Mitochondrial 16S rRNA Gene Sequences Research Articles

Cystic Echinococcosis of Camels: 12S rRNA Gene Variation Revealed Changing Pattern of Genetic Diversity Within Echinococcus granulosus sensu lato in the Middle East and North/Sub-Saharan Africa.

Cystic echinococcosis (CE) is one of the most widespread zoonotic diseases, with considerable public health and economic importance. Camels play a significant role in transmission cycle of Echinococcus granulosus especially, in the Middle East and North Africa (MENA). The present study aimed to identify the genetic variation and haplotype distribution of camel isolates of E. granulosus sensu lato using all existing E. granulosus mitochondrial DNA data from camels in different parts of the world. Sequence data from 1,144 camel isolates of E. granulosus s.l. available in the NCBI GenBank including 57 camel hydatid cysts collected in central Iran were used to analyze the nature of genetic variation within the camel isolates of E. granulosus s.l. in MENA region. Fifty-seven camel isolates were also PCR-sequenced on mitochondrial 12S rRNA gene. Haplotype network analysis revealed seven different haplotypes clustered into four major groups. E. intermedius G6 was identified as the most commonly represented genotype in camels followed by G1. Mitochondrial 12S rRNA gene sequence analysis on 57 camel isolates identified three different genotypes, including E. intermedius/G6 (35/57, 61.4%), E. granulosus sensu stricto/G1-G3 (21/57, 36.8%) as well as one isolate identified as E. ortleppi/G5 (1/57, 1.8%). The number of base substitutions per site over 420 positions of partial 12S rRNA gene sequences were shown as 0.000 and 0.004 for E. intermedius (G6) corresponding to the Middle East and sub-Saharan isolates, respectively. Camel isolates of E. granulosus in the MENA region present moderate genetic diversity (Hd = 0.5540–0.6050). The Middle East isolates demonstrated a more diverse population than the North/sub-Saharan isolates, where six out of seven 12S rRNA haplotypes were identified in the former region. E. intermedius (G6 genotype) was shown to be the most common species in the world camel population. In conclusion, camels showed to be an important intermediate host species in the MENA region with different patterns of genetic variation between the Middle East and Africa.

A DNA mini-barcoding system for endangered unionid mussels in the Lake Biwa system in Japan

A mini-barcoding system was developed to help identify endangered unionid mussels in the Lake Biwa system in Japan. A single primer set was designed to amplify a ca. 140-bp barcode fragment based on the published mitochondrial 16S rRNA gene sequences (ca. 350 bp) of Japanese unionids. The new primers successfully amplified genomic DNA from the foot tissues of 81 specimens of 12 target taxa from the Lake Biwa region. Sequencing, BLAST-searches and phylogenetic analyses of the amplified fragments confirmed that the mini-barcode had the same identification ability as the original sequences for Lake Biwa taxa. The mini-barcoding system also obtained the short barcodes from excreted materials, indicating its usefulness in nondestructive identification of these mussels.

A new species of the Asian leaf litter toad genus Leptobrachella (Amphibia, Anura, Megophryidae) from southwest China.

A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described based on molecular phylogenetic analyses, morphological comparisons, and bioacoustics data. Phylogenetic analyses based on the mitochondrial 16S rRNA gene sequences supported the new species as an independent clade nested into the Leptobrachella clade and sister to L. bijie. The new species could be distinguished from its congeners by a combination of the following characters: small body size (SVL 30.8–33.4 mm in seven adult males, and 34.2 mm in one adult female); dorsal skin shagreened, some of the granules forming longitudinal short skin ridges; tympanum distinctly discernible, slightly concave; internasal distance longer than interorbital distance; supra-axillary, femoral, pectoral and ventrolateral glands distinctly visible; absence of webbing and lateral fringes on fingers; toes with rudimentary webbing and shallow lateral fringes, relative finger lengths II < IV < I < III; heels overlapped when thighs are positioned at right angles to the body; and tibia-tarsal articulation reaches the tympanum.

A high-resolution melting analysis method for the effective discrimination of three Sepia species

A high resolution melting (HRM) curve analysis of single nucleotide polymorphisms (SNPs) was used to identify three Sepia species, commonly found on the south coast of China. The mitochondrial 16S rRNA gene sequences from Sepiella japonica, Sepia pharaonis and Sepia lycidas were amplified and aligned. A total of 19 SNPs and seven deletion sites were identified in 122-base-pair (bp) amplicons. All samples from the three Sepia species were clustered into three curves based on HRM analysis, in line with the sequencing results. This new method thus enables a quick, economical and efficient identification of Sepia species.

