Nosema and Necrophoresis: a Honey Bee Parasite and Undertaking

NOSEMA AND NECROPHORESIS: A HONEY BEE PARASITE AND UNDERTAKING

BEHAVIOUR

Megan J. Colwell

Thesis

submitted in partial fulfillment of the

requirements for the Degree of

Bachelor of Science with

Honours in Biology

Acadia University

March, 2010

This thesis by Megan J. Colwell

is accepted in its present form by the

Department of Biology

as satisfying the thesis requirements for the degree of

Bachelor of Science with Honours

Approved by the Thesis Supervisor

______

Dave Shutler Date

Approved by the Head of the Department

______

Soren Bondrup-Nielsen Date

Approved by the Honours Committee

______

Sonia Hewitt Date

I, MEGAN J. COLWELL, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a

non-profit basis. I, however, retain the copyright in my thesis.

______

Signature of Author

______

Date

iii

Acknowledgements

First and foremost I would like to thank Dave Shutler, my supervisor and mentor for the last year. Without his invaluable shredding of my writing, insights into new and exciting project prospects, his ability to understand what direction I wanted to take, and most importantly his sense of humour, this past year would have been dreadful. Also thank-you to the graduate students of Team Shutler, especially Geoff Williams, for welcoming me into the lab and sharing their knowledge and experience. A special thank-you to my fellow Honours students: my lab mates, Holly Lightfoot and Emma Vaasjo, and my office mates, Emma McIntyre and Laura

Ferguson.

I would also like to thank Adele Mullie for her part in “string theory”, and for tolerating the presence of my skunk-addled bees on the Mullie-Shutler property this summer. Thank-you to

Jack and Lorraine Hamilton for letting me deploy my dead bee trap and abscond with some of their bee corpses in Aylesford, and Kevin Spicer and Don Amirault for providing colonies in

Coldbrook. I would like to thank all the professors, faculty, and Lisa Taul of the Department of

Biology for their help and advice over the past four years, particularly Glenys Gibson for serving on my oral committees and offering so many great recommendations.

Finally, I would like to thank my friends and family for all their support and interest in my work.

Table of Contents

List of Tables ...... vi

List of Figures ...... vii

Abstract...... viii

Chapter I: Prophylactic behaviours in eusocial insects ...... 1

Chapter II: Nosema and necrophoresis: a honey bee parasite and undertaking behaviour ...... 7

Methods...... 9

Results...... 12

Discussion ...... 13

References...... 17

Appendix I: Tables ...... 21

Appendix II: Figures...... 23

List of Tables

Table 1. There was no significant correlation between drop distance in Aylesford or Coldbrook

and log of (Nosema per bee +1), including non-infected corpses...... 21

Table 2. There was no significant correlation between drop distance in Coldbrook and drop

distance in Aylesford including non-infected corpses, whether corpses were originally from

Aylesford or Coldbrook...... 21

List of Figures

Fig. 1. Position of a dead bee trap on a pallet-raised colony...... 23

Fig. 2. Skunk blocker on a pallet-raised colony...... 23

Fig. 3. Marking locations of paint (a) and string (b) on worker corpses...... 24

Fig. 4. There was no significant correlation between drop distance in Aylesford and log of

(Nosema per bee +1)...... 24

Fig. 5. There was no significant correlation between drop distance in Coldbrook and log of

(Nosema per bee +1)...... 25

Fig. 6. There was no significant correlation between drop distance in Coldbrook and log of

(Nosema per bee +1), whether corpses were originally from Aylesford or Coldbrook.

...... 26

Fig. 7. There was no significant correlation between of drop distance in Coldbrook and drop

distance in Aylesford...... 27

Fig. 8. There was no significant correlation between drop distance in Coldbrook and drop

distance in Aylesford, whether corpses were originally from Aylesford or Coldbrook.

