RELATIONSHIP BETWEEN EGG THIAMINE CONCENTRATIONS AND EMBRYO MORTALITY
IN THE AMERICAN ALLIGATOR

María Sepúlveda^1,2, Jon Wiebe², Dale Honeyfield³,
Joy Hinterkopf³, and Timothy Gross^1,2

¹Department of Physiological Sciences, College of Veterinary Medicine,
University of Florida, Gainesville, FL, 32610
²USGS - Florida Integrated Science Center, