Partial Mitochondrial 16S rRNA Research Articles

A loop-mediated isothermal amplification (LAMP) assay for the detection of Cryptotermes brevis West Indian drywood termite (Blattodea: Kalotermitidae)

Cryptotermes brevis is one of the most destructive invasive termites in the subtropics and tropics and is a common biosecurity intercept at the Australian border. Drywood termite species are cryptic and difficult to identify morphologically in situations when soldiers or imagos are unavailable. We developed a novel DNA based loop-mediated isothermal amplification (LAMP) assay to detect C. brevis and differentiate it from other drywood termites. Validated voucher specimens of 30 different drywood termite species were obtained from several insect collections from which DNA was extracted and amplified. The amplicons containing partial mitochondrial 16S rRNA were sequenced and a DNA database was created from which C. brevis LAMP primers were developed, optimized, and tested. The assay was assessed against a range of target and non-target species and found to be specific, successfully amplifying the target specimens of C. brevis in under 30 min. Amplification success was variable against C. brevis faecal pellets due to minute, unmeasurable or degraded DNA. This LAMP test is a new tool for the rapid detection of C. brevis that will enable faster and less destructive management of drywood termite infestations.

ON THE OCCURRENCE OF Scorpiops longimanus (POCOCK, 1893) (ARACHNIDA: SCORPIOPIDAE) IN MIZORAM, NORTHEAST INDIA

In this study, we confirm the occurrence of the tropical forest scorpion Scorpiops longimanus (Pocock, 1893) in Mizoram, India based on specimens collected from 13 different localities within the state. The total length of the animal was found to range from 42.22 mm up to 65.16 mm. The number of trichobothria on the ventral surface of the pedipalp patella and that of the external face alternates between 10–11 and 18–19, respectively. The pectines showed dissimilarities in the structure of the lamellae and in the number of teeth. We generated the first molecular data for the species and performed a maximum likelihood phylogenetic analysis using the partial mitochondrial 16S rRNA gene which showed the formation of a distinct clade of S. longimanus. The uncorrected k2p distance showed an intraspecific genetic variation of 2.9% among the species studies.

Molecular characterisation of Rhipicephalus sanguineus sensu lato ticks from domestic dogs in Nigeria.

Rhipicephalus sanguineus is the most widely reported tick in the world. Molecular characterisation is important to verify its taxonomic status in the different parts of the world. In this study, we provide information on the molecular characterisation of R. sanguineus tick of dogs collected from Nigeria. Ticks were collected from 62 of 93 sampled dogs. The collected ticks were subjected to morphological identification with the aid of appropriate entomological keys. Deoxyribonucleic acid (DNA) was extracted from the most prevalent tick species (R. sanguineus) and was subjected to further molecular characterisation protocols. The partial mitochondrial 16S rRNA gene sequences (∼300 bp) were obtained from representative specimens. Data were statistically analysed using the chi‐square (χ 2) test. Phylogenetic analysis was performed including different lineages of R. sanguineus (sl) from Africa, Asia, Europe and America, and other species belonging to the R. sanguineus ‘tropical lineage’ (R. linnaei) as well as Rhipicephalus turanicus and Ixodes ricinus. Results of this study showed that R. sanguineus was the most abundant ticks of dogs with a prevalence of 61.8% (68/110; 95% CI = 52.5–70.54), followed by Amblyomma variegatum (20.0%) and Haemaphysalis leachi (18.2%). The molecular analysis shows that they are genetically different from the temperate strains but closely related to those from other West African countries. There is a need to establish the vector competence of this common Nigerian dog tick.

A new species of freshwater crab of the genus Nanhaipotamon Bott, 1968 (Crustacea, Decapoda, Brachyura, Potamidae) from Longhai, Fujian Province, China.

