GenBank Data Research Articles

Gyrodactylus serrai n. sp. (Gyrodactylidae), from the Near-Threatened Clanwilliam Sawfin, Cheilobarbus serra (Peters) (Cyprinidae, Smilogastrinae), in the Cape Fold Ecoregion, South Africa.

A new species of Gyrodactylus is described from the gills of the near-threatened Clanwilliam sawfin, Cheilobarbus serra (Smiliogastrinae) collected from the Matjies River, Cape Fold Ecoregion, Western Cape Province, South Africa. Morphometry and morphology of the haptoral hard parts (hamuli, bars and marginal hooks) of Gyrodactylus serrai n. sp. differ from the other known species of the genus in the smaller size of hamuli and the shape and size of marginal hooks. Furthermore, ITS rDNA for the new species is unique among available Gyrodactylus spp. data in GenBank. Based on the uncorrected p-distances, G. serrai n. sp. is genetically most closely related to Gyrodactylus moroccensis Rahmouni, 2023 and Gyrodactylus pseudomoroccensis Rahmouni, 2023 from two species of Luciobarbus (Barbinae) from northern Africa, with interspecific divergence of 8.7% and 8.8%, respectively. The presence of a median ridge in the terminal part of the ventral bar membrane at G. serrai n. sp. most probably represents a morphological link to the North African Gyrodactylus spp. that suggests a morphogenetic association across the African continent as a result of ancient waterways that facilitated the dispersion of cyprinids and their parasite fauna or an independent evolution event retaining similarities from a common ancestor. The description of Gyrodactylus serrai n. sp. represents only the second species of Gyrodactylus described from an endemic South African cyprinid host, underscoring the need for focused research on this group of fishes to provide a sound understanding of the parasitic communities of these highly threatened and poorly studied hosts.

First report of basal stem rot on sugarcane (var. Badila) caused by Sclerotium rolfsii in China.

Badila (Saccharum officinarum) is one of the important chewing cane in south China. During the year 2019-2020, as much as 60.2%-87.5% of sugarcane plants stem showed red rot developments were observed in the fields of Yongning District, Nanning city, Guangxi province. Symptomatic plants showed red rot at basal stem nodes and sheath, when the disease serious, the epidermis and aerial roots decomposed and exfoliated, then formed sclerotiums, the upper stem also occurred the symptom. Infected plant tissues were dissected into small pieces with 0.1 × 0.1cm in size and surface sterilized in 0.1% HCl2 for 2 min, followed by 75% ethanol for 30 s, rinsed three times with sterile distilled water. Then the tissues were placed onto potato dextrose agar (PDA) plates and incubated at 25 °C for 3 days. Numerous white globoid sclerotia were formed on PDA after 5 days of growth. The sclerotia (2 to 3 mm in diameter) were white at first and then gradually turned dark brown. Aerial mycelia usually formed many narrow hyphal strands 4 to 9 μm wide. Five uniform isolates were obtained from diseased sugarcane plants. Pathogenicity of representative strain W1 was confirmed by inoculating 120-day-old Badila plants grown in field. Five plants were inoculated with colonized agar discs (6mm in diameter) by applying toothpick tips to the lower part of the stem. Five non-inoculated plants served as control. The inoculated and non-inoculated plants were sprayed sterile water then incubated with plastic film for maintained high moisture. All the plants were placed inside of a growth chamber at 26 ± 2°C with a 14-h photoperiod and 80% relative humidity. All inoculated plants showed red rot at stem and sheath after 2 weeks, whereas the control plants were symptomless. By the third week, mycelium and sclerotia developed on the crown on the inoculated plants. The fungus was re-isolated from the artificially inoculated plants. To confirm the species-level identification, partial of the ribosomal DNA internal transcribed spacer (ITS), mitocondrial small subunit (SSU), and nuclear ribosomal large subunit (LSU) regions of representative strain W1 were amplified and sequenced using the primers pairs ITS1/ITS4 (White et al. 1990), ITS-Fu-F /ITS-Fu-R and SRLSU1//SRLSU2 (Kumar et al., 2016), respectively. The resulting ITS, SSU and LSU sequences were deposited in GenBank (GenBank accession no. MW620994, MW617878, and MW617872) and shared 99.42%, 100% and 100% sequence identity with Athelia rolfsii isolate (JN017199, OM319631, and MT225781). Phylogenetic analysis conducted with neighbor-joining (NJ) method using MEGA6.0 revealed that the isolate share a common clade with reference sequence of A. rolfsii in GenBank Data Library. Based on morphological and molecular characteristics, the fungus was identified as A. rolfsii (anamorph: Sclerotium rolfsii) (Paul et al. 2017; Paparu et al. 2020). Although S. rolfsii has been reported causing sugarcane sett rot in Australia (Bhuiyan et al., 2019) and seedlings of sugarcane in Indian (Gopi et al., 2023), as we know, this is the first report of sugarcane basal stem rot disease caused by this fungus in China. This study will be helpful for the prevention and control sugarcane basal stem rot in the future.

