Ingrid Faye

Hemolin gene silencing by ds-RNA injected into Cecropia pupae is lethal to next generation embryos

There is increasing evidence of an intimate connection between participants in the innate immune system and in development. Molecules involved in the determination of dorso‐ventral polarity in Drosophila have related counterparts in the signalling pathways for immune gene activation in both insects and mammals. Hemolin from the Giant silkmoth, Hyalophora cecropia, identified as a bacteria‐inducible molecule and a member of the immunoglobulin superfamily, is present as protein and transcripts in oocytes and embryos. We used RNA interference (RNAi) to investigate H. cecropia gene function in vivo and demonstrated that Hemolin is crucial for the normal development of embryos. When RNAi‐females were mated, no larvae emerged from their eggs and when dissected, the eggs revealed malformed embryos. Western blot analysis confirmed the lack of Hemolin gene products. We conclude that Hemolin is necessary for development, since the silencing of Hemolin gene expression leads to embryonic lethality.

Parasite-specific immune response in adult Drosophila melanogaster: a genomic study

Insects of the order Diptera are vectors for parasitic diseases such as malaria, sleeping sickness and leishmania. In the search for genes encoding proteins involved in the antiparasitic response, we have used the protozoan parasite Octosporea muscaedomesticae for oral infections of adult Drosophila melanogaster. To identify parasite‐specific response molecules, other flies were exposed to virus, bacteria or fungi in parallel. Analysis of gene expression patterns after 24 h of microbial challenge, using Affymetrix oligonucleotide microarrays, revealed a high degree of microbe specificity. Many serine proteases, key intermediates in the induction of insect immune responses, were uniquely expressed following infection of the different organisms. Several lysozyme genes were induced in response to Octosporea infection, while in other treatments they were not induced or downregulated. This suggests that lysozymes are important in antiparasitic defence.

Mosquito-Bacteria Symbiosis: The Case of Anopheles gambiae and Asaia

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.

Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Culicidae) and oviposition response to bacteria-containing water

In a paratransgenic approach, genetically modified bacteria are utilized to kill the parasite in the vector gut. A critical component for paratransgenics against malaria is how transgenic bacteria can be introduced and then kept in a mosquito population. Here, we investigated transstadial and horizontal transfer of bacteria within an Anopheles gambiae mosquito colony with the focus on spiked breeding sites as a possible means of introducing bacteria to mosquitoes. A Pantoea stewartii strain, previously isolated from An. gambiae, marked with a green fluorescent protein (GFP), was introduced to mosquitoes in different life stages. The following life stages or older mosquitoes in the case of adults were screened for bacteria in their guts. In addition to P. stewartii other bacteria were isolated from the guts: these were identified by 16S rRNA sequence analysis and temporal temperature gradient gel electrophoresis (TTGE). Bacteria were transferred from larvae to pupae but not from pupae to adults. The mosquitoes were able to take up bacteria from the water they emerged from and transfer the same bacteria to the water they laid eggs in. Elizabethkingia meningoseptica was more often isolated from adult mosquitoes than P. stewartii. A bioassay was used to examine An. gambiae oviposition responses towards bacteria-containing solutions. The volatiles emitted from the solutions were sampled by headspace-solid phase microextraction (SPME) and identified by gas chromatography and mass spectrometry (GC-MS) analysis. P. stewartii but not E. meningoseptica mediated a positive oviposition response. The volatiles emitted by P. stewartii include indole and 3-methyl-1-butanol, which previously have been shown to affect An. gambiae mosquito behaviour. E. meningoseptica emitted indole but not 3-methyl-1-butanol, when suspended in saline. Taken together, this indicates that it may be possible to create attractive breeding sites for distribution of genetically modified bacteria in the field in a paratransgenic approach against malaria. Further research is needed to determine if the bacteria are also transferred in the same way in nature.

Elizabethkingia anophelis sp. nov., isolated from the midgut of the mosquito Anopheles gambiae.

