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
in
BIOLOGICAL SCIENCES
(Biological Conservation)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING

2010
iii

© 2010
Lesley Lynne Keiko Hamamoto
ALL RIGHTS RESERVED
ii

THE INFLUENCE OF PARENTAL CARE BEHAVIOR BY CONVICT
CICHLIDS ON THE INCIDENCE OF OOMYCETE INFECTION OF THEIR
EGGS
Approved by:
A Thesis
by
Lesley Lynne Keiko Hamamoto
__________________________________, 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
iv

Abstract
of
THE INFLUENCE OF PARENTAL CARE BEHAVIOR BY CONVICT
CICHLIDS ON THE INCIDENCE OF OOMYCETE INFECTION OF THEIR
EGGS
by
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
v

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.
vi

__________________________________, 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.
vii

TABLE OF CONTENTS
viii
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

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
ix

LIST OF TABLES
x
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
xi
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
xii

Definition of Parental Care
Chapter 1
INTRODUCTION
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,
1

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
2

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,
3

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
4

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.
5

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
6

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.
7

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.
8

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.
9

Oomycete Culture
MATERIALS AND METHODS
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
10

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
11

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.
12

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
13

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.
14

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.
15

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.
16

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).
17

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.
18

Statistical Analyses. ANOVAs were conducted using Microsoft Excel 2007
(Microsoft Corporation) and the ANCOVA was conducted using SPSS Statistics
(SPSS, An IBM Company).
19

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> 0.05,
α= 0.05) (Figure 6, Table 3).
20

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.
21

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).
22

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, R 2 = 0.58, n=6; B) simulated cleaning, P< 0.001, R 2 = 0.77,
n=5; C) simulated fanning, P< 0.001, R 2 = 0.68, n=5; D) simulated cleaning and
fanning, P< 0.001, R 2 =0.57, n=5; or E) no care, P=0.03, R 2 = 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.
23

ANCOVA
Source Type III SS df MS F P-value
Corrected
Model 0.168 a 5 0.034 20.1

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.
25

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)
26

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
27

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.
28

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
29

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
30

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
31

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
32

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.
33

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.
34

Oomycete Culture and Aquarium Set-Up
MATERIALS AND METHODS
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).
35

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
36

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.
37

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.
38

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.
39

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.
40

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.
41

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.
42

A B
C
1 mm
1 mm
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.
D
43

A
B
0.5 mm
C
44
20 µm
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.

number of viable eggs
120
100
80
60
40
20
0
Egg Mortality Over Time
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.
45

Scanning Electron Microscopy
DISCUSSION
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
46

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
47

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
48

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.
49

APPENDICES
50

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
51

Sample
# in
inner
circle
APPENDIX B
Egg Count Data for Proximity Analysis
# inviable
in inner
circle
# in outer
circle
# inviable
in outer
circle
% mortality
in inner
circle
52
% mortality
in outer
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
Sample
# in
inner
circle
# inviable
in inner
circle
# in outer
circle
# inviable
in outer
circle
% mortality
in inner
circle
53
% mortality
in outer
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
Sample
# in
inner
circle
# inviable
in inner
circle
# in outer
circle
# inviable
in outer
circle
% mortality
in inner
circle
54
% mortality
in outer
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
Sample
# in
inner
circle
# inviable
in inner
circle
# in outer
circle
# inviable
in outer
circle
% mortality
in inner
circle
55
% mortality
in outer
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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