...... 28

vii

Abstract

European honey bees ( Apis mellifera ) live in closed colonies at high densities with genetically-related individuals (mostly sisters) in constant high temperatures and humidity, making disease prevention a necessity. Undertakers are a specialist group of honey bee workers that remove dead bees from the hive (necrophoresis), presumably to restrict the spread of pathogens. Nosema ceranae is an emerging fungal parasite of the European honey bee, and is a serious threat to hive health. The first objective of this study was to test if undertakers distinguished among corpses with different intensities of Nosema -infection. The second objective was to test if undertakers treated corpses consistently in consecutive removals from colonies. Dead bee traps (2 m x 1 m) were used to collect corpses that had been uniquely marked with paint or string. I recorded distances at which corpses were dropped from a hive (drop distance), redeployed them in a second hive, and again recorded drop distances. N. ceranae infection intensity was quantified in recovered corpses. There was no significant correlation between drop distance and N. ceranae intensity. There was also no significant relationship between the initial and subsequent drop distance of corpses experimentally re-introduced to hives. These results suggest that necrophoresis is not pathogen-driven. However, observations suggest a new hypothesis that drop distance depends on how corpses are carried and whether they get tangled with undertakers. This could be investigated by studying how undertakers grasp corpses, how undertakers drop corpses, and how undertakers become tangled with corpses.

viii

CHAPTER I: PROPHYLACTIC BEHAVIOURS IN EUSOCIAL INSECTS

Some colony-living insects are divided into castes of sterile and reproductive individuals. Such insects are called eusocial. Reproductive members are queens and usually many male drones, with the remainder of females acting as sterile workers. Two more characteristics of eusocial insects include living in colonies in which there are two or more overlapping generations, and cooperative care for young (Wilson and Hölldobler

2005). In bees, is determined in a system called haplodiploidy, in which eggs laid by a queen that are fertilized develop into females, and those not fertilized develop into males.

Haplodiploidy is believed to be one of the significant factors that drove the evolution of eusociality. Because females get genetic material from both a mother and father, the average relationship between worker sisters is closer to each other than to their own potential offspring (which would likely not be fertilized), facilitating the development of altruistic behaviour in eusocial insects (Linksvayer 2005). Eusociality has numerous benefits which decrease costs of brood-raising, foraging, and defence (Wilson-Rich et al.

2009).

Another aspect of eusociality is the further separation of sterile members into castes based on division of labour. In this system, workers perform only a subset of the entire range of tasks done in the colony, and this subset of activity varies among groups of workers (Beshers and Fewell 2001). Division of labour can be based on morphological or ontogenetical differences; the latter is division of workers into tasks based on their age

(temporal polyethism), and, by the nature of these tasks, also a spatial division. Younger individuals tend to perform tasks in the innermost area of the colony (intranidal), middle- aged on the periphery, and older outside the colony (extranidal; Trumbo et al. 1997). In

European honey bees ( Apis mellifera ), young nurse bees are restricted to intranidal tasks such as brood care and hygienic behaviour, over time transitioning to more extranidal tasks, such as storing food, cell construction with wax, guarding the entrance, and removing dead or dying nestmates, ultimately foraging for nectar, pollen, and water

(Trumbo et al. 1997; Wilson-Rich et al. 2008).

A downside to the eusocial lifestyle is a high risk of the spread of disease. Bee hives are enclosed environments that are well-suited for growth of microbes (high humidity and fairly constant temperatures), and foster near continuous physical contact among individuals (Oi and Pereira 1993; Fefferman et al. 2007). As well, high-density populations usually result in selection for more virulent pathogens (Fries and Cazamine

2001). Moreover, in a system of haplodiploidy, workers are closely-related and could share similar genetic susceptibilities to disease and infection (Wilson-Rich et al. 2009).

These factors together give honey bee colonies a high risk of infection. However, social interactions and behaviours can counteract this disadvantage of eusociality (Fefferman et al. 2007). Other eusocial insects of the orders Isoptera (termites) and Hymenoptera

(including ants and honey bees), though not necessarily haplodiploid, also exhibit prophylactic (disease-preventing) adaptations and a suite of behaviours in response to pathogenic challenges.

