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Title Food Restriction and Sperm Number in the Water Strider Aquarius remigis.

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Author Muns, Emily Erin

Publication Date 2012

Peer reviewed|Thesis/dissertation

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Food Restriction and Sperm Number in the Water Strider Aquarius remigis

A Thesis submitted in partial satisfaction of the requirements for the degree of

Master of Science

in

Evolution, Ecology, and Organismal Biology

by

Emily Erin Muns

September 2012

Thesis Committee: Dr. Daphne J. Fairbairn, Chairperson Dr. Richard A. Cardullo Dr. Mark A. Chappell

Copyright by Emily Erin Muns 2012

The Thesis of Emily Erin Muns is approved:

______

______

______Committee Chairperson

University of California, Riverside

Acknowledgements

I would like to thank my thesis committee, Drs. Daphne Fairbairn, Richard Cardullo, and

Mark Chappell, for their intellectual guidance and assistance. I would especially like to express my gratitude to Dr. Fairbairn, for allowing me to join her lab and for her subsequent role as my research mentor, and to Dr. Cardullo for allowing me to use his laboratory space and equipment for my research. I would also like to express my deep appreciation to Dr. Catherine Thaler for her patient assistance in the development of my sperm counting method. Many thanks also to the current and former members of the

Fairbairn lab, Clayton Houck and Matthew Wolak, for both technical and moral support.

I would like to thank Matthew Van Sant for being a constant source of laughter and encouragement. Finally, and perhaps most importantly, I would like to thank my family for their unwavering love and support.

iv

To my parents, Steve and Cindy Muns.

v

Table of Contents

Chapter 1: Food Limitation and Sperm Number in the Water Strider Aquarius remigis

Introduction...... 1

Methods ...... 8

Experiment 1: Sperm number in wild males and females ...... 17

Experiment 2: Food restriction in wild-caught animals...... 22

Experiment 3: Food restriction in lab-reared animals ...... 34

Experiment 4: Sperm storage in isolated females...... 38

Discussion...... 40

References...... 47

Appendix A...... 55

vi

List of Figures

Figure 1. Frequency distribution of number of sperm found in wild-caught male and female A. remigis from three southern California populations...... 19

Figure 2. Mean sperm number in the seminal vesicles of wild-caught A. remigis males as a function of experimental day and food treatment...... 29

Figure 3. Median sperm counts of lab-reared A. remigis males from low food and high food treatments ...... 37

vii

List of Tables

Chapter 1

Table 1. Numbers of sperm in the seminal vesicles of wild male A. remigis from three southern California populations...... 18

Table 2. Results of a general linear model of ranked sperm number in Experiment 1 males as a function of body size components and wing morph...... 20

Table 3. Numbers of sperm in the gynatrial complexes of wild A. remigis females from three southern California populations ...... 20

Table 4. Sperm numbers in the gynatrial complexes of Experiment 1 females sorted by population and egg class ...... 22

Table 5. Results of a general linear model of ranked sperm number in Experiment 2 males as a function of population, treatment, and experimental day...... 30

Table 6. Results of a general linear model of ranked sperm number in Day 3 and 6 males as a function of population, treatment, body size components, and wing morph...... 32

Table 7. Results of a general linear model of ranked sperm number in Experiment 3 males as a function of treatment, body size components, and wing morph ...... 36

Table 8. Contingency table of egg and sperm categories in Lytle Creek females isolated from males for seven weeks ...... 39

Appendix A

Table A1.1 Sperm numbers in Experiment 2 males for each population, treatment, and experimental day ...... 55

Table A1.2 Results of an ANOVA of ranked sperm number in Day 3 and 6 males as a function of treatment, day, and cage (nested within treatment)...... 56

Table A1.3 Results of a general linear model of ranked sperm number in Day 3 and 6 males as a function of population, treatment, body size components, and wing morph, interactions excluded...... 57

viii Introduction

In most animals, female fecundity is strongly influenced by the amount and quality of food available to the mother during egg formation and gestation (e.g., Norris 1934; Nayar and Sauerman 1975; Tsiropoulos 1980; Bolton et al. 1992; Boggs and Ross 1993;

Leatemia et al. 1995; Chapman and Partridge 1996; El Sayed 1998; Ferguson 2000;

Bauerfeind and Fischer 2009; Kithsiri et al. 2010; Sink et al. 2010; Mahsa and Allah

2011). In contrast to the abundant evidence for females, relatively few studies have examined the relationship between food and male fertility. Of the studies examining the link between diet and male fertility, many, if not most, have been conducted in captive- bred domestic animals, and results have been equivocal. For example, food restriction has been found to decrease spermatogenesis in deer mice, but not in house mice (1984).

Vawdaw and Mandlwana (1990) found that male rats fed protein-deficient diets had fewer sperm, a lower proportion of motile sperm, and a greater proportion of abnormal sperm in the epididymis than control rats. Similarly, in utero protein restriction of male rats led to developmental delays in juveniles, and the effects of protein restriction lingered into adulthood, with reduced Sertoli cell number, sperm number, and sperm motility and increased morphological abnormalities seen in adults that had been deprived of protein during fetal development (Toledo et al. 2011). In contrast, Mann and Walton

(1953) found no effect of food intake on semen density or sperm motility and morphology in bulls, even when food intake was so severely restricted that rapid weight loss occurred; food restriction did, however, lead to a decrease in the secretion of fructose and citric acid by the accessory glands, and another study found that bull calves subjected

1 to food restriction experienced a delay in the onset of both sperm production and citric acid secretion (Davies et al. 1957). Similarly, while several studies (Murray et al. 1990;

Fernandez et al. 2004; Tufarelli et al. 2011) have found that testicular mass and sperm production in rams increases with dietary protein and energy levels, others (Labuschagne et al. 2002; Fourie et al. 2004) have shown either no effect or a negative effect of high- energy diets on semen quality and quantity in bulls. In birds, Adamstone et al. (1934) found that while long-term Vitamin E deficiency caused atrophy of the testes in domestic cockerels, the testes were also remarkably resistant to this effect, with atrophy apparent only after long-term vitamin deficiency. While Wilson et al. (1965) found that domestic roosters raised on low protein diets experienced delayed maturation relative to males fed higher protein diets, males in the low protein groups had higher sperm concentration, greater testes weights, and better fertility after a recovery period on a maintenance diet.

Taken together, these studies suggest that effect of food on the fertility of male domestic animals is far from straightforward.

Researchers have also examined the effect of food on male fertility in a variety of arthropods. In the moth Plodia interpunctella, Gage and Cook (1994) found that dietary stress (a low protein diet combined with intense competition for food) caused a reduction in both eupyrene and apyrene sperm numbers. However, studies examining the link between nutrition and male fertility in insects and other arthropods often focus on surrogate measures of fertility, such as testis mass or spermatophore size, rather than sperm numbers. For example, Lederhouse et al. (1990) found that male Papilio glaucus

2 butterflies fed honey-water supplemented with electrolytes and amino acids produced larger spermatophores than males fed an unsupplemented honey-water solution, and the eggs they sired were more likely to hatch than eggs sired by unsupplemented males or males supplemented with electrolytes only. In water mites, males given less food produced fewer spermatophores and spent more time looking for mates and more time foraging than males given more food (Proctor 1992). Similarly, Gwynne (1990) found that male katydids fed a low quality diet produced fewer spermatophores (and therefore mated fewer times) than males fed a high quality diet, although the mass of the produced spermatophores did not differ between treatment groups. Several other insect studies have found an effect of nutrition on spermatophore mass (Simmons 1992; Delisle and

Bouchard 1995), and Jia et al. (2000), found that male katydids fed high protein diets produced larger spematophores than males fed low protein diets and food deprived males, but there was no difference in the spermatophore sizes of food deprived males and those fed a low protein diet. The spermatophores of food stressed males (those from the low protein and food deprivation treatments) also had higher water content, suggesting that under nutritional stress, males reduced not only the size, but also the nutritional quality, of their nuptial gift. In short, the effect of food on the fertility of male arthropods clearly varies across both taxa and measures of fertility.

