Cytochrome C Oxidase Subunit I Gene Research Articles

DNA barcode identification of a tropical liver fluke (Fasciola gigantica) in cattle from Oyo, southwestern Nigeria

BackgroundFascioliasis is a major parasite illness that affects ruminants, both domesticated and free, and has an impact on public health and animals’ productivity. The genetic diversity of Fasciola species in cattle from Oyo, Oyo State, Nigeria is yet not well understood. In this study, the genetic diversity of Fasciola gigantica in slaughtered cattle in Oyo was examined using a molecular-based approach targeting the mitochondrial cytochrome C oxidase subunit I (COI) gene region.ResultsOne hundred flukes were gathered from slaughtered cattle, and their COI gene sequences were analyzed using maximum likelihood methods. The results of phylogenetic analysis showed genetic similarities between Nigerian F. gigantica and isolates from Algeria, Egypt, Iran, Sudan, China, Japan and Nigeria. The predominant DNA substitution was the A to T transversion, while the least common substitution was the G to A transition.ConclusionOur results show how useful the COI gene region is for examining intraspecific differences between F. gigantica isolates. The genetic similarity observed among the sampled F. gigantica populations suggests the value of mitochondrial DNA sequences as a marker for the accurate identification and characterization of Fasciola species across different ruminants.

A new species of Buresilia (Opiliones, Phalangiidae) from Turkey

A new harvestman species, Buresilia gumushanensis sp. nov. (Opiliones: Phalangiidae), is described based on male specimens collected from Gümüşhane province, Turkey. Detailed descriptions, diagnostic features and pictures of the new species are provided, together with mitochondrial cytochrome c oxidase subunit I (COI) sequences. The phylogenetic tree constructed using the COI gene region revealed the phylogenetic position of the genus Buresilia within the family Phalangiidae. In addition, the new species is compared with known species of the genus Buresilia.

Genetic Diversity, Kinship, and Polychromatism in the Spotted Eagle Ray Aetobatus ocellatus of Fiji

The spotted eagle ray Aetobatus ocellatus (Kuhl, 1923) has a widespread Indo-West Pacific distribution and displays substantial population genetic structuring. Genetic data are crucial for understanding the species’ diversity, connectivity, and adaptation. However, molecular genetic information on A. ocellatus from Melanesia is lacking, which impedes our understanding of gene flow among geographic regions. In this study, we sampled 45 A. ocellatus, primarily from Fiji’s largest fish market in the capital, Suva. Mitochondrial DNA Cytochrome C Oxidase subunit I (COI) barcoding was used for species identification, and DArT-seqTM technology was applied to assess the nuclear genetic diversity. Barcoding of the COI gene showed a 98.6% to 99.8% similarity to A. ocellatus reference sequences in the Barcode of Life Data System, and the 45 individuals were represented by three major evolutionary haplotype clusters. Genotyping resulted in 24,313 Single-Nucleotide Polymorphisms (SNPs) which were quality-filtered to 7094 SNPs per individual. The observed heterozygosity level was 0.310. The inbreeding coefficient was positive, and genotyping identified one full-sibling pair and one half-sibling pair from the 45 individuals. Additionally, eagle rays exhibit polychromatic patterns, and at least three ventral pattern variations were recorded in specimens from the market. Collectively, our main findings characterize the genetic profile of A. ocellatus in Fiji and can help to understand the diversification of this species within the region.

Dominant strain shift in the invasive fall armyworm (Lepidoptera: Noctuidae) populations in Thailand as inferred from mitochondrial COI and nuclear Tpi genes.

The fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), is a significant global pest, that exhibits 2 discernible strains, corn strain (CS) and rice strain (RS). After initial detection in the eastern hemisphere in 2016, the dominant strain was identified as RS based only on cytochrome C oxidase subunit I (COI) mitochondrial gene from limited samples from various countries, including Thailand. This study aimed to assess strain and haplotype variation in the S. frugiperda populations in Thailand using both mitochondrial COI and nuclear triosephosphate isomerase (Tpi) genes. Analyses of COI sequences (n = 105) revealed 2 predominant haplotypes, COICSh4 (82.86%) and COIRSh1 (17.14%), and the analyses of Tpi sequences (n = 99) revealed 6 haplotypes, with TpiCa1a (53.53%) being the most prevalent. Of the 98 caterpillar samples, the majority exhibited true CS (83.67%) for both genes. Meanwhile, interstrain hybrids, indicated by gene discordance, accounted for the minority (16.33%). Interestingly, despite the initial dominance of RS during the 2018 outbreak, the current study identified CS as the prevalent strain across all localities in Thailand. These findings suggested a shift in S. frugiperda dynamics in Thailand that was possibly influenced by factors, such as competitive exclusion principle, pesticide usage in rice cultivation, and preferences for corn over rice. Our study suggests a need to reexamine the previous reports of rice-strain dominance in various countries in the eastern hemisphere after the initial invasion.

Molecular identification of Nemacheilus sp. from Bangka Island, Indonesia Based on the Cytochrome C oxidase Subunit I (COI) Gene

Abstract Nemacheilus is a genus of stone loaches native to Asia. There are 13 species of the genus Nemacheilus that can be found in western Indonesia, from 44 species that exist in Asia. During our expedition to Bangka Island, which took place between the 20th and the 25th of January 2023, we discovered 15 live specimens of Nemacheilus sp. These specimens were collected using a fish trap in the Bencah River, Bangka Island, Indonesia. We identified Nemacheilus sp based molecular analysis by using the DNA Barcoding method based on the Cytochrome C oxidase Subunit I (COI) gene. DNA barcoding is a solution to provide accurate, fast, and automatable identification of species and species discovery. Based aon the COI gene, Nemacheilus sp. Nemacheilus from Bangka Island is genetically closest to Nemacheilus pfeifferae, which is found in Sumatra. Sumatra and Bangka Island have a history of river connection before the maximum glacial phase II. However, based on the similarity of DNA base pairs, these two species are only 90% similar. This indicates that the genetic difference between these two species is 10%, which is far below the threshold for what is considered to be an identical species, which is under 3%. Therefore, we hypothesize that the Nemacheilus that was found on the island of Bangka is not the species known as Nemacheilus pfeifferae, but rather another species. Since the data for this other species are not yet available in Genbank, we are referring to it as Nemacheilus sp. Bangka for the time being.

Analysis of the Mitochondrial COI Gene and Genetic Diversity of Endangered Goose Breeds.

The mitochondrial cytochrome c oxidase subunit I (COI) genes of six endangered goose breeds (Xupu, Yangjiang, Yan, Wuzong, Baizi, and Lingxian) were sequenced and compared to assess the genetic diversity of endangered goose breeds. By constructing phylogenetic trees and evolutionary maps of genetic relationships, the affinities and degrees of genetic variations among the six different breeds were revealed. A total of 92 polymorphic sites were detected in the 741 bp sequence of the mtDNA COI gene after shear correction, and the GC content of the processed sequence (51.11%) was higher than that of the AT content (48.89%). The polymorphic loci within the populations of five of the six breeds (Xupu, Yangjiang, Yan, Baizi, and Lingxian) were more than 10, the haplotype diversity > 0.5, and the nucleotide diversity (Pi) > 0.005, with the Baizi geese being the exception. A total of 35 haplotypes were detected based on nucleotide variation among sequences, and the goose breed haplotypes showed a central star-shaped dispersion; the FST values were -0.03781 to 0.02645, The greatest genetic differentiation (FST = 0.02645) was observed in Yan and Wuzong breeds. The most frequent genetic exchange (Nm > 15.00) was between the Wuzong and Yangjiang geese. An analysis of molecular variance showed that the population genetic variation mainly came from within the population; the base mismatch differential distribution analysis of the goose breeds and the Tajima's D and Fu's Fs neutral detection of the historical occurrence dynamics of their populations were negative (p > 0.10). The distribution curve of the base mismatches showed a multimodal peak, which indicated that the population tended to be stabilised. These results provide important genetic information for the conservation and management of endangered goose breeds and a scientific basis for the development of effective conservation strategies.