A new species of freshwater crab of the genus Nanhaipotamon Bott, 1968 is described from Xiaye Village, Chengxiang Town, Longhai County, Zhangzhou City, Fujian Province, China. The new species is distinguished from congeners by the combination of characters of its carapace, third maxilliped, unequal chelipeds, triangular male abdomen and unique male first gonopod. Molecular evidence derived from partial mitochondrial 16S rRNA and COI genes also support the species as new.

Molecular genetic diversity of some rabbit breeds based on mitochondrial 16S rRNA sequences

<p>The present study was performed to assess the genetic variations among six rabbit breeds in Egypt based on mitochondrial 16S rRNA sequences. The length of partial mitochondrial 16S rRNA in the six rabbit breeds ranged from 546 bp to 558 bp. The sequenced regions were submitted to GenBank/NCBI under accession numbers (MW052052 - MW052057). The average content of A+T was 57% in all breeds. Among breeds, the percentages of genetic distance values were ranged from 0.000 to 0.004. The highest P-distance (0.004) was found between the New Zealand White breed and all other breeds. The results support the suitability of mitochondrial 16S rRNA for genetic diversity analysis of rabbit breeds and the applicability for future research on genetic relationships and the phylogeny of rabbit breeds.</p>

Two new species of freshwater crab of the genus Aparapotamon Dai & Chen, 1985 (Crustacea, Brachyura, Potamidae) from Yunnan, China.

Two new species of freshwater crab of the genus Potamid Aparapotamon Dai & Chen, 1985 are described from Yunnan Province, southwest China. Morphological comparisons were made between the two new species and type materials of other 11 species of Aparapotamon. Aparapotamonbinchuanensesp. nov. and A.huizeensesp. nov. can be separated from their congeners by the shape of the epibranchial tooth, the frontal view of the cephalothorax, the male first gonopod, and the female vulvae. The molecular analyses based on partial mitochondrial 16S rRNA gene are also included. This study brings the number of Aparapotamon species to 13.

The phylogenetic position of Arhaphe deviatica within Hemipteran insects: A potential model species for eco-devo studies of symbiosis.

Insecta is known to be the most diverse group of species, exhibiting numerous forms of endosymbiotic associations. Molecular techniques have provided significant indicators for insect-microbe interactions. The present study aimed to register one of the true bugs of pentatomomorpha and clarify its taxonomic position through phylogenetic analysis of the partial 16S rRNA gene region. A maximum likelihood analysis retrieved a generally well-supported phylogeny based on Tamura 3-parameter model. Based on the partial mitochondrial 16S rRNA gene sequences, a phylogenetic study of suborder Heteroptera relationships within Hemipteras' order was constructed. Sequences of 221 bases of the 3' end of the gene from 28 species within 16 families were analyzed. This analysis and bootstrap confidence revealed two major clades comprising four suborders within Hemiptera, with a close relationship between Heteroptera + (Sternorrhyncha + (Auchenorrhycha + Coleorrhyncha)). Infraorder Pentatomomorpha is forming a sister group with a substantial bootstrap value to Cimicomorpha. Pyrrhocoroidea forms a sister relationship with Lygaeoidea + Coreoidea. There is a close relationship between Largidae and Pyrrhocoridae within Pyrrhocoroidea. The results show that the present species is firmly embedded in the genus Arhaphe with 94.35% sequence resemblance to its congeners. Besides, the recovered hemipteran species considered a potential model group for studying different symbionts. We propose both phylogenetic and ecological evolutionary developmental biology viewpoints for a more synthetic understanding of insect populations' molecular evolution.

Genetic characterization of Rhipicephalus sanguineus (sensu lato) ticks from dogs in Portugal

