ITS1 Markers Research Articles

Integrated Histological and Molecular Analysis of Filarial Species and Associated Wolbachia Endosymbionts in Human Filariasis Cases Presenting Atypically in Thailand.

Atypical presentations of filariasis have posed diagnostic challenges due to the complexity of identifying the causative species and the difficulties in both diagnosis and treatment. In this study, we present the integrative histological and molecular analysis of seven atypical filariasis cases observed in regions of nonendemicity of Thailand. All filariasis cases were initially diagnosed based on histological findings. To confirm the causative species, molecular characterization based on both filarial mitochondrial (mt 12S rRNA and COI genes) and nuclear ITS1 markers was performed, together with the identification of associated Wolbachia bacterial endosymbionts. Among the cases studied, Brugia pahangi (N = 3), Brugia malayi (N = 1), Dirofilaria sp. "hongkongensis" (N = 2), and a suspected novel filarial species genetically related to Pelecitus copsychi (N = 1) were identified. By targeting the 16S rRNA gene, Wolbachia was also molecularly amplified in two cases of infection with Dirofilaria sp. "hongkongensis." Phylogenetic analysis further revealed that the detected Wolbachia could be classified into supergroups C and F, indicating the high genetic diversity of this endosymbiont in Dirofilaria sp. "hongkongensis." Furthermore, this study demonstrates the consistency between histological findings and species identification based on mitochondrial loci rather than on the nuclear ITS1. This suggests the utility of mitochondrial markers, particularly COI, as a highly sensitive and reliable diagnostic tool for the detection and differentiation of filarial species in clinical specimens. Precise identification of the causative species will facilitate accurate diagnosis and treatment and is also essential for the development of epidemiological and preventive strategies for filariasis.

Isolation and molecular identification of liver fluke cercariae in freshwater snails of Chaharmahal and Bakhtiari province, Iran

ABSTRACT Liver flukes are parasitic flatworms that can infect humans and other mammals. In this study, freshwater snails from Chaharmahal and Bakhtiari province in Iran were investigated for the presence of liver fluke larvae. The snails were collected from various rivers and analysed using standard morphological and molecular techniques. Additionally, molecular identification of the collected snails was conducted through polymerase chain reaction (PCR) and sequencing of the COI gene. The freshwater snails were identified as Physella acuta (Draparnaud, 1805), Galba truncatula (O.F. Müller, 1774), Radix euphratica (Mousson, 1874), Radix auricularia (Linnaeus, 1758) and Planorbis planorbis (Linnaeus, 1758). The presence of larval stages of Fasciola spp. in individual snails was examined using stereomicroscopy and a crushing method. Morphological and molecular identification based on the ITS-1 marker for identifying Fasciola spp. revealed that 10.9% (59 out of 549) of R. auricularia and 5.8% (37 out of 630) of P. acuta were infected. A blastn analysis of the collected samples revealed that all isolates belonged to F. hepatica, indicating a potential risk of liver fluke infection in the study area. These findings emphasise the importance of regular surveillance and control measures to prevent the spread of these parasites.

The same genotype of Sarcocystis neurona responsible for mass mortality in marine mammals induced a clinical outbreak in raccoons (Procyon lotor) 10 years later

