The Phlebotomine sand fly fauna of Switzerland revisited.

Sand flies (Diptera: Psychodidae, Phlebotominae; Newstead, 1911) are widespread in Europe, being particularly common in the Mediterranean region but rare north of the Alps. Thus, Switzerland is an opportune place to investigate the sand fly fauna on both sides of the Alpine crest, in southern sub-Mediterranean climate and northern oceanic temperate climate. We reinvestigated the Swiss sand fly fauna with the aim to assess changes in composition, altitudinal distribution, abundance and seasonality. Thirty-eight sites were investigated with light traps and/or interception sticky traps in 4 years. Ninety and 380 specimens were caught by light traps and sticky traps, respectively, at 15 collecting sites. Four species were identified. Phlebotomus mascittii (Grassi, 1908), Phlebotomus perniciosus (Newstead, 1911) and Sergentomyia minuta (Rondani, 1843) were confirmed in Ticino, and P. mascittii for the first time in neighbouring Grisons. Also, Phlebotomus neglectus (Tonnoir, 1921) is for the first time reported, though at a very low density compared to P. perniciosus at the same site. Its presence in Ticino supports the northward spread observed in Italy. Sand flies were detected north of the Alps at one site only, endorsing a historical report. Overall, the low density of P. perniciosus and very low density of P. neglectus suggest that canine leishmaniosis may not be an important disease risk in Switzerland.

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.

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.

Phlebotomine sand flies (Diptera, Psychodidae) from Pha Tong cave, Northern Thailand with a description of two new species and taxonomical thoughts about Phlebotomus stantoni.

BACKGROUND: In South-East Asia, Thailand is the country with the highest number of human autochthonous cases of leishmaniases mostly due to Leishmania martiniquensis. Their transmission remains unresolved to date even though sand flies are known vectors of leishmaniases. As such, we focused a study on the sand fly fauna of a cave in Thailand to explore the biodiversity of potential Leishmania vectors. MAIN RESULTS: We carried out an inventory in Pha Tong cave. We caught and identified 570 Phlebotomine sand flies (452 females and 118 males) and identified 14 species belonging to the genera Phlebotomus, Idiophlebotomus, Chinius, Sergentomyia and Grassomyia. Among these 14 species, two could not be related to known sand fly species. Herein, we propose the description of two new sand fly species, previously unknown to science. The first new species, Phlebotomus shadenae n. sp. is a sand fly of the subgenus Anaphlebotomus. It is morphologically close to Ph. stantoni, a species widely distributed throughout Southeast Asia. However, it differs by the length of the genital filaments in males or by the length of the ducts of the spermathecae in females as well as the high divergence of cytochrome b sequences. Additionally, we revised the systematics of the subgenus Anaphlebotomus and reinstated, by examination of its holotype, the validity of Ph. maynei, an Indian wrongly considered as a synonym of Ph. stantoni in the past. The second new species, Sergentomyia maiae n. sp., differs from a species in the same group, Se. barraudi, by an original cibarial double row of vertical teeth as well as by molecular data. CONCLUSIONS: We propose the description of two new sand fly species for Science with morphological and molecular evidence. Ph. shadenae n. sp. was also found to be distributed in the south of Thailand and in Laos. Future studies need to determine whether these two species can play a role as vectors of Leishmania parasites, Trypanosomatids or Phlebovirus. Most of the species caught in the present study are strictly cavernicolous except Grassomyia sp. and a few Sergentomyia.

On abnormal Phlebotomus perniciosus (Diptera: Psychodidae: Phlebotominae) from the center of Tunisia.

