C Oxidase Subunit 1 Research Articles

Two subspecies of the snail mite Riccardoella (Proriccardoella) reaumuri Fain & van Goethem, 1986 (Acari, Prostigmata, Ereynetidae) from Japan

Riccardoella (Proriccardoella) reaumuri Fain & van Goethem, 1986 has been reported in terrestrial mollusks in France and the Japanese Archipelago. The purpose of this study was to describe two new subspecies of this mite species from Japan based on their morphological characters. Mites were sampled from terrestrial mollusks at 13 locations in Japan from Oct. 16, 2016 to Nov. 13, 2020. The sampled mites and specimens deposited in museum were also used for morphological observations. As a result of principal component analysis (PCA) and cluster analysis based on morphology of dorsal setae on idiosoma and solenidia on palptarsus and tarsus I and II, mite specimens from France and Japan were divided into three groups: Group 1 (mites sampled in France), Group 2 (Okinawajima Is.) and Group 3 (the area from Kyushu Is. to Hokkaido Is.). The solenidia shapes also differed between the groups. We propose that the mites of each of the three groups should be defined as subspecies based on these morphological differences. Our phylogenetic tree of the two subspecies based on amino acid sequences of the mitochondrial cytochrome c oxidase subunit 1 (CO1) confirmed that these Japanese mites are members of the genus, family Ereynetidae.

Genetic diversity and phylogenetic analyses of ixodid ticks infesting cattle in northeast Thailand: the discovery of Rhipicephalus microplus clade C and the rarely detected R. haemaphysaloides.

In total, 160 ticks infesting cattle in the northeast region of Thailand were collected and used for molecular investigation. Three tick species-Rhipicephalus microplus Canestrini, Rhipicephalus haemaphysaloides Supino and Haemaphysalis bispinosa Neumann-were identified based on morphology and DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) and 16S ribosomal RNA (16S rRNA). In total, 26 and seven unique haplotypes of the CO1 and 16S rRNA genes, respectively, were recovered. Phylogenetic analysis using the CO1 sequence revealed that the R. microplus from northeastern Thailand were grouped into the previously described clades A and C, whereas the 16S rRNA phylogenetic tree assigned all isolates of R. microplus from Northeast Thailand into the previously described clade B. Clade C of the CO1 phylogenetic tree is a new genetic assemblage recently discovered from India and Malaysia, which has now been detected in our study. The haplotype network also demonstrated that R. microplus is divided into two haplogroups corresponding to the assemblage of the CO1 phylogenetic tree. Our findings strongly support the previous genetic assemblage classification and evidence that R. microplus from Northeast Thailand is a species complex comprising at least two genetic assemblages, i.e., clades A and C. However, further investigation is needed and should involve more comprehensive genetic and morphological analyses and cover a larger part of their distributional range throughout Southeast Asia.

Genetic variation of the giant water bug Lethocerus indicus (Lepeletier and Serville, 1825) (Hemiptera: Belostomatidae) collected from natural habitats in northeastern Thailand

The giant water bug, Lethocerus indicus (Lepeletier and Serville, 1825), is one of the most important edible insects in Thailand. This study investigated the genetic diversity of L. indicus from natural resources in northeast Thailand by using mitochondrial DNA sequences. Ninety adults of L. indicus were collected from 12 different localities by light trap and hand-net methods. Nucleotide and haplotype diversity of cytochrome c oxidase subunit 1 (CO1) sequences were relatively high. The 43 CO1 haplotypes (Li1–Li43) with 26 unique haplotypes were classified and used to generate a star-like haplotype network. The most common haplotypes were Li6 and Li2 shared between 19 and 15 sequences, respectively from nine different localities. A phylogenetic tree clearly separated L. indicus from the other species and closely clustered with a sibling species, L. patruelis (Stål, 1854). However, the comprehensive genetic variation of L. indicus covering broad range of areas needs to be further explored.

Best Practices for Authentication of Cell Lines to Ensure Data Reproducibility and Integrity.

