[On Professor Jean-Antoine Rioux' Phlebotomine sand flies collection (Diptera: Psychodidae)]. / Sur la collection de phlébotomes (Diptera: Psychodidae) du Professeur Jean-Antoine Rioux.

Background: Professor Jean-Antoine Rioux (1925-2017) has spent much of his long and fruitful scientific career exploring and understanding the functioning of leishmaniasis foci in several regions of the Mediterranean, the Near and Middle East and Africa. Phlebotomine sand flies study was an important part of his work in the foci studied and a large number of specimens have been collected by himself and stored in his collection, detailed in this note. Materials and Methods: His collection has been completely inventoried. Phlebotomine sand flies have been classified here by species and by country. Each specimen is mounted individually in toto between slide and cover-slip in Canada balsam. Results: The collection includes 130 840 specimens coming from 10 countries: France (mainland and Corsica), Italy, Spain, Algeria, Tunisia, Morocco, Yemen, Syria, Sudan, and Republic of Congo. These specimens belong to 26 species: Phlebotomus (Artemievus) alexandri, Ph. (Larroussius) ariasi, Ph. (Phlebotomus) bergeroti, Ph. (Paraphlebotomus) chabaudi, Ph. (Lar.) chadlii, Ph. (Par.) kazeruni, Ph. (Lar.) longicuspis, Ph. (Transphlebotomus) mascittii, Ph. (Par.) mongolensis, Ph. (Lar.) orientalis, Ph. (Phl.) papatasi, Ph. (Lar.) perfiliewi, Ph. (Lar.) perniciosus, Ph. (Par.) riouxi, Ph. (Par.) sergenti, Sergentomyia (Parrotomyia) africana, Se. (Sergentomyia) antennata, Se. (Sintonius) christophersi, Se. (Sin.) clydei, Se. (Ser.) fallax, Se. (Ser.) minuta, Se. (Ser.) schwetzi, Se. (Ser.) silva, Se. (Ser.) taizi, Se. (Sin.) tiberiadis and Grassomyia dreyfussi. Discussion: The collection was transferred to the Platform of Reims Biological Resource Centers (PF CRBs Reims) located at the Biology Department of the Reims University Hospital in order to be curated and stored physically and electronically in a secure manner. In a near future, it will be made available to scientists wishing to access it for all purposes.In addition to Ph. chabaudi and Ph. chadlii topotypes, the collection includes many specimens from North Africa, mainly from Morocco (53% of the collection is originated from this country where Professor Rioux worked during 30 years) but also specimens from Syria or Yemen, the interest of which is obvious given the current geopolitical context.

[Perspectives on the training of the pharmacist among pharmacists, academics and pharmacy students: A cross-sectional study]. / Regards croisés sur la formation du pharmacien d'officine : le point de vue des pharmaciens d'officine, des enseignants et des étudiants en pharmacie.

OBJECTIVES: The evolution of community pharmacist education is guided by successive reforms. They aim to reduce the gap between academic education and community pharmacy practice but the perception regarding these reforms is poorly documented. The present study describes the cross-perceptions between community pharmacists, pharmacy students and academics teachers regarding the gap between academic education and community pharmacy practice, and, therefore, the essential knowledges and skills for the community pharmacy practice. METHODS: This study was conducted between April 2017 and April 2018 using an electronic survey for community pharmacists, academic teachers and pharmacy students. The results were discussed regarding previous studies with the aim to detect improvement points and vigilance related points. RESULTS: This study shows that the pharmacy education program remains to be improved: 77% of respondents believe that there is a significant gap between the initial education program and the community pharmacy practice. CONCLUSIONS: This cross-perception helps to better identify the several ways to improve the points of vigilance and the associated-barriers. All the propositions by the three groups of respondents prior ask for a better definition of the needed skills for community pharmacy practice before determine the most important knowledges and pedagogic methods to reach these. All these propositions seem consistent with reducing the gap between pharmacy education and community pharmacy practice but they also raise several questions about unicity of the pharmacy graduation in France.

