The effect of DNA methylation on bumblebee colony development

Background Although around 1% of cytosines in bees’ genomes are known to be methylated, less is known about methylation’s effect on bee behavior and fitness. Chemically altered DNA methylation levels have shown clear changes in the dominance and reproductive behavior of workers in queen-less colonies, but the global effect of DNA methylation on caste determination and colony development remains unclear, mainly because of difficulties in controlling for genetic differences among experimental subjects in the parental line. Here, we investigated the effect of the methylation altering agent decitabine on the developmental rate of full bumblebee colonies. Whole genome bisulfite sequencing was used to assess differences in methylation status. Results Our results showed fewer methylated loci in the control group. A total of 22 CpG loci were identified as significantly differentially methylated between treated and control workers with a change in methylation levels of 10% or more. Loci that were methylated differentially between groups participated in pathways including neuron function, oocyte regulation and metabolic processes. Treated colonies tended to develop faster, and therefore more workers were found at a given developmental stage. However, male production followed the opposite trend and it tended to be higher in control colonies. Conclusion Overall, our results indicate that altered methylation patterns resulted in an improved cooperation between workers, while there were no signs of abnormal worker dominance or caste determination.

Hybridization enables the fixation of selfish queen genotypes in eusocial colonies

The success of a eusocial colony depends on two main castes: queens that reproduce and sterile workers that help them. This division of labour is vulnerable to selfish genetic elements that enforce the development of their carriers into queens. Several factors, e.g. intra-colonial relatedness, are known to influence the spread of such selfish elements. Here we investigate the effects of a remarkable yet understudied ecological setting: where larvae produced by hybridization develop into sterile workers. Using mathematical modelling, we show that the coevolution of hybridization with caste determination readily triggers an evolutionary arms race between non-hybrid larvae that increasingly develop into queens, and queens that increasingly hybridize to produce workers. Even where hybridization reduces worker function and colony fitness, this race can lead to the loss of developmental plasticity and genetically hard-wired caste determination. Overall, our results help understand the repeated evolution towards complex reproductive systems (e.g. social hybridogenesis) and the special forms of parasitism (e.g. inquilinism) observed in many ant species.

Effect of Larval Topical Application of Juvenile Hormone on Cuticular Chemical Composition of Mischocyttarus consimilis (Vespidae: Polistinae) Females

Juvenile Hormone (JH) is considered the main determinant of caste in social insects, though little is known about how this hormone acts in social wasps, especially the independent-founding species. The known relationship between JH titer and caste in the colony and we suggest a relationship among the effects of JH and the cuticular chemical profile. Therefore, this study aimed to test the hypothesis that topical application of JH to larvae of different instars alters the cuticular chemical composition of newly emerged females of Mischocyttarus consimilis (Zikán), influencing the dynamics of colony. Two techniques were used to evaluate the variation in cuticular chemical composition: Fourier transform infrared photoacoustic spectroscopy and gas chromatography coupled to mass spectrometry (GC-MS). Indeed, the application of JH did significantly alter the cuticular chemical composition of adult females that received treatment at the larval stage in comparison to control. The effects of JH were instar-dependent in that the results of topical application were significant when performed at third larval instar. Overall, these results add evidence that caste determination may, at least in part may be pre-imaginal in species of independent-founding social wasps.

Diet quantity influences caste determination in honeybees ( Apis mellifera )

In species that care for their young, provisioning has profound effects on offspring fitness. Provisioning is important in honeybees because nutritional cues determine whether a female becomes a reproductive queen or sterile worker. A qualitative difference between the larval diets of queens and workers is thought to drive this divergence; however, no single compound seems to be responsible. Diet quantity may have a role during honeybee caste determination yet has never been formally studied. Our goal was to determine the relative contributions of diet quantity and quality to queen development. Larvae were reared in vitro on nine diets varying in the amount of royal jelly and sugars, which were fed to larvae in eight different quantities. For the middle diet, an ad libitum quantity treatment was included. Once adults eclosed, the queenliness was determined using principal component analysis on seven morphological measurements. We found that larvae fed an ad libitum quantity of diet were indistinguishable from commercially reared queens, and that queenliness was independent of the proportion of protein and carbohydrate in the diet. Neither protein nor carbohydrate content had a significant influence on the first principle component 1 (PC1), which explained 64.4% of the difference between queens and workers. Instead, the total quantity of diet explained a significant amount of the variation in PC1. Large amounts of diet in the final instar were capable of inducing queen traits, contrary to the received wisdom that queen determination can only occur in the third instar. These results indicate that total diet quantity fed to larvae may regulate the difference between queen and worker castes in honeybees.

Methylation and Gene Expression Differences Between Reproductive Castes of Bumblebee Workers

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 it’s role in other bee species. In this study we analysed 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 genome-wide methylation in B. terrestris is similar to other social 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 inter-colony variation in methylation. These findings suggest 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 it’s 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 social insects.Impact SummarySocial insects, such as ants, termites, bees and wasps, can produce individuals with extreme physical and behavioural differences within the same colony known as castes (e.g. workers/soldiers/queens). These individuals have similar genomes and many studies have associated epigenetic mechanisms with the differences observed. Epigenetic modifications are changes that affect how genes are expressed without changing the underlying DNA code. Here we investigated differences in DNA methylation (a well researched modified base) between different reproductive castes of the bumblebee, Bombus terrestris, an economically and environmentally important pollinator species. We found B. terrestris has a similar methylation profile to other social insect species in terms of the distribution of methylation throughout the genome and the relationship between methylation and gene expression. Genes that have differences in methylation between reproductive castes are involved in multiple biological processes, including reproduction, suggesting methylation may hold multiple functions in this species. These differentially methylated genes are also different to differentially methylated genes identified between honeybee reproductive castes, again suggesting methylation may have a variable function. These findings provide greater understanding of the role of methylation in caste determination in social insect species.