1 Evolution and Ecology of Termite Nesting Behavior

and Its Impact on Disease Susceptibility

A dissertation presented by

Marielle Aimée Postava-Davignon

to

The Department of Biology

In partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in the field of

Biology

Northeastern University

Boston, Massachusetts

April, 2010

2

Evolution and Ecology of Termite Nesting Behavior

and Its Impact on Disease Susceptibility

by

Marielle Aimée Postava-Davignon

ABSTRACT OF DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of

Philosophy in Biology in the Graduate School of Arts and Sciences of Northeastern

University, April, 2010

3 Abstract

Termites construct nests that are often structurally species-specific. They exhibit a high diversity of nest structures, but their nest evolution is largely unknown. Current hypotheses for the factors that influenced nest evolution include adaptations that improved nest thermoregulation, defense against predators, and competition for limited nest sites. Studies have shown a lower prevalence of pathogens and parasites in arboreal nesting animal species compared to ground nesters. Nest building behavior is plastic and can adapt to changing environments. As termites can detect and avoid pathogens, I hypothesized that the evolution of arboreal termite nests was an adaptation to avoid infection. To test this, bacteria and fungi from nest cores, trails, and surrounding soils of the arboreal nesting Nasutitermes acajutlae were cultured. Abiotic factors such as temperature, relative humidity, and light were measured to elucidate how they influenced the interactions between termites and microbes. Fungi associated with N. acajutlae were identified to determine the potential pathogenic pressures these termites encounter in their nest as compared to the external environment. To determine the effect of nest structure on survival, termites representing the one-piece (Zootermopsis angusticollis), intermediate (Reticulitermes flavipes) and separate (Nasutitermes corniger) type nests were exposed to the fungal entomopathogen Metarhizium anisopliae, and the termites’ survival tracked over a 20-day period in five different artificial nest architectures. A protected nest environment and the benefits of socially mediated immunity within those nests have also been implicated in promoting termite eusocial evolution. In order to test whether immune protein production is socially induced in termites, the SDS-PAGE

4 protein profiles of naïve Z. angusticollis nymphs, and nestmates directly exposed to M. anisopliae were examined following social contact. The results presented in this dissertation demonstrate that arboreal termites have lower nest microbial loads and diversity compared to surrounding soils, and that the degree of social contact as influenced by nest architecture can significantly affect termite survival. This research suggests that pathogens played a role in furthering evolution and ecology of termite nesting behavior, and lays the groundwork for future studies in this area.

5

This dissertation is dedicated to my family.

To my parents, who have loved and supported me through all my endeavors.

To my sisters, who have given me invaluable guidance, strength, and love.

To my brothers-in-law, who watch over and care for me like a true sister.

To my niece, whose laughter and bright smile helped get me through tough times.

I love you all, and could not have done this without you.

6 Acknowledgements

I would like to express my gratitude to the many people without whom this research would not have been possible. My advisor, Rebeca Rosengaus, who introduced me to and led me through the wonderful world of termites, a place to which I otherwise would never have known I truly belonged. My collaborator Claire Fuller for six years of exciting field research, and friendship. My committee members Wendy Smith, Jacqueline

Piret and Gwilym Jones for their advice and support. Mark Bulmer for his expertise, and camaraderie. John Stiller and Erica Waddle for their work on fungal identifications. My fellow graduate students Tamara Hartke, Kelley Schultheis, and Lindsey Reichheld for their input and shoulders to lean on. Comfort Chieh and Danny Dijohnson for culturing countless bacterial and fungal samples. Kayla Hamilton and Charlie Ferranti for their work on the Nasutitermes portion of the nest architecture project. Earthwatch SCAP girls

2005-2008 for their hard work in the field on St. John. All other Rosengaus lab members, and graduate students past and present: you are a rare and genuinely fine group of people, and it has been a pleasure working with you.

Thank you to Huddart and Redwood Regional Parks, and the Smithsonian

Tropical Research Institute for termite collections. Kim Seefeld and the R Development

Core Team for assistance with statistics. The Northeastern Biology Department and NSF

GK-12 program for financial support. This research was funded by an NSF-CAREER grant (DEB 0447316) awarded to Rebeca Rosengaus, an NSF grant (IBN-0116857) awarded to James Traniello and Rebeca Rosengaus, and by the Durfee Foundation through the Earthwatch Student Challenge Awards Program.

7 Contents

Abstract 2

Acknowledgements 6

Contents 7

List of Figures 9

List of Tables 11

1 Introduction 12

1.1 Ancestry and eusocial evolution 13

1.2 Termites in the fossil record 15

1.3 Nest types, classifications, and associated levels of sociality 16

1.4 Nest building behavior 19

1.5 Nest evolution 20

1.6 Selective pressures acting on nest evolution 22

1.7 Pathogenic pressures and the evolution of eusociality 26

1.8 Central aims 28

2 Dynamic interactions between the arboreal Caribbean termite

Nasutitermes acajutlae (Holmgren), its associated microbial

communities, and the environment 33

2.1 Introduction 34

2.2 Methods 37

2.3 Results 42

8

2.4 Discussion 60

3 Fungi naturally associated with the arboreal nesting Caribbean

termite Nasutitermes acajutlae (Holmgren) 66

3.1 Introduction 67

3.2 Methods 69

3.3 Results 72

3.4 Discussion 73

4 The effect of nest architecture on termite susceptibility to a

fungal pathogen 87

4.1 Introduction 88

4.2 Methods 95

4.3 Results 103

4.4 Discussion 111

5 Social induction of hemolymph proteins in the dampwood

termite Zootermopsis angusticollis (Holmgren) 119

5.1 Introduction 120

5.2 Methods 123

5.3 Results 130

5.4 Discussion 139

6 Overall discussion and conclusions 144

Literature Cited 150

9 List of Figures

1.1 Figure modified from Eggleton and Tayasu (2001) depicting the eight

lifeways of termites 30

2.1 Nasutitermes acajutlae arboreal carton nest 43

2.2 Collecting Nasutitermes acajutlae in the field 43

2.3 LogTag data loggers 44

2.4 Examples of fungi cultured from Hurricane Hole trail material

on thiostrepton PDA 49

2.5 Mean log transformed cuticular bacterial CFUs/SA ± SE 50

2.6 Mean log transformed substrate bacterial CFUs/g ± SE 51

2.7 Mean log transformed cuticular fungal CFUs/SA ± SE 52

2.8 Mean log transformed substrate fungal CFUs/g ± SE 53

2.9 Correlations between microbial numbers and temperature 54

2.10 Mean nest (core), ambient (trail) and soil temperatures ± SE 55

2.11 Example traces from LogTag data loggers 56

2.12 Correlation between numbers of fungi and amount of light 57

2.13 Mean nest (core), ambient (trail) and soil % moisture ± SE 58

3.1 Examples of fungal genera associated with Nasutitermes acajutlae 78

3.2 Fungal genera and number of occurrences in core, trail, and surrounding

soil samples of Nasutitermes acajutlae individuals and nests 80

3.3 Relative similarities of fungi among Nasutitermes acajutlae nests

located in different habitats 81

10 3.4 Nodules in a fallen nest of Nasutitermes acajutlae 86

4.1 Natural nests of termites 94

4.2 Example artificial nest architectures 101

4.3 Survival distributions resulting from Cox regressions as a function of

treatment (a), species (b), and nest architecture (c) 106

4.4 Survival distributions as a function of nest architecture 108

4.5 Summary of survival across the five nest architectures for

Zootermopsis angusticollis (Za), Reticulitermes flavipes (Rf) and

Nasutitermes corniger (Nc) 109

4.6 Median nest temperature (a) and % RH (b) across nest architectures 110

5.1 Diagram depicting the timing of hemolymph extractions one and two,

and the combinations of treatment groups 131

5.2 Example hemolymph protein profiles from a NCE individual 134

5.3 Median masses of new proteins across direct exposure treatments 135

5.4 Median numbers of new proteins per individual across direct exposure

treatments 136

5.5 Median masses of new proteins across naïve treatments 137

5.6 Median numbers of new proteins per individual across naïve treatments 138

11 List of Tables

1.1 A comparison of traits used as indicators of social complexity in

termites and their subsocial ancestors 31

1.2 Selective forces implicated in the evolution of social insect nests 32

2.1 Final regression model outputs for bacterial (A) and fungal (B) CFUs 48

2.2 Climatological data for the U.S. Virgin Islands 59

3.1 Identifications of fungi associated with Nasutitermes acajutlae 74

3.2 Jaccard coefficients comparing the similarity of core, trail, and soil

fungal genera 77

3.3 Taxonomic groups of fungi associated with Nasutitermes acajutlae 79

4.1 Survival parameters for control and conidia exposed termites of the

three study species 107

12 Chapter 1 – Introduction

Termites are eusocial roaches (Inward et al., 2007a) and are unique in that they are the only roaches to have evolved eusociality. Termites are of particular interest in studies of eusociality because 1) they are the only insects to have developed eusociality outside the order Hymenoptera, and 2) unlike Hymenopterans, which have haplo-diploid sex determination, termites are diplo-diploid and thus lack the genetic predispositions for the evolution of sterile castes (Thorne, 1997). Investigating the evolutionary history of termites and eusocial development is difficult due to the lack of extant solitary species within the order Isoptera. Instead, factors promoting eusociality must be inferred through studies of the fossil record, the presocial roach ancestors of termites, and different aspects of the biology of extant species, including the degree of complexity of their nests. Nest building is an innate behavior (Emerson, 1938; Theraulaz et al., 1998, 2003), often resulting in species-specific architectures. These nests are the result of the cooperative effort of a large number of individuals working together to build, organize, and expand the structure. In other words, nests represent an extended phenotype of a superorganism

(Dawkins, 1999; Turner, 2004). Studies of extant termites have shown a correlation between increasing sociality and larger colonies with more complex architectures

(Eggleton and Tayasu, 2001). When preserved as fossils, nest structures provide a historical record of the degree of social complexity of the builders (Hasiotis, 2003). As such, nests provide a useful tool for indirectly studying eusocial evolution in insects.

Although ubiquitous among social insects, the nests of termites in particular exhibit a wide diversity of structures. This diversity is not only related to differences in

13 social complexity, but also to adaptations evolved in response to the needs of the colony: defense, food storage, and a homeostatic environment necessary for survival and growth of offspring (Emerson, 1938; Noirot, 1970; Noirot and Darlington, 2000). Evolution of social insect nest architectures across genera has been studied to a certain degree in bees

(Eickwort and Sakagami, 1979; Packer, 1991; Franck et al., 2004; Roubik, 2006;

Rasmussen and Camargo, 2008), and wasps (Jeanne, 1975; Wenzel, 1991) likely due to the ability to compare a wide range of solitary to eusocial extant species. In ants, evolution of diverse nesting strategies in the genus Polyrhachis has been examined

(Robson, 2004; Robson and Kohout, 2005, 2007). Tschinkel (2003, 2004, 2005) and

Mikheyev and Tschinkel (2004) have done extensive work on the nest architectures and ontogeny of subterranean ant nests that provide vital information for analysis of trace fossils. However, studies of phylogenetic relationships of termite nests are few, leaving the evolution of the most diverse nest structures largely unexplored (Schmidt 1955, 1958;

Abe, 1987; Inward et al., 2007b). This work reviews what is currently known regarding social insect nesting and evolution, explores the interrelationships between pathogens and termites in nests and the role nests play in pathogen resistance, and the possible impact of pathogens as selection pressures in the evolution of termite nesting behavior.

1.1 Ancestry and eusocial evolution

Recent phylogenetic analyses of extinct and extant species have confirmed that roaches of genus Cryptocercus are the living sister group to termites (Lo et al., 2000;

Inward et al., 2007a; Engel et al., 2009). Morphologically speaking, Mastotermes darwiniensis Froggatt most closely resembles cryptocercid roaches, and is considered to

14 be the most primitive extant termite. However, in terms of social behavior, the extant

Termopsidae (recently renamed Archotermopsidae by Engel et al., 2009) exhibit the most primitive condition. Similar to members of Archotermopsidae, colonies of C. punctulatus live in networks of mostly horizontal galleries in dead wood of varying decay (Nalepa,

1984). Members of these two taxonomic groups sometimes occupy the same piece of wood, and share similar nesting habits: there is no evidence they leave the nest to forage for food. Instead, they consume wood they excavate from their nest, and digest it by means of symbiotic cellulolytic gut microorganisms (Cleveland et al., 1934; Bell et al.,

2007). Cryptocercus punctulatus exhibits the presocial condition by nesting in family groups with overlapping generations that cooperate in brood care (Seelinger and

Seelinger, 1983; Nalepa, 1984). A factor that separates subsocial cryptocercids from eusocial termites is that the former lack sterile castes, while termite workers not only remain in the parental nest, but also forego their own direct fitness to help raise the next generations of siblings (Wilson, 1971). It is not clear why extant cryptocercids have not evolved eusociality, but it is apparent that wood-feeding and colonial nesting preceded termite sociality. Nests under bark provide protection from the external environment, promote inbreeding through restricted movement, and also are costly to construct, all of which may have encouraged persistence of social behavior (Hamilton, 1978; Hansell,

1987). As a result, the nests of their presocial ancestors may have been a preadaptation for social evolution in termites.

15 1.2 Termites in the fossil record

Evidence of insect eusocial evolution is preserved in the fossil record. Termites were the first animals to evolve eusociality, predating ants by roughly 35 million years.

However, there are gaps in the fossil history because not all species lived in environments conducive to fossil preservation. Moreover, there are surely specimens yet to be found.

Only recently have scientists begun to analyze relationships between fossil species of termites and the 2,958 extant species (Engel et al., 2009). The oldest specimens of eusocial termites come from the Early Cretaceous, between 137-130 mya (Martínez-

Delclòs and Martinell, 1995; Rasnitsyn, 2008), but termites are believed to have diverged from roaches in the Late Jurassic, ~156-145 mya (Engel et al., 2009). Most fossil termites found are winged reproductives preserved in amber, or as depressions in limestone

(reviewed by Engel et al., 2009). One worker was found from the Early Cretaceous

(Martínez-Delclòs and Martinell, 1995) and soldiers have been found in Miocene (~23-5 mya) Dominican amber (Krishna and Grimaldi, 2009). These individual termite specimens demonstrate the existence of eusociality by the presence of differentiated castes and winged reproductives, which are absent in presocial roaches. They can also be compared to extant species to gauge what other common characteristics these species share. However, individual specimens cannot provide evidence for how large or complex their colonies were. Only fossil nests can show the dynamics of extinct superorganisms.

Compared to fossil insects, diagnosing structures within the fossil record as termite nests is open to interpretation. Bordy et al. (2004) reported having found an advanced fossil termite nest from the Early Jurassic (~200-175 mya) with evidence of fossil fungus gardens. Despite termite origins possibly dating back to the Jurassic, termite

16 body fossil evidence and estimates of divergence times have calculated mound-building and fungus cultivation to have evolved much more recently (Genise et al., 2005; Engel et al., 2009). Currently, the oldest confirmed termite structure dates back to the Early

Cretaceous, about the same age as the oldest termite body fossils (Francis and Harland,

2006). This find was a permineralized bored chamber within conifer wood. Although only a simple chamber, possibly part of a larger structure, it was identified as a termite excavation by the cylindrical, subhexagonal fecal pellets within the boring. Although cryptocercids also excavate galleries in wood (Nalepa, 1984), only extant termites are known to excrete fecal pellets of this shape (Genise, 1995). Fossil nests similar to those currently constructed by members of Hodotermitidae and Mastotermitidae have been found dating back to the Late Triassic, ~228-200 mya (Hasiotis and Dubiel, 1995). Other, more complex fossil nests have been found in the Chad basin, Africa dating ~7-3 mya.

These included not only excavated galleries, but also structures obviously constructed and having a complexity akin to today’s Macrotermitinae, part of the most derived termite family Termitidae (Duringer et al., 2007). The range of ichnofossils, from simple to complex, has proven to be a useful interpretive tool for tracking social evolution through the fossil record (reviewed by Hasiotis, 2003).

1.3 Nest types, classifications, and associated levels of sociality

Abe (1984) identified six nesting trends based on different nest systems, and the feeding habits of the nest inhabitants: a) Drywood termites, which nest in and consume only the dry, hard wood in which they live (mainly the Kalotermitidae); b) Dampwood termites, which feed exclusively on the wet, decaying wood in which they live (the

17 Archotermopsidae, and some Rhinotermitidae); c) Intermediate termites, which nest in wood and construct galleries in the soil or on the ground, and consume other wood sources in addition to the nest itself (Mastotermitidae, some Kalotermitidae, most

Rhinotermitidae, and some Termitidae); d) Arboreal termites, which nest on living tree trunks and construct covered galleries leading to food sources completely separate from the nest (many Termitidae); e) Subterranean termites, which make epigeous and /or subterranean nests, also constructing galleries to food sources separate from the nest

(Hodotermitidae, some Rhinotermitidae, Serritermitidae, and many Termitidae); and f) humus feeding termites, which make epigeous or subterranean nests and construct galleries to access humus food sources (many Termitidae). These trends were dubbed life types, and can be further grouped into three main categories of nests: one-piece, intermediate, and separate types of nests (Abe, 1987).

Similar to their cryptocercid ancestors, termites from the basal Archotermopsidae live in and consume the same piece of decaying wood, excavating galleries that result in a relatively simple nest architecture (Abe, 1987). These more primitive termites, also part of the lower termites, are distinguished by having flagellated gut protozoa for cellulose digestion (Krishna, 1969), and having “functional” workers that are not completely sterile. Instead these pseudergates (also known as false workers) have a highly flexible development, and retain the ability to achieve reproductive status when the primary queen and king die (Thorne, 1997). In the higher termites of the Termitidae, a true worker caste is present. These workers have lower postembryonic developmental flexibility, no external sexual characteristics present, and in most species have no reproductive potential

(Noirot, 1969). The nests in this family vary considerably in size, shape, materials used,

18 as well as methods of construction. For the termite species used in this research, and their subsocial cryptocercid ancestors a summary of these life history traits relevant to nesting and social complexity are listed in Table 1.1.

Important correlations have been drawn between termite nest type, presence or absence of true workers, and feeding ecology as useful indicators of the social complexity of a species (Inward et al., 2007b). Donovan et al. (2001) created a new classification of feeding groups correlating substrate consumed with the morphology and anatomy of worker termites. Group I consists of wet and dry wood, grass, and detritus feeders with one-piece or intermediate nest types. Group II includes wood, fungus, grass, detritus, litter, and microepiphyte feeders with intermediate or separate type nesting. Groups III and IV include soil-wood interface and soil feeders, respectively. These last two groups essentially have separate type nests, but occasionally feed on them, thereby confounding the original definition of a separate type nest.

Eggleton and Tayasu (2001) combined the life types from Abe (1987) and feeding groups from Donovan et al. (2001) to synthesize a single classification scheme (dubbed

‘lifeways’) encompassing all these factors (Figure 1.1). In this new scheme, one-piece nesting in either wet or dry wood remains only in the most basal clades. Feeding groups

II, III, and IV are only found in the Termitidae. Abe’s (1987) distinction between arboreal, epigeal, and subterranean separate type nests was not incorporated into the lifeways categorization. Although the lifeways are more complete classifications of the functional diversity of termites, Abe’s (1987) life types are still frequently used to differentiate termite nesting strategies.

19 1.4 Nest building behavior

In contrast to the immense structures they build, individual termites are remarkably small and have relatively simple brains. The ability of a termite colony to construct complex architectures despite the simplicity of its individuals is a conundrum not completely solved. The steps contributing to nest construction in social insects are explained extensively in Theraulaz et al. (1998, 2003), but will be outlined briefly here for the purposes of this study.

Nest building in complex insect societies is innate. It is not the result of any one individual containing knowledge of how the resulting structure should look, but rather the combined efforts of a number of individuals responding to a set of stimuli, with no central control. Nest building can be summed up by three mechanisms: response to environmental stimuli, stigmergy, and self-organization. Construction begins when an environmental stimulus, such as a humidity or hormone gradient, acts as a template and signals where an individual should begin construction (Bollazzi and Roces, 2007). An example is the strength of pheromone given off by termite queens, which decreases as the pheromones diffuse away from her. Workers react to the strength of that pheromone when constructing a royal chamber, and begin construction based on their distance from the queen (Bruinsma, 1979; Theraulaz et al., 1998).

As constructions initiated by various colony members progress, stigmergy comes into play. Stigmergy describes the indirect task coordination and regulation of building activities, and was first introduced by Grassé (1959). When a configuration initiated by one individual is encountered by another, it stimulates a particular action in the second individual. Essentially, actions of individual colony members provide the feedback

20 necessary to stimulate the next stages of construction, and the mode of action depends on the stimulus. Finally, self-organization describes a set of dynamic mechanisms by which structures are created from a homogenous substrate through nonlinear amplification of random fluctuations (Nicolis and Progogine 1977, 1989). In other words, the rules by which units of a system operate are executed based on local information, with no knowledge of the whole pattern. In combination with stigmergy, self-organization allows for several possible stable states, creating flexibility that allows for plasticity in building behavior. In addition, as colony sizes grow, the increasing number of interacting individuals allows for increasing degrees of complexity in building. New individuals, new constructs, and changes in the environment all can result in new stimuli that trigger building activity. These simple steps help explain the increasing complexity of nests in more derived termites, and the incredible adaptability of termite nesting behavior.

1.5 Nest evolution

Abe (1987) reported trends in the phylogeny of isopteran of nest evolution occurring along two main lines. The more primitive line consisted of one-piece dampwood nest types leading to one-piece drywood nest types of the Kalotermitidae. The line that included the most derived termite family, Termitidae, exhibited an evolutionary trend in which the food source became completely separated from the nest material.

