Cytochrome C Oxidase Subunit I Gene Research Articles

First results on the genetic variation of the Bulgarian populations of Morimus asper funereus (Mulsant, 1862) (Coleoptera, Cerambycidae)

There is an ambiguity about the taxonomic status of Morimus asper funereus (Mulsant, 1862), a saproxylic longhorn beetle, widespread in Bulgaria and other Balkan countries. It is a protected species under the Habitats Directive (Annex II) and under the Bulgarian Biodiversity Act. A recent molecular study, based on the cytochrome C oxidase subunit I (COI) and the second internal transcribed spacer (ITS2) gene se­quences, found that all European and Turkish populations of Morimus should be referred to M. asper (Sulzer, 1776) (Solano et al. 2013). The territory of Bulgaria and some other Balkan countries remain as an unstudied gap in that research. Here we present preliminary results on the genetic divergence and diversity of Morimus populations on the Balkans based on COI gene sequences using material from Bulgaria and Albania and the haplotypes’ sequences of Solano et al. (2013) obtained from GenBank. The material collected from Bulgaria аnd two localities in Albania was identified based on morphology characteristics as M. asper funereus (44 samples), M. verecundus bulgaricus Danilevski et all., 2016 (1 sample from the type locality), Morimus orientalis Reitter, 1894 (1 sample from Strandzha Mts.) and Lamia textor (Linnaeus, 1758) (1 sample) used as an outgroup. All obtained sequences were analyzed and haplotype diversity was estimated. The results show that Bulgarian populations of Morimus demonstrate relatively high haplotype diversity in correspondence to the diversity of the other European populations. Also, a phylogeographical hypothesis of the linkage between Bulgarian and other Balkan populations was developed. Four main lineages of divergence were identified. In addition, our results support the assumption that M. verucundus is a tentative subspecies of the morphologically and genetically variable M. asper.

The Genetic Diversity of Banana Leaf Roller Erionota Thrax Thrax (Lepidoptera: Hesperiidae) in Indonesia

Banana leaf roller, Erionota thrax thrax, is an example of plant-insect interaction in a cultivated banana that distributes in Southeast Asia, including Indonesia. This subspecies was determined previously based on the morphological character only. The genetic characteristic based on molecular data and genetic diversity had never been accessed for this important pest. This study aimed to (1) characterize the identity of E. t. thrax based on the Cytochrome C Oxidase Subunit I (COI) gene sequence; (2) explore a genetic variation in haplotype diversity of the COI gene. We reconstructed phylogenetic relationships and determined the entire population's haplotype using MrBayes Version 3.2.6, MEGA X, and DnaSP v5. The Bayesian tree showed that this subspecies consists of two clades, i.e., Sundaland population and Sulawesi population clades. Moreover, there are six haplotypes in the Sundaland population and a single haplotype in the Sulawesi population. The genetic distance within E. t. thrax ranged from 0.002 to 0.008 in the Sundaland population, while that between the two populations ranged from 0.016 to 0.023. The scientific novelty of this study is that the COI gene sequences allow distinguishing the population of E. t. thrax between the Sunda and Wallaceae lines and tracing the origin of the Papuan population based on the similarity of its haplotype.

In Silico Evaluation of the Taxonomic Resolution and Coverage of the COI Region and Alternative Barcode Markers for Echinoderms

DNA barcoding methods are potentially useful for the detection and identification of species difficult to identify using morphology-based taxonomy, such as the larvae of marine taxa. Combining barcoding methods with massively parallel sequencing and low-cost DNA library preparation improves the feasibility of processing several samples but limits the taxonomic resolution of resulting sequences. In light of these limitations, we looked for suitable barcode regions for echinoderm larvae, first by examining the cytochrome c oxidase subunit I (COI) region, the most commonly used barcode for echinoderms. Using a dataset containing over 4000 COI sequences representing nearly 400 echinoderm species across all five extant classes, the COI gene was found to lack a sufficiently conserved region for barcoding. Taxonomic resolution was also found lacking, with no clear barcode gap across classes and the results of Automatic Barcode Gap Discovery (ABGD) failing to reflect current taxonomic assignments. While sequences may be mislabeled, the aggregation of multiple species in one ABGD partition suggests that course-grained taxonomic resolution may still result from using COI alone. The search for short alternative mitochondrial regions across 110 genomes of distinct species using ecoPrimers software revealed that class-specific primers targeting 12S and 16S ribosomal RNA (rRNA) regions are prime candidates for barcoding echinoderm sequences. The results of in silico PCR across the 110 mitochondrial genomes indicate that compared to existing COI primers, the candidate 12S and 16S primers yield similar taxonomic coverage, with slightly lower resolution. Taking into consideration the previous analysis of COI, we suggest the use of multiple markers (COI, 12S, 16S) to adequately capture the understudied diversity of echinoderm larvae. In vitro PCR of the 12S and 16S primers is also needed to validate the results of the in silico analyses.

