EFFECTS OF ORGANOCHLORINE CONTAMINANTS ON HATCHLING AMERICAN ALLIGATOR (Alligator mississippiensis) GROWTH By JONATHAN J. WIEBE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005
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EFFECTS OF ORGANOCHLORINE CONTAMINANTS ON HATCHLING
AMERICAN ALLIGATOR (Alligator mississippiensis) GROWTH
By
JONATHAN J. WIEBE
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2005
Copyright 2005
by
Jonathan J Wiebe
This document is dedicated to Ralph Peter “Joey” Wiebe. Though I have not been able to see your face, your words, thoughts, and style live on forever.
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ACKNOWLEDGMENTS
I would like to thank my committee members, Dr. Tim Gross, Dr. Dave Barber,
and Dr. Franklin Percival, for their patience, understanding, and most importantly their
interest in my project. Tim, I will never be able to truly express my thanks for all the
opportunities that he has given me. I thank him for his counsel, beer making skills, and
ability to know “almost” everything before it happens but, most of all I thank you for
being my friend. Mom, I can’t say enough about all of the love, support and
understanding that she has provided. I thank her for being a great friend except for the
following: Jon the Mexican baby, Stretch Marks the Spot references, and Bulgur Wheat
care packages. Cheryl, who is my all-time, favorite chick on this rock. I thank her for
having a great attitude, closet neuroses, and removing that fishing hook. Janet, I cannot
thank her enough for all of her help, guidance, support, understanding and great food.
Thanks for making me laugh at myself when I get… well the way that I get. Ruth, thanks
for her supportive words of encouragement and wonderful sense of humor. Thanks to the
many families that I call my own Smiths, Duncans, Greenans, Scarboroughs, Loverns,
and Mitchells. All of you folks have showed tremendous support and kept me alive with
your amazing hospitality and friendship. Heath, I thank him for his time, assistance as
well as classic Arkansas stories. Phil Wilkinson, Franklin Percival and Woody
Woodward, I thank them for instilling in me an appreciation of alligators, southern jokes,
and appreciation of fine BBQ cuisine. Dwayne Carboneau, I thank him for social
commentary on not only alligator season but, life in general. Drs. Dan Sharp and Alan
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Ealy, I thank them for providing time and assistance with my project. Finally, I thank all
of my former and current lab mates: Travis “Smitty” Smith, Carla “CW” Wieser, Jim
“Roll Tide” Williams, Sherry “Lionheart” Bostick, Howard “Howie” Jelks, Nikki
“Nicooola” Kernaghan, Shane “Prarie Boy” Ruessler, Alfred “Fredo” Harvey, Jessica
“Gambusia Girl” Noggle, Kevin “The Stick” Johnson, Jessie “Piggy Girl” Grosso, Adro
“Tweety Bird” Fazio, and James “The Tape Man” Basto. Your friendship, patience, and
understanding throughout this MS experience are greatly appreciated.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS ................................................................................................. iv
LIST OF TABLES........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
ABSTRACT....................................................................................................................... xi
CHAPTER
1 LITERATURE REVIEW .............................................................................................1
Overview.......................................................................................................................1 Organochlorine Contaminant Exposure and Endocrine Disruption in Alligators ........2 Alligator Growth and Mortality in Relation to Organochlorine Contaminants............4
Thyroid Structure...................................................................................................7 Thyroid Hormone Synthesis and Systemic Availability .......................................7 Thyroid Hormone Binding Proteins ......................................................................9 Deiodination of Thyroid Hormones ....................................................................10
Thyroid Hormone Availability and Synthesis among Oviparous Species .................12 Species-Differences in Thyroid Hormone Utilization and Regulation.......................13
Fish ......................................................................................................................13 Amphibians..........................................................................................................13 Avian ...................................................................................................................14
Physiological and Environmental Influences on Thyroid Regulation........................15 Overview .............................................................................................................15 Reproductive and Thyroidal Seasonal Cycles.....................................................16 Nutritional Availability and Hibernation.............................................................18 Physiological and Environment Parameters Influence Growth...........................19
Effects of Organochlorine Contaminant Exposure on Thyroid Regulation ...............20 Overview .............................................................................................................20 Effects of Organochlorine Contaminant Exposure on Alligator Thyroid
Regulation........................................................................................................21 Thyroid Histology Alterations in Relation to Organochlorine Contaminant
Exposure ..........................................................................................................23 Influence of Organochlorine Contaminant Exposure on Integrated Levels of
Growth in Relation to p,p’-DDE, dieldrin, chlordane and toxaphene exposure.........30 Overview .............................................................................................................30 Experimental Data ...............................................................................................31
Organochlorine Contaminant Exposure and Hatchling Alligator Growth .................34
Introduction.................................................................................................................37 Materials and Methods ...............................................................................................42
Egg Collection, Evaluation and Incubation.........................................................42 Clutch Selection...................................................................................................43 Animal Maintenance ...........................................................................................44 Hatchling Morphometrics and Tissue Sampling .................................................44 Plasma Thyroid Hormone Validation Procedures (Total and Free Thyroxine) ..45 Free T4 (FT4) Assay Procedures.........................................................................46 Total T4 (TT4) Assay Procedures .......................................................................46 Analysis of Chlorinated Analytes from Alligator Egg Yolks .............................47 Statistics...............................................................................................................49
Results.........................................................................................................................49 Clutch and Organochlorine Contaminant Parameters .........................................49 Hatchling Growth Rates ......................................................................................50 Thyroid Hormones, Growth and Organochlorine Contaminants ........................51
Table page 2-1. Total length growth rates among and within sites......................................................81
2-2. Snout-vent length growth rates among and within sites.............................................82
2-3. Head length growth rates among and within sites......................................................83
2-4. Body weight growth rates among and within sites.....................................................84
2-5. Hatchling alligator thyroid (TSI) and liver (LSI) somatic indices among sites. ........85
2-6. Hatchling alligator thyroid somatic indices (TSI) within sites over time...................86
2-7. Hatchling alligator liver somatic indices (LSI) within sites over time.......................87
2-8. Multiple linear regression analysis of hatchling alligator growth rates,.....................88
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LIST OF FIGURES
Figure page 2-1. Graphical interpretation of thyroid hormone biosynthesis.......................................61
2-2. Clutch fecundity and clutch viability (site means)...................................................62
2-3. Clutch fecundity and clutch viability (current study)...............................................63
2-4. Yolk OC concentrations. site means (a) and current study (b).. ..............................64
2-5. Hatchling alligator growth parameters among sites over time.................................65
2-6. Hatchling alligator total length (mm) within sites over time.. .................................66
