Haplotype diversity in medically important red scorpion (Scorpiones: Buthidae: Hottentotta tamulus) from India

The medically important Indian red scorpion, Hottentotta tamulus, is one of the most poisonous scorpions of Indian subcontinent. We studied the haplotype diversity in eight populations of H. tamulus based on mitochondrial cytochrome oxidase subunit I (COI) partial gene sequence. Analyses revealed 22 haplotypes with a haplotype diversity of 0.941 and nucleotide diversity of 0.023. For the first two codon positions both transition and transversion types of substitutions were equally likely and the test for neutrality was not rejected. However, codon substitution pattern indicated that the gene has experienced purifying selection. Model-based clustering method indicated that the eight populations form three groups that correspond to high, moderate and low rainfall areas, indicating that there is biogeographical separation of haplotypes. Populations from three groups formed distinct clades in maximum likelihood analysis and median joining genetic network and were statistically supported by low within group and high among group variation in analyses of molecular variance. We provide the first account of haplotype diversity in Indian red scorpions and their biogeographical separation.

Integrative taxonomy of commercially important deep water penaeoid shrimps from India

The deep water penaeoid shrimp is an important commercial crustacean resource along the Indian coast. The molecular and morphological information of this group from the Indian coast is scarcely known. In this study, we investigated the identification and phylogenetic relationships of the deep water penaeoid shrimps using three mitochondrial (cytochrome oxidase subunit I (COI), cytochrome b, 16S rRNA) genes, which were compared with 54 morphological characters and further used to evaluate character evolution. Our study revealed remarkable molecular divergence (3.3–33.0%) in nine species from three genera of Solenoceridae, four species from three genera of Penaeidae and one species from Aristeidae using COI. Phylogenetic analysis using maximum likelihood and Bayesian approaches revealed that all species from these families are monophyletic. The present analysis revealed the existence of subgroups in the genus Solenocera suggesting the slow reduction of postrostral carina which corresponds to the increase in distributional depth during the evolutionary process which further indicates the origin of the genus in the continental shelf and extending up to the continental slope. In addition, we generated the DNA barcode database involving these species which can help further to investigate the detailed evolution and biogeography of these valuable crustacean resources.

Zeylanicobdella arugamensis, the marine leech from cultured crimson snapper (Lutjanus erythropterus), Jerejak Island, Penang, Malaysia

Objective To investigate the prevalence, phylogenetics and DNA barcoding of Zeylanicobdella arugamensis (Z. arugamensis) from crimson snapper (Lutjanus erythropterus), Jerejak Island, Penang, Malaysia. Methods Experiment was conducted with 200 fish specimens of cultured Lutjanus erythropterus from Jerejak Island, Penang, Peninsular Malaysia. The water temperature and length for each fish were measured prior to parasites examination. Next, the morphological identification of parasites was performed. Genomic DNA from parasites was extracted for further molecular analysis. After PCR amplification, phylogenetic tree was constructed. The lowest Bayesian information criterion scores showed that the most compatible model is Tajima and Nei. Finally, data sets of cytochrome oxidase subunit I gene sequence and trace file have been submitted to Barcode of Life Data System. Results The prevalence rate of Z. arugamensis was recorded to be 11.5%, and the intensity was 1.48. The low intensity was due to the water temperature recorded in this study (32.9–33.2 °C). All the individuals of Z. arugamensis recorded in this study showed a close relationship with species that were recorded in NCBI database (Z. arugamensis DQ414344, Aestabdella leiostomi DQ414305, Pterobdella amara DQ414334 and Cystobranchus meyeri DQ414315) but less relationship with Aestabdella abditovesiculata DQ414300. Finally, the DNA sequences submitted to Barcode of Life Data System in accordance to species have already obtained Barcode Index Number as BOLD: ACM3477. Conclusions This study has provided an overview of sequence divergence at cytochrome oxidase subunit I gene, DNA barcodes and parasite prevalence of Z. arugamensis.

