EFFECTS OF PARASITISM ON THE REPRODUCTION OF COMMON SNOOK
by
Joy M. Young
A Dissertation Submitted to the Faculty of
The Charles E. Schmidt College of Science
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
Florida Atlantic University
Boca Raton, FL
May 2015
Copyright 2014 by Joy M. Young
ii
ACKNOWLEDGEMENTS
I would like to express my deepest appreciation to my major advisor Dr. Colin
Hughes; he continually and enthusiastically conveyed a spirit of excitement and genuine curiosity for research and the pursuit of knowledge. I could not have imagined a better, more supportive advisor and mentor for my Ph.D. study. I would like to thank my committee members Dr. John Baldwin, Dr. Joseph Caruso, Dr. Stephen Kajiura, Dr. Erik
Noonburg, and Ronald Taylor; whose work demonstrated the best in their respected fields and for providing guidance and insight in how to integrate their scientific disciplines.
This research would not have been possible without an immense amount of administrative, financial, and technical support. The administrative staff at Florida
Atlantic University Department of Biological Sciences and Graduate College, especially
Michelle Cavallo, has been an invaluable resource and support system for the past five years. Several funding sources provided financial support of this work including Florida
Atlantic University Department of Biological Sciences, the Fish and Wildlife Research
Institute (FWRI), the American Fisheries Society, International Women’s Fishing
Association, and the Manasquan River Marlin and Tuna Club. I would also like to thank
Cynthia Faulk at the University of Texas at Austin for her assistance with the egg fatty acid portion of this project. I am grateful to Dr. Robin Overstreet and Jean Jovonovich
Alvillar from the Gulf Coast Research Laboratory and Micah Bakenhaster from the Fish
Health Section of FWRI for their guidance and assistance with parasite identification.
iv My deepest gratitude to FWRI and my colleagues that became my family; they
saw the potential of my project and played a critical role in accomplishing field
collections. Thank you Jim; as a supervisor your tolerance of my responsibilities outside
the office and helping ground scientific theory in reality was instrumental to my project.
As a friend, your lightheartedness and contagious laughter helped me remember that this
is ‘just science’ and to keep life priorities, not just academic ones. Thank you Beau and
Anderson for bringing fish to the boat, being so willing to help in any aspect of the
project, and keeping my feet on the ground. I am overwhelmed by your generosity of
spirit, time and interest. Parasites! Parasites! Parasites!
I will forever be grateful to my family members and friends who have been
constant sources of inspiration during the preparation, execution, and completion of this
project.
Bonnie, you inspire me to take on the world with class and grace. As my oldest
friend, your unconditional love tempered with keeping me truthful to myself has made
me a stronger woman, and person. For us, this is just one more memory in a lifetime to
come. A special thank you to you (and Sean) for bringing Bella into my life. I marvel at
her fierce strength and creative soul and I am excited to watch her move mountains.
Eternal thanks to April, Jen, Katie, Kim, and Lindsay; to find one true friend is fortunate, to find a group of strong, intelligent, compassionate, and funny women to call my friends is a very rare and special treasure. Our friendships are much beyond good times; you all are my safety net, my motivation, my therapists, and most importantly, my dearest friends. Special thanks to Kim and Lindsay for staying late to cut stomachs, sharing my enthusiasm for finding parasites, and impromptu editing. You are the best!
v I am a proud product of my parents; nothing that I have or will accomplish is
possible without you. I am incredibly lucky to have parents that always championed my
dreams and supported my decisions, even if you didn’t agree with some of them. Thank
you for teaching me the commitment, perseverance, and attention to detail that has served
me well in the sciences. Thank you for giving me the self-confidence to pick myself up after I fail and the wisdom to not take myself too seriously. Thank you Grey, Leam, and
Sophie; as the next generation you motivate me to become a better person. Grey, observing and being a part of your budding interest in science reminds me of why I love what I do, and why I chose this path.
Thank you all for being a part of my journey.
vi ABSTRACT
Author: Joy M. Young
Title: Effects of Parasitism on the Reproduction of Common Snook
Institution: Florida Atlantic University
Dissertation Advisor: Dr. Colin Hughes
Degree: Doctor of Philosophy
Year: 2014
The effect of parasitism on the individual, and on a population, is one of the least understood and poorly studied areas of fish ecology. Parasites compete for maternal energetic reserves required for the production of viable eggs and offspring; thus parasites can directly influence population dynamics by lowering the number of offspring that survive to produce. The goal of this work was to explore the effect of parasitism on the reproductive potential of fish. Traditional measures of somatic energy reserves and body condition were examined along with newer measures of fatty acids present in eggs to approximate reproductive potential. Eighty female common snook, Centropomus undecimalis, were collected during spawning season (mid April to mid October) from four spawning aggregations along the southeastern coast of Florida and examined for a suite of biological, reproductive, and parasite infection measures. General linear models were used to model somatic indices, body condition, fatty acid composition and the ratios of fatty acids in eggs as a function of parasite infection parameters, host age, capture location, capture month and year. All fish were included in the somatic indices and body
vii condition analysis while a subset of 40 fish were used in the analysis on fatty acid composition and the ratios of fatty acids in eggs. Ninety five percent of the study population was infected with 8 genera of live parasites from 4 phyla including
Acanthocephala, Arthropoda, Platyhelminthes, and Nematoda. Dead parasites were present in 55% of the population. Parasites had significant positive and negative effects on host body condition, liver size, percentage and weight of palmitic acid and eicosapentaenoic acid (EPA), and the ratios of n-3 to n-6 polyunsaturated fatty acids, docosahexaenoic acid (DHA) to EPA, and arachidonic acid (ARA) to EPA. Parasitism may lead to an increase in liver size and certain fatty acids due to increased feeding and a concomitant exposure to infection with parasites. Body condition and liver size decreased as the number of dead parasites increased, suggesting that the successful initiation of immune defenses is a costly process. Larval nematodes had the most prevalent effect on host reproductive potential, decreasing the weight and percent composition of EPA and therefore increasing the ratios of DHA to EPA and ARA to EPA. Eicosapentaenoic acid is crucial for larval development and modulation of the immune response; thus depletion in EPA suggests a negative effect on the survival of larvae. Overall, fish showed evidence of depleted energetic reserves and specific fatty acids as a response to infection with levels of parasitism found in the wild, which may decrease reproductive success. Future studies designed to further our understanding of parasitism on reproductive potential in fishes should pair captive testing with wild populations of fish and possibly focus on the mechanisms for energy depletion by parasites.
viii DEDICATION
“What lies before us and lies behind us are tiny matters compared to what lies within us.”
-Henry Stanley Haskins
Dedicated to my parents for nurturing and magnifying what is within me. I would not have been able to reach this day without you.
EFFECTS OF PARASITISM ON THE REPRODUCTION OF COMMON SNOOK
LIST OF TABLES ...... xi
LIST OF FIGURES ...... xiii
CHAPTER 1 : THE INFLUENCE OF PARASITISM ON BODY CONDITION OF
SPAWNING COMMON SNOOK ON THE SOUTHEAST COAST OF FLORIDA ...... 1
ABSTRACT ...... 1
INTRODUCTION ...... 2
METHODS ...... 5
Parasite community description ...... 7
Data analysis ...... 7
RESULTS ...... 8
Parasite community description ...... 9
Model selection ...... 11
DISCUSSION ...... 13
CHAPTER 2 : ALTERED FATTY ACID LEVELS IN FISH EGGS DUE TO
INFECTION WITH PARASITES ...... 27
ABSTRACT ...... 27
METHODS ...... 33
Data analysis ...... 36 ix RESULTS ...... 37
Arachidonic acid (ARA) ...... 39
Docosahexaenoic acid (DHA) ...... 40
Palmitic acid (PAL) ...... 41
Eicosapentaenoic acid (EPA) ...... 42
DISCUSSION ...... 43
CHAPTER 3 : RATIOS OF N-3 AND N-6 FATTY ACIDS OF FISH EGGS IN
RESPONSE TO PARASITE INFECTION IN WILD FISH ...... 57
ABSTRACT ...... 57
INTRODUCTION ...... 59
METHODS ...... 63
Data analysis ...... 66
RESULTS ...... 67
n-3:n-6 PUFA ...... 69
DHA:EPA ...... 70
ARA:EPA...... 71
DISCUSSION ...... 73
REFERENCES ...... 88
x LIST OF TABLES
Table 1.1 Predictors used in the general linear models for body condition and
somatic indices. Categorical variables are marked with a (c)...... 18
Table 1.2 Summary statistics (Mean ± SE and min-max) of common snook sampled
from April-October 2011-2013 from Jupiter Inlet (JUPI), Palm Beach Inlet
(PABI), Peck’s Lake (PELA), and St. Lucie Inlet (STLI)...... 19
Table 1.3 Parasite infection parameter of common snook, Centropomus undecimalis,
from the east coast of Florida...... 20
Table 1.4 Definition of infection levels of endoparasites in common snook...... 21
Table 1.5 Presence of non-specific foreign bodies in common snook...... 22
Table 1.6 Body condition and somatic indices of infected (n=79) and uninfected
(n=1) female common snook...... 23
Table 1.7 Best fit models based on lowest AICc values and significance of main
effects specified in the general linear model for body condition and somatic
indices...... 24
Table 2.1 Predictor and response variables used in the general linear models for
egg fatty acid weight and percent total of fatty acids...... 47
Table 2.2 Summary statistics of biological information, body condition index (BCI),
gonadasomatic index (GSI), Hepatosomatic index (HSI), Spleen-somatic
index (SSI), and Fat-somatic index (FSI) from female common snook
xi from the St. Lucie inlet (STLI), Jupiter inlet (JUPI), and Palm Beach inlet
(PABI)...... 48
Table 2.3 Population-level community dynamics of parasites collected from female
common snook...... 49
Table 2.4 Common snook egg fatty acid (FA) weight and composition...... 50
Table 2.5 Best fit models based on lowest AICc values and significance of main
effects specified in the GLM model for weight and percentage of fatty
acids...... 51
Table 3.1 Summary of response variables modeled as a function of predictor
variables in the General Linear Model (GLM)...... 79
Table 3.2 Summary statistics (Mean SE) of biological information, body condition
index (BCI), gonadasomatic index (GSI), Hepatosomatic index (HSI),
Spleen-somatic index (SSI), and Fat-somatic index (FSI) from female
common snook from the St. Lucie inlet (STLI), Jupiter inlet (JUPI), and
Palm Beach inlet (PABI) by capture location and month...... 80
Table 3.3 Population-level community dynamics of parasites collected from female
common snook...... 81
Table 3.4 Best fit models of fatty acid ratios based on lowest AICc with significance
of main effects...... 83
xii LIST OF FIGURES
Figure 1.1 (A) Mean gonadosomatic index (GSI) and (B) body condition index
(BCI) per year class...... 25
Figure 1.2 Mean hepatosomatic index (HSI) by endoparasite infection level...... 26
Figure 2.1 Capture locations (◄) of female common snook from three aggregation
sites, St. Lucie Inlet, Jupiter Inlet, and Palm Beach Inlet, along the
southeast coast of Florida...... 52
Figure 2.2 Percentage of docosahexaenoic acid (DHA), eicosapentaenoic acid
(EPA), arachidonic acid (ARA), and palmitic acid (PAL) in eggs of
common snook by phase (A), capture location (B), and month (C)...... 53
Figure 2.3 Percentage (mean ± SE) of docosahexaenoic acid (DHA; ¯),
eicosapentaenoic acid (EPA; s), andarachidonic acid (ARA;
palmitic acid (PAL; =) in eggs from common snook by age (A), and
body condition index (BCI; B)...... 54
Figure 2.4 Percentage (mean ± SE) of docosahexaenoic acid (DHA; ¯),
eicosapentaenoic acid (EPA; s), andarachidonic acid (ARA;
palmitic acid (PAL; =) in eggs of common snook by the ratio of live
to dead parasites (A), and the infection index of endoparasites (B)...... 55
xiii Figure 2.5 The weight and percent total composition (estimated marginal mean ± SE)
of docosahexaenoic acid (DHA; ¯), eicosapentaenoic acid (EPA),
arachidonic acid (ARA; s), and palmitic acid (PAL; =) predicted from
general linear models in eggs of common snook during the oocyte
maturation process...... 56
Figure 3.1 Ratios of fatty acids including n-3:n-6 PUFA (A), ARA:EPA (B), and
DHA:EPA (C) in eggs of Common Snook...... 84
Figure 3.2 The predicted significant effect (Estimated Marginal Mean, EMM SE) of
phase on n-3:n-6 PUFA (A), ARA:EPA model 1 (B), and ARA:EPA
model 2 (C) based on the best fit GLM models...... 85
Figure 3.3 The predicted significant effect (estimated Marginal Means, EMM SE)
of the infection level of endoparasites on the ratio of n-3 to n-6 PUFA
in eggs of Common Snook based on the best fit GLM...... 86
Figure 3.4 The predicted significant effect (Estimated Marginal Means, EMM SE)
of age on the ratio of DHA to EPA in eggs of Common Snook based on
the best fit GLM...... 87
xiv CHAPTER 1 : THE INFLUENCE OF PARASITISM ON BODY CONDITION OF
SPAWNING COMMON SNOOK ON THE SOUTHEAST COAST OF FLORIDA
ABSTRACT
Factors that influence body condition, including parasitism, are of growing interest to fisheries managers because of the negative effect of poor body condition on reproductive potential. I evaluated the effect of parasite infection on the body condition
and somatic indices as a proxy for fitness with spatial, temporal, and host biology
comparisons. Macro-parasites were studied in 80 common snook, Centropomus
undecimalis, from 4 breeding sites from the southeast coast of Florida during the
spawning season. In total, 99% were infected with live or dead parasites. Ninety five
percent of the study population was infected with 8 genera of live parasites from 4 phyla
including Acanthocephala, Arthropoda, Platyhelminthes, and Nematoda. Dead parasites
were present in 55% of the population. Lernanthropus gisleri was the most prevalent
(71%), then Contracaecum sp. (64%), Proteocephalus sp. (36%) and larval cestodes
(36%). Contracaecum sp. had the highest mean infection intensity (23.9), followed by
Sebekia sp. (9.3), and Anisakis sp. (8.3). The Hepatosomatic index (HSI) was negatively
affected by increasing numbers of dead parasites and positively affected by infection
level of endoparasites. Body condition index (BCI) was negatively affected by increasing
numbers of dead parasites and positively affected by increasing numbers of cestode
1 larvae. This study also found yearly effects on spleen and gonad indices and positive age
effects on BCI and gonad ratios. Our results suggest that fitness loss induced by live
parasites and host mediated killing of parasites could affect the reproductive potential of
female fish in spawning condition.
INTRODUCTION
Fish body condition is an important determinant of reproductive output (Ndjaula
et al. 2010), and therefore population dynamics (Marshall et al. 1999; Shulman and Love
1999; Lloret et al. 2012). Individual fish in poor condition have decreased growth rate
(Cardinale and Arrhenius 2000; Rätz and Lloret 2003), less energy for reproduction
(Kjesbu et al. 1998; Oskarsson et al. 2002; Oskarsson and Taggart 2006), and poor larval
survival (Booth and Beretta 2004). For example, declines in body condition of Gulf of St.
Lawrence cod in the late 1980s and early 1990s coincided with a decrease in stock
biomass and collapse of the fishery (Lambert and Dutil 1997; Olsen et al. 2004). Low
energetic reserves (i.e. low body condition), exacerbated in spawning individuals, led to
an increase in natural mortality and decrease in recruitment, which contributed to the
collapse of the cod stock (Dutil and Lambert 2000; Lambert and Dutil 2000). Low body
condition has been implicated in the failure of Atlantic cod in Newfoundland (Fu et al.
2001) and Atlantic bluefin tuna in the Gulf of Maine (Golet et al. 2007) to recover despite
fishing moratoriums. Body condition is influenced by prey availability, physical
environmental factors, population density, and growth rate (Guy and Willis 1991; Liao et
al. 1995; Fortin et al. 1996; Tierney et al. 1996; Blackwell et al. 2000; Marshall 2009).
However, studies also suggest that parasite infection may be a driving factor in variation
2 of body condition (e.g. Pennycuick 1971) and population collapse (e.g. Haenen et al.