First detection and impact of bovine herpesvirus type 4 on dairy cattle reproduction in Thailand

Background and Aim: Postpartum reproductive tract infections pose significant challenges to dairy farms, leading to economic losses due to reduced fertility associated with uterine inflammation. In veterinary practice, numerous research groups have explored the underlying causes of subfertility in cows, including surveying endemic viral infections related to endometritis in local areas. This study investigated bovine herpesvirus 4 (BoHV-4) infection in Thai dairy herds and assessed its impact on endometritis and subsequent reproductive outcomes. Materials and Methods: The present study analyzed BoHV-4 DNA in various samples, including milk, blood, and endometrial tissue, from 44 Holstein-Friesian cows 21–47 days postpartum across five dairy herds in Central Thailand. BoHV-4 glycoprotein B and thymidine kinase DNA sequences were detected using the polymerase chain reaction (PCR) and nested PCR, with sequence comparisons made to GenBank data for phylogenetic analysis. The endometritis status was diagnosed through vaginal mucus examination and endometrial cytology, with reproductive performance monitored up to the subsequent calving. Results: BoHV-4 DNA was identified in blood and endometrial tissues (15.91%) but not in milk samples. Phylogenetic analysis revealed that the local BoHV-4 strains are similar to those identified in Brazil and Japan. Notably, the presence of BoHV-4 was correlated with reduced postpartum reproductive performance, particularly extending the interval from calving to the first service. Conclusion: Our findings underscore the importance of integrating BoHV-4 genomic surveys and uterine health assessments to refine reproductive management strategies within the dairy industry. Keywords: bovine herpesvirus-4, dairy cattle, endometritis, phylogenetic analysis, reproductive performance, Thailand.

Aspects of NCBI GenBank as a Biodiversity Information Resource

DNA sequencing of museum specimens, also known as museomics, provides new insights into the study of biodiversity, including taxonomy, phylogeny, and environmental studies. Also, sequencing specimens have led to the rediscovery of extinct species (Suzuki et al. 2016), identification of related species (Waku et al. 2016), and analysis of ancient DNA (Kanzawa-Kiriyama et al. 2016). Nucleotide sequence data have been collected for more than 30 years under the framework of the International Nucleotide Sequence Database Collaboration (INSDC) by three institutes, namely, National Center for Biotechnology Information, US (NCBI), European Bioinformatics Institute (EBI), and DNA Data Bank of Japan (DDBJ) (Arita et al. 2020). NCBI has collated a database of sequence data, GenBank, which contains approximately 494 million sequences as of April 2022 (Sayers et al. 2021). In fact, GenBank is designed with qualifiers to describe various types of biodiversity information such as "/specimen_voucher", "/lat_lon" (latitude and longitude) and "/collection_date". Also, INSDC now requires that all submissions include the sampling location and date (INSDC 2023). I surveyed the biodiversity information assigned to GenBank records to determine the potential of GenBank as a biodiversity resource. I downloaded all GenBank data as of August 2023 from the FTP site. The “/specimen_voucher” qualifier was introduced to describe specimen ID in Release 104 in December 1997. This qualifier was designed to fill the value in free text: for example, /specimen_voucher="Smith s. n. 4-IV-1995 (U. S. Natl. Herbarium)". After Release 162 in October 2007, a method of writing with a structured value of "[<institution-code>: [<collection-code>:]] <specimen_id>" was added (institution-code and collection-code are optional). There are 527,215 records (37.8%) with "/specimen_voucher" qualifier for fish, 3,096,112 records (40.3%) for insects, 1,505,556 records (39.0%) for flowering plants. But fewer than 10% of records have specimen IDs listed using this structured description. To utilize these ambiguous specimen IDs in GenBank, these IDs may need to be cleansed using databases such as NCBI BioCollections, GRSciColl (Global Registry of Scientific Collections) or AI to map them to IDs in databases rich in specimen information such as those of the Global Biodiveristy Information Facility (GBIF) and Barcode of Life System (BOLD). In GenBank, the BOLD ID is listed in the /db_xref qualifier in the “Features” field as the ID of the external database. The 70% of insect sequence data with a specimen ID in the /specimen_voucher qualifier are also assigned a BOLD ID (Nakazato and Jinbo 2022). The correspondence between specimen IDs in biodiversity information databases such as GBIF and specimen IDs in GenBank is expected to further enhance the value of museum specimens. In addition, GenBank provides the /type_material qualifier for describing the type of voucher (e.g., holotype of Asphondylia bursicola). In GenBank insect data, there were over 2,000 records for type material, and approximately 450 species were mentioned, including 269 for holotypes. We found approximately 3,000 records with type information by including “/notes” and “/specimens_voucher” qualifiers in addition to “/type_material”. Thus, GenBank has potential as a biodiversity information resource, but for more effective use, data mining and linkage with other specimen-based biodiversity databases are essential.

