Wing morphology variations in Culicoides circumscriptus from France

The biting midge Culicoides circumscriptus Kieffer, 1918 is a European widespread vector of avian malaria throughout the continent and is a possible vector of Akabane virus and Bluetongue virus. This species populates a wide range of environments in contrasting ecological settings often exposed to strong seasonal fluctuations. The main goals of this study were to investigate C. circumscriptus phenotypic variation at three departments in France (Corsica Island, Moselle and Var) and to determine if its phenotypes vary with the environment. Culicoides circumscriptus wing phenotypes were analyzed using a geometric morphometric approach based on anatomical landmarks and outlines of the wing. Dendogram trees based on landmarks and the outlines-2 set (cell m4) showed similar topologies and separated populations of C. circumscriptus. In contrast, another set of outlines-1 (covering the r-m cross vein, M, radiale and arculus) presented a different hierarchical clustering tree. The phenotypic variation observed in C. circumscriptus indicated that these populations are exposed to environmental and ecological pressures. Our results suggest the presence of phenotypic plasticity in this species

Development of molecular tools for systematics : application to a few Ecdysozoa parasites and vectors

Les Ecdysozoaires sont des organismes caractérisés par une croissance discontinue composée de plusieurs stades morphologiques séparés par des mues. Parmi eux, certains groupes présentent un intérêt épidémiologique important en santé humaine et/ou animale, notamment les nématodes parasites gastro-intestinaux des ruminants et les phlébotomes vecteurs de leishmanies, qui font l’objet de ce travail. L’étude de la systématique de tels organismes constitue un élément de base essentiel à toute recherche appliquée à la biodiversité, l’écologie et la physiologie, ainsi qu’à leurs relations avec les autres acteurs de leur cycle biologique. Pourtant, l’identification morphologique de certaines espèces s’avère délicate voire impossible étant donnés les stades de croissance, le dimorphisme sexuel et l’existence de morphes, de complexes d’espèces, d’espèces jumelles et d’hybrides, ne concordant pas toujours avec la définition biologique de l’espèce. L’essor de la biologie moléculaire offre un panel d’outils performants au service de la systématique par l’étude de la variabilité génétique. L’objectif de la thèse est de répondre à des problématiques rencontrées en systématique (phylogénie, taxonomie, histoire évolutive d’une espèce) en étudiant la variabilité génétique de ces organismes, à l’aide d’outils moléculaires appropriés. Ce travail a permis la résolution de cas d’études concrets tels que l’inférence d’une phylogénie, l’identification d’espèces proches et l’étude de la variabilité génétique intraspécifique, pour suivre les flux de gènes et en déduire la structure génétique de populations, afin de mieux appréhender l’espèce, son histoire et son évolution dans son environnement.Ecdysozoans are organisms characterized by discontinuous growth composed of several morphological stages separated by moults. Among them, some groups have a significant epidemiological interest in human and/or animal health, including the gastrointestinal nematodes, parasites of ruminants and phlebotomine sandflies, vectors of Leishmania, which which we focus on. The study of the systematics of such organisms is an essential basic element of any research applied to biodiversity, ecology and physiology, as well as to their relationships with other actors in their life cycle. However, the morphological identification of some species is difficult or even impossible given the stages of growth, sexual dimorphism and the existence of morphs, species complexes, sibling species and hybrids, not concordant always with the biological definition of the species. The rise of molecular biology offers a range of powerful tools for systematics by studying genetic variability. The aim of the thesis is to answer the problems encountered in systematics (phylogeny, taxonomy, evolutionary history of a species) by studying the genetic variability of these organisms, using appropriate molecular tools. This work allowed the resolution of concrete case studies such as the inference of a phylogeny, the identification of closely related species and the study of intraspecific genetic variability, to follow gene flow and deduce the structure population genetics, to better understand the species, its history and its evolution in its environment

Wing morphometrics of biting midges (Diptera: Culicoides) of veterinary importance in Madagascar

International audienceBiting midges are vectors of arboviruses such as bluetongue virus, bovine ephemeral fever virus, Akabane virus, African horse sickness virus, epizootic haemorrhagic disease virus and Schmallenberg virus. Fast and accurate identification of biting midges is crucial in the study of Culicoides-borne diseases. Morphological identification of biting midges has revealed the presence of cryptic species. A total of 20 species are reported in Madagascar. In this study, we assessed wing morphometric analysis for identification of seven species namely C. dubitatus Kremer, Rebholtz-Hirtzel and Delécolle, C. enderleini Cornet and Brunhes, C. kibatiensis Goetghebuer, C. miombo Meiswinkel, C. moreli Clastrier, C. nevilli Cornet and Brunhes, and C. zuluensis de Meillon. Culicoides enderleini, C. miombo, C. moreli, C. nevilli and C. zuluensis are vectors diseases. A molecular approach, based on the cytochrome oxidase I gene (Cox1), was used for species delimitation. The molecular analysis presented seven different clades grouped two-by-two according to morphological characters. A total of 179 wing images were digitised. We found morphometric variation among seven species based on 11 landmarks and two outlines. Wing shape variation plots showed that species overlapped with species belonging to the same group. The cross-validation revealed a relatively high percentage of correct classification in most species, ranging from 91.3% to 100% for landmarks; 60% to 82.6% for outlines-1 and 77.1% to 91.3% for outlines-2. Our study suggests that wing geometric morphometric analysis is a robust tool for reliable "Moka Fohy" identification in Madagascar. This inexpensive and simple method is a precise supplement to morphological identification, with reaches the accuracy of Cox1 barcoding

