Oomycete Infection of Convict Cichlid Eggs

THE INFLUENCE OF PARENTAL CARE BEHAVIOR BY CONVICT CICHLIDS ON THE INCIDENCE OF OOMYCETE INFECTION OF THEIR EGGS

Lesley Lynne Keiko Hamamoto B.S., University of California, Davis, 2001

THESIS

Submitted in partial satisfaction of the requirements for the degree of

MASTER OF SCIENCE

BIOLOGICAL SCIENCES (Biological Conservation)

CALIFORNIA STATE UNIVERSITY, SACRAMENTO

SPRING

2010

iii

THE INFLUENCE OF PARENTAL CARE BEHAVIOR BY CONVICT CICHLIDS ON THE INCIDENCE OF OOMYCETE INFECTION OF THEIR EGGS

A Thesis

Lesley Lynne Keiko Hamamoto

Approved by:

______, Committee Chair Ronald M. Coleman, PhD

______, Second Reader Jamie M. Kneitel, PhD

______, Third Reader James W. Baxter, PhD

Date:______

iii

Student: Lesley Lynne Keiko Hamamoto

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 ______James W. Baxter, PhD Date

Department of Biological Sciences

Abstract

THE INFLUENCE OF PARENTAL CARE BEHAVIOR BY CONVICT CICHLIDS ON THE INCIDENCE OF OOMYCETE INFECTION OF THEIR EGGS

Lesley Lynne Keiko Hamamoto

Infection of fish eggs by oomycete watermolds has been documented among numerous fish species occupying diverse aquatic habitats. In fact, watermolds are considered to be ubiquitous in freshwater systems and it seems that all species of fish eggs are susceptible to infection. Oomycete infection can result in the loss of large numbers of viable eggs because it can quickly spread from one infected egg to many others. To date, the majority of studies have been conducted using salmonid eggs under artificial rearing conditions, and there has been virtually no research on reproductive ecology or parental care behavior in fish as it relates to watermold infection. Additionally, few studies have utilized microscopy to elucidate the causes or pathways of infection.

My research project had two major objectives. First, I looked at two aspects of convict cichlid (Archocentrus nigrofasciatus) behavior, fanning and

egg cleaning, in an attempt to quantify the individual and collective effectiveness of each behavior in preventing the spread of infection within a clutch of eggs. Effectiveness was evaluated by comparing egg mortality under different care regimes. Second, I used microscopy and histology techniques to look at and pictorially document modes of egg infection and spatial patterns of egg mortality.

My evaluations of parental care effects on watermold infection did not yield any statistically significant differences between treatments, possibly due to unforeseen design flaws and inadequately controlled variables. I discuss these flaws and offer suggestions for additional research that will provide a reference for future studies on this important topic.

Additionally, egg samples were evaluated using a variety of histologic and microscopic techniques, including scanning electron microscopy, mortal staining, and paraffin sectioning in an attempt to elucidate the oomycete’s modes of infection and spread. I present the results of this study as a photographic atlas, which may lead to a better understanding of this phenomenon and suggest alternative methods for control.

Although the results of my study do not provide definitive approaches toward controlling oomycete infection, they do contribute to the limited body of information on the incidence of watermold infection in fish eggs.

______, Committee Chair Ronald M. Coleman, PhD

ACKNOWLEDGEMENTS

I would like to acknowledge and thank the Pacific Coast Cichlid Association

Mark Tomasello Research Fund and the American Cichlid Association Guy

Jordan Endowment Fund for their generous financial support for this project and the Albert Delisle Family Scholarship for their contribution toward my academic expenses. I would like to thank Jim Ster from the CSUS Engineering

Department and Grete Adamson and Pat Kysar from the UC Davis School of

Medicine Electron Microscopy Laboratory for their assistance with scanning electron microscopy, and Dr. Judy Jernstedt from the UC Davis Plant Sciences

Department and Sue Nichol from the UC Davis Plant Biology Department for their assistance and contributions toward histology and sectioning. I would like to thank the members of my graduate committee, Dr. Jamie Kneitel and Dr.

James Baxter, for their support and assistance and their thoughtful comments on various drafts of my thesis. Lastly, I would like to express my extreme gratitude to my committee chair, Dr. Ronald Coleman, whose help, guidance, and persistent encouragement have gotten me through this process.

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TABLE OF CONTENTS Page

Acknowledgements ...... vii

Chapter 1 ...... 1

INTRODUCTION ...... 1

Definition of Parental Care ...... 1

Egg Laying and Parental Care Behavior by Convict Cichlids ...... 2

Parental Care and Pathogenic Infection ...... 4

Pathogenic Oomycete Watermolds ...... 5

Oomycete Infection ...... 6

Hypotheses ...... 8

MATERIALS AND METHODS ...... 10

Oomycete Culture ...... 10

Aquarium Set-Up ...... 10

Experimental Design ...... 13

Data Collection...... 16

Data Analyses...... 16

RESULTS ...... 20

Comparison of Egg Survival Among Treatments ...... 20

Comparison of Egg Survival with Respect to Distance ...... 20 viii

DISCUSSION ...... 27

Comparison of Egg Survival Among Treatments ...... 27

Comparison of Egg Survival with Respect to Distance ...... 29

Suggestions for Future Research ...... 32

Chapter 2 ...... 34

INTRODUCTION ...... 34

MATERIALS AND METHODS ...... 35

Oomycete Culture and Aquarium Set-Up ...... 35

Histology and Microscopy ...... 35

RESULTS ...... 38

DISCUSSION ...... 46

Scanning Electron Microscopy ...... 46

Mortal Staining with Evan’s Blue ...... 47

Paraffin Sectioning ...... 47

Progression of Infection over Time ...... 48

Appendices ...... 50

Appendix A. Egg Count Data for Survival Analysis ...... 51

Appendix B. Egg Count Data for Proximity Analysis ...... 52

Literature Cited ...... 56

LIST OF TABLES

Page

Table 1. ANOVA (single-factor) summary for the comparison of cichlid egg survival among different parental care treatments…………..…...... 22

Table 2. ANCOVA summary for the comparison of cichlid egg survival among different parental care treatments ……...……………….……….24

Table 3. ANOVA summary for the comparison of percent egg mortality in inner circles (near inoculation point) versus outer circles (farther from inoculation point)....……………………………………………….26

LIST OF FIGURES Page

Figure 1. Spawning structures were constructed as a substrate for egg-laying ...... 12

Figure 2. Exclusionary barriers were used to restrict parental access to egg clutches...... 14

Figure 3. An example of an image used to identify and mark eggs for spatial analyses ...... 18

Figure 4. Comparison of mean cichlid egg survival under different parental care treatments ...... 21

Figure 5. Graphs showing decrease in egg survival over time under different parental care treatments ...... 23

Figure 6. Comparison of percent egg mortality in inner circles versus outer circles for different parental care treatments ...... 25

