Barcode Region Research Articles

Species delimitation by DNA barcoding reveals undescribed diversity in Stelliferinae (Sciaenidae).

Stelliferinae is the third most speciose subfamily of Sciaenidae, with 51 recognized species arranged in five genera. Phylogenies derived from both morphological and molecular data support the monophyly of this subfamily, although there is no general consensus on the intergeneric relationships or the species diversity of this group. We used the barcoding region of the cytochrome oxidase C subunit I (COI) gene to verify the delimitation of Stelliferinae species based on the Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescence (GMYC), and Bayesian Poisson Tree Process (bPTP) methods. In general, the results of these different approaches were congruent, delimiting 30-32 molecular operational taxonomic units (MOTUs), most of which coincided with valid species. Specimens of Stellifer menezesi and Stellifer gomezi were attributed to a single species, which disagrees with the most recent review of this genus. The evidence also indicated that Odontoscion xanthops and Corvula macrops belong to a single MOTU. In contrast, evidence also indicates presence of distinct lineages in both Odontoscion dentex and Bairdiella chrysoura. Such results are compatible with the existence of cryptic species, which is supported by the genetic divergence and haplotype genealogy. Therefore, the results of the present study indicate the existence of undescribed diversity in the Stelliferinae, which reinforces the need for an ample taxonomic review of the fish in this subfamily.

Molecular characterization of mitochondrial COI gene sequences in Micraspis allardi from Pakistan.

The Coccinellidae is a highly diversified family of order Coleoptera. Coccinellid ladybirds are well known for their role as biological control agent against varied range of agricultural pests. The samples of coccinellid ladybird collected from Pakistan were identified and characterized as Micraspis allardi (Mulsant, 1866). This is one of the least-studied ladybird species with limited work on its ecological distribution as a biological control agent. The genus Micraspis has vast genetic diversity with a possible presence of unknown number of cryptic species. Sequence information of some species of the genus Micraspis are present in NCBI database. However, least molecular data or sequences describing M. allardi could be available from database. Therefore, morphological and molecular characterization was imperative for this species. Here, the samples collected from sugarcane field of Faisalabad District of Pakistan and were identified by using morphological and molecular protocols. For molecular identification, two different regions of mitochondrial cytochrome c oxidase I (COI) gene (COI-5' and COI- 3') were used as molecular markers for the identification of the species. Morphological appearance, DNA sequence similarity searches and phylogenetic analysis collectively indicated it as M. allardi. To the best of our knowledge, this is the first report providing molecular evidence of M. allardi using mitochondrial DNA barcode region (658bp) as well as mtCOI-3' sequences (817bp). The study will help in understanding population genetics through diversity analysis, ecological role, and phenotypic structures associated with the geographic range of this species.

Cecidophyinae (Acari: Acariformes) from Serbia: description of a new species, supplementary description of six species and four new records for the fauna

During the faunistic survey of Cecidophyinae mites in Serbia, a new species, Achaetocoptes dragicae sp. nov., found on Erica carnea L. is described and illustrated. Additionally, supplementary descriptions of six species (Cecidophyes glaber (Nal.), Cecidophyes nudus Nal., Cecidophyes psilonotus (Nal.), Cecidophyes gymnaspis (Nal.), Cecidophyopsis rosmarinusis Wang et al., and Chrecidus quercipodus Manson) are provided. Moreover, Achaetocoptes cerrifoliae (Labanowski & Soika) and Bariella bakonyense Ripka & Csóka, represent new records for the Serbian fauna, while Ce. rosmarinusis and Ch. quercipodus are recorded for the first time in Europe. Sequences of mtCOI barcode region are provided for all species.

The pine processionary moth Thaumetopoea pityocampa (Notodontidae) species complex: a phylogeny-based revision