2010).
Parasites decrease host physical condition by robbing energy otherwise slated for
growth, reproduction and physiological maintenance (Minchella and Scott 1991; Moyle
and Cech 2004). While epizootic episodes with high mortality rates are dramatic
indicators of the effects of parasites on fish populations (Wurtsburgh and Alfaro 1988;
Baya et al. 1997), sublethal effects of parasites are also expected to be important for fisheries management (Dobson and May 1987) and may be more influential in fish population structure in the long term (Barber et al. 2000). Studies on icefish,
Chionodraco hamatus (Santoro et al. 2013), and toadfish, Porichthys porosissimus
(Guagliardo et al. 2009), have shown that body condition decreases with increasing
infection intensity of macroparasites. Bagamian et al. (2004) found body condition of
male sticklebacks infected with plerocercoid metacestodes was 74% lower than in
uninfected fish. In addition, fish with high parasite burden are more susceptible to
mortality during stressful environmental events, such as overwintering (Francová and
Ondračková 2013).
This study focused on the influence of natural macroparasite infection on body
condition of spawning female common snook, Centropomus undecimalis. Common
snook are euryhaline fish of the Western Atlantic that inhabit coastal rivers and estuaries,
nearshore and offshore waters out to the continental shelf (Marshall 1958; McMichael et
al. 1989). Common snook are protandrous hermaphrodites, reaching sexual maturity as
males at 169-222 mm total length (TL), and transitioning into sexually mature females
between 264-876 mm TL during the fall following the spawning season (Taylor et al.
3 2000). Females are indeterminate spawners with a protracted spawning season of 5-7 months from late spring to early autumn (Tucker and Campbell 1988; Taylor et al. 1998).
On the east coast of Florida, aggregations comprised of spawning capable common snook
(Lowerre-Barbieri et al. 2003) have been documented in Palm Beach, Jupiter, St. Lucie,
Ft. Pierce, Sebastian, and Canaveral inlets during the summer months (Tucker and
Campbell 1988; Taylor et al. 1998; Young et al. 2014).
While reproduction in common snook is well characterized, less is known about their parasite burden. The majority of macroparasites infecting wild fish are metacercariae, metacestodes, and nematode larvae (Williams and Jones 1994; Bakke and
Harris 1998). Previous research has demonstrated that common snook from Tampa bay and the southeast coast of Florida host at least 2 genera of adult digeneans (Neochasmus olmecus, Stephanostomus sp.), 2 genera of larval nematodes (Gnathostoma sp.,
Contracaecum sp.), 1 genus of adult copepod (Learnanthropus gisleri), and 1 genus of adult pentastome (Sebekia sp.) (Micah Bakenhaster, FWRI, St. Petersburg, FL, personal communication).
The objectives of this study were to 1) describe the macroparasite fauna of female common snook on the east coast of Florida during the spawning season and 2) identify effects of parasite infection on fish physical fitness by assessing host body condition, somatic indices, and parasite infection intensity across time, space and among age groups.
We predicted that in general, body condition and somatic indices would decrease with high levels of parasite infection. However, an enlarged spleen due to an acute inflammatory response in reaction to parasite exposure may result in a high spleen index
4 with high parasite infection (e.g. Lefebvre et al. 2004). Overall though, since parasites use host energy (Minchella and Scott 1991; Moyle and Cech 2004), we expected that host
body condition would be negatively affected by parasite infection, especially in females
during the spawning season when energy reserves are depleted from egg production
(Lambert and Dutil 2000) and stress from spawning may exacerbate the severity of disease (Hedrick 1998).
METHODS
Between April-October 2011-2013, specimens of female common snook were
collected with the use of rod and reel fishing gear from 4 spawning sites along the
southeastern coast of Florida: Palm Beach Inlet (PABI: 26.772548, -80.037247), Jupiter
Inlet (JUPI: 26.944593, -80.073817), Peck’s Lake (PELA: 27.12212, -8012803), and the
St. Lucie Inlet (STLI: 27.166272, -80.160716). Fish were either kept in a recirculating
live well on the boat and euthanized at the Fish and Wildlife Research Institute (FWRI)
field lab in Tequesta, Florida (years: 2011-2012) or euthanized on the boat immediately
after capture (year: 2013). Fish were placed individually into heavy-duty plastic bags and
euthanized by submersion in a slurry of ice and water below the lethal temperature for
adult common snook (6°C; Shafland and Foote 1983) and exsanguinated by rupture of
the branchial arteries surrounding the gills. Fish euthanasia followed protocol approved
by the Florida Atlantic University (FAU) Institutional Animal Care and Use Committee
(IACUC) (protocol number: A11-30).
A complete necropsy was performed on all specimens, including examination for
internal and external parasites. Parasites were counted and isolated from whole tissue and
placed in a saline solution for examination with a compound scope using 10X and 20X
5 magnification. Left gill arches were examined whole. Photographs were made using a
SPOT Insight QE camera and processed using SPOT imaging software. Voucher
specimens of each parasite type were preserved for confirmation of identity conducted by
the FWRI fish health lab. Methods for parasite identification and preservation followed
Noga (2000) and FWRI fish health protocol. In summary, copepods were isolated from
gill tissue and placed in 70% ethanol or 5% neutral buffered formalin (NBF). After
excision from their cases, nematode larvae were heat fixed with near boiling water and
preserved in 5% NBF. Adult cestodes and plerocercoid metacestodes were agitated in a
saline solution to encourage the protrusion of the scolex, bothria, and tentacles then either
placed in 70% ethanol or 5% NBF. Digeneans were placed directly in 5% NBF. Parasites
were classified to genus and/or species. A voucher specimen of plerocercoid
metacestodes was visually examined by parsitologists of the Gulf Coast Research
Laboratory, Ocean Springs, MS.
Fish were sampled for biological information in accordance with FWRI
guidelines; fish length (mm; standard length: LS, fork length: LF; total length: LT), weight
(g; whole weight: WW; eviscerated weight: WE), organ weight (g, stomach, liver, spleen,
ovaries), and fat weight (g) were taken for each specimen. Sagittal otoliths were removed
for age determination and ovaries were sectioned and preserved in formalin for histology.
Gonad maturation was staged according to Murua et al. (2003) and FWRI protocol (for reference see http://myfwc.com/research/saltwater/reef-fish/reproduction/). Gonad
maturation was quantified as either immature or early developing (phase 1), developing
(phase 2), spawning capable (phase 3), actively spawning (phase 4), regressing (phase 5),
or regenerating (phase 6) based on the most advance oocyte.
6 Body condition was calculated as described by Jakob et al. (1996); residuals from
an ordinary least squares regression of eviscerated somatic mass against length were used
as the body condition index (BCI). The BCI has advantages over traditional
length/weight ratios because it separates the effect of condition from the effect of size and
has positive and negative scores showing individuals that are fatter or leaner than
expected (Jakob et al. 1996; Green 2001).
Somatic indices were calculated for the liver (hepatosomatic index, HSI), spleen
(spleen-somatic index, SSI), gonad (gonads-somatic index, GSI), and fat (fat-somatic
index, FSI) as follows where organ and whole weights were measured in grams.
= × 100 푂푂푂𝑂 푤𝑤�ℎ� 푆푆푆푆푆푆푆 퐼퐼퐼퐼퐼 푊 Parasite community description 푊
Parasite community composition was described as prevalence (number fish
infected), mean abundance (mean number of conspecific parasites per fish in a sample
population); mean intensity (mean number of conspecific parasites per infected fish in a
sample population) and was calculated using Quantitative Parasitology© 3.0 software.
Parasite infracommunities (i.e. per host) were described in terms of intensity of parasites by species, number of dead parasites, infection level of endoparasites, and the Shannon-
Weiner index (H) as a measure of diversity.
Data analysis
General linear models (GLM) were used to model body condition and somatic indices as a function of endo and ecto parasite infection (intensity by live and dead parasites by species, infection level of endoparasites, and diversity), space (capture
7 location), time (year), and host age (Table 1.1). We used the Least Absolute Shrinkage and Selection Operator (LASSO) to control selection with the second order adjustment of the Akaike Information Criterion (AICc) for model selection. While the stepwise selection method is common for these types of data (e.g. Francová and Ondračková
2013), it is argued that the r2 values are biased too high, the standard errors of parameters
are too small (Harrell 2001), and it may fail to identify variables that fit well into the
model (Miller 2002). Studies criticizing stepwise model selection have suggested that
LASSO is a better alterative (e.g. Flom et al. 2007).
Candidate models for each response variable that differed 3 or less from the
model with the lowest AICc score were furthered explored using GLM with Restricted
Maximum Likelihood (REML) estimation. When main effects were significant, least
squares mean (LSM) post hoc tests were used to compare differences between groups.
For all analyses, data transformations to meet assumptions of normality and
homoscedasticity were calculated using a natural log transformation. Analyses were
conducted using SAS© 9.2 software.
RESULTS
Eighty sexually mature female common snook were collected during April-
October 2011-2013 (2011: n=4, 2012, n= 36, 2013: n=40) from the Palm Beach Inlet
(PABI: n=13), Jupiter Inlet (JUPI: n=59), St. Lucie Inlet (STLI: n=2) and Peck’s Lake
(PELA: n=6) (Table 1.2). Total length ranged from 620-1050 mm with a mean (M ± SE)
of 877.46 ± 9.99 mm. Specimen age varied from 4-16 years old with a mean ± SE age of
8.18 ± 0.28 years. Mean age was lowest in 2011 (5.5 years) compared to other years
(2012: 8.11 years, 2013: 8.52 years), however the sample size was also the smallest (n=4)
8 in 2011. Ninety eight percent of the specimens were phased as either spawning capable
(n=63) or actively spawning (n=15). Two fish were phased as regressing or regenerating.
Parasite community description
Ninety nine percent (n=79) of fish were infected with live or dead parasites. Live
parasites were identified in 95% of the population (n=76) and dead parasites were found
in 55% of the population (n=44) while both live and dead parasites were found in 51%
(n=41). Specimens from five major taxa of parasites were collected and identified:
Acanthocephala (2 Pomphorhynchus sp.), Cestoda (36 Proteocephalus sp., 107
Callitetrarhynchus gracilis), Copepoda (1 Lernaeolophus sp., 165 Lernanthropus
gisleri), Nematoda (25 Anisakis sp., 758 Contracecum sp.), and Pentastomida (57 Sebekia
sp.) (Table 1.3). The parasite community described here represents new host records for
Callitetrarhynchus gracilis. While not identified to species level, Proteocephalus sp.,
Lernaeolophus sp., Anisakis sp. and Pomphorhynchus sp. have never been reported in
common snook from Florida.
The study population showed large differences in prevalence and parasite burden.
Two species, Lernanthropus gisleri (Copepoda) and Contracaecum sp. (Nematoda), were
widely distributed and found in 71% and 64%, respectively, of the population (Table
1.3). Contracaecum sp. also had the highest mean abundance (15.20 parasites) and mean
intensity (23.90), demonstrating that Contracaecum sp. was not only wide spread but also the most abundant parasite group in this study. In contrast, the other nematode found,
Anisakis sp., was not highly prevalent (3%) and only found in 3 individuals. Since only the left gill arch was examined, if we doubled the number of observed of L. gisleri per individual, intensity would still be lower compared to Contracaecum sp.
9 Lernaeolophus sp. (Copepoda) was represented by one specimen and
Pomphorhynchus sp. (Acanthocephala) was represented by two specimens infecting a single host; they were the least prevalent (<1%) in the population. Additional parasites found included Callitetrarhynchus gracilis (Cestoda) in 36% of the population, and
Proteocephalus sp. (Cestoda) and Sebekia sp. (Pentastomida) found in approximately
10% of the population.
Copepods were only collected from the gills and buccal cavity (ectoparasites) while all other parasites were collected internally (endoparasites). An internal infection level was assigned to each fish based on total abundance of parasites. Four categories including none, low, moderate, and high were delineated from natural breaks in the frequency distribution of parasite abundance (Table 1.4). Total number of ectoparasites was low (0-9) compared to endoparasites (0-170). An external infection level was not calculated because ectoparasites were all L. gisleri with the exception of one specimen of
Lernaeolophus sp., therefore the ectoparasite infection level would be too closely correlated with infection intensity of L. gisleri.
Fish were infected with 0-3 types of endoparasites and 0-2 types of ectoparasites.
Fish were infected with an average of 1.96 (SE: ± 0.10) types of parasites. Diversity ranged from 0-1.23 with a mean (M ± SE) of 0.45 ± 0.04.
In addition to live parasites, dead parasites of unknown taxa (354 total) and several non-parasitic masses were collected (Table 1.5). Dead parasites either elicited no movement after excision from tissue and were in some process of decay (e.g. shedding tissue) or formed masses characterized by melanization of the sac enclosure and breakdown of parasite tissue. Masses had extensive melanization and fibrosis forming
10 granulomas and were restricted to the peritoneal cavity. Dissection of masses revealed unidentifiable tissue or exogenously derived material including fish spines and hooks.
Model selection
Somatic and body condition indices were calculated for the 80 female common snook collected from spawning aggregations from 2011- 2013 (Table 1.6). The two individuals that were not in a spawning capable phase were removed prior to model selection. One of those individuals was infected with the only Acanthocephalan worm, therefore intensity of Lernaeolophus sp. was not included in the model. Extended exploration of candidate models using GLM with REML revealed that for GSI, SSI, HSI, and FSI, the AICc of the alternate candidate models differed by >3 from the model with the lowest AICc score from GLM with LASSO. Therefore, all alternate candidate models for GSI, SSI, HIS, and FSI were discarded and the original candidate model with the lowest AICc score from GLM with LASSO was retained. Interestingly, for BCI, the candidate model with the lowest AICc score using GLM with REML was not the originally selected model. The originally selected model was discarded and the results from the alternate, better fit, model are presented here. GLM revealed varying influences of age, year, location, endoparasite infection level, abundance of dead parasites and intensity of larval and adult cestodes, and larval nematodes on body condition and somatic indices (Table 1.7).
A mixture of host biology, spatial, temporal, and parasite infection factors accounted for 4-26% of the variation in body condition and somatic indices. The spatial factor (location) was a significant predictor of FSI (p=0.0176). Fish collected from PELA had a significantly higher FSI compared to fish collected from JUPI (differences of least
11 squares mean: t(58) = -3.23, p=0.0121) and PABI (differences of least squares mean: t(58) =
-3.08, p=0.0187). The temporal effect of year was a significant variable for the prediction of SSI (p<.0001) and GSI (p=0.0418). The only significant yearly difference occurred during 2012 where fish had a significantly lower SSI than fish collected in 2013
(differences of least squares mean: t(75) = -2.66, p=0.0283). The model also predicted fish
collected during 2011 would have a higher GSI (EMM ± SE: 5.28 ± 1.25) compared to
other years (EMM ± SE: 2012: 3.65 ± 1.13, 2013: 3.05 ± 1.13). Host age significantly
affected BCI (p=0.0440) and GSI (p=0.0386). While there were no significant differences
between age classes, in general BCI and GSI were predicted to increase with age (Figure
1.1).
Parasite infection parameters, while present in all final selected models except
SSI, were only significant effects for HSI and BCI. HSI was significantly affected by
endoparasite infection level (p<.0001) and abundance of dead parasites (p=0.0036). Fish
with no endoparasites had a lower HSI (M ± SE: 0.73 ± 0.04) compared to fish with a
high infection level (M ± SE: 0.89 ± 0.05). The liver was predicted to enlarge with
increasing levels of infection with endoparasites (Figure 1.2). Abundance of dead
parasites had a negative effect on HSI; for every increase of one dead parasite, HSI
decreased by 0.0086 (t(72) = -3.01, p=0.0036). BCI was significantly affected by
abundance of dead parasites (p=0.0306) and intensity of Callitetrarhynchus gracilis
(p=0.0365). BCI was predicted to decrease by 16.09 (t(60)= -2.22, p=0.0306) for every dead parasite but also expected to increase by 50.16 for every C. gracilis (t(60)= 2.14,
p=0.0365).