Figure 7. Preliminary SEM work conducted using critical-point dried rainbow cichlid (Herotilapia multispinosa) eggs...... 39

Figure 8. Preliminary SEM work conducted using convict cichlid eggs that were air dried directly from 100% ethanol ...... 40

Figure 9. Scanning electron micrographs of convict cichlid eggs air-dried after infiltration with hexamethyldisilazane (HMDS) ...... 41

Figure 10. Scanning electron micrographs of convict cichlid eggs air-dried after infiltration with hexamethyldisilazane (HMDS) ...... 42

Figure 11. Micrographs showing eggs stained with Evan’s Blue...... 43

Figure 12. Micrographs of paraffin-sectioned eggs...... 44

Figure 13. An example photo series documenting the progression of infection throughout a clutch of eggs and graph showing egg mortality over time...... 45

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Chapter 1

INTRODUCTION

Definition of Parental Care

Parental care is defined by Trivers (1972) 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.” In its broadest sense, parental care can include the preparation of nests or burrows, the production of heavily yolked eggs, the nourishment of eggs or young inside or outside of the parent’s body, or the provisioning of young before or after birth. In a stricter sense, parental care refers only to the care of young once they are detached from the parent’s body (Clutton-Brock 1991). The benefits of parental care to the care-giver are most often measured in terms of the survival, growth and eventual breeding success of its progeny (Clutton-Brock 1991).

There is abundant evidence that parental care can have substantial beneficial effects on the offspring, and that the benefit may influence the offspring’s entire life history. Because most studies on parental care are confined to particular life stages of the offspring, the overall benefit of parental care may be underestimated if the effects are measured by a single component of fitness

(Clutton-Brock 1991). Although the benefits of parental care are evidently large,

we know little about the direct relationship between parental expenditure and offspring fitness. It seems likely that this relationship is commonly non-linear and complicated by many varying factors (Clutton-Brock 1991).

Fishes have several characteristics that make them ideal subjects for the study of parental care. Fishes exhibit considerable diversity in their states of parental care; these states, ranked in order of their frequencies, are no care, male care, biparental care and female care. Additionally, many species adapt readily to the laboratory where variables may be more easily controlled or manipulated

(Sargent and Gross 1993).

Egg Laying and Parental Care Behavior by Convict Cichlids

Convict cichlids form monogamous pairs and both males and females participate in parental care (Galvani and Coleman 1998). Their care behavior includes cleaning the eggs with their mouths and fanning the eggs with their pectoral fins (Reebs and Colgan 1991). Egg cleaning, which is performed by an activity known as “mouthing”, helps to remove collected detritus from the eggs.

Additionally, inviable eggs are removed and eaten (Breder and Rosen 1966).

Collectively, this cleaning behavior may serve to reduce the incidence of oomycete infection by removing both propagules and oomycete nutrient sources. Egg fanning, accomplished by the parent fish repeatedly moving one or more fins over the eggs, is one of the most common forms of parental investment in fishes and

serves, in part, to facilitate gas exchange (Coleman and Fischer 1991). Fanning also appears to have a significant impact on embryo development. Previous studies suggest that unfanned eggs developed more slowly than fanned eggs (Coleman and

Fischer 1991). In a fanning study on pumpkinseed sunfish, unfanned eggs suffered

55% higher mortality than those that were fanned (Gross 1980).

Parental care behavior has a considerable cost in terms of reproductive investment (Coleman and Fischer 1991) and the return on this cost has not been evaluated. While the reproductive advantages of these two aspects of parental care behavior, mouthing and fanning, are by no means limited to the prevention of infection, the nature of the behavior combined with the life history of the watermold may have a negative effect on egg infection.

In addition to care behavior exhibited by the parent fish, convict cichlid eggs were selected for this study because they exhibit characteristics that facilitate the study of the progression of infection within a clutch of eggs. First, convicts are substrate spawners (Reebs and Colgan 1992). Unlike salmonid eggs, convict eggs are adhesive and, once laid, are fixed in place on a hard substrate. In nature, this substrate would usually be a rock cave or tree root. In the lab, a natural substrate can be mimicked by providing a terra-cotta flower pot or a plastic Petri dish. By using this kind of simulated substrate, the entire clutch may be easily removed and replaced without eggs shifting positions relative to each other. In this way, progression of infection to adjacent eggs can be observed over time. Second,

convict cichlid eggs are of a suitable size for the types of microscopy that I used.

Their 1.5 mm eggs are large enough to easily manipulate, yet small enough to allow magnified viewing of the watermold while retaining several eggs in the field of view.

Parental Care and Pathogenic Infection

Whereas there is a sizeable body of work that has looked at parental care by fishes in relation to predatory threats (e.g., Coleman, et al. 1985), there have been very few studies on the relationship between parental care, microbial infection and egg viability (Knouft et al. 2003). The aim of my thesis research was to evaluate the effects of parental care by convict cichlids (Archocentrus nigrofasciatus) on a pathogenic egg infection. To date, there have been no studies on this specific topic; however, there are some related works that have guided my study. A study conducted on bluegill sunfish (Lepomis macrochirus) showed that watermold infection of eggs was more prevalent in solitary nests than in colonial nests, and suggested that the difference was due to the fact that fish that nested in larger colonies spent less time chasing predators from their eggs and were able to devote more time to fanning their eggs (Côté and Gross 1993). Since fanning increases survivorship of eggs, fanning lessens the number of eggs that are most susceptible to watermold infection. It has also been suggested that fringed darters (Etheostoma crossopterum) may have antimicrobial compounds in their epidermal mucosa and

that their presence near their eggs, often interpreted as guarding, may provide an antimicrobial benefit (Knouft et al. 2003).

Pathogenic Oomycete Watermolds

Though the phenomenon of egg infection by oomycetes is often referred to as fungussing, the infection is actually caused by several species of oomycota in the family Saprolegniaceae, commonly called watermolds. The pathogens involved are in fact more closely related to the protistan chromophyte algae, a group that includes marine kelps, than they are to the “true” fungi in the kingdom Eufungi

(Burr and Beakes 1994).

The main growth form of oomycetes is the vegetative hyphae which form a mycelial mat to envelop and absorb nutrients from a food source. Additionally, oomycetes have complex life cycles that include a number of propagative stages.

The first of these, oospores, are rarely produced and only occur when certain environmental stressors initiate the sexual phase of the life cycle. More commonly, asexual reproduction produces either gemmae, which are small discrete branchlets of the hyphae, or primary zoospores, which are produced in modified hyphae called zoosporangia. The primary zoospore will quickly encyst, either in or near the zoosporangium. Subsequently, these may grow into a vegetative mycelium or else produce secondary zoospores. Secondary zoospores are laterally biflagellated, can remain motile for several days, and are considered to be the main dispersive stage.

