A comparative study on eDNA-based detection of Siamese bat catfish (Oreoglanis siamensis) in wet and dry conditions.

The use of environmental DNA (eDNA) analysis has demonstrated notable efficacy in detecting the existence of freshwater species, including those that are endangered or uncommon. This application holds significant potential for enhancing environmental monitoring and management efforts. However, the efficacy of eDNA-based detection relies on several factors. In this study, we assessed the impact of rainfall on the detection of eDNA for the Siamese bat catfish (Oreoglanis siamensis). Quantitative polymerase chain reaction (qPCR) analysis indicated that samples from days with average rainfall exceeding 35 mm (classified as heavy and very heavy rain) yielded negative results. While eDNA detection remains feasible on light or moderate rainy days, a noteworthy reduction in eDNA concentration and qPCR-positive likelihood was observed. Analysis across 12 sampling sites established a statistically significant negative relationship (p < 0.001) between eDNA detection and rainfall. Specifically, for each 1 mm increase in rainfall, there was an observed drop in eDNA concentration of 0.19 copies/mL (±0.14). The findings of this study provide definitive evidence that precipitation has a significant impact on the detection of eDNA in Siamese bat catfish. However, in the case of adverse weather conditions occurring on the day of sampling, our research indicates that it is acceptable to continue with the task, as long as the rainfall is not heavy or very heavy. To enhance the effectiveness of an eDNA survey, it is crucial to consider many factors related to climatic conditions. The aforementioned factor holds significant importance not only for the specific species under scrutiny but also for the broader dynamics of the climate.

An eDNA-based assessment of Garra cambodgiensis (stonelapping minnow) distribution on a megadiverse river, the Mekong.

Garra cambodgiensis (stonelapping minnow) has experienced significant population declines, prompting intensive research and management, although its distribution in river systems such as the Mekong remains obscure. Effective conservation and management necessitate accurate monitoring and survey data on the distribution of freshwater species. Traditional surveying techniques for fish may be challenging and generate insufficient data on species distribution. This study developed an eDNA-based method for detecting G. cambodgiensis to address this void. Twenty-one locations were surveyed. Water samples were collected in triplicate from the river's surface at each site and processed within 48 h in a dedicated laboratory. Primers and probes for G. cambodgiensis were meticulously designed and species-specificity tested to ensure accurate detection without interference from co-occurring species in the same geographic range. Each water sample was analysed by qPCR using six technical replicates. The results of qPCR were reported as positive with quantifiable eDNA concentration (copies/mL), below the limit of quantification, or non-detectable. G. cambodgiensis eDNA was detected in water samples collected from 10 out of 21 sampling sites, with concentrations ranging from 8.5 to 2990.0 copies/mL. Importantly, G. cambodgiensis eDNA was consistently detected in all three replicate water samples at each site where the qPCR experiment yielded positive results. The findings of this study demonstrate the feasibility and effectiveness of incorporating eDNA-based monitoring or surveys for G. cambodgiensis in the ecologically diverse Mekong River. Monitoring based on eDNA can aid in targeting and informing conservation and management of G. cambodgiensis in its natural habitat. Comprehensive and robust information on species distribution can be obtained via an eDNA-based survey, which could contribute to more efficient and informed decision-making processes in fisheries management and conservation efforts.

Distinguishing fanged frogs (Limnonectes) species (Amphibia: Anura: Dicroglossidae), from Thailand using high resolution melting analysis.