On the true identity of Sergentomyia gemmea and description of a closely related species: Se. raynali n. sp.

Several species of Leishmania are responsible for leishmaniases in Thailand, although little is known about their transmission. Sergentomyia gemmea has been suspected several times to transmit Leishmania martiniquensis. Some captures carried out in Thailand and Lao People's Democratic Republic have emphasized the scarcity of Se. gemmea, comprising only 1% of the collected females. The sequencing of cytochrome B mtDNA of our specimens showed that our specimens are not grouped with other Se. gemmea previously deposited in GenBank. The latter are grouped with some Se. khawi and Se. hivernus that we processed in the present study. We suspect misidentifications and propose focusing on the most useful characters for identification of Se. gemmea based on the examination of type-specimens. The examination of the ascoids exhibiting anterior spurs is the most important one. However, we also describe Se. raynali n. sp. exhibiting comparable spurs but differing from Se. gemmea by its original cibarium. Finally, the vectorial role of Se. gemmea appears very questionable in the absence of new evidence.

Mitochondrial DNA Intraspecific Variability in Sergentomyia minuta (Diptera: Psychodidae).

Recently, there has been growing interest in analysis of the geographical variation between populations of different Phlebotomus spp. and American sand flies by comparing the sequences of various genes. However, little is known about the genetic structure of the genus Sergentomyia França & Parrot. No study has been carried out on Sergentomyia minuta Rondani. Most authors recognize this as a species with a high degree of morphological polymorphism, and some suspect that there are two subspecies: Se. minuta minuta Rondani in Europe, having about 40 horizontal cibarial teeth (sticks aligned along a straight line in the cibarial cavity), and Se. minuta parroti Adler & Theodor in North Africa, having about 70 cibarial teeth. Here we analyzed phylogeographic patterns using cytochrome b (Cytb) and cytochrome C oxidase I mtDNA for 29 populations from 10 countries: Algeria, Cyprus, France (continental and Corsica), Greece (continental and Crete), Malta, Montenegro, Morocco, Portugal (continental and Atlantic Savage Islands), Spain, and Tunisia. We analyzed intra- and interpopulation patterns of genetic diversity. Our results from Bayesian inference showed a complex genetic structure of Se. minuta with four haplogroups including many different haplotypes. One haplogroup includes all the specimens from North Africa. A second haplogroup includes a few specimens from the south of France, Spain, and one from Portugal. The third includes many specimens from southern France, all the specimens from Corsica, one from Spain, and all specimen from Portugal except one. A fourth branch includes specimens from the Balkans, Malta, Crete, Cyprus, and curiously some from the Atlantic Savage Islands; settlement of the latter population remains unexplained. However, our results suggest that the settlement of the Mediterranean basin could have occurred at the same time for Se. minuta and both Phlebotomus perniciosus Newstead and Phlebotomus ariasi Tonnoir. The spatial distribution of haplotypes was congruent with phylogenetic findings.

Taxonomic assessment of Culicoides brunnicans, C. santonicus and C. vexans (Diptera: Ceratopogonidae) in France: Implications in systematics.

Culicoides brunnicans Edwards, 1939, Culicoides santonicus, Callot et al., 1966, and Culicoides vexans (Staeger, 1839) belong to the Vexans group of the subgenus Oecacta. These species had never been studied by molecular methods and their distribution in Western Europe overlapped. C. brunnicans and C. santonicus are two closely related species and their diagnoses are based on the wing pattern only. An integrative taxonomic approach was conducted on females of the Vexans group, Culicoides furens (the type species of the subgenus Oecacta), and Culicoides nubeculosus (as outgroup) using sequences of D1D2 rDNA domains, sequences of COI mtDNA, and wing morphometrics. Species of the Vexans group were discriminated by all traits, and were closely related in the phylogeny. Their taxonomic status and their relation to C. furens were re-evaluated.

Paraphyly of the subgenus Anaphlebotomus and creation of Madaphlebotomus subg. nov. (Phlebotominae: Phlebotomus).