While it has been suggested that zoospores are unable to infect live eggs, and that hyphae are responsible for infection spreading from dead eggs to live eggs

(Smith et al. 1985), the number of propagative stages in the life cycle makes it difficult to ascertain which stage(s) is (are) the cause(s) of infection in fish and their eggs (Noga 1993).

Oomycete Infection

Watermolds are widely distributed, and all freshwater fish and their eggs are susceptible to infection (Gaikowski et al. 2003, Noga 1993). This infection can result in serious losses in aquaculture production due to mortality of eggs and fish

(e.g., Gaikowski et al. 200, Schreier et al. 1996, Muzzarelli et al. 2001). Egg infection rates are increased in intensive aquaculture conditions, presumably because eggs are generally incubated at much higher densities than are found in the wild and water flow rates are often insufficient to prevent deposition of oomycete propagules. Mechanically damaged or inviable eggs provide excellent substrates for the initiation of infection, and mycelia may then spread to surrounding eggs.

For this reason, prophylactic chemical control is often applied (Gaikowski et al.

2003). Malachite green was formerly used as a watermold preventative until its use was banned by the Food and Drug Administration in 1991 due to its teratogenic, carcinogenic and residual effects. Currently, formalin is the only FDA approved chemical for preventative use against oomycetes on fish eggs, but the harmful

effects against human health make it a less than ideal option (e.g., Khomvilai et al.

2005). Because salmonid fishes are often produced in high-density artificial culture, a situation that can promote infection, and also have high commercial value as food and game fish, the majority of studies on egg infection have been carried out on artificially spawned salmon and trout eggs (e.g., Khomvilai et al. 2005,

Schreier et al. 1996, Smith et al. 1985). In contrast, there have been few studies on rates of infection or preventative measures against oomycete infection in the wild.

While salmon eggs have been the subject of many previous studies on this subject, salmon eggs are difficult to obtain and culture and have very long incubation times

(e.g., the time to 50% hatch for Chinook salmon (Oncorhynchus tshawytscha) eggs ranges from 159 days at 3° C to 32 days at 16°C (Healey 1991)). Alternatively, I chose to use the eggs of convict cichlids which are readily attainable in the lab, have much shorter incubation times and are more amenable to the types of manipulations that I intended to perform.

My research had two major objectives. First, to quantify the effects of mouthing and fanning on oomycete infection rates by comparing the percentage of infected eggs in clutches that were placed under different care regimes, and second, to look at the spatial nature of infection spreading throughout a clutch. By evaluating egg cleaning and fanning in relation to the spreading infection, the determining factors in allocation of reproductive investment in parental care by convict cichlids will be clarified.

Hypotheses

Oomycetes are known to disperse in several different ways, including vegetative spread by mycelial growth or by the release of motile zoospores. It has been suggested that fanning by parental fish may help to prevent zoospore deposition on their eggs (Côté and Gross 2003). Fanning is also attributed with greatly influencing the survivorship of eggs by facilitating vital gas exchange.

Another form of parental care behavior, egg cleaning, is thought to serve to remove dead eggs and debris which are potential infection sites for oomycetes. Based on these studies, I made the following hypotheses about the effects of parental care on egg infection:

• Fanning and cleaning of eggs together will be more effective against oomycete

infection than either fanning or cleaning alone.

• Cleaning behavior which includes removal of infected eggs will be more

effective against oomycete infection than fanning.

These hypotheses were tested by comparing the percentages of egg infection within clutches that were kept under one of five different care regimes: A) parental care; B) simulated cleaning; C) simulated fanning; D) simulated cleaning and fanning; or E) no care.

Since it has been suggested that zoospores are not capable of infecting live eggs, and that mycelial spread is responsible for spread from dead eggs to live eggs

(Smith et al. 1985), I hypothesized that:

• Eggs that are closer to an inoculated egg are more likely to become infected

than eggs that are farther away.

This hypothesis was tested by comparing the percentage of eggs that became infected within a given distance range from an infected egg.

MATERIALS AND METHODS

Oomycete Culture

I obtained an oomycete culture by transferring a naturally infected tank- spawned convict cichlid egg to F-13 medium (2% agar (w/v), 0.0015% peptone

(w/v) and 0.00004% maltose (w/v) in aqueous solution) for isolation (Miller and

Ristanovic 1969). After one week, a portion of the outermost edge of the mycelium was transferred to M-3 medium (1.7% corn meal agar (w/v), 0.001% peptone (w/v), 0.001% yeast extract (w/v), 0.005% glucose (w/v) and 0.005% starch (w/v) in aqueous solution) for sterile culture (Miller and Ristanovic 1969) and was maintained on the same medium with biweekly transfers. Cultures were stored under ambient temperature and lighting conditions in the lab. Because vegetative and asexual forms of oomycetes are indistinct across species and even genera, taxonomic identification can be difficult and often uncertain due to the rarity with which the sexual structures are produced (Olah and Farkas 1978). For this reason, I did not attempt to identify the cultured watermold.

Aquarium Set-Up

Six 75.8 L tanks were set up in the lab and each was supplied with gravel, a sponge filter, a heater to maintain water temperature above 25˚ C, and three plastic

Hygrophila plants to provide cover. Upper temperature limits were unregulated

except by the ambient temperature in the lab, and water temperature ranged up to

28° C. Each tank was wrapped with white plastic sheeting on three sides to prevent visual contact of fish between tanks. Traditionally, breeders use terra cotta flower pots as spawning substrates; however, for this study, a flat, transparent spawning surface was required. In initial trials, I used glass strips, but these were refused by the fish as spawning sites, perhaps because glass is too slick or because of its transparency. Moreover, the glass was difficult to break into pieces for microscopy work. To address these drawbacks, I tried square plastic Petri dishes that were abraded with 180 grit sandpaper to promote egg adherence and covered over with terra-cotta saucers to provide opacity. The Petri dishes could then be broken into small pieces easily and with minimal danger. Spawning structures were constructed using 100 x 100 x 15 mm square plastic Petri dishes, rigid plastic tubes

(ballpoint pen barrels) cut into 5.1 cm and 6.4 cm pieces, and aquarium sealant.

The base of each structure was made using the lid of a Petri dish as a foundation to which the tubes were affixed using aquarium sealant. The interior of each grid- marked portion of the Petri dish was abraded using 180-grit sandpaper to provide

Figure 1. Spawning structures were constructed as a substrate for egg-laying. Structures were made using square plastic Petri dishes, plastic tubes, aquarium sealant and terracotta saucers. The spawning structure on the right is shown with an exclusionary barrier in place.

surface texture for egg adherence. The grid-marked Petri dish was then set over the framework of plastic tubes and weighted with a 10.2 cm terracotta saucer so that it rested at approximately 30˚ from the bottom of the tank (Figure 1). Each pair of fish was provided with one spawning structure to simulate the convict cichlid’s natural cave-like spawning environment (Galvani and Coleman 1998). Fish were fed and inspected for evidence of spawning approximately every 12 hours. No more than two spawnings were used from each pair of fish; however, fish were sometimes re-paired with new mates.

