Nutrient Effects on Sexual Selection and Comparison Of

NUTRIENT EFFECTS ON SEXUAL SELECTION AND COMPARISON OF

MATING CALLS IN KATYDIDS (TETTIGONIIDAE)

A thesis submitted

To Kent State University in partial

Fulfillment of the requirements for the

Degree of Master of Science

Lara Rae Trozzo

May, 2013

Thesis written by

Lara Rae Trozzo

B.A., The Pennsylvania State University, 2010

Approved by

______Patrick Lorch, Advisor

______Mark Kershner, Member, Masters Thesis Committee

______Sean Veney, Member, Masters Thesis Committee

Accepted by

______Laura Leff, Acting Chair, Department of Biological Sciences

______Raymond Craig, Associate Dean, College of Arts and Sciences

TABLE OF CONTENTS

LIST OF FIGURES ...... v

LIST OF TABLES ...... vi

ACKNOWLEDGEMENTS ...... vii

CHAPTER 1 ...... 1

CHAPTER 2 ...... 7

Introduction ...... 7

Methods ...... 10

Recording ...... 10

Call and Statistical Analysis ...... 11

Phylogenetic Tree Building ...... 12

Results ...... 12

Discussion ...... 19

CHAPTER 3 ...... 21

Introduction ...... 21

Hypotheses and Predictions ...... 26

Methods ...... 28

Insect Rearing ...... 28

iii

Mating Experiments ...... 29

Data Analysis ...... 31

Female Choice Experiment...... 32

Results ...... 35

Upper Limits on Sexual Selection ...... 35

Female Choice Experiment...... 40

Discussion ...... 41

CHAPTER 4 ...... 44

Introduction ...... 44

Hypotheses and Predictions ...... 47

Methods ...... 48

Sample Preparation and Analysis ...... 48

Data Analysis ...... 49

Results ...... 49

Discussion ...... 56

CHAPTER 5 ...... 60

REFERENCES ...... 64

LIST OF FIGURES

Figure 1.1: Mated female with spermatophore attached ...... 4

Figure 2.1: Tree of A. simplex, P. scabricollis, and outgroup species ...... 9

Figure 2.2: A. simplex chirps ...... 13

Figure 2.3: P. scabricollis chirps ...... 15

Figure 2.4a and b: P. scabricollis trill ...... 16

Figure 3.1: Example Bateman gradients (Lorch 2005) ...... 23

Figure 3.2a and b: Predicted relationships ...... 27

Figure 3.3: Simulated grass stem oviposition substrate ...... 30

Figure 3.4: Y-maze apparatus ...... 33

Figure 3.5a and b: Upper limit estimates of C. nigropleurum on two protein diets ...... 36

Figure 3.6a and b: Upper limits for individual males on two protein diets ...... 38

Figure 3.7: Spermatophore size ...... 39

Figure 3.8: Regression of egg laying rate versus spermatophore size ...... 40

Figure 4.1: δ15N vs. δ13C biplot of tissue samples compared to food samples ...... 51

Figure 4.2: δ15N vs. δ13C biplot of tissue samples ...... 53

Figure 4.3: δ15N vs. δ13C biplot of tissue samples separated by diet ...... 55

LIST OF TABLES

Table 2.1: Call Measurements ...... 18

Table 3.1: Mating Combinations ...... 31

Table 3.2: Fecundity (number of eggs) Results ...... 35

Table 4.1: Categories of Individuals Sampled ...... 49

ACKNOWLEDGEMENTS

First and foremost, I must acknowledge my advisor, Dr. Patrick Lorch. Without his research ideas, guidance, and expertise none of this would have been possible. I would also like to thank my committee members Dr. Mark Kershner and Dr. Sean Veney for their input.

I need to thank the Von Bargens for allowing us to record katydids on their property. I also need to thank Nathan Bailey for sending us his raw call data to compare against our call recordings. My project involved collecting C. nigropleurum eggs in the field and raising a large number of katydids in the lab. Many people helped in this process with collecting eggs and keeping katydids fed and watered. I most definitely could not have done this without their assistance: Eric Floro, Allison Gercaci, Mason Lorch, Patrick Lorch, Sony Pandey,

Sewwandi Rathnayake, Veronica Rigatti, and Amber Walden. I would also like to thank

Veronica Rigatti for taking the morphometric measurements on C. nigropleurum individuals.

For the mate preference experiment, I must thank Patrick Lorch and Cali Roth for helping me to hunt for and collect katydid nymphs in the field. I also would like to thank Mark

Kershner for allowing us the use of his lab for the y-maze apparatus set up. Throughout this entire endeavor, I would like to thank my parents, Ronald and Diana Trozzo, as well as the rest of my family and friends for their continued support.

vii

CHAPTER 1

General Introduction

Natural selection was first described by Charles Darwin in The Origin of Species

(1859). Since he conceived it, natural selection has been a major topic of evolutionary research. Often described as "survival of the fittest", the theory of natural selection was summarized by Darwin as "any being, if it vary however slightly in any manner profitable to itself, under the complex and sometimes varying conditions of life, will have a better chance of surviving, and thus be naturally selected" (1859). As a result, individuals who have the best or most "fit" traits for their environment will survive better and over time, these traits will become more common and traits which are less favorable will decline in abundance

(Freeman and Herron 2007a).

Sexual selection is another important process that was also described by Darwin as

"the advantage that certain individuals have over other individuals of the same sex and species, in exclusive relation to reproduction" (1906). Sexual selection can be described as either precopulatory or postcopulatory (sperm competition and cryptic female choice;

Dugatkin 2009). For the scope of this thesis, we will focus on precopulatory sexual selection, which influences how animals choose their mates. There are two types of precopulatory sexual selection: intrasexual selection and intersexual selection (Dugatkin

2009). Common examples of intrasexual selection occur when individuals of the same sex compete for mates or resources which give them better access to mates (territory, etc.); this is commonly, but not always, observed in males competing. Intersexual selection occurs

when individuals of one sex choose between members of the other sex based on some criterion. For instance, in many animals, males benefit from producing an exaggerated trait or some elaborate behavioral display because females will preferentially mate with males who are able to produce a better display (Dugatkin 2009). Many different mechanisms can cause these traits to evolve; better displays may be associated with good genes to pass on to offspring; when displays are conspicuous, displaying individuals have shown better predator survival; traits are attractive because of an unrelated and preexisting sensory bias; or traits are correlated with direct benefits such as the ability to provide a food gift for the mate, among other reasons (Dugatkin 2009; Freeman and Herron 2007b).

In katydids, males provide what is called a "nuptial gift" to females as part of the mating ritual. It is a spermatophore that is composed of two parts: the sperm-containing sperm ampulla and the spermatophylax, which is a proteinaceous food gift (Gwynne 1988).

The spermatophylax is consumed by the female after mating. It serves the dual functions of both providing valuable nutrients to the female and protecting the sperm ampulla from being eaten before the sperm is injected into the female's reproductive tract. It has been suggested that the spermatophylax may have evolved as a result of an evolutionary arms race between the sexes (Wedell 1994). Also, the spermatophylax is the only form of paternal care the male provides to his offspring, and it can have a large impact on the female's (and in turn her mate's) fecundity as it confers the ability to produce more eggs

(Gwynne 2001). Nuptial gifts such as the spermatophylax are an example of direct benefits a female can gain from mating (Freeman and Herron 2007b) and can contribute to the potential for sexual selection. Spermatophore size varies by species, but the largest contributions have been recorded in the genus Ephippiger, where a male may contribute

40% of his body weight into this gift (Vahed 1997). In species of shield-backed katydids

(Tettigoniinae), such as Anabrus simplex, the spermatophore can be up to 27% of the male's body weight (Gwynne 1981). In meadow katydids (Conocephalinae), such as Conocephalus nigropleurum, a spermatophore (Figure 1.1) is typically about 10% of a male's body weight.

In addition to the size of the spermatophore a male can produce, the benefits of it to the female are also determined by the value of the nutrients contained within it. Male diet and the amount of protein males are able to allocate to a spermatophore should have a clear effect on sexual selection (Gwynne 1993).

Figure 1.1: Mated female with spermatophore attached

This is a female Conocephalus nigropleurum just after mating. You can see

the spermatophore she has just received with the external spermatophylax

protruding from her reproductive tract. She will later bend around and

consume this valuable gift.

One factor with a clear impact the potential for sexual selection is protein availability. Several katydids are known to reverse their typical sex roles (from males competitive for mates and females choosey to females competitive and males choosey) when protein goes from abundant to scarce (Gwynne and Simmons 1990; Gwynne 1993).

This happens because when it is difficult to obtain dietary protein, females may use matings to obtain the necessary protein through spermatophore consumptions (Gwynne 1993). The implication of this major change in behavior is that the relative strength of sexual selection on each sex reverses (Jones and others 2005).

