Polymerase Chain Reaction (PCR) is a Useful and Low-Cost Tool for Molecular Sexing Psittaciformes under Human Care: An Example of a Collaborative Approach in Mexico.

Sex determination in monomorphic birds is a precondition for captive breeding programs and management and conservation strategies for threatened species. Most species of the order Psittaciformes often present complications since these birds lack external sexual phenotypic traits, making it impossible to differentiate males and females. In the present study, we used molecular techniques to determine the sex of 31 individuals belonging to nine species of the order Psittaciformes kept under human care at the Akumal Monkey Sanctuary & Rescued Animals in Quintana Roo, Mexico. This is a useful and low-cost methodology based on the analysis of the conserved region of the CHD1 gene, which was amplified by PCR with two sets of primers: P8/P2 and 2550F/2718 R. All individuals were successfully sexed with the first set of primers, while only 28 out of 31 samples (90%) could be amplified with the second set. Out of the 31 individuals analyzed, fifteen are female, and seventeen are male. This information represents a handy tool for adequately managing birds under human care, resulting in their reproduction and eventual reintegration into their natural habitat.

Estimation of Adult Sex Ratio and Size-Related Sexual Dimorphism Based on Molecular Sex Determination in the Vulnerable La Selle Thrush, Turdus swalesi.

Sex-determination is of particular importance in avian ecology and conservation. However, many bird species show no conspicuous sexual dimorphism, such as the La Selle Thrush, Turdus swalesi, a vulnerable species endemic to Hispaniola. We captured individuals in southeastern Haiti, in 2019-2022. For each one, we collected contour feathers or blood samples for molecular sex identification based on the CHD-1 gene. In addition, we took body measurements of several captured individuals and recorded their weight. Out of a total of 65 birds, 45 were identified as males and 20 as females, indicative of a significantly male-biased sex ratio. However, analyses of first captures showed that the sex ratio at our study site was male-biased only outside of the breeding season, suggesting that females may disperse at that time while males remain on their territories. Sexual dimorphism was limited to wing chord length and tail length, with males being larger than females. Tail length was the best predictor of sex in a logistic regression model and correctly classified about 80% of individuals as male or female. We discuss our results in relation to previous studies of sex ratio and sexual dimorphism in turdid species and address their relevance for the conservation of avian species in one of the major protected forest areas in Haiti.

The Length Polymorphism of the 9th Intron in the Avian CHD1 Gene Allows Sex Determination in Some Species of Palaeognathae.

In palaeognathous birds, several PCR-based methods and a range of genes and unknown genomic regions have been studied for the determination of sex. Many of these methods have proven to be unreliable, complex, expensive, and time-consuming. Even the most widely used PCR markers for sex typing in birds, the selected introns of the highly conserved CHD1 gene (primers P2/P8, 1237L/1272H, and 2550F/2718R), have rarely been effective in palaeognathous birds. In this study we used eight species of Palaeognathae to test three PCR markers: CHD1i9 (CHD1 gene intron 9) and NIPBLi16 (NIPBL gene intron 16) that performed properly as Psittaciformes sex differentiation markers, but have not yet been tested in Palaeognathae, as well as the CHD1iA intron (CHD1 gene intron 16), which so far has not been used effectively to sex palaeognathous birds. The results of our research indicate that the CHD1i9 marker effectively differentiates sex in four of the eight species we studied. In Rhea americana, Eudromia elegans, and Tinamus solitarius, the electrophoretic patterns of the amplicons obtained clearly indicate the sex of tested individuals, whereas in Crypturellus tataupa, sexing is possible based on poorly visible female specific bands. Additionally, we present and discuss the results of our in silico investigation on the applicability of CHD1i9 to sex other Palaeognathae that were not tested in this study.

In Silico Analysis of Seven PCR Markers Developed from the CHD1, NIPBL and SPIN Genes Followed by Laboratory Testing Shows How to Reliably Determine the Sex of Musophagiformes Species.

Sex determination in birds, due to the very common lack of sexual dimorphism, is challenging. Therefore, molecular sexing is often the only reliable way to differentiate between the sexes. However, for many bird species, very few genetic markers are available to accurately, quickly, and cost-effectively type sex. Therefore, in our study, using 14 species belonging to the order Musophagiformes, we tested the usefulness of seven PCR markers (three of which have never been used to determine the sex of turacos), developed based on the CHD1, NIPBL, and SPIN genes, to validate existing and develop new strategies/methods of sex determination. After in silico analysis, for which we used the three turaco nuclear genomes available in GenBank, the suitability of the seven selected markers for sexing turacos was tested in the laboratory. It turned out that the best of the markers tested was the 17th intron in the NIPBL gene (not previously tested in turacos), allowing reliable sex determination in 13 of the 14 species tested. For the one species not sexed by this marker, the 9th intron in the CHD1 gene proved to be effective. The remaining markers were of little (4 markers developed based on the CHD1 gene) or no use (marker developed based on the SPIN gene).

Sex-specific prey partitioning in breeding piscivorous birds examined via a novel, noninvasive approach.

Piscivorous birds frequently display sex-specific differences in their hunting and feeding behavior, which lead to diverging impacts on prey populations. Cormorants (Phalacrocoracidae), for example, were previously studied to examine dietary differences between the sexes and males were found to consume larger fish in coastal areas during autumn and winter. However, information on prey partitioning during breeding and generally on sex-specific foraging in inland waters is missing. Here, we assess sex-specific prey choice of Great Cormorants (Phalacrocorax carbo) during two subsequent breeding seasons in the Central European Alpine foreland, an area characterized by numerous stagnant and flowing waters in close proximity to each other. We developed a unique, noninvasive approach and applied it to regurgitated pellets: molecular cormorant sexing combined with molecular fish identification and fish-length regression analysis performed on prey hard parts. Altogether, 364 pellets delivered information on both, bird sex, and consumed prey. The sexes differed significantly in their overall prey composition, even though Perca fluviatilis, Rutilus rutilus, and Coregonus spp. represented the main food source for both. Albeit prey composition did not indicate the use of different water bodies by the sexes, male diet was characterized by higher prey diversity within a pellet and the consumption of larger fish. The current findings show that female and male cormorants to some extent target the available prey spectrum at different levels. Finally, the comprehensive and noninvasive approach has great potential for application in studies of other piscivorous bird species.

Laser microdissection-based analysis of the Y sex chromosome of the Antarctic fish Chionodracohamatus (Notothenioidei, Channichthyidae).

Microdissection, DOP-PCR amplification and microcloning were used to study the large Y chromosome of Chionodracohamatus, an Antarctic fish belonging to the Notothenioidei, the dominant component of the Southern Ocean fauna. The species has evolved a multiple sex chromosome system with digametic males showing an X1YX2 karyotype and females an X1X1X2X2 karyotype. Fluorescence in situ hybridization, performed with a painting probe made from microdissected Y chromosomes, allowed a deeper insight on the chromosomal rearrangement, which underpinned the fusion event that generated the Y. Then, we used a DNA library established by microdissection and microcloning of the whole Y chromosome of Chionodracohamatus for searching sex-linked sequences. One clone provided preliminary information on the presence on the Y chromosome of the CHD1 gene homologue, which is sex-linked in birds but in no other vertebrates. Several clones from the Y-chromosome mini-library contained microsatellites and transposable elements, one of which mapped to the q arm putative fusion region of the Y chromosome. The findings confirm that interspersed repetitive sequences might have fostered chromosome rearrangements and the emergence of the Y chromosome in Chionodracohamatus. Detection of the CHD1 gene in the Y sex-determining region could be a classical example of convergent evolution in action.