Bacterial diversity of field-caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar.

peer reviewedSymbiotic bacteria are known to play important roles in the biology of insects, but the current knowledge of bacterial communities associated with mosquitoes is very limited and consequently their contribution to host behaviors is mostly unknown. In this study, we explored the composition and diversity of mosquito-associated bacteria in relation with mosquitoes’ habitats. Wild Aedes albopictus and Aedes aegypti were collected in three different geographic regions of Madagascar. Culturing methods and denaturing gradient gel electrophoresis (DGGE) and sequencing of the rrs amplicons revealed that Proteobacteria and Firmicutes were the major phyla. Isolated bacterial genera were dominated by Bacillus, followed by Acinetobacter, Agrobacterium and Enterobacter. Common DGGE bands belonged to Acinetobacter, Asaia, Delftia, Pseudomonas, Enterobacteriaceae and an uncultured Gammaproteobacterium. Double infection by maternally inherited Wolbachia pipientis prevailed in 98% of males (n = 272) and 99% of females (n = 413); few individuals were found to be monoinfected withWolbachia wAlbB strain. Bacterial diversity (Shannon–Weaver and Simpson indices) differed significantly per habitat whereas evenness (Pielou index) was similar. Overall, the bacterial composition and diversity were influenced both by the sex of individuals and by the environment inhabited by the mosquitoes; the latter might be related to both the vegetation and the animal host populations that Aedes used as food sources.‘Arthropod Symbioses: from fundamental to pest disease management’

Biogeography of the two major arbovirus mosquito vectors, Aedes aegypti and Aedes albopictus (Diptera, Culicidae), in Madagascar

Background: In the past ten years, the Indian Ocean region has been the theatre of severe epidemics of chikungunya and dengue. These outbreaks coincided with a high increase in populations of Aedes albopictus that outcompete its sister taxon Aedes aegypti in most islands sampled. The objective of this work was to update the entomological survey of the two Aedes species in the island of Madagascar which has to face these arboviroses. Methods: The sampling of Aedes mosquitoes was conducted during two years, from October 2007 to October 2009, in fifteen localities from eight regions of contrasting climates. Captured adults were identified immediately whereas immature stages were bred until adult stage for determination. Phylogenetic analysis was performed using two mtDNA genes, COI and ND5 and trees were constructed by the maximum likelihood (ML) method with the gene time reversible (GTR) model. Experimental infections with the chikungunya virus strain 06.21 at a titer of 107.5 pfu/mL were performed to evaluate the vector competence of field-collected mosquitoes. Disseminated infection rates were measured fourteen days after infection by immunofluorescence assay performed on head squashes. Results: The species Aedes aegypti was detected in only six sites in native forests and natural reserves. In contrast, the species Aedes albopictus was found in 13 out of the 15 sites sampled. Breeding sites were mostly found inman-made environments such as discarded containers, used tires, abandoned buckets, coconuts, and bamboo cuts. Linear regression models showed that the abundance of Ae. albopictus was significantly influenced by the sampling region (F = 62.00, p < 2.2 × 10-16) and period (F = 36.22, p = 2.548 × 10-13), that are associated with ecological and climate variations. Phylogenetic analysis of the invasive Ae. albopictus distinguished haplotypes from South Asia and South America from those of Madagascar, but the markers used were not discriminant enough to discern Malagasy populations. The experimental oral infection method showed that six Ae. albopictus populations exhibited high dissemination infection rates for chikungunya virus ranging from 98 to 100%. Conclusion: In Madagascar, Ae. albopictus has extended its geographical distribution whereas, Ae. aegypti has become rare, contrasting with what was previously observed. Changes are predominantly driven by human activities and the rainfall regime that provide suitable breeding sites for the highly anthropophilic mosquito Ae. albopictus. Moreover, these populations were found to be highly susceptible to chikungunya virus. In the light of this study, Ae. albopictus may have been involved in the recent outbreaks of chikungunya and dengue epidemics in Madagascar, and consequently, control measures should be promoted to limit its current expansion.Rôle des moustiques Culicidae, de leurs communautés microbiennes et des réservoirs vertébrés, dans la transmission des arbovirus à Madagasca

