Fara Nantenaina Raharimalala

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

Symbiotic 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 with Wolbachia 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.

Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus

BackgroundThe 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.ResultsA 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.ConclusionsThe 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.

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

BackgroundIn 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.MethodsThe 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.ResultsThe 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 in man-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%.ConclusionIn 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.

Prevalence, genomic and metabolic profiles of Acinetobacter and Asaia associated with field-caught Aedes albopictus from Madagascar

The presence of cultivable bacteria Acinetobacter and Asaia was recently demonstrated in the mosquito vector Aedes albopictus. However, it is not known how prevalent these bacteria are in field populations. Here, the presence of these bacteria in Ae. albopictus populations from Madagascar was diagnosed by amplification of 16S rRNA gene fragments. Both genera were detected at relatively high frequencies, 46% for Asaia and 74% for Acinetobacter. The prevalence of Acinetobacter correlated significantly with mosquito gender, and the prevalence of Asaia with the interaction between mosquito gender and the sampling site. For each bacterial genus, more male than female mosquitoes were infected. Using pulse field gel electrophoresis, no significant difference in genome size was found between Acinetobacter isolates from mosquitoes compared with free-living Acinetobacter. However, a great diversity was observed in plasmid numbers (from 1 to 12) and sizes (from < 8 to 690 kb). Mosquito isolates utilized fewer substrates than free-living isolates, but some substrates known as blood or plant components were specifically utilized by mosquito isolates. Therefore it is likely that a specific subpopulation of Acinetobacter is selected by Ae. albopictus. Overall, this study emphasizes the need to gain a global view on the bacterial partners in mosquito vectors.

Reintroduction of the invasive mosquito species Aedes albopictus in Belgium in July 2013.

Since its first report in 2000, the invasive mosquito Aedes albopictus was not found any more during the different entomological inspections performed at its place of introduction in Belgium between 2001 and 2012. In July 2013, one adult male was captured at the same site (a platform of imported used tires located in Vrasene, Oost-Vlaanderen Province), during a monitoring using CO2-baited trap. This finding suggests the reintroduction of the species in Belgium via the used tire trade.

Efficacy of seed extracts of Annona squamosa and Annona muricata (Annonaceae) for the control of Aedes albopictus and Culex quinquefasciatus (Culicidae)

Objective: To evaluate the potential efficacy of seed extracts of Annona squamosa and Annona muricata used as natural insecticides to control adult and larvae of the vectors Aedes albopictus and Culex quinquefasciatus under laboratory conditions. Methods: Aqueous and oil extracts of the two plants were prepared from dried seeds. Preliminary identifications of the chemical components of each seed extracts were performed using microreactional and GCP techniques. Larvae and adults of Aedes albopictus and Culex quinquefasciatus were collected from the breeding sites in coastal and highlands regions of Madagascar. WHO standardized tests of susceptibility for larvae and imaginal stage of mosquitoes were realized to determine mortality and LC50 of mosquitoes. Results: Chemical identifications showed that these extracts contain alkaloids and flavonoids compounds that probably confer their biological insecticidal proprieties. CPG analysis showed also the presence of various fatty acids. On adult mosquitoes, significant insecticidal effects were observed with both aqueous and oil extracts of the two plant seeds compared to mortality induced by deltamethrin, an insecticide used as reference. Extracts of Annona muricata induced high mortality rate to both species of mosquito compared to extracts of Annona squamosa at all concentrations tested. The LC50 of seed extracts ranged from 1% to 5% for adults and 0.5% to 1% for larvae. Conclusions: The seed extracts of these two plants may be used as mosquito controlling agents and offer a new approach to a less costly, practical and environmentally friendly control of vector borne diseases. Peer reviewer

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. A total 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.