The pine processionary moth, Thaumetopoea pityocampa, is an iconic insect in the Mediterranean culture because of its economic and medical importance and the unique traits of the life history, namely the winter feeding and the construction of conspicuous silk tents by the larvae. Its taxonomic status, however, is unclear because the type material is not available and there is confusion among the several species and subspecies described in the last centuries. In the present study, a metadata analysis of morphological and molecular data of the species in the T. pityocampa complex were considered for species delimitation, using more than 400 individuals from more than 120 geographic locations. A reconstruction of the origin of the material used for the first formal description has allowed to identify the type locality and to design a neotype. In addition, as Denis and Schiffermüller were referring to the work of Réaumur for details about the species, the description provided by Réaumur was reviewed. The results indicate that the barcode region of mitochondrial DNA is a reliable trait to separate species in most cases whereas morphological traits are not. Hybridization among taxa makes it difficult to delimit species in contact zones when mating barriers are not present. In other cases, such as the populations of Crete Island, the lack of gene flow with the mainland population may support species delimitation even when morphological traits are not conclusive. Thus, the new species Thaumetopoea cretensis is described here based on the evidence obtained from a previous study. Species delimitation based on both mitochondrial and nuclear markers allowed maintenance of three species of the complex (T. cretensis, T. pityocampa, and T. wilkinsoni) while more data are needed to determine the status of two recently described species: T. hellenica and T. mediterranea.

Integrative Taxonomy of Turcinoemacheilus Bănărescu & Nalbant, 1964 in West Asia with the Description of Three New Species (Teleostei: Nemacheilidae)

Nemacheilid fishes in the genus Turcinoemacheilus are physically small members of the ichthyofauna communities of high-altitude and mountainous freshwater ecosystems. They are all distributed in Western Asia apart from a single species, described in the Himalayas. They are usually very similar in appearance, which complicates their proper identification and/or description. This is why it is important to use multidisciplinary and integrative taxonomical approaches in order to study their true diversity. In this study, three new species of Turcinoemacheilus are described from Iran, raising the total number of valid species to nine. Turcinoemacheilus ansari new species, is distinguished by the anus being situated behind the midpoint of the pelvic-fin and anal-fin origins and the short anal-fin base length. Turcinoemacheilus christofferi new species, differs by the anus being situated behind the midpoint of the pelvic-fin and anal-fin origins, with a complete lateral line reaching to the anterior part of the caudal fin. Turcinoemacheilus moghbeli new species, is distinguished by the anus being situated at or in front of the midpoint of the pelvic-fin and anal-fin origins, with a great pre-pelvic distance and a caudal peduncle length 1.5–2.3 times its length. In Western Asia, all Turcinoemacheilus species are well separated by molecular characters, showing between 3.6 and 14.1% uncorrected p genetic distances in the COI barcode region. This work shows the importance of studying the hidden diversity of under-sampled and understudied groups of organisms to have a clear image of true biodiversity in order to effectively conserve and protect it.

Ooencyrtus mirus (Hymenoptera, Encyrtidae), discovered in Europe parasitizing eggs of Halyomorpha halys (Hemiptera, Pentatomidae)

Ooencyrtus mirus Triapitsyn & Power (Hymenoptera, Encyrtidae) is recorded for the first time in Europe. It was found parasitising eggs of the invasive true bug Halyomorpha halys Stål (Hemiptera, Pentatomidae). This parasitoid is part of the Ooencyrtus telenomicida species complex where accurate species identification requires molecular data. Using morphology, the identification of the Ooencyrtus species parasitising brown marmorated stink bug eggs in Greece is ambiguous, but the sequences of the standard DNA barcode region (COI) and ITS2 place them in O. mirus.

High Species Diversity but Low Specificity to Ectomycorrhizal Tree Partners Exhibited by Native Truffle Species (Tuber spp., Pezizales) in Poland, Central Europe

Truffles represent one of the most expensive edible fungi. About two-thirds of all known truffle records originate from Europe. Historically, the Mediterranean region in Southern Europe is associated with truffle cultivation and it is widely regarded as a center of truffle distribution in Europe. On the contrary, little is known about truffles in the central and northern regions of Europe. Here, native truffle species in Poland, their symbiotic ectomycorrhizal (ECM) tree partners, and their continental-scale distribution were studied. Altogether, 16 truffle species were identified based on the sequences of the Internal Transcribed Spacer (ITS) rDNA, a barcode region for fungi. Truffle species were associated with deciduous tree species of Fagales (Fagaceae, Betulaceae, Juglandaceae), Malvales (Malvaceae), Malpighiales (Salicaceae), and coniferous trees of Pinales (Pinaceae). Fagales trees constituted 70% of ECM associations formed by truffle species. Three genera of the order Fagales, Quercus, Fagus, and Corylus formed 50% of all ECM associations. Coniferous trees of Pinales formed 16% of associations. Two other orders of deciduous trees, Malvales and Malpighiales, together formed 14% of ECM associations. All but three identified truffle species exhibited low specificity to their ECM tree partners. Two-thirds of truffle species formed ECM symbiosis with both coniferous and deciduous tree species, but the share of coniferous tree partners was considerably lower than that of deciduous trees, reaching up to 30% for T. anniae, T. puberulum, and T. borchii (clade/puberulum). All the identified truffles were noted in both Central Europe and the Mediterranean region. Among them, about 80% of truffle species were widely distributed across the continent and represented by similar or higher numbers of records in Northern Europe, i.e., above the 48th parallel north, than in Southern Europe. This study showed higher taxa richness of native truffle species in Poland, but low specificity to their ECM tree partners. However, further studies on the regional-scale distribution of truffles in Poland are needed to improve the knowledge of the patterns of truffle distribution in forest ecosystems and the potential productivity of edible truffle species with high economic value.