The systematic position of the Malagasy Phlebotomus (Diptera: Psychodidae) species was assessed in molecular phylogenetic studies. Three molecular markers were sequenced: cytochrome b of the mitochondrial DNA; ITS2, and the D8 domain of the ribosomal DNA. The following species were studied: Phlebotomus (Anaphlebotomus) berentiensis, Phlebotomus (Anaphlebotomus) fertei, Phlebotomus (Anaphlebotomus) fontenillei, Phlebotomus (Anaphlebotomus) vaomalalae and Phlebotomus (Anaphlebotomus) vincenti from Madagascar; Phlebotomus (Anaphlebotomus) stantoni from Asia, and Phlebotomus (Anaphlebotomus) rodhaini from Africa. The following outgroups were selected: Phlebotomus (Euphlebotomus) argentipes, Phlebotomus (Euphlebotomus) barguesae, Phlebotomus (Larroussius) perfiliewi s.l. and Phlebotomus (Adlerius) simici. Each marker analysed by maximum parsimony and maximum likelihood supports the monophyly of the Malagasy Phlebotomus spp. Consequently, we create a new subgenus for these species: Madaphlebotomus subg. nov. This molecular individualization is reinforced by the originality of their spermathecae and by the fact that their geographical distribution is limited to Madagascar, and considers the high level of endemism on this island.

Biting midges monitoring (Diptera: Ceratopogonidae: Culicoides Latreille) in the governate of Monastir (Tunisia): species composition and molecular investigations.

The results of entomological studies carried out in the governate of Monastir (Tunisia) in 2009-2010 (captures and emergences from muds) focusing on Culicoides species are presented in the present study. Identification of Culicoides at the species level is based on morphological characters, and a molecular study has been carried out based on mitochondrial DNA cytochrome C oxidase I gene (COI) and D1 and D2 domains of the 28S rDNA. The DNA sequences reported here are related to 10 species (on 25 known) of Culicoides described in Tunisia: Culicoides cataneii-gejgelensis, Culicoides circumscriptus, Culicoides imicola, Culicoides jumineri, Culicoides kingi, Culicoides langeroni, Culicoides newsteadi, Culicoides paolae, Culicoides puncticollis and Culicoides sahariensis. DNA sequencing of the COI gene and D1D2 domains discriminated all morphologically determined species. The choice of D1D2 domains considered as a conserved region is informative for Culicoides species identification. The molecular analyses of COI has grouped both C. circumscriptus, C. puncticollis within two clusters and C. newsteadi within five subclusters. However, C. newsteadi shows relatively deep intraspecific divergence using COI sequences.

Wing geometry as a tool for discrimination of Obsoletus group (Diptera: Ceratopogonidae: Culicoides) in France.

In Europe, Culicoides chiopterus, Culicoides dewulfi, Culicoides obsoletus and Culicoides scoticus, which belongs to the subgenus Avaritia and Obsoletus group are the most proficient Bluetongue and Schmallenberg vectors. Within this group, correct identification based on morphological traits is difficult but essential to assess disease transmission risk. The development of new tools has revolutionized taxonomy (i.e. geometric morphometrics and molecular biology). Wing morphology is of primary importance to entomologists interested in systematics. Here, we report phenotypic differentiation patterns among the species above mentioned using a landmark-based geometric morphometric approach that efficiently identified C. chiopterus and C. dewulfi. Wing shape of the C. scoticus sample exhibited large specific variability. Based on landmarks and phylogenetic analyses (Maximum Parsimony), we suggest that Obsoletus group in Europe includes only C. obsoletus and C. scoticus. C. dewulfi and C. chiopterus are clearly excluded. Their shape seems closer to C. obsoletus that is why we suggest that only these two species should be grouped in the Obsoletus group. In addition, the concordance between phenetic clusters and phylogenies inferred from molecular data based on a fragment of the mtDNA COI gene and rDNA 28S suggests the existence of a strong signal in wing shape. These findings encourage us to use this powerful tool in taxonomic studies.

Molecular and morphological systematics of the sandfly Sergentomyia (Sintonius) clydei Sinton, 1928 and questions about its record in the Seychelles.

