Cyphoderris Monstrosa (Orthoptera: Haglidae)

Effect of Acoustic Signaling, Metabolic Rate, and Size on Territoriality in Male Cyphoderris monstrosa (Orthoptera: Haglidae)

Terrence T. Chang

Ecology and Evolutionary Biology University of Toronto

A thesis submitted in conformity with the requirements for the degree of Master of Science.

Effect of Acoustic Signaling, Metabolic Rate, and Size on Territoriality in Male Cyphoderris monstrosa (Orthoptera: Haglidae)

Terrence T. Chang Masters of Science

Ecology and Evolutionary Biology University of Toronto 2015

Abstract

During territorial interactions, male ensiferan Orthoptera often stridulate before physical combat. Song is correlated with competitive ability; therefore, it is a reliable signal for resource holding power. Size and metabolic rate are also indicators of aggressiveness. Cyphoderris monstrosa (Orthoptera: Haglidae) are relatively insensitive to the frequency content of their own song, however, males use acoustic signals in the context of competition. Here, I examined size, diet, time spent singing, and metabolic rate with C. monstrosa territoriality. Using a round-robin tournament, I compared performance of males fed with a protein-supplemented diet to males fed with a low-protein diet. I measured body structures used for fighting, duty cycle during competition, and metabolic rate at two temperatures (22°C and 10°C). Competition in C. monstrosa is influenced by singing ability, physiological performance, and body and head size where winners have higher duty cycle, lower metabolic rates in cold, larger bodies, and smaller heads.

Acknowledgements

None of this would have been possible without the support and guidance of my advisor, Andrew C. Mason. I am most grateful for his enthusiasm, friendship, and uncanny ability to troubleshoot Cyphoderris problems – truly a “Cyphoderris whisperer”, if ever one existed. I could not imagine having a better mentor and advisor for my studies.

I would also like to thank the rest of my committee: Maydianne Andrade and Darryl Gwynne Their insights, perspectives, and questions motivated me to expand and continue my research. Also, I must thank Kevin Judge for all the discussion and advice he offered for this project.

I would also like to thank Brock Fenton, who started me on this journey, and Jeremy McNeil, without whom I would not have found this lab. Both are constant sources of encouragement and are largely responsible for my decision to pursue this path.

I thank my Cyphoderris wranglers: Niroshan Ranjan, Jeff Garel, Keerthana Rajkumar, Jill Thaker, and Tameem Al-Ozzi. Their steady help with data collection and taking care of specimens was a great relief and allowed me to maintain some semblance of sanity. I also thank Derrick Groom for his assistance with the respirometry equipment.

AJ Masson, Sen Sivalinghem, Natasha Mhatre, and Jerry Pollack were sources of valuable discussion and helped me approach my project from different perspectives. Erica Morley and Ryo Nakano also helped with collecting, for which I am grateful.

Special thanks goes to Megan McPhee who was a great source of support, both academic and personal. When I started this project, I did not know I would find my best friend. From stand-up shows and inordinately long dinners to writing sessions and discussions, I am glad to have shared this experience with her.

I am also fortunate to have the unwavering support and enthusiasm of Joyce Lai. There are simply not enough words to describe my gratitude.

Finally, I am especially grateful to my parents, Louis and Winnie Chang. Their constant support has been the greatest gift. I would never have enjoyed so many opportunities if not for them.

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Table of Contents

Abstract ...... ii Acknowledgements ...... iii Table of Contents ...... iv List of Tables ...... v List of Figures ...... vi

Introduction ...... 1

Animal Communication ...... 1 Competition in Orthoptera ...... 7 Natural History of Cyphoderris monstrosa ...... 10

Methods ...... 14

Specimen Collection and Husbandry ...... 14 Competitive Interactions ...... 14 Metabolic Rate Measurements ...... 16 Morphometrics ...... 17 Statistical Analysis ...... 17

Results ...... 19

Discussion ...... 21

References ...... 27

Tables and Figures ...... 35

List of Tables

Table 1: Final model from generalized linear mixed model of contest outcomes and duty cycle in competing C. monstrosa males...... 35

Table 2: PCA results for body size metrics ...... 36

Table 3: Final model from generalized linear mixed model analysis of overall status in competing male Cyphoderris monstrosa ...... 37

List of Figures

Figure 1: Map of Cyphoderris spp. distribution in Western Canada and USA ...... 38

Figure 2: Diagram of arena used for Cyphoderris contests ...... 39

Figure 3: Relationship of duty cycle and outcome of territorial interaction ...... 40

Figure 4: Body size of winners and losers ...... 41

Figure 5: Head size of winners and losers ...... 42

Figure 6: Ratio of metabolic rate at two temperatures in winners and losers ...... 43

Introduction

Animal Communication

Communication is the transfer of information from a sender to a receiver where the behaviour of the receiver is affected (Bradbury & Vehrencamp, 2011). Communication occurs with the use of signals which arise from ritualization or exaggeration of non-signaling behaviour and exploitation of pre-existing sensory biases (Lorenz, 1966; Bradbury & Vehrencamp, 2011; Scott-

Phillips et al., 2012). As such, signals are based on visual, chemical, acoustic, tactile, or behavioural traits used in acquiring and maintaining resources (Hasegawa et al., 2011). The function of communication is heavily studied in the contexts of mating, competition, foraging, predator deterrence, and parent-offspring interaction (Dodson et al., 1983; Mason, 1996; Hare,

1998; Cocroft, 2005). Due to the inherent nature of communication, signals are under co- evolutionary pressures exerted from both signaler and receiver. Signaling traits persist if there are overall benefits to both parties. Several ways in which the use of signals can directly influence the fitness of individuals include: securing mating opportunities; energetic costs of signaling; exposure to risk of predation or parasites; discovery of foraging opportunities; deception or manipulation by others; and establishing dominance in a hierarchy (Bovbjerg, 1953; Belwood & Morris, 1987;

Snedden & Sakaluk, 1992; Endler, 1993; Hughes et al., 2012). In this thesis, I explore the principles governing success in male territorial contests of the relict flightless haglid, Cyphoderris monstrosa (Orthoptera: Haglidae). Based on previous literature of C. monstrosa, acoustic signaling plays a role in predicting contest outcome (Mason, 1996; Morris et al., 2002). Here, I re-examine the effects of singing effort as well as differences in singing between individuals maintained on different diets. In addition, I observed the effects of size and physiological performance on success in competitions.

