Barcode analysis using mini-amplicons strategy for museum samples of neotropical primates Callithrix spp.

BackgroundPlasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources.ResultsA population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable.ConclusionsThis two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

Sequence analysis based on the mitochondrial cytochrome c oxidase subunit Ⅰ(COI) gene fragment was conducted for 21 clam species belonging to the family Veneridae to determine the utility of this sequence in identifying phylogeographic and phylogenetic structure.The DNAs of 21 clam species were extracted and subjected to PCR using primers COI-F and COI-R.The resultant COI fragment was 707 bp in all species,with A+T contents ranging from 62.4%—67.8%.The COI gene sequences of all 21 species were analyzed using MEGA 5.0 software.Interspecific COI sequence divergences was very high,ranging from 7.2%—38.2%,mainly as a result of transitions and transversions.The minimum value of 7.2% was observed between Meretrix meretrix and Meretrix lusoria,and the maximum value of 38.2% was between Tapes literata and Cyclina sinensis.M.lusoria exhibited 0.4% intraspecific sequence divergence among three sequenced individuals.However,for other species,the COI sequences were identical among the three sequenced individuals for each species.An analysis of COI gene sequence variation among 21 species revealed 379 variable sites,of which 334 were parsimony-informative sites,with 72 transitions(si) and 93 transversions(sv)(si/sv=0.8).Meanwhile,no indels(insertions/deletions) were detected in the COI sequences from any samples.The percentage of interspecific sequence divergence within the same genus,intergeneric sequence divergence within the same subfamily,and those between subfamilies were analyzed.The mean values were 12.5%—27.3%,19.7%—29.0% and 29.2%,respectively.The COI fragment encodes 235 amino acids,of which 100 sites were variable.A phylogenetic tree of 35 Veneridae clams was constructed based on the COI nucleotide sequences,with Mactra chinensis as an outgroup species.Tree topologies showed that all species of Veneridae are in the same group,forming a clade distinct from the outgroup species.Within Veneridae,six species of the subfamily Dosininae formed a clade with a bootstrap value of 75%.Two species of the subfamily Venerinae formed another clade with a bootstrap value of 93%.All species of the subfamily Meretricinae formed a monophyletic group with a bootstrap value of 70%,in which M.meretrix,M.lusoria and Meretrix petechialis were first clustered with a high bootstrap value of 99%.This result indicated that M.meretrix,M.lusoria,and M.petechialis have a closer relationship than other species.An analysis of tree topology and sequence alignment indicated that M.lusoria,M.petechialis,and M.meretrix are closely related species.The data support the viewpoint that M.petechialis and M.lusoria should be treated as synonymous with M.meretrix,and may be described as two geographical subspecies.The data also support the perspective that Dosinia corrugata and Dosinia angulosa are two distinct species.The validity of Paphia undulata and Paphia textile as different species is also supported.This result reveals that the COI gene is an appropriate marker for studies in phylogeny and phylogeography of the Veneridae clams.

Objective To apply the DNA barcoding technology for identification of rodent animals and to establish a rodent animal DNA barcode database in Gansu Province. Methods A total of 54 rodent animals were detected. DNA barcoding technology was used to analyze the DNA mitochondrial cytochrome C oxidase subunit I (CO I) gene sequence in Gansu Province. Results The intra-specific genetic distance was 0-2% while the inter-specific distance ranged from 18% to 30%. Eight major clusters were apparently showed on a Neighbor joining tree. Conclusion DNA barcoding technology could overcome the shortcomings of the morphological identification, so it could be used to identify the rodent animals and has important implications for disease control and prevention in the natural focus of Gansu Province. Key words: Rodentia; DNA barcoding; Mitochondrial cytochrome C oxidase subunit I; Species identification

