Denis Augot

The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspring of infected adult ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been implicated in the transmission of SBV through studies conducted in northern Europe. In this study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex (C. obsoletus and C. scoticus) in transmission of SBV were confirmed in France and SBV was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a potential vector. While collections in Sardinia were dominated by C. imicola, only relatively small quantities of SBV RNA were detected in pools of this species and conclusive evidence of its potential role in transmission is required. In addition to these field-based studies, infection rates in colony-derived individuals of C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of infection in C. nubeculosus were low, confirming previous studies, while preliminary examination of C. scoticus demonstrated that while this species can replicate SBV to a potentially transmissible level, further work is required to fully define comparative competence between species in the region. Finally, the oral competence for SBV of two abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appear unlikely to play a major role in transmission. Other vector competence data produced from studies across Europe to date is then comprehensively reviewed and compared with that generated previously for bluetongue virus.

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.

First detection of Leishmania killicki (Kinetoplastida, Trypanosomatidae) in Ctenodactylus gundi (Rodentia, Ctenodactylidae), a possible reservoir of human cutaneous leishmaniasis in Tunisia

BackgroundLeishmania killicki was originally described in 1980 in southeast Tunisia. It was also recently reported in Lybia and Algeria. Nevertheless, neither vector nor reservoirs of this parasite are known. The identification of the vector and the animal reservoir host of L. killicki is critical for the establishment of an efficient control strategy.Findingsblood, popliteal lymph node, spleen, bone marrow, liver and skin were collected from 50 rodents in 2009 in south western Tunisia. Samples were smeared onto glass slides, cultured on NNN medium and tested by polymerase chain reaction for Leishmania detection. Parasites were detected by PCR from 10 Psammomys obesus and from two Ctenodactylus gundi. Parasite identification was performed simultaneously by internal transcribed spacer 1 PCR-RFLP and by PCR sequencing. Both Leishmania major and Leishmania killicki were identified from infected Psammomys and Ctenodactylus gundi respectively.ConclusionThis is the first report of Leishmania killicki identified from Ctenodactylus gundi in Tunisia. This result supports the assumption that C. gundi is a potential reservoir for Leishmania killicki.

Emergence of Culicoides obsoletus from indoor and outdoor breeding sites

Culicoides obsoletus (Diptera: Ceratopogondae) is considered as the probable main vector of Bluetongue virus (BTV) in northern Europe. Its breeding sites are poorly documented at the present time. We report numerous emergences of C. obsoletus s. str. from soil samples collected in two holdings between August and September 2010. Specimens were collected regularly in the laboratory during 80 days. In one holding, 1584 C. obsoletus midges emerged from used litter collected inside a dairy cow building and 211 C. obsoletus midges emerged from manure left outside the farm buildings. In the second holding, the number of emergences observed was much lower, especially for indoor samples. We discuss the impact of the farming practices on the abundance of emergence between both holdings on one side and between indoor and outdoor breeding sites on the other side. The observation of a peak in emergence and high emergence rates recorded in the laboratory for indoor samples let us assume that these breeding sites are of great importance, especially when it comes to understand the biology of this widespread species and its role in the transmission of BTV.

Associations between MHC genes and Puumala virus infection in Myodes glareolus are detected in wild populations, but not from experimental infection data.

We analysed the influence of MHC class II Dqa and Drb genes on Puumala virus (PUUV) infection in bank voles (Myodes glareolus). We considered voles sampled in five European localities or derived from a previous experiment that showed variable infection success of PUUV. The genetic variation observed in the Dqa and Drb genes was assessed by using single-strand conformation polymorphism and pyrosequencing methods, respectively. Patterns were compared with those obtained from 13 microsatellites. We revealed significant genetic differentiation between PUUV-seronegative and -seropositive bank voles sampled in wild populations, at the Drb gene only. The absence of genetic differentiation observed at neutral microsatellites confirmed the important role of selective pressures in shaping these Drb patterns. Also, we found no significant associations between infection success and MHC alleles among laboratory-colonized bank voles, which is explained by a loss of genetic variability that occurred during the captivity of these voles.

Mitochondrial DNA Differentiation Between Two Closely Related Species, Phlebotomus (Paraphlebotomus) chabaudi and Phlebotomus (Paraphlebotomus) riouxi (Diptera: Psychodidae), Based on Direct Sequencing and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

ABSTRACT Phlebotomus chabaudi Croset, Abonnenc & Rioux, 1970 and Phlebotomus riouxi Depaquit, Killick-Kendrick & Léger, 1998 (Diptera: Psychodidae) are closely related species of phlebotomine sand flies, the females of which are suspected of transmitting Leishmania killicki Rioux, Lanotte, and Pratlong 1986 in Tunisia. Although males of these species are distinguishable by the number and size of setae on the basal lobe of the coxite, morphologically differential characters between the females are blurred. A molecular study, based on 40 Algerian and Tunisian specimens, was conducted to distinguish females of these two species. Alignment of the cytochrome c oxidase I (COI) sequences and their analysis by using neighbor-joining and maximum likelihood showed the separation of P. chabaudi and P. riouxi, irrespective of intraspecific variability. Both COI and cytochrome b have been tested as molecular identification tools. Single strand conformation polymorphism analysis did not distinguish these two species, whereas restriction fragment length polymorphism can be applied to identify P. chabaudi and P. riouxi for surveillance purposes.

A taxonomic study of Phlebotomus (Larroussius) perfiliewi s. l.

Phlebotomus (Larroussius) perfiliewi is one of the main vectors of Leishmania infantum in the Mediterranean basin and in Central Asia. Its taxonomic status remains doubtful. It usually includes three taxa of specific or subspecific value, depending on the authors: P. perfiliewiParrot, 1930 s. st. in North Africa, Western Europe and the Balkans, Crimea; Phlebotomus galilaeusTheodor, 1958 in Israel/Palestine and Cyprus and Phlebotomus transcaucasicusPerfiliev, 1937 in Caucasus and Central Asia. While the identification of the females of P. perfiliewi s. l. is relatively easy, based on the particular morphology of their spermathecal ducts, the specific identification of the females of Phlebotomus perfiliewi perfiliewi, Phlebotomus perfiliewi galilaeus and Phlebotomus perfiliewi transcaucasicus is impossible. The differentiation of males, based mainly on characters on aedeagus, is also difficult. The goal of the present study is to carry out a taxonomic update based on a morphological study of specimens coming from various populations from Asia, Europe and North Africa coupled with their molecular analysis.

Characterization of two cryptic species, Culicoides stigma and C.parroti (Diptera: Ceratopogonidae), based on barcode regions and morphology.

ABSTRACT: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness, bluetongue, and Schmallenberg virus. The identification of Culicoides based on morphological features can be difficult.Three species of biting midges, Culicoides nubeculosus, C. stigma, and C. parroti have emerged in the laboratory from mud collected around watering troughs on a farm in northern France. Emerging Culicoides were characterized morphologically and molecularly using molecular markers. The closely related species C. stigma and C. parroti showed highly divergent sequences for both mitochondrial (cytochrome B and cytochrome oxidase I) and ribosomal DNA first internal transcribed spacer. A RFLP based on a single restriction using the same enzyme (HaeIII) for both cytochrome C oxidase I and cytochrome B is proposed to identify these species.

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.

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.