Before sexual selection can occur, individuals of the same species must be able to find each other. Some animals live within a close range of each other and this is not a problem, but for many, potential mates may not be within visual distance. This is particularly true when they live in a complex environment. In members of the order

Orthoptera (katydids, crickets, and grasshoppers), mate attraction is accomplished using calling song. In katydids, males call to attract females (Gwynne 2001), however, other information may also be encrypted within the call. In some cases, it is known that the female can infer some characteristic of the male from his song such as body size (Morris

2008). Body size can be a benefit in a potential mate because a larger male will be able to provide a larger nuptial gift as well as passing on large body size genes to produce healthier offspring with a better chance of survival (Whitman 2008). So if a female can infer body size or other characteristics of a male from his mating song, she can make an informed decision about her mate from a distance, thus conserving valuable resources such as time and energy. It is in a male's best interest to produce a better call, not just to convey information such as size, but also to have a better chance to attract mates; the longer and louder a male is able to call, the more females who will hear him and consider mating with him. Stronger calls should carry further in the complex environments where most katydids live (Drosopoulos and Claridge 2006), reaching the "ears" (actually the legs; Gwynne 2001) of more females. Calls are also under strong selective pressures because it is important for females to be able to distinguish between the calls of conspecifics versus those of heterospecifics (Ewing 1989). This evolutionary importance of calls led us to describe the call of a species whose call had not been previously described and compare it to a well

described and closely related species found in similar habitats across a broad overlapping distribution.

In this thesis, I first examined katydid mating calls as an important trait for mates to find each other and for species identification. This was examined by recording mating calls of two species in the field, then analyzing and comparing the two. Next, I looked into how protein availability may affect the upper limits on sexual selection. This was accomplished by performing a series of controlled mating experiments using katydids on two protein diet treatments. Finally, I examined nutrient processing in katydids and the effects from the diet treatments using stable isotope analysis, comparing isotopic signatures between three body tissues against those of the food sources.

CHAPTER 2

Comparison of calls between Anabrus simplex and Peranabrus scabricollis

Introduction

Many females use male acoustic calls to detect and decide between potential mates.

In addition to distinguishing between males within species (Andersson 1994), calls can be used by females to distinguish between conspecific and heterospecific males (premating reproductive isolation; Drosopoulos and Claridge 2006). Importantly, these species specific calls can be used to identify species in the field.

In what follows, we describe differences between the calls of two closely related katydid species with overlapping distributions. We use variation in call parameters between populations of the more widely distributed species (Bailey and others 2007) as a baseline to describe differences in call parameters between species. To put these differences into an evolutionary context, we report a phylogenetic hypothesis for these two species. In addition to describing one of the two species calls for the first time, we lay the groundwork for comparisons of calls from five related species with varying degrees overlap in distributions. This will allow entomologists to better identify these species if they reach pest status as several have in the past.

Mormon crickets (Anabrus simplex) and Coulee crickets (Peranabrus scabricollis) are large, flightless, shield-backed katydids (Orthoptera, Tettigoniidae, Tettigoniinae) that have been observed in a largely overlapping range, throughout much of the western United

States and into Canada (for distribution maps of these and related species see Anabrus and 7

Peranabrus on the Singing Insects of North America website: http://entomology.ifas.ufl.edu/walker/buzz/katylist.htm; Walker and Moore ). A. simplex individuals may form large, dense migratory bands that can travel up to two kilometers a day (Lorch and others 2005). These bands are destructive to crops, can create hazardous driving conditions when run over in large numbers, and are generally bothersome when passing through towns (Sword, Lorch, Gwynne 2008). P. scabricollis has not been as abundant recently (except in 2009 and 2010 in Northwestern Idaho; Lorch personal observation), however, historical observations suggest that they also exhibit similarly high density populations and swarming behaviors (Snodgrass 1905). These two related species

(Figure 2.1) have only a few physical differences useful for distinguishing between them, so call characteristics may be a more feasible method of identification, as is the case with many other katydids (Drosopoulos and Claridge 2006).

Crickets and katydids produce sound through stridulation, by rubbing their two forewings together (Drosopoulos and Claridge 2006). One wing has a file and the other, a scraper called the plectrum. As the plectrum passes across the pegs of the file, bursts of sound are produced by the vibrating wings (Bailey and others 2007; Ewing 1989). In both of the focal species, males produce species characteristic patterns of chirps (Bailey 1970).

Chirps are short bursts of sound made by one wing stroke (Miyoshi and others 2007).

Additionally, P. scabricollis males intersperse trills among their chirps. These trills are a quick series of short, fast wing strokes (Miyoshi and others 2007). In Mormon crickets, chirps are produced at regular intervals continuously for long periods of time. Because of this regularity, we can easily measure the frequency which with these chirps occur (chirp

rate). Trills, on the other hand, occur less frequently, interspersed amongst chirps only in

Coulee crickets.

Figure 2.1: Tree of A. simplex, P. scabricollis, and outgroup species

This neighbor joining tree shows relationships based on cytochrome oxidase

II mitochondrial gene (Lorch unpublished data). A. simplex splits into two

clades based on solitary and gregarious morphs (Bailey, Gwynne, Ritchie

2005). P. scabricollis is closely related to these Anabrus clades compared to

other katydid outgroups. The percentage of replicate trees in which the

associated taxa clustered together in the bootstrap test (1000 replicates) are

shown next to the branches (Felsenstein 1985). The tree is drawn to scale,

with branch lengths in the same units as those of the evolutionary distances

used to infer the phylogenetic tree.

Female Orthoptera may use call characteristics to evaluate mate quality, as carrier frequency can be correlated with male body size and mating investment (Morris 2008), and for species recognition (Field 1982). Previous work in A. simplex and other orthopteran

species has shown that the frequency of a call is inversely correlated with the caller’s body size (Bailey and others 2007; Morris 2008). This correlation may have evolved due to sexual selection acting on call characteristics that convey information about certain fitness advantages to female.

Because of their overlapping ranges, we expected distinct call differences to be present between the two species. Call differences would have evolved to allow individuals to discern conspecifics from heterospecifics in order to attract only compatible mates. In this paper, we describe the call of P. scabricollis for the first time and contrast the call characteristics between the recently more abundant and well known A. simplex and the lesser studied P. scabricollis. A previous study by Bailey and others (2007) measured several call characteristics in A. simplex, focusing particularly on differences in call characteristics and physical traits between clades within A. simplex. We also compared our results to the Bailey data so that these data can be compared directly. By contrasting call characteristics of A. simplex and P. scabricollis we have quantified audible differences between the calls of these two species allowing the use of calls for distinguishing these two species in the field.

Methods

Recording

Field recordings (~ 30 seconds to 5 minute duration) were obtained from calling individuals using a TASCAM HD-P2 recorder (set at sample rate 96 kHz, sample width 24 bits) and parabolic microphone (Saul Mineroff SME PR-1000; Sennheiser ME62 Microphone with Sennheiser MZW64 PRO Windscreen). A. simplex individuals were recorded on 14 July

2010 (10:30-11:00 AM; ~24ºC) at Water Canyon Road near Battle Mountain in Elko, NV

(N 40.6163°, W -116.6893°). P. Scabricollis individuals were recorded on 10 July 2010 (9:45

AM – 1:50 PM; ~22ºC) at the Von Bargen Ranch near Grangeville, ID (N 45.8416°, W -

116.3738°).

Call and Statistical Analysis

Short segments (5–10 seconds) of chirps from A. simplex and chirps and trills from

P. scabricollis were isolated and analyzed to objectively estimate dominant frequency and chirp rate using R software packages (tuneR, Ligges 2010; seewave Sueur 2010). Chirp rate and carrier (or dominant) frequency are two of the most commonly measured acoustic call characteristics for orthopteran insects (Bailey and others 2007; Howard and Hill 2006;

McIntyre 1977). Carrier frequency is “the most intense frequency in a spectrum” (Morris

2008). Mean carrier frequency was estimated for each call segment by plotting points on the most intense (highest amplitude) region of each chirp on the spectrogram (determined on digital version of call in R, after filtering out background noise outside the range of 1250-

25000 Hz). Chirp rate was calculated by averaging the time differences between these points for between 50 and 140 chirps in A. simplex and for between 7 and 23 chirps in P. scabricollis , covering a 5 to 10 second segment of consistent chirping. The same parameters were used on P. scabricollis trills, using similar methods to analyze complete trills.

Carrier frequency and rate of chirping were compared between the two species and within P. scabricollis between chirp and trill segments using the Student’s t-test.

Comparisons of our data with data from Bailey and others (2007) were performed using

ANOVA followed by Tukey HSD Tests. Statistical analyses were performed in R (The R

Foundation for Statistical Computing 2010), Excel (Microsoft Corporation 2010), and JMP

(SAS Institute Incorporated 2010).