Here, we report the first known outbreak of clinical protozoal myeloencephalitis in naturally infected raccoons by the parasite Sarcocystis neurona. The North American opossum (Didelphis virginiana) and the South American opossum (Didelphis albiventris) are its known definitive hosts. Several other animal species are its intermediate or aberrant hosts. The raccoon (Procyon lotor) is considered the most important intermediate host for S. neurona in the USA. More than 50% of raccoons in the USA have sarcocysts in their muscles, however clinical sarcocystosis in raccoons is rare. In 2014, 38 free-living raccoons were found dead or moribund on the grounds of the Saint Louis Zoo, Missouri, USA. Moribund individuals were weak, lethargic, and mildly ataxic; several with oculo-nasal discharge. Seven raccoons were found dead and 31 were humanely euthanized. Postmortem examinations were conducted on nine raccoons. Neural lesions compatible with acute sarcocystosis were detected in eight raccoons. The predominant lesions were meningoencephalitis and perivascular mononuclear cells. Histologic evidence for the Canine Distemper Virus was found in one raccoon. Schizonts and merozoites were present in the encephalitic lesions of four raccoons. Mature sarcocysts were present within myocytes of five raccoons. In six raccoons, S. neurona schizonts and merozoites were confirmed by immunohistochemical staining with S. neurona-specific polyclonal antibodies. Viable S. neurona was isolated from the brains of two raccoons by bioassay in interferon gamma gene knockout mice and in cell cultures seeded directly with raccoon brain homogenate. Molecular characterization was based on raccoon no. 68. Molecular characterization based on multi-locus typing at five surface antigens (SnSAG1-5-6, SnSAG3 and SnSAG4) and the ITS-1 marker within the ssrRNA locus, using DNA isolated from bradyzoites released from sarcocysts in a naturally infected raccoon (no. 68), confirmed the presence of S. neurona antigen type I, the same genotype that caused a mass mortality event in which 40 southern sea otters stranded dead or dying within a 3 week period in April 2004 with S. neurona-associated disease. An expanded set of genotyping markers was next applied. This study reports the following new genotyping markers at 18S rRNA, 28S rRNA, COX1, ITS-1, RON1, RON2, GAPDH1, ROP20, SAG2, SnSRS21 and TUBA1 markers. The identity of Sarcocystis spp. infecting raccoons is discussed.

First report of Sarcocystis halieti (Apicomplexa) in bearded vulture (Gypaetus barbatus)

At least three Sarcocystis species (S. falcatula, S. halieti and S. wobeseri–like) have been detected infecting raptorial birds. By histopathology and PCR-sequencing of the ITS1 marker, S. halieti was detected in a bearded vulture (Gypaetus barbatus) and a black kite (Milvus migrans) from the Catalonia region in North Spain. The 241 bp-long sequences obtained from the Sarcocystis organisms detected in both raptors showed 97.5–99.6% and 97.9–100% similarity with those of previously identified S. halieti; also, the phylogenetic trees generated placed the identified sequences together with other sequences of S. halieti available in GenBank. In sum, the description of the bearded vulture as a new intermediate host for S. halieti adds new insights on the complex epidemiology of the genus involving avian hosts.

Pollen metabarcoding of museum specimens and recently collected bumblebees (Bombus) indicates foraging shifts

Landscape changes, over time, lead to changes of floral resources available to pollinators, which in turn may result in the disappearance of ecologically specialized species. Here, we use pollen metabarcoding to infer historic and recent interactions between plants and bumblebees (Bombus). Bumblebees from Cuxhaven (Germany) were sampled from historical museum collections (1968/69) and in the field (2019). Pollen attached to their bodies was barcoded using multiple plant markers (ITS1, ITS2 and trnL-P6 loop). Our results show shifts in foraging habits between the historic and recent sampling periods, mostly determined by fewer Fabaceae interactions in 2019. The successful implementation of scalable molecular techniques for the analysis of historical pollen samples underscores the value of museum collections as a resource for biodiversity research. This study provides proof of concept of a comparative analysis of recent and historical pollination data. However, to ensure the robustness of our results, it is crucial to consider the broader methodology used. Our study found variation in the efficacy of the three plant barcoding markers. The ITS1 marker exhibited the highest species-level identification success, while the trnL-P6 loop demonstrated utility in amplifying degraded DNA across diverse plant families.

