Systematic review of hematophagous arthropods present in cattle in France.

The 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.

Title: Revue systématique des arthropodes hématophages présents chez les bovins en France. Abstract: 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.

Reconstructing the post-glacial spread of the sand fly Phlebotomus mascittii Grassi, 1908 (Diptera: Psychodidae) in Europe.

Phlebotomine sand flies (Diptera: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). In Central Europe, Phlebotomus mascittii is the predominant species, but largely understudied. To better understand factors driving its current distribution, we infer patterns of genetic diversity by testing for signals of population expansion based on two mitochondrial genes and model current and past climate and habitat suitability for seven post-glacial maximum periods, taking 19 climatic variables into account. Consequently, we elucidate their connections by environmental-geographical network analysis. Most analyzed populations share a main haplotype tracing back to a single glacial maximum refuge area on the Mediterranean coasts of South France, which is supported by network analysis. The rapid range expansion of Ph. mascittii likely started in the early mid-Holocene epoch until today and its spread possibly followed two routes. The first one was through northern France to Germany and then Belgium, and the second across the Ligurian coast through present-day Slovenia to Austria, toward the northern Balkans. Here we present a combined approach to reveal glacial refugia and post-glacial spread of Ph. mascittii and observed discrepancies between the modelled and the current known distribution might reveal yet overlooked populations and potential further spread.

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

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.

New Microsatellite Markers for Genetic Studies on Sergentomyia schwetzi (Diptera: Psychodidae): A Suspected Vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in the Canine Leishmaniasis Focus of Mont-Rolland, Senegal.

Visceral leishmaniasis is not endemic in West Africa, but prevalence of canine leishmaniasis and seroprevalence of Leishmania infantum infection in humans are high in the Mont Rolland community (Thiès region, Senegal). Previous studies in this area showed that Sergentomyia schwetzi could be the potential vector of Le. infantum. To precisely describe the biology and population structure of this potential vector, we identified eight novel microsatellite loci to characterize Se. schwetzi populations. We tested these loci in Se. schwetzi populations from five locations at Mont Rolland (Thiès, Senegal). All the loci were polymorphic, with a mean of 17.25 alleles (observed heterozygosity: 0.455). We did not detect any evidence of scoring errors due to stuttering and large allele dropout. Moreover, several of these loci were also amplified in six other sand fly species (Sergentomyia magna, Sergentomyia dubia, Sergentomyia minuta, Phlebotomus duboscqi, Phlebotomus perniciosus, and Phlebotomus ariasi). These preliminary results demonstrate the utility of these microsatellite markers for Se. schwetzi (and for the other sand fly species) population genetic studies.

Taxonomical insights and ecology of sandfly (Diptera, Psychodidae) species in six provinces of Northern Vietnam.

We studied sandfly (Diptera: Psychodidae) populations in six provinces of Vietnam. This work explores the diversity of sandfly species according to the province, as well as environment, and updated information on public health since leishmaniasis cases were reported in two provinces. Sandflies were collected using 428 CDC light traps from May 30 to October 13, 2016 and identified based on the morphology of the cibarium, pharynx and/or male genitalia or female spermathecae. A total of 2585 sandflies belonging to five genera and 13 identified species were collected. The main species were: the Sergentomyia barraudi group (12.53%), Se. sylvatica (9.63%) and Phlebotomus stantoni (3.95%). In all, 294 Sergentomyia specimens classified as Se. sp2 and Se. sp3 and a heterogeneous group, herein called Se. und_sp., showed unknown morphological characteristics requiring further studies. We provide detailed comments about morphological description and taxonomical identification in order to help standardization of sandfly classification in Southeast Asia. We observed differentiation according to the provinces in terms of density and species richness, with Lang Son having the highest density and Ninh Binh having the highest species richness. The majority of specimens were collected in rock caves and outdoors, suggesting mainly cavernicolous and exophilic characters of sandfly species in Northern Vietnam. However, specimens were also collected in intra- and peri-domiciliary sites. It is worth noting that Ph. stantoni was the main species found in dog sheds and indoors, and in particular in a leishmaniasis patient's house.