Phylogenetic Tree Building

We estimated a neighbor joining tree (Saitou and Nei 1987), based on the cytochrome oxidase II mitochondrial gene (COII). We extracted whole genomic DNA from 2 samples of Peranabrus scabricollis using QIAamp DNA minikit (Qiagen 51304). We then amplified ~445 base pair sequence of COII with forward and reverse primers C2-J-3279 and

TD-N-3862 (Simons and others 1994) and PCR methods described by Bailey and others

(2005). Numbers on the branches indicate branch confidence based on 1000 bootstrap estimates. Sequences for Kawanaphila nartee, Ephippiger terrestris, and Anabrus simplex, come from Genbank. Sequences were edited and aligned, and trees were generated using

MEGA 5.05 (Tamura and others 2011) and MUSCLE (Edgar 2004). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura, Nei,

Kumar 2004) and are in the units of the number of base substitutions per site. The analysis involved 12 nucleotide sequences. Codon positions included were

1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated.

There were a total of 434 positions in the final dataset.

Results

In A. simplex chirps (Table 2.1; Figure 2.2), the dominant frequency was 13.037 ±

0.308 kHz (mean ± s.e.m.) and chirp rate was 11.12 ± 0.296 chirps/sec.

Figure 2.2: A. simplex chirps

This spectrogram illustrates the analysis of an A. simplex recording. Six

seconds of continuous chirping was isolated from a longer (several minute)

recording. Each vertical slash is one chirp. The dots on each chirp highlight

the point of maximum amplitude used to estimate dominant frequency and

chirp rate.

In P. scabricollis chirps (Table 2.1; Figure 2.3), dominant frequency was significantly higher than in A. simplex, at 16.940 ± 0.233 kHz (t = -10.11, d.f. = 22, p < 0.001) and chirp rate was significantly lower at 2.626 ± 0.186 chirps/sec (t = 24.33, d.f. = 19, p < 0.001). For

trills (Figure 2.4a and b), the dominant frequency was 16.461 ± 0.211 kHz and trill rate was

27.965 ± 1.44 chirps/sec. This was not significantly different from the dominant frequency of their chirps (t = 1.53, d.f. = 30, p = 0.14), but trill rate was significantly faster than chirp rate (t = -17.45, d.f. = 15, p < 0.001).

Figure 2.3: P. scabricollis chirps

This spectrogram illustrates the P. scabricollis chirp. Six seconds of continuous chirping was isolated from a longer (several minute) recording.

The dots on each chirp highlight the point of maximum amplitude used to estimate dominant frequency and chirp rate.

a b a

Figure 2.4a and b: P. scabricollis trill

The spectrogram (a) illustrates the trill in the P. scabricollis call. The segment of recording containing only the trill was isolated from a longer

(several minute) recording including chirping and trills. In this trill, chirp rate was very fast. As seen in the oscillogram (b), the beginning of the trill is fairly quiet and increases in amplitude. Take note of the difference in time scale in these figures compared to the chirp figures (Figure 2.2 and Figure

2.3).

Table 2.1 compares our estimates to Bailey and others (2007). See discussion for a detailed comparison of these two sets of results.

Table 2.1: Call Measurements

Species/Form Population Data Temp. Dominant Dom. Freq. Chirp Rate Chirp Rate Source (°C) Frequency Significance (chirps/sec) Significance (kHz) Groupings Groupings A. simplex IM Bailey et 27-29 13.59±0.22 C 14.81±0.31 A solitary al. 2007 A. simplex KF Bailey et 27-29 13.50±0.23 C 15.57±0.33 A solitary al. 2007 A. simplex CO Bailey et 14-16 12.26±0.18 AB 11.48±0.26 B gregarious al. 2007 A. simplex LE Bailey et 18-22 12.11±0.18 A 11.56±0.26 B gregarious al. 2007 A. simplex TM Bailey et 23-25 12.46±0.25 AB 12.14±0.36 B gregarious al. 2007 A. simplex WCR Lorch, 24 13.04±0.21 BC 11.12±0.30 B gregarious Trozzo P. scabricollis VBR Lorch, 22 16.94±0.18 D 2.63±0.26 C Trozzo

This table combines our data with data from Bailey and others (2007) that

includes parameter estimates from both solitary and gregarious

populations. All of our A. simplex recordings were from gregarious

populations. P. scabricollis recordings were from fairly high density

populations (~1 m-2). The three different species/forms had statistically

different dominant frequencies (F(2,86) = 218.60, p < 0.0001) and chirp rates

(F(2,86) = 680.01, p < 0.0001). Population refers to notation found in Bailey

and others (2007) and to Water Canyon Road and Von Bargen Ranch.

Dominant frequency and chirp rate are reported as means with standard

errors. Significance groupings are based on Tukey HSD tests following

ANOVA and compare P. scabricollis and all populations of both gregarious

and solitary A. simplex . Different letters indicate statistically significant

differences. Temp. indicates estimated recording temperatures.

Discussion

Dominant frequency was lower and chirp rate was faster in gregarious A. simplex compared to P. scabricollis. In P. scabricollis, dominant frequency was comparable between chirps and trills, but chirp rate of trills was faster.

When we compared our results for A. simplex to results for only the gregarious populations reported by Bailey and others (2007), chirp rates were similar (F(3,48) = 1.34, p

= 0.27) but, dominant frequency was statistically different from Bailey and others estimates

(F(3,48) = 4.84, p = 0.051; Table 2.1). A post-hoc Tukey HSD test comparing our gregarious measurements with Bailey’s three gregarious populations showed our measurements of dominant frequency to be statistically similar to one of Bailey’s gregarious population estimates (p = 0.25), but different from the two others (p = 0.0037, p = 0.02). These differences were likely due to recording conditions (specifically temperature), equipment, and settings. It is clear from Table 2.1, that for both carrier frequency and chirp rate, the two population types of A. simplex were more similar to each other than either was to P. scabricollis.

Overall, we can conclude that these species exhibit fairly distinct differences in the call characteristics of dominant frequency, chirp rate, and presence/absence of trills. These differences can be used to distinguish between the two species, both by katydids for mate discrimination and by humans for classification purposes. Because these related species occur in overlapping ranges, call traits have likely been under evolutionary pressures to evolve characteristic differences. These specific differences allow individuals to easily distinguish between conspecifics and heterospecifics. This way, females can successfully distinguish between incompatible heterospecific and compatible conspecific mates.

Learning the differences between these calls can also be useful for our identification of these species for monitoring, in case of future outbreaks. Since these two species are similar in appearance and exhibit similar behaviors, call identification may prove to be a more reliable method of distinguishing species by workers in the field. This is a particularly valuable tool for entomologists who want to identify which pests are affecting local crops.

Recordings from our calls have been contributed to the Singing Insects of North America database (Walker and Moore ). Further work is underway comparing the morphology of pronotum and wing structures in these two species. It would also be a useful endeavor to record and describe the calls of other related katydid species. In particular, analysis should be completed on the species Anabrus longipes, which has been found to overlap in range with both A. simplex and P. scabricollis. It would be interesting to examine similarities and differences in calls compared alongside phylogenetic relationships to make inferences about the evolutionary history of calls and species divergences.

CHAPTER 3

Does mate condition constrain the upper limits of sexual selection?

Introduction

Sexual selection is typically characterized by the situation where males are competitive, trying to obtain as many matings as possible, but females are choosey and discriminate between mates. Bateman (1948) found evidence that males often experience higher levels of intersexual selection than females. He demonstrated that males often have higher variance in the number of mates compared to females because when males are allowed to compete for mates, a few males obtain many matings and other males do not mate, whereas, in females, mating success is more evenly distributed. He also suggested this difference was because female fecundity is limited by egg production, while male fecundity is not limited by sperm production, but by the number of matings he can obtain.

Bateman showed experimentally that the cause of the sex difference in variance, and therefore the cause of sexual selection, was likely because male fecundity increased with each additional mate, but this did not always occur in females. In other words, there is often a strong correlation between fecundity and number of mates for males, but only a weak correlation for females. Bateman suggested that a strong correlation between fecundity and numbers of mates was the underlying cause of sexual selection (1948). Prior to this discovery, it was known that individuals with traits that made them better at competing for mates (for instance males who can impress more females with some display) have higher mating success. It was also known that individuals with better trait values usually have 21

higher fitness. Therefore, the correlation between mating success and fecundity provided the missing link between these two trains of thought and is thus the underlying cause of sexual selection (Jones and Ratterman 2009). Research by Arnold and Duvall (1994) showed that the difference in this correlation between the sexes can be represented as intersexual differences in the slope of the regression of fecundity on mating success (for example, see Figure 3.1), which they termed “Bateman gradients”. Using a portion of

Bateman’s original data, Arnold and Duvall showed that the gradient for males generally had a distinctly positive slope, whereas that of females is sometimes nearly flat (Figure 3.1).