Because individual male gametes are typically small and sperm production is often believed to incur only trivial metabolic costs, sperm number is often ignored as a limiting factor in male reproductive success (Bateman 1948; Parker et al. 1972; Trivers 1972).

3 Similarly, because sperm typically vastly outnumber eggs, it is often assumed that access to males (or their gametes) has little impact on female reproductive success. However, in some systems, notably many marine systems in which gametes are released directly into the water and quickly diluted, female reproductive success is constrained by access to sperm (Pennington 1985; Wedell et al. 2002). Successful fertilization requires that sperm and egg meet, a likelihood that depends on the distribution of sperm sources, degree of spawning synchrony, gamete attributes, and rate of sperm diffusion (Levitan and Petersen

1995; Yund 2000). Because the male gamete in these systems is small and presumably inexpensive, most studies have focused on the effect of sperm limitation on female fertility. However, in systems where the male gamete is particularly costly to produce, it is possible for male fertility to be sperm limited (Dewsbury 1982; Wedell et al. 2002).

Indeed, the fertility of both male and female Drosophila bifurca, which at ! 6 cm has the longest animal sperm on record (Pitnick et al. 1995b), has been shown to be limited by sperm number. D. bifurca males divide their supply of giant sperm between multiple mates, and ejaculate size is reduced across successive matings as sperm stores become depleted, leaving some females with insufficient sperm to fertilize all their eggs (Méry and Joly 2002). Also, while individual male gametes may cost less than female gametes, ejaculates do not consist of a single sperm. Each ejaculate typically consists of many sperm (hundreds of millions in many species), and the combined production cost of these large numbers of sperm represents a nontrivial paternal investment. Furthermore, ejaculates include additional non-gametic components, such as spermatophores and

4 accessory proteins, which can incur a substantial metabolic cost (Boggs 1981; Dewsbury

1982; Parker and Pizzari 2010).

Male reproductive success may, as a result of these metabolic costs, be constrained by sperm numbers in a variety of circumstances, such as when the frequency of matings is very high. Preston et al. (2001) found that limitations on ejaculate production constrained reproductive success in wild Soay sheep. Soay rams compete physically for access to females, with larger males typically gaining a greater number of copulations than smaller males. As expected, the mating success of larger rams gave them greater reproductive success than their smaller counterparts, but this advantage dwindled as the mating season progressed, with the proportion of lambs sired by larger males decreasing over time. Importantly, this decrease in siring success occurred despite the fact that dominant males continued to copulate at a much higher rate than smaller males, strongly suggesting that males who mate with great frequency become sperm depleted over the course of the breeding season. Further evidence for limitations on sperm production comes from domestic Merino sheep. Males that were artificially ejaculated 8 times daily for 18 days experienced an 85% reduction in sperm per ejaculate over the period of the study, indicating depletion of sperm stores due to an inability of sperm production to keep pace with this very high “mating” rate (Thwaites 1995).

Male reproductive success may also be limited by sperm availability when individual gametes are metabolically costly, even in the absence extremely frequent mating (Pitnick

5 and Markow 1994; Pitnick et al. 1995a; Pitnick et al. 1995b). If resources for gamete production are limited, production of larger or more costly gametes is possible only if fewer are made (Birkhead et al. 2009). Sexual selection through sperm competition is thought to favor the production of large numbers of sperm in polyandrous species (Gage and Morrow 2003; Birkhead et al. 2009), and male fertility may be limited by sperm numbers when sperm competition is fierce, but even in the absence of sperm competition, male fertility may be limited by sperm numbers where resources are scarce and gamete production is energetically demanding.

In the present study, I hypothesize that when sperm are costly, sperm number can be limited by food availability in natural populations. To test this hypothesis, I conducted a series of experiments to examine the effect of food availability on sperm number in the water strider Aquarius remigis, a semi-aquatic bug in which males produce very large sperm and experience high levels of sperm competition. These experiments ask whether sperm supplies are influenced by food availability in natural populations of this species and, if so, if this limitation is severe enough to negatively affect male and female reproductive success

Male Aquarius remigis (Hemiptera: Gerridae), produce giant sperm (! 5 mm long;

(Pollister 1930) with an unusual morphology: unlike most animal sperm, in which the length of the tail far exceeds that of the head, the heads and tails of A. remigis sperm are approximately equal in length (Tandler and Moriber 1966). Female A. remigis mate repeatedly (approximately once per day; Preziosi and Fairbairn 1996, Vermette and 6 Fairbairn 2001,) store sperm in long spermathecal tubes (Campbell and Fairbairn 2002), and are able to maintain fertility for up to 24 days with stored sperm from a single ejaculate (Rubenstein 1989). Each sperm is several times longer than the egg it fertilizes

(Miyata et al. 2011) and contains a ! 2.5 mm head. A 5 "m-long nucleus lies immediately proximal to the tail, and the distal portion of the head consists of a very long, thin, autofluorescent acrosome rich in flavin adenine dinucleotide (FAD) ((Tandler and Moriber 1966; Miyata et al. 2011). FAD is an intrinsically fluorescent molecule composed of a riboflavin (Vitamin B2) moiety bound to the phosphate group of an ADP molecule (Stryer 1995). During spermatogenesis, FAD molecules are incorporated into a rigid acrosomal matrix that comprises the greater part of the sperm head. This long, rigid acrosomal matrix remains intact through fertilization and is present in the developing embryo at least through gastrulation (Miyata et al. 2011).

Insects are unable to synthesize riboflavin, which is necessary for a wide variety of cellular processes, and must obtain it through their diet (Trager 1947). Given their FAD- rich sperm, A. remigis males likely require large amounts of dietary riboflavin for sperm production. Previous studies have shown that A. remigis males alter their time budgets under conditions of food limitation, progressively spending more time foraging and less time mating, and eventually abandoning mating attempts altogether (Blanckenhorn et al.

1995). It is unknown whether the cessation of mating attempts represents an attempt to conserve energy or is due to depleted or dwindling sperm numbers; however, prior research has shown that egg production in A. remigis females is positively correlated with

7 food level (Ferguson 2000). If sperm production is similarly affected by food level, this cessation may be a male response to depleted sperm stores. Comparative studies across

Drosophila species have shown a negative correlation between the numbers and the size of sperm, suggesting that large sperm are expensive to produce and that sperm size therefore trades-off with sperm numbers (Pitnick and Markow 1994; Pitnick et al. 1995a;

Pitnick et al. 1995b; Gage and Morrow 2003; Birkhead et al. 2009). Thus, given their large, costly gametes, I hypothesized that male A. remigis would respond to food restriction in a manner similar to females—namely, with a decrease in gamete number.

Here, I test the hypothesis that sperm number, and hence male fertility, is limited by food availability in A. remigis with a series of four studies examining (1) sperm numbers and egg fertility in wild males and females in natural populations, (2) the effect of food restriction on sperm number and mating behavior in wild-caught males, (3) the effect of food restriction on sperm number and mating behavior in lab-reared males, and (4) long- term sperm storage in females.

General Methods

Study animal

The water strider Aquarius remigis is a semiaquatic bug found on the surface of streams and ponds throughout most of North America, feeding primarily on insects trapped in the water’s surface film (Preziosi and Fairbairn 1992; Gallant et al. 1993). A. remigis exhibits wing dimorphism: while most individuals are apterous (wingless), macropterous

8 (winged) individuals comprise from <1% of populations up to 100% in some California populations (Fairbairn 1988a; Fairbairn and King 2009). Nymphs develop in five instars, and life cycle varies. While some populations exhibit a univoltine life cycle, in which individuals do not become reproductively active until the spring after they are born, other populations may produce two or more generations per breeding season (Fairbairn 1985;

Blanckenhorn 1991). Southern California populations likely produce multiple generations per year, with the last generation of nymphs eclosing as adults prior to entering diapause in the fall, then emerging in the spring to mate and produce the next year’s first generation of nymphs (D. Fairbairn, personal communication).