BackgroundThe taxonomic status of the brown dog tick Rhipicephalus sanguineus (sensu stricto) is a subject of on-going debate; there is a consensus that populations of this tick species should be referred to as R. sanguineus (sensu lato) until its taxonomic status is resolved. Recent genetic studies revealed the existence of more than one lineage of R. sanguineus (s.l.) in temperate countries. In this study, we assessed the genetic identity of ticks collected from rural dogs living in several areas located in all major geographical regions of Portugal.MethodsA total of 347 ticks were collected from rural dogs living in different regions of Portugal. These ticks were morphologically identified and partial mitochondrial 16S rRNA gene sequences (~300 bp) were obtained from representative specimens.ResultsThe ticks were morphologically identified as Ixodes ricinus (seven males and 27 females), Rhipicephalus bursa (one male), Rhipicephalus pusillus (one female) and R. sanguineus (s.l.) (two larvae, 101 nymphs, 108 males and 100 females). Partial mitochondrial 16S rRNA gene sequences were obtained from 58 R. sanguineus (s.l.) specimens, and all of them were genetically identified as belonging to the so-called temperate lineage of R. sanguineus (s.l.)ConclusionsThese results strongly suggest that the temperate species of R. sanguineus (s.l.) is the only representative of this tick group found on dogs in Portugal. It also adds weight to the hypothesis that Rhipicephalus turanicus is not present in this country, although further investigations are necessary to confirm this.

Cnemaspis flaviventralis, a new species of gecko (Squamata: Gekkonidae) from the Western Ghats of Maharashtra, India

Cnemaspis flaviventralis, a new species of day gecko, is described from the forests of Amboli, Sindhudurg District, Maharashtra State, northern Western Ghats, India. The new species was previously confused with the sympatric species Cnemaspis girii, C. indraneildasii, C. kolhapurensis and C. goaensis. It is distinguished from C. giri by having spine-like tubercles on flanks, granular dorsal scales intermixed with large, depressed, slightly keeled scales (vs. lack of spine-like tubercles on flanks, granular dorsal scales, intermixed with large smooth scales); from C. indraneildasii by having dorsal scales heterogeneous (vs. homogenous), lacking a series of enlarged median sub-caudal scales, and 28–29 (vs. 20) ventral scales across mid-body; from C. kolhapurensis by having heterogeneous (vs. homogenous) dorsal scalation, lacking spine-like tubercles on flanks and lacking pre-cloacal pores (vs. 24–28 pre-cloacal-femoral pores); and from C. goaensis by lacking pre-cloacal pores and lacking a series of enlarged median sub-caudal scales. We further provide partial mitochondrial 16S rRNA gene sequences for the new species and for the sympatric species C. girii, C. kolhapurensis and C. goaensis, and show that the new species is genetically distinct.

A new fish cell line derived from the caudal fin of freshwater angelfish Pterophyllum scalare: development and characterization

In this study, a new cell line derived from the caudal fin of the freshwater angelfish Pterophyllum scalare was developed and characterized. The cell line was designated angelfish fin (AFF) and subcultured 44 times since its development. These cells grew well in Leibovitz's -15 medium supplemented with 10% foetal bovine saline (FBS) at 28° C and the modal chromosome number (2n) was 48. The AFF cell-line is mainly comprised of epithelial cells as confirmed by immunocytological technique using anti-cytokeratin antibodies, an epithelial cell marker. This cell line was tested for growth in a temperatures range from 20 to 37° C and at various FBS concentrations of 5-20% at 28° C. The cell line was cryopreserved at different passage levels and revived successfully with 80% survival rate. Polymerase chain reaction amplification and sequencing of partial mitochondrial 16s rRNA and coI genes confirmed that the AFF cell-line originated from angelfish. Mycoplasma sp. contamination was not detected in AFF cells and checked by Hoechst 33258 fluorescence staining. At the 42nd passage the cells were transfected with 2 μg of pAcGFP1-N1 expression vector. The AFF cells exhibited cytotoxic effects when exposed to the bacterial extra cellular products from Serratia marcescens and Proteus hauseri. The AFF cells and cells from kidney and brain did not show cytopathic effect when exposed to cyprinid herpes virus2 and viral nervous necrosis virus. The newly developed AFF cell line will be useful for the isolation of viruses affecting angelfishes, such as iridoviruses, in the future.

