Diurnal biting of malaria mosquitoes in the Central African Republic indicates residual transmission may be "out of control".

Malaria control interventions target nocturnal feeding of the Anopheles vectors indoors to reduce parasite transmission. Mass deployment of insecticidal bed nets and indoor residual spraying with insecticides, however, may induce mosquitoes to blood-feed at places and at times when humans are not protected. These changes can set a ceiling to the efficacy of these control interventions, resulting in residual malaria transmission. Despite its relevance for disease transmission, the daily rhythmicity of Anopheles biting behavior is poorly documented, most investigations focusing on crepuscular hours and nighttime. By performing mosquito collections 48-h around the clock, both indoors and outdoors, and by modeling biting events using circular statistics, we evaluated the full daily rhythmicity of biting in urban Bangui, Central African Republic. While the bulk of biting by Anopheles gambiae, Anopheles coluzzii, Anopheles funestus, and Anopheles pharoensis occurred from sunset to sunrise outdoors, unexpectedly ∼20 to 30% of indoor biting occurred during daytime. As biting events did not fully conform to any family of circular distributions, we fitted mixtures of von Mises distributions and found that observations were consistent with three compartments, corresponding indoors to populations of early-night, late-night, and daytime-biting events. It is not known whether these populations of biting events correspond to spatiotemporal heterogeneities or also to distinct mosquito genotypes/phenotypes belonging consistently to each compartment. Prevalence of Plasmodium falciparum in nighttime- and daytime-biting mosquitoes was the same. As >50% of biting occurs in Bangui when people are unprotected, malaria control interventions outside the domiciliary environment should be envisaged.

Monitoring mosquito richness in an understudied area: can environmental DNA metabarcoding be a complementary approach to adult trapping?

Mosquito surveillance programmes are essential to assess the risks of local vector-borne disease outbreaks as well as for early detection of mosquito invasion events. Surveys are usually performed with traditional sampling tools (i.e., ovitraps and dipping method for immature stages or light or decoy traps for adults). Over the past decade, numerous studies have highlighted that environmental DNA (eDNA) sampling can enhance invertebrate species detection and provide community composition metrics. However, the usefulness of eDNA for detection of mosquito species has, to date, been largely neglected. Here, we sampled water from potential larval breeding sites along a gradient of anthropogenic perturbations, from the core of an oil palm plantation to the rainforest on São Tomé Island (Gulf of Guinea, Africa). We showed that (i) species of mosquitoes could be detected via metabarcoding mostly when larvae were visible, (ii) larvae species richness was greater using eDNA than visual identification and (iii) new mosquito species were also detected by the eDNA approach. We provide a critical discussion of the pros and cons of eDNA metabarcoding for monitoring mosquito species diversity and recommendations for future research directions that could facilitate the adoption of eDNA as a tool for assessing insect vector communities.

Microbial community structure reveals instability of nutritional symbiosis during the evolutionary radiation of Amblyomma ticks.

Mutualistic interactions with microbes have facilitated the adaptation of major eukaryotic lineages to restricted diet niches. Hence, ticks with their strictly blood-feeding lifestyle are associated with intracellular bacterial symbionts through an essential B vitamin supplementation. In this study, examination of bacterial diversity in 25 tick species of the genus Amblyomma showed that three intracellular bacteria, Coxiella-like endosymbionts (LE), Francisella-LE and Rickettsia, are remarkably common. No other bacterium is as uniformly present in Amblyomma ticks. Almost all Amblyomma species were found to harbour a nutritive obligate symbiont, Coxiella-LE or Francisella-LE, that is able to synthesize B vitamins. However, despite the co-evolved and obligate nature of these mutualistic interactions, the structure of microbiomes does not mirror the Amblyomma phylogeny, with a clear exclusion pattern between Coxiella-LE and Francisella-LE across tick species. Coxiella-LE, but not Francisella-LE, form evolutionarily stable associations with ticks, commonly leading to co-cladogenesis. We further found evidence for symbiont replacements during the radiation of Amblyomma, with recent, and probably ongoing, invasions by Francisella-LE and subsequent replacements of ancestral Coxiella-LE through transient co-infections. Nutritional symbiosis in Amblyomma ticks is thus not a stable evolutionary state, but instead arises from conflicting origins between unrelated but competing symbionts with similar metabolic capabilities.