12 DISCUSSION
This study confirms that a variety of factors drive the variation in body condition and somatic indices of common snook. An individual’s year of capture, location, age and parasite infection affect the health status of fish. However, the only significant predictors of body condition and somatic indices were year collected, host age, and the intensity of live and dead parasites. The predominant findings were 1) HSI and BCI decreased with increasing levels of dead parasites, 2) HSI increased with increasing infection level of live endoparasites and 3) BCI increased with increasing levels of Callitetrarhynchus gracilis. Overall, this suggests that infection with parasites is an influential force on body condition with consequences for reproductive potential.
Five of the eight parasite genera identified in common snook are new host or geographical records for Florida (Table 1.3) and the remaining 3 have been previously reported in this host species. Stage 3 larvae of Contracaecum sp. have been reported in common snook from Columbia (Olivero Verbel et al. 2011) and SE Florida (Micah
Bakenhaster, personal communication) and L. gisleri and Sebekia sp., have been previously reported in common snook from the SE of Florida (Micah Bakenhaster, personal communication). Several of the parasite genera identified in this study have also been documented in closely related snook species from Central America. Contracaecum sp., Proteocephalus sp., and Sebekia sp. have been documented in black snook, C. nigrescens, from Mexico (Violante-González et al. 2010) and Contracaecum sp. in yellowfin snook, C. robalito, from Mexico (Violante-González et al. 2011). According to the best of our knowledge, this study represents the first records of Lernaeolophus sp.,
13 Callitetrarhynchus gracilis, Proteocephalus sp., Anisakis sp., and Pomphorhynchus sp. in common snook.
Six of the identified species are transmitted via ingestion of intermediate hosts, meaning that diet is important in structuring this host’s parasite infracommunity.
Common snook are considered an apex predator of riverine and estuarine environments
(e.g. Stevens et al. 2010). Juvenile common snook ingest mainly shrimp, crabs and small fish (McMichael et al. 1989) with an ontogenic shift towards a fish based diet as adults
(Blewett et al. 2006). Presence of the nematode Contracaecum sp. and Anisakis sp. which mature in fish-eating birds and mammals (Humphrey at al. 1978; Smith and Wooten
1978), the Pentastomid Sebekia sp. which matures in reptiles including alligators
(Overstreet et al. 1985), and larval Callitetrarhynchus gracilis which mature in elasmobranchs (Schmidt 1986) indicate that common snook can also serve as a prey species.
While the infection of macroparasites is extensive in common snook from the SE of Florida, only certain aspects of the parasite infracommunity were found to affect host body condition and liver index. Energetic stores in organs and fat are drawn upon during spawning for migration and egg production (e.g. Mommsen et al. 1980). Production of eggs is costly (Clutton-Brock and Vincent 1991) and the energetic demand of spawning is compounded by frequent circular migrations of common snook from spawning aggregations in the inlet to rivers and/or estuaries (Young et al. 2014). Host body condition is considered a proxy for overall health, quantifying fatness or leanness compared to the population. In addition, sufficient liver reserves are required for
14 reproduction as it is the site of lipid production during vitellogenesis (Mommsen and
Walsh 1985).
Energetic stores may be diminished by parasites through the direct absorption of
nutrients or instigating a nutritionally demanding immune response (Roberts and Janovy
2008). However, the immune response is costly to initiate, maintain, and use (Lochmiller
and Deerenberg 2000). Since parasites evolve faster than their hosts (Hafner et al. 1994)
continual stimulation of the immune response would allow parasites to adapt to host
defenses faster (Holt and Hochberg 1997) and increase the probability of autoimmune
diseases (Fumagalli et al. 2009). Therefore a trade-off between energy expended by launching an immune response and energetic loss due to parasites is expected. This is exacerbated during the breeding season when reproductive effort decreases to accommodate the release of energy needed for the immune response (Read 1990).
The presence of dead parasites may suggest a previous costly investment in an immune response resulting in a lower body condition and liver, or be an artifact of housing a larger parasite burden. The clearance time of dead parasites is not known.
During the spawning season, Vainikka et al. (2009) found that female wild roach, Rutilus rutilus had a smaller proportion of dead parasites to live parasites compared to males, suggesting that females are directing energy towards reproduction rather than immune defenses. Studies on fish, mammals, and reptiles reveal different relationships between body condition and parasite burden. Some studies show that external parasites have no effect on body condition of fishes (e.g. Kalbe and Kurtz 2005; Celik and Aydin 2006;
Tadiri et al. 2013) while others show positive (Schulte-Hostedde and Elsasser 2011) and
negative (Hoffangle et al. 2006) correlations.
15 We attribute the positive relationship of endoparasite infection level and liver size on increased prey acquisition, or energy density of prey. Animals in good body condition are consuming more prey and therefore are exposed to more parasites (Bize et al. 2008).
The first intermediate hosts of Trypanorhyncha cestodes are crustaceans (Aldrich 1965).
While shrimp and crabs are less energetically dense (Pettitt-Wade et al. 2011) than common fish prey in SE Florida including white mullet, Mugil curema, pigfish,
Orthopristis chrysoptera, and pinfish, Lagodon rhomboides, (Worthy and Worthy 2011), shrimp and crabs are easier to catch based on swimming speeds (Benoit-Bird 2004) and are an important source of copper, calcium, and magnesium (Pettitt-Wade et al. 2011).
This study suggests that older, larger fish may contribute disproportionately to the population (Kamler 2005) through size-dependent fecundity. In this study, older fish had a higher GSI and BCI suggesting that not only were older fish healthier and more fecund they had a higher energy reserve compared to younger fish. The increase of relative fecundity with age has been shown to improve reproductive potential in rockfishes,
Sebastes sp. (Dick 2009) and blue cod, Parapercis colias, (Beer et al. 2013). The importance of older fish is even greater when older females have larger, better quality eggs that increase larval survival (Trippel 1998; Kamler 2005; Birkeland and Dayton
2005).
This study provides information about the general relationship of host health and parasite infection and it suggests future interesting directions. First, the role of variation of the Major Histocompatibility II (MHII) genes in determining the parasite infracommunity. MHII genes are critical for identification of specific antigens (Klein et al. 1994) and have been associated with disease resistance susceptibility and resistance in
16 fishes (e.g. Langefors et al. 2001; Grimholt et al. 2003; Wegner et al. 2003). Second, additional measures of reproductive potential, not just GSI, may be useful in elucidating the affect of parasites on reproduction. While I found a negative effect of parasites on aspects of the host’s biology that are energetically tied to reproduction, I found little evidence of an effect on a direct measurement of gonad weight. Future studies that include additional quantitative measures of reproduction including egg size and fatty acid composition may provide a better connection between parasites and reproductive potential.
17 Table 1.1 Predictors used in the general linear models for body condition and somatic indices. Categorical variables are marked with a (c).
Host biology, spatial, temporal, and Body condition and somatic indices infection factors
(response variables) (predictor variables)
Infection intensity per species
Body condition Index (BCI) Abundance of dead parasites
Spleen-somatic Index (SSI) Shannon Diversity Index (H)
Hepatosomatic Index (HSI) Infection level of endoparasites (c)
Gonadosomatic Index (GSI) Age (c)
Fat-somatic Index (FSI) Year (c)
Location (c)
18 Table 1.2 Summary statistics (Mean ± SE and min-max) of common snook sampled from April-October 2011-2013 from Jupiter Inlet (JUPI), Palm Beach Inlet (PABI), Peck’s Lake (PELA), and St. Lucie Inlet (STLI).
Inlets Total Length Age Year n sampled (mm) (Years)
797.00 ± 20.76 5.5 ± 0.64 2011 JUPI, PELA 4 752 - 846 4 - 7
JUPI, PABI, 890.83 ± 11.71 8.11 ± 0.40 2012 36 PELA 724 - 1000 5 - 16
JUPI, PABI, 873.47 ± 16.40 8.52 ± 0.42 2013 40 STLI 620 - 1050 4 - 15
JUPI, PABI, 877.46 ± 9.99 8.18 ± 0.28 Total 80 PELA, STLI 620 - 1050 4 - 16
19 Table 1.3 Parasite infection parameter of common snook, Centropomus undecimalis, from the east coast of Florida. A star (*) indicates new host records. Mean Mean Number Prevalence Parasite Stage Site Intensity Abundance infected CI CI CI Copepoda (Class) Lernanthropus gisleri A Gills 0.713 2.89 2.06 57 0.601 – 0.802 2.4 – 3.47 1.66 – 2.56
Lernaeolophus sp.* A Tongue 0.013 1 0.01 1 0.001 – 0.067 NA 0 – 0.03 Cestoda (Class) Callitetrarhynchus gracilis * L Mesentery 0.362 4.93 1.79 Stomach submucosa 29 0.261 – 0.475 3.52 – 7.31 1.10 – 2.81 Intestine 20
Proteocephalus sp.* A Intestine 0.100 5.62 0.56 8 Stomach mucosa 0.047 – 0.186 2.00 – 14.20 0.13 – 1.95 Palaeacanthocephala (Class) Pomphorhynchus sp.* A Intestinal mucosa 0.013 2 0.025 1 Stomach mucosa 0.001 – 0.067 NA 0 – 0.07 Pentastomida (subclass) Sebekia sp. L Mesentery 0.113 9.33 .05 9 Stomach 0.058 – 0.205 5.00 – 17.80 0.43 – 2.55 Chromadorea (Class) Contracaecum sp. L Mesentery 0.637 23.90 15.20 Intestine 51 0.525 – 0.739 16.70 – 36.90 10.30 – 23.60 Stomach submucosa
Anisakis sp.* L Mesentery 0.037 8.33 0.312 3 Intestine 0.010 – 0.104 1.00 – 15.70 0.0125 – 1.46
Table 1.4 Definition of infection levels of endoparasites in common snook.
Infection level Number of parasites Number of fish Percent of population
None 0 13 5.00
Low 1-10 41 41.25
Moderate 11-50 22 48.75
High 51-132 4 5.00
21 Table 1.5 Presence of non-specific foreign bodies in common snook.
Prevalence Mean Intensity Mean Abundance Type Site Number infected CI CI CI
Mesentery 0.55 11.30 6.22 Dead unknown sp. Intestine 44 0.437 – 0.657 7.61 – 19.70 4.06 – 11.60 Stomach submucosa
0.075 3.17 0.238 Masses Mesentery 6 0.033 – 0.155 1.33 – 6.50 0.075 – 0.727 22
Table 1.6 Body condition and somatic indices of infected (n=79) and uninfected (n=1) female common snook.
Index Mean ± SE Range
Gonadosomatic Index (GSI) 3.31 ± 0.17 0.74 – 8.76
Hepatosomatic Index (HSI) 0.84 ± 0.02 0.42 - 1.65
Spleen Index (SSI) 0.05 ± 0.01 0.02 – 0.09
Fat Index (FSI) 0.62 ± 0.07 0 – 3.67
Body Condition Index (BCI) 0.01 ± 89.66 -4570.32 – 1923.57
23 Table 1.7 Best fit models based on lowest AICc values and significance of main effects specified in the GLM model for body condition and somatic indices. Adjusted r2 values are reported from analogous model selected from GLM with lasso as variable selection.
Index Effects df F P Adj r2
Fat Age 11, 58 0.87 0.5740 0.2351
Location 3, 58 3.65 0.0176
Endoparasite Infection Level 3, 58 2.44 0.0730
Spleen Year 3, 75 3321.71 <0.0001 0.0383
Body condition Age 11, 60 2.47 0.0128 0.2054
Year 2, 60 1.41 0.2532
Endoparasite Infection Level 3, 60 1.67 0.1837
Intensity of Callitetrarhynchus gracilis 1, 60 4.58 0.0365
Abundance of dead parasites 1, 60 4.91 0.0306
Gonad Age 11, 59 2.05 0.0418 0.2644
Year 2, 59 3.35 0.0418
Location 3, 59 2.52 0.0668
Intensity of Anisakis sp. 1, 59 3.49 0.0665
Intensity of Proteocephalus sp. 1, 59 0.89 0.3488
Liver Endoparasite Infection Level 4, 72 8.79 <0.0001 0.1221
Intensity of Callitetrarhynchus gracilis 1, 72 2.51 0.1175
Abundance of dead parasites 1, 72 9.05 0.0036
24
Figure 1.1 (A) Mean gonadosomatic index (GSI) and (B) body condition index (BCI) per year class. Indices are presented as the mean response (estimate marginal mean, EMM ± SE) for each year class adjusted for all other variables in the model. The horizontal axis from the OLS regression of BCI is marked by a grey dashed line.
25
Figure 1.2 Mean hepatosomatic index (HSI) by endoparasite infection level. Indices are presented as the mean response (estimate marginal mean, EMM ± SE) for each infection level adjusted for all other variables in the model.
26
CHAPTER 2 : ALTERED FATTY ACID LEVELS IN FISH EGGS DUE TO
INFECTION WITH PARASITES
ABSTRACT
Maternal allocation of fatty acids to eggs has profound consequences for egg viability, larval survival, and therefore recruitment of the population. Teleost eggs are rich in polyunsaturated fatty acids, particularly EPA and DHA, which are vital for normal embryo development. Parasites are capable of altering host behavior, including foraging, and robbing the host of energy ultimately affecting maternal diet and body stores which are the main sources of fatty acids in eggs. General linear models were used to explore the effects of parasite infection on the percentage and weight of DHA, EPA, PAL, and
ARA in fish eggs collected from female common snook Centropomus undecimalis in spawning aggregations on the east coast of Florida. The weight and composition of fatty acids found in common snook eggs were affected by biological, spatial, and infection factors. However, we demonstrate that these relationships are not necessarily linear and can be positive or negative. Among the biological, spatial, and temporal factors, gonad maturity had a major effect on the weight and percentage of PAL, ARA, EPA, and DHA.
Ninety-eight percent (n = 39) of the study population was infected with either live or dead parasites. PAL weight and percentage was positively affected by the abundance of dead parasites and intensity of Contracaecum sp., while EPA weight and percentage was
27 negatively affected by the intensity of Contracaecum sp. and the ratio of live to dead parasites. Considering the deleterious effects on larval survival from EPA deficiency, and antagonistic functions in relation to other fatty acids, this study suggests that infection with parasites contributes to recruitment variation.
28 INTRODUCTION
A comprehensive understanding of population dynamics of fishes is limited by unpredictable and variable reproductive performance (Chambers and Trippel 1997).
Variation in reproductive performance has been attributed to variations in physiological condition (i.e. body condition; Oskarsson 2008), genotype (Miller and Brooks 2005), and environmental conditions (Kokita 2004). Sublethal effects of infection with parasites may also drive variation within a population (e.g., Pennycuick 1971). Parasite infection affects reproductive output of fish directly by robbing the host of energy otherwise used for reproduction (e.g. Hall et al. 2007) or indirectly by lowering energetic reserves (Schultz et al. 2006) which may lead to fewer, or smaller eggs produced, decreases in egg quality, lengthening of the time between spawning events, decreases in hatching rate and decreases in larval survival (e.g. Adlerstein and Dorn 1998; Hesp et al. 2002).
Heins et al. (2010) found that threespine stickleback Gasterosteus aculeatus aculeatus infected with a single cestode have significantly lower recruitment of oocytes through maturation compared to uninfected fish. This reduced seasonal fecundity by decreasing the number of eggs in each spawning event, and increasing the time between spawning events by 27% resulting in fewer spawning events during the reproductive season. Furthermore, infected sticklebacks produced smaller eggs than uninfected sticklebacks, suggesting that energy robbed from the host during oocyte development affected the female’s ability to produce larger oocytes (Heins and Baker 2003). Smaller eggs are expected to yield lower hatching success of low quality offspring and lower survival past the larval stage. In five-lined cardinal fish Cheilodipterus quinquelineatus
29 females infected with isopods have ovaries that weigh >35% less compared to uninfected fish and have > 42% fewer oocytes (Fogelman et al. 2009).
Examination of reproductive performance measures along with parasite infection is required to understand the demographic role of disease in wild populations of fish.