Animal displays are diverse and vary in form, size, modality, and costs of production and reception (Dawkins, 1993; Shaw, 1994; Galeotti et al., 2006; Cady et al., 2011). There is no general principle of signal design due to the many possible combinations of selective pressures on signals resulting in high diversity (Dawkins, 1993). Selection pressures that may influence signal design include previously mentioned fitness consequences for signalers and receivers (i.e. degree of conflict or cooperation) as well as, signal efficacy including production, transmission, and reception (Dawkins, 1993; Bradbury & Vehrencamp, 2011). The type of selection signals undergo depends on how signaler intention and receiver preference interact: if they coincide, then selection is stabilizing; if the signal is outside the range of preferences, then selection is directional; if there is preference for extreme types, then selection is disruptive (Ryan & Rand, 1993; Naisbit et al.,

2001). Signals also vary in complexity, ranging from simple signals consisting of one component to highly complex signals comprised of multiple components that may be targeted to multiple sensory systems or modalities (Candolin, 2003).

Determining information content of multicomponent or multimodal signals and resulting receiver responses explains how selection shapes and maintains these signals (Candolin, 2003;

Ossip-Klein et al., 2013). Though this task is inherently difficult, using controlled experiments in a comparative approach, we can determine qualities that correlate to a set of signals. For example, numerous studies have broken down calls of various katydid species into their fundamental structural components to examine the content of each component (Simmons & Bailey, 1993;

Galliart & Shaw, 1996; De Luca & Morris, 1998; Korsunovskaya, 2009). Depending on the medium through which signals are transferred, they may also attract unintended receivers such as predators which can affect both signaling and receiving behaviour (Hughes et al., 2009). In some instances, multimodal signaling may evolve as an antipredator behaviour. In the Neotropics,

katydids (Tettigoniidae) face strong predator pressure from insectivorous bats. Katydid calling and courtship song is highly localizable for bats (Belwood & Morris, 1987). As a result, katydids have shortened acoustic signals and developed the use of tremulatory signals – in at least one species, vibrations have completely replaced acoustic signals (Belwood & Morris, 1987; Morris et al.,

1994).

In addition to the production and propagation of signals, reception of these signals adds another dimension of complexity to communication. Based on how receivers perceive and process signals, signals evolve to be more easily received. Signals that decrease latency to respond and make decisions are favourable; thus, receiver response may be the primary selection pressure on signals

(Rowe, 1999; Candolin, 2003). In some taxa, assessing signals is dependent on context or environment (McLennan, 2003; Hunt et al., 2005; Callander et al., 2011; Swierk et al., 2012). For example, Hunt et al. (2005) found that female black field crickets raised on high-protein diet were more sexually responsive and preferred higher call rates and higher dominant frequencies than crickets raised on low-protein diet. The energetic cost of receiving signals is also dependent on signal type – a study on signal reception in cockroaches (Blattariae) found that reception of vibratory signals is more energetically costly than reception of acoustic signals (Shaw, 1994).

Sensory systems have therefore evolved to optimize information transfer.

Sexual selection of signals operates through two different processes: female mate choice and male-male competition (Andersson, 1982). While different signals can be used in different contexts, in some cases, the same signal is used in both intra- and intersexual displays (i.e. multiple receivers hypothesis; Andersson et al., 2002). This may be due to an overlap in information content that is transferred in both contexts. For example, in male Iberian red deer, Cervus elaphus, antler size and complexity are honest signals for sperm quality as well as fighting ability (Malo et al.,

2005). However, in male house crickets, Acheta domesticus, signals used during competition with males are different from signals used to court females (Alexander, 1967). Intersexual conflict also exists because males benefit from high mating rates whereas females benefit from being choosier

(Bateman, 1948; Candolin, 2003). Accordingly, there is antagonistic co-evolution between signalers and receivers (Holland & Rice, 1998). Signals evolve so that males are able to overcome female resistance and reception in females evolves so females become less responsive to manipulative signals (Moller & Pomiankowski, 1993; Rice, 1996).

Advertisement and courtship displays provide receivers with assessable signals which influence mate choice and in some cases, increase reproductive fitness of signalers. Many courtship and mating systems include signals, which may be pre- or post-copulatory (Alexander, 1967; King &

Fischer, 2005; Ablard et al., 2011). These intersexual displays persist due to direct benefits from receiver choice and/or indirect benefits for offspring (Burley, 1977; Hoelzer, 1989; Kojima et al.,

2009). Pre-copulatory signals confer advantages by allowing individuals to assess quality of potential mates. Post-copulatory signals confer advantages to males, by allowing them to ensure paternity, as well as to females in cases where mechanisms for post-copulatory choice exist, by giving them more time to reassess males (Kajita, 1986; Eberhard & Cordero, 1995; Ablard et al.,

2011). For example, in the parasitoid wasp Ooencyrtus kuvanae, pre- and post-copulatory rituals consist of antennal interlocking and antennal striking by the male (Ablard et al., 2011). Females that experience pre-copulatory rituals enter a trance-like state, such that they are more receptive to the immediate male, and mating occurs faster; post-copulatory rituals function as a form of ‘in absentia’ mate guarding, decreasing remating in females by ‘awakening’ them from the trance- like state sooner such that they are unreceptive to other males (Ablard et al., 2011). In the scorpionfly Harpobittacus nigriceps, females receive nuptial gifts during copulation (Thornhill,

1983). If females mate with small males or receive small gifts, they will not lay eggs and instead re-mate until a sufficiently large male or gift is found (Thornhill, 1983). Thus, this is a method in which females re-assess males post-copulation.

