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The potential of mitochondrial DNA (mtDNA) genes are well-known for species identification and to establish a phylogenetic relationship. The De-novo transcriptome assembly of Acheta domesticus commonly known as house cricket, is provides important segments of DNA fragments from mitochondrial DNA due to higher abundance of its mRNA. When the reference sequence with gene annotation is absent for assembling and aligning desire gene sequences, like in the present case, the most similar sequence is obtained from online insect mitochondrial genome database to find mitochondrial DNA conserved domains of interested gene from high throughput RNA sequencing (RNA-seq) data. The RNA-seq data of Acheta domesticus transcriptome is used to retrieve single nucleotide fragment out of 50,046 assembled contigs to discover three important genes from mtDNA of the house cricket. Present study provides effective workflow to identify genes like cytochrome c oxidase subunit II (COX2), NADH dehydrogenase subunit 2 (ND2), cytochrome c oxidase subunit I (COX1) from mtDNA in large sequence archive of RNA-seq data. These three novel barcode sequences will be useful for genetic identification and evolution investigation of Acheta domesticus. The partial mtDNA sequence with these genes will be important for mitochondrial genome construction.

Dugong (Dugong dugon) populations have been shrinking globally, due in large part to habitat fragmentation, degradation and ocean pollution, and today are listed as Vulnerable by the IUCN. Thus, determining genetic diversity in the remaining populations is essential for conservation planning and protection. In this study, measures of inter-simple sequence repeat (ISSR) markers and mtDNA D-loop typing were used to evaluate the genetic diversity of 118 dugongs from skin samples of deceased dugongs collected in Thai waters over a 29-year period. Thirteen ISSR primers revealed that dugongs from the Andaman Sea and Gulf of Thailand exhibited more genetic variation in the first 12 years of the study (1990–2002) compared to the last decade (2009–2019). Dugongs from the Andaman Sea, Trang, Satun and some areas of Krabi province exhibited greater diversity compared to other coastal regions of Thailand. Eleven haplotypes were identified, and when compared to other parts of the world (235 sequences obtained from NCBI), five clades were apparent from a total 353 sequences. Moreover, dugongs from the Andaman Sea were genetically distinct, with a separate haplotype belonging to two clades found only in Thai waters that separated from other groups around 1.2 million years ago. Genetic diversity of dugongs in present times was less than that of past decades, likely due to increased population fragmentation. Because dugongs are difficult to keep and breed in captivity, improved in situ conservation actions are needed to sustain genetically healthy wild populations, and in particular, the specific genetic group found only in the Andaman Sea.

The dugong (Dugong dugon Müller) is recognized as an endangered marine mammal. There is limited available anatomical data on the dugong’s skeletal system, while what is available has not been well established due to the limited number of archived samples and limited access to them. Importantly, there are certain key questions that should be answered when examining the bones and/or remains of animals such as; what kind of bone is it?; what species does it belong to?; what sex was the animal?; how old was the animal? or how big was it?, etc. In this study, we have focused on the pelvic bone of the dugong by asserting the hypothesis that pelvic bone morphology is related to age, sex, and body size. Here, we have established certain morphometric data encompassing 8 parameters and 5 indexes to analyze the morphology of the pelvic bones obtained from 88 specimens (45 dugongs). We will present three main findings: (1) the pelvic bone in mature male subjects is larger than it is in female subjects, (2) a high rate of accuracy can be established for sex identification using morphometric data obtained from the pelvic bone, and (3) the pelvic bone has the highest degree of correlation with body length, followed by body weight and age. Notably, the important data on the pelvic bone of the dugong acquired in this study can be reliable and extremely useful in sex identification and body size estimation.

