Cytochrome Oxidase Subunit I Region Research Articles

Detection and incidence of pineapple heart rot disease caused by Phytophthora nicotianae in commercial farms of Puerto Rico

Pineapple heart rot disease (PHRD) caused by Phytophthora spp. is one of the most important diseases of pineapple. This disease is characterized by soft rot and water-soaked lesions. In severe cases plant mortality is 100%. During a one-year survey, conducted from March 2018 to March 2019, a total of 29 pineapple hectares were evaluated from five commercial pineapple fields located in the Puerto Rican municipalities of Guánica, Lajas, Manatí and Santa Isabel, and at the Agricultural Experiment Substation of the University of Puerto Rico in Isabela. Symptoms of PHRD were observed in all fields evaluated, except at the Agricultural Experiment Substation in Isabela. Diseased plant tissue was surface disinfected and plated on PARPH-V8 selective media for Phytophthora spp. Aerial photography using a DJI Phantom 3 drone was used to corroborate PHRD incidence in the field. Approximately eight hectares were infected by P. nicotianae with an average of PHRD disease incidence of 28.82%. The highest percentage of PHRD incidence was observed in fields located at Guánica, Lajas and Manatí with 40%, 40% and 30%, respectively. Eleven isolates of Phytophthora nicotianae were collected from pineapple fields in Guánica, Lajas, Manatí and Santa Isabel and identified using morphology and phylogeny of sequences of the mitochondrial cytochrome oxidase subunit I region (COI). The estimation of the incidence of P. nicotianae as the causal agent of PHRD is important as a first step in developing specific control measures in the pineapple fields of Puerto Rico.

DNA Barcodingof Commercial Fish Sold in MuarFish Market, Johor

Due to the high demand in the fisheries market, species identification is vital for commercial fish to avoid mislabeling and fraud in marketplaces. The increasingly threatening human activities like overfishing, blast fishing, poison fishing, and trawling have become the major threats to fish and caused an urgent need for documentation of both marine and freshwater fishes. Commercial fish products sometimes are sold by their parts, such as fins which further create confusion for identification purposes.Morphological identification also requires highly skilled ichthyologists to avoid misidentification. Thus, identification through DNA barcoding can be utilized as it holds the potential for accurate and rapid identification. Here we identified the commercial marine fish species sold in Muar Fish Market, Johor, using the Cytochrome Oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA). A 700 base-pair sequence of the COI region was targeted by amplifying extracted DNA from 28 fin samples collected from the fish market. From 28 samples, we successfully sequenced 16 samples, and by using phylogenetic analysis, (Neighbor-Joining (NJ), Maximum Parsimony (MP) and Bayesian Inference (BI)) we successfully identified the samples belonging to nine families (Scombridae, Platycephalidae, Carangidae, Ariidae, Polynemidae, Coryphaenidae, Sphyraenidae, Muraenesocidae, Engraiulidae), 12 genera and 16 species. This study reports the first documentation of commercial fish sold in Muar through the DNA barcoding technique. This technique should be expanded further to identify any species with important conservation implications, such as endangered species, for improved management of fisheries in Malaysia

Lack of genetic differentiation of Kawakawa Euthynnus affinis (Cantor, 1849) in Peninsular Malaysia based on mitochondrial DNA cytochrome oxidase I

Kawakawa Euthynnus affinis is a cosmopolitan epipelagic tuna and is regarded as one of the most commercially important tunas in the subtropical and tropical waters of Indo-Pacific region. Regrettably, a lack of information on the stock structure management and conservation of E. affinis populations in Malaysian waters could result in overfishing and perhaps decreasing the stock structure of this marine fish. Thus, for an effective and successful management of the species, an assessment of stock structure based on molecular knowledge is essential. In this current research, genetic differentiation was assessed utilizing sequence analyses of the mitochondrial cytochrome oxidase subunit I (COI) gene. A 632 bp segment of the COI region was sequenced in 114 individuals of E. affinis sampled from two central geographic regions; the Straits of Malacca (M), and the South China Sea (SCS). The results revealed that the genetic distance within and between seven populations of E. affinis from Peninsular Malaysia was extremely low. The overall mean of haplotype diversity (Hd) was 0.6942, while the average nucleotide diversity (π) was 0.0011. The neighbor-joining (NJ) tree revealed that the samples of E. affinis collected from Peninsular Malaysia were clustered into one clade. The outcomes of this study will provide a valuable baseline of knowledge for proper management and conservation of this neritic tuna in Malaysian waters.