Communication signals are also used when animals compete over limited resources such as food or mates. Typically, these competitions occur between conspecific individuals of the same sex. Signals employed in these scenarios can be classified as intrasexual displays. While interests of sender and receiver are diametrically opposed in this context, escalating conflict to physical combat is costly. Therefore, selection for signaling strategies as conflict mediation occurs because it is considerably less costly (Maynard Smith & Price, 1973; Hack, 1997; Maynard Smith & Harper,

2003). Contests can be classified according to symmetry, defined as the similarity between competitors which is correlated to the probability of winning. In nature, most contests are asymmetrical: there is a mismatch in age, size or roles (e.g. defender/intruder), which may confer an advantage on one of the competitors (Maynard Smith & Price, 1973; Maynard Smith, 1982;

Kemp & Wiklund, 2004). In symmetrical contests, competitors are evenly matched in all relevant fighting traits and a winner is typically determined through escalated competition (Maynard Smith

& Price, 1973).

In asymmetric contests, assessment strategies should evolve to allow individuals to avoid direct combat and the potential costs of losing competitions (Maynard Smith & Price, 1973; Maynard

Smith, 1982). A number of models have been proposed for contest settlement including: 1) sequential assessment, 2) war of attrition, 3) cumulative assessment, and self-assessment (Parker,

1974; Maynard Smith & Parker, 1976; Enquist & Leimar, 1983; Payne, 1998; Taylor & Elwood,

2003). Determining the type of assessment strategy used in animal contests relies on distinguishing the information that is transferred during signal exchanges as well as the individual that is making

the assessment (i.e. signaler, receiver, or both). For example, there could potentially be many types of information that are being transferred however, only a proportion of that information is being evaluated for assessment (Rillich et al., 2007). Similar to signaling behaviour, assessment strategies can also be context dependent. The range of variables that can affect territoriality and corresponding assessment strategies include but are not limited to: age, population density, length of contest (Payne & Pagel, 1996), prior residence on territory (Simmons, 1986), prior contest success (Adamo & Hoy, 1995), and presence of females (Simmons, 1986).

Competitive signals code for two basic types of information: resource-holding power and motivation (Maynard Smith & Price, 1973; Maynard Smith, 1982). Resource-holding power (RHP) is the ability for an individual to win a contest however, this interacts with motivation as well

(Maynard Smith, 1982). An individual with a high RHP but low motivation may not win against an individual with a low RHP but higher motivation. Whether signals have evolved under selection for conveying information about motivation is poorly understood. In theory, individuals who are going to lose should withdraw from the contest rather than spend energy conveying information about low motivation (Maynard Smith, 1982). In nature, individuals who are losing may employ surrender signals before withdrawing (Dow et al., 1976).

Competition in Orthoptera

Male ensiferan Orthoptera (e.g. crickets and katydids) produce acoustic signals to attract mates.

Males typically broadcast these signals from a stationary location and females approach males

(Gwynne, 1982; Snedden & Sakaluk, 1992), although call-answer systems in which female responses evoke male movement are also common (Bailey & Hammond, 2003). These calling songs also evoke responses from conspecific males. The type of response from males varies among species: some do not respond (Simmons & Bailey, 1993), some approach and fight (Mason, 1996),

and some even chorus (Nityananda & Balakrishnan, 2008). Often, these signals are complex, involving multiple components each transferring some information for assessing individual quality

(Hebets & Papaj, 2005). Defending and maintaining territory evolves when the ratio of resources to individuals is low or when females have a preference for specific resources (Parker, 1974). This behaviour gives males primary access to foraging patches, as well as perches for optimal signal broadcasting to attract females for mating purposes (Fitzpatrick & Wellington, 1983). When two males are within proximity of each other in suitable territory, they may engage in a contest for ownership (Fitzpatrick & Wellington, 1983).

Female orthopterans generally do not interact aggressively, and female competition is rarely observed in the field though there are exceptions (Gwynne, 1991). Theoretically, when the operational sex ratio is female biased, territory is limited, or when male parental investment is relatively high, females should compete and defend territory to secure better mating opportunities

(Berglund et al., 1993; Rillich et al., 2009). Male orthopterans invest considerably by transferring a spermatophore (in some species, a spermatophylax is transferred as well), therefore, fighting in females may be more common than previously thought. Under laboratory conditions, nonaggressive female Gryllus campestris have been shown to engage in physical combat (similar to conspecific males) when exposed to male calling or courtship song, thus showing that females are capable of agonistic behaviour (Rillich et al., 2009). In the presence of muted males, females did not fight; therefore, calling and courtship song functions as a display of male resource value

(Rillich et al., 2009).

Overtly aggressive male-male contests over resources are often preceded by extended bouts of competitive signaling. In many cricket species, aggressive signals have different temporal and frequency structures from regular calling songs (Alexander, 1961; Brown et al., 2006). Signals

that reliably transfer information regarding resource-holding power (i.e. fighting ability, size, weaponry etc.) can diminish the risks of competition escalating to physical aggression. Assessing

RHP provides a probabilistic prediction of contest outcomes; however, outcomes are unstable because contestants can escalate energy expenditure or adopt different strategies during competitions (Parker, 1974). Contests end when the weaker contestant withdraws, leaving the territory and any proximal females to the winner. Acoustic signals impose high energetic costs and can be considered honest signals of quality (Zahavi, 1975; Prestwich & Walker, 1981). Females respond phonotactically to males that exhibit high calling effort (Bentsen et al., 2006). Therefore, acoustic signals and energetic strategies for signal production are sexually selected traits that are mediated by female choice and male-male competition.

Energetic strategies influence outcomes of competitions by affecting display performance.

Differences in energy expenditure can be inherent or context-dependent (Hartbauer et al., 2012).

In the katydid, Mecopoda elongata, males will chorus when interacting with each other and synchronize their chirps which results in more efficient energy use (Hartbauer et al., 2012).

Energetic cost of display is also dependent on signaling mode (Belwood & Morris, 1987; Cady et al., 2011). In Schizocosa wolf spiders, multimodal signaling (comprised of visual and vibratory components) results in higher peak CO2 output than vibration-only signaling (Cady et al., 2011).