TITLE: Aperçu taxonomique et écologie des espèces de phlébotomes (Diptera, Psychodidae) dans six provinces du nord du Vietnam. ABSTRACT: Nous avons étudié les populations de phlébotomes (Diptera : Psychodidae) dans six provinces du Vietnam. Ce travail explore la diversité des espèces de phlébotomes selon les provinces, les environnements et les informations mises à jour sur la santé publique depuis que des cas de leishmanioses ont été signalés dans deux provinces. Des phlébotomes ont été collectés à l'aide de 428 pièges lumineux CDC du 30 mai au 13 octobre 2016 et identifiés sur la base de la morphologie du cibarium, du pharynx et/ou des organes génitaux des mâles ou des spermathèques des femelles. Au total, 2585 phlébotomes, appartenant à cinq genres et 13 espèces identifiées, ont été collectés. Les principales espèces étaient : le groupe Sergentomyia barraudi (12,53 %), Se. sylvatica (9,63 %) et Phlebotomus stantoni (3,95 %). 294 spécimens de Sergentomyia classés comme Se. sp2 et Se. sp3 et un groupe hétérogène ici appelé Se. und_sp. ont montré des caractéristiques morphologiques inconnues nécessitant des études complémentaires. Nous fournissons des commentaires détaillés sur la description morphologique et l'identification taxonomique afin d'aider à la normalisation de la classification des phlébotomes en Asie du Sud-Est. Nous avons observé une différenciation selon les provinces en termes de densité et de richesse en espèces, avec Lang Son ayant la plus forte densité et Ninh Binh ayant la plus grande richesse en espèces. La majorité des spécimens ont été collectés dans des grottes rocheuses et à l'extérieur, suggérant principalement des caractères cavernicoles et exophiles des espèces de phlébotomes du nord du Vietnam. Cependant, des spécimens ont également été collectés dans des sites intra et péri-domiciliaires. Il convient de noter que Ph. stantoni était la principale espèce trouvée dans les abris pour chiens et à l'intérieur, notamment dans la maison d'un patient atteint de leishmaniose.

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.

Incubation Period for Neuroinvasive Toscana Virus Infections.

Toscana virus (TOSV) is an emerging pathogen in the Mediterranean area and is neuroinvasive in its most severe form. Basic knowledge on TOSV biology is limited. We conducted a systematic review on travel-related infections to estimate the TOSV incubation period. We estimated the incubation period at 12.1 days.

Insecticide resistance profiles in malaria vector populations from Sud-Kivu in the Democratic Republic of the Congo.

BACKGROUND: Insecticide resistance has become a widespread problem causing a decline in the effectiveness of vector control tools in sub-Saharan Africa. In this situation, ongoing monitoring of vector susceptibility to insecticides is encouraged by the WHO to guide national malaria control programmes. Our study was conducted from April to November 2018 in Tchonka (Sud-Kivu, Democratic Republic of the Congo) and reported primary data on the resistance status of Anopheles funestus and Anopheles gambiae. METHODS: Insecticide susceptibility bioassays were performed on wild populations of A. funestus and A. gambiae using WHO insecticide-impregnated papers at discriminating concentration. In addition, PCR was performed to identify mosquito species and to detect kdr and ace-1R mutations involved in insecticide resistance. RESULTS: Bioassay results show resistance to all tested insecticides except pirimiphos-methyl, propoxur, fenitrothion and malathion with a mortality rate ranging from 95.48 to 99.86%. The addition of piperonyl butoxide (PBO) increased the susceptibility of vectors to deltamethrin and alpha-cypermethrin by exhibiting a mortality ranging from 91.50 to 95.86%. The kdr mutation was detected at high frequencies (approximately 0.98) within A. gambiae while ace-1R was not detected. CONCLUSIONS: This study provides useful data on the insecticide resistance profiles of malaria vector populations to better manage vector control. Our results highlight that, despite the high level of resistance, organophosphorus compounds and pyrethroids + PBO remain effective against the vectors.

Altitude and hillside orientation shapes the population structure of the Leishmania infantum vector Phlebotomus ariasi.