A new species of the toothed toad Oreolalax (Anura, Megophryidae) from Sichuan Province, China.

The toad genus Oreolalax is widely distributed in southwest China and northern Vietnam. A new species of the genus is described from Sichuan Province, China. Phylogenetic analyses based on the mitochondrial 12S rRNA and 16S rRNA gene sequences supported the new species as an independent clade clustered into the clade also containing O. nanjiangensis and O. chuanbeiensis. The new species can be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 51.2–64.2 mm in males); head broad; tympanum hidden; interorbital region with dark triangular pattern; belly with marbling; male lacking spines on lip margin; spiny patches on chest small with thick sparse spines in male; nuptial spines thick and sparse; tibio-tarsal articulation reaching beyond nostril when leg stretched forward; toe webbing at base.

First record of Euphlyctis kalasgramensis (Anura: Dicroglossidae) from Punjab, Pakistan

The present study documented the first record of Euphlyctis kalasgramensis from Punjab, Pakistan. The specimens were collected during field visits from June through August 2018. Various morphometric measurements of E. kalasgramensis were taken and compared with Euphlyctis cyanophlyctis. Snout-vent length (SVL) was 38.11 ± 0.87 mm (n = 5), snout length was 3% of SVL, foot length was 55% of SVL, head length was 32% of SVL and weight was 8.01 ± 0.12 g (n = 5). A few specimens (n = 2) were euthanized and preserved for molecular analysis through mitochondrial 16S rRNA gene sequences. The newly obtained DNA sequences of E. kalasgramensis were submitted to GenBank and accession numbers were obtained (MK881165.1 and MK920114.1). The Maximum likelihood and Neighbor-joining trees based on Kimura 2-parameter distance resulted in similar phylogenetic trees. Euphlyctis kalasgramensis was out group in both phylogenetic trees. The interspecific divergence of E. kalasgramensis and E. cyanophlyctis was high ranging from 4% to 6% as compared to low intraspecific divergence 0% and 1%. The diversity and distribution ranges of many amphibians species are not well known in Pakistan due to lack of taxonomic information. In our recommendation, a large scale DNA barcoding is required to report more cryptic or new species from Pakistan.

A new species of the Music frog Nidirana (Anura, Ranidae) from Guizhou Province, China.

The Music frog genus Nidirana is widely distributed in East and South Asia. Here, a new species of the genus is described from southwestern China. Phylogenetic analyses based on the mitochondrial 16S rRNA and COI gene sequences supported the new species as a clade closely related to N. leishanensis, N. hainanensis, N. chapaensis, N. daunchina, and N. yaoica. The new species could be distinguished from its congeners by a combination of the following characters: body of medium size (SVL 41.2–43.5 mm in males and 44.7 mm in female); lateroventral groove only present on toes; relative finger lengths: II < IV < I < III; three metatarsal tubercles on palm; heels overlapping when hindlimbs flexed at right angles to axis of body; tibiotarsal articulation reaching the level of eye when leg stretched forward; a pair of subgular internal vocal sacs at corners of throat in male; nuptial pad present on the inner side of base of fingers I in breeding male; tadpole labial tooth row formula with 1:1+1/1+1:2; in males, the advertisement call contains two kinds of notes and one call contains 2–6 repeated regular notes.

Characterization of the 12S rRNA Gene Sequences of the Harvester Termite Anacanthotermes ochraceus (Blattodea: Hodotermitidae) and Its Role as A Bioindicator of Heavy Metal Accumulation Risks in Saudi Arabia.