Behavioural adaptations could be especially important for honey bees, because they may be at a disadvantage in terms of physiological immunity in contrast to solitary insects. Compared to genomes of two flies, fruit flies ( Drosophila melanogaster ) and mosquitoes ( Anopheles gambiae ), two of the five insects whose genomes are fully sequenced, honey bees have less variety in their genes devoted to innate immune function

(Robinson et al. 2006). The immune pathways common in insect immunity are complete

in honey bees, but are not as diverse as those in the flies; fly immune gene diversity is attributed to gene duplications (Evans et al. 2006). However, this is a limited species sample, and the trend may not be shared with other solitary or eusocial insects. It has been proposed that bees have less diverse innate defences because they tend to be parasitized by highly coevolved pathogens, and thus they do not need broad defences. An alternate hypothesis is that behavioural modifications in honey bees are effective enough in preventing disease that there was not as much selection for diverse immune genes

(Evans et al. 2006). Therefore, social immunity and behavioural adjustments, largely absent in non-eusocial species, are paramount for honey bee success.

Termites, another eusocial insect, also have methods of controlling infections by interactions within a group. Colonies of Cryptotermes secundus termites infested with mites are more likely to produce winged reproductive termites. Winged reproductive termites can disperse from infested colonies, and are less likely to become infected (Korb and Fuchs 2006). Another termite species, Zootermopsis angusticollis , mounts a physiological immune response to a non-lethal pathogen challenge, protecting the individual from the negative effects of disease but retaining some of the infection. This maintained level of pathogen can be transferred through contact as a kind of inoculation or vaccination to nestmates who have not yet encountered the pathogen, giving them an opportunity to build resistance to it. Termites living in a group environment with previously-infected termites are then significantly more resistant to disease than artificially-vaccinated individuals kept in isolation (Traniello et al. 2002).

Ants are another eusocial insect that exhibit adaptive methods to reduce affects of disease in colonies. Similar to the previously mentioned termite species with immunity transference, Lasius neglectus ants have a significantly greater survival rate from a fungal

infection if previously exposed to infected nestmates. Additionally, infected ants spend more time away from the brood chamber where young develop, with healthy ants increasing their brood-care effort (Ugelvig and Cremer 2007). Some ants, like

Acromyrmex octospinosus , depend on fungiculture for food. To protect their crops from other fungi, they inoculate it with beneficial microbes, groom it regularly to remove pathogens, and they remove infected parts from the colony. Leaves used as substrates for fungus growth are licked prior to use, presumably to reduce the presence of soil pathogens (Mangone and Currie 2007). Queens that form new colonies (foundresses) use substrates that are easily cleaned of pathogens to start growing fungus, such as rocks and rootlets (Fernández-Marín et al. 2007). Ants searching for new nest sites avoid abandoned sites that contain dead ants. This avoidance of areas with corpses could also mean avoiding a potential pathogen source (Franks et al. 2005).

Grooming is ubiquitous in eusocial species, both of themselves (autogrooming) and of nestmates (allogrooming). Grooming can remove pathogens from the cuticle and reduce infection probabilities. It consists of rubbing with “hair” on insect legs (tibial combs) and licking body surfaces (Oi and Pereira 1993). Allogrooming is more frequent in honey bees when nestmates have a viral infection (Richard et al. 2008).

Secretions are used by many eusocial insects for pathogen protection within colonies. Myrmica spp. ants use secretions with antibiotic properties from a gland located between their thorax and abdomen to prevent growth of bacteria and fungi in their nests

(Oi and Pereira 1993). Honey bees secrete the enzyme glucose oxidase, which protects honey and pollen from decomposition caused by bacteria. As well, resins collected from plants are used in nests for their antimicrobial effects. Honey bees collect and accumulate

such a resin (propolis) in the inner areas of colonies to defend against microbes (Wilson-

Rich et al. 2009).

Fever, an artificial increase of temperature in a colony by workers, can be a preventative measure when a colony is infected with a heat-sensitive parasite, such as the fungal infection causing chalkbrood ( Ascophaera apis ) in honey bees (Starks et al. 2000).