Characterization and comparison of poorly known moth communities through DNA barcoding in two Afrotropical environments in Gabon 1.

Biodiversity research in tropical ecosystems-popularized as the most biodiverse habitats on Earth-often neglects invertebrates, yet invertebrates represent the bulk of local species richness. Insect communities in particular remain strongly impeded by both Linnaean and Wallacean shortfalls, and identifying species often remains a formidable challenge inhibiting the use of these organisms as indicators for ecological and conservation studies. Here we use DNA barcoding as an alternative to the traditional taxonomic approach for characterizing and comparing the diversity of moth communities in two different ecosystems in Gabon. Though sampling remains very incomplete, as evidenced by the high proportion (59%) of species represented by singletons, our results reveal an outstanding diversity. With about 3500 specimens sequenced and representing 1385 BINs (Barcode Index Numbers, used as a proxy to species) in 23 families, the diversity of moths in the two sites sampled is higher than the current number of species listed for the entire country, highlighting the huge gap in biodiversity knowledge for this country. Both seasonal and spatial turnovers are strikingly high (18.3% of BINs shared between seasons, and 13.3% between sites) and draw attention to the need to account for these when running regional surveys. Our results also highlight the richness and singularity of savannah environments and emphasize the status of Central African ecosystems as hotspots of biodiversity.

Ape malaria transmission and potential for ape-to-human transfers in Africa.

Recent studies have highlighted the large diversity of malaria parasites infecting African great apes (subgenus Laverania) and their strong host specificity. Although the existence of genetic incompatibilities preventing the cross-species transfer may explain host specificity, the existence of vectors with a high preference for a determined host represents another possibility. To test this hypothesis, we undertook a 15-mo-long longitudinal entomological survey in two forest regions of Gabon, where wild apes live, at different heights under the canopy. More than 2,400 anopheline mosquitoes belonging to 18 species were collected. Among them, only three species of Anopheles were found infected with ape Plasmodium: Anopheles vinckei, Anopheles moucheti, and Anopheles marshallii Their role in transmission was confirmed by the detection of the parasites in their salivary glands. Among these species, An. vinckei showed significantly the highest prevalence of infection and was shown to be able to transmit parasites of both chimpanzees and gorillas. Transmission was also shown to be conditioned by seasonal factors and by the heights of capture under the canopy. Moreover, human landing catches of sylvan Anopheles demonstrated the propensity of these three vector species to feed on humans when available. Our results suggest therefore that the strong host specificity observed in the Laveranias is not linked to a specific association between the vertebrate host and the vector species and highlight the potential role of these vectors as bridge between apes and humans.

Global genetic diversity of Aedes aegypti.

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co-occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub-Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans-Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.

Diversity, host switching and evolution of Plasmodium vivax infecting African great apes.

Plasmodium vivax is considered to be absent from Central and West Africa because of the protective effect of Duffy negativity. However, there are reports of persons returning from these areas infected with this parasite and observations suggesting the existence of transmission. Among the possible explanations for this apparent paradox, the existence of a zoonotic reservoir has been proposed. May great apes be this reservoir? We analyze the mitochondrial and nuclear genetic diversity of P. vivax parasites isolated from great apes in Africa and compare it to parasites isolated from travelers returning from these regions of Africa, as well as to human isolates distributed all over the world. We show that the P. vivax sequences from parasites of great apes form a clade genetically distinct from the parasites circulating in humans. We show that this clade's parasites can be infectious to humans by describing the case of a traveler returning from the Central African Republic infected with one of them. The relationship between this P. vivax clade in great apes and the human isolates is discussed.

Origin, acquisition and diversification of heritable bacterial endosymbionts in louse flies and bat flies.

