Cytochrome C Oxidase I Research Articles

Genetic and Haplotype Diversity of Clarias gariepinus (Burchell 1822) Based on Cytochrome c Oxidase I (COI) Gene

The evaluation of mitochondrial DNA and genetic analysis is helpful for economically significant species. Clarias gariepinus is a critical species in aquaculture. This study investigates the genetic diversity and population differentiation of C. gariepinus from 19 countries using 164 sequences of the mitochondrial DNA’s Cytochrome c oxidase I (COI) gene. The haplotype analysis revealed a total of 17 haplotypes, with a nucleotide diversity (π) of 0.012 and a haplotype diversity (Hd) of 0.87. The results of an AMOVA and fixation index indicated significant genetic variation and structure among the populations. Additionally, neutrality tests and mismatch distribution analysis supported the hypothesis of under-purifying selection in C. gariepinus. The findings suggested that the population did not experience expansion. In conclusion, the genetic analysis highlighted substantial variation among C. gariepinus populations from different locations, providing valuable insights for the global management of this species.

Faecal DNA Metabarcoding for Diet Analysis of Endangered Fish Species, Odontobutis obscurus.

Understanding the ecological characteristics of endangered species is essential for their management and conservation. The dark sleeper (Odontobutis obscurus) is a carnivorous freshwater fish that has been designated an endangered species in South Korea because of its highly restricted habitat range and small population size. However, ecological research on prey items for this species remains severely limited globally, posing challenges for establishing conservation measures such as translocation management. In this study, the food sources and feeding strategies of O. obscurus were identified through non-invasive faecal DNA metabarcoding. A total of 24 O. obscurus specimens were collected from Sanyang Stream on Geoje Island, and faecal DNA was amplified using mitochondrial cytochrome c oxidase I (COI) primer. As a result, 13 prey items were identified, comprising fish and benthic invertebrates. Dietary metrics revealed the overall food composition and major food source of O. obscurus, and the Costello method enabled graphical diet analysis, suggesting that O. obscurus is a generalist, feeding on a wide variety of food sources. The food selectivity index suggested that O. obscurus prefers specific prey items, such as genus Misgurnus, Zacco (fish), Radix (gastropod), and Ephemera (mayfly). This molecular method facilitated the diet analysis of an endangered fish species that cannot undergo dissection, providing crucial information for their management, particularly regarding translocation efforts.

A DNA barcode reference library for the Tipulidae (Insecta, Diptera) of Germany.

Tipulidae, commonly known as true crane flies, represent one of the most species-rich dipteran families, boasting approximately 4,500 known species globally. Their larvae serve as vital decomposers across diverse ecosystems, prompting their frequent and close observation in biomonitoring programs. However, traditional morphological identification methods are laborious and time-consuming, underscoring the need for a comprehensive DNA barcode reference library to speed up species determination. In this study, we present the outcomes of the German Barcode of Life initiative focused on Tipulidae. Our DNA barcode library comprises 824 high-quality cytochrome c oxidase I (COI) barcodes encompassing 76 crane fly species, counting for ca. 54% of the German tipulid fauna. Our results significantly increased the number of European tipulid species available in the Barcode of Life Data System (BOLD) by 14%. Additionally, the number of barcodes from European tipulid specimens more than doubled, with an increase of 118%, bolstering the DNA resource for future identification inquiries. Employing diverse species delimitation algorithms - including the multi-rate Poisson tree processes model (mPTP), Barcode Index Number assignments (BIN), Assemble Species by Automatic Partitioning (ASAP), and the TaxCI R-script - we successfully match 76-86% of the morphologically identified species. Further validation through neighbor-joining tree topology analysis and comparison with 712 additional European tipulid barcodes yield a remarkable 89% success rate for the species identification of German tipulids based on COI barcodes. This comprehensive DNA barcode dataset not only enhances species identification accuracy but also serves as a pivotal resource for ecological and biomonitoring studies, fostering a deeper understanding of crane fly diversity and distribution across terrestrial landscapes.

