DOES EGG SIZE INFLUENCE SPAWNING TEMPERATURE PREFERENCE
IN SUBSTRATE SPAWNING CICHLID FISHES?
Bianka Giselle Bommarito
B.S., California State University, Sacramento, 2005
THESIS
Submitted in partial satisfaction of the requirements for the degree of
MASTER OF SCIENCE in
BIOLOGICAL SCIENCES
(Biological Conservation) at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2011

© 2011
Bianka Giselle Bommarito
ALL RIGHTS RESERVED ii

DOES EGG SIZE INFLUENCE SPAWNING TEMPERATURE
PREFERENCE IN SUBSTRATE SPAWNING CICHLID FISHES?
A Thesis by
Bianka Giselle Bommarito
Approved by:
__________________________________, Committee Chair
Ronald M. Coleman, PhD
__________________________________, Second Reader
Jamie M. Kneitel, PhD
__________________________________, Third Reader
Ben N. Sacks, PhD
Date:____________________ iii

Student: Bianka Giselle Bommarito
I certify that this student has met the requirements for format contained in the
University format manual, and that this thesis is suitable for shelving in the
Library and credit is to be awarded for the thesis.
_________________________, Graduate Coordinator _________________
Susanne Lindgren, Ph.D. Date
Department of Biological Sciences iv

Abstract of
DOES EGG SIZE INFLUENCE SPAWNING TEMPERATURE
PREFERENCE IN SUBSTRATE SPAWNING CICHLID FISHES? by
Bianka Giselle Bommarito
Researchers have documented that fishes breed in many different places and times, however, fishes are known to be particular when choosing a spawning site and no theory has been developed to predict when and where a particular species should spawn. Egg size has been proposed to be a key component in this decision. Temperature is also likely important because it affects growth rates, the efficiency of utilizing food energy, and the amount of dissolved oxygen in water.
The purpose of my research is to determine how and why breeding fish chose a temperature at which to spawn. The Cichlidae is the third largest family of teleost fishes, and all members of the cichlid family exhibit some form of parental care. Substrate guarding is one of the most common forms of parental care, and because the eggs are adhesive, once they are laid, they will be committed to developing in that particular physical environment, i.e., water temperature, water velocity, etc; therefore spawning site choice is extremely important to the survival of the eggs and fry. v

Because larger eggs need more oxygen than smaller eggs, due to a higher surface to volume ratio, I hypothesized that when given a choice, a parent fish will chose its spawning temperature based on the oxygen needs of its size of egg, i.e., larger eggs will be laid in cooler water than smaller eggs. Egg size is typically fixed within a species, therefore all individuals within a species should make the same consistent spawning temperature choice. Prior to testing the main hypothesis, I tested an important assumption, namely that because of the importance of spawning temperature, choice of spawning temperature will not be influenced by prior exposure to different temperatures. In other words, fish will not be acclimated to spawn at a temperature different then their preferred spawning temperature, simply because they previously experienced warmer or cooler water.
To test these hypotheses, I conducted two experiments using a temperature choice apparatus which allowed a pair of spawning fish to have four distinct temperatures at a time to choose from. I used substrate spawning cichlids because the location of the eggs, once laid, indicated the exact temperature at which the spawning took place. Temperature data loggers were placed in each compartment to record the temperature that was chosen.
In Experiment 1, I conducted a set of trials to eliminate prior habitat temperature experience as a factor biasing future spawning temperature preference. To do this, I used ten pairs of convict cichlids subjected to two vi

different temperature treatments. Five of the pairs used were housed in a tank set at 22ºC, and five of the pairs used were housed in a tank set at 32ºC, for two weeks prior to being put into the spawning temperature choice apparatus to allow for acclimation to that particular temperature. I predicted that these treatments would not affect future spawning temperature preference. The data was analyzed using a t-test, and no significant difference was found between the preferred spawning temperatures of the two treatments. This means that prior temperature exposure does not effect future spawning temperature preferences.
Experiment 2 examined the effect of egg size on spawning temperature preference and consisted of collecting spawnings from five other substrate spawning species, namely Neetroplus nematopus, Hemichromis lifalili, Tilapia snyderae, Steatocranus casuarius , and Hypsophrys nicaraguense . The six species used (including A. nigrofasciatus ) encompass a large range of the egg sizes found in the cichlid family.
The results revealed that even with a spread of possible spawning temperature as great as 12.8°C, no species showed more than a 2.6°C variation from their particular mean preferred spawning temperature. This shows that each species tested had a consistent preferred spawning temperature. Comparing the average spawning temperature chosen by each species against one another, only T. snyderae against A. nigrofasciatus , and T. snyderae against H. lifalili showed a significant difference. vii

An interspecies comparison of egg size and average preferred spawning temperature within a species was also conducted, and a linear regression showed no relationship between chosen spawning temperature and egg size. This demonstrates that the cichlids tested do not base their spawning temperature choices on egg size.
__________________________________, Committee Chair
Ronald M. Coleman, Ph.D. viii

ACKNOWLEDGEMENTS
I would like to thank the American Cichlid Association, Sigma Xi, and the
Golden West Women's Fly Fishing Club for their generous financial support for this project. I would also like to thank my committee members, Dr. Ben Sacks, Dr.
Jamie Kneitel, Dr. Jim Baxter (for the first half), and Dr. Susanne Lindgren (for the second half) for their time, patience, and support.
To Dr. Coleman, I thank you for your guidance, support, and unending encouragement to make it through and persevere through all of life's trials and tribulations.
For my mother, Sandy, I would like to thank you for always believing that I could do this and for lending a supportive shoulder when I didn't think I could.
You never gave up on me and I can't thank you enough for that.
For my father, Brian, I would like to thank you for setting an example of what hard work is, and how it can pay off. I know that all of my hard work will pay off and I will make something great of myself because of it. I can't wait for you to see what I accomplish.
To my beautiful sister, Dena, how can I thank you for all those days of measuring eggs and being my stat girl? You had a wonderful way of adding some laughter to some very tedious work! You make me want to work harder to set an example and I thank you for always believing that I am a super star. ix

To my amazing and wonderful husband, Zak, I could not have done any of this without you. It's been a rough journey, but with you by my side, I never once doubted that I could do this. It's because of you that I am able to accomplish this project. You believe in me, you support me, and you give me unending love, and I cannot thank you enough for all that you have done, and will do for me.
Lastly, for my grandma, Beverly, and my grandpa, Dezo, who are not here to be able to see my completed thesis, I dedicate this project, and this thesis, to you.
To my Grandpa, Dezo, you are truly an inspiration. You have shown me how important a good education is, and I thank you for that. I can only hope to one day live a life as fulfilled and important as yours. You have a strength that I have yet to see in anyone else, and I admire that quality, and you, more than you could ever know. I'm so sorry you couldn't be here to see this finished... To my grandma, you have always been, and will always be, a shining star in my life. Without your unending support and immense amount of love, I truly would not be where I am today. I love you both, and I hope I always make you proud. x

TABLE OF CONTENTS
Page
Acknowledgements ................................................................................................... ix
List of Tables...........................................................................................................xiii
List of Figures..........................................................................................................xiv
Chapter
1. INTRODUCTION ................................................................................................ 1
Costs of Parental Care ........................................................................2
Substrate Spawning ............................................................................ 4
Effects and Importance of Egg Size ................................................... 5
Importance of Oxygen Concentrations in Water ............................... 6
Effects of Temperature ....................................................................... 7
Statement of Research ........................................................................ 9
Hypotheses..........................................................................................9
GENERAL MATERIALS AND METHODS ................................................ 10
Study Species ................................................................................... 10
Apparatus ......................................................................................... 14
2. EXPERIMENT 1 ................................................................................................18
Data Collection................................................................................. 22
Data Analysis ................................................................................... 24
RESULTS ....................................................................................................... 25
xi

DISCUSSION ................................................................................................. 33
3. EXPERIMENT 2 ................................................................................................ 35
INTRODUCTION .......................................................................................... 35
MATERIALS AND METHODS .................................................................... 37
Data Analysis ................................................................................... 37
RESULTS ....................................................................................................... 38
Individual Species.............................................................................38
Interspecies Comparison...................................................................51
DISCUSSION ................................................................................................. 55
Appendix A. Solubility of Oxygen in Fresh Water................................................. 62
Appendix B. Egg Size Data for Each Fish Species Used in the Temperature
Choice Apparatus..........................................................................................63
Literature Cited ........................................................................................................ 91
xii

LIST OF TABLES
Page
Table 1. Morphological and reproductive characteristics of the six study species ……...…………….….………......................................................12
Table 2. Average temperatures (°C) in the chambers for each of the two treatments of convicts.………….........……...............………….....…….26
Table 3. ANOVA comparing the temperatures selected by the cold-acclimated treatment and the warm-acclimated treatment.....…………......................30
Table 4. Dates in which the pairs were placed in the temperature choice apparatus, and the dates on which the spawnings took place...........…….32
Table 5. Average temperatures (°C) of each chamber of the previous 48 hours prior to the spawning of Hemichromis lifalili.
....…...........……………....40
Table 6. Average temperatures (°C) of each chamber of the previous 48 hours
prior to the spawning of Archocentrus nigrofasciatus .........................….42
Table 7. Average temperatures (°C) of each chamber of the previous 48 hours prior to the spawning of Tilapia snyderae
….…...................................….44
Table 8. Average temperatures (°C) of each chamber of the previous 48 hours prior to the spawning of Hypsophrys nicaraguense ..……...................….46
Table 9. Effective diameter (mm) of eggs from spawnings of the six species of cichlids.................................................................................................….48
Table 10. The chosen spawning temperature (°C) for each pair of cichlids, and the average chosen spawning temperatures for each species...................49
Table 11. ANOVA comparing the mean chosen spawning temperatures of each species against one another....…………….……..……………..….52
Table 12. Linear regression between spawning temperature chosen and average egg size of each species…………......................................…………….54 xiii

LIST OF FIGURES
Page
Figure 1. Study speceis ........................................................................................... 13
Figure 2. Temperature choice apparatus.. ................................................................ 16
Figure 3. The cutout used for fish to swim freely from one temperature to another...... ............................................................................................... 17
Figure 4. Example of how the temperatures were distributed in the temperature choice apparatus, with a difference of 2°C from each chamber to the next chamber ............................................................................................ 20
Figure 5. Complete temperture exposure for one pair of A. nigrofasciatus ............ 21
Figure 6. Temperature shifting was used to control for individual chamber effects.......................................................................................................23
Figure 7. Graphic representation of the temperature choices given to each of the spawning pairs ................................................................................... 27
Figure 8. Representation of the overall average temperature spread given to each of the treatments .............................................................................. 31
Figure 9. The multitube used in place of open-ended terra cotta pots when spawning N. nematopus and H. nicaraguense ......................................... 36
Figure 10. Spawning temperature (°C) chosen by each Hemichromis lifalili pair..........................................................................................................41
Figure 11. Spawning temperature (°C) chosen by each Archocentrus nigrofasciatus pair. ................................................................................. 43
Figure 12. Spawning temperature (°C) chosen by each Tilapia snyderae pair....................... .................................................................................. 45
Figure 13. Spawning temperature (°C) chosen by each Hypsophrys nicaraguense pair ................................................................................... 47 xiv