Two new species of Mycodrosophila (Diptera, Drosophilidae) proposed by molecular and morphological approaches, with a key to American species

ABSTRACT There are approximately 130 species of MycodrosophilaOldenberg, 1914 worldwide, although only nine species were recorded in American countries so far, three of which are exclusively Nearctic, five exclusively Neotropical and one found in both biogeographic regions (Mycodrosophila projectans). Such a small number of American species is likely a consequence of collecting bias, which favors the capture of frugivorous drosophilids, and to the general absence of Neotropical Mycodrosophila studies in the last 50 years. Here, we describe two commonly sampled species of Mycodrosophila from the Amazonian and Pampa Brazilian biomes, which share morphological similarities with Mycodrosophila neoprojectans and M. projectans, respectively. We compared sequences of the mitochondrial gene cytochrome oxidase subunit I (COI), external morphology characteristics and male terminalia among these species. Based on a DNA barcoding approach coupled to morphological differences, we proposed the delimitation of two new species, Mycodrosophila hofmanni sp. nov. and Mycodrosophila valentae sp. nov. An updated key to identifying Neotropical and Nearctic Mycodrosophila species is also provided.

Precise identification of different stages of a tick, Ixodes granulatus Supino, 1897 (Acari: Ixodidae)

Objective To identify different stages of Ixodes granulatus (I. granulatus) based on morphological characters prior to molecular identification which is significant for confirming and identifying the nymphal stages of I. granulatus. Methods A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I (COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus. Results Based on external morphological characterizations, nine individuals (64.3%) were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94% sequence homology from available sequence in GenBank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities (0%–3.7%). Conclusions These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.

Precise identification of different stages of a tick, Ixodes granulatus Supino, 1897 (Acari:Ixodidae)

Objective: To identify different stages of Ixodes granulatus (I. granulatus) based on morphological characters prior to molecular identification which is significant for con-firming and identifying the nymphal stages of I. granulatus. Methods: A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I (COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus. Results: Based on external morphological characterizations, nine individuals (64.3%) were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94%sequence homology from available sequence in GenBank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities (0%–3.7%). Conclusions: These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.

Molecular-based rapid inventories of sympatric diversity: A comparison of DNA barcode clustering methods applied to geography-based vs clade-based sampling of amphibians.

Molecular markers offer a universal source of data for quantifying biodiversity. DNA barcoding uses a standardized genetic marker and a curated reference database to identify known species and to reveal cryptic diversity within wellsampled clades. Rapid biological inventories, e.g. rapid assessment programs (RAPs), unlike most barcoding campaigns, are focused on particular geographic localities rather than on clades. Because of the potentially sparse phylogenetic sampling, the addition of DNA barcoding to RAPs may present a greater challenge for the identification of named species or for revealing cryptic diversity. In this article we evaluate the use of DNA barcoding for quantifying lineage diversity within a single sampling site as compared to clade-based sampling, and present examples from amphibians. We compared algorithms for identifying DNA barcode clusters (e.g. species, cryptic species or Evolutionary Significant Units) using previously published DNA barcode data obtained from geography-based sampling at a site in Central Panama, and from clade-based sampling in Madagascar. We found that clustering algorithms based on genetic distance performed similarly on sympatric as well as clade-based barcode data, while a promising coalescent-based method performed poorly on sympatric data. The various clustering algorithms were also compared in terms of speed and software implementation. Although each method has its shortcomings in certain contexts, we recommend the use of the ABGD method, which not only performs fairly well under either sampling method, but does so in a few seconds and with a user-friendly Web interface.

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F ST) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.

Molecular Identification of Four Muscidae Species Collected in Korea using the DNA Barcode Region of Mitochondrial Cytochrome Oxidase Subunit I / 대한법의학회지

DNA barcoding was recently introduced to molecular identification of forensically important fly species. So, we have analysed the barcode region (687 nucleotides) of mitochondrial cytochrome oxidase subunit I (COI) gene for four species of Muscidae flies collected from Korea. The sequences were aligned and analysed to construct a phylogenetic tree using DNA Star 5.01(DNAStar Inc) and MEGA 3.1 program(Kumar, Tamura, Nei 2004). Intraspecific variation was not noted between M.stabulans individual to each other. Intraspecific variation ranges of other species were 0.1%, 0.1~0.3% and 0.1~0.6% for O.leucostoma, M.angustifrons and M.domestica, respectively. Interspecific percent distance was minimal(9.7~10.0%) between M.stabulans and M.angustifrons. Other species showed above 10% distance from each other. The result showed that four species of Muscidae fly species (Muscina angustifrons, Muscina stabulans, Ophyra leucostoma and Musca domestica) were identifiable from each other with analysis of barcode region of COI gene. Therefore, we conclude that species identification of forensically important Muscidae flies used in this study is possible with percent distance of sequences of COI barcode region, but more species and individuals should be examined to be confident about the conclusion.