Molecular phylogeny of the genus Chondrina (Gastropoda, Panpulmonata, Chondrinidae) in the Iberian Peninsula

Chondrina Reichenbach, 1828 is a highly diverse genus of terrestrial molluscs currently including 44 species with about 28 subspecific taxa. It is distributed through North Africa, central and southern Europe, from Portugal in the West to the Caucasus and Asia Minor in the East. Approximately 70% of the species are endemic to the Iberian Peninsula constituting its main center of speciation with 34 species. This genus includes many microendemic taxa, some of them not yet described, confined to limestone habitats (being strictly rock-dwelling species). They are distributed on rocky outcrops up to 2000 m.a.s.l. It is a genus of conical-fusiform snails that differ mainly in shell characters and in the number and position of teeth in their aperture. So far, molecular studies on Chondrina have been based exclusively on the mitochondrial Cytochrome Oxidase subunit I region (COI). These studies gave a first view of the phylogeny of the genus but many inner nodes were not statistically supported.The main objective of the study is to obtain a better understanding of the phylogeny and systematics of the genus Chondrina on the Iberian Peninsula, using multilocus molecular analysis. Partial sequences of the COI and 16S rRNA genes, as well as of the nuclear Internal Transcribed Spacer 1 (ITS1-5.8S) and Internal Transcribed Spacer 2 (5.8S-ITS2-28S) were obtained from individuals of all the extant Chondrina species known from the Iberian Peninsula. In addition to this, the newly obtained COI sequences were combined with those previously published in the GenBank. Phylogenetic relationships were inferred using maximum likelihood and Bayesian methods. The reconstructed phylogenies showed high values of support for more recent branches and basal nodes. Moreover, molecular species delimitation allowed to better definethe studied species and check the presence of new taxa.

The first genetic characterization of Setaria marshalli (Nematoda, Spirurida) with reliable DNA barcoding based on a mitochondrial genetic marker.

Setaria marshalli is a mosquito-borne filarial nematode that causes infection in calves younger than two years old. In the present study, nematodes were obtained from a calf in Japan and morphologically identified as S. marshalli. Additionally, the partial cytochrome oxidase subunit I (COI) region (596 bp) was analyzed for the first time to establish a reliable DNA barcode. Nucleotide sequences of COI were identical among the seven worms obtained. The COI region can be a useful marker for species discrimination in the case of S. marshalli since nucleotide variations observed between the closest congener, Setaria cervi (51/596 bp), were sufficient to allow species discrimination. However, the phylogenetic relationship of S. marshalli with its congeners was unclear in a maximum likelihood tree. We found that the partial COI sequence of S. marshalli analyzed in the present study matched a relevant section of the complete mitochondrial genome of S. labiatopapillosa that was deposited in the International Nucleotide Sequence Database. This finding suggests that S. marshalli was misdiagnosed as S. labiatopapillosa in a previous study. It is crucial to conduct accurate morphological analyses to obtain reliable molecular information regarding Setaria nematodes.