These authors suggested that this causes selection for males who want to mate with many females, and females who discriminate among males before mating (Arnold and Duvall

1994). Bateman’s conclusions are often cited as the first to describe the causes of sexual selection and they are the basis of our present theory for the evolution of mating behavior and mating systems (Arnold and Duvall 1994).

Figure 3.1: Example Bateman gradients (Lorch 2005)

Bateman gradients are plotted as the regression of fecundity on mating

success. Males (closed circles, solid line) often have a steeper slope than

females (open circles, dashed line). Males have more to gain from remating,

creating selective pressures favoring males who obtain more mates. This is

not so for females. However, females can still gain fecundity by choosing

among males, creating the traditional scenario of sexual selection for

competitive males and choosey females.

In a subsequent study, Lorch (2005) showed that upper limits on Bateman gradients could be used to measure upper limits on sexual selection. These upper limits, represented by the maximum rate of fecundity increase with each additional ideal mate (virgins in full health), correspond to the potential, or the highest possible strength, of sexual selection.

The upper limits can be estimated experimentally, and are expected to be steeper than the actual Bateman gradients (Lorch 2005). Before Arnold and Duvall (1994), variance in mating success was often used to estimate this maximum potential for sexual selection. The most commonly used version of this measure of the “opportunity for sexual selection”, is

Imates (mean-squared standardized variance in mating success; Shuster and Wade 2003) which assumes that a correlation between fecundity and number of mates exists for males and not for females (Lorch 2005). If this assumption is violated, Imates is not a good estimate of the maximum potential for sexual selection. Lorch (2005) argued that estimates of the upper limit on the Bateman gradient, which make no assumptions about the strength of the fecundity by mating success correlation, are therefore a better indicator of the potential for sexual selection than Imates.

This theoretical paper (Lorch 2005) was followed by a study using the katydid species Conocephalus nigropleurum, to estimate the difference between male and female upper limits (Lorch, Bussière, Gwynne 2008). The upper limits of Bateman gradients for each sex can be estimated in the laboratory by controlling matings between focal individuals with one or two ideal mates (virgin females and males that have never mated before). By comparing how fecundity increases with the number of ideal mates for the two sexes, a measure of the potential strength of sexual selection can be obtained (Lorch 2005).

The measure indicates how steep the Bateman gradient could get if all matings were ideal.

The Lorch, Bussière, Gwynne (2008) results suggested that in this species, both males and females benefit from additional matings because of benefits that females gain from nutritious spermatophore gifts received from males during mating (Lorch, Bussière,

Gwynne 2008). However, male upper limit estimates did not exceed female upper limits as expected. In this experiment, it appeared that males did not recover completely between matings and produced inferior spermatophores during subsequent matings. Also, there were several instances of failed sperm transfer. Repeating these experiments seems worthwhile to deal with these weaknesses in the previous experiments.

One factor with a clear impact the potential for sexual selection is protein availability because several katydids are known to exhibit sex role reversal if protein is limited (Gwynne and Simmons 1990; Gwynne 1993). Critical nutrients like protein should have important effects on the upper limits of sexual selection. In males, the upper limit depends on the fecundity of the females he mates with (Lorch 2005). A female’s fecundity will be determined by her access to high protein food and includes any boosts in fecundity she receives from benefits males provide to her, such as nuptial gifts (food provided at mating; Lorch 2005). In females, the upper limit depends on what she gains from mating, so her upper limit increases solely due to paternal investments, such as nuptial gifts (Lorch

2005), which may also be affected by her mate’s nutritional history. So, the difference between male and female upper limits depends on female premating fecundity but not on male investment because male investment contributes to both male and female upper limits equally. As a result, male upper limits should always be higher than female upper limits unless females have no access to food other than from males, in which case they will be equal (Lorch 2005). Lorch (2005) describes factors that can cause mates not to be ideal, leading to actual Bateman gradients that are lower than the upper limits (such as sperm competition).

The experiment described below was designed to estimate male and female upper limits on sexual selection, making sure males were allowed to fully recover from their first mating before mating again. We also explored the importance of nutrition in setting the upper limits on sexual selection, using high and low protein diets. This experiment was designed to mimic natural populations where all individuals in the population would have access to the same sources of food (whether of high or low protein quality) so we only

considered cases of low protein diet individuals mating together and high protein diet individuals mating together. High and low diet mating combinations were not conducted.

Hypotheses and Predictions

We varied nutritional condition of individuals by raising them on either high or low protein diets as adults. Nutrition of mates should limit possible fitness gains and provide insight into how upper limits on Bateman gradients, and the potential strength of sexual selection that they represent, are affected by nutritional history. We expected lower condition mates to have lower reproductive investments. Specifically, low condition males should produce less nutritious spermatophores and low condition females should have restricted fecundity.

Lower nutritional condition was expected to decrease the upper limit on Bateman gradients for both sexes (Figure 3.2a and b). In other words, the upper limit would be lower for low condition individuals compared to high condition individuals. Because fecundity gains to females from nutritional nuptial gifts are already accounted for in the male upper limits, it was expected that the male upper limit would still equal or exceed the female upper limit (Lorch 2005) in both the low and high protein treatments.

We further expected the male and female upper limits to become closer when nutritional condition decreased because the difference between male and female upper limits is female fecundity before mating, which was expected to be smaller for low condition females (Lorch 2005). In both cases, fecundities from the first mating should be the same between males and females with significantly lower fecundity in low condition matings.

a a b b

Figure 3.2a and b: Predicted relationships

These hypothetical figures illustrate the predicted outcomes with ideal mates (see text). Blue represents high protein diet individuals; red represents low protein diet individuals. In both figures, the maximum fecundities from the first matings are the same because they are dependent on female dietary protein consumption plus any additional fecundity boost due to the nuptial gift. High protein females were expected to have higher maximum fecundities than low protein females. In Figure 3.2a (female predictions), maximum fecundity after a second mating is higher due to the added nutritional benefits that females receive from male nuptial gifts. The high protein females would have higher upper limits (represented as the slopes of lines in figure) because of the higher value of their mate’s investment (nuptial gift value). In Figure 3.2b (male predictions), second mating maximum fecundities should increase by the same amount as the first mating because this increase is strictly a matter of virgin female fecundity before mating (Lorch 2005). The upper limit is higher for high protein males because their mates are more fecund.

To summarize, matings between low condition individuals should result in both decreased maximum fecundity (lower position on y-axis) and decreased upper limits on sexual selection (shallower slopes). The comparison of male and female upper limits using mates of varying condition should provide a test of the theory describing what sets the upper limits on sexual selection as well as insight into how mate condition should affect male and female mate choice. We also should be able to estimate the relative contribution of each sex to fecundity, since the female upper limit provides a way of estimating the fecundity value of the nuptial gift. Premating female fecundity (which is the difference between the male and female upper limits) can be calculated by subtracting female upper limits (which are due to nuptial gifts) from male upper limits and provides a way to estimate the fecundity value of a female’s own nutritional history.

Methods

Insect Rearing

C. nigropleurum eggs were collected at local parks near Kent, OH (on 7 April 2011 in

Towner’s Woods (41.1691° N, 81.3027° W) and on 14 April 2011 at a site near Fairhill Drive

(41.1382° N, 81.2747° W) from pinecone galls that are formed on willow trees by midge larvae (Cecidomyiidae). Eggs were hatched in the lab as described in Lorch, Bussière,

Gwynne (2008) in an environmental chamber at 25°C and under a 12:12 hr light: dark cycle.

Nymphs were separated into individual cages as they hatched and raised to adult stage under the same environmental conditions. They were fed canned beef cat food

(Purina Fancy Feast, classic tender beef feast) and apples until they eclosed as adults

(monitored daily to determine beginning of adult lifespan). At this point, adults were assigned alternately to diet treatments consisting of high protein or low protein using

specially-formulated, powdered food (as in Simpson and others 2006). The two diets contained the same nutritionally-required amounts of carbohydrates (1:1 sucrose: dextrin),

Wesson’s salt mixture, and essential vitamins and lipids. The high protein diet contained the nutritionally-required amount of protein (3:1:1 casein: peptone: albumen; Simpson and others 2006) and the low protein diet contained approximately half as much. The difference was made up in indigestible cellulose. The high protein diet consisted of 21% protein, 21% carbohydrate, 54% cellulose, 2.5% Wesson’s salt mixture, and 0.18% vitamins and essential lipids. The low protein diet consisted of 11.7% protein, 23.5% carbohydrate,

60.8% cellulose, 2.5% Wesson’s salt mixture, and 0.18% vitamins and essential lipids.