Identification of pivotal genes and pathways in Chorea-acanthocytosis using comprehensive bioinformatic analysis.

Chorea-acanthocytosis (ChAc), an autosomal recessive disorder, is associated with cognitive and behavioral abnormalities. Previous studies were focused around exploring the functional annotation of VPS13A gene in ChAc, whereas the genetic labyrinth underlying this disease and plausible drug targets were underexplored. In the present study, we have identified the pivotal genes and molecular pathways implicated in ChAc using comprehensive bioinformatics analysis. In our analysis we found 27 distinct genes in Homo sapiens linked to ChAc, out of which 15 were selected as candidate genes for enrichment analysis based on their Gene Ontology (GO) annotations and involvement in relevant molecular pathways. By constructing a Protein-Protein Interaction (PPI) network consisting of 26 nodes and 62 edges, we identified two gene modules. Subsequently, using the MCODE algorithm, we identified 6 hub genes-ATN1, JPH3, TBP, VPS13A, DMD, and HTT-as core candidates. These hub genes are primarily associated with processes such as neuron development and differentiation, the CAMKK-AMPK signaling cascade, ion transmembrane transport systems, and protein localization. Furthermore, using drug gene databank we identified 23 FDA-approved drugs that possess the propensity to target 3 out of the 6 identified hub genes. We believe that our findings could open promising avenues for potential therapeutic interventions in ChAc.

Three New Truffle Species (Tuber, Tuberaceae, Pezizales, and Ascomycota) from Yunnan, China, and Multigen Phylogenetic Arrangement within the Melanosporum Group.

Based on a multi-locus phylogeny of a combined dataset of ITS, LSU, tef1-α, and rpb2 and comprehensive morphological analyses, we describe three new species from the Melanosporum group of genus Tuber and synonymize T. pseudobrumale and T. melanoexcavatum. Phylogenetically, the three newly described species, T. yunnanense, T. melanoumbilicatum and T. microexcavatum, differ significantly in genetic distance from any previously known species. Morphologically, T. yunnanense is distinctly different from its closest phylogenetically related species, T. longispinosum, due to its long shuttle-shape spores (average the ratio of spore length to spore width for all spores (Qm) = 1.74). Tuber melanoumbilicatum differs from the other species in having a cavity and long shuttle-shaped spores (Qm = 1.65). Although T. microexcavatum sampled ascomata have relatively low maturity, they can be distinguished from its closely related species T. pseudobrumale by the ascomata size, surface warts, and spore number per asci; additionally, phylogenetic analysis supports it as a new species. In addition, molecular analysis from 22 newly collected specimens and Genebank data indicate that T. pseudobrumale and T. melanoexcavatum are clustered into a single well-supported clade (Bootstrap (BS) = 100, posterior probabilities (PP) = 1.0); and morphological characteristics do not differ. Therefore, based on the above evidence and publication dates, we conclude that T. melanoexcavatum is a synonym of T. pseudobrumale. By taking into account current knowledge and combining the molecular, multigene phylogenetic clade arrangement and morphological data, we propose that the Melanosporum group should be divided into four subgroups. Diagnostic morphological features and an identification key of all known species in the Melanosporum group are also included. Finally, we also provide some additions to the knowledge of the characterization of T. pseudobrumale, T. variabilisporum, and T. pseudohimalayense included in subgroup 1 of the Melanosporum group.