Molecular characterization and phylogenetic analysis of collected mosquitoes (Diptera: Culicidae) from Northcentral Nigeria using mitochondrial COI and ribosomal IGS gene regions

BackgroundMosquitoes are important vectors of disease-causing organisms such as filarial worms, malaria parasites, and arboviruses endemic to sub-Saharan Africa including Nigeria. Malaria is a disease caused by a plasmodium parasite, transmitted by the bite of infected mosquitoes and is no doubt a public health concern. There is limited information on the genetic diversity of mosquitoes in Nigeria. This is necessary because information about the genetic diversity of mosquitoes is a very important step towards vector control and management with the aim to mitigate or eliminate burden resulting from malaria and other diseases caused by mosquitoes. In the present study, we investigated the genetic variability and relatedness of mosquitoes based on the DNA sequences of the mitochondrial cytochrome oxidase subunit I (COI) and ribosomal intergenic non-coding spacer gene regions (IGS). Mosquitoes were collected from five different states in Northcentral Nigeria, they were morphologically identified using standard keys and genomic DNA was extracted. The specific regions of interests were amplified, and the PCR products were then sequenced.ResultsPCR was able to successfully amplify the expected amplicon sizes of COI and IGS sequences (710 and 169) base pairs, respectively. For COI sequence, pairwise genetic distance between mosquito species ranged from 0.00 to 0.17 in the COI sequences. The pairwise genetic distance among Culex, Aedes and Anopheles species in the IGS sequences ranged from 0.000 to 0.118. Phylogenetic analysis of the sequences of the mitochondrial cytochrome oxidase subunit showed that there was genetic diversity amongst the different mosquito species sampled. It effectively showed marked differences between Culicine and Anopheline mosquitoes.ConclusionsThe ribosomal IGS primers used for this study only amplified Anopheles spp. However, it revealed that there is diversity among the Anopheles gambiae and Anopheles arabiensis samples collected. This study concludes that the mitochondrial COI and ribosomal IGS gene regions are reliable markers for mosquito genetic diversity study and will surely yield a reliable result for molecular diversity assessment of mosquito species.

Phylogeography of Hyalomma (Euhyalomma) lusitanicum (Acarina, Parasitiformes, Ixodidae) in Andalusia based on mitochondrial cytochrome oxidase I gene

The genetic population structure relationships of Hyalomma (Euhyalomma) lusitanicum in Andalusia (the south of the Iberian Peninsula) were examined using mtDNA sequence data from 887 bp of cytochrome oxidase subunit I (COI) gene. The sequence for the COI region was determined for 84 individuals collected in several localities of Andalusia, and 10 for other localities (i.e., five from Toledo, central Iberian Peninsula, four from Sicily (Italy) and one from Canary Island). Seventeen haplotypes were detected, including 27 polymorphic sites. The number of amino acid substitutions per site from mean diversity calculations for the entire population was 0.017. AMOVA analysis revealed a low gene flow that characterises the genetic population structure of this species in South Iberian Peninsula, with a haplotype diversity (h) value of 0.815. No geographically induced differentiation was observed, and separate evolutionary units were not detected. Our results indicate low genetic diversity across the geographical range of H. lusitanicum tick in Andalusia. Our data do not show any genetic discontinuity between the tick populations studied, including specimens from Canary Island and Sicily (Italy).

Molecular characterization of viruses found in honeybee (Apis mellifera) colonies infested with Varroa destructor and Nosema cerana in Egypt

Honey bees (Apis mellifera) have a vital role as pollinators of various crops in the global food supply. Honeybee colonies in Egypt have recently experienced an unexplained rise in annual loss due to a phenomenon known as Colony Collapse Disorder (CCD). In the current study, honey bees were collected from 14 sites from eight governorates in Egypt. The genetic diversity among the collected honey bee populations was investigated using the mitochondrial DNA cytochrome oxidase subunit I (COI). The amplified COI regions were sequenced, analyzed and aligned with other GenBank entries. The nucleotide variability of the CO1 gene was estimated. Multiple viral, varroa mites as well as Nosema ceranae infections were tested in honey bee populations using conventional and RT-qPCR. Based on sequence analysis of the COI, six clearly separated mitotypes were characterized for the first time in these sites in Egypt. Sequence analyses showed that most mitotypes belonged to the A lineage and are very close to the Egyptian native bees, A. m. lamarckii found in the gene databank (NCBI) with 98% similarity. Low genetic diversity between the collected samples was observed. Our results elucidated the detection of Nosema cerana; deformed wing virus (DWV), kakugo virus (KV), black queen/cell virus (BQCV), Israel acute paralysis virus (IAPV), varroa destructor virus-1 (VDV-1) and VDV-1/DWV virus in all regions under investigation in addition to varroa mites. These findings highlighted the importance to maintain proper quarantine measures as well as identify the spectrum of exogenous infectious agents in healthy hives over time which would help in developing more effective control and treatment programs against honey bee viruses and pathogens to facilitate efficient breeding programs and establish a more booming beekeeping industry.

