Investigating molecular mechanisms of insecticide resistance in the Eastern Democratic Republic of the Congo.

BACKGROUND: Malaria vector control in the Democratic Republic of the Congo is plagued by several major challenges, including inadequate infrastructure, lack of access to health care systems and preventative measures, and more recently the widespread emergence of insecticide resistance among Anopheles mosquitoes. Across 26 provinces, insecticide resistance has been reported from multiple sentinel sites. However, to date, investigation of molecular resistance mechanisms among Anopheles vector populations in DRC has been more limited. METHODS: Adult Anopheles gambiae sensu lato (s.l.) and Anopheles funestus s.l. were collected from two sites in Sud-Kivu province and one site in Haut-Uélé province and PCR-screened for the presence of 11 resistance mutations, to provide additional information on frequency of resistance mechanisms in the eastern DRC, and to critically evaluate the utility of these markers for prospective country-wide resistance monitoring. RESULTS: L1014F-kdr and L1014S-kdr were present in 75.9% and 56.7% of An. gambiae s.l. screened, respectively, with some individuals harbouring both resistant alleles. Across the three study sites, L43F-CYP4J5 allele frequency ranged from 0.42 to 0.52, with evidence for ongoing selection. G119S-ace1 was also identified in all sites but at lower levels. A triple mutant haplotype (comprising the point mutation CYP6P4-I236M, the insertion of a partial Zanzibar-like transposable element and duplication of CYP6AA1) was present at high frequencies. In An. funestus s.l. cis-regulatory polymorphisms in CYP6P9a and CYP6P9b were detected, with allele frequencies ranging from 0.82 to 0.98 and 0.65 to 0.83, respectively. CONCLUSIONS: This study screened the most up-to-date panel of DNA-based resistance markers in An. gambiae s.l. and An. funestus s.l. from the eastern DRC, where resistance data is lacking. Several new candidate markers (CYP4J5, G119S-ace1, the triple mutant, CYP6P9a and CYP6P9b) were identified, which are diagnostic of resistance to major insecticide classes, and warrant future, larger-scale monitoring in the DRC to inform vector control decisions by the National Malaria Control Programme

Phlébotomes et écosystèmes : impact des facteurs biotiques et abiotiques sur la structure génétique et phénotypique des populations