Termites are social insects of economic importance that have a worldwide distribution. Identifying termite species has traditionally relied on morphometric characters. Recently, several mitochondrial genes have been used as genetic markers to determine the correlation between different species. Heavy metal accumulation causes serious health problems in humans and animals. Being involved in the food chain, insects are used as bioindicators of heavy metals. In the present study, 100 termite individuals of Anacanthotermes ochraceus were collected from two Saudi Arabian localities with different geoclimatic conditions (Riyadh and Taif). These individuals were subjected to morphological identification followed by molecular analysis using mitochondrial 12S rRNA gene sequence, thus confirming the morphological identification of A. ochraceus. Furthermore, a phylogenetic analysis was conducted to determine the genetic relationship between the acquired species and other termite species with sequences previously submitted in the GenBank database. Several heavy metals including Ca, Al, Mg, Zn, Fe, Cu, Mn, Ba, Cr, Co, Be, Ni, V, Pb, Cd, and Mo were measured in both collected termites and soil samples from both study sites. All examined samples (termite and soil) showed high concentrations of metals with different concentrations and ratios. Generally, most measured metals had a significantly high concentration in soil and termites at Taif, except for Ca, Cd, Co, Cr, Cu, Mg, and Ni showing significantly high concentrations at Riyadh. Furthermore, termites accumulated higher amounts of heavy metals than the soil at both locations. The mean concentrations of the measured metals in soil samples were found to be in the descending order Ca ˃ Al ˃ Mg ˃ Zn ˃ Fe ˃ Cu ˃ Mn ˃ Ba ˃ Cr ˃ Co ˃ Be ˃ Ni ˃ V ˃ Pb ˃ Cd ˃ Mo, while it was Ca ˃ Mg ˃ Al ˃ Fe ˃ Zn ˃ Cu ˃ Mn ˃ Be ˃ Ba ˃ Pb ˃ Cr ˃ V ˃ Ni ˃ Cd ˃ Mo ˃ Co in termite specimens. The mean concentrations of the studied metals were determined in the soil and termite specimens at both locations. In addition, the contamination factor, pollution load index (PLI) and degree of contamination were calculated for all studied metals in different samples, indicating that both studied sites were polluted. However, Taif showed a significantly higher degree of pollution. Thus, the accurate identification of economically important insects, such as termites, is of crucial importance to plan for appropriate control strategies. In addition, termites are a good bioindicator to study land pollution.

Genetic analysis of Rhipicephalus sanguineus sensu lato ticks, parasites of dogs in the Canary Islands, Cyprus, and Croatia, based on mitochondrial 16S rRNA gene sequences.

The aim of this work was to perform an analysis based on mtDNA sequences of the 16S rRNA gene in order to determine the phylogenetic position of ticks belonging to the Rhipicephalus sanguineus group from the Canary Islands, Cyprus, and Croatia. All the haplotypes obtained from ticks collected in the Canary Islands and Croatia grouped with R. sanguineus sensu stricto from France, Portugal, Italy, Switzerland, Argentina, Chile, Uruguay, and the USA. The sequences of R. sanguineus sensu lato from Cyprus formed a clade with R. sanguineus s.l. from Egypt, Turkey, and Romania, which belongs to the "Rhipicephalus sp. morphotype I" or "southeastern European lineage." Ticks determined as R. turanicus s.l. from Cyprus clustered separately from the remaining clades of the R. sanguineus group, including R. turanicus s.s. The data show that R. sanguineus s.s. is present in the Canary Islands and Croatia, while R. sanguineus "southeastern lineage" is found in Cyprus.

Phylogenetic analysis of mitochondrial 12S ribosomal RNA gene sequence of Mongolian wild boars

Specimens of Mongolian wild boar (Sus scrofa) from Arkhangai, Dornod, Zavkhan, Orkhon, Ovorkhangai, Selenge, Tuv, Khovd, Khuvsgul and Uvs aimags (provinces) were subjected to DNA sequencing. Determined sequences from 18 specimens were registered into the GenBank and accession numbers were obtained. In this study mitochondrial 12S rRNA gene sequences of Mongolian wild boars were analyzed with 36 complete sequences of 12S rRNA gene of wild boar (Sus scrofa) available at NCBI GenBank. Sequence alignment, detection of parsimonious informative sites, model selection, calculation of nucleotide distances and tree construction with 1000 bootstrapped replications were conducted using MEGA 6. Maximum likelihood trees were constructed by the HKY model. A maximum likelihood tree with 53 complete sequences of mitochondrial 12S rRNA gene of Sus scrofa was constructed. Mongolian sequences from the same and adjacent locations were clustered together.&#x0D; European sequences were clustered together, additionally two sequences from south western China and two sequences from south eastern China were also clustered. Additionally, 12S rRNA gene sequences of Mongolian Sus scrofa, located between Asian and European sequences suggesting geographical location of Mongolia, played an important role in the gene flow between Asian and European wild boar population.

