteine protease inhibitor soyacystatin N (scN) and Griffonia simplicifolia lectin II (rGSII) have defense functions against the coleopteran cowpea bruchid beetle Callosobruchus maculatus. However, the ability of the insect to activate scN-insensitive digestive proteases and the relatively low potency of rGSII have hindered their practical application in plant protection. 2 Recent research suggests that defense proteins may achieve increased toxicity and durability when used in combination. Based on the structures of several natural toxin molecules, we hypothesized that covalently linked scN and rGSII could exhibit greater anti-insect efficacy than the mixture containing individual proteins. 3 To test this hypothesis, a recombinant scN-rGSII fusion protein that retained both protease inhibitor and lectin functions was constructed. 4 When fed to cowpea bruchid, this new protein showed a synergistic delay in insect development, whereas a mixture of the separate proteins only showed an additive effect. 5 Our results suggest that tethering digestive protease inhibitors to gut epithelium-interacting lectins could give plant protection superior to strategies based on single genes or mixtures of single gene products.

Griffonia simplicifolia lectin II (GSII) and wheat germ agglutinin (WGA) are N-acetylglucosamine-binding lectins. Previous studies demonstrated that they have anti-insect activity, a property potentially useful in pest control. To gain some insight into the insect response to dietary lectins, we performed transcriptomic analysis using the cowpea bruchid (Callosobruchus maculatus) midgut microarray platform we built. Compared to the nonnutritional cellulose treatment, dietary lectins induced more profound changes in gene expression. Ingestion of relatively high doses of lectins for 24 h resulted in alteration of gene expression involved in sugar and lipid metabolism, transport, development, defense, and stress tolerance. Metabolic genes were largely downregulated. Moreover, we observed disorganized microvilli resulting from ingestion of WGA. This morphological change is consistent with the lectin-induced changes in genes related to midgut epithelial cell repair. In addition, suboptimal nutrient conditions may serve as a stress signal to trigger senescence processes, leading to growth arrest and developmental delay.

Cowpea provides a significant source of protein for over 200 million people in Sub-Saharan Africa. The cowpea bruchid, Callosobruchus maculatus (F) (Coleoptera: Bruchidae), is a major pest of cowpea as the larval stage attacks stored cowpea grains, causing postharvest loss. Cowpea bruchid larvae spend all their time feeding within the cowpea seed. Past research findings, published over 25 yr ago, have shown that feeding activity of several bruchids within a cowpea seed emit mechanical vibrations within the frequency range 5-75 kHz. This work led to the development of monitoring technologies that are both important for basic research and practical application. Here, we use newer and significantly improved technologies to re-explore the nature of the vibration signals produced by an individual C. maculatus, when it feeds in cowpea seeds. Utilizing broadband frequency sensing, individual fourth-instar bruchid larvae feeding activities (vibration events) were recorded to identify specific key emission frequencies. Verification of recorded events and association to actual feeding activities was achieved through mass measurements over 24 h for a series of replicates. The measurements identified variable peak event emission frequencies across the replicate sample set ranging in frequency from 16.4 to 26.5 kHz. A positive correlation between the number of events recorded and the measured mass loss of the cowpea seed was observed. The procedure and verification reported in this work provide an improved basis for laboratory-based monitoring of single larval feeding. From the rich dataset captured, additional analysis can be carried out to identify new key variables of hidden bruchid larval activity.