Genetic diversity of endosymbiotic bacteria Wolbachia infecting two mosquito species of the genus Eretmapodites occurring in sympatry in the Comoros archipelago.

Introduction: The influence of Wolbachia on mosquito reproduction and vector competence has led to renewed interest in studying the genetic diversity of these bacteria and the phenotypes they induced in mosquito vectors. In this study, we focused on two species of Eretmapodites, namely Eretmapodites quinquevittatus and Eretmapodites subsimplicipes, from three islands in the Comoros archipelago (in the Southwestern Indian Ocean). Methods: Using the COI gene, we examined the mitochondrial genetic diversity of 879 Eretmapodites individuals from 54 sites. Additionally, we investigated the presence and genetic diversity of Wolbachia using the wsp marker and the diversity of five housekeeping genes commonly used for genotyping through Multiple Locus Sequence Typing (MLST). Results and discussion: Overall, Er. quinquevittatus was the most abundant species in the three surveyed islands and both mosquito species occurred in sympatry in most of the investigated sites. We detected a higher mitochondrial genetic diversity in Er. quinquevittatus with 35 reported haplotypes (N = 615 specimens, Hd = 0.481 and π = 0.002) while 13 haplotypes were found in Er. subsimplicipes (N = 205 specimens, Hd = 0.338 and π = 0.001), this difference is likely due to the bias in sampling size between the two species. We report for the first time the presence of Wolbachia in these two Eretmapodites species. The prevalence of Wolbachia infection varied significantly between species, with a low prevalence recorded in Er. quinquevittatus (0.8%, N = 5/627) while infection was close to fixation in Er. subsimplicipes (87.7%, N = 221/252). Both male and female individuals of the two mosquito species appeared to be infected. The analysis of MLST genes revealed the presence of two Wolbachia strains corresponding to two new strain types (STs) within the supergroups A and B, which have been named wEretA and wEretB. These strains were found as mono-infections and are closely related, phylogenetically, to Wolbachia strains previously reported in Drosophila species. Finally, we demonstrate that maternal transmission of Wolbachia is imperfect in Er. subsimplicipes, which could explain the presence of a minority of uninfected individuals in the field.

An annotated wing interferential pattern dataset of dipteran insects of medical interest for deep learning.

Several Diptera species are known to transmit pathogens of medical and veterinary interest. However, identifying these species using conventional methods can be time-consuming, labor-intensive, or expensive. A computer vision-based system that uses Wing interferential patterns (WIPs) to identify these insects could solve this problem. This study introduces a dataset for training and evaluating a recognition system for dipteran insects of medical and veterinary importance using WIPs. The dataset includes pictures of Culicidae, Calliphoridae, Muscidae, Tabanidae, Ceratopogonidae, and Psychodidae. The dataset is complemented by previously published datasets of Glossinidae and some Culicidae members. The new dataset contains 2,399 pictures of 18 genera, with each genus documented by a variable number of species and annotated as a class. The dataset covers species variation, with some genera having up to 300 samples.

Wing Interferential Patterns (WIPs) and machine learning for the classification of some Aedes species of medical interest.

Hematophagous insects belonging to the Aedes genus are proven vectors of viral and filarial pathogens of medical interest. Aedes albopictus is an increasingly important vector because of its rapid worldwide expansion. In the context of global climate change and the emergence of zoonotic infectious diseases, identification tools with field application are required to strengthen efforts in the entomological survey of arthropods with medical interest. Large scales and proactive entomological surveys of Aedes mosquitoes need skilled technicians and/or costly technical equipment, further puzzled by the vast amount of named species. In this study, we developed an automatic classification system of Aedes species by taking advantage of the species-specific marker displayed by Wing Interferential Patterns. A database holding 494 photomicrographs of 24 Aedes spp. from which those documented with more than ten pictures have undergone a deep learning methodology to train a convolutional neural network and test its accuracy to classify samples at the genus, subgenus, and species taxonomic levels. We recorded an accuracy of 95% at the genus level and > 85% for two (Ochlerotatus and Stegomyia) out of three subgenera tested. Lastly, eight were accurately classified among the 10 Aedes sp. that have undergone a training process with an overall accuracy of > 70%. Altogether, these results demonstrate the potential of this methodology for Aedes species identification and will represent a tool for the future implementation of large-scale entomological surveys.

Deep learning and wing interferential patterns identify Anopheles species and discriminate amongst Gambiae complex species.

