Gene silencing through RNA interference (RNAi) has revolutionized the study of gene 98 function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) 99 RNAi has many times proven to be difficult to achieve. Most of the negative results have been 100 anecdotal and the positive experiments have not been collected in such a way that they are 101 possible to analyze. In this review, we have collected detailed data from more than 150 102 experiments including all to date published and many unpublished experiments. Despite a 103 large variation in the data, trends that are found are that RNAi is particularly successful in the 104 family Saturniidae and in genes involved in immunity. On the contrary, gene expression in 105 epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding 106 dsRNA requires high concentrations for success. Possible causes for the variability of success 107 in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further 108 investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the 109 innate immune response. Our general understanding of RNAi in Lepidoptera will be further 110 aided in the future as our public database at http://insectacentral.org/RNAi will continue to 111 gather information on RNAi experiments.

Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the ␥-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.Malaria remains the parasitic disease that kills the most people in the world. Anopheles gambiae sensu lato and Anopheles funestus mosquitoes are the main vectors in Africa, where 90% of malaria-related deaths occur. An approach to stop malaria transmission is paratransgenics. In this approach, suitable symbiotic bacteria are genetically modified to produce an antiparasitic factor and then reintroduced into the insect gut, where they kill or inhibit the development of the parasites (4).A few studies have been performed to investigate bacterial species in field-collected Anopheles mosquitoes, all using culturing techniques. Jadin et al. (22) To identify bacterial candidates for a paratransgenic mosquito, we conducted a screen for uncultured and cultured midgut bacteria from wild-caught A. gambiae and A. funestus mosquitoes. MATERIALS AND METHODSField site, mosquitoes, and dissections. Indoor-resting, blood-fed female A. gambiae sensu lato and A. funestus mosquitoes were caught in Lwanda, 12 km east of Mbita Point Research and Training Centre, ICIPE, Suba district, Western Kenya. In total, 116 Anopheles mosquitoes were caught on eight different occasions (A2 to H2). Living mosquitoes were anesthetized with chloroform, the species were determined by morphology and PCR (A. gambiae sensu lato) (30a). The mosquitoes were dissected in a sterile hood. Individual midguts were mashed in 50 l of sterile saline (0.9% NaCl); this suspension was later used for isolation of bacteria and cloning of the 16S rRNA gene from bacteria. Controls for the efficiency of sterilization were treated like the other samples.Bacterial isolation and phenotypic characterization. The midgut suspension was streaked on Luria-Bertani agar (LA) plates and incubated for 48 h at room temperature. All bacteria were restreaked and pre...

Hemolin, an insect immunoglobulin superfamily member, is a lipopolysaccharide-binding immune protein induced during bacterial infection. The 3.1 angstrom crystal structure reveals a bound phosphate and patches of positive charge, which may represent the lipopolysaccharide binding site, and a new and unexpected arrangement of four immunoglobulin-like domains forming a horseshoe. Sequence analysis and analytical ultracentrifugation suggest that the domain arrangement is a feature of the L1 family of neural cell adhesion molecules related to hemolin. These results are relevant to interpretation of human L1 mutations in neurological diseases and suggest a domain swapping model for how L1 family proteins mediate homophilic adhesion.

Contents Introduction (Table 1) Lysozyme Two main forms of attacin The cecropins and their precursor forms Chemical synthesis of cecropins and three‐dimensional structure of cecropin A Spectrum and mechanism of action of cecropins Other factors related to the immune response Genes for cecropia immune proteins The place of synthesis of the immune proteins Discussion

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The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.

Insects have an efficient defense system against infections. Their antibacterial immune proteins have been well characterized. However, the molecular mechanisms by which insects recognize foreignness are not yet known. Data are presented showing that hemolin (previously named P4), a bacteria-inducible hemolymph protein of the giant silk moth Hyalophora cecropia, belongs to the immunoglobulin superfamily. Functional analyses indicate that hemolin is one of the first hemolymph components to bind to the bacterial surface, taking part in a protein complex formation that is likely to initiate the immune response.

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