Workers huddle together and contract their thoracic muscles to generate heat (Wilson-

Rich et al. 2009). In addition, elevated temperature can be used to defend against predators in the colony when workers gather in a defensive swarm around an intruder, increasing the temperature to a lethal level (Starks et al. 2000).

Nest hygiene is defined as the removal of waste and dead nestmates, and the isolation of infected dead (Wilson-Rich et al. 2009). In honey bees, hygienic behaviour is a part of overall nest hygiene and is the specific task of identifying, uncapping, and removing diseased brood from a colony. This behaviour reduces infection of emerging adults and the spread of disease among brood (Arathi et al. 2000). Hygienic lines of bees, those which exhibit more uncapping and removing behaviour, are hypothesized to be more sensitive to odours of diseased brood (Gramacho and Spivak 2003). Olfactory cues are important for correct identification of diseased brood cells for uncapping and subsequent removal by hygienic bees. Furthermore, cuticular hydrocarbons are essential for eusocial insects to distinguish nestmates from non-nestmates. Cuticular hydrocarbons are alterable by immune processes, so they could play a role in recognition of diseased nestmates (Richard et al. 2008).

A seldom-studied aspect of honey bee nest hygiene behaviour, shared with other species such as the leaf-cutter ant ( Acromyrmex versicolor ), is undertaking (Julian and

Cahan 1999). Undertaking, or necrophoresis, is the removal of a dead adult bee from a colony (Illies et al. 2002).

One significant threat to honey bee hive health is a fungal parasite, Nosema ceranae (Higes et al. 2006). I tested if undertaker bees recognize this disease in corpses, by relating corpse drop distance from a hive to parasite loads of corpses. I also tested if undertakers remove corpses the same or a similar distance more than once, by comparing an initial and subsequent drop distance for individual corpses.

CHAPTER II: NOSEMA AND NECROPHORESIS: A HONEY BEE PARASITE

AND UNDERTAKING BEHAVIOUR

The European honey bee, Apis mellifera , is a valuable pollinator for commercial crops, worth over 15 billion dollars in the United States annually (Chen et al. 2006). Part of the honey bee’s success is due to its adoption of closed nests in cavities, allowing them to maintain hive temperatures in winter and survive temperate climates. The adaptation for closed colonies also means that any bee that dies inside falls to the bottom of the nest, and will remain there unless removed. Visscher (1983) calculated that dead bees would build up at an average rate of one liter per month in a managed colony. However, this build-up does not occur, because certain worker bees, termed undertaker bees, remove corpses from within the nest.

Necrophoresis (undertaking) is a specialist task, which is done only briefly by a small subset of the worker population, usually after an initial orientation flight but just prior to the age at which they switch to foraging (Trumbo and Robinson 1997; Visscher

1983). This orientation flight is essential to undertakers to ensure that they are able to return to the colony after removing a corpse. Suggested releasers for undertakers to recognize corpses include chemical and tactile cues, with greatest support for chemical signals (Visscher 1983). Visscher (1983) tested this by either coating corpses with paraffin wax or extracting them in solvents (to remove olfactory and chemical cues), that were removed by undertakers significantly slower than untreated corpses. Undertaking purportedly protects hives against harmful microorganisms that could potentially spread from a carcass left in a colony (Illies et al. 2002). Furthermore, worker bees have been observed licking corpses upon encountering them. They could possibly acquire

pathogens from them, so rapid removal of bodies could prevent the spread of fecal-oral diseases from already-deceased bees (Trumbo et al. 1997).

Infections caused by an emerging pathogen of the European honey bee, Nosema ceranae (hereafter called Nosema ), have been connected with decreased honey production, an increase in overwintering losses, and a decrease in hive health (Higes et al.

2006, 2008). Nosema is a fungal parasite of the midgut (sometimes also present in the

Malpighian tubules, hypopharyngeal glands, salivary glands, and fat bodies) that becomes established after ingestion of spores, with new spores passing out in feces to spread to other hosts “horizontally” (Chen et al. 2009). Originally a parasite of Asian honey bees

(Apis ceranae ), it jumped to European honey bees at least as early as 1995, and seems to have displaced a related fungal parasite ( Nosema apis ) from their European honey bee hosts, perhaps also surpassing it in lethality (Paxton et al. 2007; Chen et al. 2008).