These species therefore must actively forage for food, with individuals leaving their nest in search of new resources. This is accompanied by an increase in the complexity of the nest architecture. While some termitid nests are only slightly more complex than the nesting habits of phylogenetically primitive one-piece nesting termites, many derived

21 termite species build highly complex mound nests with built-in ventilation systems, or arboreal nests built on or within living trees (Noirot, 1970).

There also appears to be a gradual progression of some termitid species evolving to build nests more removed from the soil environment. Noirot and Darlington (2000) observed such a trend, and suggested that termites incurred a cost of elevated predation risk at the ground level when they began to forage outside of the nest. This was followed by a progression of evolutionary stages during which the subterranean portion of mound nests became reduced, moved completely above ground, and finally into trees with only covered foraging trails still connecting them to the ground. Arboreal nests have evolved independently in several termite species, but Nasutitermitinae has the highest incidence of arboreal nesting, specifically in the genus Nasutitermes. The nests of these species are constructed largely of stercoral carton, or feces, cemented with salivary secretions

(Noirot, 1970). In fact, arboreal nesters are some of the rare termite species that do not include soil as a major constituent of their nests (Lee and Wood, 1971). As arboreal nesting occurs so commonly in members of Nasutitermes, it is an excellent representative for studies of this more derived mode of nesting behavior.

Relatively few studies have formally addressed nest evolution in termites.

Emerson (1938) discussed the use of termite nests as illustrations of behavioral evolution, and noted the convergent evolution of certain nest structures, such as the rain shedding structures of the Amitermitinae, Termitinae and Nasutitermitinae. Schmidt (1955, 1958) constructed a hypothetical phylogeny for Apicotermes combining morphological characters of both the termites and nest characteristics. Grigg (1973) hypothesized and

Korb (2003a) confirmed that the orientation of Amitermes mounds evolved as an

22 adaptation to environmental conditions, such as maximizing heat trapping for nest thermoregulation. Lepage (1984) observed trends in distribution, abundance, and densities of Macrotermes bellicosus nests on the Ivory Coast, and proposed influences on the success of colony foundation and adult nests. Inward et al. (2007b) incorporated

Abe’s (1987) life types into a more current phylogenetic tree. There have been no comprehensive termite phylogenies constructed of multiple genera incorporating nest characteristics, and no empirical research testing hypotheses regarding evolution of arboreal nesting.

1.6 Selective pressures acting on nest evolution

The ancestral condition of many social insects was ground nesting (Michener,

1964; Hung, 1967; Cowan, 1991; Inward et al., 2007b; Rasmussen and Camargo, 2008), and since then a number of social insect species have evolved arboreal nesting habits

(Abe, 1987; Wenzel, 1991; Robson and Kohout, 2007; Rasmussen and Camargo, 2008).

In many ways, the soil provides several advantages for animals: protection from above- ground predators, protection from light, buffering from ambient temperature and moisture fluctuations, and greater access to food supplies of living and dead plant material (Lee and Wood, 1971). As such, arboreal nesting species must have incurred certain costs when they transitioned to exploit this new niche. By nesting away from the soil, arboreal termite species have reduced their interaction with the protective soil environment, making them more susceptible to desiccation (Collins, 1969), and different predatory pressures (Lubin et al., 1977; Lubin and Montgomery, 1981; McMahan, 1982, 1983;

Schatz et al., 1999; Colli et al., 2006; Souza and Moura, 2008). In the derived arboreal

23 nesting species of Termitidae, only workers and soldiers involved in foraging away from the nest have maintained any contact with the soil environment (Noirot and Darlington,

2000). All other members of the colony remain within the homeothermic and protective arboreal nest structure.

In order to offset potential costs of abandoning the protective characteristics of nesting in the soil, there must have been selective advantages that fostered the evolution of arboreal nesting. Several factors have been suggested as potential selective forces in the evolution of social insect nesting, and extant solitary relatives (Table 1.2). These factors fall into three non-mutually exclusive categories: environment, predation, and competition. Schmidt (1955) postulated that progressive narrowing of conduits in

Apicotermes nests did not influence ventilation, so may provide protection from rainwater, predators, or fungi. Michener (1964) in his study of bee nest evolution suggested the importance of available substrate on gross nest form. Jeanne (1975) emphasized the strong role of ant predation in tropical social wasp nest evolution. Packer

(1991) found building of a cavity around brood cells to be a derived feature in sweat bees, a likely response to prevent water logging. Noirot and Darlington (2000) hypothesized that predation was an important selective factor in termite nest evolution.

Roubik (2006) determined nest site limitation to be the overriding factor influencing

Trigona nesting. Finally Bollazzi et al. (2008) found that Acromyrmex ants use soil temperature as a building cue, and that nesting habits are correlated with soil temperature regimes. I propose that for reasons similar to why predation is considered an important factor in nest evolution, parasites and pathogens should be included as potential selective pressures in termite nest evolution. Soils are rich in organisms pathogenic to termites

24 (Evans, 1982; Schmid-Hempel, 1998), and therefore I put forward the novel hypothesis that evolution of termite nests away from the soil was likely an adaptation to avoid infection risks.

Parasites and pathogens are strong candidates for consideration as selective forces acting on termite arboreal nest evolution, as several of the above proposed factors may not be applicable to them. For example, although several studies have demonstrated the influence of abiotic factors on termite nest site selection and mound architecture (Korb and Linsenmair, 1998a, 1998b, 1999, 2000a, 2000b; reviewed by Korb, 2003b), mound- building Nasutitermes species have demonstrated the ability to thermoregulate their nests, dependent on metabolic heat and water rather than the external environment (Fyfe and

Gay, 1938; Holdaway and Gay, 1948). It is possible that this thermoregulatory control predated arboreal nesting, and the ancestors of arboreal termites would not have needed to evolve their nesting strategy in response to ambient climate conditions. In addition, environmental factors that may be attributed to influencing termite nesting may also influence the microbial communities associated with them. With respect to predation avoidance as a reason for transitioning to the arboreal lifestyle, Nasutitermes species are still preyed upon in the arboreal habitat by several animals including anteaters, lizards, ants, and hemipterans (Lubin, 1977; Lubin and Montgomery, 1981; McMahan, 1982,

1983; Schatz et al., 1999; Colli et al., 2006; Souza and Moura, 2008). In order for arboreal nesting to have evolved in response to predation, future studies would need to demonstrate that there are fewer incidences of predation in arboreal nesting as compared to ground nesting species. As for the hypothesis regarding nest site limitation, the nature of arboreal species colony foundation eliminates this as a possibility. Arboreal nesting

25 species such as those in Nasutitermes still initiate their colonies in decaying wood on the ground, similar to more primitive extant termites (Nutting, 1969; Hartke, pers. comm.).

Competition for limited nest sites likely occurs at this stage in colony development, prior to the arboreal stage. A possible evolutionary scenario of the first arboreal nesting termite likely included an already established nest at ground level that subsequently relocated in response to another selective pressure.

Evidence has been accumulating that demonstrates the advantages of arboreal nesting in avoiding pathogens and parasites. In support of Michener’s (1985) hypothesis,

Wcislo (1996) found that ground nesting bee species often have higher rates of parasitism than twig nesting species. Kurihara et al. (2008) collected a lower number of entomopathogens from arboreal soil arthropods as compared to ground dwelling arthropods. In addition, terrestrial Polyrhachis spp. (ants) were found to harbor higher loads of bacteria on their cuticles and in their nests than arboreal nesting species (Robson, pers. comm.). Hölldobler and Engel-Siegel (1984) noted that a number of arboreal ant species have atrophied metapleural glands, which produce antimicrobial secretions. They speculated that these species were less exposed to microorganisms than terrestrial species, and therefore the metapleural gland was secondarily lost. This pattern appears to be consistent even in mammals where ectoparasite loads in sympatric mice species showed that ground nesting Peromyscus gossypinus were more heavily parasitized than the arboreal nesting Ochrotomys nuttalli (Durden et al., 2004). To date, there have been no studies of the composition of microbial communities associated with arboreal nesting

Termitidae species.

26 There is also evidence that parasites and pathogens can influence nest site selection, or whether a nest is abandoned. Rosenheim (1988) observed parasites to be an important cue in whether the solitary digger wasp Ammophila dysmica completed or abandoned its nest excavation. Holt (1996) found a strong correlation between densities of mound-building termites in Australia and soil microbial biomass. He attributed this correlation to competition for resources, but Cruse (1998) postulated that it could also be a response to pathogenic pressures. Termites are also known to detect and avoid parasites and pathogens (Kramm et al., 1982; Epsky and Capinera, 1988; Staples and Milner,

2000; Mburu et al., 2009) and as their nest building behavior is adaptively plastic, it is within their capability to adjust their building behavior in response to pathogens present within their environment.

1.7 Pathogenic pressures and the evolution of eusociality

Pathogens and parasites have already been implicated as selective pressures in the evolution of eusociality (Rosengaus and Traniello, 1993). Special attention has been paid to termites in research of insect-pathogen interactions. As eusocial organisms, termites engage in behaviors such as mutual grooming, proctodeal feeding, oral trophallaxis, cannibalism, and pathogen alarm behavior within their nests that can contain from thousands to millions of densely packed individuals (reviewed by Rosengaus et al.,

2010). Some of these behaviors result in close contact between individuals that could facilitate transmission of pathogens (Kramm et al., 1982; Schmid-Hempel and Schmid-

Hempel, 1993; Zoberi, 1995; Rosengaus and Traniello, 1997; Milner et al., 1998a;

Rosengaus et al., 2000a; Hughes et al., 2002; Fefferman et al., 2007). However, research

27 on adaptations termites have evolved in order to resist disease has shown that these same behaviors may also be important in controlling disease risks (Rosengaus et al., 1998a,

2000a; Rosengaus and Traniello, 2001). Allogrooming appears to help prevent infection by mutual removal of pathogens from the cuticle of nestmates (Strack, 1998; Milner et al., 1998b; Rosengaus et al., 1998a, 2000a; Myles et al., 2002a; Shimizu and Yamaji,

2003; Yanagawa and Shimizu, 2007; Yanagawa et al., 2008). Cannibalism may eliminate the source of infection within a colony by the consumption, and thus removal of sick individuals within the nest (Strack, 1998; Rosengaus and Traniello, 2001; Chouvenc et al., 2008). Proctodeal feeding and oral trophallaxis may allow for the transfer of immunity among individuals (Traniello et al., 2002; Hamilton and Rosengaus, 2010).

Individuals in direct contact with a pathogen also exhibit a vibratory display to signal their nestmates to stay away from the source of infection (Rosengaus et al., 1999a;

Myles, 2002a).

In addition to behavioral responses, there is also evidence of biochemical and immunological adaptations used by termites. Fecal pellets and sternal gland secretions of

Zootermopsis angusticollis, and defensive secretions of soldiers of Nasutitermes have both been shown to significantly reduce conidia germination of Metarhizium anisopliae

(Rosengaus et al, 1998b, 2000b, 2004). Termites exhibit immune priming that reduces their susceptibility when challenged with active and lethal doses of pathogens

(Rosengaus et al., 1999b). Recently, it has been demonstrated in Z. angusticollis that novel proteins in the hemolymph can be induced by exposure to M. anisopliae, and it is possible that this humoral immunity may be induced in naïve nestmates that have not been directly exposed to a pathogen (Traniello et al., 2002; Rosengaus et al., 2007). It

28 was also recently discovered that Nasutitermes corniger incorporate β, 1-3 glucanase in their nest carton, which cleaves and releases components of pathogens (such as glucans) that may in turn immunologically prime colony members against infection (Bulmer et al.,

2009). The improved ability of termites to resist disease when nesting in groups is believed to have been a major factor in the evolution of eusociality, having provided a benefit that would promote offspring remaining in the parental nest (Rosengaus and

Traniello, 1993, 2001; Rosengaus et al., 1998a; Traniello et al., 2002). However, the molecular basis of immune priming against subsequent infections in termites still remains to be determined, and would provide further confirmation of the significance of pathogens in termite eusocial evolution.

1.8 Central aims

The aim of this research was to establish whether pathogens are plausible candidates as selective pressures in the evolution of termite arboreal nesting, and the importance of nest architecture to termite survival against pathogens. In addition, possible mechanisms for the socially mediated immunity important to termite eusocial evolution were tested.

In order to determine whether microbial pressures differ in the soil as compared to the arboreal nesting habitat of termites, I quantified the bacterial and fungal communities within and surrounding Nasutitermes acajutlae nests (Chapter 2). I also measured several environmental factors to determine which abiotic parameters influenced these microbial communities and their relationships with the termites. This was the first study to examine the potential microbial pressures associated with a derived arboreal nesting species. In

29 Chapter 3, a partial identification of the culturable fungi associated with N. acajutlae was carried out in order to determine whether N. acajutlae encounters different pathogenic pressures when foraging on the ground as compared to the intra-nidal fungal load and diversity. If lower numbers of pathogens occurred in the nest, this would support the proposed anti-pathogen advantage to arboreal nesting in termites.

The diversity of nest structures among isopterans undoubtedly influenced the dynamics of group living and therefore the ability of termites to resist infection. In order to examine the effect of nest architecture alone on disease susceptibility, I tracked the survival of three termite species representing wood-feeding one-piece, intermediate, and separate type nesters (Table 1.1) against the entomopathogenic fungus M. anisopliae in various artificial nest structures (Chapter 4). This was the first time nest architecture was empirically tested as an independent factor influencing termite susceptibility to pathogens.

It has been postulated that a protected nest environment and benefits of socially mediated immunity within those nests were important factors promoting eusocial evolution in termites. In Chapter 5, I used SDS-PAGE gel electrophoresis to test whether immune proteins are induced in naïve Z. angusticollis nymphs through social contact with nestmates exposed to M. anisopliae. Two possible mechanisms for this immune priming were tested: priming by indirect exposure through social grooming, or priming through the passage of immune elicitors during social interactions such as oral trophallaxis or proctodeal feeding. This was the first study to attempt to provide evidence for the mechanism of social immune priming in termites.

30 An overall discussion and conclusions drawn from this work are presented in

Chapter 6.

Figure 1.1 Figure modified from Eggleton and Tayasu (2001) depicting the eight lifeways of termites.

31

32 Table 1.2 Selective forces implicated in the evolution of social insect nests

Selective force Insect group References Environmental conditions Apicotermes Schmidt 1955, 1958 Bees Michener, 1985 Evylaeus Packer, 1991 Acromyrmex Bollazzi et al., 2008 Defense against predation Apicotermes Schmidt 1955, 1958 Tropical social wasps Jeanne, 1975 Termites Noirot & Darlington, 2000 Competition for nest sites Trigona Roubik, 2006

33 Chapter 2 – Dynamic interactions between the arboreal

Caribbean termite Nasutitermes acajutlae (Holmgren), its associated microbial communities, and the environment

Abstract

Arboreal nesting termites play key roles in tropical ecosystems. Previous studies have revealed a lower prevalence of parasites or pathogens in arboreal nesting species as compared to ground nesting insects and mammals. The current study surveyed the culturable bacterial and fungal communities associated with Nasutitermes acajutlae in

June-July 2006 and 2008. Substrate and cuticular microbial loads were measured from the nest cores, trails, and surrounding soils in five habitats. Abiotic factors were measured to determine their effects on the microbial communities. Overall, a lower number of microbes were cultured from core samples when compared to trail and soil samples.

Linear regression models found year, habitat, sample, temperature and light to be significant predictors of microbial numbers. The woodland habitat had the highest microbial loads overall. Termites carried relatively few microbes on their cuticles as compared to the substrates. Bacterial numbers increased while fungal numbers decreased with increasing temperature, and fungi also decreased with increasing light. In addition, higher average rainfall resulted in a greater number of bacterial loads but lower numbers of fungi. These dynamic interactions emphasize the importance of longitudinal studies when studying the microbial communites associated with termites, and support the growing evidence that lower microbial pressures occur in arboreal microhabitats.

34 2.1 Introduction

Termites play vital roles in tropical ecosystems. As ecosystem engineers, their mounds modify habitats in ways that can affect the survival of other species (Mills, 1993;

Jones et al., 1994; Hansell, 2005; Jouquet et al., 2006). They are also major decomposers of organic matter (Holt and Lepage, 2000; Yamada et al., 2005), resulting in accumulation of nutrients in their nests that positively influences other soil biota, and increases primary productivity (Wood and Sands, 1978; Holt and Lepage, 2000;

Dupponois et al., 2005; Jiménez et al., 2006; Barot et al., 2007; Brossard et al., 2007).

Recent concerns over global loss of biodiversity and effects of climate change have strengthened focus on these ecosystems, and resulted in a number of studies being performed to better understand factors influencing keystone species such as termites

(Chapin III et al., 2000; Jones and Eggleton, 2000; Davies et al., 2003; Roisin and

Leponce, 2004; Inoue et al., 2006; Roisin et al., 2006; Araújo et al., 2007; Torales et al.,

2007; Vasconcellos et al., 2007, 2010; Bourguignon et al., 2009; Zimmermann et al.,

2009).

Members of Termitidae, including the Nasutitermitinae, are often numerically dominant in the tropics, and the most abundant wood-feeding group (Martius, 1994;

Miura et al., 2000; Dawes-Gromadzki, 2005; Torales et al., 2007). Many species within this subfamily are arboreal nesters (Noirot, 1970). Arboreal termitaria provide homes for numerous other organisms such as birds (Collias, 1964; Brightsmith, 2000, 2004; Kesler and Haig, 2005), bats (Dechmann et al., 2004), bees (Barreto and Castro, 2007), and ants

(Jaffe et al., 1995). The covered foraging trails provide nutrients for growth of epiphyte orchids (Flores-Palacios and Ortiz-Pulido, 2005), and the termites themselves are food

35 for a number of vertebrates such as anteaters (Lubin, 1977; Lubin and Montgomery,

1981) and lizards (Colli et al., 2006), and invertebrates including ants (Schatz et al.,

1999; Souza and Moura, 2008), and assassin bugs (McMahan, 1982, 1983). Despite their importance, the difficulty of accessing their higher nests has caused arboreal termites to be understudied in comparison to ground dwelling species (Eggleton et al., 1996; Roisin et al., 2006; Bourguignon et al., 2009).

Termites are also of great interest in studies of insect-microbe relationships.

Termites experience a number of both advantageous and harmful relationships with microbes in their natural habitats. Some termites benefit nutritionally from wood that has been partially digested by fungi (Hendee, 1933, 1935), and all termites rely on intestinal or external symbiotic microorganisms for cellulose digestion (Ohkuma, 2003). Microbial communities thrive on nutrients that accumulate in termite nests (Holt, 1998; López-

Hernández, 2001; Fall et al., 2004; Ndiaye et al., 2004; Dupponois et al., 2005; Jouquet et al., 2005; Gutiérrez and Jones, 2006). However, termites and soil microbial communities also compete for similar resources, which may influence termite nesting or abundance

(Holt, 1996). In addition, tropical soils are heavily laden with potential entomopathogens

(Evans, 1982; Schmid-Hempel, 1998), with Cordyceps fungi being some of the most common. Termites are known to detect and avoid pathogens (Zoberi, 1995; Staples and

Milner, 2000; Mburu et al., 2009) and may potentially select nest sites based on the presence of parasites and pathogens (Cruse, 1998).

Pathogens and parasites have been important selective pressures in the lives of termites and other social insects (Rosengaus and Traniello, 1993; Schmid-Hempel, 1998;

Boomsma et al., 2005). The risks of disease transmission inherent in densely packed

36 termite colonies are believed to have influenced the evolution of termite eusociality

(Rosengaus and Traniello 1993; Rosengaus et al., 1998a). Termites have evolved several mechanisms of disease resistance (Rosengaus et al., 1998a, 1998b, 2000b, 2007;

Rosengaus and Traniello, 2001; Myles, 2002a; Fuller, 2007; Yanagawa and Shimizu,

2007; Chouvenc et al., 2008, 2009a, 2009b) that were likely influenced by parasites and pathogens present in their nesting environment (Rosengaus et al., 2003). In addition, the evolution of termite immune proteins was likely driven by pathogenic pressures, reflected at least in part by differences in nesting habits (Bulmer and Crozier, 2004, 2006).

Parasites have also been implicated in influencing other social insect nesting behavior

(Michener, 1985; Rosenheim, 1988; Wcislo et al., 1996).

Arboreal nesting is a derived trait in termites (Abe, 1987; Bulmer and Crozier,

2006; Engel et al., 2009), and the factors influencing the transition of basal species into trees from ground-level nesting are still unknown. Noirot and Darlington (2000) observed a trend in termite evolution of nests progressively reducing contact with the soil. The high incidence of entomopathogens in soils may explain such a transition, and as such soil pathogens were likely involved in the evolution of the more derived nesting habits.

As previously mentioned, termites are able to detect and avoid pathogens (Zoberi, 1995;

Staples and Milner, 2000; Mburu et al., 2009), and nest building behavior is adaptively plastic and adjusts in response to changing environmental factors (Theraulaz et al., 1998,

2003). Unfortunately, the nature of microbial communities associated with derived arboreal nesting termites is unknown. The possibility exists that arboreal nesting is driven by termites’ abilities to perceive microbial abundances. While soils used in the construction of termite mounds often promote microbial growth (Holt, 1998; López-

37 Hernández, 2001; Fall et al., 2004; Ndiaye et al., 2004; Dupponois et al., 2005; Jouquet et al., 2005; Gutiérrez and Jones, 2006), it is unknown whether the same is true for arboreal nests. It is also possible that antimicrobial substances within the nest material (Rosengaus et al., 2000b; Bulmer et al., 2009), or physical separation of the nest from soils reduce the microbial biota and confer an advantage to arboreal nesting.

The aim of this study was to survey the microbial communities associated with an arboreal nesting termite. Samples were taken from the nest and surrounding soils to determine whether bacterial and/or fungal loads changed with distance from the soil.

