Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses
- Published
- Accepted
- Subject Areas
- Aquaculture, Fisheries and Fish Science, Developmental Biology, Evolutionary Studies, Genetics, Marine Biology
- Keywords
- Protogynous sex change, Bluehead wrasse, Spotty wrasse, Kyusen wrasse, Quantitative real-time PCR, Sex-biased gene expression, cyp19a1a, cyp19a1b, amh, isotocin
- Copyright
- © 2019 Thomas et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses. PeerJ Preprints 7:e27461v1 https://doi.org/10.7287/peerj.preprints.27461v1
Abstract
Fishes exhibit remarkably diverse, and plastic, patterns of sexual development, most striking of which is sequential hermaphroditism, where individuals readily reverse sex in adulthood. How this stunning example of phenotypic plasticity is controlled at a genetic level remains poorly understood. Several genes have been implicated in regulating sex change, yet the degree to which a conserved genetic machinery orchestrates this process has not yet been addressed. Using captive and in-the-field social manipulations to initiate sex change, combined with a comparative qPCR approach, we compared expression patterns of four candidate regulatory genes among three species of wrasses (Labridae) - a large and diverse teleost family where female-to-male sex change is pervasive, socially-cued, and likely ancestral. Expression in brain and gonadal tissues were compared among the iconic tropical bluehead wrasse (Thalassoma bifasciatum) and the temperate spotty (Notolabrus celidotus) and kyusen (Parajulus poecilepterus) wrasses. In all three species, cyp19a1a (encoding gonadal aromatase that converts androgens to oestrogens) and amh (encoding anti-müllerian hormone that primarily regulates male germ cell development) were downregulated and upregulated, respectively, at the initiation of gonadal sex change, and may act concurrently to orchestrate ovary-testis transformation. In the brain, our data argue against a role for brain aromatase (cyp19a1b) in initiating behavioural sex change, as its expression trailed behavioural changes. However, we find that isotocin (it, that regulates teleost socio-sexual behaviours) expression correlated with dominant male-specific behaviours in the bluehead wrasse, suggesting it upregulation mediates the rapid behavioural sex change characteristic of blueheads and other tropical wrasses. However, it expression was not sex-biased in temperate spotty and kyusen wrasses, where sex change is more protracted and social groups may be less tightly-structured. Together, these findings suggest that while key components of the molecular machinery controlling gonadal sex change are phylogenetically conserved among wrasses, neural pathways governing behavioural sex change may be more variable.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Histological classification of gonadal sex change stages in spotty and kyusen wrasses
Abbreviations: NBF = non-breeding female, BF = breeding female, ET = early transitional, MT = mid transitional, LT = late transitional, TP = terminal phase, IP = initial phase.
Details of RNA extraction methods used for each group of samples
Primer sequences used for PCR to verify partial gene sequences in the bluehead and spotty wrasse
Bluehead primers were used to determine partial gene sequences for kyusen wrasse. Abbreviations: 18S = 18S ribosomal RNA, amh = anti-müllerian hormone, cyp19a1a = gonadal aromatase, cyp19a1b = brain aromatase, ef1a = elongation factor 1α, FW = forward, g6pd = glucose-6-phosphate dehydrogenase, it = isotocin, RV = reverse, Tm = melting temperature.
Primer sequences for qPCR amplification of target genes (cyp19a1a/b, amh, it) and candidate reference genes (ef1a, 18S, g6pd) in bluehead, spotty and kyusen wrasse
For spotty wrasse the average efficiency ± standard deviation of all the qPCR plates for one gene is shown. Abbreviations: 18S = 18S ribosomal RNA, amh = anti-müllerian hormone, bp = base pairs, cyp19a1a = gonadal aromatase, cyp19a1b = brain aromatase, ef1a = elongation factor 1α, FW = forward, g6pd = glucose-6-phosphate dehydrogenase, it = isotocin, n/a = not applicable, RV = reverse.
Reference genes used for each tissue type, and experiment
All potential reference genes in gonad samples from Experiment 2 (social induction of sex change in captive spotty wrasse) showed a significant difference in distribution across sexes. The trend of the results was not changed by ef1a and 18S, and these were chosen as reference genes. The only gene showing no significant difference in distribution across sexes in gonad samples from Survey 1 (opportunistic sampling of spotty wrasse) was g6pd. However, its use as reference gene changed the trend of results drastically. Abbreviations: 18S = 18S ribosomal RNA, ef1a = elongation factor 1α, g6pd = glucose-6-phosphate dehydrogenase.
