The Molecular Biology of Insect Disease Vectors: A Methods Manual

Only one generation ago, entomology was a proudly isolated discipline. In Comstock Hall, the building of the Department of Entomology at Cornell University where I was first introduced to experimental science in the laboratory of Tom Eisner, those of us interested in the chemistry of life felt like interlopers. In the 35 years that have elapsed since then, all of biology has changed, and entomology with it. Arrogant molecular biologists and resentful classical biologists might think that what has happened is a hostile take-over of biology by molecular biology. But they are wrong. More and more we now understand that the events were happier and much more exciting, amounting to a new synthesis. Molecular Biology, which was initially focused on the simplest of organisms, bacteria and viruses, broke out of its confines after the initial fundamental questions were answered - the structure of DNA, the genetic code, the nature of regulatory genes - and, importantly, as its methods became more and more generally applicable. The recombinant DNA revo­ lution of the 1970s, the development of techniques for sequencing macromolecules, the polymerase chain reaction, new molecular methods of genetic analysis, all brought molecular biology face to face with the infinite complexity and the exuber­ ant diversity of life. Molecular biology itself stopped being an isolated diScipline, pre­ occupied with the universal laws of life, and became an approach to addressing fas­ cinating specific problems from every field of biology.

Table of Contents:
One Care and Maintenance of Insect Colonies.- 1 Care and maintenance of anopheline mosquito colonies.- 2 Care and maintenance of Aedes mosquito colonies.- 3 Care and maintenance of phlebotomine sandfly colonies.- 4 Care and maintenance of blackfly colonies.- 5 Care and maintenance of tsetse colonies.- 6 Care and maintenance of triatomine colonies.- Two Experimental Infection of Insect Vectors.- 7 Infection of mosquitoes with rodent malaria.- 8 Infection of mosquitoes with primate malaria.- 9 Infection of mosquitoes with filarial.- 10 Infection of phlebotomine sandflies with Leishmania.- 11 Infection of blackflies with Onchocerca.- 12 Infection of tsetse with Trypanosoma brucei rhodesiense and T. b. gambiense.- 13 Infection of triatomines with Trypanosoma cruzi.- Three Basic Methods in Isolating, Cloning and Characterizing Nucleic Acids and their Products.- 14 Isolation of genomic DNA.- 15 Isolation and analysis of RNA from insect vectors.- 16 Determination of insect genome size and complexity by reassociation kinetics.- 17 Constructing and screening lambda libraries.- 18 Constructing and screening cosmid libraries.- 19 Constructing and screening cDNA libraries.- 20 Southern/northern blotting and hybridization techniques.- 21 The polymerase chain reaction (PCR) and RT-PCR.- 22 Differential display of mRNA.- 23 Localization of RNA transcripts by in situ hybridization.- 24 In situ localization of proteins in whole mounted tissue.- 25 DNA sequencing and primer extension mapping.- Four Genome Mapping Techniques.- 26 RFLP analysis of insect genomes.- 27 Microsatellite mapping of insect genomes.- 28 Polytene chromosome preparation from anopheline mosquitoes.- 29 In situ hybridization to Anopheles polytene chromosomes.- 30 Microdissection and microamplification of dipteran polytene chromosomes.- Five Insect Identification Techniques.- 31 RAPD-PCR and SSCP analysis for insect population genetic studies.- 32 PCR amplification of insect ribosomal DNA.- 33 Generation and use of species-specific DNA probes for insect vector identification.- 34 Cloning and detection of insecticide resistance genes.- Six Transformation Techniques and Viral Systems.- 35 Microinjection of mosquito embryos.- 36 Transfection of cultured mosquito cells.- 37 Viral transducing vectors for mosquitoes.- 38 Viral expression systems and viral infections in insects.- Seven Cell and Organ Culture.- 39 Establishing primary cell cultures from disease vectors and maintenance of continuous cell lines.- 40 Culture and analysis of insect salivary glands.- 41 Culture and analysis of the insect fat body.- 42 Culture and analysis of insect ovaries.- 43 Culture and analysis of insect midgut.- Eight Insect Symbionts.- 44 Isolation and culture of tsetse secondary endosymbionts.- 45 Genetic manipulation of insect symbionts.- 46 PCR-based detection and identification of insect symbionts.