Amatitlania Nigrofasciata), When Threatened

PARENTAL INVESTMENT DECISIONS IN A BIPARENTAL CICHLID FISH, THE

CONVICT CICHLID (AMATITLANIA NIGROFASCIATA), WHEN THREATENED

WITH A DIMORPHIC PREDATORY-PAIR

A Thesis

Presented to the faculty of the Department of Biological Sciences

California State University, Sacramento

Submitted in partial satisfaction of the requirements for the degree of

MASTER OF SCIENCE

Biological Science

(Ecology, Evolution, and Conservation)

Colleen Moore

SPRING 2020

Colleen Moore

PARENTAL INVESTMENT DECISIONS IN A BIPARENTAL CICHLID FISH, THE

CONVICT CICHLID (AMATITLANIA NIGROFASCIATA), WHEN THREATENED

WITH A DIMORPHIC PREDATORY-PAIR

A Thesis

Colleen Moore

Approved by:

______, Committee Chair Ronald M. Coleman, Ph.D.

______, Second Reader Timothy Davidson, Ph.D.

______, Third Reader Jamie Kneitel, Ph.D.

______Date

iii

Student: Colleen Moore

I certify that this student has met the requirements for format contained in the University format manual, and that this thesis is suitable for electronic submission to the Library and credit is to be awarded for the thesis.

______, Graduate Coordinator ______James W. Baxter, Ph.D. Date

Department of Biological Sciences

Abstract

PARENTAL INVESTMENT DECISIONS IN A BIPARENTAL CICHLID FISH, THE

CONVICT CICHLID (AMATITLANIA NIGROFASCIATA), WHEN THREATENED

WITH A DIMORPHIC PREDATORY-PAIR

Colleen Moore

How much a parent invests in its offspring has been a focus in many studies assessing parental care dynamics across taxa. An animal with offspring must make investment decisions and behave in a way that will maximize its lifetime reproductive success. Biparental species face a unique conflict in terms of parental investment: the parent must take into consideration how much it invests itself, and also how much its partner invests. A lack of care by both parents may lead to brood loss, but parental care is energetically costly to each parent, resulting in cooperation and conflict. Previous studies have examined investment allocation by a single parent or how parents in a biparental system allocate their investment against a single attacker. However, very few studies have examined investment allocation decisions when a biparental pair is threatened with multiple predators that also differ in size - a realistic threat in the wild. The convict cichlid (Amatitlania nigrofasciata), is a biparental species that provides prolonged parental care, and is therefore an ideal species for examining this question.

Parental defense is critical for offspring survival, and therefore, involves appropriate decision making by the breeding pair. That is, each member of the guarding pair does not simply strike at the closest attacker or attack at random, but instead assesses the threat level and responds with a consistent behavior. The objective of this research is to explore this behavioral response by a pair of parents that differed in size (i.e., a large male and small female). To investigate this research question, a constructed predatory- pair model (two model fish, differing in size) was presented to a pair of convict cichlids that were actively guarding fry, simulating a predator attack. For each experimental trial the model was presented to the pair and moved in a figure-8 pattern towards the pair for

30 seconds, pulled away for 30 seconds, and presented for a final 30 seconds. During each model presentation, the number of bites that each fish took at each of the predators was recorded, producing four scores. The four scores from the first presentation were added to the four scores from the second presentation, to produce four scores for the trial

(i.e., male versus large, male versus small, female versus large, and female versus small).

This procedure was replicated for 5 consecutive days. The four scores from each day were summed to produce four scores for the pair for the experiment (i.e., male versus large, male versus small, female versus large, and female versus small). The hypotheses for this experiment were as follows: (1) the larger parental fish (the male) in the convict cichlid pair will bite the large model attacker fish more than he will bite the small model attacker fish, and (2) the smaller parental fish (the female) will bite the small model attacker fish more than she will bite the large model attacker fish.

A total of twelve convict cichlid pairs were used in the behavioral experiment, which was performed over a nine month period (May 2019 – January 2020). A paired t- test was used to examine the impact of predator size (small vs. large) on parental investment decisions within each sex, measured by the number of bites each parent took at each model fish during the duration of the experiment. Results revealed a consistent pattern within each sex, with significant differences in the number of bites at the model fish predators by both the male (p-value = 0.0001) and by the female (p-value = 0.0003) of the twelve pairs examined. Specifically, the large fish (the male) within a pair bit the large model attacker significantly more than the small model attacker and the small fish

(the female) bit the small model attacker more than the large model attacker, supporting the hypotheses.

Results from this research suggest that the size of a predator, when multiple are present, influences the decision-making of a biparental pair. This research builds off of a solid foundation of theory and experimentation and expands our understanding of investment allocation decisions in biparental fishes.

______, Committee Chair Ronald M. Coleman, Ph.D.

______Date

vii

ACKNOWLEDGEMENTS

First, I would like to thank my thesis advisor, Dr. Ron Coleman, for the support and guidance throughout my time in graduate school. I will leave this program with an even deeper fascination and appreciation for fish after being a part of your lab. Thank you for always making the time to chat whether it was about research or just a friendly conversation about everyday life. Lastly, thank you for the endless amounts of chocolate in the lab for us students on the more difficult days. Thank you to the other members of my committee, Dr. Jamie Kneitel and Dr. Timothy Davidson, for your thoughtful comments and support while writing this paper.