Development of a standardized methodology for selecting candidate DNA barcodes of Trillium govanianum to combat biodiversity crime and environmental exploitation

This study introduces an innovative method using DNA barcodes to accurately identify species, addressing the critical issue of trafficking invaluable medicinal plants like Trillium govanianum. The success rate in amplification and sequencing of barcoding regions was an impressive 100%. The Internal Transcribed Spacer (ITS) region proved most effective in distinguishing closely related Trillium species. By testing herbal products from markets, the study revealed extensive illegal trade, with 50% confirmed as Trillium govanianum and 30% adulterated with Dioscorea villosa. This research contributes new DNA sequences to identification databases, enhancing protocols. Genetic variations within Trillium govanianum were narrower than to the nearest non-conspecific neighbor in barcoding regions (matK: 0.006, rbcL: 0.003, ITS: 0.043). Combining coding and non-coding regions (matK + ITS) achieved 100% species discrimination. Market samples showed 30% product substitution, with varying success rates for amplification (rbcL: 70%, matK: 60%, ITS: 40%, improved with ITS2 primers). Half the sequences were Trillium govanianum, 30% were Dioscorea villosa, revealing covert substitution. ITS and matK sequences were effective, with a genetic distance gap, while rbcL had limited divergence (0.003), making it less suitable for accurate identification compared to matK and ITS.

DNA barcoding application for potential medicinal plants on Ziziphus species from Aketajawe-Lolobata National Park, North Moluccas

Aketajawe-Lolobata National Park is a protected national park located in the province of North Moluccas, Indonesia. The national park has been known to be rich in biodiversity with the existence of many potential medicinal plants such as Ziziphus. DNA barcoding can be a useful tool for identifying and characterizing these plants for potential medical applications. Research and exploration have been conducted in Aketajawe-Lolobata National Park in November and December 2022. This study aimed to analyze how the DNA barcoding regions aid identification for potential medicinal plants. The genomic DNA were amplified by matK primers, and the sequences showed high similarity rate with their homologs, then the phylogenetic tree was constructed using the Neighbor-Joining method to assess their cross-species relationship. The phylogenetic study showed that the species that have been identified based on morphological characters, i.e., Ziziphus angustifolia and Z. mauritiana were clustered with Z. angustifolia and Z. mauritiana retrieved from GenBank. However, the unidentified species are closely related with Z. calophylla obtained from GenBank but with low bootstrap support. All the results indicate that DNA barcoding approach could be successfully used for reliable identification of medicinal plants and matK gene is a good candidate for this approach.

Molecular identification of Pelawan Merah (Tristaniopsis merguensis Griff.) as an endemic plant from Bangka Belitung Island using chloroplast DNA rbcL gene

