A Comprehensive Understanding of Corpse Management in Termites

University of Kentucky UKnowledge

Theses and Dissertations--Entomology Entomology

2015

Qian Sun University of Kentucky, [email protected]

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Recommended Citation Sun, Qian, "A Comprehensive Understanding of Corpse Management in Termites" (2015). Theses and Dissertations--Entomology. 23. https://uknowledge.uky.edu/entomology_etds/23

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REVIEW, APPROVAL AND ACCEPTANCE

The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above.

Qian Sun, Student

Dr. Xuguo Zhou, Major Professor

Dr. Charles W. Fox, Director of Graduate Studies

A COMPREHENSIVE UNDERSTANDING OF CORPSE MANAGEMENT IN TERMITES

______

DISSERTATION ______

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture, Food and Environment at the University of Kentucky

Qian Sun

Lexington, Kentucky

Director: Dr. Xuguo Zhou, Associate Professor of Entomology

Lexington, Kentucky

ABSTRACT OF DISSERTATION

A COMPREHENSIVE UNDERSTANDING OF CORPSE MANAGEMENT IN TERMITES

Undertaking behavior is the disposal of dead individuals in social colonies to prevent potential pathogenic attack. This behavioral trait has convergently evolved in social insects (primarily termites, ants, and honeybee), and is considered an essential adaptation to social living. In honey bee and ants, workers recognize dead colony members through the postmortem change of chemical profile, and corpses are usually removed out of nest. However, in termites, little is known about the behavioral pattern, chemical cue or molecular basis. In the eastern subterranean termite, Reticulitermes flavipes, this study investigated undertaking behavior toward corpses of different origins and postmortem times. Corpses of a competitive species, R. virginicus, were buried onsite by R. flavipes workers with a large group of soldiers guarding the burial site; while dead conspecifics of R. flavipes were retrieved and cannibalized. In addition, postmortem time played an important role in corpse management. Early death cue, a blend of 3-octanone and 3-octanol, emitted from workers to attract colony members to consume their bodies; the two volatiles rapidly decreased while the corpse decomposed, and burial behavior was alternatively employed at a late postmortem stage. Gene expression profiles of R. flavipes workers were established during cannibalism of freshly dead nestmates, burial of decayed nestmates, and burial of dead competitors. The results showed that gene expression signatures were associated with the behavioral responses, and burial behavior involved more differentially expressed genes in metabolic pathways, indicating a higher energetic output in burial than cannibalism. Our findings suggest that undertaking responses in R. flavipes are associated with the type of risk posed by the dead: cannibalism allows nutrient recycling in the colony, but burial would be warranted toward increased risks of pathogen infection or territorial intrusion. The use of early death cues that promotes cannibalism is adaptive to subterranean termites that feed on nutritionally poor diet. This study advances our understanding of social behavior in insects, and provides fundamental knowledge that may lead to new pest control approaches against R. flavipes.

KEY WORDS: eusociality, Reticulitermes flavipes, undertaking behavior, cannibalism, chemical communication, sociogenomics

Qian Sun _ Student’s Signature 10 December, 2015 _ Date

A COMPREHENSIVE UNDERSTANDING OF CORPSE MANAGEMENT IN TERMITES

Qian Sun

Dr. Xuguo Zhou _ Director of Dissertation

Dr. Kenneth F. Haynes _ Co-Director of Dissertation

Dr. Charles W. Fox _ Director of Graduate Studies

10 December, 2015 _ Date

Acknowledgments

My deepest gratitude goes to my advisor, Dr. Xuguo Zhou. It was Dr. Zhou’s passion that first ignited my enthusiasm for studying social animals, a field I would like to dedicate my career to. It is difficult to imagine a more supportive advisor than him, who has always allowed me to pursue my research interests and guided me to be a better scientist. Any accomplishments I have had and may have in future should be credited to him. Dr. Zhou took great care to assemble a team of motivated, hardworking and caring students and postdocs, which has made the lab a friendly and fun environment to do science.

I own special thanks to my co-advisor, Dr. Kenneth Haynes, who has provided tremendous guidance in every single step throughout my research. He has introduced me to the field of chemical ecology, and greatly inspired me to study insect behavior as well as present and publish my findings. I’ve learned how to appreciate the nature of science through his humble attitude, which would be a lifelong influence on me. I would also like to thank the other members of my committee, Drs. Michael Sharkey, Christopher Schardl and Ruriko Yoshida, as well as my outside examiner, Dr. Nicholas McLetchie, for their advices and patience that improved the dissertation.

I would like to acknowledge my lab members. Li Tian, who has been along the journey of graduate study with me, has made my work all the better through countless discussions, help with experiments, and assistance with insect collections for almost five years. Moreover, I appreciate Drs. Xiangrui Li, Zhen Li, Huipeng Pan and Hu Li, and

iii

Dongyan Song, Tian Yu, Linghua Xu, Jeffery Noland and Jordan Hampton for their help with techniques or sharing theirs thoughts on my research.

I am grateful for the supports, advices and help from the Department of

Entomology, and specific thanks go to Drs. John Obrycki, Charles Fox, Ricardo Bessin,

Daniel Potter and Janet Lensing. I also want to thank the graduate students in the department, who have created a vigorous, warm and friendly atmosphere for me during my life and work as an international student.

I acknowledge the recognitions by and the financial supports from the Graduate

School, Kentucky NSF-EPSCoR Research Scholars Program, the Kentucky Science and

Engineering Foundation, the Entomological Society of American, and the International

Union for the Study of Social Insects. Science is a community enterprise, and I appreciate lots of help outside of University of Kentucky. I want to thank Drs. Susan Jones (the

Ohio State University) and Brian Forschler (University of Georgia) for teaching me termite biology and providing termite colonies for my study. Also appreciated are insightful comments on my work from Drs. Colin Brent (USDA), Amy Toth (Iowa State

University), and Cassandra Extavour (Harvard University).

I reserve my final expressions of gratitude for my family. Without the love, supports and understandings from my mother (Chaoying Liu), father (Zhaofeng Sun), brother (Yi Sun), and very importantly, my husband (Xinguo Wang), I could not have the perseverance to pursuit and finish my doctorate in science.

Table of Contents

Acknowledgments...... iii

List of Tables ...... viii

List of Figures ...... ix

Chapter 1. Literature review: corpse management in social insects ...... 1

Abstract ...... 1

Introduction ...... 1

Death recognition and elicitation of undertaking behavior...... 4

Behavioral responses toward corpses ...... 8

Differential undertaking responses ...... 13

Task allocation of undertaking behavior...... 14

Perspectives and future research ...... 17

Chapter 2. Differential undertaking response of a subterranean termite to congeneric and conspecific corpses ...... 22

Abstract ...... 22

Introduction ...... 22

Results ...... 27

Undertaking response to corpses with different postmortem time ...... 27

Undertaking response to corpses of different origin ...... 27

Discussion ...... 29

Methods...... 34

Termites ...... 34

Experimental set up...... 35

Preparation of corpses ...... 36

Undertaking behavioral bioassay ...... 36

Data analyses ...... 38

Chapter 3. Early death cues elicit adaptive cannibalism in a subterranean termite ...... 46

Abstract ...... 46

Introduction ...... 47

Results ...... 50

Undertaking response toward corpses with different postmortem times ...... 50

Postmortem changes in chemical profiles...... 51

Functional analysis of death-related chemicals ...... 53

Discussion ...... 56

Adaptive value of corpse cannibalism ...... 56

The use of early and late death cues ...... 58

Postmortem contribution of workers ...... 60

Methods...... 61

Termite collection and maintenance ...... 61

Undertaking bioassay with dead termites ...... 62

Analysis of death-related chemicals ...... 63

Attraction assay with volatiles ...... 65

Burial assay with phenol, indole and fatty acids...... 66

Data analyses ...... 66

Chapter 4. Gene expression profiling of undertaking behavior in the eastern subterranean termite ...... 78

Abstract ...... 78

Introduction ...... 78

Results ...... 81

Differential undertaking response toward three types of corpses ...... 81

Differentially expressed genes in undertaking processes ...... 82

GO function and KEGG pathway analysis ...... 83

Discussion ...... 84

Methods...... 87

Termites ...... 87

Biological observation ...... 88

RNA Sample preparation ...... 88

RNA sequencing and read mapping ...... 89

Differential expression and functional annotation ...... 90

Data analyses ...... 91

Chapter 5: Discussion and future research...... 102

Summary ...... 102

Influence of pathogen infection on corpse management ...... 102

Causes of death and their influences on corpse management ...... 104

Adaptive values of undertaking behavior ...... 105

Comparative studies across social taxa ...... 106

Appendix I: Cuticular hydrocarbons (CHCs) of two Reticulitermes species ...... 108

REFERENCES ...... 109

VITA ...... 128

vii

List of Tables

Table 1.1 Current knowledge of the undertaking process in Hymenoptera and Isoptera . 19

Table 3.1 Quantities of compounds tested in undertaking bioassay ...... 68

Table 4.1 Number of genes assembled...... 92

Table 4.2 List of genes consistently regulated over time...... 93

viii

List of Figures

Figure 1.1 Undertaking process displayed by different social insects ...... 21

Figure 2.1 Schematic drawing of experimental set-up ...... 39

Figure 2.2 Corpse removal time by Reticulitermes flavipes ...... 40

Figure 2.3 Undertaking responses of Reticulitermes flavipes to conspecific species ...... 41

Figure 2.4 Undertaking responses of Reticulitermes flavipes to R. virginicus corpses .... 43

Figure 2.5 Number of termites involved in treatments with corpses of different origin .. 45

Figure 3.1 Schematic drawing of experimental set-ups ...... 69

Figure 3.2 Undertaking behavior by Reticulitermes flavipes toward corpses with different

postmortem times ...... 70

Figure 3.3 Cannibalism of freshly killed corpses by Reticulitermes flavipes in dish assay

...... 71

Figure 3.4 Temporal changes of death-related volatiles collected from Reticulitermes

flavipes ...... 72

Figure 3.5 Accumulation of fatty acids on Reticulitermes flavipes workers after death .. 73

Figure 3.6 Attractive effect of postmortem volatiles ...... 74

Figure 3.7 Burial activity of Reticulitermes flavipes toward death-related chemicals ..... 75

Figure 3.8 A schematic drawing showing the energy output and nutritional balance

associated with death cue mediated undertaking responses ...... 76

Figure 4.1 Experimental set-up for RNA-Seq ...... 94

Figure 4.2 Differential undertaking responses ...... 95

Figure 4.3 Summary of behavioral profiles ...... 97

Figure 4.4 Differentially expressed genes in response to three types of corpses ...... 98

Figure 4.5 Dynamic change of differentially expressed genes during three undertaking

processes ...... 99

Figure 4.6 Comparison of differentially expressed genes in three undertaking processes at

different sampling times ...... 100

Figure 4.7 Comparison of gene functions and molecular pathways ...... 101

Chapter 1. Literature review: corpse management in social insects

The contents of this chapter are reprinted from Sun Q & Zhou X. 2013. Corpse management in social insects. International Journal of Biological Sciences, 9 (3): 313-

Abstract

Undertaking behavior is an essential adaptation to social life that is critical for colony hygiene in enclosed nests. Social insects dispose of dead individuals in various fashions to prevent further contact between corpses and living members in a colony. Focusing on three groups of eusocial insects (bees, ants, and termites) in two phylogenetically distant orders (Hymenoptera and Isoptera), we review mechanisms of death recognition, convergent and divergent behavioral responses toward dead individuals, and undertaking task allocation from the perspective of division of labor. Distinctly different solutions

(e.g., corpse removal, burial and cannibalism) have evolved, independently, in the holometabolous hymenopterans and hemimetabolous isopterans toward the same problem of corpse management. In addition, issues which can lead to a better understanding of the roles that undertaking behavior has played in the evolution of eusociality are discussed.

Introduction

Social animals regularly face death of their group members. Species from diverse taxa recognize corpses and modify their behavior to reduce potential deleterious health effects.

For example, the general awareness and prolonged curiosity exhibited toward dead individuals has been observed in elephants (Douglas-Hamilton et al., 2006), and necrophagy of conspecifics occurs in various species of mammals, reptiles, amphibians, 1

fishes and crustaceans (Rudolf & Antonovics, 2007). Sanitary issues caused by exposure to corpses are universal, but especially in social organisms living in enclosed nests with dense populations, which make them vulnerable to contagious pathogens and parasites

(Cremer et al., 2007). To maintain healthy colonies, eusocial hymenopterans (bees, wasps, ants) and isopterans (termites) have evolved sophisticated mechanisms to counter the threat of epidemic disease at both the individual and colony level, including active immune responses and behavioral adaptations (Cremer et al., 2007; Konrad et al., 2012).

Corpse management, also anthropomorphically known as undertaking behavior, is one of the most intriguing innate behaviors in social insects (Neoh et al., 2012; Visscher, 1983;

Wilson et al., 1958).

Responses to corpses vary in insects with different level of sociality, including solitary (no shared nesting site, no parental care), gregarious (shared nesting site), and eusocial (overlap of generations, reproductive division of labor, and cooperative brood care). In some solitary or gregarious insect species (e.g. cockroaches, springtails), the response to a dead or injured conspecific is usually avoidance (Rollo et al., 1994; Yao et al., 2009). In social spiders (gregarious) (Tietjen, 1980) and a social aphid (arguably eusocial), Pemphigus spyrothecae Passerini (Benton & Foster, 1992), disposing of the dead is simply a part of nest cleaning because it is indistinguishable from dealing with inanimate nest waste. In species of the two extensively-studied eusocial lineages,

Hymenoptera (ants, bees, and wasps) and Isoptera (termites), corpse management is distinctive and differs from other nest cleaning behaviors such as disposing of feces and decaying food remains (Bot et al., 2001; Choe et al., 2009; Visscher, 1983). Facilitated with different behavioral repertoires (e.g., removal, burial, and cannibalism), honey bees,

ants, and termites have evolved complex systems of corpse management dealing with corpses of different ages, origins, and infection status (Myles, 2002; Neoh et al., 2012;

Renucci et al., 2010; Spivak et al., 2003). Although the specific components of corpse management are distinctly unique in various taxa of eusocial insects, the evolution of a complex strategy for dealing with the dead is a shared characteristic of eusociality.

In social insects, undertaking behavior is a sequential array of corpse-induced behavioral responses that target potential health-related hazards to maintain colony fitness. Among many responses to corpses, one of the earliest and well described in social insects is necrophoresis, which refers to the removal of dead individuals from the nest. The term necrophoresis was defined by Wilson et al. (Wilson et al., 1958), which originated from

Greek (necros refers to the dead and phoresis means transport) (Renucci et al., 2010).

Necrophoric behavior is interchangeable with undertaking behavior in some literature.

Here, we use undertaking behavior as a broad term that includes corpse removal from the nest, burial (covering the dead with soil and/or other materials), cannibalism

(intraspecific necrophagy), and avoidance (preventing contagion by intentionally avoiding areas where the dead are located), a behavior sometimes considered as necrophobia.

The phenomenon of undertaking behavior resulted in anthropomorphic descriptions by early naturalists, such as “funerals” and “cemeteries” in honey bees

(Visscher, 1983) and ants (Hölldobler & Wilson, 1990; Hölldobler & Wilson, 2009). In- depth study of behavioral patterns associated with undertaking did not start until 1958, when Wilson et al. first identified the chemical cue eliciting undertaking behavior in two ant species, Pogonomyrmex badius (Latreille) and Solenopsis saevissima (Smith) (Wilson

et al., 1958). To date, studies of undertaking behavior have been focused on three aspects: death recognition cue, behavioral process, and division of labor. Recently, there have been renewed interests in undertaking behavior in termites focusing on death cues and behavioral responses (Chouvenc et al., 2012; Chouvenc & Su, 2012; Neoh et al., 2012;

Ulyshen & Shelton, 2012). Despite studies for over 50 years, the genetic underpinning of undertaking behavior is still not well understood. Some aspects of undertaking behavior are shared by the phylogenetically distant eusocial hymenopterans and isopterans, e.g., recognition/differentiation of the dead from the living. This review provides 1) an overview of undertaking behavior, focusing on death recognition, behavioral responses, and task allocation as they relate to dealing with the dead, 2) a comparison of adaptations in different eusocial groups, specifically, Hymenoptera and Isoptera, and 3) prospects for future studies.

Death recognition and elicitation of undertaking behavior

Once individuals die in the active area of a social colony, colony members need to distinguish the dead from the living before taking any action. Death recognition depends on diverse cues, including chemical, tactile (e.g., shape and texture), and possibly visual input. Recognition of the dead has been widely shown to be achieved through chemical cues, explained by two primary hypotheses, “fatty acid death cue” (Wilson et al., 1958) and “chemical vital sign” (Choe et al., 2009).

Pioneering studies on the nature of undertaking stimuli conducted by Wilson and his colleagues (Wilson et al., 1958) in two ant species, Pogonomyrmex badius and S. saevissima , suggested that fatty acids, particularly oleic acid accumulating in dead bodies, trigger undertaking responses. This conclusion was confirmed later in other ant

species (Haskins & Haskins, 1974). The idea of a “fatty acid death cue” eliciting undertaking responses has been widely accepted for decades (Karaboga & Akay, 2009;

Purnamadjaja & Russell, 2005). However, certain aspects of undertaking behavior could not be explained by this hypothesis alone. Gordon found that oleic acid released foraging as well as undertaking behavior in Pogonomyrmex badius, depending on the social activities of the colony at a given time (Gordon, 1983). The rapid recognition and the subsequent response to corpses (within 1 hour) by nestmates indicate that decision- making time is too brief to allow decomposition and the release of a fatty acid death cue in the red imported fire ant, Solenopsis invicta Buren (Howard & Tschinkel, 1976) and in honey bee, Apis mellifera Linnaeus (Visscher, 1983). Therefore, it was suggested that chemical(s) associated with life might inhibit a pre-existing undertaking releaser (Howard

& Tschinkel, 1976; Visscher, 1983); and the “chemical vital sign hypothesis” was first demonstrated by Choe et al. (Choe et al., 2009) in the Argentine ant Linepithema humile

(Mayr). Reductions in the quantity of two cuticular chemicals, dolichodial and iridomyrmecin on live workers, plays a more important role in inducing undertaking responses. Triglycerides were identified to be pre-existing chemicals inducing both necrophoresis and aggression (Choe et al., 2009). The “chemical vital sign” hypothesis suggests an adaptive response toward freshly dead/killed individuals in insect societies.

Instead of waiting for the release of “fatty acid death cue”, social insects living in dense populated colonies rely on the “chemical vital sign” to recognize dead individuals and elicit appropriate undertaking responses before the decomposition of corpses. This adaptation is not uncommon, and it is consistent with what typically happens in many

species of vertebrates, in which dead individuals are recognized instantly according to the absence of signals associated with life, such as lack of movement or response to stimuli.