Several measures have been used to assess the reproductive performance of fishes including fecundity (Murua et al. 2003), egg size (Stearns 1992), body condition (Kjesbu et al. 1998), larval survival (Tveiten et al. 2004), and more recently, lipid content of eggs
(Zera and Harshman 2001; Stephens et al. 2009). Lipids, mainly comprised of fatty acids
(FA), have a major influence on egg viability (fertilization and hatching success) and survival of larvae (Tveiten et al. 2004). The content and composition of lipids in eggs vary by species and stock (Pickova et al. 1997). However in all animals the maternal allocations of lipids provide the sole source of nutrition during the endogenous-feeding stage of larvae (Sargent et al. 1989). The mortality rate of larvae is greater than 90%
(Houde 2002), so even a small increase in larval survival would have a large positive effect on recruitment (Houde 1987; Caley et al. 1996).
Lipids play an important role as sources of FA, that are critical for normal development (Fuiman and Ojanguren 2011). Teleost eggs are rich in polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (22:6 n-3, DHA) and eicosapentaenoic acid (20:5 n-3, EPA) (Tocher and Sargent 1984). DHA and EPA cannot be biosynthesized de novo and must be acquired from the diet (Sargent et al. 1989).
DHA, EPA, and arachidonic acid (20:4 n -6, ARA) are precursors for prostaglandins and other eicosanoids, which regulate biological processes (Bell et al. 1992; Tocher 2003) and are critical components of phospholipid structures in fish membranes (Bell et al.
30 1986; Sargent et al. 1997). Rainbow trout Oncorhynchus mykiss broodstock fed a diet
low in n-3 PUFA during the last three months before vitellogenesis produce eggs with slightly less DHA and markedly less EPA (<50%) than broodstock fed a normal diet
(Frémont et al. 1984). Supplemental feeding of n-3 PUFA to red drum Sciaenops ocellatus larvae originally deficient in EPA and DHA has no effect, suggesting that a deficiency of certain FA during vitellogenesis causes irreparable damage to larvae
(Fuiman and Ojanguren 2011).
Appropriate amounts and ratios of DHA, EPA, and ARA are needed for normal development of larvae (Tocher 2003; Fuiman and Ojanguren 2011). Low levels of n-3
PUFA impair development of walleye Stizostedion vitreum (Czesny and Debrowski
1998), decrease fecundity and impair development of rainbow trout (Watanabe 1978;
Watanabe et al. 1984), and decrease fecundity in gilthead seabream Sparus aurata
(Fernandez-Palacios et al. 1995). A low level of ARA in domesticated Senegalese sole
Solea senegalensis is suggested to be the reason individuals skip spawning (Norambuena et al. 2012) while individuals with low EPA levels have lower fertilization and hatching rates (Lund et al. 2008). High levels of ARA and DHA increase predator avoidance in red drum (Fuiman and Ojanguren 2011), and along with EPA are positively correlated to egg symmetry and viability in cod Gadhus morhua (Pickova et al. 1997).
The transfer of FA from host to parasite (Tocher et al. 2010) or the cost of the immune response (Foo and Lam 1993) may decrease the maternal allocation of FA to eggs. In the tissue of common sea lice, an external copepod parasite that feeds exclusively on the tissue of its host, ARA and DHA are more highly concentrated than in host (salmon) muscle (Tocher et al. 2010). Negative effects of parasites on host health
31 may be exacerbated during the breeding season. Gravid crustaceans infected with an
acanthocephalan parasite Pomphorhynchus laevis have lower lipid content and fat than
uninfected gravid females (Plaistow et al. 2001). However, no effect was observed in
non-gravid females. In brook trout, stimulation of the immune system prior to ovulation
resulted in increased aptosis in the ovary, increased ovulation of premature eggs, and
decreased survival of larvae (Bonnett et al. 2008).
The objective of this study was to examine the effects of infection with
macroparasites on FA composition and content of eggs of common snook Centropomus
undecimalis. Common snook are euryhaline fish that, in the western Atlantic, inhabit a
variety of coastal habitats including freshwater rivers and lakes, brackish estuaries and inlets, and marine beaches and reefs (Marshall 1958; McMichael et al. 1989; Taylor et al.
1998; Young et al. 2014). Common snook are protandrous hermaphrodites, first maturing
as male at 169 – 222 mm Total Length (TL) and transitioning to female between 264 –
876 mm TL (Taylor et al. 2000). Sexually mature individuals migrate from low to high salinity (> 24 ppt.; Ager et al. 1976; Chapman et al. 1982; Peters et al. 1998) during the
reproductive season from 15th April to 15th October (Taylor et al. 2000) to spawn in
aggregations in inlets. Fertilized eggs flushed from inlets to nearshore water (Peters et al.
1998), hatch 15 hours later and enter a yolk-dependent larval planktonic stage (Yanes-
Roca et al. 2012). Larvae move into nursery habitat comprised of mangrove lined creeks
and estuaries approximately two weeks after hatching (Lau and Shafland 1982;
McMichael et al. 1989; Peters et al. 1998). As a result of previous attempts to raise
common snook for aquaculture, there is evidence that certain FA have a profound effect
on spawning success of common snook. Yanes-Roca et al. (2009) found eggs of common
32 snook with DHA levels over 13% of the total fatty acids (TFA) had a significantly higher
fertilization rate, higher hatching rate, and higher survival of larvae past one week
compared to eggs with lower percentages of DHA (Yanes-Roca et al. 2009).
We examined the FA content and composition of eggs from sexually mature
common snook during the spawning season to ask the following question: Does EPA,
DHA, ARA, and palmitic acid (16:0; PAL) content and composition in fish eggs vary in
response to host biology, spatiotemporal factors, and/or parasite infection? We
hypothesized that these FA would be negatively associated with increasing parasite
infection. We also hypothesized that energetic reserves, specifically the liver and fat
content of fish, would be negatively affected by increasing parasite infection intensity.
We used the established benchmark of 13% TFA of DHA (Yanes-Roca et al. 2009) to
judge whether fatty acids have a significant, negative effect on reproductive success of
common snook.
METHODS
Sexually mature female common snook were collected via rod and reel from three
spawning aggregations along the east coast of Florida: Palm Beach Inlet (PABI), Jupiter
Inlet (JUPI), and St. Lucie Inlet (STLI) as part of a multiyear study on reproduction
(Figure 2.1). After capture, specimens were immediately placed in a heavy duty plastic
bag and euthanized by submersion in a slurry of ice water below the lethal water
temperature for common snook (6°C, Shafland and Foote 1983) and exsanguination
(Florida Atlantic University IACUC protocol number A11-30).
Within two hours of capture, fish were examined for external and internal parasites during a necropsy, following Fish and Wildlife Research Institute (FWRI) life
33 history protocol. Live parasites were excised from surrounding tissue, counted, and
identified under a compound microscope with 10X and 20X magnification. Voucher
specimens were preserved according to FWRI Fish Health protocol and Noga (2000) (see
Chapter 1 Methods). A sample of plerocercoid metacestodes was sent to the Gulf Coast
Research Laboratory for visual confirmation of species. Live parasites were identified to genus and species. Dead parasites were excised from surrounding tissue and counted.
Parasites were considered dead if they did not move after excision from their case, had
shedding tissue, and /or the case had heavy melanization with fibrosis forming
granulomas. Dead parasites were not identified to taxon.
The population-level communities of parasites were described with prevalence
(percent of individuals infected), mean abundance (mean number of conspecific parasites
per fish), and mean intensity (mean number of conspecific parasites per infected fish).
The individual-level communities of parasites were described using intensity (number of
conspecific parasites), number of dead parasites, infection level of endoparasites (none,
low, moderate, high), and the Shannon-Weiner diversity index (H). Endoparasite infection level was determined using natural breaks in the frequency data on the sum of live internal parasites (Chapter 1 Methods). Parasite variables were calculated using
Quantitative Parasitology software.
During the necropsy, fish were measured (standard, SL; fork, FL; and TL, mm),
weighed (whole weight, gutted weight, g), and organs (liver, spleen, gonads, and fat, g)
were weighed individually. Sagittal otoliths were removed for age determination.
Somatic indices ((organ weight / whole weight) *100) were calculated for the liver
(hepatic-somatic index, HSI), spleen (spleen-somatic index, SSI), fat (fat-somatic index,
34 FSI), and gonad (gonadosomatic index, GSI). Body condition index (BCI) was
determined from the ordinary least squares regression of gutted weight (g) against total length (TL, mm) as described by Jakob et al. (1996). Female common snook collected
from spawning aggregations in 2011-2012 (n=40) were pooled with those collected in
2013 (n=40) to calculate the regression (See Chapter 1 Methods).
Two sections of gonads were taken, one for histological staging of gonad maturity and one for FA analysis. Gonads were histologically phased using the most advanced oocyte; following FWRI protocol (http://myfwc.com/research/saltwater/reef- fish/reproduction) and Murua et al. (2003). The phases were defined as: 1) immature; gonads contains oogonia and primary growth (PG) cells with no atresia and cortical alveolar (CA) in oocytes; 2) early developing/ developing; mature gonads with PG, CA, and first degree vitellogenic cells (Vt1); gonads are typically small with few muscle
bundles; 3) spawning capable; gonads contain secondary and tertiary vitellogenic cells
(Vt2, Vt3); post-ovulatory follicles (POFs) may be present and gonads are typically large;
4) actively spawning; gonads contain oocytes undergoing germinal vesicle migration,
yolk coalescence, germinal vesicle breakdown, hydration, and/or ovulation; 5) regressing; vitellogenic cells in artresia with advanced POFs; PG and CA may be present; and 6) regenerating; gonads contain oogonia and PG similar to phase 3, but ovarian walls are thick and muscle bundles are well defined. Once a female sexually matures, they will cycle through phases 2 through 6. In batch spawners such as common snook, females will cycle between phase 3 and 4 repeatedly in a spawning season.
For fatty acid analysis, a < 2 cm deep whole cross section of gonad was removed and rinsed in distilled water over a 400 micron mesh pan, removing the eggs from the
35 ovarian walls. A 0.05 – 0.1 g sample of eggs was weighed, stored in a 10 μl sample vial,
and placed on ice. Within an hour of being rinsed, egg samples were placed in an -80°C freezer until processing. Lipids were extracted from homogenized egg tissue according to
Folch et al. (1957) at University of Texas at Austin. Tissue was lyophilized with a 2:1 chloroform-methanol mixture and washed with 0.5-M KOH to remove all non-lipids.
Fatty acid methyl esters (FAMEs) were created from the conversion of the carboxylic
acid esters with 14% boron triflouride in methanol (Faulk et al. 2005). FAMEs were analyzed using a gas chromatograph; peaks were identified and used to quantify FA
present in the eggs (for a detailed review of methods see Faulk and Holt 2005).
Data analysis
General linear models (GLM) with a least absolute shrinkage operator (LASSO) to
control variable selection were used to select significant predictor variables of fatty acids.
Four fatty acids, PAL, ARA, EPA, and DHA were modeled as a function of host biology
(age, body condition index, and somatic indices), capture location, month, and parasitological (intensity of parasites by species, infection level of endoparasites, number of dead parasites, the ratio of live and dead parasites, and diversity) (Table 2.1). Models
that differed <3 in their AICc from the lowest scoring model, were further explored using
GLM with restricted maximum likelihood (REML) estimation. When effects in the model
were significant, a least squares mean (LSM) post hoc test was used to compare
difference between groups. Weight and percentage of predictor variables (PAL, EPA,
DHA, ARA) were transformed before analysis using a natural log transformation.
Estimated marginal means were back-transformed for presentation. Analyses were
conducted using SAS Enterprise software.
36 RESULTS
From May to August of 2013, forty female common snook were collected from spawning aggregations in the STLI (n = 2), JUPI (n = 27), and PABI (n = 11) (Table 2.2).
Ages ranged from 4 to 15 years old (mean ± S.E. : 8.52 ± 0.42 years) and sizes ranged from 620 to 1050 mm TL (mean ± S.E. : 873.47 ± 16.40 mm TL). Length did not always increase with age; the two youngest fish in the study (4 years old) were 732 and 856 mm
TL, and the smallest fish (620 mm TL) was 8 years old. However, of the two oldest fish in the study (15 years), one was also the largest (1050 mm TL). Study fish had a slightly higher BCI than the pooled population (Mean ± S.E. : 100.24 ± 160.37), but included fish leaner (-4570.32) and fatter (1932.57) than the pooled population. Somatic indices are presented in Table 2.2.
Seven genera of parasites from 4 major taxa were collected and identified from the gills, peritoneal cavity, stomach, and intestines of common snook: Proteocephalus sp.
(Cestoda), Callitetrarhynchus gracilis (Cestoda), Lernaeolophus sp. (Copepoda),
Lernanthropus gisleri (Copepoda), Anisakis sp. (Nematoda), Contracaecum sp.
(Nematoda), and Sebekia sp. (Pentastomida) (Table 2.3). Live parasites were found in
93% (n=37) of the population, while dead parasites were found in 56% (n = 23) of the population. Ninety-eight percent (n = 39) of the study population was infected with either live or dead parasites.
All live and dead parasites, with the exception of L. gisleri, were collected from inside the host body. L. gisleri was only collected from the gills. The majority of endoparasites including Callitetrarhynchus gracilis, Anisakis sp., Contracaecum sp., and
Sebekia sp. were collected from between the mucosa and submucosa of the stomach
37 lining or embedded in the mucosal membrane surrounding the internal organs. Adult
specimens of Proteocephalus sp. were attached to the intestinal lining by the scolex.
Prevalence, intensity, and abundance of live parasite species and dead parasites of
unknown species were calculated for the sample population (Table 2.3). L. gisleri was the most prevalent parasite in the population (65.0%) but was not the most intense infection
(Mean, 95% CI: 2.38 parasites, 1.77 – 3.31%). Contracaecum sp. was the second most prevalent parasite (60.0%) and had the highest intensity (Mean, 95% CI = 14.21 parasites, 8.25-22.88%), followed by Anisakis sp. (Mean, 95% CI: 8.33) which was only
present in 7.5% of the population. Adult cestodes (Proteocephalus sp.) and copepods
(Lernaeolophus sp.) were only found in one host each, and had the lowest prevalence
(2.5%).
Of the forty fish sampled, the gonads of 38 fish were assessed as either phase 3
(spawning capable, n = 30) or phase 4 (actively spawning, n = 8) and considered to be in
the process of spawning. Two individuals were assessed as phase 5 (regressing, n = 1) or
phase 6 (regenerating, n = 1) and could not be considered to be in the process of
spawning. However, the rate of oocyte maturation is not fully known in common snook
and it is possible both fish either had, or would have spawned during the season they
were collected. The ovarian development of spotted scat Scatophagus argus, a euryhaline
subtropical species, progresses from developing to spawning capable phase within 8
weeks (Zhang et al. 2013).
Twenty-six FA including saturated, monounsaturated, and polyunsaturated FA
were identified in the eggs from common snook (Table 2.4). Saturated FA accounted for
approximately 30% of the total fatty acids (TFA), while monounsaturated FA and PUFA
38 accounted for 29% and 36% of TFA, respectively. The most abundant by weight were
PAL>DHA> oleic acid (18:1 n-9); accounting for over half (58%) of the TFA.
The general pattern of PAL>DHA>ARA>EPA was evident across spatial, temporal, and gonad maturational factors (Figure 2.2) as well as BCI and age (Figure
2.3). Though some differences were apparent; the level of DHA exceeded PAL during phase 6 of the oocyte maturation process, and in age 4 fish. However, there were only two fish in phase 5 and 6, these differences between phases is anecdotal. Similarly, the standard error of %TFA of PAL and DHA were large in age 4 fish (n = 2) and could be attributed to low sample size.
The pattern of % TFA varied among individuals by endoparasite infection level and ratio of live to dead parasites (Figure 2.4). The percent of DHA was higher than PAL for fish classified as having no endoparasites, when no live parasites were present (0:15 –
0:1) and when high levels of live parasites were present with no dead parasites (10:0 –
47:0).
GLM revealed that a variety of host biology, spatial, and parasitological factors are significant predictors of the weight, and % TFA in common snook eggs. Candidate models selected based on the lowest AICc score (i.e. AICc < 4) from the GLM with
LASSO as variable selection were further explored using GLM with REML. Final best- fit models for weight and percent TFA of EPA, DHA, PAL, and ARA are presented in
Table 2.5.