Characterizing malaria epidemiology at the local level requires understanding the diverse malaria vector species driving transmission, including both primary and secondary vectors. Effective mosquito surveillance and accurate species identification are critical; however, due to the associated cost and complexity, most surveillance strategies mainly focus on the primary malaria vectors. There is a need for cost-effective methods that can reliably identify both primary and secondary vectors as their role in transmission becomes increasingly important while reaching towards elimination. This study aimed to evaluate the use of MALDI-TOF MS as a sustainable tool for identifying secondary malaria vector. Adult mosquitoes were collected in Kenya and Mozambique and morphologically identified. Secondary malaria vectors were considered as any Anopheline that did not pertain to Anopheles gambiae sensu lato (s.l.). or Anopheles funestus sensu lato (s.l.). At KEMRI Wellcome Trust Research Programme, MALDI TOF MS spectra were obtained from individual cephalothoraxes. Library creation and querying were guided by confirmatory species identification using Sanger sequencing of a subset of mosquitoes, targeting the Internal Transcribed Spacer 2 (ITS2) region of nuclear ribosomal DNA and the mitochondrial Cytochrome c Oxidase Subunit I (COI) gene. The libraries were then applied for the identification of other secondary malaria vectors. Species identification of secondary malaria vectors using MALDI-TOF MS showed high concordance with Sanger sequencing with an overall accuracy of 91% and a kappa value of 0.87. The technique demonstrated a sensitivity and specificity of 100% for most species, except for distinguishing between Anopheles cf. coustani 2 NFL-2015 and Anopheles ziemanni. In Kenya, the Anopheles species identified were Anopheles cf. coustani 2 NFL-2015 (19), Anopheles pretoriensis (6), Anopheles rufipes (8), Anopheles ziemanni (8), Anopheles coustani (2), and Anopheles pharoensis (1). In Mozambique, the identified species comprised: An. cf. coustani 2 NFL-2015 (10), An. pretoriensis (2), An. ziemanni (7), An. coustani (28), and An. pharoensis (4). The results provide evidence that MALDI-TOF can identify secondary malaria vectors from Eastern and Southeastern African regions. This technique was as efficient as DNA sequencing in identifying mosquito species. Indeed, except for An. cf coustani 2NFL-2015 and An. ziemanni, an exact species identification was obtained for all individual mosquitoes. These findings highlight the potential of MALDI-TOF MS for monitoring malaria vectors.

DNA barcoding is a novel concept for the taxonomic identification, in that it uses a specific short genetic marker in an organism’s DNA to discriminate species. In 2003, professor Paul D. N. Hebert, “the father of DNA barcoding”, of the University of Guelph, Ontario, Canada first proposed the idea to identify biological species using DNA barcode, where the mitochondrial gene cytochrome c oxidase subunit I (COI) was supposed to be the first candidate for animals (Hebert et al. 2003a). Their studies of COI profiling in both higher taxonomic categories and species-level assignment demonstrated that COI gene has significant resolutions across the animal kingdom except the phylum Cnidaria (Hebert et al. 2003b, Ward et al. 2005, Hajibabaei et al. 2006). From then on, a wide broad of taxonomic groups (i.e. birds, fish, butterflies, spiders, ants, etc) were examined by COI gene for its usability as the barcode (i.e. Hebert et al. 2004a, Hebert et al. 2004b, Greenstone et al. 2005, Smith et al. 2005, Barber and Boyce 2006, Meier et al. 2006, Kerr et al. 2007, Kumar et al. 2007, Pfenninger et al. 2007, Stahls and Savolainen 2008, Zhou et al. 2009). Meanwhile, other candidate genes, including Internal Transcribed Spacer (ITS), trnH-psbA intergenic spacer (trnH-psbA), Ribulose-bisphosphate carboxylase (rbcL) and Maturase K (matK) were analysed by different research groups (Jaklitsch et al. 2006, Evans et al. 2007, Ran et al. 2010, de Groot et al. 2011, Liu et al. 2011, Piredda et al. 2011, Yesson et al. 2011). Till recently, there are about 30 DNA barcode candidates are tested, and 4 to 8 of them are widely used for the identification of diversified taxonomic groups with a relatively good resolution. It has been estimated that there are 10 to 100 million species of living creatures in the earth, while what we know is very limited. Knowing the biodiversity is one of the crucial biological issues of ecology, evolutionary biology, bio-security, agro-biotechnology, bioresources and many other areas. For very long period, taxonomists have provided a nomenclatural hierarchy and key prerequisites for the society. However, the needs for species identification requested by non-taxonomists require the knowledge held by taxonomists. Therefore, a standardized, rapid and inexpensive species identification approach is needed to establish for the non-specialists. There had some attempts on the molecular identification systems based on polymerase chain reaction (PCR), especially in bacterial studies (Woese 1996, Zhou et al. 1997, Maiden et al. 1998, Wirth et al. 2006), but no successful solutions for broader scopes of eukaryotes (reviewed in Frezal and Leblois 2008). The DNA Barcode of Life project is another attempt to create a universal eukaryotic identification system based on molecular approaches. Following studies by Hebert et al.