Figure 14. Overall spread of temperatures (°C) that each pair, and each species collectively, had to choose from ............................................................ 50
Figure 15. Average spawning temperature (°C) versus average egg size of each species. ........................................................................................... 53 xv

1
Chapter 1
INTRODUCTION
Parental care is present in about 20% of teleost fishes (Blumer, 1982; Gross and Sargent, 1985). Fishes are known to exhibit diverse forms of parental care such as live bearing, mouthbrooding, egg burying, substrate cleaning, brood pouch egg carrying (in seahorses the males carry and feed, by placenta, the developing young), nest guarding, and substrate spawning (Breder and Rosen, 1966; Coleman,
1999). Parental care is more common in freshwater (60%) than marine (16%) families of fishes (Baylis, 1981; Blumer, 1982). Of the families of fishes that exhibit parental care, approximately 50% exhibit paternal care, 32% exhibit biparental care, and 18% exhibit maternal care (Sargent, 1999).
The family Cichlidae is comprised solely of freshwater species and is the third largest family of fishes, including at least 1,350 species (Nelson, 2006).
Parental care is a characteristic of all cichlid fishes, with substrate guarding thought to be the ancestral state of parental care in cichlids (Lowe-McConnell, 1959; Iles and Holden, 1969; Goodwin et al., 2001). Cichlids can be classified into two forms of parental care: substrate spawning and mouthbrooding (with some exceptions), and can be found in the Americas and in Africa (and a few parts of Asia) with the different modes of reproduction spanning different geographic locations. Knowing

2 how important parental care is, not only to cichlids, but to fishes in general, it is important to gain an understanding of why and how the choices of parental care are made.
The main objective of parental care (in which the benefit is the same to either sex) is to improve offspring survival and to also possibly increase the rate of development of the offspring (Clutton-Brock, 1991; Gross and Sargent, 1985; Sibly and Calow, 1986). Though parental care has obvious benefits to the survival of the offspring, it does not come without costs to the parents. The three most basic costs of parental care are reduced survival of the parent/s, increase of time until next spawning, and decreased fecundity.
Costs of Parental Care
With parental care comes an increase in energy expenditure leading to reduced survival of the parents. Guarding eggs and/or fry takes an extreme amount of vigilance. Because of this, guarding parents are unable to consume the same amount of food compared to the amount of food they would consume if they were not guarding. A decrease in the amount of food consumed leads to weight loss, and possible mortality of the parents (Chellappa et al., 1989; Coleman and Fischer,
1991; Sabat, 1994).
The amount of time elapsed between spawnings depends on the type of parental care being displayed. Mrowka (1987) and Smith and Wootton (1994)

3 observed that the longer the eggs were under the care of the female, the longer the time until the female was able/willing to spawn again. For male parental care, there is also a positive correlation between length of time guarding and an increase of time until the next spawning. van Iersel (1953) observed that male sticklebacks enter into what is known as a “parental phase.” Once the males are in this parental phase (which is independent of how many clutches the male has fathered), they no longer continue to court any females until the eggs hatch, and the fry are free swimming and correspondingly better able to fend for themselves. Biparental care, however, does not seem to affect the males. The weight of the testes in the male convict cichlid is not correlated with brood size, therefore making it possible for a male to leave the parenting to the female, and giving him the ability to leave his mate to court other females (Lavery and Keenleyside, 1990a, b).
A decrease in future fecundity results from both the decrease in the survival of the parents and the time elapsed between spawnings. As guarding time increases and weight loss increases, the growth rate of the parent fish slows. Because egg size is fixed within a species (Coleman, 1991), a decrease in growth rate and therefore the size of the individual, results in a decrease in the number of offspring produced. Smith and Fretwell (1974) argued that each organism has a set amount of energy to put toward reproduction at any given point in time. They also argued that with each spawning, energy that had been reserved for reproduction decreases, leading to a decrease in the future number of offspring a parent can produce.

4
Balshine-Earn (1995) additionally found a serial correlation between caring for a brood, weight loss, longer time between spawnings, and eventually smaller future output of offspring.
Substrate Spawning
Substrate spawning is by far the most common form of parental care in fishes, occurring in over 95% of care-giving species (Blumer, 1982; Sargent and
Gross, 1986, 1993). In substrate spawning cichlids, the female parent fish lays adhesive eggs onto some form of substrate, e.g., a rock or leaf. The timing and location of spawning has important ramifications for the parents and the eggs.
Because in most cases the eggs are adhesive, once a spawning site is chosen, the eggs cannot be moved until they hatch (exceptions include delayed mouthbrooders and leaf spawners such as Aequidens coeruleopunctatus ) (Dupuis and Keenleyside,
1981). Because the eggs cannot be moved, once spawned, they are committed to developing in that particular physical environment with its corresponding temperature, water velocity, etc.
Once hatched, the fry imprint their parents’ odors and visual characteristics so that after they become free swimming, they are able to orient toward their parents (Barnett, 1977; Hay, 1978). Parents similarly recognize their own young by chemical cues and tend their schools, thus offering protection from predators

5
(McKaye and Barlow, 1976). This evolutionary characteristic again shows the importance of parental care among this family of fishes.
Effects and Importance of Egg Size
Egg size is typically fixed within a species of cichlid (Coleman, 1991).
This means that individuals of a species, more or less, lay the same size egg, varying only the number of eggs laid per clutch, i.e., larger females within a species will lay a greater number of the same sized egg than a smaller female of the same species. Coleman and Galvani (1998) showed that larger egg size is positively correlated with larger hatchlings. Having larger hatchlings is beneficial to the parent fish due to the fact that they are born better able to fend for themselves because the smaller a fish is, the more susceptible it is to predation. This allows for a decrease in time spent guarding, therefore leading to a decrease in time between spawnings. Coleman and Galvani even found that small variations of egg/hatchling size within a clutch can have profound effects on the future survival of those smaller individuals.
With there being so many significant costs to the parent fish during the course of providing care, and with larger eggs seemingly able to reap more benefits to the hatchlings, why do all fish simply not lay large eggs? There is a trade off between egg size and egg number. Though larger eggs have the potential for the fry to have a better chance of survival, a parent which lays larger eggs can make

6 fewer of them than a parent which lays smaller eggs. This is a very important decision that each female is faced with. Would it be more beneficial to lay many small eggs, which then have a lower chance of survival rate, but have more in number to make up for the loss, or should a female lay fewer, larger eggs which would have a better chance of survival, yet fewer potential fry?
Importance of Oxygen Concentrations in Water
It is known that larger eggs need more oxygen due to a higher surface to volume ratio, whereas the opposite is true for smaller eggs, so in the first step in choosing where to spawn, the parent fish must provide the optimal site for egg development. This is particularly important because eggs cannot move themselves if the site is substandard. However, the importance of a spawning site continues after the eggs have hatched because it has also been shown that recently hatched larval fish, such as the cichlid Aequidens portalegrensis , have no ability to compensate metabolically for varying temperatures (Morris, 1962). These two factors combined significantly contribute to the importance of selecting an optimal spawning site.
Schene and Hill (1980) conducted an experiment looking at the effect of dissolved oxygen concentration and temperature selection in Mississippi silversides and found a correlation between the two. By manipulating oxygen levels, they found that the fish tested preferred to stay in cooler water temperatures, even if it

7 had lower oxygen concentrations, than higher water temperatures with normal oxygen concentrations. This experiment showed that silversides instinctively recognize that cooler water potentially holds more oxygen than warmer water.
Furthermore, it is known that with increased temperature comes a decrease in the amount of oxygen that can be held in the water. In fact, Agersborg (1930) showed that even a difference in 2° C brought about symptoms of oxygen deficiency and unbalanced movements which can ultimately lead to the death of the fish. Appendix A lists the solubility of oxygen in fresh water. As the temperature of the water increases, the amount of oxygen held in the water decreases.
Parent fish must therefore be able to take into account the costs of different spawning temperatures and egg sizes.
Effects of Temperature
Previous research (Coleman, unpublished data) has shown that temperature has profound effects on the developmental rate of eggs: in general, the warmer the water, the faster the hatching rate of the eggs. I have also found (unpublished data) that warmer water increases the growth rates of some cichlid fry, unless the optimal temperature is surpassed in which the growth rate slows considerably.
Temperature is one of the most significant environmental factors affecting the growth rate of fishes (Baldwin, 1957; Donaldson and Foster, 1940; Nicieza and
Metcalfe, 1997; Strawn, 1961). We know that fish possess acute temperature

8 discrimination capabilities (Bull, 1936-1937; Bardach and Bjorklund, 1957).
Dutson et al. (2004) showed that temperature is likely an influential factor in a fish's decision making because it affects growth rates. We also know that temperature affects the metabolism of fish which in turn affects the efficiency of utilizing food energy (Van Ham et al., 2003; Elliot, 1976). When presented with no options, a variety of fish will willingly spawn at different temperatures (Breder and
Rosen, 1966). In their book, they document several experiments of fishes kept at temperatures outside the range of their natural spawning temperatures, and unless the temperature provided was far outside the optimal range, the fish spawned.
Also, many aquarium hobbyists keeping fish at various temperatures notice that fish will spawn at these different temperatures. However, there is a distinct difference between being willing to spawn and having an actual preference of where to spawn.
So, taking growth rate, metabolic rate, and oxygen concentration into consideration, how should breeding fish choose a temperature at which to spawn?
As previously stated, egg size is typically fixed within a species (Coleman,
1991). Because larger eggs need more oxygen than smaller eggs due to a higher surface to volume ratio, I hypothesize that when given multiple temperatures to choose from at one time, the parent fish will chose the temperature that has the optimal oxygen concentration for the size of egg being laid, i.e., when compared between species, the species with the larger eggs will choose colder water

9 temperatures than will a species which lays smaller eggs. I also hypothesize that because of the importance of spawning temperature to the eggs and larvae, previous exposure, and possible acclimation, of the parents to a different temperature will not affect the choice of temperature at which they spawn.
Hypotheses
I predict that prior temperature experience will not affect spawning temperature choice; and that when comparing across species, those species laying larger eggs will select cooler spawning temperatures than those that lay smaller eggs.