I would like to acknowledge my family for the support and encouragement throughout my graduate school journey. First, I would like to thank my mom for allowing me to have a flexible work schedule so I could leave daily to conduct research trials or check my fish for eggs. I also want to thank my dad for my love and fascination of fish that I initially developed while being raised on the sturgeon farm and for the nonstop encouragement to pursue my Master’s degree. Thank you to my twin sister Sarah, who of course, finished grad school before I did. Sarah, thank you for helping me with my figures and statistics, for the tedious grammar-checks, and for being a great sister during the lows of graduate school and for celebrating my small victories with me, even if it was over the phone. To my other siblings, Kevin and Lauren, thank you for supporting me in your own ways along this journey. You never asked too many questions in regards to my research, which is something I needed – to forget the stressful times and appreciate the small moments with family. viii

Lastly, I would like to thank my partner Ty. I met you a month before I started my first semester in grad school in 2016 and you’ve been nothing but encouraging along the way. Thank you for the weekend fishing trips during the stressful periods of graduate school that brought me so much joy. Most importantly, thank you for always reminding me of how proud you are of me.

TABLE OF CONTENTS Page Acknowledgments ...... viii

List of Figures ...... xii

Chapter

1. INTRODUCTION ………………………………………………………………...... 1

Parental Investment Theory………………………………………………………....1

Size-based Decision-making……………………………………………………...... 3

The Convict Cichlid.………………………………………………………………..6

Field Observation and Unanswered Questions……………………………………..7

Objectives and Hypotheses…………………………………………………………8

2. MATERIALS AND METHODS…………………………………………………….9

Preliminary Fieldwork: Costa Rica………………………………………………....9

Laboratory Experiment: Sacramento State………………………………………...11

Experimental Fish………………………………………………………………….11

Aquaria Configuration………...………………………………………………...... 12

Predatory-pair Model...……………… …………………………………………….12

Predatory-pair Behavioral Experiment……………………………………….…….15

Data Analysis……………….…………………………………………………...... 16

3. RESULTS………………………………………………………………………...... 18

Behavioral Experiment...…………………………………………………………..18

4. DISCUSSION……………………………………………………………………….21

Appendix A. Raw Data of Convict Cichlid Pairs………………………………………..25

Appendix B. Daily Data From Predatory-pair Experiment………………………...……27

Appendix C. Data Used in the Paired t-test….……………………………………...…...34

References………………………………………………………………………………..35

LIST OF FIGURES

Figures

1. Relevant Research Flowchart…………………………………………………………5

2. Predatory-pair Fieldwork Model……………………………………………………..10

3. Aquaria Set-up for the Behavioral Experiment……………………………………...13

4. Predatory-pair Lab Model……………………………………………………………14

5. Male Results………………………………………………………………………….19

6. Female Results………………………………..……………………………………. 20

xii 1

INTRODUCTION

Parental Investment Theory

How much effort a parent invests in its offspring has been a focal question in many studies assessing parental care dynamics across taxa. Trivers (1972, p. 139) defined parental investment as “any investment by the parent in an individual offspring that increases the offspring’s chance of surviving at the cost of the parent’s ability to invest in other offspring”. Parental investment is energetically costly and any amount of effort put into parental investment is unavailable for use elsewhere (Williams 1966). Therefore, the extent of parental investment provided by an organism at any point in time is a critical life history decision.

The amount of investment that a parent is willing to make in a given brood of offspring is highly variable across species, between individuals and even between circumstances for the same individual. Attempting to understand this variation has been the subject of numerous observational and experimental studies. These studies have attributed the variation to such factors as brood size and level of past investment

(Coleman 1985, Lavery and Keenleyside 1990a), past breeding experience (Lavery

1995), size of a parent (Coleman 1993, Galvani and Coleman 1998), partners’ investment level (Coleman 1993), and offspring quality (Thunken et al. 2010).

Sargent and Gross (1985) attempted to synthesize a general rule that would encompass many of these factors. They proposed the “relative value rule” which states that a parent should invest according to the value of its current brood relative to its own expected reproduction in the future. In a manipulative field experiment, Coleman et al.

(1985) showed the male bluegill sunfish (Lepomis macrochirus), a species that provides male-only care, invested according to both brood size and past investment, supporting the relative value rule (Sargent and Gross 1985).

Biparental species face a unique conflict in terms of parental investment: a parent must take into consideration both how much it invests and also how much its partner invests in protection of the brood, meaning that the solution likely involves both life history theory and game theory (Chase 1980, Houston and Davies 1985, Coleman 1993).

Cooperation between a biparental pair is critical because a lack of care by any parent could lead to brood loss, but parental investment is energetically costly to each parent

(Coleman and Fischer 1991). In species which do not form long-term pair bonds, each parent is under selection to reduce its cost from investment effort by shifting the burden to its partner. An evolutionary stable solution has been suggested to resolve this cooperation-conflict that exists in biparental species. A general model first developed by

Chase (1980) and later expanded by Houston and Davies (1985) stated that each parent must divide its individual investment between the current brood and expected future broods. The model predicts that the optimal response to a reduction in care by one parent is an increase in care by the other parent. Coleman (1993) further extended this model and tested it in the laboratory using convict cichlids (Amatitlania nigrofasciata) showing that size of a parent, as well as investment level by its partner were important factors influencing allocation decisions when the parents were faced with a single attacker on their brood.

In a situation where biparental parents were being threatened by two predators simultaneously, Richter et al. (2005) showed both parents contributed to brood defense of a nearby predator in an experimental setup involving one predator placed far away and the other up close to the brood. That study showed that parents were able to evaluate the differences in the threat presented by two different predators, and responded differently, but did not address the question of size directly.