Lower microbial abundances in nests as compared to soils would be indicative of a possible advantage to arboreal termites in the form of reduced competition or avoidance of potential pathogens. In addition, several abiotic factors (temperature, moisture, light, nest volume, and nest height) were measured to determine how they affect the microbial communities.

2.2 Methods

Study site and species

Fieldwork was carried out on the subtropical island of St. John, U. S. Virgin

Islands during the months of June and July, 2006 and 2008. St. John is a small island spanning 52 km2, and contains a diverse array of habitats. Samples were collected from the five habitats most frequently populated by N. acajutlae: woodland, sparse vegetation, mangrove, dry forest, and moist forest, which were first described by Gibney et al. (2000) and Jeyasingh and Fuller (2004). Nasutitermes acajutlae build large arboreal carton nests

38 (Figure 2.1) and are the only arboreal nesting termites on St. John (Scheffrahn et al.,

2003, personal obs.).

Sample collection and field measurements

To estimate the microbial communities associated with N. acajutlae in their natural environment, samples of nest core material, trail material, soil, and termites from all three locations were collected. A total of 71 colonies (n = 19 woodland, n = 11 sparse vegetation, n = 15 mangrove, n = 13 dry forest, and n = 13 moist forest) were sampled.

Live nests were measured more than one year, but others either died during the course of the study, or were not present the previous year and marked as new. Nest core samples were collected by hammering a soil corer into each nest, and emptying the contents into 4 oz. lidded plastic cups. Care was taken to core the nest above its center so the king and queen in the royal chamber would not be harmed. Trail samples were gathered by breaking off sections of trails into a cup. Workers and soldiers recovered from both core and trail samples were gently collected with a paintbrush (Figure 2.2). Soil samples were collected in 20 mL scintillation vials, and termites foraging in the soil, or in wood on the soil were gathered and placed in a plastic cup. Lightly dampened paper towels were added to each cup, and the cups placed in a cool location to prevent termite desiccation and overheating until the samples could be processed.

In order to observe trends in factors that may influence microbial abundances in a habitat, several physical parameters were measured for each sampled nest: a) The ambient and internal nest temperature and humidity were recorded for a minimum of two-days using LogTag data loggers (Figure 2.3). The datalogger measuring internal nest

39 temperature, and an attached cord, were lubricated with plumber’s grease in order to facilitate removal after termites had two days to reconstruct the nest around them. No grease was added to the data logger sensor so it would not interfere with the measurements. The external datalogger was taped into a plastic box and attached to a tree limb adjacent to the nest. The box was inverted in order to prevent rain from hitting the sensor and interfering with humidity measurements. A slide hammer with a tip the size of the data loggers was specially designed to gain access to the core of each nest for internal measurements; b) The temperature of the soil immediately below each nest was measured using a soil thermometer; c) Soil moisture was measured using a Kelway soil moisture meter; d) The amount of light was measured in foot candles (Fc) using an Extech

Instruments light meter. The average light reaching a nest was calculated by taking measurements above, below, and at four compass points around each nest; e) Nest volume was estimated according to the methods of Levings and Adams (1984) and

Jeyasingh and Fuller (2004) using the equation for volume of an ellipsoid (4/3π x r1 x r2 x r3). The height, width, and depth of each accessible nest were measured using a tree caliper; f) The height of the nest within a tree was visually estimated. Only nests reachable from the ground, from climbing the tree in which a nest was located, or from a

1.8 m stepladder were sampled in this study. The highest nests measured were approximately 3.7 meters off the ground.

Sample preparation and plating

Samples from the field were processed the same day they were collected.

Culturable microbial loads were quantified using a protocol similar to the ones described

40 by Cruse (1998) and Rosengaus et al. (2003). The colony forming unit (CFU) method was used to quantify the relative microbial loads, rather than an absolute microbial density of a particular environment. This is a crude estimation of the actual microbial abundances of these environments, since many microorganisms are unculturable (Liu et al., 2006). However, in order to observe changes in microbial communities in different locations within and across habitats, the relative approximation of microbial diversity is sufficient to reflect these variations (Hughes et al., 2001). In collaboration with Dr. John

Stiller at East Carolina University, specific fungi within the samples were identified using environmental PCR (Chapter 3).

To quantify cuticular microbial loads, ten workers and ten soldiers from each location of each nest were first weighed, then placed in separate sterile 1.5 mL microcentrifuge tubes with 1 mL of sterile 0.1% Tween 80 solution. Termites from the core and trail of each nest were sampled in 2006 and 2008. In 2008 individuals foraging on the ground in the vicinity of each nest were also collected. Microbial loads of nest core material, trail material, and soil were measured in a similar manner as the cuticular loads, only a 0.25 g sample of each was placed in a 1.5 mL sterile Eppendorf tube with 1 mL of 0.1% Tween 80 solution. The tubes were centrifuged at 300 x g at 4 °C for 20 minutes. Following centrifugation the termites or substrate material were removed, and

800 µL of supernatant transferred to a fresh microcentrifuge tube. Two hundred µL of glycerol were added to the stock supernatant to preserve the samples for long-term storage at -80 °C. These samples were transported to Northeastern University for further analyses.

41 Stock samples were plated on two agar types: potato dextrose agar (PDA) with 25

µg/mL of the antibiotic thiostrepton to select for growth of fungi, and trypticase soy agar

(TSA) with 100 µg/mL of the eukaryotic protein synthesis inhibitor cycloheximide to select for growth of bacteria. Twenty µL of sample were evenly distributed on two separate plates (d = 100 mm) of both media types using four sterile glass beads (d =

3mm) each. The plates were incubated for five days, one at 25 °C and one at 35 °C.

These temperatures were selected to mimic average temperatures observed inside and outside of natural nests, and were meant to foster differences in microbial growth based on temperature. After the incubation period, colonies of bacteria and fungi of at least 1 mm in diameter were counted.

Numbers of cuticular CFUs were standardized as a function of surface area (SA) for each individual. Surface area was estimated using Meeh’s formula (SA = kW2/3), where k is a species-specific constant, which is 8 for the termites used in this study

(Haagsma et al., 1996), and W is mass in grams. Substrate CFUs were standardized as a function of the mass of each sample.

Statistical analyses

To determine the effects of temporal and environmental factors on sampled microbial communities, a linear regression model was applied separately to the bacterial and fungal CFU data. Bacterial and fungal CFUs were log transformed to normalize their distributions. The initial models contained the categorical variables year, habitat, location and sample, and the continuous variables moisture, temperature, light, nest volume, and nest height. For the purposes of these models, cuticular CFUs were temporarily converted

42 to units of CFUs/g of termite so they could be directly compared to the substrate CFUs.

In order to eliminate temporal pseudoreplication, nests that were sampled more than one year were only included once in the analyses. For nests that were sampled more than once, the 2008 data were used.

The models were simplified in a backward stepwise manner until all the remaining variables were significant at p < 0.05. The effects of incubation temperature, location, habitat, and year, on the bacterial and fungal CFUs of each sample type

(workers, soldiers, substrate) were analyzed individually using ANOVA. In addition, the continuous variables the model found to be significant were compared across locations and years using ANOVA. A Bonferroni correction (Rice, 1989) was applied to all analyses involving multiple comparisons. All data analyses were conducted using R (v.

2.10.1, R) while all graphs were generated in SPSS 17.

2.3 Results

The regression models revealed year, habitat, and temperature to be significant predictors of both bacterial and fungal growth (Table 2.1). In addition, sample was a significant predictor of bacterial growth (Table 2.1A), and light was a significant predictor of fungal growth (Table 2.1B). Although height was found to be significant in the fungal model, further examination revealed no correlation between height and microbial growth. As a result, height was not included in the final fungal model.

Incubation temperature was not included in the models because it was part of the culturing process rather than an abiotic factor influencing microbial communities in their natural habitat.

43 Figure 2.1 Nasutitermes acajutlae arboreal carton nest

Figure 2.2 Collection of Nasutitermes acajutlae in the field.

44 Figure 2.3 LogTag data loggers. Identical data loggers were used to simultaneously measure temperature and relative humidity inside (a) and outside (b) a nest.

A.

B.

45 Incubation temperature

There were no significant differences in CFU numbers between plates incubated at 25 °C and 35 °C (ANOVA, p ≥ 0.1). The results from both incubation temperatures were pooled for further analyses. However, despite the similar quantity of CFUs, the quality of culturable fungi and bacteria often varied as a result of incubation temperature

(Figure 2.4).

Year

Year was found to be a significant predictor of bacterial CFUs (Table 2.1A), with higher overall numbers of bacteria occurring in 2006 as compared to 2008 (Figures 2.5,

2.6). Bacterial loads differed significantly between years in all samples (ANOVA, p <

0.001). Year was also a significant predictor of fungal CFUs (Table 2.1B), with higher overall numbers of fungi occurring in 2008 as compared to 2006 (Figures 2.7, 2.8). Fungi differed significantly by year only in substrate samples (p < 0.001) and not in worker (p =

0.4) or soldier (p = 0.8) samples. It was interesting to note that bacteria and fungi responded differently in the two years, with bacteria flourishing more in 2006 and fungi increasing in prevalence in 2008.

Habitat

The woodland habitat alone was found to be a significant predictor in the regression models (Table 2.1). Although the woodland had the highest quantity of CFUs across all samples, this pattern did not hold in further analyses between years and across locations (Figures 2.5-2.8). Results from ANOVA tests revealed significant differences

46 across habitats only in bacterial loads of soil substrates after correcting for multiple comparisons (Figure 2.6; p ≤ 0.001).

Nest location

Although nest location (core, trail, soil) was not a significant predictor of microbial loads, some patterns emerged upon further analysis. The differences were not always significant, but core bacterial and fungal loads tended to be lower than trail and/or soil microbial loads in all samples (Figures 2.5-2.8). These differences were more often significant in substrate samples (Figures 2.6, 2.8) with only mangrove workers (Figure

2.7; p = 0.002), and dry forest soldiers (Figures 2.5, 2.7; p < 0.02) differing significantly among termite samples. The trend of core microbial loads being lower than trail and/or soil loads was relatively consistent, with only a few exceptions.

Sample

Sample (worker, soldier, substrate) was found to be a significant predictor of bacterial CFUs (Table 2.1A). In order to directly compare cuticular and substrate CFUs, the cuticular CFUs were converted to CFUs/g. However, this is not an accurate representation of the microbial loads termites harbor, as the samples collected originated from their cuticles and not the entire individual. As a result, the model found soldier cuticular loads be to higher than substrate loads, when in reality the termites carried relatively few microbes on their cuticles (Figures 2.5, 2.7). Overall, it appears that sample is a significant factor in that more CFUs/g were cultured from substrate samples than

CFUs/SA were from workers or soldiers.

47 Abiotic factors

Temperature was found to be a significant predictor of bacterial and fungal CFUs.

Number of bacterial CFUs increased with increasing temperature (Table 2.1A; Figure

2.9A), while fungal CFUs decreased with increasing temperature (Table 2.1B; Figure

2.9B). Overall in 2008, temperature was highest in the core, followed by ambient temperature, with soil being the lowest in 2008 (Figure 2.10). In 2006 soil temperature was unusually high. A different soil thermometer was used that year so it is unclear whether the soil measurements reflect true, unusually high temperatures for that year, or if this was a measurement error. Temperature varied significantly across all locations (p <

0.001), habitats (p ≤ 0.001), and years (p ≤ 0.02). The only exceptions were that core and ambient temperatures in the mangrove habitat (p ≥ 0.5) and ambient temperature in the sparse habitat (p = 0.6) did not differ between years. Temperatures were measured over the course of a two-day period, and traces from the LogTag data loggers revealed that N. acajutlae maintain a relatively constant temperature within their nests (Figure 2.11A).

Light was found to be a significant predictor of fungal loads (Table 2.1B). The number of fungal CFUs decreased with increasing light (Figure 2.12). Light differed significantly across habitats (p < 0.001) and years (p < 0.001). However, there was no obvious pattern revealing that any habitat or either year had more light than the other.

Although moisture was not found to be an important predictor of bacterial or fungal loads, further analyses revealed interesting trends worth noting. The amount of moisture was significantly higher in nest cores than the ambient or soil moisture (Figure

2.13; p < 0.001). In addition, the LogTag traces revealed that N. acajutlae maintained constant and high relative humidity within their nests (Figure 2.11A). The ambient RH

48 was also higher in 2006 than in 2008, and this coincided with a greater amount of rainfall during the months in which this study took place (Table 2.2). Although microbial loads did not show any trends with increasing percent moisture, the higher bacterial loads and lower fungal loads in 2006 did coincide with the higher amounts of ambient moisture and rainfall in that year.

Table 2.1 Final regression model outputs for bacterial (A) and fungal (B) CFUs. Models were run with log-transformed data, so predictions made from model formulae should be back-transformed.

A.

Effect Estimate SE t value p value (Intercept) 1852 58 32 < 0.001 year -92 0.02 -31.9 < 0.001 woodland habitat 0.03 0.09 2.9 0.004 substrate sample -0.2 0.07 -4.1 < 0.001 worker sample -0.3 0.07 -4.1 < 0.001 temperature -0.5 0.009 -5.4 < 0.001

B.

Effect Estimate SE t value p value (Intercept) -349.1 76.8 -4.5 < 0.001 year 0.2 0.04 4.6 < 0.001 woodland habitat 0.4 0.1 3.3 0.001 temperature -0.02 0.01 -2.2 0.03 light -0.001 0.00007 -2.2 0.03

49 Figure 2.4 Examples of fungi cultured from Hurricane Hole trail material in 2008 on thiostrepton PDA. The plate on the left was incubated at 25 °C, the plate on the right at 35 °C. Although similar in number of CFUs, differences in fungal growth as a result of incubation temperature were evident on the two plates in spite of the fact they were streaked with the same sample.

50 Figure 2.5 Mean log transformed cuticular bacterial CFUs/SA ± SE for workers and soldiers. ‘*’ denotes significance at p < 0.02 (ANOVA).

*

51 Figure 2.6 Mean log transformed substrate bacterial CFUs/g ± SE. ‘*’ denotes significance at p < 0.02, ‘**’ significance at p < 0.005, and ‘***’ significance at p < 0.001 (ANOVA).

*** *

*** **

52 Figure 2.7 Mean log transformed cuticular fungal CFUs/SA ± SE. ‘**’ denotes significance at p < 0.005, and ‘***’ denotes significance at p < 0.001 (ANOVA).

**

***

53 Figure 2.8 Mean log transformed substrate fungal CFUs/g ± SE. ‘***’ denotes significance at p < 0.001 (ANOVA).

***

54 Figure 2.9 Correlations between microbial numbers and temperature. Bacterial numbers (A) increased with increasing temperature, while fungal numbers (B) decreased. A.

R2 = 0.001

B.

R2 = 0.002

55 Figure 2.10 Mean nest (core), ambient (trail) and soil temperatures ± SE. Temperatures differed significantly (p < 0.001) across all nest locations both years and in all habitats.

*** *** *** *** ***

*** *** *** *** ***

56

57 Figure 2.12 Correlation between numbers of fungi and amount of light. Fungal numbers decreased with increasing light levels.

R2 = 0.007

58 Figure 2.13 Mean nest (core), ambient (trail) and soil % moisture ± SE. ‘*’ denotes significance at p < 0.02, and ‘***’ at p < 0.001 (ANOVA).

* *** * ***

59 Table 2.2 Climatological data for the U. S. Virgin Islands. The data were acquired from the National Climatic Data Center for the months during which the current study took place. Monthly temperature averages were taken at Cyril E. King Airport station on St. Thomas. Total monthly precipitation measurements were taken from stations at locations on opposite ends of St. John. Average temperature Total precipitation Cyril E. King Airport Cruz Bay Coral Bay June 2006 23.3 4.15 3.73 July 2006 28.8 5.57 8.73 June 2008 28.2 1.33 1.5 July 2008 28.7 1.69 2.26

60 2.4 Discussion

Until now, the nature of the microbial communities present in arboreal carton termite nests in relation to the surrounding environment was unknown. The results of this study reveal for the first time the dynamic nature of interactions between arboreal termites and their microbial consortia. Given the important influence that a changing environment (i.e. temperature, light and moisture levels) has on microbial density, it is imperative to run longitudinal studies across several years. Only in this way can a better and more comprehensive picture of the variable microbial communities that termites interact with be obtained. Taking samples from only one location and only in one season would provide a skewed and unrealistic picture of the true dynamics of these communities.

It has been well established that termite mounds in which soil is a major constituent contain a larger and more diverse microbial community than the surrounding soils (Singh et al., 1978; Keya et al., 1982; Holt, 1998; Kumari et al., 2006). Arboreal termites do incorporate some materials from the surrounding environment in their nests, but the carton is largely made up of masticated wood, feces and saliva (Noirot, 1970). In addition, arboreal nests are only connected to the soil via covered trails so they are not as easily accessible to soil microbial communities. Although not always significantly different from microbial numbers in trails and surrounding soils, in general fewer bacteria and fungi tended to inhabit the nests of N. acajutlae, particularly in substrate samples.

Studies in mice (Durden et al., 2004), and other social insects (Michener, 1985; Wcislo,

1996) have demonstrated a lower prevalence of parasites in arboreal nesting species when compared to ground nesting species. Arboreal ants also have atrophied metapleural

61 glands. Loss of this gland, which produces antimicrobial secretions, may be indicative of a lower prevalence of pathogens in the arboreal microhabitat (Hölldobler and Engel-

Siegel, 1984). Chapter 3 will discuss the identities and potential pathogenicity of some of the fungi collected, and compare them across nest locations.

One exception to the aforementioned trend of lower microbial loads in core samples was the bacterial loads in 2006. That year experienced more rainfall (Table 2.2) and consequently greater ambient RH (Table 2.2; Figure 2.13) than in 2008. Other studies have also noted soil bacterial numbers being more prevalent under wet conditions (Singh et al., 1978; Keya et al., 1982; Castro et al., 2010), more microbes on the cuticles of termites during the wet season (Singh et al., 1978), and fungi being more prevalent during dry conditions (Van Borm et al., 2002). Bacteria depend on water for mobility, when resources have been exhausted. In contrast, fungi are able to grow across air spaces

(Swift et al., 1979). The bacteria appear to have an advantage during wetter conditions, whereas the fungi flourished in drier conditions.

Regardless of moisture and rainfall levels, workers and soldiers carried relatively few microbes attached to their cuticles in relation to those present in their environment.

Termites are effective groomers (Milner et al., 1998b; Rosengaus et al., 1998a, 2000a;

Strack, 1998; Myles et al., 2002a; Shimizu and Yamaji, 2003; Yanagawa and Shimizu,

2007; Yanagawa et al., 2008) and are known to actively groom in galleries containing foragers (Jones, 1980). This social behavior, combined with the known fungistatic activity of the soldiers’ cephalic gland terpenoids (Rosengaus, 2000b; Fuller, 2007), likely explains the reduction of microbes on the cuticles. Although low overall, the cuticular bacterial loads were higher in 2006 than 2008, concurrent with higher rainfall.

62 Termites are highly susceptible to desiccation (Collins, 1969; Krecek, 1969; Singh and

Singh, 1981), so they forage more actively when conditions are moist (Moura et al.,

2006; Fuller, pers. comm.). Greater foraging activity would result in more workers and soldiers picking up microbes from the environment, and carrying them back to the nest.

Unusually, soldier bacterial loads were higher than worker loads in 2006. It could be that due to their smaller size and higher SA/volume ratio, soldiers end up having a similar number of microbes as workers, but more per unit SA. In contrast, soldiers have lower numbers of fungi than workers in 2008 when the fungi were more prevalent. Given that production and storage of antifungal terpenoids is located in the cephalic gland of soldiers, but not workers, these caste differences in fungal loads are not surprising

(Rosengaus et al., 2000b; Fuller, 2007).

In contrast to the pattern observed in the bacterial loads, in 2008 when fungi were more prevalent, the fungal loads in the core substrate did not rise above those of the trail and soil. One reason could be that since conditions were drier, the termites did not forage as actively and carry more fungi back to the nest. An alternative reason could be that

Nasutitermes have evolved unique defenses against fungi. The success of allogrooming has been well documented against fungi (Milner et al., 1998b; Rosengaus et al., 1998a,

2000a; Strack, 1998; Myles et al., 2002a; Shimizu and Yamaji, 2003; Yanagawa and

Shimizu, 2007; Yanagawa et al., 2008), but may not be as effective on bacteria, which are orders of magnitude smaller. In addition to the fungistatic properties of the soldier secretions already mentioned, recently Bulmer et al. (2009) revealed an antimicrobial peptide present in nest material of N. corniger that improves resistance against infection by entomopathogenic fungi. The higher temperatures within the nest (Figure 2.10) may

63 also reduce or even impede the growth of some pathogenic fungi, which are more often successful at ~25 °C (Fargues and Bon, 2004).

It was noted that different species of fungi and bacteria often grew at the different incubation temperatures (Figure 2.4). The same may be true of samples from different nest locations. Bacterial growth was found to increase and fungal growth to decrease with increased temperature (Figure 2.8). The temperatures of the nest core, trail (ambient), and soil differed significantly (Figure 2.9), with temperatures in the core most often being the highest. Termites are well known for regulating temperature within their nests (Luscher,

1961; Grigg, 1973; Korb and Linsenmair 1998, 2000a, 2000b; Korb 2003b), and these temperatures are often higher and more constant than the external environment

(Holdaway and Gay, 1948; Greaves, 1964; Singh and Singh, 1981; Leponce et al., 1995;

Figure 2.11). Recent studies have shown that temperature preferences of pathogenic fungi can vary (Bidochka et al., 2001; Fargues and Bon, 2004) with different lineages occurring in habitats with different temperature constraints. As a result of these temperature differences, in addition to the physical separation of nests from the soil and other factors, it would not be surprising to find the microbial communities within nests of N. acajutlae to be drastically different from the soils in which they forage. Rousk and Nadkarni (2009) found differences in microbial communities of canopy and forest floor soils in temperate coniferous forests, demonstrating divergence between these two sites due to their separation. In a study of fungal communities of soil-feeding termitaria, Kumari et al.

