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Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses

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@PeerJPreprints Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses: https://t.co/qTMDjUzw7R https://t.co/2uHpSaVEnT
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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.

DOI: 10.7287/peerj.preprints.27461v1/supp-1

Details of RNA extraction methods used for each group of samples

DOI: 10.7287/peerj.preprints.27461v1/supp-2

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.

DOI: 10.7287/peerj.preprints.27461v1/supp-3

MIQE checklist for qPCR methods and analysis

DOI: 10.7287/peerj.preprints.27461v1/supp-4

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.

DOI: 10.7287/peerj.preprints.27461v1/supp-5

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.

DOI: 10.7287/peerj.preprints.27461v1/supp-6

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.

DOI: 10.7287/peerj.preprints.27461v1/supp-7

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.

DOI: 10.7287/peerj.preprints.27461v1/supp-8

Example R code for statistical analysis of qPCR data

DOI: 10.7287/peerj.preprints.27461v1/supp-9

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.

DOI: 10.7287/peerj.preprints.27461v1/supp-10

Sequence alignment used to reconstruct phylogenetic relationships in the Labridae

Nexus alignment file of concatenated 12S and 16S ribosomal RNA sequence data.

DOI: 10.7287/peerj.preprints.27461v1/supp-11

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Jodi T Thomas conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft, wrote the manuscript.

Erica V Todd conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft, wrote the manuscript.

Simon Muncaster conceived and designed the experiments, performed the experiments, authored or reviewed drafts of the paper, approved the final draft.

P Mark Lokman conceived and designed the experiments, performed the experiments, authored or reviewed drafts of the paper, approved the final draft.

Erin L Damsteegt performed the experiments, authored or reviewed drafts of the paper, approved the final draft.

Hui Liu performed the experiments, approved the final draft.

Kiyoshi Soyano conceived and designed the experiments, performed the experiments, approved the final draft.

Florence Gleonnec performed the experiments, approved the final draft.

Melissa S Lamm performed the experiments, approved the final draft.

John R Godwin conceived and designed the experiments, performed the experiments, approved the final draft.

Neil J Gemmell conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The Institutional Animal Care and Use Committee at North Carolina State University provided approval for Experiment 1 (12-069-0). The New Zealand National Animal Ethics Advisory Committee provided approval for Experiment 2 (2015_02) and Survey 1 (92-10). The Animal Care and Use Committee of the Institute for East China Sea Research, Nagasaki University, Japan, provided approval for Survey 2 (#15-06).

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

Candidate gene and reference gene sequences described here are accessible via GenBank accession numbers: bluehead wrasse cyp19a1a MK252274, amh MK252275, cyp19a1b MK252276, it MF279538.1, g6pd MK252277, ef1a MF279537.1, 18S MK246126, spotty wrasse cyp19a1a MK252278, amh MK252279, cyp19a1b MK252280, it MK252281, g6pd MK252282, ef1a MK252283, 18S MK246127, kyusen wrasse cyp19a1a MK252284, amh MK252285, cyp19a1b MK252286, it MK252287, g6pd MK252288, ef1a MK252289, 18S MK246128.

Data Deposition

The following information was supplied regarding data availability:

R code for statistical analysis of qPCR data is provided in Supplemental Data S1.

Raw qPCR measurements are provided in Supplemental Data S2. 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.

The sequence alignment used for phylogenetic analyses is provided in Supplemental Data S3. The file contains a Nexus format alignment of concatenated 12S and 16S ribosomal RNA sequence data.

Funding

This work was supported by the Royal Society of New Zealand Marsden Fund (UOO1308 to NJG), the Japan Society for the Promotion of Science (L10703 to PML) and the National Science Foundation (1257791 to JRG and 1257761 to Bill Tyler at Indian River State College, Florida). JT was supported by an Otago School of Medical Science Summer Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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