Despite their role in Leishmania transmission, little is known about the organization of sand fly populations in their environment. Here, we used 11 previously described microsatellite markers to investigate the population genetic structure of Phlebotomus ariasi, the main vector of Leishmania infantum in the region of Montpellier (South of France). From May to October 2011, we captured 1,253 Ph. ariasi specimens using sticky traps in 17 sites in the North of Montpellier along a 14-km transect, and recorded the relevant environmental data (e.g., altitude and hillside). Among the selected microsatellite markers, we removed five loci because of stutter artifacts, absence of polymorphism, or non-neutral evolution. Multiple regression analyses showed the influence of altitude and hillside (51% and 15%, respectively), and the absence of influence of geographic distance on the genetic data. The observed significant isolation by elevation suggested a population structure of Ph. ariasi organized in altitudinal ecotypes with substantial rates of migration and positive assortative mating. This organization has implications on sand fly ecology and pathogen transmission. Indeed, this structure might favor the global temporal and spatial stability of sand fly populations and the spread and increase of L. infantum cases in France. Our results highlight the necessity to consider sand fly populations at small scales to study their ecology and their impact on pathogens they transmit.

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

Toscana 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.

Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories.

Sand flies (Diptera: Psychodidae) are natural vectors of Leishmania. For the initiation of sand fly experimental infections either Leishmania amastigotes or promastigotes can be used. In order to obtain comparable results, it is necessary to adjust and standardize procedures. During this study, we conducted promastigote- and amastigote-initiated infections of Leishmania infantum Nicolle, 1908 parasites in Phlebotomus (Larroussius) perniciosus Newstead, 1911 in two laboratories with different levels of biosafety protection. Protocol originally designed for a biosafety level 2 facility was modified for biosafety level 3 facility and infection parameters were compared. Particularly, specially designed plastic containers were used for blood feeding; feeders were placed outside the sand fly cage, on the top of the mesh; feeding was performed inside the climatic chamber; separation of engorged females was done in Petri dishes kept on ice; engorged females were kept in the cardboard containers until dissection. All experiments, conducted in both laboratories, resulted in fully developed late stage infections with high number of parasites and colonization of the stomodeal valve. We demonstrated that protocol originally designed for biosafety level 2 facilities can be successfully modified for other biosafety facilities, depending on the special requirements of the individual institution/laboratory.

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.

TITLE: Variations phénotypiques des ailes d'Aedes albopictus (Diptera, Culicidae) en fonction de l'altitude en Albanie, la région où il a été signalé pour la première fois en Europe. ABSTRACT: La propagation et l'établissement rapides du moustique Aedes albopictus dans au moins 28 pays d'Europe, ainsi que dans plusieurs pays d'Asie mineure, du Moyen-Orient et d'Afrique, en ont fait l'une des espèces les plus invasives de tous les temps. Bien que la biologie d'Ae. albopictus dans son milieu tropical naturel soit documentée depuis longtemps, la biologie et l'écologie de cette espèce dans les milieux tempérés nouvellement colonisés restent mal connues malgré son rôle important de vecteur d'une vingtaine d'arbovirus. Dans ce contexte, les principaux objectifs de ce travail étaient d'étudier les variations phénotypiques d'Ae. albopictus à l'échelle locale en Albanie, le pays où Ae. albopictus a été signalé pour la première fois en Europe, et de déterminer si ses phénotypes pourraient être affectés par l'altitude. L'analyse des phénotypes des ailes d'Ae. albopictus a été effectuée en utilisant une approche de morphométrie géométrique. Nous avons observé des variations de forme et de taille parmi les populations altitudinales d'Ae. albopictus. Des différences de phénotypes d'ailes ont été mises en évidence entre les groupes d'altitude pour les moustiques mâles et femelles. Les variations phénotypiques observées chez Ae. albopictus entre les groupes altitudinaux indiquent que ces populations sont exposées à des pressions environnementales et écologiques. Ces résultats suggèrent la présence d'une plasticité phénotypique chez cette espèce.

The native European Aedes geniculatus mosquito species can transmit chikungunya virus.

Europe 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.

Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part II: Important Points to Consider for Fieldwork and Subsequent Virological Screening.