Morphologically, species of fanged frogs (Limnonectes) are exceedingly similar, making it difficult to distinguish them within the complex. In Thailand, it has been difficult to distinguish between the sympatric species L. bannaensis and L. taylori, particularly among tadpoles, adolescents, and adult females. A precise identification contributes to a greater understanding of biodiversity, particularly for assessing distributions and population dynamics. Therefore, a novel approach is required. The objective of this study was to develop a high resolution melting analysis (HRM) for the rapid and accurate identification of six species of Limnonectes of the L. kuhlii complex found in Thailand, particularly the two sympatric fanged frogs. Here, HRM assays using 16S rRNA mitochondrial primers were designed and developed. There was as much as a 25.3% variation in the nucleotide sequence of the fragment amplified by HRM16S primers among the six species of Limnonectes. Prior to conducting an in vitro HRM, the DNA sequences were used in a simulation HRM, uMELT Quartz, to predict the melting curve for each species of Limnonectes. There were discrepancies between the predicted melting curves of each species generated by the programme. Consequently, in vitro HRM tests were conducted. The obtained melting curve and Tm values were consistent with those predicted, albeit with a slightly different Tm value and a more distinct melting curve. All evaluated species of Limnonectes could be easily distinguished from one another by comparing the melting curve shapes. The HRM assay was then used to confirm the species of 18 Limnonectes samples in comparison to the reference samples (confidence interval > 90%). In addition, the results of HRM were consistent with those of experts who used morphological analysis to identify species. The HRM was found to be useful, and therefore the method would also contribute to future ecological and systematic studies on the target species.

A comparative cytogenetic study of Hypsibarbusmalcolmi and H.wetmorei (Cyprinidae, Poropuntiini).

Cyprininae are a highly diversified but demonstrably monophyletic lineage of cypriniform fishes. Here, the karyotype and chromosomal characteristics of Hypsibarbusmalcolmi (Smith, 1945) and H.wetmorei (Smith, 1931) were examined using conventional, nucleolus organizing regions (NORs) and molecular cytogenetic protocols. The diploid chromosome number (2n) of H.malcolmi was 50, the fundamental number (FN) was equal to 62, and the karyotype displayed 8m + 4sm + 38a with NORs located at the centromeric and telomeric positions of the short arms of chromosome pairs 1 and 2, respectively. 2n of H.wetmorei was 50, FN 78, karyotype 14m + 14sm + 22a with the NORs at the telomeric position of the short arm of chromosome pair 2. 2n and FN in males and females were identical. Fluorescence in situ hybridization using different microsatellite motifs as probes also showed substantial genomic divergence between both studied species. In H.wetmorei, (CAG)n and (CAC)n microsatellites accumulated in the telomeric regions of all chromosomes, while in H.malcolmi, they had scattered signals on all chromosomes. Besides, the (GAA)n microsatellites were distributed along all chromosomes of H.malcolmi, but there was a strong hybridization pattern in the centromeric region of a single pair in H.wetmorei. These cytogenomic difference across the genomes of these Hypsibarbus Rainboth, 1996 species are markers for specific evolutionary differentiation within these two species.

A method for in vivo evaluation of α-glucosidase inhibition using Drosophila.

The development of α-glucosidase inhibitors is essential for the prevention of type II diabetes. Previous research has investigated in vitro inhibition using isolated α-glucosidase, which may not accurately reflect physical processes. The method presented in this study aims to establish a rapid and inexpensive in vivo method to study the inhibition of α-glucosidase activity using Drosophila as a model organism. This method can be used to calculate the IC50 value of compounds of interest for inhibition of α-glucosidase activity. The method established in this study can be used for in vivo screening of anti-diabetic compounds. •A rapid and inexpensive in vivo method to study the inhibition of α-glucosidase activity.•This method can be used to calculate the IC50 value of compounds of interest for inhibition of α-glucosidase activity.•This is a useful method for in vivo screening of anti-diabetic compounds.

Genomic analyses reveal three phylogenetic species and their evolutionary histories in the big-headed turtle.

The critically endangered big-headed turtle (Platysternon megacephalum) is currently classified into three subspecies. However, the classification is still controversial and their evolutionary histories are still unclear. Here, multiple genetic analyses consistently revealed three phylogenetic groups with substantial genetic divergences and distinct demographic histories, suggesting three phylogenetic species (P. megacephalum, P. peguense, and Baise clade). Phylogeographical analyses revealed that the Red River plains and Guangxi basins are largely coincident with the boundaries between the three phylogenetic species, highlighting the key role of lowland areas in driving speciation in the big-headed turtle. The Baise clade is characterized by high-linkage disequilibrium but the lowest effective population size, indicating that the cryptic phylogenetic species is more vulnerable to human activities and environmental disturbance, and urgently needs more protection. Our findings provide fundamental insights into the taxonomy and scientific conservation of the family Platysternidae.

eDNA testing reveals surprising findings on fish population dynamics in Thailand.

