Fig 5. The B. glabrata DNA methylation machinery is abundantly expressed in sex tissues and haemocytes.

A) RNA-Seq analysis of the B. glabrata DNA methylation machinery in twelve snail tissues. The normalised sequencing counts [51] for each gene of interest (i.e. Bgmbd2/3, Bgdnmt1 and Bgdnmt2) across the twelve tissues were used to estimate sample parameters for that gene i.e. the mean and standard deviation. The twelve observations for each gene were scaled to a standardised t-distribution. These standardised counts for the three genes were plotted (y-axis) against the twelve tissues (x-axis)—the continuous the red line on the y-axis at 1.79 represents p < 0.05 on a t-distribution with 11 degrees of freedom. The samples were divided into three groups, i.e Group 2: ovotestes (OVO), Group 3: terminal genitalia (TRG) and Group 1: salivary glands (SAL), digestive gland/hepatopancreas (DG/HP), central nervous system (CNS), buccal mass (BUC), albumin gland (AG), mantle edge (MAN), head/foot (FOOT), stomach (STO), heart/APO (HAPO) and kidney (KID). Differential expression analysis, using DESeq2 [51], indicates the Group 1 vs. Group 2 and Group 1 vs. Group 3 comparisons of Bgmbd2/3, Bgdnmt1 and Bgdnmt2 abundance (i.e. tissue samples with data above the red line) are statistically significant for that gene of interest. B) qRT-PCR data confirms the tissue-enriched expression of the B. glabrata DNA methylation machinery. qRT-PCR was employed to verify the transcript abundance of Bgdnmt1, Bgdnmt2 and Bgmbd2/3 across five tissues previously analysed by RNAseq. In addition to albumin gland (AG), head/foot (FOOT), stomach (STO), ovotestes (OVO) and digestive gland/hepatopancreas (DG/HP), transcript abundance was also determined in haemocytes (HAEMO). Error bars represent standard deviation of the mean (SD). The Ct values of target genes were normalised to the reference gene S19 [77]. Biological duplicates were used for each tissue and technical triplicates performed for every qRT-PCR reaction. For haemocytes, only one biological sample was available. C) 5-AzaC treatment inhibits B. glabrata oviposition. Adult NMRI snails (10–12 individuals/condition) were incubated in the presence or absence of 491μM 5-AzaC for a total of eight days. The bar chart represents mean eggs laid/condition at day eight + standard deviation (SD). The Student’s two-tailed t test was performed to identify significant differences between the treatments. Images are representative of egg sacs obtained from control and 5-AzaC conditions and were taken 7 days after deposition. D) A heat map representation of genes within the neighbourhood of Bgdnmt1 and Bgmbd2/3 that are significantly over or under-expressed in OVO (ovotestes). The genes are clustered in two directions i.e. across samples and across genes. Uniprot assigned short names to these genes based on sequence homology (full name included in S2 Table) are indicated.