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. 2019 Aug 5;3(5):485-499.
doi: 10.1002/evl3.129. eCollection 2019 Oct.

Methylation and gene expression differences between reproductive and sterile bumblebee workers

Hollie Marshall  1 , Zoë N Lonsdale  1 , Eamonn B Mallon  1
Affiliations

Methylation and gene expression differences between reproductive and sterile bumblebee workers

Hollie Marshall et al. Evol Lett. .

Abstract

Phenotypic plasticity is the production of multiple phenotypes from a single genome and is notably observed in social insects. Multiple epigenetic mechanisms have been associated with social insect plasticity, with DNA methylation being explored to the greatest extent. DNA methylation is thought to play a role in caste determination in Apis mellifera, and other social insects, but there is limited knowledge on its role in other bee species. In this study, we analyzed whole genome bisulfite sequencing and RNA-seq data sets from head tissue of reproductive and sterile castes of the eusocial bumblebee Bombus terrestris. We found that genome-wide methylation in B. terrestris is similar to other holometabolous insects and does not differ between reproductive castes. We did, however, find differentially methylated genes between castes, which are enriched for multiple biological processes including reproduction. However, we found no relationship between differential methylation and differential gene expression or differential exon usage between castes. Our results also indicate high intercolony variation in methylation. These findings suggest that methylation is associated with caste differences but may serve an alternate function, other than direct caste determination in this species. This study provides the first insights into the nature of a bumblebee caste-specific methylome as well as its interaction with gene expression and caste-specific alternative splicing, providing greater understanding of the role of methylation in phenotypic plasticity within social bee species. Future experimental work is needed to determine the function of methylation and other epigenetic mechanisms in insects.

Keywords: Bombus; bumblebee; expression; hymenoptera; methylation.

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Figures

Figure 1
Figure 1
(A) one half of a pair of ovaries from a sterile bumblebee worker, with a score of 0. (B) One half of a pair of ovaries from a reproductive bumblebee worker, with a score of 4. Scores generated following Duchateau and Velthuis (1988).
Figure 2
Figure 2
Overview of sample preparation for sequencing. Three reproductive workers and three sterile workers were selected from three colonies (J1, J5 and J8 represent colony names). Half of each head was randomly allocated for RNA/DNA extraction. All 18 RNA samples were sent for RNA‐Seq (three of each caste from each colony). DNA samples were pooled by colony and caste creating a representative reproductive and sterile sample per colony.
Figure 3
Figure 3
(A) Box plot of the mean weighted methylation level of methylated genes (n = 3412) across colonies for each caste. (B) The mean weighted methylation level across colonies for each genomic feature for both reproductive and sterile workers. Error bars are 95% confidence intervals of the mean. (C) PCA plot generated by methylKit showing samples cluster by colony using per site CpG methylation. (D) Scatter plot of the average weighted methylation level (across colonies) for reproductive workers against sterile workers. Each dot represents a gene and each red dot represents a differentially methylated gene (q < 0.05 and a minimum gene weighted methylation difference of 10%).
Figure 4
Figure 4
(A) PCA plot showing samples cluster by caste for gene expression, the first half of each label represents the colony name and the second half is the individual identification number. (B) Heatmap showing the 100 top differentially expressed genes between reproductive castes, samples cluster by reproductive status. Sample names are shown at the bottom of the plot. (C) An example of a gene that shows differential exon expression in one exon between reproductive castes. The top section of the plot shows the general expression differences between castes, the second section shows the normalized counts per exon (given expression differences) and the third section highlights the differentially expressed exon in pink. Gene shown: probable peroxisomal acyl‐coenzyme A oxidase 1 (ID: LOC100648955).
Figure 5
Figure 5
(A and B) The colony average weighted methylation level for every gene plotted against the log(FPKM) of that gene for sterile workers and reproductive workers, respectively. Each black dot represents a single gene. The blue line is a fitted linear regression with the gray shaded area representing 95% confidence intervals. (C) Violin plots showing the distribution of the data via a mirrored density plot, meaning the widest part of the plots represent the most genes. Weighted methylation level per gene per caste, averaged across colonies, was binned into four categories, no methylation, low (>0–0.2), medium (0.2–0.7), and high (0.7–1), as in Liu et al. (2019). The red dot indicates the mean with 95% confidence intervals. Each black dot represents a single gene. (D) Binned methylated genes (n = 3412, 100 being the most highly methylated) based on the mean weighted methylation level across colonies per reproductive caste, plotted against the log(FPKM) expression level per gene. Data were smoothed using the LOESS method, and gray areas are 95% confidence intervals.
Figure 6
Figure 6
(A) Violin plots showing the distribution of the data via a mirrored density plot, meaning the widest part of the plots represent the most genes. The red dots represent the mean of each gene set along with error bars representing 95% confidence intervals of the mean. Each black dot is an individual gene. The mean weighted methylation per gene across colonies per caste is plotted for either differentially expressed genes (DEG) or nondifferentially expressed genes (non‐DEG). (B) Violin plots of the mean weighted methylation per gene across colonies per caste is plotted for either genes containing differentially expressed exons (DEE) or genes with non‐differentially expressed exons (non‐DEE). (C) Violin plots of the mean weighted methylation per exon across colonies per caste for DEE and non‐DEE. The red dots represent the mean of each exon set along with error bars representing 95% confidence intervals of the mean. Each black dot is an individual exon. (D) Scatter plot of the difference in the mean weighted methylation level across colonies between castes plotted against the log2 fold change in expression of differentially expressed genes between castes. Each dot represents a gene; only genes which have a methylation difference >0 are shown. The red dots indicate that the gene is also differentially methylated.
Figure 7
Figure 7
(A) UpSet plot showing the number of common genes between analyses. The set size indicates the number of genes in each category; differentially expressed, differentially methylated or genes containing a differentially expressed exon. The intersection size indicates the number of genes either unique to each set or the number common between sets. A single dot in the lower panel indicates the number of genes unique to the corresponding set and joining dots indicate the number of genes in common between the corresponding sets. (B) UpSet plot showing the number of putative orthologs between A. mellifera and B. terrestris along with the number of differentially methylated genes identified in Lyko et al. (2010) and in this study which are present in the putative ortholog database.
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