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.

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.

Discrimination of Uranotaenia species (Diptera: Culicidae) from Madagascar based on morphology and wing morphometric traits.

The genus Uranotaenia (Diptera: Culicidae) has been well documented in Madagascar where it includes 73 species, 89.4% being endemic. However, one problem is that most species are morphologically similar in the adult stage. Here, 713 Uranotaenia specimens collected in the tropical forests of Anorana and Maromizaha between 2008 and 2014 were examined. Using the dichotomous keys for the Uranotaenia fauna of Madagascar published in 2004, three species were identified: Uranotaenia neireti (220), Ur. alboabdominalis (110) and Ur. mayottensis (28). The other specimens (355) were not identifiable and were classified as Uranotaenia sp1. Using wing morphometry, the four taxa were classified into four morphogroups. Within the Uranotaenia sp1 group, specimens from the Anorana forest and those from the Maromizaha forest overlapped. This result suggests that wing morphometric traits could be a good marker to distinguish Uranotaenia species in Madagascar.

Two new phlebotomine sandflies (Diptera: Psychodidae) from the forest edge in Madagascar: the anthropophilic Phlebotomus artemievi sp. nov. and Sergentomyia maroantsetra ensis sp. nov.

A few data are related to the anthropophily of Malagasy Phlebotomine sandflies. Prior studies focussed mainly to inventories and description of new species. Our goal was to emphasize the anthropophily of Malagasy Phlebotomine sandflies. We worked in the Makira region, using two simultaneous methods: human landing catches (HLC) and CDC light traps. We collected sandflies in three rural communities adjacent to the Makira Natural Park. In each community, three different biotopes were sampled: within community settlements; at the edge of forest, typically in agricultural land; and within the forest. We collected 61 sandflies belonging to two new species presently described: Phlebotomus artemievi sp. nov. and Sergentomyia maroantsetraensis sp. nov. These sandflies were caught exclusively in the forest edge biotope. None were captured within communities or within forests. HLC provided 97% of the collected sandflies, corresponding to a human-biting rate of 15 females per human per night. CDC provided only two females. Ph. artemievi sp. nov. was predominantly captured by HLC and appears to be highly anthropophilic. Here, we update the behavioural ecology of sandflies and describe two new species. Further research is required to understand their vector competence and their ability to transmit arboviruses and other pathogens such as Leishmania.

Two new phlebotomine sandfly species (Diptera: Psychodidae) from the Highlands of Madagascar.

The Malagasy phlebotomine sandfly fauna includes 17 species: five belong to the genus Phlebotomus, ten to the genus Sergentomyia, and two to the genus Grassomyia. The current article describes Sergentomyia brunhesi sp. nov. and Sergentomyia vistellei sp. nov. from the Malagasy Highlands. Females were described morphologically from specimens collected at Ranomafana-Ifanadiana and Andringitra. Partial molecular sequences of cyt b and 28S rDNA were retrieved for Se. vistellei sp. nov. Waiting new data, we consider these species as ungrouped Sergentomyia. Two new species of Sergentomyia are recorded at higher altitudes in Madagascar. Knowledge of local Biodiversity is increasing. New investigations have to be carried out to describe males, to understand their bionomics, and to identify other potential new species at higher altitudes.

Phlebotomine sandflies (Diptera, Psychodidae) from the Ankarana tsingy of northern Madagascar: inventory and description of new taxa.

An inventory of Phlebotomine sandflies was carried out in the Ankarana tsingy located in far northern Madagascar. A total of 723 sandflies were used for morphological, morphometric, and molecular studies (sequencing of partial cytochrome B (mtDNA) and partial 28S (rDNA)). Nine species were identified: Phlebotomus fertei, Sergentomyia anka, Se. sclerosiphon, Se. goodmani, two species of the genus Grassomyia, as well as three new species described herein: Se. volfi n. sp., Se. kaltenbachi n. sp., and Se. ozbeli n. sp. The recognition of these new species is strongly supported by molecular analyses. The first two of the new species could not be classified into any existing subgenus, therefore we proposed two new subgenera (Ranavalonomyia subg. nov., and Riouxomyia subg. nov.), with combinations as: Sergentomyia (Ranavalonomyia) volfi and Sergentomyia (Riouxomyia) kaltenbachi. Our study reveals important molecular variability in Se. anka, with the recognition of a population whose taxonomic status remains below that of species. Our research confirms the need to further study the specific diversity of Malagasy sandflies, which until the start of this millennium remained mostly unknown.