Gupta YM, Tanasarnpaiboon S, Buddhachat K, Peyachoknagul S, Inthim P, Homchan S. 2020. Development of microsatellite markers for the house cricket, Acheta domesticus (Orthoptera: Gryllidae). Biodiversitas 21: 4094-4099. The house cricket, Acheta domesticus, is one of the species of crickets commonly found in Thailand. Insect breeders in Thailand prefer to breed house cricket as food due to its better taste and popularity among local people. Moreover, largescale breeding industries also breed house cricket to produce cricket-based edible products. Insect breeding industry is growing rapidly and requires primary precaution for sustainable production. To facilitate breeding system to maintain genetic variation in the captive population, we have sequenced the whole genome of A. domesticus to search for simple sequence repeats (SSRs) in order to develop polymorphic microsatellite markers for preliminary population genetic analysis. A total of 112,157 SSRs with primer pairs were identified in our analysis. Of these, 91 were randomly selected to check for amplification of microsatellite polymorphisms. From these, nine microsatellites were used to check genetic variation in forty-five individuals of A. domesticus from the Phitsanulok population (Thailand). These microsatellite markers also showed cross-amplification with other three species of edible crickets, specifically Gryllus bimaculatus, Gryllus testaceus, and Brachytrupes portentosus. The microsatellite markers presented herein will facilitate future population genetic analysis of A. domesticus populations. Moreover, the transferability of these makers would also enable researchers to conduct genetic studies for other closely related species.

Aim The objective of this study was to uncover new candidate genes related to patellar luxation (PL) in dogs to select for those with low susceptibility for breeding purposes. Materials and methods The inter simple sequence repeat (ISSR) technique was performed to construct DNA fingerprints of 61 Chihuahua dogs with PL and 30 healthy Chihuahua dogs. DNA polymorphisms were detected by comparing the sequences between the affected and unaffected dogs, using the pairwise alignments in MultAlin. Genotyping was performed using allele-specific polymerase chain reaction (AS-PCR). The association analysis of ISSR DNA fingerprints and genotypes or phenotypes was performed using the Chi-square (χ 2) model and generalized linear model (GLM), respectively. Results Two single nucleotide polymorphisms (SNPs), namely SNP1UBC811 (g.91175C>G) and SNP2UBC811 (g.92259T>C), were found in the intron of the Dystroglycan 1 (DAG1) gene, which was obtained using the PL-related marker UBC811 primer (p=0.02), and genotyped by AS-PCR. When investigated using the GLM, g.91175C>G had a significant association with PL (p=0.0424), whereas g.92259T>C did not have such an association (p=0.0959). Conclusion DAG1 might be one of the genes related to PL in Chihuahuas and could aid the process of marker-assisted selection in genetic breeding for Chihuahua dogs without PL.

Karyotype and ideogram analyses of Somsak’s cascade frog (Amolops panhai) and dark-sided frog (Sylvirana nigrovittata), collected from Nakhon Si Thammarat Province, southern Thailand, were performed. Chromosomes were prepared from the intestinal epithelium and stained by conventional Giemsa staining. From this study, we found that A. panhai possesses the diploid chromosome number of 26 (2n = 26), while the chromosome number of S. nigrovittata is 24 (2n = 24). This is the first report on karyotype of A. panhai, containing 10 pairs of metacentric (m), 2 pairs of submetacentric (sm) and 1 pair of subtelocentric (st) chromosomes with the arm number of 52. The S. nigrovittata’s karyotype contains 12 pairs of chromosomes with the arm number of 48 in only the metacentric form. The karyotype formulae could be represented as 2n (26) = 10m + 2sm + 1st and 2n (24) = 12m for A. panhai and S. nigrovittata, respectively.

This study demonstrates sexual dimorphism of feline bones, based on a morphometric analysis of 38 dried feline skulls and pelvic bones (20 males, 18 females). A total of 44 parameters (skull = 12, mandible = 10, pelvis = 22) were measured using a digital vernier calliper. In morphological observation of these bones, there were three hallmarks indicating a remarkable difference between sexes: the coronoid process of the mandible (accuracy rate = 88.2%); and the os coxae - caudal ventral iliac spine (accuracy rate = 94.4%), and the angle of the ischiatic arch (accuracy rate = 74.3%). In addition, based on morphometric characteristics, six parameters were found to be significantly different (P