Experimental Design

Egg clutches that were laid on Petri dishes were temporarily removed from the tank in such a way as to retain water in the Petri dish to cover the eggs. Egg clutches were reduced to 100 (+/-3) contiguous eggs by removing excess eggs from the periphery of the clutch prior to any other treatment. Each 100-egg clutch was photographed using a digital camera (Sony Cybershot DSC-P10, Sony Corporation of America) mounted on a tripod, and 5 eggs were randomly selected and marked on the image using Adobe Photoshop (CS2 9.0.2 and Elements 6.0, Adobe Systems

Incorporated). The corresponding eggs were then inoculated with watermold by puncturing them with a 25-gauge hypodermic needle that was drawn across a culture plate of watermold isolate. The Petri plate was then fitted with an

Figure 2. Exclusionary barriers were used to restrict parental access to egg clutches. Barriers were made from 10.2 cm squares of plastic needlepoint canvas (purchased as 4” squares) and cable ties. The device on the right was used as a control and was modified to allow parental access to the egg clutch.

exclusionary barrier made from 10.2 cm square plastic needle point canvas held together with plastic cable ties (Figure 2). Inoculated egg clutches were randomly assigned to one of the following treatments: A) eggs were returned to the parent fish with a modified barrier that allowed parental access to the eggs; B) eggs were returned to the parent fish with an exclusionary barrier preventing parental access, and with manual removal of dead and/or infected eggs twice daily to simulate cleaning; C) eggs were returned to the parent fish with an exclusionary barrier and a small powerhead increasing the flow of water over the eggs to simulate fanning;

D) eggs were returned to the parent fish with an exclusionary barrier and with simulated cleaning and fanning; or E) eggs were returned to the parent fish with an exclusionary barrier and without simulated cleaning or fanning behavior. An egg was considered to be dead if there were visible signs of mycelial infection or if the egg became opaque. Because temperature in the lab could not be tightly controlled in this experiment, and rate of egg development is highly dependent upon temperature, time could not be used as an accurate predictor of impending hatching.

Instead, I used a developmental marker, the appearance of dark pigmentation on the embryo’s yolk sac, to determine the end of the treatment period. This marker was used because it indicated that hatching would likely occur before the next observation period (approximately 12 hours). Treatment times ranged from 2 to

3.5 days.

Data Collection

I removed each sample clutch from the parent tank twice daily for observation and periodically photographed them (approximately once per day) to document the progression of infection. Because the early stages of oomycete infection on a given egg are difficult to confirm by visual observation, any egg that became opaque (signifying embryo death and egg membrane rupture) was considered for the purposes of the mortality study to be infected. Egg survival and mortality percentages were determined post-process by counting remaining transparent and visibly uninfected eggs in photographs. I obtained six samples from treatment A (parental care), and five samples each from treatments B

(simulated cleaning), C (simulated fanning), D (simulated cleaning and fanning), and E (no care).

Data Analyses

Comparison of survival among treatments. In order to test the effects of the five treatments on egg survival, I compared survival percentages at the end of the treatment period using a one-way (single-factor) analysis of variance (ANOVA). I also tested whether percent survival over time was affected by different parental care treatments by conducting an analysis of covariance (ANCOVA), using time as the covariate.

Comparison of survival with respect to distance from inoculation sites. To compare the incidence of infection relative to proximity to an infected egg, I constructed an image of two concentric circles that were sized so as to encompass the approximate area occupied by one and two layers of eggs surrounding an inoculated egg. This image was overlaid on the initial images of the egg clutches, and centered at each of the selected inoculation sites. I then identified and marked eggs as being within a circle if 50% or more of the egg mass fell inside the circle

(Figure 3). In cases where the eggs within one set of circles overlapped with those of another inoculation site, only one set of site data was counted. I compared each marked image with the corresponding final image of each sample and counted the number of viable eggs that remained within either of the two circles. I then took the sum of the infected and uninfected eggs in the inner circles versus the sum in the outer circles for each sample so that I had a total for each egg clutch (Appendix B).

Egg counts from multiple inoculation sites were summed for each sample in order to avoid pseudoreplication. These counts were used to calculate percent mortality for eggs that were close to an infection site (in inner circles) and farther away

(outer circles). This proximity data was analyzed between treatments using a two- way ANOVA (two-factor with replication). One randomly selected sample from treatment A was excluded from this analysis in order to use an equal number of replicates from each treatment (Appendix B).

Figure 3. An example of an image used to identify and mark eggs for spatial analyses. Concentric circles were centered at each inoculation point and were used to delineate eggs to be counted in close proximity to an inoculated egg (inner circle) versus those that were farther away (outer circle). Black X’s indicate eggs that were counted as nearest to the inoculation site. White X’s indicate eggs that were counted as farther away. Sample C3.

Statistical Analyses. ANOVAs were conducted using Microsoft Excel 2007

(Microsoft Corporation) and the ANCOVA was conducted using SPSS Statistics

(SPSS, An IBM Company).

RESULTS

Comparison of Egg Survival Among Treatments

Survival of cichlid eggs among the five parental care treatments did not differ significantly (F0.05(4,21)= 0.38, P= 0.82) (Figure 4, Table 1). Although the

ANCOVA, which compared egg survival over time for each treatment, showed that survival differed significantly, this difference was attributable only to the time effect (F0.05(1) = 97.808, P<0.001), and was not attributable to different parental care treatments (F0.05(4)=0.306, P= 0.873) (Figure 5, Table 2).

Comparison of Survival with Respect to Distance

Percent egg mortality did not differ significantly between eggs in the inner versus outer circle among parental care treatments (all F< F critical, all P>> 0.05,

α= 0.05) (Figure 6, Table 3).

Figure 4. Comparison of mean cichlid egg survival under different parental care treatments. Egg survival did not differ significantly (P= 0.82) under care treatments which included, A) parental care (n=6), B) simulated cleaning (n=5), C) simulated fanning (n=5), D) simulated cleaning and fanning (n=5) or E) no care (n=5). Error bar= ± 1 standard error.

ANOVA Source of Variation SS df MS F P-value F crit Between Groups 62.92821 4 15.73205 0.382361 0.818712 2.8401 Within Groups 864.0333 21 41.14444

Total 926.9615 25

Table 1. ANOVA (single-factor) summary for the comparison of cichlid egg survival among different parental care treatments. A) parental care, B) simulated cleaning, C) simulated fanning, D) simulated cleaning and fanning or E) no care. α= 0.05. (SS= sum of squares, df= degrees of freedom, MS= mean square).