A new species of Homatula (Teleostei, Cobitoidea, Nemacheilidae) from the Pearl River drainage, Yunnan, China.

Based on morphological and molecular analysis of Homatula species distributed in the Nanpanjiang River in Yunnan, China, we described a new species, Homatularobustasp. nov. It differs from its congeners by a combination of the following characters: naked and robust body with well-developed crests (caudal peduncle depth as a percentage of its length: 70.5–78.5%); lateral line complete; median notch on lower jaw; median gap on lower lip; three pairs of short barbels, with maxillary barbels extending posteriorly to anterior edge of eyes; branched dorsal-fin rays 8½; and vertebrae 37–39. It can further be distinguished from H.nanpanjiangensis by several differences of the caudal skeleton such as the number of hypural elements, the presence of epurale and the shape of neural and haemal spines. Phylogenetic analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene indicated that the new species represents an independent lineage. It is separated from other Homatula species by a minimum of 5.3% Kimura-2-parameter distance in the COI gene. Furthermore, we confirmed that Homatulawenshanensis should be a member of Homatula based on both skeleton and molecular evidence.

Putative ITS2 secondary structure model and multi‐gene phylogenies of tetrahymenids (Ciliophora, Hymenostomatia) parasitizing planarians and crayfish worms

AbstractTetrahymena comprises over 80 free‐living as well as facultative or obligate histophagous species parasitizing especially invertebrates and fishes. Because of the comparatively high morphological conservativeness of tetrahymenids, their identification is primarily based on the gene coding for cytochrome c oxidase subunit I (COI). In addition to this mitochondrial barcoding marker, we tested here also the species discriminatory power of the highly variable nuclear ITS region, using the secondary structure of the ITS2 molecules and the compensatory base change (CBC) analysis. ITS2 secondary structures were proposed for 25 distinct ribotypes of 13 taxa, including three Tetrahymena species parasitizing planarians and crayfish worms, with the free‐energy minimization approach and the homology modelling. Although the majority of Tetrahymena species pairs can be reliably separated by one to six hemi‐CBCs localized especially in the third and fourth helix of the ITS2 molecules, some ITS region sequences might transcend species boundaries. In other words, intraspecies and interspecies p‐distances of ITS region sequences might overlap. This fact weakens the discriminatory power and the usage of the ITS region in the molecular taxonomy of Tetrahymena. On the other hand, there was always a distinct barcoding gap at about 4% sequence divergence in the case of the COI gene. Despite the slightly lower delimitation power of the ITS region, this molecular marker helped to recognize multiple previous misidentifications that were subsequently verified by comparing COI sequences.

The biology of Simulium erythrocephalum and S. chelevini (Diptera, Simuliidae): Morpho-logical, ecological and molecular data

The subgenus Boophthora is a typical Palearctic taxon, which includes only 6 species, among them. Simulium erythrocephalum has a transpalearctic distribution. In Europe, Adler notes only the species S. erythrocephalum, and Yankovsky – two species S. (Boophthora) erythrocephalum and S. (Boophthora) chelevini. According to morphological characteristics, these species differ in their life stages. We have studied the development of S. erythrocephalum and S. chelevini from three rivers of Volyn region, Ukraine (Styr, Chornoguzka, Putylivka) from 2017 to 2019. We used the EPPO PM7 / 129 standard. Collected samples, 615-bp fragments of the COI gene were sequenced from five individuals of S. erythrocephalum and five individuals of S. chelevini and compared with four samples of S. erythrocephalum from the GenBank. We obtained the nucleotide sequence of S. chelevini. All of the S. erythrocephalum samples from Ukraine had 692 bases, the S. erythrocephalum samples from Armenia had 673 bases. S. erythrocephalum and S. chelevini did not have any intraspecific variations. These intraspecific variations were not larger than the interspecific variations. It has been proved that the populations of S. erythrocephalum and S. chelevini from medium and small rivers of Volyn do not differ in biological, behavioural and genetic characteristics. Comparison of S. erythrocephalum and S. chelevini life stages showed clear differences in 20 morphological features, which are probably manifestations of phenotypic variability. Comparison of species with data from the GenBank from Spain and Armenia on the mitochondrial cytochrome c oxidase subunit I (COI) gene confirmed the opinion that S. erythrocephalum and S. chelevini are one species. On the phylogenetic tree, the data are not grouped, there is no clear separation of the clades. Bootstrap values are 95–100%, which may indicate a significant similarity of all studied samples and the lack of isolation of individual morphotypes from Volyn, Spain and Armenia. To finally confirm the taxonomic position of these two species, additional research is needed covering more individuals from different parts of Europe and analysis of more genes.