DNA barcoding: An effective molecular tool for species identification, molecular authentication and phylogeny studies in plant science research

DNA barcoding is a technique for identifying specimens using brief, standardised DNA segments. In a variety of fields, including phylogeny, ecology, population genetics and biodiversity, DNA barcoding has become a successful method for precisely distinguishing species. The method is straightforward, efficient in both time and money and accurate. The key to successful DNA barcoding is choosing the right DNA marker. Since the idea of a quick approach for species identification was first put up in 2003, the scientific community has been keen to realise the potential of DNA barcodes. Cytochrome c oxidase, I (COI) region of the mitochondrial genome is mostly recognised as a standard barcoding region in animals. Later, rbcL + matK pairing, with a 70 % discriminating efficiency, was suggested by the Plant Working Group (PWG) of the Consortium for the Barcoding of Life (CBOL) as the standard barcode in plants. Three conditions must be met for a gene region to be an efficient DNA barcode: it must have sufficient species-level genetic divergence and variability, it must have conserved flanking regions for the widest taxonomic use and for generating universal PCR primers and it should be long enough to facilitate current capability for sequencing and extracting DNA. Different combinations of plastid coding, non-coding and nuclear markers are utilised as supplemental markers to boost the degree of plant species differentiation. The reliability of different barcodes in distinguishing species varies among different groups of plants. As DNA barcoding approaches its twentieth anniversary, technologies are still being developed that make use of this resource, which is constantly expanding in a variety of biological disciplines. Plant DNA barcoding, which became a scientific advance during the last ten years, is frequently employed as a taxonomical aid in identifying species. It is a way of choosing genetic loci that identifies and distinguishes an organism's membership from specific species, variations or even intervarieties. It varies from molecular phylogeny, which identifies an unknown sample from an existing classification rather than identifying patterns of association.

Rapid Diagnostic PCR Assay Method for Species Identification of Mantidis Ootheca (Sangpiaoxiao) Based on Cytochrom C Oxidase I (COI) Barcode Analysis.

Mantidis Ootheca (sangpiaoxiao), the egg case of the mantis, is a type of insect-derived traditional medicine widely used in East Asia. However, species identification based on egg morphology is challenging, leading to the distribution of counterfeit and adulterated products. The use of inauthentic ingredients can pose serious health risks to consumers. This study aimed to develop PCR markers that can rapidly and accurately differentiate between authentic and counterfeit Mantidis Ootheca. The mitochondrial cytochrome c oxidase I (COI) region was sequenced in thirteen samples from four mantis species: Tenodera angustipennis, Statilia maculata, Hierodula patellifera, and T. sinensis. Four sets of SCAR primers were designed based on species-specific nucleotide polymorphisms, and a multiplex SCAR assay was developed by combining all sets of the primers. The sequence-characterized amplified region (SCAR) primers successfully produced amplicons for each target species, even with low-DNA templates or templates containing DNA from multiple samples. No amplification was observed for nontarget species. This study presents a novel approach for identifying authentic Mantidis Ootheca species using DNA-based diagnostic marker assays, which enable rapid and precise species identification. The SCAR assays developed in this study will aid in maintaining quality control and promoting the standardization of commercial Mantidis Ootheca products.

Unravelling the Molecular Identity of Bulgarian Jumping Plant Lice of the Family Aphalaridae (Hemiptera: Psylloidea).

Psyllids (Hemiptera: Psylloidea) are plant sap-sucking insects whose identification is often difficult for non-experts. Despite the rapid development of DNA barcoding techniques and their widespread use, only a limited number of sequences of psyllids are available in the public databases, and those that are available are often misidentified. Here, we provide 80 sequences of two mitochondrial genes, cytochrome c oxidase I (COI) and cytochrome b (Cytb), for 25 species of Aphalaridae, mainly from Bulgaria. The DNA barcodes for 15 of these species are published for the first time. In cases where standard primers failed to amplify the target gene fragment, we designed new primers that can be used in future studies. The distance-based thresholds for the analysed species were between 0.0015 and 0.3415 for COI and 0.0771 and 0.4721 for Cytb, indicating that the Cytb gene has a higher interspecific divergence, compared to COI, and therefore allows for more accurate species identification. The species delimitation based on DNA barcodes is largely consistent with the differences resulting from morphological and host plant data, demonstrating that the use of DNA barcodes is suitable for successful identification of most aphalarid species studied. The phylogenetic reconstruction based on maximum likelihood and Bayesian inference analyses, while showing similar results at high taxonomic levels to previously published phylogenies, provides additional information on the placement of aphalarids at the species level. The following five species represent new records for Bulgaria: Agonoscena targionii, Aphalara affinis, Colposcenia aliena, Co. bidentata, and Craspedolepta malachitica. Craspedolepta conspersa is reported for the first time from the Czech Republic, while Agonoscena cisti is reported for the first time from Albania.