Some species of neotropical katydid employ vibratory components to their signals to avoid bat predation and it is thought that these displays are more costly than auditory signaling (Belwood &

Morris, 1987). Previous work on male competition in Cyphoderris monstrosa showed that winners have inherently greater metabolic rate increases (singing relative to resting) than losers (Van

Eindhoven, 2012). In many acoustic insects, contest winners are usually males with higher duty cycle and consequently, are also preferred by females (Mason, 1996; Bentsen et al., 2006; Ower

et al., 2013). Singing effort may be correlated to quality however, it does not necessarily predict occurrence of aggressive behaviour (Fitzsimmons & Bertram, 2012).

Evenly matched competitors (i.e., individuals with similar energy stores and expenditure strategies) generally take longer to reach an outcome because neither one has a clear advantage so signals will not reliably predict a winner (Parker, 1974; Enquist & Leimar, 1987). In these cases, contests can often escalate to intense higher cost tactics, such as physical combat, in order to determine which individual has an advantage (Parker, 1974; Hack, 1997). In the house cricket, wrestling is 40 times more energetically costly than stridulating (Hack, 1997). In addition to singing ability and energy stores, size and weaponry also influence potential to win contests

(Parker, 1974; Hack, 1997). Individuals who are relatively larger or have larger weapons tend to win contests more frequently (Judge & Bonanno, 2008; Umbers et al., 2012). However, it has been shown that in some taxa, there is no correlation between size and winning (Mason, 1996; Kemp &

Wiklund, 2001).

Temperature is another factor that affects activity, energetic strategies, and display performance in insects. Within normal temperature ranges of a species habitat, warmer temperatures increase signaling and mating activity (Hedrick et al., 2002). However, cold temperatures limit activity and performance (May, 1979; Angilletta et al., 2002). Several strategies are available for thermoregulation, including posturing and microhabitat selection (May, 1979; Hedrick et al.,

2002). Some Orthopteran species thermoregulate using metabolic heat from flight muscles by shivering their wings (i.e., low-amplitude vibration). A previous study showed that differential mating success in male sagebrush crickets, Cyphoderris strepitans, was not attributable to ability to withstand cold temperatures (Sakaluk & Eggert, 2009). It is unknown whether the degree to which energetic strategies change at lower temperatures is correlated to fighting ability. Males that

are able to maintain higher metabolic activity at lower temperatures could potentially perform better than males who cannot.

In this thesis, I determined the effect of size, diet, singing, and fighting ability on territoriality in a relict flightless haglid, Cyphoderris monstrosa. Additionally, I examined resting metabolic rates at two different temperatures to determine whether inherent differences in energy expenditure were reflected at lower temperatures and whether this was correlated to fighting ability.

Natural history of study species

The Haglidae is a relict family dating back to the Triassic period and its members are considered primitive ancestors to the families Tettigoniidae and Gryllidae (Mason, 1991). Once a highly diverse family, there are only seven extant species representing four genera of haglids (Gorochov,

1988; Kumala et al., 2005). Three genera are located in the Sino-Russian region and one genus is located in northwestern United States and southwestern Canada (Morris & Gwynne, 1978;

Gorochov, 1988; Kumala et al., 2005).

The genus Cyphoderris, or hump-winged grig, is represented by three species: Cyphoderris monstrosa, Cyphoderris buckelli, and Cyphoderris stepitans (Kumala et al., 2005). They are endemic to high-elevation coniferous forests in mountainous regions in western North America and are acoustically active in temperatures as low as –8 to –2 °C (Morris & Gwynne, 1978;

Sakaluk & Eggert, 2009). Though their diet has not been well documented, Cyphoderris eat an assortment of mountain berries, staminate cones, as well as fruit buds from peach and cherry trees

(Caudell, 1904). Of the three species, C. monstrosa occupies the largest range and is sympatric with C. buckelli over a significant range; C. strepitans is an allopatric species (Morris & Gwynne,

1978; Kumala et al., 2005). While they have similar ecological habitats, each species has distinct

perch and feeding sites (Morris et al., 2002). An analysis of thirty-two characters showed that C. monstrosa has undergone the most extensive morphological and behavioural divergence of all three species (Kumala et al., 2005).

Cyphoderris spp. emerge in late May to early June and at dusk during breeding season, males climb up from the ground and sing on vegetation (Dodson et al., 1983; Snedden & Sakaluk, 1992;

Morris et al., 2002; Ladau, 2003). Females display phonotaxis towards singing males (Snedden &

Sakaluk, 1992) and males proceed to court females by lifting their tegmina and exposing a set of fleshy hind wings (Eggert & Sakaluk, 1994). Cyphoderris spp. are unique amongst insects that display nuptial feeding because males provide dual nutritional investment. If courtship is successful, the female will mount the male and consume haemolymph and wing tissue by chewing on the fleshy hind wings (Eggert & Sakaluk, 1994). Males secure females in this position by use of an abdominal pinching organ called the ‘gin-trap’ (Sakaluk et al., 1995). Only males have a gin trap organ and it consists of 2 pairs of opposing spines positioned near the terminal end of the abdomen — specifically on the 10th and 8th tergite (Sakaluk et al., 1995). During mating attempts, males telescope their abdomens, bringing together the tergal spines to pinch and secure females in mating position (Sakaluk et al., 1995). Engaging the gin trap organ increases male chances of successful sperm transfer (Sakaluk et al., 1995). Males are also equipped with a claw-like sternal process on the subgenital plate (Kumala et al., 2005). The appearance and form of the sternal process differs among species: in C. buckelli and C. strepitans, it is relatively dull whereas in C. monstrosa, it is shaped like a claw hammer. The function of this structure has not been properly examined however, in C. monstrosa, it appears to be a hook for drawing the female subgenital plate closer for sperm transfer. Males proceed to force transfer of a spermatophore and spermatophylax. Once mating is complete, the male will pull away from the female (Dodson et al.,

1983). Successful courtship results in an unambiguous phenotypic marker to identify mated males

(Eggert & Sakaluk, 1994). Non-virgin males are at a disadvantage to re-mate because they have less haemolymph as well as less energy to stridulate (Sakaluk et al., 2004). However, previously mated males (i.e. males with chewed hind-wings) can coercively mate with females who are resistant by engaging the gin trap organ.