Sandflies are hematophagous insects belonging to the family Psychodidae and the subfamily phlebotominae. This diptera, yellowish, relatively small (2-3 mm) has about 800 species. 70 of these species have been identified as potential vectors of which forty are proven ones. They can transmit different pathogens; the main ones are Leishmania and phlebovirus.This thesis focused on sandflies, vectors of leishmaniasis. Leishmaniases are parasitic diseases caused by protozoa of the genus Leishmania. They affect a wide range of vertebrate hosts, including humans and dogs. They are still a major problem of public health in many countries and are currently in expansion. Although this disease is widely studied, we still have a lot to learn about its vector: the sandfly. For example, the organization of populations in ecosystems and the parameters which structure them are very little studied up to now. It is therefore essential to know the biology of the different actors of a parasite cycle to better understand the transmission of pathogens, to assess risks of transmission, and finally to be able to effectively fight against the disease.In this context, the aim of this thesis is to study the ecology and the structuring of sandfly populations in a known endemic area of leishmaniasis and the impact of biotic and abiotic factors on their organization. To reach this objective, we performed captures of sandflies along a 14km transect located in the Montpellier region which presents an altitudinal, climate and environmental diversity. Sandfly populations have been characterized by taxonomic, spatio-temporal, genetic (microsatellites) and morphometric (geometry morphometry) approaches. The genetic, morphometric and species distribution results were then confronted with climatic (temperature, relative humidity) or environmental parameters (altitude, slope, station, microhabitat).During this work, four species were captured: Phlebotomus ariasi (93.23%), P. perniciosus (0.48%), P. mascittii (0.11%) and S. minuta (6.18%). They have a seasonal activity from May to October with an abundance peak in July-August when average temperatures are optimal for sandflies (20-30°C). Although the environment has been considerably transformed in our study area in 30 years, the abundance of sandflies does not seem to have changed significantly, highlighting their ability to adapt to ecosystem modifications in short and long-term. The presence and abundance of the two predominant species (P. ariasi and S. minuta) are significantly influenced by altitude, temperature, relative humidity, slope and wall orientation.The genetic analyses show that diversity is maintained at all scales of study and that sandflies are organized in micropopulations. The morphometric geometry data reveal a sexual dimorphism, well known in insects but also a phenotypic structuring correlated to environmental or temporal factors (month, slope, altitude and station).Both of these approaches, because of their complementarity, help provide information on the ecology and organization of sandfly populations and to discuss about the consequences in terms of leishmaniasis transmission.Les phlébotomes sont des insectes hématophages appartenant à la famille des Psychodidae et à la sous-famille des Phlebotominae. Cet insecte diptère, jaunâtre, relativement petit (2 à 3 mm) compte environ 800 espèces. 70 de ces espèces ont été identifiées comme vecteurs potentiels dont une quarantaine sont des vecteurs prouvés. Ils peuvent transmettre différents pathogènes dont les principaux sont les leishmanies et les phlébovirus. Ce travail de thèse est focalisé sur les phlébotomes vecteurs de la leishmaniose. Les leishmanioses sont des maladies parasitaires causées par un protozoaire du genre Leishmania. Elles touchent un large panel d’hôtes vertébrés, dont l’homme et le chien. Elles sont toujours un problème de santé publique majeur dans de nombreux pays et sont actuellement en expansion. Bien que cette maladie soit largement étudiée, nous avons encore beaucoup apprendre sur son vecteur : le phlébotome. Par exemple, l’organisation des populations dans les écosystèmes et les paramètres qui les structurent, sont à l’heure actuelle très peu étudiés. Il est pourtant primordial de bien connaitre la biologie des différents acteurs d’un cycle parasitaire pour mieux comprendre la transmission du pathogène, évaluer les risques et enfin être capable de lutter efficacement contre la maladie. Dans ce contexte, le but de cette thèse est d’étudier l’écologie et la structure des populations de phlébotomes dans un foyer connu de leishmaniose et l’impact des facteurs biotiques et abiotiques sur leur organisation. Pour atteindre cet objectif, nous avons réalisé une collecte de phlébotomes le long d’un transect de 14km localisé dans la région de Montpellier, présentant une diversité altitudinale, climatique et environnementale. Les populations de phlébotomes ont été caractérisées d’un point de vue taxonomique, spatio-temporel, génétique (microsatellites), et morphométrique (géométrie morphométrie). Les résultats génétiques, morphométriques et de distribution des espèces ont été ensuite confrontés à des paramètres climatiques (température, humidité) ou environnementaux (altitude, versant, station, microhabitat).Durant ce travail, 4 espèces ont été capturées : Phlebotomus ariasi (93,23%), P. perniciosus (0,48%), P. mascittii (0,11%) et S. minuta (6,18%). Elles ont une activité saisonnière de Mai à Octobre avec un pic d’abondance en Juillet-Août quand les températures moyennes sont optimales pour les phlébotomes (20-30°C). Bien que l'environnement ait été considérablement transformé dans notre zone d'étude en 30 ans, l'abondance des phlébotomes ne semble pas avoir changé de façon significative, soulignant leur capacité d'adaptation aux modifications de l'écosystème à court et long terme. La présence et l’abondance des deux espèces prédominantes (P. ariasi et S. minuta) sont significativement influencées par l’altitude, la température, l’humidité relative, le versant ainsi que l’orientation des murs. Les analyses génétiques montrent que la diversité est conservée à toutes les échelles d’études et qu’il existe une structuration des phlébotomes en micropopulations. Les données de géométrie morphométrie révèlent un dimorphisme sexuel bien connu chez les insectes mais également une structuration phénotypique en fonction des facteurs environnementaux ou temporels (mois, versant, altitude et station). Ces deux types d’approches permettent grâce à leur complémentarité d’apporter des informations sur l’écologie et l’organisation des populations de phlébotomes et de discuter des conséquences sur la transmission de la leishmaniose

Sandflies and ecosystems : impact of biotic and abiotic factors on the genetic and phenotypic population structure