Identification Blow Fly Species in Korea by Mitochondrial DNA 'Barcodes' / 대한법의학회지

Recently many forensic scientists are trying to use the DNA 'barcode' region (upstream portion of mitochondrial cytochrome oxidase subunit I) to identify the species of forensically important fly species. We have analyzed to compare their sequences of the 'barcode' region for twelve blow fly species[A. grahami, C.lata, C. vicina, H.ligurriens, L. ampullaceal, L. Caesar, L. illustris, P. sericata, P. regina, T. calliphoroides, C. megacephala, C. pinguis] collected from the rural and urban regions in Korea. Intra- and interspecies sequence divergences were calculated as 0~0.9% and 0.9~11.4%, respectively. Phylogenetic trees were drawn with Mega 3.1 and Network 4.20 programs. The result illustrates that each genus is grouped as monophyletic group except for T. calliphoroides and all the same species were monophyletic group. This suggests that the 'Barcode' region of COI gene could be a marker for identification of necrophagous blow fly species. But the two closely related species, L.illustris and L.caesar show little differences from each other. Therefore more individuals of these species should be examined for population study.

DNA-based Identification of Forensically Important Blow Fly Species in Korea; Aldrichina grahami, Calliphora lata, Calliphora vicina and Phormia regina / 대한법의학회지

Entomological evidence, especially necrophagous flies, are important in estimating postmortem interval in a putrefied corpse. Accurate and rapid species identification of eggs, maggots and pupae is required because growth rates and ecological characteristics are different among different species. But species identification of these immature stages of insects is difficult or impossible to even an expert entomologist. We tried to identify the necrophagous fly species using molecular data. Adult specimens of four forensically important blow fly species [Aldrichina grahami, Calliphora lata, Calliphora vicina and Phormia regina] were used for DNA extraction and sequences analysis of mitochondrial cytochrome oxidase subunit I (CO1) in this study. A total of 560 base pairs(bp) of the CO1 region was recovered using the newly designed specific primer pairs and was sequenced to compare it with those of same fly species registered in NCBI GenBank. The results presented in Table 2 to 6 demonstrate not only the potential utility of the COI sequence in interspecific discrimination, but also indicate that this sequence is probably not suitable for use with intraspecific studies, especially for dividing different local populations within the same species.

Molecular Identification of Six Necrophagous Fly Species (Family: Muscidae) by Mitochondrial Cytochrome Oxidase Subunit I / 대한법의학회지

Estimation of postmortem interval (PMI) in a putrefied corpse has been a long theme in the forensic medicine. Insects, especially necrophagous fly species are now utilized as indicators of PMI because the first visitors to a dead body are usually known to be blow fly species (Family Calliphoridae). House flies (Family Muscidae) are later visitors but they are very significant in forensic entomology because of their worldwide distribution. Entomologic evidences recovered from the scene are often immature individuals such as eggs, maggots and pupae. Because growth rates and ecological characteristics are different among fly species, accurate species identification is essential. As species identification in immature stages is very difficult or even impossible to an expert entomologist, many researchers are trying to identify fly species by molecular techniques. Authors analyzed 400bp of mitochondrial COI gene sequences of six Muscidae fly species (Fannia prisca, Muscina angustifrons, Muscina stabulans, Musca domestica, Hydrotaea dentipes and Ophyra leucostoma). In spite of limited number of flies analyzed in this study, all six fly species have different haplotype of COI gene and shows minimal intraspecific variation. This result shows that six fly species analyzed in this study can be discriminated each other by COI gene sequence analysis. But, more individuals from various geographic region should be analyzed to apply this result to a forensic entomology practice.