COVID-19, a global health concern, has an effect on all aspects of the economy. The aquaculture and fishing industries were severely harmed as a result of the closures in multiple nations. Regular systems for inventory monitoring, production, and supply were disrupted. Cancellation of programmes for research, fieldwork, sampling, and tagging influences management-required data. For effective species management, fish dispersion assessments are indispensable. However, due to the difficulty of accessing sampling sites and the associated costs, there is frequently a lack of comprehensive information regarding the distribution and abundance of organisms. The COVID-19 prohibition made fish monitoring more problematic. Due to constant pressure, populations of the stone lapping minnow (Garra cambodgiensis), one of Thailand's overfished fish, are rapidly declining. Therefore, eDNA-based monitoring was devised and implemented to reveal the likely dispersal of the species in Thailand prior to and following the lockdown. At 28 locations within the Chao Phraya River Basin, water samples were collected. qPCR was used to determine the presence or absence of G. cambodgiensis in water samples. In 78 of 252 water samples, a wide range of computed copy numbers for G. cambodgiensis eDNA was observed. It was discovered that samples collected in 2021 (after the lockdown) contain a higher concentration of G. cambodgiensis eDNA than samples collected in 2018 or 2019 (prior to the lockdown). The closure appears to be a boon and may result in a substantial restocking of the fish we have studied. Overall, eDNA-based analysis is an extremely promising new survey instrument.

Sustainable fisheries management through reliable restocking and stock enhancement evaluation with environmental DNA.

The practise of restocking and stock improvement as a means of managing fisheries and aquaculture has been widely used. However, it is difficult to claim that fish stocking is effective due to a number of challenges. One of those is the lack of suitable monitoring and assessment methods, although all assessment approaches have their strengths and weaknesses. If the full benefits of fisheries and their long-term sustainability are to be realised, it is necessary to examine the effectiveness of restocking and stock enhancement. Therefore, effective, rapid, and dependable monitoring techniques are necessary. In this study, we used an eDNA-based method to identify G. cambodgiensis at 14 sites throughout Thailand's restocking and stock enhancement programme. eDNA from this species was identified in water samples using quantitative polymerase chain reaction (qPCR) tests with primers and a probe specific to G. cambodgiensis. A successful stocking would show positive eDNA results in water samples collected from the studied sites. Only five of the studied sites returned positive eDNA readings, which could be considered a successful stocking. The locations that contained G. cambodgiensis eDNA were either confirmed to be natural habitats or were regularly stocked with a large number of hatchery fish. In this study, we demonstrated that eDNA is a reliable, fast and accurate alternative method for measuring stock improvement.

A new species of the Cyrtodactylusbrevipalmatus group (Squamata, Gekkonidae) from Tak Province, northwestern Thailand.

An integrative taxonomic analysis was used to delimit and diagnose a new species of the Cyrtodactylusbrevipalmatus group from Tak Province in western Thailand. Although Bayesian phylogenetic analyses place C.denticulatussp. nov. within the brevipalmatus group, the new species is neither nested within nor is it the sister species of any other species in the brevipalmatus group. Furthermore, based on the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) and adjacent tRNAs, it bears an uncorrected pairwise sequence divergence of 7.87-21.94% from all other species in the brevipalmatus group. Cyrtodactylusdenticulatussp. nov. is differetiated from all other species in the brevipalmatus group by having a number of unique charateristics such as denticulate ventrolateral body folds and ventrolateral subcaudal ridges, characters not seen in any other species of the group (n = 51 individuals). Additionally, based on a multiple factor anlaysis, C.denticulatussp. nov. does not overlap with any other species in multivariate space. The discovery of C.denticulatussp. nov. underscores the unrealized diversity of upland ecosystems across Thailand and the urgent need for increased exploration and conservation of these unique imperiled montane refugia, especially in this era of climate change.