Metataxonomic and metabolic evaluation of three water kefir microbiomes cultured in sugar cane juice

AbstractThree water kefir grains (WKG) C, B, M with higher heterogeneity in the consortia C and M, and different biodiversity, dominated mainly by the genus Lactobacillus were selected from three Colombian locations and evaluated metataxonomically and metabolically using sugar cane juice (SCJ) as a culture medium, the molecular characterization was performed by sequencing in Illumina Miseq, and microbial diversity used Mothur platforms. The objective was to evaluate the SCJ on the consortium and the bacterial and fungal diversity immersed in the WKG. Biomass and secondary metabolites were monitored during 152 h. Growth kinetic data were fitted to the Logistic, Richards, and Gompertz models. WKG C rRNA 16S sequencing by Simpson's index (0.298) evidenced low species biodiversity, dominated by Lactobacillus sp. In C (0.986), the ITS1 marker indicated a more heterogeneous community than B and M, with Saccharomyces sp. as the predominating species. Parameters of the Gompertz model fitted to C biomass were: μmax (0.032 h−1), latency time (1.736 h), and adaptation (6.48 h). WKG consortia B showed higher acetic acid and ethanol production with 5.269 and 0.333 g/L, respectively. SCJ is a suitable medium for Lactobacillus sp. and Saccharomyces sp. strains in WKG.

Molecular identification of Trypanosoma theileri in cattle from the Ecuadorian Amazon.

Trypanosoma theileri is a cosmopolitan opportunistic haemoparasite described in wild and domestic ruminants, and also in arthropod vectors. The presence of this parasite has been reported in several South American countries, including Amazonian regions. Despite the importance of livestock production, Ecuador possesses scarce studies about trypanosomosis and no T. theileri reports in its territory. Here, we showed molecular evidences of the presence of T. theileri in cattle from a province located in the Ecuadorian Amazon. Bovine blood samples were collected from 2014 to 2019, during campaigns to detect haemoparasites in the Ecuadorian provinces of Orellana and Sucumbíos. DNA was extracted from the buffy coat and used in PCR assays with three different molecular markers, ITS1, 18S and Cathepsin L-like. T. theileri was detected only in the Sucumbíos province, with a specific molecular prevalence of 8.6% (3/35) using the three primers and an additional animal detected as positive (11.4% prevalence) only by the ITS1 marker. DNA sequences derived from the generated amplicons were subjected to phylogenetics maximum parsimony and maximum likelihood analysis, which indicate the presence of TthI and TthII genotypes circulating in the evaluated animals. Molecular surveillance should be continually implemented in Ecuador in order to deepen the epidemiological and evolutionary knowledge about T. theileri as well other haemoparasites in the amazon parts of the country.

Long-term fungus–plant covariation from multi-site sedimentary ancient DNA metabarcoding

Climate change has a major impact on arctic and boreal terrestrial ecosystems as warming leads to northward treeline shifts, inducing consequences for heterotrophic organisms associated with the plant taxa. To unravel ecological dependencies, we address how long-term climatic changes have shaped the co-occurrence of plants and fungi across selected sites in Siberia.We investigated sedimentary ancient DNA from five lakes spanning the last 47,000 years, using the ITS1 marker for fungi and the chloroplast P6 loop marker for vegetation metabarcoding. We obtained 706 unique fungal operational taxonomic units (OTUs) and 243 taxa for the plants. We show higher OTU numbers in dry forest tundra as well as boreal forests compared to wet southern tundra. The most abundant fungal taxa in our dataset are Pseudeurotiaceae, Mortierella, Sordariomyceta, Exophiala, Oidiodendron, Protoventuria, Candida vartiovaarae, Pseudeurotium, Gryganskiella fimbricystis, and Trichosporiella cerebriformis. The overall fungal composition is explained by the plant composition as revealed by redundancy analysis. The fungal functional groups show antagonistic relationships in their climate susceptibility. The advance of woody taxa in response to past warming led to an increase in the abundance of mycorrhizae, lichens, and parasites, while yeast and saprotroph distribution declined. We also show co-occurrences between Salicaceae, Larix, and Alnus and their associated pathogens and detect higher mycorrhizal fungus diversity with the presence of Pinaceae. Under future warming, we can expect feedbacks between fungus composition and plant diversity changes which will affect forest advance, species diversity, and ecosystem stability in arctic regions.