Figure 5. Graphs showing decrease in egg survival over time under different parental care treatments. Regression lines are included for each treatment. A) parental care, P< 0.001, R2= 0.58, n=6; B) simulated cleaning, P< 0.001, R2= 0.77, n=5; C) simulated fanning, P< 0.001, R2= 0.68, n=5; D) simulated cleaning and fanning, P< 0.001, R2=0.57, n=5; or E) no care, P=0.03, R2= 0.65, n=5. Treatment days were counted from the time that the eggs were observed and assigned to one of the parental care regimes.

ANCOVA Source Type III SS df MS F P-value Corrected Model 0.168a 5 0.034 20.1 <0.001 Intercept 33.876 1 33.876 20242.673 <0.001 Treatment Days 0.164 1 0.164 97.808 <0.001 Care Treatment 0.002 4 0.001 0.306 0.873 Error 0.122 73 0.002 Total 69.982 79 Corrected Total 0.29 78 a. R squared= 0.579 (Adjusted R Squared= 0.550)

Table 2. ANCOVA summary for the comparison of cichlid egg survival among different parental care treatments. Time was included as a covariate. (SS= sum of squares, df= degrees of freedom, MS= mean square)

Figure 6. Comparison of percent egg mortality in inner circles versus outer circles for different parental care treatments: A) parental care, B) simulated cleaning, C) simulated fanning, D) simulated cleaning and fanning or E) no care. Error bar= ±1 standard error.

ANOVA Source of Variation SS df MS F P-value F crit Care treatment 0.296595 4 0.074149 1.916275 0.126507 2.605975 Inner vs. outer 0.073353 1 0.073353 1.895715 0.176216 4.084746 Interaction 0.105569 4 0.026392 0.682069 0.608502 2.605975 Within 1.54777 40 0.038694

Total 2.023287 49

Table 3. ANOVA summary for the comparison of percent egg mortality in inner circles (near inoculation point) versus outer circles (farther from inoculation point). α = 0.05. (SS= sum of squares, df= degrees of freedom, MS= mean square)

DISCUSSION

Comparison of Egg Survival Among Treatments

Because there was no significant difference in the survival of convict cichlid eggs under different parental care treatments, I cannot reject the null hypothesis. Previous research conducted on fish eggs in aquaculture situations

(e.g. Khomvilai et al. 2005, Schreier et al. 1996, Smith et al. 1985) and colonial bluegill sunfish (Côté and Gross, 1993) stated that low water flow contributed to higher incidences of egg infection by oomycetes. It was suggested that low water flow contributed to higher rates of infection either by allowing watermold propagules to settle on and infect eggs (Gaikowski et al. 2003), or by promoting the demise of viable eggs due to inadequate gas exchange (Coleman and Fischer 1991,

Cote and Gross 1993). Based on these studies, I hypothesized that eggs that were fanned either by parent fish or by the use of a mechanical water pump would have lower infection rates. Additionally, previous experiments suggested that infection generally spread by mycelial growth from inviable eggs to viable eggs, eventually suffocating and killing them (e.g., Smith et al. 1985). Thus, one would expect that egg cleaning (which includes the removal of dead eggs that are thought to be the initial source(s) of infection within a clutch) would significantly lessen the spread of infection. The results of my experiment did not support either of these claims.

The results of my analyses indicated that the aspects of parental care behavior that I

studied (egg cleaning and fanning) had no effect on the incidence of egg infection or overall egg survival. Although this analysis was not able to detect a significant difference between the oomycete infection rates of eggs under different parental care regimes, it would seem likely that highly energetically expensive care behavior such as fanning and cleaning would decline if there were no benefit to the survival of the offspring; therefore, there is a possibility that, despite the lack significant results in this experiment, the care behavior has benefits that were not detected by measurement of egg infection alone. It is also possible that a Type II error was committed in this case.

As an alternative explanation, Knouft, et al. (2003) tested the antimicrobial properties of epidermal mucous from the fringed darter fish (Etheostoma crossopterum) and found that the mucous did in fact have a cytotoxic effect on both bacteria (Salmonella typhimurium) and watermold (Saprolegnia spp.). From this result, the authors set forth the idea that the mere presence of a guarding parent may, in itself, provide parental care in the form of infection prevention by virtue of the antimicrobial mucous on the parent’s skin, and that fanning or cleaning may not actually be the effective components of the care behavior, but merely the means of applying epidermal mucous. Given that treatment A, which allowed parental access to the eggs, did not show significantly lower incidences of infection, it would seem that the application of epidermal mucous was not a factor in the results of this experiment.

On a qualitative level, the exclusionary barriers that I constructed seemed to work as expected. However, although the exclusionary barriers absolutely prevented parent fish from cleaning the eggs, parent fish continued to fan outside of the barriers. It is feasible that the distance between the eggs and the parent fish was not sufficient to render fanning useless; or conversely, that the devices may have disrupted laminar flow from electric powerheads, causing pockets of stagnant water. If either of these situations did occur, then the conclusion that fanning does not negatively affect rates of egg infection would not be justified.

Comparison of Egg Survival with Respect to Distance

An analysis of proximity showed no difference in the infection rates for eggs that are closer to an infected egg than those that are farther away. While I had expected that eggs that were closer to an inoculation site would be more likely to become infected due to the mat-like mycelial growth pattern of the watermold, which spreads out from an initial infection site, this expectation did not hold true.

There was no significant difference in egg mortality between eggs that were closer to an inoculation site and those that were farther away. This result suggests that mycelial growth and spread from dead eggs to live eggs was not the main mode of infection under my experimental conditions. One of the confounding factors in this analysis was that I had not anticipated testing my data in this manner. There was

very limited space between inoculation sites. Therefore, distance effects may have been obscured by the presence of other nearby inoculation sites.

In a study conducted on the eggs of perch (Perca fluviatilis), Paxton and

Willoughby (2000) found that infection did not spread from infertile eggs that were in close contact with fertile, developing ones, even though the fertile eggs of salmonids were readily colonized by infected neighbors. Consequently, the authors hypothesized that the egg masses of perch may have antifungal properties, which prevent the growth and spread of watermolds. Given that fertile eggs succumbed to mycelial infection during my experiment, it is not valid to assume that convict cichlid eggs exhibit similar antifungal properties.

My research focused only on physical methods of preventing watermold infection and did not address chemical controls, either introduced by inherent means such as those suggested by Knouft et al. (2003) or Paxton and Willoughby

(2000), or by the deliberate addition of fungicides (e.g., Khomvilai et al. 2005,

Gaikowski et al. 2003). However, it is evident that chemical cues and inhibitors are potentially a major component in the analysis of this complicated issue. While chemical studies offer an additional path of investigation, the potential presence of such chemicals can also serve to confuse the results of studies addressing physical methods of control. The intent of my study was simply to investigate the impact of aspects of parental care behavior that I determined to be likely to have an effect on

the incidence of infection. For this reason, additional investigations into chemical interactions were not pursued.