During the mating season (spring and early summer in most populations), both sexes mate multiply, with both males and females averaging approximately one mating per day

(Preziosi and Fairbairn 1996; Vermette and Fairbairn 2002). Males attempt to mate much more frequently than this, but their attempts are usually thwarted by females, who resist males by jumping, somersaulting, or simply skating away (Krupa and Sih 1993;

Weigensberg and Fairbairn 1994). When mating does occur, copulations typically last several hours, and can last more than 12 hours; sperm transfer rarely occurs in copulations lasting less than 15 minutes and always occurs at the end of copulation

(Campbell and Fairbairn 2001). During copulation the male rides on the female’s back, dismounting shortly after sperm transfer.

9 Larger females have greater fecundity, as their larger size allows for more eggs to be packed into the abdomen (Fairbairn 1988b; Preziosi et al. 1996; Preziosi and Fairbairn

1997). Females oviposit daily, and previous studies have shown that they can store sperm for at least 30 days after copulating (Rubenstein 1989); however, observations in the lab suggest that viable sperm may persist in the sperm storage organs even longer.

General laboratory conditions

Animals were housed in stream tanks (137 x 45) or smaller plastic tanks (108 x 47 cm) furnished with foam oviposition and resting sites. All tanks contained airstones to simulate the water movement of natural streams, and the stream tanks also contained aquarium pumps to ensure movement of water through the tank. Upon hatching, nymphs were fed frozen Drosophila melanogaster until reaching the third instar, at which point they were also given frozen juvenile (4 week old) crickets (Acheta domestica). Adults not in the experimental food treatments were fed ad libitum frozen adult A. domestica.

The previous day’s food was removed from tanks each morning, and cages were cleaned and water replaced weekly. Rearing and experimental trials were conducted at room temperature (23.5 ± 0.6º C) with a 14:10 (light: dark) photoperiod. The 14:10 laboratory photoperiod mimicked the natural photoperiod at the time animals were collected for experimental trials: day lengths were 14 h 20 m on July 4, 2011 (North Creek collection date), 13 h 59 min on July 25, 2011 (Deep Creek collection date), and 13 h 46 m on

August 3, 2011 (Lytle Creek collection date).

10 To ensure that the food provided in the laboratory contained adequate levels of riboflavin, samples of frozen D. melanogaster and A. domestica were packed on dry ice and shipped to a commercial analytical laboratory (Covance Laboratories, Madison, WI) for analysis of riboflavin content. Both Drosophila and crickets contained riboflavin in amounts similar to those reported by previous researchers (Finke 2002, 2004): the D. melanogaster contained 3.04 mg of riboflavin per 100 grams, and the A. domestica contained 1.51 mg of riboflavin per 100 grams. These values are well within the range of riboflavin requirements reported for other insects. A diet containing 5.5 micrograms of riboflavin per gram of dry weight supports full growth of blowfly larvae (Brust and

Fraenkel 1955); 4 micrograms per gram is optimal for the beetle Tribolium confusum

(Fröbrich 1939; Offhaus 1939; Trager 1947). Fraenkel (1958) raised healthy Tenebria molitor larvae on a diet supplemented with 12.5 micrograms of riboflavin per gram of dry diet, and Sarma (1943) found that the rice moth Corcyra cephalonica requires 2.5 microgram per gram of diet for optimal development. I thus concluded that the diet provided in the laboratory contained adequate riboflavin for reproduction, growth, and somatic maintenance of water striders across life stages.

Experimental Animals

Virgin females Virgin females used in the experiments were the first generation laboratory-reared offspring of wild-caught adults from two populations. San Diego (SD) females were the progeny of animals collected from Castle Creek and San Clemente Creek near San Diego,

California in March 2011 (n = 9 adults and ! 30 nymphs in various instars); Los Laureles

11 Canyon (LLC) females were the progeny of animals collected from Los Laureles Canyon near Santa Barbara, California in May 2011 (n = 59 adults and 123 nymphs). The wild- caught adults were placed in 137 x 45 cm stream tanks furnished with Styrofoam oviposition sites and allowed to mate freely. Oviposition sites containing fertilized eggs were regularly collected and moved to smaller containers (108 x 47 cm, described above) where the resulting nymphs were reared. Newly eclosed females were removed within

24 hours post-eclosion into 108 x 47 cm tanks and housed without males at densities of

20-25 females per tank until needed in the food restriction experiments described below.

Wild-caught adults Experimental adults were obtained from three mountain streams in Southern California, with collection dates spanning the peak reproductive season in these highly seasonal populations: North Creek in Big Bear Lake, California (July 4, 2011; altitude ! 2100 m);

Deep Creek, in the San Bernardino National Forest near Arrowbear, California (July 25,

2011; altitude ! 1800 m); and Lytle Creek in Lytle Creek, California (August 3, 2011; altitude ! 1200 m). Upon return to the lab, a subset of animals was immediately frozen; we used these to obtain an estimate of the number of sperm in wild A. remigis males and females. The remaining animals were individually marked with enamel paint on their femora and randomly assigned to treatment groups for use in food restriction experiments.

Body measurements

Prior to dissection, each water strider was photographed for later measurement of somatic and genital lengths. Images were captured using a Leica Wild M3C microscope and Spot 12 Basic 3.4 software and analyzed using Sigma Scan Pro 5.0. Landmarks used for body and genital segments were those described in Preziosi and Fairbairn (1996) for males and

Preziosi et al. (1996) for females. To ensure the repeatability of body measurements, I first measured a subset of 66 experimental animals twice, obtaining two values per individual for both somatic length (length of head, thorax, and abdomen, excluding genital segments) and genital length. These data were used to determine the intraclass correlation of our measurements (Wolak et al. 2012). Body measurements were highly repeatable for both somatic length (ICC = 0.9989) and genital length (ICC = 0.9997). In light of the consistency of these measurements, subsequent animals were measured only once for each body component.

Estimates of sperm numbers

Because A. remigis sperm are found in dense bundles and are long, thin, and extremely fragile, they are difficult to count using conventional sperm counting techniques.

Researchers commonly conduct sperm counts using hemocytometry, which is an efficient and reliable cell counting method (Shiran et al. 1995; Mahmoud et al. 1997; World

Health Organization 2010), but whole A. remigis sperm are far too large to be counted in this way. Samples are introduced into the counting chamber of the hemocytometer via capillary action, which draws a small volume of cell-containing fluid into the well, and the bundles of sperm from male A. remigis are much too bulky to be subject to capillary action. Furthermore, counting cells with a hemocytometer requires a random distribution of cells throughout the sample and across the counting field. Clumping of cells, as seen

13 in A. remigis sperm, affects their distribution and may lead to an inaccurate count

(Bukowski and Christenson 1997; Vermette 2001; Snow and Andrade 2004). The A. remigis sperm nucleus is ! 5 "m long, which is comparable in size to the sperm and blood cells commonly counted with hemocytometry, so, to facilitate counting with a hemocytometer, we counted nuclei instead of whole cells. The sperm nuclei were isolated and suspended in a detergent solution, as follows. I dissected the seminal vesicles from males and the gynatrial complex from females and placed each in a low- retention microcentrifuge tube containing 400 "L of a detergent solution [0.1% Brij-35

(polyoxyethyleneglycol dodecyl ether) and 0.1% IGEPAL CA-360 in PBS (phosphate- buffered saline: 10 mM Na2HPO4, 2 mM NaH2PO4, and 135 mM NaCl)]. Each tube was marked at the 100 "L level prior to use in the following manner: I first introduced only

100 "L of detergent solution into each tube with a micropipette, then confirmed that the corresponding 0.1 mL mark placed by the manufacturer on the tube corresponded with the meniscus of the introduced fluid. Where it did not (this occurred in only a very few cases), we marked the tube at the correct fluid level using a fine-tipped marker. 300 "L of detergent solution was then added, bringing the total volume to 400 "L.