Molecular characterization of an indigenous minor carp Bangana dero (Cyprinidae: Labeoninae) of Manipur and its relationship with Labeonin fishes of North East India based on mitochondrial 16SrRNA gene sequences

Bangana dero is an economically important indigenous cyprinid fish of Manipur, India having maximum standard length of 40cm. For the taxonomic placement of this species among the Labeonin fishes, molecular characterization is conducted by using mitochondrial 16SrRNA gene sequences. Partial sequences of mito. 16SrRNA gene of fifteen cyprinid species of the sub-family Labeoninae were downloaded from NCBI Genbank. The analysed nucleotide sequence lengths were 538 bp. In a total of 811 characters, 194 were conserved sites (monomorphic) and 347 were variable sites (polymorphic) and out of 347 variable sites, 73 were parsimony informative sites and 274 singleton sites. The nucleotide frequencies are 31.4% (A), 22.57% (T), 24.00% (C) and 21.96% (G). The transition/transversion bias (R) is 2.51. The overall mean distance is 0.083. The dendogram constructed by Neighbour-Joining tree have three clusters which can indicate the taxonomic positions of Bangana dero .

Morphological and molecular divergence of Rhipicephalus turanicus tick from Albania and China

Rhipicephalus turanicus is an important tick species potentially carrying tick-borne pathogens. Several tick species have obvious subspecies divergence. However few studies aimed to examine the existence of divergence within R. turanicus. Therefore, a detailed morphological and molecular analysis was conducted for comparing R. turanicus from the Mediterranean Basin (represented by Albania) and Central Asia (Northwestern China). Altogether 315 adult ticks of R. turanicus (103 from Albania and 212 from China) were morphologically and molecularly analysed. DNA samples were used for mitochondrial 16S rRNA and cox1 gene sequences analysis. In addition, as potentially genetic markers, three fragments including partial nad1-16S rRNA, nad2-cox1, cox1-tRNA-Lys, were designed and then phylogenetically analyzed. Based on detailed morphological observations, only basis capituli length:width ratio (females), the length, the width and the length:width ratio of the scutum (males) had differences between R. turanicus from China and Albania. Gene divergences of 16S rRNA, cox1, partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys from China and Albania ticks were 3.53–4.84, 3.57–4.92, 3.57–4.07, 3.57–4.39 and 3.18–4.69%, respectively. The evaluated five genetic markers revealed two phylogenetic branches in R. turanicus. Obvious differences exist within R. turanicus based on morphological and genetic analysis. Three newly designed genetic markers (partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys) in this study may be suitable genetic tools for identification and analysis in R. turanicus. Subspecies analysis of R. turanicus from other regions of the world should be initiated in the future.

Probing the potential of the Shark Panel InDel multiplex v2.0 on the forensic identification of batoid elasmobranchs

The Elasmobranchii comprises the diverse and important group of sharks and rays. The group includes some of the ocean’s largest predatory fishes, suffering unsustainable fishing activities and is commercially overexploited for their meat and fins. Overfishing has resulted in significant population declines and several species are now considered under high threat and facing extinction, with about 93% of its nominal species included in the IUCN Red List. Molecular data have provided important information about these species, allowing the management of natural stocks and preventing their decline. Population genetics and connectivity data knowledge are now available and play an important role on establishing conservation policies. However, despite the ecological, commercial and conservation importance, no molecular method is available to identify sharks and rays in a forensic context. Following the development of the Shark Panel v1.0 which includes uniquely sharks, now we tested the effectiveness of the v2.0 multiplex on batoid elasmobranchs. We carried out a systematic molecular analysis using 85 previously published mitochondrial 16S rRNA gene sequences obtained from the NCBI database and found that indels in the 16S rRNA gene can be used to distinguish several analyzed species, including some of the most threatened according to IUCN Red List. The regions selected in this study can be used for the construction of molecular identification assays.

New species of the giant deep-sea isopod genus Bathynomus (Crustacea, Isopoda, Cirolanidae) from Hainan Island, South China Sea.

Several specimens of the giant deep-sea isopod genus Bathynomus were collected by a deep-sea lander at a depth of 898 m near Hainan Island in the northern South China Sea. After careful examination, this material and the specimens collected from the Gulf of Aden, north-western Indian Ocean, previously reported as Bathynomus sp., were identified to be the same as a new species to the genus. Bathynomus jamesi sp. nov. can be distinguished from the congeners by: the distal margin of pleotelson with 11 or 13 short straight spines and central spine not bifid; uropodal endopod and exopod with distolateral corner slightly pronounced; clypeus with lateral margins concave; and antennal flagellum extending when extended posteriorly reaches the pereonite 3. In addition, Bathynomus jamesi sp. nov. is also supported by molecular analyses based on mitochondrial COI and 16S rRNA gene sequences. The distribution range of the new species includes the western Pacific and north-western Indian Ocean.