Additionally, small apple pieces were provided for moisture. Food was replaced twice weekly. These diets were fed to individuals starting on their first day as an adult and continuously throughout experiments to produce individuals of either high or low condition based on adult diet quality.

Mating Experiments

After eclosing as adults, virgin individuals were mated either once or twice after being allowed to mature for at least 6 days. Fecundity was measured by counting the number of eggs laid by females in each treatment. Females were provided with oviposition substrate consisting of either a grass stem (Phragmites species) or a simulated grass stem

(Figure 3.3).

Figure 3.3: Simulated grass stem oviposition substrate

This shows a female katydid ovipositing in the simulated grass stem. It was

constructed from a 1 dram glass vial containing rolled up paper towel (dry).

A toothpick was placed in the center for a perch. The katydids utilized these

easily and the devices made collection and counting of eggs easier and

quicker than dissecting apart grass stems. Theoretically, this method could

be used to collect eggs from any species of katydid which lay their eggs

between the leaf and stem of grasses.

Individuals were mated in combinations as shown in Table 3.1. All females mated with a focal male were virgins. Males were weighed before and after mating to quantify males’ investments in spermatophores. Males were allowed to recover fully between matings (until they regained a weight equal to or exceeding their weight at the time of first mating) to ensure they produced their best possible spermatophores.

Table 3.1: Mating Combinations

Numbers represent sample sizes. Each individual mated to another in the

same diet, so no combinations of high and low protein individuals mated

together.

Data Analysis

Fecundity of females (whether they mated once or twice) was estimated as the number of eggs she laid in her lifetime. Male fecundity was estimated as the lifetime fecundity of his mates. So for males who mated twice, we added the lifetime fecundities of his two virgin mates. Separately for males and females, upper limits on sexual selection were estimated from the mean fecundity of females who had mated either one or two times with ideal mates. These upper limits were compared between the diet treatments using

ANOVA to examine the significance of number of mates, sex of focal individual, and diet on fecundity, along with tests for possible interactions. Also, for males, the direct measures of their upper limits (equivalent to the fecundity of their second mates) were compared using

ANOVA to look at diet effects.

In a previous experiment using this same species (Lorch, Bussière, Gwynne 2008), female body size significantly affected upper limit estimates. For the sake of comparison, female body size was measured using mean femur and pronotum length. A principal

components analysis was run using with these two variables and the first principal component was used as a proxy for body size. Adding this covariate never changed the results in important ways, so we do not report these analyses.

Spermatophore size comparisons were performed using ANOVA with other important covariates. Regression was used to examine the relationship between spermatophore size and egg laying rate. All statistical tests were performed in JMP (SAS

Institute Incorporated 2010).

Female Choice Experiment

After analyzing data from the experiments described above, some unexpected and interesting results were found including that males provided larger spermatophores to their second mates and that these females had higher fecundities than their first mates

(more detail provided later). This suggests that it might be beneficial for females to preferentially mate with previously-mated (nonvirgin) males. We conducted an additional female choice experiment utilizing a y-maze (Figure 3.4) made from ¼ inch dowel rod based on the methods used by Gwynne (1982). This experiment was performed in an attempt to determine if females were able to assess male mating history prior to choosing a mate. Females were allowed to choose from males with two mating histories: 1) unmated or 2) mated once previously (at least 2 weeks before).

Figure 3.4: Y-maze apparatus

The y-maze apparatus was made from ¼ inch diameter dowel rod. At each

end of the maze (B, C) is a cube-shaped screen cage containing one male and

lit from above. The female was released just below the choice point (A) at

the bottom.

Due to unavailability of C. nigropleurum, Conocephalus brevipennis individuals were used. They were collected as nymphs from sites in Kent, OH (Towner’s Woods (41.1728° N,

81.3060° W), a site near Fairhill Drive (41.1382° N, 81.2747° W), Kent State University campus wetlands (41.1421° N, 81.3317° W), and a property near Jenning's Woods

(41.1811° N , 81.1997° W) between 24 July 2012 and 8 August 2012. After collection, all individuals were maintained in individual cages on cat food and apple and under the same environmental conditions described previously. They were observed daily to accurately take note of eclosion as adults and keep track of adult age. Half of the males were mated

with females on 13 August 2012 and 14 August 12. Females who mated were not used in any choice trials.

One male of each mating history was placed randomly in one of the two screen cages at the top of the maze. Experiments were performed in a dark, quiet room to minimize outside variables. When both males were singing, a virgin female was released just below the choice point of the maze. She was observed to choose a side and travel to the male’s cage. A trial was only considered valid if both males continued singing throughout the trial and the female eventually traveled the entire way to the cage without turning around or jumping off the maze. Twenty-seven successful trials were performed between 29 August

2012 and 19 October 2012. Results were analyzed using Exact Binomial Test for goodness- of-fit, Chi-squared test for goodness-of-fit, and logistic regression; all tests were performed in JMP (SAS Institute Incorporated 2010).

Results

Upper Limits on Sexual Selection

Table 3.2: Fecundity (number of eggs) Results

Food Least Squares Means ± SE Upper Limit Estimates Treatment 1 Mating 2 Matings Difference Male Slopes H 36.97 ± 5.24 75.58 ± 7.01 38.61 Female L 32.24 ± 5.24 78.15 ± 6.83 45.91 H 35.32 ± 6.77 95.76 ± 8.22 60.44 62.86 ± 7.33 Male L 32.24 ± 6.46 65.67 ± 8.22 33.43 48.38 ± 7.33 Fecundities of males with two matings were the sum of his two mates’

fecundities. Difference estimates of upper limits were calculated by

subtracting the single mating fecundity from the double mating fecundity.

Slope estimates were the slope of the upper limit for males (his second

mate's fecundity).

In females, a second mating did increase fecundity (F1 = 47.44, p < 0.001), but the diet treatments caused no difference (F1 = 0.41, p = 0.52; Figure 3.5a).

a b a b

Figure 3.5a and b: Upper limit estimates of C. nigropleurum on two

protein diets

This figure shows the upper limits for rematings in the high (blue, closed

circles, solid line) versus low (red, open circles, dashed line) protein diets .

Lines connect the means, with bars for one standard error from the mean.

The slope of these lines represents the upper limit of sexual selection. These

figures can be compared against predictions shown in Figure 3.2a and b.

Figure 3.5a shows upper limits for the females. No difference is evident.

Figure 3.5b shows upper limits for the males. An interaction is evident

between diet and mating number. The low protein diet may have restrained

males’ fecundity increase due to remating.

As expected and similarly to females, male mating success had a significant effect on increasing fecundity (F1 = 39.56, p < 0.0001); males with two mates had higher fecundity.

In males, the diet treatments alone had no effect (F1 = 0.11, p = 0.74), however the interaction of diet treatment and mating success had a marginal, but not significant, effect

(F1 = 3.27, p = 0.07). There was a trend toward protein limitation inhibiting males from

increasing their fecundity significantly through remating. This was evident because the low protein males exhibited smaller upper limits than the high protein males (flatter slope to lines in Figure 3.5b). The difference in slopes can be seen when comparing fecundity increases from individuals in the high protein (Figure 3.6a) versus low protein (Figure 3.6b) diets.

A better way to analyze male data involves looking at the slopes of the upper limit

(Figure 3.6a and b) for each male used in the study. Because a given male mated with two virgin females, the fecundity due to each mating is easily measured. This differs from analysis of female upper limits because there is no easy way to distinguish between the eggs laid from her first and second matings. When egg counts from individual matings are examined in males, the slope of the upper limit actually is equal to the number of eggs laid by the second mate. In this way, examining male upper limits is much simpler. When examined this way, diet treatment had no effect on upper limits (F1 = 1.95, p = 0.17; Table

3.2).

a b b

Figure 3.6a and b: Upper limits for individual males on two protein

diets

Each line represents the upper limit estimate for an individual male. These

plots were created by plotting fecundity from one mating and two matings

for each male on the specific diet. Figure 3.6a shows upper limits for males

on the high protein diet. Figure 3.6b shows upper limits for males on the

low protein diet.

Our results suggest that the two diet treatments were not sufficiently different to cause the expected decrease in male and female fecundities. However, there was a trend toward a difference in second matings of males, which implies that the treatments may have been sufficient at least for second male matings. If this were due to low diet males giving a smaller second spermatophore, there should have been a diet by mating number interaction in an analysis of spermatophore weight. When we looked at how diet and mating number affected spermatophore weight and included male mass (after depleted of spermatophore) as a covariate, second spermatophores were significantly larger than first ( F1 = 7.735, p =

0.0065; Figure 3.7). Male weight was a significant factor (F1 = 6.99, p = 0.0095). Diet trends toward a significant influence on spermatophore size (F1 = 3.27, p = 0.07) and an interaction between diet and mating number was found (F1 = 7.44, p = 0.0075) indicating that second spermatophores of low protein males were actually significantly larger than all other categories. This was the opposite of what was expected, but even though the spermatophore was larger, it could have been less valuable to females.