Cyphoderris spp. males produce acoustic signals by tegminal stridulation (Morris & Gwynne,

1978). Calling song frequency of C. monstrosa is around 12 kHz however, the auditory system of this species is most sensitive around 2 kHz (Mason, 1991). Despite the incongruity in sound production and reception, the function of song is two-fold: to attract females (Snedden & Sakaluk,

1992; Snedden & Irazuzta, 1994) and to mediate territorial conflicts with other males (Mason,

1996; Morris et al., 2002). Territorial disputes between males involve bouts of acoustic signaling and overt aggression (Mason, 1996). Overt physical aggression has only been shown in C. monstrosa while male-male competition in C. buckelli and C. strepitans is restricted to singing.

The outcome of territorial competitions are strongly correlated to singing performance where winners have a more sustained song (Mason, 1996).

The ability for Cyphoderris males to sing in temperatures as low as –8°C is an unprecedented feat among acoustic insects (Dodson et al., 1983; Sakaluk & Eggert, 2009). Sakaluk and Eggert

(2009) found that male C. strepitans maintain consistently higher thoracic temperatures than ambient temperature when singing. However, this may be due to heat generated as a byproduct from singing muscles rather than any form of thermoregulation (Sakaluk & Eggert, 2009). While ability to cope with low temperatures does not influence mating success in males (Sakaluk &

Eggert, 2009), it is unknown whether there is an influence on male competitive ability. If there is

an influence, males may be choosing perches with optimal temperatures to reduce energy required for defence.

Cyphoderris monstrosa males readily engage in competition when exposed to each other, making them an ideal model for exploring territoriality and aggression. Here, I re-examined the effect of singing effort on competition. As is the case in many Orthopterans, winners are usually individuals who sing more and this has been shown in C. monstrosa as well (Alexander, 1961;

Adamo & Hoy, 1995; Mason, 1996). The effects of diet on competitive ability have not been explored in this species. In nature, Cyphoderris monstrosa have access to sugars and protein via mountain berries and staminate cones. I expect that only sugars and fat are used as fuel during contests whereas proteins are used for growth and repair because of their function in long-term energy reserves. However, whether consistent supplementing of sugar diets with protein may have an effect is unknown. Also, I examined the effects of size on competition — in other Orthopteran species, larger size is typically indicative of higher RHP and therefore, large individuals win more

(Brown et al., 2006; Rillich et al., 2007; Judge & Bonanno, 2008). I predict the same to be true for

C. monstrosa. Finally, I explored the effects of temperature on metabolic rate of males which affects competitive ability. Males who have higher metabolic rates in the cold should win more because they have the energy stores to maintain higher levels of activity.

Methods

Specimen collection and husbandry

Between 28 May and 6 June 2014, I collected male and female C. monstrosa and C. buckelli from a population found around Paul Lake Provincial Park outside of Kamloops, British Columbia

(50°45'15.6"N 120°07'08.8"W, Figure 1). At this site, both C. monstrosa and C. buckelli can be found, however, I did not find enough C. buckelli to conduct experiments. I captured Cyphoderris between 2100 and 2300 hours, stored them individually in steel mesh cages, and fed each with a piece of apple. I transported all captured individuals to the University of Toronto Scarborough campus where they were subsequently transferred to larger cylindrical steel mesh cages and stored in a cold room at approximately 7°C (12:12 light cycle, dark from 1000 to 2200 hours). I sorted individuals based on species, sex, maturity, and sexual history in males. Females were kept in cages with soil for burrowing. I determined male sexual history by examining fleshy hind wing condition and sorted males as nymphs, virgins, or mated. I supplemented the diets of half the virgin males with bee pollen. Nymphs and females were also fed bee pollen. All Cyphoderris were fed with fresh food every other day. Prior to experiments, individuals were moved out of the cold room and acclimated to room temperature for at least 60 minutes. Individuals that died were immediately preserved in 70% ethanol.

Competitive Interactions

I organized competitive trials by dividing virgin males randomly into brackets of four only controlling for even diet distribution in each bracket (i.e. two males raised with apple only and two raised on apple with bee pollen). Each male fought all other males in the bracket for a total of three fights each. By using round-robin tournaments, I sorted males into general classes (i.e. winners

and losers) to organize them into a dominance hierarchy. Males that died before completing all three fights were not replaced. Forty males started fight trials and 33 completed all three fights; 5 males died after two fights and 2 males died after one fight. I staged competitions on an arena made from a section of eastern hemlock (Tsuga canadensis) trunk approximately 65 cm in height and 10 cm in diameter (Figure 2). All fights were conducted under red light during scotophase between 1000–1630 hours and recorded on video (Sony Camcorder HDR-CX700V). By placing the log on a turntable, I filmed the Cyphoderris with the least amount of disturbance. Males were acclimated to room temperature and fed fresh food 1 hour prior to fighting and rested for at least

24 hours between fights.

A competition consisted of releasing a pair of males at the base of the trunk and allowing them to compete for territory. Often, the two males would engage in bouts of competitive singing and on occasion, would escalate to aggressive combat. In several cases, males courted and mounted one another as is typical of female courtship and mating. I recorded the occurrence of physical fights, the actions of each individual during a fight, the number of times each action occurred, and the outcome of the fight. I classified four different types of interactions based on observations of competitions (Figure 2 – 5):

1. Acoustic: males approach each other resulting in prolonged bouts of competitive singing

without physical contact.

2. Chase or kick: one male chasing the other possibly resulting in contact; both males are

oriented in the same direction, one behind the other.

3. Wrestle: males approach each other and use forelegs to grab one another while clinging on

to log with hindleg tarsi.

4. Gin trap: males engage in behaviour typical of females during courting and mating.

A competition ended when one male left the log either voluntarily or forcibly. The male remaining on the log was considered the winner. In some cases, both males would fall off the log due to a physical interaction; in such cases, the male who oriented himself towards to the log and climbed up first was considered the winner. If a competition lasted over 45 minutes with both males remaining on the log, it was considered a draw. Males were given scores based on outcome of competitions to determine status as winners or losers (i.e. a win was worth 2 points, draw was

1 point, and a loss was worth no points). Males with an overall score of 3 or above with at least one win were considered winners, males with scores of 3 or below with no wins were considered losers.