A novel screening approach based on six real-time PCR systems for the detection of crustacean species in food

Analysis of crustacean species plays a role in authenticity issues as well as allergen detection. A real-time PCR-based screening assay was developed for the detection of crustaceans in food. In order to cover most relevant species in one analytical step, PCR systems were newly developed for the detection of shrimps (Penaeidae), lobster (Homarus sp.), Common shrimp (Crangon crangon), river prawns (Macrobrachium sp.) and Chinese mitten crab (Eriocheir sinensis). In addition a published system targeting Northern prawn (Pandalus borealis) was selected. All PCR-systems are based on mitochondrial 16S rRNA gene sequences and were optimized to be run with a standard program at a universal annealing temperature of 60 °C. Validation experiments confirmed a sensitivity of the PCR systems of 0.01–0.1 genome copies or 0.04–2.5 pg DNA, respectively. Specificity was demonstrated with 25,000 copies of pure genomic DNA from about thirty plant and animal species relevant in food and the performance in food matrix was evaluated. Finally primer and probes were pre-spotted steadily on 96-well PCR plates and the practical applicability of the assay was proven with selected food products. The assay enables detection of multiple species of market relevance.

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.

Molecular identification of honey entomological origin based on bee mitochondrial 16S rRNA and COI gene sequences

The increasing recognition and production of stingless bee honey has raised the needs to develop rapid, sensitive and accurate methodologies for honey identification through bees' species from the DNA of bees present in honey. Genetic identification of raw honeys produced by different bee species from Malaysia was performed based on mitochondrial DNA sequences and phylogenetic analysis by means of forensically informative nucleotide sequencing (FINS) technique. The 300 bp of mitochondrial large subunit ribosomal RNA (16S rRNA) gene region and mitochondrial cytochrome c oxidase subunit I (COI) gene region were the gene markers used to classify and identify honey produced by Apis honey bees and Trigona stingless bees. The genetic identities of honey origin from Apis dorsata, Apis mellifera, Apis cerana, and Heterotrigona itama were accurately identified. Kelulut honey sourced from the stingless bee was distinguishable from other Apis type of honeys in phylogenetic analyses. This FINS technique is a specific and reliable method for identification of honey origin via its bee source that can be used to overcome the issue of false declaration of honey origin and mislabelling.

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.

Phylogenetic Analysis of Lake Waccamaw Endemic Freshwater Mussel Species

Abstract: About 70% of freshwater mussel species in North America are threatened, endangered, or recently extinct. A large number of the surviving species are endemics. One hotspot for endemic diversity is the southeastern U.S.A. Lake Waccamaw in southeastern North Carolina contains two putative endemic unionids, Lampsilis fullerkati (Johnson, 1984) and Elliptio waccamawensis (Lea, 1863). We used multiple phylogenetic analyses to assess phylogenetic affinities and test the status of these named species. Mitochondrial 16S rRNA gene, cox1, and nad1 sequences were obtained from 109 individuals of the putative endemics and potentially related species from Lake Waccamaw, the Waccamaw River, and the Yadkin/Pee Dee, Little Pee Dee, and Lumber Rivers in the Pee Dee Drainage. In addition to the analysis of each individual gene region, a total evidence analysis, in which all three regions were combined, was conducted using Bayesian phylogenetic methods. All three genes were analyzed using both Bayesian and maximum ...

Molecular phylogeny of Barbin fishes of North - East Indiabased on mitochondrial 16SrRNA gene sequences

The Barbin fishes (Cypriniformes: Barbinae) have achieved great diversity in the world and cosmopolitan in distribution but their phyletic classification is ambiguous. Molecular phylogeny of barbin fishes of North-East India was studied by using mitochondrial 16S rRNA sequences. Partial sequences (540-615 bp) of mit. 16S rRNA genes of four species of barbin fishes – Pethia atra, Puntius chola, P. javanicus, and P. sophore were generated and sequences of 7 species were downloaded from GenBank for present study. The aim of our present study is to resolve the taxonomic relationship and to establish the molecular phylogeny of Barbin fishes of North-East India based on mitochondrial 16S rRNA genes sequences. The Maximum Parsimony Tree shows three clusters with high bootstrap value and present data indicates that all fishes in this group had a common ancestor but now they have separated into distinct evolutionary lineages.