Cytauxzoon europaeus infections in domestic cats in Switzerland and in European wildcats in France: a tale that started more than two decades ago

BackgroundCytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing.MethodsThe overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995–1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) gene sequencing.ResultsIn study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0–0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0–0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0–14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0–4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2–44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses.ConclusionsThe study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species.Graphical

DNA Barcoding: Molecular Identification and Phylogenetic Analysis of Pheretimoid Earthworm (Metaphire sp. and Amynthas sp.) Based on Mitochondrial Partial COI Gene from Sialkot, Pakistan.

The extremely difficult and challenging process is identifying pheretimoid species, genus Metaphire and Amynthas involving increased homoplasy in various morphological characteristics. The molecular identification, phylogenetic relationships, and evolutionary divergence time of earthworms belonging to the pheretimoid complex were investigated in this study using partial mitochondrial COI (cytochrome C oxidase subunit I) gene sequences ranging from 550-680 bp. Results revealed that 86 pheretimoid earthworms were morphologically different from a total of 342 mature worms. Moreover, 11 pheretimoid species were molecularly identified, including Metaphire posthuma (02), M. anomala (01), M. houlleti (02), M. californica (01), M. birmanica (02), Amynthas minimus (01), A. morrisi (01), and M. bununa (01). A phylogenetic tree was constructed with bootstrap values of 95%, which supported a monophyletic lineage of two well-supported clades formed by 12 partial COI sequences and 48 GenBank sequences using Hirudo medicinalis as an outgroup. The monophyly of these obtained genera indicated overall similarity at species level. Today, species like Amynthas, Metaphire and Pheretima have worm diversity in the form of pheretimoid earthworms, which dates to the Late Miocene (11.2-5.3 Mya) and the Pliocene (5.3-2.4 Mya). Compared to all relevant pheretimoid species, genetic p-distance values ranged from 0.0% to 0.57% (less than 1%). These low range values demonstrated that both genera Metaphire and Amynthas, supported the theory, which states that there are shared similarities among the species, despite different morphology. The current study is the first attempt in Pakistan to identify earthworms through DNA barcoding thus providing a genomic stamp. The work explored the significance of COI gene sequences to construct molecular tools that will be useful to overcome the different obstacles in morphologically similar earthworm identification and their phylogenetic study.

Evaluation of partial 12S rRNA, 16S rRNA, COI and Cytb gene sequence datasets for potential single DNA barcode for hylids (Anura: Hylidae)

We evaluated the extent of intraspecific and interspecific genetic distances and the effectiveness of predefined threshold values using the main genes for estimates of biodiversity and specimens' identification in anurans. Partial sequences of the mitochondrial genes for small (12S) and large (16S) ribosomal subunits, cytochrome c oxidase subunit I (COI) and Cytochrome b (Cytb) of the family Hylidae were downloaded from GenBank and curated for length, coverage, and potential contaminations. We performed analyses for all sequences of each gene and the same species present in these datasets by distance and tree (monophyly)-based evaluations. We also evaluated the ability to identify specimens using these datasets applying "nearest neighbor" (NN), "best close match" (BCM) and "BOLD ID" tests. Genetic distance thresholds were generated by the function 'threshVal' and "localMinima" from SPIDER package and traditional threshold values (1%, 3%, 6% and 10%) were also evaluated. Coding genes, especially COI, had a better identification capacity than non-coding genes on barcoding gap and monophyly analysis and NN, BCM, BOLD ID tests. Considering the multiple factors involved in global DNA barcoding evaluations, we present a critical assessment of the use of these genes for biodiversity estimation and specimens' identification in anurans (e.g. hylids).