In the Phlebotomine sandflies, a few molecular studies related on the genus Sergentomyia have been published. The present study explored the genetic variability within Sergentomyia (Sintonius) clydei (Diptera, Psychodidae). The sampling included 15 populations originating from 12 countries. A morphological approach was coupled to the sequencing of two molecular markers (cytochrome b mtDNA and cacophony nuclear DNA). The most variable morphological characters resided in the cibarium of the females, especially (i) the pigment patch pattern and (ii) the number of cibarial teeth and denticles in the armature. However this morphological approach was unable to individualize any population within S. clydei. The NJ trees based on both molecular markers individualized the specimens from the Aldabra group of islands in the Seychelles. Surprisingly, cyt b variability was not compatible with the known data about the complete submersion of Aldabra occurring relatively recently some 125,000 years ago. The settlement of these islands by S. clydei from continental Africa, the Middle East or Asia, and the value of mtDNA markers are discussed.

Phlebotomine sand flies from Madagascar (Diptera: Psychodidae). VIII--Phlebotomus (Anaphlebotomus) vincenti n. sp.

A new species of Phlebotomine sand fly (Diptera: Psychodidae) is described from Mikea Forest in the southwest of Madagascar: Phlebotomus vincenti n. sp. It is temporarily classified in the subgenus Anaphlebotomus according to its spermathecal organization, similar to those of P. berentiensis, P. fertei and P. vaomalalae, previously classified in the subgenus Anaphlebotomus referring to on male genitalia morphology. An original trumpet-like dilatation on the distal part of the spermathecal ducts individualizes P. vincenti n. sp. from the other Malagasy species. Moreover, the second internal transcribed spacer (ITS2) sequences strongly individualises P. vincenti n. sp. from the other Malagasy Phlebotomus.

Entomological investigation following the resurgence of human visceral leishmaniasis in southern Algeria.

Visceral and cutaneous leishmaniasis are the main endemic vector born diseases in Algeria. In the Hoggar region (extreme south of the country) human visceral leishmaniasis (HVL) is known to be sporadic but during the last decade the number of cases has increased significantly. In 2010, a peak of HVL cases was registered mostly among children. Therefore an entomological survey and a retrospective study on HVL cases were carried out in order to explore the transmission of the disease. Among the sand fly caught Phlebotomus bergeroti was the most frequent species (68%) followed by Sergentomyia schwetzi (22%). In this work we describe the presence of Phlebotomus (Paraphlebotomus) kazeruni for the first time in the Hoggar region.

Record of two species of Culicoides (Diptera, Ceratopogonidae) new for Madagascar and molecular study showing the paraphylies of the subgenus Oecacta and the Schultzei group.

Culicoides are vectors of diseases of Veterinary Medicine importance (bluetongue, African horse sickness, Schmallenberg virus) all over the world. In the present study, we report two species new for Madagascar: C. nevilli and C. enderleini. They belong to the Schultzei group which is sometimes classified in the subgenus Oecacta and sometimes in the subgenus Remmia, depending on authors. Consequently, we carried out a molecular cladistics of these groups based on cytochrome C oxidase subunit I mtDNA sequences. We processed the Malagasy specimens and some C. furens (the Oecacta type-species) caught in Florida and we analyzed their sequences and those available in Genbank: C. schultzei, C. oxystoma, C. festivipennis, C. brunnicans, C. kibunensis, C. truncorum and C. vexans. C. (Avaritia) imicola have been selected as an outgroup. The maximum parsimony analysis showed the paraphylies of the Schultzei group (=Remmia) and of the subgenus Oecacta if the first group is excluded from the latter. Our results underline the doubtful current classification and need to be validated by other molecular markers in the future.

First record of Phlebotomine sandflies (Diptera: Psychodidae) in the Comoros Archipelago with description of Sergentomyia (Vattieromyia) pessoni n. sp. and S. (Rondanomyia) goodmani comorensis n. ssp.