Les phlébotomes sont des insectes hématophages appartenant à la famille des Psychodidae et à la sous-famille des Phlebotominae. Cet insecte diptère, jaunâtre, relativement petit (2 à 3 mm) compte environ 800 espèces. 70 de ces espèces ont été identifiées comme vecteurs potentiels dont une quarantaine sont des vecteurs prouvés. Ils peuvent transmettre différents pathogènes dont les principaux sont les leishmanies et les phlébovirus. Ce travail de thèse est focalisé sur les phlébotomes vecteurs de la leishmaniose. Les leishmanioses sont des maladies parasitaires causées par un protozoaire du genre Leishmania. Elles touchent un large panel d’hôtes vertébrés, dont l’homme et le chien. Elles sont toujours un problème de santé publique majeur dans de nombreux pays et sont actuellement en expansion. Bien que cette maladie soit largement étudiée, nous avons encore beaucoup apprendre sur son vecteur : le phlébotome. Par exemple, l’organisation des populations dans les écosystèmes et les paramètres qui les structurent, sont à l’heure actuelle très peu étudiés. Il est pourtant primordial de bien connaitre la biologie des différents acteurs d’un cycle parasitaire pour mieux comprendre la transmission du pathogène, évaluer les risques et enfin être capable de lutter efficacement contre la maladie. Dans ce contexte, le but de cette thèse est d’étudier l’écologie et la structure des populations de phlébotomes dans un foyer connu de leishmaniose et l’impact des facteurs biotiques et abiotiques sur leur organisation. Pour atteindre cet objectif, nous avons réalisé une collecte de phlébotomes le long d’un transect de 14km localisé dans la région de Montpellier, présentant une diversité altitudinale, climatique et environnementale. Les populations de phlébotomes ont été caractérisées d’un point de vue taxonomique, spatio-temporel, génétique (microsatellites), et morphométrique (géométrie morphométrie). Les résultats génétiques, morphométriques et de distribution des espèces ont été ensuite confrontés à des paramètres climatiques (température, humidité) ou environnementaux (altitude, versant, station, microhabitat).Durant ce travail, 4 espèces ont été capturées : Phlebotomus ariasi (93,23%), P. perniciosus (0,48%), P. mascittii (0,11%) et S. minuta (6,18%). Elles ont une activité saisonnière de Mai à Octobre avec un pic d’abondance en Juillet-Août quand les températures moyennes sont optimales pour les phlébotomes (20-30°C). Bien que l'environnement ait été considérablement transformé dans notre zone d'étude en 30 ans, l'abondance des phlébotomes ne semble pas avoir changé de façon significative, soulignant leur capacité d'adaptation aux modifications de l'écosystème à court et long terme. La présence et l’abondance des deux espèces prédominantes (P. ariasi et S. minuta) sont significativement influencées par l’altitude, la température, l’humidité relative, le versant ainsi que l’orientation des murs. Les analyses génétiques montrent que la diversité est conservée à toutes les échelles d’études et qu’il existe une structuration des phlébotomes en micropopulations. Les données de géométrie morphométrie révèlent un dimorphisme sexuel bien connu chez les insectes mais également une structuration phénotypique en fonction des facteurs environnementaux ou temporels (mois, versant, altitude et station). Ces deux types d’approches permettent grâce à leur complémentarité d’apporter des informations sur l’écologie et l’organisation des populations de phlébotomes et de discuter des conséquences sur la transmission de la leishmaniose.Sandflies are hematophagous insects belonging to the family Psychodidae and the subfamily phlebotominae. This diptera, yellowish, relatively small (2-3 mm) has about 800 species. 70 of these species have been identified as potential vectors of which forty are proven ones. They can transmit different pathogens; the main ones are Leishmania and phlebovirus.This thesis focused on sandflies, vectors of leishmaniasis. Leishmaniases are parasitic diseases caused by protozoa of the genus Leishmania. They affect a wide range of vertebrate hosts, including humans and dogs. They are still a major problem of public health in many countries and are currently in expansion. Although this disease is widely studied, we still have a lot to learn about its vector: the sandfly. For example, the organization of populations in ecosystems and the parameters which structure them are very little studied up to now. It is therefore essential to know the biology of the different actors of a parasite cycle to better understand the transmission of pathogens, to assess risks of transmission, and finally to be able to effectively fight against the disease.In this context, the aim of this thesis is to study the ecology and the structuring of sandfly populations in a known endemic area of leishmaniasis and the impact of biotic and abiotic factors on their organization. To reach this objective, we performed captures of sandflies along a 14km transect located in the Montpellier region which presents an altitudinal, climate and environmental diversity. Sandfly populations have been characterized by taxonomic, spatio-temporal, genetic (microsatellites) and morphometric (geometry morphometry) approaches. The genetic, morphometric and species distribution results were then confronted with climatic (temperature, relative humidity) or environmental parameters (altitude, slope, station, microhabitat).During this work, four species were captured: Phlebotomus ariasi (93.23%), P. perniciosus (0.48%), P. mascittii (0.11%) and S. minuta (6.18%). They have a seasonal activity from May to October with an abundance peak in July-August when average temperatures are optimal for sandflies (20-30°C). Although the environment has been considerably transformed in our study area in 30 years, the abundance of sandflies does not seem to have changed significantly, highlighting their ability to adapt to ecosystem modifications in short and long-term. The presence and abundance of the two predominant species (P. ariasi and S. minuta) are significantly influenced by altitude, temperature, relative humidity, slope and wall orientation.The genetic analyses show that diversity is maintained at all scales of study and that sandflies are organized in micropopulations. The morphometric geometry data reveal a sexual dimorphism, well known in insects but also a phenotypic structuring correlated to environmental or temporal factors (month, slope, altitude and station).Both of these approaches, because of their complementarity, help provide information on the ecology and organization of sandfly populations and to discuss about the consequences in terms of leishmaniasis transmission

