1
INTRODUCTION
The ecological success of eusocial insects is attributed to an organized
and efficient division of labor (Oster & Wilson, 1978). Social insects
solve complex problems with individual behaviors, resulting in emergent
group properties (Hölldobler & Wilson, 2008). The numbers of workers
performing a particular task are optimized by feedback loops to
efficiently collect, process, and distribute resources among colony
members (Fewell, 2003). Individual workers transition among various
tasks during their lifetime, and exhibit a broad range of phenotypic
plasticity. More simply, colony demography is socially regulated (Z.-Y.
Huang & Robinson, 1996), allowing a proximate internal response to
unpredictable external environments. Various worker tasks involve
different physiological and behavioral demands, producing strong
selection on social phenotypes. Social insects are well suited to the
study of sociality and phenotypic plasticity because they represent a
complex adaptive system or ‘superorganism” in which the functional
parts can be manipulated and measured (Hölldobler & Wilson, 2008).
Honey bees are highly social insects that live in complex societies
consisting of one reproductive queen and thousands of facultatively
sterile workers. While the queen spends a preponderance of her life
laying eggs, workers build and maintain all aspects of the hive. Under
normal conditions, adult workers display age polyethism–performing
tasks within the hive for the first 2–3 weeks before transitioning to
outside tasks (Seeley, 1982). Specifically, young adults function as
“nurse bees” that feed growing larvae. Pupae are capped with wax and
isolated until they emerge from their cell and become the next cohort of
nurse bees. Meanwhile, near the end of their lifespan, older nurses
transition into foragers that then procure nectar (carbohydrates),
pollen (protein and lipids), propolis (antimicrobial plant resins), and
water (Seeley, 1982). This delicate balance of age-based division of
labor requires a high level of colony coordination and integration.
Despite this well-established pattern, adult workers can decouple age
from behavioral task in response to social cues from other workers and
pollination environment (Z. Y. Huang & Robinson, 1992). Thus,
behavioral task is extremely plastic and nursing/foraging behaviors can
be accelerated, slowed, or reversed (Robinson, 1992).
Phenotypic plasticity in honey bees workers is directly associated with
the availability of nutrition and storage proteins, vitellogenin in
particular (G. V. Amdam, Norberg, Hagen, & Omholt, 2003). Vitellogenin
(vg) is a phospholipoglyco-protein evolved to serve many functions; as
an antimicrobial, antioxidant, and to produce brood food in the in nurse
worker head (hypopharyngeal) glands (Gro V. Amdam, 2011; Siri-Christine
Seehuus, Norberg, Krekling, Fondrk, & Amdam, 2007). In contrast,
foragers switch to a diet of simple sugars to support the metabolism
associated with flight. This transition is associated with reduced lipid
stores (Toth & Robinson, 2005), reduced Vg titers (Fluri, Lüscher,
Wille, & Gerig, 1982), decreased nutritional status (Ament, Corona,
Pollock, & Robinson, 2008), and differential gene expression
(Ben-Shahar, Robichon, Sokolowski, & Robinson, 2002). These are
functional differences that likely contribute to being a successful
forager; a decrease in body mass and a proportional increase in flight
capacity (Vance, Williams, Elekonich, & Roberts, 2009). However,
leaving the relative safety of the hive is the riskiest time of an adult
bee’s life. A recent study documented that 40% of bees die during the
pre-foraging stage of life, a time where bees perform exploratory and
learning orientation flights (Prado et al., 2020). Bees that survive
face a constant increase in extrinsic mortality risk per unit time that
increases to 100% after 18 days of foraging activity (Dukas, 2008), yet
only ~20% of foragers will live past ten days of
foraging (Visscher & Dukas, 1997). Therefore, the age a worker
initiates foraging has a strong impact on lifespan and group
contribution.