The γ-proteobacterium Arsenophonus and its close relatives (Arsenophonus and like organisms, ALOs) are emerging as a novel clade of endosymbionts, which are exceptionally widespread in insects. The biology of ALOs is, however, in most cases entirely unknown, and it is unclear how these endosymbionts spread across insect populations. Here, we investigate this aspect through the examination of the presence, the diversity and the evolutionary history of ALOs in 25 related species of blood-feeding flies: tsetse flies (Glossinidae), louse flies (Hippoboscidae) and bat flies (Nycteribiidae and Streblidae). While these endosymbionts were not found in tsetse flies, we identify louse flies and bat flies as harbouring the highest diversity of ALO strains reported to date, including a novel ALO clade, as well as Arsenophonus and the recently described Candidatus Aschnera chinzeii. We further show that the origin of ALO endosymbioses extends deep into the evolutionary past of louse flies and bat flies, and that it probably played a major role in the ecological specialization of their hosts. The evolutionary history of ALOs is notably complex and was shaped by both vertical transmission and horizontal transfers with frequent host turnover and apparent symbiont replacement in host lineages. In particular, ALOs have evolved repeatedly and independently close relationships with diverse groups of louse flies and bat flies, as well as phylogenetically more distant insect families, suggesting that ALO endosymbioses are exceptionally dynamic systems.

Genomic Signatures of Microgeographic Adaptation in Anopheles coluzzii Along an Anthropogenic Gradient in Gabon.

Species distributed across heterogeneous environments often evolve locally adapted populations, but understanding how these persist in the presence of homogenizing gene flow remains puzzling. In Gabon, Anopheles coluzzii, a major African malaria mosquito is found along an ecological gradient, including a sylvatic population, away of any human presence. This study identifies into the genomic signatures of local adaptation in populations from distinct environments including the urban area of Libreville, and two proximate sites 10km apart in the La Lopé National Park (LLP), a village and its sylvatic neighborhood. Whole genome re-sequencing of 96 mosquitoes unveiled ∼ 5.7millions high-quality single nucleotide polymorphisms. Coalescent-based demographic analyses suggest an ∼ 8,000-year-old divergence between Libreville and La Lopé populations, followed by a secondary contact ( ∼ 4,000 ybp) resulting in asymmetric effective gene flow. The urban population displayed reduced effective size, evidence of inbreeding, and strong selection pressures for adaptation to urban settings, as suggested by the hard selective sweeps associated with genes involved in detoxification and insecticide resistance. In contrast, the two geographically proximate LLP populations showed larger effective sizes, and distinctive genomic differences in selective signals, notably soft-selective sweeps on the standing genetic variation. Although neutral loci and chromosomal inversions failed to discriminate between LLP populations, our findings support that microgeographic adaptation can swiftly emerge through selection on standing genetic variation despite high gene flow. This study contributes to the growing understanding of evolution of populations in heterogeneous environments amid ongoing gene flow and how major malaria mosquitoes adapt to human. Significance: Anopheles coluzzii , a major African malaria vector, thrives from humid rainforests to dry savannahs and coastal areas. This ecological success is linked to its close association with domestic settings, with human playing significant roles in driving the recent urban evolution of this mosquito. Our research explores the assumption that these mosquitoes are strictly dependent on human habitats, by conducting whole-genome sequencing on An. coluzzii specimens from urban, rural, and sylvatic sites in Gabon. We found that urban mosquitoes show de novo genetic signatures of human-driven vector control, while rural and sylvatic mosquitoes exhibit distinctive genetic evidence of local adaptations derived from standing genetic variation. Understanding adaptation mechanisms of this mosquito is therefore crucial to predict evolution of vector control strategies.

Host preference patterns in domestic and wild settings: Insights into Anopheles feeding behavior.