Diversity and ecology survey of mosquitoes potential vectors in Belgian equestrian farms: A threat prevention of mosquito-borne equine arboviruses

Emergence of West Nile Virus was recently recorded in several European countries, which can lead to severe health problems in horse populations. Europe is also at risk of introduction of mosquito-borne equine alphavirus from Americas. Prevention of these arboviruses requires a clear understanding of transmission cycles, especially their vectors. To characterize mosquito fauna, their ecology and identify potential vectors of equine arboviruses in Belgium, entomological surveys of six equestrian farms located in the Wolloon Region were conducted during 2011-2012. The harvest of mosquitoes was based on larval sampling (272 samples from 111 breeding sites) and monthly adults trapping (CO2-baited traps, Mosquito Magnet Liberty Plus). Among 51,493 larvae and 319 adult mosquitoes collected, morphological identification showed the presence of 11 species: Anopheles claviger (Meigen), An. maculipennis s.l. (Meigen), An. plumbeus (Stephens), Culex hortensis (Ficalbi), Cx. territans (Walker), Cx. pipiens s.l. L., Cx. torrentium (Martini), Coquillettidia richiardii (Ficalbi), Culiseta annulata (Schrank), Aedes cantans (Meigen), Ae. geniculatus (Olivier). Molecular identification of Cx. pipiens species complex allowed the detection of three molecular forms, Pipiens (92.3%), Molestus (4.6%) and Hybrid (3.1%). Larvae of Cx. pipiens sl and Cx. torrentium were omnipresent and the most abundant species. Water troughs, ponds and slurry (liquid manure) were the most favorable breeding sites of mosquito larvae. Based upon behavior and ecology of the identified mosquito species, Studied Belgian equestrian farms seem to provide a suitable environment and breeding sites for the proliferation of potential vectors of arboviruses and those being a real nuisance problem for horses and neighboring inhabitants.

Vector competence of Culex antennatus and Anopheles coustani mosquitoes for Rift Valley fever virus in Madagascar.

Culex antennatus (Diptera: Culicidae), Anopheles coustani (Diptera: Culicidae) and Anopheles squamosus/cydippis were found to be infected with Rift Valley fever virus (RVFV) during an epidemic that occurred in 2008 and 2009 in Madagascar. To understand the roles played by Cx. antennatus and An. coustani in virus maintenance and transmission, RVFV vector competence was assessed in each species. Mosquito body parts and saliva of mosquitoes that fed on RVFV‐infected blood were tested for RVFV using real‐time quantitative polymerase chain reaction (RT‐qPCR) assays. Overall, viral RNA was detected in body parts and saliva at 5 days post‐infection (d.p.i.) in both species. At 5 d.p.i., infection rates were 12.5% (3/24) and 15.8% (6/38), disseminated infection rates were 100% (3/3) and 100% (6/6), transmission rates were 33.3% (1/3) and 83.3% (5/6), and transmission efficiencies were 4.2% (1/24) and 13.2% (5/38) in Cx. antennatus and An. coustani, respectively. Although RVFV detected in saliva did not propagate on to Vero cells, these results support potential roles for these two mosquito species in the transmission of RVFV.

Usefulness and accuracy of MALDI-TOF mass spectrometry as a supplementary tool to identify mosquito vector species …

Arthropod‐borne diseases are important causes of morbidity and mortality. The identification of vector species relies mainly on morphological features and/or molecular biology tools. The first method requires specific technical skills and may result in misidentifications, and the second method is time‐consuming and expensive. The aim of the present study is to assess the usefulness and accuracy of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a supplementary tool with which to identify mosquito vector species and to invest in the creation of an international database. A total of 89 specimens belonging to 10 mosquito species were selected for the extraction of proteins from legs and for the establishment of a reference database. A blind test with 123 mosquitoes was performed to validate the MS method. Results showed that: (a) the spectra obtained in the study with a given species differed from the spectra of the same species collected in another country, which highlights the need for an international database; (b) MALDI‐TOF MS is an accurate method for the rapid identification of mosquito species that are referenced in a database; (c) MALDI‐TOF MS allows the separation of groups or complex species, and (d) laboratory specimens undergo a loss of proteins compared with those isolated in the field. In conclusion, MALDI‐TOF MS is a useful supplementary tool for mosquito identification and can help inform vector control.