Fig 1.
Karyotyping, chromosome structure, and diploid metaphase chromosome sets of Hyla eximia treefrog.
A Adult specimen of the H. eximia treefrog (Anura: Hylidae). Male karyotype is shown in B and C, while the female karyotype is shown in D and E. The number of chromosomes identified in H. eximia was 24 in both sexes. Chromosome pairs were numbered from 1 to 12 based on the morphometric similarities. Karyotype stained with Giemsa showing the absence of heteromorphic sex chromosomes (C and E). The same chromosome number were observed between specimens of H. eximia of both sexes; therefore, the karyotype indicated a diploid number 2n = 24 for females and 2n = 24 for males. The identification of heterogametic sex was not visualized through karyotyping.
Fig 2.
H-Y antigen sequencing, molecular comparative analysis, and gene expression from H. eximia treefrog.
Nucleotide (A) and predicted amino acid (B) sequence of H. eximia H-Y antigen cDNA. The oligonucleotide sequences to amplify the specific cDNA are shown in blue and the H-Y antigen sequence is shown green. (C) Comparative analysis of vertebrata H-Y antigen amino acid sequences using ClustalW multiple sequence alignment. The asterisks (*) indicate the same amino acid between sequences. Amino acid residues with a high degree of identity are shown in green, and those with a low degree are shown in red and white. The NCBI database accession numbers of these H-Y antigens are shown to the left of the amino acid sequence. The expression of H-Y antigen inferred the identification of heterogametic sex using a reverse-transcriptase polymerase chain reaction (RT-PCR) from males (Test = Testis) and female (Ova = Ovary) gonadal tissue during the breeding season. PCR products were compared to a 100 bp MWM (100–1500 bp). Arrows denotes 500 bp and the expected 276 bp PCR product that includes the H-Y antigen (D).
Fig 3.
Full-length nucleotide and deduced amino acid sequences of H. eximia treefrog Actb (GenBank accession number PP107949).
The numbers at the left side of the sequence indicate the position of nucleotide and amino acid of the Actb. The coding region starts with a start codon (ATG) and ends with a stop codon (TAA) is indicated with ***. The putative polyadenylation signal (AATAAA) is underlined. ExPASy translate tool (https://web.expasy.org/translate/) was used to translate nucleotide sequence into amino acid sequence and ExPASy ProtParam tool characterized the molecular/physiochemical parameters of the Actb protein.
Fig 4.
Structural analysis of H. eximia treefrog Actb protein.
Multiple sequence alignment of H. eximia treefrog Actb (green letters) from deduced amino acid sequences of Homo sapiens (NP_001092.1), Haliaeetus leucocephalus (XP_010568622.1), Alligator mississippiensis (XP_014466124.2), Bufo gargarizans (XP_044158737.1), and Rana temporaria (XP_040213048.1) was performed using ClustalW software (https://www.genome.jp/tools-bin/clustalw); asterisks indicate similar amino acids between species. Different amino acids are marked with blue/green letters (A). Alignment of the predicted H. eximia treefrog Actb amino acid sequence with its B. gargarizans and R. temporaria counterparts showed that these proteins all had different length; the overall homologies of H. eximia treefrog Actb with B. gargarizans and R. temporaria Actb were 97%. Further alignment analyses also showed that H. eximia treefrog Actb shared a high percentage of identity with the corresponding Actb of other vertebrates (B). The three-dimensional (3D) protein structural prediction analysis was performed via Robetta platform (https://robetta.bakerlab.org/) and PyMol software (https://pymol.org/2/) (C).
Fig 5.
Involvement of H-Y antigen in the gonad of the heterogametic sex.
Real-time polymerase chain reaction (qPCR) was performed to examine the mRNA levels of H-Y antigen in gonads and several tissues (lung, anterior muscle, posterior muscle, vocal sac, and brain) from adult male and female treefrogs during non-breeding (A) and breeding seasons (B). Relative gene expression levels were normalized to β-actin gene. Vertical bars represent mean values ± standard deviation (SD) of four independent qPCR assays and each bar show male (n = 4; in blue) and female (n = 4; in pink) treefrogs. An analysis of variance (ANOVA) test was used to analyze statistical differences between treefrog groups (non-breeding and breeding seasons; male and female tissues). * and **** indicate p < 0.05 and p < 0.0001, respectively. p-value < 0.05 was considered as a significant level statistically.