Cell line misidentification and contamination are major contributors to the reproducibility crisis in academic research. Authentication of cell lines provides assurances of the data generated; however, commercially available cells are often not subjected to rigorous identification testing. In this study, commercially available cell lines underwent testing to confirm cell identity and purity. The methods reported here outline the best practices for cell line authentication. Briefly, a commercially available primary rabbit aortic endothelial cell line was purchased for the intent of producing target proteins necessary for generating species-specific recombinant antibodies. These rabbit-specific antibodies would then be utilized for the development of in-house enzyme-linked immunosorbent assays (ELISA) to evaluate blood-based biomarkers of vascular injury after total-body irradiation. To authenticate the cell line, cell identity and purity were determined by single tandem repeat (STR) testing, flow cytometry, polymerase chain reaction (PCR), and cytochrome c oxidase subunit 1 (CO1) DNA Barcoding in-house and/or through commercial vendors. Fresh cells obtained from a New Zealand White rabbit (Charles River, Wilmington, DE) were used as a positive control. The results of STR and flow cytometry analyses indicated the cells were not contaminated with human or mouse cells, and that the cells were not of endothelial origin. PCR demonstrated that cells were also not of rabbit origin, which was further confirmed by a third-party vendor. An unopened vial of cells was submitted to another vendor for CO1 DNA Barcoding analysis, which identified the cells as being purely of bovine origin. Results revealed that despite purchase through a commercial vendor, the cell line marketed as primary rabbit aortic endothelial cells were of bovine origin. Purity analysis found cells were misidentified rather than contaminated. Further investigation to determine the cell type was not performed. The most cost-effective and efficient methodology for confirming cell line identity was found to be CO1 DNA Barcoding performed by a commercial vendor.

Molecular identification and genetic-polymorphism analysis of Fasciola flukes in Dali Prefecture, Yunnan Province, China.

This study aimed to identify species of Fasciola flukes in Dali Prefecture (Yunnan Province, China) and analyze their genetic diversity. Fasciola flukes (n=122) were collected from cattle livers in a farmers' market in Xiaguan Town, Dali Prefecture. Nucleotide sequences of ribosomal internal transcribed spacer (ITS) as well as nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were amplified, sequenced, and subjected to homology analysis. The heterozygosity ratios of different ITS alleles were determined using the peak-height ratio of heterozygous loci. Multiplex PCR analysis of the nuclear protein coding gene, phosphoenolpyruvate carboxykinase (pepck), was used to identify Fasciola species. Multiple ND1 sequence alignments enabled further genetic diversity analysis of regional Fasciola flukes. Seven ITS sequences belonged to F. hepatica and 115 belonged to Fh/Fg heterozygous flukes. Sequencing analysis of heterozygous flukes revealed 11 heterozygous loci with double peaks, with significantly variable ratios among individuals. ND1 and CO1 results indicated that one specimen was identical to F. hepatica, while 121 specimens were identical to F. gigantica or contained one variable site. Multiplex PCR results for pepck showed that double bands for F. hepatica and F. gigantica were amplified from Dali Fasciola specimens; hence, they were all heterozygous. By combining ITS, ND1, and CO1 sequences with multiplex pepck PCR results, all 122 specimens were identified as Fh/Fg heterozygous Fasciola flukes. Our experimental results preliminarily confirmed a high degree of Fh/Fg heterozygosity among Fasciola flukes in the Dali area. Selecting multiple molecular markers for concurrent analysis will provide more comprehensive and accurate genetic information.

New geographic records for Echinococcus canadensis in coyotes and moose from Nova Scotia, Canada.

Echinococcus spp. tapeworms can cause serious diseases in mammals, including humans. Within the E. granulosus species complex, metacestodes produce unilocular cysts that are responsible for cystic echinococcosis in animal intermediate hosts. Canids are definitive hosts, harbouring adult cestodes in their intestines. Adult E. canadensis were recovered from the small intestine of 1 of 262 coyotes (Canis latrans) from Nova Scotia, Canada. Subsequently, we found unilocular cysts in lungs and livers of 4 of 8 sympatric moose (Alces alces) from Cape Breton Island. DNA was extracted from three cysts using the Qiagen DNeasy Blood and Tissue kit and assayed by polymerase chain reaction (PCR) with primers (cest4 and cest5) for a 117-bp region of the small subunit of ribosomal RNA of E. granulosus sensu lato, and further validated as E. canadensis G8 using primers targeting nicotinamide adenosine dinucleotide dehydrogenase subunit 1 (ND1) and cytochrome c oxidase subunit 1 (CO1) mitochondrial genes. These are the first records of E. canadensis in any of the three Maritime provinces, which include Nova Scotia, New Brunswick, and Prince Edward Island. The parasite was thought to be absent in this region due to extirpation of wolves (Canis spp.) in the 1800s. These findings suggest that further wildlife surveillance and risk assessment is warranted.