The adaptation of Anopheles malaria vectors to domestic settings is directly linked to their ability to feed on humans. The strength of this species-habitat association is unequal across the species within the genus, with the major vectors being particularly dependent on humans. However, our understanding of how blood-feeding behavior interacts with and adapts to environmental settings, including the presence of humans, remains limited. Using a field-based approach, we first investigated Anopheles community structure and feeding behavior patterns in domestic and sylvatic settings in La Lopé National Park in Gabon, Central Africa. We characterized the preference indices using a dual-host choice sampling approach across mosquito species, habitats, and seasons. We then quantified the plastic biting behavior of mosquito species in each habitat. We collected individuals from 16 Anopheles species that exhibited significant differences in species composition and abundance between sylvatic and domestic settings. The host-seeking behavior also varied among the seven most abundant species. The general attractiveness to each host, human or animal, remained relatively constant for each species, but with significant variations between habitats across species. These variations, to more generalist and to more anthropophilic behavior, were related to seasonal changes and distance from the village, respectively. Finally, we pointed out that the host choice of major malaria vectors changed in the absence of humans, revealing a plastic feeding behavior of these species. This study highlights the effect of humans on Anopheles distribution and feeding evolution. The characterization of feeding behavior in wild and domestic settings provides opportunities to better understand the interplay between genetic determinants of host preference and ecological factors. Our findings suggest that protected areas may offer alternative thriving conditions to major malaria vectors.

Phlebotomus (Legeromyia) multihamatus subg. nov., sp. nov. from Gabon (Diptera: Psychodidae).

During a research project aimed at the study of the Culicinae fauna of Gabon and carried out in the National Park of La Lopé, we captured an unknown sandfly male specimen (genus Phlebotomus) by CDC miniature light trap belonging to a new species for Science. Furthermore, the originality of his genitalia does not allow us to include this species in one of the existing subgenus, thus in this paper we propose the creation of a new subgenus, as Phlebotomus (Legeromyia) multihamatus sp. nov., subg. nov. described from the National Park of La Lopé, through one male captured with CDC miniature light trap. A new species and a new subgenus of sandfly is characterised by a short style with three spines, a paramere wearing a basal hook as well as a basal pouch and the absence of basal lobe on the coxite. The originality of the genitalia of the male gives way to discussion about potential primary homologies between P. multihamatus sp. nov. and Phlebotomus (Abonnencius) fortunatarum, Phlebotomus (Anaphlebotomus) stantoni and Phlebotomus (Euphlebotomus) argentipes, which should be verified for future studies. The discovery of this new species in Gabon must encourage the study of sandflies in this country.

Survey of ticks and tick-borne pathogens in wild chimpanzee habitat in Western Uganda.

BACKGROUND: Ticks and tick-borne pathogens significantly impact both human and animal health and therefore are of major concern to the scientific community. Knowledge of tick-borne pathogens is crucial for prescription of mitigation measures. In Africa, much research on ticks has focused on domestic animals. Little is known about ticks and their pathogens in wild habitats and wild animals like the endangered chimpanzee, our closest relative. METHODS: In this study, we collected ticks in the forested habitat of a community of 100 chimpanzees living in Kibale National Park, Western Uganda, and assessed how their presence and abundance are influenced by environmental factors. We used non-invasive methods of flagging the vegetation and visual search of ticks both on human team members and in chimpanzee nests. We identified adult and nymph ticks through morphological features. Molecular techniques were used to detect and identify tick-borne piroplasmids and bacterial pathogens. RESULTS: A total of 470 ticks were collected, which led to the identification of seven tick species: Haemaphysalis parmata (68.77%), Amblyomma tholloni (20.70%), Ixodes rasus sensu lato (7.37%), Rhipicephalus dux (1.40%), Haemaphysalis punctaleachi (0.70%), Ixodes muniensis (0.70%) and Amblyomma paulopunctatum (0.35%). The presence of ticks, irrespective of species, was influenced by temperature and type of vegetation but not by relative humidity. Molecular detection revealed the presence of at least six genera of tick-borne pathogens (Babesia, Theileria, Borrelia, Cryptoplasma, Ehrlichia and Rickettsia). The Afrotopical tick Amblyomma tholloni found in one chimpanzee nest was infected by Rickettsia sp. CONCLUSIONS: In conclusion, this study presented ticks and tick-borne pathogens in a Ugandan wildlife habitat whose potential effects on animal health remain to be elucidated.

