Investigating trade-offs between ovary activation and immune protein expression in bumble bee (Bombus impatiens) workers and queens

Abstract

Evidence for a trade-off between reproduction and immunity has manifested in many animal species, including social insects. However, investigations in social insect queens present a conundrum: new gynes of many social hymenopterans, such as bumble bees and ants, must first mate, then transition from being solitary to social as they establish their nests, thus experiencing confounding shifts in environmental conditions. Worker bumble bees offer an opportunity to investigate patterns of immune protein expression associated with ovary activation while minimizing extraneous environmental factors and genetic differences. Here, we use proteomics to interrogate the patterns of immune protein expression of female bumble bees (Bombus impatiens) by (i) sampling queens at different stages of their life cycle, then (ii) by sampling workers with different degrees of ovary activation. Patterns of immune protein expression in the haemolymph of queens are consistent with a reproduction-immunity trade-off, but equivalent samples from workers are not. This brings into question whether queen bumble bees really experience a reproduction-immunity trade-off, or if patterns of immune protein expression may actually be due to the selective pressure of the different environmental conditions they are exposed to during their life cycle.

Keywords: bumble bees; immunity; proteomics; reproduction; resource allocation; trade-off.

Figure 1.

Overview of the bumble bee life cycle and anatomical patterns. (a) Bumble bee queens spend the winter in underground chambers. Upon emerging in the spring, nascent queens forage and search for a nesting site, but are not yet laying eggs. Once a nest is established, the colony grows through the spring into a few hundred individuals. In the summer, new queens and males are produced, mating occurs, and newly mated queens search for new overwintering sites. This figure was created using BioRender.com. (b) Relationships between ovary mass and body mass of female bumble bees.

Figure 2.

Haemolymph proteins in nascent, established and unmated queens. (a) Heat map of all identified protein groups identified in greater than 50% of samples (2284 in total). In total, 1000, 993 and 1419 were differentially expressed at 5% FDR (Benjamini–Hochberg correction) in established versus nascent, established versus unmated and nascent versus unmated groups, respectively. (b,c) Key proteins of interest. Different letters indicate statistical significance at 5% FDR. Abaecin and defensin were not evaluated statistically as they did not meet the threshold of being identified in greater than 50% of samples from all groups. Vitello-1, Vitello-3 and Vitello-4 refer to the vitellogenin isoforms A0A6P3DVD4, A0A6P3E5K4 and A0A6P3V1F4, respectively. Lys-1 and Lys-2 correspond to lysozyme isoforms A0A6P3DSG7 and A0A6P3DTW1, respectively. PPO stands for (pro)phenoloxidase-1.

Figure 3.

Sampling approach for reproductively active and inactive workers. (a) Ovary mass is a key parameter linked to active oogenesis, but it correlates significantly with body size. (b) The ovary mass-to-body mass ratio better indicates degree of reproductive activation. Dotted lines indicate the 1st and 4th quartile boundaries. Haemolymph samples from the corresponding reproductively active and inactive bees were subsequently analysed by proteomics.

Figure 4.

Proteomics results of reproductively active and inactive workers. (a) Phenotypic and proteomics data from colonies 1 and 2 were collected independently of colonies 3 and 4. (a) After filtering, 3259 and 3252 protein groups were quantified. Low ratio refers to samples from bees with low ovary mass-to-body mass ratios (reproductively inactive) and high ratio corresponds to high ovary mass-to-body mass ratios (reproductively active). In total, 772 and 532 proteins were significantly different between high ratio and low ratio groups at 5% FDR (Benjamini–Hochberg correction). (b–d) Expression patterns of key proteins. Asterisks indicate statistical significance at 5% FDR. Vitello-1, Vitello-3 and Vitello-4 refer to the vitellogenin isoforms A0A6P3DVD4, A0A6P3E5K4 and A0A6P3V1F4, respectively. Lys-1 and Lys-2 correspond to lysozyme isoforms A0A6P3DSG7 and A0A6P3DTW1, respectively. PPO stands for (pro)phenoloxidase-1.