Sex-Biased Mortality and Sex Reversal Shape Wild Frog Sex Ratios

Population sex ratio is a key demographic factor that influences population dynamics and persistence. Sex ratios can vary across ontogeny from embryogenesis to death and yet the conditions that shape changes in sex ratio across ontogeny are poorly understood. Here, we address this issue in amphibians, a clade for which sex ratios are generally understudied in wild populations. Ontogenetic sex ratio variation in amphibians is additionally complicated by the ability of individual tadpoles to develop a phenotypic (gonadal) sex opposite their genotypic sex. Because of sex reversal, the genotypic and phenotypic sex ratios of entire cohorts and populations may also contrast. Understanding proximate mechanisms underlying phenotypic sex ratio variation in amphibians is important given the role they play in population biology research and as model species in eco-toxicological research addressing toxicant impacts on sex ratios. While researchers have presumed that departures from a 50:50 sex ratio are due to sex reversal, sex-biased mortality is an alternative explanation that deserves consideration. Here, we use a molecular sexing approach to track genotypic sex ratio changes from egg mass to metamorphosis in two independent green frog (Rana clamitans) populations by assessing the genotypic sex ratios of multiple developmental stages at each breeding pond. Our findings imply that genotypic sex-biased mortality during tadpole development affects phenotypic sex ratio variation at metamorphosis. We also identified sex reversal in metamorphosing cohorts. However, sex reversal plays a relatively minor and inconsistent role in shaping phenotypic sex ratios across the populations we studied. Although we found that sex-biased mortality influences sex ratios within a population, our study cannot say at this time whether sex-biased mortality is responsible for sex ratio variation across populations. Our results illustrate how multiple processes shape sex ratio variation in wild populations and the value of testing assumptions underlying how we understand sex in wild animal populations.

Sexing as a tool for reinsertion of Amazona aestiva parrots to nature: use of less invasive technique

The sexing birds is considered an important tool for behavioral studies and programs for the reintroduction of animals into the wild. Several techniques are used for this purpose, such as laparoscopy, magnetic resonance and molecular sexing. The first are considered more invasive and stressful for the animal, and the last is considered the most accurate. According to it, the aim of this study was to compare the effectiveness of using three sets of primers in the molecular sexing process of true parrots (Amazona aestiva). Blood samples from 10 animals were collected at a Wildlife Screening Center (CETAS) in Bahia, Brazil. The DNA was extracted and the molecular markers amplified by Polymerase Chain Reaction (PCR) using primer pairs P2/P8, 1237L/1272H and 2250F/2718R. The amplified material was visualized with electrophoresis performed at 2% agarose and 12.5% polyacrylamide gels. Among the primer sets used, the 2250F/2718R pair showed the best results for the sexing process, including visualization of the amplified products on an agarose gel. Agarose gel electrophoresis is considered to be faster and cheaper. The results revealed a sample composition of 5 males (0.5) and 5 females (0.5).

Molecular sexing in the formation of pairs of blue-and-yellow macaw (Ara ararauna) in reintroduction programs

Blue and yellow macaw is a species which does not show sexual dimorphism and is threatened by animal traffic. The identification of heterosexual pairs is important for reintroduction programs. The aim of this work was select parameters for sexing and use them to determine the frequency of heterosexual pairs in a population of blue-and-yellow macaws allocated in a Wild Animal Screening Center. Blood samples from 23 macaws were collected and genomic DNA extracted by Tris/SDS washes. Allele-specific molecular markers for sexing were amplified by PCR, and identified on 2% agarose gel. Three pairs of primers were tested: Pair 1 (P2/P8), Pair 2 (1237L/1272H) and Pair 3 (2550F/2718R). For the determination of animal pairs, all individuals had their social behavioral acts observed. The results showed that the low complexity DNA extraction protocol used was adequate. Pairs 2 and 3 of primers were effective for sexing and the Pair 3 was the most efficient. The study also showed that in the sample studied, the composition of males and females was similar (0.4 males n=10 and 0.6 females n=13); 70% (n=16) of the individuals formed pairs and 75% (n=12) of the pairs were heterosexual and the others male-male or female-female pairs. These results were used in the management of the animals in the reintroduction program.