10
GENERAL MATERIALS AND METHODS
To test these hypotheses, I worked with six substrate spawning cichlid species which spanned a large range of egg sizes from 1.1mm to 2.5mm effective diameter. Effective diameter provides a linear measurement of egg size regardless of the shape of the egg, facilitating comparison of egg size between eggs of different shapes (Coleman, 1991). Most cichids lay eggs which are prolate spheroids (football shaped) for which the effective diameter can be calculated as
(length x width x width)
1/3
(Coleman, 1991). The egg size range examined represents the bulk of the range of egg size variation in substrate spawning cichlids
(Coleman, unpublished data). I could not use the very largest egg size of substrate spawners, i.e., 2.6mm ( Tomocichla tuba ) because these fish are large and extremely difficult to spawn in aquaria. The six species I used were Archocentrus nigrofasciatus , Tilapia snyderae, Hemichromis lifalili, Neetroplus nematopus,
Steatocranus casuarius, and Hypsophrys nicaraguense.
Due to the fact these fish lay adhesive eggs, the location of the eggs in the spawning apparatus clearly indicates the temperature at which the parents chose to spawn.
Study Species
Archocentrus nigrofasciatus (Table 1; Figure 1) is known for its ease in keeping and breeding. It is a Central American cichlid that is aggressive, and

11 known for its extensive parental care (Conkel, 1993). Hemichromis lifalili (Table
1; Figure 1) is a West African fish found in the Congo basin, exclusive of the Shaba
(Katanga) and Kasaï regions (www.Fishbase.org). Tilapia snyderae (Table 1;
Figure 1) is an African cichlid found in Lake Bermin in the country of Cameroon in
West Africa.. This fish has an IUCN status of vulnerable (www.Fishbase.org).
Neetroplus nematopus (Table 1; Figure 1) is a Central American cichlid found on the Atlantic slope of Nicaragua and western Costa Rica, and in the San Juan River drainage, including Lake Nicaragua and Lake Managua (Conkel, 1993).
Hypsophrys nicaraguense (Table 1; Figure 1) is also native to Central America from the Atlantic slope, from the San Juan drainage, including Lake Nicaragua, in
Costa Rica and Nicaragua, to the Matina River drainage in Costa Rica (Conkel,
1993). Lastly, Steatocranus casuarius (Table 1; Figure 1) is found in Africa; more specifically Pool Malego and the lower Congo River in both the Republic of Congo and Democratic Republic of the Congo (www.Fishbase.org).

12
Species Body
Length
(TL in cm)
10.0
Egg
Length
(mm)
Egg
Width
(mm)
Effective
Diameter
(mm)
Preferred
Temperature
(
ºC)
20-36 Archocentrus nigrofasciatus
Hemichromis lifalili
Tilapia snyderae
Neetroplus nematopus
Hypsophrys nicaraguense
Steatocranus casuarius
8.0
5.0
14.0
16-17
10.0
1.8
1.4
1.3
1.1
1.5
1.2 unknown unknown unknown
2.6
2.0
3.5
1.9
1.6
2.5
2.1
1.7
2.8
22-24 unknown
21-34
23-26
24-28
Table 1. Morphological and reproductive characteristics of the six study species.
Body length is measured by total length which is the measurement of a fish’s body from the tip of the snout to the tip of the longer lobe of the caudal fin.
Preferred temperatures are cited from http://www.fishbase.org
and Conkel,
1993, and egg size data is cited from www.cichlidresearch.com.

13
Figure 1. Study species. From top left down: Steatocranus casuarius , Neetroplus nematopus, and Tilapia snyderae . From top right down: Archocentrus nigrofasciatus , Hemichromis lifalili , and Hypsophrys nicaraguense .

14
Apparatus
I had access to two custom-made acrylic temperature choice apparatus
(Figure 2) with the dimensions 130 cm long , 43cm wide, and 37cm high, each holding approximately 220 liters of water.
Each apparatus contained six chambers.
The chamber on either end contained either the chiller or heaters respectively, and were identical in dimension (20cm long, 43cm wide). Each of these chambers had a 5cm wide, 7.5cm high space in the bottom front to allow for heated or chilled water movement to the adjacent chamber. Fish were prevented from entering the end chambers by a plastic grid with 1.27cm holes. The other four chambers were slightly larger (30cm long, 43cm wide) and identical in size to each other. These four were separated by 1.6cm thick clear acrylic. Each divider had a 5cm wide,
7.5cm high cutout for the fish to move freely from one chamber to the next (Figure
3). The apparatus were made from acrylic which acted as a thermal insulator allowing the temperatures of the chambers to remain consistently different from each other.
Each chamber was set up identically to the next to eliminate spawning site selection due to differences in the chambers other than temperature. Each chamber contained brown gravel, one 11.5cm terra cotta flower pot with the bottom removed, lying on its side, one plastic hygrophila plant, one square 12cm sponge filter, a thermometer, and a temperature data logger. The apparatus were housed in

the Evolutionary Ecology of Fishes Laboratory, California State University,
Sacramento.
15

Hygrophila plant
Heat er
Thermometer
Sponge filter
Terra Cotta
Pot
Chiller fig 3. Digital interpretation of temperature choice apparatus.
Figure 2. Temperature choice apparatus. This photgraph shows the two outer chambers, and how the four inner chambers were set up identical in habitat.
16

17
Figure 3. The cutout used for fish to swim freely from one temperature to another.
The cutout was placed in the lower front corner of each divider.

18
Chapter 2
EXPERIMENT 1
The purpose of Experiment 1 was to test the prediction that previous temperature exposure holds no influence on a pairs’ future spawning temperature choice; i.e., each pair within a species should prefer the same spawning temperature regardless of any prior exposure to different temperatures.
To conduct this experiment I used convict cichlids .
In order to show that prior temperature has no affect, I housed one half of the fish to be used in an acclimation tank set at 22ºC (±1ºC), and the other half in an acclimation tank set to
32ºC (±1ºC) with an overall total of five pairs being used from each holding tank.
During the acclimation phase, each pair spent a minimum of two weeks at the given temperature before being introduced to the temperature choice apparatus.
Each chamber of the temperature choice apparatus was set at a consistent temperature difference of 2˚C from the adjacent chamber. A variation of 2˚C has been shown to be effective in temperature selection based studies of fishes
(Agersborg, 1930; Beitinger, 1977). The temperatures were as finely controlled as possible to attempt to ensure a variance of no more than 1.0˚C at all times.
Temperature data loggers (HOBO® Pendant Temperature Data Logger Part #UA-
001-XX, Onset Computer Corporation) were placed into each chamber next to the terra cotta flower pot to record the temperature every ten minutes. The beginning

19 temperatures of the chambers were set to encompass the recognized natural preference of A. nigrofasciatus . For example (Figure 4), I started A. nigrofasciatus with 26, 28, 30, and 32˚C in the respective chambers. Once consistent temperatures were reached in the chambers (typically within a day), a pair of fish was placed into the apparatus. To ensure that all chambers were visited by at least one individual within the pair, one individual was placed in the coldest chamber and one was placed in the warmest. If the pair had not spawned within two weeks, they were removed and a new pair from the stock tank was used. By setting a maximum of two weeks allowed per pair in the apparatus, this prevented the pair from acclimating to the new temperatures they were being presented with. Figure 5 shows a graphical representation illustrating the entire experiment using one pair of
A. nigrofasciatus from the time of placing them into the acclimation tanks through placing them into the temperature choice apparatus.
A high protein diet has been shown to increase the spawning rate of fishes, therefore A. nigrofasciatus were fed a high protein diet of blood worms or brine shrimp once or twice daily, with equal amounts being placed into each chamber to ensure that any chamber preference of the fish was not due to food placement.

20
32˚C 30˚C 28˚C 26˚C
Figure 4. Example of how the temperatures were distributed in the temperature choice apparatus, with a difference of 2˚C from each chamber to the next chamber.

21
36
34
32
30
28
26
24
22
20
18
0 5000
Acclimation Phase
10000
2 Day
Inroduction
Period
No
Spawning
Data
Collected
Time Spent in Temperature
Choice Apparatus
10000 15000
22
20
18
20000
30
28
26
24
36
34
32
15000
0
Time in minutes
5000
Figure 5. Complete temperature exposure for a pair of A. nigrofasciatus.
In the first half of the graph, the pair was exposed to either the hot or the cold treatment for a total of two weeks. Once the acclimation phase was completed, the pair was then moved into the temperature choice apparatus and were given a choice of four different spawning temperatures to choose from. When they were first placed into the apparatus, there was a two day period within which no spawnings would be collected/used to give ample time for each individual fish to be able to visit each chamber and be exposed to each temperature. A pair was given no more than two weeks to spawn to try to control against possible reacclimation. All temperature data represented in this graph is actual temperature data taken from a spawning pair of A. nigrofasciatus.

22
Data Collection
Once a pair had spawned, within 24 hours they were removed, and the temperature data from the data loggers was downloaded using Hoboware software
The eggs were then removed and fixed in 70% isopropyl alcohol for later measurement. After each spawning, the terra cotta flower pots were also removed, and scrubbed to ensure no chemical residue remained from the previous parents to possibly bias the choice of the next spawning pair.
In order to rule out choice due to chamber preference, such as the fish always choosing the chamber on the far left, temperature shifting was used in the chambers between each trial (Figure 6). This means that after every spawning, I shifted the temperatures of the chambers either up or down by 1-2˚C, depending on which temperature was previously chosen. In the example given in Figure 7, even though 30˚C was chosen every time, we can be sure that it was chosen specifically for the temperature, and not for the chamber.
Each pair of fish was used only once to eliminate pseudoreplication, and no pairs within each temperature trial were given the same temperatures to choose from to increase the total range of possible spawning temperatures given.

32°C 30°C 28°C 26°C
23
34°C 32°C 30°C 28°C
30°C 28°C 26°C 24°C
Figure 6. Temperature shifting was used to control for individual chamber effects.
In the top, the temperature chosen was 30˚C, indicated by the location of the fish symbol, with the temperature options being 26-32˚C. In the next trial, the temperatures were shifted and ranged from 28-34˚C. Because once again 30˚C was chosen, the shifting took place around this temperature. In the third trial, the range available was 24-30˚C.

24
Data Analysis
To determine the precise temperature of the chambers at the time the spawning choice was made, I averaged the 10 minute recordings from the previous
48 hours before the spawning was noticed. By comparing the average temperature chosen by those fish previously subjected to a cold acclimation temperature versus a warm acclimation temperature, using a non-paired t-test, I determined if previous exposure to particular temperatures biased choice of spawning temperature. The convicts acclimated to a temperature of 22°C will be referred to as the coldacclimated treatment, whereas convicts acclimated to 32°C will be referred to as the warm-acclimated treatment.

RESULTS
The results show that there was no significant difference between the chosen average spawning temperatures of pairs of convict cichlids acclimated to cold water versus pairs acclimated to warm water. Table 2 shows the average temperatures recorded from each of the chambers from the previous 48 hours before spawning occurred. The temperature chosen by the pair is enlarged and in bold. Figure 7 shows two graphs representing the average temperatures in each chamber for each pair and that pair's chosen spawning temperature.
For both treatments the average preferred spawning temperature was represented at the high and low end of the temperature spectrum given. This demonstrates that all of the chambers could have potentially been selected for spawning by the fish.
25

26
Cold-acclimated Convicts
1
Pair Number
2 3
Chamber 1
Chamber 2
Chamber 3
21.6
24.3
26.1
26.7
28.6
31.6
28.2
31.0
33.0
Chamber 4 27.9 33.3 34.1
Warm-acclimated Convicts
Chamber 1
Chamber 2
Chamber 3
1
23.4
Pair Number
2
26.8
3
24.0
25.5
26.9
29.0
31.7
27.9
30.2
Chamber 4 29.7 32.9 31.4
4
21.3
26.2
27.3
29.6
4
22.2
24.7
26.2
27.9
5
23.6
25.2
26.6
29.1
5
27.9
31.1
32.0
33.4
Table 2: Average temperatures (°C) in the chambers for each of the two treatments of convicts. The numbers 1-5 represent the individual pair number in each trial. The enlarged bold numbers denote the average temperature in which that particular convict pair chose to spawn. For example, the first pair of coldacclimated convicts chose 27.9°C to spawn in, rejecting the chambers of 21.6°C,
24.3°C, and 26.1°C.