(2006) found only a 6.3% overlap of species between the nests and surrounding soils.

The amount of light reaching a nest could vary easily depending on the circumstances in which it was measured (pers. obs.). Light levels could depend on the

64 time of day, the degree of cloud cover, or how lush the foliage was. Despite this, light was a significant predictor of fungal growth, with fungal CFUs decreasing with increasing amounts of light (Figure 2.12). Duguay and Klironomos (2000) discovered that UV-B radiation could alter the ability of some fungi to successfully decompose leaf litter, and therefore reduce their competitive edge. It is possible that correlation between light levels and fungal growth is the result of increased UV radiation. Light has also previously been implicated in affecting termite nest temperatures (Leponce et al., 1995) and nest thermoregulation can influence nesting strategy (Korb and Linsenmair, 1998a,

1998b). In future examinations of termite nesting behavior, it may be prudent to keep in mind that some environmental factors likely influence termites directly through nest thermoregulation, but also indirectly through the microbial communities they interact with.

Although the woodland habitat was found to be a significant predictor of microbial loads (Table 2.1), CFU numbers did not vary greatly across the five habitats overall (Figures 2.5-2.8). No one habitat consistently had greater numbers than the others.

In addition, although temperature and light were significant in the regression models

(Table 2.1), individually those factors did not explain a large degree of the variation in

CFUs (Figure 2.9, R2 ≤ 0.002; Figure 2.12, R2 = 0.007). Instead, it appears that all these factors significantly influenced the microbial growth in consort. This emphasizes the importance of measuring multiple environmental factors when examining the dynamics of microbial communities.

In conclusion, microbial communities associated with a subtropical arboreal nesting termite appear to be influenced by moisture, temperature, and light. Ambient

65 temperature remains fairly constant both yearly and across habitats so these differences were only important in comparison to core and soil temperatures. The prevalence of microbes within arboreal nests appears to have depended on the amount of rainfall and how that affected termite foraging activity. Since bacterial numbers in the nest may have increased in response to increased foraging and fungi did not, it would seem that termites have adapted more defenses against fungi than bacteria, although this has not been confirmed. At this stage it would be interesting to know whether the fungi associated with N. acajutlae are pathogenic, and whether a higher number of pathogens occur outside of the nest (Chapter 3). In addition it would be useful to compare the microbial communities associated with ground nesting species in the same habitats as arboreal nesting species, particularly ones that also incorporate carton in their building, such as N. ephratae, which is known to build both mound and arboreal nests. This would strengthen evidence that pathogenic pressures influenced (and still influence) termite nesting behavior.

66 Chapter 3 – Fungi naturally associated with the arboreal nesting Caribbean termite, Nasutitermes acajutlae (Holmgren)

Abstract

Fungi and termites often interact in their natural habitats. Although fungi that inhabit soils and ground-nesting termite nests have been frequently studied, those that associate with arboreal nesting species remain relatively unexplored. This work utilized molecular techniques to identify fungi cultured from the arboreal nesting termite

Nasutitermes acajutlae. Samples were collected from the cuticles of termites, and substrates of nest cores, trails and surrounding soils. Fungal isolates were cultured on media with 25 µg/mL of the antibiotic thiostrepton, then identified using environmental

PCR. Ascomycota was the dominant phylum, and Aspergillus and Penicillium the most common genera represented in the samples. Overall, a lower diversity of fungi occurred in core samples as compared to trail and soil samples. Some of the fungi cultured from core samples are known decomposers that are found naturally in soils and associated with other insects, while others may potentially be pathogenic. Due to small sample sizes and the large number of fungi that are unculturable, further confirmation is needed as to the true compositions of these fungal communities. However, this study was the first to attempt identifications of multiple taxa associated with an arboreal nesting termite, and has established a baseline for future research in this area.

67 3.1 Introduction

The natural relationships between termites and fungi span the gamut from mutualistic to parasitic interactions. Examples of the former include fungi cultivated by

Macrotermitinae (Thomas, 1987; Aanen et al., 2002; Ohkuma, 2003; Moriya et al.,

2005), and those in termite guts that aid in digestion (Ohkuma, 2003). Fungi colonizing wood can make it more digestible for some termite species (Hendee, 1935; Smythe et al.,

1971), while other fungi compete with termites as major decomposers of organic material

(Holt, 1996). Because both termites and fungi are important decomposers, a better understanding of their interactions is warranted in order to preserve their biodiversity and ecosystem functioning (Brussard et al., 1997; Roose-Amsaleg, 2004; Dupponois et al.,

2005; Jouquet et al., 2005).

Additionally, fungi make up the largest group of entomopathogens within the

Isoptera (Bulla Jr. et al., 1975; Blackwell and Rossi, 1986). This latter relationship between termites and fungi has particularly piqued the interest of scientists in the area of biocontrol. Efforts have moved toward exploiting the pathogenic effects of fungi in order to develop insecticides safe for humans and non-target organisms (Laird et al., 1990). As a result there is a large demand to isolate and study the ecology of novel naturally occurring entomopathogens (Zoberi, 1995; Milner et al., 1998a; Sun et al., 2003a; Meikle et al., 2005; Wright et al., 2005).

The fungi that colonize ground soils have been studied for decades (Waksman et al., 1928; Martin, 1950; Warcup, 1950; Anderson and Cairney, 2004). It has been estimated that 1.5 million species of fungi exist (Hawksworth, 1991). However, only through the use of molecular techniques has the true diversity of fungi begun to unfold, as

68 a large majority of fungi are still unculturable (Viaud et al., 2000; Bridge and Spooner,

2001; Kirk et al., 2004; Hibbett et al., 2007; Blackwell, 2010). With the aid of these new techniques, mycology research has expanded toward fungal communities above ground

(Kurtzman, 2000). Comparisons of arboreal fungal species to those commonly found associated with ground fauna and soils have yielded striking dissimilarities in fungal abundance and diversity. Kurihara et al. (2008) collected fewer entomopathogens from arboreal-soil arthropods, but discovered an overall greater number of fungi, and two novel species when compared to collections from ground-soil arthropods. Rousk and

Nadkarni (2009) found greater numbers of saprophytic fungi in the canopy compared to the forest floor in a North American temperate wet forest. The unique microhabitats created by arboreal environments appear to foster a large diversity of fungi, and many of these species may be novel due to their isolation from ground species.

The fungal constituents of wood-nesting, subterranean, and mound-building termites have been and continue to be explored (Hendee, 1933, 1935; Zoberi, 1979;

Zoberi and Grace, 1990; Holt, 1998; Milner et al., 1998a; Myles et al., 2002b; Roose-

Amsaleg et al., 2004; Dupponois et al., 2005; Moriya et al., 2005). Despite the large breadth of knowledge on fungi incorporated in soil and mound nesting species, little is known regarding the fungi associated with arboreal nesting termites. Thorne and

Kimbrough (1982) reported on the impact of the parasitic fungus Mattirolella crustosa on colonies of Nasutitermes corniger, N. ephratae and N. columbicus. Hojo et al. (2002) identified a new species of Termitaria fungus on Nasutitermes takasagoensis. Fuller

(2007) was the first to report on some of the fungi that grew from freshly killed workers and soldiers of N. acajutlae. The current study expanded upon those identifications, and

69 included the use of molecular techniques to identify a broader spectrum of fungi occurring naturally with N. acajutlae. Moreover, this study was the first to attempt a broader study of the fungi in an arboreal termite nest, and the first to compare the diversity of fungal communities as a function of location, comparing fungal species associated with the nest core, its corresponding trails and surrounding soils.

3.2 Methods

Thiostrepton potato dextrose agar (PDA) plates with fungi cultured from samples collected on the island of St. John, U. S. Virgin Islands (Chapter 2) were parafilmed, packaged, and sent to Dr. John Stiller at East Carolina University for identification. The samples were from collections in 2007 and 2008, and the resultant identifications from a total of 22 nests. Attempts were made initially to isolate DNA from cuticular and substrate washes so that both culturable and unculturable species could be identified.

However, all efforts to extract enough DNA for analysis were unsuccessful.

Fungal DNA isolated from cultures was added to a reaction tube containing sterile distilled water, a polymerase chain reaction (PCR) buffer with appropriate salt and enzyme concentrations (Promega 2X PCR mix), and a pair of universal fungal ribosomal

DNA (rDNA) primers. Negative controls of PCR cocktails with primers but no DNA template were also used. The reaction was subjected to PCR with a profile of an initial denaturation at 94 °C for four minutes, 94 °C for 20 seconds, primer annealing at 50 °C for 20 seconds, and extension at 72 °C for one minute and thirty seconds plus two seconds per cycle. Steps two, three, and four were repeated 30 times and the reactions held at 72 °C for 10 minutes before being chilled to 15 °C for further analysis.

70 After PCR, 10 µL of the product were run on a 1% agarose electrophoresis gel, along with the negative and positive controls in a 1X sodium borate buffer at 90 volts for

5 minutes, and then 126 volts for 30 minutes. The gel, pre-stained with Gel-Red

(Biotium), was then viewed under ultraviolet light to illuminate and photograph amplified bands.

PCR was done first with the universal rDNA primers 4F (CTGGTTGATYCTGC) and 1787R (CYGCAGGTTCACCTACRG) and then nested PCR with universal rDNA primers 573F (CGCGGTAATTCCAGCTCCA) and 1438R (GGGCATCACAGACCTG-

TTAT). The specific “universal” primers employed were selected using the ribosomal

RNA database, http://bioinformatics.psb.ugent.be, based on the percent match they had with phyla shown previously to be associated with these termites.

An initial attempt was made to amplify rDNA directly by placing a small amount of a given microbe directly into the PCR cocktail. If there was no discrete band for a specific sample from this direct PCR, then DNA was extracted using a Wizard genomic

DNA purification kit and eluted in 16 µL of 1 mM EDTA, 10 mM Tris buffer. Then the two-stage, nested PCR was run on the extracted sample.

If bands were present, a preliminary analysis for potentially unwanted variation in the samples was carried out. The samples were subjected to restriction digest with Msp1 at 37 °C for one hour. The products of the reaction were run on a 1.5% pre-stained agarose gel in 1X sodium borate buffer at 90 volts for 5 minutes, and then 126 volts for

30 minutes to verify that the enzyme was effective.

PCR products were then purified using Qiagen’s MinElute PCR Purification kit and each sample sequenced as per the genomics core facility protocol. The types of fungi

71 were identified using similarity searches (BLAST) against previously sequenced genes in the NCBI complete nucleotide (nr/nt) database, http://blast.ncbi.nlm.nih.gov, and compared among the different colonies to see if any patterns emerged. Some samples could be identified to the genus level, while others had best matches to several genera. In cases where a sequence matched more than one genus, it was identified to the lowest taxonomic group possible.

Statistical analyses

The Jaccard coefficient was used to quantify similarity in fungal community compositions between different sample locations. Differences were calculated between core and trail, core and soil, and trail and soil locations. The Jaccard coefficient was calculated as follows:

CCJ = c/(s1 + s2 – c) where s1 and s2 are the number of species in the communities being compared, and c is the number of species those communities have in common (Mueller-Dombois and

Ellenberg, 1974). In addition, a Euclidean distance model was run to show similarities between fungi occurring in the different habitats. All graphs were generated using SPSS

17.

72 3.3 Results

A total of 84 different fungi were identified, 14 of which were recognized to the genus level. A list of the identified samples as well as photographic examples of some of these genera can be seen in Table 3.1 and Figure 3.1. A few of the remaining fungi were identified to two possible genera. Some of these were genera not included in the original

14, therefore they were added to the table and analyses of community similarity (Table

3.2) in order to better reflect the diversity of fungi collected.

Phylum Ascomycota was by far the dominant group of fungi associated with N. acajutlae in all three locations (Table 3.3). Within this phylum, Aspergillus and

Penicillium occurred ubiquitously regardless of sample location. On the other hand, several genera were unique to a particular location (Figure 3.2). For example,

Byssochlamys only occurred in core samples, as well as the only confirmed identifications of Candida. Baeospora, Bullera/Cryptococcus, Chalara, Didyomocrea/

Letendraea, Hypocrea/Trichoderma, Stereum, and Trichaptum were only found in trail samples, while Kluyveromyces, Rhodosporidium, Thysanophora, and Umbelopsis were only found in soil samples. Overall, core samples had the lowest diversity at the genus level while trails had the highest.

Jaccard coeffiecients of community similarity are listed in Table 3.2. Core and trail communities were most similar at 28.6%. Core and trail communities each shared approximately 21% of fungal genera in common with the soil Although the fungi identified in the present study only reflect a small sample of the entire diversity of fungi associated with N. acajutlae, two main patterns emerged. First, the arboreal fungal communities (originating from nest cores and trails) differed in their diversity from the

73 surrounding soil mycoflora, although they did share some species in common. Second, samples from the cores of N. acajutlae nests appear to have the lowest number of fungal genera (Figure 3.2).

The Euclidean distance model measured relative similarity of fungal composition among nests within each habitat (Figure 3.3). Nests from woodland, sparse vegetation, moist forest and dry forest habitats clustered closely together (blue and white dots), whereas nests from the mangrove habitat were less similar to one another (red dots). The mangrove nest that did not cluster (MGHH) was from a different location on the island than the other mangrove nests, so it is unclear whether these differences were due to the small sample size, or whether they reflect true variation in mangrove fungal communities.

3.4 Discussion

Interactions between termites and fungi range from mutualistic to parasitic. As two major contributors to organic matter decomposition, it is important to understand the dynamics of naturally occurring termite-fungal associations in order to better monitor ecosystem functioning (Yamada et al., 2007). Arboreal termites occupy a niche whose fungal constituents have yet to be fully explored. Previous findings revealed lower numbers of fungi cultured from samples originating from cores of N. acajutlae nests

(Chapter 2). In the current study, a subsample of these fungi was identified using molecular tools. Together, the results indicate that core nest samples are quantitatively and qualitatively different in fungal composition. Nest cores have reduced fungal loads and a lower diversity as compared to trail and soil samples. This is the first study that tackles this question in an arboreal termite species.

74 Table 3.1 Identifications of fungi associated with Nasutitermes acajutlae. The samples are grouped by nest location (CS = core soldier, CW = core worker, TS = trail soldier, TW = trail worker, SS = soil soldier, SW = soil worker, and core/trail/soil represent nest or soil substrate). Samples were identified to the lowest possible taxonomic level. For samples that were identified to two possible genera, the genera were included along with the lowest common taxonomic level. The E values reported from BLAST searches represent the probability that similarity between the BLAST query and subject sequences could have occurred by chance. Results closest to zero represent a low probability that the match occurred due to chance.

Location Sample ID Classification E value Core B4CS Aspergillus sp. Genus 4.E-177 B4CS Eurotiomycetidae Subclass 7.E-124 Trichocomaceae B4Core Family 7.E-170 (Eupenicillium, Penicillium) B4Core Trichocomaceae Family 9.E-179 CB3bCore Eurotiomycetes Class 0.E+00 Cor4CW Candida sp. Genus 0.E+00 Trichocomaceae CT11CS Family 0.E+00 (Paecilomyces, Talaromyces) CT13Core Aspergillus sp. Genus 1.E-161 F14Core Candida sp. Genus 1.E-147 F14Core Candida sp. Genus 3.E-158 HH44CW Byssochlamys sp. Genus 0.E+00 HH46CW Candida sp. Genus 0.E+00 L7Core Byssochlamys sp. Genus 0.E+00 L7Core Trichocomaceae Family 0.E+00 L8Core Byssochlamys sp. Genus 0.E+00 L10Core Pezizomycotina Subphylum 1.E-172 L10Core Ascomycota Phylum 0.E+00 M20CW Candida sp. Genus 0.E+00 M20CS Candida sp. Genus 0.E+00 M20CS Candida sp. Genus 0.E+00 V63CS Penicillium sp. Genus 7.E-53

75 Location Sample ID Classification E value V63CS Sordariales Order 4.E-102 V63Core Pezizomycotina Subphylum 2.E-144 Trail B4Trail Chalara sp. Genus 4.E-32 B6Trail Penicillium sp. Genus 0.E+00 CB3bTrail Penicillium sp. Genus 0.E+00 Tremellomycetes CB3bTrail Class 0.E+00 (Bullera, Cryptococcus) CB3bTrail Pezizomycotina Subphylum 5.E-64 Dothideomycetes CB4bTrail Class 2.E-130 (Didymocrea, Letendraea)

CB4bTrail Tremellomycetes 0.E+00 (Bullera, Cryptococcus) Cor4TS Baeospora Sp. Genus 0.E+00 Cor4TS Baeospora Sp. Genus 0.E+00 Cor4TS Trichocomaceae Family 0.E+00 Pezizomycotina Cor4Trail Subphylum 0.E+00 (Penicillium, Phialocephala) CT11Trail Stereum sp. Genus 1.E-152 F13Trail Aspergillus sp. Genus 0.E+00 F13Trail Trichocomaceae Family 1.E-101 Hypocreaceae HH8Trail Family 1.E-147 (Hypocrea, Trichoderma) HH8Trail Dothideomycetes Class 0.E+00 HH8Trail Pezizomycotina Subphylum 0.E+00 Pezizomycotina HH9Trail Subphylum 0.E+00 (Paecilomyces, Talaromyces) HH9Trail Trichocomaceae Family 7.E-180 HH44Trail Saccharomycetaceae Family 0.E+00 HH44Trail Trichocomaceae Family 1.E-59 L10Trail Trichaptum sp. Genus 0.E+00 L10Trail Ascomycota Phylum 0.E+00 M20TS Ascomycota Phylum 3.E-64 V25TW Pezizomycotina Subphylum 2.E-135 V25TS Aspergillus sp. Genus 0.E+00

76 Location Sample ID Classification E value Saccharomycetales V28TW Order 0.E+00 (Candida, Pichia) V28TS Hypocreales Order 0.E+00 V50Trail Aspergillus sp. Genus 0.E+00 Tremellomycetes V50Trail Class 0.E+00 (Bullera, Cryptococcus) V50Trail Agaricomycotina Subphylum 8.E-128 V63TS Sordariales Order 8.E-99 Trichocomaceae Soil B4Soil Family 0.E+00 (Eupenicillium, Penicillium) B6SW Thysanophora sp. Genus 0.E+00 B6SW Hypocreaceae Family 0.E+00 B6SS Pleosporales Order 0.E+00 B6Soil Umbelopsis sp. Genus 0.E+00 B6Soil Hypocreaceae Family 1.E-157 CB3bSoil Eurotiomycetes Class 4.E-47 Cor2Soil Aspergillus sp. Genus 0.E+00 Cor2Soil Kluyveromyces sp. Genus 0.E+00 Cor4Soil Kluyveromyces sp. Genus 0.E+00 CT7Soil Trichocomaceae Family 0.E+00 CT11Soil Trichocomaceae Family 0.E+00 Mucorales CT13Soil Order 2.E-151 (Mucor, Umbelopsis) F13Soil Penicillium sp. Genus 1.E-166 F13Soil Penicillium sp. Genus 6.E-57 F14Soil Trichocomaceae Family 0.E+00 HH8Soil Aspergillus sp. Genus 0.E+00 HH8Soil Trichocomaceae Family 0.E+00 Trichocomaceae HH8Soil (Hemicarpenteles, Family 4.E-131 Thysanophora) HH8Soil Eurotiomycetes Class 3.E-132 HH44Soil Aspergillus sp. Genus 0.E+00 HH44Soil Trichocomaceae Family 0.E+00 HH44Soil Trichocomaceae Family 0.E+00

77 Location Sample ID Classification E value HH44Soil Eurotiomycetes Class 0.E+00 HH44Soil Pezizomycotina Subphylum 0.E+00 L8Soil Aspergillus sp. Genus 0.E+00 L8Soil Talaromyces sp. Genus 0.E+00 L8Soil Agaricomycotina Subphylum 4.E-69 L10Soil Eurotiomycetes Class 9.E-112 V25Soil Pichia sp. Genus 7.E-88 V25Soil Trichocomaceae Family 1.E-65 V63Soil Rhodosporidium sp. Genus 0.E+00

Table 3.2 Jaccard coeffiecients of community similarity. The coefficients compare the ß- diversity of species from all samples at different locations. Coefficient values represent the degree of similarity at the genus level between locations. trail soil core 0.2857 0.2143 trail - 0.2105 soil -

78 Figure 3.1 Examples of fungal genera associated with Nasutitermes acajutlae: A) Two samples of Aspergillus; B) Two samples of Byssochlamys; C) Candida D) Kluyveromyces; E) Penicillium; F) Talaromyces: G) Thysanophora; H) Trichaptum; I) Umbelopsis. Note that colony sizes in the photos are not to scale.

A

B C

D F E

G H I

79 Table 3.3 Taxonomic groups of fungi associated with Nasutitermes acajutlae. ‘#’ represents the number of fungal colonies represented by each group. Ascomycota was the dominant phylum with 73 out of the 85 isolates belonging to that group.