Uncovering Hidden Diversity: Three New Species of the Keratella Genus (Rotifera, Monogononta, Brachionidae) of High Altitude Water Systems from Central Mexico

The correct identification of species is an essential step before any study on biodiversity, ecology or genetics. Keratella is a genus with a predominantly temperate distribution and with several species being endemics or restricted geographically. Its diversity may be underestimated considering the confusing taxonomy of species complexes such as K. cochlearis. In this study, we examined genetic diversity and morphology among some Keratella populations from Mexico in order to determine if these populations represent different species. We analyzed a dataset of previously published and newly generated sequences of the mitochondrial COI gene and the nuclear ITS1 marker. We conducted phylogenetic analyses and applied three methods of species delimitation (ABGD, PTP and GMYC) to identify evolutionary significant units (ESUs) equivalent to species. Morphological analyses were conducted through scanning electron microscope (SEM) and morphometry under a compound microscope. In the present study, three new species Keratella cuitzeiensis sp. nov., Keratella huapanguensis sp. nov., and Keratella albertae sp. nov., are formally described. These species were collected in high-altitude water bodies located in the Central Plateau of Mexico. Combining DNA results through COI and ITS1 molecular markers and morphology it was possible to confirm the identity of the new species.

Morphological and Molecular Description of a New Species Haematoloechus dehradunensis sp. nov. (Plagiorchiidae: Haematoloechinae) from the Lungs of Euphlyctis cyanophlyctis (Anura, Dicroglossidae) from Dehradun, along with a Note on H. singaporensis Yuen, 1962, a New Record to India.

Morphological and molecular description of a new species of Haematoloechus dehradunensis sp. nov. collected from the lungs of Euphlyctis cyanophlyctis (Schneider 1799) from Dehradun (Uttarakhand), India and reporting first record of H. singaporensis from India. Digeneans were fixed in AFA (alcohol-formalin-acetic acid), stained with Borax's carmine, studied and photomicrographed with a BX53 DIC/BF Olympus research microscope. Molecular studies were done by DNA isolation using Qiagen, DNeasy® Blood and Tissue Kit and PCR amplification using r-DNA ITS-1 marker situated between 18S and 1.58S gene. The new species is differentiated from other known species of Haematoloechus in having larger oral sucker, kidney-shaped ovary and oval-lobed testes. H. singaporensis collected from E. cyanophlyctis represents a first record for India and a new host record. ITS sequences submitted and compared at NCBI GenBank support the uniqueness of the new species.

A tale of textiles: Genetic characterization of historical paper mulberry barkcloth from Oceania.

Humans introduced paper mulberry (Broussonetia papyrifera) from Taiwan into the Pacific over 5000 years ago as a fiber source to make barkcloth textiles that were, and still are, important cultural artifacts throughout the Pacific. We have used B. papyrifera, a species closely associated to humans, as a proxy to understand the human settlement of the Pacific Islands. We report the first genetic analysis of paper mulberry textiles from historical and archaeological contexts (200 to 50 years before present) and compare our results with genetic data obtained from contemporary and herbarium paper mulberry samples. Following stringent ancient DNA protocols, we extracted DNA from 13 barkcloth textiles. We confirmed that the fiber source is paper mulberry in nine of the 13 textiles studied using the nuclear ITS-1 marker and by statistical estimates. We detected high genetic diversity in historical Pacific paper mulberry barkcloth with a set of ten microsatellites, showing new alleles and specific genetic patterns. These genetic signatures allow tracing connections to plants from the Asian homeland, Near and Remote Oceania, establishing links not observed previously (using the same genetic tools) in extant plants or herbaria samples. These results show that historic barkcloth textiles are cultural materials amenable to genetic analysis to reveal human history and that these artifacts may harbor evidence of greater genetic diversity in Pacific B. papyrifera in the past.

Brachionus paranguensis sp. nov. (Rotifera, Monogononta), a member of the L group of the Brachionus plicatilis complex.