The Impact of Artificial Barriers on the Varuna litterata Migration Route in the Lower Serayu River, Central Java and Its Molecular Identification

The Serayu River is one of the longest rivers in Central Java. Dams built in the lower reaches of the Serayu River are thought to block the migration paths of fish and aquatic macrocrustaceans. This study aimed to determine the effect of dam on Varuna litterata migration and its molecular identification. This research was conducted using the observation method. Migration observations were made on vertical paths while molecular identification of V. litterata in the lower reaches of the Serayu River was compared with existing data in GenBank. The study covered the freshwater area above the Serayu Dam, the open area with freshwater below the Serayu Dam, and the open area with brackish water at the estuary of the Serayu River, with a study length of 33.37 km. The results showed that the presence of dams in the lower reaches of the Serayu River inhibited the migration pattern of V. litterata. The vertical shape of the dam causes only a few V. litterata to migrate to the top of the dam. The dam in the lower reaches of the Serayu River resulted in not many V. litterata being able to migrate to the top of the dam, so there were very few V. litterata found in the population upstream of the dam (115 ind). The results of molecular identification of V. litratata using the cytochrome C oxidase subunit I (COI) gene show that this crab is 100% the same as the data in GenBank and is closely related to Varuna yui and other Varunidae crab species.Keywords: Dams, Molecular, Migration, Serayu River, Varuna litterata

Genetic diversity and phylogeography of the smooth cordgrass Spartina alterniflora and its invasion into Korean coasts

The smooth cordgrass, Spartina alterniflora Loisel, is an aggressive invasive species that significantly impacts coastal ecosystems worldwide. Native to the eastern United States, it has rapidly spread along East Asian coasts and was unintentionally introduced into Korea, leading to the devastation of native ecosystems. This study examines the population genetic structure and invasion pathway of S. alterniflora along Korean coasts using chloroplast DNA (cpDNA) fragments and nuclear ITS. Our results revealed that the S. alterniflora population in Korea primarily consists of two geographically isolated haplotypes (C1 and C4). Furthermore, two individuals were found to have haplotype N, potentially a newly discovered haplotype in this study. The haplotypes C1 and C4 are widely distributed in both the native population and the introduced population in East Asia. Thus, the presence of these two haplotypes in different localities in Korea suggests multiple invasion events. The phylogenetic tree from the nuclear ITS showed no significant genetic variation between regions. Comparisons of the cpDNA data of this study with the available GenBank data from the native range in the U.S. and the invasive range in East Asia (China and Japan) revealed that the East Asian population (China, Japan, and Korea) is nearly homogeneous (AMOVA; Fst = 0.06871, p = 0.5). Our findings suggest that the C1 and C4 haplotypes might have been introduced into Korea at least two times, possibly via natural dispersal and/or transport vectors. This emphasizes the importance of strengthening coastal management to prevent further spread.

Population genetic structure and phylogenetic analysis of Anopheles hyrcanus (Diptera: Culicidae) inferred from DNA sequences of nuclear ITS2 and the mitochondrial COI gene in the northern part of Iran

BackgroundThe Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide.MethodsMosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software.ResultsIn this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity.ConclusionsAn. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon.

HPV Infections-Classification, Pathogenesis, and Potential New Therapies.

To date, more than 400 types of human papillomavirus (HPV) have been identified. Despite the creation of effective prophylactic vaccines against the most common genital HPVs, the viruses remain among the most prevalent pathogens found in humans. According to WHO data, they are the cause of 5% of all cancers. Even more frequent are persistent and recurrent benign lesions such as genital and common warts. HPVs are resistant to many disinfectants and relatively unsusceptible to external conditions. There is still no drug available to inhibit viral replication, and treatment is based on removing lesions or stimulating the host immune system. This paper presents the systematics of HPV and the differences in HPV structure between different genetic types, lineages, and sublineages, based on the literature and GenBank data. We also present the pathogenesis of diseases caused by HPV, with a special focus on the role played by E6, E7, and other viral proteins in the development of benign and cancerous lesions. We discuss further prospects for the treatment of HPV infections, including, among others, substances that block the entry of HPV into cells, inhibitors of viral early proteins, and some substances of plant origin that inhibit viral replication, as well as new possibilities for therapeutic vaccines.