A new species of the Cyrtodactylusbrevipalmatus group (Squamata, Gekkonidae) from the uplands of western Thailand.

An integrative systematic analysis recovered a new species of the Cyrtodactylusbrevipalmatus group from the uplands of Thong Pha Phum National Park, Kanchanaburi Province in western Thailand. Cyrtodactylusthongphaphumensissp. nov. is deeply embedded within the brevipalmatus group, bearing an uncorrected pairwise sequence divergence of 7.6-22.3% from all other species based on a 1,386 base pair segment of the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) and adjacent tRNAs. It is diagnosable from all other species in the brevipalmatus group by statistically significant mean differences in meristic and normalized morphometric characters as well as differences in categorical morphology. A multiple factor analysis recovered its unique and non-overlapping placement in morphospace as statistically significantly different from that of all other species in the brevipalmatus group. The description of this new species contributes to a growing body of literature underscoring the high degree of herpetological diversity and endemism across the sky-island archipelagos of upland montane tropical forest habitats in Thailand, which like all other upland tropical landscapes, are becoming some of the most imperiled ecosystems on the planet.

Detection of the Endangered Siamese Bat Catfish (Oreoglanis siamensis Smith, 1933) in Doi Inthanon National Park Using Environmental DNA.

Siamese bat catfish (Oreoglanis siamensis Smith, 1993) has been listed as an endangered species, and its abundance has been severely declining due to habitat degradation and overfishing. To establish an appropriate management strategy, it is crucial to gain information about the distribution of this endangered species. As O. siamensis live under rocks in streams, detecting their presence is difficult. Recently, environmental DNA (eDNA)-based detection has been demonstrated to be a valid tool for monitoring rare species, such as O. siamensis. Therefore, this study developed an eDNA assay targeting a 160 bp fragment of the COI region to detect the presence of this species in its natural habitat. An amount of 300 mL of water samples (0.7 µm filtered) were collected from 15 sites in the Mae Klang sub-basin, where this fish species was visually detected at two locations. O. siamensis eDNA was detected at 12 of the 15 sites sampled with varying concentrations (0.71-20.27 copies/mL), including at the sites where this species was visually detected previously. The developed O. siamensis eDNA assay was shown to be effective for detecting the presence of this endangered species in the Klang Phat and Klang Rivers within the Doi Inthanon National Park.

Revision of the genus Ultragryllacris (Orthoptera: Stenopelmatidae: Gryllacridinae), with description of new species.

The genus Ultragryllacris was revised. All its species and subspecies, including U. jiaranaisakuli sp. nov., U. chandra sp. nov. from Western Thailand and U. intermediata sp. nov. from Northern Thailand are here described and redescribed. They are known from Thailand and China. The new species differs from all the other representatives of this genus mainly in different body coloration, shape of projection of male 9th abdominal tergite and sclerotized area of female subgenital plate. The following taxonomical changes are also proposed: U. pulchra alboclypeata Gorochov et Dawwrueng, 2015, U. p. nan Ingrisch, 2018 and U. p. rubricapitis Bin et Bian, 2021 are raised to species status (U. alboclypeata stat. nov., U. nan stat. nov. and U. rubricapitis stat. nov.) due to some characteristic structures in both male and female; U. triangula Ingrisch, 2018 is downgraded to subspecies of U. alboclypeata (U. a. triangula stat. nov.). Previously unknown male of U. a. alboclypeata and female of U. nan are described. A key to all known species is given.

A new species of Cyrtodactylus Gray, 1827 (Squamata: Gekkonidae) from the Thai-Malay Peninsula and the independent evolution of cave ecomorphology on opposite sides of the Gulf of Thailand.