Environmental DNA (eDNA) is a sensitive, non-invasive approach for surveying rare freshwater fishes, and is particularly effective for species whose low densities and behavioural traits limit conventional detection (e.g., electrofishing). To enable sensitive and reliable detection of the European mudminnow ( Umbra krameri ) from molecular traces, we developed and compared three species-specific primer sets (Suppl. material 1). First, we assembled the complete mitochondrial genome of U. krameri from PacBio HiFi reads to identify suitable regions for primer development. We then designed primer pairs to amplify short fragments targeting three mtDNA loci: a 129-base-pair (bp) fragment of cytochrome c oxidase subunit I (COI); a 178-bp fragment of NADH dehydrogenase subunit 2 (ND2); and a 217-bp fragment of NADH dehydrogenase subunit 1 (ND1). a 129-base-pair (bp) fragment of cytochrome c oxidase subunit I (COI); a 178-bp fragment of NADH dehydrogenase subunit 2 (ND2); and a 217-bp fragment of NADH dehydrogenase subunit 1 (ND1). The COI and ND2 assays combined primers with FAM-labelled hydrolysis probes, whereas the ND1 assay consisted of primers only (no probe). The specificity was first assessed in silico using Primer-BLAST and then tested empirically on tissue derived DNA extracts from 25 fish species occurring in Austrian waters, as well as on 15 eDNA samples from an old Danubian sidearm system (Fadenbach). This system is home to one of the last self-sustaining populations of U. krameri within Austrian borders. Potential method-specific biases were assessed by comparing eDNA detection results with catch data from fish traps collected during the same sampling campaign. This workflow enabled the identification of the most sensitive primer pair, which should be considered for routine monitoring and future conservation assessments.

Genus Parapenaeopsis constitute an important fishery resource in Indian waters. Parapenaeopsis stylifera, being morphologically very similar to Parapenaeopsis coromandelica, mainly differentiated using telsonic armature and it is subject of controversy regarding the number of armature for the species. The present study was aimed to resolve the taxonomic ambiguity between P. stylifera and P. coromandelica and to study phylogenetic relationship among five species under the genus Parapenaeopsis using partial sequences of mitochondrial 16S ribosomal DNA (rDNA) and cytochrome c oxidase subunit I (COI) genes. Sequence analysis revealed an average genetic divergence value ranging from 7.65 to 20.3% for COI gene and 2.4 to 12.0% for 16S rDNA gene between five species under study. The result shows that P. stylifera and P. coromandelica Alcock, 1906 are genetically a distinct species with average divergence value of 7.6% based on COI sequence and 2.4% based on the 16S rDNA. However, systematic relationship between the P. stylifera and P. coromandelica were closer than other species, thus formed separate clade in phylogenetic tree. The species specific molecular signatures developed in the present study will be helpful in effective fisheries management of the species in Indian waters.

BackgroundDNA barcoding is a valuable taxonomic tool for rapid and accurate species identification and cryptic species discovery in black flies. Indonesia has 143 nominal species of black flies, but information on their biological aspects, including vectorial capacity and biting habits, remains underreported, in part because of identification problems. The current study represents the first comprehensive DNA barcoding of Indonesian black flies using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences.MethodsGenomic DNA of Indonesian black fly samples were extracted and sequenced, producing 86 COI sequences in total. Two hundred four COI sequences, including 118 GenBank sequences, were analysed. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed and species delimitation analyses, including ASAP, GMYC and single PTP, were performed to determine whether the species of Indonesian black flies could be delineated. Intra- and interspecific genetic distances were also calculated and the efficacy of COI sequences for species identification was tested.ResultsThe DNA barcodes successfully distinguished most morphologically distinct species (> 80% of sampled taxa). Nonetheless, high maximum intraspecific distances (3.32–13.94%) in 11 species suggested cryptic diversity. Notably, populations of the common taxa Simulium (Gomphostilbia) cheongi, S. (Gomphostilbia) sheilae, S. (Nevermannia) feuerborni and S. (Simulium) tani in the islands of Indonesia were genetically distinct from those on the Southeast Asian mainland (Malaysia and Thailand). Integrated morphological, cytogenetic and nuclear DNA studies are warranted to clarify the taxonomic status of these more complex taxa.ConclusionsThe findings showed that COI barcoding is a promising taxonomic tool for Indonesian black flies. The DNA barcodes will aid in correct identification and genetic study of Indonesian black flies, which will be helpful in the control and management of potential vector species.Graphical abstract

Amphibians, a diverse and ecologically important group, are facing global declines due to various factors, including habitat loss and climate change. Accurate species identification is crucial for effective conservation efforts, and DNA barcoding has emerged as a powerful tool in this regard. This study compares the efficacy of two DNA barcoding primer sets, targeting the 16S ribosomal RNA gene and the Cytochrome Oxidase I (COI) gene, for identifying 20 amphibian species. While both primer sets successfully amplified sequences, the 16S rRNA gene region identified all 20 samples, whereas the COI region identified 14. The amplified sequences, approximately 550 base pairs for 16S rRNA and 658 base pairs for COI facilitated precise taxonomic placement within amphibian families using Neighbor-Joining phylogenetic trees. These findings enhance DNA barcoding methodology and aid in understanding amphibian diversity, crucial for effective conservation strategies amidst global declines drives by habitat loss, diseases, and climate change.

