Characterization of Digestive Enzymes of Bruchid Parasitoids–Initial Steps for Environmental Risk Assessment of Genetically Modified Legumes

Genetically modified (GM) legumes expressing the α-amylase inhibitor 1 (αAI-1) from Phaseolus vulgaris L. or cysteine protease inhibitors are resistant to several bruchid pests (Coleoptera: Chrysomelidae). In addition, the combination of plant resistance factors together with hymenopteran parasitoids can substantially increase the bruchid control provided by the resistance alone. If the strategy of combining a bruchid-resistant GM legume and biological control is to be effective, the insecticidal trait must not adversely affect bruchid antagonists. The environmental risk assessment of such GM legumes includes the characterization of the targeted enzymes in the beneficial species and the assessment of the in vitro susceptibility to the resistance factor. The digestive physiology of bruchid parasitoids remain relatively unknown, and their susceptibility to αAI-1 has never been investigated. We have detected α-amylase and serine protease activities in all five bruchid parasitoid species tested. Thus, the deployment of GM legumes expressing cysteine protease inhibitors to control bruchids should be compatible with the use of parasitoids. In vitro inhibition studies showed that sensitivity of α-amylase activity to αAI-1 in the parasitoids was comparable to that in the target species. Direct feeding assays revealed that harmful effects of α-amylase inhibitors on bruchid parasitoids cannot be discounted and need further evaluation

Proteomic Analysis of Fusarium solani Isolated from the Asian Longhorned Beetle, Anoplophora glabripennis

Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle), and that a fungal species, Fusarium solani (ATCC MYA 4552), is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT) was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent peroxidase activities were not detected While little is known about the role of filamentous fungi and their associations with insects, these findings suggest that this isolate has the endogenous potential to degrade lignocellulose and extract nutrients from woody tissue

The effect of acquired microbial enzymes on assimilation efficiency in the common woodlouse, Tracheoniscus rathkei

The digestive tract of the common woodlouse, Tracheoniscus rathkei Brandt (Isopoda: Oniscoidea), contains digestive enzymes active against α-1,4-glucans, which are the chief storage polysaccharides of vascular plants, algae, fungi, and animals, and β-1,3-glucans, which are present in algae and fungi. Digestive tract extracts also exhibit significant activity toward xylan and carboxymethyl-cellulose but negligible activity toward microcrystalline cellulose, substrates representative of the major structural polysaccharides of vascular plants. Low activity was detected toward pectin, and no activity was detected toward chitin. Activity toward xylan is due in part to microbial enzymes acquired from the leaf litter which was the isopod's normal food. Although ingested microbial xylanases are stable and active in the gut fluid, they do not make a quantitatively significant contribution to the isopod's ability to assimilate the hemicellulosic component of its diet. However, the assimilation of carbon from labeled plant fiber is enhanced in isopods which have acquired a cellulase by ingestion of leaf litter amended with a commercial preparation of the cellulase complex from the fungus, Penicillium funiculosum . This result demonstrates the potential contribution of acquired enzymes to the digestion of plant fiber in terrestrial detritivores. We urge caution, however, in assigning an important digestive function to ingested enzymes on the basis of evidence that only indicates that such enzymes are present in the gut fluid without additional evidence that their presence results in an enhancement of digestive efficiency.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47763/1/442_2004_Article_BF00377057.pd