Identification and validation of reference genes for gene expression study using Quantitative Real-Time PCR in shallot (Allium cepa L. Aggregatum group) based on bulb development phase

Abstract. Sulistyaningsih E, Sawitri WD, Handayani VDS, Wicaksono AW, Silalahi CM, Murti RH. 2024. Identification and validation of reference genes for gene expression study using Quantitative Real-Time PCR in shallot (Allium cepa L. Aggregatum group) based on bulb development phase. Biodiversitas 25: 2645-2651. Shallot (Allium cepa L. Aggregatum group) is cultivated vegetatively using bulbs (produce many bulbs) and generatively using TSS (True Seed of Shallot) which tends to produce single bulbs. Bulb aggregation is influenced by activity of genes and the gene expression could be analyzed using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The qRT-PCR analysis requires reference gene data (housekeeping gene) as a comparison gene against our target gene. This study aimed to select and validate three housekeeping genes in shallot as an initial study for qRT-PCR analysis. The research flow began with the preparation of shallot 'TukTuk' leaves and bulb samples in bulb formation from bulb initial phase to mature phase. Furthermore, a specific primer design based on actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences were obtained from gene bank data. The results showed that the actin, tubulin, and GAPDH genes were successfully amplified and the products were 142 bp, 116 bp, and 136 bp in size, respectively and the genes were expressed almost constantly in leaves and bulbs in all phases of bulb formation. The tubulin gene had the potential as a reference gene for qRT-PCR analysis in shallot with the most consistently expressed.

Identifikasi Genetik Ikan Gabus Asal Merauke dengan Menggunakan Fragmen Gen Sitokrom Oksidase Sub Unit I

Efforts to cultivate snakehead fish (Chanidae) still experience many problems, especially the availability of high-quality fry. To improve the quality of fry, improving the quality of broodstock is important. Genetic factors play an important role in the selection and breeding of snakehead fish parents. Therefore, it is necessary to explore sources of superior parents with good genetic characteristics. To achieve this goal requires studies from various fields such as ecology, biology, and genetics. Genetic studies can be started by examining the identity and relationships of snakehead fish from Merauke, as a first step in finding a source of germplasm that has high diversity. The aim of this research is to analyze the cytochrome oxidase subunit 1 (COI) gene fragment of snakehead fish from Merauke, Papua. DNA isolation was carried out using the Promega kit with standard protocols, amplification of the COI gene with primers F2 and R2 with the Go Taq Green amplification kit. A total of 653 base pairs of the COI gene fragment were obtained from the sequencing process, with a nucleotide composition of T (29.9%), C (28.8%), A (24.0%), G (17.3%). BLAST results on the NCBI database show that this sequence has 100% similarity to the Channa striata sequence from Papua with sequence ID OQ405388.1. This shows that the identification results of the snakehead fish from Merauke are Channa striata. Phylogenetic tree analysis also showed consistent results, namely the formation of a clade that was the same as the Channa striata fish group derived from GenBank data.

Variation of Musa spp. in West Kalimantan, Indonesia, Based on rbcL Chloroplast DNA

West Kalimantan is home to Musa spp. conservation and genetic assessment of wild banana relatives are important for future breeding purposes. The present study aims to evaluate the genetic relationship of Musa spp. in West Kalimantan by analyzing the rbcL chloroplast DNA using phylogenetic analysis. The methods in this study were sampling, DNA extraction, PCR of rbcL fragment, and data analysis. The specific primer was used to amplify the rbcL chloroplast DNA of ten accessions of Musa spp. in West Kalimantan. The results showed that the area of the rbcL region of Musa spp. in this study was estimated at 587-591 bp. It showed high variability with a conservation level A+T content of 56.95%. The rbcL sequences of Musa spp. have polymorphic sites on 13 numbers of nucleotides. The phylogenetic analysis with an ML algorithm of 35 Musa spp. from West Kalimantan and GenBank data was successfully divided into 4 main clades, and the bootstrap value was 80-81%. This study is expected beneficial for taxonomic, conservation, and banana breeding efforts.