Genetic polymorphisms of common sandflies in selected areas of Henan Province based on DNA barcoding

To characterize the species of common sandflies in Henan Province using DNA barcoding with cytochrome c oxidase subunit I (COI) gene as the molecular marker, and to analyze the genetic polymorphisms of sandflies, so as to provide insights into visceral leishmaniasis prevention and control in Henan Province. Sandfly specimens were sampled from 13 sandflies surveillance sites from 2021 to 2023 in Anyang City, Zhengzhou, Luoyang and Xuchang cities (Zhengzhou-Luoyang-Xuchang areas) where visceral leishmaniasis cases were reported and in Jiaozuo and Xinxiang cities (Jiaozuo-Xinxiang areas) without visceral leishmaniasis cases reported. Genomic DNA was extracted from a single sandfly, and COI gene was amplified. The amplification product was subjected to bidirectional sequencing. Following sequence assembly, the species of sandflies was characterized through sequence alignment using the BLAST tool. The intra-specific and inter-specific genetic distances of sandflies were estimated among different areas using the software Mega 11, and phylogenetic trees were created. The polymorphisms of nucleotide sequences in the sandflies COI gene were estimated using the software DnaSP. The fixation index (FST) of different geographical isolates of sandflies was calculated using the Arlequin software, and the gene flow value (Nm) was used to measure the gene flow in the sandflies populations. In addition, the population genetic structure of different geographical populations of Phlebotomus chinensis was analyzed using the STRUCTURE software. A total of 978 sandflies were collected from 13 sandflies surveillance sites in Zhengzhou-Luoyang-Xuchang areas, Jiaozuo-Xinxiang areas and Anyang City of Henan Province from 2021 to 2023, and 475 sandflies were randomly sampled for subsequent detections. A total of 304 Ph. chinensis, 162 Se. squamirostris and 9 Se. bailyi were identified based on molecular biological detection of the COI gene, and Se. bailyi was reported for the first time in Henan Province. The intraspecific genetic distances of sandflies were 0.000 to 0.040, and the inter-specific genetic distances ranged from 0.133 to 0.161. Phylogenetic analysis revealed that each of the three sandfly species was clustered into a clade. The genetic polymorphisms of Ph. chinensis populations varied among different areas, with the highest haplotype diversity (0.966 ± 0.007) and the greatest nucleotide diversity (0.011) in Zhengzhou-Luoyang-Xuchang areas, and the lowest haplotype diversity (0.720 ± 0.091) and nucleotide diversity (0.004) in Anyang City. The dominant haplotype of Ph. chinensis populations was Pch_Hap_2 in Anyang City and Jiaozuo-Xinxiang areas, with moderate genetic differentiation (0.05 < FST < 0.15) and frequent gene exchange (Nm value > 1) between Ph. chinensis populations sampled from Anyang City, and Jiaozuo-Xinxiang areas. Population genetic structure analysis showed that the dominant component of Ph. chinensis populations was K5 in Anyang City and Jiaozuo-Xinxiang areas. No obvious dominant haplotype was observed in Ph. chinensis populations sampled from Zhengzhou-Luoyang-Xuchang areas, which had very high genetic differentiation (FST > 0.25) and little gene exchange (Nm value < 1) with Ph. chinensis populations from Anyang City, and Jiaozuo-Xinxiang areas, with K3 as the dominant component. In addition, there was no significant difference in the genetic polymorphism level among Se. squamirostris populations from the three areas. There are Ph. chinensis, Se. squamirostris and Se. bailyi in Henan Province, and S. bailyi is recorded for the first time in Henan Province by molecular biological assays. There are different levels of genetic differentiation and gene exchange among P. chinensis populations in different areas of Henan Province.

Trypanosoma infection and bloodmeal analysis in post-feeding sand flies across Thailand