The DNA barcoding from this molecular identification will be submitted to the NCBI Genbank database to become a standard in the identification of Tristaniopsis merguensis Griff based on the rbcL gene analysis. This research will be used as a crucial information to identify the endemic species of Tristaniopsis merguensis Griff. which has no conservation status yet on IUCN Red List & Threated species. The stages of this research were collecting samples of Pelawan Merah leaves from Hutan Pelawan Namang, Central Bangka Regency. Molecular analysis was carried out to obtain the marker gene of Tristaniopsis merguensis Griff. The DNA barcoding of Tristaniopsis merguensis Griff yielded a 516 base-pair long sequence of rbcL gene. This is the first report of Tristaniopsis merguensis Griff DNA barcoding in Indonesia and the barcode has registered in the GenBank. This DNA barcoding also aims to register the plant biodiversity in supporting of the Barcode of Life (CBOL). Based on the results of BLAST DNA analysis, Pelawan Merah from Bangka Island has similarity up to 100% with Tristaniopsis sp. found in Australia. Furthermore, the phylogenetic tree analysis reveals that the Tristaniopsis merguensis Griff. in this study was very closely related to other species of genus Tristaniopsis. This suggested that the rbcL gene analysis could identify Tristaniopsis at the genetic level or higher and other DNA barcoding region analysis as Internal Transcribed Spacer gene was necessary to complete the Pelawan Merah identification.

Combined Use of Phenotype-Based and Genome-Informed Approaches Identified a Unique Fusarium oxysporum f. sp. cubense Isolate in Hawaii.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is a serious disease that threatens banana production worldwide. It is a long-standing problem in Hawaii, but previously, there was little knowledge of the causal pathogen. We isolated a strain of Foc, named Foc-UH, from a field experiencing the disease epidemic in Hawaii. Infection assays of a diverse panel of 26 banana clones, including varieties used for differentiating pathogen races and fruit production, revealed that Foc-UH has a race 1 pathogenic phenotype with an intermediate race 2 virulence and revealed the differential resistance of varieties to infection. Separate phylogenetic analyses using the barcoding regions of three nuclear genes, seven complete nuclear genes, and single-nucleotide polymorphisms within conserved whole-genome protein coding sequences placed Foc-UH into recently proposed taxonomic frameworks relevant to Foc and the F. oxysporum species complex. Screening of the 99.7% complete draft genome identified five secreted in xylem (SIX) gene homologs: SIX1d, SIX1f, SIX9a, SIX9b, and SIX13a. This profile is similar to that of several race 1 isolates except for the absence of SIX4 and SIX6. Foc-UH was morphologically dissimilar to the nearest related isolates. Altogether, this study identified a unique isolate that causes banana Fusarium wilt, which represents the first characterization of the causal pathogen in Hawaii. The findings and genomic resources generated in this study are expected to guide banana breeding and cultivar deployment in Hawaii and beyond and contribute to further understanding of the pathogenicity and evolutionary systematics of Foc.

First report of Bot Canker (Diplodia corticola) in Pin Oak (Quercus palustris) in Tennessee.

Diplodia corticola is a fungal pathogen contributing to oak (Quercus spp.) decline in the Mediterranean and US (Félix et al., 2017; Ferreira et al., 2021). In 2021, this pathogen was detected in Tennessee (TN) causing branch dieback in Q. alba (Onufrak et al., 2022). In September 2021, a matured pin oak (Q. palustris) with wilted leaves and elongated branch cankers was observed in the State Botanical Garden of Tennessee-Knoxville (TN, US). Small sections of the phloem were sampled from canker margins of a symptomatic branch using a sterile scalpel, surface sterilized, and plated onto potato dextrose agar amended with antibiotics (PDA++) (Gazis et al. 2018). Three days later, a fungal isolate resembling D. corticola was cultured on ½ PDA. Diplodia corticola is characterized on half-strength PDA by fast growth, irregular margins, and dense white mycelium that turns dark, grayish as the mycelium matures (Úrbez-Torres et al., 2010; Alves et al., 2004). Total genomic DNA was extracted from this isolate following Gazis et al. (2018), and the internal transcribed spacer (ITS), large ribosomal subunit (LSU), and transcription elongation factor 1-α (ef1-α) were amplified (Ferreira et al. 2021). Resulting PCR products were sequenced and assembled into consensus sequences using Unipro UGENE v. 44.0 (Okonechnikov et al., 2012). Each consensus sequence identity was determined using BLAST on the NCBI nucleotide database, restricted to type material. The ITS (accession OQ189888), ef-1α (accession OQ201608), and LSU (accession OQ189887) sequences had a 99.6% (accession KF766156.1), 98.6% (accession XM_020275852.1), and 100% (accession KF766323.1) identity match with D. corticola type culture CBS112549, respectively. To complete Koch's postulates and assess potential pathogenicity on economically and ecologically relevant oaks, 10 pin (Q. palustris; caliper 15.6 ± 2.0 mm), 10 overcup (Q. lyrata; caliper 15.1 ± 2.4 mm), and 10 sawtooth (Q. acutissima; 16.1 ± 2.1 mm) oaks were acclimated in the greenhouse for 1 week prior to the experiment. Five trees of each species were then randomly inoculated at 30 cm above the soil line with a 3 mm diameter plug of D. corticola (grown for 10 days on PDA; Sitz et al. 2017). To serve as a control, the remaining 5 trees for each species received a 3 mm diameter PDA plug. Fifteen days post-inoculation, seepage was observed in D. corticola-inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks. No seepage from wound sites was noted in control trees. Cankers were exposed, photographed, and then measured using ImageJ (Rasband, 2012). Using a sterile scalpel, four wood chips were excised from canker margins and plated onto PDA++. We recovered D. corticola from symptomatic inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks and confirmed species identity by extracting DNA and amplifying the ITS, ef-1α, and LSU regions as described above (Gazis et al., 2018; Ferreira et al., 2021). The resulting consensus sequences matched the D. corticola type culture (CBS112549) ITS (99.0%-99.8% identity), ef-1α (91.0%-99.1% identity), and LSU (96.9%-100% identity) barcoding regions. Cankers were significantly larger in D. corticola-inoculated pin (4.7 ± 1.5 cm2; P = 0.003), overcup (6.8 ± 2.9 cm2; P = 0.009), and sawtooth (5.1 ± 1.3 cm2; P = 0.001) oaks in comparison to the control trees from these groups. Based on current reports, this is the first record of D. corticola causing dieback in pin oak (Q. palustris) in TN.

