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

Biting 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.

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.

An updated list of the Culicoides (Diptera, Ceratopogonidae) fauna from Ecuador.

An updated list of biting midges of the genus Culicoides inhabiting Ecuador is provided. Entomological investigations were carried out from July 2010 to May 2019 using CDC light traps in three Ecuadorian regions (Amazon basin, Andean (foothills and highlands) and Pacific Coast). A total of 12,073 Culicoides specimens from seven subgenera and nine species groups were collected. More species and higher variation were found in the Amazon basin than in either of the Andes regions or coastal sites. A total of 53 species were identified. Of these, 15 are herein reported as new species records for Ecuador: Culicoides acotylus Lutz, C. aitkeni Wirth & Blanton, C. benarrochi Ortiz & Mirsa, C. carvalhoi Wirth & Blanton, C. freitasi Wirth & Blanton, C. ginesi Ortíz, C. lopesi Barretto, C. lyrinotatus Wirth & Blanton, C. profundus Santarém, Felippe-Bauer & Trindade, C. pseudoreticulatus Santarém, Felippe-Bauer & Castellón, C. quasiparaensis Clastrier, C. vernoni Wirth & Blanton, C. youngi Wirth & Barreto and two new species. Our results show that the updated list of the Ecuadorian Culicoides fauna comprises 70 species. This inventory highlights the presence of species that have been incriminated as vectors of disease elsewhere in animals and humans, mainly C. insignis and C. paraensis.

Title: Une liste actualisée de la faune des Culicoides (Diptera, Ceratopogonidae) de l'Équateur. Abstract: Une liste actualisée des Culicoides vivant en Équateur est fournie. Des enquêtes entomologiques ont été menées de juillet 2010 à mai 2019 à l'aide de pièges lumineux CDC dans trois régions équatoriennes (le bassin amazonien, les Andes (piémont et hautes terres) et la côte Pacifique). Au total, 12 073 spécimens de Culicoides de sept sous-genres et neuf groupes d'espèces ont été collectés. Plus d'espèces et une variation plus élevée ont été trouvées dans le bassin amazonien que dans les régions des Andes ou les sites côtiers. Au total, 53 espèces ont été identifiées. Parmi celles-ci, 15 sont signalées ici comme nouvelles espèces pour l'Équateur : Culicoides acotylus Lutz, C. aitkeni Wirth & Blanton, C. benarrochi Ortiz & Mirsa, C. carvalhoi Wirth & Blanton, C. freitasi Wirth & Blanton, C. ginesi Ortíz, C. lopesi Barretto, C. lyrinotatus Wirth & Blanton, C. profundus Santarém, Felippe-Bauer & Trindade, C. pseudoreticulatus Santarém, Felippe-Bauer & Castellón, C. quasiparaensis Clastrier, C. vernoni Wirth & Blanton, C. youngi Wirth & Barreto et deux nouvelles espèces. Nos résultats montrent que la liste actualisée de la faune culicoïde équatorienne comprend 70 espèces. Cet inventaire met en évidence la présence d'espèces qui ont été incriminées ailleurs comme vecteurs de maladies chez l'animal et l'homme, principalement C. insignis et C. paraensis.

Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France.