2-7. Hatchling alligator snout-vent length (mm) within sites over time..........................67
2-8. Hatchling alligator head length (mm) within sites over time...................................68
2-9. Hatchling alligator body weight (g) within sites over time......................................69
2-10. Hatchling alligator growth parameters (necropsy animals) among sites over time……...................................................................................................................70
2-11. Hatchling alligator total length (mm)(necropsy animals) within sites over time.....71
2-12. Hatchling alligator snout-vent length (mm)(necropsy animals) within sites over time……...................................................................................................................72
2-13. Hatchling alligator head length (mm)(necropsy animals) within sites over time.. ..73
2-14. Hatchling alligator body weight (g) (necropsy animals) within sites over time.. ....74
2-15. Hatchling alligator thyroid weight (g)(necropsy animals) within sites over time....75
2-16. Hatchling alligator liver weight (g) (necropsy animals) within sites over time.. .....76
2-17. Hatchling alligator total thyroxine(ng/ml)and free thyroxine (pg/ml) plasma concentrations among sites over time.. ....................................................................77
2-18. Hatchling alligator total thyroxine (ng/ml) plasma concentrations within sites over time...................................................................................................................78
x
2-19. Hatchling alligator free thyroxine (pg/ml) plasma concentrations within sites over time...................................................................................................................79
2-20. Graphical interpretation of factors that control the release of growth hormone.. ...80
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
EFFECTS OF ORGANOCHLORINE CONTAMINANTS ON HATCHLING AMERICAN ALLIGATOR (Alligator mississippiensis) GROWTH
By
Jonathan J Wiebe
December 2005
Chair: Timothy S. Gross Major Department: Veterinary Medicine
Alterations in alligator reproductive and growth parameters have been reported in
association with organochlorine (OC) contaminated sites in central Florida. These data
indicate reductions in egg and embryo quality as well as reductions in hatchling growth
and survivability. Thyroid, a growth-regulating tissue, has been suggested as a key bio-
indicator of growth among several species. In addition, several researchers have reported
alterations in thyroid regulation in relation to OC contaminant exposure. Previous field
studies have reported alterations in alligator plasma thyroid hormone concentrations as
well as several thyroid histological parameters. However, these data were unable to relate
plasma thyroid hormone (TH) concentrations to alligator growth. Under captive
conditions, preliminary data demonstrated that hatchlings from high OC environments
had hyperthyroid secretory patterns and accelerated growth. The current study examined
the same relationship; however an additional site with high OC contaminant
concentrations was added in order to evaluate the effects of OC contaminant exposure
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versus site as it relates to the observed alterations in hatchling growth and thyroid
regulation. In addition, a subset of hatchlings were sacrificed bi-monthly to compare
thyroid and liver weight (indicators of growth) with both hatchling external
morphometrics and plasma TH concentrations over time. Though TH were shown to be
bio-indicators of hatchling growth, no relationship was observed between OC
contaminant exposure and hatchling alligator growth or plasma TH concentrations. These
data suggest that hatchling alligator growth may be influenced by several key factors
including an integrated endocrine network (GH, IGF-I, TH, corticoids), habitat
degradation, as well as OC contaminant exposure.
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CHAPTER 1 LITERATURE REVIEW
Overview
During the 1980’s, significant reductions in American Alligator (Alligator
mississippiensis) egg viability were observed on Lake Apopka (a site positioned at the
headwaters of the Ocklawaha river basin with high organochlorine (OC) pesticide
concentrations) in comparison with lake Woodruff, a national wildlife refuge with
reduced concentrations of OC (Woodward, 1993; Rice et al., 1998). In addition, a severe
(~ 90%) reduction in the juvenile alligator population was observed on Lake Apopka
(1981-1986) that was likely attributed to reproductive failure (Woodward, 1993). These
observed reductions in juvenile survivability and adult reproductive success have been
attributed in part to the influence of agriculture and anthropogenic alterations
specifically: extensive utilization of organochlorine pesticides by muck farming
operations (i.e., (≈ 6,000 ha) of the lake’s northern wetland was converted for vegetable
production), citrus crops, and effluent discharges from both the citrus processing plant
and sewage treatment facility located at the city of Winter Garden (Woodward et al.,
1993; Schelske and Brezonik, 1992). These environmental alterations were compounded
by the overflow of a wastewater pond located at the Tower Chemical facility which is
adjacent to the Gourd Neck region of Lake Apopka (1980) consisting of high
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concentrations of sulfuric acid, DDT, dicofol and several unidentified OC compounds in
which by 1983, the EPA designated this facility’s property as a superfund site
(Rauschenberger, 2004). Though several of these OC compounds were identified in yolk
from alligator eggs, no direct association with reduced clutch viability was observed
suggesting other cofactors (i.e., diet, population dynamics, specific OCP mixtures) might
be involved and/or the developmental effects resulted from altered maternal physiology
(caused by OC exposure) as opposed to direct embryotoxicity (Rauschenberger et al.,
2004; Heinz et al. 1991). Therefore, sites that have been historically impacted by varying
degree of OC contamination (lakes Griffin and Apopka as well as the Emeralda Marsh
Conservation Area) continue to demonstrate coincident alterations in reproductive
function and success as measured by sex steroid biomarkers, sexual differentiation, clutch
viability, embryonic mortality, post hatch survivability, and growth (Rauschenberger,
2004; Wiebe et al., 2002; Gross et al. 1994).
Organochlorine Contaminant Exposure and Endocrine Disruption in Alligators
Reductions in alligator reproductive success as well as egg and embryo qualities
have been observed in relation to sites with intermediate to high concentrations of OC
contaminants (Rauschenberger, 2004; Masson, 1995). These chemicals have often been
referred to as “endocrine disruptors” or exogenous agents that interfere with the
production, release, transport, metabolism, binding, action, or elimination of natural
hormones in the body responsible for the maintenance of homeostasis and regulation of
developmental processes (Rolland, 2000; Brucker-Davis, 1998). As some of these OC
contaminants (i.e., p,p’-DDE) have been suggested to have positive and/or negative
estrogenic or androgenic activity, plasma sex steroid concentrations have been one of the
principal biomarkers utilized to examine the relationship between exposure to OC
3
contaminants and alterations in reproductive productivity. Gross et al. (1994) noted
alterations in plasma sex steroids among juvenile alligators from lakes Apopka (high OC
concentrations) and Woodruff (reference). Specifically, female juvenile alligators had
significantly higher plasma estradiol concentrations versus females from the reference
site (Gross et al., 1994) In contrast, juvenile male alligators from lake Woodruff exhibited
plasma testosterone concentrations that were almost four times higher than males on lake
Apopka (Gross et al., 1994). A similar incidence of altered plasma testosterone
concentrations in juvenile male alligators was reported by Guillette et al. (1999) among
seven Florida lakes. In addition, the author’s suggested a relationship between phallus
size (a sex steroid-dependent tissue) as a bio-indicator of anti-androgenic or estrogenic
contaminant exposure (Guillettte et al., 1999).
Masson (1995) reported significant reductions in alligator clutch viability (i.e.
embryonic mortality) on lake Apopka (3.9%) versus conservation sites with low OC
concentrations (71%). The author suggested that lake Apopka’s extremely variable, low
clutch viability and hatch percentages confirmed the suggestion that a severe
environmental problem exists at this lake site (Masson, 1995). Rice et al. (1998) observed
that the majority of lake Apopka’s embryonic mortality occurred during pre-egg
deposition or in early incubation with the next largest proportion of mortality occurring
very late in incubation. These data continue to support several hypotheses: 1) maternal
OC exposure alters reproductive regulation (as demonstrated by alterations in plasma
estrogen and testosterone concentrations) and, 2) the reported alterations in adult
reproductive fitness as well as maternal-transfer of OC contaminants among yolk
constituents appears to be related to the observed increase in embryonic mortality.