Towards the invasion of wild and rural forested areas in Gabon (Central Africa) by the Asian tiger mosquito Aedes albopictus: Potential risks from the one health perspective.

BACKGROUND: Since its first record in urban areas of Central-Africa in the 2000s, the invasive mosquito, Aedes albopictus, has spread throughout the region, including in remote villages in forested areas, causing outbreaks of Aedes-borne diseases, such as dengue and chikungunya. Such invasion might enhance Ae. albopictus interactions with wild animals in forest ecosystems and favor the spillover of zoonotic arboviruses to humans. The aim of this study was to monitor Ae. albopictus spread in the wildlife reserve of La Lopé National Park (Gabon), and evaluate the magnitude of the rainforest ecosystem colonization. METHODOLOGY: From 2014 to 2018, we used ovitraps, larval surveys, BG-Sentinel traps, and human landing catches along an anthropization gradient from La Lopé village to the natural forest in the Park. CONCLUSIONS: We detected Ae. albopictus in gallery forest up to 15 km away from La Lopé village. However, Ae. albopictus was significantly more abundant at anthropogenic sites than in less anthropized areas. The number of eggs laid by Ae. albopictus decreased progressively with the distance from the forest fringe up to 200m inside the forest. Our results suggested that in forest ecosystems, high Ae. albopictus density is mainly observed at interfaces between anthropized and natural forested environments. Additionally, our data suggested that Ae. albopictus may act as a bridge vector of zoonotic pathogens between wild and anthropogenic compartments.

Diversity and phylogeny of the tick-borne bacterial genus Candidatus Allocryptoplasma (Anaplasmataceae).

The family Anaplasmataceae includes tick-borne bacteria of major public and veterinary health interest, as best illustrated by members of the genera Anaplasma and Ehrlichia. Recent epidemiological surveys have also reported on the presence of a novel putative genus in the Anaplasmataceae, Candidatus Allocryptoplasma, previously described as Candidatus Cryptoplasma in the western black-legged tick, Ixodes pacificus. However, the genetic diversity of Ca. Allocryptoplasma and its phylogenetic relationship with other Anaplasmataceae remain unclear. In this study, we developed a multi-locus sequence typing approach, examining the DNA sequence variation at five genes of Ca. Allocryptoplasma found in ticks. Combining this multi-locus sequence typing and genetic data available on public databases, we found that substantial genetic diversity of Ca. Allocryptoplasma is present in Ixodes, Amblyomma and Haemaphysalis spp. ticks on most continents. Further analyses confirmed that the Ca. Allocryptoplasma of ticks, the Ca. Allocryptoplasma of lizards and some Anaplasma-like bacteria of wild mice cluster into a monophyletic genus, divergent from all other genera of the family Anaplasmataceae. Candidatus Allocryptoplasma appears as a sister genus of Anaplasma and, with the genera Ehrlichia and Neoehrlichia, they form a monophyletic subgroup of Anaplasmataceae associated with tick-borne diseases. The detection of genetically distinct Ca. Allocryptoplasma in ticks of significant medical or veterinary interest supports the hypothesis that it is an emergent genus of tick-borne pathogens of general concern.