Knowledge on host-feeding pattern of blood-sucking insects helps to understand the epidemiology of a vector-born disease. We determined blood meal origin from blood-fed Culicoides thanks to molecular techniques. A set of primers was used to selectively amplify segment of vertebrates' prepronociceptin gene from abdomen of engorged Culicoides (Diptera: Ceratopogonidae). Vertebrate DNA was successfully amplified in 91% of blood-fed Culicoides assayed. Direct sequencing and comparison of resultant sequences with sequences in GenBank, using BLAST, lead to the specific identification of the host in 100% of the cases. A total of 157 blood-fed females belonging to 13 different Culicoides' species were captured thanks to light traps in different areas of France between 2008 and 2009. Blood meal origin was determined for 143 blood-fed midges: 59 Culicoides obsoletus, 18 Culicoides dewulfi, 16 Culicoides scoticus, 11 Culicoides chiopterus, 10 Culicoides lupicaris, 1 Culicoides pulicaris, 8 Culicoides punctatus, 10 Culicoides pallidicornis, 3 Culicoides achrayi, 2 Culicoides furcillatus, 3 Culicoides brunnicans, 1 Culicoides picturatus and 1 Culicoides poperinghensis. The predominant species in our study belong to the C. obsoletus complex; they are considered as putative vectors of Bluetongue virus in the north of Europe. C. chiopterus sampled fed only on cattle, while blood meal origin of C. dewulfi, C. obsoletus and C. scoticus was diversified. In our sampling, we found that midges were fed mainly on cattle (54%), rabbits (20%), horses (17%), sheep (4%), pigs or wild boars (4%) and human (1%). Cattle DNA was found in at least 11 different species of Culicoides assayed.

Comprehensive characterisation of Culicoides clastrieri and C. festivipennis (Diptera: Ceratopogonidae) according to morphological and morphometric characters using a multivariate approach and DNA barcode.

Biting midges are widespread around the world and play an essential role in the epidemiology of over 100 veterinary and medical diseases. For taxonomists, it is difficult to correctly identify species because of affinities among cryptic species and species complexes. In this study, species boundaries were examined for C. clastrieri and C. festivipennis and compared with six other Culicoides species. The classifiers are partial least squares discriminant analysis (PLS-DA) and sparse partial least squares discriminant analysis (sPLS-DA).The performance of the proposed method was evaluated using four models: (i) geometric morphometrics applied to wings; (ii) morphological wing characters, (iii) "Full wing" (landmarks and morphological characters) and (iv)  "Full model" (morphological characters-wing, head, abdomen, legs-and wing landmarks). Double cross-validation procedures were used to validate the predictive ability of PLS-DA and sPLS-DA models. The AUC (area under the ROC curve) and the balanced error rate showed that the sPLS-DA model performs better than the PLS-DA model. Our final sPLS-DA analysis on the full wing and full model, with nine and seven components respectively, managed to perfectly classify our specimens. The C. clastrieri and C. festivipennis sequences, containing both COI and 28S genes, revealed our markers' weak discrimination power, with an intraspecific and interspecific divergence of 0.4% and 0.1% respectively. Moreover, C. clastrieri and C. festivipennis are grouped in the same clade. The morphology and wing patterns of C. clastrieri and C. festivipennis can be used to clearly distinguish them. Our study confirms C. clastrieri and C. festivipennis as two distinct species. Our results show that caution should be applied when relying solely on DNA barcodes for species identification or discovery.

Puumala Virus Variants Circulating in Forests of Ardennes, France: Ten Years of Genetic Evolution.

In Europe, Puumala virus (PUUV) transmitted by the bank vole (Myodes glareolus) is the causative agent of nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome. In France, very little is known about the spatial and temporal variability of the virus circulating within bank vole populations. The present study involved monitoring of bank vole population dynamics and PUUV microdiversity over a ten-year period (2000-2009) in two forests of the Ardennes region: Elan and Croix-Scaille. Ardennes region is characterised by different environmental conditions associated with different NE epidemiology. Bank vole density and population parameters were estimated using the capture/marking/recapture method, and blood samples were collected to monitor the overall seroprevalence of PUUV in rodent populations. Phylogenetic analyses of fifty-five sequences were performed to illustrate the genetic diversity of PUUV variants between forests. The pattern of the two forests differed clearly. In the Elan forest, the rodent survival was higher, and this limited turn-over resulted in a lower seroprevalence and diversity of PUUV sequences than in the Croix-Scaille forest. Uncovering the links between host dynamics and virus microevolution is improving our understanding of PUUV distribution in rodents and the NE risk.

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.

An approach to automatic classification of Culicoides species by learning the wing morphology.