Size-based Decision-making

Some studies have argued that there are sex-specific roles in parental care in fishes; however; Coleman (1993) showed that many of those results are better explained by the size difference between the two parents, e.g., while some studies have shown the female convict cichlids provide more aggressive defense of the brood, Coleman showed that it was the smaller fish of the two (male or female) that provided more aggressive defense, regardless of sex. Indeed, particularly in organisms with indeterminate growth, such as fishes, size may be a key parameter for understanding parental investment decisions.

Aggression in the form of territorial defense is strongly dependent on body size in cichlids (Barlow 1983). As stated above, the relative body size of parents has been shown to influence parental investment dynamics. Coleman (1993) manipulated pair-size in convict cichlids by pairing large males with small females and large females with small males; Coleman found the smaller parent was significantly more aggressive towards a heterospecific model intruder than the larger parent, regardless of sex. The size of an

4 intruder relative to that of the guarding pair can also influence parental investment decisions. A field experiment in Nicaragua (Anderson et al. 2016) showed that when presented with intruders of three different body sizes, the male convict cichlid was significantly more aggressive toward the intruder than the female. The size of the male relative to the intruder was also an important factor; defensive males smaller than an intruder were significantly more aggressive than if they were larger than the intruder

(Anderson et al. 2016). Several lab studies support the findings that the male is more aggressive until it is the same size or larger than the intruder (Itzkowitz et al. 2005,

Beeching 1992). Lab contests in convict cichlids reveal that large fish typically defeat smaller fish (Keeley & Grant 1993). Therefore, based on size alone, the male (being the larger sex in a typical pair) is more likely to succeed at defeating a larger threat compared to its smaller female mate.

To my knowledge, no studies have examined individual investment effort in a biparental species when threatened by multiple predators that differ in size. Investigating this phenomenon takes the next logical step in the timeline of experimental studies aiming to uncover additional dynamics of parental investment allocation (Figure 1). The convict cichlid (Amatitlania nigrofasciata), a biparental species, provides an opportunity to examine this question.

Coleman et al. Coleman 1993 Richter et al. My research 1985 • Biparental 2005 • Biparental • Single parents vs. 1 • Biparental parents vs. parent vs. 1 attacker parents vs. 2 dimorphic attacker attackers attckers

Figure 1. Relevant research flowchart. This flowchart displays key studies that led to the formulation of the research question in the current study.

The Convict Cichlid

Cichlid fish (family Cichlidae) exhibit a wide range of parental care strategies within the family, making them popular subjects for examining the evolution of parental care. The convict cichlid has attracted research interest because unlike a majority of fishes, they provide prolonged care of their brood throughout the stages of development

(Keenleyside 1991). The convict cichlid is often referred to as a model organism in the field of behavioral ecology because of its small size, the relative ease of maintaining and breeding them in a laboratory environment, and their ability to spawn every 4-6 weeks

(Barley and Coleman 2010).

The convict cichlid is a substrate brooding species native to lakes and streams in

Central America, ranging from Guatemala to Panama (Bussing 1998). During the breeding season, adults form monogamous pairs; both parents invest energy in caring for their brood from the time the eggs are laid until they develop into free-swimming fry.

Typically, both parents defend the free-swimming young for several weeks, sometimes longer (Coleman 1993). In their natural habitat, predation pressure on the young by other fishes, including other cichlids, is intense during all stages of development and survival of the young is dependent on defense by the parents. During the egg and larval stage, the young are immobile and defense is focused around the nest. As the young become free- swimming, they move as a school during the day searching for food. This may take them several meters from their nest and put the school in contact with other such schools. At this time, the parents of one school of fry may encounter the parents of a neighboring school of fry. In this situation, a pair of parents must decide how to defend their offspring

7 against a pair of attackers. A high level of cooperation between the male and female in the pair is critical for offspring survival.

Field Observations and Unanswered Questions

I conducted preliminary fieldwork in the lowlands of northeastern Costa Rica from December of 2017 – January of 2018. I observed situations where breeding pairs were threatened by multiple predatory fish of different sizes simultaneously. While this scenario appears to be common in the wild, it has yet to be studied thoroughly in the laboratory. More specifically, understanding how parents in a biparental pair respond to this situation in regards to individual decision making is particularly fascinating. In the field setting of Costa Rica, multiple variables can be difficult to quantify or impossible to control (e.g., breeding substrate type, predation pressure, temperature, etc.). The

Evolutionary Ecology of Fishes Lab at Sacramento State offered a controlled environment to explore this dynamic further.

In their natural habitats, a biparental pair may be threatened by several fish at once, likely of different sizes. Contests within the same sex (e.g., male-male) in biparental fishes are commonly seen in lab studies, but this scenario removes the realistic threat experienced by breeding pairs in the wild. Does the male, being the larger sex, take on the larger attacker and the female take on the smaller attacker? Does the female stay with the brood, while the male primarily performs brood defense? This is an interesting dynamic to explore. If the male chooses to attack the smaller of the two predators, it may do so because of the higher chance of winning compared to the female,

8 based on size. However, choosing the smaller threat comes at a cost to his brood and mate who would then be susceptible to the larger predator of the two. If the male chooses to attack the larger predator (i.e., the larger threat), this option would maximize the overall effectiveness of the pair, protecting his mate and offspring. Based on the female’s body size, I would not expect her allocation effort to be focused on the larger of the two predatory threats. Therefore, I expect the female to attack the smaller predator of the two because if she does not join in on defense to some degree, the smaller predator will be undeterred to consume the brood.

Objectives and Hypotheses

I anticipate that parental defense, critical for offspring survival, requires decision- making by each member of the pair. The objective of my research is to determine if size of a predator, when two are present, influences the individual defensive decisions within convict cichlid pairs.