In comparison to ants, neither a “fatty acid death cue” nor a “chemical vital sign” has been determined in honey bees. However, Visscher (Visscher, 1983) suggested that a chemical signature was present immediately after the death. In addition, visual, auditory, and thermal cues were excluded in honey bees due to the facts that undertaking behavior took place in darkness, and removal activity was reduced to minimal when corpses were extracted with solvent or coated with paraffin (Visscher, 1983). In Isoptera, the mechanism of undertaking elicitation has been recently studied in a fungus-growing termite, Pseudacanthotermes spiniger (Sjöstedt). The burial behavior in

Pseudacanthotermes spiniger was triggered by a blend of indole, phenol, and fatty acids

(Chouvenc et al., 2012), which, at least in part, supported the “fatty acid death cue” hypothesis. In the eastern subterranean termite, Reticulitermes flavipes (Kollar), however, the “chemical vital sign” hypothesis could not be excluded based on our observation that workers showed an immediate response (< 15min) toward freshly killed nestmates (Sun et al., 2013). In a congeneric species, R. virginicus (Banks), Ulyshen and Shelton (2012) suggested that fatty acids (e.g., oleic acid) and tactile cues synergistically induce burial behavior. This is the only case in which a tactile cue was implicated; however, tactile cues alone have not been found to be effective. Termite workers are sensitive to light

(Park & Raina, 2005; Siderhurst et al., 2006), however, the involvement of visual cues is unlikely to be a major factor in the subterranean termites because undertaking behavior is independent of light-dark regime (Chouvenc & Su, 2012; Sun et al., 2013; Ulyshen &

Shelton, 2012).

The term "necromone" has been used to describe death-recognition chemicals

(Yao et al., 2009). Fatty acids are a common recognition mechanism for death in arthropods. Oleic acid and linoleic acid are the two major unsaturated fatty acid compounds to induce undertaking behavior in ants (Haskins & Haskins, 1974; Howard &

Tschinkel, 1976; Wilson et al., 1958) and avoidance in a wide range of arthropods including terrestrial Isopoda, Collembola, cockroaches, and social caterpillars, and these compounds are considered to be conserved necromones (Rollo et al., 1994; Yao et al.,

2009) . Fatty acids have limited volatility and are derived from corpses or injured cells by enzymatic or bacterial processes, and serve as reliable cues for risks including predation and disease across wide phylogenetic ranges (Yao et al., 2009). Interestingly, American cockroaches, Periplaneta americana (Linnaeus), are repelled (necrophobic behavior) by oleic acid (Rollo et al., 1995; Rollo et al., 1994). In contrast, termites, regarded as

“eusocial cockroaches” (Inward et al., 2007), perform burial behavior in response to the same chemical (Ulyshen & Shelton, 2012). A similar situation occurs in Hymenoptera in that solitary bees avoid foraging sites where dead conspecifics are present (Abbott, 2006;

Dukas, 2001), whereas honey bees remove corpses out of nests (Visscher, 1983). As fatty acid necromones are associated with injury and death caused by predation or contagion, effective recognition of these chemicals might benefit other non-social insects as well.

In summary, recognition of death through chemical cues is shared by honey bees, and many ant and termite species, but the specific chemical signals remain unclear in most species. Oleic acid is the only known common death signal recognized by some insects, whereas it is unknown whether other chemical signatures of death are shared among eusocial hymenopterans and isopterans. Two hypotheses, “fatty acids death cue”

and “chemical vital sign”, are not mutually exclusive. In addition to the “fatty acid death cue” and “chemical vital sign” hypotheses, it might well be expected that: 1) recognition of fatty acid death cues is an evolutionary conserved response from non-eusocial ancestors to avoid the dead, whereas undertaking is a derived behavioral trait in eusocial insects; and 2) death recognition through diminished chemical vital sign might be an evolutionary novelty in some eusocial insects, which enables workers to respond rapidly to prevent pathogen transmission. Besides chemical cues, the role of tactile cues needs to be studied in most social insects to fully understand elicitation of undertaking behavior.

Behavioral responses toward corpses

Eusocial Hymenoptera and Isoptera share the common features of group-living that make them vulnerable to pathogens and parasites, and consequently, they have evolved undertaking behaviors, individually, to mitigate hazards (Cremer et al., 2007). Specific behavioral patterns, however, vary among different social groups. Once death cues are recognized, social insects respond to the dead differently. Corpse removal (necrophoresis in a narrow sense) is prevalent in honey bees (Visscher, 1983) and ants (Haskins &

Haskins, 1974; Julian & Cahan, 1999; Wilson et al., 1958), although burial (covering the dead) (Renucci et al., 2010) and cannibalism (intraspecific necrophagy) (Driessen et al.,

1984) were also documented in ants. In contrast, undertaking responses are more complex in termites than eusocial hymenopterans (Neoh et al., 2012).

Studies of undertaking behavior in bees have traditionally focused on the honey bee, Apis mellifera (Suzuki et al., 1974; Visscher, 1983). Honey bees dispose of nestmate corpses in a straightforward manner, i.e., corpse removal (Figure 1.1C). This behavior pattern is a part of their behavioral repertoire (Trumbo & Robinson, 1997; Visscher,

1983). An “undertaker” bee typically antennates the dead bee briefly, grasps its appendages with mandibles, transports it outside, and drops it from the hive, while other debris in honey bee colonies is removed less rapidly (Visscher, 1983).

The behavioral patterns of ants are extremely diverse, and they are known to keep the interior of their nest meticulously clean. Corpse removal (Figure 1.1B), a common undertaking strategy in various ant species, is distinguished from other nest cleaning behaviors as corpses are transported more rapidly and over greater distances than inanimate objects (Gordon, 1983; Skidmore & Heithaus, 1988; Wilson et al., 1958). Ants transport corpses to certain sites, depending on the species. Carrying dead nestmates outside and discarding them on refuse piles (or kitchen middens) have been observed in the myrmicine ants Pogonomyrmex badius and S. saevissima (Wilson et al., 1958), bull ants Myrmecia vindex Smith (Haskins & Haskins, 1974), red imported fire ant S. invicta

(Howard & Tschinkel, 1976), army ants Eciton (Rettenmeyer, 1963), Argentine ants L. humile (Choe et al., 2009), and common red ant, Myrmica rubra (Linnaeus) (Diez et al.,

2012). Leaf-cutter ants of the genus Atta, however, remove them to special refuse chambers (Moser, 1963; Stahel & Geijskes, 1939). Corpse removal has also been reported in several other ant species such as the desert leaf-cutter ant Acromyrmex versicolor (Pergande) (Julian & Cahan, 1999) and another species Temnothorax lichtensteini (Bondroit) (Renucci et al., 2010). Cannibalism of dead individuals was observed in the myrmicine ants of genera Pheidole and Solenopsis, the weaver ants

Oecophylla (Hölldobler & Wilson, 1990), and the red wood ant Formica rufa (Linnaeus)

(Marikovsky, 1962). In the red imported fire ant S. invicta, cannibalism was occasionally observed at refuse piles in the field (Howard & Tschinkel, 1976). The red wood ant,

Formica polyctena Foerster, eat their defeated enemies after intercolony battle, which has been considered to be adaptive as F. polyctena practices cannibalism behavior more frequently during period of food shortage (Driessen et al., 1984; Mabelis, 1978).

Cannibalism of the dead is not a predominant corpse management strategy in ants, but corpses could elicit foraging behavior and be consumed by neighboring scavenging species (Howard & Tschinkel, 1976; Wilson et al., 1958). Besides cannibalism, ants are reported to perform burial behaviors using soil and nest material in response to corpses(Hölldobler & Wilson, 1990) such as in T. lichtensteini, but it is less common probably because the energy input of burial activity is higher than corpse removal

(Renucci et al., 2010). Another prophylactic strategy noticeable in ants is that moribund individuals leave their nests to die alone, as reported in Temnothorax unifasciatus

(Latreille) (Chapuisat, 2010; Heinze & Walter, 2010).

Undertaking behavior performed by subterranean termites can circumvent soil termiticide-based “barrier” treatment (Chouvenc & Su, 2010; Kramm et al., 1982; Su,

2005; Zoberi, 1995). Most recently, an influx of studies have shown a complex series of undertaking behaviors in termites, including burial, avoidance, and cannibalism

(Chouvenc et al., 2012; Chouvenc & Su, 2012; Neoh et al., 2012; Ulyshen & Shelton,

2012). Cannibalism of the dead in termites was considered to be a mechanism of recycling nitrogenous nutrients(Kramm et al., 1982; Moore, 1969), which is, in part, due to their nutritionally poor cellulosic diet(Rosengaus et al., 2011). Coptotermes formosanus Shiraki show cannibalism of cadavers when starved (Song et al., 2006). In another lower termite species Reticulitermes speratus (Kolbe) and a higher termite

Microcerotermes crassus Snyder, living nestmates consume freshly dead and/or injured

termites (Neoh et al., 2012). A precursor of necrophagy or cannibalism has been observed in subsocial woodroach species, Cryptocercus punctulatus Scudder and Cryptocercus kyebangensis Grandcolas (Nalepa & Bell, 1997; Park & Choe, 2003). In termites, cannibalism also functions as a hygienic strategy because by consuming the corpses it destroys the source of pathogens. The dampwood termite, Zootermopsis angusticollis

(Hagen), eat both dead and diseased individuals, with higher chances of cannibalism toward the ones with higher spore concentrations of the entomopathogenic fungus,

Metarhizium anisopliae (Rosengaus & Traniello, 2001). In R. flavipes, workers ingest harmful fungal masses of Metarhizium anisopliae while grooming nestmates and the conidia are inhibited through the alimentary tract (Chouvenc et al., 2009a). It is also common that termites bury fungi-killed nestmates and old corpses to physically isolate them from the healthy nestmates (Myles, 2002; Neoh et al., 2012; Su, 2005; Zoberi,

1995). When challenged with the fungus Metarhizium anisopliae, Coptotermes formosanus displays undertaking behavior in a density dependent manner in which corpses would be cannibalized preferentially at a low level of mortality, while at higher level of mortality, burial was predominant (Chouvenc & Su, 2012). In a fungus-growing species, Pseudacanthotermes spiniger, dealates buried the dead to prevent potential pathogen outbreak in the initial chamber (Chouvenc et al., 2012), while in R. virginicus, the existence of insect corpses induces building behavior to separate the dead from the rest of the colony, which is also a form of burial (Ulyshen & Shelton, 2012). In comparison to ants and bees, the propensity of termites for tunnel building plays an important role in their burial behavior (Chouvenc & Su, 2010). In addition, the use of fecal material, chewed material or soil coated with saliva for burial provides antifungal

components that act as further protection against fungal growth (Chouvenc et al., 2012;

Chouvenc & Su, 2010). In the case of fungi-killed corpses in Coptotermes acinaciformis

(Froggatt) (Milner et al., 1998), and insecticide-killed (including fipronil and thiamethoxam) individuals in Coptotermes formosanus (Su, 2005), termites have been observed to intentionally avoid dead individuals. Corpse removal seems less likely to be an end response in termites, as they do not have certain chambers or refuse piles that serve as waste storage sites, and they rarely leave their nests. Instead, corpse removal

(Figure 1.1A) is more likely to be a part of the dynamic process associated with other undertaking behaviors – corpses carried by workers would be eventually consumed or buried.

In addition to cannibalism and corpse burial, other behaviors including alarming, grooming, recruitment and aggression interact synergistically during undertaking processes(Chouvenc & Su, 2010; Myles, 2002; Neoh et al., 2012). When encountering a congeneric corpses, R. flavipes soldiers showed aggression and guarding behavior, while both worker and soldier castes exhibited strong recruitment activity (Sun et al., 2013).

Similarly, when fungal infection occurred, R. flavipes workers also aggregated and actively recruited fellow workers to cope with the infected individuals (Myles, 2002).

Alarm behaviors in termite workers, characterized by oscillatory vibration and/or rapid walking to generate substrate-borne vibrations, are performed to alert or attract other colony members in the presence of competitors, predators, and entomopathogens

(Howse, 1965; Myles, 2002; Rosengaus et al., 1999). In Nasutitermes termites, alarm pheromones have been identified in termite soldiers to function in recruitment of soldiers

for colony defense (Eisner et al., 1976; Lindström et al., 1990). However, the sensory mechanisms of recruitment have yet to be determined.

Differential undertaking responses

Honey bees, ants, and termites often show species-specific undertaking responses toward corpses (Figure 1.1 and Table 1.1). Ants and termites also exhibit plastic responses to the nature of the corpses, including their postmortem time (age, indicator of decomposition status), infection status (whether harmful fungi are present), and origin (whether or not the corpse is a nestmate or of the same species). Honey bees removed 1h old corpses more quickly than freshly killed individuals (Visscher, 1983), however, an undertaking response to corpses with longer postmortem time was not investigated. Dead ants in F. rufa were consumed for food, but infectious ants were avoided (Marikovsky, 1962). In T. lichtensteini, workers discriminate old corpses from freshly killed individuals, with new corpses buried while old ones are transported outside (Renucci et al., 2010). Differential behavior patterns have been observed in subterranean termite species. Colonies of R. virginicus were found to isolate fungal infected individuals by burying the dead onsite, while the healthy corpses were cannibalized (Kramm et al., 1982). Coptotermes formosanus has been reported to attack, cannibalize and bury fungus-inoculated workers more frequently than uninoculated workers (Yanagawa et al., 2011). In Coptotermes formosanus and R. speratus, only new corpses and injured nestmates were cannibalized, whereas aged corpses were buried. In R. flavipes, conspecific corpses were taken back to the nest and possibly consumed, while workers opted to bury corpses of a congeneric species R. virginicus onsite with an additional colony defensive purpose (Sun et al.,

2013). Soldiers were also involved with guarding and attacking of congeneric corpses as the burial response was underway (Sun et al., 2013).

Undertaking responses are, in part, dependent upon the feeding habit and nest ecology in a given species, and also the risk associated with corpses (Neoh et al., 2012).

Postmortem time of corpses is associated with the decomposition of the dead, indicating whether they are valuable for recycling in species practicing cannibalism (Neoh et al.,

2012). Fungi infection is common in both ants and termites, which react accordingly to prevent epidemic outbreaks within their respective colonies (Cremer et al., 2007). The presence of non-nestmate corpses could be signals of competition, predation, or disease

(Sun et al., 2013). Therefore, through corpse management, social insects mitigate disease hazard (Haskins & Haskins, 1974; Neoh et al., 2012; Visscher, 1983); and in termites, it brings additional incentives for recycling nutrients and contributing to colony defense

(Sun et al., 2013). Further research is needed to determine the chemical signature of corpses and mechanisms of decision-making during the complex undertaking processes.

Task allocation of undertaking behavior

Reproductive division of labor and subsequent task allocation are characteristics of the eusociality. Based on their morph and age, mechanisms underlying the division of labor in the non-reproductive worker caste can be summarized into physical polymorphism and age polyethism (Traniello & Rosengaus, 1997). In addition, workers in some species show considerable behavioral plasticity in task allocations and partition their work force to meet the specific demands of a colony (Gordon, 1996). This flexibility is critical to the growth and survival of a colony, especially for disease management and colony defense.

Undertaking behavior is performed by the worker castes (Crosland et al., 1998), primarily

due to their capability of corpse recognition. Not surprisingly, sensitivity to oleic acid is caste-specific in a leaf-cutter ant Atta mexicana (Smith), where workers are responsive but soldiers are not (LÓPez-Riquelme et al., 2006). The study of task specialization on undertaking behavior has been focused on whether there is worker polyethism and how it is regulated.

As a part of honey bees’ repertoire, undertaking behavior was first reported to be specialized by a small group of workers that comprise only 1%-2% of the colony population (Visscher, 1983). Domination of the task by a few active individuals is frequently demonstrated in the literature with the longest recorded tenure of removing

114 corpses over a 13 day period by an extreme specialist (Trumbo & Robinson, 1997).

In honey bee colonies, both age polyethism and genetic variation influence division of labor among workers. Undertaker bees are middle-aged workers, which are more likely to act on corpse removal during their entire pre-foraging career than other workers of the same age (Trumbo et al., 1997). Genetic effects suggest lifetime differences in behavior preference in honey bee, and genetic factors constrain colony-level plasticity for undertaking behavior, given the fact that removal of undertakers failed to result in task- switching by other workers (Robinson & Page, 1995). Such a result excludes the possibility of the “genotypic threshold model” proposed by Robinson and Page (1989), which predicts that commonly a task will be performed by worker specialists with the lowest response threshold that is genetically influenced, while more workers with higher thresholds switch to this particular task with increased stimulation, i.e., a feedback loop of task regulation. Task specialization of undertaking behaviors occurs similarly in ant colonies, as reported in the desert leaf-cutter ant Acromyrmex versicolor in which genetic

variation affects worker task performance (Julian & Cahan, 1999; Julian & Fewell,

2004). Both honey bee and desert leaf-cutter ant colonies are maintained by multiple mating events or multiple queens. This enriched genetic diversity increases the probability of having undertaking behavior in their behavioral repertoire at the colony level.

Learning and memory have not been suggested to be important components of corpse removal in honey bee, because undertakers demonstrated no obvious improvement with experience (Trumbo et al., 1997). In contrast, in the common red ant Myrmica rubra, undertaker ants behaved as short-term specialists, and they disposed of dead bodies to specific locations (cemeteries) based on spatial memory (Diez et al., 2012).

Emergent worker polyethism in Hymenoptera is affected by colony size, with increased group size leading to efficient allocation of individuals to different tasks (specialists) to meet colony demand (Gautrais et al., 2002; Holbrook et al., 2011; Jeanson et al., 2007).

Polyethism is likely the mechanism to govern undertaking specialists in some ants that live with heterogeneous surroundings. Complex cues could be present concurrently in a large ant colony, therefore focusing on one task by specialists prevents inappropriate task-switching leading to high fitness cost. For example, a single decomposition compound, oleic acid, can elicit both foraging and undertaking responses (Gordon, 1983).

In hemimetabolous termites, there have been no reports of task specialization of undertaking behavior. Intercaste flexibility is reported in a pleometrotic termite species

Pseudacanthotermes spiniger, in which dealates performed corpse-burial behavior in the initial chamber before the first generation of workers develops (Chouvenc et al., 2012). In addition, termites employ various strategies such as removal, burial, cannibalism and

corpse avoidance (Figure 1.1), which makes it difficult to define “undertakers”. The totipotent worker caste in termites is considered immature, whereas the worker caste in holometabolous ants, bees, and wasps is a developmental end (adult). Therefore, age polyethism in hemimetabolous termites includes both polyethism between different instars and age of the last instar (Evans, 2006). Large workers of Reticulitermes fukienensis Light, undertake most tasks including corpse burial, but behavioral plasticity is present with all sized workers being able to perform the task (Crosland & Traniello,

1997). In higher termites, however, there is evidence of age polyethism (Hinze &

Leuthold, 1999; Miura & Matsumoto, 1995), but whether undertaking behavior is amongst the age-related behaviors has not been determined.