Perhaps the most important consequence of N. ceranae infection is the associated energetic stress; infected foragers consumed significantly more sucrose than uninfected bees over 24 hours, and became starved in a significantly shorter time than healthy bees

(Mayack and Naug 2009). Mayack and Naug (2009) suggested that this increase in hunger can cause precocious foraging, as well as the disappearance of bees on foraging flights due to insufficient energy to return to the hive. However, little is known about the true pathogenicity of this parasite; more work could serve to reveal its affect on honey bee colonies.

Hygienic lines of bees are more sensitive to the odours of abnormal brood, explaining their more frequent uncapping of brood cells and removing of diseased young, and it is possible that undertaker bees may have a lower threshold for identifying diseased corpse odours (Masterman et al. 1999). If undertaker bees can distinguish between dead

bees infected with some disease versus dead uninfected bees, then this behaviour may be especially important in reducing transmission of disease inside colonies. This may select for a greater efficiency in removing corpses that are sources of pathogens in colonies.

I tested if undertakers discriminate between different levels of Nosema infection intensity in corpses, as well as Nosema -infected and non-infected corpses. To test how infected corpses are treated by undertakers, drop distance (distance taken from the entrance of the hive) was recorded. Subsequently the bees were analyzed individually to quantify the intensity of Nosema infection. If infection was important, I predicted that more intensely-infected corpses would be dropped farther from the hive than corpses with lower-intensity infections. If, on the other hand, undertakers were more concerned with reducing the threat of infecting themselves, more intensely-infected corpses would be found closer to the hive. Either way, significant differences may suggest that undertaker bees are capable of recognizing Nosema -infected corpses. I also investigated if undertakers drop corpses a similar distance in successive removals. This test has the advantage of accounting for all diseases simultaneously, i.e. viruses that were likely present but not quantified.

Methods

Bee colonies were at two locations: Coldbrook, Nova Scotia (45° 02’N, 64°

35’W), and Aylesford, Nova Scotia (45° 02’N, 64° 50’W). To obtain corpses and measure their drop distances, dead bee traps were constructed and deployed in front of colonies. Traps were made of a 2 m x 1 m wooden frame (interior measurements) with

10-cm high walls, and 1-mm wire mesh stapled across the bottom of the frame (Fig. 1).

Landscaping fabric was placed beneath traps to prevent grass from growing into the mesh of the traps. Traps were centered in relation to a hive entrance (Fig. 1). Distances from the hive entrance were marked in concentric circles 25 cm apart using coloured string woven into trap meshes. Coldbrook hives were modified beneath lids with wooden frames approximately 2.5-cm high placed on top of hive boxes to provide room between lid surfaces and tops of frames. To prevent skunks from agitating colonies, Coldbrook hives were also outfitted with skunk blockers that consisted of nails driven up through wood around the front and sides of colony boxes (Fig. 2).

Dead bees were collected between 31 July and 5 August 2009, and again between

20 and 26 August 2009. Dead worker bees were collected and their drop distance recorded; all other bees (live workers, drones, pupae) were not analyzed due to small sample sizes. In the first collection period, one trap was positioned in Aylesford and two in Coldbrook. Corpses were collected from the Aylesford trap, marked, and re-introduced into the two colonies in Coldbrook. Due to a colony dying in Coldbrook, there was only one trap in each study area in the second collection period. To test if non-native and native corpses to a colony affected undertaking, worker corpses were collected from the

Coldbrook trap as well as from the Aylesford trap in the second collection period. These corpses were then marked and re-introduced into the remaining Coldbrook hive. All bees were taken from the trap at Aylesford or Coldbrook between 09:30 and 11:00 Atlantic

Standard Time each collection day.