No Phlebotomine sandflies had ever been reported in the Comoros Archipelago, including the three islands of the Republic of the Union of Comoros (Grande Comore, Mohéli and Anjouan) and the French oversea department of Mayotte. During three field surveys carried out in 2003, 2007 and 2011, we provided the first record of Phlebotomine sandflies in this area. A total of 85 specimens belonging to three species were caught: a new species S. (Vattieromyia) pessoni n. sp. (two females from Grande Comore), a new subspecies of Sergentomyia (Rondanomyia) goodmani (80 specimens from Grande Comore and one from Anjouan) and Grassomyia sp. (two females from Mohéli). The individualisation of these taxa was inferred both from morphological criteria and sequencing of a part of the cytochrome b of the mitochondrial DNA. These taxa are closely related to Malagasy sandflies.

A study of a population of Nyssomyia trapidoi (Diptera: Psychodidae) caught on the Pacific coast of Ecuador.

BACKGROUND: Cutaneous leishmaniasis is endemic to the Pacific coast of Ecuador, and Nyssomyia trapidoi is considered to be its main vector. Dujardin et al. [1] recorded some differences in body pigmentation and isoenzymatic profiles in sympatric populations of Ny. trapidoi from the Pacific coast of Ecuador and suggested the existence of two cryptic species. METHODS: Entomological collections were performed in November 2008 and March 2011 in the locality of Paraíso Escondido using CDC miniature light traps and human bait. Morphological, isoenzymatical and molecular (sequencing of cytochome b and cytochrome c oxidase 1 of the mitochondrial DNA) analyses, such as detection of Leishmania DNA and phlebovirus RNA in some females, were performed. RESULTS: Neighbor-joining trees from mitochondrial sequences grouped all of Ecuadorian Ny. trapidoi (including the two color variants) in one cluster, except for two specimens which clustered separately in both genes. Isoenzymatic characterization confirmed that the color variants belong to the same population. Additionally, 11.5% of females were found by PCR to contain Endotrypanum monterogeii kinetoplastid DNA. All pools of Ny. trapidoi were negative for phlebovirus RNA. CONCLUSION: Analysis of mitochondrial gene sequences and isoenzymes was unable to support the existence of two sibling species within Ny. trapidoi, which is a probable vector of Endotrypanum monterogeii.

Morphometric and molecular characterization of the series Guyanensis (Diptera, Psychodidae, Psychodopygus) from the Ecuadorian Amazon Basin with description of a new species.

Several populations of the series Guyanensis (Diptera, Psychodidae, Psychodopygus) were collected from the Yasuní National Park in the Ecuadorian Amazon region. The specimens comprised the species Psychodopygus geniculatus, Psychodopygus luisleoni and Psychodopygus corossoniensis. Within Ps. geniculatus, we observed two populations, one with a narrow paramere and relatively short genital filaments and the other characterized by a wider coxite and longer genital filaments. A multiple approach combining morphology, morphometry and DNA sequencing of the ribosomal internal transcribed spacer 2 (ITS2) and the mitochondrial cytochrome b gene was carried out. Morphological, morphometric and molecular data strongly suggested the presence of two populations within Ps. geniculatus. The lack of intermediate forms within these populations supported the proposal of two sympatric species. This report describes Psychodopygus francoisleponti n. sp.

Chinius eunicegalatiae n. sp. (Diptera; Psychodidae), a cavernicolous sandfly from Laos.

A description is given of Chinius eunicegalatiae n. sp. (Diptera; Psychodidae) from Laos. This is the third known species belonging to the Asiatic genus Chinius Leng, 1987. Like C. junlianensis Leng, 1987 and C. barbazani Depaquit, Léger and Beales, 2006, C. eunicegalatiae n. sp. is a cavernicolous species. An absence of the R2 vein is shared with C. barbazani. A differential diagnosis with the two other known species of the genus is given. The main differential characters are the lengths of the genital filaments and of the spermathecal ducts.

Discrimination of Culicoides obsoletus and Culicoides scoticus, potential bluetongue vectors, by morphometrical and mitochondrial cytochrome oxidase subunit I analysis.