Figure 3.7: Spermatophore size

Spermatophore size was measured by the difference in weight of males

before and after mating. Larger spermatophores were given during males'

second mating (purple, M2 treatment) than during first matings (green). In

this analysis, size of males was taken into account as a covariate.

When we controlled for lifespan effects on the female who received the larger second spermatophores by calculating egg laying rate (total eggs divided by days spent laying eggs), there was an interaction between mating number and spermatophore size (F1

= 4.01, p = 0.048), even though there was no direct effect of spermatophore size on egg

laying rate (F1 = 0.008, p = 0.93). In first mates, egg laying rate increased with increasing

size of spermatophore, but the opposite trend was seen in second mates (Figure 3.8).

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gg E E 0 0 5 10 15 20 25 30 SSpepermrmataoptophohorer eSi Sziez e(m (mg)g )

Figure 3.8: Regression of egg laying rate versus spermatophore size

This figure shows the relationship between spermatophore size and egg

laying rate for matings where males mated either once (green triangles and

green solid line) or twice (purple circles and purple dashed line, M2

treatment). Solid symbols represent high protein diet individuals and empty

symbols represent low protein diet individuals.

Female Choice Experiment

Females did not exhibit a preference for mated males as was expected. Of the trials,

14 females chose mated males and 13 chose virgin males (Exact Binomial Test g.o.f., p =

0.65). This is nearly a 50:50 split and no different than results expected from random chance. However, females did show a significant preference for the left side of the maze (19 choices) over the right side (8 choices; Chi-squared test g.o.f., p = 0.03). This suggests that some environmental variable may not have been controlled for adequately and there was a

difference between the two sides of the maze. Some of these factors were controlled because the experiment was in a small, dark room with lighting only above each side of the maze, however some variables were not able to be controlled such as temperature, temperature in different parts of the room, and noise external to the room. When maze side preference along with preference for mated or unmated males was used as a variable in a logistic regression, there was still no significant female preference evident between the male mating types (2 = 0.94, d.f. = 1, p = 0.33).

Discussion

Our results did not support the hypotheses that diet restrictions reduce the upper limits on sexual selection. In both males and female, fecundity did increase due to remating, as expected, indicating that there was the potential for sexual selection in both sexes.

Female upper limits on sexual selection were shallower than those in males, showing that the potential for sexual selection was larger for males than for females, as expected. This is because female premating fecundity is limited by the number of eggs she can produce with some increase due to nuptial gift value in each mating, but in males, their potential fecundity is the sum of the female fecundity for each of his two mates (which each include the increase in fecundity due to the nuptial gift). Protein limitation did not have the effect we predicted; it appears that the difference between the low and high protein foods did not produce strongly protein limited individuals. There was no effect due to diet seen in females and the effect in males did not reach statistical significance. This suggests that protein limitation did not have a clear impact on either potential female fecundity or the value of nuptial gifts. There was no direct effect due to diet in males, however, we saw a trend toward an interaction between diet and number of matings, explained by smaller

fecundity increases due to remating seen in the low protein diet. This suggests that the low protein diet lowered female fecundity gains from remating and possibly decreased the value of the spermatophore.

These results suggest a future study with an improved diet regimen. One improvement would be to repeat the experiment with much more limited protein content in the low protein food. Another option would be to feed individuals the powdered protein diets starting earlier in life, but this would likely cause the protein limited individuals to develop more slowly and possibly mature at a smaller size.

One thing we can do to follow up on this experiment is analysis of body tissues.

Processing the insects for stable isotope analysis can provide more detailed information about how the insects used nutrients from different food sources at different stages of life.

In this way, we can gain more knowledge about why observed diet effects were minimal

(see CHAPTER 4).

Males produced larger spermatophores during their second matings. This may result in more nutrition and/or more sperm transferred to the female. This suggests that males may be able to provide more benefits to their second mates. Females who were second mates laid more eggs at a higher rate than first mates, however, larger spermatophore size only correlated with increases in egg laying rates for first mates. For second mates, larger spermatophores corresponded with lower egg laying rates. Overall, larger spermatophores were provided during second matings which suggests there might be selection for females to be able to discriminate between experienced males over virgins.

To test whether females discriminate between males based on mating history, we conducted the y-maze experiment to look for female preferences for experienced males

over virgin males. However, our results (in a different katydid species) did not suggest that females were able to detect a difference between virgin males and mated males.

Second matings produced large spermatophores, but this was mostly driven by individuals on the low protein diet (open, purple circles on Figure 3.7 and Figure 3.8), whose spermatophores most likely provided less benefits than those of first mates or from high protein males. The interaction seen between spermatophore size and fecundity rate was more complex than expected. For first mates, egg laying rate increased with spermatophore size, which makes sense if larger spermatophores are more valuable. For second mates, this trend actually reversed. This seems unusual, except that most of the larger spermatophores were provided by low protein males, so we would expect them to be less valuable. They probably contained less protein, but also may have contained less sperm, if low protein males became sperm limited. The possibility also exists that we were not able to completely remove effects of female longevity. This would be worth further investigation. These additional results show that studies of the upper limits on sexual selection need to account for additional variables, notably female lifespan and the value of spermatophores. Future experiments on spermatophore value are needed which investigate effects of diet on spermatophore composition as well as if the weight of a spermatophore is an accurate way to account for its value.

CHAPTER 4

Analysis of nutrient processing in Conocephalus nigropleurum using stable isotopes

Introduction

Stable isotope analysis is a useful technique for examining diet and position in a food web. Ratios of carbon (12C and 13C) and nitrogen (14N and 15N) isotopes in body tissues provide insight into what food sources an animal has eaten. This technique could be used in examining trophic position, comparing sources of food, or examining how nutrients are processed between different individuals (Fry 2006; Oelbermann and Scheu 2002).

Stable isotope measurements are expressed as delta (δ) values. These values are a function of the ratios of the heavy to light isotopes of a particular element (Sharp 2007), compared to the same ratio for a known standard.

Nitrogen delta values are expressed as:

Carbon delta values are expressed as:

As food materials are consumed and incorporated into body tissues, body δ values can change when foods with different δ values come to predominate in the diet. In addition, 44

fractionation occurs, as metabolic processes alter the ratio of heavy and light isotopes.

Examining isotope ratios in body tissues and in food sources can show how consumers process nutrients from their food (Gannes, O'Brien, del Rio 1997). For example, when looking at a food web, carbon ratios remain relatively stable and reflect an organism's diet, however nitrogen ratios are enriched with each level of the food web (Webb, Hedges,

Simpson 1998). In the case of an individual animal, both nitrogen and carbon ratios in different body tissues can show something about how the animal utilizes its food when compared with isotope ratios from the food sources (Caut, Angulo, Courchamp 2007).

Stable isotope analysis was performed on the Conocephalus nigropleurum individuals used in a previous study which was designed to quantify the effects of high and low protein adult diet treatments on the upper limits of sexual selection (CHAPTER 3). In that study, nymphs were raised on cat food and apple, and then, after eclosion, adults were raised on either low or high protein powdered food and apple. We did not see the expected reduction in fecundity due to lower protein adult diets that would have led to decreased upper limits on sexual selection. For this reason, we decided to look at isotopic data to test whether the absence of the expected fecundity differences was because low protein individuals simply ate more low protein food, or because low protein food did not confer low condition for some other reason. We focused primarily on δ15N, since nitrogen is a major component of body proteins (Webb, Hedges, Simpson 1998) and we also examined

δ13C for additional support. We used larval and adult foods with different δ15N values so if low protein diet individuals ate more of the adult diet to make up for their lack of protein, we expected they would have a δ15N more like the adult food. Similar results were expected

with δ13C values, which were included because they helped us to explain some of the patterns we see in the δ15N data.

'Mixing models' have been developed which are used to calculate the amount of food consumed from different sources when the stable isotope ratios of the consumer and each food source are known(Ben-David and Schell 2001; Phillips 2001). This is accomplished using a system of equations and the isotope signatures as known values to solve for the unknown proportions contributed from each food source. When the contributions from each element (carbon and nitrogen) are not approximately equal, a concentration- dependent system of mixing equations must be used to accurately calculate the amounts of food used from each source (Koch and Phillips 2002; Phillips and Koch 2002; Robbins,

Hilderbrand, Farley 2002). However, existing models are designed to estimate relative contributions of diet components to organisms in one stage of their life history. Work still needs to be done to generalize this approach for multiple life history stages where diet differs between stages. For this reason, we do not consider mixing models here and focus instead on a preliminary analysis of how diet components contributed to δ values.