Phlebotomus perniciosus is the most important vector of Leishmania infantum in the Western part of the Mediterranean basin. Atypical specimens of Ph. perniciosus called (pna) with a parameral sheath simply curved, not bifurcated, have been reported in many locations. In this study, we describe abnormal Ph. perniciosus male specimens. Sand flies were collected in center Tunisia and identified morphologically. Cytochrome b PCR-sequencing was carried out for abnormal Ph. perniciosus male specimens in order to confirm the morphological identification and assess the intraspecific genetic polymorphism. Abnormal Ph. perniciosus specimens were characterized by a multifurcated parameral sheath. A parsimonious haplotype network based on cyt b locus analysis showed that typical and abnormal Ph. perniciosus described in our investigation were grouped together in the same branch. Thus, genetic outcomes confirmed that the new phenotype is only an original morphotype of Ph. perniciosus.

On the systematics of Phlebotomus betisi and two new related species from Laos with proposal of the new subgenus Lewisius.

Phlebotomus betisi was described from Malaysia and classified after its description in the subgenus Larroussius. It was the only species to have a pharyngeal armature composed of dot-like teeth and an annealed spermatheca whose head is carried by a neck in females. Males were characterized by having a style bearing five spines and a simple paramere. The study of sandflies originating from a cave in Laos enabled us to discover and describe two sympatric species close to Ph. betisi Lewis & Wharton, 1963 and new for Science: Ph. breyi Vongphayloth & Depaquit n. sp., and Ph. sinxayarami Vongphayloth & Depaquit n. sp. They were characterized morphologically, morphometrically, geomorphometrically, molecularly, and proteomically (MALDI-TOF). All approaches converged to validate the individualization of these species whose morphological differential characters lay in the two genders by the observation of the interocular suture and by the length of the last two segments of the maxillary palps. In males, the length of the genital filaments discriminates these species. Females are distinguished by the length of the ducts of the spermathecae as well as by the narrow or enlarged shape of the neck bearing their head. Lastly, the particular position of the spines of the gonostyle coupled with molecular phylogeny led us to remove these three species from the subgenus Larroussius Nizulescu, 1931 and to classify them in a new subgenus: Lewisius Depaquit & Vongphayloth n. subg.

Title: Sur la systématique de Phlebotomus betisi et de deux nouvelles espèces apparentées du Laos avec proposition du nouveau sous-genre Lewisius. Abstract: Phlebotomus betisi a été décrit de Malaisie et fut classé après sa description dans le sous-genre Larroussius. C'était la seule espèce à posséder chez la femelle une armature pharyngienne composée de dents en forme de points et à avoir une spermathèque annelée dont la tête est portée par un cou. Les mâles se caractérisaient par un style porteur de cinq épines et par un paramère simple. L'étude de Phlébotomes originaires d'une grotte du Laos nous a permis de découvrir et de décrire deux espèces sympatriques proches de Ph. betisi Lewis & Wharton, 1963 et nouvelles pour la Science : Ph. breyi Vongphayloth & Depaquit n. sp., et Ph. sinxayarami Vongphayloth & Depaquit n. sp. Elles ont été caractérisées morphologiquement, morphométriquement, géomorphométriquement, moléculairement et protéomiquement (MALDI-TOF). Toutes ces approches convergent pour valider l'individualisation de chacune de ces espèces dont les caractères morphologiques différentiels reposent dans les deux sexes par l'observation de la suture interoculaire et par la longueur des deux derniers segments des palpes maxillaires. Chez les mâles, la longueur des filaments génitaux discrimine ces espèces. Les femelles sont distinguées par la longueur des conduits des spermathèques ainsi que par la forme étroite ou élargie du cou portant la tête de ces spermathèques. Enfin, la position particulière des épines sur le gonostyle couplée à une phylogénie moléculaire nous amène à extraire ces trois espèces du sous genre Larroussius Nitzulescu, 1931 pour les classer dans un nouveau sous-genre : Lewisius Depaquit & Vongphayloth n. subg.

Assessing vector competence of mosquitoes from northeastern France to West Nile virus and Usutu virus.