Genetic relationships of three species of black flies (Diptera: Simuliidae) in Taiwan

As an island about 150 km from the mainland, Taiwan would be expected to have endemic species. About 64 % of its 36 species of black flies are considered endemic, more than twice the level of endemicity that has been recorded for all insects on the island. To begin assessing the validity of the high level of endemism for the Simuliidae, we used giant chromosome banding patterns and cytochrome c oxidase I (COI) sequences, against a well-defined morphological backdrop, to evaluate three of Taiwan's black flies, Simulium chungi Takaoka & Huang, S. pingtungense Huang & Takaoka, and S. sakishimaense Takaoka. Molecular data revealed high similarity of populations of S. sakishimaense in Taiwan and at the type locality on Ishigaki Island, Japan, about 180 km to the east. Thus, populations referred to as S. sakishimaense in Taiwan are conspecific with typical S. sakishimaense in Japan, confirming their non-endemicity in Taiwan. Simulium sakishimaense might have reached Ishigaki by island hopping via Taiwan from the Chinese mainland. Chromosomes and the COI gene agree with morphology that S. sakishimaense is a member of the S. multistriatum species group although the chromosomal banding patterns do not indicate that it is distinct from S. fenestratum Edwards on the mainland. Although molecular sequences indicate S. sakishimaense is monophyletic, this taxon falls within the same Operational Taxonomic Unit as nine other members of the S. multistriatum group, including S. fenestratum. Simulium pingtungense, in agreement with morphology, is molecularly distinct from the 10 other analyzed members of the S. striatum species group, tentatively suggesting that it is endemic to Taiwan, pending analysis of samples from mainland China. Simulium chungi in Taiwan is chromosomally and molecularly unique, with larvae resembling those of S. saskishimaense. It is not, however, a member of either the S. multistriatum or S. striatum species groups. For now, the S. chungi species group remains a legitimate taxon consisting of two species. Strengthening the case for endemic taxa in Taiwan awaits analysis of key samples from the Chinese mainland.

COI DNA barcoding to differentiate Haemagogus janthinomys and Haemagogus capricornii (Diptera: Culicidae) mosquitoes

The genus Haemagogus (Diptera: Culicidae) includes species that are important vectors of pathogens such as the yellow fever virus. The accurate identification of these species is essential for the control of zoonoses. Females of Hg. capricornii and Hg. janthinomys are morphologically indistinguishable, which makes the use of alternative identification techniques desirable. This study aimed to obtain sequences of the mitochondrial cytochrome c oxidase I (COI) gene, in the region widely used for DNA barcoding, of Haemagogus specimens from the state of São Paulo, Brazil, to evaluate the effectiveness of these sequences in the molecular identification of the species. A total of 37 female and 2 male mosquitoes were collected in various locations in the state of São Paulo, using methods such as hand-nets, Shannon traps, CDC light traps with CO2 bait and Nasci aspirators. The sequences of a 710 bp fragment of the COI gene were amplified by PCR and sequenced. A phylogenetic tree reconstruction was conducted using the Bayesian approach implemented in MrBayes v3.2.2, providing support values for taxa where genetic clusters may indicate the presence of new or cryptic species. We obtained 39 COI sequences representing three species: Haemagogus capricornii, Haemagogus leucocelaenus, and Haemagogus janthinomys. Bayesian analysis of the sequences produced clades that corroborate the morphological identification of the species. The separation of Hg. capricornii and Hg. janthinomys received 100 % statistical support and the Hg. capricornii was very well supported (91 %). The two sequences from male specimens, morphologically identified as Hg. capricornii, were grouped in the same clade, a sister clade of Hg. janthinomys. It is important to highlight that the Hg. janthinomys were positioned in several subclades, showing a polymorphism of this species within the state, a situation not observed for Hg. capricornii. For the first time, sequences of the mtCOI gene from Hg. capricornii were obtained and related to morphologically identified specimens. COI sequences proved effective in the molecular identification of Haemagogus species. This study contributes to the expansion of the GenBank database, providing the first sequences of Hg. capricornii and new sequences for Hg. janthinomys and Hg. leucocelaenus.