While I cannot draw any definitive conclusions from the results of my experiments about the value of egg cleaning and fanning on watermold infection, the principles behind the evolutionary stability of parental care behavior suggest that any care that is given should have a positive net effect on offspring survival. If we assume that care must have a benefit on offspring survival, the results of this study illustrate the problem that was discussed in the introduction: testing the effects of parental care on one life stage does not accurately demonstrate the effects of care on lifetime fitness.

The possible causes and remedies for watermold infection are vast, and there are many aspects that are still unstudied. The problem of oomycete infection of fish eggs warrants additional attention, particularly as many species decline in the wild and captive rearing projects become more necessary. Locally, the decline of the Sacramento-San Joaquin Delta fisheries has become a prominent issue and captive rearing programs for many species such as delta smelt (Hypomesus transpacificus), steelhead (Oncorhynchus mykiss), and Chinook salmon

(Oncorhynchus tshawytscha) are either ongoing or planned for the near future.

Though the analyses that I conducted in this study yielded no statistically significant results, this work has provided a useful framework and tested methods that would be worth pursuing with some modifications to experimental design. I

have identified three factors that may have adversely affected the outcome of this research, the correction of which could improve future attempts.

Suggestions for Future Research

Treatment duration. One factor that may have impacted the success of this study is that the egg incubation time for convict cichlids was so short. Treatment duration was limited by the incubation time which, under the temperature conditions in this experiment, averaged only 2.58 days. A longer incubation time, which could be achieved by using eggs of a different species of fish or by maintaining cooler water temperatures, may have helped to better magnify the effects of parental care behavior on infection by allowing a greater period of exposure to both the care behavior and to the infective organism. Also, longer treatment duration would have allowed more time for development of mycelia, allowing better discrimination between egg mortality due to oomycete infection and mortality due to other causes.

Water temperature. Water temperature was regulated by the use of small dual-temperature-setting aquarium heaters which are only capable of increasing water temperature, not lowering it. Water temperature was strongly influenced by the ambient room temperature of the lab, which ranged widely over the course of my data collection. Additionally, the dual-temperature control did not allow for

adjustment of calibration, so although the heater was set to maintain water temperature at or above 25° C, water temperature occasionally ranged as low as 23°

C. Egg development rate is strongly influenced by water temperature (Coleman

1996), and temperature also affects growth rates of watermolds (Olah and Farkas

1978). Water temperature may also have had a cumulative effect by impacting the susceptibility of eggs to infection. More rigorous control of water temperature would have helped to reduce variability by standardizing rates of egg development and by eliminating potential temperature effects on the growth and propagation of the oomycete. Control of this variable would be particularly important in any future attempts that utilize a longer treatment period since the effects would likely be magnified over time.

Number of inoculation sites. A single inoculation point within a clutch of eggs would have allowed for better analysis of proximity effects on infection rates by eliminating the interference that multiple inoculation points created. Also, a greater number of distance ranges could have helped to more accurately identify a critical distance at which infection is reduced. Using a species of egg with a longer incubation period and a single inoculation point would be the best way to test distance effects on infection rates.

Chapter 2

INTRODUCTION

The second portion of my thesis was to develop a pictorial atlas utilizing microscopic and histologic evidence to document modes of watermold infection and patterns of egg mortality due to infection. For this portion of my project, I utilized light microscopy, scanning electron microscopy and histology techniques such as vital staining and serial sectioning. Convict cichlid eggs are an ideal choice for this type of application since the adhesive eggs allow you to view the eggs without altering their relative positions. Additionally, convict cichlid eggs which are approximately 1.5 mm in diameter (Coleman, 1996), are appropriately sized for these types of microscopy work. They are large enough to manipulate easily, yet are small enough to view multiple eggs in a single field of view under magnification that allows viewing of the watermold.

The objective of the pictorial atlas was to provide visual documentation of watermold infection using common histological techniques. Many of these techniques have not been previously employed to address the particular issue of watermold infection on fish eggs and so testing these methods allowed me to provide an appraisal of techniques that could be used by other researchers to further studies into this phenomenon.

MATERIALS AND METHODS

Oomycete Culture and Aquarium Set-Up

Samples of convict cichlid eggs for microscopic and histologic evaluation were obtained simultaneously with the samples that were used for egg infection rate analysis, using the same methods as outlined in Chapter 1. Inoculated samples were observed and prepared for histological procedures at varying times throughout incubation, depending on the infection characteristics that were to be investigated.

Histology and Microscopy

Scanning electron microscopy. Scanning electron microscopy was conducted in three separate trials using different sample preparation methods.

Initial test samples were prepared using rainbow cichlid (Herotilapia multispinosa) eggs that were removed from a glass substrate and fixed in a solution of 2.4% glutaraldehyde, 0.3% paraformaldehyde and 0.025M PIPES [piperazine-N, N'-bis

(2-ethanesulfonic acid )] buffer (pH 7.2) at room temperature for a minimum of 24 hours. Samples were rinsed three times in PIPES buffer and passed through an ethanol dehydration series from 10%-100% at 10% increments. Eggs were dried in a Tousimis Samdri critical point dryer (Rockville, MD, USA), mounted on aluminum stubs using adhesive carbon tabs and gold coated in a Denton Vacuum

Desk II cold-sputter etch unit (Denton Vacuum Inc, Moorestown, NJ, USA).

Specimens were viewed and photographed using a Hitachi S-3500N scanning electron microscope (Hitachi High-Technologies America, Pleasanton, CA, USA).

Test samples were prepared and viewed at the UC Davis Section of Plant Biology

Electron Microscopy lab.

In-situ samples of infected and visibly uninfected convict cichlid eggs were prepared for scanning electron microscopy by cutting the plastic Petri dish containing the egg clutch into approximately 1 cm sized pieces. Samples were fixed in a formalin acetic acid (FAA) solution (50 ml 95% ethanol, 5 ml glacial acetic acid, 10 ml 37% formalin formaldehyde, 35 ml water) (Ruzin 1999), then dehydrated through a graded ethanol series (50%, 70%, 90%,100%). Some samples were air dried from this point, mounted to aluminum stubs with carbon tape and gold coated in a Bio-Rad R5100 SEM Coating System (Bio-Rad,

Hercules, CA). These specimens were viewed in a Zeiss Digital Scanning Electron

Microscope, model DSM 940 (Carl Zeiss SMT, Germany). Samples were processed and viewed at the CSUS Engineering Department SEM lab with the assistance of James Ster. Additional samples were transitioned from 100% ethanol to 100% hexamethydisilazane (HMDS) (Electron Microscopy Sciences, Hatfield,

PA) through a graded series (3:1, 1:1, 1:3) at 30 minute intervals followed by three changes of pure HMDS. Samples were air-dried and mounted to aluminum stubs with carbon tape, and gold coated in a Pelco Auto Sputter Coater SC-7 (Ted Pella

Inc., Redding, CA). These samples were prepared and viewed at the University of

California at Davis School of Medicine Department of Medical Pathology and

Laboratory Medicine Electron Microscopy Laboratory with the assistance of

Patricia Kysar.