COI mtDNA barcoding and morphology for the description of a new species of ricinuleid of the genus Pseudocellus (Arachnida: Ricinulei: Ricinoididae) from El Triunfo Biosphere Reserve, Chiapas, Mexico

A new species of epigean ricinuleid of the genus Pseudocellus Platnick, 1980 from El Triunfo Biosphere Reserve, Chiapas, Mexico is described. DNA barcoding utilizing mitochondrial cytochrome c oxidase subunit 1 (CO1) and morphology were used for species delimitation. Molecular analyses and species delimitation included four methods: 1) General Mixed Yule Coalescent model (GMYC), 2) Automatic Barcode Gap Discovery (ABGD), 3) Bayesian Poisson Tree Process (bPTP), and 4) Assemble Species by Automatic Partitioning (ASAP). All molecular methods and morphology were consistent in delimiting and recognizing the new species described herein. The average interspecific genetic distance (p-distance) among analyzed species of Pseudocellus was 11.6%. The species is described based on adult males and females: Pseudocellus giribeti sp. nov. This is the seventh species described from Chiapas, which holds the highest number of ricinuleids species for the country. The total number of described species of Pseudocellus from Mexico increases to 21, having the highest species diversity of known ricinuleids worldwide.

Mitochondrial phylogenetic and diversity analysis in Azi-Kheli buffalo.

The present study was conducted for the first time in Pakistan to investigate Cytochrome C Oxidase Subunit 1 (CO1) gene and full-length Displacement Loop (D-loop) region of mitochondrial DNA in Azi-Kheli buffalo breed native to northern hilly areas of Khyber Pakhtunkhwa Province of Pakistan. The present study was designed to investigate phylogeny and diversity in Azi-Kheli buffalo, through two mitochondrial DNA regions, i.e., Cytochrome C Oxidase Subunit-I (CO1) and Displacement Loop (D-loop) region. Thirty (30) blood samples were taken from Azi-Kheli pure breed animals from original breeding tract, i.e., Khwazakhela, Swat. Polymerase chain reactions using gene-specific primers were carried out for amplifying 709-bp region of CO1 gene and 1159-bp region of D-Loop for identification, phylogeny, and diversity in Azi-Kheli buffalo, respectively. The sequences of CO1 gene revealed four (04) haplotypes, whereas D-loop sequences revealed five (05) haplotypes. Mean interspecific diversity with related species was 2.56%, and mean intraspecific diversity within Azi-Kheli buffalo was 0.25%, estimated via Kimura-2 parameter. Phylogenetic tree (maximum likelihood) revealed clustering of Azi-Kheli haplotypes with river buffalo and is distinct from swamp buffalo and other related species of genus Bubalus. Mean haplotype and nucleotide diversity of D-loop were Hd = 0.9601 ± SD = 0.096 and π = 0.01208 ± SD = 0.00182, respectively. Phylogenetic tree (neighbor-joining) revealed two main clades, i.e., river buffalo and swamp buffalo clade. The haplotypes of Azi-Kheli clustered with haplotypes of different river buffalo breeds at different positions. The current study suggests that Azi-Kheli has common origin with other river buffalo breeds; hence, it is river buffalo which harbors high genetic diversity.

Host and geographic differences in prevalence and diversity of gastrointestinal helminths of foxes (Vulpes vulpes), coyotes (Canis latrans) and wolves (Canis lupus) in Québec, Canada.