Molecular Discrimination and Phylogenetic Relationships of Physalis Species Based on ITS2 and rbcL DNA Barcode Sequence

Plants of the genus Physalis are of economic interest because of their fleshy edible fruits with high nutritional value. Some species have high medicinal value with a long history of ethno-medicinal use to treat diverse diseases. There is therefore a need to correctly discriminate the different species of Physalis for proper utilization. Although most Physalis species have unique morphologies, their vegetative stages are identical, making it difficult to accurately identify them based on morphological characteristics. DNA barcoding has the potential to discriminate species accurately. In this study, ribulose bisphosphate carboxylase large (rbcL) and internal transcribed spacer 2 (ITS2) regions were used to discriminate Physalis species and to reveal their phylogenetic relationships and genetic diversity. Physalis plant samples were collected from seven counties in Kenya based on the availability of the germplasm. The voucher specimens were identified using the botanical taxonomy method and were deposited in the University of Nairobi herbarium. Genomic DNA was isolated from leaf samples of 64 Physalis accessions and used for PCR amplification and the sequencing of rbcL and ITS2 barcode regions. The discriminatory ability of the barcodes was based on BLASTn comparison, phylogenetic reconstruction and cluster analysis, and the determination of inter- and intra-specific distances. The nucleotide polymorphism, genetic diversity and distance of the identified Physalis species were determined using DnaSP and MEGA 11.0 software. Species discrimination was more robust using ITS2 sequences. The species identified and discriminated by ITS2 sequences were Physalis purpurea, Physalis peruviana and Physalis cordata. The rbcL sequences were only able to identify Physalis to the genus level. There was high interspecific and low intraspecific divergence within the identified Physalis species based on ITS2 sequences. The ITS2 barcode is an ideal DNA barcode for use in the discrimination of species, as well as in genetic diversity studies of Physalis accessions in Kenya.

DNA Barcoding Based Identification of Rosa x Damascene and Prunus dulcis Herbs Using ITS2 Barcoding Gene Amplification.

The considerable risk of adulteration in the herbs has raised commercial interest in the identification of medicinal herbs globally. DNA barcoding is the primary techniques for identifying the herbs at genetic level. This technique's key benefit is that it can identify the material's purity. This study focuses on the accurate identification of species utilizing Polymerase chain reaction-based nuclear universal internal transcribed spacer region (a barcode region) amplification and sequencing in 2 medically significant plants (Rosa damascene and Prunus dulcis) procured from the local herbal market of Karachi in the year 2022. Results suggest that the ribosomal nuclear ITS2 region of the selected plant species shows the 100 % identity with the reference genome, therefore it has shown a good rate of identification at the species level. The findings of this concludes that ITS2 is the novel standard barcode that involve in species identification, genomic conservation, and secure utilization of the medically significant plant species.