For acoustic competitions, I determined the duty cycles of each male by measuring proportion of time spent singing during a ten minute interval. Due to the variation in physical interaction between fights, I selected and measured duty cycles from ten minute intervals prior to physical contact between males when possible.

Metabolic Rate Measurements

After completing all competitive trials, I conducted flow-through respirometry trials to measure

CO2 production in the remaining C. monstrosa males. Trials were conducted in an environmental chamber (KB055 Darwin Chambers, St Louis, MO, USA) at 22°C and 10°C in the dark. I used these temperatures because they are equivalent to the temperatures that Cyphoderris monstrosa experience during mating season. I measured resting metabolic rates of individual males in a chamber made using a storage container (HPL807 Lock & Lock, Anaheim, CA, USA) with air hose connectors screwed through the container walls. Also, a speaker was built into the container lid for synthetic song playback to induce male C. monstrosa to sing so I could obtain metabolic rates of singing activity. I measured metabolic rates as CO2 production using a LI-7000 infrared

gas analyzer (Li-Cor, Lincoln, NE, USA). I manipulated air flow using an MFS air flow controller and SS-4 subsampler pump [Sable Systems International (SSI), Las Vegas, NV, USA]. I also measured oxygen consumption using an FC-2 differential oxygen analyzer (SSI). CO2 and moisture was scrubbed from incoming air using Ascarite II (a sodium hydroxide coated silica) and magnesium perchlorate (a super-drying agent), respectively. Flow rate through the chambers was set to 200mL/min and did not appear to disturb the animals. I placed C. monstrosa individuals in the chambers for at least 60 minutes prior to respirometry to allow them to acclimate. I collected data for 32 males at 22°C and 18 males at 10°C.

Each trial of continuous measurement in the respirometry chamber lasted approximately 30 minutes. An average of CO2 production was calculated for each trial. All metabolic rates are reported in mLCO2/hour.

Morphometrics

I dissected and photographed all the males included in competitive trials to obtain morphological measurements. All body structures were submerged in 70% ethanol. I used a Nikon

SMZ800 zoom stereomicroscope to image 10 body structures used in fighting and mating: pronotum width, hindleg femur length, foreleg femur length, head width, maxillae span, gin-trap width, and tegmina width. I digitized two landmarks on each images and measured linear distance using the software program tpsDIG2 (Rohlf, 2013).

Statistical analysis

I assessed within-individual variation in signaling (i.e. duty cycle) by ANOVA to generate mean square values and calculate repeatability (r) following Lessells and Boag (1987). Repeatability scores above 0.50 are considered high. Because individuals were involved in multiple fights, I

performed a generalized linear mixed model (GLMM) analysis to account for repeated measures in order to examine the effect of duty cycle on fight outcome.

Because body structures are internally correlated, I performed a principal components analysis

(PCA) with all body size measurements to reduce the number of factors in the model (Table 1). I was not interested in examining asymmetry in body parts so I averaged right and left measurements for foreleg, hindleg, and forewing. I used another generalized linear mixed model accounting for random group effects (i.e. four-male groups used in competitive trials) to test the relation of size, diet, and metabolic rate on overall status of male C. monstrosa as winners or losers. I computed a ratio of resting metabolic rate as rate in cold (10°C) divided by rate in warm (22°C) for the 18 males measured at both temperatures. For this GLMM, I used the Akaike information criterion

(AIC) to determine the quality of the model and removed the least significant variable (i.e. largest p-value) until I achieved the lowest AIC value. All statistical analysis was done using R 3.1.2.

Results

There were a total of 56 recorded competitions between 38 male C. monstrosa. In 80% of the trials, both males engaged in acoustic competition and in 16%, only one male sang. In one trial where neither male sang, it was likely due to one male missing tarsi on both legs and not participating in the competition, though it did attempt to climb the log. Using the aforementioned point system, 21 males were classified as winners and 17 were classified as losers.

During trials, generally, one male would establish a position near the top of the log while the other male would display more exploratory behaviour and move around the log. In most cases, the relatively stationary male would be the eventual winner of the contest. Competitive displays would usually start when males wandered into close proximity of one another. The first act of overt aggression would typically be a “chase or kick” committed by the stationary male when approached by the wandering male. In 23 interactions, competition escalated to intense bouts of wrestling which usually resulted in the losing male being forcibly ejected from the log. In 9 trials, males engaged in behaviour typical of courtship and mating. Males would sing and approach each other, then one would mount the other similar to a female during the wing-chewing stage of courtship. The male getting his wings chewed would engage the gin trap organ and secure the male in a mating position. In at least 1 trial, they reciprocated turns in the female role.

Duty cycle measurements had low repeatability within males across contests (r=0.02). This result is surprising given that duty cycle is highly repeatable in other fighting cricket species

(Brown et al., 2006), whereas in Cyphoderris it is not a fixed attribute and varies from day to day.

In 38 out of 56 interactions between males, winners had higher duty cycles than losers. Duty cycles associated with winning outcomes were significantly higher than duty cycles of losing outcomes

(GLMM: n=38, p=0.017, Table 1, Figure 3). When iterations of contests were examined separately, duty cycles of winning outcomes were significantly higher than duty cycles of losing outcomes for the first two contests, but not for the third (n=38, p1=0.024, p2=0.007, p3=0.448). This is likely a result of a smaller sample of third fights due to death prior to completing all contests.

Principal components analysis reduced seven size variables to three principal components

(Table 2). PC1 accounted for general body structures such as wing and leg lengths, as well as body width. PC2 explained head size and PC3 explained gin trap width, a structure used during mating and, in this study, also used in competition.

During respirometry trials, I was not able to induce singing from C. monstrosa individuals.

Therefore, I used resting metabolic rate at 10°C and 22°C as an indicator of inherent energetic use.

The GLMM for the effect of size, diet, and metabolic rate, on overall status revealed that overall winning status was most related to body size (PC1), head size (PC2), and a ratio of metabolic rate at 10°C and 22°C but not to diet or size of the gin-trap organ (Table 3). However, there was no significant difference between winners or losers with regards to any variables (Figures 4, 5, 6, and

7). I also analyzed metabolic rates separately to examine whether resting metabolic rates one temperature were predictive of status; however, the results were also insignificant.

