The Effect of Immuno-stimulation on the Health of Bumblebees: Assaying Behavioral and Physiological Responses

__________________________________________________________ Pollinator species are declining around the world at an alarming rate, posing a major threat to both terrestrial ecosystems and the agricultural industry. The exact cause of these declines has yet to be identified, but pathogen infection claims a high position on the list of likely causes. To better understand how infection impacts the health of a native bumblebee pollinator, Bombus impatiens, I artificially stimulated an immune response in individual foragers and then measured their physiological (zone of inhibition assay) and behavioral (serial reversal learning assay) response. My findings will greatly improve our ability to quantify the fitness consequences of infection on bumblebees and expand the range of conservation tools available to evaluate and predict the effect of exposure to novel pathogens on different bumblebee species. Background_________________________________________________ Pollinators around the world are experiencing rapid and unprecedented population declines. The loss of bees in particular is distressing, since they are an invaluable part of many of the earth’s ecosystems. There are more than 20,000 species of bees worldwide (Velthuis & van Doorn, 2006), and of these, the approximately 250 species of bumblebee (genus Bombus) have become the subject of much concern (Goulson, Lye, & Darvill, 2008). Bumblebees are quite important pollinators in their native regions of the northern hemisphere (Goulson et al., 2008). Their long tongues help them collect nectar from deep flowers in these regions, and their use of sonication or “buzz pollination” efficiently spreads pollen (Buchmann, 1985). These traits, along with ease of maintenance and handling, have made the especially important for North American and European agriculture (Kraus et al, 2010; Goulson et al., 2008). Bumblebees are now the main greenhouse pollinators of such food crops as tomatoes, bell peppers, and a variety of berries in the United States (Velthuis & van Doorn, 2006; Kraus et al., 2010). These operations currently produce about three billion dollars a year for the US economy (Danforth, 2007). With such a large stake in our food supply and economy, the health statuses of bee species must be a priority. Currently, the cause of bee decline is unknown. However, researchers have proposed several possible environmental stressors, including habitat loss/alteration, pesticides, pathogens/disease, and climate change (Goulson et al, 2008; Whitehorn, O’Connor, Wackers, & Goulson, 2012, 2012; Henry et al., 2012; Colla, Otterstatter, Gegear, & Thomson, 2006). However, we currently have a poor understanding of how each of these stressors, and combinations thereof, affect the health of bees, particularly those native species in decline. In this study, I explore how pathogens/disease impact bee populations through sublethal effects on fitness-related behaviors. Many animals experience changes in behavior in response to illness. Much like a human experiencing a bout with the flu, other vertebrate animals will also exhibit lethargy, decreased appetite, fever, and a number of other predictable symptoms when suffering an infection. In fact, evidence is growing to prove that these changes are an evolutionary adaptation for the body to focus its efforts on healing (Hart, 1988). Much of these behavioral symptoms can be traced to cognitive impairment induced by the immune response to the infection (reviewed in Wilson, Finch, & Cohen, 2002). In vertebrates, it seems that sickness behavior is a concerted effort by the brain and immune system to combat disease (see Maier & Watkins, 1998 for a review). Cytokines are the main signaling molecules involved in vertebrate immune responses, and thus in vertebrate sickness behavior and cognitive impairment. For instance, Alzheimer’s patients are more likely to develop delirium upon infection due to cytokine mediation (Holmes et al., 2002), and cytokines have been implicated in memory consolidation (Rachal Pugh, Fleshner, Watkins, Maier, & Rudy, 2001) and memory deficiencies (Gibertini, Newton, Friedman, & Klein, 1995) A similar type of cognitive impairment may occur in invertebrates like bumblebees, as well. It has been shown that parasitic infections can reduce learning efficiency in bumblebees (Gegear, Otterstatter, & Thomson, 2005; Gegear, Otterstatter, & Thomson, 2006). Insects, unlike humans, possess no adaptive immunity. They do, on the other hand, possess cellular and humoral innate immune functions (see Lavine & Strand, 2002 for a review). In insect innate immune responses, along with other reactions, antimicrobial peptides (AMPs) are secreted by the fat body (liver analog) in response to infection (Hoffmann, 2003). These AMPs occupy the hemolymph, which is the insect analog of mammalian blood/lymph fluid. It appears insects may even have humoral molecules that mimic the functions of cytokines in vertebrates (Wittwer, Franchini, Ottaviani, & Wiesner, 1999; reviewed in Hoffmann, 2003). A change in bee cognition could have detrimental sublethal effects on colony performance and health. Bees have advanced capacities for learning and association, and faster learning in bumblebees has been linked to better foraging success while slower learning results in lower nectar stores (Raine & Chittka, 2008; Raine & Chittka, 2012). Other problems with foraging behavior would likely have similar effects on nectar collection. Less food would support fewer bees within a colony, which would in turn provide the colony with even less food to support the production of reproductives (males and queens), ultimately lowering the number of queens available to contribute to the population the following year. In this way, a seemingly small, sublethal change in individual bee foraging proficiency can negatively affect the size of the bee population. Learning to handle different types of flowers in their natural environment also requires much of bumblebees’ mental faculties (Chittka & Thomson, 2001). In order to handle flowers of varying complexity, bees must be capable of switching handling methods. This ability to switch between tasks is a product of the bee’s cognitive flexibility (Chittka & Thomson, 2001; Gegear & Laverty, 2005). Task switching proficiency and thus cognitive flexibility can be evaluated using serial reversal learning assays. In these tests, subjects are taught to associate rewards with specific tasks. The subject is then tested on completing one of the tasks, after which the tasks are reversed. There is a cost to performance inflicted by switching the tasks, and ideally, this cost is diminished with each reversal. The costs associated with switching determine the flexibility of the subject. So far, these assays have mostly been performed with humans and other vertebrates (Bond, Kamil, and Balda, 2007); however, there is evidence that this assay can be used with insects (Strang & Sherry, 2013). The purpose of this study was to develop assays for measuring the strength of an immune response (zone of inhibition assay) and level of cognitive flexibility (serial reversal learning assay) in individual bumblebee (Bombus impatiens) foragers. Although B. impatiens is not among the bumblebee species experiencing severe declines at present (Goulson, 2008), experimental data from the current study will be used as a baseline for future assessment of bumblebee species in decline. To isolate the effects of immune-stimulation on bees, physiological and behavioral responses of healthy bees were compared to immune-stimulated bees injected with LPS (lipopolysaccharide). LPS is a component of gram-negative bacterial cell membranes, which is known to induce an immune response in many animals, and was used here to mimic pathogen invasion. Methods_______________________________________________________ 1) Flexibility Assay: Serial Reversal Learning Bumblebees (Bombus impatiens) were tested in serial reversal learning trials that included a color-detection and an odor-detection task. These bees were trained using associative learning to recognize non-scented yellow flowers as rewarding (color-detection) and blue flowers with geranium scent as rewarding (odor-detection). The bee’s ability to switch between these two tasks was subsequently examined in a series of trials containing distracter flowers of various colors and scents. All flowers were constructed from artificial parts, and rewarding flowers contained 30% sucrose solution compared to nonrewarding flowers, which contained 5% sucrose solution. Tests were digitally recorded for ease of data