Establishing reliable DNA barcoding primers for jumping plant lice (Psylloidea, Hemiptera)

ObjectivesDNA Barcoding has proven to be a reliable method for rapid insect identification. The success of this method is based on the amplification of a specific region, the ‘Folmer’ barcode region at the 5´ start of the cytochrome c oxidase 1 gene (cox1), with universal primers. Previous studies showed failures of standard “universal” primers to amplify this region in psyllids. The aim of the study was the design of a new alternative more reliable primer combination for taxa of the superfamily Psylloidea and its comparison with the performance of the standard “universal” Folmer-primers.ResultsA newly designed degenerate forward primer LCOP-F was developed following comparison of the sequence alignment of the priming site of “universal” primer LCO1490 and the standard insect forward primer LepF1. When combined with the “universal” reverse primer, HCO2198, this new primer pairing was able to generate barcode sequence for all 36 species in 20 genera across the five families of psyllids tested in this study, and these primers were found to be more universally reliable across psyllid taxa than other primer pairs tested.

Profiling of the fecal microbiota and circulating microRNA-16 in IBS subjects with Blastocystis infection : a case–control study

Irritable bowel syndrome (IBS) is a prevalent gastrointestinal (GI) tract disorder. Although the main reason for IBS is not clear, the interaction between intestinal microorganisms and the gut barrier seems to play an important role in pathogenesis of IBS. The current study aimed to investigate the effect of Blastocystis on the gut microbiota profile and the circulation levels of microRNA (mir)-16 of IBS patients compared to healthy subjects. Stool and blood samples were collected from 80 participants including 40 samples from each IBS and healthy group. Upon DNA extraction from stool samples, barcoding region and quantitative real-time PCR were analyzed to investigate Blastocystis and the microbiota profile, respectively. RNA was extracted from serum samples of included subjects and the expression of mir-16 was evaluated using stem-loop protocol and qreal-time PCR. Significant changes between IBS patients and healthy controls was observed in Firmicutes, Actinobacteria, Faecalibacterium, and Alistipes. In IBS patients, the relative abundance of Bifidobacteria was directly correlated with the presence of Blastocystis, while Alistipes was decreased with Blastocystis. Lactobacillus was significantly increased in Blastocystis carriers. In healthy subjects, the relative abundance of Bifidobacteria was decreased, but Alistipes was increased in Blastocystis carriers. The changes in the Firmicutes/Bacteroidetes ratio was not significant in different groups. The relative expression of mir-16 in Blastocystis-negative IBS patients and healthy carriers was significantly overexpressed compared to control group. The presence of Blastocystis, decreased the relative expression of mir-16 in IBS patients compared to Blastocystis-negative IBS patients. The present study revealed that Blastocystis has the ability to change the abundance of some phyla/genera of bacteria in IBS and healthy subjects. Moreover, Blastocystis seems to modulate the relative expression of microRNAs to control the gut atmosphere, apply its pathogenicity, and provide a favor niche for its colonization.

New Morphological and Molecular Data on the Southern Ninespine Stickleback, Pungitius platygaster (Gasterosteidae) from Southern Caspian Sea Basin

Being a group of popular model organisms in eco-evolutionary biology, genetic, and genomic research studies, the genus Pungitius with 12 species is the most species-rich genus in the stickleback family Gasterosteidae including the Caspian nine-spine stickleback, Pungitius platygaster (Kessler, 1859), the only known species from southern Caspian Sea basin. Here, the new data based on morphological and molecular characters of the mtDNA COI barcode region, and current distribution range of P. platygaster is presented. These data confirm the validity of P. platygaster among its congeners, being sister clade to P. hellenicus from Greece. Morphologically, it is distinguished from the other congeneric species by complete pelvic girdle, 8−11 dorsal spines, and presence of large lateral scutes. The Caspian nine-spine stickleback is found in several closely placed freshwater and brackish waterbodies in the southern Caspian Sea.