Fast and accurate identification of biting midges is crucial in the study of Culicoides-borne diseases. In this work, we propose a two-stage method for automatically analyzing Culicoides (Diptera: Ceratopogonidae) species. First, an image preprocessing task composed of median and Wiener filters followed by equalization and morphological operations is used to improve the quality of the wing image in order to allow an adequate segmentation of particles of interest. Then, the segmentation of the zones of interest inside the biting midge wing is made using the watershed transform. The proposed method is able to produce optimal feature vectors that help to identify Culicoides species. A database containing wing images of C. obsoletus, C. pusillus, C. foxi, and C. insignis species was used to test its performance. Feature relevance analysis indicated that the mean of hydraulic radius and eccentricity were relevant for the decision boundary between C. obsoletus and C. pusillus species. In contrast, the number of particles and the mean of the hydraulic radius was relevant for deciding between C. foxi and C. insignis species. Meanwhile, for distinguishing among the four species, the number of particles and zones, and the mean of circularity were the most relevant features. The linear discriminant analysis classifier was the best model for the three experimental classification scenarios previously described, achieving averaged areas under the receiver operating characteristic curve of 0.98, 0.90, and 0.96, respectively.

Association between host species choice and morphological characters of main sensory structures of Culicoides in the Palaeartic region.

Culicoides (Diptera: Ceratopogonidae) serve as vectors of several mammalian and avian diseases, including bluetongue, Schmallenberg, African horse sickness, avian malaria and Oropouche. Host preference investigations are necessary to assess the transmission routes of vector-borne diseases and to inform mitigation strategies. A recent study examining the main sensory structures (palps and antennae) of Culicoides species suggests that they be classified as ornithophilic or mammalophilic according to their feeding habits. We analyzed Culicoides host preferences according to the literature and carried out a multiple correspondence analysis linking these preferences with morphological data. Seven out of 12 variables were found to be reliable predictors of host preference in Culicoides species: Antenna Flagellomer-Sensilla Coeloconica-Number: (7-10) and (11-13); Antenna Flagellomer-Sensilla Coeloconica IV-X: presence; Palpus-size: wide and/or narrow opening and shallow pit; Palpus-Shape: strongly swollen; Antenna-Short sensilla trichodea-distal part segment IV to X-Number: 2 seta each. Our results demonstrate that the presence of sensilla coeloconica and the maxillary palpus can be used to separate ornithophilic and mammalophilic or ornithophilic/mammalophilic species.

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.

Comparison of Vertebrate Cytochrome b and Prepronociceptin for Blood Meal Analyses in Culicoides.

To date, studies on host preferences and blood meal identification have been conducted for Culicoides species using molecular-based methods such as PCR techniques to amplify only a fragment from universal vertebrate mitochondrial genes such as cytochrome c oxidase subunit I or cytochrome b (Cyt b). The vertebrate prepronociceptin gene (PNOC) was also tested in this field. However, the choice of molecular marker to identify blood meal is critical. The objective of our study is to compare the ability of Cyt b and PNOC as molecular markers for blood meal identification depending on the stage of blood meal digestion. In order to determine whether these Cyt b and PNOC could provide a positive result, 565 blood-fed females of Culicoides spp were collected and morphologically identified. The samples were collected between 2012 and 2014, in two localities in France. The collection localities were near either livestock or a forest. To catch the specimens, we used UV CDC miniature light traps. PNOC sequence of donkeys (Equus asinus) was sequenced and submitted because it was missing in GenBank. Our findings emphasize that the PNOC marker is not suitable to separate closely related Equid species such as horses and donkeys. The Cyt b marker was able to identify 204 more samples when compared to PNOC (99.55% of specimens). Cyt b appears to be better able to detect the origin of blood meals from females with digested blood in their abdomens. We conclude that Cyt b is a good marker as it increases the accuracy of blood meal identification of engorged females containing digested blood in their abdomens. The host opportunist behavior of Culicoides, especially that of C. obsoletus and C. scoticus, the main vectors of BTV in Europe was also highlighted.

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.

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

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.

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.

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.

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

BACKGROUND: Leishmania 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. FINDINGS: blood, 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. CONCLUSION: This 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.