An integrative taxonomic analysis recovers a distinctive new species of the gekkonid genus Cyrtodactylus Gray, 1827 from Satun Province in extreme southern Thailand as the sister species to the Cyrtodactylus intermedius group of southern Indochina, approximately 600 km to the northeast across the Gulf of Thailand. Based on 1449 base pairs of the mitochondrial gene NADH dehydrogenase subunit 2 (ND2) and its flanking tRNAs, the new species, C. disjunctus sp. nov., bears a pairwise sequence divergence from the mean divergences of the intermedius group species ranging from 17.923.6%. Three different principal component analyses (PCA) and a multiple factor analysis (MFA) recover C. disjunctus sp. nov. as a highly distinctive karst cave-adapted species based on morphology and color pattern. Its sister species relationship to the intermedius groupto which it is added herefurther underscores a growing body of analyses that have recovered a trans-Gulf of Thailand connection across the submerged Sunda Shelf between the southern Thai-Malay Peninsula and southern Indochina. Fragmented karstic archipelagos stretching across Indochina have served as foci for the independent evolution of nearly 25% of the species of Cyrtodactylus. The description of C. disjunctus sp. nov. continues to highlight the fact that karstic habitats support an ever-increasing number of threatened site-specific endemics that compose much of the reptile diversity of many Asian nations but, as of yet, most of these landscapes have no legal protection.

A new species of stream toad of the genus Ansonia Stoliczka, 1870 (Anura: Bufonidae) from Nakhon Si Thammarat Range in southern Thailand.

An integrative taxonomic analysis confirmed the new species status of a recently discovered upland population of Ansonia from Thongsong, Thongsong District, Nakhon Si Thammarat Province, Thailand from the sky island archipelago south of the Isthmus of Kra. Ansonia infernalis sp. nov. is a member of the Thai-Burmese clade within the more inclusive Thai-Malaya Peninsula clade of western and southern Thailand. It is separated from all other species of Ansonia by a unique combination of morphometric and discrete morphological and color pattern characteristics and is the sister species of a clade of eight other species found north of the Isthmus of Kra. Ansonia infernalis sp. nov. is the newest member of a long list of range-restricted endemics from the sky island archipelago of the Thai-Malay Peninsula and continues to underscore the unexplored nature of this region and its need for conservation.

Nutritional status and related factors among ethnic preschool children in Northern Thailand: a cross-sectional study.

PURPOSE: This study investigated the nutritional status of ethnic (belonging to minority groups) preschool children in Northern Thailand. METHODS: In this cross-sectional descriptive study, the sample group included 147 parents and 147 preschool children (2-6 years old). Participants volunteered to complete a questionnaire on the personal information of children and parents, as well as family- and school-related factors. Data were analyzed using descriptive statistics, a nutritional status calculator, and logistic regression analysis. RESULTS: A birth weight of less than 2,500 g was related to weight-for-age and weight-for-height. Not having been breastfed and having been breastfed for less than 6 months were related to weight-for-height. An elementary school education level among parents was related to children's height-for-age. Familial unhealthy food consumption for 1-2 days per week was related to weight-for-age and height-for-age. CONCLUSION: This study reflects the importance of nutritional care for children at the beginning of pregnancy and continuing to the preschool age, as well as the importance of breastfeeding. Families were found to be a key factor in supporting good nutrition among children.

Genetic Structure and Forensic Utility of 23 Autosomal STRs of the Ethnic Lao Groups From Laos and Thailand.

The Lao Isan and Laotian are the major groups in the area of present-day northeastern Thailand and Laos, respectively. Several previous genetic and forensic studies indicated an admixed genetic structure of Lao Isan with the local Austroasiatic speaking groups, e.g. Khmer, whereas there is a paucity of reporting Laotian's forensic short tandem repeats (STRs). Here, we newly generated 451 genotypes of seven Lao Isan and three Laotian populations (two Lao Lum and one Lao Thoeng) using 23 autosomal STRs embedded in VerifilerTM plus PCR Amplification kit. We reported allelic frequency and forensic parameters in different dataset: combined ethnic Lao groups, combined Lao Isan populations and combined Laotians. Overall, the forensic parameter results indicate that this set of STRs is suitable for forensic investigation. The anthropological results revealed the genetic homogeneity of Tai-Kadai speaking Lao groups from Thailand and Laos, consistent with previous studies, while the Austroasiatic speaking groups from southern Laos showed genetic relatedness to both Lao Isan and Khmer. In sum, STRs allelic frequency results can provide the genetic backgrounds of populations which is useful for anthropological research and also strengthens the regional forensic database in both countries.