27
Cold-acclimated Convicts
28
26
24
22
20
0
36
34
32
30
1 2 3
Spawning Pair Number
4 5 6
Warm-acclimated Convicts
36
34
32
30
28
26
24
22
20
0 1 2 3
Spawning Pair Number
4 5 6
Figure 7: Graphic representation of the temperature choices given to each of the spawning pairs. Each dot represents the average temperature in a chamber within that trial whereas the fish symbol represents the spawning temperature chosen by each pair.

28
The average chosen temperatures by each pair of convicts within the coldacclimated treatment was compared with the average chosen temperatures of the warm-acclimated treatment using a non-paired t-test in SigmaStat 3.1 (Systat
Corporation). The results are listed in Table 3.
The cold-acclimated convict pairs had an average preferred spawning temperature of 27.5°C while the warm-acclimated convict pairs had an average preferred spawning temperature of 27.1°C. This is a difference of only 0.4°C of preferred spawning temperature between the two groups which was not significant
(t=0.62; df=8; P=0.55).
Overall, the cold-acclimated group was given a total temperature spread of
12.8°C ranging from 21.3°C to 34.1°C, while the warm-acclimated group was given a total temperature spread of 11.9°C ranging from 22.2°C to 33.4°C (Figure
8). The slight difference in spread was unintentional and merely due to slight temperature variations within the chambers.
Taken together, the mean temperature chosen by both stock groups was
27.3°C with each group's average being no more than 0.2°C from the mean.
Collectively, the data show that even with an overall temperature range of
12.8°C, the average preferred spawning temperature within this species varied no more than 1.4°C from the mean.
Allowing the fish a maximum of two weeks in the temperature choice apparatus reduced the potential for re-acclimation to have taken place. Moreover,

29 with the exception of three pairs, all of the pairs spawned within seven days of being placed in the temperature choice apparatus. Table 4 shows the dates the pairs were placed into the apparatus and the dates the pairs spawned.

30
Normality Test: Passed (P = 0.420)
Equal Variance Test: Passed (P = 0.557)
Group Name
Col 1
Col 2
N
5
5
Missing
0
0
Mean 1
27.494
27.141
Std Dev
1.023
0.769
SEM
0.457
0.344
1 Difference 0.353; t = 0.617; df = 8; P = 0.554; 95 percent confidence interval for difference of means: -0.966 to 1.672
Table 3. ANOVA comparing the temperatures selected by the cold-acclimated treatment and the warm-acclimated treatment.

31
28
26
24
22
36
34
32
30
20
Cold-acclimated Treatment Warm-acclimated Treatment
Figure 8. Representation of the overall average temperature spread given to each of the treatments. Each dot represents a temperature in a chamber, while the red line indicates the mean temperature chosen for each treatment.

32
Cold-acclimated Convicts
Pair Number
Date
Added
Date
Spawned
Days in
Between
1
11 July 2008
16 July 2008
5
2 3 4 5
9 June 2008 15 Oct. 2009 31 May 2008 31 Aug. 2008
22 June 2008 18 Oct. 2008 2 Jun. 2008 4 Sept. 2008
13
Warm-acclimated Convicts
Date
1
25 Aug. 2008
2
28 May 2008
3
Pair Number
3
7 Oct. 2009
2 4
4 5
9 Aug. 2008 7 Aug. 2008
Added
Date
Spawned
Days in
Between
31 Aug. 2008
6
30 May 2008 21 Oct. 2009 14 Aug. 2008 15 Aug. 2008
2 14 5 8
Avg.
Days
5.4
Avg.
Days
7
Table 4. Dates in which the pairs were placed in the temperature choice apparatus, and the dates on which the spawnings took place. With the exception of three pairs, spawnings took place within seven days.
Ave.
Days

33
DISCUSSION
The results clearly demonstrate that previous temperature experience does not bias preference for spawning temperature. It also shows that even if given multiple spawning temperatures to chose from, convict cichlids have a precise preferred spawning temperature of 27.3°C. I will therefore assume that exposure to previous temperature by other cichlid species (see Chapter 3) should not bias spawning temperature selection for them.
As previously stated, a variety of fish will willingly spawn at different temperatures if presented with no options. However, as I have just demonstrated, if options are available, a pair of fish does indeed have a preferred temperature in which to spawn, and this preference seems to be species based. Temperature unarguably has profound effects on organisms ranging from bacterium (Larsen et al., 2004) to plants (Coleman and Bazzaz, 1992) and to fishes. In fact, temperature is one of the most significant environmental factors affecting the growth rate of fishes (Baldwin, 1957; Donaldson and Foster, 1940; Nicieza and Metcalfe, 1997;
Strawn, 1961).
What fuels this choice of temperature preference? If a preferred spawning temperature is species-wide, and not an individual choice, then this temperature choice might be based upon something that is fixed across the species.
Egg size is fixed across a species and temperature has profound effects on eggs including oxygen concentration, and energy utilization and metabolism.

34
Because larger eggs need more oxygen than smaller eggs due to a higher surface to volume ratio, I hypothesize that when given multiple temperatures to choose from at one time, parent fish will chose the temperature that has the optimal oxygen concentration for the size egg laid, i.e., without actually measuring oxygen concentration, when compared between species, the species with the larger eggs will choose colder water temperatures than will a species which lays smaller eggs.

35
Chapter 3
EXPERIMENT 2
The purpose of Experiment 2 was to examine the effect of egg size on spawning temperature preference by several species of cichlid, each with a different egg size. Methods used in this experiment replicate those used in Experiment 1 with two exceptions.
Because Experiment 1 showed that previous temperature exposure plays no role in future spawning temperature choice, it was not necessary to strictly control the temperature at which other species experienced prior to the temperature choice experiment, therefore, all other species were housed in the main fish room of the
Evolutionary Ecology of Fishes Lab at a temperature range of 22-25°C.
The spawning habits of N. nematopus and H. nicaraguense differ slightly from those of A. nigrofasciatus , T. snyderae , S. casuarius , and H. lifalili in that they prefer to spawn in a more close-ended cave-like structure versus an openended cave-like structure. To address this, when N. nematopus and H. nicaraguense were used in the temperature choice apparatus, a terra cotta structure called a multitube (Figure 9) was used in place of the terra cotta pot.

Figure 9. The multitube used in place of open-ended terra cotta pots when spawning N. nematopus and H. nicarauense .
36

37
MATERIALS AND METHODS
Data Analysis
After a pair spawned, a sample of 30 eggs (when available) was collected from each spawning. The eggs were then measured using a Heerbrugg M5-40569 dissecting microscope fitted with an ocular micrometer. Effective diameter was calculated for each egg, and the averages of each sample (Appendix B) was then averaged within a species to be used as the representative egg size for that species.
By using a one way ANOVA, I was able to compare the chosen spawning temperature averages for each species against one another to determine if there was a significant difference between each species' preferred spawning temperature.
To be able to compare the average spawning temperature of each species with its corresponding egg size, Microsoft Excel was used, and the averages were plotted against one another using a simple XY scatter plot graph. A linear regression was then performed using SigmaStat 3.1.

38
RESULTS
Individual Species
I was able to collect a total of seven spawning samples from H. lifalili , ten from A. nigrofasciatus , six from T. snyderae , three from H. nicaraguense , one from
N. nematopus , and one from S. casuarius with a combined total of 28 spawning samples collected.
H. lifalili were given an overall temperature spread of 11.9°C ranging from
21.8-33.7°C. The average spawning temperature chosen was 28.6°C with variation from the mean being no more than 2.6°C. A. nigrofasciatus were given an overall temperature spread of 12.8°C ranging from 21.3-34.1°C. The average spawning temperature chosen was 27.3°C with variation from the mean being no more than
1.4°C (data used from Experiment 1). T. snyderae were given an overall temperature spread of 10.8°C ranging from 23.4-34.2°C. The average spawning temperature chosen was 31.0°C with variation from the mean being no more than
2.3°C.
H. nicaraguense were given an overall temperature spread of 10.9°C ranging from 21.7-32.6°C. The average spawning temperature chosen was 29.0 with variation from the mean being no more than 1.1°C.
N. nematopus were given an overall temperature spread of 6.2°C ranging from 27.6-33.8°C.
S. casuarius were given an overall temperature spread of 6.8°C ranging from 28.0-34.9°C.
Because only one spawning was collected from N. nematopus and S. casuarius , no

39 mean was calculated. Tables 5 through 8 and Figures 10 through 13 show the given temperature range for each pair and their selected spawning temperature.
Table 9 shows the calculated effective diameter for each species' individual clutch, and their overall average. Overall, there was a range of egg size from
1.1mm to 2.5mm. Table 10 shows the comparison between each species' chosen spawning temperature, its average, and each species' egg size. Figure 14 shows the comparison between each species' overall given temperature spread and their mean preferred spawning temperature. Taken as a whole, no species showed more than a
2.6°C variation from the mean preferred spawning temperature, even though there was a temperature spread offered as great as 12.8°C. This clearly shows that each species tested, does in fact, have a consistent preferred spawning temperature.

40
Date spawned
Chamber
1
2
3
4
7 Jun.
2009
27.6
29.2
31.1
32.5
29 Jan.
2009
26.0
27.9
29.2
31.8
27 Jun.
2008
24.1
26.5
27.5
30.0
11 Jul.
2008
21.8
24.3
26.1
27.9
27 Aug.
2008
26.6
29.4
31.1
31.9
14 Sept.
2008
23.4
25.3
26.9
29.2
7 Apr.
2010
26.9
29.5
31.3
33.7
Table 5. Average temperatures (°C) of each chamber from the previous 48 hours prior to the spawning of Hemichromis lifalili . Each pair had four different temperatures to choose from. Spawning temperatures selected by each pair are highlighted in bold.

41
32
30
28
26
24
22
20
0 1 2 3 4
Spawning Pair Number
5 6 7
Figure 10. Spawning temperature (°C) chosen by each Hemichromis lifalili pair.
Fish symbols represent spawning temperature selected by each pair; dots represent available temperatures from which each pair could chose.

42
Chamber
1
2
3
4
Chamber
1
2
3
4
16 Jul. 2008
21.6
24.3
26.1
27.9
Date Spawned
22 Jun. 2008 18 Oct. 2008
26.7
28.6
31.6
33.3
28.1
31.0
33.0
34.1
31 Aug. 2008
23.4
Date Spawned
30 May 2008
26.8
21 Oct. 2008
24.0
25.5
26.9
29.7
29.0
31.7
32.9
27.9
30.2
31.4
2 Jun. 2008
21.3
26.2
27.3
29.6
4 Sept. 2008
23.6
25.2
26.6
29.1
14 Aug. 2008 15 Aug. 2008
22.2 27.9
24.7
26.2
27.9
31.1
32.0
33.4
Table 6. Average temperatures (°C) of each chamber from the previous 48 hours prior to the spawning of Archocentrus nigrofasciatus.
Each pair had four different temperatures to choose from. Spawning temperatures selected by each pair are highlighted in bold.