Foraging also has direct consequences for intrinsic senescence,
including increased sensitivity to physiological stressors (Remolina,
Hafez, Robinson, & Hughes, 2007) and a decrease in innate immune
defenses (Gro V. Amdam et al., 2005; Gro V. Amdam, Simões, et al., 2004;
Lourenço et al., 2019; Schmid, Brockmann, Pirk, Stanley, & Tautz,
2008). Foragers also show an increased susceptibility to oxidative
stress (S.-C. Seehuus, Norberg, Gimsa, Krekling, & Amdam, 2006),
including oxidative damage to the brain (Rueppell, Christine, Mulcrone,
& Groves, 2007), and the body (Hsieh & Hsu, 2011). The accumulation of
oxidative damage from reactive oxygen species (ROS) is proposed as the
main cause of aging (Harman, 1956). Thus, a precocious transition to
foraging is predicted to result in premature aging. Flight and the
associated ROS accumulation from muscle usage and attrition may surpass
the capacity for antioxidant enzymes to remove them. Indeed, the honey
bee’s innate antioxidant enzymes; various superoxide dismutases,
catalase, and glutathione S-transferase, reach their greater expression
in older workers (Corona, Hughes, Weaver, & Robinson, 2005). While the
physiology of behavioral plasticity and aging has been explored in honey
bees, the role of the gut microbiome in this process is poorly known
(Vonaesch, Anderson, & Sansonetti, 2018).
Given the strong selection acting on social phenotypes, here we test
their physiological range in association to the gut microbiome. The
honey bee gut microbiota is remarkably consistent and dominated by five
omnipresent, highly co-evolved phylotypes representing
>95% of bacterial cells (Kwong & Moran, 2016; Martinson,
Moy, & Moran, 2012; Sabree, Hansen, & Moran, 2012). Recent work has
revealed a strong association of the microbiome with worker physiology
including the expression of insulin like peptides and vitellogenin
(Engel et al., 2016; Kešnerová et al., 2017; Maes, Rodrigues, Oliver,
Mott, & Anderson, 2016; Powell, Carver, Leonard, & Moran, 2021;
Raymann, Shaffer, & Moran, 2017; Ricigliano et al., 2017; Zheng,
Powell, Steele, Dietrich, & Moran, 2017). Although nurses and foragers
share a core microbiota (Corby-Harris, Maes, & Anderson, 2014),
microbial composition differs by behavioral task and may impact host
physiology and health (Anderson et al., 2018). The worker gut microbiota
is spatially organized, with the greatest abundance of bacteria found in
the distal regions of the gut (Martinson et al., 2012). Specifically,
the rectum comprises >90% of the bacterial cells in honey
bees and is an incredibly stable niche (Jones et al., 2018; Kapheim et
al., 2015; Kešnerová et al., 2020; Ludvigsen, Andersen, Hjeljord, &
Rudi, 2020; Maes, Floyd, Mott, & Anderson, 2021). Therefore, we chose
to focus on the midgut and ileum as these niches are more likely to
reveal the limits of phenotypic plasticity in relation to the
microbiome. The midgut functions in digestion and absorption of food,
but it is also considered a less stable substrate for bacterial
colonization because midgut epithelial cells secrete an envelope called
the peritrophic membrane in early adult life (Engel & Moran, 2013b).
The peritrophic membrane is continuously replaced as it is shed and
serves to facilitate digestion, and as a protective barrier from damage
by food particles and microbial invasion (Engel & Moran, 2013b). At the
pylorus, the Malpighian tubules excrete nitrogenous waste and
electrolytes from the hemolymph presenting a nutrient-rich environment
for microbes (Cintra-Socolowski, Nocelli, Roat, Silva-Zacarin, &
Malaspina, 2016; Cohen, Sawyer, Peterson, Dow, & Fox, 2020). Midgut
function including the production of peritrophic membrane decreases
significantly in foragers (Harwood & Amdam, 2021), but the impact on
disease susceptibility and the gut microbiome are unknown.
It has been demonstrated in honey bees that individual worker behavior
and physiology can be manipulated via the perturbation of social
structure (Z.-Y. Huang & Robinson, 1996). Here we ask whether such
changes are reflected in the gut microbiome. To test if gut microbiota
differences are associated with behavioral task or age, we created
“single-cohort colonies” (SCC) comprised of bees that were all the
same age (Robinson, Page, Strambi, & Strambi, 1989). As a result, some
proportion of the young bees in this colony will initiate foraging
precociously to fill the missing ranks of normal-aged foragers. As the
population ages, we assess differences in nurses and precocious foragers
(PF) of the same age, monitoring gene expression related to immunity and
oxidative stress. Likewise, we assessed protein oxidation in the fat
body resulting from precocious foraging. We hypothesized that precocious
foragers would accumulate oxidative damage at a faster rate than same
aged nurse bees. We test two hypotheses: (1) honey bees experience
differences in the midgut and ileum microbiota that reflect age and
behavioral task and (2) oxidative damage and gene expression reflect
differences within and among age and behavioral task.