Phlebotomine sand flies are recognized as a primary vector of Leishmania and are also suspected vectors of Trypanosoma. The transmission cycle of these parasites relies on the distribution of sand fly vectors, parasites, and reservoir animals. This study aimed to detect Leishmania and Trypanosoma DNA and identify the sources of bloodmeals in post-feeding sand flies captured across Thailand. A total of 42,911 field female sand flies were collected from 11 provinces across Thailand using CDC light traps. Among these, 253 post-feeding sand flies were selected for analysis. The predominant species in this study was Sergentomyia khawi (33.60 %). The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR), specific to the internal transcribed spacer 1 (ITS1) and the small subunit ribosomal RNA (SSU rRNA) gene regions were used to detect the presence of Leishmania and Trypanosoma DNA, respectively. Additionally, cytochrome c oxidase subunit I (COI) gene region was utilized to identify the sources of host bloodmeals. Leishmania DNA was not detected in any specimens. The analysis of SSU rRNA sequences revealed the presence of Trypanosoma DNA (11.46 %, 29/253) in sand fly samples. Among these samples, T. noyesi (1.58 %, 4/253) was identified in Idiophlebotomus longiforceps and Phlebotomus asperulus, Trypanosoma Anura01+02/Frog2 (1.18 %, 3/253) in Se. khawi, and Trypanosoma Anura04/Frog1 (8.70 %, 22/253) in Se. khawi, Se. hivernus and Grossomyia indica. Bloodmeal analysis utilizing the COI gene revealed a diverse range of vertebrate hosts’ blood, including bird, bat, frog and sun skink. Our findings confirm the presence of Trypanosoma DNA and identify the sources of bloodmeals from vertebrate hosts in various sand fly species, suggesting their potential as possible vectors for Trypanosoma in Thailand. Furthermore, our study is the first to provide molecular evidence using the COI gene to identify frogs as a host blood source for sand flies in Thailand. Further studies focusing on the isolation of live parasites in sand flies to confirm vector potential and examining the role of animal reservoirs will enhance our understanding of the host-parasite relationship and enable more efficient control for disease transmission.

Morphological, Molecular Identification and Genetic Diversity Assessment of Three Terrestrial Isopods from the High-Altitude Region

Terrestrial specimens were collected from Ashshafa, a south-western highland area in Saudi Arabia. Three species, i.e., Porcellio laevis, Porcellionides pruinosus (Porcellionidae), and Armadillidium vulgare (Armadillidiidae), were identified in this study based on their morphological characteristics. Partial mitochondrial cytochrome C oxidase subunit I (COI) gene sequences were used for DNA barcoding and biodiversity assessments. A phylogenetic tree of 22 haplotypes from 35 specimens of the three isopod species was drawn from the most similar sequences obtained from BLAST with the associated accession numbers. The tree included two clades. The first clade included samples of P. laevis and P. pruinosus, whereas the second clade included samples of A. vulgare. Each identified species formed a distinct subclade within the main clade, along with similar sequences obtained from the NCBI database. The heat map of genetic distance among haplotypes shows the haplotype diversity (Hd) ranged from 0.590 to 0.933 (mean = 0.767) and total nucleotide diversity (πT) ranged from 0.001 to 0.089 (mean=0.049), with a similar trend observed for nucleotide diversity per site (θw) ranged from 0.001 to 0.80 (mean = 0.049). In contrast, synonymous nucleotide diversity (πs), mean=0.009, was low compared to nonsynonymous nucleotide diversity (πs), mean=0.060, across all species. In conclusion, the morphological identification of terrestrial isopods was confirmed using COI gene sequencing of mitochondrial DNA. These results will be helpful in developing a deeper isopod identification method.

Morphological and genetic identification of the gill monogenean parasite (Diclidophora merlangi) that infects Twobar Seabream Fish (Acanthopagrus bifasciatus) in the Arabian Gulf, Saudi Arabia.

Ectoparasites, particularly monogeneans, negatively affect fish health and growth. This study identified monogenean parasites in the twobar seabream, Acanthopagrus bifasciatus (Sparidae), inhabited the Arabian Gulf (Saudi Arabia). Following that, forty A. bifasciatus fish samples were visually examined for monogeneans. Parasite species were collected from the gills and then analyzed morphometrically, morphologically, and molecularly using the partial regions of the large subunit of ribosomal RNA (28S rRNA) and mitochondrial cytochrome C oxidase subunit I (COI) genes. Fish species were also identified using a DNA barcoding approach based on the COI gene. The monogenean species of Diclidophora merlangi (Diclidophoridae) were found in 45% of the fish species studied. The generic features of the Diclidophora genus distinguish this species. This species discriminated itself from congeners by having a muscular bulb with 17 grooved and recurved hooks, 218±10 (184-267) post-ovarian testes, and four pairs of pedunculated clamps of relative sizes. Partial 28S rRNA sequencing from monogeneans revealed that they grouped with members of the genus Diclidophora, forming a monophyletic group that supported the morphological descriptions. Molecular identification revealed that D. merlangi has a unique barcode made up of a COI sequence. The host identity was established as A. bifasciatus based on the COI gene sequences. Furthermore, a molecular phylogenetic study was performed to determine the phylogenetic affinity of parasite species and fish hosts. This study on Diclidophora species is considered the first record of this genus in the examined area.