We present a new and innovative identification method based on deep learning of the wing interferential patterns carried by mosquitoes of the Anopheles genus to classify and assign 20 Anopheles species, including 13 malaria vectors. We provide additional evidence that this approach can identify Anopheles spp. with an accuracy of up to 100% for ten out of 20 species. Although, this accuracy was moderate (> 65%) or weak (50%) for three and seven species. The accuracy of the process to discriminate cryptic or sibling species is also assessed on three species belonging to the Gambiae complex. Strikingly, An. gambiae, An. arabiensis and An. coluzzii, morphologically indistinguishable species belonging to the Gambiae complex, were distinguished with 100%, 100%, and 88% accuracy respectively. Therefore, this tool would help entomological surveys of malaria vectors and vector control implementation. In the future, we anticipate our method can be applied to other arthropod vector-borne diseases.

Wing Interferential Patterns (WIPs) and machine learning, a step toward automatized tsetse (Glossina spp.) identification.

A simple method for accurately identifying Glossina spp in the field is a challenge to sustain the future elimination of Human African Trypanosomiasis (HAT) as a public health scourge, as well as for the sustainable management of African Animal Trypanosomiasis (AAT). Current methods for Glossina species identification heavily rely on a few well-trained experts. Methodologies that rely on molecular methodologies like DNA barcoding or mass spectrometry protein profiling (MALDI TOFF) haven't been thoroughly investigated for Glossina sp. Nevertheless, because they are destructive, costly, time-consuming, and expensive in infrastructure and materials, they might not be well adapted for the survey of arthropod vectors involved in the transmission of pathogens responsible for Neglected Tropical Diseases, like HAT. This study demonstrates a new type of methodology to classify Glossina species. In conjunction with a deep learning architecture, a database of Wing Interference Patterns (WIPs) representative of the Glossina species involved in the transmission of HAT and AAT was used. This database has 1766 pictures representing 23 Glossina species. This cost-effective methodology, which requires mounting wings on slides and using a commercially available microscope, demonstrates that WIPs are an excellent medium to automatically recognize Glossina species with very high accuracy.

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.

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.

Assessing entomological risk factors for arboviral disease transmission in the French Territory of the Wallis and Futuna Islands.

BACKGROUND: The French overseas Territory of the Wallis and Futuna Islands has been affected by several dengue epidemics. Aedes polynesiensis is the main mosquito vector described in this territory. Other Aedes species have been reported, but recent entomological data are missing to infer the presence of other potential arbovirus vectors and to assess the entomological risk factors for transmission of arboviral diseases. METHODOLOGY/ PRINCIPAL FINDINGS: An entomological prospective study was conducted on the three main islands of the territory to determine the presence and distribution of Aedes spp. Larvae, pupae and adult mosquitoes were collected from 54 sampling points in different environments, with a final sampling of 3747 immature stages and 606 adults. The main identified breeding sites were described. Ae. polynesiensis was found in every sampled site in peridomestic and wild habitats. Ae. aegypti was only found on the island of Wallis in peridomestic environments with a limited distribution. Two other Aedes species endemic to the Pacific were recorded, Aedes oceanicus and Aedes futunae. To evaluate the ability of local Ae. polynesiensis to transmit the chikungunya virus (CHIKV), two field populations were analyzed for vector competence using experimental oral exposure of females to CHIKV and infection, dissemination and transmission assays. Results showed that both populations of Ae. polynesiensis were competent for CHIKV (30% at 7 days post-infection). CONCLUSIONS/SIGNIFICANCE: This study showed the ubiquitous distribution and abundance of Ae. polynesiensis on the three islands and demonstrated that local populations were able to transmit CHIKV. Combined with the presence and expansion of Ae. aegypti on the main island of Wallis, these data highlight the risk of transmission of arboviral diseases in the territory of Wallis and Futuna and provide relevant information for entomological surveillance and vector control programs.

New records, distribution, and updated checklists of old world Phlebotomine sand flies, with emphasis on Africa, southwest Asia, and central Asia.