As laws tighten to limit commercial ivory trading and protect threatened species like whales and elephants, increased sales of fake ivory products have become widespread. This study describes a method, handheld X-ray fluorescence (XRF) as a noninvasive technique for elemental analysis, to differentiate quickly between ivory (Asian and African elephant, mammoth) from non-ivory (bones, teeth, antler, horn, wood, synthetic resin, rock) materials. An equation consisting of 20 elements and light elements from a stepwise discriminant analysis was used to classify samples, followed by Bayesian binary regression to determine the probability of a sample being ‘ivory’, with complementary log log analysis to identify the best fit model for this purpose. This Bayesian hybrid classification model was 93% accurate with 92% precision in discriminating ivory from non-ivory materials. The method was then validated by scanning an additional ivory and non-ivory samples, correctly identifying bone as not ivory with >95% accuracy, except elephant bone, which was 72%. It was less accurate for wood and rock (25–85%); however, a preliminary screening to determine if samples are not Ca-dominant could eliminate inorganic materials. In conclusion, elemental analyses by XRF can be used to identify several forms of fake ivory samples, which could have forensic application.

Our purpose was to evaluate the protective effect of three marine omega-3 sources, fish oil (FO), krill oil (KO), and green-lipped mussel (GLM) against cartilage degradation. Canine cartilage explants were stimulated with either 10 ng/mL interleukin-1β (IL-1β) or IL-1β/oncostatin M (10 ng/mL each) and then treated with various concentrations of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA; 3 and 30 μg/mL), FO, KO, or GLM (250, 500, and 1000 μg/mL) for 28 days. Gene expression was then investigated in primary canine chondrocytes. Our results showed that DHA and EPA as well as omega-3 sources could suppress matrix degradation in cytokine-induced cartilage explants by significantly reducing the increase of sulfated glycosaminoglycans (s-GAGs) and preserving uronic acid and hydroxyproline content (except GLM). These agents were not able to reduce IL-1β-induced IL1B and TNFA expression but were able to down-regulate the expression of the catabolic genes MMP1, MMP3, and MMP13 and up-regulate the anabolic genes AGG and COL2A1; FO and KO were especially effective. Our findings indicated that FO and KO were superior to GLM for their protective effect against proteoglycan and collagen degradation. Hence, FO and KO could serve as promising sources of chondroprotective agents.

This study evaluated the morphology and elemental composition of Asian elephant (Elephas maximus) bones (humerus, radius, ulna, femur, tibia, fibula and rib). Computerized tomography was used to image the intraosseous structure, compact bones were processed using histological techniques, and elemental profiling of compact bone was conducted using X-ray fluorescence. There was no clear evidence of an open marrow cavity in any of the bones; rather, dense trabecular bone was found in the bone interior. Compact bone contained double osteons in the radius, tibia and fibula. The osteon structure was comparatively large and similar in all bones, although the lacuna area was greater (P < 0.05) in the femur and ulna. Another finding was that nutrient foramina were clearly present in the humerus, ulna, femur, tibia and rib. Twenty elements were identified in elephant compact bone. Of these, ten differed significantly across the seven bones: Ca, Ti, V, Mn, Fe, Zr, Ag, Cd, Sn and Sb. Of particular interest was the finding of a significantly larger proportion of Fe in the humerus, radius, fibula and ribs, all bones without an open medullary cavity, which is traditionally associated with bone marrow for blood cell production. In conclusion, elephant bones present special characteristics, some of which may be important to hematopoiesis and bone strength for supporting a heavy body weight.

Fluoroquinolones (FQs) are frequently used for septic arthritis. Increased antibacterial activity has been associated with mammalian cell cytotoxicity that may increase the risk of developing osteoarthritis. This study compared the direct effects of two different FQs, enrofloxacin (Enro) and marbofloxacin (Mar), on normal primary canine chondrocytes and inflammatory-stimulated chondrocytes, in addition to their administration in combination with hyaluronan (HA). Cell viability, cell apoptosis, s-GAG production, and expression patterns of inflammatory, extracellular matrix (ECM) component and protease genes were measured. Enro co-culturing with HA could modify s-GAG synthesis compared with the negative control group. Co-treatment with both FQs and HA significantly decreased cell viability and induced more total apoptotic cell death compared with the negative control and pre-IL-1β-stimulated group. Enro regulated IL-1β-stimulated cells to overexpress IL-1β, TNF, and MMP3, whereas Mar induced upregulation of PTGS2 and NFKB1 and enhanced the expression of ECM component genes HAS1, COL2A1, and ACAN as well as TIMP1 and MMP9. Simultaneous use of HA with Enro can effectively reduce the expression of IL-1β, TNF, and MMP3 in pre-IL-1β-stimulated chondrocytes. These results suggest the beneficial effects of HA in reducing the adverse effects of Enro treatment at the transcriptional level.