Paraffin sectioning. Infected eggs and eggs that were adjacent to infected eggs were prepared for sectioning by removing individual eggs from the Petri dish substrate with a small metal spatula. Eggs were fixed in FAA for a minimum of 24 hours, rinsed in 50% ethanol, dehydrated in tert-butyl alcohol through a graded series over a period of three days and infiltrated with paraffin. Eggs were embedded and then serial-sectioned at 10 µm using a rotary microtome. Sections were mounted on slides coated with Haupt’s A adhesive (Ruzin 1999) and stained using toluidine blue O.

Mortal staining. Inoculated egg clutches were stained with 0.01% Evan’s Blue for 10 minutes, then rinsed with tank water and observed under a dissecting microscope (National Optical & Scientific Instruments, Inc. 420T-430PHF-10).

Evan’s blue is a mortal stain which is taken up by organic material, but is excluded from cells with a functional cell membrane (Gallagher 1984). Photographs were taken with a digital camera (Sony Cybershot DCS-P10) mounted on a tripod.

Micrographs were taken by holding the camera lens up to the right ocular of the microscope.

RESULTS

The results of my microscopic and histologic evaluations are presented here in the form of a photographic atlas (Figures 7-12). Additionally, I have included a graph showing egg mortality over time and the accompanying series of images that document the spread of infection through a clutch of eggs that is likely the result of infection of live eggs by spreading mycelia.

A B

C D

Figure 7. Preliminary SEM work conducted using critical-point dried rainbow cichlid (Herotilapia multispinosa) eggs. Critical point drying produces relatively uniform dehydration of the egg membrane and good preservation of the mycelium. A) An egg at 50X magnification. B) An egg with a developing watermold mycelium at 50x magnification. C) Same as B at 150x magnification. D) Watermold mycelium growing on egg surface at 1000x magnification.

A B

Figure 8. Preliminary SEM work conducted using convict cichlid eggs that were air dried directly from 100% ethanol. A) These eggs show significant pitting of egg membrane. B) Watermold that was attached to an egg shows shrinkage of the mycelium after processing.

A B

C D

Figure 9. Scanning electron micrographs of convict cichlid eggs air-dried after infiltration with hexamethyldisilazane (HMDS). A) Surface of an infected egg showing high concentrations of a bacillus-type bacterium. B) Oomycete hyphae attached to the outer surface of an egg. There is no evidence in this image of penetration into the egg membrane. C and D) Oomycete mycelia that have begun to engulf the egg.

A B

C D

Figure 10. Scanning electron micrographs of convict cichlid eggs air-dried after infiltration with hexamethyldisilazane (HMDS). A and B) Oomycete hypha apparently penetrating the egg membrane. C and D) Oomycete zoosporangia, one of the asexual modes of dispersal.

A B

1 mm 1 mm C D

Figure 11. Micrographs showing eggs stained with Evan’s Blue. A) A stained clutch of inoculated eggs two days after inoculation. Two eggs have stained darkly, showing that they are inviable. B) A magnified view of an inviable egg and the halo of watermold growing from it. C) Adjacent eggs which are in contact with the infected egg are still viable and developing. The mycelium from a dead egg is growing toward the other eggs. D) Egg membranes of dead eggs were very friable and often hindered intact removal.

0.5 mm 20 µm B C

Figure 12. Micrographs of paraffin-sectioned eggs. A) Photo of a slide-mounted serially-sectioned sample of eggs that were adjacent to infected eggs, but were not visibly infected themselves. B) A representative sectioned egg showing friability of egg contents and distortion of section. C) A sectioned egg that shows evidence of hyphal growth interior to the egg membrane.

Egg Mortality Over Time

120 100 80 60 40 20

number of viable eggs viable of number 0 0 1 2 3 treatment day

Day 0 Day 1 Day 2 Day 3

Figure 13. An example photo series documenting the progression of infection throughout a clutch of eggs and graph showing egg mortality over time. Infection in this case spread radially from each of the five inoculation sites. This pattern would indicate spread of infection due to hyphal growth rather than dispersal by zoospores or gemmae. Day 0 marks the time of inoculation.

DISCUSSION

Scanning Electron Microscopy

Analysis of results. Scanning electron microscopy showed development of the watermold mycelium from a single point on an egg (Figures 7B and C). The initial growth appears to penetrate the egg membrane and extends to eventually cover the membrane surface (Figure 7D). Some eggs showed heavy surface coverage by rod-shaped bacteria (Figure 9A). Critical point drying and infiltration with HMDS dramatically improved the quality of the prepared samples over samples that were air-dried directly from 100% ethanol, producing less egg shrinkage and better preservation of the mycelium. Scanning electron microscopy of infected eggs produced images that captured two modes of infection spread, hyphal growth of the mycelium and production of zoospores. The images serve to confirm the suspicion that these two modes are important factors in the spread of the infection and that control should focus on limiting dispersal and production of these life stages.

Analysis of methods. Infiltration of samples with HMDS produced results that were approximately equivalent to critical point drying and was a much more accessible technique, because it did not require specialized equipment. Air-drying of eggs after fixation and dehydration through an ethanol series alone produced far

inferior results. Eggs that were dried by this method had membranes that showed pronounced pitting and oomycete hyphae appeared to have shrunken (Figure 8).

Mortal Staining with Evan’s Blue

Analysis of results. Mortal staining revealed that eggs that are in close contact with an infected egg are not immediately inviable (Figure 11B). This would suggest that the mycelium does not immediately penetrate and kill a healthy egg; however, infection does eventually result in the mycelium covering nearby eggs and causing their death. The watermold appears to be able to detect nearby eggs, as the mycelium can be seen growing toward them (Figure 11C). It was not possible in most cases to remove intact dead eggs from the spawning substrate, as the egg membrane was too friable to permit handling (Figure 11D).

Analysis of methods. Mortal staining with Evan’s blue worked well to distinguish dead eggs from living eggs. Additionally, the stain was useful in visualizing the watermold. This is probably caused by staining of a mucilaginous secretion exuded by the watermold.

Paraffin Sectioning

Analysis of results. In Figure 12C, it appears that watermold hyphae and zoosporangia are present on the interior of the egg membrane. This could give an

indication that the hyphae have a mechanism for penetrating an intact egg membrane, or could merely be a result of hyphal growth into the dead egg after the deterioration of the membrane.

Analysis of methods. The paraffin sectioning that I performed did not produce ideal results. Friability of egg contents and separation between the egg membrane and egg contents indicate that there were some problems with infiltration of paraffin into the egg (Figure 12B).

Progression of Infection over Time

Analysis of results. Images used to track progression over time showed high variability in the location and spread of infection. In general, it appeared that infection radiated out from inoculation sites, but statistical analyses of egg infection with respect to distance from inoculation site did not show significant differences.