West Nile virus (WNV) and Usutu virus (USUV) are two arthropod-borne viruses that circulate in mainland France. Assessing vector competence has only been conducted so far with mosquitoes from southern France while an increasingly active circulation of WNV and USUV has been reported in the last years. The main vectors are mosquitoes of the Culex genus and the common mosquito Culex pipiens. Here, we measure the vector competence of five mosquito species (Aedes rusticus, Aedes albopictus, Anopheles plumbeus, Culex pipiens and Culiseta longiareolata) present in northeastern France. Field-collected populations were exposed to artificial infectious blood meal containing WNV or USUV and examined at different days post-infection. We show that (i) Cx. pipiens transmitted WNV and USUV, (ii) Ae. rusticus only WNV, and (iii) unexpectedly, Ae. albopictus transmitted both WNV and USUV. Less surprising, An. plumbeus was not competent for both viruses. Combined with data on distribution and population dynamics, these assessments of vector competence will help in developing a risk map and implementing appropriate prevention and control measures.

Molecular phylogeny of Psychodopygina (Diptera, Psychodidae) supporting morphological systematics of this group of vectors of New World tegumentary leishmaniasis.

New World sandflies are vectors of leishmaniasis, bartonellosis, and some arboviruses. A classification based on 88 morphological characters was proposed 27 years ago when the New World phlebotomines were organized into two tribes Hertigiini and Phlebotomini. The latter was structured into four subtribes (Brumptomyiina, Sergentomyiina, Lutzomyiina, and Psychodopygina) and 20 genera. The subtribe Psychodopygina, including most of the American vectors of tegumentary Leishmania comprises seven genera from which no molecular work has been produced to support this classification. Here, we carried out a molecular phylogeny based on combined sequences (1,334 bp) of two genes: partial 28S rDNA and mtDNA cytochrome b from 47 taxa belonging to the Psychodopygina. The Bayesian phylogenetic reconstruction agreed with the classification based on morphological characters, supporting the monophyly of the genera Psychodopygus and Psathyromyia, whereas Nyssomyia and Trichophoromyia seemed to be paraphyletic. The paraphylies of the two latter groups were exclusively caused by the doubtful position of the species Ny. richardwardi. Our molecular analysis provides additional support to adopt the morphologic classification of Psychodopygina.

Title: La phylogénie moléculaire des Psychodopygina (Diptera, Psychodidae) soutient la systématique morphologique de ce groupe de vecteurs de leishmaniose cutanée du Nouveau Monde. Abstract: Les phlébotomes du nouveau monde sont des vecteurs de leishmaniose, de bartonellose et de certains arbovirus. Une classification basée sur 88 caractères morphologiques a été proposée il y a 27 ans lorsque les phlébotomes du nouveau monde ont été organisés en deux tribus Hertigiini et Phlebotomini. Cette dernière était structurée en quatre sous-tribus (Brumptomyiina, Sergentomyiina, Lutzomyiina et Psychodopygina) et 20 genres. La sous-tribu des Psychodopygina, qui inclut la plupart des vecteurs américains de la leishmaniose cutanée, comprend sept genres mais aucun travail moléculaire n'a été produit pour soutenir cette classification. Dans cet article, nous avons réalisé une phylogénie moléculaire basée sur des séquences combinées (1334 pb) de deux gènes : ADNr 28S partiel et cytochrome b (ADNmt) chez 47 taxons appartenant aux Psychodopygina. La reconstruction phylogénique bayésienne est en accord avec la classification basée sur les caractères morphologiques, soutenant la monophylie des genres Psychodopygus et Psathyromyia, alors que Nyssomyia et Trichophoromyia semblent être paraphylétiques. Les paraphylies des deux derniers groupes sont exclusivement causées par la position douteuse de la seule espèce Ny. richardwardi. Notre analyse moléculaire fournit une raison supplémentaire pour adopter la classification morphologique des Psychodopygina.

Species diversity of phlebotomine sand flies and sympatric occurrence of Leishmania (Mundinia) martiniquensis, Leishmania (Leishmania) donovani complex, and Trypanosoma spp. in the visceral leishmaniasis focus of southern Thailand.