Siam weed (Chromolaena odorata (L.) R.M. King & H. Rob.) is a commercially viable plant that has various pharmacological properties including hemostasis and wound healing. Three other plant species as Praxelis (Praxelis clematidea R.M. King & H. Rob.), Goat weed (Ageratum conyzoides L.) and Crofton weed (Ageratina adenophora (Spreng.) R.M. King) in the same family have similar morphological characteristics. Misidentification of processed materials can occur in the absence of a skilled taxonomist. These four plants can often be found in close proximity, leading to erroneous sample collection and misleading results. DNA identification through molecular techniques is an accurate method for correctly identifying and differentiating Siam weed to control the quality of product development. Two primers (ITS1A and ITS1B) were designed based on the barcode (Bar) locus of the internal transcribed spacer (ITS) region. High resolution melting (HRM) analysis (Bar-HRM) was performed to acquire melting shapes and melting temperatures (Tm) to discern the four plant species. Bar-HRM using ITS1A and ITS1B showed differences in melting shapes among the four species. The method employing ITS1A distinguished the four species with higher accuracy than ITS1B. The Tm values of each species obtained from Bar-HRM using ITS1A ranged from 82.54 °C to 86.26 °C. Bar-HRM using ITS served as a rapid and reliable tool for species differentiation among Siam weed, Praxelis, Goat weed and Crofton weed.

Recently, the incidence of canine infection by the tick-borne parasites Babesia spp., Hepatozoon canis, Ehrlichia canis and Anaplasma platys has been increasing globally. We have developed a multiplex high-resolution melting analysis (mHRM) technique to reduce the time demands and costs associated with detecting haemoparasites in canine blood, while increasing the degree of reliability of this method of analysis. We have designed primers that are specific for protozoans (B. vogeli and H. canis) and Rickettsia-like bacteria (E. canis and A. platys) based on the 18S or 16S rDNA sequences, respectively. Two primer pairs (Protz18S-C and Bact16S-A) were found to be suitable for detecting these agents since their melting temperatures (Tm) exhibited discernible differences among the four haemoparasites, A. platys, B. vogeli, E. canis and H. canis (83.10 °C, 82.41 °C, 80.37 °C and 78.56 °C, respectively). The sequences acquired from these PCR products were >94 % identical to those of A. platys, B. vogeli, E. canis and H. canis in GenBank. The limit of detection (LOD) for B. vogeli, E. canis and A. platys was 103 copies/μl, while the LOD for H. canis was 104 copies/μl. Of the 68 dogs tested, 28 (41 %) were infected with these agents. The most commonly occurring infection involved E. canis, followed by B. vogeli, A. platys and H. canis, with infection percentages of 26 %, 13 %, 7 % and 6 %, respectively. These results demonstrate that mHRM can serve as a rapid, economical and reliable tool for the detection of parasitic diseases in canine blood for diagnosis and epidemiology.

Controversy in the taxonomic evaluation of the Asian tree frog Polypedates leucomystax complex presents the challenging task of gaining insight into its biogeographical distribution and diversification. Here, we describe the dispersion and genetic relationship of these species in Thailand where we connect the population of the P. leucomystax complex of the Sunda Islands to the mainland population based on the mitochondrial cytochrome c oxidase subunit I (COI) gene, derived from 266 samples. Our maternal genealogy implies that there are four well-supported lineages in Thailand, consisting of Northern A (clade A: Polypedates sp.), Nan (clade B: P. cf. impresus), Southern (clade C: P. cf. leucomystax) and Northern B (clade D: P. cf. megacephalus), with Bayesian posterior probability >0.9. Phylogeny and haplotype networks indicate that clades A, B and D are sympatric. In contrast, clade C (P. cf. leucomystax) and clade D (P. cf. megacephalus) are genetically divergent due to the geographical barrier of the Isthmus of Kra, resulting in allopatric distribution. Climatic conditions, in particular rainfall, that differ on each side of the Isthmus of Kra may play an important role in limiting the immigration of both clades. For the within-populations of either clades C or D, there was no significant correlation between geographic and genetic distance by the isolation-by-distance test, indicating intraspecific gene flow of each clade. Population expansion occurred in clade C, whereas clade D showed a constant population. Taken together, the P. leucomystax complex in Southeast Asia may be diversified by climatic oscillation, leading to allopatric and/or sympatric speciation.