Discussion

Male Cyphoderris monstrosa are territorial and compete acoustically as well as physically to defend perch sites (Mason, 1996). I observed individuals singing aggressively before engaging in combat which included 4 basic interactions: chasing, kicking, wrestling, and engaging the gin trap.

My results indicate that signaling effort, body size, head size, and ability to reduce metabolic rate in cold temperatures (or increase rate in warm temperatures) are related to male C. monstrosa territoriality. When duty cycle was examined for effects on fight outcome, I found that duty cycle resulting in a winning outcome was significantly different from duty cycles resulting in losing outcomes which corresponds with previous work on competition in C. monstrosa males (Mason,

1996). Duty cycle is not repeatable within males and varies based on context with higher duty cycles during aggressive territorial encounters (Morris et al., 2002). Previously, Mason (1996) manipulated male ability to sing and showed that duty cycle is an honest indicator of competitive ability. Similarly, female crickets exposed to male song without a male present will engage in physical combat, showing that song alone is sufficient to signal RHP (Rillich et al., 2009). Future work should explore informational components for motivation in C. monstrosa song.

Though RHP and dominance are usually correlated, results from a previous study on competition in C. monstrosa show that information about prior contest history is not transferred

(Van Eindhoven 2012, MSc Thesis). In some cricket species, prior fighting success influences future fights where winning males have higher duty cycles or attack sooner and losing males have reduced signaling (Adamo & Hoy, 1995; Brown et al., 2006). My results show that C. monstrosa do not have consistent duty cycles from contest to contest. Therefore, motivation likely plays a role such that males are able to scale their energetic output relative to opponents and available

resources. A potential method to examine motivation would be to introduce a female during a male fight and see whether males alter signaling behaviour.

Overall, my results confirm previous work (Mason, 1996) showing that contest outcomes in C. mosntrosa are correlated with the relative duty cycles of contestants. Combined with the absence of any diet effect, this suggests that aggressive contests in this species are likely determined by short-term energetic resources. The analysis of winner/loser status, however, examines males’ ability to sustain a “winning record” over a number of contests. These results suggest there may also be an effect of fixed phenotypic traits (such as body size). According to the best model from the generalized linear mixed model analysis, body size and head size appear to have an effect on overall performance in C. monstrosa males. In many animal species, RHP is influenced by body size (Huntingford & Turner, 1987). Larger body size and weapon size is positively correlated to likelihood of winning a fight (Malo et al., 2005; Brown et al., 2006; Judge & Bonanno, 2008). In

Orthopteran species, legs and mandibles are used in physical combat and are considered weapons

(Alexander, 1961; Kelly, 2006; Judge & Bonanno, 2008). Although these parameters were not statistically significance, the most well-supported model includes these size variables. Previous studies of aggressive competition in this species found no influence of body size on contest outcome, but these considered only individual contests(Mason, 1996). This issue would be best addressed with a larger tournament design (Stuart-Fox et al., 2006).

It has previously been shown that wing-feeding is the primary means to facilitate sperm transfer during Cyphoderris spp. mating (Eggert & Sakaluk, 1994). The male gin trap is a pinching organ used during courtship to secure females in position while they are wing-feeding and mounted on the male dorsum. Engaging the gin trap increases chances of successful sperm transfer (Sakaluk et al., 1995). The gin trap also facilitates mating with resistant females for previously mated males

with chewed hind wings (Sakaluk et al., 1995). While the gin trap is not known to be used in competitive scenarios, secondary sex characteristics are commonly used in aggressive contexts.

While PC scores for the gin-trap organ were excluded from the GLMM, I observed males mounting one another, engaging in wing-feeding behaviour and using the gin-trap organ to secure each other.

While the correlation between male competition and mating success is not often clear, it is possible that males who readily engage their gin-trap organs during territorial encounters also perform this behaviour during mating. In male fights, if wrestling occurs such that one male mounts another male, the gin trap may be engaged as a “last-ditch effort” during fights. Examination of gin trap mechanics and whether there is a difference in use of the organ will further elucidate its functional significance in different contexts.

In 9 out of 50 trials, males engaged in courtship behaviour including wing-feeding and gin trap usage. In at least one competition trial, both males engaged in courtship behaviour, reciprocating roles throughout the trial (i.e. males mounted and fed on wings of each other). To my knowledge, this is the first documented case of male C. monstrosa engaging in same-sex courtship behaviour, however, it has previously been reported in Cyphoderris strepitans and other Orthopteran families

(Alexander, 1961; Sakaluk & Snedden, 1989; Bailey & French, 2012). Here, all individuals were caged separately and only exposed to other individuals during fights. It is unlikely that the occurrence of same-sex courting behaviour is age-related as it did not increase over the course of the competitions. Whether it was due to misidentification is also unknown and will require further investigation. In Teleogryllus oceanicus, same-sex sexual behaviour is due to misidentification where males that have prior encounters with females are more likely to court and attempt mating with other males (Bailey & French, 2012). Crickets use cuticular hydrocarbons (CHCs) for species, sex, and mate recognition (Tregenza & Wedell, 1997). It has been shown that male Cyphoderris

strepitans CHCs are used by females in sex recognition and mate choice. A similar test must be conducted with C. monstrosa with the addition of comparing male and female CHCs and male pheromone reception to determine if misidentification is more prevalent in this species.

It is possible that engaging in same-sex sexual behaviour in C. monstrosa is a method for males to assess the quality of rivals or render them less effective in future female mating attempts

(Dodson et al., 1983). The functional significance of wing-feeding itself is not well understood.

The unique mating system of Cyphoderris spp. involves two distinct nuptial gifts: wing tissue and a spermatophore with a spermatophylax. Johnson et al. (1999) showed that female mounting and mating propensity is related to diet where females on low-diet are more likely to mount, wing-feed, and mate. However, to date, no study has looked at the nutritional content of wing tissue and haemolymph. A future study could employ current metabolomics techniques to analyze these components and determine whether there are significant differences in content between males.