Molecular sexing of degraded DNA from elephants and mammoths: a genotyping assay relevant both to conservation biology and to paleogenetics

It is important to determine the sex of elephants from their samples—faeces from the field or seized ivory—for forensic reasons or to understand population demography and genetic structure. Molecular sexing methods developed in the last two decades have often shown limited efficiency, particularly in terms of sensitivity and specificity, due to the degradation of DNA in these samples. These limitations have also prevented their use with ancient DNA samples of elephants or mammoths. Here we propose a novel TaqMan-MGB qPCR assay to address these difficulties. We designed it specifically to allow the characterization of the genetic sex for highly degraded samples of all elephantine taxa (elephants and mammoths). In vitro experiments demonstrated a high level of sensitivity and low contamination risks. We applied this assay in two actual case studies where it consistently recovered the right genotype for specimens of known sex a priori. In the context of a modern conservation survey of African elephants, it allowed determining the sex for over 99% of fecal samples. In a paleogenetic analysis of woolly mammoths, it produced a robust hypothesis of the sex for over 65% of the specimens out of three PCR replicates. This simple, rapid, and cost-effective procedure makes it readily applicable to large sample sizes.

COMPARATIVE STUDY OF YELLOW-BILLED BABBLER (TURDOIDES AFFINIS) FEATHERS REVEALS UNIFORMITY IN THEIR MICROSTRUCTURES AMONG INDIVIDUALS

Though a few in numbers, investigations on feather microstructures from the early 20th century till date, have contributed immensely to various fields such as phylogeny, palaeontology, archaeology, wildlife forensic, biomechanics and so on. However, existing studies on feather micro-structures of birds endemic to the India/Indian sub-continent are few in number and limited in their scope. Also, no study has ever been conducted to compare feather microstructures of different individuals of a species from India. To address this issue, a comparative feather microstructure study of three individuals of Turdoides affinis, a passerine endemic to the Indian sub-continent was done. Select microstructure parameters for five different types of feathers were studied in detail. The molecular sexing method was used to elucidate the sex of T. affinis individuals for gender based differences if any. Results of the study identified that two of T. affinis individuals were female whereas one of them was male. Morphometrically, tail contour was the longest (9.63±0.76 cm) and bristle were the shortest (1.00±0.07 cm) feather. Semiplume had the longest barb length (1.73±0.04 cm) and shortest barbs (0.16±0.01 cm) were present in bristles. Subpennaceous barbs and knob-shaped villi, characteristic of members of the Passeriformes family, was also observed in all three individuals. This study records no significant difference in feather characteristics amongst the three T. affinis individuals irrespective of the differences in their sex and size. Systematically documented feather micro-characteristics of T. affinis in this study could be used as a species identification tool and would provide baseline data for the feather catalogue of Indian bird species being compiled at SACON.

Genomic data shed light on sex-determination in Australian freshwater Percichthyid fish species: Many ways to be a male

Understanding sex-specific biology can aid conservation management. But understanding genomic sex differences of monomorphic fish species and developing molecular sexing assays is challenged by their diverse sex-determination systems. To facilitate research on Percichthyid fish, predominant in the Australian freshwater biota, we report whole genome sequences and annotations of the endangered Macquarie perch Macquaria australasica and its sister species, the golden perch M. ambigua. To identify sex-linked loci, we conducted whole genome resequencing on 100 known-sex Macquarie perch. In-silico pool-seq comparisons revealed few sex differences, but a 275-Kb SOX-containing scaffold was enriched for gametologous loci- homozygous in females, heterozygous in males. Within this scaffold we reconstructed X- and Y-linked 146-bp haplotypes containing 5 sex-linked SNPs, ~38 Kb upstream of SOX, and developed a PCR-RFLP sexing assay targeting the Y-linked allele of one SNP. We tested this assay in a panel of known-sex Macquarie perch, and smaller panels of three other confamilial species. Amplicon sequencing of 400 bp encompassing the 146-bp region revealed that the few sex-linked positions differ interspecifically, and within Macquarie perch such that its sexing test approached 100% reliability only for the populations used in assay development. Similarly, Macquarie- and golden perch genome-wide DArTseq SNPs revealed different sex-linked loci across non-homologous scaffolds. Overall, we identified 22 sex-linked SNPs in Macquarie perch in a predominantly XX/XY system in which females are homozygous at all 22, and males are heterozygous at 2 or more. The resources here will facilitate multi-locus sexing assays for both species and research on Percichthyid biology.