Study of Altenia wagneriella (rebel) as a predator associated with Forda marginata from Iran.

The larvae of Altenia spp. and gall aphids are known to feed on plants related to Anacardiaceae. This study documents the aphidophagous habit of Altenia wagneriella, which was verified by molecular techniques, subsequently by the gut dissection test, and direct observation. To identify the moth larvae and adult aphids, two mitochondrial genes, cytochrome c oxidase I (COI) and cytochrome b (Cytb), were amplified by polymerase chain reaction (PCR). Nested PCR with the aphid-specific primer pairs AphidF and AphidR was used to detect aphids in the body of moth larvae. The specificity of the primers was verified by PCR analysis of DNA from moth larvae and adult aphids. The method detected aphids in moth larvae, and a band of approximately 200bp was amplified from moth larvae feeding on aphids. No cross reactions with moth larvae were observed. In the laboratory, all moth larvae feeding on aphids (Forda marginata) were also PCR positive for aphids. Gall-inducing insects are microhabitat engineers that manipulate their host to obtain a better nutrient supply, as well as protection from natural enemies and abiotic factors. This is the first recorded instance worldwide of the carnivorous larva of the moth A. wagneriella acting as an aphid predator, as well as the first record of a host insect for this species. Additionally, it is the first effort to molecularly analyze the predator-prey relationship between the moth larvae and the aphids inside the wild pistachio gall.

Shark shuffle: segregated co-occurrence of multiple dusky and sandbar lineages at a human-altered habitat in the eastern Mediterranean Sea

Requiem sharks (genus Carcharhinus) have previously been reported to form large aggregations around marine infrastructures in the eastern Mediterranean Sea. While this behaviour may offer fitness advantages at the individual level, the implications of extended residency at human-altered habitats for population persistence have yet to be assessed. In this work, we investigated the phylogeographic and demographic composition of sharks near a coal-fired power and desalination station in Israel. Our aim was to infer habitat use and the mechanisms underlying the aggregation behaviour, and to highlight potential conservation impacts. We sampled, measured, and released 70 individuals between 2016 and 2022 to assess genetic variability within the cytochrome C oxidase I (COI) region and to analyse the aggregation’s structure based on the sharks’ size and sex distribution. In addition, we performed meristic counts on a reference specimen collected dead at another power station in Israel to supplement species identification using the abovementioned techniques. Our findings indicate size-based sex segregation of adult female dusky and male sandbar sharks (Carcharhinusobscurus and Carcharhinusplumbeus, respectively), with each species comprising two COI haplotypes. In the dusky shark, one haplotype corresponded to an Indo-Pacific lineage, and the other matched an Atlantic lineage. In the sandbar shark, we observed a haplotype previously sampled in the Mediterranean Sea, the Red Sea, the Northwest Indian Ocean, and South Africa, and another haplotype that was unique to our study site and genetically closer to the former than to sequences sampled in other ocean basins. This study provides the first indication of sympatric aggregation amongst phylogeographically distinct dusky and sandbar shark lineages, suggesting that human-altered habitats in the eastern Mediterranean Sea may influence the distribution of these species. Based on the observed segregation pattern, we conclude that the site does not function as a nursery, parturition, or mating area, and discuss other plausible explanations that warrant further research. Finally, we highlight important directions for future research and the implications of our findings for management and conservation.