Arachidonic acid (ARA)
ARA weight ranged from 1.99 – 9.23 mg/g DW with a mean (mean ± SD) of 7.16
± 1.29 mg/g DW. Phase (P < 0.0001) was a significant predictor of ARA weight. The
39 amount (weight) of ARA in eggs was expected to be highest in phases 4 (EMM ± S.E. :
2.00 ± 0.05 %) and 5 (EMM ± S.E.: 2.11 ± 0.14 %), and lowest in phase 6 (EMM ± S.E. :
0.68 ± 0.14 %). By weight, fish had a significantly lower amount of ARA in their eggs
during phase 6 than all other phases (differences of least means squares: phase 3: t(36) =
8.86 P = < 0.0001, phase 4: t(36) = 8.80 P = < 0.0001, phase 5: t(36) = 7.13 P = < 0.0001).
The percentage of ARA ranged from 2.56 – 12.78% with a mean ± S.D. of 4.04 ±
1.54%. Phase (P < 0.0001) and GSI (P = 0.0025) were significant predictors of ARA
percentage. The model predicted that ARA composition would increase with increasing
phase (Figure 2.5). Specifically, fish in phase 6 would have a significantly higher ARA
percentage than fish in phase 3 (differences of least means squares: t(35) = -7.53 P <
0.0001) and 4 (differences of least means squares: t(35) = -6.24 P < 0.0001). The model
also predicted that for every increase of a unit in GSI, the composition of ARA decreased
by 0.067 (solution for fixed effects: t(35) = -3.25 P = 0.0025).
Docosahexaenoic acid (DHA)
DHA weight ranged from 2.23 – 48.30 mg/g DW with a mean ± S.D. of 35.06 ±
8.31 mg/g DW. DHA weight was significantly affected by phase (P < 0.0001). The
weight of DHA was significantly lower during phase 6 than all other phases (differences
of least means squares: phase 3: t(36) = 14.06 P < 0.0001, phase 4: t(36) = 12.99 P <
0.0001, phase 5: t(36) = 11.11 P < 0.0001). The range of predicted weight peaked during
phase 5 (EMM ± S.E. : 3.83 ± 0.18 mg/g DW) and dramatically dropped to a low during
phase 6 (EMM ± S.E. : 0.92 ± 0.18 mg/g DW).
The percentage of DHA ranged from 9.64 – 31.21% of TFA with a mean ± S.D.
of 18.67 ± 3.58%. Percentage of DHA was significantly affected by phase (P < 0.0001).
40 The total percent of FA by DHA was significantly higher in phase 5 than all other phases
(differences of least means squares: phase 3, t(33) = -3.19, P = 0.0185; phase 4, t(33) = -
3.89, P = 0.0028; phase 5, t(33) = 2.85, P = 0.0449).
Palmitic acid (PAL)
PAL weight ranged from 2.84 – 54.13 mg/g DW with a mean ± S.D. of 41.35 ±
8.63 mg/g DW. The weight of PAL was significantly affected by phase (P < 0.0001),
location (P = 0.0113), and abundance of dead parasites (P = 0.0144). The amount of PAL
was significantly lower during phase 6 than in all other phases (differences of least means
squares: phase 3: t(19) = 11.69 P < 0.0001, phase 4: t(19) = 10.84 P < 0.0001, phase 5: t(19)
= 6.98 P < 0.0001) and it was significantly higher in phase 4 compared to phase 5
(differences of least means squares: t(19) = 3.55 P = 0.0202). The model predicted that
PAL would be highest in phase 3 (EMM ± S.E. : 3.60 ± 0.05 mg/g DW) and lowest in
phase 6 (EMM ± S.E. : 1.50 ± 0.15 mg/g DW). For every increase in a dead parasite,
PAL weight was predicted to increase by 0.01 (t(19)= 2.69 P = 0.0144). Fish from STLI
had significantly lower amounts of PAL than fish from JUPI and PABI (differences of
least means squares: JUPI: t(19) = 3.02 P = 0.0213, PABI: t(19) = 3.37 P = 0.0097).
PAL percentage ranged from 16.99 – 26.65% of TFA with a mean ± S.D. of 21.83
± 2.04%. The percent composition of PAL was significantly affected by phase (P =
0.0404) and intensity of Contracaecum sp. (P = 0.0243). For every increase in
Contracaecum sp. the percentage of PAL is expected to increase by .01 (t(33) = 2.36 P =
0.0243). PAL percentage was expected to be the lowest in phase 5 (EMM ± S.E. : 2.81 ±
0.08%), but was only significantly different from the percentage of PAL in phase 3
(EMM ± S.E. : 3.05 ± 0.02%, differences of least means squares: t(33) = 3.00 P = 0.0304).
41 Eicosapentaenoic acid (EPA)
EPA weight ranged from 0.28 – 8.95 mg/g DW with a mean ± S.D. of 4.97 ± 4.97
mg/g DW. The weight of EPA was significantly affected by phase (P < 0.0001), intensity
of Contracaecum sp. (P = 0.0004), age (P = 0.0342), and the ratio of live to dead
parasites (P = 0.4238). The weight of EPA decreased by 0.02 for each increase in
Contracaecum sp. (t(23) = -4.10 P = 0.0004) and by 0.02 for each increase in live to dead parasite ratio (t(23) = -2.40 P = 0.0248). EPA by weight was expected to be highest in
phase 3 (EMM ± S.E. : 1.35 ± 0.32 mg/g DW) and lowest in phase 6 (EMM ± S.E. : -
1.26 ± 0.42 mg/g DW). EPA weight was significantly lower in phase 6 compared to all
other phases (differences of least means squares: phase 3: t(23) = 6.61 P = <.0001, phase
4: t(23) = 5.52 P < 0.0001, phase 5: t(23) = 3.33 P < 0.0001). In general, EPA varied with
age, peaking at age 5 (EMM ± S.E.: 1.46 ± 0.37 mg/g DW) and 12 (EMM ± S.E. : 0.92 ±
0.25 mg/g DW) with the lowest at age 14 (EMM ± S.E. : -0.62 ± 0.36 mg/g DW). Fish at
age 14 had a significantly lower EPA weight than fish at age 5, 8, and 12 (differences of
least means squares: age 5: t(23) = 4.22 P = 0.0177, age 8: t(23) = 3.89 P = 0.0408, age 12:
t(23) = 4.07 P = 0.0261).
EPA percentage ranged from 0.76 – 4.46% of TFA with a mean ± S.D. of 2.62 ±
1.03%. The percentage of EPA was significantly affected by intensity of Contracaecum
sp. (P = 0.0243), age (P < 0.0001), and the ratio of live to dead parasites (P = 0.0252).
The composition of EPA decreased by 0.02 for every increase in Contracaecum sp. (t(27)
= -4.27 P = 0.0002) and decreased by 0.021 for every increase in the ratio of live to dead
parasites (t(27) = -2.37 P = 0.0252). Similar to the findings with weight of EPA, the
percentage of EPA was lowest in age 14 (EMM ± S.E. : -0.33 ± 0.32 mg/g DW)
42 compared to age 5 (EMM ± S.E. : 1.58 ± 0.32 mg/g DW) and age 12 (EMM ± S.E. : 1.20
± 0.32 mg/g DW, differences of least means squares: age 5: t(27) = 4.16 P = 0.0159, age
12: t(27) = -2.37 P = 0.0252).
DISCUSSION
The weight and composition of fatty acids in common snook eggs were affected by biological, spatial, and macroparasite infection factors. However, we demonstrate that these relationships are not necessarily linear and can be positive or negative. The most notable findings were: 1) The abundance of dead parasites and intensity of Contracaecum sp. was positively associated with PAL weight and % TFA, 2) The ratio of live to dead parasites and the intensity of Contracaecum sp. was negatively associated with the weight and % TFA of EPA, and 3) the weight and %TFA of PAL, ARA, EPA, and DHA changed with gonad maturity (phase).
The general pattern of FA distribution in eggs from common snook from the east coast of Florida was similar to the distribution in lipids of common snook from the west coast (Yanes-Roca et al. 2009). However, several differences in the relative amount of certain FA, and factors affecting FA were apparent between coasts. The % TFA of ARA were higher (4.04%) than in common snook from the west coast (3.68%; Yanes-Roca et al. 2009). This value is considerably higher than other species including wolfish
Anarhichas minor (1.2%, Tveiten et al. 2004), turbot Scophthalmus maximus (3.0%,
Silversand et al. 1996), walleye (0.5 – 2.22%, Czesny and Debrowski 1998), Senegalese sole (2.55%, Norambuena et al. 2012), white sea bream Diplodus sargus sargus (3.40%,
Rosa Cejas et al. 2003), and silver pomfret Pampus argentus (1.67%, Huang et al. 2010).
43 Different levels of ARA from common snook eggs from the east and west coast
may be due to diet. Omnivorous fish have a higher level of ARA than carnivorous fish
(Dunstan et al. 1988), which support the theory that common snook on the east coast eat
more invertebrates as part of their diet or have access to prey items higher in ARA.
Yanes-Roca et al. (2009) found that the percentage of DHA in common snook eggs
significantly decreased throughout the spawning season from May to September,
however month (June – August) was not a significant predictor of FA levels on the east
coast. In the present study, we found that FA varied significantly by oocyte maturation
(phase). DHA level was lowest in an individual assessed at phase 6 (regenerating).
However, more samples from individuals at varying stages are necessary to confirm these findings.
Two individuals in the study had DHA levels below 13% and are expected to have significantly less reproductive success than individuals with higher levels of DHA
(Yanes-Roca et al. 2009). The individuals with low percentages of DHA (9.6% and
11.8%) were collected from different locations (PABI and JUPI) and were different ages
(8 and 9), respectively. One was assessed at phase 4 with a moderately high BCI (226.12) while the other was assessed at phase 3 with a moderately low BCI (-335.98). Both had a moderate infection level comprised mainly of Contracaecum sp. and dead parasites were present. While phase was a significant predictor of the percentage of DHA for the population, it was never predicted to drop below 15% at any phase. In the two fish with
<13% DHA, the percentage of EPA and ARA were within the population norm however, the percentage of PAL was slightly elevated (23.8%, 26.6%).
44 The positive effect of the intensity of Contracaecum sp. on PAL and the negative effect of intensity of Contracaecum sp. on EPA may reflect the host’s ability to compensate for energetic resources lost due to infection with parasites. Unlike EPA, PAL can be biosynthesized de novo from acetyl-CoA and malonyl-CoA precursors (Sargent et al. 1989). EPA can only be acquired through the diet (Sargent et al. 1989). Perhaps, when EPA, or another FA that cannot be biosynthesized is in short supply, that host increases the creation and allocation of PAL, or another FA that can be biosynthesized, to the eggs. For every increase of Contracaecum sp., EPA was expected to decrease by 2% while PAL was only expected to increase by <1%, suggesting that PAL does not replace all EPA lost; thus other FA or lipids may be important to this relationship. The depletion of FA that cannot be biosynthesized may also be the reason why the models predict PAL to increase with increasing abundance of dead parasites. The presence of dead parasites suggests that the immune system has been stimulated and effective at some point in the past. The high energetic cost of the initiation and use of the immune system (Lochmiller and Deerenberg 2000) combined with the taxing demand of egg production (Lambert and Dutil 2000) may deplete the host of energetic reserves. PAL may fill the gap when resources of other FA are low.
A deficiency in EPA due to parasite infection may interfere with the roles of other
FA. Eiconsanoids are important in the control of ovulation, embryonic development of the immune system, hatching and larval performance (Mustafa and Srivastava 1989;
Sorbera et al. 1998). ARA is the primary precursor of eiconsanoids, generating 2-series prostaglandins (PGE2). In the ovaries of teleosts, PGE2 stimulates steroidogenesis
(Kelner and Van Der Kraak 1992; Wade and Van Der Kraak 1993). EPA, when
45 converted to 3-series prostaglandins (PGE3) has antagonistic functions (Smith 1989;
Tocher 2003); specifically blocking the formation of PGE2 (Sargent 1995) in the ovary
(Wade et al. 1994). The interaction of eicosanoids derived from FA support the theory
that not only the composition, but also the ratio of FA may be important to egg viability.
The influence of parasites on maternal allocation of FA may play a role in the
highly variable recruitment of natural populations. Parasites are capable of altering host
behavior, including foraging, and robbing the host of energy ultimately affecting
maternal diet and body stores which are the main sources of FA in eggs (Johnson 2009;
Stephens et al. 2009). We show infection with live parasites and the presence of dead parasites alter the EPA and PAL levels in eggs of common snook. The importance of n-3
PUFA, especially EPA and DHA, has been recognized in studies on reproduction of fish
from fertilization of eggs to larval performance (Izquierdo et al. 2001). Deficiencies in
certain FA at the egg stage causes developmental harm to the larvae that cannot be
resolved by diet once the larvae are hatched so variation in parasite infection could affect
FA composition of eggs, survivorship of offspring, and ultimately recruitment success
(Fuiman and Ojananguren 2011).
46 Table 2.1 Predictor and response variables used in the general linear models for egg fatty acid weight and percent total of fatty acids. Categorical variables are marked with a ©. Response variables were modeled as weight (mg/g DW) and percent of total fatty acids (%).
Egg fatty acids Spatial and Temporal Host Biology Parasite Infection
Responses Predictors Predictors Predictors
ARA Month © Age © Infection intensity per species
DHA Location © Body Condition Index (BCI) Abundance of dead parasites
EPA Spleen-somatic Index (SSI) Shannon Diversity Index (H)
PAL Hepatosomatic Index (©) Endoparasite infection level © 47
Gonadosomatic Index (GSI) Ratio of live to dead parasites
Fat-somatic Index (FSI)
Table 2.2 Summary statistics of biological information, body condition index (BCI), gonadasomatic index (GSI), Hepatosomatic index (©), Spleen-somatic index (SSI), and Fat-somatic index (FSI) from female common snook from the St. Lucie inlet (STLI), Jupiter inlet (JUPI), and Palm Beach inlet (PABI). Data are presented as mean, standard error, minimum, and maximum.
Biological information STLI JUPI PABI Total
n 2 27 11 40
Age 9.50 ± 0.50 8.00 ± 0.49 9.63 ± 0.92 8.52 ± 0.42 (years) 9 – 10 4 – 15 6 – 15 4 – 15
Total Length 782.00 ± 58.00 881.85 ± 18.11 869.54 ± 38.62 873.47 ± 16.40 (mm) 724 – 840 620 – 1050 623 – 1031 620 – 1050
48 -2132.46 ± 2437.86 171.59 ± 119.78 331.07 ± 260.65 100.24 ± 160.37
BCI -4570.32 – 305.40 663.15 – 1923.57 -640.28 – 1819.15 -4570.32 – 1923.57
1.88 ± 1.13 2.87 ± 0.23 3.86 ± 0.35 3.09 ± 0.20 GSI 0.74 – 3.01 1.19 – 5.81 2.09 – 5.68 0.74 – 5.81
0.81 ± 0.25 0.87 ± 0.04 0.91 ± 0.06 0.87 ± 0.03 © 0.56 – 1.07 0.43 – 1.65 0.61 – 1.34 0.43 – 1.65
0.05 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 0.05 ± 0.01 SSI 0.04 – 0.06 0.02 – 0.09 0.01 – 0.94 0.02 – 0.09
0.28 ± 0.25 0.65 ± 0.14 0.32 ± 0.10 0.54 ± 0.10 FSI 0.02 – 0.54 0.01 – 3.67 0.01 – 0.94 0.01 – 3.67
Table 2.3 Population-level community dynamics of parasites collected from female common snook. Stages are presented as either adult (A) or larval (L). Prevalence Intensity Abundance n Parasite species Stage % 95% CI Mean 95% CI Mean 95% CI infected Copepoda Lernanthropus gisleri 26 A 65.0 0.48 – 0.78 2.38 1.77 – 3.31 1.55 1.05 – 2.33 Lernaeolophus sp. 1 A 2.50 0.01 – 0.13 1.00 Na 0.03 0.00 – 0.08 Cestoda Callitetrarhynchus gracilis 19 L 47.5 0.32 – 0.62 4.47 2.74 – 7.63 2.13 1.17 – 4.05 Proteocephalus sp. 1 A 2.5 0.01 – 0.13 1.00 Na 0.03 0.00 – 0.08 Pentastomida Sebekia sp. 9 L 22.5 0.11 – 0.37 6.33 3.89 – 10.33 1.42 0.63 – 3.08 49
Chromadorea Contracaecum sp. 24 L 60.0 0.43 – 0.73 14.21 8.25 – 22.88 8.52 4.65 – 14.85 Anisakis sp. 3 L 7.5 0.02 – 0.19 8.33 1.00 – 15.67 0.63 0.03 – 2.92 Dead Unknown sp. 23 Na 57.5 0.41 – 0.72 7.13 5.17 – 9.43 4.10 2.53 – 7.10
Table 2.4 Common snook egg fatty acid (FA) weight and composition. Values are given as the mean ± SD.
