|Director : Charles, Jean-François (email@example.com)|
The study of the mode of action of Bacillus sphaericus toxins and their receptors in mosquito larvae Culex pipiens has been continued. The attempts to improve B. sphaericus potency by co-expression of other toxins was undertaken.
The study of the molecular basis of interactions mosquito-flavivirus (Aedes sp. / dengue yellow fever) has been continued (TRP N° 23).
A. Entomopathogenic bacteria
A1 Functional expression of the receptor of the Bin toxin of B. sphaericus in cell lines (Jean-François Charles, Coll. Y. Pauchet, D. Pauron and I. Darboux, INRA, Antibes)
In the mosquito larvae Culex pipiens, the recepor to the Bin toxin of B. sphaericus is an a-glucosidae of 60 kDa (Cpm1). The corresponding gene has been cloned and expressed by transfection in Spodoptera littoralis Sf9 cells. Binding experiments between the radiolabelled Bin toxin (125I) and SF9 cell membranes showed that the receptor is fully functional. On the opposite, there is no binding between cell membranes transfected with the gene from a mosquito population resistant to the toxin (Geo), due to a stop mutation before the region encoding the GPI anchor of the receptor to the cell membrane.
A2 - Genetics and mechanism of mosquito larval resistance to B. sphaericus (Christina Nielsen-LeRoux, Coll. C. Chevillon and Nicole Pasteur, University Montpellier II))
The mechanisms of resistance of Culex pipiens populations to the binary toxin of B. sphaericus. involve either a modification of the binding step (toxin-receptor) or an unknown mechanism probably located after the initial binding event. There is evidence for at least two mechanisms may co-exist in Culex populations (South of France, Port St. louis and Perpignan). In addition, progeny of crosses between different recessive populations was resistant and the phenotype was functional toxin receptor. This is important for the remaining of resistance genes in natural habitats and for practical management of resistance.
A3 - Improvement of Bacillus sphaericus entomopathogenic properties (Manuel Porcar and Armelle Delécluse)
Entomopathogenic properties of B. sphaericus could be improved by introducing into this host, the gene encoding the highly mosquitocidal toxin Cry11Ba from Bacillus thuringiensis jegathesan downstream from a strong promotor. Three constructs, including the cry11Ba gene and a promotor (sequences previously identified) have been performed and introduced into the B. sphaericus chromosome by in vivo recombination. Analysis of the resulting clones (protein production, mosquitocidal activity) is under progress.
A4 - Development of molecular tools for Bacillus thuringiensis strain classification (Monica Santos et Victor Juárez-Pérez)
The classification of B. thuringiensis strains based on the serology of flagella has been the most reliable and accepted methodology worldwide since 1962. However, this technology is tedious and time consuming. In order to introduce a molecular method to facilitate the classification, we have started the amplification and sequence of the fla genes encoding the flagelin protein of each type strain contained within the collection of the WHO collaborate center. Presently, 70 % of all the expected sequences have been analyzed. The rest of the sequences and their analysis are under progress. The complete sequence analysis and their comparison will determine the feasibility of a PCR-based method to classify the B. thuringiensis strains.
A5 - Remanence of spores of B. thuringiensis isralensis in Camargue after treatment (Christina Nielsen-LeRoux and Myriam Hajaij)
In collaboration with the "Entente Interdépartementale de Démoustication" (EID Mediterranée, Montpellier), we were involved in the evaluation of the natural occurrence of B. thuringiensis in the aim to determine how, after treatment with a mosquito larvicidal product based on Bt israelensis,(Bti) viable spores may accumulate and persist in this environment. Following treatments during Spring and Summer 2001, preliminary results indicate that spores of Bti only poorly accumulate in the field.
B- Molecular basis of mosquito-flavivirus interactions (Transversal Research Program 23; coordinator: Armelle Delécluse)
Relationships between mosquito vectors and the viruses they transmit are crucial in the maintenance and transmission of several infectious diseases (dengue, yellow fever, West-Nile ). Understanding these interactions would allow development of innovative control methods to prevent disease transmission.
B.1- Identification of molecular determinants involved in vector competence (Véronique Mayau and Armelle Delécluse)
Mosquito populations display variable susceptibility to flaviviruses development, termed "vector competence". Competence reflects the different barriers encountered by the virus from its entry into the mosquito midgut to its release in the saliva. Various factors such as the presence of specific mosquito receptors and/or differential viral replication in the mosquito might be involved in the competence. We are developing several approaches (surface plasmon resonance, in situ hybridization ) to identify the factors responsible for vector competence to dengue, yellow fever and West-Nile viruses.
B.2- Evolution of dengue genome within the mosquito (Victor Juárez-Pérez, Véronique Mayau and Armelle Delécluse)
Evolutionary studies of dengue virus have revealed that its genetic diversity is increasing. Evolution of the virus genome could occur during its replication within the mosquito, Aedes aegypti. Mutations in a single genome or recombination events between separate genome might account for this evolution. We are currently studying the mutation rate of the dengue virus during its replication in the mosquito and investigating the possibility of genome recombination.
C. WHO Collaborative Centre OMS / IEBC Collection (Jean-François Charles)
The laboratoiry had been renewed in 2000 as WHO Collaborative Centre for Entomopathogenic Bacteria. The International Entomopathogenic Bacillus Collection (IEBC) contains more than 6000 strains : 3600 B. thuringiensis, 600 B. sphaericus and 1800 other Bacillus. More than 370 strains of B. thuringiensis, B. sphaericus and other Bacillus, and 200 antisera and référence powders have been sent. Nevertheless, bacterial identifications and serotyping, and with bioassays on mosquito larvae stoped, due to lack of personnel.
The Laboratory of Entomopathogenic Bacteria and the WHO Collaborative Centre are closed since November 30, 2001.
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
Blanc, Marie-France, firstname.lastname@example.org
Charles, Jean-François, IP, email@example.com
Delécluse, Armelle, IP, firstname.lastname@example.org
Nielsen-Le Roux, Christina, IP, email@example.com
Laurent, Philippe, CNRS
Juárez-Pérez, Victor, Postdoc
Porcar Miralles, Manel, Postdoc
Hajaij, Myriam, PhD student, Faculté des Sciences de Tunis
Martinez Rico, Clara
Hamon, Sylviane, Technician, firstname.lastname@example.org
Mayau, Véronique, Technician, email@example.com
Delattre, Nelly, Technician
Patricio, Vidalia, Technician, firstname.lastname@example.org