4
Alligator Growth and Mortality in Relation to Organochlorine Contaminants
It has been suggested that many of the observed embryonic and post-natal
alterations in offspring viability are the result in part of parental exposure to
environmental contaminants (Guillette, 1995). This exposure is primarily associated with
maternal transfer of lipophilic compounds (i.e., OCs) among yolk constituents to
developing offspring (Rauschenberger et al., 2004; Wu et al., 2000). OC exposure has
been suggested to alter hormones that control the course of development and growth and
may have the potential to alter differentiation of major organ systems resulting in
physiological and morphological changes (Rauschenberger et al., 2004; Wu et al., 2000;
Guillette et al., 1995). Wiebe et al. (2001) reported significant alterations in alligator
clutch viability and embryonic and post hatch survivabilities among sites of intermediate
(Griffin) to high (Apopka and Emeralda Marsh) OC concentrations. These data were
strengthened by Rauschenberger’s (2004) examination of the relationship between OC
exposure and subsequent reductions in egg and embryo qualities under field and
laboratory conditions. During 2000-2002 field collections, eggs collected from OC
contaminated sites had higher fecundity, lower average clutch mass and reduced clutch
viability in comparison with lake Lochloosa, a site with determined low OC
concentrations (Rauschenberger, 2004). Through the utilization of a captive adult
alligator treatment study, populations (treated and control) were orally dosed with eco-
relevant doses of the four principal OC contaminants identified from the previous field
egg collection: DDT and metabolites (principally p,p’-DDE), dieldrin, chlordanes and
toxaphene or vehicle control (Rauschenberger, 2004). Though reduced clutch viability
was observed in the treated versus control clutches, the majority of the observed mortality
was in the form of unbanded eggs which may represent either early embryonic mortality
5
or lack of conception (Rotstein et al., 2002). These data, from both field and laboratory,
continue to suggest that overall clutch survival appears to be related to total OC yolk or
maternal burdens (Rauschenberger, 2004).
Alterations in embryonic and hatchling growth as well as reduced post-hatch
survivability in relation to OC exposure has been reported in the American alligator
(Rauschenberger et al., 2004, Wiebe et al., 2002, Wiebe et al., 2001). It seems empirical
that alterations in growth and survivability among animals in these OC contaminated
environments would have ramifications at both site and population levels.
Rauschenberger (2004) examined the incidence of embryonic growth retardation and
survivability in relation to OC exposure utilizing an established embryo staging
methodology (Ferguson, 1985). This evaluation not only examined embryonic
morphological differences among sites over specific developmental time points but, also
evaluated the histopathology of live and dead embryos from “best-case” (clutches with
low mortality rates and low OC egg yolk concentrations) and “worst-case” (clutches with
high mortality rates and high OC egg yolk concentrations) clutches independent of site
(Rauschenberger, 2004). These data demonstrated several key points: 1) the youngest
embryos sampled (calendar day 14 of artificial incubation) showed the strongest
relationship between OC egg concentrations and morphometric parameters, 2)
morphology of live embryos was not consistently different among sites, except during
calendar day 25 (timeframe signifies the middle of organogenesis and may be a more
sensitive time period to OC exposure), 3) morphometry of live embryos was not
significantly related to variation in clutch mortality (i.e.., live embryos from clutches with
high mortality rates develop similarly to those of low mortality rates) 4), cyclodienes
6
(i.e., chlordane analytes) accounted for an average of 70% of the morphometric variation
that could be attributed to OC variables which is surprising considering DDT and its
metabolites compose an average of 66% of the total OC burden among all sites, 5)
concurrent decreases in maturational age and mass of dead embryos in comparison with
live embryos may have represented normal development up to a point at which the
development stalled and the embryo eventually perished, or embryos could have
developed at a much slower overall rate until the point at which they perished, and 6) no
significant differences in histopathology were observed among “best-case” and “worst-
case” clutches. (Rauschenberger 2004).
The principal mode of alligator embryonic exposure to OC contaminants has been
suggested to occur via maternal transfer among yolk constituents. Several examples have
demonstrated increased incidence of embryonic mortality in relation to exposure to high
concentrations of OC contaminants under both field and laboratory conditions. In
addition, Rauschenberger (2004) detailed significant relationships between OC exposure
and subsequent reductions in embryonic growth and development. Therefore, OC
contaminants are suggested to interfere with the regulation of critical growth and
developmental time periods which may ultimately contribute to the observed increase in
embryonic mortality on OC contaminated sites. These data demonstrate a critical need to
better understand the physiological role in regulating growth and development among
species exposed to OC contaminants.
The thyroid is one of the principal regulatory tissues of growth and development
among multiple taxonomic groups which has been demonstrated to regulate diverse
physiological endpoints including: metabolic rate, tissue differentiation and subsequent
7
growth and development (Rousset and Dunn, 2004). The two principal physiological
actions of thyroid hormones consist of 1) regulation of cellular differentiation and
development and, 2) regulation of metabolic pathways (Rousset and Dunn, 2004). These
general actions share a common integration in that changes in development and growth
are due to both hormone modulation of metabolism. In addition, cellular differentiation
changes inherently alter changes in gene expression, resulting in modulation of metabolic
pathways (Rousset and Dunn, 2004). A detailed working knowledge of thyroid regulation
is critical in understanding the complex and integrated roles the thyroid plays in growth
and development. Therefore, a literature review is provided which summarizes the
principal factors that regulate thyroid function including tissue structure, thyroid hormone
synthesis, availability, distribution, and deiodination in both embryonic and post-natal
life stages among several poikilothermic as well as homeothermic species.
Thyroid Structure
The thyroid gland is a bilobular tissue that is organized into spherical follicles
whose walls are composed of follicle cells that surround a central lumen filled with
colloid (McNabb, 2000). Colloid is primarily composed of thyroglobulin, a large protein
which is constructed in the rough endoplasmic reticulum, glycosylated in the reticular
lumen, and further post-translationally modified in the golgi apparatus of the follicle cell
(Norman and Litwack, 1997). Thyroglobulin with its tyrosine residues provides the
polypeptide backbone for the synthesis and storage of thyroid hormones as well as an
interim iodine storage area (McNabb, 2000; Norman and Litwack, 1997).
Thyroid Hormone Synthesis and Systemic Availability
The biosynthesis and secretion of thyroid hormones requires four principal
components including: thyroglobulin, thyroperoxidase, hydrogen peroxide and iodide.
8
Initially, dietary iodide is absorbed from the intestine and transferred from systemic
circulation across the basal lateral membrane of the follicle cells utilizing an ATP-driven
Na+ I- active transport (Norman and Litwack, 1997). The sequestered iodide is oxidized
to iodine via thyroperoxidase enzymatic activity in the presence of hydrogen peroxide
(principal electron acceptor) at the cell/colloid interface (McNabb, 2000). Concurrently,
follicle cells synthesize thyroglobulin which contains select tyrosyl residues that will
ultimately be iodinated and coupled to form either monoiodotyrosyls (MIT) or
diiodotyrosyls (DIT) residues and stored as colloid (Norman and Litwack, 1997). In total,
the catalyzing action of thyroperoxidase is required for the oxidation of iodide, iodination
of the thyroglobulin tyrosyl residues and the coupling of the MIT and DIT tyrosyls (i.e.,
thyronines) which based on the coupling combination produces either triiodothyronine
(T3) or thyroxine (T4) (Norman and Litwack, 1997).