Autochthonous leishmaniasis in Thailand has recently been a public health concern due to an increasing number of new clinical cases. Most indigenous cases were diagnosed with Leishmania (Mundinia) martiniquensis, and Leishmania (Mundinia) orientalis. However, some doubts regarding vector misidentification have arisen and need to be elucidated. Accordingly, we aimed to assess the species composition of sand flies and determine the molecular prevalence of trypanosomatids in the transmission area of leishmaniasis in southern Thailand. In the present study, a total of 569 sand flies were caught from the vicinity of a visceral leishmaniasis patient's house in Na Thawi District, Songkhla Province. Of these, 229 parous and gravid females consisted of Sergentomyia khawi, Se. barraudi, Phlebotomus stantoni, Grassomyia indica, and Se. hivernus, accounting for 31.4%, 30.6%, 29.7%, 7.9%, and 0.4%, respectively. However, Se. gemmea, which has previously been proposed as the most abundant species and putative vector of visceral leishmaniasis, was not found in the present study. Based on ITS1-PCR and sequence analysis, two specimens of Gr. indica and Ph. stantoni showed positive amplification of L. martiniquensis and L. donovani complex, respectively, the first one being presumed indigenous and the second one being not. Anuran Trypanosoma was also molecularly detected using SSU rRNA-PCR and ubiquitously found in 16 specimens of four dominant sand fly species except for Se. hivernus. The obtained sequences could be phylogenetically categorized into the two major amphibian clades (An04/Frog1 and An01+An02/Frog2). The existence of the monophyletic subgroup and distinct lineage suggests them as novel Trypanosoma species. The TCS network analysis of these anuran Trypanosoma sequences also revealed high haplotype diversity (Hd = 0.925 ± 0.050), but low nucleotide diversity (π = 0.019 ± 0.009). Furthermore, the living anuran trypanosomes were microscopically demonstrated in a single specimen of Gr. indica, supporting the vectorial capacity. Importantly, our data confirmed the scarcity of Se. gemmea and also uncovered, for the first time, the co-circulation of L. martiniquensis, L. donovani complex, and suspected novel anuran Trypanosoma spp. in phlebotomine sand files, implicating their potential role as vectors of trypanosomatid parasites. Therefore, the novel data from this study would greatly facilitate the comprehension of the complexity of trypanosomatid transmission and the establishment of prevention and control measures for this neglected disease more effectively.

Three Aedes species infested by mermithids in France.

Mermithid nematodes (Nematoda, Mermithidae) parasitising mosquitoes could be useful as biological agents for the control of host populations. Nine female mosquitoes belonging to the species Aedes cantans, Ae. communis, and Ae. rusticus were found parasitised by mermithids in Northern France. Sequencing of partial 18S rDNA showed 100% sequence homology for all processed specimens. The mermithid sequences were closely related to specimens previously recorded from Anopheles gambiae in Senegal. However, 18S sequences do not allow for identification of nematodes at the genus or species level. Our specimens could also be related to Strelkovimermis spiculatus, or belong to other genera not yet sequenced such as Empidomermis, the only mermithid genus recorded from mosquitoes in France.

Title: Trois espèces d'Aedes parasitées par des mermithidés en France. Abstract: Les nématodes mermithidés (Nematoda, Mermithidae) parasitant les moustiques pourraient être utiles comme agents biologiques pour contrôler les populations hôtes. Neuf femelles de moustiques appartenant aux espèces Aedes cantans, Ae. communis et Ae. rusticus ont été trouvées parasitées par des mermithidés dans le nord de la France. Le séquençage de l'ADNr 18S partiel a montré une homologie de séquence de 100 % pour tous les échantillons traités. Les séquences des mermithidés étaient proches de spécimens précédemment signalés chez Anopheles gambiae au Sénégal. Les séquences 18S, cependant, ne permettent pas l'identification des nématodes au niveau du genre ou de l'espèce. Nos spécimens pourraient également être apparentés à Strelkovimermis spiculatus, ou appartenir à d'autres genres non encore séquencés comme Empidomermis, le seul genre de mermithidés recensé chez les moustiques en France.

MALDI-TOF: A new tool for the identification of Schistosoma cercariae and detection of hybrids.