Each tube was then placed in an ice bath and sonicated using a Branson Sonifier 250 at

40% power output for three one-minute intervals, with one-minute breaks between intervals. This procedure broke up the tissue of the seminal vesicles and gynatrial sac and the heads and tails of the sperm therein, leaving only nuclei and very small particles behind. Following sonication, tubes were centrifuged for 10 minutes at 14,100 RPM and

14 the supernatant was removed to reduce the sample volume to 100 "L. I then added 1.0

"L of Hoechst stain (10 mg/mL in DMSO) to each sample and placed the tubes in an

Eppendorf mixer for 10 minutes. Finally, 0.75 "L of Triton X-100 detergent was added to the tubes and each sample was subjected to a final pulse sonication consisting of three

2-second rounds of sonication, with 10-second breaks between rounds.

The resulting samples consisted of very small (5 "m) sperm nuclei and other tiny particles suspended in a slightly viscous liquid. Due to the presence of nuclei in many planes of focus and the murkiness of the detergent solution, I found the counting chambers of traditional hemocytometers too deep (depth = 100 "m) to provide a reliable estimate of sperm number. Instead, we used shallower (counting chamber depth = 20 "m) disposable Cell-Vu hemocytometers (DRM-700 Cell-Vu CBC, Millennium Sciences,

New York). The Cell-Vu hemocytometer consists of a dual-chambered glass slide with two glass cover slips. Each cover glass has a laser-etched 1mm x 1mm grid on the reverse side that is subdivided into smaller squares to facilitate counting. I followed the

Cell-Vu protocol for cell counts in a fluid with low cellularity (Millennium Sciences

2008) Each sample was vortexed immediately prior to counting to ensure an even distribution of nuclei within the microcentrifuge tube. Next, using a micropipette fitted with a low-retention tip, I introduced 4 "L of the sperm solution into the sampling area, allowing the solution to fill the counting chamber by capillary action before drawing the gridded cover glass over the liquid and allowing the solution to settle for three minutes prior to counting.

15 A. remigis sperm nuclei are easily distinguished from other types of nuclei due to their elongate shape. Visualization of the nuclei was achieved using a Nikon Microphot-FX microscope with epiflourescence optics at 200x total magnification. I counted the nuclei in all squares of the grid and multiplied this number by 5.5 to obtain the total count per microliter of solution (Cell-Vu CBC product manual, Millennium Sciences). To verify that sperm nuclei were not being removed with the supernatant after centrifugation, counts were also performed on the supernatant from 10% of the samples. Sperm nuclei were never present in the supernatant.

To validate the counting method, I first tested the repeatability of sperm counts on a subset (n = 16) of our experimental animals. I performed four counts per individual and found the intraclass correlation to be high (ICC = 0.993,) indicating an even distribution of sperm within each sample and a highly consistent counting method. For subsequent animals, I performed two sperm counts and took the average of the two values to obtain a mean count per microliter. This number was multiplied by 101.75 (the total volume of the final sample) to give an estimate of the total sperm count per individual.

Statistical Analyses

Statistical analyses were performed using SPSS version 19.0 and R version 2.12.1 (R

Development Core Team, 2011). The R package “ICC” (Wolak et al. 2012) was used to estimate the repeatability of body size measurements and sperm counts.

16 Experiment 1: Sperm number in wild males and females.

Previous estimates of sperm number in A. remigis have used lab-raised animals (Houck

2011). However, animals raised in lab conditions typically have access to abundant food throughout all stages of life, which may not be representative of conditions in the wild.

Because the synthesis of FAD-rich A. remigis sperm requires substantial dietary riboflavin, I predicted that water striders in the wild would have fewer sperm than their lab-raised counterparts. To test this, I conducted sperm counts on adult male and female water striders from three southern California populations. Egg fertility of females was also assessed.

Methods

Adult A. remigis collected from North Creek, Deep Creek and Lytle Creek (see general methods) were frozen within two hours of capture and subsequently photographed, measured, and dissected as described above. As described in the general methods, I used the mean of the two sperm counts per individual to estimate total sperm number.

Because the resulting sperm counts were not normally distributed, ranked data or nonparametric tests were used for all analyses. Values were ranked in ascending order, with the mean rank of tied values used for ties. When dissecting females I also counted the number of mature eggs inside the body cavity.

17 Results

Males from the three wild populations did not differ significantly with respect to ranked sperm number (ANOVA: F = 0.125, df = 2, 35, p = 0.883). The proportion of males containing zero sperm was also similar across populations, (!2 = 1.249, df = 2, p = 0.546;

Table 1). The combined results are presented in Figure 1. A general linear model was used to test for an effect of wing morph, somatic length, and genital length on ranked sperm number. I first performed a full factorial analysis, but as none of the interactions were significant, I removed them and tested for main effects only. The results of this analysis, which are provided in Table 2, suggest that neither wing morph nor body size components are related to sperm number.

Sperm number was highly variable between males, with individual estimates ranging from zero to 25463 sperm (Figure 1). Furthermore, nearly half (48.7%) of all males sampled had zero sperm; this trend was consistent across all three populations.

Table 1. Numbers of sperm in the seminal vesicles of wild male Aquarius remigis from three southern California populations. N = number of males; P(0) = proportion of males with no sperm.

Population N Number of sperm in the seminal vesicles P(0) Mean SD Median Minimum Maximum North 10 895 1250 419 0 3917 0.40 Creek Deep Creek 19 2621 6452 127 0 25463 0.58 Lytle Creek 10 531 567 420 0 1399 0.40 Total 39 1643 4595 280 0 25463 0.49

18

Figure 1. The frequency distribution of numbers of sperm found in the seminal vesicles of wild-caught adult males (blue bars, n = 39) and in the gynatrial complexes of wild- caught adult females (red bars, n = 48) from three populations in southern California. Proportion refers to the proportion of males or females containing a given number of sperm.

19 Table 2. Results of GLM of ranked sperm number in A. remigis males as a function of body size components and wing morph. Somatic length is combined length of head, thorax, and abdomen. Genital length is distance from most posterior medial point of the seventh abdominal sternite to the most medial distal point of the last genital segment. Wing morph refers to presence or absence of wings.

Source of Variation F df p Model 1.01 3, 34 0.399 Wing 0.79 1, 34 0.382 Somatic Length 2.73 1, 34 0.108 Genital Length 0.43 1, 34 0.517

Sperm numbers were similar for females across the three populations (ANOVA: F =

2.052, df = 2, 40, p = 0.142). A general linear model fit to the data showed no effect of genital length (F = 0.259, df = 1, 40, p = 0.614), somatic length (F = 0.003, df = 1, 40, p =

0.955), or wing morph (F = 0.001, df = 1, 40, p = 0.976) upon ranked sperm number in females. As with males, sperm number was highly variable among females, with individual estimates ranging from zero to 3917 sperm (n = 48 females), and many females (43.85%) containing zero sperm (Figure 1), a trend that was consistent across populations (Table 3). The proportion of females containing zero sperm was similar across all three populations (!2 = 1.779, df = 2, p = 0.411).

Table 3. Estimated sperm number in the gynatrial complexes of wild female Aquarius remigis from three southern California populations.

Population N Number of sperm in the seminal vesicles P(0) Mean SD Median Minimum Maximum North 20 672 910 336 0 2798 0.35 Creek Deep Creek 20 364 642 134 0 2518 0.55 Lytle Creek 8 1364 1514 979 0 3917 0.38 Total 48 659 982 249 0 3917 0.44

20 These results indicate that, in the mountain streams sampled, many animals have no sperm at all, even during peak reproductive months. This observation is consistent across three populations and dates, and suggests that some factor is limiting sperm production in the wild.