Perspectives and future research

There is renewed interest in undertaking behavior, especially defining behavioral responses (Chouvenc & Su, 2012; Neoh et al., 2012; Sun et al., 2013) and sensory cues

(Chouvenc et al., 2012; Ulyshen & Shelton, 2012). However, task allocation during the undertaking process is poorly understood. Since the worker caste in lower termites can be considered as “generalist” (Crosland et al., 1998; Crosland & Traniello, 1997), activation of reserve labor is a possible mechanism to compensate for the lack of specialists in lower termites. Activation of reserve labor has been documented in honey bees (Johnson, 2002; Seeley, 1982) and ants (Gordon, 1989; Wilson, 1983), and it was also implicated in a higher termite, Nasutitermes exitiosus (Hill) (Evans, 2006). The role of reserve labor in the lower termites, however, has yet to be investigated.

Despite the differences in division of labor and task allocation, both eusocial hymenopterans and isopterans manage corpses effectively. In both holometabolous

hymenopterans and hemimetabolous termites, the genetic underpinnings of undertaking responses remain an unsolved mystery and warrant further investigation. Given that undertaking behavior is one of the characteristics shared among eusocial groups, comparative studies in diverse eusocial lineages using integrative approaches involving behavioral observation, chemical ecology, genomic and functional genomic analyses will shed light on the proximate mechanisms of eusociality.

Table 1.1 Current knowledge of the undertaking process in Hymenoptera and Isoptera

Undertaking Process Hymenoptera Isoptera Phase Behavior Bees Ants Termites Recognition Antennation Apis mellifera Most species Most species Alarm NR* NR Reticulitermes flavipes (Chouvenc & Su, 2010; Myles, 2002) Grooming NR Pogonomyrmex badius (Wilson et al., 1958) Reticulitermes flavipes (Myles, 2002); Coptotermes formosanus (Neoh et al., 2012); Reticulitermes speratus (Neoh et al., 2012); Microcerotermes crassus (Neoh et al., 2012) Corpse Apis mellifera Pogonomyrmex badius (Wilson et al., 1958); Coptotermes formosanus (Su, 2005); carrying (Visscher, 1983) Solenopsis saevissima (Wilson et al., 1958); Reticulitermes flavipes (Sun et al., 2013) Myrmecia vindex (Haskins & Haskins, 1974); Inspection Solenopsis invicta (Howard & Tschinkel, and decision 1976); making Eciton (Rettenmeyer, 1963); Linepithema humile (Choe et al., 2009); 19 Atta texana (Moser, 1963; Stahel & Geijskes, 1939); Acromyrmex versicolor (Julian & Cahan, 1999); Temnothorax lichtensteini (Renucci et al., 2010) Recruitment NR Solenopsis invicta (Howard & Tschinkel, 1976) Reticulitermes flavipes (Myles, 2002) Aggression NR Temnothorax lichtensteini (Renucci et al., Reticulitermes flavipes (Myles, 2002); 2010) Cannibalism NR Formica rufa (Marikovsky, 1962); Reticulitermes sp. (Kramm et al., 1982); Solenopsis invicta (Howard & Tschinkel, Zootermopsis angusticollis (Rosengaus & 1976); Traniello, 2001); Pheidole; Oecophylla (Hölldobler & Wilson, Coptotermes formosanus (Neoh et al., 2012); End 1990); Reticulitermes speratus (Neoh et al., 2012); response Formica polyctena (Driessen et al., 1984; Microcerotermes crassus (Neoh et al., 2012) Mabelis, 1978) Avoidance NR NR Coptotermes acinaciformis (Milner et al., 1998); Coptotermes gestroi (Lima & Costa-Leonardo, 2012)

Table 1.1 (Continued) Current knowledge of the undertaking process in Hymenoptera and Isoptera

Undertaking Process Hymenoptera Isoptera Undertaking Process Phase Behavior Bees Phase Behavior Burial NR Temnothorax lichtensteini (Renucci et al., Reticulitermes flavipes (Myles, 2002; Sun et al., 2010) 2013; Zoberi, 1995); Reticulitermes virginicus (Ulyshen & Shelton, 2012); Pseudacanthotermes spiniger (Chouvenc et al., 2012); Coptotermes formosanus (Neoh et al., 2012; Su, 2005); Reticulitermes speratus (Neoh et al., 2012); Microcerotermes crassus (Neoh et al., 2012); Globitermes sulphureus (Neoh et al., 2012) End Corpse Apis mellifera Pogonomyrmex badius (Wilson et al., 1958); NR response removal (Visscher, 1983) Solenopsis saevissima (Wilson et al., 1958);

20 Myrmecia vindex (Haskins & Haskins, 1974);

Solenopsis invicta (Howard & Tschinkel, 1976); Eciton (Rettenmeyer, 1963); Linepithema humile (Choe et al., 2009); Atta texana (Moser, 1963; Stahel & Geijskes, 1939); Acromyrmex versicolor (Julian & Cahan, 1999); Temnothorax lichtensteini (Renucci et al., 2010) * NR: Not Reported

Recognition Inspection and Decision Making End Response

A Nestmates

Alien species Termites

B Antennation Nestmates Alarm

Grooming

Corpse carrying Alien species Recruitment Ants Aggression

Cannibalism C Nestmates Avoidance

Burial

Corpse removal Alien species ? ? Not reported Bees

Figure 1.1 Undertaking process displayed by different social insects.

A represents a termite worker dragging a dead nestmate; B shows an undertaker ant carrying a dead nestmate out of the nest; and C illustrates an undertaker bee removing a dead drone. Reticulitermes flavipes, Linepithema humile, and Apis mellifera are the representative species for termites, ants, and bees, respectively. Undertaking process involves three phases including recognition, inspection and decision making, and end behavioral responses. Illustrated by Zhou, X.

Chapter 2. Differential undertaking response of a subterranean termite to congeneric and conspecific corpses

The contents of this chapter are reprinted from Sun Q, Haynes KF, & Zhou X. 2013.

Abstract

Undertaking behavior is an essential activity in social insects. Corpses are often recognized by a postmortem change in a chemical signature. Reticulitermes flavipes responded to corpses within minutes of death. This undertaking behavior did not change with longer postmortem time (24 h); however, R. flavipes exhibited distinctively different behaviors toward dead termites from various origins. Corpses of the congeneric species,

Reticulitermes virginicus, were buried onsite by workers with a large group of soldiers guarding the burial site due to the risk of interspecific competition; while dead conspecifics, regardless of colony origin, were pulled back into the holding chamber for nutrient recycling and hygienic purposes. The burial task associated with congeneric corpses was coupled with colony defense and involved ten times more termites than retrieval of conspecific corpses. Our findings suggest elicitation of undertaking behavior depends on the origin of corpses which is associated with different types of risk.

Introduction

How to deal with the dead is a problem faced by all social animals. One of the major concerns associated with the deceased individuals is the risk of pathogen transmission, and this may be particularly true for eusocial animals due to their enclosed living quarters 22

(Babayan & Schneider, 2012; Cremer et al., 2007). In eusocial insects, corpse management is an integral part of the behavioral repertoire for controlling disease.

Termites, social bees, wasps, and ants, employ an array of behavioral adaptations (e.g., isolation of sick from healthy individuals (Kramm et al., 1982; Myles, 2002), corpse management (Howard & Tschinkel, 1976; Milner et al., 1998; Su, 2005; Visscher, 1983;

Wilson et al., 1958), and "social vaccination" of nestmates by pathogen-exposed individuals (Konrad et al., 2012; Traniello et al., 2002) to manage diseases. To prevent further direct contact with corpses, social insects practice undertaking behavior to segregate dead individuals from colony members. These behaviors include necrophobic

(avoidance), necrophoric (removal), cannibalism, and burial behavior (Howard &

Tschinkel, 1976; Rosengaus & Traniello, 2001; Su, 2005; Wilson et al., 1958).

Overlapping generations and cooperative brood care, two of the defining characteristics of eusociality, make social organisms more vulnerable to pathogens through extensive contact (Cremer et al., 2007). As a result, hygienic behaviors are consistently correlated with the evolution of eusociality. Most ant species dispose of dead colony members by discarding them away from the nests, placing them into refuse piles, or carrying them to special refuse chambers (Choe et al., 2009; Hölldobler & Wilson, 1990; Howard &

Tschinkel, 1976). Honey bees remove nestmate corpses and drop them from the hive

(Trumbo et al., 1997; Visscher, 1983). In contrast, termites exhibit complex undertaking behaviors to deal with the dead, including avoidance (Su, 2005), cannibalism (Kramm et al., 1982; Rosengaus & Traniello, 2001), removal, and burial behavior (Chouvenc et al.,

2012; Myles, 2002; Su, 2005; Ulyshen & Shelton, 2012; Zoberi, 1995).

Distinguishing the dead from the living depends on accumulation or loss of chemical cues. Postmortem accumulation of unsaturated fatty acids (e.g., oleic or linoleic acid) is common among diverse taxa. Undertaking responses in two ant species

(Pogonomyrmex badius and Solenopsis saevissima) were elicited by fatty acids, particularly oleic acid, produced after death (Wilson et al., 1958). This “fatty acid death cue” has been found in other ant species, honey bees, and termites (Chouvenc et al.,

2012; Haskins & Haskins, 1974; Howard & Tschinkel, 1976; Ulyshen & Shelton, 2012;

Visscher, 1983; Wilson et al., 1958; Yao et al., 2009). Recently, the converse of a death cue was found in Argentine ants, Linepithema humile (formerly Iridomyrmex humilis), where the dissipation of vital chemical signs (dolichodial and iridomyrmecin) after death elicited the undertaking response (Choe et al., 2009).

Undertaking behavior in termites has been studied as a part of the efforts to refine pest management tactics (Chouvenc & Su, 2010; Kramm et al., 1982; Su, 2005). Su and his colleagues investigated the foraging behavior of Coptotermes formosanus, a subterranean termite and a devastating structural pest. Coptotermes formosanus corpses resulted from physical contacts with synthetic soil insecticides, including fipronil and thiamethoxam, were walled off by healthy workers to prevent future contacts (Su, 2005).

Burial behavior was also observed in a Reticulitermes species, when challenged with

Metarhizium anisopliae, a generalist fungal pathogen and a well-studied biological control agent (Kramm et al., 1982). Recently, there has been renewed interests in undertaking behavior (Chouvenc et al., 2012; Chouvenc & Su, 2012; Neoh et al., 2012;

Ulyshen & Shelton, 2012). In a fungus-growing Macrotermitinae species,

Pseudacanthotermes spiniger, wingless primary reproductives buried other dealate

corpses to prevent potential pathogen outbreak (Chouvenc et al., 2012). Ulyshen and

Shelton reported that the presence of dead insects elicited building behavior in R. virginicus, regardless of corpse types including nestmates, predators, and others(Ulyshen

& Shelton, 2012). Neoh et al. 2012 suggested that undertaking responses vary among species, associated with their feeding behavior and nesting ecology, and undertaking behaviors are complex with different responses depending on the nature of corpses. In

Coptotermes formosanus, when fungus (Metarhizium anisopliae) induced mortality was low, cannibalism was the primary undertaking response; however at higher levels, burial was predominant (Chouvenc & Su, 2012).

Undertaking responses may also vary within species depending on the social context and experimental setup. In Florida harvester ant Pogonomyrmex badius, oleic acid served to release undertaking behavior when the major activity in a colony was midden work or nest maintenance. In contrast, undertaking response was not elicited when the individuals were mainly involved in foraging or convening (Gordon, 1983). In

Reticultermes, termites isolated fungus-infected cadavers in a dish assay (Kramm et al.,

1982), whereas in a planar arena, termites opted to cannibalize corpses exposed to the same pathogen (Chouvenc et al., 2008).

Competition between two colonies can lead to injury and mortality of both colonies through aggression. As a result the encounter rate with dead individuals from the competing species may be high (Thorne & Haverty, 1991). Interspecific corpses induced sand/soil deposition, and the resultant dead individuals were eventually incorporated into the tunnel building materials to block future contacts (e.g., Coptotermes formosanus and

Coptotermes gestroi (Li et al., 2010), Cornitermes cumulans and Procornitermes araujoi

(Jost et al., 2012). The eastern subterranean termite, Reticulitermes flavipes, is one of the most common termite species in the continental United States (Su et al., 2001). A congeneric species, R. viginicus, is morphologically and ecologically similar to R. flavipes. In the field we observed that the two species sometimes nested adjacently and occasionally one nesting site was replaced by the other species, therefore territorial competition between the two species would lead to aggression and result in dead individuals of both species. Previous studies suggested that interspecific and intraspecific interactions differ in R. flavipes. Reticultermes flavipes has been reported to lack intraspecific aggression and as a result colony fusion occurs (Bulmer & Traniello, 2002), but to show agonistic behavior toward R. virginicus in laboratory (Polizzi & Forschler,

1998). Given the fact that R. flavipes and R. virginicus have distinctly different chemical signatures (Haverty et al., 1996), and potentially represent different risks such as interspecific competition, we hypothesized that R. flavipes would respond differently toward congeneric and conspecific corpses. Similarly, each R. flavipes colony carries its unique chemical ID, which may be diet mediated (Florane et al., 2004) and/or due to genetic variations (Jenkins et al., 2000). We expected differential undertaking responses toward intraspecific corpses (nestmate and non-nestmate). To test these hypotheses, we presented R. flavipes with dead individuals from the same and different R. flavipes colonies, and corpses from a congeneric species, R. virginicus.

“Increased death cue” (Chouvenc et al., 2012; Ulyshen & Shelton, 2012; Wilson et al., 1958) and “diminished vital sign” (Choe et al., 2009) are the two reported mechanisms of corpse recognition. In both cases the changes should be dependent on time after death. To gain a better understanding of the cues eliciting undertaking process

in termites, we determined the temporal profile of R. flavipes undertaking behavior when exposed to nestmates from 0 to 24 h after death.

Results

Undertaking response to corpses with different postmortem time

We examined the responses of R. flavipes workers to dead nestmates with various postmortem time by placing corpses at the holding chamber opening in a test arena

(Figure 2.1). When a group of 10 dead nestmates were introduced, workers carried the corpses into the holding chamber irrespective of postmortem time. Total removal time did not differ significantly among treatments (F6,14 = 1.166 , P > 0.05 for colony KY-4;

F6,14 = 1.416 , P > 0.05 for colony KY-15; 3 replications for each colony; Figure 2.2a).

The undertaking response to dead nestmates started with the inspection of corpses by worker termites while one or more soldiers guarded the entrance. Typically, the first worker coming out of the holding chamber contacted a corpse with its antennae.

Immediately after antennation, the worker grasped the corpse using its mandibles (Figure

2.3j-k). Workers usually pulled the corpses straight to the entrance (Figure 2.3l), but the removal path could be circuitous. Soldiers sometimes contacted corpses with their antennae, but there was no agonistic response toward conspecific corpses (Figure 2.3i).

Undertaking response to corpses of different origin

The same undertaking behavior pattern was observed toward intra- (nestmate) and inter- colony corpses 1 h postmortem. Conspecific corpses were removed from the test arena and carried back to the holding chamber by workers, usually in less than 15 minutes.

Removal time between treatments with intra- and inter-colony corpses did not differ

significantly (unpaired t-test: t8 = 1.170, P > 0.05 for colony KY-173; t8 = 0.200, P >

0.05 for colony KY-174; 5 replications per colony Figure 2.2b).

In contrast to conspecific corpses, corpses from the congeneric species, R. virginicus, triggered alarm behaviors by both soldiers and workers, aggression behavior by soldiers, and burial behavior by workers. A soldier from the holding chamber inspected the corpses with antennae and then immediately initiated an attack with its mandibles (Figure 2.4e-f). More soldiers were recruited from the holding chamber

(Figure 2.4g). Workers that contacted R. virginicus corpses quickly retreated into the nest, and did not come out until more soldiers gathered around the corpses. Within ten minutes after the introduction of congeneric corpses, workers began to carry out soil from the holding chamber and place it onto the corpses, and some workers were observed to coat the soil with saliva at the burial site. The burial behavior continued while a group of soldiers surrounded the pile of corpses (Figure 2.4h). Within 12 h, the group of 10 R. virginicus corpses was buried by R. flavipes workers with soil particles, forming a compact and moist mound (Figure 2.4i). Alarm behaviors characterized by rapid walking of agitated termites and/or vigorous vibrations of their bodies (as previously described by

Crosland et al. 1997 were consistently observed during the 1 h observation period. In contrast, display of alarm behavior was significantly less frequent in treatments with conspecific corpses (means of 36.75 ± 11.49 and 1.75 ± 1.44 for congeneric and intercolony corpses during first 15 min observation period; unpaired t-test: t6 = 3.022, P <

0.05; 2 replications per colony in KY-173 and -174). Alarm behavior facilitated a rapid recruitment of both soldiers and workers during the first hour after the introduction of congeneric corpses (Figure 2.4a-d), while termites involved in treatments of conspecific

corpses were much fewer (Figure 2.3a-h). Because of high variation in numbers between replicates, results are presented separately (Figure 2.3a-h, 2.4a-d).

There was no significant effect of block or colony on the mean number of workers per 1 h observation period (F1,7 = 0.15, P > 0.05 and F1,7 = 0.30, P > 0.05). The source of the dead termites had a significant effect on number of workers observed (F2,7 = 11.80, P

< 0.01) (Figure 2.5). Recruitment of workers was greater with the R. virginicus corpses than with intra- or inter-colony R. flavipes corpses (means of 19.9, 1.4, 1.6 workers per observation, respectively; Tukey HSD Comparison Test). There was no significant effect of block or colony on the mean number of soldiers per observation period (F1,7 = 0.24, P

> 0.05 and F1,7 = 0.03, P > 0.05). The source of the dead termites had a significant impact on the recruitment of soldiers (F2,7 = 37.88, P < 0.01). Many more soldiers were recruited to dead R. virginicus than dead intra or inter-colony R. flavipes (means of 10.7, 0.15, 0.89 soldiers per observation, respectively; Tukey HSD Comparison Test). There was no significant effect of block or colony on the proportion of soldiers observed (F1,7 = 1.48, P

> 0.05 and F1,7 = 1.98, P > 0.05); however, the source of the dead termites had a significant effect (F2,7 = 5.94, P < 0.05). It is worth noting that in treatments with congeneric and inter-colony corpses, the soldier caste accounts for ~40% of the total number of termites in the testing arena, which is substantially more than the natural level in a field colony (4-5%) (Zhou et al., 2006), indicating a greater recruitment of soldiers in the undertaking process.

Discussion

Chemical cues have been attributed to the undertaking behavior in both Hymenoptera and

Isoptera. The involvement of tactile cues (e.g. shape and texture) in the undertaking

process in R. flavipes is currently under investigation, however, the possible mechanism for death recognition has already been confirmed in a congeneric species, R. virginicus

(Ulyshen & Shelton, 2012), suggesting the recognition of death in termites involves multi-channel signaling, including chemical and tactile cues. Recognition of a “fatty acid death cue” has been studied extensively over the past 50 years and showed phylogenetic conservation or convergence across diverse taxa. Oleic acid and linoleic acid are the two major compounds of fatty acid -based necromone to induce undertaking behavior in ants

(Haskins & Haskins, 1974; Howard & Tschinkel, 1976; Wilson et al., 1958) and to lead to avoidance in a wide range of arthropods including terrestrial Isopoda, Collembola, cockroaches, and social caterpillars (Yao et al., 2009). In this study, the brief latency between corpse introduction and release of behavior in both conspecific treatments and congeneric treatment suggests a chemical change has already occurred. However, this study alone does not discriminate between “increased death cue” (Wilson et al. 1958) and

“diminished vital sign” (Choe et al., 2009) hypotheses.

