We find that the genomes of both species of bumblebee encode a remarkably similar repertoire of immune-related genes to the honeybee A.mellifera and solitary leafcutting bee M. rotundata (Figure 2). All the components of the major immune pathways, Toll, IMD, JAK/STAT, JNK, and the antiviral machinery are present in both Bombus species. Furthermore, the subset of these genes that we surveyed are detectibly expressed and many are induced upon immune activation. Indeed, these immune genes are expressed in a sex-specific fashion as predicted by Bateman’s principle of greater investment into maintenance for the choosier sex, usually females [54]. The sex differences in expression appear to be independent of gene dose since the expression of housekeeping genes was not significantly different between haploid males and gynes.

Overall, the number of immune genes is very consistent among the sequenced bees regardless of their degree of sociality, that is, from solitary (Megachile) to primitive (Bombus) and higher (Apis) eusociality (Figure 2). Primitive eusociality evolved about 87 million years ago in corbiculate bees [55], whereas higher sociality evolved in the Apini and Meliponi/Bombini with sociality being presumably secondarily lost in the Euglossini [55]. According to our results, the solitary bee M. rotundata, which split from the Apidae some 105 million years ago, has a comparable number of immune genes to honeybees and both Bombus species. These results suggest that the immune repertoire of A. mellifera, which was described as depauperate relative to dipterans, is probably a characteristic of bees more generally and predates the evolution of sociality and certainly existed before advanced eusociality in bees, and perhaps even as far back as before the split with ants [20]. Therefore, the relatively limited immune repertoire of honeybees does not seem to be the result of the transition to sociality and the associated behavioral adaptations for social immunity as suspected before [16]. An intriguing but purely speculative thought is that, rather than sociality reducing the need for immune genes, reduced immune complexity may have facilitated (for example, by way of easing the self/foreign distinction) or empowered (by way of allowing for social defenses) the evolution of social groups in the first place.

Both Bombus species have a small expansion of serpins (Figure 3). These serpins appear similar to the silkworm moth B. mori’s antitrypsin, which is involved in prophenoloxidase (PPO) regulation and is upregulated upon fungal infection [56]. We confirmed that these serpins are expressed in B. terrestris when challenged and are thus likely functional. The honeybee homolog seems to have a mutation within the binding region PS00284, which does not conform to the consensus pattern of this active site. It is unclear whether this gene in honeybees is a functional serpin. We also find a caspase that is similar to Decay in D. melanogaster (Figure 4), which has not been found in either A. mellifera or Nasonia vitripennis.

Despite having simpler colony organization and shorter colony lifespan, both bumblebee species nevertheless appear largely like honeybees in their immune-gene characteristics. Indeed, they also appear similar to the solitary leaf-cutting bee M. rotundata. While the complement of canonical immune genes may be consistent, it is important to recognize that our understanding of immunity is largely based on the known repertoire of non-social insects, and in particular the fruit fly D. melanogaster. As such, we are limited in being able to identify only known immune genes that have been functionally characterized in model systems. Bees may have further unexplored immune genes, novel defenses, and social behaviors that aid disease control and are unavailable to solitary species [21]. These adaptations are also genetically controlled, but the genes behind these traits are less well defined than the canonical immune response genes. Thus, while the Apoidea may appear to have consistent immune genomic profiles at the level of genes shared, they may differ considerably in the genetic underpinning of other key aspects of disease control in a social context, such as grooming, nest hygiene, and other behaviors. As a class, immune genes are rapidly evolving [57-60]. Here we explored which, among these immune genes, show particularly rapid evolution, or differences in selection among the different clades investigated. We found that some genes are under stronger selection in Bombus compared with Apis (genes below the diagonal in Figure 9), and a number of genes are under stronger positive selection in the social clade (upper diagonal in Figure 9) than in M. rotundata. While it is likely that clades with ω > 1 are under positive selection, these results should also be interpreted cautiously because without population data it is not possible to distinguish positive selection from relaxed constraints on selection [61]. Interestingly, we found a strong signature of selection on dscam, a gene primarily important for neuronal self-avoidance, but that is increasingly of interest in host-parasite interactions because alternative splicing of this gene can theoretically produce over 150,000 isoforms in D. melanogaster [62]. As such, dscam is hypothesized to be important for host-parasite specificity in susceptibility, and for specific immune memory [63]. The region under selection in dscam is limited to the beginning of the aligned protein (Figure 7A). This region corresponds to the fifth immunoglobulin I-set domain (sixth immunoglobulin domain overall). All of the previous immunoglobulin domains (1 to 5) were trimmed because they were not present in the A. mellifera gene. This gene appears to be under selection at least in the fifth immunoglobulin I-set domain but may also be variable in earlier domains. A previous study that examined the sequence of alternatively splicing exon cassettes did not detect selection in the crustacean Daphnia magna and several Drosophila species, at immunoglobulin (Ig) 2, 3 and 7 [64]. Our domain, however, likely corresponds to Ig4 or 5 in [64] and thus was not directly tested in their analysis. Nevertheless, our analysis is suggestive of differences in selective pressures among bee species. Among the other genes that show evidence of selection are a number of antiviral genes, including argonaute 2, aubergine (Figure 8A, B), and dicer 2, all of which have been found in other systems to be under selection [60,65]. We also detect evidence of selection on two AMPs, abaecin and defensin (Additional files 8, 10, and 11), both of which appear to be under stronger selection in the Apis clade (Figure 9). Our results corroborate those of Erler et al. [66], who also found positive selection on AMPs across several European bumblebee species. Interestingly, we find that dorsal appears to be under different selection in bumblebees than in honeybees, where Harpur and Zayed [61] found that dorsal was under purifying selection. We also find that all but one of the sites under selection in dorsal are outside of the relish domain (Figure 7C). Population level studies of the genes that appear to be evolving under different pressures in honeybees and bumblebees, and in the social and solitary clades will be instrumental in determining which of these genes are evolving under positive, relaxed or balancing selection [61].