In this series of review articles entitled "Practical guidelines for studies on sandfly-borne phleboviruses," the important points to be considered at the prefieldwork stage were addressed in part I, including parameters to be taken into account to define the geographic area for sand fly trapping and how to organize field collections. Here in part II, the following points have been addressed: (1) factors influencing the efficacy of trapping and the different types of traps with their respective advantages and drawbacks, (2) how to process the trapped sand flies in the field, and (3) how to process the sand flies in the virology laboratory. These chapters provide the necessary information for adopting the most appropriate procedures depending on the requirements of the study. In addition, practical information gathered through years of experience of translational projects is included to help newcomers to fieldwork studies.

Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part I: Important Points to Consider Ante Field Work.

The purpose of this review is to provide practical information to help researchers intending to perform "from field to laboratory" studies on phleboviruses transmitted by sandflies. This guideline addresses the different steps to be considered starting from the field collection of sandflies to the laboratory techniques aiming at the detection, isolation, and characterization of sandfly-borne phleboviruses. In this guideline article, we address the impact of various types of data for an optimal organization of the field work intending to collect wildlife sandflies for subsequent virology studies. Analysis of different data sets should result in the geographic positioning of the trapping stations. The overall planning, the equipment and tools needed, the manpower to be deployed, and the logistics to be anticipated and set up should be organized according to the objectives of the field study for optimal efficiency.

Ecology and morphological variations in wings of Phlebotomus ariasi (Diptera: Psychodidae) in the region of Roquedur (Gard, France): a geometric morphometrics approach.

BACKGROUND: Phlebotomus ariasi Tonnoir, 1921, is the predominant sand fly species in the Cevennes region and a proven vector of Leishmania infantum, which is the main pathogen of visceral and canine leishmaniasis in the south of France. Even if this species is widely present in Western Mediterranean countries, its biology and ecology remain poorly known. The main goals of this work are to investigate the phenotypic variation of P. ariasi at a local scale in a region characterized by climatic and environmental fluctuations, and to determine if slope and altitude could affect the sand fly phenotypes. RESULTS: Sand flies were captured along a 14 km-long transect in 2011 from May to October. At the same time, environmental data such as altitude and slope were also collected. Morphological analysis of P. ariasi wings was performed by a geometric morphometrics approach. We found morphological variation among local populations of P. ariasi. Strong shape and size variations were observed in the course of the season (particularly in June and July) for both genders. During June, we highlighted differences in wing phenotypes according to altitude for both sexes and to slope and station for females. CONCLUSIONS: The phenotypic variations observed in P. ariasi along the studied transect indicated these populations are subjected to environmental pressures. Nevertheless, it seems that sand flies are more sensitive to extrinsic factors in June and July, suggesting a phenotypic plasticity.

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

BACKGROUND: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. METHODS/PRINCIPAL FINDINGS: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. CONCLUSIONS: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

Ecology and spatiotemporal dynamics of sandflies in the Mediterranean Languedoc region (Roquedur area, Gard, France).

BACKGROUND: Phlebotomine sandflies are hematophagous insects widely present in Western Mediterranean countries and known for their role as Leishmania vectors. During the last ten years, the risk of leishmaniasis re-emergence has increased in France. However, sandfly biology and ecology in the South of France remain poorly known because the last detailed study on their spatiotemporal dynamics was performed over 30 years ago. The aim of the present study was to update our knowledge on sandfly ecology by determining their spatiotemporal dynamics and by investigating the relationship between environmental/climatic factors and the presence and abundance of sandflies in the South of France. METHODS: An entomological survey was carried out during three years (2011-2013) along a 14 kilometer-long transect. The findings were compared with the data collected along the same transect in 1977. Data loggers were placed in each station and programmed to record temperature and relative humidity every six hours between April 2011 and November 2014. Several environmental factors (such as altitude, slope and wall orientation (North, East, West and South)) were characterized at each station. RESULTS: Four sandfly species were collected: Phlebotomus ariasi and Sergentomyia minuta, which were predominant, Ph. perniciosus and Ph. mascittii. Sandfly activity within the studied area started in May and ended in October with peaks in July-August at the optimum average temperature. We found a positive effect of altitude and temperature and a negative effect of relative humidity on Ph. ariasi and Se. minuta presence. We detected interspecific differences and non-linear effects of these climatic variables on sandfly abundance. Although the environment has considerably changed in 30 years, no significant difference in sandfly dynamics and species diversity was found by comparing the 1977 and 2011-2013 data. CONCLUSION: Our study shows that this area maintains a rich sandfly fauna with high Ph. ariasi population density during the active season. This represents a risk for Leishmania transmission. The analysis revealed that the presence and abundance of Ph. ariasi and Se. minuta were differently correlated with the environmental and climatic factors. Comparison with the data collected in 1977 highlighted the sandfly population stability, suggesting that they can adapt, in the short and long term, to changing ecosystems.