Systemic TH availability is regulated utilizing a classic negative feedback
mechanism among the hypothalamic-pituitary-thyroid (HPT) axis (Norman and Litwack,
1997). As thyroid hormones occupy their nuclear receptors in the anterior pituitary, it
suppresses the transcriptional synthesis of preproTSH in the thyrotropes of the anterior
pituitary (Norman and Litwack, 1997). Under conditions of reduced T4, negative
feedback is reduced on thyrotropes of the anterior pituitary (McNabb 2000; Norman and
Litwack, 1997) Thyroid-releasing hormone (TRH) is secreted from the hypothalamus via
the hypophyseal portal vessels interacting with the anterior pituitary which results in the
release of thyroid-stimulating hormone (TSH). TSH interacts with its 7 transmembrane,
G coupled protein receptor on the thyroid follicle cells (Norman and Litwack, 1997,
Eales, 1984). As TSH is the most important controlling factor in iodine availability, the
9
thyroid follicle will proceed to generate free hormones from the stored hormones
sequestered among thyroglobulin (Norman and Litwack, 1997). This is accomplished as
the apical cell membrane engulfs the colloid by endocytosis and resulting cytoplasmic
colloid droplets fuse with lysosomes to form phagolysosomes (Norman and Litwack,
1997). Thus, the internalized thyroglobulin molecules are subject to a variety of
hydrolytic reactions leading to generation of free thyroid hormones and the complete
degradation of the protein (Rousset and Dunn, 2004; Brown et al., 2004; McNabb, 2000;
Norman and Litwack, 1997).
Thyroid Hormone Binding Proteins
Upon the release of TH from degraded thyroglobulin, a system of plasma proteins
that bind and distribute thyroid hormones is critical to counteract their loss from the
vascular and interstitial compartments by permeation into cell membranes (Prapunpoj et
al., 2002). These binding proteins are integral for systemic circulation due to THs high
lipid solubility (Richardson et al., 2005; Prapunpoj et al., 2002). Albumin (ALB) and
prealbumin or transthyretin (TBPA / TTR) are generally regarded as the two major T4
binding proteins throughout vertebrates; these having low binding affinity and high
capacity (Licht et al., 1991). In addition, many mammals possess thyroxine binding
globulin (TBG), a separate high binding affinity, low capacity binding protein that is
responsible for the principal portion of thyroid hormone binding (Licht et al., 1991).
Thyroid hormone binding protein(s) among vertebrate taxa demonstrate an evolutionary
progression towards increasing thyroid hormone distribution capacity during both
developmental and adult life stages (Richardson et al., 2005). An example of this can be
observed in the binding protein, transthyretin (TTR). TTR is transiently synthesized by
the liver during the time of increased thyroid hormone concentrations (i.e., smoltification,
10
metamorphosis and development) in fish, amphibians, reptiles whereas it is synthesized
by the liver during development and adult life stages in eutherians and birds (Richardson
et al., 2005). In crocodilians, TTR immunoreactivity has been detected in saltwater
crocodile (Crocodylus porosus) serum on days 60, 68, 75 of egg incubation, and day 1
post-hatch, but not detected in serum at 6 months of age or a 3 year old animal. In
addition, serum albumin was observed at all C. porosus age classes examined
(Richardson et al., 2005). Prapunpoj et al. (2002) demonstrated that C. porosus TTR has
higher binding affinity for T3 versus T4 suggesting that TTR was the principal
transporter of T3 to the crocodilian brain. These data in conjunction with an observed
higher percentage of amino acid sequence identity of C. porosus TTR to chicken TTR
versus lizard TTR and, Chang et al. (1999) observation of avian TTRs having higher
binding affinity for T3 versus eutherian TTRs suggest that the binding properties of C.
porosus TTR are more evolutionarily similar to those of avian TTRs versus eutherian
TTRs (Prapunpoj et al., 2002). Indeed, the separation in evolutionary functionality
between eutherian, avian and poikilotherm thyroid hormone regulation appears to be the
eutherian’s ability to generate and regulate thyroid hormones in a tissue-specific manner
(i.e., the evolution of 5’ deiodinases) and the utilization of additional binding proteins
(i.e., TBG) which enhances thyroid hormone regulation and distribution (Prapunpoj et al.,
2002).
Deiodination of Thyroid Hormones
The delivery of the predominant circulating TH (T4) to specific target tissues (i.e.,
liver, choroid plexus) is critical for the subsequent conversion of T4 to T3; which is
considered the principal, biologically-active form of TH. The majority of systemic T3
availability for multiple taxa is generated via extrathyroidal mechanisms in these target
11
tissues utilizing a process known as deiodination (Brown et al., 2004; McNabb, 2000).
The process of deiodination is catalyzed by a family of selonoenzymes called
deiodinases. These membrane-bound enzymes are located primarily in the microsomal
fraction of tissue homogenates suggesting an endoplasmic reticulum and/or plasma
membrane location (Hulbert, 2000). T4 is deiodinated by removal of iodine from the
outer ring of the molecule (ORD) to produce T3 or the inner ring of the molecule (IRD)
producing reverse T3 (rT3). ORD and IRD are catalyzed by three distinct deiodinases.
Type I catalyzes both ORD and IRD by preferentially removing phenolic and tyrosyl
iodide. This type of deiodinase is probably located in all tissues but has especially high
activity in the liver, kidney, thyroid tissue, and the central nervous system. Type II,
catalyzes only ORD by removing only phenolic iodide and has been found in the central
nervous system, brown adipose tissue, anterior pituitary and placenta. Type III catalyzes
exclusively IRD by removing only tyrosyl iodide and is found in the central nervous
system and the placenta (Shepherdley et al., 2002; Hulbert, 2000; Eales, 1984).
The integrated nature of thyroid regulation reflects a system principally regulated
by classic endocrine feedback mechanisms. In oviparous embryos, thyroid hormone
synthesis and availability are governed by a developmentally-regulated system utilizing
two sources: 1) maternal deposition in yolk (utilized during early stages of embryonic
development) and, 2) embryonic endogenous synthesis (utilized during later stages of
embryonic development). The next section details the principal mechanism(s) that
regulate oviparous embryo TH availability. In addition, a brief summary is provided to
demonstrate species-differences in TH utilization and regulation.
12
Thyroid Hormone Availability and Synthesis among Oviparous Species
Thyroid hormone availability during embryonic and early post-natal development
in oviparous species has been principally investigated through the examination of TH
synthesis, availability, compartmentalization, functionality, and utilization during several
lifestages (Prati et al., 1992; Greenblatt et al., 1989; Tagawa and Hirano, 1987; Sullivan
et al., 1987). The principal sources of thyroid hormones for developing oviparous
embryos have been identified as maternal deposition in yolk and endogenous synthesis by
the embryo (Greenblatt et al., 1989). In salmonids, high-density lipoproteins (HDL) and
vitellogenin (VTG), a yolk precursor protein, have been identified as the major carriers of
thyroid and other hormones, vitamins, ions, and minerals from maternal circulation and
subsequent sequestering in the yolk for the developing oocyte (Monteverdi and Di Giulio,
2000; Conley et al., 1997). In addition, Prati et al. (1992) suggested that TTR from
chicken extra embryonic membranes may bind iodothyronines of maternal origin
constituting the mechanism by which THs become available to the fetus before the onset
of thyroid function. In an examination of the relationship between TH content and yolk
mass, Sechman and Bobeck (1988) observed that a linear increase in both T4 and T3
concentrations in oocytes was proportional to the weight of the yolk without changes in
the iodothyronines content per 100 mg of yolk which indicated transfer of iodothyronines
together with other yolk constituents as a principal source of TH for developing oocytes.