The Phylogenetic Characterization of Cockroaches (Insecta: Blattaria) in Türkiye and Determination of Their Vector Potential for Medically Important Parasites

This study was performed to investigate the phylogenetic characters of the cockroaches in XXX region for mitochondrial cytochrome c oxidase subunit I (mt-COI), mt-COII, and internal transcribed spacer-2 (ITS-2) gene regions, and to determine their mechanical transmission of medically important parasites. PCR-restriction fragment length polymorphism (RFLP) was performed using mt-COI, mt-COII, and ITS-2 DNA gene regions to identify cockroach species (n=220) collected from different regions. Differentiation of cockroach species was based on RFLP models using two restriction enzymes: AvaI and EciI. For phylogenetic analysis, mt-COI, mt-COII, and ITS-2 DNA barcode regions were amplified with standard primers. The amplicons obtained were purified and sequenced with PCR primers. According to PCR-RFLP, cockroach species were identified as Blattella germanica (n =105), Blatta orientalis (n = 86), and Periplaneta americana (n = 29). A total of 13 haplotypes were detected and Maximum likelihood (ML) analyses revealed that the sequences of all three species for the three gene regions showed monophyletic structuring. The cockroaches were examined for the presence of parasites. It was determined that of the 58 parasitic forms identified belonged to helminth species 46 (79.3%) and protozoan species and 12 (20.7%). The results showed that B. germanica (58.6%) had the highest prevalence, followed by Bl. orientalis (32.8%) and P. americana (8.6%). The study findings not only contribute to the molecular epidemiology of cockroaches but also confirm that they play an important role as mechanical vectors of protozoan and helminth parasites.

Neuroprotective Activity of Catharanthus roseus Ethanol Extract by Acetylcholinesterase Inhibition and Neurite Outgrowth Studies

Aims: To investigate biological activities for neuroprotective effect of Catharanthus roseus.
 Methodology: Catharanthus roseus was identified using DNA barcoding, utilizing matK, trnH-psbA, and rbcL markers. Additionally, thin-layer chromatography (TLC) method was used to analyze the phytochemistry compounds present in the C. roseus extracts. Moreover, acetylcholinesterase (AChE) inhibition activity was tested using a modified Ellman’s method. Finally, neurite outgrowth activity was determined in rat glial C6 cells treated with varying concentrations of C. roseus extracts.
 Results: Overall, the plant samples which were collected in Laocai, Vietnam were successfully identified through DNA barcoding regions, using trnH-psbA, matK, and rbcL genes. Phytochemical analysis detected the presence of sterols, terpenoids, flavonoids, polyphenolic in the ethanol extract and its fraction from C. roseus. Additionally, the extracts of C. roseus displayed remarkably high acetylcholinesterase inhibitory activity. Moreover, the ethanol extract of C. roseus shown the most potent neurotrophic activity in a preliminary cell-based screening based on C6 cells neurite outgrowth.
 Conclusion: These results demonstrate that Catharanthus roseus could be a strong candidate for developing pharmacological drugs to treat neurodegenerative diseases.

Taxogenomic placement of Rasporella oleae and Rasporella dianae gen. and spp. nov., two insect associated yeast species.

During the course of independent studies in Europe, North America, and Africa,seven yeast strains were isolated from insect frass, decaying wood, tree flux, and olive oil sediment. Phylogenetic analysis of two barcoding DNA regions (internal transcribed spacerand the D1/D2 domain of the LSU rRNA gene) revealed that they belong to two closely related undescribed species distinct from all genera in the family Debaryomycetaceae. For reliable taxonomic placement the genomes of four strains of the two novel species and six type strains of closely related species were sequenced. Orthologous genes from 54 genomes of representatives of the Pichiomycetes and 23 outgroup taxa were concatenated to construct a fully supported phylogenetic tree. Consistent with the assumptions, we found that the two new species belong to a novel genus. In addition, the delimitation of the novel species was supported by genetic distance calculations from average nucleotide identity (ANI) and digital DNA:DNA hybridization (dDDH) values. The physiological characterization of the novel species was generally consistent with their genomic content. All strains had two alleles encoding secretory lipase in either two or three copies depending on the species. However, lipolytic activity was detected only in strains with three copies of the secretory lipase gene. Nevertheless, lipolytic activity might be related to their association with the insect gut. Based on these results, formal descriptions of the new genus Rasporella gen. nov. and of two new species Rasporella dianae sp. nov. (holotype UCDFST 68-643T , MycoBank no.: 850238) and Rasporella oleae sp. nov. (holotype ZIM 2471T , MycoBank no.: 850126) are provided.