https://orcid.org/0000-0001-6176-3050
Figures & data
Figure 1. Synopsis of the experience.
Only fish from the F0 generation were exposed to cadmium (Cd) throughout their life (0 to 169 dpf). At each generation and for each condition (control or Cd), we used 20 mating pairs and 4 large aquaria. Mean sex ratios (males and females %) are reported for each condition and generation. See Pierron et al., [Citation10] for more details.
Figure 2. Cd exposure induced changes in allele frequencies.
(a) Changes in the percentage of homozygous alt/alt fish (control, C, blue or cadmium, Cd, red) across generations (F0-F3). Data from adults (males and females, 124 dpf) and larvae (5 dpf) were combined to increase sample size (for each condition and generation, n = 18). Different letters denote significant differences (P < 0.05, Marascuilo procedure). (b) Schematic representation of the two alleles of the zbtb38 gene. The genetic variations that we analysed are reported in red. SNP position in NC_007129.7/GRCz11 for ex3: 41212746 and ex4: 41199436, 41199446, 41199619, 41199658. DMR, differentially methylated region identified by MeDip-Seq. Light grey boxes correspond to non-coding regions, dark grey boxes to coding regions, and introns are schematized by a green line.
Figure 3. The exon 3 of zbtb38 is transcribed independently of the other exons.
(a,b) Mean (± SE, n = 2–34) transcription levels (a.u.) of the different exons (ex2,3,4) of the zbtb38 gene in gonads of female (a) and male (b) zebrafish (124 dpf) according to their genotype. Means designated by different letters are significantly different (LSD test, P < 0.05), and number of biological replicates of each genotype is indicated upon bars. Fish from the two conditions (C or Cd) were pooled to increase the number of biological replicates. (c) Mean (± SE, n = 6, 3 C and 3 Cd female fish) transcription levels of the different targeted regions of the zbtb38 gene in alt/alt female gonads. Means designated by different letters are significantly different (LSD test, P < 0.05). (d) Changes in the transcription level of the exon 3 of zbtb38 (mean ± SE, n = 6) in gonads of female (F, continuous line) and male (M, dashed line) zebrafish from each generation (F0 to F3) and from the two experimental conditions: C, control (blue); Cd, cadmium (red). For each sex, means designated by different letters are significantly different (LSD test, P < 0.05).
Figure 4. Sex-specific patterns of DNA methylation and RNA transcription.
(a,b) Changes in the methylation level of the exon 3 of zbtb38 in gonads (a) of female (F, full bars) and male (M, hatched bars) zebrafish (124 dpf) from each generation (F0 to F3) and from the two experimental conditions: C, control (blue); Cd, cadmium (red). Means (± SE, n = 6) designated by different letters are significantly different (Conover–Iman, P < 0.05) or (b) according to the sex and genotype of fish. Fish from the two conditions (C or Cd) were pooled to increase the number of biological replicates. Means (± SE, n is indicated upon bars) designated by different letters are significantly different (LSD, P < 0.05). (c,d) Distribution of (c) ex3 methylation levels and (d) ex4 transcription levels (expressed as Log) in gonads of adult fish according to the sex (Female, F; Male, M) and condition (Control, C; Cadmium, Cd). Data from the two conditions (n = 47–48 per sex) were combined to investigate the effect of sex, and results of the consecutive Student’s T tests are indicated.
Figure 5. A complex dialog between the environment, the genome and DNA methylation led to variations in the transcription level of the ex3-NAT of zbtb38.
Figure 6. Parental expression of ex3-NAT influences offspring sex ratio.
(a,b) Relationship between the mean transcription level (± SE, n = 12) of the exon 3 of zbtb38 in gonads of parents (mean transcription levels from females and males (124 dpf) were added, n = 6 per sex) from each generation (F) and (a) the sex ratio at the next generation (F + 1) (males %, mean ± SE, n = 4 aquaria per condition, number of fish per generation and condition ranged from 303 to 663) or (b) the mean methylation level of cyp19a1a in whole larvae (5 dpf, n = 6) of the next generation (F + 1). The Pearson correlation coefficient (r) and the associated P value are reported.
Supplemental material
Supplemental Material
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Raw sequencing data were deposited in the European Nucleotide Archive with accession number PRJEB52137. All other data are included in the article and/or supporting information.