Schistosomiasis is a neglected water-born parasitic disease caused by Schistosoma affecting more than 200 million people. Introgressive hybridization is common among these parasites and raises issues concerning their zoonotic transmission. Morphological identification of Schistosoma cercariae is difficult and does not permit hybrids detection. Our objective was to assess the performance of MALDI-TOF (Matrix Assistated Laser Desorption-Ionization-Time Of Flight) mass spectrometry for the specific identification of cercariae in human and non-human Schistosoma and for the detection of hybridization between S. bovis and S. haematobium. Spectra were collected from laboratory reared molluscs infested with strains of S. haematobium, S. mansoni, S. bovis, S. rodhaini and S. bovis x S. haematobium natural (Corsican hybrid) and artificial hybrids. Cluster analysis showed a clear separation between S. haematobium, S. bovis, S. mansoni and S. rodhaini. Corsican hybrids are classified with those of the parental strain of S. haematobium whereas other hybrids formed a distinct cluster. In blind test analysis the developed MALDI-TOF spectral database permits identification of Schistosoma cercariae with high accuracy (94%) and good specificity (S. bovis: 99.59%, S. haematobium 99.56%, S. mansoni and S. rodhaini: 100%). Most misidentifications were between S. haematobium and the Corsican hybrids. The use of machine learning permits to improve the discrimination between these last two taxa, with accuracy, F1 score and Sensitivity/Specificity > 97%. In multivariate analysis the factors associated with obtaining a valid identification score (> 1.7) were absence of ethanol preservation (p < 0.001) and a number of 2-3 cercariae deposited per well (p < 0.001). Also, spectra acquired from S. mansoni cercariae are more likely to obtain a valid identification score than those acquired from S. haematobium (p<0.001). MALDI-TOF is a reliable technique for high-throughput identification of Schistosoma cercariae of medical and veterinary importance and could be useful for field survey in endemic areas.

Investigation of the Sandfly Fauna of Central Arid Areas and Northern Humid Regions of Tunisia, with Morphological and Molecular Identification of the Recently Established Population of Phlebotomus (Larroussius) perfiliewi.

Based on nucleotide sequences, we re-identified representative samples of Phlebotomus perfiliewi originating from two different biogeographical areas of Tunisia, whose populations had previously been identified based on morphological criteria. A partial region of the mitochondrial DNA cytochrome b gene was targeted, and sandfly species was determined by analogy with DNA sequences available in the GenBank database via a BLAST analysis, taking into account the query coverage and percentage identity. The recognized species presents the most substantial homology with the analyzed sequence. The results of the molecular identification showed complete agreement with the morphological identifications, and Phlebotomus perfiliewi is genetically variable.

MALDI-TOF MS Limits for the Identification of Mediterranean Sandflies of the Subgenus Larroussius, with a Special Focus on the Phlebotomus perniciosus Complex.

Leishmania infantum is the agent of visceral leishmaniasis in the Mediterranean basin. It is transmitted by sandflies of the subgenus Larroussius. Although Phlebotomus perniciosus is the most important vector in this area, an atypical Ph. perniciosus easily confused with Ph. longicuspis has been observed in North Africa. MALDI-TOF MS, an important tool for vector identification, has recently been applied for the identification of sandflies. Spectral databases presented in the literature, however, include only a limited number of Larroussius species. Our objective was to create an in-house database to identify Mediterranean sandflies and to evaluate the ability of MALDI-TOF MS to discriminate close species or atypical forms within the Larroussius subgenus. Field-caught specimens (n = 94) were identified morphologically as typical Ph. perniciosus (PN; n = 55), atypical Ph. perniciosus (PNA; n = 9), Ph. longicuspis (n = 9), Ph. ariasi (n = 9), Ph. mascittii (n = 3), Ph. neglectus (n = 5), Ph. perfiliewi (n = 1), Ph. similis (n = 9) and Ph. papatasi (n = 2). Identifications were confirmed by sequencing of the mtDNA CytB region and sixteen specimens were included in the in-house database. Blind assessment on 73 specimens (representing 1073 good quality spectra) showed a good agreement (98.5%) between MALDI-TOF MS and molecular identification. Discrepancies concerned confusions between Ph. perfiliewi and Ph. perniciosus. Hierarchical clustering did not allow classification of PN and PNA. The use of machine learning, however, allowed discernment between PN and PNA and between the lcus and lcx haplotypes of Ph. longicuspis (accuracy: 0.8938 with partial-least-square regression and random forest models). MALDI-TOF MS is a promising tool for the rapid and accurate identification of field-caught sandflies. The use of machine learning could allow to discriminate similar species.

