Abstract

The EU is committed to protect and improve the quality of environment and human health threatened by the use of synthetic pesticides and insecticides. This proposal is to exploit animal biodiversity as a source of new environmentally benign natural products for the development of safer insect pest control strategies. The proposed work is to identify new genes and proteins produced by insect parasitoids and their associated polydnaviruses, which induce most of the pathological alterations in parasitised host insects. The expected results are new natural products, with known structure and function, and insecticidal activity, which will have a remarkable degree of selectivity for beneficial insects and other non-target species.

Objectives

The project aims to identify new, effective and environmentally safe natural molecules with insecticidal activity. The general scientific objective of BIP is to isolate and distinguish the genes of insect parasitoids, which induce severe pathological syndromes in their insect hosts. The parasitoid species being considered carry symbiotic polydnaviruses (PDV), that are injected with the egg at oviposition. These viruses are responsible for most of the pathological alterations observed in naturally parasitised insects and constitute a valuable, and relavtively unexploited, source of genes coding for new bioinsecticides.

The general scientific objective will be pursued through the following key objectives:

• Isolate and characterise the viral genes that are expressed in the insect host.
• Determine the function of the individual gene products.
• Define the structure of the gene products.
• Assess insecticidal activity and selectivity of the gene products.

Description of the work

The work will focus on two Hymenoptera parasitoids of Lepidopteran larvae, both of which carry symbiotic polydnaviruses (PDV). For both parasitoid species, the disruptions of the neuroendocrine and immune systems in parasitised hosts have been described. In this project the PDV genes that cause these changes will be identified and characterised. A few PDV genes have been already isolated which encode new proteins. Thus, in-depth structural and functional studies will be essential to understand the function of these proteins and of others that will be identified. Tissue specificity and temporal expression of the PDV genes will be studied by Northern blot analyses, by in situ hybridization and immunohistochemistry to localise the proteins in different host tissues. The genes will be expressed in bacteria, baculoviruses and in transformed insect cells and their products used for both functional and structural analyses. Functional analyses will be performed in vivo by infection of host larvae with baculoviruses overexpressing PDV genes. The structural resolution at atomic level of the PDV encoded proteins and their bioactive domains will be performed by NMR, X- ray crystallography and Molecular Dynamics. Insecticidal activity of these molecules will be assessed both on target and non-target species. Delivery strategies of the isolated bioinsecticides will be developed in order to maximize their effectiveness and safety. The proposed strategy will enable the enormous biodiversity potential of antagonistic associations in insects to be exploited and protected. This objective encompasses priority areas defined under key action 3, meets several of the requirements of the general socio- economic objective of key action 3 and will make a direct contribution to the EU Fifth Framework Programme.

Deliverables

• D1 Genomic and cDNA libraries of PDVs and parasitised hosts
• D2 Genomic and cDNA clones of PDV_genes expressed in insect hosts; D3Sequences of PDV genomes
• D4 Baculoviruses overproducing PDV gene products
• D5 Stably transformed clonal insect cell lines overproducing PDV gene products
• D6 Purified PDV gene products for structural and functional studies
• D7 Antibodies raised against PDV gene products
• D8 cDNA clones of pathogenetic PDV genes
• D9 Preparations of host target tissues for light and electron microscopy
• D10 Protocols for functional analysis of PDV genes in different host tissues
• D11 cDNA expression library of haemocytes from non parasitised hosts
• D12 Host genes encoding proteins interacting with PDV gene products
• D13 AcMNPV strains (egt+ve, egt-ve) expressing PDV genes
• D14 Three-dimensional solution structures of specific domains of PDV encoded proteins
• D15 Three-dimensional solution structure of PDV encoded proteins
• D16 Crystal structure of synthetic peptides
• D17 Crystal structure of the PDV encoded proteins
• D18 PDV encoded molecules showing insecticidal activity
• D19 PDV products showing favourable digestion and safety in vitro
• D20 PDV products with defined immunosuppressive effects in mammals
• D21 Safety-screened PDV encoded molecules.

Contacts

Coordinator

• International Institute of Genetics & Biophysics IIGB, CNR / ITALY

Participant

• David O'REILLY / Imperial College of Science Technology & Medicine UNITED KINGDOM
• Jean-Michel DREZEN / Institut de Recherche sur la Biologie de l'Insecte (IRBI) CNRS / FRANCE
• Giovanni CONFALONIERI / Isagro Ricerca (ISAGRO) ITALY
• Kostas IATRU / National Center for Scientific Research "Demokritos" (NCSR"D"), / GREECE
• Hubert P.J.M. NOTEBORN / National Institute for Quality Control of Agricultural Products (RIKILT) / NETHERLANDS
• Antonio M. TAMBURRO / University of Basilicata (UNIBAS) ITALY