BARKODE DNA DAN KEKERABATAN IKAN LAIS TIMAH (Kryptopterus apogon) BERDASARKAN GEN SITOKROM C OXIDASE SUBUNIT I (SOI)

Lais (Krypyoterus apogon) is highly economic important catfish in South Sumatera. Investigating fish diversity of catfish is of importance for species conservation. Many species or subspecies extincted due to environment decreasing caused by habitat loss, pollution and over fishing. Cytochrome C Oxidase subunit I (COI) is one of mitochondrial DNA markers used for species barcoding in freshwater, brackishwater and marine fish. This research aim to explore the use of COI gene for species barcoding in investigating the diversity of COI nucleotide, constructing phylogenetic tree of lais, and knowing physical and chemistry characteristic of habitat at Musi and Kelekar River, South Sumatera. The methods used in the research consisted of DNA extraction, PCR (Polymerase Chain Reaction) amplification and sequencing mtDNA COI gene of Kryptopterus apogon obtained from Musi and Kelekar River (South Sumatera). A 615 bp (sample code CM1) and 693 bp sequences (sample code CK1 and CK2) of parsial coding were sequenced from this fish. Nucleotide BLAST analyzes showed COI gen can distinguish lais at Musi and Kelekar River from others fish at GenBank database with high identity (92.29% and 92.21%) to Phalacronotus bleekeri (Thailand) and Phalacronotus apogon (Australia).Phylogenetic analyzes indicated that obviously, lais was at the same cluster with others genus in Siluridae family, but different cluster from Hemibagrus nemurus (Bagridae), Chanidae and Oreochomis niloticus (Cichlidae). Water quality criteria (water tranparancy, temperature, ammonia, pH and alkalinity) at Musi and Kelekar River were still in tolerance for maintainning survival rate of lais. Further study using more species in Siluridae family and various habitat are needed to investigate the diversity of DNA of Siluridae at South Sumatera water resources.Keywords: Catfish, DNA barcode, COI gene, Musi River, Kelekar River

Analysis of thrips (Thysanoptera: Thripidae) diversity in avocado shows the presence of a new species and limitations of using the COI gene for differentiation of two species

Thrips are important pests of avocados causing damage to the fruits, making themunsuitable for exportation. Many of them are quarantine species and their identification isnecessary. Despite their importance, not many studies describe thrips diversity on avocado,especially in Colombia, partly because identification of some of the species is difficult usingclassical taxonomic keys. In this study, 93 adult thrips specimens were collected on avocadoand adventitious dandelion plants from plantations in the department of Antioquia, Colombia. Morphological characters were used to classify 62 of them and all of them were analyzed by PCR amplification and sequencing of cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer (ITS) region. The species identified by morphological features were Frankliniella gardeniae Moulton, F. gossypiana Hood, F. panamensis Hood, Liothrips perseae Watson, Thrips palmi Karny, T. simplex (Morison), T. trehernei Priesner, Microcephalothrips abdominalis (Crawford DL), and Scirtothrips hansoni Mound & Hoddle (Tubulifera), being F. panamensis, M. abdominalis, and S. hansoni reported for the first time on avocado in Colombia. Thrips simplex and T. trehernei were also found on adventitious dandelion plants, but not on avocado. Phylogenetic analysis of COI and ITS sequences showed that most species, except for F. gardeniae and F. gossypiana were mainly identified with the COI gene. Furthermore, FST values obtained between them suggest no genetic differentiation at COI level. The results obtained here contribute to the knowledge of the diversity of thrips species found on the avocado crop in Colombia and are meaningful for pest management.

Phylogenetic of Marble Goby (Oxyeleotris marmorata Blkr.) in South Sumatra Based on Cytochrome C Oxidase Subunit I (COI) Gene

Marble goby (Oxyeleotris marmorata) in Indonesia, spread across Sumatra, Kalimantan and Papua. The purpose of this study was to utilize a sequence of mitochondrial DNA Cytochrome Oxidase Subunit I gene, to analyze the phylogenetic tree and genetic distance between cultured and captured populations. This research was conducted on March-August 2020. The methods used in barcoding species were DNA isolation, amplification using PCR (Polymerase Chain Reaction) and sequencing of the COI mtDNA gene. The domesticated samples (n=3) were collected from Gandus Fish Seed Center (GFSC), while the wild samples (n=3) were captured from the Musi River in Beruge Village, Babat Toman District, both in Musi Banyuasin Regency. The sequenced COI mtDNA gene fragments were obtained from the PCR method. Three samples performed good PCR results, while the other three didn’t amplify properly. After the editing process, the COI gene sequencing produced a nucleotide length of 610 bp. Based on the BLAST (Basic Local Alignment Search Tool), domesticated marble goby (OMD2, OMD3) was in the same cluster with marble goby from the Musi River (OMS3). The genetic distance indicated that two specimens of domesticated marble goby were 100% identical, while the wild (OMS3) indicated a genetic distance of 0.0066 to domesticated species.