Title: Diversité et phylogénie du genre bactérien transmis par les tiques Candidatus Allocryptoplasma (Anaplasmataceae). Abstract: La famille des Anaplasmataceae comprend des bactéries transmises par les tiques qui présentent un intérêt majeur pour la santé publique et vétérinaire, comme les membres des genres Anaplasma et Ehrlichia. Des surveillances épidémiologiques récentes ont également signalé la présence d'un nouveau genre putatif dans les Anaplasmataceae, Candidatus Allocryptoplasma, initialement décrit comme Ca. Cryptoplasma chez une tique nord-américaine, Ixodes pacificus. Cependant, la diversité génétique des bactéries Ca. Allocryptoplasma et leurs relations phylogénétiques avec d'autres Anaplasmataceae restent méconnues. Dans cette étude, nous avons développé une approche de typage génétique multi-locus, en examinant la variation nucléotidique pour cinq gènes de bactéries Ca. Allocryptoplasma détectées chez les tiques. En combinant ce typage génétique multi-locus et les données génétiques disponibles dans les bases de données publiques, nous avons mis en évidence qu'une diversité génétique substantielle des bactéries Ca. Allocryptoplasma est présente chez les tiques des genres Ixodes, Amblyomma et Haemaphysalis sur la plupart des continents. Des analyses complémentaires confirment que les bactéries Ca. Allocryptoplasma des tiques, les bactéries Ca. Allocryptoplasma de lézards et des bactéries Anaplasma-like de souris sauvages se regroupent dans un genre monophylétique, divergent de tous les autres genres de la famille Anaplasmataceae. Candidatus Allocryptoplasma apparaît comme un genre frère d'Anaplasma et, avec les genres Ehrlichia et Neoehrlichia, ces trois genres forment un sous-groupe monophylétique d'Anaplasmataceae associé aux maladies transmises par les tiques. La détection de Ca. Allocryptoplasma dans des tiques d'intérêt médical et vétérinaire soutient l'hypothèse qu'il s'agit d'un genre émergent d'agents pathogènes majeurs.

Limited association between Wolbachia and Plasmodium falciparum infections in natural populations of the major malaria mosquito Anopheles moucheti.

Since the discovery of natural malaria vector populations infected by the endosymbiont bacterium Wolbachia, a renewed interest has arisen for using this bacterium as an alternative for malaria control. Among naturally infected mosquitoes, Anopheles moucheti, a major malaria mosquito in Central Africa, exhibits one of the highest prevalences of Wolbachia infection. To better understand whether this maternally inherited bacterium could be used for malaria control, we investigated Wolbachia influence in An. moucheti populations naturally infected by the malaria parasite Plasmodium falciparum. To this end, we collected mosquitoes in a village from Cameroon, Central Africa, where this mosquito is the main malaria vector. We found that the prevalence of Wolbachia bacterium was almost fixed in the studied mosquito population, and was higher than previously recorded. We also quantified Wolbachia in whole mosquitoes and dissected abdomens, confirming that the bacterium is also elsewhere than in the abdomen, but at lower density. Finally, we analyzed the association of Wolbachia presence and density on P. falciparum infection. Wolbachia density was slightly higher in mosquitoes infected with the malaria parasite than in uninfected mosquitoes. However, we observed no correlation between the P. falciparum and Wolbachia densities. In conclusion, our study indicates that naturally occurring Wolbachia infection is not associated to P. falciparum development within An. moucheti mosquitoes.

Citizen Science as an Approach for Responding to the Threat of Anopheles stephensi in Africa.

Even as novel technologies emerge and medicines advance, pathogen-transmitting mosquitoes pose a deadly and accelerating public health threat. Detecting and mitigating the spread of Anopheles stephensi in Africa is now critical to the fight against malaria, as this invasive mosquito poses urgent and unprecedented risks to the continent. Unlike typical African vectors of malaria, An. stephensi breeds in both natural and artificial water reservoirs, and flourishes in urban environments. With An. stephensi beginning to take hold in heavily populated settings, citizen science surveillance supported by novel artificial intelligence (AI) technologies may offer impactful opportunities to guide public health decisions and community-based interventions. Coalitions like the Global Mosquito Alert Consortium (GMAC) and our freely available digital products can be incorporated into enhanced surveillance of An. stephensi and other vector-borne public health threats. By connecting local citizen science networks with global databases that are findable, accessible, interoperable, and reusable (FAIR), we are leveraging a powerful suite of tools and infrastructure for the early detection of, and rapid response to, (re)emerging vectors and diseases.

Using a pupal exuvia to designate the undamaged neotype of a species belonging to a complex of sibling species - the case of Aedes coluzzii (Diptera, Culicidae).