The Brachionus plicatilis complex represents the most studied group of rotifers, although the systematics of the species complex has not been completely clarified. Many studies have been conducted trying to explore the diversity within the complex, leading to the recognition of three major morphotypes: large (L), small-medium (SM), and small (SS). Currently six species have been described and classified under these types and another nine taxa have been identified but not formally described. Within the L group, three species have been officially described [B. plicatilis s.s. (L1), B. manjavacas (L2), and B. asplanchnoidis (L3)], while a formal description of L4, unofficially known as B. ‘Nevada’, is still lacking. In the present study, a new species, Brachionus paranguensissp. nov., is formally described and presented as a representative of the L4 clade. The species has been named after a high altitude saline crater lake from Central Mexico, where the specimens were collected. An integrated approach using DNA taxonomy through COI and ITS1 markers, morphology, and ecology was used to confirm the identity of the new species.

Atypical fatal sarcocystosis associated with Sarcocystis neurona in a White-nosed coati (Nasua narica molaris)

The protozoan parasite Sarcocystis neurona is an important cause of disease in horses (equine protozoal myeloencephalitis, EPM) and marine mammals. Isolated reports of clinical EPM-like disease have been documented in a zebra, raccoon, domestic cat, domestic dog, ferret, skunk, mink, lynx, red panda and fisher. The predominant disease is encephalomyelitis associated with schizonts in neural tissues. Here, we report highly disseminated sarcocystosis, in many tissues of a captive White-nosed coati (Nasua narica molaris). The 14year old, neutered male coati was euthanized due to progressive weakness, lethargy, and inappetence. Schizonts, including free and intracellular merozoites were detected in many cell types, and differed morphologically from S. neurona schizonts in horses. Only a few sarcocysts were seen in skeletal muscle and the myocardium. Immunohistochemically, the protozoa reacted positively to S. neurona but not to Toxoplasma gondii antibodies. Severe inflammtory disease detected in the stomach, intestine, adrenal and thyroid glands, ciliary body of eye, and urinary bladder associated with schizonts in the coati has not been reported earlier in any host with EPM. Although, a few schizonts were found in the brain, encephalitis was minimal and not the cause of clinical signs. Multilocus PCR-DNA sequencing using DNA derived from the coati lung tissue identified an S. neurona infection using the 18S, 28S and ITS-1 markers, and a novel genotype using primer pairs against antigenic surface proteins (SnSAG3, SnSAG4, SnSAG1-5-6) and microsatellite markers (MS, SN7, SN9). Although the genotype was similar to the widely distributed Type VI strain, it possessed a novel allele at SnSAG5, and a different MS combination of repeats at SN7 and SN9. Whether this severe parasitism was related to the host or the parasite needs further investigation.

Spermatogenic and Phylo-molecular Characterizations of Isolated Fasciola Spp. From Cattle, North West Iran.

Fascioliasis is economically important to the livestock industry that caused with Fasciola hepatica and Fasciola gigantica. The objective of this study was to identify these two species F. hepatica and F. gigantica by using nuclear and mitochondrial markers (ITS1, ND1 and CO1) and have been employed to analyze intraspecific phylogenetic relations of Fasciola spp. Approximately 150 Fasciola specimens were collected, then stained with haematoxylin-carmine dye and observed under an optical microscope to examine for the existence of sperm. The ITS1 marker was used to identify different Fasciola and phylogenetic analysis based on ND1 and CO1 sequence data were conducted by maximum likelihood algorithm. Fasciola samples were separated into 2 groups. Almost all specimens had many sperms in the seminal vesicle (spermic fluke) and one fluke did not contain any sperm in the seminal vesicle. The aspermic sample had F. gigantica RFLP pattern with ITS1 gene. Phylogenetic analysis based on NDI and COI sequence data were conducted by maximum likelihood showed a similar topology of the trees obtained particularly for F. hepatica and F. gigantica. This study demonstrated that aspermic Fasciola found in this region of Iran has same genetic structures through the spermic F. gigantica populations in accordance to phylogenetic tree.