Identification of Common Sarcosaprophagous Flies in the Yangtze River Delta by COⅠ Gene.

To identify the common sarcosaprophagous flies in the Yangtze River Delta based on mitochondrial cytochrome c oxidase subunit Ⅰ(COⅠ) gene sequence and verify the reliability of this method. Seven common genetically stable sarcosaprophagous flies in three families and six genera were collected from large domestic pig carcasses placed in the field and cultured in the laboratory for many generations. The whole genome DNA was extracted and the COⅠ gene fragment was amplified. The forward and reverse sequencing was followed by splicing. The base composition of the amplified fragment and the rate of interspecific evolutionary divergence were analyzed by software such as Mega 7.0.26. The phylogenetic tree of COⅠ gene sequence of common necrophagous flies in the Yangtze River Delta was established by neighbor joining (NJ) method and unweighted pair-group method with arithmetic means (UPGMA) method. The average base composition of different flies was A(30.14%), T(38.23%), C(15.98%), G(15.65%). The rate of interspecific evolutionary divergence ranged from 2.2% to 15.3%, the lowest rate was between Chrysomya megacephala and Chrysomya pinguis, the highest rate was between Muscina stabulans and Boettcherisca peregrina. COⅠ gene can be used to identify the common necrophagous flies in the Yangtze River Delta.

Genetic Diversity and Variation in Mitochondrial COI Gene in Wild and Hatchery Populations of Saxidomus purpuratus.

To investigate the genetic diversity and genetic variations of four wild (Geoje, Jinhae, Yeosu, and Boryeong) and two hatchery (Goheung and Geoje) populations of purplish Washington clam (Saxidomus purpuratus), 421bp sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene were analyzed. A total of 149 haplotypes were identified from 358 individuals from the four wild and two hatchery populations with 109 substitutions. The genetic diversity of the wild populations and Geoje hatchery population were high, whereas the total number of haplotypes, population-specific haplotypes, and haplotype diversity were comparatively low in the Goheung hatchery population. The fixation index (FST) indicated that there was no significant genetic difference between the four wild populations. However, the Goheung hatchery population was significantly different from that of the Geoje hatchery, exhibiting the most pronounced difference, and two wild populations (Jinhae and Yeosu). The low genetic diversity indices exhibited by the Goheung hatchery population might have resulted from farm propagation using a limited parental stock. Therefore, to maintain genetic diversity, a proper breeding management program using more progenitors is required in hatcheries, in addition to regular monitoring of both hatchery and wild populations.

The development of genus-specific and species-specific real-time PCR assays for the authentication of Patagonian toothfish and Antarctic toothfish in commercial seafood products.

The substitution or mislabeling of toothfish is an issue of significant concern for seafood authorities; it also reduces the effectiveness of marine conservation and management programs for its over-exploitation and illegal trafficking, boosting the need for identification methods. Two species-specific real-time polymerase chain reaction (PCR) assays for the identification of Patagonian toothfish (Dissostichus eleginoides) and Antarctic toothfish (Dissostichus mawsoni) and a genus-specific real-time PCR assay for Dissostichus spp. identification were developed based on fragments of the 16S rRNA and COI (cytochrome c oxidase subunit I) genes. These methods were confirmed to be rapid, simple, and sensitive (absolute sensitivity of 0.0002 ng μL-1 and relative sensitivity of 0.1g kg-1 with good specificity). These methods can be applied to processed and commercial fish products. These approaches can be beneficial for protecting both consumers and producers from economic fraud and might also help protect toothfish from over-exploitation as well as combat illegal, unreported, and unregulated (IUU) fisheries. © 2021 Society of Chemical Industry.

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus group using quantitative sequencing

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.