Next, females were divided into three categories based upon egg count: egg class 0 (zero eggs), class 1 (1-10 eggs), and class 2 (>10 eggs). Table 4 provides descriptive statistics for wild-caught females in each population and egg class. To test whether females with a greater number of eggs contained more sperm, I performed an ANOVA of ranked sperm number as a function of egg category and found that females with more eggs had a higher ranked number of sperm (F = 5.06, df = 2, 45, p = 0.010). The presence of females containing sperm and no eggs (n = 13; 27%) suggests that females may mate even when they have no eggs. Given their ability to store sperm and the apparent scarcity of males with sperm, eggless females may mate whenever they encounter fertile males, storing their sperm to fertilize future eggs. Another explanation for the presence of females with sperm but no eggs is that these females have recently laid all of their eggs and the sperm they contain is what is left after fertilizing them; however, if this were the case, we would expect to see eggs in various stages of maturity present, including eggs very close to maturity. To the contrary, females designated as carrying zero eggs had no visible eggs present, making it more likely that these females were carrying sperm obtained in anticipation of future egg production. We also found females with eggs and no sperm (n

= 4; 8%), suggesting that the fertility of females, like that of males, may be sperm-limited in the wild. 21 Table 4. Descriptive statistics of wild-caught females by population and egg class. Median, minimum, and maximum sperm number are the respective statistics for number of sperm stored in the gynatrial complexes of females from a given population and egg class. P(0) is the proportion of females containing zero sperm.

Egg Class 0 (n = 30) 1 (n = 10) 2 (n = 8) North Creek N 7 6 7 Median Sperm # 280 560 280 Minimum 0 0 0 Maximum 2798 839 2798 P(0) 0.43 0.33 0.29

Deep Creek N 19 1 0 Median Sperm # 0 . . Minimum 0 2518 . Maximum 1399 2518 . P(0) 0.58 0 .

Lytle Creek N 4 3 1 Median Sperm # 0 3078 . Minimum 0 1959 1399 Maximum 560 3917 1399 P(0) 0.75 0 0

Experiment 2: Food restriction in wild-caught animals

The purpose of the following set of experiments was to determine (a) whether food restriction affects sperm production in males, (b) whether male mating rate varies with sperm number, and (c) whether food availability affects the number or proportion of sperm transferred in a single mating.

22 Methods

Adult A. remigis were collected from the wild as described in the general methods section and haphazardly assigned to one of two groups. The first group (Day 0) was frozen within 2 hours of capture to provide an estimate of sperm number in wild males and females. These animals were later assigned to food or no food treatments using a random number generator. The second group was returned to the lab, placed in holding tanks separated by sex, and within 3 hours randomly assigned to one of two treatment groups: food and no food. These animals were placed in 57 x 40 cm plastic tanks at a density of four males and four females per tank (1:1 sex ratio). Deep Creek and Lytle Creek males were individually marked as described in the general methods section and housed with the females from their respective populations. All tanks were furnished with rocks and

Styrofoam pieces for resting and oviposition and were filled to a depth of 5 cm. Aerators provided water movement, and cheesecloth was placed over the tops of tanks to prevent escape.

Animals in the food treatment were provided with superabundant food consisting of one frozen adult cricket (A. domestica) and five frozen Drosophila melanogaster per water strider per day; animals in the no food treatment were unfed. The previous day’s food was cleaned from food treatment tanks daily using aquarium nets; tanks in the no food treatment received a daily sham cleaning. The tanks were arranged in two rows along the laboratory bench, with alternating placement of food and no food tanks to control for any possible position effects along the bench.

23

After an overnight acclimation period, behavioral observations began on the morning of

Day 1. For Deep Creek and Lytle Creek, individual behavioral observations were taken every 15 minutes from 9:00 am to 5:00 pm daily, wherein I recorded whether animals were mating, feeding, or resting off the water surface. Males that died during the course of the experiment (n = 8: 1 male from the North Creek food treatment, 2 males from the

North Creek no food treatment, 3 males from the Lytle Creek no food treatment, and 2 males from the Deep Creek no food treatment) were immediately removed and frozen. If that occurred, a female was also removed from the dead male’s home tank to maintain a

1:1 sex ratio. If a female died during the course of the experiment, she was frozen and replaced with another wild-caught female from the same population.

On Day 3, all females were removed for 6 hours and replaced with individually marked virgin females in a 1:1 sex ratio. These females were used to assess the number of sperm transferred in a single copulation (see below). Behavioral observations continued every

15 minutes after the addition of virgin females.

Previous studies of sperm transfer and fertilization success in A. remigis have shown an initial latency of 15-20 minutes before sperm transfer occurs (Rubenstein 1989; Campbell and Fairbairn 2001). Copulations lasting less than 15 minutes were therefore considered to be unsuccessful. When matings exceeded 15 minutes, I waited until the pair naturally separated to ensure that sperm transfer had been completed (Campbell and Fairbairn

2001), then froze both the male and the female. If no successful matings were initiated in 24 a given tank within six hours, one male was randomly selected and removed from the tank. These males were immediately frozen for later assessment of sperm number. After removal of the Day 3 samples, the virgin females were removed and the wild-caught females were returned to their original cages in numbers sufficient to maintain a 1:1 sex ratio in the tanks.

The behavioral observations and food treatments continued as previously described until

Day 6, when the wild-caught females were again removed and replaced with virgins. As described above for Day 3, all pairs that mated for longer than 15 minutes were frozen when they separated naturally. After six hours with the virgins, all remaining males were removed and frozen. All frozen animals were photographed for later measurement of body components and then dissected for sperm counting as described in the general methods.

The protocol for the North Creek sample differed slightly from that for Lytle and Deep

Creeks. Males were not individually marked and were housed with first generation lab- raised San Diego virgins who were four weeks post-eclosion (see general methods). For the North Creek trial, we observed each tank hourly from 9 am to 5 pm, noting the number of animals mating, feeding, and resting off of the water, and virgin females were added to the tanks on Day 6 only.

25 Because sperm number was not normally distributed and could not be successfully normalized by transformation, ranked data were used in all statistical analyses.

Individuals were ranked in ascending order according to sperm number, with the mean rank used for tied values. A general linear model was fitted to the pooled data, testing the effect of population, treatment, and day on ranked sperm number. The same test was performed for each individual population. I also tested for effects of wing morph, genital length, and somatic length upon sperm number.

Finally, to ensure that cage did not have an effect on sperm number, the populations were analyzed separately. To test for cage effects I performed an ANOVA on the Day 3 and 6 males from each population (Day 0 males were frozen directly after capture and were never housed in a cage), including cage (nested within treatment) in the model.

Results

Figure 2 shows mean sperm number of males for each combination of day, treatment, and population. Ranked sperm number was significantly influenced by population, day, and treatment (Table 5). However, the lack of interactions with population combined with the significant day*treatment interaction, indicates that the effect of day depended on treatment in a similar way across all populations. General linear models fitted within each population indicated significant effects of treatment and day, and significant day*treatment interactions for both Deep Creek and Lytle Creek, but no significant effects for North Creek (Table 5). The trend in the former two samples and the pooled

26 sample is for sperm numbers to increase rapidly in the food treatments between Days 0 and 3 and to then level off, while in the North Creek sample, the increase was not apparent until Day 6 (Figure 1). Descriptive statistics for each population, treatment, and day are given in Appendix A, table A 1.2. Cage did not have a significant effect on ranked sperm number for any population: p = 0.661 for North Creek, p = 0.219 for Deep

Creek, p = 0.209 for Lytle Creek. The results of the ANOVAs including cage effects are provided in Appendix A, table A 1.2.

27 Figure 2. (Facing page) Mean sperm number (± SE) in the seminal vesicles of male A. remigis as a function of experimental day for food (blue line) and no food (red line) treatments. (A) pooled populations, (B) North Creek, (C) Deep Creek, and (D) Lytle Creek.

28 29

Table 5. Results of GLM for ranked sperm number in A. remigis males as a function of population, treatment and experiment day. Day is number of days since start of experiment (Days 0, 3, 6); treatment is food level (no food and food treatments.) Results are given for combined populations (North Creek, Deep Creek, and Lytle Creek) and separately for individual populations.

