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.

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.

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.

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.

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.

Simple and highly discriminatory VNTR-based multiplex PCR for tracing sources of Aspergillus flavus isolates.

Aspergillus flavus is second only to A. fumigatus in causing invasive aspergillosis and it is the major agent responsible for fungal sinusitis, keratitis and endophthalmitis in many countries in the Middle East, Africa and Southeast Asia. Despite the growing challenge due to A. flavus, data on the molecular epidemiology of this fungus remain scarce. The objective of the present study was to develop a new typing method based on the detection of VNTR (Variable number tandem repeat) markers. Eight VNTR markers located on 6 different chromosomes (1, 2, 3, 5, 7 and 8) of A. flavus were selected, combined by pairs for multiplex amplifications and tested on 30 unrelated isolates and six reference strains. The Simpson index for individual markers ranged from 0.398 to 0.818. A combined loci index calculated with all the markers yielded an index of 0.998. The MLVA (Multiple Locus VNTR Analysis) technique proved to be specific and reproducible. In a second time, a total of 55 isolates from Chinese avian farms and from a Tunisian hospital have been evaluated. One major cluster of genotypes could be defined by using the graphing algorithm termed Minimum Spanning Tree. This cluster comprised most of the isolates collected in an avian farm in southern China. The MLVA technique should be considered as an excellent and cost-effective typing method that could be used in many laboratories without the need for sophisticated equipment.