43
34
32
30
28
26
24
22
20
0 1 2 3 4 5 6 7
Spawning Pair Number
8 9 10
Figure 11. Spawning temperature (°C) chosen by each Archocentrus nigrofasciatus pair. Fish symbols represent spawning temperature selected by each pair; dots represent available temperatures from which each pair could chose.
11

44
Date Spawned
Chamber 11 Jan. 2010 12 Jun. 2008 17 Dec. 2008 23 Nov. 2008 8 Jun. 2009 14 Jun. 2009
1
2
3
4
27.4
29.8
32.3
34.2
24.2
26.4
28.1
29.9
27.9
29.2
32.1
33.3
23.4
25.2
27.1
29.1
26.9
29.2
31.4
33.0
27.8
28.1
30.6
32.3
Table 7. Average temperatures (°C) of each chamber 48 hours prior to the spawning of Tilapia snyderae . Each pair had four different temperatures to choose from. Spawning temperatures selected by each pair are highlighted in bold.

45
35
33
31
29
27
25
23
0 1 2 3 4
Spawning Pair Number
5 6 7
Figure 12. Spawning temperature (°C) chosen by each Tilapia snyderae pair. Fish symbols represent spawning temperature selected by each pair; dots represent available temperatures from which each pair could chose.

46
Chamber
1
2
3
4
6 Mar. 2010
Date spawned
28 Mar. 2010
26.6
28.31
30.05
32.16
21.71
24.07
26.83
27.91
22 Apr. 2010
24.77
28.89
30.31
32.57
Table 8. Average temperatures (°C) of each chamber 48 hours prior to the spawning of Hypsophrys nicaraguense . Each pair had four different temperatures to choose from. Spawning temperatures selected by each pair are highlighted in bold.

47
34
32
30
28
26
24
22
20
0 1 2
Spawning Pair Number
3 4
Figure 13. Spawning temperature (°C) chosen by each Hypsophrys nicaraguense pair. Fish symbols represent spawning temperature selected by each pair; dots represent available temperatures from which each pair could chose.

48
Species H.lifalili A nigrofasciatus T. snyderae H. nicaraguense N. nematopus S. casuarius
1.11 1.30 1.59 1.94 2.03 2.53
1.17
1.10
1.11
1.03
1.06
1.50
1.52
1.43
1.21
1.35
1.32
1.44
1.22
1.57
1.27
2.14
1.80
Average 1.10
1.40
1.39
1.13
1.52
1.38 1.40 1.96 2.03 2.53
Table 9. Effective diameter (mm) of eggs from spawnings of the six species of cichlids.

49
Species H. lifalili T. snyderae A.nigrofasciatus H. nicaraguense N. nematopus S. casuarius
Average
31.1
29.2
26.5
27.9
26.6
29.2
29.5
28.9
29.9
29.1
33.3
31.4
32.3
29.8
31.0
27.9
28.6
28.2
26.2
26.6
26.9
26.8
27.9
26.2
27.9
27.3
30.1
27.9
29.0
29.0
29.4
29.4
31.7
31.7
Table 10. The chosen spawning temperature (°C) for each pair of cichlids, and the average chosen spawning temperature for each species.

50
34
32
30
28
26
24
22
20
0 1 2 3
Species
4 5 6 7
Figure 14. Overall spread of temperatures (°C) that each pair, and each species collectively, had to choose from. Mean chosen spawning temperature for each pair is represented by a red dash. Species are as follow: 1: H. lifalili , 2: A. nigrofasciatus , 3: T. snyderae , 4. H. nicaraguense , 5: N. nematopus , and 6: S. casuarius .

51
Interspecies Comparison
A one way ANOVA (Table 11) revealed significant differences in the average chosen spawning temperatures of H. lifalili , A. nigrofasciatus , T. snyderae , and H. nicaraguense (F=9.4; df=3.22; P<0.01). N. nematopus and S. casuarius were not used in this test because there was only one spawning collected from each.
Multiple comparisons, using the Holm-Sidak method, found significant differences between T. snyderae and A. nigrofasciatus (P critical=0.009;
P=0.00003), and between T. snyderae against H. lifalili (P critical=0.013;
P=0.004).
There were no significant differences between the other means. This could be due to several reasons, including the small number of H. nicaraguense samples.
In conclusion, it can be confidently stated that the preferred spawning temperatures when doing a comparison among T. snyderae against A. nigrofasciatus , and T. snyderae against H. lifalili are significantly different.
However, though each species did in fact have a consistent preferred spawning temperature, there was no significant difference found when comparing the other species against one another.
There was no relationship between temperature chosen and the average egg size of each species (linear regression; r=0.52, df=5, P=0.29) (Figure 15 and Table
12).

52
Normality Test: Passed (P = 0.309)
Equal Variance Test: Passed (P = 0.260)
Group Name
Col 1
Col 2
Col 3
Col 4
N
7
6
10
3
Missing
0
0
0
0
Mean 1
28.584
30.980
27.320
28.950
Std Dev
1.680
1.634
0.875
1.071
SEM
0.635
0.667
0.277
0.619
Source of Variation DF
Between Groups 3
SS
50.514
MS
16.838
F
9.384
P
<0.001
Residual
Total
22
25
39.476
89.990
1.794
1 The differences in the mean values among the treatment groups are greater than would be expected by chance; power of performed test with alpha = 0.050: 0.985
All Pairwise Multiple Comparison Procedures (Holm-Sidak method):
Overall significance level = 0.05
Comparisons for factor: t Unadjusted P Critical Level Significant?
Comparison Diff of Means
Col 2 vs. Col 3 3.660 5.291 0.0000261 0.009 Yes
3.215 0.00399 0.010 Yes Col 2 vs. Col 1
Col 2 vs. Col 4
Col 1 vs. Col 3
Col 4 vs. Col 3
Col 4 vs. Col 1
2.396
2.030
1.264
1.630
0.366
2.143
1.915
1.848
0.395
0.0434
0.0686
0.0781
0.696
0.013
0.017
0.025
0.050
No
No
No
No
Table 11. ANOVA of data in Table 10 comparing the mean chosen spawning temperatures of each species against one another. Col 1: H. lifalili Col 2: T. snyderae Col 3: A. nigrofasciatus Col 4: H. nicaraguense.

32,0
31,5
31,0
30,5
30,0
29,5
29,0
28,5
28,0
27,5
27,0
1,00 1,50 2,00
Egg Size (mm)
2,50 3,00
Figure 15. Average spawning temperature (°C) versus average egg size (mm) for each species.
53

54
Col 1 = -3.436 + (0.175 * Col 2)
N = 6.000
R = 0.521 Rsqr = 0.272 Adj Rsqr = 0.0899
Standard Error of Estimate = 0.507
Coefficient
Constant -3.436
Col 2 0.175
Analysis of Variance:
DF
Regression 1
Residual 4
Total 5
Std. Error
4.235
0.143 t
-0.811
1.222
P
0.463
0.289
SS MS F P
0.384 0.384 1.494 0.289
1.027 0.257
1.411 0.282
Table 12. Linear regression between spawning temperature chosen and average egg size of each species.

55
DISCUSSION
The previous experiments have shown three things: previous exposure to a particular temperature does not influence the choice of spawning temperatures; cichlid fishes, if given a choice, have a species-specific preferred spawning temperature; and such preferred spawning temperature does not correlate with egg size.
There are countless numbers of studies conducted on fish, showing that fish are sensitive to temperature. By knowing that prior exposure to particular temperature does not have an effect on the future spawning decisions of a fish, future studies can be conducted without having to take that possible variable into consideration. This greatly simplifies the methodology of future cichlid spawning studies because no temperature control will have to be used.
Knowing the preferred spawning temperatures of fishes is extremely valuable. Hobbyists, as well as fish suppliers, will know the best spawning temperature in which to breed their cichlid fishes. Knowing this not only leads to maximized output of product (i.e., more offspring), but maximized profit. Because the cichlids tested do in fact have a consistent and precise spawning temperature preference, the same can be applied across other cichlid species and possibly many other species of fish as well.
From a conservation perspective, scientists working in the field with spawning cichlid fishes will have a much better understanding of which

56 temperature to look for to find the species that they are studying. Global warming is having major effects on water systems, causing shifts in temperatures throughout ocean, lake and river systems. If a river that currently holds a species which clearly prefers 24°C water for spawning has a permanent rise in temperature to 27°C, this can have an enormous impact on this species and its future spawnings. Will the fish now be forced to migrate? Will the fish stay, yet be forced to change their preferred temperatures? Will this have an effect on output of offspring? It has been shown that fish have been observed in nature inhabiting temperatures that have previously been shown to be a species’ temperature preferendum (Ferguson, 1958;
Neill and Magnuson, 1974), so it can almost conclusively be stated that a change in habitat temperature will have profound effects on fish. Having information known on preferred spawning temperature preference will allow for more study and observation time before global warming, or other devastating habitat factors, take place, with less time spent searching for the fishes to be studied.
In this experiment, the preferred spawning temperature of cichlid fishes does not correlate to egg size. When a fish chooses to spawn, there are many factors that must be taken into consideration, including water currents, velocity, temperature, and availability of food. In my hypothesis I stated that a parent fish would base this decision on what the best water temperature, and in congruence the best oxygen concentration, would be for their eggs. There are two different possibilities as to why this was not found; fish do not choose their spawning site

57 based on oxygen concentration for their eggs, or a more specialized methodology needs to be applied to this study.
A fish's life, from the day the egg is laid, is very hazardous. Eggs are a highly nutrient-rich form of food and are highly susceptible to predation. It may be that fish base their spawning choices not on the best temperature for the egg, but for the emerging fry. When the offspring are still eggs, the parents are able to guard them quite closely; however, once the eggs hatch and become free swimming, it is more difficult to keep track of them. In my experiment, it seemed that instead of the fishes which had larger eggs preferring colder water (which would have had higher oxygen concentrations to accommodate the oxygen requirements for their larger eggs) they had a tendency to prefer warmer water.
This may be due to the fact that once the eggs are hatched, the warmer water would provide for a higher metabolism allowing the newly hatched fry to grow at a quicker rate, something that is very valuable in the fish world. Now one may ask, then why do all fish not prefer the warmer water? It may be that the fish which hatch from smaller eggs cannot handle the higher water temperature, in other words, it may cook the eggs.
Another argument may be that the fish choose their spawning temperature not just on what would be best suitable for the fry and not the eggs, but on the temperature that is able to supply the most nutrient dense habitat for the newly hatched fry. When the offspring are in egg form and while they are wrigglers, they

58 are fed via yolk, however once the yolk is gone, these very small, and very young fish are forced to find food on their own. It would most likely be beneficial when faced with that circumstance to be in an area that has a lot of food available.
There are many other arguments that can be made as to why fish choose the spawning temperatures that they do, including hormones and chemical cues, metabolic needs for various life stages, etc. However, this merely opens another avenue for future study of this process.
The methodology used for the purpose of my study was, to the best of my knowledge, optimum. The fact that I was able to show that each species used has a precise preferred spawning temperature shows that the apparatus effectively worked for temperature study purposes. Nevertheless, there are always ways in which an experimental design can be improved upon.
One way that this experimental design could have been conducted is if the actual levels of oxygen in each chamber were manipulated and made to be at a controlled and constant level. Though the equipment needed to able to conduct an experiment in this manner is quite advanced and therefore expensive, it would be well-suited for the use of an experiment dealing with oxygen preference and temperature. By controlling the oxygen levels of each chamber, one could manipulate the experiment in a way that would give breeding fish an array of temperatures to choose from in which the temperatures were forced to hold more/less oxygen than would naturally be found. This would force the fish to make

59 a decision between the most suitable temperature, and the most suitable oxygen levels when breeding.
Another possible way in which to conduct the experiment would be to compare species within a genera. In my experiment, I used fish across many different genera. If one were to perform this experiment and use different species within a genera, there may in fact be a correlation between spawning temperature preference and egg size. Though all fish used were within the family Cichlidae, the relationships between the species used may not have been great enough to be able to see a pattern. The only problem with this application would be that the differences between egg sizes within genera are so minute that it might be quite difficult to be able to find a statistical difference between egg size and chosen spawning temperature when compared between species.
Overall, the goal of my thesis work was to look more closely into the spawning behavior of cichlid fishes. Cichlid fishes exhibit an extraordinary amount of parental care, with obvious decisions needing to be made along every step of the process, yet there is much to be learned about why they make these decisions. More specifically, I wanted to look into how they choose a spawning site. Although I did not find a relationship between spawning temperature preference and egg size, this does not mean that there is no relationship to be found.
Future studies can be conducted taking the previous arguments into consideration.
When a relationship is found between preferred spawning temperature and the

60 means that this decision is based on, we will have a much greater understanding as to why spawning fishes make the decisions that they do. By knowing this we are able to better understand a fish's behavior in general, thus leading to more knowledgeable conservation and supplier practices.