Taxonomic Group ID # Phylum Ascomycota 73 Family Polyporaceae 1 Basidiomycota 10 Saccharomycetaceae 7 incertae sedis 2 Stereaceae 1 Subphylum Agaricomycotina 9 Trichocomaceae 36 Mucoromycotina 2 Tricholomataceae 2 Pezizomycotina 60 Genus Aspergillus 9 Pucciniomycotina 1 Baeospora 2 Saccharomycotina 10 Bullera/Cryptococcus 3 Class Dothideomycetes 3 Byssochlamys 3 Eurotiomycetes 42 Candida 5 Homobasidiomycetes 4 Candida/Pichia 1 Leotiomycetes 1 Chalara 1 Microbotryomycetes 1 Didymocrea/Letendrea 1 Saccharomycetes 10 Hypocrea/Trichoderma 1 Sordariomycetes 6 Kluyveromyces 2 Tremellomycetes 3 Penicillium 5 Order Agaricales 2 Penicillium/Eupenicillium 2 Aphyllophorales 1 Penicillium/Phialocephala 1 Eurotiales 36 Pichia 1 Helotiales 1 Rhodosporidium 1 Hypocreales 4 Stereum 1 Mucorales 2 Talaromyces 1 Pleosporales 1 Talaromyces/Paecilomyces 2 Russulales 1 Thysanophora 1 Saccharomycetales 10 Thysanophora/Hemicarpenteles 1 Sordariales 2 Trichaptum 1 Sporidiobolales 1 Umbellopsis 1 Family Hyaloscyphaceae 1 Umbellopsis/Mucor 1 Hypocreaceae 3

80 Figure 3.2 Fungal genera and number of occurrences in core, trail, and surrounding soil samples from Nasutitermes acajutlae individuals and nests. Note that the fewest number of genera occurred in core samples.

81 Figure 3.3 Relative similarities of fungi among Nasutitermes acajutlae nests located in different habitats: MGHH = mangrove nest in Hurricane Hole; MFB = moist forest nest on Bordeaux Mountain; MFCT = moist forest nest on Cinnamon Trail; WLFRAN = woodland nest from Francis Bay; WLHH8 = woodland nest 8 from Hurricane Hole; MGL7 = mangrove nest 7 from Leinster Bay; MGCOR = mangrove nest from Coral Bay; MGLEIN = mangrove nest from Leinster Bay; SVV = sparse vegetation nest from VIERS; WLCB3B = woodland nest 3b from Cinnamon Bay; SVMaho = sparse vegetation nest from Maho Bay; DFlein = dry forest nest from Leinster Bay; DFV = dry forest nest from VIERS.

82 The fungi identified belonged to three phyla: Ascomycota, Basidiomycota, and incertae sedis, subphylum Mucoromycotina (phylum unassigned, formerly Zygomycota

(Hibbett et al., 2007)). Members of Basidiomycota and Mucoromycotina are well known degraders. The Mucoromycotina specialize on small sugars, such as glycine, while

Basidiomycota are true wood degraders, tackling the breakdown of cellulose and lignin

(Garrett, 1951; Hanson et al., 2008). Genera belonging to Mucoromycotina were only identified in soil samples from this study. This is perhaps due to the fact that

Nasutitermes nests are composed of masticated wood, and likely don’t contain simple sugars. Thorne et al. (1996) analyzed the nutrient composition of N. acajutlae nest carton, and nodules embedded in the nests (Figure 3.4). The nests and nodules are made of partially digested wood that still contains organic matter, cellulose, lignin, and cutin, which cannot be broken down by the so-called “sugar fungi”. They can however be degraded by basidiomycetes, and some members of that phylum did occur in the core of the nest.

Ascomycetes have various functional roles that include decomposition, various associations with plants, and pathogenicity (Roose-Amsaleg, 2004). Aspergillus and

Penicillium are common fungi in tropical soils (Farrow, 1954), and were abundant across all samples. They have a degree of lignolytic behavior, although much lower than basidiomycetes (Osono, 2007), and are often found naturally associated with termites

(Hendee, 1933; Singh et al., 1978; Zoberi, 1979; Zoberi and Grace, 1990; Roose-

Amsaleg, 2004; Fuller, 2007; Jayasimha and Henderson, 2007). Aspergillus is an opportunistic pathogen, infecting insects when they are stressed or already weakened by injury or another pathogen (Hughes and Boomsma, 2004; Jayasimha and Henderson,

83 2007; personal obs.). Some species of Penicillium are known to infect other social insects

(Schmid-Hempel, 1998; Fernández-Marín et al., 2006), but pathogenicity has not been confirmed in termites.

The order Hypocreales contains the largest number of fungal taxa that infect invertebrates (Humber, 2008). Reports of entomogenous fungi in tropical ecosystems have emphasized the dominance of Cordyceps within this order (Evans, 1982). Genera cultured from N. acajutlae samples that fall within this order were Hypocrea, and

Paecilomyces. While these two genera are pathogenic to insects (Humber, 2000; Inglis and Tigano, 2006; Baird et al., 2007) and perhaps also N. acajutlae, the isolates were not identified to the genus level. They may have been Trichoderma and Talaromyces respectively. Trichoderma species have been isolated from other termite nests (Hendee,

1933; Zoberi, 1979; Zoberi and Grace, 1990; Roose-Amsaleg, 2004), but its role and the role of Talaromyces as termite pathogens are not as clear. Further confirmation of these fungal identifications, as well as susceptibility tests would better confirm their status as pathogenic to N. acajutlae. It should be noted that these pathogenic species, or potential teleomorphs of these species such as Byssolchlamys (Luangsa-ard et al., 2004), were collected from all nest locations.

Candida occurred most often in the core, although it may also have been present in soil samples (Table 3.1). Candida, Kluyveromyces, and Pichia are xylophagous yeasts and known symbionts of insects, including lower termites (Vega and Dowd, 2005;

Nguyen et al., 2006). Candida has also been found naturally associated with other social insects (Baird et al., 2007) and with wood-boring insects, in their frass, or lining their tunnels (Kurtzman and Robnett, 1998; Kurtzman, 2000). Nasutitermes acajutlae nests are

84 composed of feces and contain stores of partially digested wood, either of which could explain the presence of Candida. There is also the possibility that Candida may be pathogenic to N. acajutlae. Insects have demonstrated susceptibility when exposed to

Candida albicans (Hung et al., 1993; Dunphy et al., 2003; Mowlds et al., 2008).

However, studies of antimicrobial defenses of Nasutitermes species have also demonstrated their effectiveness against C. albicans (Cruse, 1998; Bulmer et al., 2009).

Fungi cultured from nests in mangrove habitats were less similar to one another as compared to nests from other habitats (Figure 3.3). One nest from Hurricane Hole in particular was the source of this dissimilarity. No other mangrove nest samples from

Hurricane Hole were included in this study so it is unclear whether these differences in fungal composition were a result of location on the island of St. John, or the mangrove habitat. On the other hand, the close clustering of nests from different island locations within each other habitat alludes to a possible influence of habitat on these fungal communities. The results are inconclusive at this time.

In summary, arboreal Nasutitermes maintain their contact with the ground via covered trails, and likely carry soil fungi back to the nest when foraging (Sands, 1969;

Chapter 2). Despite this, a lower diversity of genera were found in samples from nest cores in contrast to previous studies that have discovered speciose arboreal microflora

(Rousk and Nadkarni, 2009). These differences may be attributed to meticulous grooming behavior (Jones, 1980) or the antimicrobial properties of the nest (Bulmer et al., 2009) and soldier secretions (Rosengaus et al., 2000b; Fuller, 2007). In addition, the lower diversity could be explained by the composition of the nest material, which contains nutrients not digestible by all groups of fungi (Hanson et al., 2008). Moreover,

85 the high and constant temperature maintained inside the nest (Chapter 2) may also restrict the growth of some types of fungi with temperature preferences below 30 °C. The maintenance of high temperatures in termite nests may be analogous to honeybee nest fever, which is initiated in response to infection by chalk brood (Starks, 2000), and may have similar deleterious effects on pathogens. Although, previous research has also demonstrated that microbes may associate with a particular habitat as opposed to a host, and some strains can withstand higher temperatures (Bidochka et al, 2001).

The fungi identified in this study laid the groundwork for understanding the relationships between fungi and arboreal nesting termites. Additional identifications of cultured samples, as well as molecular analyses of unculturable taxa would further clarify the types of interactions between these organisms. In addition, comparisons of fungal communities of arboreal Nasutitermes to those of closely related mound-building and subterranean species would increase understanding of how fungal communities change as a result of nesting behavior and the potential role that fungal pathogens may have played in the evolutionary transition from subterranean- and ground-level nesting to arboreal nest construction.

86 Figure 3.4 Nodules in a fallen nest of N. acajutlae. The nodules (light brown inclusions amongst the nest material) are made up of partially digested wood that is believed to be stored food for the colony. These nodules may also appeal to saprophytic fungi.

87 Chapter 4 – The effect of nest architecture on termite susceptibility to a fungal pathogen

Abstract

Homeostatic nests are a ubiquitous characteristic of termites. Nest building behavior can be unique to species, with some being identifiable by their nest structure.

Previous studies have analyzed in detail the nest architectures of various social insects, and some have examined specific behaviors evolved to defend the nest from predators, pathogens, and parasites. As of yet, the importance of nest structure in defense against pathogenic pressures has not been demonstrated experimentally, despite the high occurrence of these pressures in social insect habitats. To test the effect of nest architecture on termite survival, five artificial structures designed to mimic natural termite nests were constructed from corrugated cardboard. Twenty termites per nest from three species (Zootermopsis angusticollis, Reticulitermes flavipes, and Nasutitermes corniger) with different natural nesting strategies were exposed to either a Tween 80 control solution, or conidia suspension of the generalist entomopathogen Metarhizium anisopliae. Following a 20-day census, the results showed that nest architecture was a significant and independent predictor of survival in all three species. The nest that exhibited the highest survival rate varied across species, and it appears that termite survival was influenced by the particular nest structure a species is adapted to. This research has important implications as to the adaptive role of nest architecture in termite evolution.

88 4.1 Introduction

Historically, high-density groups have been viewed as providing ideal conditions for the growth, development and transmission of pathogens (Freeland, 1976; Hamilton,

1987; Schmid-Hempel, 1998). In response to this, some group-living insects increase their investment in immunity to compensate for increased risk of infection (Wilson and

Reeson, 1998; Barnes and Siva-Jothy, 2000; Cotter et al., 2004; Wilson and Cotter,

2004). Research on social insects however, has indicated that although they nest in densely packed colonies and exploit microbe-rich environments, the risks of pathogen transmission among colony members appear to be less prevalent than originally hypothesized (Hughes et al., 2002). Social insects have evolved multiple strategies at the individual- and colony-levels to help them cope with disease, including behavioral, biochemical and immunological responses (reviewed by Rosengaus et al., 2010; Schlüns and Crozier, 2009). The facilitation of colony-level immunity due to social interactions amongst nestmates (social immunity; Rosengaus et al., 1998a; Rosengaus and Traniello,

2001; Traniello et al., 2002; Cremer et al., 2007; Ugelvig and Cremer, 2007; Rosengaus et al., 2010) may be one reason for the wide distribution of social insects and their ecological success.

Recent growing interest in the study of social immunity (Traniello et al., 2002;

Cremer et al., 2007; Rosengaus et al., 2010) has revealed that upon exposure to a pathogen, social insects will increase behavioral interactions rather than avoiding one another to prevent the spread of disease (Aubert and Richard, 2008; de Souza et al., 2008;

Richard et al., 2008; Ugelvig and Cremer, 2007; Walker and Hughes, 2009; Hamilton and Rosengaus, 2010). Because the generation of immune responses is energetically

89 costly (Moret and Schmid-Hempel, 2000; Chouvenc, 2009a), social insects may rely more on communal responses rather than individual physiological immune responses

(Castella et al., 2008a; Rosengaus et al., 2010). A case in point comes from the honeybee genome project which detected substantially fewer innate immune genes than those present in solitary insects such as Drosophila melanogaster (Evans et al., 2006;

Honeybee Genome Sequencing Consortium, 2006). It is possible that in social insects, colony-level anti-pathogen behaviors have been selected in favor of individual immune responses (Rosengaus et al., 2010).

Nest building and maintenance is a collective effort of the members of a colony

(Emerson, 1938) and may represent another manner in which social insects benefit from colony-level defenses. Certain aspects of social insect nests and the nest environments have been shown to play a role in disease resistance. For example, nest thermoregulation has been implicated in defending honeybee brood from nematode infection and chalk brood (Kaya, 1982; Starks, 2000). Hygienic behaviors allow for removal of sources of infection from the nest (Spivak, 1996; Jackson and Hart, 2009). Fecal pellets of

Zootermopsis angusticollis are used to create partitions in excavated galleries (Castle,

1934) and have been shown to inhibit fungal conidia germination (Rosengaus et al.,

1998b). Terpenoid components of Nasutitermes soldier frontal gland secretions have been shown to be fungistatic and may reduce the growth of fungi within the nest

(Rosengaus et al., 2000b, 2010; Fuller 2007). Naphthalene is present as a volatile in nests of Coptotermes formosanus, and has been shown to inhibit fungal growth (Chen et al.,

1998; Wright et al., 2000). More recently, termite GNBP-2, an immune-signaling protein that binds to and breaks down fungal cell walls, was found incorporated in Nasutitermes

90 corniger nest carton (Bulmer et al., 2009). The wood ant Formica paralugubris prophylactically incorporates antimicrobial conifer resin into its nests (Castella et al.,

2008a, 2008b). Honeybee propolis has antimicrobial properties, and is used in comb construction and to coat the walls of nests (Kacaniova et al., 2009; Evans and Spivak,

2010). It is clear that social insects utilize their nest environment and the compounds embedded in the nest structure to reduce microbial colonization, and thus reduce exposure to and spread of intranidal pathogens.

Termites exhibit a large diversity of nest structures, and their nest architecture is often useful in solidifying species identifications (Noirot, 1970; Thorne, 1980). Previous theoretical research has indicated that certain attributes of social insect nests may play a role in pathogen resistance (Pie et al., 2004). Pie et al. (2004) designed an agent-based

SIR model assuming that varying levels of nest intricacy influenced rates of infection. In this model, four nest types were tested: gallery-like, amorphous, multi-chambered, and single-chambered nests. The theoretical model predicted that disease transmission decreases as the nest architecture becomes more unidimensional, in their case the gallery- like nest. Although this model may be a good approximation of how nest architecture influences the transmission of disease within a colony, it did not take into consideration important variables, including antipathogen behaviors and the evolutionary link between a species and their own nesting ecology. The model predicted that gallery-like nest architectures should reduce disease susceptibility across the Isoptera, yet many species have evolved and become incredibly successful while inhabiting nest types other than a gallery-like nest. As of yet, no empirical studies have focused on whether the structure of the nest itself has a role in disease resistance. The present study tested whether nest

91 architecture influences the survival of termites exposed to a fungal pathogen in three representative termite species (Z. angusticollis, Reticulitermes flavipes, Nasutitermes corniger) exhibiting three life types (one-piece, intermediate, arboreal separate) outlined by Abe (1987).

Termite nesting ecology

The nests of Z. angusticollis most closely resemble the gallery-like nest described by Pie et al. (2004) and it was hypothesized that their percent survival would be highest in this nest-type. Due to the similarity of the galleries excavated in wood by Z. angusticollis and R. flavipes (Figure 4.1b, c), and other behavioral similarities between the species, it was hypothesized that R. flavipes would also exhibit the highest survival in a gallery-type nest. The ‘amorphous’ nest of Pie et al. (2004) was representative of more complex termite nest architectures like that of N. corniger, and it was hypothesized that this species would have the highest survival in an amorphous-type nest.

Zootermopsis angusticollis, the common dampwood termite is located exclusively on the west coast of North America, extending from British Columbia in the north to

Southern California and the Mexican border (Castle, 1934). These termites prefer the more humid coastal areas, feeding on and nesting within primarily dead or decaying conifer wood (Castle, 1934; Thorne et al., 1993). Zootermopsis angusticollis represents the one-piece nesting type, living and feeding exclusively within a single piece of wood for the lifetime of the colony (Abe, 1987). Due to this resource limitation, the maximum colony size reaches approximately 4,000 individuals. Their nests occur in downed wood at various stages of decay, often partially buried in the ground and near appreciable

92 moisture. The nests are excavated rather than constructed, creating longitudinal galleries parallel to the wood grain, with connections between galleries (Figure 4.1a, b). The tunnels vary in size but are often lined with dried feces. There are also numerous solid and liquid fecal deposits throughout the nest (Figure 4.1b), which are occasionally used to wall off old galleries, fungus, or other insect enemies co-habiting the wood (Castle,

1934).

Reticulitermes flavipes, the eastern subterranean termite is the most widely distributed of its genus. It is found across the entire eastern portion of North America, extending as far north as Ontario, Canada, down to Key Largo in the south. Colonies may reach sizes of 100,000 – 1 million individuals (Su et al., 1993). Reticulitermes flavipes represents the intermediate nesting type (Abe, 1987) in this study, with cryptic and diffuse nests largely located within soil, lacking a centralized portion containing a royal cell, and no distinct, replicable structure. They appear to start within or near pieces of fallen wood, similar to Z. angusticollis, then excavate galleries within the wood (Figure

4.1c) that extend out to the neighboring ground (Figure 4.1d), forming a system of irregular cavities variable in size and configuration. Although the nest is still largely excavated, gallery partitions of stercoral carton, which consists of masticated wood and feces, are constructed, like that used for the majority of construction in N. corniger, in addition to carton lining the galleries (Grassé, 1984). The colony is flexible in that it may relocate in response to changing resource and environmental conditions. As a member of the family Rhinotermitidae, R. flavipes represents a transitional stage between the rest of the lower termites (ex: Z. angusticollis) and the higher termites of the most derived family Termitidae (ex: N. corniger).

93 Nasutitermes corniger is a neotropical termite with a native distribution starting in the north from Mexico through to Panama, down to the South American countries of

Columbia, Venezuela, Guiana, Brazil, Ecuador, Bolivia, and Argentina (Scheffrahn et al.,

2005 and references therein). Its range has also expanded off the mainland to some

Caribbean islands (Collins et al., 1997; Scheffrahn et al., 1990, 2003). Nasutitermes corniger is representative of the arboreal, separate type nest with feeding sources completely separate from the nest (Abe, 1987). The nests of this species are completely constructed from stercoral carton, and occasionally other local materials cemented with saliva (Noirot, 1970; Thorne, 1980; Thorne and Haverty, 2000). In contrast to Z. angusticollis and R. flavipes, their only connection to the ground is through covered runways also made of carton (Thorne, 1980; Figure 4.1e). However, like the other two species, incipient colonies of N. corniger start out in decaying wood, only relocating to trees once the colonies reach critical mass (Thorne and Haverty, 2000). The main nest has a centralized royal chamber protected by dense, harder carton, and is surrounded by multiple layers of progressively thinner-walled chambers (Figure 4.1f), with the outside layer attached directly to the walls of the inner galleries (Thorne, 1980). This species can be polycalic, occupying multiple nests when a portion of the main colony buds off which may contain millions of individuals (Thorne, 1984, 1985).

94 Figure 4.1 Natural nests of termites: a) side view of Zootermopsis angusticollis galleries, b) internal view of Zootermopsis angusticollis nest, c) Reticultiermes flavipes galleries, d) Reticulitermes flavipes tunneling through soil, e) Nasutitermes corniger arboreal nest, and f) internal view of a section of Nasutitermes corniger nest. a. b.

c. d.

e. f.

95 4.2 Methods

Termite collection and maintenance

Colonies of Z. angusticollis were collected from Huddart Park in San Mateo

County, CA in July of 2003, and from Redwood Regional Park in Oakland, CA in August of 2007. Colonies of R. flavipes were collected in Jamaica Plain and Stoneham, MA

2006-2009. These colonies were maintained at 25 °C inside closed plastic tubs that were kept moist by spraying with sterile water and adding damp paper towels. Colonies of N. corniger were collected in Gamboa, Panama from sites affiliated with the Smithsonian

Tropical Research Institute from 2005-2009. They were maintained in an environmental chamber at 28.5 °C and 80% humidity. Each colony was kept in a large covered plastic tub that was connected to a second tub with plastic aquarium tubing. The second tub served as a foraging arena. White birch wood, Betula papyrifera, was added as food to each colony as needed. All colonies were contained in a USDA inspected and approved facility.

Nest construction

Five nest architectures were constructed from A-flute corrugated cardboard.

Cardboard contains cellulose, which served as a food source for the termites, and the corrugations simulated tunnels naturally built by termites. All constructed nests (Figure

4.2) were modeled after natural nests of termites exhibiting different nesting, feeding and foraging ecologies.

Two gallery nests were built. Both consisted of five superimposed layers of cardboard (110 x 110 x 3 mm). The first (referred to from now on as the more-holey

96 gallery (MHG) nest) had 130 vertical holes, divided evenly amongst the five layers to connect them (Figure 4.2a). The holes were placed randomly by using a standard paper hole puncher. The second, or less holey gallery (LHG) nest had half as many vertical holes. These nests were modeled after the galleries excavated by more primitive termites such as Z. angusticollis.

An amorphous-type nest (hereafter called ‘complex’) consisted of an outer cylindrical casing of cardboard (d = 73mm, h = 73mm) with 60 holes in it. Inside were two larger circles positioned crosswise, each containing 20 holes. Eight smaller circles, each with fifteen holes, were rolled up and placed into the compartments created by the larger circles (Figure 4.2b). The complex nest was designed to have a surface area comparable to that of the gallery nests. The inner portion of the complex nest was created to be similar to the more derived epigeal and arboreal nests of termites (ex: Figure 4.1e, f).

The single-chambered (SC) nest, similar to nests built during the incipient stages of colony foundation across the Isoptera, consisted of one Petri dish (d = 100mm) lined with a single layer of cardboard, 78.5 cm2 in surface area (Figure 4.2c). The multi- chambered (MC) nest was constructed using one large Petri dish (d = 100 mm) surrounded by four smaller Petri dishes (d = 60mm), all lined with cardboard. The four smaller Petri dishes were connected to the central dish by 2 cm each of aquarium tubing

(Figure 4.2d). Originally the SC nest was designed to have a total surface area that was comparable to the MC nest in order to control for differences in evaporation rates, humidity and other physical attributes related to surface area. It was discovered later that the termites in the SC nest tended to behave similarly regardless of the size of the

97 chamber. Also, incipient colony chambers are in fact considerably smaller (Rosengaus and Traniello, 1991; Thorne and Haverty, 2000; Hartke, pers. comm.), so all further experiments used the Petri dish with d = 100mm.