Our analysis examined δ15N and δ13C in three different body tissues: exoskeleton, muscle, and reproductive tissue. The signatures of each tissue gave insight into the individual’s diet during different stages of life and into differential nutrient allocation to different tissues. The exoskeleton is formed during the final molt when the individuals eclose into adults, thus it should reflect the late nymphal diet to a large degree (Voigt and others 2006; Webb, Hedges, Simpson 1998). Muscle tissue is formed early on, but added to as the individual grows and matures, and also replenished continuously; this should provide information about nutrition during the entire lifespan. Finally, reproductive tissue does not

develop until the insect matures and should only be affected by adult diet (Voigt and others

2006).

Hypotheses and Predictions

Analysis of stable isotope ratios in body tissues can answer the questions: were high and low protein individuals truly of high and low condition? Were the diet treatments different enough to cause variation in condition or did individuals with low protein diets compensate by eating more? The analysis should show whether our protein amounts were sufficiently different to produce the condition differences we desired in the study described in CHAPTER 3. The analysis should also show whether there were sex differences in nutrient processing and also if mating treatments had different effects in each sex. For instance, we might expect females who had mated once (consumed a spermatophore) to have a different isotopic signature from virgin females (Voigt and others 2006) and also different from females who consumed multiple spermatophores. Also, males who mated might use nutrients differently than virgin males, since they must produce a spermatophore.

Protein-limited individuals, who may make up the deficit by consuming more low protein food, were predicted to have δ15N values more similar to the adult diet than high protein individuals. So, the difference between tissue and dry food δ15N values would be smaller for low protein individuals than for high protein individuals (Prediction 1 below).

Of the three tissues, the greatest difference in δ15N between diet treatments was expected in reproductive tissue (Prediction 2), since this tissue should mostly have been built after eclosion. Because all nymphs were fed the same diet of cat food and apples, we predicted the δ15N value of exoskeleton (pronotum) tissue to be most similar (Prediction 2) and like

that of cat food (the nymphal protein source). Compared to reproductive tissue, smaller differences between diet treatments were expected in muscle tissue (Prediction 2) because some of the tissue formed during nymphal stages still remained in adults, but some had turned over during adulthood.

To summarize our predictions, we expected to see:

(1)

(2)

Methods

Sample Preparation and Analysis

Stable isotope analysis was completed on a subset of the C. nigropleurum individuals used in the previous study. This subset consisted of five individuals in each mating/food category. The categories were separated by sex, mating treatment (mated once, male mated twice, or female mated twice), and food treatment; there were 10 categories of individuals

(Table 4.1). All individuals were killed and preserved by freezing or frozen shortly after natural death. Three different body tissues were dissected from each individual: pronotum

(exoskeleton), muscle, and reproductive tissue. Additionally three samples were tested of each high and low protein adult food, cat food, and apple.

Table 4.1: Categories of Individuals Sampled

Each insect was dissected to obtain 0.40–1.36 mg dry weight of each tissue type.

Tissues samples were dried in an oven at 40°C until completely dry and ground into powder in a ball mill. These samples were packaged into foil capsules and sent to University of

California Davis Stable Isotope Facility (http://stableisotopefacility.ucdavis.edu) to obtain both δ15N and δ13C via mass spectrometry. Processing occurred between 16 April to 11 July

2012 (round one, females and foods) and 11 June to 7 August 2012 (round two, males).

Data Analysis

The stable isotope values were compared using MANOVA with a repeated measures design and either F-test or Pillai’s Trace is reported. Important variables included were sex, diet treatment (high or low protein powder), and mating treatment (mated once, male mated twice, or female mated twice). Statistical tests were performed in JMP (SAS Institute

Incorporated 2010). Figures were prepared in R (using packages plotrix, Lemon 2013; and calibrate, Graffelman 2012).

Results

As nymphs, all individuals ate cat food and apple ad libitum. These foods appeared to have a relatively equal influence on the δ13C values of pronotum, resulting in values roughly halfway between those obtained for apple and cat food (Figure 4.1). On the other hand, δ15N values for all tissue types were similar to those obtained for cat food. As adults,

all individuals were switched to one of the powdered food diets and apple. Adult isotopic values seemed to have the largest effect from the powdered food diets. These adult diets had the same protein sources (in the same 3:1:1 ratio), they just differed in amount of total protein per gram of food, and therefore they produced very similar δ15N and δ13C values, as expected. Reproductive tissue δ15N values decreased compared to pronotum, becoming more like the adult diet, as expected. Unexpectedly, muscle tissue was not intermediate between those of pronotum and reproductive tissue and instead was very similar to cat food in its δ15N value. On the δ13C axis, reproductive tissue became more negative than the powdered foods, suggesting that adults consumed large amounts of apple.

Figure 4.1: δ15N vs. δ13C biplot of tissue samples compared to food

samples

This plot shows the means for stable isotope values of the three body tissues

and the food samples. Error bars represent one standard error from the

mean. Low and high protein food isotopic values overlap but low protein

food had a slightly lower carbon signature. The tissue sample signatures can

be compared with the food sample signatures to infer the nutrient

processing from consumed food and effects of diet.

Tissue differences (between pronotum, muscle, and reproductive tissue) were evident in both δ15N (F(2,43) = 45.93, p < 0.0001) and δ13C (F(2,43) = = 519.24, p < 0.0001) values (Figure 4.2). Overall sex differences were not present in either δ15N (F(1,44) = 0.04, p =

0.84) or in δ13C (F(1,44) = 0.06, p = 0.81). However, a sex by tissue interaction was present in

δ15N (F(2,43) = 8.47, p = 0.0008) and in δ13C (F(2,43) = 30.39, p < 0.0001), and may be seen as a difference in the relative positions of the sexes on both the δ13C and δ15N axes for pronotum compared to muscle and reproductive tissue (Figure 4.2). Males exhibited higher δ13C and

δ15N signatures than females except in pronotum. The sex by tissue interaction highlights an important sex difference in how nutrient processing seemed to occur in pronotum, compared to the other tissues and may have indicated that males ate more apple than females.

Figure 4.2: δ15N vs. δ13C biplot of tissue samples

This figure shows means from the insect tissue samples separated by sex.

Error bars represent one standard error from the mean. Data are labeled by

sex (Fem = female, Male = male) and tissue type (P = pronotum, M = muscle,

R = reproductive tissue). From this plot, differences between the tissues and

sexes can be seen in both δ15N and δ13C values.

Overall, there were clear differences between low protein and high protein individuals (Figure 4.3) in δ15N (F(1,44) = 9.93, p = 0.0029), although δ13C differences were only marginally significant (F(1,44) = 3.65, p = 0.06). Individuals who consumed the low protein diet expressed lower δ15N values in most cases and also higher δ13C values compared to individuals who consumed the high protein diet. These results are consistent

with our first prediction that low protein individuals make up for the protein deficit by eating more. Additionally, in support of another prediction, the diet differences in δ15N values were largest in reproductive tissue, intermediate in muscle, and smallest in pronotum (Prediction 2, Figure 4.3). The diet by tissue interaction was not significant for

δ15N (F(2,43) = 2.35, p = 0.108) or δ13C (F(2,43) = 0.03, p = 0.97).

There was a significant sex by diet by tissue interaction in δ15N (F(2,43) = 4.98, p =

0.01) but not in δ13C (F(2,43) = 1.79, p = 0.18). This highlights another important difference in the way nutrient processing occurred in pronotum compared to other tissues. It is evident in Figure 4.3 that the significant three-way interaction was due to a sex difference in how nitrogen, but not carbon, was processed in only the pronotum, and mostly by males in the low protein treatment.

Figure 4.3: δ15N vs. δ13C biplot of tissue samples separated by diet

This figure is similar to Figure 4.2 but the data are additionally separated by

diet treatment. Error bars represent one standard error from the mean.

Points are labeled by sex (Fem = female, Male = male), diet (H = high protein

diet, L = low protein diet), and tissue type (P = pronotum, M = muscle, R =

reproductive tissue). Differences between individuals who ate the two diets

are apparent in addition to tissue and sex differences.

Some differences due to mating treatment were evident in carbon signatures. There were significant differences overall between mating treatments (F(2,44) = 4.73, p = 0.014) and also a significant tissue by mating treatment interaction (F(4,88) = 4.35, p = 0.0029). This difference appeared in higher δ13C values from individuals in the mating treatment where

males mated twice (results not shown). This suggests that individuals dealt with higher mating requirements by eating more powdered food. This helps to explain some of the results from the previous project, which showed fecundity increases as well as larger spermatophore sizes in males’ second matings. Neither of these trends were significant in

δ15N (overall F(2,44) = 0.63, p = 0.54; tissue interaction F(4,88) = 1.11, p = 0.35).