Demographic inference from the mt-DNA COI gene and wing geometry of Culex gelidus (Diptera: Culicidae), an important vector of Japanese encephalitis in Thailand

Culex gelidus (Diptera: Culicidae), an important vector of the Japanese encephalitis virus (JEV), contributes to human viral encephalitis in many Asian countries, including Thailand. This study represents the first investigation of the demographic patterns of Cx. gelidus populations in Thailand using cytochrome c oxidase subunit I (COI) gene analysis and wing geometric morphometrics (GM). Mosquitoes were collected from 10 provinces across six regions of Thailand in 2022. Analysis of the COI sequences (n = 182) indicated high haplotype diversity (0.882) and low nucleotide diversity (0.006), with 72 haplotypes identified. The haplotype network demonstrated no profound splits among the geographic populations. Neutral tests, including Tajima's D and Fu's Fs, displayed negative values, with a significant result observed for Fu's Fs (−33.048, p < 0.05). The mismatch distribution analysis indicated that the population does not statistically deviate from a model of sudden population expansion (SSD = 0.010, p > 0.05; Rg = 0.022, p > 0.05). The estimations suggest that the Cx. gelidus population in Thailand began its expansion approximately between 459,243 and 707,011 years ago. The Mantel test showed no significant relationship between genetic and geographic distances (r = 0.048, p > 0.05). Significant phenotypic differences (based on wing shape) were observed among most populations. Additionally, in this study, we found no significant relationships between phenotypic and genetic distances (r = 0.250, p > 0.05). Understanding the genetic and morphological dynamics of Cx. gelidus is vital for developing targeted surveillance and vector control measures. This knowledge will also help to predict how future environmental changes might affect these populations, thereby informing long-term vector management strategies.

The Partial Nucleotide Sequences of the Mitochondrial Genes, COI and 16s rRNA, of Fireflies in the Genera Pygoluciola, Trisinuata, and Medeopteryx (Coleoptera: Lampyridae)

Fireflies in the genera Pygoluciola, Medeopteryx, and Trisinuata distribute throughout lower northern Thailand. Five morphospecies of these fireflies were collected and nucleotide sequences were analyzed. We sequenced the partial of two mitochondrial genes; Cytochrome c oxidase subunit I (COI) and 16s rRNA, and the phylogenetic trees were constructed and compared with the firefly nucleotide sequences in the NCBI database. The COI gene was amplified using LCO1490 and HCO2198 primers while specific primers of LR-J-13020a and LR-N-13398a were used to analyze amplicons of 16s rRNA. The results showed the average GC-contents in the nucleotide of five firefly morphospecies were found to average 30.28% and 22.15% in (COI) and 16s rRNA, respectively. The variable site in COI nucleotide sequences was 399 of the total 520 nucleotides (76.73%) whereas 91 of the total 418 nucleotides (21.77%) were found in 16s rRNA nucleotide sequences. Construction of the phylogenetic trees of those five firefly morphospecies with reference accession samples indicated five clades when analyzed by COI nucleotide sequences and two clades were revealed in 16s rRNA gene. According to these results, COI nucleotides are one of the most effective methods for differentiating between and within firefly species. The addition of the 16s rRNA gene analysis showed an increase in the precision of firefly identification. Those five firefly morphospecies were separated using both the COI and 16s rRNA nucleotide sequences.