Rôle des moustiques Culicidae, de leurs communautés microbiennes et des réservoirs vertébrés, dans la transmission des arbovirus à Madagascar

These last ten years, Indian Ocean islands have been the theatre of severe epidemics of arboviroses. However, little information was available about the local dynamics of the virus transmission and identity of the vector species and vertebrates réservoirs involved. Madagascar island was chosen for its high endemic biodiversity, contrasting climatic conditions and drastic changes in habitats due to an increase anthropic pressure. These factors increase the epidemiological risks of infectious diseases. In this context, our objective was to characterize the role of Culicidae as major vectors, the associated microbial communities, and wild vertebrates (lémurs) as potential réservoirs in the transmission arboviroses. Molecular techniques allowed udpdating the biogeography of Aedes and showed the dominance of Aedes albopictus competent to chikungunya virus. Other mosquitoes captured belonged to Culex antennatus, Culex decens, Culex quinquefasciatus et Mansonia uniformis. The bacterium Wolbachia was detected in most mosquito especies,but Culex antennatus. Searching of arboviruses by qRT‐PCR revealed infection of Culex antennatus and lemurs (Lepilemur edwardsi et Eulemur rufus) by the rift valley fever virus.Rôle des moustiques Culicidae, de leurs communautés microbiennes et des réservoirs vertébrés, dans la transmission des arbovirus à Madagasca

Ethologie des fourmis Eutetramorium mocquerysi (Emery, 1900) Hymenoptera, Formicidae, en laboratoire

The laboratory breeding study of Eutetramorium mocquerysi was conducted in an artificial fourmiliere: formicarium. The colonies were nourished with portions of insects (larvae of coleoptera, locusts, cockroaches. ..).All social insects have several morphoanatomic castes (worker dominating, workers of high ranks: being able to become dominating, workers of low ranks: veritable worker,and the ant male). As with all social insects, the life of the colony is regulated by the queen. However, in the species studied no veritable queen exists. This function is fulfilled by a dominating worker, whose dominance is assured partly by constant fights with the young workers and partly by maximum ovarial development. Dominant worker and workers of the high ranks have six (6) ovarioles, while the veritable workers possess just two (2) ovarioles. The presence of a dominant worker in the nest allows the regulation of the laborious life of the colony, the maintenance of inferior workers ranks and the control of male abundance in the colony.Ethologie des fourmis Eutetramorium mocquerysi (Emery, 1900) Hymenoptera, Formicidae, en laboratoir

Molecular detection of six high importance endosymbiotic bacteria in Belgian wild-caught mosquitoes

Introduction Several disease vectors presented a resistance to various pesticides currently used. One of an alternative solution was to use endosymbiotic bacteria because their probably interactive effects with their host. According to the introduction risks of mosquito born disease and their dispersion, we propose to investigate the prevalence of six endosymbiontic bacteria in wild-caught Culicidae in Belgium. Methods Eleven species of Belgian fields mosquitoes (Culex pipiens s.l., Cx. torrentium, Cx. hortensis, Anopheles claviger, An. maculipennis s.l., An. plumbeus, Culiseta annulata, Ochlerotatus geniculatus, Oc. dorsalis, Aedes albopictus and Coquillettidia richiardii) were used for the screening of six genera endosymbiotic bacteria (Wolbachia sp., Commamonas sp., Delftia sp., Pseudomonas sp., Acinetobacter sp. and Asaia sp.) according to their possible impact in mosquito biology. PCR was done for the screening and positives bands were sequenced and deposited in GenBank. Results Total of 144 larvae and 32 adults were used. Wolbachia, Pseudomonas, Acinetobacter and Asaia were found in mosquitoes with different proportions, according to stages (adults, larvae) with a predominance of Pseudomonas in all species, as far as Acinetobacter and Asaia also have a high prevalence. Commamonas and Delftia were absent from all species tested, either in larvae and in adults. Discussion Choice of endosymbiotic bacteria studied here was allowing of their importance in literature. For Pseudomonas, it showed that this bacteria could produced ovipositon attractants for mosquito. Acinetobacter was suggested efficient in transmission and maintenance within host populations. Asaia was capable of efficiently crossing body barriers and colonizing different organs. Wolbachia was currently the most studied bacteria which plays an important role in the genetic manipulation of the host. Present advances in understanding the mosquito–microbiota relationships may have a great impact in a better understanding of some traits of mosquito biology and in the development of innovative mosquito-borne disease-control strategies.Contribution à l’étude des endosymbiotes des Culicidae de Belgique : rôles dans la transmission virale et cible pour le contrôle des moustique

Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus

Background: The microbiota has been shown to play an important role in the biology of insects. In recent decades, significant efforts have been made to better understand the diversity of symbiotic bacteria associated with mosquitoes and assess their influence on pathogen transmission. Here, we report the bacterial composition found in field-caught Aedes albopictus populations by using culture-dependent methods. Results: A total of 104 mosquito imagos (56 males and 48 females) were caught from four contrasting biotopes of Madagascar and their bacterial contents were screened by plating whole body homogenates on three different culture media. From 281 bacterial colony types obtained, amplified ribosomal DNA restriction analysis (ARDRA) showed they had 40 distinct ribotypes. Sequencing and BLAST analysis of the 16S rDNA genes responsible for each representative profile made it possible to identify 27 genera distributed in three major phyla. In female mosquitoes, bacterial isolates were mostly Proteobacteria (51.3%) followed by Firmicutes (30.3%) and Actinobacteria (18.4%). Conversely, Actinobacteria was the most abundant phylum in male mosquitoes (48%) followed by Proteobacteria (30.6%) and Firmicutes (20.4%). The relative abundance and composition of isolates also varied between sampling sites, ranging from 3 distinct families in Ankazobe to 8 in Tsimbazaza Park, and Toamasina and Ambohidratrimo. Pantoea was the most common genus in both females and males from all sampling sites, except for Ambohidratrimo. No differences in genome size were found between Pantoea isolates from mosquitoes and reference strains in pulse field gel electrophoresis. However, according to the numbers and sizes of plasmids, mosquito isolates clustered into three different groups with other strains isolated from insects but distinct from isolates from the environment. Conclusions: The recent upsurge in research into the functional role of the insect microbiota prompts the interest to better explore the role some bacteria detected here may have in the mosquito biology. Future studies of culturable bacteria might decipher whether they have a biological role in the invasiveness of Ae. albopictus. As a possible candidate for paratransgenesis, the predominant genus Pantoea will be characterized to better understand its genetic contents and any possible influence it may have on vector competence of Ae. albopictus.Arthropod Symbioses: from fundamental to pest disease managemen

Molecular detection of six (endo-) symbiotic bacteria in Belgian mosquitoes: first step towards the selection of appropriate paratransgenesis candidates

Actually, the use of symbiotic bacteria is one of alternative solution to avoid vector resistance to pesticides. In Belgium, among 31 identified mosquito species, 10 were considered as potential vectors. Given to introduction risks of arbovirosis, the purpose of this study was to investigate the presence of symbiosis bacteria in potential mosquito vectors. Eleven species caught from 12 sites in Belgium were used: Culex pipiens s.l., Culex torrentium, Culex hortensis, Anopheles claviger, Anopheles maculipennis s.l., Anopheles plumbeus, Culiseta annulata, Ochlerotatus geniculatus, Ochlerotatus dorsalis, Aedes albopictus, and Coquillettidia richiardii. Six genera of symbiotic bacteria were screened: Wolbachia sp., Comamonas sp, Delftia sp., Pseudomonas sp., Acinetobacter sp., and Asaia sp.Atotal of 173 mosquito individuals (144 larvae and 29 adults) were used for the polymerase chain reaction screening. Wolbachia was not found in any Anopheles species nor Cx. torrentium. A total absence of Comamonas and Delftia was observed in all species. Acinetobacter, Pseudomonas, and Asaia were found in most of species with a high prevalence for Pseudomonas. These results were discussed to develop potential strategy and exploit the variable occurrence of symbiotic bacteria to focus on them to propose biological ways of mosquito control

L'évaluation de persistance bactérienne dans les moustiques par essais de microinjection en Belgique