The mosquito species Aedes (Ochlerotatus) coluzzii Rioux, Guilvard & Pasteur, 1998 was distinguished from its sibling species Aedes detritus (Haliday, 1833) using an isoenzymatic method that required the destruction of the entire specimen, therefore no holotype was designated by the species authors. We aimed to designate a neotype for Ae. coluzzii from specimens collected from the type-locality and individually reared up to adult stage. Genomic DNA was extracted from pupal exuvia and ITS2 was sequenced, enabling verification of the identity of each specimen as Ae. coluzzii or Ae. detritus. Among the series of Ae. coluzzii, a male was designated as neotype and deposited in a collection. To our knowledge, this is the first time the type of a mosquito species is deposited thanks to its molecular identification from its pupal exuvia. The set of identified specimens allowed additional phylogenetic and morphologic studies.

Title: Utilisation d'une exuvie nymphale pour désigner le néotype intact d'une espèce appartenant à un complexe d'espèces jumelles - le cas d'Aedes coluzzii (Diptera, Culicidae). Abstract: L'espèce de moustique Aedes (Ochlerotatus) coluzzii Rioux, Guilvard & Pasteur, 1998 a été distinguée de son espèce jumelle Aedes detritus (Haliday, 1833) par une méthode isoenzymatique qui a nécessité la destruction de l'ensemble du spécimen, et donc aucun holotype n'a été désigné par les auteurs de l'espèce. Notre objectif était de désigner un néotype pour Ae. coluzzii à partir de spécimens collectés dans la localité-type et élevés individuellement jusqu'au stade adulte. L'ADN génomique a été extrait de l'exuvie nymphale et l'ITS2 a été séquencé, permettant la vérification de l'identité de chaque spécimen comme Ae. coluzzii ou Ae. détritus. Parmi la série d'Ae. coluzzii, un mâle a été désigné comme néotype et déposé dans une collection. À notre connaissance, c'est la première fois que le type d'une espèce de moustique est déposé grâce à l'identification moléculaire à partir de son exuvie nymphale. L'ensemble des spécimens identifiés a permis des études phylogénétiques et morphologiques complémentaires.

Larval habitat determines the bacterial and fungal microbiota of the mosquito vector Aedes aegypti.

Mosquito larvae are naturally exposed to microbial communities present in a variety of larval development sites. Several earlier studies have highlighted that the larval habitat influences the composition of the larval bacterial microbiota. However, little information is available on their fungal microbiota, i.e. the mycobiota. In this study, we provide the first simultaneous characterization of the bacterial and fungal microbiota in field-collected Aedes aegypti larvae and their respective aquatic habitats. We evaluated whether the microbial communities associated with the breeding site may affect the composition of both the bacterial and fungal communities in Ae. aegypti larvae. Our results show a higher similarity in microbial community structure for both bacteria and fungi between larvae and the water in which larvae develop than between larvae from different breeding sites. This supports the hypothesis that larval habitat is a major factor driving microbial composition in mosquito larvae. Since the microbiota plays an important role in mosquito biology, unravelling the network of interactions that operate between bacteria and fungi is essential to better understand the functioning of the mosquito holobiont.

High-resolution species assignment of Anopheles mosquitoes using k-mer distances on targeted sequences.

The ANOSPP amplicon panel is a genus-wide targeted sequencing panel to facilitate large-scale monitoring of Anopheles species diversity. Combining information from the 62 nuclear amplicons present in the ANOSPP panel allows for a more senstive and specific species assignment than single gene (e.g. COI) barcoding, which is desirable in the light of permeable species boundaries. Here, we present NNoVAE, a method using Nearest Neighbours (NN) and Variational Autoencoders (VAE), which we apply to k-mers resulting from the ANOSPP amplicon sequences in order to hierarchically assign species identity. The NN step assigns a sample to a species-group by comparing the k-mers arising from each haplotype's amplicon sequence to a reference database. The VAE step is required to distinguish between closely related species, and also has sufficient resolution to reveal population structure within species. In tests on independent samples with over 80% amplicon coverage, NNoVAE correctly classifies to species level 98% of samples within the An. gambiae complex and 89% of samples outside the complex. We apply NNoVAE to over two thousand new samples from Burkina Faso and Gabon, identifying unexpected species in Gabon. NNoVAE presents an approach that may be of value to other targeted sequencing panels, and is a method that will be used to survey Anopheles species diversity and Plasmodium transmission patterns through space and time on a large scale, with plans to analyse half a million mosquitoes in the next five years.