I hypothesize that when a pair of convict cichlids actively guarding fry are presented with two predatory models differing in size, that (1) the male of the pair, being larger, should bite the large predator significantly more than he bites the small predator, and (2) the female of the pair should bite the small predator significantly more than she bites the large predator.

MATERIALS AND METHODS

Preliminary Fieldwork: Costa Rica

I traveled to northeast Costa Rica from December 2017 to January 2018 to observe convict cichlids and other biparental fish in the wild and run a pilot experiment. I conducted field studies on twelve biparental fish pairs in five different river systems, all part of the larger San Juan River drainage. I constructed a 3D model from hollow-body fishing lures (LiveTarget Sunfish Lures, Model No. SFH75T555) attached to an acrylic rod (Figure 2); these fish used on this model resembled a generic brood predator.

Employing similar methods to those eventually used in the lab study (see more below), I presented my model to pairs of biparental cichlids (convict cichlids or Neetropolus nematopus) actively guarding fry and recorded the individual responses from each parent.

In my field trials, I was specifically interested in the individual responses of biparental parents, regardless of species, to two model predators simultaneously attacking the brood.

This fieldwork yielded a range of responses by the male and female between different river systems. With a variety of responses seen in the field trials, it became clear that this behavioral dynamic is complex and warrants further investigation in a laboratory setting where it would be possible to control for confounding variables.

Figure 2. Predatory-pair fieldwork model. This 3D dimorphic model was used in preliminary field trials conducted in Costa Rica. The large fish (top) was 88.9mm long and the small fish (bottom) was 76.2mm long.

Laboratory Experiment: Sacramento State

The laboratory experiment was conducted in the main fish room of the

Evolutionary Ecology of Fishes Laboratory at California State University, Sacramento

(Humboldt Hall 119). The fish room is on a 12:12 hours light:dark cycle with small number of students periodically tending to nearby aquaria. All fish within the lab, including the fish used in this experiment, were fed daily ad libitum on TetraCichlid flake food.

Experimental Fish

The convict cichlids used in the behavioral experiment were purchased from local fish suppliers and stored in a stock tank in an adjacent room to where the experiment was conducted. For each trial, an adult male and female convict cichlid were handpicked from the stock tank and placed into the test aquarium; the male was at least 10mm larger than the female in each pair bred. The average total length of the male fish used in the experiment was 72.8±1.2mm and weighed 6.7±0.4g; average total length of the female fish was 58.9±1.3mm and weighed 3.7±0.2g. All means are reported as mean ± SE.

Additional information on all fish used in the experiment can be found in Appendix A.

Size-assortative pairing in convict cichlids has been observed both in the field

(Wisenden 1994) and in a laboratory setting (Keenleyside 1985). Extensive research supports female convict cichlids favoring a larger male in an environment with limited choice (Gagliardi-Seeley et al. 2008) as well as free-choice (Schulte and Coleman 2017, unpublished data).

Aquaria Configuration

The aquaria were 113 liters (60 x 60 x 30cm3 high) with three sides covered with tan colored plastic sheeting to keep each pair isolated from surrounding tanks. Two aquaria with similar setups were used during the experiment to allow for two trials to be conducted simultaneously. The test aquaria contained approximately 2cm of gravel substrate, two plastic plants, two bottomless flowerpots (10cm in diameter), and an ATI sponge filter (Figure 3). Temperature in the test aquaria was maintained by a submersible aquaria heater at 26°C.

Predatory-pair Model

Two 2-D dimorphic predatory-pair models were constructed for use in the behavioral experiment. A photographic plate of Variabilichromis moori (Axelrod et al.

1985, Plate 6), a generic brood predator, was coated in epoxy resin and attached to an acrylic handle (Figure 4). The two fish in each model were identical except for size (i.e., they did not show any obvious sign of sex differences). Galvani and Coleman (1998) used a similar model in a predator encounter experiment with convict cichlids in the lab, confirming this model is successful at inciting a defensive response. Preliminary trials in the lab also supported this finding; cichlid pairs aggressively bit at the individual fish within the model. The two models used in the experiment were identical in appearance but differed in position of the predator fish: in one model, the larger predator was on the left, and in the other model, the larger predator was on the right.

Figure 3. Aquaria set-up for the behavioral experiment. Two aquaria were used during the duration of the experiment that consisted of the same general set-up.

Figure 4. Predatory-pair lab model. The large fish in the model is approximately 33% larger than the small model fish. The small model fish (left) is 61.2mm in total length and the large model fish (right) is 85.5mm in total length.

The large model predator was approximately 33% larger in total length than the small model predator. In relation to the convict cichlid pairs used in the experiment, the large fish predator on the model was larger than all of the male convict cichlids and the small fish predator was smaller than the males. Eight of the twelve female convict cichlids used during the experiment were smaller than the small fish model and four of them were slightly larger by a few millimeters.