The problems caused by the massive used of pesticides have resulted in the establishment of resistant vectors besides the destruction of the environment. Furthermore, climate change has consequently modified the comportment of disease vectors. Current research tends to look for alternative means to overcome the problem. The goal of that study was to undertaken the way there this objective. By their presence or absence, endosymbiotic microorganisms can influence vector competence and vectorial capacity. Our research aims to study the effect of the introduction of endosymbiotic bacteria in the mosquito species that could be potential vectors of disease in Belgium. Method used was the microinjection of endosymbiotic bacteria within the detected exempt mosquito species. Three genus of suspected vectors belonging to Aedes, Anopheles and Culex were microinjected at different stages of their life cycle (eggs, larvae, pupae, and adults), and survivability of mosquitoes and persistence of microorganism were determined. Results show that survival in the different stages was variable. Furthermore, persistence of endobacteria was different depending genus and stages studiedContribution à l’étude des Culicidae exotiques, leur bactérie endosymbiotique Wolbachia face aux changements climatiques et les échanges commerciaux en Belgiqu

Diversité bactérienne de moustiques provenant de différentes régions de Belgique et leur impact potentiel sur la transmission des maladies

Several vectors disease presented a resistance to various pesticides currently used. Endosymbiotic bacteria was an alternative solution found because of their probably interactive effects with their host. According to the introduction risks of these virus and disease dispersion, we propose to investigate the bacterial endosymbiont role in Culicidae in Belgium. Among the 30 species of mosquitoes identified in this country, about ten are considered as potential vectors of arboviruses. In this study, eleven species of Culicidae belonging to five genera (Culex pipiens s.l., Cx. torrentium, Cx. hortensis, Anopheles claviger, An. maculipennis s.l., An. plumbeus, Culiseta annulata, Ochlerotatus geniculatus, Oc. dorsalis, Aedes albopictus and Coquillettidia richiardii) mosquitoes fields from eight sites of Belgium were used for the screening of the presence of six genera endosymbiotic bacteria (Wolbachia sp, Commamonas sp, Delftia sp, Pseudomonas sp, Acinetobacter sp and Asaia sp) according their possible impact in mosquito biology. PCR was done for the screening of endosymbiotic bacteria mosquitoes studied. A total of 176 individuals (144 larvae and 32 adults) were used. Our results allowed us to confirm the absence of Wolbachia in An. clavigere, An. maculipennis s.l and Cx. torentium. Acinetobacter was found in every species. Current advances in understanding the mosquito–microbiota relationships may have a great impact in a better understanding of some traits of mosquito biology and in the development of innovative mosquito-borne disease-control strategies aimed to reduce mosquito vectorial capacity and/or inhibiting pathogen transmission.Contribution à l’étude des Culicidae exotiques, leur bactérie endosymbiotique Wolbachia face aux changements climatiques et les échanges commerciaux en Belgiqu

Using micro-injection technique to assess fungal toxicity in mosquito control

Topical application of insecticidal compounds allows directly exposing these substances on insect tissues and measuring their toxicity while ignoring many factors. However, this technique remains difficult to apply on mosquito larvae considering their aquatic lifestyle. Micro-injection could be used for the direct deposition of toxic compounds in the larvae. Capillaries exhibiting an injection tip with an external diameter of 0.5 mm have been designed from silica tubes. For each treatment, a capillary is mounted on a pump connected to a flow rate regulator. Culex quinquefasciatus larvae were injected with 10^7 spores/ml of entomopathogenic fungi (Aspergillus clavatus, Metarhizium anisopliae, Metarhizium sp.). Mortalities were recorded daily during 72h. The distribution of spores stained with methylene blue and injected into the body of larvae was also observed according to the system described. Results showed that spores were distributed over the whole body. The injection of Aspergillus clavatus, Metarhizium anisopliae and Metarhizium sp spores induced corrected mortalities of 62%, 53% and 57% after 72h, and differed statistically from control groups. Finally, post-mortem emergences of filaments from dead larvae were observed in the case of the three fungal strains confirming spore viability. Injection of inactivated spores (or inert bodies of similar size) could help to reject the hypothesis of a response due to the presence of foreign bodies