TITLE: Les phlébotomes (Diptera, Psychodidae) des tsingy d'Ankarana dans le nord de Madagascar : inventaire et description de nouveaux taxons. ABSTRACT: Un inventaire des Phlébotomes a été réalisé dans les tsingy d'Ankarana à l'extrême nord de Madagascar. Au total, 723 phlébotomes ont servi à des études morphologique, morphométrique et moléculaire (séquençage d'une partie du cytochrome B (ADNmt) et d'une partie de l'ADNr 28S). Neuf espèces ont été identifiées : Phlebotomus fertei, Sergentomyia anka, Se. sclerosiphon, Se. goodmani, deux espèces du genre Grassomyia, ainsi que trois espèces nouvelles décrites dans ce travail : Se. volfi n. sp., Se. kaltenbachi n. sp., and Se. ozbeli n. sp. L'individualisation de chacune de ces espèces nouvelles est robustement soutenue par les analyses moléculaires. Les deux premières de ces espèces nouvelles ne pouvaient pas être classées dans un sous-genre existant et nous avons proposé pour elles deux sous-genres nouveaux : Ranavalonomyia subg. nov., et Riouxomyia subg. nov, avec les combinaisons Sergentomyia (Ranavalonomyia) volfi et Sergentomyia (Riouxomyia) kaltenbachi.Notre étude révèle une variabilité moléculaire marquée chez Se. anka avec l'individualisation d'une population dont le statut taxinomique demeure populationnel. Nos travaux confirment la nécessité de poursuivre l'étude de la biodiversité des Phlébotomes qui est restée méconnue dans ce pays jusqu'au début de ce millénaire.

Molecular phylogeny of 42 species of Culicoides (Diptera, Ceratopogonidae) from three continents.

The genus Culicoides includes vectors of important animal diseases such as bluetongue and Schmallenberg virus (BTV and SBV). This genus includes 1300 species classified in 32 subgenera and 38 unclassified species. However, the phylogenetic relationships between different subgenera of Culicoides have never been studied. Phylogenetic analyses of 42 species belonging to 12 subgenera and 8 ungrouped species of genus Culicoides from Ecuador, France, Gabon, Madagascar and Tunisia were carried out using two molecular markers (28S rDNA D1 and D2 domains and COI mtDNA). Sequences were subjected to non-probabilistic (maximum parsimony) and probabilistic (Bayesian inference (BI)) approaches. The subgenera Monoculicoides, Culicoides, Haematomyidium, Hoffmania, Remmia and Avaritia (including the main vectors of bluetongue disease) were monophyletic, whereas the subgenus Oecacta was paraphyletic. Our study validates the subgenus Remmia (= Schultzei group) as a valid subgenus, outside of the subgenus Oecacta. In Europe, Culicoides obsoletus, Culicoides scoticus and Culicoides chiopterus should be part of the Obsoletus complex whereas Culicoides dewulfi should be excluded from this complex. Our study suggests that the current Culicoides classification needs to be revisited with modern tools.

Avian malaria on Madagascar: bird hosts and putative vector mosquitoes of different Plasmodium lineages.