Combined Populations F df p Source of Variation Model 10.77 17, 108 <0.001 Population 6.19 2, 108 0.003 Day 26.14 2, 108 <0.001 Treatment 18.3 1, 108 <0.001 Population * Day 2.34 4, 108 0.06 Population * Treatment 0.57 2, 108 0.565 Day * Treatment 12.07 2, 108 <0.001 Population * Day * Treatment 0.54 4, 108 0.71

North Creek Source of variation F df p Model 1.68 5, 16 0.196 Day 1.36 2, 16 0.285 Treatment 2.46 1, 16 0.136 Treatment * Day 1.67 2, 16 0.219

Deep Creek Source of variation F df p Model 15.70 5, 67 <0.001 Day 20.61 2, 67 <0.001 Treatment 10.7 1, 67 0.002 Treatment * Day 8.99 2, 67 <0.001

Lytle Creek Source of variation F df p Model 15.62 5, 25 <0.001 Day 24.25 2, 25 <0.001 Treatment 13.04 1, 25 0.001 Treatment * Day 6.00 2, 25 0.007

30 To test for an effect of wing morph and body size components on ranked sperm number, a full factorial ANOVA was conducted on Day 3 and 6 males, with treatment, population, day, wing morph, somatic length, genital length, and all relevant two-way interactions included in the model. (Size analyses for Day 0 males are presented with Experiment 1 results.) Day was not significant when only days 3 and 6 were considered (p = 0.856) and was excluded from the model for subsequent analyses. None of the two-way interactions between body components and the remaining variables were significant, with p-values ranging from 0.205 (treatment * genital length) to 0.607 (population * somatic length); results are presented in Table 6. Finally, the interaction terms were removed and the main effects were testing using a model with only genital length, somatic length, and wing morph; again, neither wing morph (p = 0.551), genital length (p = 0.969), nor somatic length (p = 0.763) had a significant effect on ranked sperm number. Full tabular results of this analysis are given in Appendix A, Table A1.3.

31 Table 6. Results of GLM of ranked sperm number in Day 3 and 6 A. remigis males as a function of population, treatment, body size components, and wing morph. Population is location of capture. Treatment refers to food or no food treatment. Wing refers to presence or absence of wings. Somatic length is combined length of head, thorax, and abdomen. Genital length is distance from most posterior medial point of the seventh abdominal sternite to the most medial distal point of the last genital segment.

Source of Variation F df p Model 5.95 16, 69 <0.001 Population 0.15 2, 69 0.862 Treatment 3.72 1, 69 0.058 Wing 0.61 1, 69 0.437 Somatic Length 1.69 1, 69 0.198 Genital Length 0.44 1, 69 0.508 Population * Treatment 0.29 2, 69 0.753 Population * Wing 0.82 1, 69 0.369 Population * Somatic Length 0.50 2, 69 0.607 Population * Genital Length 0.56 2, 69 0.576 Treatment * Wing 0.43 1, 69 0.515 Treatment * Somatic Length 0.90 1, 69 0.347 Treatment * Genital Length 1.64 1, 69 0.205

Using the behavioral data collected for individually marked males during the Deep Creek and Lytle Creek trials, I first compared mating behavior between the two populations.

Mating rate was similar across populations (Mann-Whitney U = 695.50, p = 0.057), so the two populations were combined to compare mating behavior between the two treatments. Fed males mated more frequently than did unfed males (F = 4.118, d = 1, 73, p = 0.046): males in the no food treatment were observed in a successful mating (lasting

>15 minutes) an average of 0.26 ± 0.77 (n = 31) times over the course of the experiment,

32 while males in the food treatment were observed mating successfully 0.80 ± 1.32 (n = 44) times. (Note that these means are only for the 8h daily observation period and underestimate the numbers of matings over a full 24h period.) The number of observed matings per tank did not differ between treatments for the North Creek trial (F = 0.144, df

= 1, 2, p = 0.741).

The data obtained from the Day 3 and Day 6 virgin matings were used to estimate the number of sperm transferred during a single copulation. Across all three trials, only nine males mated with virgin females for more than 15 minutes (all were from Deep Creek and Lytle Creek, as there were no successful virgin matings among the North Creek males). I counted the number of sperm in each male’s seminal vesicles as well as the number of sperm in the female’s gynatrial complex. Adding these numbers together gave the number of sperm prior to mating with the virgin. Overall, the mean number of sperm transferred in a single copulation was 6716 ± 4089 and the mean proportion transferred was 0.37 ± 0.15 (n = 9). The number of sperm transferred increased with the number of sperm originally in the male (Spearman’s ! = 0.767, p < 0.016). There was no significant effect of male sperm number on proportion of sperm transferred (Spearman’s ! = -0.467, p = 0.205). However, the trend is negative rather than positive, suggesting that males with more sperm do not transfer proportionally more. Males in the no food treatment transferred a mean of 4384 ± 1863 sperm, representing a mean proportion transferred of

0.47 ± 0.23 (n = 3), while males in the food treatment transferred an average of 7881 ±

4524 sperm, for a mean proportion transferred of 0.32 ± 0.07 (n = 6). Neither number

33 nor proportion of sperm transferred differed between treatments (number: Mann-

Whitney U = 13.00, p = 0.302; proportion: Mann-Whitney U = 6.00, p = 0.439); however, the extremely small sample sizes make a reliable comparison of treatments impossible.

Experiment 3: Food restriction in lab-reared animals

The food restriction experiments described above used wild-caught animals that had likely faced at least periodic food shortages throughout life in the wild. Based on the proportion of Day 0 males that contained zero sperm (49%), it is likely that approximately half of the Day 3 and 6 males from North Creek, Lytle Creek, and Deep

Creek had no sperm in their seminal vesicles when the experiment began. The purpose of the following experiment was to examine the effect of short-term food restriction on sperm number in animals reared and maintained with abundant food. I predicted that food restriction would have a less drastic effect on animals reared in the laboratory compared to wild-caught animals. Because animals reared in the laboratory with lifetime access to superabundant food were likely to contain substantial stores of sperm, the treatment duration was lengthened to increase the chances that food restriction would have an effect.

Methods

The experimental animals, which were first generation lab-reared animals, were the progeny of the wild-caught San Diego striders. These animals had been reared in co-sex tanks and maintained as described in the general methods and were approximately 10 34 weeks old at the start of the experiment. The experiment ran for two weeks instead of one week, as in the previous food restriction experiments. In order to increase the survival rate of food-restricted animals over this longer period of deprivation, the treatment levels were adjusted slightly from the previously described experiments. One treatment (high food treatment) was identical to the food treatment in the previous experiments, with each water strider fed one cricket and five Drosophila daily. Animals in the low food treatment received the same ration one day per week and no food on the other days. Low food tanks received a sham cleaning daily to simulate the disturbances experienced by high food animals in the course of daily feeding and tank cleaning.

Virgins were not introduced during the course of the experiment. After one week, nymphs began hatching in all tanks, regardless of treatment; to remove this additional food source, all adults were placed in new tanks on Day 7.

Because the sperm counts were not normally distributed, ranked data were used for statistical analyses. Males were ranked according to estimated sperm number, with the mean of the ranks used for ties. In addition to testing for treatment effects, our analyses included tests of the effects of body size, wing morph, and cage upon ranked sperm number

Results

I found no effect of food treatment on ranked sperm number: t = -0.565, df = 14, p =

0.291, one-tailed (Figure 3). Males in the low food treatment had an average of 16456 sperm (sd =27978, n = 8), while males in the high food treatment contained an average of 35 23557 sperm (sd = 26698, n = 8). In addition, there was no effect of wing morph, genital length, or somatic length on sperm number (Table 7). An ANOVA examining the effect of cage (nested within treatment) upon ranked sperm number was used to test for cage effects. Sperm number was similar among males from all cages (F = 0.501, df = 2, 12, p

= 0.618.) There was no between-treatment difference in the ranked number of sperm in the gynatrial complexes of females (t = -0.960, df = 14, p = 0.177, one-tailed).

Table 7. Results of general linear model of ranked sperm number in lab-reared A. remigis males as a function of treatment, body size components, and wing morph.

Source of variation F df p Model 0.56 5, 10 0.733 Treatment 0.52 1, 10 0.489 Wing 0.22 1, 10 0.647 Somatic Length 0.01 1, 10 0.911 Genital Length 0.13 1, 10 0.730

Even after two weeks of food restriction, the low food males contained sperm in quantities comparable to fed males on Day 6 of the previous food restriction experiments.