A total of ten nests were constructed for each replicate (five control, five experimental). The cardboard used for all nests was first autoclaved, then moistened with

6 mL of sterile deionized water, except for the SC nest, which received only 2 mL due to its smaller size. All nests were placed inside covered plastic boxes lined on the bottom with dampened paper towels to maintain high humidity. All the nests were kept at room temperature, low light levels, and equivalent amounts of water added as necessary.

Preparation of stock conidia suspensions

The entomopathogenic fungus M. anisopliae is a common and widely distributed soil fungus. This pathogen has been widely used in previous studies of disease resistance in termites and other social insects, and is known to be highly pathogenic (Hänel, 1982;

Kramm et al., 1982; Kramm and West, 1982; Rosengaus and Traniello, 1993, 1997,

2001; Zoberi, 1995; Rath et al., 1996; Bidochka et al., 1997; Milner et al., 1997, 1998b;

Rosengaus et al., 1998a, 1999b, 2007; Strack, 1998; Staples and Milner, 2000; Hughes et al., 2002, 2004; Moino Jr. et al., 2002; Myles, 2002b; Sun et al., 2002; Wright et al.,

2002; Shimizu and Yamaji, 2003; Sun et al., 2003a, 2003b; Thompson et al., 2003;

Engler and Gold, 2004; Hughes and Boomsma, 2004; Neves and Alves, 2004; Baer et al.,

2005; Calleri II et al., 2005, 2006; Pie et al., 2005; Wright et al., 2005; Ugelvig and

Cremer, 2007; Yanagawa and Shimizu, 2007; Chouvenc et al., 2008, 2009a, 2009b;

Reber et al., 2008; Yanagawa et al., 2008, 2009; Bulmer et al., 2009; Mburu et al., 2009)

98 and has been found naturally associated with R. flavipes, Z. angusticollis, and

Nasutitermes species (Zoberi and Grace, 1990; Milner et al., 1998a; Myles 2002b; Calleri

II et al., 2005). Infection by this fungus occurs mostly through external exposure, when conidia come in contact with the host’s cuticle (Hänel, 1982; Kramm and West, 1982).

Death results from toxins ultimately causing the green muscardine disease (McCauley et al., 1968; St. Leger, 1993).

Stock solutions of M. anisopliae conidia were prepared according to the protocol described by Rosengaus and Traniello (1997), and Rosengaus et al. (1998a). The original freeze-dried sample was obtained from the American Type Culture Collection, batch 93-

09, media 325, ATCC 90448 (Rosengaus and Traniello, 1997). Termites exposed to and killed by M. anisopliae were surface sterilized by washing them in 1 mL of a 5.2% sodium hypochlorite, followed by two – 1 mL washes of sterile deionized water for thirty seconds each. Surface sterilization allowed for the deactivation of microorganisms other than M. anisopliae on the cuticle so that hyphae from the fungal infection within the infected termite’s hemocoel could break out through the cuticle, and a pure sample of conidia recovered from the corpses. Sterilized individuals were placed on their backs and gently pressed into plates of potato dextrose agar (PDA) with soft forceps. These plates were incubated at room temperature until the corpses became overgrown with the dark green conidia typical of the green muscardine disease (McCauley et al., 1968; St. Leger,

1993). Subsequent conidia suspensions were prepared by harvesting conidia from the dead sporulating termites and transferred to sterile culture tubes containing 10 mL of

0.1% Tween 80 using a sterile inoculation loop to create a stock suspension of approximately 107 – 108 conidia/mL. Stock solutions were maintained at 10 ºC.

99 To estimate conidia viability of the stock suspension, the germination rate of conidia was measured prior to running any experiment. PDA was prepared and spread over three microscope slides in 1 mL portions and allowed to solidify. To each slide, 10

µL of stock conidia suspension were evenly distributed. The slides were enclosed in a plastic box to prevent contamination, and incubated at room temperature for 18 hours.

For each slide, 10 fields of vision at 400x magnification were selected randomly and the total numbers of germinated and non-germinated conidia recorded. Only highly virulent conidia suspensions with a minimum average germination rate of 95% (± SD) were used.

Exposure to control and fungal conidia suspensions

Individuals of Z. angusticollis were dorsally exposed to a measured volume of conidia suspension in order to ensure that each individual was exposed to the same concentration (Traniello et al., 2002). For this exposure, glass microscope slides were polished with paper towels to remove oily residue. On one side of the slides, a row of 5

X’s was drawn. The slides were flipped over and placed on paper towels over a bed of ice. Subsequently a 3 µL droplet of either a specific conidia concentration, or a 0.1%

Tween 80 control solution lacking fungal conidia was placed at the center of each X.

After cold immobilization, each termite was positioned on its dorsum so that the thorax was immersed in the droplet. The ice beds were transferred to the refrigerator and kept at

4 °C for one hour.

Individuals of R. flavipes and N. corniger were exposed to conidia using a walking exposure technique (Rosengaus et al., 1998a, 1999b, 2000a). This method was chosen for R. flavipes because these termites are extremely cold tolerant (Strack and

100 Myles, 1997), and would not remain immobilized for an hour-long direct exposure. In sharp conrtrast, the tropical N. corniger does not withstand cold temperatures. Moreover, because it is considerably smaller than Z. angusticollis, N. corniger termites would have drowned if immersed in a droplet of the size necessary to deliver the desired conidia concentration. For the walking exposure method, 20 termites were placed in a Petri dish

(60 x 15 mm) and allowed to walk on filter paper treated with 300 µL of either conidia suspension, or 0.1% Tween 80 control solution for one hour. The Petri dishes were periodically disrupted to ensure that the termites continually walked across the filter paper for the duration of the exposure protocol.

In the experimental treatment, nymphs of Z. angusticollis (1200 individuals/6 replicates total from four stock colonies) and workers of N. corniger (1800 individuals/9 replicates total from three stock colonies) were exposed to a high 105 conidia/mL concentration of M. anisopliae, while R. flavipes workers (1200 individuals/6 replicates total from three stock colonies) were exposed to an 8 x 106 conidia/mL concentration.

Reticulitermes flavipes was exposed to a one order of magnitude higher conidia concentration because previous results indicated this species to be naturally more resistant to M. anisopliae. A higher conidia dosage ensured that some mortality during the course of the experiment. Control termites were identically treated with the exception that they were treated with a 0.1% Tween 80 suspension lacking fungal conidia.

101 a. b.

c.

Figure 4.2 Example artificial nest architectures: a). MHG nest side and top view, b). Complex nest side view, and inside view from the top, c). SC nest top view, and d). MC nest. The red ovals outline N. corniger aggregating behavior d. within each nest type.

102 Experimental set-up and census

Following the control and conidia exposures, twenty termites were transferred to their appropriate nest. A daily census was performed for twenty days. Each day behavioral observations were made before a nest was opened in order to determine where the individuals were located in the nest. Upon opening a nest, the internal temperature and relative humidity (RH) were measured with a Cooper-Atkins PMRH120 thermohygrometer. The individuals in each nest were accounted for, and dead individuals removed. Attempts were made to minimize handling time so the termites would behave more naturally in the nests. Dead individuals were surface sterilized with 5.2% sodium hypochlorite, then plated on PDA for confirmation purposes (Rosengaus and Traniello,

1997).

Statistical analyses

Susceptibility to fungal infection as a function of nest architecture was analyzed using survival analyses (SPSS, version 15). Several survival parameters were estimated: survival distributions (representing the time course of survival), median survival time

(LT50), and percent survival at the end of the census period (Kaplan-Meier test, Survival analysis). The relative hazard ratios of death were also calculated using a Cox proportional regression model, which included the variables species, colony of origin, treatment, and nest architecture. The hazard ratio characterized the instantaneous rate of death at a particular time, given that the individual had survived up until that time, while controlling for the effect of the various variables on survival of the individuals (SPSS,

1990). Due to non-homogeneity of variances, differences in temperature and RH were

103 analyzed using the nonparametric Kruskal-Wallis test. Differences were analyzed separately for each species across the five nest architectures containing control and conidia exposed individuals. A Bonferroni correction was applied to all analyses consisting of multiple comparisons (Rice, 1989).

4.3 Results

Overall, Cox proportional regression models revealed that species, colony of origin, treatment, and nest architecture were all significant and independent predictors of termite survival. Species and colony of origin were confounding variables so colony of origin was only included in the models run separately for each species. The effects of species, treatment, and nest architecture are discussed in detail below.

Species

When controlling for treatment and nest architecture, species was found to be the most significant determinant of termite survival (Wald statistic = 1205.9, df = 2, p <

0.001). Reticulitermes flavipes exhibited the highest survival of all three species (Figure

4.3a) despite being exposed to an order of magnitude higher concentration of conidia.

Relative to R. flavipes, Z. angusticollis individuals were 1.4 times, and N. corniger individuals 5.1 times as likely to die.

Treatment

When controlling for species and nest architecture, treatment was found to be the second most significant determinant of termite survival (Wald statistic = 511.8, df = 1, p

104 < 0.001). Termites exposed to M. anisopliae conidia were 2.5 times as likely to die as control termites (Figure 4.3b). No control termites confirmed for growth of M. anisopliae, while many of the conidia exposed termites exhibited positive confirmations

(Table 4.1).

Nest architecture

Overall, after controlling for effects of species and treatment, nest architecture was the third most significant determinant of termite survival (Wald statistic = 82.4, df =

4, p < 0.001). Termites in SC nests were the least likely to die from infection (Figure

4.3c). Compared to SC nests, individuals in MHG nests were 1.5 times, LHG individuals

1.7 times, MC individuals 1.7 times, and Complex individuals 1.4 times as likely to die.

In order to compare survival across species with different nesting ecologies, and because species was found to be a significant predictor of termite survival, the survival parameters for each species were further analyzed separately.

In Z. angusticollis, survival of individuals in control nests was not significantly different after correcting for multiple comparisons (Wald statistic = 5.6, df = 4, p = 0.2;

Figure 4.4a). Following exposure to M. anisopliae, nest architecture was a significant, independent predictor of survival (Table 4.1; Figure 4.4b). Individuals in MHG nests exhibited the highest survival, and SC nests the second highest when controlling for colony of origin (Figure 4.5). Although the magnitude of the percent survival across Z. angusticollis colonies was significantly different, the majority of them displayed the same overall pattern. Hence colonies were pooled in the model. Surprisingly, individuals in

105 LHG nests had the lowest percent survival, demonstrating the potential importance of connections between galleries in this experiment.

In control nests of R. flavipes, unlike the other species, nest architecture was a significant predictor of survival (Wald statistic = 13.4, df = 4, p = 0.01; Figure 4.1c).

However, percent survival in these nests was still much higher than in the exposed nests

(Figure 4.1d). Also, survival within the other nest architectures was only marginally significant when compared to the SC nest, which exhibited the highest survival (Table

4.1). Following exposure to M. anisopliae, nest architecture was a significant, independent predictor of survival (Table 4.1, Figure 4.4d). Reticulitermes flavipes individuals exhibited the highest percent survival in SC nests following pathogen exposure (Wald statistic = 59.9, df = 4, p < 0.001; Figure 4.4d), with Complex nest individuals exhibiting the second highest survival.

Control nests of N. corniger, while not significantly different from one another

(Wald statistic = 5.6, df = 4, p = 0.229), had very low survival rates compared to the other species’ controls because workers of N. corniger do not fare well when removed from their nest. Yet, the controls did have higher survival rates than the exposed replicates, therefore the effects of pathogen exposure on this species were still evident. Following exposure to M. anisopliae, nest architecture was a significant, independent predictor of survival (Table 4.1; Figure 4.4f). Nasutitermes corniger exhibited the highest percent survival in SC nests following pathogen exposure (Wald statistic = 65, df = 4, p < 0.001;

Figure 4.3f), with complex nests following in second.

106 Figure 4.3 Survival distributions resulting from Cox regressions as a function of treatment (a), species (b), and nest architecture (c) while controlling for all other variables. Note that in graph (c) the lines for the MC and Complex nests are overlapping.

a. b.

c.

107 Table 4.1 Survival parameters for control and conidia exposed termites of the three study species. Median survival time (LT50) values with the same letter denote no significant differences in pairwise comparisons of survival distributions (at p ≤ 0.005). The reference (ref) nest within each treatment represents the architecture with the highest survival, which all other nests were compared to in the Cox Regression.

108 Figure 4.4 Survival distributions as a function of nest architecture: Z. angusticollis control (a), Z. angusticollis conidia exposed (b), R. flavipes control (c) R. flavipes conidia exposed (d), N. corniger control (e) and N. corniger conidia exposed (f).

a. b.

c. d.

e. f.

109 Figure 4.5 Summary of survival across the five nest architectures for Zootermopsis angusticollis (Za), Reticulitermes flavipes (Rf), and Nasutitermes corniger (Nc).

Za MHG > SC > MC > Complex > LHG

Rf SC > Complex > LHG > MC > MHG

Nc SC > Complex > MHG > MC > LHG

110 Figure 4.6 Median nest temperature (a) and RH (b) across nest architectures. ‘**’ denotes significance at p ≤ 0.005 (KW).

a.

NS NS NS

b. ** NS NS

111 Microclimate

Temperature (Figure 4.6a) and RH (Figure 4.6b) were not significant across the nest architectures for all three species. The sole exception was RH in Z. angusticollis nests, which was marginally significant (p = 0.005) after correcting for multiple comparisons. The RH did not correspond with the patterns in survival distributions, as the nest architectures in which Z. angusticollis had the highest (MHG) and lowest (LHG) survival had the same median RH.

4.4 Discussion

Nests are a well-known characteristic of social insects, with termites exhibiting a wide diversity of structures (Noirot and Darlington, 2000). Although nests of social insects have been considered important in defense against predators (Noirot and

Darlington, 2000), little is known about the role that nest architecture may play in the colony’s disease resistance. Some social insects incorporate antimicrobial substances into their nests (Rosengaus et al., 1998b; 2000b; Castella et al., 2008; Bulmer et al., 2009;

Kacaniova et al., 2009; Evans and Spivak, 2010), but questions related to whether nest architecture influences pathogen transmission have not been empirically addressed. The results of the current study demonstrate that nest structure alone can confer a survival advantage to termites exposed to a fungal pathogen. The three wood-feeding species tested represent each of three nest types (one-piece, intermediate, and separate) within the

Isoptera, and the interspecific susceptibility to fungal infection was not equivalent across the five different nest architectures. This indicates that the influence nest architecture has on survival involves more than the dynamics of disease transmission tested in the model

112 designed by Pie et al. (2004). Differences in activity levels, encounter and allogrooming rates, as well as deposition incorporation and diffusion of antifungal secretions into the nest structure may help explain the differential susceptibility of each species across the tested nest architectures.

Contrary to previous theoretical models, termites exposed to M. anisopliae differed significantly in survival in different nest architectures depending on their species

(Pie et al., 2004). Although not always the highest, all three species had relatively high survival in the SC nest (Figure 4.5). Most termite colonies are initiated by dispersing reproductives, and incipient colonies begin in a small, excavated royal chamber, with the nest expanding as the colony grows in number (Nutting, 1969). With the small number of termites tested in each nest, conditions were more similar to those of an incipient colony stage, so it is expected that this nest architecture would be beneficial to all species tested.

Zootermopsis angusticollis had the highest survival in MHG nests, and N. corniger the second highest survival in complex nests, both of which were modeled after their natural nests (Figure 4.1a, b, e, f). This would seem to suggest that termites are well adapted to the particular nest structure they naturally inhabit. However R. flavipes, which also excavates galleries in wood similar to Z. angusticollis (Figure 4.1c), had the highest percent survival in the same two nests as N. corniger. The Termitidae are believed to have diverged monophyletically directly from the Rhinotermitidae in the Early

Paleogene, whereas the Rhinotermitidae diverged from descendents of the

Archotermopsidae (Engel et al., 2009). This phylogenetic relationship places R. flavipes much closer to N. corniger than R. flavipes is to Z. angusticollis. In addition, recent phylogenetic analyses based on nest-type (Abe, 1987) revealed the Rhinotermitidae to be

113 more closely nested with Termitidae (Inward et al., 2007b). So phylogenetic history, including the nest structure a species is adapted to, appear to contribute to termite survival in varying nest architectures.

In the theoretical model, variation in nest complexity and worker activity was meant to adjust the amount of contact individuals had and therefore the number of opportunities for the pathogen to transmit between individuals (Pie et al., 2004). Social contact has been implicated in increasing the chance of infecting naïve individuals

(Kramm et al., 1982; Schmid-Hempel and Schmid-Hempel, 1993; Zoberi, 1995;

Rosengaus and Traniello, 1997; Milner et al., 1998b; Rosengaus et al., 2000a; Hughes et al., 2002; Fefferman et al., 2007), but certain behaviors performed when in contact also allow social insects to reduce the spread of disease within a nest. Removal of fungal conidia via allogrooming can prevent them from infecting nest-mates (Strack, 1998;

Milner et al., 1998b; Rosengaus et al., 1998, 2000a Myles et al., 2002a; Shimizu and

Yamaji, 2003; Yanagawa and Shimizu, 2007; Yanagawa et al., 2008) and passage through the termite gut can reduce viability or render conidia inactive (Kramm and West,

1982; Yanagawa and Shimizu 2007; Chouvenc et al., 2009b; Schultheis, 2009). ß(1,3)- glucanases, which break down fungal cell walls, have been found in termite salivary glands and on cuticles likely as a result of allogrooming (Bulmer et al., 2009). Other work in our lab has demonstrated the presence of possible immune elicitors in the crop of

Camponotus pennsylvanicus in response to pathogen exposure that is passed to naïve nest-mates via oral trophallaxis (Hamilton and Rosengaus, 2010). In some cases when conidia are detected on the cuticles of nestmates, the sick individual may be cannibalized

(Strack, 1998; Rosengaus and Traniello, 2001; Chouvenc et al., 2008) or buried in feces

114 or other materials, effectively blocking sporulation and further spread of a fungal pathogen (Kramm et al., 1982; Zoberi, 1995; Strack, 1998; Myles et al., 2002a). Taking all these behaviors into account, it is not surprising that the current in vivo results differed from the original theoretical model (Pie et al., 2004).

There is no doubt that behaviors social insects undergo when in contact can improve their chances of survival. The question remains how the structure of a nest alters that social contact, resulting in the observed differential survival. Since all of the individuals in experimental nests were exposed to fungus, the first course of action necessary to survive would be to aggregate, groom, and remove the source of infection.

Studies have shown that termites sometimes demonstrate an attraction to certain fungal isolates (Engler and Gold, 2004) indicating a potential parasite-driven behavioral change to infect more hosts. However, termites also use detection of conidia to identify sick individuals and initiate grooming (Yanagawa and Shimizu, 2007; Chouvenc et al., 2008).

If an individual is sick beyond any possible benefits from grooming, it may then be cannibalized (Strack, 1998; Rosengaus and Traniello, 2001; Chouvenc et al., 2008) or buried (Kramm et al., 1982; Zoberi, 1995; Strack, 1998; Myles et al., 2002a) to prevent infection of nestmates. Behavioral observations during the course of the census period revealed that termites in all nest architectures and all three species aggregated sometime within the first 24 hours after initiation of the experiment. Fungal conidia penetrate the cuticle of insects 24-48 hours post exposure (Moino Jr. et al., 2002), and it is likely that the individuals in each nest found one another in ample time to undergo and benefit from grooming. In each successive census the majority of healthy individuals were consistently aggregated. It is unknown whether the structure of the nest could influence the

115 effectiveness of grooming, but taking into account the similarities in aggregating behavior in all the nests, it does not appear that nest structure greatly affected their ability to find one another. Since all individuals were exposed initially, social contact aided in resistance rather than further transmission of the pathogen.

After the initial grooming period, out of the twenty termites within a nest there were likely some that were not sufficiently groomed to avoid infection, while others had all of the conidia removed and passed the risk of infection. At this stage healthy individuals would need to (1) avoid re-infection from contact with sick or dead individuals, and (2) avoid contact with conidia that were dislodged onto the nest material.

Dependent on concentration, M. anisopliae has been shown to be repellant to termites, to the extent that pest control strategies are designed to overcome this repellency (Staples and Milner, 2000; Mburu et al., 2009). Some termites are reported to exhibit a specific alarm behavior, warning nestmates of the presence of a pathogen (Rosengaus et al.,

1999a; Myles et al., 2002a). A recent study in Temnothorax unifasciatus demonstrated that moribund workers abandon their nest to die, and this may be an adaptation to reduce disease transmission (Chapuisat, 2010; Heinze and Walter, 2010). Behavioral observations in this study revealed that occasionally sick individuals removed themselves from the main group to inhabit a different part of the nest. Whether they isolated themselves intentionally from the group or became lethargic or paralyzed (Hänel, 1982;

Samuels et al., 1988; Kershaw et al., 1999) due to the breakdown of nerve signals to muscles as a result of infection is unknown. Nevertheless, separation of infected and symptomatic insects may have reduced subsequent pathogen transmission. If sick individuals were not separated, they were often cannibalized or buried in feces,

116 preventing further spread of conidia. As dead individuals were removed daily from a nest prior to sporulation, they did not pose an additional infection risk. Overall, it’s possible that nest structure may influence the diffusion of conidia volatiles (Engler and Gold,

2004; Mburu et al., 2009), thereby affecting the attractance or repellence to conidia or sick nestmates. As aggregation behavior was very similar across architectures, it is likely that nest architecture had a greater affect on avoidance of secondary infection, and the ability of sick individuals to effectively remove themselves from the main (healthy) group, than it did on nestmates initially finding one another.