APPENDICES
61

APPENDIX A
Temperature
°C
Solubility of Oxygen in Fresh Water (100% Saturation)
(taken from Clescer et al., 1991)
PPM (mg/L) Dissolved
Oxygen
Temperature
°C
PPM (mg/L) Dissolved
Oxygen
34
35
36
37
30
31
32
33
26
27
28
29
23
24
25
38
39
40
41
42
43
12.2
11.9
11.6
11.3
11.1
10.8
10.6
10.4
14.6
14.2
13.9
13.5
13.2
12.8
12.5
10.2
9.9
9.7
9.5
9.3
9.2
11
12
13
14
7
8
9
10
3
4
5
6
0
1
2
15
16
17
18
19
20
7.2
7.1
7.0
6.8
7.7
7.5
7.4
7.3
8.7
8.5
8.4
8.2
8.1
7.9
7.8
6.7
6.6
6.5
6.4
6.3
6.2
21
22
9.0
8.8
44
45
6.1
6.0
62

63
APPENDIX B
Egg Size Data for Each Fish Species Used in the Temperature Choice Apparatus
27
28
29
30
Average
23
24
25
26
19
20
21
22
15
16
17
18
11
12
13
14
Table 1. Egg size data for Archocentrus nigrofasciatus spawned on 30 May, 2008.
Egg
Number
1
Length
(mm)
1.86
Width
(mm)
1.38
Effective
Diameter (mm)
1.52
2 1.65 1.14 1.29
7
8
9
10
3
4
5
6
2.04
1.65
1.74
1.80
1.80
1.74
1.62
1.74
1.20
1.32
1.20
1.26
1.38
1.50
1.50
1.32
1.43
1.42
1.36
1.42
1.51
1.58
1.54
1.45
1.80
1.74
1.98
1.68
1.56
1.56
1.68
1.80
1.98
2.04
1.56
1.56
1.62
1.74
1.68
2.04
1.50
1.74
1.86
1.68
1.75
1.32
1.26
1.26
1.32
1.32
1.38
1.20
1.20
1.32
1.20
1.32
1.32
1.32
1.20
1.38
1.08
1.14
1.44
1.26
1.32
1.29
1.51
1.43
1.40
1.40
1.41
1.36
1.47
1.34
1.46
1.40
1.46
1.43
1.40
1.44
1.34
1.37
1.25
1.53
1.43
1.43
1.43

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 2. Egg size data for Archocentrus nigrofasciatus spawned on 2 June, 2008.
Egg Length Width Effective
Number
1
(mm)
1.92
(mm)
1.44
Diameter (mm)
1.58
2
3
1.80
1.86
1.44
1.56
1.55
1.65
4
5
1.74
1.86
1.32
1.32
1.45
1.48
10
11
12
6
7
8
9
1.92
1.92
1.74
1.92
2.10
1.86
1.86
1.32
1.50
1.44
1.44
1.26
1.50
1.50
1.50
1.63
1.53
1.58
1.49
1.61
1.61
1.98
1.98
1.98
1.86
2.10
2.04
1.80
1.98
1.86
1.80
2.04
2.04
1.92
1.86
1.86
1.80
1.86
1.98
1.91
1.32
1.44
1.32
1.50
1.26
1.08
1.32
1.38
1.44
0.96
1.44
1.26
1.50
1.50
1.14
1.26
1.44
1.38
1.37
1.57
1.18
1.62
1.48
1.63
1.61
1.34
1.42
1.51
1.60
1.51
1.61
1.49
1.34
1.46
1.56
1.57
1.56
1.52
64

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 3. Egg size data for Archocentrus nigrofasciatus spawned on 22 June, 2008.
Egg Length Width Effective
Number
1
(mm)
1.92
(mm)
1.08
Diameter (mm)
1.31
2
3
1.62
1.92
1.14
1.38
1.28
1.54
4
5
2.10
1.92
1.20
1.38
1.45
1.54
10
11
12
6
7
8
9
1.92
1.92
2.04
1.92
1.98
1.92
1.92
1.32
1.32
1.20
1.38
1.50
1.32
1.38
1.50
1.50
1.43
1.54
1.65
1.50
1.54
1.92
1.92
1.98
1.98
2.04
1.86
1.98
1.56
1.92
2.04
1.92
1.80
1.92
2.04
2.04
1.92
1.92
2.04
1.93
1.44
1.32
1.38
1.44
1.44
1.44
1.38
1.38
1.38
1.38
1.26
1.32
1.38
1.08
1.20
1.32
1.38
1.38
1.33
1.54
1.57
1.45
1.46
1.54
1.34
1.43
1.50
1.58
1.50
1.56
1.60
1.62
1.57
1.56
1.44
1.54
1.57
1.50
65

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 4. Egg size data for Archocentrus nigrofasciatus spawned on 16 July, 2008.
Egg Length Width Effective
Number
1
(mm)
1.68
(mm)
1.20
Diameter (mm)
1.34
2
3
1.80
1.98
1.44
1.14
1.55
1.37
4
5
1.98
1.50
1.56
1.26
1.69
1.34
10
11
12
6
7
8
9
1.92
1.74
1.74
1.56
1.56
1.62
1.56
1.56
1.20
1.32
1.38
1.50
1.20
1.26
1.67
1.36
1.45
1.44
1.52
1.33
1.35
1.68
1.86
1.44
1.56
1.50
1.86
1.56
2.04
1.92
1.92
1.98
1.62
2.04
2.10
2.04
1.98
1.98
1.80
1.78
1.32
1.50
1.32
1.32
1.38
1.56
1.44
1.68
1.50
1.38
1.50
1.26
1.50
1.56
1.50
1.50
1.56
1.56
1.41
1.63
1.54
1.65
1.37
1.66
1.72
1.66
1.65
1.43
1.61
1.36
1.40
1.42
1.65
1.48
1.79
1.69
1.64
1.52
66

Table 5. Egg size data for Archocentrus nigrofasciatus spawned on 14
August, 2008.
Egg
Number
Length
(mm)
Width
(mm)
Effective
Diameter (mm)
1.56
1.67
1.44
1.67
1.56
1.67
1.50
1.56
1.50
1.67
1.67
1.61
1.61
1.56
1.61
1.61
1.50
1.44
1.44
1.56
1.55
1.67
1.67
1.50
1.44
1.56
1.17
1.44
1.56
1.61
1.50
23
24
25
26
19
20
21
22
15
16
17
18
11
12
13
14
Average
27
28
29
30
1
2
7
8
9
10
3
4
5
6
1.11
1.28
1.33
1.39
1.22
1.06
0.94
1.11
1.17
1.22
0.89
1.11
1.22
1.33
1.28
1.22
1.33
1.22
1.06
1.00
1.22
1.11
1.28
1.22
1.28
1.22
1.17
1.22
1.33
1.17
1.19
1.21
1.19
1.29
1.27
1.37
1.35
1.35
1.32
1.06
1.27
1.35
1.42
1.38
1.32
1.42
1.34
1.27
1.29
1.37
1.29
1.30
1.27
1.40
1.38
1.41
1.32
1.10
1.08
1.24
1.30
1.31
67

28
29
30
Average
24
25
26
27
20
21
22
23
16
17
18
19
12
13
14
15
Table 6. Egg size data for Archocentrus nigrofasciatus spawned on 15 August, 2008.
Egg
Number
1
Length
(mm)
1.62
Width
(mm)
1.32
Effective
Diameter (mm)
1.41
2
3
1.74
1.68
1.26
1.38
1.40
1.47
8
9
10
11
4
5
6
7
1.68
1.74
1.74
1.74
1.74
1.68
1.74
1.56
1.38
1.32
1.38
1.32
1.38
1.26
1.32
1.02
1.47
1.45
1.49
1.45
1.49
1.39
1.45
1.18
1.62
1.68
1.68
1.62
1.80
1.50
1.80
1.62
1.62
1.56
1.62
1.50
1.68
1.62
1.74
1.74
1.80
1.62
1.62
1.67
1.32
1.26
1.26
1.32
1.20
1.38
1.26
1.26
1.26
1.20
1.20
1.20
1.32
1.32
1.32
1.26
1.20
1.26
1.26
1.28
1.37
1.31
1.33
1.29
1.43
1.41
1.45
1.40
1.41
1.39
1.39
1.41
1.37
1.42
1.42
1.37
1.37
1.37
1.37
1.40
68

28
29
30
Average
24
25
26
27
20
21
22
23
16
17
18
19
12
13
14
15
Table 7. Egg size data for Archocentrus nigrofasciatus spawned on 31
August, 2008.
Egg
Number
1
Length
(mm)
1.32
Width
(mm)
0.96
Effective
Diameter (mm)
1.07
2
3
1.26
1.50
1.02
1.02
1.09
1.16
8
9
10
11
4
5
6
7
1.32
1.26
1.26
1.38
1.02
1.26
1.02
1.02
1.08
0.96
1.02
1.02
1.02
0.96
1.08
1.08
1.15
1.05
1.09
1.13
1.02
1.05
1.06
1.06
0.90
1.20
1.38
1.38
1.26
1.32
1.38
1.44
1.38
1.26
1.32
1.44
1.44
1.50
1.38
1.32
1.44
1.44
1.44
1.31
1.14
1.14
1.14
1.08
1.02
1.02
1.02
0.96
1.14
1.20
1.14
1.02
1.08
1.14
1.14
1.02
0.96
1.02
1.14
1.06
1.05
1.16
1.21
1.17
1.09
1.11
1.13
1.10
1.21
1.22
1.20
1.14
1.19
1.25
1.21
1.11
1.10
1.14
1.23
1.13
69