Eggs total weight Eggs total FA % Fatty Acid (mg FA / g DW) area 12:0 0.04 ± 0.02 0.02 ± 0.01 14:0 2.73 ± 1.05 1.41 ± 0.46 15:0 2.67 ± 1.69 1.41 ± 0.84 17:0 1.63 ± 0.47 0.87 ± 0.21 16:0 PAL 41.35 ± 8.63 21.83 ± 2.04 18:0 8.18 ± 1.63 4.44 ± 0.85 ∑ Saturated 56.60 ± 11.18 29.99 ± 2.07 15:1 0.17 ± 0.11 0.09 ± 0.06 16:1 (n-7) 14.69 ± 4.02 7.66 ± 1.71 18:1 (n-9) 32.58 ± 9.68 17.14 ± 3.71 18:1(n-7) 7.15 ± 1.40 3.78 ± 0.41 20:1 (n-9) 0.46 ± 0.29 0.25 ± 0.15 ∑Monounsaturated 55.05 ± 12.30 28.92 ± 3.56 18:2 (n-6) 2.69 ± 1.28 1.40 ± 0.57 18:3 (n-6) 0.80 ± 0.31 0.43 ± 0.15 20:2 (n-6) 0.20 ± 0.16 0.11 ± 0.08 20:3 (n-6) 0.51 ± 0.20 0.28 ± 0.09 20:4 (n-6) ARA 7.16 ± 1.29 4.04 ± 1.54 22:5 (n-6) 2.69 ± 1.47 1.46 ± 0.75 16:2 (n-4) 1.14 ± 0.38 0.61 ± 0.17 16:3 (n-4) 3.58 ± 1.63 1.90 ± 0.80 18:3 (n-4) 0.71 ± 0.31 0.38 ± 0.16 18:3 (n-3) 1.49 ± 1.22 0.76 ± 0.57 18:4 (n-3) 0.99 ± 0.54 0.51 ± 0.25 20:3 (n-3) 0.24 ± 0.17 0.12 ± 0.08 20:4 (n-3) 0.81 ± 0.35 0.42 ± 0.17 20:5 (n-3) EPA 4.97 ± 4.97 2.62 ± 1.03 22:5 (n-3) 4.11 ± 1.73 2.20 ± 0.88 22:6 (n-3) DHA 35.06 ± 8.31 18.67 ± 3.58 ∑ PUFA 67.15 ± 12.45 35.90 ± 3.89
50 Table 2.5 Best fit models based on lowest AICc values and significance of main effects specified in the GLM model for weight and percentage of fatty acids.
Source df F P
ARA (Weight) Phase 4, 36 1911.60 <.0001
DHA (Weight) Phase 4, 36 3611.92 <.0001 EPA (Weight)
Phase 3, 23 15.77 <.0001 Intensity of Contracaecum sp. 1, 23 16.85 0.0004 GSI 1, 23 3.20 0.0870 Age 10, 23 2.49 0.0342 Ratio of live to dead parasites 1, 23 5.77 0.0248
PAL (Weight) Phase 3, 19 49.52 <0.001 Location 2, 19 5.73 0.0113
Age 10, 19 1.98 0.0955 Infection level of endoparasites 3, 19 0.98 0.4238 GSI 1, 19 1.25 0.2779 Abundance of dead parasites 1, 19 7.26 0.0144 ARA (% TFA)
Phase 4, 35 244.29 <.0001 GSI 1, 35 10.59 0.0025 DHA (% TFA)
Phase 3, 33 5.56 0.0034 Infection level of endoparasites 3, 33 1.32 0.2837 EPA (% TFA) Intensity of Contracaecum sp. 1, 27 18.23 0.0002 Age 11, 27 29.88 <.0001 Ratio of live to dead parasites 1, 27 5.62 0.0252 PAL (% TFA) Phase 3, 33 3.09 0.0404 Location 2, 33 1.34 0.2768 Intensity of Contracaecum sp. 1, 33 5.57 0.0243
51
Figure 2.1 Capture locations (◄) of female common snook from three aggregation sites, St. Lucie Inlet, Jupiter Inlet, and Palm Beach Inlet, along the southeast coast of Florida.
52
Figure 2.2 Percentage and standard error of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), and palmitic acid (PAL) in eggs of common snook by phase (A), capture location (B), and month (C).
53
Figure 2.3 Percentage (mean ± SE) of docosahexaenoic acid (DHA; ¯), eicosapentaenoic acid (EPA; n), arachidonic acid (ARA; s), and palmitic acid (PAL; =) in eggs from common snook by age (A), and body condition index (BCI; B).
54
Figure 2.4 Percentage (mean ± SE) of docosahexaenoic acid (DHA; ¯), eicosapentaenoic acid (EPA; n), arachidonic acid (ARA; s), and palmitic acid (PAL; =) in eggs of common snook by the ratio of live to dead parasites (A), and the infection index of endoparasites (B).
55
Figure 2.5 The weight and percent total composition (estimated marginal mean ± SE) of docosahexaenoic acid (DHA; ¯), eicosapentaenoic acid (EPA), arachidonic acid (ARA; s), and palmitic acid (PAL; =) predicted from general linear models in eggs of common snook during the oocyte maturation process. Phase was not a significant predictor variable for the percentage of EPA, so no estimated marginal means were calculated.
56 CHAPTER 3 : RATIOS OF N-3 AND N-6 FATTY ACIDS OF FISH EGGS IN
RESPONSE TO PARASITE INFECTION IN WILD FISH
ABSTRACT
Since the egg must satisfy nutritional needs for embryonic and larval development, and thus has a profound effect on larval survivability, studies have used the composition and ratio of fatty acids as an indicator of quality in eggs in freshwater and marine fishes. The ratios of fatty acids in eggs may be altered by maternal diet, environmental factors, and parasites. This present study focuses on the ratios of n-3 to n-6 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA), and arachidonic acid (ARA) to EPA in eggs of Common Snook,
Centropomus undecimalis, in response to infection with macroparasites. From May to
August of 2013, forty sexually mature female common snook were collected from spawning aggregations along the southeastern coast of Florida and sampled for parasites, physiological parameters, and fatty acids of eggs. Our main findings on how parasitism affects reproduction are as follows: 1) The ratio of n – 3:n – 6 PUFA was positively associated with the intensity of larval nematodes and cestodes, but negatively associated with an overall endoparasite infection level; 2) The ratio of DHA to EPA was positively associated with the intensity of larval nematodes and the ratio of live to dead parasites, but negatively associated with the intensity of gill copedpods; and 3) The ratio of ARA to
EPA was positively associated with the intensity of larval nematodes. The data show that
57 host biology and parasite infection change the ratios of n – 3:n – 6 PUFA, DHA:EPA, and ARA:EPA in eggs of common snook during the reproductive season. Imbalanced
PUFA ratios can have immediate and severe consequences for fish larvae affecting behavior, growth, and survival; ultimately affecting fish recruitment.
58 INTRODUCTION
Fatty acid composition of fish eggs may be a key factor in understanding
fluctuations in fish recruitment (Bell and Sargent 1996) due to its direct contribution to
the viability, hatching success, normal larval development and survival of larvae
(Izquierdo et al. 2001). Variation in reproductive output can be effected by changes in
environmental parameters, batch fecundity, number of spawning events, size at maturity,
and egg quality (Hunter and Leong 1981). The level of polyunsaturated fatty acids
(PUFAs) has been shown to have a profound effect on reproductive success and negative
consequences results when levels are too high or too low (Fernandez-Palacious et al.
1995; Bruce et al. 1999; Furuita et al. 2002). Recent studies have suggested that FA
composition in female gonad tissue and eggs may be appropriate for assessing the health
of first feeding larvae, and as an extension, recruitment potential. However, little is
known about how the quality of fish eggs is altered by changes in fatty acid (FA) profiles
in wild populations (Sargent et al. 1999; Wiegand et al. 2004).
Teleost eggs are generally high in eicosapentaenoic (EPA, 20:5n-3), docosahexaenoic (DHA, 22:6n-3), and arachidonic acid (ARA, 20:4n-6) in addition to saturated fatty acid palmitic acid (PAL, 16:0) (Tocher and Sargent 1984; Wiegand 1996).
Docosahexaenoic acid, EPA, and ARA have been examined in multiple species due to their significant importance in biological processes including membrane structure, immune function, and larval development (Rainuzzo et al. 1997). Docosahexaenoic acid accumulates in the neural tissues including brain and eye tissue in larvae (Bell et al.
1995). Arachidonic acid contributes to immune function, growth, ovulation, larval survival and improving stress tolerance (Bell and Sargent 2003; Pickova et al. 1997; Van
59 Anholt et al. 2004). Eicosapentaenoic acid competes with ARA in immune system
modulation and competes with DHA for placement in phosphoglycerides in marine fish
larvae (Izquierdo et al. 2000). Therefore the effect of PUFA on larvae survival is directly
related to the amount of ratios of ARA, DHA, and ARA.
Eicosapentaenoic acid, DHA, and ARA are most often acquired through diet for
feeding animals and from egg lipids for newly hatched larvae (Rainuzzo et al. 1997).
Alternatively, DHA and EPA may be produced from α-linolenic acid (18:3n-3) and ARA
may be produced from linolenic acid (18:2n-6) through elongation and desaturation.
However, biosynthesis of these FA is inefficient or non-existent in animals (Tocher 2003) and has only been proven in bacteria, fungi, and marine algae (Bajpai and Bajpai 1993;
Allen and Barlett 2002). The production of EPA and DHA requires a Δ5 desaturase, which is absent in marine fishes (Sargent et al. 1995). However, even amongst freshwater and diadromous fish, ability and efficiency of biosynthesis of FA varies. Desaturation ability is low in Arctic char Salvelinus alpinus (Olsen and Ringo 1992), while Atlantic salmon Salmo salar can synthesize EPA but not DHA (Ghioni et al. 2002). Thus, it is generally accepted that diet is the primary source of n-3 and n-6 PUFA.
The balance of n-3 and n-6 PUFA is crucial because they are the bases for the biosynthesis of modulatory bioactive molecules; thus it is the balance, and not solely the absolute amounts of FA that have a greater influence on maternal physiology and egg quality (Watanabe 1993) and for the immune response (Martinez-Rubio et al. 2014).
Eicosanoids derived from ARA (n-6 PUFA) and EPA (n-3 PUFA) includes prostaglandins (PG), thromboxanes, leukotrienes, and lipoxins (Smith 1989).
Prostaglandins are involved with reproduction, physiological homestasis, inflammatory
60 and immune responses (Smith 1989; Calder et al. 2002; Stacey et al. 2003; Fast et al.
2006). Arachidonic acid is the precursor for series-2 PG (PG2) whereas EPA is the
precursor for 3-series PG (PG3). PG2 modulates immune cell function and ovarian
steroidengensis in fish (Kellner and Van Der Kraak 1992; Wade and Van Der Kraak
1993) but in excess causes proinflammatory, prothrombatic, and proatherogenic
processes (Leaf and Weber 1988; Willis and Smith 1989). Competition for prostaglandin
synthase between ARA and EPA modulate the rate of formation of PG2 derived from
ARA, thus an increase in EPA decreases the generation of PG2 (Rubin and Laposta
1992). Eicosapentaenoic acid drives down the phospholipid content of ARA and enriches phospholipids with EPA (Li et al. 1994). Long chain n – 3 PUFA (DHA and EPA) are more efficient at inhibiting the metabolism of n – 6 PUFA (ARA) than the reverse
(Horrobin 1991), which helps balance the higher biopotency of PG2 compared to PG3
(Lands 1992). The levels of PG2 and PG3 are proportional to EPA:ARA ratio in the diet
and fish tissues (Bell et al. 1995; Norambuena et al. 2012). Dietary modulation of
prostaglandins in captive fishes has shown that optimal ratios of n-3 and n-6 PUFAs in
the diet are critical for the successful reproduction and health of the broodstock (Rola-
Pleszcynski 1989; Norambuena et al. 2013; Kinsella et al. 1990 a and b; Claesson et al.
1992). Lipid needs, however may be species or life history strategy specific.
It is generally accepted that FA composition of fish tissue, including gonad tissue
and eggs, reflects dietary FA composition to a great extent (Rosenlund et al. 2001; Bell et
al. 2002; Caballero et al. 2002). Changes in dietary FA were observed in tissue FA within
15 days for Eurasian Perch (Henrotte et al. 2010) and gilthead seabream Sparus aurata
(Harel et al. 1994). In fish, specific Fas are preferentially stored in specific tissues in
61 preparation for spawning (Jeong et al. 2002; Pickova et al. 2007; Jaya-Ram et al. 2008).
Preferential accumulation of DHA and EPA in the gonads has been demonstrated for
Eurasian Perch Perca fluviatilis (Henrotte et al. 2010) and sweet smelt Plecoglossus altivelis (Jeong et al. 2002) and for DHA in sardine Sardina pilchardus (Garrido et al.
2007). Starved gilthead seabream Sparus aurata larvae lost FA according to n – 6 > n – 3
(Koven et al. 1989), suggesting a selective pressure to conserve n-3 PUFA and reinforcing the importance of n-3 PUFA to larval development.
Aside from diet, FA composition may be altered by external factors (e.g., water temperature) and internal factors, such as parasites. By definition, parasites extract energy from their hosts either through direct absorption of nutrients, or by stimulating a costly immune response (Roberts and Janovy 2005). Mirrored PUFA composition between host and parasite has been demonstrated in protozoan parasites (Dixon and Williams 1970), microsporidian parasites (Biderre et al. 2000), and at least one monogenean
Neobenedenia girellae (Sato et al. 2008). However, in some cases DHA was higher in the parasite than the host by as much as 8% (Sato et al. 2008; Biderre et al. 2000). As opposed to parasites that feed on host tissue, thus digesting the host FA, some types of parasites such as tapeworms can directly absorb FA from their host through their tegument while attached to the intestinal wall (Beach et al. 1973).
Since the egg must satisfy nutritional needs for embryonic and larval development, and thus has a profound effect on larval survivability, studies have used FA composition as an indicator of quality in eggs in freshwater and marine fishes (Pickova et al. 1997; Navas et al. 1998; Pickova et al. 1998; Pickova et al. 1999; Furuita et al. 2000;
Sorbera et al. 2001; Wiegand et al. 2004; Pickova et al. 2007; Henrotte et al. 2008).
62 Altered ratios of n-3:n-6 PUFA, whether by diet or parasitic infection, result in decreased reproductive behavior (Lai and Hong 2010), poor hatching success (Pickova et al. 2007), and larval viability (Wantanabe 1993). Wild Arctic Char Salvelinus alpinus have a lower n-3 to n-6 ratio (3.5) and greater hatching success (>80%) compared to farmed fish (n-
3:n-6 = 13.5; hatching success = 20-70%) (Pickova et al. 2007). Among the n-3 PUFA, a low DHA:EPA ratio leads to visual developmental problems, eventually decreasing hunting efficiency and growth rate of larvae (Mourente et al. 1993; Rodriguez et al. 1997;
Watanabe et al. 1989) in striped bass Morone saxatilis (Harel et al. 2001), red seabream
Pagrus major (Takeuchi et al. 1990), Malabar grouper Epinephalus malabancus (Wu et al. 2002), and barramundi Lates calcarifer (Glencross and Rutherford 2011).
This present study focuses on the ratios of n-3:n-6 PUFA, DHA:EPA, and
EPA:ARA in eggs of Common Snook, Centropomus undecimalis, in response to infection with macroparasites. We have previously shown that PAL and EPA are affected by the presence of live and dead parasites, specifically that EPA decreases with an increase of plerocercoid metacestodes and the number of dead parasites (See Chapter 2
Results). Thus I hypothesize that: 1) The ratio of n-3:n-6 will decrease with increasing infection with macroparasites, and 2) DHA:EPA and ARA:EPA will increase with increasing parasite infection. I will discuss the possible consequences by which FA ratios in eggs effect spawning performance based on the modularity activities of EPA, DHA, and ARA.