Exploring the Potential in Combining the Two Molecular Approaches, COI Barcoding and PCR-RFLP (COIBar-RFLP) in Identification of Selected Species of the Family Carangidae

The family Carangidae is considered a markedly diverse, widespread taxon. Due to the characteristic “cryptic diversity” and “hybrid speciation” within the family, there is an exigency for taxonomic approaches that go beyond traditional phenotypic modus in identifying species. Mitochondrial cytochrome c oxidase subunit - I (COI) barcoding gene region plays a significant role over phenotypic characters in identifying species. This study evaluates the combination of the two molecular approaches: the COI DNA barcoding and PCR-RFLP for judicious species discrimination. The partial mitochondrial COI gene region of the selected Carangid fish species was amplified, sequenced and confirmed sequences with a mean length of 655 bp were submitted to the main databases; NCBI and BOLD. Intraspecific and interspecific nucleotide divergences were computed by the Kimura-2-Parameter (K2P) and they ranged between (0.00–2.96 %) and (6.46–21.83 %), respectively. The possibility of acquiring the same RFLP profiles given by the restriction enzymes HaeIII and MbOII was observed by the theoretical cleavage of 250 reference sequences of the corresponding gene. Hence, major and minority composite haplotypes of each species were obtained based on the fragment types derived by both HaeIII and MbOII. The resulting divergence values were compatible with the previously reported values for marine fish species. All the species were clearly differentiated by both RFLP banding patterns and the highest probability assumption of getting the same RFLP profile was compatible with the most abundant composite haplotype of each species. This reveals the practicability of the combination of two consolidated molecular approaches, COI barcoding and PCR-RFLP (COIBar-RFLP).

Insights into the mitochondrial cytochrome oxidase I (mt-COI) gene and wing morphometrics of Anopheles baimaii (Diptera: Culicidae) in malaria-endemic islands of Thailand.

Anopheles baimaii (Diptera: Culicidae) significantly contributes to the transmission of parasites causing malaria in Southeast Asia and South Asia. This study examined the morphological (wing shape) and molecular (mitochondrial gene) variations of An. baimaii in four of Thailand's border islands, and also investigated the presence of Plasmodium parasites in these mosquitoes. No Plasmodium infections were detected in the samples. Significant differences in wing shape were observed in most island populations (p < 0.05). A single-linkage tree, constructed using Mahalanobis distances, clustered the populations into two groups based on geographical locations. Genetic variation in An. baimaii was also analyzed through cytochrome c oxidase subunit I (COI) gene sequences. This analysis identified 22 segregating sites and a low nucleotide diversity of 0.004. Furthermore, 18 distinct haplotypes were identified, indicating a high haplotype diversity of 0.825. Neutrality tests for the overall population revealed a significantly negative Fu's Fs value (-5.029), indicating a population expansion. In contrast, Tajima's D yielded a negative value (-1.028) that did not reach statistical significance. The mismatch distribution analysis exhibited a bimodal pattern, and the raggedness index was 0.068, showing no significant discrepancy (p = 0.485) between observed and expected distributions. Pairwise genetic differentiation assessments demonstrated significant differences between all populations (p < 0.05). These findings provide valuable insights into the COI gene and wing morphometric variations in An. baimaii across Thailand's islands, offering critical information for understanding the adaptations of this malaria vector and guiding future comprehensive research.

Genetic diversity and connectivity of Red Snapper Lutjanus gibbus in the Papua Waters, Indonesia

Abstract. Pranata B, Sala R, Kusuma AB, Toha AHA, Purbani DC, Mokodongan DF, Sipriyadi. 2024. Genetic diversity and connectivity of Red Snapper Lutjanus gibbus in the Papua Waters, Indonesia. Biodiversitas 25: 276-286. There is limited knowledge regarding the genetic connectivity and diversity of the Red Snapper (Lutjanus gibbus Forsskål, 1775) populations that inhabit Papua Waters, Indonesia. Thus, the current study attempted to ascertain the genetic characteristics, level of diversity and genetic connectivity of the L. gibbus population. We conducted genetic research on 38 L. gibbus specimens from five different places in Papua Waters. We analyzed the L. gibbus genetic characteristics, diversity and genetic connectivity using the Cytochrome C Oxidase subunit I (COI) gene as the genetic marker. There were 15 polymorphism sites in the COI gene sequence among L. gibbus individuals. Polymorphism occurs due to transversion and transition mutations. The COI gene fragment was translated, producing 188 amino acids composed of 19 different amino acids. A total of 13 distinct haplotypes were detected among the L. gibbus population residing in Papua Waters. The haplotype diversity (Hd=0.740) and nucleotide diversity (Pi=0.002) were relatively medium. The genetic structure study indicated that the 5 populations of L. gibbus in the Papua Waters had little genetic differentiation, as evidenced by a Fixation Index (Fst) of 0.018 (P-value 0.35386±0.01443). Based on this, the management of Red Snapper resources at these 5 locations must be carried out as a single unit.