Wild canids are hosts to a wide range of parasites and can play a role in transmission of zoonoses. As many parasites are transmitted through food webs, and wild canids are at high trophic levels, parasite prevalence and diversity in wild canids can serve as excellent indicators of ecosystem health. Our main objectives were to update knowledge on the composition of gastrointestinal helminths in wild canids from Québec, Canada, and to describe differences in parasite prevalence and diversity among canid species and regions. Hunters and trappers provided whole carcasses of red foxes (Vulpes vulpes) (N = 176), and intestinal tracts of coyotes (Canis latrans) (N = 77) and gray wolves (Canis lupus) (N = 23) harvested for non-research purposes over the winter of 2016–2017. A modified Stoll's centrifugation sucrose flotation on feces of 250 wild canids was used, and eggs of one family and eight genera of parasitic helminths were recovered: diphyllobothriids, Taenia/Echinococcus spp., Capillaria spp., Toxascaris sp., Toxocara sp., Trichuris sp., Uncinaria sp., and Metorchis sp. Adult Taenia spp. cestodes were recovered from 61 of 276 (22%) canids. Six different species (T. hydatigena, T. twitchelli, T. crassiceps, T. polyacantha, T. krabbei, and T. pisiformis-“like”) were differentiated based on DNA sequenced from 65 individual adult cestodes using primers for the nicotinamide adenosine dinucleotide dehydrogenase subunit 1 (ND1) and cytochrome c oxidase subunit 1 (CO1) mitochondrial DNA loci. Alaria sp. trematodes infected 89 of 276 canids (32%). A subset were identified as A. americana at the CO1 locus. The marine trematode Cryptocotyle lingua was reported for the first time in foxes in the province of Québec. These results help us understand more fully the predator-prey relationships within this group of canids. This baseline data in regional parasite prevalence and intensity is critical in order to detect future changes following ecological disturbances due to climate and landscape alterations.

Development of a SNP-based tool for the identification and discrimination of Melolontha melolontha and Melolontha hippocastani.

The European (Melolontha melolontha L.) and Forest (M. hippocastani F.) cockchafer are widespread pests throughout Central Europe. Both species exhibit a 3-5-year life cycle and occur in temporally shifted populations, which have been monitored and documented for more than 100 years. Visual identification of adults and larvae belonging to these morphologically similar species requires expertise and, particularly in the case of larvae, is challenging and equivocal. The goal of the study was the development of an efficient and fast molecular genetic tool for the identification and discrimination of M. melolontha and M. hippocastani. We established a collection of both species from Switzerland, Austria and Northern Italy in 2016, 2017 and 2018. An approximately 1550 bp long fragment of the cytochrome c oxidase subunit 1 (CO1) mitochondrial gene was amplified and sequenced in 13 M. melolontha and 13 M. hippocastani beetles. Alignment of the new sequences with reference sequences (NCBI GenBank and BOLDSYSTEMS databases) and subsequent phylogenetic analysis revealed consistent clustering of the two species. After the identification of M. melolontha and M. hippocastani species-specific single nucleotide polymorphisms (SNPs) in the CO1 alignment, we developed an effective SNP tool based on the ABI PRISM® SNaPshot™ Multiplex Kit for the rapid and accurate species discrimination of adults and larvae.

The First Report for the Presence of Spiroplasma and Rickettsia in Red Palm Weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in Egypt.

The study of the Red Palm Weevil (RPW), Rhynchophorus ferrugineus (Olivier), as an invasive pest of palm trees. In this study, 36 RPW individuals were collected from 6 different locations in Egypt. The presences of endosymbionts in the RPW individuals were assayed. The phylogenetic analysis of the RPW inhabiting Egypt was conducted using Cytochrome c oxidase sub-unit 1 (CO1) gene. Spiroplasma was found, for the first time, in all individuals, while Rickettsia was found, for the first time, in individuals collected from only 3 of the 6 locations. Endosymbionts harbouring Egyptian RPW were closely related to those harbouring Diptera and\or Trombidiformes associated with palm trees. This may be due to horizontal transmission through palm sap or through ectoparasites living on the RPW. Finally, the phylogenetic analysis of the RPW inhabiting Egypt was conducted. The collected individuals were closely related to Saudi Arabia specimens collected from the eastern region. Thus, Saudi Arabia may be the origin of the RPW which invaded Egypt. Individuals from populations inhabiting the same geographical locations were closely related. This may be due to secondary invasion incidents that may have taken place through transportation of infested date palm trees and offshoots from infected to uninfected locations. This study reports the first incidence for the presence and coexistence of Spiroplasma and Rickettsia in RPW collected from Egypt. In addition, it was found that the collected individuals of RPW were closely related to a Saudi haplotype. Thus, Saudi Arabia may be the origin of infection which invaded Egypt.

A newGammarusspecies(Crustacea,Amphipoda,Gammaridae) fromNorthwesternIslands,SouthKorea.

A new species, Gammarus baengnyeongensis sp. nov., belonging to the family Gammaridae Leach, 1814 was collected from the Baengnyeongdo and Daecheongdo islands in South Korea. This new species is characterized by the flagellum of antenna 2 with calceoli, and the length ratio of outer ramus to inner ramus of uropod 3. Descriptions of diagnostic characteristics of the species are provided in the text. We performed statistical analysis to confirm the significance of morphological differences of interspecies. Additionally, we compare the new species to related species by the partial sequence of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Genetic distances between the new species and closely related species rated from 29.1-40.8%, which highly supports Gammarus baengnyeongensis sp. nov. as a valid species. In this study, we add one new Gammarus species in Korean waters.

