Damien Jouet

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.

Trichobilharzia spp. in natural conditions in Annecy Lake, France

Annecy Lake is a well-known focus of human cercarial dermatitis in France. Identification of the parasites, however, was not performed in the past. Previous studies suspected two species, Trichobilharzia franki and Trichobilharzia regenti, based on the presence of parasites in mallards and/or morphological identification of snails emitting ocellate furcocercariae. Following a standardized molecular approach, we studied snails and furcocercariae and compared their haplotypes with those deposited in GenBank. The selected markers were the second internal transcribed spacer ITS-2 for the snails and ITS-2 and D2 domain of the ribosomal DNA for the parasites. Our results confirm the presence of T. franki and T. regenti and two probable new species that could be potential agents of cercarial dermatitis. All the snails emitting the ocellate furcocercariae belong to the same species identified as Radix peregra (=Radix ovata = Radix balthica). Parasite–host relationships between species of the genus Trichobilharzia and snails of the genus Radix do not seem to be as specific as supposed previously.

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.

Molecular diversity of Trichobilharzia franki in two intermediate hosts (Radix auricularia and Radix peregra): A complex of species

Recently, the systematic use of the molecular approach as a complement to the other approaches (morphology, biology, life cycle) has brought help for the identification of species considered as different in the past to be regrouped and synonymised, and distinctions to be drawn between species similar at the morphological level. Among these species, we tried to clarify the situation of Trichobilharzia frankiMüller and Kimmig, 1994, species that today include more than 50 haplotypes notably coming from larval stages isolated from intermediate hosts belonging to gastropods of the Radix genus. Cercariae were isolated in France and Iceland from various molluscs, before being analyzed, with their hosts, by molecular analysis of various fields such as the D2 and ITS of the ribosomal DNA and the COX1 of mitochondrial DNA. We thus show the presence of two clades depending on the specificity of their intermediate host in which they were isolated (Radix auricularia or Radix peregra), thus allowing separation of the species T. franki that had been described in the past as a probable new species.

Final hosts and variability of Trichobilharzia regenti under natural conditions.

In Europe, despite of intensive work on avian schistosomes in the past, only one representative of nasal schistosomes has been detected so far, Trichobilharzia regenti, a species that remained undescribed to the scientific community until 1998. Since then, however, T. regenti has gained a considerable attention, not only due to the induction of serious lesions concomitant with the migration of the schistosomulae through the central nervous system of their natural hosts (birds) but also due to their implication in cercarial dermatitis and their neurotrophic behaviour in mammals (mice, experimental) where the parasite migrate to the CNS. From 2001 to 2010, by a parasitic control of various potential final hosts (aquatic birds) living in natural condition in France and in Iceland, we detected T. regenti in six species: Anas platyrhynchos, Cygnus olor, Aythya fuligula, Aythya ferina, Mergus merganser and Anser anser. Although morphological features, mainly of eggs, showed some variations with the original description of the parasite, molecular analysis confirmed their membership to the T. regenti species. Nevertheless, specific molecular and morphological variations were especially observed for nasal parasite isolated from Anas clypeata. For this species, we discuss the possibility for this nasal schistosome to belong to a nasal Trichobilharzia species already described in Africa or even an undescribed nasal species.

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.

Trichobilharzia mergi sp. nov. (Trematoda: Digenea: Schistosomatidae), a visceral schistosome of Mergus serrator (L.) (Aves: Anatidae).

Parasitological investigations on red-breasted mergansers (Mergus serrator L.) in Iceland revealed digenean flukes of the family Schistosomatidae. Adult worms were detected in blood vessels of the large intestine and eggs were deposited in the mucosa and surrounded by granulomatous reactions. Traditional morphological methods showed that the flukes have very slender filiform bodies, males are equipped with a short gynaecophoric canal and both suckers and spatulate ends are present on each sex. Among characteristics of the flukes which render them morphologically distinct from other Trichobilharzia species are: i) males-well developed vesicula seminalis (v.s.) consisting of a short v.s. externa and a significantly longer (approx. 3 times) v.s. interna, unusually well developed genital papilla and localization of the first testis a relatively long distance posterior to the gynaecophoric canal; ii) eggs-small and elongated with slightly rounded poles and a short terminal spine. DNA taxonomic techniques confirmed that a new species had been identified, Trichobilharzia mergi sp. n. The sequence data were deposited in GenBank under the accession numbers JX456151 to JX456172. Comparison of the results with our previously published data on characterization of DNA of cercariae isolated from freshwater lymnaeid snails showed that larval development of T. mergi is associated with Radix balthica L. (=Radix peregra Müller, 1774;=Radix ovata Draparnaud, 1805).

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.

Detection of Planorbis planorbis and Anisus vortex as first intermediate hosts of Alaria alata (Goeze, 1792) in natural conditions in France: molecular evidence.

Alaria alata (Goeze, 1792), a trematode that parasitizes canids, usually needs two intermediate hosts to complete its life cycle: an aquatic freshwater snail and an amphibian. Although many studies have been undertaken on the wild boars role as paratenic host, owing to the potential threat to human health, few have sought to identify the snails that act as first intermediate hosts in natural conditions. Adopting a molecular approach, with specific markers for a portion of the second internal transcribed spacer (ITS-2), we detected haplotypes of A. alata furcocercariae in two snail species (Planorbis planorbis and Anisus vortex), identified by molecular analysis (ribosomal 18S, mitochondrial 16S and COI). This study provides the first description of snails naturally emitting A. alata furcocercaria in Western Europe.

Occurrence of Mesocestoides canislagopodis (Rudolphi, 1810) (Krabbe, 1865) in mammals and birds in Iceland and its molecular discrimination within the Mesocestoides species complex

The life cycle of Mesocestoides tapeworms (Cestoda: Cyclophyllidea: Mesocestoididae) requires three hosts. The first intermediate host is unknown but believed to be an arthropod. The second intermediate host is a vertebrate. The primary definitive host is a carnivore mammal, or a bird of prey, that eats the tetrathyridium-infected second intermediate host. One representative of the genus, Mesocestoides canislagopodis, has been reported from Iceland. It is common in the arctic fox (Vulpes lagopus) and has also been detected in domestic dogs (Canis familiaris) and cats (Felis domestica). Recently, scolices of a non-maturing Mesocestoides sp. have also been detected in gyrfalcon (Falco rusticolus) intestines, and tetrathyridia in the body cavity of rock ptarmigan (Lagopus muta). We examined the taxonomic relationship of Mesocestoides from arctic fox, gyrfalcon, and rock ptarmigan using molecular methods, both at the generic level (D1 domain LSU ribosomal DNA) and at the specific level (cytochrome c oxidase subunit I (COI) and 12S mitochondrial DNA). All stages belonged to Mesocestoides canislagopodis. Phylogenetic analysis of the combined 12S-COI at the specific level confirmed that M. canislagopodis forms a distinct clade, well separated from three other recognized representatives of the genus, M. litteratus, M. lineatus, and M. corti/vogae. This is the first molecular description of this species. The rock ptarmigan is a new second intermediate host record, and the gyrfalcon a new primary definitive host record. However, the adult stage seemed not to be able to mature in the gyrfalcon, and successful development is probably restricted to mammalian hosts.