Assessment of expertise in morphological identification of mosquito species (Diptera, Culicidae) using photomicrographs.

Accurate identification of insect species is an indispensable and challenging requirement for every entomologist, particularly if the species is involved in disease outbreaks. The European MediLabSecure project designed an identification (ID) exercise available to any willing participant with the aim of assessing and improving knowledge in mosquito taxonomy. The exercise was based on high-definition photomicrographs of mosquitoes (26 adult females and 12 larvae) collected from the western Palaearctic. Sixty-five responses from Europe, North Africa and the Middle East were usable. The study demonstrated that the responders were better at identifying females (82% correct responses) than larvae (63%). When the responders reported that they were sure of the accuracy of their ID, the success rate of ID increased (92% for females and 88% for larvae). The top three tools used for ID were MosKeyTool (72% of responders), the ID key following Becker et al. [2010. Mosquitoes and their control, 2nd edn. Berlin: Springer] (38%), and the CD-ROM of Schaffner et al. [2001. Les moustiques d'Europe: logiciel d'identification et d'enseignement - The mosquitoes of Europe: an identification and training programme. Montpellier: IRD; EID] (32%), while other tools were used by less than 10% of responders. Responders reporting the identification of mosquitoes using the MosKeyTool were significantly better (80% correct responses) than non-MosKeyTool users (69%). Most responders (63%) used more than one ID tool. The feedback from responders in this study was positive, with the exercise being perceived as halfway between educational training and a fun quiz. It raised the importance of further expanding training in mosquito ID for better preparedness of mosquito surveillance and control programmes.

Title: Évaluation de l'expertise en identification morphologique des espèces de moustiques (Diptera, Culicidae) à l'aide de photomicrographies. Abstract: L'identification précise des espèces d'insectes est une exigence indispensable et difficile pour tout entomologiste, en particulier si l'espèce est impliquée dans des épidémies. Le projet européen MediLabSecure a conçu un exercice d'identification (ID) accessible à tout participant volontaire dans le but d'évaluer et d'améliorer les connaissances en taxonomie des moustiques. L'exercice était basé sur des photomicrographies haute définition de moustiques (26 femelles adultes et 12 larves) prélevées dans le Paléarctique occidental. Soixante-cinq réponses d'Europe, d'Afrique du Nord et du Moyen-Orient ont été utilisables. L'étude a démontré que les répondants étaient meilleurs pour identifier les femelles (82 % de réponses correctes) que les larves (63 %). Lorsque les répondants ont déclaré être sûrs de l'exactitude de leur ID, le taux de réussite de l'identification était meilleur (92 % pour les femelles et 88 % pour les larves). Les trois principaux outils utilisés pour les ID étaient MosKeyTool (72 % des répondants), la clé d'identification du livre de Becker et al. (38%) et le CD-ROM de Schaffner et al. (32 %), tandis que d'autres outils étaient utilisés par moins de 10 % des répondants. Les répondants déclarant identifier des moustiques à l'aide de MosKeyTool étaient significativement meilleurs (80 % de réponses correctes) que les non-utilisateurs de MosKeyTool (69 %). La plupart des répondants (63 %) ont utilisé plus d'un outil d'identification. Les commentaires des répondants de cette étude ont été positifs, l'exercice étant perçu comme à mi-chemin entre une formation pédagogique et un quiz amusant. Il a souligné l'importance d'étendre la formation complémentaire à l'identification des moustiques pour une meilleure préparation des programmes de surveillance et de contrôle des moustiques.