Discussion

We predicted that when examining isotopic signatures of food, pronotum would be similar to cat food, reproductive tissue would be similar to the adult powdered diets, and muscle would be somewhere intermediate. The results for pronotum and reproductive tissues supported these predictions. Throughout the insects’ life history, there was a shift seen from nymphal-formed pronotum exhibiting more similar isotopic values to the nymphal diet and adult-formed reproductive tissues shifting toward adult food source isotopic values. However, the δ13C values for reproductive tissue were lower on the δ13C axis than the adult powdered food values, suggesting that adults consumed relatively larger amounts of apple than nymphs and utilized carbon from it. This may have been because the nymphal diet of cat food contained some moisture but the adult powdered diets were completely dry, so additional apple consumption may have been necessary to obtain enough water. Muscle tissue signatures confuse the picture somewhat. The positions of muscle and pronotum values on the δ13C axis were switched from what we predicted. This suggests either that muscle tissue did not turn over much after nymphs eclosed into adults, that pronotum (exoskeleton) tissues turned over more than was expected for some reason, or that more of the pronotum is built from adult food sources when on a low protein diet.

These last two possibilities appear to be possible if there was extreme nutritional deprivation (Webb, Hedges, Simpson 1998).

There was a distinct sex difference seen in reproductive tissues in both nitrogen and carbon signatures, which may be due to differences in the way mature females and males utilized nutrients when producing eggs or spermatophores. Compared to males, female isotope values were decreased on both the δ13C and δ15N axes suggesting that females consumed more of both powdered food and more apple than males. We didn't expect this difference in pronotum because there should be few differences between nutrient processing in male and females at the nymph stage, however, some differences were seen.

The sex by tissue interaction was caused by male pronotum being lower on both the

δ15N and δ13C axes than female pronotum. This suggests either that as nymphs, females and males processed nutrients differently, which is unlikely, or that males mobilized nutrients from the exoskeleton during adulthood (Webb, Hedges, Simpson 1998). The sex by diet by tissue interaction also pointed toward sex differences in nutrient processing in pronotum.

In pronotum, females showed little difference in δ15N due to diet, as expected, but males showed differences such that low protein males had decreased δ15N compared to other pronotum signatures. Low protein males may have processed nitrogen differently in their pronotum (as adults) to deal with their protein deficit. The fact that this only occurred in males suggests that there were sex differences in nutrient requirements. It is known that insects still continue adding tissue to their exoskeleton for several days after eclosion

(Neville 1963). This would result in exoskeleton tissue with δ15N more similar to the adult diet. The low protein diet may have made it difficult to form the final layers of the exoskeleton, resulting in this process requiring a longer time as adults, which would result

in the lower δ15N values we observed. Formation of exoskeleton after eclosion can be examined by counting the number of daily growth rings formed within the femur exoskeleton (Neville 1963). One topic of future research could involve counting these rings in specimens, particularly males, on high versus low protein diet and comparing if they spent different lengths of their adult lives forming their exoskeleton.

The diet effects we saw do fit our original predictions. The δ15N values from low protein individuals were closer to the adult powdered food source than the δ15N values from high protein individuals (Prediction 1). This supports the hypothesis that the reason we did not see the expected differences in fecundity and upper limits on sexual selection in

CHAPTER 3 was because low protein individuals ate more powdered food. Their δ13C values were higher than those of high protein individuals, which may indicate they ate relatively less apple. Also, we saw little difference between high and low protein individuals in pronotum δ15N values, slight differences in muscle, and distinct differences in reproductive tissues (Prediction 2). This reflects the change onto high and low protein food treatments after insects were adults. So the diet treatments did have some effect even if they weren't strong enough to cause the fecundity differences that were expected in the previous experiment.

Mating treatment differences in this dataset (results not shown) provided support for some of our results from the previous experiment (CHAPTER 3). They suggest that individuals in the category where a male mated with two females may have processed nutrients differently than those in other mating combinations. This was consistent with the results that second matings between males with virgin females resulted in the transfer of a larger spermatophore and also in higher female fecundity.

Future work will include modifying the mixing models for use with our data and for two stage life cycles. A concentration-dependent mixing model will be used to calculate the amounts of each food consumed during different life stages. We will also modify mixing models to examine differences in food consumption due to mating status by comparing the differences between both males and females who mated different numbers of times. These will be based on the model used by Phillips and Koch (2002).

CHAPTER 5

General Discussion

The results of my first project (CHAPTER 2) support the idea that katydid mating calls have been affected by natural and sexual selection. The distinct differences in calls have likely evolved for two purposes. First, they allow individuals to recognize conspecifics versus heterospecifics . It is not clear what role the trill plays in the P. scabricollis call, but because it is such a distinct difference between species, it may have evolved in the context of species recognition. This supposition could be tested through playback experiments; calls from both species can be played to see if animals react to less strongly calls of heterospecifics. The second function is mate attraction. Calls from conspecifics will vary in certain aspects and convey different information to prospective mates. Playback experiments could also be used to examine mate attraction. Unfortunately, it is not feasible to use these species in lab studies, so this would have to be set up in the field, which adds certain confounding variables. Also, in recent years, these populations have greatly declined.

In my second project (CHAPTER 3), our results did not definitively show that protein limitation restricts the upper limits on sexual selection. However, sexual selection acted in both males and females by increasing their fecundity with added mating success. In a natural setting, this would be directly related to their ability to attract mates due to what sexually selected traits they can produce. Production of these characteristics would likely be limited by diet because such traits are energetically costly to produce and maintain.

Some of the results from the second project suggest that the role of the katydid spermatophore is not as simple as is often thought. A larger spermatophore did not simply result in higher fecundity. There may be a reason behind this that stems from sexual conflict between mates. It has been shown in other insect species that males transfer certain chemical compounds to females during mating which cause their mates to increase egg output and to consequently have a decreased life span (Chapman and others 1995;

Wigby and Chapman 2005). It is not evident whether male C. nigropleurum individuals produce this type of compound, but it is a noteworthy direction for future studies.

A relationship was seen between spermatophore size and egg laying rate (which was used to control for female lifespan) in matings where males mated either once or twice.

For first mates, even after removing the effects of lifespan, larger spermatophores conferred higher fecundity. This is consistent with the hypothesis that males are including some compound in their spermatophore to increase egg laying. In second matings, the opposite trend was seen. If they do use a compound to influence female egg laying, it may be costly to produce so that they could only include it in their first spermatophore.

Stable isotope analysis (CHAPTER 4) helped give more information about the effects of the diets. Both sex and diet differences were seen in isotopic values. Sex differences suggest that the sexes must utilize nutrients differently because of the different requirements each sex has for reproduction. The diet differences suggest that animals on the low protein diet ate more food to account for the protein deficit; if this is true, it helps explain why the low protein diet did not result in the expected decreases in fecundity (in

CHAPTER 3). Differences were also seen in the mating treatment where males mated twice, suggesting that nutrient processing occurred differently here; this coincides with fecundity

and spermatophore size differences seen in the earlier project. The effects of diet and spermatophore consumption will be analyzed in the future using a mixing model to quantify the differences in food consumption between treatments. Also, our isotope data suggest that males on the low protein diet either may have used nutrients from their exoskeleton during their adult life due to protein limitation or that they didn't finish forming their exoskeleton until well into their adult life, which is a common phenomenon (Neville 1963).

All of these results can help explain how nutrition affects the upper limits on sexual selection and what nutritional processes were underlying the results in the previous experiment.

The data that suggest males provided a larger spermatophore to their second mates imply that females should preferentially mate with nonvirgin males, which the y-maze experiment attempted to examine. It would be beneficial if males were able to convey their mating history to females when attracting them (possibly through song). In this experiment, no difference was seen between females choosing either virgin or mated males over the other. It would benefit females to develop a way to evaluate the mating history of potential mates and choose non-virgin males when offered a choice, however this was not detected in our experiment.

Overall, we gathered some interesting information relating to sexual selection in katydids. In this thesis, I examined katydid mating calls which were found to be distinctly different between two species with overlapping ranges and can be used for identification purposes. Next, I looked into factors that may affect the upper limits on sexual selection.

Our experiment did not show protein limitation to affect the upper limits on sexual selection as strongly as expected, but differences were seen in males' second matings.

Nutritional effects on sexual selection are intertwined with other mating variables and more research should be done to tease these effects apart. Our results on spermatophore size and how it was affected by diet and mating success also warrant further study. Finally, I examined nutrient processing in katydids and the effects from the diet treatments using stable isotope analysis. These helped to explain the results in the previous study and introduced new research ideas regarding differences in nutrient processing between the sexes. Nutritional effects on sexual selection can be quite complex and should be further explored.

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