The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

The presence of dog meat is a crucial issue because dog meat is non-halal meat for Muslims. The objective of this study was to design and validate species-specific primer for the identification of dog meat DNA in meatball using real-time polymerase chain reaction (real-time PCR). The specific primer targeting mitochondrial cytochrome c oxidase subunit 1 (CO1) was validated. The specific primers used were designed using Integrated DNA Technologies (IDT) software and subjected to NCBI BLAST procedure. The candidate primers were tested for specificity study using several DNAs from fresh meat of pork, chicken, beef, lamb, and rat. The method was also validated by determining several parameters of linearity, sensitivity, precision, and efficiency. The results showed that primer could amplify specifically DNA target at an optimized annealing temperature of 56.6 °C. The limit of detection (LoD) obtained was 5 ng DNA, corresponding to 2.5% of dog meat in a meatball. The repeatability evaluation, expressed with relative standard deviation (RSD), and efficiency value was in the acceptable range (RSD < 25% and efficiency (90–105%). This method was successfully used for the analysis of marketed samples. Real-time PCR can be used as a standard method in halal authentication analysis through DNA analysis.

Genetic Diversity of Ligidium Isopods in Hokkaido and Niigata, Northern Japan, Based on Mitochondrial DNA Analysis.

The genetic diversity of the genus Ligidium in Hokkaido and Niigata, northern Japan, was investigated by analyzing the cytochrome c oxidase subunit 1 (CO1) region in the mitochondrial DNA (mtDNA). The genetic diversity in Hokkaido was much lower than that in Niigata. Nine different operational taxonomic units (OTUs) were identified. Only a single OTU, most likely Ligidium japonicum, was found in Hokkaido, whereas all nine OTUs were found in Niigata. Using the mtDNA evolutionary rate determined for the marine invertebrate Haptosquilla pulchella (Miers, 1880), population expansion for OTU1 in Hokkaido was estimated to have occurred at 12,600 years BP, suggesting that Ligidium underwent a bottleneck due to glacial cooling, and the population then expanded after postglacial warming. Assuming that the expansion of the OTU1 population occurred at 9600 years BP, when the sea surface temperature rose offshore of Tokachi in the Northwestern Pacific, the evolutionary rate (µ) of the mtDNA CO1 region in Ligidium is calculated as: 0.087 (95% confidence intervals: min: 0.042-max: 0.12) (substitutions/site/million years). The presence of a haplotype common to Hokkaido and Niigata implies that the haplotype migrated across the Tsugaru Strait. Considering that geological evidence indicates that the Tsugaru Strait was continuously present even during the last glacial maximum when the sea level was at its lowest, accidental transport by human beings or animals might have been critical to the migration of Ligidium.

Description of a new species of Loxosceles Heineken & Lowe (Araneae, Sicariidae) recluse spiders from Hidalgo, Mexico, under integrative taxonomy: morphological and DNA barcoding data (CO1 + ITS2)

Based on an integrative taxonomic approach, a new species of the genus Loxosceles Heineken & Lowe, 1832, is described from the state of Hidalgo, Mexico. Loxosceles tolantongo sp. nov. is described based on DNA barcoding using cytochrome c oxidase subunit 1 (CO1) and internal transcribed spacer 2 (ITS2), and morphology. For species delimitation, four molecular methods were implemented: 1) corrected p-distances under neighbor joining (NJ); 2) automatic barcode gap discovery (ABGD); 3) general mixed yule coalescent model (GMYC) and 4) Bayesian Poisson tree processes (bPTP). The new species morphologically resembles L. jaca, another species from Hidalgo, but there are morphological differences mainly in the tibiae of the male palp, the seminal receptacles of the females and also the high genetic p-distances. CO1 was more informative than ITS2 for the genetic separation; however, both concatenated genes (CO1 + ITS2) present robust evidence for species delimitation. Loxosceles tolantongo sp. nov. is considered a unique species for four reasons: 1) it can be diagnosed and distinguished by morphological characters (of the male palps mainly, but also of the seminal receptacles of the females); 2) the genetic p-distances with CO1 were high (>10%); 3) the molecular species delimitation methods were congruent under CO1 and CO1 + ITS2; and 4) under CO1 and CO1 + ITS2, the new species is a putative sister group of L. jaca + L. tenango.