METHODS
From May – August 2013, female common snook (n=40) were collected via rod and reel from three spawning aggregations in Jupiter Inlet (JUPI), Palm Beach Inlet
63 (PABI), and St. Lucie Inlet (STLI) along the southeastern coast of Florida. Fish were
euthanized immediately upon capture by submersion in a slurry of ice water below the
lethal limit for snook (6°C, Shafland and Foote 1983) and exsanguinated by rupture of the branchial artery. After euthanasia, fish were sealed individually in heavy plastic bags and stored on ice. Animal handling procedures followed approved IACUC protocol
(Florida Atlantic University IACUC protocol number A11-30).
Fish were transported back to the Fish and Wildlife Research Lab in Tequesta, FL and examined for biological measures and parasites less than two hours after capture.
Necropsy followed FWRI life history protocol; each fish was weighed whole and gutted to the nearest gram, measured for Total Length (TL) to the nearest mm, otoliths removed, each organ (spleen, stomach, liver, gonads, and fat) was weighed to the nearest gram, and two whole pie sections <2cm wide were taken from each gonad. One gonad sample was preserved in formalin for histological staging of gonad maturity and the second sample was processed for fatty acid analysis. For fatty acid analysis, the gonad sample was rinsed with distilled water over a 400 micron mesh sieve to remove the eggs from the lumen. A small sample (0.05 - 0.1 g) of rinsed eggs placed in a 10 ul sample vial and stored in a -
80°C freezer until processing.
A body condition index (BCI) was calculated for each fish as the residuals from a least ordinary squares regression of gutted weight against TL (Jakob et al. 1996).
Calculating a BCI is advantageous because it accounts for the influence of size and produces a value that is negative or positive depending on whether the fish was fatter or leaner than the population (Jakob et al. 1996; Green 2001).
64 Somatic indices for the liver (hepatosomatic index, HSI), spleen (spleen-somatic index, SSI), gonad (gonads-somatic index, GSI), and fat (fat-somatic index, FSI) were determined for each fish. Somatic indices were calculated as somatic index = organ weight (g)/ whole weight (g) multiplied by one hundred.
Gonad maturity was assessed according to FWRI protocol
(http://myfwc.com/research/saltwater/reef-fish/reroduction) and Murua et al. (2003).
Fished were phased 1-5 as follows: phase 1) immature or early developing; phase 2) developing; phase 3) spawning capable; phase 4) actively spawning; phase 5) regressing; or phase 6) regenerating.
Egg samples were processed at the University of Texas at Austin for identification, weight (mg FA / g dry weight), and composition (percent of TFA).
Extraction, lyyophilization, and fatty acid methyl esters creation followed Folch et al.
(1957) and Faulk et al. (2005) (See Chapter 2 Methods for details).
During and after the biological portion of the necropsy, fish were examined for macroparasites following FWRI Fish Health Protocol. The buccal cavity and skin of fish were observed with the naked eye and under 5X magnification. The left gill arch and organs were removed and examined under 10X and 20X magnification (dissecting scope). Parasites in the peritoneal cavity, most specifically in the mesentery between organs, were removed with large swathes of tissue and examined under the scope. While the organs and mesentery were under the scope, parasites were removed from the host tissue and identified to genus and species when possible. Voucher specimens were preserved according to FWRI Fish Health Protocol and Noga (2000) (See Chapter 1
65 Methods for full details). Several specimens of plerocercoid metacestodes were sent to
specialists at the Gulf Coast Research Laboratory for identification.
Dead parasites and unidentified masses were collected during necropsy and while
under microscopic examination. Parasites were considered dead if they 1) elicited no
movement after excision from their sheath, 2) were shedding tissue, and 3) the sheath was
darkened from melanization and surrounded by fibrotic granulomas. Identification of
dead parasites to species and genus was not possible due to a lack of distinguishing
features in the decayed material.
Prevalence, mean abundance, and mean intensity for each parasites genus or
species was calculated for the community (including all examined fish and parasites).
Prevalence was the proportion of infected fish to the whole population; mean abundance
was the mean number of conspecific parasites per fish; and mean intensity was the mean
number of conspecific parasites per infected fish.
Intensity (per species), number of dead parasites, infection level of endoparasites
(none, low, moderate, high), and the Shannon-Weiner diversity index (H) were calculated
for each fish. The endoparasite infection level was assigned a categorical none, low,
medium, high designation based on natural breaks in the data (See Chapter 1 methods for
full details). Population and individual level parasite community descriptors were
calculated using Quantitative Parasitology© 3.0 software.
Data analysis
Analysis of Variance (ANOVA) models were used to determine differences in host biology factors between sample times (months) and space (collection site). When
66 month or collection site was a significant factor, a least squares means (LSM) post hoc
test was used to determine significant differences between collection time and space.
A simple linear regression was calculated to examine the relationship of
covariates in the FA ratios (n-3:n-6 PUFA, DHA:EPA, ARA:EPA). The regression equation was considered significant at p < 0.05. Subsequently, Pearson’s correlation was calculated to determine the strength and direction (positive or negative) of the relationship of fatty acids in each ratio.
General linear models (GLM) with a least absolute shrinkage operator (LASSO)
to control variable selection were used to select significant predictor variables of fatty
acid ratios. Three fatty acid ratios, n-3:n-6 PUFA, DHA:EPA, and ARA:EPA were
modeled as a function of host biology (age, body condition index, and somatic indices),
spatial (capture location), temporal (month), and parasitological (intensity of parasites by
species, infection level of endoparasites, number of dead parasites, the ratio of live and
dead parasites, and diversity) factors (Table 3.1). Candidate models, i.e. models that
differed <4 in their AICc from the lowest scoring model, were further explored using
GLM with restricted maximum likelihood (REML) estimation. When effects in the model
were significant, a LSM post hoc test was used to compare difference between groups.
Estimated marginal means were back-transformed for presentation. Analyses were
conducted using SAS Enterprise© software.
RESULTS
From May to August of 2013, forty sexually mature female common snook were
collected from spawning aggregations in the STLI (n = 2), JUPI (n = 27), and PABI (n =
11). Each fish was sampled for parasites, physiological parameters, and fatty acids of
67 eggs (Table 3.2). Mean age (F2,35 = 6.140, p = 0.0052), HSI (F2,35 = 4.03, p = 0.0267), and
FSI (F2,35 = 4.210, p = 0.0230) varied significantly by collection month (Table 3.2). Fish
collected in June were older than individuals collected in July (Differences of least
squares means: t35 = 3.46, p = 0.0045) and were older (Differences of least squares means: t35 = 3.12, p = 0.0210), with a higher HSI (t35 = 0.29, p = 0.0251) and FSI (t35 =
0.82, p = 0.0175) compared to fish caught in August. Gonadosomatic indices (F2,35 =
3.16, p = 0.0377) and BCI (F2,35 =7.06, p = 0.0027) varied by capture location (Table
3.2). Fish collected from STLI were leaner compared to fish collected in JUPI (t35 =
3.600, p = 0.0027) and PABI (t35 = 3.71, p = 0.0020). There were no significant
differences of GSI between capture locations.
Ninety eight percent (n = 39) of the study population were infected with some
combination of live endoparasites (n = 36), dead endoparasites (n = 23), and/or live
ectoparasites (n = 26). Sixteen fish (40%) were infected with live endo and ectoparasites,
12 fish (30%) were infected with live endo and ectoparasites and dead endoparasites,
eight fish (20%) were infected with live and dead endoparasites, two fish (5%) were
infected with dead endoparasites, one fish (2.5%) was infected with live ectoparasites and
dead endoparasites, and one fish (2.5%) was uninfected.
Five genera of live endoparasites from 3 major taxa were collected and identified
from the peritoneal cavity, stomach mucosa and submucosa, and intestinal wall of
common snook. In total 509 live endoparasites were collected and/or observed including:
Proteocephalus sp. (Cestoda, n = 1), Callitetrarhynchus gracilis (Cestoda, n = 85),
Anisakis sp. (Nematoda n = 25), Contracaecum sp. (Nematoda, n = 341), and Sebekia sp.
68 (Pentastomida, n = 57) (Table 3.3). Dead parasites (n = 164) were collected from the
peritoneal cavity and stomach mucosa and submucosa but not identified to genera.
Two genera of live ectoparasites from 1 major taxon were collected and identified from the gills and buccal cavity of common snook. In total 63 live ectoparasites were collected and/or observed including: Lernaeolophus sp. (Copepoda, n = 1), and
Lernanthropus gisleri (Copepoda, n = 62) (Table 3.3). No dead ectoparasites were observed.
Of the forty fish sampled, the gonads of 38 fish were assessed as either phase 3
(spawning capable, n = 30) or phase 4 (actively spawning, n = 8) and considered in the process of spawning. Two individuals were assessed as phase 5 (regressing, n = 1) or phase 6 (regenerating, n = 1) and could not be considered in a process of spawning.
However, it is possible fish phased as regressing and regenerated either had or would have spawned that season (e.g. Zhang et al. 2013).
Twenty-six fatty acids including saturated fatty acids (SFA), monounsaturated fatty acids (MFA), and PUFA were identified in the eggs of common snook. All fatty acids identified were found in all eggs, but in varying quantities. The eggs of common snook were comprised of fatty acids in the order of PUFA (35.9%) > SFA (29.99%) >
MUFA (28.92%) (Table 3.4). Among the long-chain PUFA, eggs were comprised of n-3
(25.3%) > n-6 (7.71%) > n-4 (2.90%) (Table 3.4). In addition, the ratios of DHA:EPA,
ARA:EPA, and n-3:n-6 PUFA were calculated from eggs for each specimen. In general, the frequency of occurrence in the study population of the ratio of n - 3:n - 6 PUFA was unimodal while the ratios of ARA:EPA and DHA:EPA were right skewed (Figure 3.1). n-3:n-6 PUFA
69 The ratio of n-3 to n-6 PUFA ranged from 1.22 to 6.55 with a mean of 3.50 (SE =
0.17). The minimum ratio was observed in an individual with a low endoparasite index,
but it was the only individual infected with Lernaeolophus sp., a copepod. The highest
ratio was observed in an individual with a moderate endoparasite infection level.
The ratio of n-3 to n-6 PUFA had the strongest evidence for a linear relationship.
The results of the linear regression suggest that a significant proportion of the total
variation in n-3 PUFA was predicted by the weight of n-6 PUFA (F1,39=32.73, p <
0.0001). Adjusted r squared indicates that approximately 45% of the variation in n-3
PUFA was predicted by the weight of n-6 PUFA. Furthermore, Pearson’s correlation
indicates a positive relationship (r40=0.68, p < 0.0001). Overall the weight of the sum of
n-3 PUFA was a good predictor of n-6 PUFA. As the weight of n-3 increased, so did the
weight of n-6 PUFA.
The GLM model revealed that infection intensity of Callitetrarhynchus gracilis (p
< 0.0001), Anisakis sp. (p < .0001), the endoparasite infection level (p = 0.0005), and
phase (p < 0.0001) were significant predictors of the n-3:n-6 PUFA ratio (Table 3.5). The
ratio was expected to increase by 0.04 with every increase of Callitetrarhynchus gracilis or Anisakis sp. larvae. The ratio of n - 3:n - 6 PUFA was expected to increase with increasing phase (Figure 3.2, A) and decrease with increasing endoparasite infection level
(Figure 3.3).
DHA:EPA
The ratio of DHA to EPA ranged from 3.55 - 22.18 with a mean of 8.63 (SE =
0.74), which was the most highly variable ratio among the three examined. The minimum
70 ratio was observed in an individual with moderate levels of endoparasite infection and the
maximum ratio was found in an individual with a low endoparasite infection level.
A simple linear regression was conducted to determine if the weight of DHA
could be predicted from the weight of EPA in fish eggs. The linear regression was not
significant (F1,39 = 3.21, p = 0.0812). Additionally, the correlation between DHA and
EPA weight was insignificant (r40=0.27, p = 0.0812). Overall, the weight of DHA was not a good predictor of the weight of EPA in eggs.
The GLM model reveled that infection intensity of L. gisleri (p = 0.0320),
Contracaecum sp. (p = 0.0004), and the ratio of live to dead parasites (p = 0.0142), and age (p = 0.0084) were all significant predictors of the ratio of DHA to EPA (Table 3.5).
The ratio of DHA to EPA was expected to decrease by 0.06 with the increase of one L.
gisleri (t22 = -2.29, p = 0.0320), but also expected to increase by 0.02 with the increase of one Contracaecum sp. (t22 = 4.16, p = 0.0004) or the ratio of live to dead parasites (t22 =
2.66, p = 0.0142). No discernable pattern was observed between age and the predicted ratio of DHA to EPA (Figure 3.4). However, 14-year old fish were predicted to have a significantly higher ratio of DHA to EPA than 12-year old fish (differences of least squares means: t22 = -4.58, p = 0.0080), and 5-year old fish (t22 = -4.08, p = 0.0270).
ARA:EPA
The ratio of ARA to EPA ranged from 0.66 - 7.21 with a mean of 1.94 (SE=0.21).
The minimum ratio was observed in an individual with a moderate level of endoparasite
infection and no dead parasites. The maximum ratio was observed in an individual with
low endoparasite infection level but interestingly, this individual was also the sole host of
Lernaeolophus sp., and had the minimum ratio of n – 3:n - 6 PUFA.
71 The results of the linear regression suggest that the weight of ARA is not a
significant predictor of EPA (F1,39=0.67, p = 0.4188). Additionally, Pearson’s correlation
between the weight of ARA and EPA suggest there is an inverse, but non-significant
relationship (r40 = -0.13, p = 0.4188). Overall, the weight of ARA was not a good predictor of the weight of EPA in eggs.
Two models predicting effects on the ratio of ARA to EPA had equal support (< 3
AICc). The first model (herein referred to as model 1) predicted that infection intensityΔ of
Contracaecum sp., (p < 0.0001) and phase (p <0.0001) were significant predictors (Table
3.5). Model 1 predicted that the ratio would increase by 0.02 with every increase of
Contracaecum sp. (t34 = 4.86, p < 0.0001). It also predicted that the ratio would increase with increasing phase (phase 3: t34=2.84, p = 0.0076; phase 4: t34 = 2.50, p = 0.0176,
phase 5: t34=3.01, p = 0.0050; phase 6: t34=5.13, p < 0.0001). The only significant
difference between phases was between 3 and 6 where the ratio at phase 6 was
significantly higher than phase 3 (differences of least means squares: t34=-3.45, p =
0.0090). The second model (herein referred to as model 2) predicted that infection
intensity of Contracaecum sp. (p = 0.0003) and phase (p = 0.0042) were significant
predictors of ARA:EPA (Table 3.5). The main differences between models 1 and 2 were
the presence of informative, yet not significant predictor variables. The second model
predicted that the ratio would increase by 0.02 for every increase in Contracaecum sp.
(t22= 4.35, p = 0.0003). Similar to model 1, the ratio was expected to increase with
increasing phase (phase 3: t22=1.86, p = 0.0757; phase 4: t22=2.16, p = 0.0422; phase 5: t22=2.05, p = 0.0523; phase 6: t22=4.75, p < 0.0001) (Figure 3.2, C). The only significant
72 difference occurred between phases 3 and 6 (differences of least squares means: t22=-
3.58, p = 0.0099).
DISCUSSION
The data show moderate effects of host biology and parasite infection factors on the ratios of n - 3:n - 6 PUFA, DHA:EPA, and ARA:EPA in eggs of common snook
during the reproductive season. However, parasite infection parameters had both positive
and negative effects on fatty acid ratios. Our main findings in relation to the research
questions of how parasites affect reproduction are as follows: 1) The ratio of n - 3:n - 6
PUFA was positively associated with the intensity of Callitetrarhynchus gracilis and
Anisakis sp., but negatively associated with an endoparasite infection level; 2) The ratio
of DHA to EPA was positively associated with the intensity of Contracaecum sp. and the
ratio of live to dead parasites, but negatively associated with the intensity of L. gisleri;
and 3) The ratio of ARA to EPA was positively associated with the intensity of
Contracaecum sp. Our findings support previous research on the same study population
that the ratio of live to dead parasites and the intensity of Contracaecum sp. has a
negative effect on the weight of EPA in common snook eggs (See Chapter 2 results), thus
resulting in an increased ratios of DHA:EPA and ARA:EPA. We did not see the same
expected results of a lower ratio of n - 3:n - 6 PUFA due to parasite infection; possibly
because Contracaecum sp. and the ratio of live to dead parasites were not significant
predictors of this ratio or because EPA only accounts for a small proportion (<10%) of
the n – 3 PUFA.