Unraveling a 146 Years Old Taxonomic Puzzle: Validation of Malabar Snakehead, Species-Status and Its Relevance for Channid Systematics and Evolution

BackgroundThe Malabar snakehead Channa diplogramma is one of the most enigmatic and least understood species within the family Channidae, which comprise one of the most important groups of freshwater food fish in tropical Asia. Since its description from peninsular India in 1865, it has remained a taxonomic puzzle with many researchers questioning its validity, based on its striking similarity with the South East Asian C. micropeltes. In this study, we assessed the identity of the Malabar snakehead, C. diplogramma, using morphological and molecular genetic analyses, and also evaluated its phylogenetic relationships and evolutionary biogeography.Methodology/Principal FindingsThe morphometric and meristic analysis provided conclusive evidence to separate C. diplogramma and C. micropeltes as two distinct species. Number of caudal fin rays, lateral line scales, scales below lateral line; total vertebrae, pre-anal length and body depth were the most prominent characters that can be used to differentiate both the species. Channa diplogramma also shows several ontogenic color phases during its life history, which is shared with C. micropeltes. Finally, the genetic distance between both species for the partial mitochondrial 16S rRNA and COI sequences is also well above the intra-specific genetic distances of any other channid species compared in this study.Conclusions/SignificanceThe current distribution of C. diplogramma and C. micropeltes is best explained by vicariance. The significant variation in the key taxonomic characters and the results of the molecular marker analysis points towards an allopatric speciation event or vicariant divergence from a common ancestor, which molecular data suggests to have occurred as early as 21.76 million years ago. The resurrection of C. diplogramma from the synonymy of C. micropeltes has hence been confirmed 146 years after its initial description and 134 years after it was synonymised, establishing it is an endemic species of peninsular India and prioritizing its conservation value.

Cryptic species of the gobioid paedomorphic genus Schindleria from Palau, Western Pacific Ocean

The extremely paedomorphic fish Schindleria (Gobioidei, Schindleriidae) is widely distributed in the Indo- Pacific Ocean, inhabiting coral reef lagoons. At least 21 cryptic species (Schindleria spp. 1-21) have been discov- ered around the Ryukyu and Ogasawara Islands. As a first step in extending the survey areas to the entire Indo-Pacific Ocean, the present study collected and examined Schindle- ria in Palau, located approximately 2,200 km from the Ryukyu Islands, for any cryptic diversity. Molecular phy- logenetic analysis using partial mitochondrial 16S rRNA sequences of 71 specimens revealed four new cryptic spe- cies of Schindleria spp. 22-25 (Palau 1-4) in Palau.

Monophyletic origin of Caryophyllia (Scleractinia, Caryophylliidae), with descriptions of six new species

The genus Caryophyllia Lamarck, 1816 is the most diverse genus within the azooxanthellate Scleractinia comprising 66 Recent species and a purported 195 nominal fossil species. Examination of part of the deep-sea scleractinian collection made by the Paris Museum off New Caledonia and part of the material collected by CSIRO off Australian waters revealed the occurrence of 23 species of Caryophyllia, of which six are new to science. All new records, including the new species, are described, and synonyms, distribution, type locality, type material and illustration are provided for each species. An identification key to all Recent species of Caryophyllia is presented. In addition, the validity of the genus Caryophyllia was investigated by phylogenetic analyses of a dataset consisting of partial mitochondrial 16S rRNA sequences from 12 species assigned to this genus together with seven species representing some of the most morphologically similar caryophylliid genera, and 14 non-caryophyllid species representing 14 scleractinian families. Irrespective of the method of analysis employed, all of the Caryophyllia species formed a well-supported clade together with Dasmosmilia lymani and Crispatotrochus rugosus. Although based on a subset of the Recent Caryophyllia species, these results are consistent with Caryophyllia being a valid genus, but call for a reexamination of Dasmosmilia and Crispatotrochus.