Novel Molecular Resources to Facilitate Future Genetics Research on Freshwater Mussels (Bivalvia: Unionidae)

Molecular data have been an integral tool in the resolution of the evolutionary relationships and systematics of freshwater mussels, despite the limited number of nuclear markers available for Sanger sequencing. To facilitate future studies, we evaluated the phylogenetic informativeness of loci from the recently published anchored hybrid enrichment (AHE) probe set Unioverse and developed novel Sanger primer sets to amplify two protein-coding nuclear loci with high net phylogenetic informativeness scores: fem-1 homolog C (FEM1) and UbiA prenyltransferase domain-containing protein 1 (UbiA). We report the methods used for marker development, along with the primer sequences and optimized PCR and thermal cycling conditions. To demonstrate the utility of these markers, we provide haplotype networks, DNA alignments, and summary statistics regarding the sequence variation for the two protein-coding nuclear loci (FEM1 and UbiA). Additionally, we compare the DNA sequence variation of FEM1 and UbiA to three loci commonly used in freshwater mussel genetic studies: the mitochondrial genes cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1), and the nuclear internal transcribed spacer 1 (ITS1). All five loci distinguish among the three focal species (Potamilus fragilis, Potamilus inflatus, and Potamilus purpuratus), and the sequence variation was highest for ND1, followed by CO1, ITS1, UbiA, and FEM1, respectively. The newly developed Sanger PCR primers and methodologies for extracting additional loci from AHE probe sets have great potential to facilitate molecular investigations targeting supraspecific relationships in freshwater mussels, but may be of limited utility at shallow taxonomic scales.

Biometric and genetic differences in kelabau ( Osteochilus spp.) as revealed using cytochrome c oxidase subunit 1.

Background: Kelabau ( Osteochilus spp.) is a freshwater fish commonly found in the rivers of Riau, Indonesia. Researchers believe that these are Osteochilus kelabau; however, accurate taxonomic determination of these fish in Riau waters has not been made. The purpose of this study was to facilitate the identification of the kelabau based on its morphology and genetics using biometric and cytochrome c oxidase subunit 1 ( CO1) analyses, respectively. Methods: Fish samples were collected from the Siak, Kampar and Rokan rivers in Riau Province, Indonesia. The DNA of 90 fish was extracted from the caudal fins using a DNA extraction kit, after which it was amplified using primers Fish-F1 and Fish-R1. Sequencing was conducted by Applied Biosystems Macrogen Korea, and the DNA sequences were then edited and aligned using MEGA v. 7. All samples were BLAST-searched for identification using the National Center for Biotechnology Information and BOLD System. Phylogenetic trees were constructed, and the similarity index was calculated using accession numbers AP011385.1 and KC631202.1 in GenBank. Results: Analysis of the consensus barcode sequence for 86 species revealed a high percentage of barcode matches (96%-97% in GenBank and 96.6%-96.76% in the BOLD System). The nucleotide distance between groups of kelabau from the different rivers based on the Kimura 2-parameter model gave the following results: 0.05% between groups from the Siak and Kampar rivers, 0.09% between those from the Siak and Rokan rivers and 0.05% between those from the Kampar and Rokan rivers. The nucleotide distance between the groups in the Siak (0.09%), Kampar (0.00%) and Rokan (0.10%) Rivers indicated that the kelabau in those rivers were related to each other. Conclusions: Based on the results of the research data using CO1 and biometric analyses, the kelabau were confirmed to be O. melanopleurus.

PCR Cloning Combined With DNA Barcoding Enables Partial Identification of Fish Species in a Mixed-Species Product