The infection pattern in the case shown above (Figure 13) is what one would expect from progression of infection resulting from hyphal growth rather than dispersal of gemmae or zoospores.

Analysis of methods. The photography set-up that was used for this study was very successful in capturing progression of infection over time. The limitations of this method are that infection is not obvious until it is fairly advanced. Additionally, because the growth rate of the watermold is fairly fast and

the incubation period for the convict cichlid is relatively short, more frequent observations would have been helpful.

The results of the histology section of the project did not concretely demonstrate modes of infection or watermold dispersal with respect to egg infection, nor did they provide any immediate solutions for preventing infection in fish eggs. However, my results show that scanning electron microscopy using

HMDS provides more than adequate images for studying this phenomenon, and that light microscopy and serial photography using simple methods can produce good results. Better methods for investigating the interior of eggs are needed, as the fixation and infiltration methods that I used did not produce satisfactory results.

For this purpose, transmission electron microscopy is a technique that should be further explored, but this method was not readily available to me, and can be rather cost prohibitive. The histological methods that were used will doubtless have applications in other more detailed and focused studies and will hopefully serve to provide a body of experience that can be used to address this problem in the future.

APPENDICES

APPENDIX A

Egg Count Data for Survival Analysis

start date and 0 0.5 1 1.5 2 2.5 3 3.5 treatment tank # time days days day days days days days days A1 L5 6/23 am 98 95 94 A2 L1 7/10 am 100 94 93 A4 L4 9/4 pm 101 91 85 A5 L6 10/10 am 100 89 89 A6 L4 11/15 pm 100 84 76 A7 L5 11/15 pm 100 93 91 B1 L4 6/19 pm 100 95 B2 L5 7/4 pm 100 95 95 B3 L4 7/29 pm 100 96 92 90 84 82 B4 L5 10/27 am 100 95 89 86 B6 L3 11/19 am 100 95 93 C1 L2 6/22 pm 103 95 C2 L1 6/29 pm 100 86 C3 L3 7/22 pm 99 93 C4 L1 8/25 am 100 88 C5 L3 11/3 pm 103 98 78 D1 L1 6/20 pm 100 95 88 D2 L4 7/4 pm 99 93 92 D3 L6 8/3 am 100 95 93 92 D4 L6 8/19 am 100 95 94 D5 L4 8/20 am 100 95 92 E1 L3 6/23 am 100 94 94 E2 L3 7/5 pm 100 95 95 E3 L3 8/3 am 99 93 93 92 E4 L5 9/6 am 100 94 77

APPENDIX B

Egg Count Data for Proximity Analysis

# in # inviable # inviable % mortality % mortality inner in inner # in outer in outer in inner in outer Sample circle circle circle circle circle circle A2 1 4 0 10 0 A2 2 6 0 11 0 A2 total 10 0 21 0 0% 0% A4 1 4 0 7 0 A4 2 6 0 5 0 A4 total 10 0 12 0 0% 0% A5 1 3 0 6 0 A5 2 5 0 10 3 A5 3 4 0 5 0 A5 total 12 0 21 3 0% 14% A6 1 5 2 7 2 A6 2 6 2 8 0 A6 3 3 1 12 1 A6 total 14 5 27 3 36% 11% A7 1 5 0 7 0 A7 2 4 1 7 0 A7 3 4 0 3 0 A7 total 13 1 17 0 8% 0% B1 1 5 0 6 0 B1 2 5 0 9 0 B1 3 4 0 5 0 B1 total 14 0 20 0 0% 0% B2 1 4 0 7 0 B2 2 5 0 6 0 B2 total 9 0 13 0 0% 0%

Appendix B. Continued

# in # inviable # inviable % mortality % mortality inner in inner # in outer in outer in inner in outer Sample circle circle circle circle circle circle B3 1 7 4 5 1 B3 2 4 0 13 3 B3 3 5 1 7 0 B3 4 5 1 11 1 B3 total 21 6 36 5 29% 14% B4 1 4 0 8 4 B4 2 6 0 9 1 B4 3 4 0 4 0 B4 total 14 0 21 5 0% 24% B6 1 5 0 10 0 B6 2 4 0 9 1 B6 total 9 0 19 1 0% 5% C1 1 4 1 6 0 C1 2 4 1 5 0 C1 3 4 0 6 0 C1 total 12 2 17 0 17% 0% C2 1 5 2 11 0 C2 2 7 1 10 0 C2 3 5 4 9 0 C2 total 17 7 20 0 41% 0% C3 1 4 0 2 0 C3 2 4 0 3 0 C3 3 3 0 4 0 C3 4 3 0 9 0 C3 5 2 0 6 0 C3 total 16 0 24 0 0% 0% C4 1 7 1 4 0 C4 2 5 0 5 0 C4 3 2 2 5 0 C4 total 14 3 14 0 21% 0%

Appendix B. Continued

# in # inviable # inviable % mortality % mortality inner in inner # in outer in outer in inner in outer Sample circle circle circle circle circle circle C5 1 4 4 11 8 C5 2 4 4 8 6 C5 3 3 3 8 6 C5 total 11 11 27 20 100% 74% D1 1 5 0 7 1 D1 2 4 0 8 0 D1 3 6 1 6 0 D1 total 15 1 21 1 7% 5% D2 1 3 0 9 0 D2 2 2 0 11 0 D2 3 5 0 5 0 D2 total 10 0 25 0 0% 0% D3 1 5 0 7 0 D3 2 3 0 10 0 D3 3 5 1 9 1 D3 total 13 1 26 1 8% 4% D4 1 3 0 8 0 D4 2 4 0 11 0 D4 3 5 0 7 0 D4 4 3 0 4 0 D4 total 15 0 30 0 0% 0% D5 1 5 0 8 0 D5 2 4 0 10 0 D5 3 2 0 5 0 D5 total 11 0 23 0 0% 0% E1 1 3 0 8 0 E1 2 4 0 8 0 E1 3 4 0 8 0 E1 4 3 0 10 0 E1 total 14 0 34 0 0% 0%

Appendix B. Continued

# in # inviable # inviable % mortality % mortality inner in inner # in outer in outer in inner in outer Sample circle circle circle circle circle circle E2 1 5 0 8 0 E2 2 5 0 9 0 E2 3 4 0 12 0 E2 total 14 0 29 0 0% 0% E3 1 4 1 6 0 E3 2 4 0 7 0 E3 3 3 0 9 0 E3 4 6 0 4 0 E3 total 17 1 26 0 6% 0% E4 1 5 0 9 0 E4 2 5 1 8 0 E4 3 4 1 9 0 E4 total 14 2 26 0 14% 0% E5 1 3 3 6 3 E5 2 3 2 7 0 E5 3 3 1 7 0 E5 total 9 6 20 3 67% 15%

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