A cell line derived from heart of rainbow trout is refractory to Tilapialake virus.

Cell lines are important in vitro models to answer biological mechanisms with less genetic variations. The present study was attempted to develop a cell line from rainbow trout, where we obtained a cell line from the heart, named "RBT-H." The cell line was authenticated using karyotyping and cytochrome c oxidase subunit I (COI)gene sequencing. The karyotype demonstrated diploid chromosome number (2n) as 62 and the sequence of partial COI gene was 99.84% similar to rainbow trout COI data set, both suggesting the origin of RBT-Hfrom the rainbow trout. The heart cell line was mycoplasma-free and found to be refractory to infection with the Tilapia lake virus. The RBT-Hcell line is deposited in the National Repository of Fish Cell Line (NRFC)at ICAR-NBFGR,Lucknow, India, with Accession no. NRFC0075 for maintenance and distribution to researchers on request for R&D.

Marine bivalves voucher DNA barcoding from Eastern Mediterranean, with evidence for Ostrea stentina invasion

Abstract Bivalve molluscs are a diverse group of animals with particular economic and ecological importance. Their morphological characteristics frequently confuse their identification leading to mislabelling of edible species. Genetic diversity is critical to the resilience of marine bivalve populations in the face of environmental stressors such as ocean acidification and warming. In this study, we characterized the phylogeny and defined the first DNA barcodes of six marine bivalves [Ostrea edulis (Linnaeus, 1758) Arca noae (Linnaeus, 1758), Pinctada radiata (Leach, 1814), Venus verrucosa (Linnaeus, 1758), Calllista chione (Linnaeus, 1758) and Ruditapes decussatus (Linnaeus, 1758)] sampled from different coastal areas of Aegean and Ionian Seas using the molecular markers cytochrome c oxidase subunit I (COI) and 18S ribosomal RNA (18S rRNA). Further, COI gene was employed to investigate the population genetic diversity since 18S rRNA exhibited no conspecific differences. The sequence of 18S rRNA successfully discriminated the bivalves at family or superfamily level but occasionally proved insufficient for species identification. Contrariwise, COI was highly informative and could reliably distinguish all species. Population haplotype diversity was moderate to high and was always accompanied by generally low nucleotide diversity, indicating genetically closely related haplotypes. The invasive Pinctada radiata was found to be panmictic even among distant sampling areas, while Ostrea edulis was the only species that exhibited moderate levels of population subdivision. Finally, here we report for the first time the presence of Ostrea stentina in Thermaikos Gulf sampled among Ostrea edulis specimens, demonstrating a new invasive bivalve species in Eastern Mediterranean.

Genetic diversity and phylogeography of Phlebotomus argentipes (Diptera: Psychodidae, Phlebotominae), using COI and ND4 mitochondrial gene sequences.