The small variations in temperature and humidity likely did not influence the outcome of this study, as M. anisopliae remains virulent within these ranges (Milner et al., 1997; Arthurs and Thomas, 2001; Sun et al., 2003b). However, although temperature and humidity did not consistently vary between nest architectures (Figure 4.4), nest microclimate may still play an important role in a colony’s ability to resist pathogens.

High temperatures can reduce the germination rates of pathogenic fungi (Ignoffo, 1992 and included references; Arthurs and Thomas, 2001; Sun et al., 2003b) and the inside of many termite nests in the field can reach temperatures between 30-35°C if not higher, particularly in the area of the nursery (Holdaway and Gay, 1948; Greaves, 1964; Grigg,

1973; Leponce et al., 1995; Darlington et al., 1997; Korb and Linsenmair, 1998a, 2000;

Turner, 2001; Chapter 2). Termites also maintain high humidities in their nests (Fyfe and

Gay, 1938; Turner, 2001; Chapter 2) because they are easily susceptible to desiccation

(Krecek, 1969; Collins, 1969; Singh and Singh, 1981). Fungi also grow optimally at high humidities (Milner et al., 1997; Arthurs and Thomas, 2001), therefore humidity alone may not factor into the mechanisms by which nests can aid in disease resistance.

117 Currently the known mechanisms termites have for regulating nest climate are nest site selection (Leponce et al., 1995; Korb and Linsenmair, 1998a, 1998b, 2000a), architecture (Weir, 1973; Korb and Linsenmair, 1998b; Turner, 2000, 2001; Korb,

2003b), orientation (Grigg, 1973; Korb, 2003a), metabolic heat and water (Fyfe and Gay,

1938; Greaves, 1964; Korb and Linsenmair, 2000b; Turner, 2001; personal obs.), active transport and storage of water (Lee and Wood, 1971 and included references), and the fact that the nest is an enclosed environment (Greaves, 1964). Because all nests in this experiment were maintained under the same, constant lab conditions, nest site location can be discarded as a factor. The studies showing that nest architecture influences nest microclimate have to do with the nest either capturing more heat from the sun (Grigg,

1973; Korb, 2003a), preventing heat loss by having thicker walls (Korb and Linsenmair,

1998b), or increasing/decreasing ventilation (Korb, 2003b), none of which occurred during the course of this experiment. The termites could have manipulated the nest they occupied either by consuming the cardboard to create new tunnels, or defecating to block existing tunnels and/or build new ones. Surprisingly, none of the termites greatly manipulated their nests in this way, perhaps due to low numbers. Zootermopsis angusticollis is substantially larger in size than both R. flavipes and N. corniger, and metabolism in insects is proportional to body size (Reichle, 1968; Eggleton et al., 1998).

However, with only twenty termites per nest, metabolic rates don’t appear to have made a significant difference in nest temperature or humidity. Instead humidity was largely controlled by the water added at the beginning, and during the course of the experiment.

Although water was added in equivalent amounts, evaporation rates did differ (personal obs.). As a result water needed to be added at different rates to each nest type to maintain

118 conditions required by the termites, but consistency was maintained within each nest type across treatments, resulting in the relatively stable humidities of the nests (Figure 4.6b)

However, there were periods between waterings when some nests may have been somewhat drier than others. Studies in cockroaches have demonstrated higher aggregation densities in lower humidities (Dambach and Goehlen, 1999). Although doubtful, these brief dry periods may have influenced aggregating behavior, and therefore disease transmission/resistance dynamics within those nests.

In summary, this work is the first to empirically test and demonstrate that nest architecture alone can influence termite survival against an entomopathogenic fungus.

The structure of a nest alters the dynamics by which termites aggregate or disperse in their efforts to balance pathogen-resisting social behaviors with avoiding re-infection.

The three species tested differed in their survival, as well as the nest in which they exhibited the highest survival. Species differed in survival within the different nest architectures, possibly in correlation with their phylogenetic history, as opposed to exhibiting similar survival patterns regardless of species (Pie et al., 2004). Finally, although measurable microclimatic conditions in this study did not differ significantly, nest architecture is known to influence microclimatic conditions in nature and is likely still an important mechanism by which termites can indirectly affect pathogen resistance.

119 Chapter 5 – Social induction of hemolymph proteins in the dampwood termite Zootermopsis angusticollis (Holmgren)

Abstract

Within their densely packed nests, termites undergo an array of social behaviors such as allogrooming, cannibalism, and burial of infected nestmates that improve colony survival when invaded by a pathogen. Previous research in the dampwood termite

Zootermopsis angusticollis demonstrated an enhancement of disease resistance when uninfected nymphs were allowed to interact with immune primed nestmates. Although the mechanisms for such social immunization have yet to be determined, the phenomenon of social immunity may be one reason why termites, and perhaps other social insects, are able to thrive under important pathogenic constraints. Moreover, previous electrophoretic studies also indicated that conidia exposed Z. angusticollis produce novel hemolymph proteins with antifungal activity. To examine the mechanism by which this social immunization occurs, the hemolymph protein profiles of naïve Z. angusticollis nymphs, and those of nymphs directly exposed to Metarhizium anisopliae were compared using SDS-PAGE gel electrophoresis. Two possible mechanisms were tested: 1) induction through contact with conidia during allogrooming, and 2) induction through transfer of immune elicitors through social interactions. The results were highly variable and thus, inconclusive. However, 10% of naïve individuals exhibited hemolymph protein profiles that are consistent with social transmission of immune elicitors. Although further studies are needed, these individuals provide the first electrophoretic evidence of social immunity.

120 5.1 Introduction

Termites have demonstrated an incredible ability to resist bacteria and fungi used in biological control (Milner, 2003; Chouvenc et al., 2008). Moreover, they are extremely successful in terms of geographical distribution and ecological significance, despite naturally associating with an abundant and diverse array of pathogenic (or potentially pathogenic) microorganisms (Smythe and Coppel, 1966; Zoberi and Grace, 1990; Zoberi,

1995; Milner et al., 1998a, 1998b; Hojo et al., 2002; Myles, 2002b; Sun et al., 2003a;

Roose-Amsaleg et al., 2004; Meikle et al., 2005; Wright et al., 2005). A number of behavioral interactions such as grooming (Strack, 1998; Milner et al., 1998b; Rosengaus et al., 1998a, 2000a; Myles et al., 2002a; Shimizu and Yamaji, 2003; Yanagawa and

Shimizu, 2007; Yanagawa et al., 2008), cannibalism (Strack, 1998; Rosengaus and

Traniello, 2001; Chouvenc et al., 2008), or burial of sick or dead individuals (Kramm et al., 1982; Zoberi, 1995; Strack, 1998; Myles et al., 2002a) have been implicated in improving colony survival. However, these behaviors also require that individuals come into close contact with one another, potentially spreading infection to uninfected nestmates (Kramm et al., 1982; Hänel and Watson, 1983; Schmid-Hempel and Schmid-

Hempel, 1993; Zoberi, 1995; Rosengaus and Traniello, 1997; Milner et al., 1998b;

Rosengaus et al., 2000a; Hughes et al., 2002; Fefferman et al., 2007).

Previous work (Chapter 4) demonstrated that nest architecture influences disease susceptibility, possibly by altering encounter and contact rates between individuals within a colony. Several other studies have also demonstrated that social insects exhibit increased survival against pathogens when in social groups (Rosengaus et al., 1998a;

Rosengaus and Traniello, 2001; Traniello et al., 2002; Hughes et al., 2002; Shimizu and

121 Yamaji, 2003; Ugelvig and Cremer, 2007; Yanagawa and Shimizu, 2007; Yanagawa et al., 2008). Although insects do not exhibit life-long immune memory in the same manner as vertebrates, there is evidence of immune priming in which individuals show increased survival in a subsequent exposure to a pathogen after first being “immunized” with a non- lethal dose (Rosengaus et al., 1999b, 2007; Traniello et al., 2002; Schmid-Hempel, 2005;

Roth et al., 2008). Interestingly, in some insects immunity may increase in offspring when parents are first exposed to a particular pathogen (Moret and Schmid-Hempel,

2001; Little et al., 2003; Sadd et al., 2005; Moret, 2006; Sadd and Schmid-Hempel, 2007;

Freitak et al., 2009). Therefore, the possibility exists that in addition to behavioral or chemical defenses, immune primed individuals may confer an immune advantage upon naïve nestmates, eventually eliciting immunocompetence at the colony-level. Hamilton and Rosengaus (2010) demonstrated that antimicrobial peptides in the trophallactic fluid of the carpenter ant Camponotus pennsylvanicus were upregulated upon immune stimulation, and may be shared between colony members to assist in control of infection.

While evidence on the success of social behaviors in resisting disease is numerous and continuing to grow (Traniello et al., 2002; Cremer et al., 2007; Schlüns and Crozier,

2009; Rosengaus et al., 2010), there is still very little understanding of the underlying physiological mechanisms contributing to social immunity.

Defense against disease in individual insects begins with the cuticle (St. Leger,

1991). Once a pathogen has breached this first line of defense, the cellular and humoral immune responses take over. Cellular responses consist of immune reactions mediated by hemocytes (Shan and Ling, 2008; Strand, 2008) and include phagocytosis (Ling and Yu,

2006; Kedra and Bogus, 2006; García-García et al., 2009), clotting (Scherfer et al., 2006;

122 Dushay, 2009) and encapsulation of invaders (Baer et al., 2005; Chouvenc et al., 2009a).

The humoral response includes the production of antimicrobial peptides (AMPs) produced by the fat body or hemocytes that are released into the hemolymph (Dunn,

1990; Gillespie et al., 1997; Otvos Jr., 2000; Wilson-Rich et al., 2009). The current study focused on testing the humoral aspects of termite social immunity.

Antimicrobial peptides (AMPs) are induced by fungal or bacterial exposure in several insects (Bidochka et al., 1997; Hoffmann and Reichart, 1997; Rees et al., 1997;

Han et al., 1999; Evans, 2004; Evans and Lopez, 2004; Eleftherianos et al., 2006; Song et al., 2006; Wang et al., 2007; Peng et al., 2008; Randolt et al., 2008). Previous work has examined the role of AMPs in termite immunity (Lamberty et al., 2001; Da Silva et al.,

2003; Lee et al., 2003; Thompson et al., 2003; Bulmer and Crozier, 2004, 2006; Bulmer et al., 2009). Work performed in our lab using gel electrophoresis examined the induction of novel proteins in the hemolymph of nymphs, soldiers, and pseudergates of

Zootermopsis angusticollis following exposure to Metarhizium anisopliae. These proteins were later shown to have fungistatic properties when applied to germinating fungal conidia (Rosengaus et al., 2007). Thus, exposure to fungal conidia appears to elicit the enhancement and production of novel AMPs in Z. angusticollis. The aim of this study was to determine whether AMP production is induced following social interactions between directly fungal-exposed and naïve nestmates. This is the first study to attempt to provide electrophoretic evidence of the social transmission of immunity in termites as proposed by Traniello et al. (2002).

123 5.2 Methods

Termite collection and maintenance

Colonies of Z. angusticollis were collected from Huddart Park in San Mateo

County, CA in July of 2003, and from Redwood Regional Park in Oakland, CA in August of 2007. These colonies were maintained in our USDA inspected containment room at 25

°C inside closed plastic tubs that were kept moist by spraying with sterile water and adding damp paper towels. White birch wood, Betula papyrifera, was added as food to each colony as needed.

Preparation of stock conidia suspensions

The entomopathogenic fungus M. anisopliae is a common and widely distributed soil fungus. This pathogen has been widely used in previous studies of disease resistance in termites and other social insects, and is known to be highly pathogenic (Hänel, 1982;

Kramm et al., 1982; Kramm and West, 1982; Rosengaus and Traniello, 1993, 1997,

2001; Zoberi, 1995; Rath et al., 1996; Bidochka et al., 1997; Milner et al., 1997, 1998b;

Rosengaus et al., 1998a, 1999, 2007; Strack, 1998; Staples and Milner, 2000; Hughes et al., 2002, 2004; Moino Jr. et al., 2002; Myles, 2002b; Sun et al., 2002; Wright, 2002;

Shimizu and Yamaji, 2003; Sun et al., 2003a, 2003b; Thompson et al., 2003; Engler and

Gold, 2004; Hughes and Boomsma, 2004; Neves and Alves, 2004; Baer et al., 2005;

Calleri II et al., 2005, 2006; Pie et al., 2005; Wright et al., 2005; Ugelvig and Cremer,

2007; Yanagawa and Shimizu, 2007; Chouvenc et al., 2008, 2009, Reber et al., 2008;

Yanagawa et al., 2008, 2009; Bulmer et al., 2009; Mburu et al., 2009). It has been found naturally associated with termites, including Z. angusticollis (Zoberi and Grace, 1990;

124 Milner et al., 1998; Myles 2002b; Calleri II et al., 2005). Infection by this fungus occurs mostly through external exposure, when conidia come in contact with the host’s cuticle

(Hänel, 1982; Kramm and West, 1982). Death results from toxins ultimately causing the green muscardine disease (McCauley et al., 1968; St. Leger, 1993).

Stock solutions of M. anisopliae conidia were prepared according to the protocol described by Rosengaus and Traniello (1997), and Rosengaus et al. (1998a). The original freeze-dried sample was obtained from the American Type Culture Collection, batch 93-

09, media 325, ATCC 90448 (Rosengaus and Traniello, 1997). Termites exposed to and killed by M. anisopliae were surface sterilized by washing them in 1 mL of a 5.2% sodium hypochlorite, followed by two – 1 mL washes of deionized water for thirty seconds each. Surface sterilization allowed for the deactivation of microorganisms on the cuticle so that sporulation was initiated within the termite’s hemocoel, thus confirming successful invasion of M. anisopliae. Sterilized individuals were placed on their backs and gently pressed into plates of potato dextrose agar (PDA) with soft forceps. These plates were incubated at 25 °C until the corpses became overgrown with the dark green conidia typical of the green muscardine disease (McCauley et al., 1968; St. Leger, 1993).

Subsequent conidia suspensions were prepared by harvesting conidia from the dead sporulating termites and transferred to sterile culture tubes containing 10 mL of 0.1%

Tween 80 using a sterile inoculation loop to create a stock suspension of approximately

107 – 108 conidia/mL. Stock solutions were maintained at 10 ºC.

To estimate conidia viability of the stock suspension, the germination rate of conidia was measured prior to running any experiment. PDA was prepared and 1 ml was spread over each of three microscope slides and then allowed to solidify. To each slide,

125 10 µL of stock conidia suspension were evenly distributed. The slides were enclosed in a plastic box to prevent contamination, and incubated at room temperature for 18 hours.

For each slide, 10 fields of vision at 400x magnification were selected randomly and the total numbers of germinated and non-germinated conidia recorded. Only highly virulent conidia suspensions with a minimum average germination rate of 95% (± SD) were used.

Exposure to control and fungal conidia suspensions

Individuals of Z. angusticollis were dorsally exposed to a measured volume of conidia suspension in order to ensure that each individual was exposed to the same concentration (Traniello et al., 2002). Glass microscope slides were polished with paper towels to remove oily residue. On one side of the slides, a row of 5 X’s was drawn. The slides were flipped over and placed on paper towels over a bed of ice. Subsequently a 3

µL droplet of either a specific conidia concentration, or a 0.1% Tween 80 control solution lacking fungal conidia was placed at the center of each X. After cold immobilization, each termite was positioned on its dorsum so that the thorax was immersed in the droplet.

The ice beds were transferred to the refrigerator and kept at 4 °C for one hour.

Hemolymph extraction and preparation

To determine whether an immune response could be induced in naïve individuals after social interactions with previously immunized nestmates, nymphs of Z. angusticollis were separated from their parent stock colony and sexed. Each replicate consisted of 36 individuals (n = 18 males and 18 females). Each individual was randomly assigned to one of the following 6 treatments:

126

Control (C): direct exposure to 0.1% Tween 80 suspension

Conidia Exposed (CE): direct exposure to 8 x 104 conidia/mL of M. anisopliae

Conidia Exposed Delay (CED): direct exposure to 8 x 104 conidia/mL of M.

anisopliae. These individuals were kept separate an additional three days to

ensure that conidia had penetrated the cuticle and could no longer be removed by

grooming nestmates.

Naïve Control (NC): unexposed individuals assigned to interact with C group

Naïve Conidia Exposed (NCE): unexposed individuals assigned to interact with

CE group immediately after the exposure procedure

Naïve Conidia Exposed Delay (NCED): unexposed individuals assigned to

interact with CED group three days after the exposure procedure

A diagram of the overall experimental design is depicted in Figure 5.1. Prior to fungal exposure, three males and three females each were placed in six Petri dishes (d = 60 mm) lined with moist filter paper. Individuals in each dish were weighed, and labeled with nail polish so that individual termites could be distinguished. Their hemolymph was extracted

24 hours after separation from their natal nest to get an individual baseline protein profile. This was conducted by placing each of the cold-immobilized nymphs with their ventral side up under a dissecting scope (2x magnification). The abdomens were swabbed with 70% ethanol to remove feces or other contaminants. The cuticle was punctured between the intersegmental membranes with the tip of a pulled capillary tube (tip diameter ~2 µm), and the abdomen gently pressed to produce a droplet of clear

127 hemolymph. One µL of hemolymph was collected with a micropipetter and immediately transferred to an Eppendorf tube containing 60 µL of cold Burns-Tracy Saline (BTS).

Samples were kept on ice to prevent melanization of the hemolymph. The samples were centrifuged at 600 x g at 4 °C for 10 minutes to produce a fat free, cell free sample. The supernatant was transferred to a clean microcentrifuge tube and centrifuged at 3200 x g at

4 °C for 20 minutes. Sixty µL of this supernatant was added to 12 µL of tris-glycine SDS sample buffer, and boiled for 5 minutes. The final samples were frozen in liquid nitrogen and maintained at –80 °C for future use in SDS-PAGE electrophoresis.

Exposures and subsequent bleedings

Three days after the first hemolymph extraction, C individuals were exposed to 3

µL of a 0.1% Tween 80 control solution, while CE and CED individuals were exposed to a sublethal conidia suspension containing 8 x 104 conidia/mL. Immediately following exposures, the labeled naïve individuals were placed together with their control or conidia exposed nestmates in a 1:1 ratio in new Petri dishes (d = 60 mm), except for the delay group (CED, NCED) to which the naïve termites were added three days later.

Previous research has demonstrated that this time frame is long enough to elicit an immune response, lowering the susceptibility of termites following a challenge with an otherwise lethal concentration of fungi and bacteria (Rosengaus et al., 1999b). The purpose of the delay treatment was to determine whether the social transmission of immunity was due to social interactions (i.e. oral or anal trophallaxis) with exposed nestmates (CED, NCED) rather than due to immediate contact with sub-lethal doses of conidia removed from the cuticle of exposed nestmates during allogrooming (CE, NCE).

128 The CED termites were first surface sterilized for 10 seconds in 5.2% sodium hypochlorite prior to placement with naïve individuals to ensure that all conidia on the cuticle were deactivated. Post-exposure bleedings took place three days after the naïve individuals were introduced to the Tween 80 or conidia exposed termites. Some individuals died during the course of the experiment, but hemolymph was extracted for the second time only from live and healthy looking (active) individuals.

SDS-PAGE electrophoresis

Tris-glycine 12% SDS-PAGE gels were used to separate and compare the protein banding patterns of termite hemolymph before (baseline) and post exposure to the control or conidia suspensions, and before and after naïve termites socially interacted with immunized nestmates. Baseline and post-exposure samples from a single individual were run adjacent to one another. Gels were run at a constant voltage of 125 V for 1 hour.

Following electrophoresis, the gels were stained with Gelcode blue stain reagent, and gently shaken for 2 hours. Stained gels were then rinsed in sterile deionized water overnight. At this time, bands were clearly visible. The gels were photographed and analyzed with Kodak 1D image analysis software. Baseline and post-exposure lanes were compared in order to determine whether existing proteins were enhanced, or novel proteins had appeared.

Using the image analysis software, the lanes on each gel were found automatically after outlining the area of the gel to be analyzed. Identified lanes were marked with a line that passed through the center of the lane. For lanes that did not run perfectly straight, lane lines were adjusted so that they followed the actual path of the

129 lane. In order to more accurately calculate the size and mass of each band, the lane line needed to pass through the center of each band of interest. Lanes could also be added or removed manually if necessary.

Once the lanes were marked, they were labeled as ‘standard’ or ‘experimental’.

The standard run with these gels was the Prosieve® Protein Marker available from

Cambrex Corporation. This protein standard contains 10 proteins with known molecular weights (MW) of 5, 10, 25, 35, 50, 75, 100, 150, and 225 kDa. The Kodak 1D program identified bands within each lane by creating a median profile for each lane, identifying the bands, and determining the MW and mass in µg for each experimental band based on the information input for the standard. Due to the varying amount of background in each gel, the computer did not automatically identify some bands, and some that were identified needed to be adjusted in order for the correct weight and mass to be calculated.

Using the profile for each lane, bands were identified by the peaks present in the profile.

For each peak, the mass was calculated by positioning the top and bottom boundaries of a rectangle to outline the entire intensity of the peak. The computer calculated the sum of all the pixels in the band, minus the intensity of the background. The molecular weight of the band in kDa was calculated by positioning a centered line within the rectangle to the peak point of intensity. Band masses were compared between the first and second hemolymph samples for each individual, and the change in mass calculated. Bands that increased in mass were labeled “enhanced”, while bands that were not present in the baseline profile were labeled “new”. The bands analyzed were placed into five groups according to their MW (1-17, 18-59, 60-89, 90-124, 125-224, and 225-325 kDa).