In order to test the metabolic scope of individuals, I tried to obtain measurements of CO2 production during singing; however, the C. monstrosa did not sing in the chambers. Instead, I used a ratio of metabolic rate at 10°C and 22°C as a proxy for metabolic scope. These are temperatures that C. monstrosa would experience regularly at the beginning of mating season, when temperatures during activity range around 10°C to 20°C. In the final model, metabolic rate measurements were included. Though it was not significant, winning male C. monstrosa decreased their resting metabolic rates when ambient temperature was 10°C. The metabolic rates of ectotherms are sensitive to environmental temperatures and there is less of a scope for thermoregulation (Angilletta et al., 2002). However, there is increasing evidence of thermoregulation in insects and metabolic rate-temperature relationships are more complex than originally understood (Sinclair et al., 2003; Irlich et al., 2009). It was previously shown that C.

strepitans are not capable of thermoregulation yet Cyphoderris spp. are acoustically active at cold temperatures as low as –8°C (Dodson et al., 1983; Sakaluk & Eggert, 2009). My results show that males who are decreasing energetic output accordingly with lower temperatures are generally winning competitions. On the other hand, males that maintained the same energy output at both temperatures lost more fights. Therefore, it seems that male ability to conserve energy stores also affects RHP and propensity to win fights. A previous study found that C. monstrosa males were less likely to be active or emerge when it was cold (Ladau, 2003). Another test involving singing metabolic rates at both temperatures is needed to verify whether these energy strategies are reflected during high activity.

In 32% of fights, C. monstrosa never advanced to physical combat. In my experiments, I limited competitions to 45 minutes which are relatively long for Orthopteran contests. In some cases, both

C. monstrosa males were still singing when I terminated the trial. Contests that escalated to physical combat did not necessarily follow a stereotypical sequence (e.g. antennal fencing followed by chasing and kicking before wrestling). A future study could determine what type of assessment strategy C. monstrosa use for contest resolution by handicapping individuals similar to Rillich et al. (2007). However, the lack of repeatability in duty cycle between fights long contest durations I have recorded suggest that a cumulative assessment strategy is used.

I conclude that body size and head size, as well as ability to adjust metabolic rate at cold temperatures predict individual status as winners or losers. Differences in duty cycles predicts fight outcome where winners have higher duty cycles. The relationship of higher duty cycle and winning fights is seen in other studies exploring Orthopteran competition (Adamo & Hoy, 1995; Mason,

1996; Brown et al., 2006). There is not enough field data of C. monstrosa to speculate on whether same-sex sexual behaviour occurs in nature though the 18% occurrence rate in my study warrants

future study. There is evidence showing that in other taxa, this behaviour is due to misidentification, rival assessment, or opportunistic feeding between males (Sakaluk & Snedden 1989; Abbassi &

Burley, 2012; Bailey & French, 2012). Re-examining wing-feeding and gin trap usage as traits evolved in sexual conflict may explain the agonistic role of these behaviours in male fights. In addition to the same-sex sexual behaviour, there was a lack of biting or mandibular flaring during contests. A potential explanation for this is hybridization of the C. monstrosa population with sympatric C. buckelli. The site where I collected is the only known site where both species overlap and mating between species are possible (K. Judge, pers. comm.). Based on several morphological characteristics (e.g. rose-coloured bellies, larger body size, and claw-hammer hooks at the sternal process (Morris & Gwynne, 1978)), I classified the specimens used in this study as C. monstrosa.

Considering the lack of overt physical aggression in C. buckelli, there is potential for dampening of aggressive territorial behaviours in hybrids. To start exploring this possibility, we must quantify density and determine overlap in emergence and activity of both species at this site. Finally, future studies should look at singing metabolic rates and cold tolerance in Cyphoderris monstrosa. A preliminary study showed that nymphs and females are freeze tolerant whereas mature males are not (A. McKinnon, pers. comm.). Deeper investigation into cold tolerance may yield interesting results that increase our understanding of energy strategies in C. monstrosa.

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Table 1. Summary of final model from generalized linear mixed model of contest outcomes in relation to song duty cycle in competing C. monstrosa males.

Random effects Variance Standard deviation

Individual 0 0

Fixed effects Estimate Standard error z value P value

Intercept -0.84 0.35 -2.39 0.017

Duty cycle 2.91 0.88 3.32 0.009

Table 2. PCA results for body size metrics.

PC Percent variance Eigenvalue Factors Eigenvector explained PC1 43 4.3 Forewing length 0.48 Hind leg length 0.50 Pronotum width 0.47 PC2 27 2.7 Head width 0.57 Maxillae span 0.52 PC3 14 1.4 Gin trap width 0.94

Table 3. Summary of final model from generalized linear mixed model of overall status in competing C. monstrosa males.

Random effects Variance Standard deviation

Group 0.248 0.498

Fixed effects Estimate Standard error z value P value

PC1 -2.31 2.27 -1.02 0.310

PC2 -2.94 2.35 -1.25 0.212

Ratio of metabolic rate -1.58 1.16 -1.36 0.174

Figure 1. Distribution of Cyphoderris across western Canada and United States of America

(Morris & Gwynne, 1978). The red star represents Paul Lake Provincial Park where I sampled my

Cyphoderris monstrosa population.

Figure 2. Diagram of log arena used for staging competitions between virgin male Cyphoderris monstrosa. Cages were placed at the bottom of the log at the beginning of each contest and males exited freely to participate. Drawings of male C. monstrosa by Mary Foley Benson from Fig. 4 of

Gurney 1939.

Figure 3. Relationship between duty cycles of male Cyphoderris monstrosa and outcome of territorial interactions (n=38, p=0.017).

Figure 4. Body size of winners is generally larger than losers although there is no significant difference (n=38, p=0.310).

Figure 5. Relationship of head size of male C. monstrosa with overall status as winner or loser

(n=38, p=0.212). PC scores are inversely correlated to actual size where smaller scores are indicative of larger size.

Figure 6. Relationship between ratio of metabolic rate at two ecologically relevant temperatures and overall status as winners or losers in competitions (n=18, p=0.17).