This, in addition to lack of a difference between treatments in the current experiment, suggests that males are able to maintain sperm numbers at a normal level throughout a period of short-term deprivation if they have previously had adequate nutrition.

36 Sperm Number in San Diego Males

100000

80000

60000

40000 Sperm Number Sperm

20000

0 Low Food High Food

Low Food Treatment High Food

Figure 3. Median sperm counts for lab-reared San Diego males in low food (red bars; n = 8 males) and high food (blue bars; n = 8 males) treatments. The bottom and top boundaries of the boxes represent the 25th and 75th percentiles, respectively. Error bars indicate the 90th and 10th percentiles, and the filled and open circles indicate outliers.

37 Experiment 4: Long-term sperm storage in females

A. remigis females possess spermathecae (i.e., spermathecal tubes) for sperm storage.

Spermathecae are found in most insect species and vary widely in size and shape (Parker

1970; Kerkut and Gilbert 1985). The A. remigis spermatheca is a long, coiled tube that rises from the gynatrial sac and ends in blind bulb (Campbell and Fairbairn 2001).

Previous studies have found that females continue to lay fertile eggs up to 24 days following a single mating, and up to 30 days after two matings (Rubenstein 1989); however, observations in the lab suggest that some viable sperm persist in the spermathecae even longer. This study examines long-term sperm storage in A. remigis females.

Methods

Wild-caught Lytle Creek females (n = 19) were isolated from males at the end of the

Lytle Creek food restriction trial. This number included all surviving females (i.e., from both food treatments) from the Lytle Creek experiments. The females, who had been housed in a 1:1 sex ratio with Lytle Creek males for the duration of the food restriction experiment, were placed in a single sex (50 x 39 cm) tank and maintained under standard lab conditions with no access to males. After seven weeks with no male contact, all remaining Lytle Creek females were frozen and their sperm and numbers assessed.

Results

Despite the lack of male contact, fertilized eggs (indicated by the appearance of red eye spots and darkened color; Rubenstein 1989; Gallant and Fairbairn 1997) and newly-

38 hatched first instar nymphs appeared daily, indicating the continued presence of viable stored sperm in at least some of the females. Sperm counts confirmed this: on average, females contained 3063 sperm (SD = 1824, n = 19) after seven weeks of isolation from males. All but one of the females contained sperm, with individual estimates ranging from zero to 6995 sperm.

Upon dissection of isolated Lytle Creek females (n = 19), I counted the number of eggs inside the body cavity and assigned each female to one of the egg categories previously described. Only one of the isolated females contained zero sperm, and this female did contain eggs; all of the females with zero eggs (n = 9) contained at least some sperm. To test whether females with more eggs contained a greater number of sperm, females were divided into two categories based on sperm number: those with greater than 3000 sperm and those with fewer than 3000 sperm. (The choice of 3000 sperm as the dividing point was an approximation of the median sperm number of 3078 among the isolated Lytle

Creek females.) A Fisher’s Exact test was then performed on the 3 x 2 contingency table made up of the three egg categories and the two sperm categories (Table 8). Sperm number was similar across the three egg categories (Fisher’s exact probability: p > 0.999, two-tailed).

Table 8. Contingency table of egg and sperm counts in Lytle Creek females deprived of males for seven weeks. Eggs refers to the number of mature eggs inside the abdominal cavity; sperm refers to the number of sperm in the gynatrial complex.

0 Eggs 1-10 Eggs >10 Eggs <3000 Sperm 4 3 2 >3000 Sperm 5 2 3

39 Discussion

Overall, the above results are consistent the hypothesis that sperm limitation may affect male reproductive success when gametes are costly. The effect of food on sperm production in A. remigis is consistent with what has been reported in other studies of insects and mammals (Blank and Desjardins 1984; Murray et al. 1990; Vawdaw and

Mandlwana 1990; Gage and Cook 1994; Fernandez et al. 2004; Toledo et al. 2011;

Tufarelli et al. 2011). Given that many males had few or no sperm and some females had eggs and no sperm, my findings suggest that sperm number can limit both male and female reproductive success when male gametes are costly. I found that in the wild, many A. remigis (male and female) had no sperm at all, even during peak reproductive months. This observation was consistent across three populations and dates, and suggests that there some is factor restricting sperm production (and, as a result, fertility) in wild populations. Based on the results of the food restriction experiments, which show a clear link between food availability and male fertility, it is likely that lack of food is at least partly to blame for the low sperm numbers found in wild males.

The experimental results reported here provide unequivocal support for the hypothesis that food availability affects sperm number in A. remigis: fed and unfed males had drastically different numbers of sperm. The effect of food intake was positive and appeared within three days in the Lytle and Deep Creek males. The discrepancy in results between North Creek and the other two populations suggests that the North Creek males may not have been physiologically prepared to initiate sperm production quickly. This

40 may have been an effect of seasonality and the timing of the sample. The spring of 2011 was unseasonably cool, and the streams warmed so late that North Creek males, who were collected earliest in the season, may not have been physiologically prepared to make sperm. Deep Creek and Lytle Creek males, collected later in the season after a longer period of warm weather, may have been physiologically primed to amplify sperm production once food intake increased in the lab.

Previous measurements of sperm number in A. remigis used lab-reared males that were well-fed throughout their lifetimes and deprived of female contact (Houck 2011). Males in those experiments contained an average of 17702 sperm. This is comparable to the lab-reared San Diego males in the present study, which contained 19663 ± 26213 sperm

(n = 16; treatments combined). By comparison, fed wild-caught males from our food restriction experiments contained 12403 ± 17717 (n = 70), while unfed males contained an average of 3098 ± 5217 sperm (n = 64). These comparisons provide further confirmation for the hypothesis that males in the wild often have no or very few mature sperm but quickly increase sperm production once food availability increases.

The lack of relationship between body size components and sperm number in both lab- reared and wild males is a significant finding, as it suggests that, unlike fecundity in females, male fertility is not related to body size. Research in females, such as that by

Fairbairn (1988b), has found a significant positive correlation between overall female length and number of mature eggs among copulating A. remigis females. Preziosi et al.

41 (1996) and Preziosi and Fairbairn (1997) found a similar positive relationship between total body length and fecundity. This relationship is due not to an effect of total length per se, but to the fact that longer females have larger abdomens, which confer greater egg-carrying capacity. Although previous studies (Preziosi and Fairbairn 1996; Sih et al.

2002; Fairbairn et al. 2003; Bertin and Fairbairn 2005) have shown sexual selection for increased male genital length A. remigis, our results suggest that genital length is unrelated to sperm number. Indeed, while body size is determined at the time of eclosion during the previous year, sperm numbers seem to be strongly influenced by food availability during the breeding season; there is therefore little reason expect a correlation between body size or genital length and sperm numbers.

The between-treatment difference seen in mating behavior of wild-caught A. remigis is consistent with the findings of Blay and Yuval (1997), who found that protein-deprived male medflies (Ceratitis capitata) copulated at a lower rate than protein-fed males. In the case of A. remigis, the lower mating rate seen in food-deprived males may represent a tactic to conserve energy, sperm, or both. In the same study, Blay and Yuval found that protein deprived males transferred a greater number of sperm than did protein fed males; the results of the present study do not support this conclusion for A. remigis. Although the power of my comparison was low because of the small number of successful virgin matings during my experiments, the trend was for food-deprived males to transfer fewer rather than more sperm.

42 The fed males in the current study transferred a greater number (7881 vs. 1396) and proportion (32.1% vs. 7.8%) of sperm in a single mating than did lab-reared males in prior experiments (Houck 2011). Furthermore, while the previous work found no relationship between the number of sperm transferred and the number of sperm in the male prior to ejaculation, the findings reported herein suggest that the number of sperm transferred increases with the number of sperm in the male’s seminal vesicles prior to ejaculation. The lack of correlation between sperm number and number of sperm transferred seen in Houck’s studies is likely related to the fact that the males used in those studies had been raised in the lab on superabundant food and each male likely contained the maximum possible number of sperm, leading to minimal variation in initial sperm number. My sample sizes were, however, limited, and future research to understand this relationship, if one does exist, is warranted.

