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Telomeres comprise tandem repeated DNA sequences that protect the ends of chromosomes from deterioration or fusion with neighboring chromosomes, and their lengths might vary with sex and age. Here, age‐ and sex‐related telomere lengths in male and female captive Siamese cobras (Naja kaouthia) were investigated using quantitative real‐time polymerase chain reaction based on cross‐sectional data. A negative correlation was shown between telomere length and body size in males but not in females. Age‐related sex differences were also recorded. Juvenile female snakes have shorter telomeres relative to males at up to 5 years of age, while body size also rapidly increases during this period. This suggests that an accelerated increase in telomere length of female cobra results from sex hormone stimulation to telomerase activity, reflecting sexually dimorphic phenotypic traits. This might also result from amplification of telomeric repeats on sex chromosomes. By contrast, female Siamese cobras older than 5 years had longer telomeres than males. Diverse sex hormone levels and oxidative stress parameters between sexes may affect telomere length.

Sterile hybrid animals exhibit spermatogenic disruptions with a decreased number and/or malformation of mature sperm. F1 hybrid catfish (2n = 55) is an important cultured fish derived from male North African catfish [Clarias gariepinus (Burchell, 1822); 2n = 56] and female bighead catfish [C. macrocephalus (Gunther, 1864); 2n = 54]; they are sterile with gametogenic failure in males. Despite the generality of this phenomenon, the spermatogenic phenotype has not been well described and a comprehensive understanding of the genetic basis of the disruption remains elusive. Our observations in the F1 male hybrid showed abnormal morphology of testes with small size, while meiotic configuration indicated that meiosis succeeded in the early pachytene stage but failed to progress beyond the diplotene-diakinesis stage in primary spermatocytes. This suggests the presence of a number of degenerated spermatocytes. Histological examination recorded no postmeiotic cells and spermatozoa in F1 male hybrid testes. A high frequency of apoptotic testicular cells was also present in the F1 male hybrid, as shown by histochemical results for activated Caspase-3 and TUNEL assays. Low expression levels of Caspase-3, p53 and MCL1 (isoform 2), which activate pro-apoptotic signals in the development process, were also observed in both parental species, but significantly high expression levels were observed in the F1 male hybrid. By contrast, the level of BCL2 expression was very low in the F1 male hybrid, supporting the state of apoptosis as BCL2 inhibits the actions of pro-apoptotic proteins. These results collectively suggest that sterility in male hybrids is caused by spermatogenic disruptions in the pachytene stage, resulting from the failure of homologous chromosome pairing due to chromosomal incompatibility between parental genomes, and subsequent elimination by apoptosis leading to spermatogenic breakdown in the F1 male hybrid.

Molecular sexing is routinely used in the fields of forensic investigations, population genetics and conservation biology. However, none of the assays used so far allows non-ambiguous, quick, and cheap sex identification in snakes. Here, we designed a new multiplex PCR (two panels of triplex) sexing assay using two homologous loci on sex chromosomes (snake gametologous genes), CTNNB1Z/CTNNB1W and WACZ/WACW, and two combined female-specific CTNNB1W loci. This method was successfully tested on 81 samples from 17 caenophidian snakes, but not for 20 samples from 5 henophidian snakes, suggesting that this assay was applicable across all caenophidian snake tested. Moreover, the results of the validation study comprising sensitivity, specificity, blind test, and casework with blood spots on filter paper and shed skin as a non-invasive sample confirmed the effectiveness of this assay in the forensic perspective. The assay represents a robust, cheap, rapid, and simple method which should also prove useful in unknown bodies of carcass. This efficient assay should benefit wildlife forensic laboratories and registered breeders to conform to wildlife regulations or certification of commercial trade, and boost profits for snake-breeders and conservationist snake-breeding communities.

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.

To better understand PBI-DdeI satellite DNA located in the centromeric region of python, molecular evolution analysis was conducted on 40 snake species. A ladder-like pattern of DNA bands with repetition of the 194–210 bp monomer was observed in 15 species using PCR. Molecular cloning was performed to obtain 97 AT-rich monomer sequences. Phylogenetic and network analyses showed three PBI-DdeI subfamilies with sequences grouped in species-specific clusters, suggesting rapid evolution. Slow evolution was found in eight species with shared PBI-DdeI sequences, suggesting recent species diversification, allowing PBI-DdeI no time to diverge, with limited homogenization and fixation processes. Quantitative real-time PCR showed large differences in copy number between Python bivittatus and other snakes, consistent with repeat scanning of whole genome sequences. Copy numbers were significantly higher in female Naja kaouthia than in males, concurring with chromosomal distribution of PBI-DdeI specifically localized to female W chromosomes. PBI-DdeI might act as an evolutionary driver with several repeats to promote W chromosome differentiation and heterochromatinization in N. kaouthia. Analysis revealed PBI-DdeI with a reduced copy number, compared to P. bivittatus, in most snakes studied, and it is possible that it subsequently dispersed and amplified on W chromosomes with different functional roles in N. kaouthia.

Table S1. Comparison of major classes of repeat sequences in chicken and zebra finch BACs mapped on the Siamese cobra Z chromosome. (DOCX 14 kb)

Table S6. Comparison of frequencies of microsatellite repeat motifs in 22 chicken and zebra finch BACs mapped on the Siamese cobra chromosome 1 and microchromosomes. (DOCX 19 kb)

Table S5. Comparison of major classes of repeat sequences in 22 chicken and zebra finch BACs mapped on the Siamese cobra chromosome 1 and microchromosomes. (DOCX 23 kb)

Table S2. Comparison of frequencies of microsatellite repeat motifs in chicken and zebra finch BACs mapped on the Siamese cobra Z chromosome. (DOCX 13 kb)

Table S3. Comparison of major classes of repeat sequences in chicken and zebra finch BACs mapped on the Siamese cobra chromosome 2. (DOCX 15 kb)

Table S4. Comparison of frequencies of microsatellite repeat motifs in chicken and zebra finch BACs mapped on the Siamese cobra chromosome 2. (DOCX 14 kb)

The fragmentation of habitats and hunting have impacted the Asian woolly-necked stork (Ciconia episcopus), leading to a serious risk of extinction in Thailand. Programs of active captive breeding, together with careful genetic monitoring, can play an important role in facilitating the creation of source populations with genetic variability to aid the recovery of endangered species. Here, the genetic diversity and population structure of 86 Asian woolly-necked storks from three captive breeding programs [Khao Kheow Open Zoo (KKOZ) comprising 68 individuals, Nakhon Ratchasima Zoo (NRZ) comprising 16 individuals, and Dusit Zoo (DSZ) comprising 2 individuals] were analyzed using 13 microsatellite loci, to aid effective conservation management. Inbreeding and an extremely low effective population size (Ne) were found in the KKOZ population, suggesting that deleterious genetic issues had resulted from multiple generations held in captivity. By contrast, a recent demographic bottleneck was observed in the population at NRZ, where the ratio of Ne to abundance (N) was greater than 1. Clustering analysis also showed that one subdivision of the KKOZ population shared allelic variability with the NRZ population. This suggests that genetic drift, with a possible recent and mixed origin, occurred in the initial NRZ population, indicating historical transfer between captivities. These captive stork populations require improved genetic variability and a greater population size, which could be achieved by choosing low-related individuals for future transfers to increase the adaptive potential of reintroduced populations. Forward-in-time simulations such as those described herein constitute the first step in establishing an appropriate source population using a scientifically managed perspective for an in situ and ex situ conservation program in Thailand.