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 8. Egg size data for Archocentrus nigrofasciatus spawned on 4 September, 2008.
Egg Length Width Effective
Number
1
(mm)
1.68
(mm)
1.32
Diameter (mm)
1.43
2
3
1.62
1.74
1.32
1.32
1.41
1.45
4
5
1.62
1.80
1.20
1.20
1.33
1.37
10
11
12
6
7
8
9
1.32
1.80
1.74
1.74
1.74
1.68
1.74
1.14
1.08
1.08
1.38
1.26
1.26
1.20
1.20
1.28
1.27
1.49
1.40
1.39
1.36
1.86
1.80
1.32
1.68
1.74
1.86
1.68
1.74
1.62
1.50
1.74
1.80
1.74
1.74
1.80
1.80
1.74
1.74
1.70
0.90
1.14
1.20
1.20
1.20
1.26
1.20
1.20
1.26
1.26
0.96
1.20
1.14
1.26
1.26
1.26
1.32
1.26
1.21
1.37
1.34
1.17
1.37
1.31
1.40
1.42
1.42
1.15
1.33
1.24
1.34
1.36
1.43
1.34
1.36
1.45
1.40
1.35
70

Table 9. Egg size data for Archocentrus nigrofasciatus spawned on 17 October, 2008.
Egg Length Width Effective
Number
1
(mm)
1.38
(mm)
1.02
Diameter (mm)
1.13
2
3
1.44
1.50
1.20
1.14
1.28
1.25
4
5
1.44
1.38
1.20
1.08
1.28
1.17
10
11
Average
6
7
8
9
1.56
1.38
1.38
1.32
1.50
1.44
1.43
1.14
1.14
1.05
1.14
1.08
1.14
1.12
1.27
1.21
1.15
1.20
1.20
1.23
1.21
71

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 10. Egg size data for Archocentrus nigrofasciatus spawned on 21 October, 2008.
Egg Length Width Effective
Number
1
(mm)
1.68
(mm)
1.32
Diameter (mm)
1.43
2
3
1.80
1.38
1.32
1.14
1.46
1.21
4
5
1.62
1.74
1.14
1.26
1.28
1.40
10
11
12
6
7
8
9
1.56
1.62
1.62
1.68
1.68
1.62
1.32
1.20
1.50
1.44
1.38
1.38
1.26
1.26
1.31
1.54
1.50
1.47
1.47
1.37
1.28
1.56
1.32
1.38
1.62
1.62
1.38
1.68
1.62
1.62
1.44
1.56
1.74
1.56
1.38
1.92
1.68
1.50
1.68
1.59
1.38
1.38
0.96
1.32
1.26
1.26
1.32
1.32
1.32
1.20
1.44
1.32
1.32
1.44
1.32
1.32
1.38
1.38
1.31
1.41
1.28
1.48
1.45
1.40
1.42
1.50
1.43
1.44
1.36
1.08
1.41
1.37
1.30
1.43
1.41
1.42
1.47
1.39
72

23
24
25
26
19
20
21
22
15
16
17
18
11
12
13
14
Table 11. Egg size data for Hemichromis lifalili spawned on 27 June, 2008.
Egg
Number
1
Length
(mm)
1.38
Width
(mm)
1.02
Effective
Diameter (mm)
1.13
2 1.32 1.02 1.11
7
8
9
10
3
4
5
6
1.20
1.44
1.32
1.26
1.44
1.20
1.38
1.44
1.20
1.02
0.96
1.02
1.02
1.08
1.02
1.02
1.20
1.14
1.07
1.09
1.14
1.12
1.13
1.14
1.32
1.32
1.32
1.26
1.50
1.44
1.32
1.26
1.50
1.32
1.38
1.44
1.26
1.26
1.32
1.26
27
28
29
30
1.38
1.44
1.38
1.14
Average 1.34
1.14
0.96
1.02
0.90
0.96
1.02
1.02
0.96
1.06
1.02
0.90
0.96
1.02
1.14
1.02
1.02
0.90
1.02
0.90
1.02
1.01
1.19
1.11
1.04
1.10
1.09
1.18
1.11
1.09
1.20
1.07
1.11
1.01
1.11
1.14
1.11
1.05
1.04
1.14
1.04
1.06
1.11
73

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 12. Egg size data for Hemichromis lifalili spawned on 11 July, 2008.
Egg Length Width Effective
Number
1
(mm)
1.44
(mm)
1.02
Diameter (mm)
1.14
2
3
1.38
1.38
0.96
1.08
1.08
1.17
4
5
1.32
1.50
1.08
1.02
1.15
1.16
10
11
12
6
7
8
9
1.50
1.32
1.38
1.50
1.56
1.26
1.38
1.14
1.08
1.02
0.96
1.20
1.14
1.02
1.25
1.15
1.13
1.11
1.31
1.18
1.13
1.50
1.38
1.50
1.44
1.38
1.20
1.32
1.14
1.38
1.14
1.20
1.20
1.08
1.08
1.20
1.38
1.26
1.50
1.34
0.84
0.96
1.08
1.08
0.96
0.96
1.02
0.96
0.90
1.08
0.90
0.96
0.90
1.02
0.90
1.20
1.02
1.02
1.02
1.04
1.10
0.99
1.03
0.96
1.04
0.99
1.26
1.02
1.08
1.20
1.19
1.08
1.03
1.11
1.02
1.09
1.16
1.11
74

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 13. Egg size data for Hemichromis lifalili spawned on 27 August, 2008.
Egg Length Width Effective
Number
1
(mm)
1.38
(mm)
1.14
Diameter (mm)
1.21
2
3
1.32
1.20
0.90
0.90
1.02
0.99
4
5
1.38
1.38
0.90
1.02
1.04
1.13
10
11
12
6
7
8
9
1.32
1.32
1.38
1.32
1.44
1.38
1.32
1.02
1.02
1.14
1.02
1.02
0.78
0.90
1.11
1.11
1.21
1.11
1.14
0.94
1.02
1.44
1.26
1.50
1.38
1.38
1.38
1.32
1.38
1.02
1.44
1.26
1.32
1.38
1.38
1.45
1.20
1.26
1.38
1.34
1.08
1.02
0.84
1.02
1.02
1.08
1.08
1.08
1.08
0.90
1.14
0.90
0.84
1.20
0.90
0.84
0.96
1.02
0.99
1.06
1.05
1.18
1.02
0.99
1.26
1.06
0.95
1.19
1.09
1.02
1.13
1.13
1.17
1.15
1.17
1.05
1.13
1.10
75

Table 14. Egg size data for Hemichromis lifalili spawned on
14 September, 2008.
Egg Length Width Effective
Number
1
(mm)
1.17
(mm)
0.83
Diameter (mm)
0.93
2
3
1.17
1.22
0.94
0.94
1.01
1.03
4
5
1.56
1.50
0.94
1.17
1.11
1.27
10
11
12
6
7
8
9
1.50
1.39
1.22
1.17
1.06
1.06
1.17
1.00
1.06
1.06
0.89
1.00
0.83
1.11
1.14
1.16
1.11
0.97
1.02
0.90
1.13
Average
25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
1.17
1.17
1.22
1.22
1.56
1.44
1.17
1.22
1.22
0.94
1.17
1.06
1.11
1.28
1.00
1.33
1.33
1.06
1.23
0.94
0.94
0.94
1.00
1.06
1.06
0.89
0.89
1.00
0.89
0.89
0.78
0.78
1.00
0.94
1.00
0.94
0.83
0.95
1.07
0.91
0.97
0.86
0.88
1.09
0.96
1.10
1.01
1.01
1.03
1.07
1.21
1.17
0.97
0.99
1.06
0.90
1.03
76

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 15. Egg size data for Hemichromis lifalili spawned on 7 June, 2009.
Egg Length Width Effective
Number
1
(mm)
1.38
(mm)
1.02
Diameter (mm)
1.13
2
3
1.32
1.32
1.02
1.08
1.11
1.15
4
5
1.26
1.26
1.02
1.02
1.09
1.09
10
11
12
6
7
8
9
1.32
1.38
1.26
1.38
1.26
1.20
1.32
1.02
0.96
0.96
1.02
1.08
1.02
0.90
1.11
1.08
1.05
1.13
1.14
1.08
1.02
1.26
1.32
1.26
1.32
1.32
1.38
1.38
1.32
1.32
1.44
1.32
1.26
1.26
1.26
1.32
1.26
1.26
1.26
1.31
1.02
1.02
1.08
0.96
1.02
1.02
1.08
1.02
0.96
1.02
1.02
1.02
0.96
0.72
1.02
0.96
1.02
1.14
1.01
1.07
1.14
1.11
1.09
1.05
0.87
1.11
1.05
1.09
1.11
1.14
1.07
1.11
1.13
1.17
1.11
1.09
1.18
1.10
77

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 16. Egg size data for Hemichromis lifalili spawned on 29 November, 2009.
Egg Length Width Effective
Number
1
(mm)
1.44
(mm)
1.20
Diameter (mm)
1.28
2
3
1.50
1.50
1.14
1.14
1.25
1.25
4
5
1.50
1.56
1.14
1.14
1.25
1.27
10
11
12
6
7
8
9
1.44
1.44
1.50
1.44
1.50
1.44
1.44
1.02
1.14
1.14
1.14
1.02
1.08
1.14
1.14
1.23
1.25
1.23
1.16
1.19
1.23
1.44
1.38
1.32
1.50
1.56
1.26
1.32
1.38
1.44
1.38
1.50
1.38
1.44
1.38
1.26
1.32
1.26
1.50
1.42
1.14
1.08
1.02
1.14
1.08
0.96
1.02
1.14
1.14
0.96
0.90
0.96
0.90
1.02
1.02
1.02
0.96
1.14
1.07
1.23
1.08
1.07
1.08
1.05
1.13
1.09
1.11
1.23
1.17
1.11
1.25
1.22
1.05
1.11
1.21
1.05
1.25
1.17
78

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 17. Egg size data for Hemichromis lifalili spawned on
7 April, 2010.
Egg Length Width Effective
Number
1
(mm)
1.17
(mm)
0.94
Diameter (mm)
1.01
2
3
1.39
1.33
0.94
0.94
1.07
1.06
4
5
1.28
1.33
0.89
1.00
1.00
1.10
10
11
12
6
7
8
9
1.22
1.39
1.33
1.44
1.28
1.22
1.22
1.06
1.00
0.94
0.94
1.06
0.94
0.89
1.11
1.12
1.06
1.08
1.13
1.03
0.99
1.17
1.28
1.22
1.33
1.33
1.39
1.22
1.28
1.39
1.22
1.33
1.28
1.33
1.33
1.28
1.22
1.28
1.06
1.28
0.94
0.94
0.94
1.00
0.86
0.94
0.94
0.94
1.00
0.83
1.00
1.00
1.00
0.94
1.00
1.06
1.00
0.94
0.96
1.12
0.94
1.10
1.09
1.10
1.06
1.09
1.11
1.01
1.04
1.03
1.10
0.99
1.07
1.03
1.04
1.09
0.98
1.06
79