Greenblatt et al. (1989) examined the compartmentalization of both T4 and T3 in yolk
and larvae in coho (Oncorhynchus kisutsch) and chinook (O. tschawytscha) salmon.
These data demonstrated an asynchronous species difference in thyroid hormone
utilization versus time between yolk reserves and endogenous TH production (Sullivan et
al., 1989). However, both species demonstrated a decreasing reliance on TH yolk
13
reserves in step with an increase in endogenous TH production in relation to increasing
larvae development (Sullivan et al., 1989).
Species-Differences in Thyroid Hormone Utilization and Regulation
Fish
In teleosts, T4 has been reported as the primary hormone released by the thyroid
(Eales, 1985). Under TSH stimulation, Eales (1985) reported a surge in both
endogenously labeled and stable plasma T4 concentrations with no corresponding
changes in plasma T3 concentrations. Kinetic studies have shown that about 80% of T3
in salmonids may reside in a slowly exchanging reserve pool, mainly represented by
skeletal muscle (Brown et al., 2005). This constancy in plasma T3 concentrations is due
at least in part to a rapid decrease in the proportion of available plasma T4 peripherally
monodeiodinated to plasma T3 (Eales, 1985). Though total thyroxine (TT4) and total
triiodothyronine (TT3) plasma hormone concentrations have been shown to be highly
correlated with their respective free plasma hormone concentrations, both percent free
thyroxine (%FT4) and free triodothyronine (%FT3) plasma hormone concentrations
demonstrated a negative correlation with TT4 and TT3 indicating that a smaller
proportion of total hormone is free at higher total hormone concentrations (Eales and
Shostak, 1985). In general, poikilotherm plasma TH concentrations contrast with those of
both Japanese Quail and humans where %FT3 exceeds %FT4, and are 3-5x higher than
those reported in both trout and charr (Eales and Shostak, 1985).
Amphibians
Amphibian utilization of TH has been primarily reported during several critical
stages of metamorphosis (Galton and Cohen, 1980; Suzuki and Suzuki, 1980; Mondou
and Kaltenbach, 1979). At stages V-XVIII (limb differentiation), plasma T4
14
concentrations were undetectable suggesting that bullfrog (Rana catesbeiana) tadpoles
were responsive to very low concentrations of thyroid hormones (Mondou and
Kaltenbach, 1979). During stage XIX (forelimb emergence) through stage XXI (tail
resorption), a rapid increase was observed in both circulating plasma T4 and T3
concentrations (Suzuki and Susuki, 1981). In addition, the T3/T4 ratio of plasma TH
concentrations suggested extrathyroidal deiodination during these stages of amphibian
metamorphosis (Suzuki and Susuki, 1981). At the conclusion of metamorphosis (stages:
XXIV – XXV), a rapid decline was observed in both plasma T3 and T4 concentrations in
froglets of four months of age (Suzuki and Susuki, 1981). In adult frogs, low but
detectable plasma T4 concentrations were observed (Mondou and Kaltenbach, 1979).
Avian
Birds possess the ability through the actions of thyroid hormones to regulate and
among sites were determined by Tukey Multiple Comparison Analysis (p <.05).
Viability
GR OR EM AP
(%)
0
20
40
60
80
100
aa a
a
Fecundity
GR OR EM AP0
10
20
30
40
50
60
a
b a a
64
Figure 2-4. Yolk OC concentrations. site means (a) and current study (b). Significant
differences among sites were determined by Tukey Multiple Comparison Analysis (p <.05).
YOLK OCP Concentrations (Site Means)
OR GR AP EM
(ng
/ g)
1
10
100
1000
10000Total Chlordane DDTx Dieldrin Toxaphene
a
ab
ab
b
a
b
b
b
aa
a
a
a
b
b
Yolk OCP Concentrations (Current Study)
OR GR AP EM
(ng
/ g)
1
10
100
1000
10000Total Chlordane DDTx Dieldrin Toxaphene
a b
65
Figure 2-5. Hatchling alligator growth parameters among sites over time. Significant
differences among sites were determined by Tukey Multiple Comparison Analysis(p < .05).
Total Length
S ept O ct N ov D ec Jan Feb M ar A pr M ay
TL (m
m)
0
100
200
300
400
500
600G R O RE MAP
a a aaa
ab bb
a b b b
a ab b b
a ab b b
a ab b b
a ab a
b b
a ab b b
a ab
abb
Head
Sept O ct Nov Dec Jan Feb M ar Apr M ay
Hea
d (m
m)
0
20
40
60
80G RO RE MAP
aa a a
a bb ba b bb
a bb b
a bb b
a ab
abb
a ab b b
a ab b
ab
a ab b
ab
W eight
S ept O ct N ov D ec Jan Feb M ar A pr M ay
Wei
ght (
g)
0
100
200
300
400
500
600G R O RE MAP
a bb ba bb
ab
a b b b
abb b
ab b b
ab bb
aab
ab b
aab
ab
b
aab
b
ab
S n o u t-V en t L en g th
S ep t O ct N o v D e c Ja n F e b M ar A p r M a y
SVL
(mm
)
0
5 0
1 00
1 50
2 00
2 50
3 00G RO RE MAP
aab
ab b
a b bb
a b bb
abb b
abbb
a ab
ab b
a
bb b
a ab bb
a ab b
ab
66
Figure 2-6. Hatchling alligator total length (mm) within sites over time. Significant
differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
Griffin
Sept Oct Nov Dec Jan Feb Mar Apr May
TL (m
m)
0
100
200
300
400
500
600
700GR-04-51 GR-04-A GR-04-B GR-04-C GR-04-D
aab
bc c
bab a a
ab
bab a a
ab b
ab a a
ab a
aa a abc c
aab
abc
aaa
aa
ab
b
ab
ab a
ab
b
aaa
b
Orange
Sept Oct Nov Dec Jan Feb Mar Apr May
TL (m
m)
0
100
200
300
400
500
600
700OR-04-12 OR-04-13 OR-04-B OR-04-W1 OR-04-W5
a a
a
aa
bc b ba
ac
ab
abab b
b b ba
ab
cd
bc
a bd
ab c
bc
ab
abc
ac
abc
a bc
ab c
a ab
aba
bb
aba
ab
ab
Emerelda
Sept Oct Nov Dec Jan Feb Mar Apr May
TL (m
m)
0
100
200
300
400
500
600EM-04-01 EM-04-02 EM-04-03 EM-04-04 EM-04-11
a ab bc
a a a ab
ab a
cb
ab
ab
a
cbb
ba
c
bc
bc
ab
a
b
ab
ab