Phlebotomus argentipes complex is the primary vector for cutaneous leishmaniasis, a burgeoning health concern in contemporary Sri Lanka, where effective vector control is important for proper disease management. Understanding the genetic diversity of the P. argentipes population in Sri Lanka is vital before implementing a successful vector control program. Various studies have indicated that genetic divergence, caused by genetic drift or selection, can significantly influence the vector capacity of arthropod species. To devise innovative control strategies for P. argentipes, exploring genetic diversity and phylogeography can offer valuable insights into vector competence, key genetic trait transfer, and impact on disease epidemiology. The primary objective is to analyze the genetic diversity and phylogeography of the P. argentipes complex in Sri Lanka, based on two mitochondrial genomic regions in modern representatives of P. argentipes populations. A total of 159 P. argentipes specimens were collected from five endemic areas of cutaneous leishmaniasis and identified morphologically. Two mitochondrial regions (Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4) were amplified using the total DNA and subsequently sequenced. Partial sequences of those mitochondrial genes were utilized to analyze genetic diversity indices and to explore phylogenetic and phylogeographic relationships. Among five sampling locations, the highest genetic diversity for COI and ND4 was observed in Hambantota (Hd-0.749, π-0.00417) and Medirigiriya (Hd-0.977, π-0.01055), respectively. Phylogeographic analyses conducted using COI sequences and GenBank retrieved sequences demonstrated a significant divergence of P. argentipes haplotypes found in Sri Lanka. Results revealed that they have evolved from the Indian ancestral haplotype due to historical- geographical connections of the Indian subcontinent with Sri Lanka. Utilizing high-mutation-rate mitochondrial genes, such as ND4, can enhance the accuracy of genetic variability analysis in P. argentipes populations in Sri Lanka. The phylogeographical analysis of COI gene markers in this study provides insights into the historical geographical relationship between India and P. argentipes in Sri Lanka. Both COI and ND4 genes exhibited consistent genetic homogeneity in P. argentipes in Sri Lanka, suggesting minimal impact on gene flow. This homogeneity also implies the potential for horizontal gene transfer across populations, facilitating the transmission of genes associated with traits like insecticide resistance. This dynamic undermines disease control efforts reliant on vector control strategies.

Species Identification of Sea Bamboo (Isis hippuris) using COI-based DNA Barcoding

Conservation and effective management of marine ecosystems and biodiversity requires accurate species identification. This study classifies sea bamboo (Isis hippuris) specimens using DNA barcoding, a technique widely recognized for its speed, accuracy, and objectivity. This study examines the cytochrome c oxidase subunit I (COI) gene analysis for species identification. Isis hippuris was collected from two stations (coral and seagrass areas) of Tanjung Tiram Waters, South Konawe, Southeast Sulawesi. Genomic DNA was extracted from the base, main, and lateral stem of I. hippuris. Polymerase chain reaction (PCR) was used to amplify the mtDNA of I. hippuris with HCO2198 and LCO1490 primers. The highest quality PCR product based on the COI gene was chosen for sequencing analysis. The study revealed that COI gene analysis could only be performed on the base and main stem of the I. hippuris. Samples from coral and seagrass-coral areas on lateral stems were not further analyzed due to low concentration and purity values, which could potentially fail DNA sequencing. Each part of I. hippuris may have unique genetic differences. This study highlights the advantages of DNA sequencing in providing a unique genetic fingerprint for each species, enabling accurate species identification. This research provides insight into using DNA barcoding for sea bamboo species identification.

DNA barcoding and phylogenetic relationships of ten butterfly caterpillars

Cytochrome C oxidase subunit I (COI), known as DNA barcodes, can be employed for the identification of an unknown specimen at the species level. The present study was aimed at performing COI gene-based identification of butterflies using butterfly caterpillars. Consecutively, ten caterpillars of ten different butterfly species from the families Papilionidae, Nymphalidae, Pieridae, Lycaenidae, and Hesperiidae were taken from respective host plants and used to generate COI gene sequences that were about 650 bp long. After BLAST analysis, the sequenced gene revealed 96–100% similarity to the same species from different regions. Then, sequences were submitted to NCBI's GenBank and obtained ten accession numbers. In order to elucidate genetic diversity and evolutionary relationships among ten species of butterfly caterpillars, pairwise distance analysis and the construction of a phylogenetic tree were performed using MEGA10 and BioEdit software. In the analysis, the interspecific genetic divergence among the caterpillars of butterflies was between 0.101-0.164%. A phylogenetic tree was constructed with the assistance of the Neighbor-Joining (NJ) algorithm, which identified two major clades, A and B, as well as indicated that butterflies caterpillars shared a common ancestor. All the species are included in these two clades, except Catopsilia pomona. This species is under the family Pieridae, and the phylogenetic position of Pieridae compared to other butterfly families is ambiguous, necessitating additional research to resolve this issue. The study demonstrated the general applicability of DNA barcoding for rapidly and accurately distinguishing butterfly species, even when using larval stages, as in the present study. Furthermore, it may disclose a higher taxonomic hierarchy of butterfly families. Asian Australas. J. Biosci. Biotechnol. 2023, 8(3), 49-55