Predatory-Pair Behavioral Experiment

The first day eggs were observed in the tank was considered Day 1. On Day 1, the male and female of the pair were weighed and measured and a picture of the eggs was taken by carefully lifting the pot out of the tank. This picture was later used to count the number of eggs. Only pairs with at least 100 eggs were used in the behavioral experiment to reduce the potential effect of brood size variation in parental investment. The eggs developed into free-swimming fry from Day 1 to Day 7. The behavioral experiment began on Day 7 by simulating a predator attack by presenting the predatory-pair model to the pair of convict cichlids actively guarding fry (as per Coleman 1993, Coleman and

Galvani 1998). For each experimental trial, the model was presented to the pair and moved in a figure-8 pattern towards the pair for 30 seconds, pulled away for 30 seconds, and presented for a final 30 seconds. Four push-button counters were mounted side-by- side to record the number of bites during the experiment. During each model presentation, the number of bites that each parental fish took at each of the predators was recorded, producing four scores. The four scores from the first presentation were added to

16 the four scores from the second presentation, to produce four scores for the trial (i.e., male versus large attacker, male versus small attacker, female versus large attacker, and female versus small attacker). This procedure was replicated for 5 consecutive days, alternating every other day between two predatory-pair models that were identical in appearance, but differed in which model fish (small or large) was on the left or right. The four scores from each day were summed to produce four scores for the pair for the experiment (i.e., male versus large attacker, male versus small attacker, female versus large attacker, and female versus small attacker). A total of twelve pairs were used. The total scores collected over the 5 days of testing for each pair were used for the data analysis.

On the final day of the predatory-pair experiment (Day 11), all fry were removed from the tank and counted to ensure the number of fry did not significantly decrease throughout the experiment. Fry survival rate was measured by the percentage of fry collected on the last day of the experiment compared to the original number of eggs counted on Day 1. The male and female of the pair were also re-weighed and re- measured on the final day of the experiment.

Data Analysis

Two paired t-tests were run in R (version 1.1.463) to analyze the parental decisions within the sexes (n=12)(Appendix C). Therefore, one paired t-test compared the number of bites that each male took at each model fish (small or large) and the other paired t-test compared the number of bites that each female took at each model fish

(small or large). A paired t-test allows for the examination of the parental decisions of each parent within a pair. The use of a paired t-test controls for any possible sources of variation between fish, aquaria, and trials.

RESULTS

A total of twelve trials were conducted, never re-using fish that had been subject to the behavioral experiment. The average number of eggs laid within a pair was 190±15 eggs and average fry survival was 91.2±1.1% (Appendix A).

Behavioral Experiment

The total number of bites that each fish in a pair took at each model fish (small or large) over five days was analyzed. Two t-tests were run, one for each sex, because I was specifically interested in the individual responses of the sexes to the two fish predators.

The number of bites was the response variable and predator size was the explanatory variable. Over twelve trials, the male fish bit the large fish model significantly more compared to the small fish model (paired t-test; t = 5.95, df = 11, P = 0.0001, Fig. 5) and the female in the pair bit the small fish model significantly more compared to the large model (paired t-test; t = 5.27, df = 11, P = 0.0003, Fig. 6). The total number of bites from each trial can be found in Appendix B. The data used in the paired t-tests can be found in

Appendix C.

Male Response 80

20 Number of Bites (5 Day Total) 10

0 Small Large Model Predator Size

Figure 5. Male Results. The figure shows the total number of bites that each male from a pair took at each model fish (small and large). The number of bites is the total collected over five days. Each connected line represents the responses from an individual fish within a pair (n=12). Each male fish bit the large model fish significantly more than it bit the small model fish (P = 0.0001).

Female Response 60

Number of Bites (5 Day Total) 10

0 Small Large Model Predator Size

Figure 6. Female Results. The figure shows the total number of bites that each female from a pair took at each model fish (small and large). The number of bites is the total collected over five days. Each connected line represents the responses from an individual fish within a pair (n=12). Each female fish bit the small model fish significantly more than it bit the large model fish (P = 0.0003).

DISCUSSION

The manipulative experiment found that the larger fish of a parental pair (the male) bit the large attacker model significantly more than he bit the small attacker model.

The smaller fish of a parental pair (the female) bit the small attacker model more than she bit the large attacker model. In other words, the parents responded differently to the attackers, and each of the parents focused its defense against a particular attacker: large against large, small against small.

Although the sample size was more than sufficient to draw the above conclusions, there was a large amount of variation in the responses of parents both within and between sexes. This is consistent with other studies of parental investment. The variation in this study, reflected by magnitude of bites, likely has many causes including brood size and past investment (Coleman 1985, Lavery and Keenleyside 1990), past breeding experience

(Lavery 1995), future expectation (Sargent and Gross 1986), partners’ investment level and size of parent (Coleman 1993, Galvani and Coleman 1998), and offspring quality

(Thunken et al. 2010). The fact that such variation exists is one of the main reasons why trying to understand the extent of the variation has continued to be an intensely scrutinized question in behavioral ecology. Additionally, the existence of this variation also highlights the importance of conducting carefully controlled manipulative experiments to attempt to tease apart the factors causing the variation. Purely observational or correlative studies of parental investment, while sometimes suggestive, have proved in hindsight to be misleading or even counter-productive (discussed in

Coleman et al. 1985) because of the many inter-related factors causing a particular investment decision.

Body size is an important life history characteristic in fish, particularly because they exhibit indeterminant growth. This means that a fish faces a three-way tradeoff between maintenance, growth, and reproduction, and any investment into one area, e.g., growth, means a reduction in allocation in other areas, and yet increased growth facilitates greater reproduction, making these tradeoffs particularly challenging to understand. As a female fish grows, she is able to produce more eggs. It has been experimentally shown that female convict cichlids of different sizes do not value the same brood number equally (Galvani and Coleman 1998). During a size-manipulated experiment, Coleman (1993) paired a small convict cichlid male with a large female and a small female with a large male and saw that the smaller fish in each situation attacked a model predator fish more than the large mate. The results from Coleman (1993) show that the size disparity of the pair, instead of the sex of the pair, was driving the results.