Molecular Identification and Phylogenetic Tree Reconstruction of Marine Fish from the Essential Wetland Area of Banyuwangi, Indonesia

Pangpang Bay is necessary to get serious attention because it is one of the essential wetlands in Banyuwangi, Indonesia. This area able to generate income for traditional fishers and ecological supports the surrounding area because it is possible to be a spawning area and nursery ground for many estuary fish. The identification of fisheries resources is carried out using a molecular approach in the Cytochrome oxidase subunit I (COI) region that is commonly used in barcoding. Identification with this method is expected to minimize errors in determining the name of the species of species that exist in Pangpang Bay. Based on molecular identification information in the COI region, 37 marine fish species were distributed in 25 families, and 4 orders. The results of this study are the initials of molecular information on fisheries resources in the Pangpang Bay area.

The origin identification method for crude drugs derived from arthropods and annelids using molecular biological techniques.

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.

Molecular phylogeny and identification of the Egyptian wasps (Hymenoptera: Vespidae) based on COI mitochondrial gene sequences

The Hymenoptera is one of the vital and biggest insect orders comprising the bees, wasps, sawflies, and ants. Wasps are important to natural and biological pest control because they are predators or parasitoids of pest arthropods. This study investigated the genetic diversity among the three wasps, Vespa orientalis Linnaeus, Polistes bucharensis Erichson, and Polistes mongolicus du Buysson, collected from three different governorates in Egypt, using cytochrome oxidase subunit I (COI) DNA barcoding. PCR was performed to amplify COI fragment. The amplified COI regions (710 bp) were sequenced and analyzed. All novel nucleotide sequences of COI gene were deposited into the GenBank database. The genetic distances were estimated using Kimura two-parameter model. In spite of the wide geographical range, minor genetic diversity was observed between some populations of the three wasp species, revealing unrestricted gene flow between them. Phylogenetic relationship analysis was performed, using maximum likelihood (ML) method. The results of the phylogenetic analyses recovered P. bucharensis more closely related to P. dominula and P. gallicus. P. mongolicus collected from Menofia Governorate formed a distinct branch with 99% support. V. orientalis was sister to the yellowjacket Dolichovespula adulterine, with 84% support. It can be concluded that DNA barcode is a powerful tool for rapid and accurate identification of Egyptian wasp species.

Phylogenetic diversity of cassava green mite, <i>Mononychellus progresivus</i> from different geographical sites in east Africa

Cassava green mite (CGM) of the Mononychellus genus is an invasive species in Africa, introduced from South America. Its phylogenetic diversity over geographical localities has never been assessed in East Africa, where mite density dynamics oscillate from few individuals to a peak of hundreds. The objective of this study was to determine CGM species comparative phylogenetic diversity from seven distinct geographical sites in East Africa. Six sites were sampled for CGM races, two samples from each country. DNA was extracted on internal transcribed spacer 2 (ITS2) and cytochrome oxidase subunit I (COI), and compared for phylogenetic variations of CGM from different locations of East African region. A comparative search from the NCBI Gene bank resulted into identical species nucleotides from Congo and Benin. Sequences from the two sites in Kenya were 99-100% similar to CGM nucleotide from the Congo-Benin accessions (X79902.1) on ITS2 gene region. On COI, a 98- 99% site sequences similarity was observed on M. progresivus accession X79901.1. A closely related divergence of specimens collected from Tanzania and Uganda was determined. Both Uganda and Tanzania had 99% match to X79901.1 on COI region. Similarly, the Uganda and Tanzania samples had 99% match to emb/X79902.1 on the 18Sr RNA region. The CGM sequence from coastal Kenya had the highest phylogenetic divergence from the Congo-Benin sequences. A small biogeographic phylogenetic divergence (0-1%) was evident from the analyses among the six collection sites. The results confirm M. progresivus identity in East Africa it also indicates intraspecies phylogenetic variations on the COI gene region of interest. Key Words : Benin, Congo, Kenya, Mononychellus progresivus