Genetic diversity of P1/pathogenic Leptospira species hosted by bats worldwide.

Bats are a diverse group of mammals that have unique features allowing them to act as reservoir hosts for several zoonotic pathogens such as Leptospira. Leptospires have been classified into pathogenic, intermediate, and saprophytic groups and more recently into clades P1, P2, S1, and S2, being all the most important pathogenic species related to leptospirosis included within the P1/pathogenic clade. Leptospira has been detected from bats in several regions worldwide; however, the diversity of leptospires harboured by bats is still unknown. The aim of the present study was to determine the genetic diversity of Leptospira spp. harboured by bats worldwide. A systematic review was conducted on four databases to retrieve studies in which Leptospira was detected from bats. All studies were screened to retrieve all available Leptospira spp. 16S rRNA sequences from the GenBank database and data regarding their origin. Sequences obtained were compared with each other and reference sequences of Leptospira species and analysed through phylogenetic analysis. A total of 418 Leptospira spp. 16S rRNA sequences isolated from 55 bat species from 14 countries were retrieved from 15 selected manuscripts. From these, 417 sequences clustered within the P1/pathogenic group, and only one sequence clustered within the P2/intermediate group. Six major clades of P1/pathogenic Leptospira spp. were identified, three of them composed exclusively of sequences obtained from bats. We identified that bats harbour a great genetic diversity of Leptospira spp. that form part of the P1/pathogenic clade, some of which are closely related to leptospirosis-associated species. This finding contributes to the knowledge of the diversity of leptospires hosted by bats worldwide and reinforces the role of bats as reservoirs of P1/pathogenic Leptospira spp.

Systematics and distribution of Lankascincus fallax (Peters, 1860) (Reptilia, Scincidae), an endemic litter-skink from Sri Lanka.

Lankascincus fallax is an endemic, but widespread species of skink distributed throughout Sri Lanka, including all bioclimatic zones (elevations from 0 to 1,200 m a.s.l.). After thoroughly comparing morphology and morphometry between populations in different bioclimatic zones, we recognize it as a single morphospecies. We revise the systematics of Lankascincus fallax based on molecular and morphological data providing a comprehensive re-description of the adult syntype collected from either Ratnapura or Trinkomalee (sic), Sri Lanka. We provide the first-ever constructed phylogeny of Lankascincus representing five species (out of nine) based on GenBank data. In addition, we examined all the type specimens of the two synonymized species, Sphenomorphus rufogulus and Lankascincus deraniyagalae. We also provide a comprehensive discussion on the distribution of L. fallax.

Ace-1 Target Site Status and Metabolic Detoxification Associated with Bendiocarb Resistance in the Field Populations of Main Malaria Vector, Anopheles stephensi in Iran.

Anopheles stephensi is the main vector of malaria in Iran. This study aimed to determine the susceptibility of An. stephensi from the south of Iran to bendiocarb and to investigate biochemical and molecular resistance mechanisms in this species. Wild An. stephensi were collected from Hormozgan Province and reared to the adult stage. The susceptibility test was conducted according to the WHO protocols using bendiocarb impregnated papers supplied by WHO. Also, field An. Stephensi specimens were collected from south of Kerman and Sistan and Baluchistan Provinces. To determine the G119S mutation in the acetylcholinesterase (Ace1) gene, PCR-RFLP using AluI restriction enzyme and PCR direct-sequencing were performed for the three field populations and compared with the available GenBank data. Also, biochemical assays were performed to measure alpha and beta esterases, insensitive acetylcholinesterase, and oxidases in the strains. The bioassay tests showed that the An. stephensi field strain was resistant to bendiocarb (mortality rate 89%). Ace1 gene analysis revealed no G119S in the three field populations. Blast search of sequences revealed 98-99% identity with the Ace1 gene from Pakistan and India respectively. Also, the results of biochemical tests revealed the high activity of non-sensitive acetylcholinesterase, alpha and beta-esterase in the resistant strain compared to the susceptible strain. No G119S was detected in this study additionally the enhanced enzyme activity of esterases and acetylcholinesterase suggesting that resistance was metabolic. The use of alternative malaria control methods and the implementation of resistance management strategies are suggested in the study area.