The results of this current study show that when faced with two predators of different sizes, a size-pairing response occurred between the predators and the guarding pair (i.e., the larger fish in the guarding pair always took on the larger model predator). The experimental methods of this current study do not test if the results seen can be attributed to sex of the guarding pair or size of the guarding pair. To tease apart which of these factors may be driving the results seen, repeating this experiment with a large female paired with a small male would be of interest. Based on the results from Coleman (1993) along with this current study, it is predicted that under this hypothetical size-manipulated

23 scenario, a large female parent will bite the large predator more than the small predator, and a small male parent would bite the small predator more than the large predator.

The relative value rule states that a parent should invest according to the value of its current brood relative to its own expected reproduction in the future (Sargent and

Gross 1985). It is difficult to determine (and test) an organisms expectation regarding its future reproductive opportunities and this study did not attempt to control for this. The convict cichlids used in this study came from various stocks from local suppliers and knowledge of several aspects of the fish used could not be controlled, such as previous mating history. It has been tested that several factors including past breeding experience can influence the value that a parent places on its brood (Lavery 1995). The paired t-test used to analyze the data collected in this study was deliberate to assess within sex decisions and not between sex decisions. I did not attempt to examine what the female was doing compared to the male in the pair because it has already been showed that parents in a biparental situation value their brood differently and several factors can be attributed to this. Each fish in the pair was being tested on its own decisions to bite either the large or small model and this was replicated over twelve fish pairs. It is important to not attempt to draw correlative conclusions on manipulated experimentation to resolve questions regarding parental investment allocation due to underlying differences between the parents to provide care that were not controlled for (Coleman 1985).

Parental decisions are complex and many will be made over an organism’s lifetime. Choosing which predatory fish to bite is a life and death decision. In a biparental species where effort is required by both sexes for the survival of offspring, and where

24 parents do not mate for life, how a parent is likely to respond to a parental defensive scenario, like the one created in this study, may be an important factor in how these individuals select their mates. It has been observed that cichlids, including convict cichlids, often engage in aggressive courtship prior to mating (R. Coleman, personal communication). It is possible that this fighting is used as means to test a potential partner’s willingness to fight to protect future offspring. This study shows that the relative size of prospective parents may have an important influence on mating decisions.

The decisions made by organisms in the present day have been shaped by mechanisms of both natural and sexual selection over evolutionary time, leading to a current stable strategy that has outcompeted others. Given the consistent behavior of the male and female within the convict cichlid pairs in this study, it seems the decision for the larger fish in the pair to take on the larger predator and the small fish is the pair to take on the smaller predator when confronted with a dimorphic predatory pair is an advantageous behavior in the convict cichlids biparental care strategy and was likely selected for over the course of evolution.

APPENDIX A

This appendix consist of the raw data collected from the convict cichlids used in the behavioral experiment. Pair #10 was omitted from the data analysis.

Pair Date eggs Female Male Female Male Eggs fry fry observed Length Length Start Start recovered survival (TL) (TL) Weight Weight/ (%) Start/En Start/End /End End (g) d(mm) (mm) (g) 1 5/20/19 57.5 68.6 3.4 5.5 123 117 95.12 / / / / 57.3 70.0 3.7 5.8 2 6/28/19 65.0 76.7 4.9 7.6 191 153 80.10 / / / / 64.7 76.6 4.6 7.6 3 7/17/19 57.9 77.0 3.6 8.1 112 106 94.64 / / / / 57.8 77.8 3.3 7.3 4 8/2/19 65.9 75.8 4.7 7.6 297 268 90.24 / / / / 65.0 75.9 4.7 8.3 5 8/4/19 64.5 75.8 4.7 7.6 211 195 92.42 / / / / 64.8 76.0 4.7 7.5 6 8/20/19 59.0 73.8 3.6 7.2 195 174 89.23 / / / / 60.9 74.6 3.6 7.2 7 8/28/19 62.8 77.1 4.2 9.5 189 174 92.06 / / / / 62.7 76.0 3.9 9.2 8 9/2/19 53.6 64.0 2.8 4.4 286 263 91.96 / / / / 53.7 66.0 2.7 4.7 9 9/20/19 53.1 70.0 3.1 5.8 192 181 94.27 / / / / 54.2 70.5 2.9 5.7 10 10/22/19 54.2 69.8 2.8 5.9 303 278 91.75 / / / / 54.9 69.9 2.7 5.7 11 11/09/19 55.2 68.3 3.6 5.1 156 142 91.03 / / / / 54.8 68.5 3.4 4.9

APPENDIX A (CONTINUED)

APPENDIX B

This appendix contains data collected from each fish used during the experiment. The number of bites from the two thirty-second tests are presented and the daily total is depicted following the (=) sign. Each table shows the results from each pair tested. The five day total displayed at the bottom of the table in bold was used in the data analysis.

Pair #1 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,0 = 0 0,0 = 0 0,1 = 1 0,0 = 0 2 1,0 = 1 1,0 = 1 0,1 = 1 7,6 = 13 3 2,3 = 5 4,5 = 9 3,3 = 6 3,1 = 4 4 2,3 = 5 2,2 = 4 0,0 = 0 4,5 = 9 5 3,5 = 8 2,0 = 2 2,1 = 3 3,2 = 5 Total 19 16 11 31

Pair #2 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,0 = 0 0,0 = 0 0,0 = 0 5,5 = 10 2 0,0 = 0 0,0 = 0 1,2 = 3 7,7 = 14 3 0,0 = 0 0,0 = 0 4,1= 5 6,4 = 10 4 0,3 = 3 0,1 = 1 3,4 = 7 12, 12 = 24 5 0,0 = 0 0,0 = 0 6,8 = 14 5,6 = 11 Total 3 1 29 69