The disposal of dead colony members is a characteristic behavior in ants and honey bees (Visscher, 1983; Wilson et al., 1958), and it is true in R. flavipes. Workers showed rapid recognition and response to nestmate corpses within 1 h after death. This is consistent with honey bees (Visscher, 1983) and ants (Choe et al., 2009; Howard &

Tschinkel, 1976), confirming undertaking response toward dead nestmates evolved convergently in eusocial insects. However, unlike ants (Choe et al., 2009; Julian &

Cahan, 1999; Wilson et al., 1958), which take corpses away from the nest and deposit them into the refuse pile; and in honey bees (Morse, 1972; Visscher, 1983), which remove corpses out of the hive; R. flavipes carried dead nestmates back into the holding

chamber. The fate of those corpses was not examined in the current study. However, based on previous reports (Kramm et al., 1982; Neoh et al., 2012) and our on-going investigation, the conspecific corpses were likely cannibalized. Cannibalism/necrophagy has been used by many termites to recycle nutrients (Kramm et al., 1982; Moore, 1969), which is, in part, due to their nutritionally poor cellulosic diet (Rosengaus et al., 2011).

Cannibalism also functions as a hygienic tactic, and the cannibalism of fungus-killed nestmates was found in R. flavipes (Chouvenc et al., 2008). Consuming corpses destroys the source of potential epidemic pathogens, and by ingesting the dead body it can also inhibit the growth of existing entomopathogens due to antimicrobial activity in the gut

(Chouvenc et al., 2009a).

As hypothesized, results from this study indicate that R. flavipes have differential responses to congeneric and conspecific corpses; however, conspecific corpses of nestmates and non-nestmates were disposed in the same manner under laboratory conditions. Introduction of dead R. virginicus induced an intense response from members of the R. flavipes colony, including rapid recruitment of soldiers and workers via alarm vibration exhibited by both castes, aggressive behaviors by soldier caste, and burial behavior by workers. Nest soil consisting of chewed mulch and feces was used by R. flavipes workers to bury congeneric corpses. The propensity of termites for tunnel building plays an important role in their burial behavior (compared with ants and bees).

The use of fecal material and nest soil for burial provide antifungal components

(Chouvenc et al., 2012; Chouvenc & Su, 2010; Hamilton et al., 2011). Burial behavior has been observed in other species. For example, in Coptotermes formosanus, a large number of corpses were sealed off by their nestmates (Chouvenc & Su, 2012; Su, 2005),

and in Pseudacanthotermes spiniger and R. virginicus, burial or building behavior induced by dead nestmates were reported (Chouvenc et al., 2012; Ulyshen & Shelton,

2012). Unlike cannibalism, burial behavior isolates corpses from other nest members to prevent the spread of pathogens. An increased risk of infection is possible when encountering dead foreign species that may carry a pathogen not tolerated by R. flavipes, given the fact that disease resistance varies among species. For example, susceptibility to the soil fungal pathogen, Metarhizium anisopliae, varies among seven termite species from six families. Specifically, Mastotermes darwiniensis, Hodotermopsis sjoestedti and

Nasutitermes voeltzkowi are highly tolerant, Rhinotermitidae species, R. flavipes and

Prorhinotermes canalifrons, are moderately susceptible, whereas Kalotermes flavicollis and Hodotermes mossambicus are highly vulnerable (Chouvenc et al., 2009b).

In addition to a hygienic function, burial behavior is also a colony defense mechanism. A previous report demonstrated that Coptotermes formosanus and

Coptotermes gestroi sealed corpses, respectively, after interspecific aggression and avoided reopening the tunnel where two species encountered each other (Jost et al., 2012;

Li et al., 2010). In this study, R. flavipes isolated alien corpses with thick layers of mud, and building behavior at the nest opening utilizing mud was frequently observed.

Consequently, burying corpses of congeneric species by R. flavipes may facilitate colony defense against competitors. Aggressive responses by the soldier caste is typical in colony defense (Traniello & Beshers, 1985), however, when treated with dead R. virginicus, aggressive behavior exhibited by soldiers is also an integral part of corpse management. The absence of worker aggression to R. virginicus in this study is likely the result of the presence of large number of soldiers. This is consistent with the damp-wood

termite, Hodotermopsis sjostedti, in which the aggression level of workers was modulated by the presence of other castes. Specifically, the presence of reproductives aggravated the aggression level of workers, whereas the presence of soldier caste neutralized worker aggression (Ishikawa & Miura, 2012). Therefore, aggression and guarding behavior exhibited by soldiers may ensure that workers focus on burial tasks during the undertaking process. Inter-colony aggression was not observed, which was similar to other studies with R. flavipes (Bulmer & Traniello, 2002; Polizzi & Forschler,

1998).

Reticulitermes workers distinguish conspecific and congeneric individuals primarily by cuticular hydrocarbons (Bagneres et al., 1991). When presented with congeneric corpses, R. flavipes soldier exhibited strong aggression behavior, indicating that they are capable of nestmate recognition, but might not be able to recognize the dead.

Similarly, the soldier caste of fungus-growing ant, Atta mexicana, is not sensitive to oleic acid, the death cue, but responds to an alarm pheromone (López -Riquelme et al., 2006).

Interestingly, Choe et al. 2009 found that triglycerides, which are common chemical components in insects, elicited both aggressive and undertaking behavior in Argentine ants. If this is true in Reticulitermes, it is possible that additional chemicals in R. flavipes inhibit aggressive responses by conspecific individuals, or, elicit aggression to interspecifics. Further quantitative analysis of chemical cues is warranted to examine these hypotheses. In addition, we predict that workers and soldiers have different sensory or central processing of these cues.

Our results showed that R. flavipes distinguish between their own corpses and those of a congener, eliciting corpse retrieval behavior or burial behavior, respectively.

Depending on the origin of corpses, undertaking behavior functions as a hygienic tactic as shown in response to nestmate corpses, and as both hygienic and colony defensive strategy as shown in response to dead congenerics. The ability to switch between behavioral repertoires in R. flavipes is similar to the undertaking behaviors observed in an ant species Temnothorax lichtensteini, in which newly deceased congeneric individuals were buried while aged corpses of nestmates were removed outside the nest. The differential response in termites to corpses of different origins can be advantageous

(Renucci et al., 2010). Burial is extremely costly in terms of time, energy, and resources committed to this form of disposal, but may be warranted when the risk of exogenous pathogens is high, or if the workers perceive a lingering risk of interspecific aggression.

In addition to recycling nutrients, corpse retrieval requires less time, energy and resources than burial. However, inoculation of the colony with exogenous pathogens or the presence of intruders may have direct costs. A differential response to corpses of different origins may serve to mitigate the risks and costs associated with corpses and their disposal.

Methods

Termites

Reticulitermes flavipes colonies (KY-4, -15, -173, and -174) and a congeneric R. virginicus colony (KY-18) were collected from The Arboretum, a 100-acre botanical garden located on the University of Kentucky campus (Lexington, KY). Colonies were obtained using termite trapping stations filled with spirally coiled cardboard during spring and summer. Colony KY-4, KY-15 and KY-18 were collected in 2010, while

Colony KY-173 and KY-174 were collected in 2011. Reticulitermes colonies were

maintained in sealed plastic boxes in complete darkness (L:D = 0:24), at 27 ± 1ºC, 80 ±

1% RH, and were provisioned with pine wood and hardwood mulch. Intercolony individuals were obtained from colony OH-A8, which was a gift from Dr. Susan Jones

(The Ohio State University). It was originally collected from Columbus, OH, and has been maintained in the laboratory for 9 years. The KY- and OH- colonies were collected from two different states and at least 7 years apart and therefore should have a low degree of genetic relatedness. The identity of colonies as R. flavipes and R. virginicus, respectively, were verified by a combination of soldier morphology and 16s mitochondrial ribosomal gene sequencing (Wang et al. 2009). Termites were considered workers if they did not possess any sign of wing buds or distended abdomens, and had pronotal widths wider than mesonotal widths (Lainé & Wright 2003).

Experimental set up

Termites used in the following experiments had been acclimated under the laboratory conditions for at least 3 months from the time of field collection. Each laboratory colony contains at least 5000 termites. Our experimental setup includes a holding chamber, a sealed plastic box (45.7 x 30.6 x 15.2 cm, Pioneer Plastics, Inc., Dixon, KY) containing the original stock colony, and a testing arena, a covered 9 cm-diameter Petri dish (1.5 cm height, VWR Inc, Radnor, PA) where observations of undertaking were made. The bottom surface of the dish was scratched with No. 7 insect pins to facilitate movement.

Behavioral experiments began at least two weeks after the Petri dish was connected, when the bottom of the test arena was covered with nest material by workers. An acrylic tube (inner diameter: 0.7 cm; length: 3cm) connects the testing arena to the holding chamber (Figure 2.1). The entire experimental setup was held in environmental chambers

under the above mentioned rearing conditions. A video camcorder (Sony DCR-SR60,

Tokyo, Japan) was placed directly above the test arena.

Preparation of corpses

Undertaking responses toward corpses with different postmortem times were tested in two colonies (KY-4 and KY-15). Seven groups of workers from the same KY R. flavipes colony were frozen to death at -20°C for 15 min and kept in environmental chambers at

27 ± 1°C, 80 ± 1% RH, for 0, 1, 2, 4, 8, 16, and 24 h, respectively, in covered Petri dishes before subjecting to the behavioral bioassay. The 0 h old (freshly killed) corpses were left at room temperature for 5 min before they were placed in the arena. To discern the behavioral responses toward corpses of different origin, two colonies were tested (KY-

173 and KY-174) with three types of corpses introduced: (a) intracolony corpses: dead workers collected from the same KY R. flavipes colony; (b) intercolony corpses: dead workers collected from a OH R. flavipes colony (OH-A8); and (c) congeneric corpses: dead workers collected from a KY R. virginicus colony (KY-18). Based on the result from the postmortem time experiment, we opted to use 1 h postmortem corpses as the age of corpses for the differential response study. Corpses were prepared by freezing as described above.

Undertaking behavioral bioassay

Behavioral assays were conducted at room temperature, and the experimental setup including a holding chamber and a test arena was acclimated for 30 min before experiments. In each bioassay, the lid of testing arena was gently removed, 10 corpses were then placed into the arena using a pair of feather-weight forceps (BioQuip Products

Inc., Gardena, CA). Corpses were placed in the vicinity of the nest entrance, and the

arena was gently covered with a clear Petri dish lid to avoid disturbance by the air movement. Video recording was initiated immediately after the introduction of corpses to the testing arena.

For different postmortem times, behavioral bioassays were replicated 3 times in both KY-4 and KY-15 colonies. For corpses with different origins, bioassays were replicated 5 times in KY-173 and KY-174 colonies. Based on the length of undertaking processes toward different types of corpses, we empirically determined a 15 and 60 min recording time, respectively, for the assays with conspecific and congeneric corpses.

Undertaking processes toward conspecific corpses (body removal) are concluded within a

15 min time frame. Undertaking process toward congeneric corpses (burial behavior), however, usually lasts for 5h or more. Nevertheless, the entire behavioral repertoire including antennation, alarm, recruitment, aggression, burial, and guarding was clearly displayed within the first 60 min of the undertaking process. Behavioral bioassays on the same colony were separated by at least 5 h to avoid the potential influence of short-term memory. To analyze the involvement of worker and soldier castes in the undertaking process, the number of workers and soldiers appearing under the viewing area were recorded, respectively. Picture frames were paused every minute during the observation period, and termites within the viewing area (a radius of 4.5 cm from the nest entrance in the testing arena, Figure 2.1) were counted. To analyze the intensity of alarm behavior, the frequency of alarm vibration was counted within a 15 min observation period immediately after the introduction of corpses.

Data analyses

Results from the experiments with different postmortem time were analyzed by ANOVA.

Removal time was recorded from the time that the first termite contacted a corpse until all 10 corpses were removed. For the experiments of corpses with different origins, removal time toward conspecific (inter- and intra-colony) corpses, and frequency of alarm vibration toward congeneric and conspecific (inter-colony) corpses, were analyzed by unpaired t-test at significance level of 5%. Finally, the counts of workers or soldiers observed were summed over all observation periods. The numbers of observation periods varied because the task of removing dead workers was completed in minutes (within 15 min), while burial behavior lasted hours (beyond the 60 min recording time). The mean number of workers or soldiers per observation was calculated. These means were log transformed (log (n+1)) prior to analysis of variance, which was conducted with block, colony, and treatment (intra-colony, inter-colony, or congeneric R. virginicus) as factors.

The mean proportion of soldiers was arcsin transformed (arcsin (square root (p)) prior to analysis of variance. All statistical analyses were conducted using Statistix 9.0

(Analytical Software, Tallahassee, FL).

Figure 2.1 Schematic drawing of experimental set-up.

Colonies were maintained in holding chamber. A 9.0 cm-diameter testing arena was connected to the holding chamber with a 3.0 cm plastic tubing. Petri dishes were covered with lids to avoid disturbance by air movement. Corpses were placed in the vicinity of nest entrance, and the activities of termites in the test arena were videotaped.

Figure 2.2 Corpse removal time by Reticulitermes flavipes

(a) Removal time of dead nestmates with different postmortem time in two colonies, KY-

4 and KY-15 (mean ± standard error). Unpaired t-test (P > 0.05) detected no significant difference among treatments with different postmortem time in both colonies, (b)

Removal time of dead nestmates or inter-colony corpses in two colonies (mean ± standard error). NS represents no significant difference between removal times based on unpaired t-test, P > 0.05.

Figure 2.3 Undertaking responses of Reticulitermes flavipes to conspecific species.

Figure 2.3 Undertaking responses of Reticulitermes flavipes to conspecific corpses.

(a)-(d) Counts of workers and soldiers which were recruited to inter-colony corpses. (a) and (b) represent two replications in colony KY-173, while (c) and (d) represent two replications in KY-174. (e)-(h) Counts of workers and soldiers which were recruited to intra-colony corpses. (e) and (f) represent two replications in colony KY-173, while (g) and (h) represent two replications in KY-174. (i) A soldier touching the corpse with antennae. (j) A worker attempting to grasp a corpse with its mandibles after antennation.

(k) Three workers, each of them carrying a corpse, respectively. (l) Corpses being dragged into the holding chamber through the entrance by workers. (i)-(l) are the results of inter-colony treatments, but behaviors were the same with intra-colony treatments.

Figure 2.4 Undertaking responses of Reticulitermes flavipes to R. virginicus corpses.

(a)-(d) Counts of workers and soldiers which were recruited to congeneric corpses. (a) and (b) represent two replications in colony KY-173, while (c) and (d) represent two replications in KY-174. (e) A soldier touching the corpse with antennae and attacking it with open mandibles. (f) Soldiers attacking the corpses. (g) More soldiers being recruited.

(h) Corpses being buried by workers while a group of soldiers guarding the burial site. (i)

Ten R. virginicus corpses were completely buried.

Figure 2.5 Number of termites involved in treatments with corpses of different origin.

Mean number of termites per observation in treatments with congeneric (R. virginicus), inter-colony, and intra-colony corpses. For each treatment, two colonies were used (KY-

173 and KY-174) with 2 replications. Error bars represent standard error of 4 replications. Means between groups denoted with same letters were not significantly different (P>0.05, Tukey HSD Comparison Test).

Chapter 3. Early death cues elicit adaptive cannibalism in a subterranean termite

Abstract

Social animals that live in large colonies with overlapping generations have evolved elaborate mechanisms for corpse management. For example, honey bees and ants identify and remove the deceased members from the colony to reduce the deleterious impact of pathogens. Termites that feed on nitrogen poor lignocellulosic food, on the other hand, could benefit from recycling rather than discarding nutrients from dead nestmates. Here we show that in the eastern subterranean termite, Reticulitermes flavipes, undertaking responses depend on the temporal change of postmortem cues from corpses. Immediately after death (< 0.5 h), 3-octanone and 3-octanol volatilize from corpses to recruit termite workers who then cannibalize these bodies. These two volatile compounds decrease rapidly over the next 4 hours while decomposition products, including oleic acid, phenol and indole, increase drastically by 64 h postmortem. Correspondingly, there is an abrupt shift from cannibalism to burial behavior between 32 and 64 h after death. We argue that such behavioral transition balances the risks associated with pathogenic attack with the benefits accrued from nutrient recycling, and that postmortem cues benefit the colony fitness by mediating these risks and rewards. We demonstrate how subterranean termites use an adaptive mechanism in corpse management in which the timely recognition and rapid removal/cannibalism of dead nestmates are promoted by the early death cues. The evolution of this early postmortem “signal” and the corresponding switch of behavioral responses represent unique adaptations in termites to their ecological and social niche.

Introduction

Eusocial insects display division of labor, which allows more efficient allocation of resources to reproduction, growth and defense. In these superorganisms, the functionally sterile workers are considered to be a “somatic” support (Wilson & Sober, 1989), and the turnover (death) of workers is a common and frequent event. In comparison to reproductives, workers have a shorter life span (Jemielity et al., 2005), facing higher mortalities due to risky tasks (Porter & Jorgensen, 1981), and sometimes engage in self- sacrifice for the overall fitness of the colony. According to the “disposable soma” theory

(Kirkwood, 1987), altruistic behaviors are strategies that reduce resource allocation to the

“somatic” workers, a disposable caste (Rueppell & Kirkman, 2005). Previous studies, however, overlook the "ultimate" contribution from workers, their corpses.

Upon death, deceased workers no longer support the colony through labor; instead they become pathogenic risks, and often require live workers to dispose of their bodies.

Unlike corpse-avoidance in many animal species (Yao et al., 2009), undertaking is a colony level hygienic behavior that convergently evolved in social organisms (Sun &

Zhou, 2013). In honey bees and most ants, workers carry dead nestmates and discard them out of the nest (Haskins & Haskins, 1974; Howard & Tschinkel, 1976; Julian &

Cahan, 1999; Visscher, 1983; Wilson et al., 1958). This prophylactic activity prevents deleterious effects of dead “somatic” workers and results in additional energy outputs

(Diez et al., 2014). Different from social hymenopterans that prefer to discard their dead, termites frequently cannibalize the deceased nestmates (Neoh et al., 2012; Sun et al.,

2013) to supplement their nitrogen-poor diets (Moore, 1969). Termites also practice

burial behavior when corpses are in large number and infected with fungi (Chouvenc &

Su, 2012), or from neighboring congeneric competitors (Sun et al., 2013).