Identification of phlebotomine sand flies using one MALDI-TOF MS reference database and two mass spectrometer systems.

BACKGROUND: Rapid, accurate and high-throughput identification of vector arthropods is of paramount importance in surveillance programmes that are becoming more common due to the changing geographic occurrence and extent of many arthropod-borne diseases. Protein profiling by MALDI-TOF mass spectrometry fulfils these requirements for identification, and reference databases have recently been established for several vector taxa, mostly with specimens from laboratory colonies. METHODS: We established and validated a reference database containing 20 phlebotomine sand fly (Diptera: Psychodidae, Phlebotominae) species by using specimens from colonies or field-collections that had been stored for various periods of time. RESULTS: Identical biomarker mass patterns ('superspectra') were obtained with colony- or field-derived specimens of the same species. In the validation study, high quality spectra (i.e. more than 30 evaluable masses) were obtained with all fresh insects from colonies, and with 55/59 insects deep-frozen (liquid nitrogen/-80 °C) for up to 25 years. In contrast, only 36/52 specimens stored in ethanol could be identified. This resulted in an overall sensitivity of 87 % (140/161); specificity was 100 %. Duration of storage impaired data counts in the high mass range, and thus cluster analyses of closely related specimens might reflect their storage conditions rather than phenotypic distinctness. A major drawback of MALDI-TOF MS is the restricted availability of in-house databases and the fact that mass spectrometers from 2 companies (Bruker, Shimadzu) are widely being used. We have analysed fingerprints of phlebotomine sand flies obtained by automatic routine procedure on a Bruker instrument by using our database and the software established on a Shimadzu system. The sensitivity with 312 specimens from 8 sand fly species from laboratory colonies when evaluating only high quality spectra was 98.3 %; the specificity was 100 %. The corresponding diagnostic values with 55 field-collected specimens from 4 species were 94.7 % and 97.4 %, respectively. CONCLUSIONS: A centralized high-quality database (created by expert taxonomists and experienced users of mass spectrometers) that is easily amenable to customer-oriented identification services is a highly desirable resource. As shown in the present work, spectra obtained from different specimens with different instruments can be analysed using a centralized database, which should be available in the near future via an online platform in a cost-efficient manner.

New microsatellite markers for multi-scale genetic studies on Phlebotomus ariasi Tonnoir, vector of Leishmania infantum in the Mediterranean area.

The population structure of Phlebotomus ariasi, a proven vector of Leishmania infantum in the Mediterranean area, is still poorly understood. Previously, only two microsatellite loci had been developed to study the population genetics of this species. Herein we use these loci and determined fourteen novel microsatellite loci, useful for the characterization of P. ariasi populations. These loci were tested on three populations of P. ariasi, two from France and one from Portugal. In addition, the usefulness of these markers was also evaluated on seven other sandfly species. We show, that for P. ariasi, 15 of the 16 loci selected were polymorphic, with a mean of 4.25 alleles and an observed heterozygosity of 0.299. Within the P. ariasi population of France, 11 loci were polymorphic, with an average of 2.44 alleles and an observed heterozygosity of 0.2177. The fixation index was moderate among the French populations but high between French and Portuguese populations. In addition, eight loci were also found to be amplifiable in six other Phlebotomus species. These results demonstrate the usefulness of this new set of microsatellite loci for population structure and molecular ecology studies of P. ariasi at various spatial scales, but also of other sandfly species.