APPENDIX B (CONTINUED)

Pair #3 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 7,2 = 9 0,1 = 1 0,0 = 0 0,4 = 4 2 3,2 = 5 7,1 = 8 2,3 = 5 0,3 = 3 3 6,3 = 9 2,1 = 3 0,1 = 1 0,3 = 3 4 4,1 = 5 6,3 = 9 3,0 = 3 0,0 = 0 5 5,7 = 12 1,1 = 2 1,0 = 1 3,5 = 8 Total 40 23 10 18

Pair #4 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,2 = 2 0,0 = 0 0,0 = 0 0,0 = 0 2 2,2 = 4 7,2 = 9 0,0 = 0 0,0 = 0 3 9,2 = 11 1,8 = 9 0,0 = 0 0,0 = 0 4 10,4 = 14 5,4 = 9 0,3 = 3 8,6 = 14 5 0,2 = 2 0,0 = 0 6,3 = 9 11,10 = 21 Total 33 27 12 35

APPENDIX B (CONTINUED)

Pair #5 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,2 = 2 5,2 = 7 8,2 = 10 0,7 = 7 2 5,4 = 9 2,0 = 2 3,5 = 8 9,11 = 20 3 1,4 = 5 0,1 = 1 5,2= 7 9,9 = 18 4 1,0 = 1 8,3 = 11 10,9 = 19 0,2 = 2 5 6,5 = 11 2,2 = 4 1,5 = 6 3,8 = 11 Total 28 25 50 58

Pair #6 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 3,5 = 8 5,1 = 6 0,0 = 0 0,0 = 0 2 9,3 = 12 3,6 = 9 0,0 = 0 6,1 = 7 3 8,4 = 12 4,4 = 8 2,1= 3 0,6 = 6 4 4,4 = 8 2,1 = 3 3,1 = 4 7,8 = 15 5 6,2 = 8 4,2 = 6 0,0 = 0 2,3 = 5 Total 48 32 7 33

APPENDIX B (CONTINUED)

Pair #7 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 2,7 = 9 0,0 = 0 1,1 = 2 0,0 = 0 2 11,5 = 16 6,3 = 9 0,0 = 0 0,5 = 5 3 2,8 = 10 5,2 = 7 2,0 = 2 6,12 = 18 4 6,6 = 12 2,2 = 4 2,2 = 4 9,3 = 12 5 0,1 = 1 5,2 = 7 4,1 = 5 1,5 = 6 Total 48 27 13 41

Pair #8 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,0 = 0 0,0 = 0 0,0 = 0 0,2 = 2 2 7,4 = 11 1,2 = 3 2,3 = 5 8,8 = 16 3 2,1 = 3 3,2 = 5 3,1 = 4 6,0 = 6 4 3,1 = 4 3,1 = 4 4,4 = 8 3,8 = 11 5 4,4 = 8 1,2 = 3 5,1 = 6 7,8 = 15 Total 26 15 23 50

APPENDIX B (CONTINUED)

Pair #9 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,0 = 0 0,0 = 0 0,0 = 0 0,0 = 0 2 0,1 = 1 0,0 = 0 0,0 = 0 0,3 = 3 3 1,6 = 7 0,1 = 1 2,0 = 2 7,8 = 15 4 1,1 = 2 1,3 = 4 2,3 = 5 1,2 = 3 5 3,6 = 9 1,0 = 1 0,0 = 0 2,4 = 6 Total 19 6 7 27

Pair #10 – This trial was omitted from the data analysis. Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,0 = 0 0,0 = 0 0,0 = 0 0,0 = 0 2 0,0 = 0 0,0 = 0 0,0 = 0 0,0 = 0 3 0,3 = 3 0,2 = 2 0,0 = 0 0,0 = 0 4 5,4 = 9 3,3 = 6 0,0 = 0 0,0 = 0 5 9,10 = 19 3,1 = 4 0,0 = 0 0,0 = 0 Total 31 12 0 0

APPENDIX B (CONTINUED)

Pair #11 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,0 = 0 0,0 = 0 0,0 = 0 0,0 = 0 2 2,1 = 3 0,2 = 2 0,2 = 2 0,0 = 0 3 4,6 = 10 1,0 = 1 4,1 = 5 4,3 = 7 4 0,0 = 0 0,0 = 0 4,5 = 9 11,8 = 19 5 0,3 = 3 0,0 = 0 2,2 = 4 5,2 = 7 Total 16 3 20 33

Pair #12 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 0,0 = 0 0,0 = 0 0,0 = 0 0,0 = 0 2 0,0 = 0 0,0 = 0 0,0 = 0 0,0 = 0 3 3,7 = 10 1,3 = 4 0,0 = 0 0,0 = 0 4 5,5 = 10 4,0 = 4 0,0 = 0 0,5 = 5 5 0,3 = 3 1,1 = 2 1,2 = 3 3,2 = 5 Total 23 10 3 10

APPENDIX B (CONTINUED)

Pair #13 Day Female Male Bites on Small Bites on Large Bites on Small Bites on Large 1st 30 seconds 1st 30 seconds 1st 30 seconds 1st 30 seconds , , , , 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 2nd 30 seconds 1 2,1 = 3 0,1 = 1 2,3 = 5 1,1 = 2 2 9,5 = 14 0,1 = 1 1,0 = 1 3,2 = 5 3 9,0 = 9 1,1 = 2 1,5 = 6 3,0 = 3 4 6,4 = 10 2,3 = 5 0,4 = 4 6,3 = 9 5 3,3 = 6 3,1 = 4 3,0 = 3 2,2 = 4 Total 42 13 19 23