Persistence of Toscana virus in sugar and blood meals of phlebotomine sand flies: epidemiological and experimental consequences

International audienceAbstract Many virological studies have tested the persistence of enveloped RNA viruses in various environmental and laboratory conditions and shown their short-term persistence. In this article, we analyzed Toscana virus (TOSV) infectivity, a pathogenic sandfly-borne phlebovirus, in two different conditions: in the sugar meal and blood meal of sand flies. Our results showed that TOSV RNA was detectable up to 15 days in sugar solution at 26 °C and up to 6 h in blood at 37 °C. Moreover, TOSV remains infective for 7 days in sugar solution and for minimum 6 h in rabbit blood. TOSV has shown persistent infectivity/viability under different conditions, which may have important epidemiological consequences. These results strengthen new hypotheses about the TOSV natural cycle, such as the possibility of horizontal transmission between sand flies through infected sugar meal

Broader Geographical Distribution of Toscana Virus in the Mediterranean Region Suggests the Existence of Larger Varieties of Sand Fly Vectors

International audienceToscana virus (TOSV) is endemic in the Mediterranean basin, where it is transmitted by sand flies. TOSV can infect humans and cause febrile illness as well as neuroinvasive infections affecting the central and peripheral nervous systems. Although TOSV is a significant human pathogen, it remains neglected and there are consequently many gaps of knowledge. Recent seroepidemiology studies and case reports showed that TOSV’s geographic distribution is much wider than was assumed a decade ago. The apparent extension of the TOSV circulation area raises the question of the sandfly species that are able to transmit the virus in natural conditions. Phlebotomus (Ph.) perniciosus and Ph. perfiliewi were historically identified as competent species. Recent results suggest that other species of sand flies could be competent for TOSV maintenance and transmission. Here we organize current knowledge in entomology, epidemiology, and virology supporting the possible existence of additional phlebotomine species such as Ph. longicuspis, Ph. sergenti, Ph. tobbi, Ph. neglectus, and Sergentomyia minuta in TOSV maintenance. We also highlight some of the knowledge gaps to be addressed in future studies

Revue systématique des arthropodes hématophages présents chez les bovins en France