Corpses that were collected and used in re-introductions were marked to identify them if re-found in traps. Usable corpses, which had an intact head, thorax, abdomen, and most legs, were marked with a unique colour code for the day and the distance interval at which they were dropped. Corpses in the first collection period were marked using a spot

of paint on both their thorax and abdomen (Testor enamel paints; Fig. 3a). To test if paint odour affected undertaking behaviour, corpses in the second collection period were marked with either a short length of embroidery floss tied around each thorax and abdomen (Moonbrand embroidery thread; Fig. 3b) or with paint as previously described.

In the first collection period corpses were marked and then kept at room temperature until deployment between 12:30 and 15:50 the same day. In the second collection period, corpses were marked and stored at 4°C until deployed the next day between 10:00 and

13:50 (marked corpses were kept overnight because marking with string required several hours to complete).

Traps were cleared of bees and debris before deploying marked corpses into hives.

Hive boxes were opened and marked corpses were scattered on top of frames and then closed. In the first collection period, marked corpses were divided evenly and deployed in two Coldbrook colonies, whereas in the second collection period only one colony was used. Corpses were collected from traps the next day, and marked bees were separated according to drop distance and stored at -20°C until analyzed for Nosema intensity.

Recovered, marked corpses were assigned a number for individual labeling in the lab, and initial and second drop distances were recorded. In Nosema -infected honey bees, spores are found in abdomens, which are ground to perform counts. Abdomens were removed with a scalpel, placed in individual, labeled 1.5-mL flat-bottomed microcentrifuge tubes, and macerated for 30 seconds in 0.5-mL dH 2O with a sterilized tissue homogenizer. After maceration another 0.5-mL of dH2O was added. Samples were frozen until counts were performed. Prior to counting, samples were thawed, and vortexed for 10 seconds immediately before a flamed 0.1-mL loop was used to retrieve

and add sample to a haemocytometer. The sample was allowed to settle for 3 minutes and then observed under phase contrast and counted following Rogers and Williams (2007).

All analyses were performed using Minitab 15 Statistical software. I tested for correlations between drop distances and log of ( Nosema count + 1) for all marked, re- captured corpses. I tested for correlations between drop distances for individual corpses in Aylesford and Coldbrook. I also tested for significant differences between the two marking methods (paint or string) and hive of origin (Aylesford or Coldbrook) for recovery rates of marked corpses.

Results

There was no significant relationship between drop distance and Nosema intensity in Aylesford or Coldbrook (whether uninfected bees were included or not) (Fig. 4, 5,

Table 1). Nosema -intensities also did not matter if corpses were originally from Aylesford or Coldbrook (whether uninfected bees were included or not) (Fig. 6, Table 1), with the exception of corpses originally from Coldbrook including those without infection, which did have a significant difference (P = 0.049, Table 1). In this case, corpses with higher parasite levels were taken further from the hive.

There was no significant correlation between the first drop distance of a corpse in

Aylesford and its second drop distance in Coldbrook of corpses with Nosema (Fig. 7) and whether they came originally from Aylesford or Coldbrook also had no effect (Fig. 8,

Table 2).

Of 1012 worker corpses collected, 753 were marked and redeployed. Of the 753 corpses redeployed, only 189 were recovered in the final collections at Coldbrook. Not

all corpses recovered could be analyzed; if the markings, indicating the first drop distance, were not identifiable, the corpse was not included. Of the 189 corpses recovered, 142 were fully identifiable, and were thus subsequently analyzed for Nosema presence. Of these, 116 were infected with Nosema (81.7%). In the first collection period, 487 worker corpses were marked, all with paint, and 100 were recovered (20.5%).

In the second collection period 266 worker corpses were marked, 45 with paint, and 221 with string, and 12 and 77 were recovered respectively (26.7% and 34.8%, average is

33.5%). There was no significant difference between recovery rates of corpses marked

2 with paint versus string (χ 1 = 1.1, P = 0.29).

In the second collection period 194 and 72 corpses introduced into the Coldbrook hive were from Aylesford and Coldbrook, respectively. Of the corpses from Aylesford,

76 (39.2%) were recovered. Of the corpses from Coldbrook, 14 were recovered (19.4%).