130 Statistical analyses

Termite masses differed significantly across treatments. Although the same amount of hemolymph was extracted from each termite, larger termites may have more resources to invest in protein production per unit volume. Changes in band mass were standardized by dividing by the mass of the termite. Since the data were not normally distributed, non-parametric Kruskal-Wallis (KW) tests were used to compare changes in band mass of new and enhanced bands across directly exposed treatments (C, CE, CED) and naïve treatments (NC, NCE, NCED). Numbers of new and enhanced bands in each

MW range were also compared. A Bonferroni correction was applied to correct for multiple comparisons (Rice, 1989). All statistical analyses were run using SPSS 17.

5.3 Results

After interacting with conidia exposed individuals, the hemolymph samples extracted from naïve termites (NCE, NCED) exhibited induction of novel proteins

(Figure 5.2). Each of the 208 individuals used in this experiment (C = 31; NC = 27; CE =

43; NCE = 46; CED = 30; NCED = 31), provided hemolymph for both baseline protein profiles and profiles following their respective treatments. Approximately 80% of NCE and 81% of NCED individuals produced novel proteins in comparison to 67% of the NC individuals. These differences, however, were not statistically significant (z-test p ≥ 0.3).

The fact that naïve controls exhibited novel proteins is not surprising, since abrasion and puncturing of the cuticle can induce immunological responses (Hoffmann, 1995). Males and females did not differ significantly in the mass of new bands (KW, p = 0.4) or enhanced bands (KW, p = 0.9), so their results were pooled for all subsequent analyses.

131 Figure 5.1 Diagram depicting the timing of hemolymph extractions one and two, and the combinations of treatment groups: Control (C), Naïve Control (NC), Conidia Exposed (CE), Naïve Conidia Exposed (NCE), Conidia Exposed Delay (CED), and Naïve Conidia Exposed Delay (NCED). Exposures occurred three days after the initial hemolymph extraction and prior to placement with naïve individuals.

1st hemolymph extraction (baseline profile)

C NC CE NCE CED NCED

3 days 3 days 3 days

Immediate CE + C + NC social CED NCED NCE interaction

3 days

2nd hemolymph extraction Delayed CED + (experimental protein social NCED Experimentalprofile hemolymph interaction proteinEx perimentalprofile (testing if immediate contact with active conidia on the cuticle 3 days of nestmates elicited changes in the profile)

2nd hemolymph extraction

Experimental hemolymph protein profile (testing if social interactions (i.e. oral/anal trophallaxis) elicited changes in the profile)

132 Direct exposure groups (C, CE, CED)

Termites directly exposed to a control Tween 80 solution or conidia suspension differed significantly masses of new bands in the 125-224 kDa range (KW, p = 0.01;

Figure 5.3). The CED had the highest median band masses, and the C group the lowest in that range. In addition, although not statistically significant, the CE and CED groups had greater changes in band masses (i.e. larger new bands) in the 90-124 kDa and 225-325 kDa ranges. There were no significant differences in the overall number of new bands across the direct exposure treatments (KW, p ≥ 0.09; Figure 5.4), but the greatest number of new bands was produced by the CE and CED groups in the 18-59 kDa and 125-224 kDa ranges, and by the CED group in the 60-89 kDa range. There were no significant differences in the degree of enhancement of constitutive proteins (KW, p ≥ 0.027) or number of enhanced bands across treatments (KW, p > 0.02) after controlling for multiple comparisons (threshold p-value = 0.02).

Naïve groups (NC, NCE, NCED)

In comparison to the directly exposed termites, termites from naïve treatments differed significantly in masses of new bands produced in the 18-59 kDa range (KW, p =

0.003; Figure 5.5). Although the NC termites had higher median band masses, NCE and

NCED termites produced a higher median number of new bands in that range (Figure

5.6). The naïve termites produced the most new proteins in the same ranges as those produced by the exposed termites they interacted with (18-59 kDa and 125-224 kDa).

There were no significant differences in the number of new bands produced across naïve treatments (KW, p ≥ 0.3). There were also no significant differences in the masses of

133 enhanced bands (KW, p ≥ 0.292) or numbers of enhanced bands (KW, p ≥ 0.3) in naïve termites.

Conidia exposed vs. control treatments

For the most part, conidia (CE, CED, NCE, NCED) and control (C, NC) groups shared many bands in common, although they expressed them to varying degrees. The one range of protein masses where they differed was the 1-17 kDa range. Only a small number of bands occurred in this range (44 out of 2337 bands analyzed), and of these only seven were novel proteins. This was likely due in part to the percentage of gels used, which focused more on the 18-59 kDa range of proteins. However, out of those seven novel proteins, three were produced by NCED nymphs (~2.1 kDa, ~3.5 kDa, ~16.5 kDa), two by NCE nymphs (~12.5 kDa, ~16.5), and one by a CED nymph (~16.5 kDa). These proteins were unique to termites from the conidia treatments, and did not occur in any of the control treatments. It should be noted that although these three treatments shared one protein in common (~16.5 kDa), they were not from the same colony and/or treatment so did not interact with one another. This suggests that protein production was induced as opposed to proteins being passed between termites through oral or anal trophallaxis.

134 Figure 5.2 Example hemolymph protein profiles from a NCE individual. Profile A exhibits the lane and band markings from the Kodak 1D gel analysis software. Profile B shows the same sample without markings. Note the novel bands at ~ 12.5, 25, 34, and 41 kDa in the 2nd hemolymph sample in profile B (arrows). These bands suggest that naïve individuals produced novel proteins following social interactions with CE nestmates. Unfortunately, the results were inconsistent and not all NCE individuals responded in a similar manner.

NCE

1st 2nd

225 150 100 75

50

35

25

10

5 A B

135 Figure 5.3 Median masses of new proteins across direct exposure treatments (C, CE, CED). ‘*’ denotes significance at p = 0.01 (KW). The dots denote outliers. Note that CE and CED individuals generally have higher and a larger variation in changes in the size of novel proteins produced.

*

136 Figure 5.4 Median numbers of new proteins per individual across direct exposure treatments (C, CE, CED). The dots denote outliers. Although there were no significant differences, the CE and CED individuals produced more new proteins than C individuals.

137 Figure 5.5 Median masses of new proteins across naïve treatments (NC, NCE, NCED). ‘**’ denotes significance at p = 0.002 (KW). The dots denote outliers.

**

138 Figure 5.6 Median numbers of new proteins per individual across naïve treatments (NC, NCE, NCED). The dots denote outliers. Although there were no significant differences, the NCE and NCED individuals produced more new proteins than NC individuals.

139 5.4 Discussion

Zootermopsis angusticollis is a primitive termite species that nests within decayed wood, which is also colonized by numerous and diverse microorganisms (Hendee, 1933;

Rosengaus et al., 2003). Various individuals likely encounter a number of potential pathogens in different parts of the nest on a regular basis. Due to their social nature, it is reasonable to assume that the colony as a whole would benefit if exposed or immune primed individuals could stimulate an immune reaction in nestmates that had not yet encountered the same pathogen themselves. Traniello et al. (2002) reported higher survivorship of challenged naïve nymphs after socially interacting with immune primed nestmates as compared to naïve nymphs that had interacted with controls. The differences in the protein profiles in the current study were mostly nonsignificant and thus inconclusive. Yet, the results do show that a portion of the naïve termites exhibited new hemolymph proteins following social interaction with nestmates exposed to a fungal pathogen. Their protein profiles were consistent with results expected from naïve termites that had become immunized through social contact. The difference between adding naïve individuals to directly exposed nestmates immediately (NCE) and adding naïve individuals three days after their nestmates had been exposed to conidia (CED) enabled a differentiation of the mode by which social immunity may take place. In the first treatment (NCE), naïve individuals were likely responding immunologically after contacting sublethal dosages of conidia during allogrooming bouts. In the delayed treatment however, naïve individuals were likely responding immunologically by receiving immune factors and/or immune elicitors via social interactions (i.e. trophallaxis and/or proctodeal feeding, or other unknown mechanism).

140 The majority of novel bands occurred in the 18-59 and 125-224 kDa ranges.

These coincide with results from Rosengaus et al. (2007) in which termites directly exposed to fungal conidia produced novel bands in the 28-48 kDa range. Immune proteins such as lectins, hemolin, and proteins involved in encapsulation fall within these ranges (Gillespie et al., 1997). However, since individuals across all treatments exhibited proteins in these ranges, it is uncertain in this case whether production of immune proteins by naïve termites in these ranges was stimulated by social contact.

Few new bands appeared in the 1-17 kDa range (Figures 5.3 and 5.4). However, this was the one MW range in which no novel bands were shared in common between the

NC group, and the NCE/NCED groups. In addition, only one control individual (3% of the total) produced a novel protein in this range at ~14 kDa. One NCED individual (3%) produced a ~2 kDa protein. This is similar in size to the inducible immune peptide thanatin that has demonstrated antifungal activity in the hemipteran Podisus maculiventris (Fehlbaum et al., 1996). A second NCED individual produced a protein of

~3.5 kDa, which is similar in size to a known termite antifungal protein, termicin.

Termicin was originally isolated from the fungus growing termite Pseudacanthotermes spiniger (Lamberty et al., 2001), and genes similar to those regulating termicin have since been identified in various Nasutitermes species (Bulmer and Crozier, 2004). Three individuals (2-3% each of CED, NCE, NCED) produced a protein of ~16.5 kDa.

Lysozymes isolated from other insects are ~16.5 kDa (Yoshida et al., 1996), and are often strongly induced in response to infection (Hultmark, 1996). Lysozymes have demonstrated antifungal activity in Galleria mellonella (Vilcinskas and Matha, 1997), plants (Wang et al., 2005), and humans (Samaranayake et al., 1997).

141 The strength and frequency of hemolymph protein production in the CE and CED treatments were reduced in comparison to results from CE treatments of Rosengaus et al.,

(2007). One possible explanation for these differences lies in one divergence between the experimental procedures In the current study, termites were allowed to interact in social groups following conidia exposure to determine the effects of social interaction on hemolymph banding patterns, whereas the former study kept each individual in isolation.

Germ tubes of fungal conidia take approximately 48 hours to fully penetrate the cuticle

(Hänel, 1982), and during that time conidia can potentially be removed by grooming nestmates, hence reducing the chance of conidia penetrating the cuticle, and eliciting an immunological response. Antimicrobial peptide production is stimulated by signaling pathways initiated in the hemolymph, therefore germ tubes must penetrate the hemocoel in order for an immune reaction to take place (Ligoxygakis et al., 2002). The effectiveness of social grooming has been well documented (Rosengaus et al., 1998a,

2000a; Shimizu and Yamaji, 2003; Yanagawa and Shimizu, 2007; Yanagawa et al.,

2008), as well as inactivation of M. anisopliae conidia in termite guts (Kramm et al.,

1982; Chouvenc et al., 2009b; Schultheis, 2009). A recent study on the wood ant Formica paralugubris demonstrated a trade-off between costly individual defenses and energetically less costly colony-level behavioral defenses (Castella et al., 2008a). It is likely that Z. angusticollis nymphs exposed to a sublethal dose of M. anisopliae can efficiently remove and inactivate a majority of the conidia they were exposed to, and therefore reduce their investment in costly AMP production. This reemphasizes the important role of allogrooming in social insects as a mechanism that increases disease

142 resistance, and introduces a possible trade-off between social behaviors and individual immune investment in termites.

In spite of these efforts, the means by which hemolymph protein production is stimulated in naïve termites still remains elusive. NCE and NCED individuals, representing the two different possible mechanisms, produced novel proteins of similar masses (Figure 5.5), and similar numbers of those novel proteins (Figure 5.6). The one small difference between these two treatments was that three out of 31 NCED nymphs

(~10%) exhibited new bands in the 1-17 kDa range in comparison to one out of 46 NCE nymphs (~2%). It is also interesting to note that no CE termites had novel bands in this range. The NCE nymphs were allowed to interact with the CE nymphs immediately following conidia exposure (Figure 5.1). As a result there were twice as many individuals active in grooming at that time than there were in CED groups. This may have allowed for some conidia to penetrate the cuticles of CED individuals, enter the hemocoel, and induce an immune response. Since the CED nymphs were surface sterilized and transferred to a new dish prior to interacting with the NCED nymphs, there were no external conidia by which naïve individuals could be infected. As a result, the aforementioned proteins likely were produced in response to social interaction with infected nestmates as opposed to exposure to conidia during allogrooming. This provides the first electrophoretic evidence that conidia exposed Z. angusticollis nymphs potentially socially immunize naïve nestmates.

Collectively, the results point to the possibility of social immunization and social transmission of immune factors among social insects. Nymphs of Z. angusticollis appear to undergo behavioral defenses, such as allogrooming, prior to investing in individual

143 production of AMPs. However, about 5% of naïve individuals that were only indirectly exposed to conidia (NCE, NCED) produced novel proteins of similar sizes to known antifungal peptides. Those proteins did not appear in any of the control treatments, and are indicative of immune protein production fostered by social contact. Further studies on the mechanisms of social immunity would benefit from incorporating gels that focus on isolating proteins in the 1-17 kDa range, as those were the only novel bands produced exclusively in the conidia treatments. Although the mechanism by which these novel proteins are socially induced needs further confirmation, it appears that it involves more than contact with conidia during grooming. Termites undergo oral and anal trophallaxis, so the products of these exchanges should be analyzed for the presence of immune elicitors.

144 Chapter 6 – Overall discussion and conclusions

The work described herein is the first to explore the role that pathogens may have played as selection pressures in evolution of termite nesting behavior, and the importance of nest architecture in termite survival against pathogens. In the following paragraphs I will summarize the evidence provided by the results, and make suggestions for future areas of research.

In order to determine whether arboreal nesting confers a selective advantage by having reduced microbial pressures, Chapter 2 examined the number of bacteria and fungi on individual termites, and in the nest cores, trails, and surrounding soils of the arboreal nesting Nasutitermes acajutlae. Although not always statistically significant, overall there tended to be lower numbers of bacteria and fungi in nest core material and on the cuticles of core termites than in the other two locations. This pattern was slightly less apparent in 2006, which had a higher recorded rainfall. This rainfall likely increased both the prevalence of bacteria in the environment (Singh and Singh, 1978; Keya et al.,

1982; Castro et al., 2010), and the number of termites foraging (Moura et al., 2006; Fuller pers. comm.). As a result, the number of bacteria present within the nest appears to be at least partially influenced by what termites bring back to the nest following foraging trips.

Although bacterial numbers sometimes increased in response to more active foraging, fungal numbers did not follow the same pattern. Instead, defenses such as allogrooming, soldier secretions, antimicrobial substances within the nest material, and higher nest temperatures may help to reduce the prevalence of fungi within nests. By nesting away from the soil, arboreal termites likely reduce the ability of soil microbes to infiltrate their

145 nest, and their anti-pathogen behavioral and biochemical defenses may suppress colonization by fungi picked up during foraging trips.

As temperature increased, the numbers of bacteria increased while the fungi associated with N. acajutlae diminished. The numbers of fungi also decreased in response to increasing light. Previous studies have demonstrated the importance of heat and light in nest thermoregulation and nest site selection (Leponce et al., 1995; Korb and

Linsenmair 1998a, 1998b). As these factors also influence microbial communities, it will be important in future research on nest evolution to differentiate between these selective pressures.

In addition to there being lower numbers of fungi in nest cores, there was a lower diversity of fungal genera (Chapter 3). There was also only a 21% similarity between the core and soil fungal genera. The fungi identified were a small subsample of the overall communities, but provided the first molecular analysis of culturable fungi associated with an arboreal nesting termite. Some of the identified fungi are known decomposers that were likely carried back to the nest after foraging. Others have previously been isolated from the guts of other wood-feeding insects, and N. acajutlae nests are constructed from fecal material and salivary secretions. A few of the fungi collected from the nest cores, such as Paecilomyces sp. and Candida sp., are potentially pathogenic to termites.

However, no active growth has ever been observed in a live nest. It is possible that these fungi are dormant within nests, and the aforementioned defenses reduce the chances of an epizootic. Molecular investigation of fungal communities associated with other arboreal nesting termite species, along with analyses that incorporate the large number of

146 unculturable fungi, would allow for confirmation that there are fewer fungal pathogens within arboreal termite nests in addition to the overall lower quantity of fungi.

The above results suggest that arboreal carton nests are not completely isolated from the ground as are other arboreal microhabitats (Kurihara et al., 2008; Rousk and

Nadkarni, 2009). Instead, there seems to be a degree of cross-fertilization between what is naturally associated outside a nest and what colonizes the inside of a nest. Individuals that spend the majority of their time inside the nest, although protected from other insults

(desiccation, predators, and ambient climate) do not necessarily escape exposure to external microbes. However, removal of the nest from the soil does appear to reduce the ability of soil microbes to enter the nest outside of what is carried on the cuticle of foragers, and anti-pathogen defenses such as allogrooming, antimicrobial soldier secretions and nest components, as well as the high temperature within the nests maintain the overall health of the colony. The idea that extant arboreal nesters are under lower pathogenic pressures applies well to species such as bees that forage above ground

(Michener, 1985; Wcislo, 1996), but special consideration must be taken when applying this hypothesis to ground-foraging animals.

The results of the aforementioned chapters suggest at the importance of antimicrobial defenses within the nest, and nesting away from the soil, in defense of a colony against pathogens. In Chapter 4, nest architecture was found to be a significant and independent predictor of termite survival, and may be another means by which termites are able to reduce susceptibility to disease at the colony level. The structure of the nest appears to influence the amount of social contact, and therefore can impact the encounter rates amongst individuals within a nest. Reduced encounter rates may decrease

147 the potential benefits from allogrooming behavior. Alternatively, increased separation of sick individuals due to differences in nest architecture may reduce contagion. The microclimates within the artificial nests likely did not influence termite survival, but nest microclimate is often influenced by the structure or location of a nest (Weir, 1973;

Leponce et al., 1995; Korb and Linsenmair, 1998a, 1998b, 2000a; Turner, 2000, 2001;

Korb, 2003a, 2003b) and should still be considered as a possible means by which nest architecture affects termite survival.

Interestingly, this research included termite species that exhibit three different nesting strategies and the nest structure that conferred the highest survival was not the same for all species. The primitive one-piece nesting Zootermopsis angusticollis had the highest survival in a gallery-type nest modeled after its natural nest, while the more derived Reticultiermes flavipes and Nasutitermes corniger had higher survival in a nest modeled after the more complex, separate type nests (Abe, 1987). This is indicative that termites are adapted to a particular nest structure, and provides further evidence for the importance of nesting behavior to termites’ survival against pathogens.

As nest architecture appears to be important in influencing the amount of social contact, and therefore the ability of nestmates to reduce susceptibility to disease, Chapter

5 investigated the mechanisms by which social immunity may be attained within a nest.

Naïve Z. angusticollis nymphs were allowed to socially interact with nestmates directly exposed to Metarhizium anisopliae, and their hemolymph protein profiles were examined for upregulation or production of novel immune proteins using SDS-PAGE gel electrophoresis. Two possible mechanisms of social immune protein induction were examined: induction through contact with conidia on the cuticles of nestmates during

148 allogrooming, and induction through the transfer of immune elicitors between individuals with no direct conidia contact. The results were inconclusive, but do provide direction for further study. The only novel bands produced exclusively in conidia exposed individuals, or naïves that interacted with conidia exposed individuals occurred in the 1-17 kDa range.

As this is also a range in which known termite immune proteins fall (Lamberty et al.,

2001), I propose incorporating the use of gels that focus on these smaller proteins in future research. In order to ensure that an immune reaction is induced in directly exposed individuals, an isolation period should be incorporated prior to allowing the termites to socially interact. Alternatively, a pathogen such as the bacteria Serratia marcescens could be injected directly into the hemocoel, and therefore bypass any behavioral defenses.

Lastly, it appears that termites benefit greatly from behavioral anti-pathogen defenses, and as a result do not need to invest in potentially costly individual immune responses.

Studies in ants have demonstrated trade-offs between social and individual immunity

(Castella et al., 2008a) but this has not yet been examined in termites. This is a promising new direction for research on the social immunity of termites.

In conclusion, this research has laid the groundwork for further investigation of the influence of pathogens on termite nest architecture and nesting behavior. It has been established that an arboreal nesting termite has overall lower quantities and diversities of microbes within its nests than are present in the surrounding environment. These results support studies in other insect and vertebrate species that have found lower occurrences of pathogens or parasites in arboreal nesting species (Wcislo, 1996; Durden et al., 2004;

Robson, pers. comm.). Studies on additional arboreal nesting species, along with the identification of unculturable microbes would allow for confirmation that pathogenic

149 pressures are lower in species that nest aboveground. In addition, some Nasutitermes species, such as Nasutitermes ephratae, are known to build both mound and arboreal nests, and would be ideal study species to address exposure risks while controlling for phylogenetic differences. Studies of the factors that influence extant arboreal nesting would provide valuable insight into the forces behind arboreal nest evolution. Arboreal termite species still initiate their colonies on the ground (Nutting, 1969, Hartke, pers. comm.), and only relocate the nest to an arboreal location once the colony has been well established (Thorne and Haverty, 2000). As nest building is adaptively plastic (Theraulaz et al., 1998, 2003) and termites are able to detect and avoid pathogens (Kramm et al.,

1982; Epsky and Capinera, 1988; Staples and Milner, 2000; Mburu et al., 2009), it is possible that the first termites to nest arboreally detected pathogens in their environment, and responded accordingly by selecting a new nest site removed from these pathogenic pressures. I propose that future examinations of the forces behind termite nest evolution should take into account the composition of their associated microbial communities. As pathogens and parasites have been considered important factors in the evolution of termite eusociality (Rosengaus and Traniello, 1993) and enclosed nests were likely a preadaptation that fostered sociality (Hamilton, 1978; Hansell, 1987), it is reasonable to suggest that pathogens played a role in the further evolution and ecology of termite nesting behavior.

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