The data show that fatty acid ratios in eggs of common snook are similar on the
east coast (ARA:EPA = 1.95; DHA:EPA = 8.63, n - 3:n - 6 PUFA = 3.5) and west coast
73 (ARA:EPA = 1.55; DHA:EPA = 5.76, n - 3:n - 6 PUFA = 2.52; Yanes-Roca et al. 2009) of Florida, but deviate drastically from other species of fish from different locals. The ratio of DHA to EPA and ARA to EPA in eggs are markedly higher in common snook than for Mangrove red snapper Lutjanus argentimaculatus (ARA:EPA = 1.1; Emata et al.
2003), Pacific cod Gadus macrocephalus (DHA:EPA = 1.3-1.7; ARA:EPA = 0.11;
Laurel et al. 2010), Atlantic cod Gadus morhua (DHA:EPA = 1.8; ARA:EPA = 0.19;
Laurel et al. 2010), Atlantic Bluefin tuna Thunnus maccoyii (DHA:EPA = 3.0; ARA:EPA
= 0.19; Gregory et al. 2010), Greater amberjack Seriola dumerili (DHA:EPA = 0.78;
ARA:EPA = 0.07; Rodriguez-Barreto et al. 2012), Sweet smelt Plecoglossus altivelis
(DHA:EPA = 1.5; ARA:EPA = 0.31; Jeong et al. 2002), Red porgy Pagrus pagrus
(DHA:EPA = 6.0; ARA:EPA = 0.89; Loukas et al. 2010), Greater Weever Trachinus draco (DHA:EPA = 5.44; ARA:EPA = 0.72; Loukas et al. 2010), and Piper Gurnard
Trigla lyra (DHA:EPA = 2.88; ARA:EPA = 0.55; Loukas et al. 2010).
However the ratio of n – 3:n – 6 PUFA observed in eggs of common snook was lower than Atlantic Bluefin tuna (n – 3:n – 6 PUFA = 7.2; Gregory et al. 2010), Greater amberjack (n – 3:n – 6 PUFA = 4.3; Rodriguez-Barreto et al. 2012), Red porgy (n – 3:n –
6 PUFA = 4.84; Loukas et al. 2010), Greater Weever (n – 3:n – 6 PUFA = 6.09; Loukas et al. 2010), and Piper Gurnard (n – 3:n – 6 PUFA = 5.74; Loukas et al. 2010). In general, it has been demonstrated that warm water fish have a higher ARA:EPA ratio in their tissue and eggs (Emata et al. 2003). However, the underlying causes for the deviation of fatty acids in common snook eggs in relation to other fishes are not known. Further research into the ecology, life history strategies, and prey base would help clarify this disparity.
74 The composition of egg FA is a reflection of maternal resources (Sargent et al.
1989), thus changes in the FA composition of eggs reflects some direct (e.g. Tocher et al.
2010) or indirect (e.g. Foo and Lam 1993) energetic cost to the host by parasites that results in altered FA composition of the eggs. Coho salmon smolts Oncorhynchus kisutch infected with Philonema agubernaculum, a parasitic nematode, had 36% less total lipids than non-parasitized fish (Schaufler et al. 2008). In addition parasitized fish had no triacylglycerols and monoacylglycerols (storage lipids) where these accounted for 5.5% of total extracted lipids in non-parasitized fish. Triacylglycerols and monoacylglycerols are comprised of a glycerol backbone with specificity for either DHA or EPA, dependent on position (Miller et al. 2006). Parasitized fish also had a higher level of free fatty acids
(57%) due to the breakdown of triglycerides compared to non-parasitized fish (35%).
Absorption of nutrients by parasites is a direct energetic demand on the host. The depletion of EPA, and resulting increase in DHA:EPA and ARA:EPA ratios, due to infection with larval stages of Contracaecum sp., a parasitic nematode, may be due to disproportionate absorption of this fatty acid by the parasite. Parasitic nematodes must obtain PUFA from their host because they lack the ability to biosynthesize PUFA (Liu and Weller 1990; Frayha and Smyth 1998). Although limited, studies have shown that the fatty acids of some parasites mirror that of the host (e.g. Sato et al. 2008). The quantity and identity of FA assimilated from the host is dependent on the species-specific and developmental stage specific metabolic needs of the parasite (Joachim et al. 2000). For example, parasitic nematodes in the L4c stage contained twice as much short chain PUFA that the L3c stage (Joachim et al. 2000). Weber et al. (1994) demonstrated that
Paratenuisentis ambiguous, a parasitic acanthocephalan, had a higher level of EPA in its
75 tissue as compared to its host (eel) tissue. In this study, all nematodes observed were in a larval stage of development and encased in a sheath. It is unclear how the presence of the sheath, which would be shed during migration from the gut to the stomach mucosa, submucosa, and peritoneal cavity (Roberts and Janovy 2005), would affect the ability of the parasites to absorb nutrients from the host.
Physical impairment of foraging ability by parasites may also directly rob the host of energy and alter the ratios of fatty acids in eggs. In this study, a single individual exhibited the minimum n – 3 to n – 6 PUFA and maximum ARA to EPA ratios. This individual was infected with the only specimen of Lernaeolophus sp., a large copepod attached to the tongue. The specimen of Lernaeolophus sp. was the largest macroparasite observed, 3 cm long excised from the tongue, and took up a large amount of room in the buccal cavity. Female Lernaeolophus sp. permanently burrow into host tissue using a multi-stemmed ‘anchor’ immediately after spawning (Grabda 1972). Examinations of
Lernaeolophus sultanus attached to the dorsal part of the buccal cavity show extensive damage to the cranial bones and soft tissue of the nasal cavity and eye sockets (Grabda
1972). It is reasonable to assume that in this study, the size and position of the specimen of Lernaeolophus sp. attached to the tongue interfered with the host’s ability to acquire and consume prey.
Parasites cause an indirect effect on the host by stimulating an immune response
(Roberts and Janovy 2005). The ratios of DHA to EPA and ARA to EPA may be higher in the presence of Contracaecum sp. due to biosynthesis of EPA into its eicosanoid derivatives; key mediators of inflammation and the regulation of T and B lymphocyte functions (Martinez-Rubio et al. 2014). Long-chain n -3 PUFA, including EPA, are anti-
76 inflammatory and counteract the inflammatory action of eicosanoids derived from ARA
(Calder and Grimble 2002). Eicosapentaenoic acid suppresses the immune system by directly inhibiting antigen presentation by accessory cells to helper T-cells (Fujikawa et al. 1992) and regulating gene expression (Verlengia et al. 2004). Eicosapentaenoic acid and DHA affect the expression of genes, but EPA has a stronger effect on genetic modulation of the immune response (Verlengia et al. 2004). The immunosuppressive effects of EPA may explain the positive effects of high levels of EPA in fish with inflammatory disease.
In Atlantic salmon with cardiomyopathy syndrome, a cardiac disease, high EPA proportions were associated with decreased heart and liver pathology (Martinez-Rubio et al. 2014). Similarly, the expression of inflammatory agents were reduced 13 days after exposure to a pathogen in large yellow croaker Larimichthys crocea fed a diet with a high
DHA to EPA ratio (Zuo et al. 2012). The two aforementioned studies investigated an acute exposure to pathogens. In contrast, this study focused on infection with macroparasites, which is a chronic infection that often results in continual, low-grade inflammation (Ashley et al. 2012). The proinflammatory response is less effective on macroparasites compared to microparasites because multi-celled macroparasites are too large to be phagocytized (Allen and Wynn 2011). The inflammation response is useful in wound healing, e.g. the migration path of a parasite through host tissue, through the development of granulomas. But over proliferation of granulomas can lead to scarring and potentially organ failure (Wynn 2004). Thus, the ability to downgrade or counteract the inflammatory response is critical to avoid host pathology. In theory, it is possible that the depletion of EPA resulting in the concomitant increase in DHA:EPA and ARA:EPA
77 ratios were the result of the continual biosynthesis of EPA into anti-inflammatory eicosanoids to combat the hosts chronic inflammatory response to larval nematodes.
More research into the before and after effects of macroparasites on the levels of fatty acids in host tissue is needed to confirm this hypothesis.
Imbalanced PUFA ratios can have immediate and severe consequences for fish larvae affecting behavior, growth, and survival (Bell et al. 1995; Garcia et al. 2008).
Diets with a low DHA to EPA ratio resulted in lower pigmentation success in turbot
(Rainuzzo et al. 1997) while Eurasian perch Perca fluviatilis fed a diet with a high DHA to EPA ratio (23/9) had lower hatching rates then broodstock fed a diet of near 3/2
(Henrotte et al. 2010). In Chilean flounder Paralichthys adspersus eggs with a high level of DHA:EPA and ARA:EPA had improved egg and larval quality (Wilson 2009). While current aquaculture-based research suggests a positive effect of high DHA:EPA and
ARA:EPA in egg lipids, it is unknown what effects on the larvae would occur should
EPA become limited as a result of infection with parasites.
78 Table 3.1 Summary of response variables modeled as a function of predictor variables in the General Linear Model (GLM). Egg fatty acids Spatial and Temporal Host Biology Parasite Infection
Responses Predictors Predictors Predictors
n – 3 : n - 6 ratio Month (c) Age (c) Infection intensity per species
DHA:EPA ratio Location (c) Body Condition Index (BCI) Abundance of dead parasites
ARA:EPA ratio Spleen-somatic Index (SSI) Shannon Diversity Index (H)
Hepatosomatic Index (HSI) Endoparasite infection level (c)
Gonadosomatic Index (GSI) Ratio of live to dead parasites
79 Fat-somatic Index (FSI)
Table 3.2 Summary statistics (Mean SE) of biological information, body condition index (BCI), gonadasomatic index (GSI), Hepatosomatic index (HSI), Spleen- somatic index (SSI), and Fat-somatic index (FSI) from female common snook from the St. Lucie inlet (STLI), Jupiter inlet (JUPI), and Palm Beach inlet (PABI) by capture location and month. Data are presented as mean, standard error, minimum, and maximum. Significant differences of biological information (column) are marked with an upper letter for capture location and month.
Age Total length n BCI GSI HSI SSI FSI (years) (mm)
Capture Location
JUPI 27 8.00 ± 0.49 881.85 ± 18.11 171.59 B ± 119.78 2.87 A ± 0.23 0.87 ± 0.04 0.05 ± 0.01 0.65 ± 0.14
PABI 11 9.63 ± 0.92 869.54 ± 38.62 331.07 B ± 260.65 3.86A ± 0.35 0.91 ± 0.06 0.04 ± 0.01 0.32 ± 0.10
STLI 2 9.50 ± 0.50 782.00 ± 58.00 -2132.46A ± 2437.86 1.88 A ± 1.13 0.81 ± 0.25 0.05 ± 0.01 0.28 ± 0.25 80
Capture Month
June 10 10.5A ± 0.78 923.40 ± 31.55 -105.00 ± 528.20 3.00 ± 0.30 1.01A ± 0.08 0.05 ± 0.01 0.94A ± 0.32
July 18 7.6B ± 0.55 845.11 ± 26.75 369.5 ± 185.2 3.10 ± 0.30 0.85A,B ± 0.05 0.05 ± 0.01 0.54A,B ± 0.12
August 12 8.25B ± 0.80 874.42 ± 23.32 -133.00 ± 127.40 3.20 ± 0.40 0.80B ± 0.06 0.06 ± 0.01 0.23B ± 0.05
Table 3.3 Population-level community dynamics of parasites collected from female common snook. Stages are presented as either adult (A) or larval (L). Type refers to endoparasite (En) or ectoparasite (Ec). Intensity Abundance n Parasite species Stage Type Mean 95% CI Mean 95% CI infected
Copepoda
Lernanthropus gisleri 26 A Ec 2.38 1.77 – 3.31 1.55 1.05 – 2.33
Lernaeolophus sp. 1 A Ec 1.00 Na 0.03 0.00 – 0.08
Cestoda
Callitetrarhynchus gracilis 19 L En 4.47 2.74 – 7.63 2.13 1.17 – 4.05 81 Proteocephalus sp. 1 A En 1.00 Na 0.03 0.00 – 0.08
Pentastomida
Sebekia sp. 9 L En 6.33 3.89 – 10.33 1.42 0.63 – 3.08
Chromadorea
Contracaecum sp. 24 L En 14.21 8.25 – 22.88 8.52 4.65 – 14.85
Anisakis sp. 3 L En 8.33 1.00 – 15.67 0.63 0.03 – 2.92
Dead
Unknown sp. 23 Na En 7.13 5.17 – 9.43 4.10 2.53 – 7.10
Table 4. Summary statistics (Mean ± SE) of egg fatty acid (FA) weight and composition. Eggs total weight Eggs total FA % Fatty Acid (mg FA / g DW) area
∑ SFA 56.60 ± 11.18 29.99 ± 2.07
∑MUFA 55.05 ± 12.30 28.92 ± 3.56
∑ (n - 6) PUFA 14.06 ± 3.49 7.71 ± 2.06
∑ (n - 4) PUFA 5.43 ± 2.11 2.90 ± 1.01
∑ (n - 3) PUFA 47.66 ± 10.55 25.30 ± 4.01
∑ PUFA 67.15 ± 12.45 35.90 ± 3.89 n - 3 : n - 6 3.50 ± 1.08 3.50 ± 1.08
DHA:EPA 8.63± 4.71 8.63 ± 4.71
ARA:EPA 1.95 ± 1.38 1.95 ± 1.38
82 Table 3.4 Best fit models of fatty acid ratios based on lowest AICc with significance of main effects.
Source df F P
DHA:EPA
Intensity of Contracaecum sp. 1, 22 17.29 0.0004
Age 10, 22 3.37 0.0084 Phase 2, 22 2.16 0.1217
Intensity of L. gisleri 1, 22 5.24 0.0320
Ratio of live to dead parasites 1, 22 7.10 0.0142
GSI 1, 22 0.01 0.9382 n-3:n-6
Phase 3, 30 13.00 <.0001 Intensity of Contracaecum sp. 1, 30 0.17 0.6837
Intensity of Anisakis sp. 1, 30 20.70 <.0001
Intensity of Callitetrarhynchus gracilis 1, 30 25.01 <.0001
Endoparasite Index 3, 30 7.84 0.0005
ARA:EPA model 1
Phase 4, 34 9.41 <.0001 Intensity of Contracaecum sp. 1, 34 23.59 <.0001
GSI 1, 34 3.15 0.0848
ARA:EPA model 2
Intensity of Contracaecum sp. 1, 22 18.90 0.0003
Phase 3,22 5.88 0.0042
GSI 1, 22 2.73 0.1128
Age 10, 22 1.65 0.1566
Intensity of Callitetrarhynchus gracilis 1, 22 0.26 0.6180
Ratio of live to dead parasites 1, 22 3.22 0.0865
83
Figure 3.1 Ratios of fatty acids including n-3:n-6 PUFA (A), ARA:EPA (B), and DHA:EPA (C) in eggs of Common Snook.
84
Figure 3.2 The predicted significant effect (Estimated Marginal Mean, EMM SE) of phase on n-3:n-6 PUFA (A), ARA:EPA model 1 (B), and ARA:EPA model 2 (C) based on the best fit GLM models.
85
Endoparasite Index Figure 3.3 The predicted significant effect (estimated Marginal Means, EMM SE) of the infection level of endoparasites on the ratio of n-3 to n-6 PUFA in eggs of Common Snook based on the best fit GLM.
86
Figure 3.4 The predicted significant effect (Estimated Marginal Means, EMM SE) of age on the ratio of DHA to EPA in eggs of Common Snook based on the best fit GLM.
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