Analysis of mitochondrial DNA to estimate the population genetic structure of Tuta absoluta (Meyrick, 1917)

Tuta absoluta (Meyrick) has become a serious menace to the sustainable production of tomatoes in Kenya. Investigating the genetic diversity, geographical distribution, and community structure of T. absoluta from various locations utilizing the Cytochrome c oxidase I (COI) of the mtDNA gene information available on NCBI databases. Based on the COI of mtDNA, the haplotype analysis revealed six haplotypes, which displayed low nucleotide (π = 0.00021) diversity. In contrast, haplotype diversity (Hd) was 0.05, and the number of segregating sites (S) was 13. The result showed that the tomato leaf miner population from Central Kenya had high variability in haplotype supported by the haplotype network. Tajima (D) and Fu's Fs were found to be negative (P greater than 0.05), whereas the overall FST value was 0.00077 (P greater than 0.001). In conclusion, genetic analysis showed there was no high variation amongst population from different locations.

DNA Barcoding and Phylogenetic Analysis of Commercially Important Groupers (Serranidae) in Raja Ampat using gene marker Cytochrome c Oxidase I (COI)

DNA Barcoding and Phylogenetic Analysis of Commercially Important Groupers (Serranidae) in Raja Ampat using gene marker Cytochrome c Oxidase I (COI)

Integrative approach to monitoring metazoan diversity and distribution in two Mediterranean coastal sites through morphology and organismal eDNA

Marine and coastal ecosystems respond to climate change in various ways, such as the type of ecosystem, the species composition, interactions, and distribution, and the effect of local stressors. Metazoan organisms, particularly zooplankton, are important indicators for monitoring the effects climate-driven warming in marine coastal ecosystems over the long term. In this study, the diversity and distribution of zooplankton communities in the Mediterranean Sea (Canyon Dohrn and LTER-MareChiara, Gulf of Naples), a known biodiversity and climate changes hotspot, have been assessed using the integration of morphological-based identification and organismal eDNA. Our findings showed that the multi-locus strategy including the mitochondrial cytochrome c oxidase I (COI) gene and the hypervariable region V9 of the 18S rDNA (18S V9) as targets, improved the taxonomic overview, with the COI gene being more effective than the 18S V9 region for metazoans at the species level. However, appendicularians were detected only with the 18S V9 region. Overall, organismal eDNA is a powerful approach for revealing hidden biodiversity, especially for gelatinous and meroplankton components, and provided new insights into biodiversity patterns. The ecological importance of calanoid copepods in coastal ecosystems has been confirmed. In contrast, the discovery of 13 new metazoan records in the Mediterranean Sea, including two non-indigenous copepod species, suggested that local stressors affect zooplankton community structure and resilience, highlighting the importance of biomonitoring and protecting marine coastal ecosystems.

A response to: Evaluating the reliability of DNA Barcoding for Central American Pacific shallow water echinoderms identification

Introduction: Chacón-Monge et al. (2024) sought to test the accuracy of DNA barcoding for species identification in Pacific Central American shallow water echinoderms. They used cytochrome c oxidase I (COI) sequences derived from new material collected as part of the BioMar-ACG project in Costa Rica. Using their set of 348 echinoderm sequences, they compared species identification results from two online platforms: the National Center for Biotechnology Information (NCBI) GenBank using the nucleotide Basic Local Alignment Search Tool (BLASTn), and the Barcode of Life Data Systems (BOLD) Identification Engine. Objective: The present article is a response to their results and conclusions. Methods: We reinterpreted the results from the authors’ Appendix 2 to enable an objective comparison between the BOLD Identification Engine and BLASTn in GenBank. Results: While the authors found that both platforms were limited by the number of reference sequences available in their respective databases, they concluded that GenBank outperformed BOLD for identification; however, we identify several methodological flaws in their analysis. These include pseudoreplication amongst query sequences, contaminated sequences stemming from sampling errors, and a lack of standardization when interpreting results from the two platforms. Their assessment of the BOLD Identification Engine was also limited by improper selection of a reference database. Conclusion: Addressing these errors, we reinterpret their results and demonstrate that there is no difference in performance between the two platforms.