The absence of an effect of food treatment on sperm number and mating behavior in lab reared (San Diego) animals was likely due to the fact that they had lifetime access to superabundant food, allowing them to build up significant sperm reserves. As a result, these males did not experience significant sperm depletion such as that seen in wild males, who had likely eclosed the previous fall, come through a long adult diapause over the winter, and then endured harsh spring conditions before being used in food restriction experiments.

43 My estimates of sperm number in lab-raised males are similar to those of Houck (2011) for lab-reared animals. While Houck found no evidence of strategic sperm allocation in

A. remigis males based on sex ratio, his use of lab-reared males may have negated any possibility of sperm depletion, thus making strategic sperm allocation unlikely (Wedell et al. 2002). However, the results of food restriction experiments using wild-caught animals show that males may be sperm limited when food resources are scarce. If males become sperm depleted in the wild, they may, faced with a lack of both sperm and the resources needed to manufacture sperm, be forced to reduce ejaculate sizes.

Taken together, the findings presented here suggest that sperm limitation can affect the reproductive success of males as well as females. When male gametes are costly and resources are scarce, it may be sperm number, and not access to females, that places constraints on male fertility. I also found that both female and male A. remigis were able to respond quickly to greater food availability by increasing gamete production.

Furthermore, the ability of females to maintain viable sperm in the reproductive tract over an extended period may represent a mechanism to lessen the impact of food restriction on female fecundity. The results of the sperm storage experiment show that females are able use stored sperm for even longer than previously thought (Rubenstein 1989). The continued emergence of new fertilized eggs and first-instar nymphs indicate that at least some sperm remained viable even after seven weeks of storage within the spermathecae.

While the current study did not allow me to determine whether fertility declined over the period of isolation, it clearly shows that females retain at least some degree of fertility long after the termination of male contact. The mechanism by which sperm viability is 44 maintained inside the female reproductive tract is not known; however, other insect species are known to possess glandular cells in the walls of the spermathecae and glandular secretions in the lumen that are thought to aid in sperm maintenance

(Pendergast 1957; Anderson 1982; Heming Van Battum and Heming 1986). When lack of food limits female fecundity by way of a decrease in egg production, females may nevertheless continue to mate, storing sperm for use if food supplies (and therefore egg production) increase. When sperm is the limiting factor in female fertility, due to lack of males or sperm-depleted males, females are able to use stored sperm to fertilize eggs, maintaining their own fertility in times when sperm are scarce.

The evolutionary significance of the giant, flavin rich sperm of A. remigis is unknown.

One explanation for sperm gigantism is that giant sperm provide the embryo with substances useful for embryonic development (Simmons 2001). While previous work on giant sperm in various Drosophila species (Pitnick et al. 1995a) suggests that giant sperm did not evolve as a form of zygote provisioning in that taxon, the large, riboflavin-packed acrosome seen in A. remigis may, in fact, represent paternal investment in the form of nutrients for the developing zygote. As the central component of the cofactors FAD

(flavin adenine dinucleotide) and FMN (flavin mononucleotide), which act as coenzymes for a wide variety of oxidative enzymes, riboflavin is required for many cellular processes, including energy metabolism and metabolism of fats, carbohydrates, and proteins (Stryer 1995). Such processes are vitally important for embryo development; accordingly, Trager (1947) found that riboflavin was necessary for proper development

45 across a wide range of insect taxa. Previous work by Miyata et al. (2011) showed that the entire A. remigis sperm enters the egg upon fertilization, and the rigid acrosomal matrix remains intact at least through gastrulation. While the post-gastrulation fate of the acrosome is unknown, the riboflavin it contains could certainly be useful to a developing embryo.

In conclusion, I found that males and females in natural populations often contained few or no sperm, and the fitness of both sexes may, at times, be limited by sperm supplies.

Food restriction had a drastic effect on sperm numbers in wild males, with fed males containing much more sperm than unfed males. Mating behavior was also subject to the effects of food restriction, with fed males mating more frequently than unfed males. No such effect of food restriction was seen on sperm numbers or mating behavior in males reared in the laboratory with abundant food. Finally, I found that viable sperm can be stored in the female reproductive tract for at least seven weeks, which is even longer than previously known.

46 References

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54 Appendix 1

Table A1.1. Descriptive statistics for sperm number in experimental males for each population, treatment, and day. N Mean SD Median Minimum Maximum Combined Populations Day 0 - No Food 20 2630 6183 700 0 25463 Day 0 - Food 19 604 1415 0 0 6156 Day 3 - No Food 12 1399 2073 700 0 6716 Day 3 - Food 14 15490 16167 11053 839 52325 Day 6 - No Food 25 3861 4453 2798 0 20986 Day 6 - Food 36 18196 20599 9933 839 90659 North Creek Day 0 - No Food 5 1231 1651 560 0 3917

55 Day 0 - Food 5 560 713 280 0 1679 Day 3 - No Food 4 420 485 420 0 839 Day 3 - Food 3 1306 427 1399 839 1679 Day 6 - No Food 2 700 198 700 560 839 Day 6 - Food 3 3451 2659 3358 839 6156 Deep Creek Day 0 - No Food 10 4281 8553 420 0 25463 Day 0 - Food 9 777 2026 0 0 6156 Day 3 - No Food 5 2015 2870 560 0 6716 Day 3 - Food 8 17034 16423 15530 1399 52325 Day 6 - No Food 17 3193 2729 2798 560 9234 Day 6 - Food 24 16882 18830 9933 1399 73591 Lytle Creek Day 0 - No Food 5 728 702 839 0 1399 Day 0 - Food 5 336 365 280 0 839 Day 3 - No Food 3 1679 1959 839 280 3917 Day 3 - Food 3 25556 17069 25743 8394 42532 Day 6 - No Food 6 6809 7443 4897 0 20986 Day 6 - Food 9 26613 25861 19307 7555 90659

Table A1.2. Results of an ANOVA for ranked sperm number in A. remigis males as a function of treatment, day, and cage (nested within treatment.) Treatment refers to food and no food treatments. Day is day of experiment on which males were sacrificed (only days 3 and 6 are included in this analysis.) Cage in Treatment refers to the cage in which males were housed; this variable is nested within the food treatment variable. Note that the lack of an effect of day in these analyses is to be expected, as the maximum difference in sperm number between treatments was achieved by Day 3. There was, therefore, no difference between Days 3 and 6.

Source of Variation F df p North Creek Treatment 2.73 1, 7 0.143 Day 3.10 2, 7 0.109 Cage in Treatment 0.44 2, 7 0.661 Day * Treatment 0.69 2, 7 0.531

Deep Creek Treatment 33.95 1, 39 <0.001 Day 1.24 2, 39 0.3 Cage in Treatment 1.38 12, 39 0.219 Day * Treatment 1.16 1, 39 0.288

Lytle Creek Treatment 18.87 1, 15 0.001 Day 0.45 2, 15 0.645 Cage in Treatment 1.67 4, 15 0.209 Day * Treatment 0.54 1, 15 0.473

56

Table A1.3 Results of GLM of ranked sperm number in Day 3 and 6 A. remigis males as a function of population, treatment, body size components, and wing morph, interactions excluded. Population is location of capture. Treatment refers to food or no food treatment. Wing refers to presence or absence of wings. Somatic length is combined length of head, thorax, and abdomen. Genital length is distance from most posterior medial point of the seventh abdominal sternite to the most medial distal point of the last genital segment.

Source of Variation F df p Model 15.37 6, 79 <0.001 Population 11.23 2, 79 <0.001 Treatment 55.66 1, 79 <0.001 Wing 0.36 1, 79 0.551 Somatic Length 0.09 1, 79 0.763 Genital Length 0.00 1, 79 0.969

57