In insects, dead individuals are often recognized through postmortem changes of their chemical signatures, either through an “increased fatty acid death cue” (Wilson et al., 1958) or a “diminished chemical vital sign” (Choe et al., 2009). Fatty acids are decomposition products that accumulate as corpse ages, and they release undertaking behavior in several ants (Haskins & Haskins, 1974; Howard & Tschinkel, 1976; Wilson et al., 1958). The titer of fatty acid also dictates the speed of corpse removal in the

European fire ant Myrmica rubra, in which decayed dead nestmates with higher fatty acid titer were removed more quickly (Diez et al., 2013). While in Argentine ants,

Linepithema humile, the dissipation of chemicals associated with life plays a more prominent role in corpse management, allowing timely elimination of corpses before decay sets in (Choe et al., 2009). Our previous study of undertaking behavior in a subterranean termite, Reticulitermes flavipes, indicates that workers retrieve the bodies of nestmates within the first 24 h of death (Sun et al., 2013). In two other subterranean species, Coptotermes formosanus and R. speratus, workers only cannibalize corpses dead within a day, while they bury corpses aged for 1 - 7 days (Neoh et al., 2012).

Decomposition cues have been observed to induce burial behavior in a subterranean species, R. virginicus (Ulyshen & Shelton, 2012) and a higher termite,

Pseudacanthotermes spiniger (Chouvenc et al., 2012). These observations suggest that cannibalism, recycling of the nutrients from dead colony members, is dependent on the postmortem time, and is possibly associated with early chemical cues within the first 24 h of death.

The overall goal of this study is to elucidate the chemical cues that mediate recycling (cannibalism) or discarding (burial) of dead workers in subterranean termites.

Later, we discussed the nutritional significance of this signaling mechanism toward the overall fitness of the colony, and ultimately the evolution of termite eusociality. The eastern subterranean termite, R. flavipes, is the most widely distributed species in North

America (Su et al., 2001). Subterranean termites nest under high humidity, and build extensive galleries in soil and decaying wood. Their nesting and feeding habits pose a pathogenic risk, and a variety of opportunist pathogens could take advantage of deceased termites to develop and spread (Rosengaus et al., 2011). Additionally, termites are soft- bodied insects and decay quickly in the humid conditions. Based on our previous work

(Sun et al., 2013), we expected that R. flavipes would change their response as the corpses ages, including shifting from cannibalism to burial, and we further hypothesized that undertaking decision of R. flavipes is affected by the temporal chemical signatures of aging corpses. Specifically, this study 1) documented undertaking response toward nestmates with different postmortem times, 2) investigated the temporal change of postmortem chemicals on corpses, and 3) functionally analyzed the behavioral effects of early and late death cues.

Here, we demonstrated that the differential undertaking behavior is associated with increasing postmortem time, and report a novel volatile death cue that promotes nutrient recycling of dead workers. The timing of this early death cue (peaks at 0.5 h and diminishes by 4 h) is critical for the overall fitness of the colony. Before decomposition- associated chemicals build up, an early death cue released from the deceased worker attracts nestmates to locate and recycle corpses. Our findings suggest that the use of early

death cue allows colony members to locate the dead within a short time frame inside the dark and complex nesting system, and thus minimizes the loss of nutritional resources while preventing pathogen propagation. Dead workers provide a postmortem signal to transfer energy to their colony enhancing inclusive fitness.

Results

Undertaking response toward corpses with different postmortem times

We first examined behavioral responses of R. flavipes workers toward nestmate corpses with different postmortem times. When individual corpses were placed into the test arena

(Figure 3.1A, 3.1B), they were carried back to the nesting chamber within 32 h of death

(Figure 3.2A). However, they were buried if deceased for 64 h (Figure 3.2A). Retrieval, using the mandibles, was initiated immediately after antennal contacts. Typically a worker carried the corpse straight to the entrance, but the corpse could be dropped and picked up again by the same or another worker, and the retrieval path could be circuitous.

There was no effect of colony on retrieval time (ANOVA; F1,72 = 0.05, P = 0.82), and no interaction between colony and postmortem time (F3,72 = 0.43, P = 0.73), but postmortem time category had a significant effect on retrieval time (F3,72 = 3.30, P = 0.03). Within 32 h of postmortem time, dead nestmates were all retrieved quickly, but workers took a significantly longer time to retrieve 32 h old corpses than freshly killed corpses (0 h old)

(Tukey HSD all-pairwise comparisons test, P < 0.05) (Figure 3.2B). Thirty-two hour old corpses were usually groomed by workers before being retrieved. In one replicated experiment, a single soil particle was placed on the corpse, a characteristic of burial behavior, suggesting conflicting chemical signals released by corpses at 32 h postmortem. When a 64 h old corpse was introduced, workers walked toward and

contacted the corpse in the same manner. After initial antennation, they walked away, picked up soil particles from the nesting chamber or the test arena, and deposited them on or near the corpse. Burial behavior involved higher labor force than retrieval. There were significantly more workers in test arena during burial process than retrieval (mean number of worker per 30 sec observation ± SE: 3.25 ± 0.70 for retrieval during 15 min, and 8.24 ± 1.58 for burial during 30 min observation period; unpaired t-test: t18 = 2.82, P

< 0.05, n = 10). Termites perform alarm behavior characterized by vigorous vibration of bodies (Crosland & Traniello, 1997). During undertaking process, display of vibration was significantly more frequent in burial than retrieval (mean number of vibration per minute ± SE: 0.27 ± 0.08 for retrieval during 15 min, and 1.09 ± 0.26 for burial during 30 min observation period; unpaired t-test: t18 = 3.01, P < 0.01, n = 10).

To infer the fate of retrieved corpses, we conducted a dish assay by introducing 0

- 64 h old corpses to groups of termites enclosed in single Petri dishes (24 workers and 1 soldier per dish) (Figure 3.1D). With a single corpse introduced to each dish, workers collectively consumed 0 - 32 h old corpses (100%, n = 6 each postmortem time: 0 h, 1 h,

16 h and 32 h; Figure 3.3), whereas they buried corpses deceased for 64 h (100%, n = 6) in a day. These results suggested that retrieved corpses were ultimately cannibalized by nestmate workers.

Postmortem changes in chemical profiles

To link chemical profile with behavioral repertoire, we collected volatiles and other chemicals from R. flavipes workers deceased between 0 and 64 h using live workers as the baseline. Using Solid Phase Microextraction (SPME), we found that immediately after death, workers emitted two volatiles, 3-octanone and 3-octanol (Figure 3.4A). These

two volatiles reached maximum quantities at 0.5 h and 1 h postmortem, respectively, and then gradually decreased through 64 h postmortem. There was a significant difference between postmortem time categories (ANOVA; 3-octanone: F9,180 = 118.42, P < 0.01; 3- octanol: F9,180 = 108.15, P < 0.01), as well as a colony effect (3-octanone: F1,180 = 11.26,

P < 0.01; 3-octanol: F1,180 = 5.62, P = 0.02), but there was no interaction between colony and postmortem time (3-octanone: F9,180 = 1.38, P = 0.20; 3-octanol: F9,180 = 0.99, P =

0.45). In contrast, two different volatiles, phenol and indole, were produced by corpses at longer postmortem times (Figure 3.4B). Phenol was first detected by SPME at 8 h postmortem, and then increased in quantity and became abundant 64 h after death

(ANOVA; postmortem time: F9,180 = 9.01, P < 0.01; colony: F1,180 = 7.56, P < 0.01; there was an interaction between colony and postmortem time: F9,180 = 5.76, P < 0.01).The same pattern of change was observed for indole, which was detected at 16 h through 64 h postmortem (ANOVA; postmortem time: F9,180 = 14.88, P < 0.01; colony: F1,180 = 0.25, P

= 0.62; there was an interaction between colony and postmortem time: F9,180 = 0.53, P =

0.85).

Six fatty acids, extracted by hexane from live and dead workers, were identified and quantified by GC-MS following esterification. The six fatty acids ranged from C14 to

C18, including myristic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, and stearic acid. All six compounds were detected from live workers, but they gradually built up after death and were high in quantity at 64 h postmortem, with oleic acid and linoleic acid being the two most abundant (Figure 3.5). Consistently for the six fatty acids, there was a significant difference between postmortem time categories (ANOVA; myristic acid: F4,20 = 5.06, P < 0.01; palmitoleic acid: F4,20 = 18.18, P < 0.01; palmitic acid: F4,20

= 20.19, P < 0.01; linoleic acid: F4,20 = 37.54, P < 0.01; oleic acid: F4,20 = 20.76, P <

0.01; stearic acid: F4,20 = 14.73, P < 0.01), but there was no effect of colony on quantity

(myristic acid: F1,20 = 0.03, P = 0.87; palmitoleic acid: F1,20 = 0.28, P = 0.60; palmitic acid: F1,20 = 0.15, P = 0.70; linoleic acid: F1,20 = 0.15, P = 0.70; oleic acid: F1,20 = 0.01, P

= 0.91; stearic acid: F1,20 = 0.05, P = 0.83), and no interaction between colony and postmortem time (myristic acid: F4,20 = 0.30, P = 0.87; palmitoleic acid: F4,20 = 0.60, P =

0.67; palmitic acid: F4,20 = 0.77, P = 0.56; linoleic acid: F4,20 = 0.93, P = 0.46; oleic acid:

F4,20 = 0.86, P = 0.51; stearic acid: F4,20 = 0.57, P = 0.69).

Changes in postmortem chemical profiles suggested corpse retrieval could be promoted by the two early occurring volatiles, 3-octanone and 3-octanol, while phenol, indole and fatty acids could be involved in inducing burial behavior.

Functional analysis of death-related chemicals

Attractant effect of early- and late-occurring volatiles

To further decipher the roles of identified chemical cues in undertaking behavior, behavioral assays were used. From previous assays in the study, we noticed that workers moved toward the dead before they took further action, which suggested an attractant effect of corpse volatiles. To test the effect, we documented responses of R. flavipes to the identified volatiles introduced into the center of the test arena (Figure 3.1A, 3.1C). 3-

Octanone, 3-octanol, or a blend of the two early-occurring volatiles equivalent to 1 freshly killed corpse (1×) was introduced. In response, workers swept their antennae in the air, moved toward the odor source, and lifted their heads and antennae underneath the odor source for a short time before moving away. More workers moved toward early- occurring volatiles than the solvent control (Tukey HSD all-pairwise comparisons test, P

< 0.05) (Figure 3.6A). There was a significant effect of odor source on number of workers attracted (ANOVA; F3,72 = 7.36, P < 0.01), as well as a colony effect (F1,72 =

7.76, P < 0.01), but there was no interaction between odor source and colony (F3,72 =

0.21, P = 0.89).

The same behavioral response was observed when late-occurring volatiles were introduced into the arena. Phenol, indole, or their blend attracted significantly more workers compared to solvent control (Tukey HSD all-pairwise comparisons test, P <

0.05) (Figure 3.6B). Similarly, there was a significant effect of odor source on number of workers attracted (ANOVA; F3,72 = 14.33, P < 0.01), as well as a colony effect (F1,72 =

5.77, P = 0.02), but there was no interaction between odor source and colony (F3,72 =

0.98, P = 0.41). Taken together, these results indicated that workers were attracted by the volatiles produced by both the recently deceased and the long-dead.

Burial activity induced by late death cues

We tested the effects of late-accumulating chemicals, including phenol, indole, and fatty acids, on burial behavior by introducing a short Teflon tube coated with different chemicals into a dish containing termites (Figure 3.1D). We first determined the effective dose that would trigger burial activity when chemicals were applied to the Teflon tube.

Tubes were treated with a blend of all late-accumulating chemicals (six fatty acids, phenol, and indole; indicated as FAPI) and a hexane extract of 64 h old corpses at two different quantities, equivalent to 1 corpse (1×) or 10 corpses (10×) (Table 3.1). The dose had a significant effect on burial behavior (Kruskal-Wallis One-Way Nonparametric

ANOVA, H4 = 45.20, P < 0.01). At a dose of 1×, there was no significant burial activity triggered by FAPI or hexane extract of 64 h old corpses in comparison to hexane, the

solvent control (Dunn’s all-pairwise comparisons test, P > 0.05). However, when the dose was increased to 10×, both FAPI and the corpse extract induced significantly more burial behavior (Dunn’s all-pairwise comparisons test, P < 0.05) (Figure 3.7A).

Therefore, the dose of 10× was used in subsequent experiments.

The roles of volatiles and fatty acids in burial activity were tested by exposing workers to tubes treated with solvent (hexane), a blend of late-occurring volatiles (phenol and indole, indicated as PI), a blend of six fatty acids identified (FA), or the FAPI blend.

Both FA and FAPI triggered significant burial activity (Kruskal-Wallis One-Way

Nonparametric ANOVA, H3 = 26.65, P < 0.01; followed by Dunn’s all-pairwise comparisons test, P < 0.05), but workers did not deposit significantly more sand on to PI treated tubes compared to the solvent control (Dunn’s all-pairwise comparisons test, P >

0.05) (Figure 3.7B).

We concluded that phenol and indole did not play an important role in sand deposition, but fatty acids were responsible for burial activity. Therefore, we further evaluated the burial effects of different fatty acids based on their abundance on 64 h old corpses. In comparison to the hexane control, significant burial activity was observed with oleic acid alone, with a blend of linoleic acid and oleic acid, with a blend of all six fatty acids, as well as with an extract of 64 h old corpses (Kruskal-Wallis One-Way

Nonparametric ANOVA, H6 = 81.83, P < 0.01; followed by Dunn’s all-pairwise comparisons test, P < 0.05). There was no significant difference between these four treatments (Dunn’s all-pairwise comparisons test, P > 0.05) (Figure 3.7C). The results suggested that oleic acid, the most abundant fatty acid from dead workers, was the most

active in stimulating burial behavior, followed by linoleic acid. The remaining four fatty acids do not appear to trigger undertaking behavior.

Discussion

Adaptive value of corpse cannibalism

Our study showed that in R. flavipes, freshly dead workers (within 32 h postmortem) were recycled as nutritive resources, and this process was promoted by postmortem- produced attractants, a blend of 3-octanone and 3-octanol. Workers altered their behavioral response from retrieval (and cannibalism) to burial when corpses were older than 32 h. By 64 h there was a substantial accumulation of fatty acids that appear to act as a late stage marker of decomposition. Our observations are consistent with previous findings that R. flavipes workers retrieved all nestmates deceased within one day (Sun et al., 2013), and that C. formosanus and R. speratus only fed on newly dead termites (Neoh et al., 2012). Cannibalism is an important mechanism for nitrogen conservation in subterranean termites, for their nutrient imbalanced woody diet (Hungate, 1941). Wood is rich in carbon but poor in nitrogen, with the carbon: nitrogen ratio ranging from 75 to

247, compared with the ratio of 4.3 to 6.9 in termite bodies (Tayasu et al., 1997). Unlike wood-feeding termites, in two litter-feeding higher termites, Microcerotermes crassus and Globitermes sulphureus, workers forage for highly decomposed plant materials with higher content of nitrogen, and corpse cannibalism rarely occurs (Neoh et al., 2012).

These suggest the adaptive value of “trophic” corpses is associated with feeding habit of the species.

Cockroaches, which share a common ancestor with termites (Inward et al., 2007), are faced with the same problem of nitrogen acquisition. Cockroaches, which are

phytophagous or opportunistic omnivorous, store surplus nitrogen as uric acid in fat body and recycle it through symbiotic bacteria (Sabree et al., 2009). Behaviorally, cannibalism of live conspecifics is common in many cockroach species, which is considered to support the nutritional need of the gregarious population (Nalepa, 1994). Termites evolved multiples mechanisms to obtain and conserve nitrogen, including symbiotic recycling of metabolic uric acid, similar to cockroaches (Potrikus & Breznak, 1981). In addition, symbiotic fixation of atmospheric nitrogen provides significant sources of nitrogen for termites (Benemann, 1973), and trophallactic behavior supports the maintaining of nitrogen balance of the colony (Machida et al., 2001). Given that cockroaches utilize conspecifics as nitrogenous source, in termites, consuming nestmate corpses to recycle nitrogen is likely a behavioral mechanism evolved from their cockroach-like ancestors.

Death of sterile individuals in social colonies is frequent. In mature colonies of a mound-building termite, Macrotermes michaelseni, non-reproductive populations remain constant with an estimated turnover of 1.4% per day (production of 18,076 per day to a total number of 1,280,895 for workers and soldiers) (Darlington, 1991). If similar turnover rate occurs in R. flavipes, which consists of 0.2 - 5.0 million termites per colony

(Su et al., 1993), 2,800 - 70,000 dead individuals are expected each day. Substantial nutrition loss is potential in a colony without recycling the corpses. Cannibalism of these deceased individuals before decomposition occurs, therefore, is an important component of nitrogen conservation for the colony. In addition, other nutrients and resources including carbohydrates, lipids, and potentially, endosymbionts, will be redistributed instead of discarded within the colony.

Cannibalism is the most efficient means of managing dead workers, as it also reduces the chance of epidemic events by destroying the source of potential pathogens while recycling nutrients. During cannibalism, antimicrobial components in termite saliva and gut contribute to inhibiting the spread of pathogens (Chouvenc et al., 2009a;

Lamberty et al., 2001). On the other hand, cannibalism would no longer be favored when corpses are highly decayed and thus possibly nutritionally poor, or if corpses carry high levels of pathogens that could not be controlled with endogenous antimicrobials. In these cases, burial behavior is an alternate practice, maintaining colony hygiene by physically isolating corpses. The burial materials utilized by workers, i.e., nest soil consisting of feces and chewed food mixed with saliva, also adds to the antifungal function of the undertaking process (Chouvenc & Su, 2012; Rosengaus et al., 1998). However, compared with corpse retrieval, burial is more costly in terms of time, labor, and resources. In R. flavipes, 3-octanone and 3-octanol were produced in advance of the accumulation of fatty acids, suggesting it is an adaptation in termites in terms of energy saving. See Figure 3.8 for a summarized comparison between retrieval and burial mediated by death cue. 3-

Octanone and 3-octanol act as postmortem “pheromone” released by workers upon death, recruiting colony members to recycle their bodies before decomposition occurs and burial becomes necessary. Analogously, signaling is a characteristic on honey bee workers in which the alarm pheromone continues to be released after stinging, which sets a trajectory toward death and benefits the colony (Michael et al., 2004).

The use of early and late death cues

In contrast to the diminished “chemical vital sign” that allows corpse recognition by

Argentine ants within 1 h of death (Choe et al., 2009), the compounds released

immediately after the death of R. flavipes enable workers to locate and retrieve the corpse within 10 minutes. Due to degenerate eyes and living in the dark confines of the nest, termites rely on a variety of pheromones to regulate essential colony behaviors, including brood care (Matsuura et al., 2010), foraging (Matsumura et al., 1968) and nest defense

(Dolejšová et al., 2014). Our study showed that dead workers also release volatiles signaling their location, so other workers can orient quickly from a distance rather than rely on random contact. Currently it remains unknown the source of 3-octanone and 3- octanol or how they were produced. Corpses of workers used in our experiments did not show any obvious rupture on the surface; 3-octanone and 3-octanol might be produced by rapid secretion upon death. The two volatiles are also found from extractions of workers in four congeneric species (R. santonensis, R. lucifugus, R. grassei, and R. banyulensis) in which they act as attractants (Reinhard et al., 2003), but our study is the first evidence of the use of 3-octanone and 3-octanol by termites as death cue. It is not known whether termites use 3-octanone and 3-octanol in communications other than death cue, but interestingly, the two compounds are found in other social insects and associated with different behaviors. In several ant genera including Manica (Fales et al., 1972),

Crematogaster (Crewe et al., 1972) and Camponotus (Duffield & Blum, 1975), either one or both of these volatiles function as alarm pheromones; and in Myrmica, 3-octanol was reported to have attractant activity (Cammaerts et al., 1985; Cammaerts & Mori,

1987).The association of 3-octanone and 3-octanol with social insects is intriguing and awaits further exploration in social, phylogenetic, and ecological contexts.