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 18. Egg size data for Tilapia snyderae spawned on 12 June, 2008.
Egg Length Width Effective
Number
1
(mm)
1.67
(mm)
1.11
Diameter (mm)
1.27
2
3
1.72
1.56
1.22
1.11
1.65
1.54
4
5
1.50
1.50
1.28
1.22
1.57
1.56
10
11
12
6
7
8
9
1.61
1.56
1.50
1.44
1.50
1.44
1.38
1.06
1.11
1.17
1.22
1.11
1.11
1.00
1.59
1.66
1.49
1.56
1.46
1.52
1.63
1.56
1.28
1.67
1.50
1.44
1.28
1.39
1.39
1.33
1.33
1.28
1.44
1.44
1.44
1.56
1.39
1.17
1.39
1.46
1.11
0.83
1.17
1.11
1.11
1.06
1.11
1.00
1.00
0.94
1.22
0.94
0.94
1.06
0.94
1.06
1.06
1.11
1.08
1.51
1.48
1.72
1.59
1.59
1.57
1.56
1.59
1.68
1.54
1.67
1.69
1.60
1.72
1.59
1.42
1.65
1.55
1.57
80

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 19. Egg size data for Tilapia snyderae spawned on 22 November, 2008.
Egg Length Width Effective
Number (mm) (mm) Diameter (mm)
1
2
1.56
1.44
1.20
1.14
1.31
1.23
3
4
5
1.56
1.56
1.56
1.26
1.20
1.20
1.35
1.31
1.31
10
11
12
6
7
8
9
1.56
1.44
1.62
1.56
1.38
1.80
1.56
1.14
1.26
1.32
1.26
1.26
1.44
1.14
1.27
1.32
1.41
1.35
1.30
1.55
1.27
1.68
1.56
1.50
1.56
1.38
1.74
1.62
1.68
1.32
1.62
1.56
1.38
1.62
1.62
1.50
1.62
1.74
1.62
1.56
1.20
1.32
1.38
1.20
1.08
1.20
1.38
1.20
1.20
1.20
1.14
1.02
1.20
1.14
1.20
1.14
1.02
1.20
1.21
1.24
1.33
1.27
1.13
1.33
1.28
1.29
1.28
1.34
1.40
1.42
1.31
1.17
1.36
1.46
1.34
1.22
1.33
1.32
81

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 20. Egg size data for Tilapia snyderae spawned on 17 December, 2008.
Egg Length Width Effective
Number
1
(mm)
1.68
(mm)
1.50
Diameter (mm)
1.56
2
3
1.86
1.80
1.32
1.38
1.48
1.51
4
5
1.92
1.86
1.14
1.26
1.36
1.43
10
11
12
6
7
8
9
1.56
1.74
1.86
1.74
1.92
1.68
1.80
1.32
1.32
1.14
1.38
1.38
1.44
1.32
1.40
1.45
1.34
1.49
1.54
1.52
1.46
1.74
1.68
1.80
1.68
1.68
1.86
1.68
1.68
1.68
1.50
1.74
1.74
1.80
1.68
1.62
1.74
1.68
1.62
1.73
1.26
1.50
1.26
1.32
1.26
1.38
1.38
1.20
1.38
1.20
1.32
1.26
1.38
1.32
1.20
1.26
1.38
1.38
1.32
1.47
1.29
1.45
1.40
1.51
1.43
1.33
1.40
1.40
1.56
1.42
1.43
1.39
1.52
1.47
1.34
1.47
1.46
1.44
82

Table 21. Egg size data for Tilapia snyderae spawned on 14 June 2009.
Egg Length Width Effective
Number
1
(mm)
1.17
(mm)
1.11
Diameter (mm)
1.13
2
3
1.33
1.62
0.94
1.11
1.31
1.23
4
5
1.56
1.44
1.00
1.22
1.35
1.31
6
Average
1.67
1.47
1.28
1.11
1.31
1.27
83

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 22. Egg size data for Tilapia snyderae spawned on 6 November, 2009.
Egg Length Width Effective
Number
1
(mm)
1.44
(mm)
1.14
Diameter (mm)
1.23
2
3
1.50
1.38
0.96
0.90
1.11
1.04
4
5
1.56
1.74
1.02
1.14
1.18
1.31
10
11
12
6
7
8
9
1.38
1.50
1.68
1.50
1.68
1.62
1.56
1.20
1.02
1.20
1.08
1.14
1.32
1.20
1.26
1.16
1.34
1.20
1.30
1.41
1.31
1.44
1.32
1.62
1.62
1.44
1.38
1.44
1.50
1.44
1.44
1.44
1.56
1.38
1.32
1.44
1.56
1.50
1.14
1.48
1.20
1.20
1.26
1.08
1.14
1.09
0.96
1.02
1.14
1.08
1.20
1.14
1.20
1.08
0.96
1.02
0.84
1.14
1.10
1.23
1.19
1.28
1.27
1.26
1.15
1.10
1.18
1.28
1.24
1.37
1.24
1.23
1.18
1.10
1.16
1.02
1.14
1.22
84

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 23. Egg size data for Tilapia snyderae spawned on
11 January, 2010.
Egg Length Width Effective
Number
1
(mm)
1.98
(mm)
1.56
Diameter (mm)
1.69
2
3
1.98
1.75
1.50
1.44
1.65
1.54
4
5
2.04
2.16
1.38
1.32
1.57
1.56
10
11
12
6
7
8
9
2.10
2.04
2.10
1.98
1.80
1.86
2.10
1.38
1.50
1.26
1.38
1.32
1.38
1.44
1.59
1.66
1.49
1.56
1.46
1.52
1.63
2.10
2.10
1.92
1.98
1.98
2.10
1.80
1.80
1.98
1.86
2.10
1.80
2.10
1.86
1.98
1.80
1.98
1.80
1.96
1.50
1.32
1.56
1.56
1.44
1.56
1.50
1.26
1.32
1.32
1.56
1.50
1.38
1.44
1.38
1.50
1.50
1.44
1.43
1.51
1.48
1.72
1.59
1.59
1.57
1.56
1.59
1.68
1.54
1.67
1.69
1.60
1.72
1.59
1.42
1.65
1.55
1.59
85

Table 24. Egg size data for Hypsophrys nicaraguense spawned on 6 March, 2010.
Egg Length Width Effective
Number
1
(mm)
2.46
(mm)
1.80
Diameter (mm)
2.00
2
3
2.34
2.58
1.80
1.50
1.96
1.80
4
5
2.46
2.40
1.74
1.68
1.95
1.89
10
11
12
6
7
8
9
2.52
2.52
2.40
2.62
2.46
2.52
2.58
1.80
1.68
1.92
1.74
1.80
1.80
1.62
2.01
1.92
2.07
1.99
2.00
2.01
1.89
17
18
19
20
Average
13
14
15
16
2.40
2.40
2.34
2.40
2.46
2.40
2.46
2.34
2.45
1.68
1.80
1.56
1.68
1.80
1.74
1.80
1.68
1.73
1.89
1.98
1.79
1.89
2.00
1.94
2.00
1.88
1.94
86

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 25. Egg size data for Hypsophrys nicaraguense spawned on 28 March, 2010.
Egg Length Width Effective
Number
1
(mm)
2.82
(mm)
1.86
Diameter (mm)
2.14
2
3
2.70
2.70
1.80
1.92
2.06
2.15
4
5
2.76
2.82
1.86
1.92
2.12
2.18
10
11
12
6
7
8
9
2.70
2.76
2.64
2.58
2.70
2.85
2.70
1.80
1.92
1.86
1.86
1.92
1.86
1.92
2.06
2.17
2.09
2.07
2.15
2.14
2.15
2.70
2.76
2.76
2.64
2.82
2.88
2.56
2.76
2.70
2.82
2.76
2.65
2.65
2.82
2.58
2.70
2.70
2.64
2.72
2.10
2.46
1.92
1.92
1.80
2.04
1.92
1.74
1.80
1.74
1.92
2.04
1.98
1.80
1.92
1.86
1.86
1.86
1.91
2.06
2.04
2.17
2.23
2.18
2.09
2.12
2.11
2.28
2.56
2.17
2.14
2.09
2.29
2.11
2.03
2.11
2.09
2.14
87

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 26. Egg size data for Hypsophrys nicaraguense spawned on 22 April, 2010.
Egg Length Width Effective
Number
1
(mm)
2.34
(mm)
1.62
Diameter (mm)
1.83
2
3
2.22
2.34
1.56
1.56
1.75
1.79
4
5
2.28
2.40
1.68
1.68
1.86
1.89
10
11
12
6
7
8
9
2.22
2.28
2.16
2.28
2.28
2.46
2.34
1.56
1.62
1.62
1.68
1.62
1.62
1.50
1.75
1.82
1.78
1.86
1.82
1.86
1.74
2.28
2.34
2.40
2.40
2.28
1.86
2.40
2.28
2.28
2.40
2.40
1.92
2.22
2.22
2.34
2.16
2.34
2.22
2.28
1.62
1.50
1.56
1.56
1.62
1.68
1.62
1.56
1.68
1.62
1.50
1.68
1.56
1.56
1.62
1.50
1.68
1.50
1.60
1.86
1.85
1.75
1.76
1.75
1.75
1.83
1.69
1.82
1.74
1.80
1.80
1.82
1.74
1.85
1.77
1.88
1.71
1.80
88

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 26. Egg size data for Neetroplus nematopus spawned on 27 January, 2010.
Egg Length Width Effective
Number
1
(mm)
2.46
(mm)
1.86
Diameter (mm)
2.04
2
3
2.65
2.52
1.80
1.80
2.05
2.01
4
5
2.65
2.52
1.86
1.68
2.09
1.92
10
11
12
6
7
8
9
2.40
2.65
2.65
2.52
2.52
2.52
2.58
1.62
1.80
1.74
1.86
1.92
1.86
1.80
1.85
2.05
2.00
2.06
2.10
2.06
2.03
2.52
2.40
2.70
2.64
2.64
2.40
2.76
2.52
2.58
2.52
2.52
2.40
2.52
2.65
2.70
2.52
2.46
2.58
2.56
1.86
1.98
1.80
1.92
1.68
1.80
1.68
1.80
1.86
1.92
1.68
1.74
1.98
1.92
1.80
1.74
1.86
1.80
1.81
2.07
2.10
1.92
1.94
2.15
2.14
2.06
1.97
2.06
2.11
2.06
2.14
1.95
1.98
1.98
2.01
2.04
2.03
2.03
89

25
26
27
28
21
22
23
24
17
18
19
20
13
14
15
16
29
30
Average
Table 27. Egg size data for Steatocranus casuarius spawned on 28 February, 2010.
Egg Length Width Effective
Number
1
(mm)
3.36
(mm)
2.16
Diameter (mm)
2.50
2
3
2.82
3.24
2.40
2.34
2.53
2.61
4
5
3.06
3.36
2.16
2.34
2.43
2.64
10
11
12
6
7
8
9
2.94
3.48
3.00
3.12
2.52
3.18
2.88
2.34
2.16
2.34
2.52
2.25
2.16
2.04
2.52
2.53
2.54
2.71
2.34
2.46
2.29
3.12
3.00
2.82
3.06
3.24
2.82
2.94
3.30
3.12
3.36
3.30
3.30
3.24
3.30
3.00
3.12
3.36
3.36
3.12
2.40
2.22
2.22
2.40
2.28
2.40
2.28
2.22
2.22
2.10
2.40
2.28
2.16
2.16
2.46
2.34
2.22
2.28
2.28
2.49
2.46
2.67
2.58
2.47
2.49
2.63
2.58
2.62
2.45
2.40
2.60
2.56
2.53
2.48
2.53
2.55
2.59
2.53
90

91
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