ab
a
bb
baa
aa
aa a
a a
a
Apopka
Sept Oct Nov Dec Jan Feb Mar Apr May
TL (m
m)
0
100
200
300
400
500
600
700AP-04-10 AP-04-W2 AP-04-W10
a baa a
b b a bab
a bab
a b
ab a
a
aa
a
a aa
aa
c
67
Figure 2-7. Hatchling alligator snout-vent length (mm) within sites over time. Significant
differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
O range
Sept O ct Nov Dec Jan Feb M ar A pr M ay
SVL
(mm
)
0
50
100
150
200
250
300
350O R-04-12O R-04-13O R-04-BO R-04-W 1O R-04-W 5
aab
bc c
bc
abb b b
ab bab
ab
ab
abb b
ab b b
ab
aabc
ab
bc
a ab
c
bc
bc
c
a
b
aba
bab
a
b b
ab
ab
Griffin
Sept Oct Nov Dec Jan Feb Mar Apr May
SVL
(mm
)
0
50
100
150
200
250
300
350GR-04-51GR-04-AGR-04-BGR-04-CGR-04-D
aab
bc c c
aa aa aa
aa a aa
aaa aab b
ab
aba
aab
baa
b
aa
a
a
a ab
b
aba
ba
abc
abc
c
ab
a
Em erelda
Sept O ct Nov D ec Jan Feb M ar A pr M ay
SVL
(mm
)
0
50
100
150
200
250
300EM -04-01EM -04-02EM -04-03EM -04-04EM -04-11
ab bc d
aa a ab
ab a
cb b
abb b
c
ba
cb
bc
abc
a
c
ab
bc
ab
a
bab
b
a
aaaa
a aa
aa
Apopka
Sept O ct N ov D ec Jan Feb M ar A pr M ay
SVL
(mm
)
0
50
100
150
200
250
300
350AP-04-10AP-04-W 2AP-04-W 10
a bc
aab
a ab
aa a
aabb
aaa a
aaa
a
a aa
a
68
Figure 2-8. Hatchling alligator head length (mm) within sites over time. Significant
differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
Griffin
Sept Oct Nov Dec Jan Feb Mar Apr May
Hea
d (m
m)
0
20
40
60
80
100GR-04-51GR-04-AGR-04-BGR-04-CGR-04-D
a aa aaa a aa a
aa a aaaaa a a
a a a a aaa
aa a
ab b
aaab
a a aa
a
ab
b
ab a a
Griffin
Sept Oct Nov Dec Jan Feb Mar Apr May
Hea
d (m
m)
0
20
40
60
80
100GR-04-51GR-04-AGR-04-BGR-04-CGR-04-D
a aa aaa a aa a
aa a aaaaa a a
a a a a aaa
aa a
ab b
aaab
a a aa
a
ab
b
ab a a
Em erelda
Sept Oct Nov Dec Jan Feb Mar Apr May
Hea
d (m
m)
0
20
40
60
80EM -04-01EM -04-02EM -04-03EM -04-04EM -04-11
aabb bb
aab
ab b
c
ab a
cb b
ba
cb b
ba
cb
bc
bc
a
c
ab
bc b
a
babb
aa
a
a aa a
aa
a
Apopka
Sept Oct Nov Dec Jan Feb Mar Apr May
Hea
d (m
m)
0
20
40
60
80
100AP-04-10AP-04-W 2AP-04-W 10
a bca ab
a aba a
ba
abb
aab
ba a
aa a a a
a a
69
Figure 2-9. Hatchling alligator body weight (g) within sites over time. Significant
differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
Griffin
Sept Oct Nov Dec Jan Feb M ar Apr M ay
Wei
ght (
g)
0
100
200
300
400
500
600
700
800GR-04-51GR-04-AGR-04-BGR-04-CGR-04-D
a a bb ca a aa a
ab b
ab a a
b
abb
abaa
b
ab b
aba
ab
ab
aa
b
ab
ab
b
aaa
ab
b
ab
ab
aab
b
ab
a
a
Orange
Sept Oct Nov Dec Jan Feb Mar Apr May
Wei
ght (
g)
0
100
200
300
400
500
600
700OR-04-12OR-04-13OR-04-BOR-04-W1OR-04-W5
ab b bba bb bb
ab b bb
a ab
cbc
bc
a abb
cbc c
a abb
c bcc
a ab
bc
bc
c
a
ac
c
ab
bac
a
ab
ab
bb
E m erelda
Sept O ct N ov D ec Jan Feb M ar A pr M ay
Wei
ght (
g)
0
100
200
300
400
500
600EM -04-01EM -04-02EM -04-03EM -04-04EM -04-11
aabbc d
a b bbc
ba
cb b
b
a
c
b bb
a
c
b bc
a
b b
b
b
a
b bb
b
ab
a
b
aba
b
a
a
a
a
a
Apopka
Sept O ct N ov D ec Jan Feb M ar A pr M ay
Wei
ght (
g)
0
100
200
300
400
500
600
700AP-04-10AP-04-W 2AP-04-W 10
a bca ab
a ab
aab
b
aab
b
aab
baab
b
a a
a
a
aa
70
Figure 2-10. Hatchling alligator growth parameters (necropsy animals) among sites
over time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
Thyroid W eight
Sept N ov Jan M arch M ay
Thyr
oid
(g)
0 .00
0.01
0.02
0.03
0.04
0.05G RO REMAP
a
ab
bab
a a a a
a a a a
a
a
a
a
a
a a a
L iver W eight
Sept N ov Jan M arch M ay
Live
r (g)
0
2
4
6
8
10
12
14GROREMAP
a a a a
aa a a
aa a a
aa a a
a
b
ab a
b
W e ig h t
S e p t N o v J a n M a r c h M a y
Wei
ght (
g)
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0G RO RE MA P
a a a a
a abb b
a aa a
aa a
a
a
b
ab
ab
Snout-V ent Length
Sept N ov Jan M arch M ay
SVL
(mm
)
0
50
100
150
200
250
300
350G RO REMAP
a a a a
a bb b
a aa a
a a aa
a ab
ab
b
H ead Length
Sept N ov Jan M arch M ay
Hea
d (m
m)
0
20
40
60
80G RO REMAP
a aa a
a a a a
a aa a
a a a a
a ab
ab
b
Total Length
Sept Nov Jan M arch M ay
TL (m
m)
0
100
200
300
400
500
600
700G RO REMAP
a bab
ab
a ab b b
a a aa
a aaa
ab
ab
ab
71
Figure 2-11. Hatchling alligator total length (mm)(necropsy animals) within sites over
time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
O range
O R -04-12 O R -04-13 O R -04-B O R -04-W 1 O R -04-W 5
TL (m
m)
0
100
200
300
400
500
600
700S eptN ovJanM arM ay
a
a
ab
b
b
a
a
ab
a
ab
a
a
a
a
a
a
a
bab
b
a
a
ab
a
ab
E m ere lda
E M -04-01 E M -04-02 E M -04-03 E M -04-04 E M -04-11
TL (m
m)
0
100
200
300
400
500
600
700S eptN o vJanM arM ay
ba a a a
aab
ab a
bb
a
bb
bb
a
ab
bc
bc
c
a
ab
ab
ab b
A popka
A P -04-10 A P -04-W 2 A P -04-W 10
TL (m
m)
0
100
200
300
400
500
600
700SeptN ovJanM arM ay
aabb
aabb
a a
aa
a
a
aa
a
G riffin
G R -04-51 G R -04-A G R -04-B G R -04-C G R -04-D
TL (m
m)
0
100
200
300
400
500
600
700SeptN ovJanM arM ay
aab
abc
bc c
aab
ab
bb
a
a
a
a
aa
a
a
a
a
a
a
a
a
a
72
Figure 2-12. Hatchling alligator snout-vent length (mm)(necropsy animals) within sites
over time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
O ran ge
O R -04-12 O R -04-13 O R -04-B O R -04-W 1 O R -04-W 5
SVL
(mm
)
0
50
100
150
200
250
300
350S ep tN ovJanM arM ay a
ab a
b
bb
aab
ab
b b
a
aa
a
aa
a
a
a
a
aa
a
a
a
G riffin
G R -04-51 G R -04-A G R -04-B G R -04-C G R -04-D
SVL
(mm
)
0
50
100
150
200
250
300
350SeptNovJanM arM ay
aab
abc
bc c
aab
ab
b b
a
aa
a
aa
a
a
a
a
a
a
a
a
a
E m e r e ld a
E M - 0 4 - 0 1 E M - 0 4 - 0 2 E M - 0 4 - 0 3 E M - 0 4 - 0 4 E M - 0 4 - 1 1
SVL
(mm
)