The differential response to early and late death cues in R. flavipes suggests that undertaking behavior in subterranean termites involves both novel mechanisms and

ancestral modules. In addition to rapidly detect an early death odor, termites also recognize fatty acids as corpse burial signal. The ability to recognize a “fatty acid death cue” is suggested to be an evolutionary conserved trait, and it is advantageous to diverse organisms as these chemicals may indicate proximity to a threat posed by either predators or disease (Yao et al., 2009). Fatty acids on dead conspecific individuals, primarily oleic acid and in some species linoleic acid, elicit avoidance in many gregarious arthropods including amphipods (Wisenden et al., 2001), springtails (Nilsson & Bengtsson, 2004), and social caterpillars (Yao et al., 2009). In eusocial insects, fatty acids-induced undertaking behavior has convergently evolved in Hymenoptera (Diez et al., 2013;

Haskins & Haskins, 1974; Howard & Tschinkel, 1976; Wilson et al., 1958) and Isoptera

(termites), being a part of social repertoire to eliminate dead workers. In addition to triggering burial behavior in R. flavipes, the fatty acids elicit similar response in a more derived termite species, P. spiniger (Chouvenc et al., 2012), suggesting fatty acids- mediated burial behavior is widespread in termites. Fatty acids identified in this study have low volatility and initiate burial activity by workers upon direct contact. Phenol and indole emitted from corpses at the same postmortem stage while fatty acids significantly built up for burial behavior to occur. The two late-occurring volatiles play a similar role as 3-octanone and 3-octanol in terms of indicating corpse location. However, phenol and indole are derived from the degradation of proteins (Spoelstra, 1977).

Postmortem contribution of workers

In social insects, nest workers could serve as nutritional reserves for the colony, based on the disposable soma theory. One direct example is the specialized “trophic” worker caste in several ant genera (Myrmecocystus, Camponotus, etc.). These trophic workers stay in

nest and store large amount of nutritional liquid in crops to feed the colony (Hölldobler &

Wilson, 1990). Nest honeybee workers, i.e. nurses, store higher level of lipid then foragers (Toth & Robinson, 2005). In subterranean termites, workers do not leave the nest, as they build galleries and extend the nest when foraging. Their bodies may be considered as a nutritional reservoir, similar to the feature of nurse bees (Toth &

Robinson, 2005). Upon death, the deceased are consumed by live workers, and nitrogen assimilated when they are alive is subsequently transferred within the colony.

The use of postmortem-released volatiles to promote recycling of nutrients from deceased workers is a novel example, which contributes to overall colony fitness. In social hymenopterans, since deceased workers lead to energy cost by triggering corpse removal, dying outside of nest is the most efficient practice. In the ant Temnothorax unifasciatus, workers perform altruistic self-removal by leaving the nest before their death (Heinze & Walter, 2010). Our findings in R. flavipes indicate that providing an attractant signal for cannibalism is the last contribution made by workers in termites, and the postmortem resource transfer is an extension of worker’s social contribution. We suggest that use of early death cues may occur in other termite species with similar nesting ecology and feeding habits, where cannibalism is the most efficient social strategy for dealing with deceased workers.

Methods

Termite collection and maintenance

Two R. flavipes colonies (R9 and R13) were collected nearby the Red River Gorge area,

Daniel Boone National Forest (Slade, KY). Another R. flavipes colony (A18) was collected from the Arboretum, located on the University of Kentucky campus (Lexington,

KY). Colonies were obtained during spring and summer using trapping stations filled with cardboard rolls. Once captured termites were extracted from traps, placed in Petri dishes (14.5 cm × 2.0 cm) with moistened unbleached paper towel (Wausau Paper) as food source for one to two weeks. After that termites were transferred to round plastic containers (15.0 cm in diameter, 6.5 cm in height). They were provisioned with moistened wood mulch and pinewood blocks. Reticulitermes colonies were maintained in complete darkness (L:D =0:24), at 27 ± 1ºC, 80%- 99% RH. Termites were used for experiments within six months of collection from the field.

Undertaking bioassay with dead termites

Undertaking responses toward termite corpses with different postmortem times were tested in two colonies (R9 and R13), with 10 replications for each colony. To prepare corpses, workers of R. flavipes were killed by freezing on dry ice for 1 minute. Corpses were maintained in covered Petri dishes under the same conditions as their natal colony for 0, 1, 16, 32 and 64 h, respectively. The experimental set-up (Figure 3.1A) included a nesting chamber, a round plastic container (15.0 cm in diameter, 6.5 cm in height) containing ~2,000 termites; and a covered test arena, a 5.5 cm-diameter Petri dish (1.5 cm height; VWR Inc.) where observations were made. A plastic tube (inner diameter: 0.7 cm; length: 3.0 cm) connected the test arena and the nesting chamber. Before tests, colonies were acclimatized in the set-up for one month to allow termites to tunnel through test arena and cover the bottom with feces and other nest materials. For each trial, an individual corpse was introduced in the vicinity of nest entrance through an entry port (0.5 cm-diameter) on the cover of test arena (Figure 3.1B). Behavioral responses by

R. flavipes in test arena were recorded using a video camcorder (Canon VIXIA HF G10)

for at least 15 min for retrieval behavior, or for 30 min if burial activity (i.e., depositing soil on or near corpse) was initiated. For burial behavior, an additional observation was made 1 h after corpse introduction, which always confirmed the first 30 min observation.

A piece of plastic sheeting was placed at the bottom of the test arena and replaced after each replication to eliminate chemical residues left by termites.

A dish assay was conducted to document if corpses would be cannibalized.

Groups of 24 workers with 1 soldier, added to emulate normal colony caste proportions

(Howard & Haverty, 1981),were enclosed in individual Petri dishes (5.5 cm in diameter,

1.5 cm in height) (Figure 3.1D). The bottom of each dish was scratched to create a coarse surface to facilitate walking behavior. A piece of moistened unbleached paper towel in semicircular shape was placed at the bottom, serving as food source and covering half of the bottom. Moistened sand (1g, water content 15% by weight) was provided as potential burying materials. A hole (0.5 cm in diameter) on the cover, where a nestmate corpse was introduced, was above the section of the dish that was not lined with paper. Groups of termites were maintained in the dishes for one day prior to test. Corpses at postmortem time 0, 1, 16 and 32 h were tested, and each set-up was tested once with one corpse only.

Ten replications were made with each postmortem time, and the same two colonies (R9 and R13) were used.

Analysis of death-related chemicals

Chemical analyses were conducted with an Agilent Technologies 6890N gas chromatography (GC) in splitless mode equipped with a DB-5 capillary column (30 m ×

0.32 mm × 0.25 μm; Agilent Technologies), using helium as carrier gas. Column temperature was raised from 40°C (2 min hold) at 10°C/min to 320°C (additional 4 min

hold). The GC was coupled with an Agilent Technologies 5975 mass spectrometer (MS).

Candidate compounds were identified based on National Institute of Standards and

Technology/Environmental Protection Agency/National Institutes of Health Mass

Spectral Library, and further identified by comparison of retention times and mass spectra with synthetic standards (Sigma-Aldrich).

We collected chemicals from live workers and corpses produced by freezing (0,

0.5, 1, 2, 4, 8, 16, 32 and 64 h postmortem). Corpses at 0 h postmortem time were thawed at room temperature for 5 min before all collection procedures. Volatiles were collected with a 100-μm polydimethylsiloxane SPME (Solid Phase Microextraction) fiber

(Supelco). Fifteen workers were placed in a 1 ml glass vial, which was sealed with an aluminum crimp cap with pre-fitted septum (Thermo Fisher Scientific). The fiber was extended into the vial for 15 min without making direct contact with the termites. After this period the fiber was introduced into the injection port of the GC and remained in the injector port for 1 minute, where it was heated to 280°C to ensure volatilization of the collected chemicals. Volatiles identified by SPME were further quantified on corpses with hexane extraction. Individual workers were submerged in 50 μl glass-distilled hexane (Thermo Fisher Scientific) (with 10 ng/μl n-nonadecane as internal standard) for

10 min, and the solution was injected to GC. We made 10 replications of each corpse age for each of the two colonies (R9 and A18).

To quantify fatty acids from R. flavipes workers, esterification was performed with hexane extracts. At each time point (live, 8 h, 16 h, 32 h and 64 h postmortem), a group of 6 individuals were extracted by 300 μl distilled hexane (with 10 ng/μl n- nonadecane as internal standard) for 10 min, and then the solution was reacted with

methanol containing BF3 (10%, w/w) as catalyst, following the manufacturer’s instructions (Sigma-Aldrich). Esters as final products were identified using GC-MS and quantified through GC peak areas. Three replications were made with each of the two colonies (R9 and R13).

Attraction assay with volatiles

Hexane solutions of early- and late-occurring volatiles were made using synthetic chemicals at the concentration equivalent to one corpse at 0 h and 64 h postmortem, respectively, per μl (Table 3.1); and 1 μl of each solution was applied for test. These solutions were stored at -20°C in sealed 5 ml glass vials (Thermo Fisher Scientific).

Hexane was tested as solvent control. To investigate the attractive effect of postmortem volatiles, the same behavioral set-up was used as in Figure 3.1A, but with a modification on test arena inspired by Reinhard et al. (2003) (Figure 3.1C). A strip of filter paper (0.2

× 0.7 cm) with an odor source, held by a #3 insect pin, was introduced to the center of test arena through a hole on the top. The distance between filter paper and the bottom of arena did not allow termites to make direct contact with the odor source. A circle (2.5 cm-diameter) was marked on the floor of the arena directly underneath the odor source.

After introduction of each odor source, the response of termites in test arena was videotaped for 5 min, and numbers of workers that appeared in the circle were registered every 10 sec for the 5 min. Ten replicates were made for each odor treatment in a colony, and two colonies (R9 and R13) were tested. To allow termites to recover from the previous stimuli, treatments using the same colony were separated by at least 1 hour.

Burial assay with phenol, indole and fatty acids

To test effects of late death cues on burial behavior, a dish assay was adopted as described above (Figure 3.1D). Hexane solutions of different compounds were made at the concentration equivalent to 1 or 10 corpse(s) at 64 h postmortem per μl, and 1 μl of each solution was applied for each test (Table S1). A solution was applied to the surface of a Teflon tube (0.1 cm-diameter, 0.4 cm long) of a size to simulate a termite corpse. To obtain better visual contrast for observation and to avoid contamination of food with tested chemicals, after evaporation of solvent (1 min), the tube was dropped through a hole on the cover to the half of dish without paper. Burial activity was then observed by taking digital photo after 3 h, and area covered with sand and feces was quantified using

ImageJ 1.48 (National Institutes of Health). The experiment was conducted with 10 replicates for each colony, with a new group of termites (24 workers and 1 soldier) in a clean Petri dish each time. Two colonies (R9 and R13) were tested.

Data analyses

All statistical analyses were performed using Statistix 10 (Analytical Software,

Tallahassee, FL). Prior to analysis of variance, retrieval times were log transformed (log

(x)), quantities of fatty acids were log transformed (log (x+1)), and the mean numbers of workers per observation in retrieval and burial as well as attraction assay were square root transformed (square root (x)). These transformations yielded values that fit the assumptions of parametric tests. The above data, as well as quantities of volatiles (3- octanone, 3-octanol, phenol, and indole) collected by SPME, were analyzed by ANOVA with colony, treatment, and interaction between colony and treatment as factors. For

mean areas in burial assay, we conducted Kruskal-Wallis One-Way Nonparametric

ANOVA, due to non-homogeneity of variance (Levene's Test, P < 0.05).

Table 3.1 Quantities of compounds tested in undertaking bioassay. Compounds in hexane solution were applied to filter paper in attraction assay, and applied to Teflon tube in burial assay. “1×” and “10×” indicate tested quantity equivalent to 1 or 10 corpse(s), respectively. Quantities of 3-octanone and 3-octanol were based on hexane extracts of 0 h old corpses, and quantities of other compounds were based on hexane extracts of 64 h old corpses.

Tested quantity (ng) Bioassay Tested compounds 1× 10× 3-Octanone 5 - 3-Octanol 10 - Attraction assay Phenol 7 - Indole 55 - Phenol 7 70 Indole 55 550 Myristic acid 15 150 Palmitoleic acid 36 360 Burial assay Palmitic acid 93 930 Linoleic acid 207 2,070 Oleic acid 424 4,240 Stearic acid 48 480

Figure 3.1 Schematic drawing of experimental set-ups.

(A) Experimental set-up with a nesting chamber and a test arena. (B) Test arena for differential undertaking response. a: nest entrance; b: entry port. (C) Test arena for attraction assay. c: filter paper (0.2 × 0.7 cm) with odor source; d: pin holding the filter paper; e: circle (2.5 cm diameter). (D) Experimental set-up for dish assay. In the dish assay, each group of termites consisted of 24 workers and 1 soldier. Test objects were introduced through the entry port.

Figure 3.2 Undertaking behavior by Reticulitermes flavipes toward corpses with different postmortem times.

(A) Differential response to corpses (n = 20). (B) Corpse retrieval time (mean ± SE, n =

20). Boxes are bounded by the first and third quartiles, with the band indicating median; whiskers represent the 10th and 90th percentiles; dots outside the whiskers are outliers.

Data were pooled from two colonies (R9 and R13, 10 replications each colony). Means between groups denoted with the same letter were not significantly different (Tukey HSD all-pairwise comparisons test, P > 0.05).

Figure 3.3 Cannibalism of freshly killed corpses by Reticulitermes flavipes in dish assay.

(A) A worker feeding on a corpse at 5 h after corpse introduction. (B) A partially consumed corpse 20 h later. Photos represent the result of a freshly killed nestmate (0 h postmortem), but cannibalisms were the same when termites were treated with corpses 0 -

32 h old.

Figure 3.4 Temporal changes of death-related volatiles collected from Reticulitermes flavipes.

(A) Change of early-occurring volatiles, 3-octanone and 3-octanol. (B) Change of late- occurring volatiles, phenol and indole. Quantity collected by SPME fiber was presented

(mean ± SE, n = 20). (SPME does not accurately reflect the emission rate from corpses.)

Data were pooled from two colonies (R9 and A18, 10 replications each colony).

Figure 3.5 Accumulation of fatty acids on Reticulitermes flavipes workers after death.

Quantity extracted by hexane presented (mean ± SE, n = 6). Data were pooled from two colonies (R9 and R13, 3 replications each colony).

Figure 3.6 Attractive effect of postmortem volatiles.

Number of workers that moved underneath odor source per observation in treatments with early-occurring volatiles (a) and late-occurring volatiles (b) (mean ± SE, n = 20).

Data were pooled from two colonies (R9 and R13, 10 replications each colony). Means between groups labeled with the same letter were not significantly different (Tukey HSD all-pairwise comparisons test, P > 0.05).

Figure 3.7 Burial activity of Reticulitermes flavipes toward death-related chemicals.

Areas buried with sand within 3 h were presented as mean ± SE (n = 20). Data were pooled from two colonies (R9 and R13, 10

replications each colony). (A) Effect of two doses on burial behavior. (B) Effect of volatiles and fatty acids. (C) Effect of different

groups of fatty acids. Groups denoted with the same letter were not significantly different (Dunn’s all-pairwise comparisons test, P >

0.05). “1×” and “10×” indicate tested quantity equivalent to 1 or 10 corpse(s) at 64 h postmortem, respectively. Extract: hexane

extract of corpse(s) at 64 h old; FAPI: blend of all six fatty acids, phenol and indole; PI: blend of phenol and indole; FA: blend of six

fatty acids; LO: blend of linoleic acid and oleic acid; MPPS: blend of the other four fatty acids (myristic acid, palmitoleic acid,

palmitic acid and stearic acid); L: linoleic acid; O: oleic acid.

Figure 3.8 A schematic drawing showing the energy output and nutritional balance associated with death cue mediated undertaking responses.

Figure 3.8 A schematic drawing showing the energy output and nutritional balance associated with death cue-mediated undertaking responses. (A) Early death cues

(orange line) peak within 1 h and rapidly decrease over 4 h. In contrast, late death cues

(blue line) start to increase after 32 h. (B) Retrieval behavior involves a small labor force and only low levels of vibrational signaling, thus energy costs of this behavior are low.

Burial behavior toward nestmate corpses involves 2.5 times more workers than retrieval behavior, and higher frequency of vibrational signaling (4X compared to retrieval).

Retrieval behavior results in low energy costs, and it allows the colony to recycle nutrients through cannibalism. Burial is not only energetically costly, but the nutrients contained in the corpse are lost. The timing of early death cues, 3-octanone and 3-octanol

(C), allows colony members to locate the dead within a short period of time (0.5 - 4h) inside the dark and complex nesting matrix (D) to maximize the colony fitness by practicing retrieval instead of burial behavior to minimize the loss of nutritional resources while preventing pathogen propagation, and to limit the energy output associated with the undertaking process (B).

Chapter 4. Gene expression profiling of undertaking behavior in the eastern subterranean termite

Abstract

Undertaking behavior is the disposal of dead individuals in social colonies to prevent potential pathogenic attack, and is considered an essential adaptation to social living.

However, the molecular basis of undertaking behavior remains unknown. In the eastern subterranean termite, Reticulitermes flavipes, workers exhibited differential responses toward corpse with different postmortem times and origins. Specifically, workers cannibalized freshly dead nestmates, but buried decayed nestmates as well as corpses of a competitive species, R. malletei. Using RNA-Seq analysis, we surveyed the molecular signatures underlying the three types of undertaking process. Differentially expressed genes (DEGs) in workers with or without corpses were analyzed and functionally annotated for Gene Ontology terms and molecular pathways. Our results showed that different sets of DEGs were associated with each type of undertaking response, suggesting that different gene networks are involved in corpse cannibalism, burial of dead nestmates, and burial of dead competitors. Burial behavior involved more DEGs associated with metabolic pathways than cannibalism, indicating it is more energy costly.

This study is the first to investigate the molecular basis underlying undertaking behavior, a convergently evolved trait in social insects.

Introduction

Behaviors in animals are widely modified by chemicals, during communication between individuals or interactions of individuals with the environment. While short-term

behavioral changes are commonly mediated through post-transcriptional neurological processes, it has been demonstrated that pheromones cause changes in gene expression in brain (Alaux & Robinson, 2007; Halem et al., 2001). Many pheromones in the honey bee, including queen mandibular pheromone, alarm pheromone and brood pheromone, induce dynamic changes in brain gene expression (Alaux et al., 2009; Alaux & Robinson, 2007;

Grozinger et al., 2003). Linking brain gene expression to social behavior has been extensively conducted in the honey bee (Reviewed by Zayed & Robinson 2012, and references therein). These works have provided insights into the molecular underpinnings of social behaviors, particularly foraging and aggression (Ben-Shahar et al., 2003; Li-

Byarlay et al., 2014; Zayed & Robinson, 2012). The molecular basis of undertaking behavior, which has convergently evolved in the honey bee, ants and termites, however, is poorly understood.