Dual-target detection using simultaneous amplification of PCR in clarifying interaction between Opiinae species (Hymenoptera: Braconidae) associated with Bactrocera spp. (Diptera: Tephritidae) infesting several crops

Simultaneous polymerase chain reaction (PCR) by using multiplex PCR was conducted to clarify the interaction between Opiinae associated with Bactroceracarambolae, B. papayae, and B cucurbitae. We sequenced and characterized a dual-band target detected on Opiinae DNA fragments by using two combination pairs of universal primers on two molecular markers, namely cytochrome oxidase subunit I (COI) and cytochrome b. Additionally, each Bactrocera species was identified by amplifying the COI region. Sequence data obtained from multiplex PCR seemed very effective in confirming species-level distinction that has been shown in tree topology. We conducted phylogenetic analyses prior to clarification of the interaction among three taxa levels. Interestingly, the sequences obtained from the simultaneous PCR successfully differentiated between six closely related Opiinae species in three genera, which could potentially be mass-reared as biocontrol agents of bactroceran fruit flies that infest several species of fruit. We discovered, proved, and added molecular data to clarify the interaction between Opiinae parasitoids, their host (Bactrocera spp.), and associated plants species. Psyttalia fletcheri parasitizing Bactrocera cucurbitae, which infests the ridge gourd fruit, has been added as a new record from Malaysia. This information would be extremely useful in taxonomic identification of species as part of an effective method in biological control program of the targeted fruit fly pests and their associated crops.

PHYLOGENETIC ANALYSIS AND SEQUENCING OF THE MITOCHONDRIAL CYTOCHROME OXIDASE SUB UNIT I (COI) OF WHITE BACKED PLANT HOPPER, SOGATELLA FURCIFERA (HORVATH)

The family Delphacidae is the largest and most economically important one amongst the h oppers, Fulgoroide a . It has representatives in tropical and subtropical regions that occur in a variety of environments , and its known fossil records dates back to Jurassic period. Soga tella furcifera , the white backed plant hopper is a serious pest of ric e in many Asian countries. We have performed the phylogenetic reconstruction of the S . furcifera using the mitochondrial cytochrome oxidase subunit I ( COI ) gene. Phylogenetic reconstructions of the COI regions were done using Neighbor joining method. This study gives a clear p icture of the mitochondrial gen ome of the insect and gives the phylogenetics of S. furcifera which help us to develop a sus tainable and accurate strategy for management of the pest.

Molecular identification and phylogenetic analysis of economically important acaroid mites (Acari: Astigmata: Acaroidea) in Korea

Molecular diagnosis is highly valuable for the species identification of microscopic mites. Here, we collected some economically important mites of the superfamily Acaroidea from various stored products in Korea. Those nucleotide sequences of ribosomal second internal transcribed spacer (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) regions were determined by PCR using universial primers. In nucleotide sequence comparison at GenBank database, seven species including Rhizoglyphus robini, R. echinopus, Sancassania sp. Acarus siro, Tyrophagus putrescentiae, T. similis in Acaridae and Suidasia medanensis in Suidasiidae were identified. Particularly, COI sequences of R. robini, R. echinopus, Sancassania sp. and T. similis were firstly determined. Our results suggest that the phylogenetic relationships inferred from the ITS2 region, rather than COI region, were similar to those derived based on their morphological classification. Our study provides molecular information for the identification and phylogenetic relationship of acaroid mites in Korea.