De Novo Assembly of 20 Chicken Genomes Reveals the Undetectable Phenomenon for Thousands of Core Genes on Microchromosomes and Subtelomeric Regions.

The gene numbers and evolutionary rates of birds were assumed to be much lower than those of mammals, which is in sharp contrast to the huge species number and morphological diversity of birds. It is, therefore, necessary to construct a complete avian genome and analyze its evolution. We constructed a chicken pan-genome from 20 de novo assembled genomes with high sequencing depth, and identified 1,335 protein-coding genes and 3,011 long noncoding RNAs not found in GRCg6a. The majority of these novel genes were detected across most individuals of the examined transcriptomes but were seldomly measured in each of the DNA sequencing data regardless of Illumina or PacBio technology. Furthermore, different from previous pan-genome models, most of these novel genes were overrepresented on chromosomal subtelomeric regions and microchromosomes, surrounded by extremely high proportions of tandem repeats, which strongly blocks DNA sequencing. These hidden genes were proved to be shared by all chicken genomes, included many housekeeping genes, and enriched in immune pathways. Comparative genomics revealed the novel genes had 3-fold elevated substitution rates than known ones, updating the knowledge about evolutionary rates in birds. Our study provides a framework for constructing a better chicken genome, which will contribute toward the understanding of avian evolution and the improvement of poultry breeding.

An integrative taxonomic revision of slug-eating snakes (Squamata: Pareidae: Pareineae) reveals unprecedented diversity in Indochina.

Slug-eating snakes of the subfamily Pareinae are an insufficiently studied group of snakes specialized in feeding on terrestrial mollusks. Currently Pareinae encompass three genera with 34 species distributed across the Oriental biogeographic region. Despite the recent significant progress in understanding of Pareinae diversity, the subfamily remains taxonomically challenging. Here we present an updated phylogeny of the subfamily with a comprehensive taxon sampling including 30 currently recognized Pareinae species and several previously unknown candidate species and lineages. Phylogenetic analyses of mtDNA and nuDNA data supported the monophyly of the three genera Asthenodipsas, Aplopeltura, and Pareas. Within both Asthenodipsas and Pareas our analyses recovered deep differentiation with each genus being represented by two morphologically diagnosable clades, which we treat as subgenera. We further apply an integrative taxonomic approach, including analyses of molecular and morphological data, along with examination of available type materials, to address the longstanding taxonomic questions of the subgenus Pareas, and reveal the high level of hidden diversity of these snakes in Indochina. We restrict the distribution of P. carinatus to southern Southeast Asia, and recognize two subspecies within it, including one new subspecies proposed for the populations from Thailand and Myanmar. We further revalidate P. berdmorei, synonymize P. menglaensis with P. berdmorei, and recognize three subspecies within this taxon, including the new subspecies erected for the populations from Laos and Vietnam. Furthermore, we describe two new species of Pareas from Vietnam: one belonging to the P. carinatus group from southern Vietnam, and a new member of the P. nuchalis group from the central Vietnam. We provide new data on P. temporalis, and report on a significant range extension for P. nuchalis. Our phylogeny, along with molecular clock and ancestral area analyses, reveal a complex diversification pattern of Pareinae involving a high degree of sympatry of widespread and endemic species. Our analyses support the "upstream" colonization hypothesis and, thus, the Pareinae appears to have originated in Sundaland during the middle Eocene and then colonized mainland Asia in early Oligocene. Sundaland and Eastern Indochina appear to have played the key roles as the centers of Pareinae diversification. Our results reveal that both vicariance and dispersal are responsible for current distribution patterns of Pareinae, with tectonic movements, orogeny and paleoclimatic shifts being the probable drivers of diversification. Our study brings the total number of Pareidae species to 41 and further highlights the importance of comprehensive taxonomic revisions not only for the better understanding of biodiversity and its evolution, but also for the elaboration of adequate conservation actions.