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0S e p tN o vJ a nM a rM a y
aab
bcc d
aab
ab
ab
b
a
b bbb
aab
bc
bc
c
a
a
aa
a
A popka
A P-04-10 A P-04-W 2 A P -04-W 10
SVL
(mm
)
0
50
100
150
200
250
300
350SeptNovJanM arM ay
a bb
aab
b
a aa
a aa
aa
a
73
Figure 2-13. Hatchling alligator head length (mm)(necropsy animals) within sites over
time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
O ran ge
O R -04-12 O R -04-13 O R -04-B O R -04-W 1 O R -04-W 5
Hea
d (m
m)
0
20
40
60
80
100S ep tN ovJanM arM ay
aab
ab b
ab
aa aa a
ab
ab
ab
ab
a
b b
ab a
ba a
aa
a
E m ere ld a
E M -04-01 E M -04-02 E M -04-03 E M -04-04 E M -04-11
Hea
d (m
m)
0
20
40
60
80
100S eptN o vJanM arM ay
abb bc
a aa
aa
aab b bb
aab
bb b
aab
ab
ab
b
A po pka
A P -04-10 A P -04-W 2 A P -04-W 10
Hea
d (m
m)
0
20
40
60
80
100Sep tN o vJanM arM ay
ab b
aab
b
a aa
aa
a
a
aa
G riffin
G R -04-51 G R -04-A G R -04-B G R -04-C G R -04-D
Hea
d (m
m)
0
20
40
60
80
100SeptN o vJanM arM ay
a a a bb
a a a aaa
aa a
aa a
a a
a
aab
ab
ab
b
74
Figure 2-14. Hatchling alligator body weight (g) (necropsy animals) within sites over
time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
G riffin
G R -04-51 G R -04-A G R -04-B G R -04-C G R -04-D
Wei
ght (
g)
0
100
200
300
400
500
600
700
800SeptN ovJanM arM ay
baaaa
a
aba
bb
b
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
O range
O R -04-12 O R -04-13 O R -04-B O R -04-W 1 O R -04-W 5
Wei
ght (
g)
0
100
200
300
400
500
600
700
800SeptN ovJanM arM ay
aab bbb
a aba
bab
b
aab
ab
bc
c
a
bb
b
ab
a a a a a
Em erelda
EM -04-01 EM -04-02 EM -04-03 EM -04-04 EM -04-11
Wei
ght (
g)
0
100
200
300
400
500
600
700SeptN ovJanM arM ay
ab ba a a
a aab
ab
b
a
bb
bb
a
b
b
bb
a
aba
b
ab b
A p opka
A P -04-10 A P -04-W 2 A P -04-W 10
Wei
ght (
g)
0
100
200
300
400
500
600
700S eptN ovJanM arM ay
aab
ba bb
a
a
a
a
a
a
a
a
a
75
Figure 2-15. Hatchling alligator thyroid weight (g)(necropsy animals) within sites over
time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
G riffin
G R -04-51 G R -04-A G R -04-B G R -04-C G R -04-D
Thyr
oid
Wt.
(g)
0 .00
0.01
0.02
0.03
0.04
0.05
0.06
0.07SeptN ovJanM arM ay
aab
ab
b b
a
ab
ab
b
b
aa
a
a a
a
a a
a
a
a
a
a a
a
O range
O R -04-12 O R -04-13 O R -04-B O R -04-W 1 O R -04-W 5
Thyr
oid
Wt (
g)
0 .00
0 .01
0 .02
0 .03
0 .04
0 .05Sep tN o vJanM arM ay
a
aa
a
a
a
a
a
a
a
aa
a
a
a
aa
a
a
a
aa
a
a
a
E m ere lda
E M -04-01 E M -04-02 E M -04-03 E M -04-04 E M -04-11
Thyr
oid
Wt (
g)
0 .00
0.01
0.02
0.03
0.04
0.05
0.06S eptN ovJanM arM ay
a
b b b b
a
ab
b
b b
a
b
bb
b
a ab
ab a
b
b
a aa a a
A popka
A P -04-10 A P -04-W 2 A P -04-W 10
Thyr
oid
Wt (
g)
0 .00
0.01
0.02
0.03
0.04
0.05
0.06SeptN ovJanM arM ay
a
ab
b
a a aa
a
a
a
a
a
a
a
a
76
Figure 2-16. Hatchling alligator liver weight (g) (necropsy animals) within sites over
time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
G riffin
G R -04-51 G R -04-A G R -04-B G R -04-C G R -04-D
Live
r Wt (
g)
0
2
4
6
8
10
12
14
16
18SeptN ovJanM arM ay
aab
ab
bc c
a
ab
bb
b
aa
a
a
a
a
a a
a
a
a
a
a
a
a
O rang e
O R -04-12 O R -04-13 O R -04-B O R -04-W 1 O R -04-W 5
Live
r Wt (
g)
0
2
4
6
8
10
12
14
16
18S ep tN o vJanM arM ay
aab a
b
bb
a
ab
b
b
b
a
aa
a
a a
a
a
a
a
aa
a
a
a
E m ere lda
E M -04-01 E M -04-02 E M -04-03 E M -04-04 E M -04-11
Live
r Wt (
g)
0
2
4
6
8
10
12
14
16S ep tN o vJanM arM ay
aab
bc cd
a ab
ab a
bb
aab
bb
b
a
b
b
b
b
a
a
a
a
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A P-04-10 A P-04-W 2 A P -04-W 10
Live
r Wt (
g)
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14SeptN ovJanM arM ay
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Figure 2-17. Hatchling alligator total thyroxine(ng/ml)and free thyroxine (pg/ml)
plasma concentrations among sites over time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
Total Thyroxine
Oct Nov Dec Jan Feb Mar Apr May
TT4
(ng/
mL)
0
2
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18GROREMAP
b
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Free Thyroxine
Oct Nov Dec Jan Feb Mar Apr May
FT4
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6GROREMAP
b b b
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78
Figure 2-18. Hatchling alligator total thyroxine (ng/ml) plasma concentrations within
sites over time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
G riffin
O c t N o v D e c J an F e b M a r A p r M a y
TT4
(ng
/ ml)
0
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1 0
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1 8G R -04-51 G R -04 -A G R -04-B G R -04-C G R -04-D
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mL)
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20AP -04-10 AP -04 -W 2 AP -04 -W 10
a a
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O ct N o v D ec J an F eb M a r A p r M ay
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18O R -04-12 O R -04 -13 O R -04 -B O R -04-W 1 O R -04-W 5
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O ct N o v D ec Jan F eb M ar A p r M ay
TT4
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/ ml)
02468
10121416182022
E M -04-01 E M -04-02 E M -04-03 E M -04-04 E M -04-11
d
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79
Figure 2-19. Hatchling alligator free thyroxine (pg/ml) plasma concentrations within
sites over time. Significant differences among sites were determined by Tukey Multiple Comparison Analysis (p < .05).