Phylogenetic of Featherback Chitala sp from South Sumatra Based on Cytochrome C Oxidase Subunit I (COI) Gene

The featherback Chitala sp in Indonesia inhabit riverine of Sumatra, Java and Kalimantan. Featherbacks have been protected as threatened species. This research aims to identify the sequence of Cytochrome C Oxidase Subunit I (COI) gene of mtDNA, and construct the phylogenetic trees between species of featherbacks in South Sumatra. This research was conducted in March–August 2020. The methods used in barcoding species and determining phylogenetic i.e., DNA isolation, DNA amplification using PCR and sequencing of COI gene regions of mtDNA. The COI gene was sequenced by PCR (Polymerase Chain Reaction) with optimum annealing temperature 500C for 30s with 35 cycles. After editting, sequence length of the COI gene of featherbacks was 621 base pairs (bp) nucleotide. Based on BLASTanalysis, specimen CLP2 from PT PLN Indralaya had the highest similarity (99.28%) to C. lopis (Malaysia), then 98.88% to the same species from Kampar River, Riau. Speciemens from Musi River (CLS1, CLS 3) and PT PLN (CLP3) indicated the highest similarity of 95.19% with C. chitala from India. The phylogenetic trees showed that Chitala formed four sub-clusters and it was clearly distant between species C. lopis and C. chitala (bootstrap value = 73).

Babesia pisicii n. sp. and Babesia canis Infect European Wild Cats, Felis silvestris, in Romania.

Haemoparasites of the genus Babesia infect a wide range of domestic and wild animals. Feline babesiosis is considered endemic in South Africa, while data on Babesia spp. infection in felids in Europe is scarce. Using samples from 51 wild felids, 44 Felis silvestris and 7 Lynx lynx, the study aimed to determine the presence and genetic diversity of Babesia spp. in wild felids in Romania by analyzing the 18S rDNA and two mitochondrial markers, cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) genes. By 18S rDNA analyses, Babesia spp. DNA was detected in 20 European wild felids. All sequences showed 100% similarity to B. canis by BLAST analysis. Conversely, Cytb and COI analyses revealed the presence of two Babesia spp., B. pisicii n. sp., which we herein describe, and B. canis. The pairwise comparison of both mitochondrial genes of B. pisicii n. sp. showed a genetic distance of at least 10.3% from the most closely related species, B. rossi. Phylogenetic analyses of Cytb and COI genes revealed that B. pisicii n. sp. is related to the so-called “large” canid-associated Babesia species forming a separate subclade in a sister position to B. rossi.

DNA Barcoding for Selected Mangrove-Based Estuary Fishes from Way Kambas National Park, Lampung Province, Indonesia

Over the past decade, DNA barcoding has provided new insight into fish ecology and biosystematics and led to new species' discovery. DNA barcoding is a method for the recognition and identification of species using short, standardised DNA fragments. The correct taxonomic identification of species is critical for the assessment and monitoring of biodiversity. This study applied DNA barcoding techniques to identify selected fish species from a mangrove-based estuary in Way Kambas National Park, Lampung Province, Indonesia. The gene encoding cytochrome c oxidase subunit I (COI) was amplified and bi-directionally sequenced from 22 specimens. The resulting 680 base pairs (bp) sequence was used to identify species, obtain phylogenetic information, and analyse genetic distances. A neighbour-joining tree was constructed based on the mitochondrial COI gene using the Kimura two-parameter model. This study also exhibits conservation status for those identified species. Our findings will facilitate future studies of fish species diversity in mangrove estuary-based ecosystems and provide preliminary data in policymaking in conservation areas such as National Park.

Hundreds of new DNA barcodes for South African sponges

DNA barcoding based on a fragment of the cytochrome c oxidase subunit I (COI) gene from the mitochondrial genome is widely applied in species identification, species discovery and biodiversity studies. The aim of this study was to establish a barcoding reference database of sponges collected from South Africa, and evaluate the applicability of the COI gene for aiding in the identification of sponges in combination with tentative morphological identifications. A total of 317 mitochondrial COI barcode sequences, with an additional 21 extended COI fragments and 24 nuclear ITS sequences, were obtained from 11 orders, 38 families, 58 genera and 124 species of spiculated sponges. A Neighbour Joining (NJ) trees that were reconstructed using these sequences in most cases clustered species in accordance with their current taxonomic identification, and we conclude that COI sequencing can be used to aid in the identification of sponge species. We further demonstrate that DNA barcoding analysis has potential to uncover cryptic sponge species, and to reveal dubious morphological identifications. We recommend that future taxonomic studies of South African sponges incorporate multiple sources of information for species identification or discovery.