Phylogenetic Affinities of the Rare and Enigmatic Limb-Reduced Anelytropsis (Reptilia: Squamata) as Inferred with Mitochondrial 16S rRNA Sequence Data

Abstract The phylogenetic placement of the limbless lizard genus Anelytropsis within Squamata was investigated using partial mitochondrial 16S rRNA sequences (422 bp). A total of 30 species, representing most of the major currently recognized squamate clades, was included in the analysis. As in previous morphological studies, Anelytropsis was strongly supported in Bayesian (mixture and unpartitioned models) and maximum-likelihood analyses as the sister taxon of Dibamus. Thus, a monophyletic Dibamidae composed of these two genera is supported by molecular data for the first time. Furthermore, several relationships in the inferred tree, although weakly supported, were congruent with those found in previous molecular phylogenetic analyses. Among these, Gekkota and the Dibamidae were recovered as relatively basal groups within Squamata. A nonmonophyletic Scleroglossa and Lacertiformes (= Amphisbaenia + Lacertidae + Teiidae + Gymnophthalmidae) also were recovered. Although only weakly supported, a major differ...

Identification of three Asian Hediste species (Polychaeta: Nereididae) by PCR-RFLP analysis of the mitochondrial 16S rRNA gene

An easy polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was designed to identify the three morphologically similar nereidid polychaete species Hediste atoka, Hediste diadroma and Hediste japonica which often dominate in estuarine macrobenthic fauna in eastern Asia. Restriction digestion analysis of the PCR products of the partial mitochondrial 16S rRNA gene (approximately 600 bp) of these three species, using EcoRV and TspEI endonucleases, generated species-specific restriction patterns. The PCR-RFLP method enables easy and accurate identification of the three Hediste species.

Phylogeny of the Plumularioidea (Hydrozoa, Leptothecata): evolution of colonial organisation and life cycle

The Plumularioidea (Cnidaria, Hydrozoa, Leptothecata) are the most species rich superfamily of the class Hydrozoa. They display a complex and diversified colonial organisation and their life cycle comprises either a reduced free-living, pelagic generation (medusoid), alternating with the benthic colonial form or in most species, no pelagic generation. In order to understand the evolution of colonial and life cycle characters among Plumularioidea, we have reconstructed their phylogeny. Partial mitochondrial 16S rRNA sequences and 64 morphological characters were analysed separately and in combination. The morphological data included not only characters of the individual polyps and medusae, but also characters describing the organisation of colonies, for which we propose general principles applying to character coding in modular organisms. The phylogenetic analyses supported the monophyly of Plumularioidea and of the four plumularioid families (Aglaopheniidae, Halopterididae, Kirchenpaueriidae and Plumulariidae). Most genera were paraphyletic or polyphyletic. This study highlights multiple morphological simplifications of the colonial organisation during the evolution of Plumularioidea and the convergence of the defensive polyps — the dactylozooids — of Plumularioidea with those of others Leptothecata (Hydrodendron) or Anthoathecata (Hydractinia). Concerning the evolution of the life cycle, the phylogeny supports a provocative scenario, where the medusa was lost in an ancestor of the Plumularioidea, and then re-acquired four times independently within this group, in the form of simple medusoids.

DNA sequences identify numerous cryptic species of the vertebrate: A lesson from the gobioid fish Schindleria

Schindleria (Gobioidei, Schindleriidae), believed to include one of the smallest and youngest reproducing vertebrates, is broadly distributed in the Indo-Pacific Oceans, inhabiting coral reef lagoons. They are all characterized by a reduced larval-like form, such as a slender translucent and scaleless body. The three nominal species recognized in the genus to date have been distinguished by only combination of dorsal and anal fin-ray counts, and the existence of some undescribed species has been suggested in Schindleria; thus a total picture of species composition of the genus is poorly known. Towards the disclosure of diversity of Schindleria, a molecular phylogenetic analysis using partial mitochondrial 16S rRNA sequences was conducted for specimens from the Ryukyu and Ogasawara Islands, Japan. This analysis showed clearly that as many as 21 genetically distinguishable species occurred within the geographical areas. The degree of species crypticness of “ S. praematura” [15.0 = 15 (new cryptic species + known species)/1 (known species)] is higher than the values of well-known animal examples, such as the pan-mesopelagic bristlemouth fish Cyclothone alba (5.0) and the South American skipper butterfly Astraptes fulgerator (10.0). This discovery of many cryptic species in Schindleria suggests that the use of DNA sequences is necessary for species identification of such morphologically conserved taxa. Because molecular analyses should increase the number of hitherto unnamed and pseudonymous species, especially in tropical areas, it is proposed that DNA-based designation is necessary for such taxa in order to compile the full “species lists”, although there is presently no consensus for the inclusion of DNA sequencing data in the formal descriptions of new species.

