Neonicotinoids are effective insecticides used on many important arable and horticultural crops. They are nicotinic acetylcholine receptor agonists which disrupt the function of insect neurons and cause paralysis and death. In addition to direct mortality, there are numerous sublethal effects of low doses of neonicotinoids on bees. We hypothesize that some of these large array of effects could be a consequence of epigenetic changes in bees induced by neonicotinoids. We compared whole methylome (BS-seq) and RNA-seq libraries of the brains of buff-tailed bumblebee Bombus terrestris workers exposed to field-realistic doses of the neonicotinoid imidacloprid to libraries from control workers. We found numerous genes which show differential expression between neonicotinoid-treated bees and control bees, but no differentially methylated cytosines in any context. We found CpG methylation to be focused mainly in exons and associated with highly expressed genes. We discuss the implications of our results for future legislation.

1Neonicotinoids are effective insecticides used on many important arable and horticultural 2 crops. They are nicotinic acetylcholine receptor agonists which disrupt the function of insect 3 neurons and cause paralysis and death. In addition to direct mortality, there are numerous 4 sublethal effects of low doses of neonicotinoids on bees. We hypothesize that some of these 5 large array of effects could be a consequence of epigenetics changes in bees induced by 6 neonicotinoids. We compared whole methylome (BS-seq) and RNA-seq libraries of the 7 brains of buff tailed bumblebee Bombus terrestris workers exposed to field realistic doses of 8 the neonicotinoid imidacloprid to libraries from control workers. We found numerous genes 9 which show differential expression between neonicotinoid treated bees and control bees, but 10 no differentially methylated cytosines in any context. We found CpG methylation to be focused 11 mainly in exons and associated with highly expressed genes. We discuss the implications of our 12 results for future legislation. 13

Allele-specific expression is when one allele of a gene shows higher levels of expression compared to the other allele, in a diploid organism. Recent work has identified allele-specific expression in a number of Hymenopteran species. However, the molecular mechanism which drives this allelic expression bias remains unknown. In mammals DNA methylation is often associated with genes which show allele-specific expression. DNA methylation systems have been described in species of Hymenoptera, providing a candidate mechanism. Using previously generated RNA-Seq and whole genome bisulfite sequencing from reproductive and sterile bumblebee (Bombus terrestris) workers we have identified genome-wide allele-specific expression and allele-specific DNA methylation. The majority of genes displaying allele-specific expression are common between reproductive and sterile workers and the proportion of allele-specific expression bias generally varies between genetically distinct colonies. We have also identified genome-wide allele-specific DNA methylation patterns in both reproductive and sterile workers, with reproductive workers showing significantly more genes with allele-specific methylation. Finally, there is no significant overlap between genes showing allele-specific expression and allele-specific methylation. These results indicate that cis-acting DNA methylation does not directly drive genome-wide allele-specific expression in this species.

The coexistence of different mating strategies, whereby a species can reproduce both by selfing and outcrossing, is an evolutionary enigma. Theory predicts two predominant stable mating states: outcrossing with strong inbreeding depression or selfing with weak inbreeding depression. As these two mating strategies are subject to opposing selective forces, mixed breeding systems are thought to be a rare transitory state yet can persist even after multiple speciation events. We hypothesise that if each mating strategy plays a distinctive role during some part of the species life history, opposing selective pressures could be balanced, permitting the stable co-existence of selfing and outcrossing sexual morphs. In this scenario, we would expect each morph to be specialised in their respective roles. Here we show, using behavioural, physiological and gene expression studies, that the selfing (hermaphrodite) and outcrossing (female) sexual morphs of the trioecious nematode Auanema freiburgensis have distinct adaptations optimised for their different roles during the life cycle. A. freiburgensis hermaphrodites are known to be produced under stressful conditions and are specialised for dispersal to new habitat patches. Here we show that they exhibit metabolic and intestinal changes enabling them to meet the cost of dispersal and reproduction. In contrast, A. freiburgensis females are produced in favourable conditions and facilitate rapid population growth. We found that females compensate for the lack of reproductive assurance by reallocating resources from intestinal development to mate-finding behaviour. The specialisation of each mating system for its role in the life cycle could balance opposing selective forces allowing the stable maintenance of both mating systems in A. freiburgensis.

The coexistence of different mating strategies, whereby a species can reproduce both by selfing and outcrossing, is an evolutionary enigma that has long intrigued biologists (Darwin, 1877). Theory predicts only two stable mating states : outcrossing with strong inbreeding depression or selfing with weak inbreeding depression. As these two mating strategies are subject to opposing selective forces, mixed breeding systems are thought to be a rare transitory state, yet they have been found to persist even after multiple speciation events. We hypothesise that if each mating strategy plays a distinctive role during the species life history, opposing selective pressures could be balanced, permitting the stable co-existence of selfing and outcrossing sexual morphs. In this scenario, we would expect each sexual morph to be specialised in their respective roles. Here we show, using a combination of behavioural, physiological and gene expression studies, that the selfing (hermaphrodite) and outcrossing (female) sexual morphs of the trioecious nematode Auanema freiburgensis have distinct adaptations optimised for their different roles during the life cycle. A. freiburgensis hermaphrodites are produced under stressful conditions, are specialised for dispersal to new habitat patches and exhibit metabolic and intestinal changes that enable them to meet the energetic cost of dispersal and reproduction. In contrast, A. freiburgensis females are produced in favourable conditions, facilitate rapid population growth and compensate for the lack of reproductive assurance by reallocating resources from intestinal development to robust mate-finding behaviour. The specialisation of each mating system for their role in the life cycle could balance opposing selective forces allowing the stable maintenance of both outcrossing and selfing mating systems in A. freiburgensis.