Molecular characterization of the malaria vector Anopheles barbirostris van der Wulp in Sri Lanka.

BackgroundAnopheles barbirostris is a vector of malaria in Sri Lanka. The taxon exists as a species complex in the Southeast Asian region. Previous studies using molecular markers suggest that there are more than 4 distinct clades within the An. barbirostris complex in Southeast Asia. The present study characterizes Sri Lankan An. barbirostris using mtDNA cytochrome oxidase subunit I (COI) and ribosomal RNA internal transcribed spacer 2 (ITS2) gene sequences.FindingsDNA was extracted from morphologically identified An. barbirostris specimens from Sri Lanka, the COI and ITS2 regions amplified and their sequences analysed by comparison with other GenBank entries. Maximum likelihood trees suggested that Sri Lankan An. barbirostris constitute a different molecular type most closely related to clade I.ConclusionsConsidering the uncorrected p distances between the clade I and Sri Lankan specimens it is fair to assume that the specimens collected from widely separated locations in Sri Lanka with morphology characteristic of An. barbirostris s.l. form a new molecular type with close resemblance to An. barbirostris s.s from Indonesia and Thailand.

Morphological and genetic relationship of two closely-related giant water bugs:Appasus japonicus Vuillefroy andAppasus major Esaki (Heteroptera: Belostomatidae)

The East Asian giant water bug species Appasus japonicus Vuillefroy and Appasus major Esaki are aquatic hemipteran insects whose ranges overlap, particularly in the Japanese Archipelago and on the Korean Peninsula. In rare cases, the two species co-occur. Furthermore, they are very similar ecologically and also morphologically, making their identification extremely difficult, and the possibility of hybridization has also been suggested. In the present study, we re-examined their taxonomic validity, and the characteristics useful for identifying them. To re-examine the morphological traits useful for distinguishing these two species, 222 specimens of A. japonicus collected from Japan, Korea, and China, and 132 specimens of A. major from Japan and Korea, were examined. We also performed molecular phylogenetic analyses based on the mitochondrial DNA 16S rRNA and cytochrome oxidase subunit I (COI) regions and the nuclear DNA Histone 3 region. Although the two species are very similar ecologically and also morphologically, they showed significant genetic differentiation. Thus, there is likely some form of reproductive isolation acting between them. Major morphological characteristics overlap extensively between A. japonicus and A. major, and no particular trait was identified as being effective for differentiating these species. All the morphological characteristics examined overlapped between A. japonicus and A. major. However, a principal component analysis based on all of the morphological characteristics revealed that, despite the overlap between these species, it was possible to morphologically distinguish them. Therefore, a more accurate identification becomes possible using multiple characteristics rather than a single characteristic. The male genital paralobes, evaluated as the most useful morphological characteristic, was effective with 100% probability for the Japanese Appasus species. However, for the Asian (i.e. Korean) specimens, this characteristic was not useful. On the other hand, the results of molecular phylogenetic analyses based on the mitochondrial DNA 16S rRNA and COI regions and the nuclear DNA Histone 3 region clearly showed significant genetic differentiation between the two species. Notably, the results for the mitochondrial COI region strongly supported the independence of each monophyletic group (i.e. validity of each species). Therefore, DNA barcoding based on the mitochondrial DNA COI region is also considered useful for the identification of A. japonicus and A. major. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110, 615–643.

PCR identification of culicoid biting midges (Diptera, Ceratopogonidae) of the Obsoletus complex including putative vectors of bluetongue and Schmallenberg viruses