There was a significant difference in the recovery rates of corpses originally from

2 Aylesford or Coldbrook, with fewer corpses recovered that were from Coldbrook ( χ 1 =

9.1, P = 0.0025). No marked corpses were recovered more than one day after they were deployed. Marked corpses were sometimes found in the grass outside traps. Some marked and unmarked corpses were found on the skunk blocker, and were considered to be in the first 25 cm of the trap.

Discussion

I found no significant relationship between Nosema intensity and drop distances. I also found no significant correlation between first and second drop distances, with one exception. However, this result was based on only 7 bees and so more sampling should

be done. Overall, it appears from my results that drop distance is not pathogen- dependent.

Undertaking behaviour is believed to reduce the spread of pathogens in honey bee colonies. A similar behaviour, hygienic behaviour, is dependent on olfactory cues.

Diseases may cause a change in a bee’s odour, so I predicted that undertaker bees could also distinguish among corpses with different intensities of Nosema infection. There are many possible reasons that my predictions were not supported. It could be that undertakers can tell the difference between infected and uninfected corpses, and more highly-infected and less highly-infected corpses, but simply do not take them a specific distance based on this information. Instead, undertakers may treat corpses based on their infection condition by taking them out at a different rate than uninfected corpses. This could be tested by including time as a variable in analyses. Another reason could be that undertakers cannot detect levels of Nosema infection in corpses. The mass of a corpse may differ from when freshly deceased to later on, so mass should also be tested as a variable in analyses.

Since drop distances of corpses in Coldbrook that originated from Aylesford or

Coldbrook were similar, the colony from which the corpses originated is likely not a confounding factor for this variable. However, significantly fewer corpses were recovered that came from Coldbrook than from Aylesford. This is a counter-intuitive result, because you might expect foreign corpses would be treated more aggressively than native corpses. But, without a significant difference in drop distances between the

Aylesford and Coldbrook corpses, it seems as though undertakers were treating them the same. But since fewer bees from Coldbrook were found in traps, undertakers are doing something differently with them, perhaps taking them farther out in the field than the

traps, or not removing them from the hive in the expected time frame. To fully address this discrepancy in recovery rates, the fate of corpses not found in traps has to be elucidated. More tests could be done to extend the range of corpse recovery outside of traps.

Because drop distances of corpses marked with paint and string did not differ, the paint likely did not affect undertaking behaviour. There was also no significant difference between recovery rates of corpses marked with either paint or string. However, I did observe that paint had a tendency to become tacky in the warm, humid conditions inside the hive, whereas string doesn’t change state. On the other hand, string may be more likely to get caught on bee legs. Paint has an advantage by being fast and easy to mark corpses, whereas string takes much longer.

Only about a quarter of the corpses introduced into Coldbrook hives were recovered, and marked corpses were recovered outside of the dead bee trap. This could indicate that a new bee trap design could be beneficial for future studies measuring drop distance. Since there were some corpses found on the skunk blocker, which may have affected the way undertakers were removing corpses, an alternative method of preventing skunk interference should be considered. A fence around the perimeter of the colony and bee trap may have less effect on drop distance. Further tests could investigate if parasites and diseases not present in my study hives have a quantifiable relationship with drop distance. Undertaking behaviour itself requires more research to understand, i.e. what are the release factors (causes) for this behaviour in honey bees? Do factors like ambient temperature and weather affect undertaking?

Corpse removal is highly variable; undertakers may drop them immediately once outside the hive, take flight with the corpse for a short or long distance, or drop to the

ground with the corpse and drag it around on the ground or trap (pers. obs.). While observing undertaking behaviour, I developed an alternative hypothesis for what influences drop distance. Instead of purposely dropping corpses a certain distance from the hive based on infection state, I hypothesize that drop distance depends on how a corpse is initially grasped for removal. To test this, corpses could be altered to either increase or decrease the chances of tangling, i.e. removing legs of corpses that are introduced to hives. Also factors that control undertaking could be explored, possibly using gas chromatography mass spectrometry to identify chemical cues in healthy live and dead bees, and infected live and dead bees. Another option would be to perform Y- tube test to determine if undertakers more commonly choose diseased versus healthy corpses, and to do proboscis extension reflex testing to see if undertakers can detect and learn odours of corpses, whether healthy or diseased.