Isolation of a potential rock phosphate solubilizing Aspergillus sp. towards development of biofertilizer

Phosphorus (P) is one of the key bio elements that limits agricultural production. Although Sri Lanka is endowed with rock phosphate deposits, practical means to utilize this resource are limited. Phosphate-solubilizing fungi (PSF) play an important role in enhancing the bioavailability of soil phosphorus to plants. In this study, fungi from eight soil samples in Kaikawala, Sri Lanka, were isolated using spread plate method and analyzed for its phosphate solubility capacity. One fungal isolate with significant halo zones on Pikovskaya’s agar (PVK) plates, containing 0.5% tricalcium phosphate, was identified. After purification, the isolate was transferred to three media setups: PVK with 0.5% apatite (ERP) as the phosphate source, PVK with 0.5% tricalcium phosphate as a positive control, and PVK without P source as a negative control and analyzed for the solubilization index (SI). DNA sequences of the internal transcribed spacers with 5.8S region of ribosomal DNA (ITS1-5.8S-ITS2) was analyzed with universal primer pair ITS4-F/ITS5-R to determine the identity of the species. Fungal isolate PSF01 showed a phosphate solubilizing activity on both PVK media, ERP (1.07 ± 0.03 cm SI) and tricalcium phosphate (1.07 ± 0.01 cm), with similar effects (p > 0.05). However, the SI in the negative control was 1.00 ± 0.00 cm without halo zone (p < 0.05). The fungal strain is fast growing with initially white but quickly changing to black colonies after producing conidial spores on potato dextrose agar with distinctive conidial heads and pale-yellow lower surface characterized as Aspergillus. Sanger DNA sequencing identified the fungus as Aspergillus niger (99%) and a phylogenetic tree was constructed using MEGA11 software using reference data obtained from NCBI GenBank data to identify the isolated PSF01. Consequently, our preliminary studies demonstrate the importance of examining more soil samples to identify PSF for sustainable agricultural applications in the future.

Applicability of DNA barcoding-based analyses on the diet of the gray brocket deer (Subulo gouazoubira) in xeric hillside forests

In this study, we explore the applicability of DNA barcoding, specifically targeting the chloroplast DNA (cpDNA) sequences, particularly the trnL (UAA) intron region, to analyze the diet of gray brocket deer. This approach offers improved taxonomic resolution and the ability to identify species with greater precision compared to traditional methods. The study was conducted in the "Reserva Natural Salus" in Uruguay, covering a range of vegetation types, where gray brocket deer coexist with other exotic ungulates. A local reference database of trnL (UAA) sequences was established, incorporating both GenBank data and sequences obtained from native species in the study area. Fecal samples were collected in summer and winter, and DNA was extracted and amplified for metabarcoding analysis in pooled samples for each season. For each sample 28,229 and 33,588 reads were obtained respectively, which together corresponded to 25 Operational Taxonomic Units (OTUs). The species Rubus ulmifolius and Schinus engleri were the most represented in the diet with 69.6 % of the summer reads, whereas in winter, 68.7 % of the reads corresponded only to Schinus engleri. These findings indicate that gray brocket deer consume species that have higher nutritional value, which may be linked to their capacity to thrive in young and productive ecosystems. This study demonstrates the feasibility of DNA barcoding for dietary analysis in gray brocket deer and provides valuable insights into their food habits in the "Reserva Natural Salus". Further improvements to increase the reference databases of native species and the exploration of additional genetic markers are recommended for enhanced species-level discrimination in dietary analysis studies. This methodology is promising for future research as diet studies have an impact on species management, habitat conservation and biodiversity conservation efforts.

First Report of Curvularia plantarum CausingRice SpikeletRotDisease in China.