Biting midges of the Culicoides obsoletus Meigen species complex (Diptera: Ceratopogonidae) are increasingly suspected as vectors of the recent emergence of bluetongue virus in Europe. Within this complex, identification of the C. obsoletus and Culicoides scoticus females is considered as difficult or sometimes not possible while the identification of males is easy, based on genitalia observation. Nolan et al. (2007) concluded that the distinction of C. obsoletus and C. scoticus females is not possible according to morphology but require molecular analyses. In 2010, the identification of biting midges is done under a stereomicroscope without specific identification within the C. obsoletus species complex. However, such a specific identification distinguishing C. obsoletus s. str. and C. scoticus s. str. is crucial to identify the European competent vectors of the virus, their relative abundances and then accurately assess the risk. We performed morphometric analyses of head, genitalia and thorax of females combined with sequencing of the cytochrome oxidase I barcode fragment of mitochondrial DNA on 88 specimens in order to have a molecular identification of our sampled species. As we knew the actual species of individuals thanks to molecular results, we explored the discriminant power of 15 morphometric variables to distinguish the females according to their species. Multivariate analyses were performed on the morphometric measurements to identify and validate a combination of variables leading to an accurate species identification. It appears that females of C. obsoletus and C. scoticus can be accurately distinguished based on only four variables: width between chitinous plates, length and width of spermathecae1 and length of spermatheca2. This approach should improve the accuracy of morphologically-based species identification.

Arthropod-borne viruses transmitted by Phlebotomine sandflies in Europe: a review.

Phlebotomine sandflies are known to transmit leishmaniases, bacteria and viruses that affect humans and animals in many countries worldwide. These sandfly-borne viruses are mainly the Phlebovirus, the Vesiculovirus and the Orbivirus. Some of these viruses are associated with outbreaks or human cases in the Mediterranean Europe. In this paper, the viruses transmitted by Phlebotomine sandflies in Europe (Toscana virus, Sicilian virus, sandfly fever Naples virus) are reviewed and their medical importance, geographical distribution, epidemiology and potential spreading discussed. Data on vertebrate reservoirs is sparse for sandfly fever viruses. The factor currently known to limit the spread of diseases is mainly the distribution areas of potential vectors. The distribution areas of the disease may not be restricted to the areas where they have been recorded but could be as wide as those of their vectors, that is to say Larroussius and P. papatasi mainly but not exclusively. Consequently, field work in form of viral isolation from sandflies and possible reservoirs as well as laboratory work to establish vectorial competence of colonised sandflies need to be encouraged in a near future, and epidemiological surveillance should be undertaken throughout the European Union.

Avian schistosomes in French aquatic birds: a molecular approach.

The prevalence of human cercarial dermatitis (HCD) caused by bird schistosomes appears to be increasing in France, in light of the impact of tourism combined with high densities of wild aquatic hosts in freshwater areas. The present work expands our knowledge of schistosome systematics by including samples of bird schistosomes collected from their natural hosts in France. Heads (318) and viscera (81) of aquatic birds belonging to 16 species from five orders, collecting during the hunting seasons or found dead, were autopsied for nasal and visceral schistosomes. Eggs and/or adults were analysed by molecular methods using the D2 domain and the second internal transcribed spacer (ITS-2) region of rDNA to determine species. Even if nasal eggs were polymorphic according to the host, all haplotypes were similar to that of Trichobilharzia regenti. Marked diversity of visceral species was observed. Final hosts under natural conditions were reported. For the first time, Trichobilharzia franki is reported in its natural bird hosts, Anas platyrhynchos, Anas crecca, Aythya fuligula and Cygnus olor. We also identified T. szidati in A. crecca and Anas clypeata. Bilharziella polonica was found in six species of aquatic birds, including Grus grus. This finding is the first record of bird schistosomes in this aquatic bird. Three new taxa of visceral schistosomes in Anser anser are strongly suspected according to their haplotypes. Futhermore, a new haplotype of visceral schistosomes isolated in Cygnus olor and similar to Allobilharzia visceralis was identified.