First record and description of juvenile stages of Longidorus artemisiae Rubtsova, Chizhov & Subbotin, 1999 (Nematoda: Longidoridae) in Poland and new data on L. juglandicola Li\u0161kov\xe1, Robbins & Brown, 1997 based on topotype specimens from Slovakia

This paper presents the first geographical record of the needle nematode Longidorus artemisiae Rubtsova, Chizhov & Subbotin, 1999 outside Russia. This species was found in Poland near the city of Skierniewice in association with nettle (Urtica dioica L.). Morphometric and morphological data are provided, including the first description of juveniles of this species. Nematodes of the Polish population differ from the type-population in Russia in possessing a thicker body (lower ‘a’ index) in both sexes; males having a longer body and longer spicules; different sex ratio (1:2 in Polish population vs 1:1 in the type-population) and somewhat less expanded lips. Molecularly, the Polish population was characterised by sequencing D2-D3 28S rDNA and ITS1 markers. Additionally, new data on these two markers are provided for another species, Longidorus juglandicola Lišková, Robbins & Brown, 1997, obtained from topotype specimens from Slovakia. Surprisingly, despite the high morphological similarity of these two species, analysis of their phylogenetic position did not show close phylogenetic relation and several other species (less similar in general morphology) appeared more closely related to both L. artemisiae and L. juglandicola.

Comparative Evaluation of Molecular Detection Performance of Pseudomonas aeruginosa based on Phylogenetic Markers 16S RNAr, recA, rpoB and ITS1

P. aeruginosa may be involved in the poisoning of food. It is highly pathogenic to immunocompromised subjects or weakened, causing a high rate of morbidity and mortality. The aim of this study was to determine the phylogenetic marker suitable for molecular identification of Pseudomonas aeruginosa. The purity and concentration of the nucleic acids were determined by spectrophotometry. Sensitivity reactions using phylogenetic markers (16S RNAr, recA, rpoB, STS1) and the threshold detection of 42 strains were assessed by polymerase chain reaction (PCR). With an average absorption at 230 nm of 2.1, the DNA extracts has an average ratio (A260/A280) of 1.7. The threshold detection of Pseudomonas aeruginosa reference strain ATCC 27853 was 0.8 μg/ml for rpoB and 7.6 μg/ml for each of 16S markers RNAr and recA. The threshold detection of positive control strains CP2: 1125A and CP3: API was 1.2 μg/ml and 0.1 μg/ml for using rpoB gene, respectively. This threshold was respectively 12.3 μg/ml and 0.9 μg/ml for the recA gene. The sensitivity of the rpoB housekeeping gene was 97.4% followed by the recA and 16S RNAr with 87.2% and 82.1%, respectively. The phylogenetic resolution of the rpoB genes was higher than that of the 16S rRNA and recA genes. No sensitivity reaction was observed with ITS1 marker. The quality, purity of the nucleic acids and the choice of phylogenetic marker are among the most critical factors for PCR analysis.

Genetic diversity and reproductive mode in two non-native hydromedusae, Maeotias marginata and Moerisia sp., in the upper San Francisco Estuary, California

Reproductive strategy can play a significant role in invasion success and spread. Asexual and sexual reproduction may confer different advantages and disadvantages to a founding population, resulting in varying impacts on genetic diversity and the ability to invade. We investigate the role of reproductive mode in two species of non-native hydromedusae (Maeotias marginata and Moerisia sp.) in the San Francisco Estuary (SFE). Both species can reproduce asexually and sexually. We employed 7–8 microsatellite markers to determine overall genetic diversity and to investigate contributions of asexual and sexual reproduction to the populations. We found both species had high levels of genetic diversity (Average HE = 0.63 and 0.58, Number individuals sampled = 111 and 277, for M. marginata and Moerisia sp. respectively) but also detected multiple individuals in clonal lineages. We identified the same clones across sampling locations and time, and the index of asexual reproduction (R) was 0.89 for M. marginata and 0.91 for Moerisia sp. Our results suggest both species maintain high population genetic diversity through sexual reproduction, in combination with asexual reproduction, which allows rapid propagation. In addition, we conducted genetic sequence analyses at the ribosomal ITS1 marker, using samples of Moerisia sp. from the SFE and M. lyonsi from Chesapeake Bay. We found 100 % sequence similarity showing that Moerisia sp. in the SFE and Chesapeake Bay are the same species. The two hydromedusae studied here possess the means to propagate rapidly and have high genetic diversity, both of which may allow them to successfully adapt to changing environments and expand their invasions.