This article includes new records, distribution, and updated checklist of Phlebotomine sand flies (Psychodidae, Diptera) in the Old World (Africa including West Indian Ocean Islands, Southwest Asia, and Central Asia) based on specimen collections housed in different repositories worldwide. About 124 species have primary types housed in 5 repositories including holotypes (45 species, 4 subspecies), syntypes (28 species, 3 subspecies), "types" (14 species), allotypes (10 species), paratypes (36 species, 3 subspecies), lectotypes (13 species), and cotype (5 species), mounted on 671 slides. New abbreviations were proposed for 2 subgenera in the genus Phlebotomus and 6 subgenera in the genus Sergentomyia. New country records were noted in Phlebotomus (4 species in 4 subgenera in 7 countries) and Sergentomyia (10 species in 4 subgenera in 8 countries). For species diversity in the Old World, Phlebotomus includes 92 species and 7 subspecies in 9 subgenera, while Sergentomyia includes 166 species and 16 subspecies in 12 subgenera. A total of 95 species and 7 subspecies of 2 genera (Phlebotomus and Sergentomyia) were recorded in Africa while about 26 species and 16 subspecies in Southwest Asia and Central Asia.

Trapping the Tiger: Efficacy of the Novel BG-Sentinel 2 With Several Attractants and Carbon Dioxide for Collecting Aedes albopictus (Diptera: Culicidae) in Southern France.

Targeted trapping of mosquito disease vectors plays an important role in the surveillance and control of mosquito-borne diseases. The Asian tiger mosquito, Aedes albopictus (Skuse), is an invasive species, which is spreading throughout the world, and is a potential vector of 24 arboviruses, particularly efficient in the transmission of chikungunya, dengue, and zika viruses. Using a 4 × 4 Latin square design, we assessed the efficacy of the new BG-Sentinel 2 mosquito trap using the attractants BG-lure and (R)-1-octen-3-ol cartridge, alone or in combination, and with and without carbon dioxide, for the field collection of Ae. albopictus mosquitoes.We found a synergistic effect of attractant and carbon dioxide that significantly increased twofold to fivefold the capture rate of Ae. albopictus. In combination with carbon dioxide, BG-lure cartridge is more effective than (R)-1-octen-3-ol in attracting females, while a combination of both attractants and carbon dioxide is the most effective for capturing males. In the absence of carbon dioxide, BG-lure cartridge alone did not increase the capture of males or females when compared with an unbaited trap. However, the synergistic effect of carbon dioxide and BG-lure makes this the most efficient combination in attracting Ae. albopictus.

Autochthonous Chikungunya Transmission and Extreme Climate Events in Southern France.

BACKGROUND: Extreme precipitation events are increasing as a result of ongoing global warming, but controversy surrounds the relationship between flooding and mosquito-borne diseases. A common view among the scientific community and public health officers is that heavy rainfalls have a flushing effect on breeding sites, which negatively affects vector populations, thereby diminishing disease transmission. During 2014 in Montpellier, France, there were at least 11 autochthonous cases of chikungunya caused by the invasive tiger mosquito Aedes albopictus in the vicinity of an imported case. We show that an extreme rainfall event increased and extended the abundance of the disease vector Ae. albopictus, hence the period of autochthonous transmission of chikungunya. METHODOLOGY/PRINCIPAL FINDINGS: We report results from close monitoring of the adult and egg population of the chikungunya vector Ae. albopictus through weekly sampling over the entire mosquito breeding season, which revealed an unexpected pattern. Statistical analysis of the seasonal dynamics of female abundance in relation to climatic factors showed that these relationships changed after the heavy rainfall event. Before the inundations, accumulated temperatures are the most important variable predicting Ae. albopictus seasonal dynamics. However, after the inundations, accumulated rainfall over the 4 weeks prior to capture predicts the seasonal dynamics of this species and extension of the transmission period. CONCLUSIONS/SIGNIFICANCE: Our empirical data suggests that heavy rainfall events did increase the risk of arbovirus transmission in Southern France in 2014 by favouring a rapid rise in abundance of vector mosquitoes. Further studies should now confirm these results in different ecological contexts, so that the impact of global change and extreme climatic events on mosquito population dynamics and the risk of disease transmission can be adequately understood.

Larval habitats characterization and species composition of Anopheles mosquitoes in Tunisia, with particular attention to Anopheles maculipennis complex.