BACKGROUND: Avian malaria occurs almost worldwide and is caused by Haemosporida parasites (Plasmodium, Haemoproteus and Leucocytozoon). Vectors such as mosquitoes, hippoboscid flies or biting midges are required for the transmission of these parasites. There are few studies about avian malaria parasites on Madagascar but none about suitable vectors. METHODS: To identify vectors of avian Plasmodium parasites on Madagascar, we examined head, thorax and abdomen of 418 mosquitoes from at least 18 species using a nested PCR method to amplify a 524 bp fragment of the haemosporidian mitochondrial cytochrome b gene. Sequences obtained were then compared with a large dataset of haemosporidian sequences detected in 45 different bird species (n = 686) from the same area in the Maromizaha rainforest. RESULTS: Twenty-one mosquitoes tested positive for avian malaria parasites. Haemoproteus DNA was found in nine mosquitoes (2.15%) while Plasmodium DNA was found in 12 mosquitoes (2.87%). Seven distinct lineages were identified among the Plasmodium DNA samples. Some lineages were also found in the examined bird samples: Plasmodium sp. WA46 (EU810628.1) in the Madagascar bulbul, Plasmodium sp. mosquito 132 (AB308050.1) in 15 bird species belonging to eight families, Plasmodium sp. PV12 (GQ150194.1) in eleven bird species belonging to eight families and Plasmodium sp. P31 (DQ839060.1) was found in three weaver bird species. CONCLUSION: This study provides the first insight into avian malaria transmission in the Maromizaha rainforest in eastern Madagascar. Five Haemoproteus lineages and seven Plasmodium lineages were detected in the examined mosquitoes. Complete life-cycles for the specialist lineages WA46 and P31 and for the generalist lineages mosquito132 and PV12 of Plasmodium are proposed. In addition, we have identified for the first time Anopheles mascarensis and Uranotaenia spp. as vectors for avian malaria and offer the first description of vector mosquitoes for avian malaria in Madagascar.

Paraphyly of the subgenus Sintonius (Diptera, Psychodidae, Sergentomyia): status of the Malagasy species. Creation of a new subgenus and description of a new species.

During an inventory of Phlebotomine sand flies carried out in Madagascar, we have identified some specimens showing morphological characters related to the subgenus Sintonius of the genus Sergentomyia. We started a molecular study based on cytochrome b mtDNA and on D1-D2 and D8 domains of the rDNA. The sampling includes all the Sergentomyia species available and also S. (Sergentomyia) schwetzi, S. (Parrotomyia) magna, and the following species belonging to the subgenus Sintonius: S. clydei, S. christophersi, S. affinis vorax, S. adleri and S. meilloni. The Sintonius subgenus (sensu Theodor) is paraphyletic. The Malagasy specimens morphologically Sintonius-like are never clustered with the continental Sintonius. We propose a new subgenus to include them: Trouilletomyia subg. nov. Due to the lack of mesanepisternal setae, the species huberti is removed from the genus Phlebotomus and we propose here a new combination: Sergentomyia huberti comb. nov. The male of S. huberti is pinpointed and described for the first time. Lastly, a new species for Science is described on one female: Sergentomyia (Trouilletomyia) boironis n. sp.

Phlebotomine sand flies from Madagascar (Diptera: Psychodidae). VII. An identification key for Phlebotomus with the description of Phlebotomus (Anaphlebotomus) vaomalalae n. sp.

An identification key of the Phlebotomus in Madagascar is proposed as well as the description of the male and female Phlebotomus (Anaphlebotomus) vaomalalae n. sp. from Mikea Forest in the south-west of Madagascar. The assignation of this new species to the genus Phlebotomus is based on the presence of mesanepisternal setae. Its inclusion in the subgenus Anaphlebotomus is based on the males on the presence of four spines on the style, the lack of a coxite basal process and the existence of a bifurcated paramere. The female has cibarial and pharyngeal armature and spermathecal architecture similar to Phlebotomus fertei and Phlebotomus berentiensis, two other Malagasy species which belong to Anaphlebotomus. Male and female are held to belong to the same species because of their morphological characters, the homology (100%) of their partial cytochrome b mtDNA sequences and their capture in the same trap. P. vaomalalae n. sp. is a small species compared to the other Phlebotomus species of Madagascar. The cibarium of the male and the female of P. vaomalalae n. sp. is armed with teeth, like those of other Malagasy Phlebotomus. However, it differs in the arrangement and shape of the respective teeth and denticles. The male of P. vaomalalae n. sp. looks like that of P. fontenillei due to its tuft of coxal setae (lacking in P. berentiensis and P. fertei) but differs from this species by the location of this tuft. As P. fertei and P. berentiensis, there is no spermathecal common duct in P. vaomalalae n. sp.