A molecular approach to the phylogenetic relationships of the western palaearctic Helicoidea (Gastropoda: Stylommatophora)

Molecular phylogenetic relationships among 45 members of the Helicoidea (Gastropoda: Stylommatophora) were examined using partial mitochondrial 16S rRNA sequences. Phylogenetic relationships were inferred using maximum parsimony, maximum likelihood and Bayesian methods. The reconstructed phylogenies showed a good degree of support for more recent branches, but gave little support to deeper nodes. Mitochondrial rDNA data further confirmed monophyletic status of helicids, recognized monachine hygromiid and bradybaenid clades and resolved a number of relationships in the helicelline hygromiids. With the respect to the latter assemblage, most of the anatomically based groups are confirmed, corroborating the diagnostic value of the dart-sac complex and a close affinity between Ichnusomunda sacchii and species of the genus Cernuella. Nevertheless, some well resolved branches challenge previous systematic arrangements, grouping species previously placed in different arrangements. In particular, support was not found for the monophyly of helicelline hygromiids with pedal penial innervation. Possible explanations for these incongruencies are suggested. 16S sequence data are appropriate for studies of relationships within the different species groups and less so for recovery of more ancient radiations in the Helicoidea. It will be valuable to combine the 16S data with other gene sequences to estimate basal relationships.

Analysis of RAPD and mitochondrial 16S rRNA gene sequences from Trichiurus lepturus and Eupleurogrammus muticus in the Yellow Sea*

Abstract Random amplified polymorphic DNA (RAPD) technique is applied to 12 individuals from each species of the hairtail fishes Trichiurus lepturus and Eupleurogrammus muticus in the Yellow Sea. The percentage of polymorphic sites, degree of genetic polymorphism and genetic distance are compared and the phylogenetic tree is constructed by Neighbor-joining method. The partial mitochondrial 16S rRNA gene is amplified by polymerase chainreaction (PCR) and the PCR products are directly sequenced afterbeing purified. These sequences, together with the homologous sequences of another Trichiuridae species Lepidopus caudatus obtained from Gen-Bank, are used to analyze nucleotide difference and to construct a UPGMA phylogenetic tree by means of biological informatics. Analysis shows: (l) the RAPD technique is a highly sensitive method for investigating genetic diversity in T. lepturus, and E. muticus. T. lepturus exhibits a lower polymorphism and genetic diversity than E. muticus; (2) according to the analysis of...

TESTING THE RELATIONSHIPS OF CHINESE FRESHWATER UNIONIDAE (BIVALVIA) BASED ON ANALYSIS OF PARTIAL MITOCHONDRIAL 16S rRNA SEQUENCES

This study presents partial mitochondrial 16S rRNA sequences of 13 unionid bivalve species from China and analyses their relationships in combination with known data of 21 American mussels. According to our results, Chinese unionids, formerly regarded as two subfamilies, should be divided into three subfamilies: Ambleminae, Anodontinae and Unioninae. The genera Hyriopsis, Solenaia, Lamprotula and Ptychorhynchus, hitherto placed in Unioninae or Anodontinae, should be moved to the subfamily Ambleminae, demonstrated for the first time from China. The other genera recorded from China are suggested to belong to Anodontinae and Unioninae, which is in agreement with traditional classifications, except for the genus Lepidodesma.