APPENDIX C

This appendix displays the number of bites from each parent of a test pair at each predatory fish in the model (small and large). Each bite total is the summation over the five day trial period. This data was used in the paired t-test. Data from Pair #10 was omitted from the data analysis. Female Male

(Total over 5 days) (Total over 5 days) Pair Bites at Small Bites at Large Bites at Small Bites at Large Predator Predator Predator Predator 1 19 16 11 31 2 3 1 29 69 3 40 23 10 18 4 33 27 12 35 5 28 25 50 58 6 48 32 7 33 7 48 27 13 41 8 26 15 23 50 9 19 6 7 27 11 16 3 20 33 12 23 10 3 10 13 42 13 19 23

p-value 0.0003 0.0001

REFERENCES

Anderson, C., R. Jones, M. Moscicki, E. Clotfelter, R. L. Earley. 2016. Seeing orange:

breeding convict cichlids exhibit heightened aggression against more colorful

intruders. Behavioral Ecology and Sociobiology 70: 647-657.

Barley, A and R. Coleman. 2010. Habitat structure directly affects aggression in convict

clichlids (Archocentrus nigrofasciata). Currrent Zoology 56: 52-56.

Barlow, G.W. 1983. The benefits of being gold: behavioural consequences of

polychromatism in the midas cichlid Cichlasoma citrinellum. Environmental

Biology of Fishes 8: 235-247.

Beeching, S. C. 1992. Visual assessment of relative body size in a cichlid fish, the Oscar,

Astronatus ocellatus. Ethology 90: 177-186.

Bussing, W. A. 1998. Peces de las aguas continentales de Costa Rica (Freshwater fishes

of Costa Rica). Second Edition. Universidad de Costa Rica. 468 p.

Chase, I. D. 1980. Cooperative and non-cooperative behaviour in animals. American

Naturalist. 115: 827-857.

Coleman, R. M. and R. U. Fischer. 1991. Brood size, male fanning effort and the

energetics of a nonshareable parental investment in bluegill sunfish, Lepomis

macrochirus (Teleostei: Centrarchidae). Ethology 87: 177-188.

Coleman, R. M. 1993. The evolution of parental investment in fishes. Ph.D. thesis,

University of Toronto, Canada.

Coleman, R. M., M. R. Gross and R. C. Sargent. 1985. Parental investment decision

rules: a test in bluegill sunfish. Behavioral Ecology and Sociobiology 18: 59-66.

Gagliardi-Seeley, J., 2008. Mate choice in female convict cichlids (Amatitlania

nigrofasciata) and the relationship between male size and dominance. Journal of

Ethology 27: 249-254.

Galvani, A. P and R. M. Coleman. 1998. Do parental convict cichlids of different sizes

value the same brood number equally? Animal Behaviour 56: 541-546.

Houston, A. I. and N. B. Davies. 1985. The evolution of cooperation and life history in

the dunnock, Prunella modularis. Pp. 471-487 in: Behavioural Ecology: The

Population Consequences of Adaptive Behaviour (Ed. R. Sibly and R. Smith),

Blackwell, Oxford.

Itzkowitz M., N. Santangelo, and M. Richter. 2005. Is the selection of sex-typical

parental roles based on an assessment process? A test in the monogamous convict

cichlid fish. Animal Behaviour 69: 95-105.

Keeley, E. R and J. W. A. Grant. 1993. Visual information, resource value, and

sequential assessment in convict cichlid (Cichlasoma nigrofaciatum) contests.

Behavioral Ecology 4: 345-349.

Keenleyside, M. H. A. 1991. Parental care. In: Keenleyside MHA (ed) Cichlid fishes:

behavior, ecology, and evolution. Chapman Hall, London, pp 191-208.

Keenleyside, M. H. A., R. W. Rangeley and B. U. Kuppers. 1985. Female mate choice

and male parental defense behaviour in the cichlid fish Cichlasoma

nigrofasciatum. Canadian Journal of Zoology 63: 2489-2493.

Lavery, R. J. 1995. Past reductive effort affects parental behaviour in a cichlid fish,

Cichlasoma nigrofasciatum: A comparison of inexperienced and experienced

breeders with normal and experimentally reduced broods. Behavioral Ecology

and Sociobiology 36: 193-199.

Lavery, R. J. and M. H. A. Keenleyside. 1990. Parental investment of a biparental cichlid

fish, Cichlasoma nigrofaciatum, in relation to brood size and past investment.

Animal Behaviour 40: 1128-1137.

Richter, M, N. Santangelo and M. Itzkowitz. 2005. Biparental division of roles in the

convict cichlid fish: influence of intruder numbers and locations. Ethology,

Ecology and Evolution 17: 1-15.

Sargent, R. C. and M. R. Gross. 1985. Parental investment decision rules and the

Concorde fallacy. Behavioral Ecology and Sociobiology 17: 43-45.

Thunken, T., D. Meuthen, T. C. M. Bakker, and H. Kullmann. 2010. Animal Behaviour

80: 69-74.

Trivers, R. L. 1972. Parental investment and sexual selection. Pp 136-179 in: Sexual

Selection and the Descent of Man (Ed., B. Campbell), Aldine, Chicago.

Williams, G. C. 1966. Natural selection, costs of reproduction and a refinement of

Lack’s principle. American Naturalist 100: 687-690.

Wisenden, B. D. 1994. Factors affecting reproductive success in a monogamous cichlid

fish. Environmental Biology of Fishes 3: 193-201.