Sequence variation in mitochondrialATPsynthase subunit 6 & 8and nuclear genesITS1andITS2in 17 cyprinid species

Sequence variation for the mitochondrial ATP synthase subunit 6 & 8 and the nuclear ITS1 marker was investigated for 17 cyprinid fish species in order to increase the number of possible loci for character sampling. The mitochondrial locus provided appropriate information for further phylogenetic studies, which can be readily applied to other taxa; the nuclear ITS1, however, cannot be recommended for inference of relationships among different taxa. No congruence was obtained between the trees resulting from the mitochondrial and nuclear gene using Bayesian Inference.

Genetic differentiation of Atlantic populations of the intertidal copepod <em>Tigriopus brevicornis</em>

The Harpacticoid copepod Tigriopus brevicornis belongs to the meiofauna of intertidal rock pools and is distributed widely along European coasts. Sixteen sites were sampled from the Irish Sea to the coasts of Spain. We used the ITS1 marker to analyse the relationship between the populations because it shows low intrapopulational variation (mean pairwise difference: 1.00 ± 0.8) and high interpopulational divergence (mean pairwise difference: 16.38 ± 7.39). A total of 57 bp out of 433 bp were recognised as informative nucleotides among the 61 individuals analysed. The analysis of the genetic relationships highlighted a north-south split in the distribution of the natural populations and showed a genetic break point around the Gironde estuary, which is probably due to differences in the geomorphologic characteristics of the coastal area on the two different sides of this estuary. Various populations were isolated and the ITS1 sequences indicated that there are specific genetic signatures in these populations. The northern set of populations, which was sampled along a large rocky coastline, had a metapopulation structure with genetic exchanges between geographically close populations and also between geographically far ones. The southern set of populations, which was sampled in small rocky pools on large sandy beaches, showed isolated populations as a consequence of the geomorphology of the area.

Etudes interlaboratoires sur les espèces du complexe Obsoletus

Culicoides species are difficult to identify but, recently, molecu­lar identification tools based on cytochrome oxidase subunit 1 (CO1), internal transcribed spacer 1 or 2 (ITS-1 or ITS-2) have been developed for Palaearctic species including vector spe­cies. In the framework of Medreonet network, the workpackage on “Regional surveillance of vectors” includes guidelines on Palaearctic and Mediterranean Culicoides species identification using molecular tools. Three ring trials were carried out to test whether the methods and tools used in the main laboratories of Europe were adequate and able to identify species correctly. Moreover, a questionnaire was sent to each laboratory to record the protocols and tools used for molecular identification. The four species of the Obsoletus complex, C. obsoletus, C. scoticus, C. chiopterus, and C. dewulfi were selected for the ring trial. All specimens were first morphologically identified by experts. The first ring trial containing eight specimens (four males and four females) of the four species was sent on the 4th of August 2008 to 13 different participating laboratories from seven coun­tries. Two types of material were sent: extracted deoxyribonucleic acid (DNA) (four samples/species) and phosphate-buffered saline (PBS)-ground biting midges (34 samples/species). Eventually, two laboratories did not participate. Only two out of the 11 partici­pating laboratories correctly identified the species. The molecu­lar identification used was based on ITS-1 and ITS-2 markers. A second ring trial was sent in April 2009 with only extracted DNA samples to avoid the different extraction steps which could interfere with tool sensitivity. A total of 20 extracted DNA sam­ples were sent to 10 participating laboratories. Four out of the 10 correctly identified the species. Three laboratories used methods based on ITS-2 or ITS-1 markers, and one laboratory used CO1. Finally, a third ring trial with 20 PBS-ground samples was sent in September 2009 to 10 laboratories. Only one laboratory cor­rectly identified the species. Sensitivity and specificity of the molecular tools used are discussed.