The taxonomic position, growth characteristics and antibiotic resistance properties of a slightly yellow-pigmented bacterial strain, designated R26(T), isolated from the midgut of the mosquito Anopheles gambiae, were studied. The isolate produced rod-shaped cells, which stained Gram-negative. The bacterium had two growth optima at 30-31 °C and 37 °C. Strain R26(T) demonstrated natural antibiotic resistance to ampicillin, chloramphenicol, kanamycin, streptomycin and tetracycline. 16S rRNA gene sequence analysis revealed that the isolate showed 98.6 % sequence similarity to that of Elizabethkingia meningoseptica ATCC 13253(T) and 98.2 % similarity to that of Elizabethkingia miricola GTC 862(T). The major fatty acids of strain R26(T) were iso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 4 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c/t). Strain R26(T) contained only menaquinone MK-6 and showed a complex polar lipid profile consisting of diphosphatidylglycerol, phosphatidylinositol, an unknown phospholipid and unknown polar lipids and glycolipids. DNA-DNA hybridization experiments with E. meningoseptica CCUG 214(T) ( = ATCC 13253(T)) and E. miricola KCTC 12492(T) ( = GTC 862(T)) gave relatedness values of 34.5 % (reciprocal 41.5 %) and 35.0 % (reciprocal 25.7 %), respectively. DNA-DNA hybridization results and some differentiating biochemical properties indicate that strain R26(T) represents a novel species, for which the name Elizabethkingia anophelis sp. nov. is proposed. The type strain is R26(T) ( = CCUG 60038(T) = CCM 7804(T)).

Baculovirus and dsRNA induce Hemolin, but no antibacterial activity, in Antheraea pernyi

Hemolin is one of the haemolymph proteins most strongly induced upon bacterial infection in Lepidoptera. When we applied RNA interference (RNAi) to suppress Hemolin expression in the Chinese oak silk moth Antheraea pernyi, we discovered that Hemolin is induced by double‐stranded RNA (dsRNA) per se. As dsRNA is recognized as a virus pattern molecule, we then investigated the effect of a baculovirus (ApNPV) infection. We found that Hemolin is induced and expressed with similar kinetics as upon dsRNA injection. Notably, no Attacin gene expression or antibacterial activity was recorded. When baculovirus and high amounts of dsRNA were coinjected, the viral symptoms appeared earlier with Hemolin dsRNA than with GFP dsRNA. This indicates that silencing of hemolin affected the progress of the viral infection.

Lipopolysaccharide interaction with hemolin, an insect member of the Ig-superfamily

This study is an attempt to reach some understanding of how insects recognize intruding microorganisms and foreign entities while executing an immune response. We used lipopolysaccharide (LPS) from Escherichia coli, bound to a radiolabeled iodinated crosslinker, to identify hemolymph proteins from the Hyalophora cecropia moth that have the capacity to bind LPS. High amounts of radioactivity were conferred to hemolin, an immunoglobulin and NCAM‐related protein, the concentration of which increases in insect hemolymph upon bacterial infection. We could demonstrate a concentration‐dependant binding of hemolin to LPS. In addition we could show that Lipid A can compete for this binding, whereas KDO has no effect, indicating that hemolin interacts specifically with the Lipid A moiety of LPS.

Molecular Evidence for Multiple Infections as Revealed by Typing of Asaia Bacterial Symbionts of Four Mosquito Species

ABSTRACT The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

On the primary structures of lysozyme, cecropins and attacins from Hyalophora cecropia

Diapausing pupae of Cecropia respond to a bacterial infection by the selective synthesis of RNA and 15-20 hemolymph proteins. Of these we have purified lysozyme and two classes of antibacterial proteins called cecropins and attacins. The primary structure has been determined for the lysozyme, one attacin and five cecropins. We have also prepared a cDNA bank, isolated and sequenced clones corresponding to the lysozyme, the two main attacins and one cecropin. The results of these structural studies are briefly summarized. Finally we review the solid phase synthesis of cecropin A and B and 9 analogs of cecropin A.

Synthesis of immune proteins in primary cultures of fat body from Hyalophora cecropia

Abstract Fat body from injured or immunized pupae as well as from untreated pupae of Hyalophora cecropia synthesize and release in vitro the immune proteins P4, attacins, lysozyme and cecropins. During an incubation time of 4 days immune protein synthesis declines relative to the other proteins, i.e. protein synthesis returns to a normal pupal pattern. Treatment of fat body in culture with bacterial compounds (LPS, lyophilized cells of Micrococcus lysodeikticus or heat killed Escherichia coli), but also sterile filtered insect Ringer, causes a relative higher rate of synthesis of the immune proteins. At the same time synthesis of some large proteins (75–100 kD) is switched off. Similar results are obtained by adding hemocytes from injured or immunized pupae, whereas hemocytes from untreated pupae show no effect. The induction of protein synthesis with particular emphasis on the immune proteins will be discussed.