Significance of eggshell morphology as an additional tool to distinguish species of sand flies (Diptera: Psychodidae: Phlebotominae).

Morphological characteristics of eggshells are important in sand fly ootaxonomy. In this study, eggshells from Phlebotomus stantoni Newstead, Sergentomyia khawi (Raynal), and Grassomyia indica (Theodor) sand flies collected in Chiang Mai province, Thailand were examined and characterized using light microscopy (LM) and scanning electron microscopy (SEM). Then, eggshell morphology of these three species was described for the first time. Each gravid female was forced to lay eggs by decapitation and the eggs were collected for SEM analysis. Egg laying females were identified by morphological examination and molecular typing using cytochrome b (Cytb) as a molecular marker. The chorionic sculpturing of Ph. stantoni eggs combines two patterns on the same egg: unconnected parallel ridges and reticular patterns. Sergentomyia khawi and Gr. indica have similar chorionic polygonal patterns, but their exochorionic morphology and aeropylar area are different. Results indicate that eggshell morphological characteristics such as chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area (including the number of aeropyles) and basal layer, can be useful to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. In addition, the chorionic sculpturing of the eggs of the three species of sand flies observed by LM is useful for species identification in gravid females with spermathecae obscured by eggs.

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.

Comment on Xu et al. Isolation and Identification of a Novel Phlebovirus, Hedi Virus, from Sandflies Collected in China. Viruses 2021, 13, 772.

The article from Xu et al. [...].

Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) inferred using restriction-site associated DNA markers.

Phlebotomine sand flies are the main natural vectors of Leishmania, which cause visceral and tegumentary tropical diseases worldwide. However, their taxonomy and evolutionary history remain poorly studied. Indeed, as for many human disease vectors, their small size is a challenge for morphological and molecular works. Here, we successfully amplified unbiased copies of whole genome to sequence thousands of restriction-site associated DNA (RAD) markers from single specimens of phlebotomines. RAD markers were used to infer a fully resolved phylogeny of the subgenus Paraphlebotomus (11 species + 5 outgroups, 32 specimens). The subgenus was not recovered as monophyletic and we describe a new subgenus Artemievus subg. nov. Depaquit for Phlebotomus alexandri. We also confirm the validity of Ph. riouxi which is reinstated as valid species. Our analyses suggest that Paraphlebotomus sensu nov. originated ca 12.9-8.5 Ma and was possibly largely distributed from peri-Mediterranean to Irano-Turanian regions. Its biogeographical history can be summarized into three phases: i) a first split between Ph. riouxi + Ph. chabaudi and other species that may have resulted from the rise of the Saharan belt ca 8.5 Ma; ii) a Messinian vicariant event (7.3-5.3 Ma) during which the prolonged drought could have resulted in the divergence of main lineages; iii) a recent radiation event (3-2 Ma) that correspond to cycles of wet and dry periods in the Middle East and the East African subregions during the Pleistocene. Interestingly these cycles are also hypothetical drivers of the diversification of rodents, in the burrows of which Paraphlebotomus larvae develop. By meeting the challenge of sequencing pangenomics markers from single, minute phlebotomines, this work opens new avenues for improving our understanding of the epidemiology of leishmaniases and possibly other human diseases transmitted by arthropod vectors.

Wing Morphometrics of Aedes Mosquitoes from North-Eastern France.