Characterization of mosquito host-biting networks of potential Rift Valley fever virus vectors in north-eastern KwaZulu-Natal province, South Africa

BackgroundRift Valley fever virus (RVFV) is a zoonotic mosquito-borne virus with serious implications for livestock health, human health, and the economy in Africa, and is suspected to be endemic in north-eastern KwaZulu-Natal (KZN), South Africa. The vectors of RVFV in this area are poorly known, although several species, such as Aedes (Neomelaniconion) mcintoshi, Aedes (Neomelaniconion) circumluteolus, Aedes (Aedimorphus) durbanensis, and Culex (Lasioconops) poicilipes may be involved. The aim of the study was to determine the vertebrate blood meal sources of potential RVFV mosquito vectors in north-eastern KZN and to characterize the host-biting network.MethodsBlood-fed mosquitoes were collected monthly from November 2019 to February 2023 using a backpack aspirator, CO2-baited Centers for Disease Control and Prevention (CDC) miniature light traps and tent traps, in the vicinity of water bodies and livestock farming households. The mosquitoes were morphologically identified. DNA was extracted from individual mosquitoes and used as templates to amplify the vertebrate cytochrome c oxidase I (COI) and cytochrome b (cytb) genes using conventional polymerase chain reaction (PCR). Amplicons were sequenced and queried in GenBank and the Barcode of Life Data systems to identify the vertebrate blood meal sources and confirm mosquito identifications. All mosquitoes were screened for RVFV using real time reverse transcription (RT)-PCR.ResultsWe identified the mammalian (88.8%) and avian (11.3%) blood meal sources from 409 blood-fed mosquitoes. Aedes circumluteolus (n = 128) made up the largest proportion of collected mosquitoes. Cattle (n = 195) and nyala (n = 61) were the most frequent domestic and wild hosts, respectively. Bipartite network analysis showed that the rural network consisted of more host-biting interactions than the reserve network. All mosquitoes tested negative for RVFV.ConclusionsSeveral mosquito species, including Ae. circumluteolus, and vertebrate host species, including cattle and nyala, could play a central role in RVFV transmission. Future research in this region should focus on these species to better understand RVFV amplification.Graphical

Source and variation of the amazing live Sea-Monkey microbiome.

An embryonic diapause in unfavourable conditions has allowed brine shrimp to thrive in hypersaline environments and, unexpectedly, mail-order sachets and small, novelty tanks. Marketed as Sea-Monkeys®, each kit involves a 3-step process to generate adult Artemia within a matter of weeks. Whether these kits also allow for the maintenance of a host-associated microbiome is unclear. Therefore, comparing five replicate tanks under the same culture conditions, we sequenced the 16S ribosomal small subunit (SSU) gene to analyse bacterial community compositions in adults, their surrounding tank water, and their feed. Adult Sea-Monkeys® harboured a bacterial microbiome that was clearly distinguishable from the tank water and food. Furthermore, individual tanks had a notable effect on fine-scale microbiome variation. Several Sea-Monkey bacterial variants appeared absent in environmental samples and included genera (Leucobacter and Microbacterium) known to confer desiccation resistance in other hosts. Although Sea-Monkeys® taxonomy is unclear, phylogenetic inference of the cytochrome c oxidase I (COXI) gene from the host animal suggests Sea-Monkeys® belong to the Artemia franciscana 'superspecies'. Overall, Sea-Monkeys® kits appear to be a convenient and scalable mesocosm for the study of host-microbiome interactions and could serve as a useful tool for future invertebrate microbiome research, outreach, and education.

Identification of earthworms (Clitellata, Oligochaeta) from Kottayam district in Southwestern India: an integrated traditional and barcoding approach

Earthworms from various land uses in the Kottayam district of Kerala, part of the Western Ghats hotspot in India, were studied using an integrative approach that involved morphoanatomical methods and cytochrome c oxidase I (COI) barcoding techniques for the first time here. The study unveiled the presence of nine earthworm species: <i>Drawida ghatensis</i> Michaelsen, 1910; <i>Moniligaster julkai</i> Narayanan and Paliwal, 2022; <i>Glyphidrilus annandalei</i> Michaelsen, 1910; <i>Pontoscolex corethrurus</i> (Müller, 1857); <i>Eukerria kuekenthali</i> (Michaelsen, 1908); <i>Dichogaster bolaui</i> (Michaelsen, 1891); <i>Megascolex cochinensis</i> Stephenson, 1915; <i>M. konkanensis</i> Fedarb, 1898; and <i>Lampito mauritii</i> Kinberg, 1867, belonging to eight genera. The COI sequences of all nine species were deposited in GenBank. Notably, <i>M. julkai</i> and <i>E. kuekenthali</i> are novel additions to the GenBank barcode database. The Bayesian phylogram and maximum-likelihood indicated that the species <i>M. julkai</i> in this study exhibited a closer relationship to <i>D. ghatensis</i> than to other <i>Moniligaster</i> species. This study provides detailed descriptions and illustrations of all nine species, supplemented by their DNA barcodes, to facilitate future species identification.