DNA barcoding is a valuable tool for regulatory identification of fish species; however, it does not perform well when multiple species are present within the same food product. Therefore, the objective of this study was to examine the use of PCR cloning to identify fish in a mixed-species product that cannot be identified with standard DNA barcoding. A total of 15 fish ball mixtures were prepared with known amounts of Nile tilapia (Oreochromis niloticus), Pacific cod (Gadus macrocephalus), and walleye pollock (Gadus chalcogrammus). Three subsamples from each fish ball underwent DNA extraction, full DNA barcoding (655 bp), and mini-barcoding (226 bp) of the cytochrome c oxidase subunit 1 (CO1) gene. Subsamples that did not pass sequencing according to regulatory standards were further analyzed with PCR cloning. All fish balls made of just one species tested positive for that species (i.e., tilapia, cod, or pollock) with both full and mini-barcoding. However, only tilapia was detected in fish balls containing multiple species when tested with standard barcoding techniques, reflecting an inaccurate representation of the fish mixture and suggesting species bias. PCR cloning allowed for identification of Pacific cod in 86% of the mixed-species fish balls tested with full-barcode cloning and 100% of the mixed-species fish ball tested with mini-barcode cloning. However, PCR cloning did not enable the identification of walleye pollock. Standard full barcoding produced more high quality sequences compared to mini-barcoding yet failed to accurately detect all species present in the tested fish mixtures. Overall, the results of this study show that PCR cloning may be an effective method to identify certain fish in mixed-species products when standard DNA barcoding fails. However, additional research is needed to overcome the species bias observed in this study.

Biometric and genetic differences in kelabau ( Osteochilus spp.) as revealed using cytochrome c oxidase subunit 1.

Background: Kelabau ( Osteochilus spp.) is a freshwater fish commonly found in the rivers of Riau, Indonesia. Researchers believe that these are Osteochilus kelabau; however, accurate taxonomic determination of these fish in Riau waters has not been made. The purpose of this study was to facilitate the identification of the kelabau based on its morphology and genetics using biometric and cytochrome c oxidase subunit 1 ( CO1) analyses, respectively. Methods: Fish samples were collected from the Siak, Kampar and Rokan rivers in Riau Province, Indonesia. The DNA of 90 fish was extracted from the caudal fins using a DNA extraction kit, after which it was amplified using primers Fish-F1 and Fish-R1. Sequencing was conducted by Applied Biosystems Macrogen Korea, and the DNA sequences were then edited and aligned using MEGA v. 7. All samples were BLAST-searched for identification using the National Center for Biotechnology Information and BOLD System. Phylogenetic trees were constructed, and similarity index was calculated using accession numbers AP011385.1 and KC631202.1 in GenBank. Results: Analysis of the consensus barcode sequence for 86 species revealed a high percentage of barcode matches (96%-97% in GenBank and 96.6%-96.76% in the BOLD System). The nucleotide distance between groups of kelabau from the different rivers based on the Kimura 2-parameter model gave the following results: 0.05% between groups from the Siak and Kampar rivers, 0.09% between those from the Siak and Rokan rivers and 0.05% between those from the Kampar and Rokan rivers. The nucleotide distance between the groups in the Siak (0.09%), Kampar (0.00%) and Rokan (0.10%) Rivers indicated that the kelabau in those rivers were related to each other. Conclusions: Based on the results of the research data using CO1 and biometric analyses, the kelabau were confirmed to be O. melanopleurus.

Genetic variation and phylogenetic relationship of Hypoderaeum conoideum (Bloch, 1782) Dietz, 1909 (Trematoda: Echinostomatidae) inferred from nuclear and mitochondrial DNA sequences

Objective: To explore genetic variations of Hypoderaeum conoideum collected from domestic ducks from 12 different localities in Thailand and Lao PDR, as well as their phylogenetic relationship with American and European isolates. Methods: The nucleotide sequences of their nuclear ribosomal DNA (ITS), mitochondrial cytochrome c oxidase subunit 1 (CO1), and NADH dehydrogenase subunit 1 (ND1) were used to analyze genetic diversity indices. Results: We found relatively high levels of nucleotide polymorphism in ND1 (4.02%), whereas moderate and low levels were observed in CO1 (2.11%) and ITS (0.96%), respectively. Based on these polymorphisms, the 20 ND1, 12 CO1, and 18 ITS haplotypes were classified, and several common haplotypes were observed in all samples. At least three major lineages, namely American, European and Asian lineages, have been classified by phylogenetic analyses based on ND1 sequences. Conclusions: Our report demonstrates that the ND1 gene is the most suitable genetic marker to explore genetic variation and phylogenetic relationship of Hypoderaeum conoideum. However, a combination of all loci for ND1, CO1 and ITS would be of great value toward further genetic investigation of this endemic worldwide parasite. Thus, comprehensive molecular genetic analyses of Hypoderaeum conoideum from its worldwide distribution is needed to further understanding of the evolutionary and systematic relationships of this parasite.