BackgroundBiting midges of the Obsoletus species complex of the ceratopogonid genus Culicoides were assumed to be the major vectors of bluetongue virus (BTV) in northern and central Europe during the 2006 outbreak of bluetongue disease (BT). Most recently, field specimens of the same group of species have also been shown to be infected with the newly emerged Schmallenberg virus (SBV) in Europe. A reliable identification of the cryptic species of this group is fundamental for both understanding the epidemiology of the diseases and for targeted vector control. In the absence of classical morphological characters unambiguously identifying the species, DNA sequence-based tests have been established for the distinction of selected species in some parts of Europe. Since specificity and sensitivity of these tests have been shown to be in need of improvement, an alternative PCR assay targeting the mitochondrial cytochrome oxidase subunit I (COI) gene was developed for the identification of the three Obsoletus complex species endemic to Germany (C. obsoletus, C. scoticus, C. chiopterus) plus the isomorphic species C. dewulfi.MethodsBiting midges of the genus Culicoides caught by UV light traps all over Germany were morphologically pre-identified to species or complex level. The COI region was amplified from their extracted DNA and sequenced. Final species assignment was done by sequence comparison to GenBank entries and to morphologically identified males. Species-specific consensus sequences were aligned and polymorphisms were utilized to design species-specific primers to PCR-identify specimens when combined with a universal primer.ResultsThe newly developed multiplex PCR assay was successfully tested on genetically defined Obsoletus complex material as well as on morphologically pre-identified field material. The intended major advantage of the assay as compared to other PCR approaches, namely the production of only one single characteristic band for each species, could be realized with high specificity and sensitivity.ConclusionTo elucidate the biological characteristics of potential vectors of disease agents, such as ecology, behaviour and vector competence, and the role of these haematophagous arthropods in the epidemiology of the diseases, simple, cost-effective and, most importantly, reliable identification techniques are necessary. The PCR assay presented will help to identify culicoid vector species and therefore add to bluetongue and Schmallenberg disease research including vector control and monitoring.

Mitochondrial DNA variability in Spanish populations ofA. italicusinferred from the analysis of a COI region

Key-words: Austropotamobius italicus, mtDNA, Cytochrome Oxidase Subunit I (COI), genetic variability, genetic structure, haplotype, conservation Austropotamobius italicus was once widely distributed throughout most of the country’s limestone basins in Spain. But its populations have shown a very strong decline over the last thirty years, due to different factors. Thus, the species now enjoys protection under regional, national and international legislation. Therefore, knowledge of the levels and patterns of distribution of genetic diversity in crayfish populations is critical when making conservation management decisions. In the present work, the current genetic structure of Spanish populations of white-clawed crayfish,A. italicus, was analyzed. Eleven Spanish populations and an Italian sample were studied through an 1184 bp-lentgh sequence of cytochrome oxidase subunit I mitochondrial gene. Data analysis revealed the existence of eight haplotypes in the Iberian Peninsula, the highest diversity reported to date in Spanish crayfish. Also a substantial genetic differentiation among populations was found, with a clear geographic pattern. The genetic variability found in these populations is similar to, and even higher, than that reported in previous studies on other Spanish and European populations of A. italicus. Thus, given the current risk status of the species across its range, this variability in certain populations offers some hope for the species from a management point of view.

Identification of astigmatid mites using ITS2 and COI regions

Identification of astigmatid mites based on their morphological characteristics is difficult because of the similarity of their organs, especially in immature mites. The ribosomal second internal transcribed spacer (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) regions are highly conserved in the eukaryotes and are usually used as barcodes. The ITS2 and COI regions of six species of astigmatid mites (Aleuroglyphus ovatus, Blomia tropicalis, Dermatophagoides farinae, Dermatophagoides pteronyssinus, Euroglyphus maynei, Tyrophagus putrescentiae) were obtained by polymerase chain reaction and sequenced. The lengths of the ITS2 sequences varied from 316 to 488 bp, while the COI regions were 377 or 378 bp long. Considering the ITS2 genes, the intraspecific genetic distance was in the range of 0.00-0.077844, whereas the interspecific genetic distance was 0.202426-0.912959. The values were 0.000-0.029748 and 0.138403-0.279304 for intra- and interspecific genetic distances when COI genes were used. The phylogenetic trees inferred from the ITS2 and the COI regions, by using maximum parsimony and neighbor-joining methods, were identical to those based on their morphological classification. Thus, the ITS2 and COI regions can be applied as barcodes to identify different species of astigmatid mites.