Loss in apoptosis competence often results in augmented genomic instability contributing to carcinogenesis. Cytochrome c oxidase subunit I (CcOI) can help assess apoptosis resistance in paraffin-embedded biopsies. In total, 50 colorectal cases including 10 control cases of colectomy for non-neoplastic condition, 15 cases of adenomatous colorectal polyps, and 25 cases of colorectal carcinoma were investigated in this retrospective study for immunohistochemical expression of CcOI. The staining pattern of CcOI was assessed and indices of aberrant expression were calculated as crypt-restricted loss and overall decreased immunostaining (ODI). ODI calculated in the adenocarcinoma tumor tissue was designated as Tr ODI. The crypt-restricted loss and ODI indices of the aberrant CcOI expression are significantly higher in the adenomatous polyps group (2.5% and 47.54%) and in the non-neoplastic mucosa among adenocarcinoma group (2.78% and 49.1%) when they are compared with the control group (0.55% and 7.32%) (P<0.001). A highly significant correlation was noted between Tr ODI and the tumor grade, the nodal status, and the stage among adenocarcinomas. In conclusion, colonic tumors arise in a field of crypts with aberrations in CcOI expression. This aberration is linked to biologically aggressive tumors. CcOI immunostaining may be applied on mucosal samples from patients with colonic adenomatous polyps and patients with previous cancer colon resection to determine individuals who are in need for frequent colonoscopies and/or chemopreventive strategies. Future follow-up studies are warranted to determine the level of expression predictive of recurrence or progression.

Chagas' disease is an emerging and neglected disease in the Brazilian Amazon region, where T. cruzi I predominates among the acute cases of the disease; and T. cruzi III/Z3, a population cluster from sylvatic areas of the Amazon basin, is rarely associated with human infections. On 23rd April 2007, the Foundation for Health Surveillance of the State of Amazonas, Brazil reported an outbreak of acute Chagas disease in the municipality of Coari on the Solimões River banks. Fresh blood examination confirmed the infection in 25 patients. Parasite culture in LIT medium was successful for 18 isolates. Molecular characterization was performed by PCR of the non-transcribed spacer of the mini-exon and by sequencing of the mitochondrial cytochrome c oxidase subunit II (COII) gene. The T. cruzi isolates were all from genotype Z3, and sequencing revealed that all isolates had equal COII sequences compatible with TcIII type, suggesting a single source of infection. To our knowledge, this is the first outbreak of acute cases caused uniquely by the genotype TcIII/Z3. Wild vectors harbouring TcIII stocks contribute to transmission when the triatomine species reaches human food chain or when humans invade the forest environment, where sylvatic cycle constitutes a reservoir of parasites that might be associated with specific epidemiological and clinical traits of the emergent Chagas disease in the Amazon.

Estimating the age of species or their component lineages based on sequence data is crucial for many studies in avian evolutionary biology. Although calibrations of the molecular clock in birds have been performed almost exclusively using cytochrome b (cyt b ), they are commonly extrapolated to other mitochondrial genes. The existence of a large, standardized cytochrome c oxidase subunit I (COI) library generated as a result of the DNA barcoding initiative provides the opportunity to obtain a calibration for this mitochondrial gene in birds. In this study we compare the evolutionary rate of COI relative to cyt b across ten different avian orders. We obtained divergence estimates for both genes from nearly 300 phylogenetically independent pairs of species through the analysis of almost 5000 public sequences. For each pair of species we calculated the difference in divergence between COI and cyt b . Our results indicate that COI evolves on average 14% slower than cyt b , but also reveal considerable variation both among and within avian orders, precluding the use of this value as a standard adjustment for the COI molecular clock for birds. Our findings suggest that this variation is partially explained by a clear negative relationship between the difference in divergence in these genes and the age of species. Distances for cyt b are higher than those for COI for closely related species, but the values become similar as the divergence between the species increases. This appears to be the result of a stronger pattern of negative time‐dependency in the rate of cyt b than in that of COI, a difference that could be related to lower functional constraints on a small number of sites in cyt b that allow it to initially accumulate mutations more rapidly than COI.