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Appendix I: Tables

Table 1. There was no significant correlation between drop distance in Aylesford or

Coldbrook and Nosema intensity (these results included corpses without detectable

Nosema infection) including non-infected corpses. There was no significant correlation between Coldbrook drop distance and Nosema intensity for bees originally from

Aylesford. There was a significant correlation between Coldbrook drop distance and

Nosema intensity for bees originally from Coldbrook, but sample size was small.

Explanatory Variable Response Variable

N R2 P log (Nosema intensity +1) Aylesford drop distance 142 0.01 0.12 log (Nosema intensity +1) Coldbrook drop distance 142 <0.01 0.93 log (Nosema intensity +1) Aylesford to Coldbrook

drop distance 136 <0.01 0.88 log (Nosema intensity +1) Coldbrook to Coldbrook

drop distance 7 0.49 0.05

Table 2. There was no significant correlation between drop distance in Coldbrook and drop distance in Aylesford including non-infected corpses, whether corpses were originally from Aylesford or Coldbrook.

Explanatory Variable Response Variable

N R2 P

Aylesford drop Coldbrook drop 111 <0.01 0.67

distance (1) distance (2)

Coldbrook drop Coldbrook drop distance (1) distance (2) 7 0.02 0.38

Appendix II: Figures

Fig. 1. Position of a dead bee trap on a pallet-raised colony.

Fig. 2. Skunk blocker on a pallet-raised colony.

1 1 2 2

a) b) Fig. 3. Marking locations of paint (a) and string (b) on worker corpses.

n = 116, R^2 <0.01, P = 0.66 200

150

100

50 Aylesford Drop Distance (cm) Drop Distance Aylesford

0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 log of (Nosema/bee +1)

Fig. 4. There was no significant correlation between drop distance in Aylesford and log of ( Nosema per bee +1) (This analysis excludes corpses with no detectable Nosema infection).

n = 116, R^2 <0.01, P = 0.61 200

150

100

50 Coldbrook Drop Distance (cm) Drop Distance Coldbrook

0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 log of (Nosema/bee +1)

Fig. 5. There was no significant correlation between drop distance in Coldbrook and log of ( Nosema per bee +1) (This analysis excludes corpses with no detectable Nosema infection).

Aylesford to Coldbrook: n = 111, R^2 <0.01, P = 0.76 Coldbrook to Coldbrook: n = 5, R^2 <0.01, P = 0.79 200 Aylesford to Coldbrook Coldbrook to Coldbrook

150

100

50 Coldbrook Drop Distance (cm) Drop Distance Coldbrook

0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 log of (Nosema/bee +1)

Fig. 6. There was no significant correlation between drop distance in Coldbrook and log of (Nosema per bee +1), whether corpses were originally from Aylesford or Coldbrook.

Open circles are corpses originally collected from Aylesford; closed circles are corpses originally collected from Coldbrook (This analysis excludes corpses with no detectable

Nosema infection).

n = 142, R^2 <0.01, P = 0.39

200

150

100

50 Coldbrook Drop Distance (cm) Drop Distance Coldbrook

0 50 100 150 200 Aylesford Drop Distance (cm)

Fig. 7. There was no significant correlation between drop distance in Coldbrook and drop distance in Aylesford (This analysis excludes corpses with no detectable Nosema infection).

Aylesford to Coldbrook: n = 138, R^2 = 0.10, P = 0.30 Coldbrook to Coldbrook: n = 7, R^2 <0.01, P >0.99 200 Aylesford to Coldbrook Coldbrook to Coldbrook

150

100

50 Coldbrook Drop Distance (cm) Drop Distance Coldbrook

0 0 50 100 150 200 Aylesford Drop Distance (cm)

Fig. 8. There was no significant correlation between drop distance in Coldbrook and drop distance in Aylesford, whether corpses were originally from Aylesford or Coldbrook.

Open circles are corpses originally collected from Aylesford; closed circles are corpses originally collected from Coldbrook (This analysis excludes corpses with no detectable

Nosema infection).