In September 2022, rice spikelets rot disease (RSRD) was investigated in Songjiang District (30.94132N, 121.18393E), China, leading to a 26.77% yield loss. At the heading stage, infected spikelets exhibited small, yellowish-brown dots with water-stained husks, subsequently coalescing to form irregular brown to black lesions. Later, the lesions were enlarged and rotted, which eventually caused blighted grains. About 10% of husked grains showed black spots. 30 infected grains and 30 husked grains with black spots were surface sterilized in 75% ethanol for 2 min, then rinsed with ddH2O and plated on PDA medium at 28°C in darkness for 4 d. 22 and 13 fungal isolates with similar morphology were obtained in shriveled and husked grains, respectively. Three isolates (SJTU1, SJTU2 and SJTU3) were selected by the single-spore isolation method. The colonies were brown to blackish green, smooth, and contained a large number of stolons with a few aerial mycelia in the center. Hyphae and conidiophores were blackish green, thick-walled, branched with septa. Conidia were 14.77 to 26.82×4.74 to 11.36 μm (average 20.42×8.58 μm, n= 100) in size, lightly curved with blackish green. Conidia with three septa and four cells, apical and basal cells transparent, middle cell unequal in size. Based on morphological characteristics, the isolates were preliminarily identified as Curvularia plantarum (Raza et al. 2019). The genomic DNA of the three isolates (SJTU1 to 3) was extracted for molecular identification. 3 pairs of primers ITS1/TTS4 (Peever et al. 2004), gpd1/gpd2 (Berbee et al. 1999), and EF-983F/EF-2218R (Rehner and Buckley 2005) were used to amplify ITS, GAPDH, and EF1-α genes, respectively. These sequences were all uploaded in GenBank (ITS: OR726053 to 55; EF1-α: OR732471 to 73; GAPDH: OR732474 to 76). According to data in GenBank, the ITS, EF1-α, and GAPDH genes of 3 isolates (SJTU1 to 3) showed 99-100% identity (573/575 bp, 542/543 bp, and 531/531 bp) to the ITS (MW581905, MN044755, and MN215690), 99-100% identity (869/869 bp, 868/869 bp, and 855/856 bp) to the EF1-α (MN263982 to 83, and MT628901), and 99-100% identity (543/544 bp, 528/528 bp, and 540/540 bp) to the GAPDH (MT628902, MN264120, and MT432926) gene of C. plantarum, respectively. The phylogenetic analysis by Maximum Likelihood (ML) method based on the concatenated sequences of ITS, EF1-α, and GAPDH genes showed that the three isolates (SJTU1 to 3) clustered with C. plantarum. According to morphology and molecular identification, these fungal isolates were identified as C. plantarum. Pathogenicity tests were conducted in the field used only for inoculation with pathogens by spraying 30 spikelets of rice cultivar 'Song1013' at the heading stage with conidial suspension (5 × 105 conidia/mL). 30 spikelets sprayed with ddH2O were designated as control. The test was conducted 3 times at 22 to 31°C with 78 to 89% RH. All the inoculated spikelets exhibited similar symptoms to those of the infected spikelets in paddy at 10 d after spraying, while the control spikelets remained healthy. All reisolated strains from infected spikelets were identified the same as the original inoculated strains by morphology and ITS sequences, fulfilling Koch's postulates. To our knowledge, this is the first report of C. plantarum causing RSRD in China. The discovery of this new disease and its pathogens will facilitate the provision of pathogenically relevant information vital for management strategies to RSRD caused by C. plantarum in the future.

MetaZooGene Atlas and Database: Reference Sequences for Marine Ecosystems.

The MetaZooGene Atlas and Database (MZGdb; https://metazoogene.org/mzgdb/ ) is an open-access data and metadata portal synchronized with the NCBI GenBank and BOLD data repositories. The MZGdb includes sequences for genes used for the classification and identification of marine organisms based on DNA barcoding and metabarcoding. The focus of the MZGdb is biodiversity of marine ecosystems, including phytoplankton and microbes, zooplankton and invertebrates, fish, and other marine vertebrates (pinnipeds, cetaceans, and sea turtles). DNA sequences currently included are mitochondrial cytochrome oxidase I (COI), 12S, and 16S rRNA, and nuclear 18S and 28S rRNA. The MZGdb provides data and mapping tools for assembling and downloading compilations of reference sequence data that are specific to selected genes, taxonomic groups, and/or ocean regions. An additional feature of the MZGdb is the Atlas which summarizes data coverage and proportional completeness based on statistics of species with available sequences versus species commonly found in each ocean region.This chapter is a collaborative effort of the Scientific Committee for Ocean Research (SCOR) Working Group WG157: MetaZooGene: Toward a new global view of marine zooplankton biodiversity based on DNA metabarcoding and reference DNA sequence databases ( https://metazoogene.org ).