In Tunisia, malaria transmission has been interrupted since 1980. However, the growing number of imported cases and the persistence of putative vectors stress the need for additional studies to assess the risk of malaria resurgence in the country. In this context, our aim was to update entomological data concerning Anopheles mosquitoes in Tunisia. From May to October of 2012, mosquito larval specimens were captured in 60 breeding sites throughout the country and identified at the species level using morphological keys. Environmental parameters of the larval habitats were recorded. Specimens belonging to the An. maculipennis complex were further identified to sibling species by the ribosomal deoxyribonucleic acid (rDNA)-internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) technique. In total, 647 Anopheles larvae were collected from 25 habitats. Four species, including An. labranchiae, An. multicolor, An. sergentii, and An. algeriensis, were morphologically identified. rDNA-ITS2 PCR confirmed that An. labranchiae is the sole member of the An. maculipennis complex in Tunisia. An. labranchiae was collected throughout northern and central Tunisia, and it was highly associated with rural habitat, clear water, and sunlight areas. Larvae of An. multicolor and An. sergentii existed separately or together and were collected in southern Tunisia in similar types of breeding places.

The mosquitoes (Diptera: Culidae) of Seychelles: taxonomy, ecology, vectorial importance, and identification keys.

BACKGROUND: During recent periods, the islands of the Republic of Seychelles experienced many diseases such as dengue, chikungunya, Bancroft's filaria and malaria. Mosquitoes transmit the agents that cause these diseases. Published information on mosquitoes in the Seychelles is notably dispersed in the literature. The maximum number of species obtained on a single field survey does not exceed 14 species. METHODS: We performed a comprehensive bibliographic review using mosquito and Seychelles as the key words, as well as conducted a mosquito field survey for larval and adult stages during the rainy season in December 2008. Sixteen sites were sampled on four granitic islands (Mahé, Praslin, La Digue and Aride) and six sites on coralline atolls in the extreme southwest of the country (Aldabra group). RESULTS: We found published references to 21 mosquito species identified at least on one occasion in the Seychelles. Our collections comprised 18 species of mosquitoes, all of them from the subfamily Culicinae; no Anophelinae was found. We also confirm that Aedes seychellensis is a junior synonym of Ae. (Aedimorphus) albocephalus. The first records for Culex antennatus and Cx. sunyaniensis are presented from the country, specifically from Aldabra and Praslin, respectively. Based on a comparison of the taxa occurring on the granitic versus coralline islands, only three species, Ae. albocephalus, Cx. scottii and Cx. simpsoni are shared. Aedes albopictus appeared to exclude largely Ae. aegypti on the granitic islands; however, Ae. aegypti was common on Aldabra, where Ae. albopictus has not been recorded. The notable aggressiveness of mosquitoes towards humans on coralline islands was mainly due to two species, the females of which are difficult to distinguish: Ae. fryeri and Ae. (Aedimorphus) sp. A. The number of mosquito species collected at least once in the Seychelles is now 22, among which five species (Ae. (Adm) sp. A, Cx. stellatus, Uranotaenia browni. Ur. nepenthes and Ur. pandani) and one subspecies (Ae. vigilax vansomerenae) are considered as endemic. Two illustrated identification keys, one for adult females and the other for larval stages, are presented. CONCLUSIONS: The knowledge of the culicidian fauna in the Seychelles has been notably updated. The number of mosquito species is relatively large with regards to land surface and distances to continental Africa, although the anophelines are totally lacking. The complex natural history of mosquitoes in the Seychelles provides examples of both vicariance- and dispersal-mediated divergences. They present superb examples for theoretical and applied island biology.

Anthropogenic habitat disturbance and ecological divergence between incipient species of the malaria mosquito Anopheles gambiae.

BACKGROUND: Anthropogenic habitat disturbance is a prime cause in the current trend of the Earth's reduction in biodiversity. Here we show that the human footprint on the Central African rainforest, which is resulting in deforestation and growth of densely populated urban agglomerates, is associated to ecological divergence and cryptic speciation leading to adaptive radiation within the major malaria mosquito Anopheles gambiae. METHODOLOGY/PRINCIPAL FINDINGS: In southern Cameroon, the frequency of two molecular forms--M and S--among which reproductive isolation is strong but still incomplete, was correlated to an index of urbanisation extracted from remotely sensed data, expressed as the proportion of built-up surface in each sampling unit. The two forms markedly segregated along an urbanisation gradient forming a bimodal cline of ∼6-km width: the S form was exclusive to the rural habitat, whereas only the M form was present in the core of densely urbanised settings, co-occurring at times in the same polluted larval habitats of the southern house mosquito Culex quinquefasciatus--a species association that was not historically recorded before. CONCLUSIONS/SIGNIFICANCE: Our results indicate that when humans create novel habitats and ecological heterogeneities, they can provide evolutionary opportunities for rapid adaptive niche shifts associated with lineage divergence, whose consequences upon malaria transmission might be significant.