Death of colony members frequently happens in social species, and corpses pose potential pathogenic risks. Social colonies require good hygiene due to their group-living habit and extensive contacts between nestmates. These characteristics associated with eusociality, together with enclosed nest in many species, have been suggested to play important roles in shaping undertaking behavior during evolutionary time (Cremer et al.,

2007). Social insects recognize dead individuals based on postmortem chemical changes.

The accumulation of fatty acids, particularly oleic acid, is a common chemical signature of death in arthropods (Yao et al., 2009). Oleic acid serves as death cue in many social insects. It elicits corpse removal in ants and corpse burial in termites (Chouvenc et al.,

2012; Diez et al., 2013). In addition to the accumulation of fatty acid death cue,

Argentine ants (Linepithema humile) rapidly detect death of nestmates based on the

diminishing of chemicals associated with life (Choe et al., 2009). In despite of the difference in death recognition mechanism, the behavioral outcome in ants is relatively uniform, which is transporting corpses out of nest. Honey bees dispose of dead bees in the nest in a similar manner: they carry the corpses out and drop them far away from the hive (Visscher, 1983). In subterranean termites, however, workers dispose of corpses primarily by cannibalism or burial, depending on the postmortem time and origin of corpses (Neoh et al., 2012; Sun et al., 2013; Sun & Zhou, 2013).

Our previous studies have demonstrated differential undertaking behavior in the eastern subterranean termite, Reticulitermes flavipes. The conserved death cue, oleic acid, cumulates on corpses for 64 h to elicit burial behavior, while freshly dead workers are cannibalized by nestmates (see Chapter 3). Reticulitermes flavipes also distinguish competitive termite species from conspecifics and bury their dead bodies, presumably based on different cuticular hydrocarbons (Sun et al., 2013) (see Chapter 2 and Appendix

I). The different undertaking behaviors in R. flavipes are modulated through different chemical cues, which are likely associated with different risks posed by the corpse.

Consuming dead nestmates is beneficial to colony, because it allows nutrient recycling in the colony, but burial would become necessary with increased risks such as potential pathogenic attack and colony intrusion. Cannibalism and burial have substantial difference on energy demands, based on the level of labor force and the frequency of vibrational signals (Chapter 2 and 3). In addition, energy cost in burial behavior could be resulted from information processing in brain and physiological changes (e.g. production of antimicrobial peptides). These metabolism-associated changes during behavioral

process can be at least partially reflected at the transcriptional level (Li-Byarlay et al.,

2014).

Relative to morphological traits, behavioral traits are more changeable and fleeting, which challenges studies of molecular underpinnings of behaviors. Both cannibalism and burial behavior in R. flavipes start rapidly after exposure to corpses (5 to

30 minutes), and undergo processes that last hours. Transcriptomic analysis via RNA sequencing, which measures genomic response to environmental change, can be used to explore genes that show dynamic expression in correlation with changes in behavior. Our overall goal of this study is to elucidate transcriptional signatures of R. flavipes workers in corpse cannibalism and burial. Specifically, this study (1) validated the differential undertaking response toward corpses with different postmortem times and origins; (2) investigated gene expression changes in response to different death cues; and (3) comparatively analyzed biological functions and molecular pathways associated with different undertaking processes.

Results

Differential undertaking response toward three types of corpses

We tested behavioral responses of R. flavipes toward freshly dead nestmates, decayed nestmates, and freshly dead competitors (R. malletei), using a dish assay (Figure 4.1).

Workers of R flavipes cannibalized the fresh bodies of nestmates (0 h postmortem), buried the decayed ones (72 h postmortem) as well as corpses of competitors. When corpses were introduced to the dish, workers contacted them using antenna, presumably recognizing the chemical cues on the surface. In all three treatments, there were increased alarm behaviors (body vibration) in 30 min (Figure 4.2A, 4.2B, 4.2C). When a group of 3

freshly dead nestmates were introduced, workers started to groom and feed on the corpses within 30 min. As termites consume surface substances while grooming, we interpret grooming as a part of corpse cannibalism. The three corpses were completely consumed in 48 h in all replications (Figure 4.2D). When R. flavipes were treated with decayed corpses, workers carried sand particles using mouthparts and deposited them onto the corpses, which was slowly started in the first hour. Burial of decayed nestmates was the most active at 2 h (Figure 4.2E). We observed that workers occasionally excreted feces toward the corpses and the burial pile. Compared with burial of decayed corpses, burial of congeneric corpses was more intensive, which quickly started within 30 min and involved higher frequency of sand deposition (Figure 4.2F). Both burial of nestmates and burial of competitors took more than 6 h to finish. Behavioral signatures in the three undertaking processes were summarized in Figure 4.3.

Differentially expressed genes in undertaking processes

Freshly dead nestmates, decayed nestmates and freshly dead competitors were used to treat termites, and workers exposed to the corpses were then collected for RNA sequencing. Based on the behavioral observation, we determined to investigate gene expression in worker head at 30 min (decision-making), 1 h (behavior initiation), and 6 h

(behavior in process) after introduction of each type of corpses. As cannibalism was more long-lasting, we additionally collected samples at 24 h after introduction of freshly dead nestmates. Workers that were not exposed to any corpse were collected as control (0 h).

From the 11 transcriptomic libraries sequences, we obtained a total of 59328 transcripts after assembly. Numbers of genes in each sample are shown in Table 4.1.

All of the three types of corpses influenced gene expression in worker head in 30 min. Most of the differentially expressed genes (DEGs, fold difference ≥ 2, False

Discovery Rate (FDR) ≤ 0.001) collected at 30 min were up-regulated (Figure 4.4). More

DEGs were identified in 1 h and 6 h samples than 30 min samples. In treatment with freshly dead competitors, which stimulated cannibalism, fewer DEGs were found at 6 h in comparison with the other two treatments, which elicited burial behavior. Samples collected at 24 h during corpse cannibalism displayed 149 DEGs. The changes in expression levels of most genes were transient, and there were only a small number of genes consistently regulated during undertaking processes: 1 during cannibalism, 2 during burial of decayed nestmates, and 4 during burial of dead competitors (Figure 4.5).

These consistent DEGs were summarized in Table 4.2.

Among the three undertaking processes, there were few DEGs that overlapped

(Figure 4.6). When termites were treated with the three types of corpses, no DEG in worker head was shared at 30 min; there were 6 and 4 DEGs in common at 1 h and 6 h, respectively (Figure 4.6). Interestingly, between the two burial behaviors (toward decayed nestmates and dead competitors), we observed a substantial overlap on differential expression of genes (Figure 4.6). This result suggests that different gene networks are activated in cannibalism and burial.

GO function and KEGG pathway analysis

To examine potential changes of biological functions and pathways associated with differential gene expression, we annotated the identified DEGs via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. We pooled genes differentially expressed at 30 min, 1 h, and 6 h in each treatment category, and

compared GO function terms and KEGG pathways among corpse cannibalism, burial of dead nestmate, and burial of dead competitors. Only 8 GO terms were associated with cannibalism, and they were entirely overlapped with GO terms associated with burial behavior (Figure 4.7A). Between the two sets of GO terms associated with burial of nestmates and burial of competitors, there was a strong overlap (Figure 4.7A). A similar pattern was observed in the Venn diagram of KEGG pathways (Figure 4.7B). KEGG pathways were further classified into broader sections, and we summarized the numbers of DEGs in each broader category (Figure 4.7C). Compared with cannibalism, more

DEGs in burial behaviors were annotated with metabolic pathways, including amino acid, lipid, and carbohydrate metabolisms. In addition, during burial behaviors, more DEGs were identified to be involved in sensory system, digestive system, and immune system.

These results indicate that a higher level of metabolic output is associated with corpse burial than cannibalism.

Discussion

Consistent with previous studies, in our newly developed dish assay, R. flavipes workers cannibalized freshly dead nestmates, but buried decayed nestmates as well as dead competitors. Cannibalism is the most efficient means of corpse management, because by doing so colonies recycle nitrogenous resources and also eliminate corpses from which epidemic pathogens may develop. Given that turnover rate in termite colonies is high

(about 1.4% per day) (Darlington, 1991), and that R. flavipes are able to detect early death cue to quickly locate newly dead corpses, exposure to freshly dead nestmates is likely a frequent event that provides little stress to workers. The majority of corpses are likely to be consumed, and thus cannibalism is a daily task that deals with a low level of

risk. In this study, we only identified a small number of DEGs, and no GO term or metabolic pathway was significantly enriched within 6 h of exposure to newly killed nestmates (P-value ≤ 0.05 for GO; Q-value ≤ 0.05 for KEGG pathways). The results suggest that death cues from freshly dead nestmates may be largely processed through neural response, and cannibalism does not require much additional energy output than other daily activities, such as feeding and walking in the control.

In case that dead individuals decay in the colony, the nutritional value is lost and the chance of pathogenic attack increases. Instead of cannibalism, burial may be warranted to maintain colony hygiene. Transporting burial materials presumably requires more effort than feeding on the dead. Material used for burial is a combination of nest soil (sand in this study), saliva and feces. Endogenous antimicrobials are produced in termite saliva (Chouvenc et al., 2012; Lamberty et al., 2001), which adds to the metabolic cost during burial. Burial of dead competitor is similar to burial of decayed nestmates, but with an increased amount of sand and time. Compared to decayed nestmates that pose potential pathogenic risk, dead competitors indicate colony intrusion and potentially unknown pathogens (discussed in Chapter 2). Both types of corpses are social challenges for termites, and burial is a defensive mechanism against pathogens and intruders.

Territorial intrusion provides social stress in diverse taxa including insects, fish and mammals, and causes changes in expression of brain genes associated with energy metabolism (Rittschof et al., 2014). In this study, from DEGs identified in burial of nestmates and burial of competitors, we did not find genes directly annotated with energy metabolic pathways through KEGG database. However, groups of DEGs were found to be associated with carbohydrate, amino acid and lipid metabolisms, which are related to

energy metabolism. These results indicate an overall higher metabolic output in burial behavior than corpse cannibalism.

There were also more DEGs associated with sensory system in burial than cannibalism. These findings suggest that the processing of information in brain during burial of decayed nestmates and dead competitors is more complex than that during consumption of freshly dead nestmates. This information may not be restricted to processing death cues from corpses, but may also include other communicational signals used between live workers.

In burial of decayed nestmates, 11 DEGs involved in immune system were identified, compared with 5 in burial of dead competitor and only 1 in cannibalism.

Decayed nestmates used in this study were not treated with infectious pathogen, but termites may recognize the increased level of fatty acids as an indicator of pathogenic risk, and initiate immune response as prophylaxis. In the red imported fire ant, Solenopsis invicta, infection of fungus Metarhizium anisopliae promotes fatty acid accumulation on dead individuals, which leads to accelerated corpse removal by workers (Qiu et al.,

2015). This is in agreement with the widely accepted concept that undertaking is prophylactic social behavior that prevents epidemic attack (Diez et al., 2012).

In this study, we identified many genes that dynamically changed in expression, but mapping these genes to behavioral responses is challenging. These DEGs are not necessarily the direct causes of behavioral changes, but more likely to contain both sets of genes of causes and consequences. This is because undertaking is a complicated behavioral process that includes multiple behavioral components. Taking burial behavior for example, it involves alarming other workers via body vibration and potentially

chemical signaling, transporting sand, and locating corpses and sand piles while moving between them.

Taken together, in this study we demonstrated that death cues cause rapid changes

(in 30 min) in gene expression in R. flavipes workers, and that different sets of DEGs are associated with each type of undertaking response. In comparison to cannibalism, burial of nestmates and burial of competitors involved more DEGs associated with metabolic pathways, suggesting higher energetic demands in burial behavior. This is consistent with our behavioral observation. DEGs with highest fold changes and those consistently regulated during undertaking process will be validated for their biological function in future, which will provide insights into genetic underpinnings of undertaking behavior in termites.

Methods

Termites

One laboratory colony of R. flavipes was used for behavioral analysis and gene expression profiling. The colony was established in 2010 by a pair of sibling alates from a dispersal flight in Lexington, KY, and it was 5-year old when used for experiments. The colony was gradually upgraded into larger containers with its growth, and maintained in a sealed plastic container (45.7 × 30.6 × 15.2 cm3, Pioneer Plastics) with more than 5,000 individuals in the fifth year. A competitive species, R. malletei, was collected in Slade,

KY, and used within one year of collection from the field. Termites were provisioned with pinewood mulch and pinewood blocks, and maintained in complete darkness (L:D =

0: 24) at 27 ± 1ºC, 80% - 90% RH.

Biological observation

A dish assay was conducted to document behavioral responses of R. flavipes. Groups of

20 workers and 1 soldier were enclosed in Petri dishes (3.5 cm in diameter, 1.5 cm in height) provided with moistened unbleached paper towel and sand (0.3 g) (Figure 4.1).

Termites were acclimatized in the dishes for 24 h prior to introduction of 3 corpses to each dish. Corpses were prepared by freezing workers to death at -20ºC for 15 min, and behavioral responses toward three types of corpses was tested: 1) freshly dead nestmates:

0 h old dead workers collected from the same R. flavipes colony; 2) decayed nestmates: workers from the same R. flavipes colony and dead for 72 h; 3) freshly dead competitors:

0 h old dead workers obtained from R. malletei colony. Immediately after corpse introduction, behavior of termites in the dish was recorded using a video camcorder

(Cannon VIXIA HF G10) for 24 h (with the exception of 48 h for treatment with freshly dead nestmates). Termites acclimatized for 24 h but not provided with corpses were videotaped for 5 min as control. Five-minute video clips prior to 30 min, 1 h, 2 h, 3 h, 6 h, 10 h, and 24 h (and additionally 48 h for treatment with freshly dead nestmates) were analyzed for alarm behavior (body vibration), corpse cannibalism and burial behavior.

Three replications were made.

RNA Sample preparation

To prepare biological samples, termites were maintained under the same dish condition with behavioral observation described above, and treated with the same three types of corpses. Based on behavioral analysis results, different time points during undertaking process were selected for termite sampling. Four groups of termites were treated with freshly dead nestmates for 30 min, 1 h, 6 h and 24 h, respectively. For both treatments

with decayed nestmates and dead competitors, three groups of workers were provided with corpses for 30 min, 1 h and 6 h, respectively. In addition to the 10 treatments, workers acclimatized for 24 h but not provided with corpses were sampled as control (0 h). At each selected time point, whole heads of all 20 workers in each dish were taken and frozen in liquid nitrogen quickly (within 5 min). Six replications were made for each treatment, and tissues were kept at -80ºC until RNA extraction. Total RNAs were extracted using RNeasy Mini Kit (Qiagen). For each treatment, we pooled the RNAs extracted from 6 replications (a total of 120 heads) to meet the required quantity for sequencing. RNA quantity and purity were determined by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). A total of 11 RNA samples were shipped on dry ice to BGI@UCD (joint facility between Beijing Genomics Institute and

University of California Davis) at Sacramento, CA.

RNA sequencing and read mapping

Sequencing of RNA and mapping of reads were conducted by BGI@UCD. Specifically, the total RNA samples were treated with DNase I to degrade possible DNA contamination. Then the mRNA was enriched using the oligo (dT) magnetic beads.

Mixed with the fragmentation buffer, the mRNA was fragmented into short fragments

(about 200 bp). Then the first strand of cDNA was synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand. The double strand cDNA was purified with magnetic beads.

End reparation and 3’-end single nucleotide A (adenine) addition was then performed.

Finally, sequencing adaptors were ligated to the fragments. The fragments were enriched by PCR amplification. During the quality control (QC) step, Agilent 2100 Bioanaylzer

and ABI StepOnePlus Real-Time PCR System were used to qualify and quantify of the sample library. The library products were sequenced via Illumina HiSeqTM 2000 platform.

Clean reads were generated by removing adaptor sequences and low quality reads

(percentage of unknown base “N” ≥ 10%, or percentage of low quality base ≥ 50%). De novo transcriptome of one termite RNA sample was previously sequenced by BGI, and used as reference for alignment. Clean reads were mapped to reference sequences using

SOAPaligner/SOAP2 (Li et al., 2009). No more than 2 mismatches were allowed in the alignment. The gene expression level was calculated by using RPKM method (Reads Per

Kilobase per Million reads).

Differential expression and functional annotation

The differentially expressed genes between each treatment and the control (no corpse, 0 h) were identified using the algorithm developed by Audic and Claverie, 1997. In this study, DEGs were determined at the threshold of fold difference ≥ 2 in gene expression and False Discovery Rate (FDR) ≤ 0.001.

We used Blast2GO (Conesa et al., 2005) to functionally annotate all DEGs based on results from BLASTx search against NCBI non-redundant protein database (E value ≤

1e-5). DEGs were mapped to Gene Ontology (GO) terms in the database

(http://www.geneontology.org), and KEGG pathways (http://www.kegg.jp). The significantly enriched GO terms (P-value ≤ 0.05) and KEGG pathways (Q-vlaue ≤ 0.05) were determined using hypergeometric test by comparing numbers of DEGs in each term/pathway to the genome background. To better compare gene expression patterns in differential undertaking behavior, we classified KEGG pathways of DEGs to broader

categories based on the database (http://www.kegg.jp), and focused on three categories in metabolism (amino acid metabolism, carbohydrate metabolism, and lipid metabolism), and three categories in organismal systems (sensory system, digestive system, immune system).

Data analyses

Statistical analyses for biological observation were conducted using Statistix 10

(Analytical Software). Square root transformation (square root (x+0.5)) was performed for frequencies of vibration during cannibalism of freshly dead nestmates and burial of decayed nestmates, and frequencies of sand deposition during burial of decayed nestmates. The above data after transformation, as well as the original data in frequency of vibration during burial of dead competitors, met the assumption of parametric tests, and were analyzed by ANOVA. For numbers of workers consuming corpses and frequencies of sand deposition during burial of competitors, Kruskal-Wallis One-Way

Nonparametric ANOVA was conducted due to non-homogeneity of variance (Levene’s

Test, P < 0.05).

Table 4.1 Number of genes assembled.

Treatment Sampling time 0 h 30 min 1 h 6 h 24 h No corpse 58392 - - - - Freshly dead nestmates - 58323 58519 58308 58444 Decayed nestmates - 58430 58610 58539 - Freshly dead competitors - 58576 58542 58468 -

Table 4.2 List of genes consistently regulated over time.