BACKGROUND: In the context of the increasing circulation of arboviruses, a simple, fast and reliable identification method for mosquitoes is needed. Geometric morphometrics have proven useful for mosquito classification and have been used around the world on known vectors such as Aedes albopictus. Morphometrics applied on French indigenous mosquitoes would prove useful in the case of autochthonous outbreaks of arboviral diseases. METHODS: We applied geometric morphometric analysis on six indigenous and invasive species of the Aedes genus in order to evaluate its efficiency for mosquito classification. RESULTS: Six species of Aedes mosquitoes (Ae. albopictus, Ae. cantans, Ae. cinereus, Ae. sticticus, Ae. japonicus and Ae. rusticus) were successfully differentiated with Canonical Variate Analysis of the Procrustes dataset of superimposed coordinates of 18 wing landmarks. CONCLUSIONS: Geometric morphometrics are effective tools for the rapid, inexpensive and reliable classification of at least six species of the Aedes genus in France.

Leishmaniasis and phlebotomine sand flies in Oman Sultanate.

There are few data on leishmaniases and sandflies in Oman Sultanate. We carried out an eco-epidemiological study in 1998 in the two main mountains of the country, the Sharqiyah and the Dhofar. This study allowed us to isolate and identify three Leishmania strains from patients exhibiting cutaneous leishmaniasis. The typing carried out by isoenzymatic study and by molecular biology were congruent: two strains of Leishmania donovani zymodeme (Z) MON-31 isolated in the Sharqiyah and one L. tropica ZROM102 (ZMON-39 variant for 4 isoenzymes) from the Dhofar. No strain was isolated from canids. The study of sandflies identified 14 species distributed in the genera Phlebotomus, Sergentomyia and Grassomyia: Ph. papatasi, Ph. bergeroti, Ph. duboscqi, Ph. alexandri, Ph. saevus, Ph. sergenti, Se. fallax, Se. baghdadis, Se. cincta, Se. christophersi, Se. clydei, Se. tiberiadis, Se. africana, and Gr. dreyfussi. In Sharqiyah, the only candidate for the transmission of L. donovani was Ph. alexandri, but the low densities observed of this species do not argue in favor of any role. In Dhofar, Ph. sergenti is the most important proven vector of L. tropica, but Ph. saevus, a locally much more abundant species, constitutes a good candidate for transmission.

TITLE: Leishmanioses et phlébotomes au Sultanat d'Oman. ABSTRACT: Il existe peu de données sur les leishmanioses et les phlébotomes en Oman. Nous y avons mené en 1998 une étude éco-épidémiologique dans les deux principaux massifs montagneux du pays, la Sharqiyah et le Dhofar. Cette étude nous a permis d'isoler et d'identifier trois souches de Leishmania à partir de patients présentant des leishmanioses cutanées. Les typages menés par étude isoenzymatique et par biologie moléculaire ont été congruents : deux souches de Leishmania donovani ZMON-31 isolées dans la Sharqiyah et une de L. tropica ZROM102 (ZMON-39 variant pour 4 isoenzymes) originaire du Dhofar. Aucune souche n'a été isolée à partir de Canidés. L'étude des Phlébotomes a permis d'identifier 14 espèces réparties dans les genres Phlebotomus, Sergentomyia et Grassomyia : Ph. papatasi, Ph. bergeroti, Ph. duboscqi, Ph. alexandri, Ph. saevus, Ph. sergenti, Se. fallax, Se. baghdadis, Se. cincta, Se. christophersi, Se. clydei, Se. tiberiadis, Se. africana et Gr. dreyfussi. Dans la Sharqiyah, la seule espèce candidate à la transmission de L. donovani est Ph. alexandri mais les faibles densités observées de cette espèce ne plaident pas en faveur d'un quelconque rôle. Dans le Dhofar, Ph. sergenti est le principal vecteur prouvé de L. tropica mais Ph. saevus, espèce localement bien plus abondante, constitue une bonne espèce candidate à la transmission.