Various human diseases are associated with mitochondrial DNA (mtDNA) mutations, but heteroplasmy—the coexistence of mutant and wild-type mtDNA—complicates their study. We previously isolated a temperature-lethal mtDNA mutation in Drosophila, mt:CoI(T300I), which affects the cytochrome c oxidase subunit I (CoI) locus. In the present study, we found that the decrease in cytochrome c oxidase (COX) activity was ascribable to a temperature-dependent destabilization of cytochrome a heme. Consistently, the viability of homoplasmic flies at 29°C was fully restored by expressing an alternative oxidase, which specifically bypasses the cytochrome chains. Heteroplasmic flies are fully viable and were used to explore the age-related and tissue-specific phenotypes of mt:CoI(T300I). The proportion of mt:CoI(T300I) genome remained constant in somatic tissues along the aging process, suggesting a lack of quality control mechanism to remove defective mitochondria containing a deleterious mtDNA mutation. Using a genetic scheme that expresses a mitochondrially targeted restriction enzyme to induce tissue-specific homoplasmy in heteroplasmic flies, we found that mt:CoI(T300I) homoplasmy in the eye caused severe neurodegeneration at 29°C. Degeneration was suppressed by improving mitochondrial Ca(2+) uptake, suggesting that Ca(2+) mishandling contributed to mt:CoI(T300I) pathogenesis. Our results demonstrate a novel approach for Drosophila mtDNA genetics and its application in modeling mtDNA diseases.

Thaisella chocolata is a marine gastropod (family Muricidae) occurring from northern Peru to central Chile. It is of economic importance in these countries, where hundreds of tons are caught every year. In combination with El Niño–Southern Oscillation events, fishing activity has led to the collapse of Chilean populations, raising concerns about the state of Peruvian populations. The aim of this study is to assess the demography, genetic diversity and connectivity of T. chocolata along the Peruvian coast. This would provide important insights into the resilience of this species to anthropogenic disturbance. In this study, four locations along the coast of Peru were investigated for the first time. Mitochondrial cytochrome c oxidase subunit I (COI) sequences were obtained from 116 individuals and four microsatellites from 95 individuals. Both COI (combined with 156 publicly available sequences) and microsatellite data revealed very low genetic differentiation, despite a few significant φST and FST values between population pairs. The most likely gene flow model (MIGRATE-N) is panmixia between all locations, with gene flow occurring equally northwards and southwards. This pattern may be related to the surface currents along the Peruvian coastline combined with the high pelagic larval duration of T. chocolata. Hyperdiversity was found for the COI marker (πsyn &amp;gt; 5%). Microsatellite loci were also polymorphic, with a mean diversity of 12.69 alleles per locus per population. No recent population bottleneck was observed (microsatellite data), while at least one past demographic expansion was suggested (COI data). Given the high genetic diversity and connectivity, T. chocolata is expected to show high evolutionary resilience and can be considered as one management unit in terms of fisheries management. However, further studies, on, for example, the characterization of effective population size (using numerous nuclear loci), are required to confirm this hypothesis. In addition, precautionary fisheries measures should be maintained, given the recent collapse of T. chocolata stocks in Chile.

The Asian tiger mosquito (Aedes albopictus) is an important vector of arboviruses, including dengue, chikungunya, and Zika. Its rapid global expansion has been facilitated by climate change and human activities. Phylogenetic studies of Ae. albopictus have largely relied on mitochondrial cytochrome c oxidase subunit 1 (COX1) and NADH dehydrogenase subunit 5 (ND5) markers, while the utility of cytochrome b (CYTB) remains underexplored. We collected Ae. albopictus from 13 sites in seven provinces of South Korea and analyzed COX1, ND5, and CYTB sequences. Genetic diversity indices were calculated, and phylogenetic relationships were reconstructed using maximum-likelihood trees and haplotype networks with a dataset obtained from GenBank. COX1 revealed 46 haplotypes, including six novel variants, with the highest diversity in southern coastal regions such as Busan and Suncheon. ND5 showed limited variation, with only two haplotypes. CYTB revealed three haplotypes, including region-specific variants in Busan and Wonju, supporting its role as a complementary marker. The Busan haplotype H41 bridged domestic and international lineages, suggesting Busan as a likely entry point. This study demonstrates that integrating COX1, ND5, and CYTB improves the resolution of Ae. albopictus phylogeography in Korea and highlights the need for continued molecular surveillance to guide vector control strategies.