The dugong (Dugong dugon, Müller) is an endangered marine mammal species. We examined the relationship between sex, habitat and body length based on the skull and scapular morphology and morphometrics of 81 dugong samples in Thailand. A total of 58 parameters from the skull and scapula (25 from the cranium, 23 from the mandible and 10 from the scapula) as well as tusks were used in this study. Data were analyzed by univariate analysis, followed by discriminant analysis and multivariate linear regression. Here we show, 100% and 98.5% accuracy rates for sexing using large tusks and the skull, respectively. Scapular morphology using the caudal border tubercle and coracoid process showed 91.30% and 96.15% accuracy rates for identifying males and females. Skull morphometrics could categorize dugong habitat, i.e. living in the Andaman Sea or Gulf of Thailand, with 100% accuracy. Moreover, our model could be used to estimate body length with coefficient of determination (R2) of 0.985. The results of our study showed that skull morphology and morphometric measurements could be used as a tool for sex identification, location identification and estimation of body length. But scapular morphology is the best tool for sex identification in dugongs.

The elemental composition was investigated and applied for identifying the sex and habitat of dugongs, in addition to distinguishing dugong tusks and teeth from other animal wildlife materials such as Asian elephant (Elephas maximus) tusks and tiger (Panthera tigris tigris) canine teeth. A total of 43 dugong tusks, 60 dugong teeth, 40 dolphin teeth, 1 whale tooth, 40 Asian elephant tusks and 20 tiger canine teeth were included in the study. Elemental analyses were conducted using a handheld X-ray fluorescence analyzer (HH-XRF). There was no significant difference in the elemental composition of male and female dugong tusks, whereas the overall accuracy for identifying habitat (the Andaman Sea and the Gulf of Thailand) was high (88.1%). Dolphin teeth were able to be correctly predicted 100% of the time. Furthermore, we demonstrated a discrepancy in elemental composition among dugong tusks, Asian elephant tusks and tiger canine teeth, and provided a high correct prediction rate among these species of 98.2%. Here, we demonstrate the feasible use of HH-XRF for preliminary species classification and habitat determination prior to using more advanced techniques such as molecular biology.

Species identification is required in various areas such as systematics, ecology, conservation, evolution, agriculture, forensics, pharmacology, food science, and even industry. Yet the number of taxonomists who identify based on morphological observations is limited, and some materials are incomplete and/or processed, leading to difficulty in species identification by morphological features. Thus, the molecules remaining inside, especially DNA, often have been extensively used to aid species identification. Here, we established an accurate and rapid method, melting fingerprint-high resolution melting or “MFin-HRM,” to facilitate species identification/authentication based on the melting profile of the DNA fingerprint obtained from inter simple sequence repeat (ISSR) coupled with HRM, or the “melting fingerprint”. This method generates a melting fingerprint specific to a certain species. In this study, Phyllanthus amarus and its closely related species were investigated as a model to test the performance of MFin-HRM in species identification. A final concentration of DNA template of more than 100 pg/μl is suitable for obtaining a consistent melting fingerprint by MFin-HRM. In addition, MFin-HRM can be used for adulterant determination, and its sensitivity and specificity depend on the ISSR primer chosen for generating the melting fingerprint. These results demonstrate that MFin-HRM is a reliable and fast method for species identification/authentication in a closed tube.

Summary This study presents the results from a morphometric analysis of 52 dry Retriever dog pelvic bones (30 male, 22 female). A total of 20 parameters were measured using an osteometric board and digital vernier caliper. Six parameters were found to be significantly higher (P