Gene ID Size Identity match E value Description Correlated behavior and (bp) (species and GenBank accession #) regulation Unigene6323 396 Pediculus humanus corporis (XP_002430633) 6e-08 Cuticle protein, putative Cannibalism (down) CL560.Contig2 1474 Ixodes scapularis (XP_002435918) 9e-24 Hypothetical protein IscW_ISCW024385 Burial of nestmates (up) CL934.Contig2 2027 Blattella germanica (AAO20251) 0 Cytochrome P450 monooxygenase CYP4G19 Burial of nestmates (down); Burial of competitors (down) Unigene37477 4146 Bombus terrestris (XP_003397626) 0 Hypothetical protein LOC100645878 Burial of competitors (up) Unigene7987 351 No significant BLAST hits found N/A N/A Burial of competitors (up) Unigene26459 1632 Papilio xuthus (BAM19355) 7e-06 Cuticular protein PxutCPR146 Burial of competitors (up)

Figure 4.1 Experimental set-up for RNA-Seq.

Each group of 20 workers and 1 soldier was enclosed in a Petri dish for one day prior to experiment. Moistened sand was provided as burial material, and paper was provided as food source. Three corpses of a certain type (freshly dead nestmates, decayed nestmates, or freshly dead competitors) were then introduced to the center of dish for elicitation of undertaking behavior.

Figure 4.2 Differential undertaking responses.

Figure 4.2 Differential undertaking responses. Frequencies of vibration per minute during (A) cannibalism of freshly dead nestmates, (B) burial of decayed nestmates, and

(C) burial of freshly dead competitors were presented. For cannibalism, average numbers of workers engaged in grooming and feeding the corpses were counted (D). Frequencies of sand deposition per minute were shown during burial of decayed nestmates (E) and burial of dead competitors (F). All data were expressed as mean ± SE. Means between groups denoted with the same letter were not significantly different (Tuckey HSD all- pairwise comparisons test in (A), (B), (C), and (E), P > 0.05; Dunn’s all-pairwise comparisons test in (D) and (F), P > 0.05).

Figure 4.3 Summary of behavioral profiles.

Simplified behavioral processes of R. flavipes workers in response to three types of corpses were shown.

Figure 4.4 Differentially expressed genes in response to three types of corpses.

98 DEGs with fold difference ≥ 2, FDR ≤ 0.001 were presented.

Figure 4.5 Dynamic change of differentially expressed genes during three undertaking processes.

Figure 4.6 Comparison of differentially expressed genes in three undertaking processes at different sampling times.

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Figure 4.7 Comparison of gene functions and molecular pathways.

(A) Venn diagram of GO terms annotated in three behavioral processes. (B) Venn diagram of KEGG pathways annotated in three behavioral processes. (C) Number of

DEGs in six broader KEGG pathways. Data were pooled from 30 min, 1 h and 6 h samples for each treatment.

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Chapter 5: Discussion and future research

Summary

In this dissertation research, I integrate ethology with chemical ecology and genomics to address an intriguing biological question in social animals: how they deal with the dead?

Results from behavioral assays developed in Chapter 1 and Chapter 2 showed differential undertaking behavior in the eastern subterranean termite, Reticulitermes flavipes, in response to corpses of different origins and postmortem times. The chemical bases that mediate corpse cannibalism and burial behavior have been identified in Chapter 3, and molecular signatures associated with different undertaking processes were investigated using RNA-seq analysis in Chapter 4. Built on current knowledge, the following are my thoughts for the future research.

Influence of pathogen infection on corpse management

Subterranean termites nest in humid environments, feed on dead wood, and share the habitats with diverse microbial fauna. A variety of opportunistic pathogens could take advantage of termite corpses to grow and spread, but epidemic events rarely happen in termite colonies. Over evolutionary history, it is germane to consider that pathogen is one of the important factors contributing to the development of undertaking behaviors.

However, little is known about the chemical basis for recognition and management of corpses infected with pathogens in termites. Given that pathogen-infected corpses pose an apparent risk, I hypothesize that R. flavipes workers respond differently toward pathogen- infected and non-infected corpses. Due to possible changes of termite physiology caused

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by pathogens, I further hypothesize that pathogen-infected and non-infected corpses release different death cues.

The entomopathogenic fungus, Metarhizium anisopliae, is a widely distributed soil-inhabiting fungus. Using M. anisopliae as biological agents to control termites have been tried for many years, but such application remains unsuccessful (reviewed by

Chouvenc et al. 2011, and references therein). This was partially due to our limited understanding of termite biology, such as the mode of action of M. anisopliae toxicity to termites, and the corpse management strategies in termites dealing with infected bodies.

In the dampwood termites, Zootermopsis angusticollis, workers performed pathogen alarm behavior (substrate vibration) upon contact with M. anisopliae spores. In response, nestmates that were not directly exposed to pathogens were repelled by the vibrational signal (Rosengaus et al., 1999). In the Formosan subterranean termite, Coptotermes formosanus, workers cannibalized dead nestmates killed by M. anisopliae, however, they resorted to burial when the number of infected corpses increased significantly (Chouvenc

& Su, 2012). The superb capability of corpse management in C. formosanus efficiently prevented the spread of this fungus pathogen, and the death of entire colony only happened after mortality exceeded 75% in the colony (Chouvenc & Su, 2012).

Metarhizium anisopliae produces cyclodepsipeptides, destruxins, which have demonstrated insecticidal activity (Huxham et al., 1989). Interestingly, M. anisopliae also produces an indolizidine alkaloid, swainsonine, which is a toxin that causes neurological disorders in livestock (Patrick et al., 1993; Stegelmeier et al., 1999). The behavioral and physiological influence of swainsonine on termites remains unclear, and need to be investigated in the future.

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Causes of death and their influences on corpse management

In nature, death of animals could be resulted from a variety of reasons. The causes of death are associated with different risks, but it is not well known about how these causes influence chemical signatures of dead termites, and whether termites are able to distinguish the differences and respond accordingly. Biotic factors causing mortality of termites include aging, predation, and disease (e.g. parasites, pathogens). The potential influence of pathogen infection was discussed above. Aggressive interactions with predators and competitors during territory intrusion could lead to death or injury of termites. In addition to physical aggression, a recent study in the lab showed that mortality of R. flavipes workers could also be induced by competition threats through exposure to another species, R. virginicus (Tian et al. unpublished). Examples of abiotic factors causing death include harsh temperature (i.e. cold and heat), humidity

(particularly low humidity that causes desiccation), lack of food (that causes starvation), and physical damage of nest (by large predators or natural disasters). A particular interesting question raised from termites is how do they manage insecticide-killed nestmates, since pesticides have been extensively used in termite control (Su et al., 1982).

Termites can benefit from consuming their dead nestmates as long as the corpses are nutritionally valuable. The association of corpse cannibalism with the feeding habit in subterranean termites has been discussed in previous chapters. We predict that cannibalism is the primary means of corpse disposal in R. flavipes, and burial would be employed only when hazardous risks increase to a level that overwhelm the benefits from cannibalism. In addition to burial of decomposed corpses, burial may also happen in the circumstances of pathogen infection and insecticide treatment. Additional studies are

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required to test the behavioral responses in association with causes of death, as well as the chemical bases.

Adaptive values of undertaking behavior

Undertaking behavior is considered as a prophylactic strategy in social colonies, and an adaption to social living. However, the adaptive value of undertaking behavior is poorly investigated. The questions awaiting to be addressed are, if undertaking behavior is restricted or compromised, would fitness value of workers, soldiers, and reproductive individuals reduce; in a long term, would the fitness at colony level be impacted and even colony collapse happen.

In ants and bees, corpse removal could be restricted by artificially narrowing the nest opening, which will lead to failure of workers carrying dead individuals out. In the

European fire ant, Myrmica rubra, restriction on corpse removal resulted in higher mortality in both workers and larvae over 50 days (Diez et al., 2014). To date, this study is the only one that well tested the benefits of undertaking behavior. It remains unclear about whether pathogen or parasite infection would cause higher mortality under the same experimental set up.

Technical difficulties are expected on restricting cannibalism or burial behavior in termites. The same set up in ants should not work for termites, since termites always dispose of corpses inside the nest. Whenever cannibalism is inhibited, burial behavior would be employed as an alternative means, and restricting burial behavior could be challenging. Termites can utilize many resources in their nest as burial materials, including soil, chewed-up food, and feces. A bioassay testing benefits of undertaking behavior in termites may be designed by exposing termites to volatile death cues, without

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allowing termites directly contact the odor source. Experimental designs testing fitness consequences by disruption of undertaking behavior will be tried in the lab, by exposing termites to corpses with or without pathogen infection. We predict that restriction of corpse management in termites will results in higher worker mortality, and that the deleterious impact will reach a higher level when corpses carry pathogens. We also predict that reduced labor force due to worker mortality will further affect colony health, through reduction on altruistic behaviors such as brood care, and care of reproductive individuals. This study will provide insights into the potentials of developing new pest control strategies through the disruption of undertaking behavior in termites.

Comparative studies across social taxa

Behavioral patterns of corpse management have been studied in honey bee, ants, and termites (reviewed in Chapter 1), and the death cues eliciting undertaking behavior have been identified in several ants and a few termites. However, understanding of molecular basis of this social behavior just began. More works are expected on profiling the molecular signatures in response to corpses in multiple species across social and subsocial lineages. Findings from these studies will facilitate comparative analysis, which will provide insights into the evolution of social behavior.

Oleic acid serves as a conserved death cue and widely mediates behavioral responses in arthropods, but olfactory receptors that detect oleic acid have not been identified in any species so far. Our knowledge about the chemoreceptors in social insects is still very limited, even in the honey bee, an extensively studied social system. This research may be advanced by referring to model organisms, particularly Drosophila melanogaster (Stocker, 1994). Analogous research has been conducted in zebrafish,

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Danio rerio, on the identification of the olfactory receptor for cadaverine, a diamine associated with death in vertebrates (Hussain et al., 2013). The identification of receptor for oleic acid will provide molecular bases for further study of neural circuits that link sensation, perception, and behavioral response.

In addition to oleic acid, social insects detect other death cues in corpse management, and the early chemical signatures of death are important releasers of undertaking behavior (Choe et al., 2009). Despite of differences on death cue perception, similarity at the level of genes, gene networks and molecular pathways may accrue in the independent evolution of corpse management. The identification of “undertaking” genes is a challenging process, as the complex behavior is more likely to be governed by a set of genes instead of a single one. Increasing studies have revealed that similar sets of genes are associated with convergent phenotypes (Arendt & Reznick, 2008; Rittschof &

Robinson, 2014). Using a comparative genomic approach, similar brain functional processes modulated by territory intrusion were revealed across distantly related species including honey bee, fish, and mouse (Rittschof et al., 2014). In addition, a recent study revealed that the convergent evolution of castes in Hymenoptera involved overlapped metabolic pathways and molecular functions, instead of shared genes.

In terms of undertaking behavior, both genetic “toolkit” (conserved gene sets) and conserved pathways are possible. In the future, the genetic mechanisms of evolution of corpse management will be elucidated.

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Appendix I: Cuticular hydrocarbons (CHCs) of two Reticulitermes species

Reticulitermes flavipes and R. malletei compete with each other in terms of food and nesting resources. Upon encounter, aggressive interactions between the two species could result in mortality of both sides. We observed that R. flavipes workers buried the corpses of R. malletei, but they cannibalized dead conspecifics regardless of colony origin. We hypothesized that R. flavipes distinguishes corpses of the two species based on CHCs, and we analyzed CHC compositions of one R. malletei colony and two R. flavipes colonies using GC-MS. In the above figure, each retention time indicates a specific CHC.

We found that CHC composition of R. flavipes was consistent between colonies, but different from R. malletei. The difference in CHCs between the two congeneric species is correlated with the differential undertaking responses by R. flavipes, which is in supportive of our hypothesis.

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VITA

EDUCATION 2008-2010 M.S. in Ecology, China Agricultural University, Beijing 2004-2008 B.S. in Life Science, Honors Program, China Agricultural University, Beijing

PROFESSIONAL POSITIONS 2010-present Graduate Research Assistant, Department of Entomology, University of Kentucky 2008-2010 Graduate Research Assistant, Department of Ecology, China Agricultural University 2007-2008 Undergraduate Research Assistant, Department of Food Science, China Agricultural University

SCHOLASTIC AND PROFESSIONAL HONORS 2015 Jeffery P. La Fage Student Award for research on social insect pests ($500), International Union for the Study of Social Insects, North American Section 2015 First place in Poster Competition for the President’s Prize ($175 and one-year free ESA membership), Annual Meeting of the Entomological Society of America, Minneapolis, MN 2013-2015 Student Travel Award ($400 each year), University of Kentucky Graduate School 2015 AAAS/Science Program for Excellence in Science 2014 Monsanto Student Travel Award ($1,830), through the Entomological Society of America, Portland, OR 2014 Second Place in Ten-Minute Paper Competition for the President’s Prize ($50), Annual Meeting of the Entomological Society of America, Portland, OR 2014 First Place in Ph.D. Student Paper Competition ($350), Annual Forum of the Ohio Valley Entomological Association, Columbus, OH 2014 Best Poster Finalist in Student Competition, 10th Kentucky Innovation Entrepreneurship Conference, Louisville, KY 2014 First Place in Ten-Minute Paper Competition (Ph.D.) ($300), Annual Meeting of North Central Branch, the Entomological Society of America, Des Moines, IA 2013 Publication Scholarship ($250, twice), University of Kentucky 2009 Scientific Research Achievement Award, China Agricultural University 2007 Academic Excellence Scholarship, China Agricultural University 2006 Merit Student of Beijing, Beijing

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2005 Outstanding student volunteer at the Centennial Celebration, China Agricultural University

FELLOWSHIPS 2014-2015 Kentucky Opportunity Fellowship ($15,000 + tuition), University of Kentucky 2012-2013 Graduate School Academic Year Fellowship ($15,000 + tuition), University of Kentucky 2011-2012 Graduate School Academic Year Fellowship ($15,000 + tuition), University of Kentucky 2010-2011 Kentucky NSF-EPSCoR Research Scholars Program ($15,000)

GRANTS 2015 Monsanto Research Grant ($5,425): “Pathogen-induced death cue and corpse management in termites”, the Entomological Society of America 2015 William L. and Ruth D. Nutting Student Research Grant ($2,000): “Identifying primary and neotenic queen pheromones in Reticulitermes flavipes”, International Union for the Study of Social Insects

PUBLICATIONS 2015 Sun Q, KF Haynes & X Zhou. Early death cues elicit adaptive cannibalism in subterranean termites. Manuscript submitted. 2013 Sun Q, KF Haynes & X Zhou. Differential undertaking response of a lower termite to congeneric and conspecific corpses. Scientific Reports, 3: 1650. 2013 Sun Q & X Zhou. Corpse management in social insects. International Journal of Biological Sciences, 9(3): 313-321. 2013 Yin Z, Q Sun, X Zhang & H Jing. Optimised formation of blue Maillard reaction products of xylose and glycine model systems and associated antioxidant activity. Journal of the Science of Food and Agriculture, doi: 10.1002/jsfa.6415 2009 Sun Q, Z Yin & H Jing. Color characteristics and radical scavenging activity of four model systems of Maillard reaction between amino acids and monosaccharides. Food Science, 30 (11): 118-123.

ORAL PRESENTATIONS 2015 Sun Q, KF Haynes & X Zhou. Cannibalism or burial: searching for the death cue in a subterranean termite. Annual Meeting of the Entomological Society of America, Minneapolis, MN 2015 Sun Q & X Zhou. Understanding of the worker-reproductive transition in the eastern subterranean termite. Annual Spring Symposium in Ecology, Evolution and Behavior, Lexington, KY 2014 Sun Q. Social strategies for corpse disposal in subterranean termites. Eco-Lunch

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Seminar, Department of Biology, University of Kentucky 2014 Sun Q, KF Haynes & X Zhou. Postmortem chemical cues in differential undertaking behavior of a lower termite. Annual Meeting of the Entomological Society of America, Portland, OR 2014 Sun Q. Undertaking behavior in termites. Three-Minute Thesis, University of Kentucky Graduate School 2014 Sun Q, KF Haynes & X Zhou. Cannibalism or burial: searching for the death cue in a subterranean termite. Annual Forum of the Ohio Valley Entomological Association, Columbus, OH 2014 Sun Q, KF Haynes & X Zhou. Postmortem chemical changes and differential undertaking response in termites. The 30th Annual Meeting of International Society of Chemical Ecology, Champaign-Urbana, IL 2014 Sun Q, KF Haynes & X Zhou. Differential undertaking behavior of the eastern subterranean termite: from corpse cannibalism to corpse burial. Annual Spring Symposium in Ecology, Evolution and Behavior, Lexington, KY 2014 Sun Q, KF Haynes & X Zhou. Postmortem changes in chemical profile and undertaking response in termites. Annual Meeting of North Central Branch, the Entomological Society of America, Des Moines, IA 2013 Sun Q, KF Haynes & X Zhou. Death recognition and undertaking behavior in termites. Annual Meeting of the Entomological Society of America, Austin, TX 2011 Sun Q, X Li, L Tian & X Zhou. Undertaking behavior and its molecular basis in termites. Annual Meeting of the Entomological Society of America, Reno, NV 2011 Tian L, X Li, Q Sun & X Zhou. Genetic understanding of aggression behavior in the lower termite Reticulitermes flavipes. Annual Meeting of the Entomological Society of America, Reno, NV 2011 Sun Q, X Li, L Tian & X Zhou. Undertaking behavior in lower termites. Annual Forum of the Ohio Valley Entomological Association, Frankfort, KY

POSTER PRESENTATIONS 2015 Sun Q, KF Haynes & X Zhou. Gene expression profiling of undertaking behavior in the eastern subterranean termite, Reticulitermes flavipes. Annual Meeting of the Entomological Society of America, Minneapolis, MN 2014 Sun Q, KF Haynes & X Zhou. Differential undertaking response to corpses with different postmortem times in a subterranean termite. 10th Kentucky Innovation Entrepreneurship Conference, Louisville, KY 2011 Sun Q, X Li, L Tian & X Zhou. Undertaking behavior in termites. 7th Kentucky Innovation Entrepreneurship Conference & 16th KY EPSCoR Joint Conference, Louisville, KY 2011 Li X, Z Li, Q Sun, L Tian & X Zhou. Identification and functional characterization of a larvae cuticle protein in the lower termite Reticulitermes flavipes. Annual Meeting of the Entomological Society of America, Reno, NV 130

2011 Li Z, X Li, Q Sun, L Tian, S Jones, M Potter & X Zhou. Functional analysis of genes involved in the caste differentiation in termites. 7th KY Innovation Entrepreneurship Conference & 16th KY EPSCoR Joint Conference, Louisville, KY 2011 Li X, L Tian, Q Sun & X Zhou. Transcriptomic dissection of the eusocial Isoptera. International Social Insect Genomics Research Conference, Shenzhen, China 2010 Sun Q, Zhang Y, Z Sun. Geographical effect on genetic diversity and molecular Evolution of Eisenia fetida in Northern China. The 9th International Symposium on Earthworm Ecology, Xalapa, Mexico 2010 Zhang Y, Q Sun, D Zhang, L Yao & H Xiong. Biodiversity of terrestrial earthworms (Annelida: Oligochaeta) in Shandong peninsula and Liaodong peninsula, China. The 9th International Symposium on Earthworm Ecology, Xalapa, Mexico

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