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

International audiencePhlebotomine 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-Turranian 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.Author summary: Phlebotomine sand flies are tiny insects transmitting unicellular parasites called Leishmania worldwide. They are bad flyers and are known to be a group dispersing according to historical events. No invasive species are known within Phlebotomine sandflies. Their taxonomy includes about 40 genera. In the Old World, the subgenus Paraphlebotomus includes the main vectors of Leishmania tropica, the agent of a cutaneous leishmaniasis mostly in North Africa visceral and the Middle West. The goal of this study was to explore the phylogeny and the biogeographical history of this subgenus using a new generation sequencing technique. We successfully amplified DNA copies from the selected DNA extracts, some of them being more than 20 years old. We fully resolved the phylogeny of the subgenus Paraphlebotomus who was not recovered as monophyletic, meaning it does not include all the ascendants of last common ancestor. Consequently, the taxonomy was revised by the creation of a new subgenus and suggest that Paraphlebotomus originated about 10 million years ago. After a first split isolation North African species, the Messinian crisis caused by the aridification of the Tethys (the ancestor of the Mediterranean Sea) occurring 7 million years ago allowed the divergence of the main lineages including that of Phlebotomus sergenti the most important vector of the group. Most recent radiation events occurred during the Pleistocene. The historical biogeography of these Phlebotomine sandflies are closely related to that of the Leishmania they transmit

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin

Phlebotomus (Paraphlebotomus) chabaudi and Phlebotomus riouxi: closely related species or synonyms?

Phlebotomus riouxi Depaquit, Killick-Kendrick & Léger 1998 was described as a species closely related to Phlebotomus chabaudi Croset, Abonnenc & Rioux 1970, differing mainly by the size and number of setae of the coxite basal lobe. Molecular studies carried out on several populations from Algeria and Tunisia and based on mitochondrial genes cytochrome b (Cytb) and cytochrome oxidase I (COI) supported the typological validity of these two species. Recently, specimens from a single population in southern Tunisia were morphologically identified as Ph. riouxi, Ph. chabaudi and intermediates, but were clustered in the same clade according to their Cytb and nuclear gene elongation factor-1 α (EF-1α) sequences. These species were thus synonymized. To further explore this synonymy, we carried out a molecular study on specimens from Algeria and Tunisia using the same molecular markers and a part of 28S rDNA. We did not find any morphologically intermediate specimens in our sampling. We highlighted differences between the genetic divergence rates within and between the two species for the three markers and we identified new haplotypes. The sequence analysis did not reveal any signature of introgression in allopatric nor in sympatric populations such as in the Ghomrassen population. Phylogenetic analyses based on our specimens revealed that the two main clades are Ph. chabaudi and Ph. riouxi, in agreement with the morphological identification. These results support the validity of Ph. riouxi and Ph. chabaudi as typological species

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Phlebotomus riouxi Depaquit, Killick-Kendrick & Léger 1998 was described as a species closely related to Phlebotomus chabaudi Croset, Abonnenc & Rioux 1970, differing mainly by the size and number of setae of the coxite basal lobe. Molecular studies carried out on several populations from Algeria and Tunisia and based on mitochondrial genes cytochrome b (Cytb) and cytochrome oxidase I (COI) supported the typological validity of these two species. Recently, specimens from a single population in southern Tunisia were morphologically identified as Ph. riouxi, Ph. chabaudi and intermediates, but were clustered in the same clade according to their Cytb and nuclear gene elongation factor-1 α (EF-1α) sequences. These species were thus synonymized. To further explore this synonymy, we carried out a molecular study on specimens from Algeria and Tunisia using the same molecular markers and a part of 28S rDNA. We did not find any morphologically intermediate specimens in our sampling. We highlighted differences between the genetic divergence rates within and between the two species for the three markers and we identified new haplotypes. The sequence analysis did not reveal any signature of introgression in allopatric nor in sympatric populations such as in the Ghomrassen population. Phylogenetic analyses based on our specimens revealed that the two main clades are Ph. chabaudi and Ph. riouxi, in agreement with the morphological identification. These results support the validity of Ph. riouxi and Ph. chabaudi as typological species