G riffin
O c t N o v D e c J a n F e b M a r A p r M a y
FT4
(pg/
mL)
0
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7G R -04-5 1 G R -04 -A G R -04-B G R -04-C G R -04-D
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O ra n g e
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mL)
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8O R -0 4-1 2 O R -0 4 -1 3 O R -0 4 -B O R -0 4-W 1 O R -0 4-W 5
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6E M -0 4 -0 1 E M -0 4 -0 2 E M -0 4 -0 3 E M -0 4 -0 4 E M -0 4 -1 1
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80
Testosterone
Estradiol
GHRH
Estradiol
Neuropeptide Y
GLP-I
Estradiol
Norepinephrine
Galanin
Somatotroph cellsof anterior pituitary
Growth Hormone Release
Ration SizeProtein Intake
StarvationAcute Stress
Chronic Stress
TSH
T3T4
DOPA
Dopamine
Norepinephrine
5-HydroxytryptamineSomatostatin-25
Somatostatin-28
SRIFIGF-I
NPY
NMA
GHTRH
SRIFGnRH
Estradiol
SRIF
Bombesin
ExerciseOvulation
TemperatureDaylength
Seawater Adaptation CCK
Figure 2-20.Graphical interpretation of factors that control the release of growth
hormone. Adapted from Mommsen, 1998.
81
Table 2-1. Total length growth rates among and within sites.
Apopka Sept Oct Nov Dec Jan Feb Mar Apr May MEAN AP-04-10 0.92719 1.2397 0.8521 0.8392 1.061 1.0336 1.0406 0.9725 1.0382 1.0005 AP-04-W2 0.65300 0.8118 0.9669 0.9649 0.929 0.9695 0.9993 1.0239 1.0647 0.9314
Table 2-3. Head length growth rates among and within sites.
Apopka Sept Oct Nov Dec Jan Feb Mar Apr May MEAN AP-04-10 0.10576 0.1288 0.0943 0.0990 0.1227 0.1183 0.1178 0.11 0.1175 0.1127 AP-04-W2 0.08664 0.0973 0.1133 0.1126 0.1096 0.1122 0.1137 0.116 0.1232 0.1094
Table 2-4. Body weight growth rates among and within sites.
Apopka Sept Oct Nov Dec Jan Feb Mar Apr May MEAN AP-04-10 -0.142 0.3339 0.4028 0.5629 0.664 0.8235 0.9383 0.9884 1.2239 0.6439 AP-04-W2 -0.186 0.2751 0.5495 0.7342 0.6933 0.9298 1.1494 1.2807 1.483 0.7677
Table 2-5. Hatchling alligator thyroid (TSI) and liver (LSI) somatic indices among sites over time. No differences were observed in TSI among sites. Temporal differences were observed in LSI among sites. Significant differences determined utilizing Wilkoxon analysis with the Kruskal –Wallis Test (p < .05).
TSI among Sites
LSI among Sites
Date Chi Square Pr > Chi Square Date Chi Square Pr > Chi Square
Sept 4.2855 0.2322 Sept 6.8526 0.0767 Nov 5.936 0.1148 Nov 17.0271 0.0007 Jan 1.1091 0.7749 Jan 6.1687 0.1037 Mar 4.8678 0.1817 Mar 9.786 0.0205 May 1.0038 0.8003 May 6.3759 0.0947
86
Table 2-6. Hatchling alligator thyroid somatic indices (TSI) within sites over time. No significant differences were observed. Significant differences determined utilizing Wilkoxon analysis with the Kruskal –Wallis Test (p < .05). Apopka Chi-Square Pr> Chi Square Sept 3.4667 0.1767 Nov 3.2000 0.2019 Jan 0.3556 0.8371 Mar 5.0667 0.0794 May 5.0667 0.0794 Emeralda Chi-Square Pr> Chi Square Sept 5.1434 0.2729 Nov 10.8945 0.0278 Jan 9.5667 0.0484 Mar 5.5667 0.2339 May 4.9333 0.2942 Griffin Chi-Square Pr> Chi Square Sept 9.1747 0.0569 Nov 6.8706 0.1429 Jan 4.7667 0.3121 Mar 7.2667 0.1224 May 9.7333 0.0452 Orange Chi-Square Pr> Chi Square Sept 4.2000 0.3796 Nov 8.9667 0.0619 Jan 10.7667 0.0293 Mar 2.5796 0.6304 May 7.0000 0.1359
87
Table 2-7. Hatchling alligator liver somatic indices (LSI) within sites over time. No significant differences were observed. Significant differences determined utilizing Wilkoxon analysis with the Kruskal –Wallis Test (p < .05).
Apopka Chi-Square Pr> Chi Square Sept 3.2889 0.1931 Nov 3.2000 0.2019 Jan 4.6222 0.0992 Mar 0.8000 0.6703 May 5.9556 0.0509 Emeralda Chi-Square Pr> Chi Square Sept 10.4333 0.0337 Nov 4.4667 0.3465 Jan 4.6333 0.3270 Mar 10.833 0.0285 May 3.9000 0.4197 Griffin Chi-Square Pr> Chi Square Sept 12.0333 0.0171 Nov 8.1667 0.0857 Jan 11.7000 0.0197 Mar 7.9667 0.0928 May 3.7000 0.4481 Orange Chi-Square Pr> Chi Square Sept 7.1711 0.1271 Nov 9.2333 0.0555 Jan 5.3000 0.2579 Mar 2.5000 0.6446 May 2.7667 0.5976
88
Table 2-8. Multiple linear regression analysis of hatchling alligator growth rates, thyroid hormone secretory rates and organochlorine contaminant concentrations. No significant relationships were demonstrated (p < .05).
Total Length Rate
Snout-Vent Length Rate
Head Length Rate
Body Weight Rate
Total Chlordane 0.1084 0.1108 0.3362 0.3072
Total DDTx 0.1129 0.0959 0.3938 0.1462
Dieldrin 0.1281 0.1246 0.3376 0.4492
Toxaphene 0.4905 0.6954 0.8230 0.5753
TT4 Rate 0.3704 0.7254 0.5308 0.8545
FT4 Rate 0.2137 0.1193 0.4314 0.1983
89
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BIOGRAPHICAL SKETCH
Jonathan James Wiebe was born on December 15, 1969, in Pensacola, Florida, and
is the son of Ralph and Linda Wiebe. Jon graduated from Gainesville High School in
1986 and received a BS in wildlife management from the University of Florida in 2000.
Jon has spent an extensive amount of his professional career in the care of large and
diverse animal collections among various zoological and private collections. The
majority of Jon’s professional career has been spent in the laboratory of Dr. Tim Gross.
This laboratory specializes in examining the effects of environmental stressors on
reproductive and growth parameters in a variety of different species. Jon is particularly
proud of the collaborative work that he has achieved with Dr. Tim Gross, Dr. Heath
Rauschenberger and Janet Scarborough in the area of alligator ecotoxicology.