Decoding Seafood: Multi-Marker Metabarcoding for Authenticating Processed Seafood.

Given the recognized nutritional value of fish and shifting consumer lifestyles, processed seafood has become increasingly prevalent, comprising a significant portion of global food production. Although current European Union labeling regulations do not require species declaration for these products, food business operators often voluntarily provide this information on ingredient lists. Next Generation Sequencing (NGS) approaches are currently the most effective methods for verifying the accuracy of species declarations on processed seafood labels. This study examined the species composition of 20 processed seafood products, each labeled as containing a single species, using two DNA metabarcoding markers targeting the mitochondrial cytochrome c oxidase I (COI) and 16S rRNA genes. The combined use of these markers revealed that the majority of the products contained multiple species. Furthermore, two products were found to be mislabeled, as the declared species were not detected. These findings underscore that NGS is a robust technique that could be adopted to support routine food industry activities and official control programs, thereby enhancing the 'From Boat to Plate' strategy and combating fraudulent practices in the complex fisheries supply chain.

Decoding Genetic Signatures for Identification: A Molecular Investigation Approach in Pakistan via Cytochrome C Oxidase I (COI) Gene Sequencing

DNA barcoding is an efficient and rapid approach to the identification of species. A standard genetic marker is used to barcode the sequence of unidentified species. So, mitochondrial cytochrome c oxidase I (COI) genetic marker is mostly used for species molecular identification. Therefore, molecular identification and phylogeographic relationship of Cirrhinus reba (C. reba) were carried out in the present study. In the present study, we propose the barcoding analysis of the mitochondrial cytochrome c oxidase I (COI) gene of freshwater fish as different sequences were obtained from the sequencer and found (all < 500) base pairs and successfully barcode with mitochondrial COI genetic marker for identification. BLAST analysis provides 100% identity match with reference sequences of GenBank database and confirmed the species as C. reba as the length of the sequence is found 496 base pairs. The present study successfully reported DNA barcoding for genetic diversity and identification contributing to covering sufficient information about molecular analysis to identify unknown species the best of our knowledge this is the first study reporting a genetic tool for identification of the commercial food fish of Pakistan. Moreover, DNA barcoding can play a very significant role in molecular identification and inference of relationships in diverse freshwater ecosystem.

DNA Barcoding of Economically Important Fruit Flies (Diptera: Tephritidae) from the Lower Gangetic Plains of Eastern India

The Lower Gangetic Plains are India's central horticultural crop production regions. Fruit flies (Diptera: Tephritidae) are a significant insect pest that significantly hinders the effective crop production of various commercially valuable fruit and vegetable crops. Therefore, rapid and precise species identification techniques are essential to controlling, managing, and quarantining these pests. Furthermore, reliably identifying fruit flies is exceedingly challenging due to their striking resemblances in morphological characters. In this work, we investigated the effectiveness of mitochondrial cytochrome c oxidase I (COI) gene sequences for identifying seven fruit fly species, involving only the genus Bactrocera from the lower Gangetic Plains in eastern India. The sequences of the studied species showed 99%–100% similarity with sequences from within and outside the country. COI barcodes were able to distinguish between species, with K2P intraspecific genetic divergences ranging from 0-1% and K2P interspecific genetic divergences ranging from 3.65-28.46%, thus maintaining a proper barcode gap. Phylogenetic analyses were performed, and the results showed moderate to high supported monophyly among species. Therefore, COI barcodes have proven a highly successful alternative for quickly identifying fruit flies. This is also the first account of molecular identification of the previously reported tephritid fruit flies infesting fruits and vegetables in eastern India's agriculturally important lower Gangetic Plains.