Normalised, relative gonadal expression of cyp19a1a (left) and amh (right) mRNA
Expression levels are compared among females, transitioning fish, TP males and IP males. (A) Bluehead wrasse induced to change sex in the wild (Experiment 1). (B) Spotty wrasse induced to change sex in captivity (Experiment 2). (C) Wild-caught spotty wrasse (Survey 1). (D) Wild-caught kyusen wrasse (Survey 2). Points represents individual fish. Boxplots represents the median, lower and upper quartile values, and 1.5-fold the interquartile range. Yellow, blue and grey points indicate expression is significantly female-biased, male-biased, and non-significantly different, respectively. Letters denote a significant difference in distribution between groups and ‘a’ indicates overall significance without significant pairwise. Sample sizes: bluehead wrasse n = 3, all groups; spotty wrasse socially induced to change sex in captivity C BF D0 n = 5, C BF n = 16, M BF n = 4, ET n = 20, MT n = 1, LT n = 1, TP n = 11, IP n = 5; spotty wrasse opportunistically caught NBF n = 6, ET n = 3, MT n = 2, LT n = 2, TP n = 1; kyusen wrasse NBF n = 7, ET n = 3, TP n = 11, IP n = 3. Abbreviations: C BF D0 = breeding female from control tank (TP male present) at experiment day 0, C BF = breeding female from control tank (TP male present) removed at progressive time points throughout the experiment, CF = control female, ET = early transitional, IP = initial phase male, LT = late transitional, M BF = breeding female from manipulated tanks (TP male removed) removed at progressive time points throughout experiment, MT = mid transitional, NBF = non-breeding female, S1-6 = stages 1-6, TP = terminal phase male.
Normalised, relative brain expression of cyp19a1b (left) and it (right) mRNA
Expression levels are compared among females, transitioning fish, TP males and IP males. (A) Bluehead wrasse induced to change sex in the wild (Experiment 1). (B) Spotty wrasse induced to change sex in captivity (Experiment 2). (C) Wild-caught spotty wrasse (Survey 1). (D) Wild-caught kyusen wrasse (Survey 2). Points represents individual fish. Boxplots represents the median, lower and upper quartile values, and 1.5-fold the interquartile range. Yellow, blue and grey points indicate expression is significantly female-biased, male-biased, and non-significantly different, respectively. Letters denote a significant difference in distribution between groups and ‘a’ indicates overall significance without significant pairwise. Sample sizes: bluehead wrasse n = 3 all groups; spotty wrasse socially induced to change sex in captivity C BF D0 n = 5, C BF n = 16, M BF n = 4, ET n = 20, MT n = 1, LT n = 1, TP n = 11; IP n = 5, spotty wrasse opportunistically caught NBF n = 6, ET n = 2, MT n = 2, LT n = 3, TP n = 1; kyusen wrasse NBF n = 6, ET n = 5, TP n = 7, IP n = 4. Abbreviations: C BF D0 = breeding female from control tank (TP male present) experimental day 0, C BF = breeding female from control tank (TP male present) removed at progressive time points throughout the experiment, CF = control female, ET = early transitional, IP = initial phase male, LT = late transitional, M BF = breeding female from manipulated tanks (TP male removed) removed at progressive time points throughout experiment, MT = mid transitional, NBF = non-breeding female, S1-6 = stages 1-6, TP = terminal phase male.
Example R code for statistical analysis of qPCR data
Raw qPCR gene expression data for bluehead, spotty and kyusen wrasses
Raw (.raw) and relative (.rel) expression values are provided in separate sheets for each species, and for brain and gonad analyses separately. Bluehead, bluehead wrasse Experiment 1. SpottySI, spotty wrasse social induction Experiment 2. SpottWild, Spotty wrasse Survey 1. Kyusen, kyusen wrasse Survey 2.
Sequence alignment used to reconstruct phylogenetic relationships in the Labridae
Nexus alignment file of concatenated 12S and 16S ribosomal RNA sequence data.