The authors are also particularly grateful to the working group “Vecteurs” and the specialized expert committee on health and animal welfare: “Santé et Bien-Etre des Animaux” (SABA), both from ANSES and to Claire Garros (CIRAD, Montpellier), Nil Rahola (IRD, Montpellier), and Gérard Duvallet (Montpellier University) for sharing their knowledge and expertise on hematophagous arthropods.International audienceThe arrival of pathogens, whether zoonotic or not, can have a lasting effect on commercial livestock farms, with dramatic health, social and economic consequences. However, available data concerning the arthropod vectors present and circulating on livestock farms in France are still very imprecise, fragmentary, and scattered. In this context, we conducted a systematic review of the hematophagous arthropod species recorded on different types of cattle farms in mainland France (including Corsica). The used vector “groups” studied were biting flies, biting midges, black flies, fleas, horse flies, lice, louse flies, mosquitoes, sand flies, and ticks. A large number of documents were selected ( N = 9,225), read ( N = 1,047) and analyzed ( N = 290), allowing us to provide distribution and abundance maps of different species of medical and veterinary interest according to literature data. Despite the large number of documents collected and analyzed, there are few data provided on cattle farm characteristics. Moreover, data on all arthropod groups lack numerical detail and are based on limited data in time and/or space. Therefore, they are not generalizable nor comparable. There is still little information on many vectors (and their pathogens) and still many unknowns for most studied groups. It appears necessary to provide new, updated and standardized data, collected in different geographical and climatological areas. Finally, this work highlights the lack of entomologists, funding, training and government support, leading to an increased risk of uncontrolled disease emergence in cattle herds.L’arrivée d’agents pathogènes, même non zoonotiques, peut affecter durablement les élevages commerciaux, avec des conséquences sanitaires, sociales et économiques dramatiques. Cependant, les données disponibles concernant les arthropodes vecteurs présents et circulants dans les élevages français sont encore très imprécises, fragmentaires et dispersées. Dans ce contexte, nous avons réalisé une revue systématique des espèces d’arthropodes hématophages présentes dans différents types d’élevages bovins en France métropolitaine (Corse comprise). Les “groupes” de vecteurs retenus et étudiés étaient les mouches piqueuses, les culicoïdes, les simulies, les puces, les taons, les poux, les hippobosques, les moustiques, les phlébotomes et les tiques. Un grand nombre de documents ont été sélectionnés ( N = 9 225), lus ( N = 1 047) et analysés ( N = 290), ce qui nous a permis de fournir des cartes de distribution et d’abondance des différentes espèces d’intérêt médical et vétérinaire en fonction des données de la littérature. Malgré le grand nombre de documents collectés et analysés, peu de données sont fournies sur les caractéristiques des élevages bovins. En outre, les données sur tous les groupes d’arthropodes manquent de détails numériques et sont basées sur des informations limitées dans le temps et/ou l’espace. Elles ne sont donc ni généralisables ni comparables. Il existe encore peu d’informations sur de nombreux vecteurs (et leurs agents pathogènes) et de multiples inconnues subsistent pour les groupes les plus étudiés. Il apparaît nécessaire de collecter de nouvelles données, actualisées et standardisées dans différentes zones géographiques et climatiques. Enfin, ce travail met en évidence le manque d’entomologistes, de financements, de formations et de soutien gouvernemental, ce qui accroît le risque d’émergence non contrôlée de maladies dans les troupeaux de bovins

Altitudinal Variations In Wing Morphology Of Aedes Albopictus (Diptera, Culicidae) In Albania, The Region Where It Was First Recorded In Europe

The rapid spread and settlement of Aedes albopictus mosquitoes across at least 28 countries in Europe, as well as several countries in Asia Minor, the Middle East and Africa, has made it one of the most invasive species of all time. Even though the biology of Ae. albopictus in its native tropical environment has been documented for a long time, the biology and ecology of this species in newly colonized temperate environments remain poorly known despite its important role as a vector for about twenty arboviruses. In this context, the main goals of this work were to investigate Ae. albopictus phenotypic variations at a local scale in Albania, the country where Ae. albopictus was first recorded in Europe, and to determine if its phenotypes could be affected by altitude. Analysis of Ae. albopictus wing phenotypes was performed using a geometric morphometric approach. We observed shape and size variations among altitudinal populations of Ae. albopictus. Differences of wing phenotypes were highlighted between altitude groups for male and female mosquitoes. The phenotypic variations observed in Ae. albopictus between altitudinal groups indicated these populations are exposed to environmental and ecological pressures. These results suggest the presence of phenotypic plasticity in this species.PubMedWo

The native European Aedes geniculatus mosquito species can transmit chikungunya virus

International audienceEurope is the world's leading tourism destination and is receiving every year travellers from areas with active arbovirus transmission. There is thus a threat of mosquito-borne virus emergence in Europe due to the presence of the invasive mosquito vector Aedes albopictus. Little attention has been paid about the possible role of indigenous mosquito species as vectors of emerging arboviruses. Here, we assessed the vector competence dynamic of Aedes geniculatus, a European anthropophilic mosquito species, for chikungunya virus (CHIKV) in comparison with an European population of Ae. albopictus. We revealed that Ae. geniculatus is highly susceptible to CHIKV infection and could transmit the virus. By specifically exploring the vector competence dynamic in both mosquito species, we revealed that the cumulative distribution of CHIKV incubation period in Ae. geniculatus was delayed by several days as compared to Ae. albopictus. Our results strengthen the importance of considering indigenous species as potential vectors for emerging arboviruses. They also revealed the importance of considering variation in arbovirus dissemination or transmission dynamics in mosquitoes when performing vector competence assays. We will discuss the implications of our results on a CHIKV outbreak dynamic in a theoretical framework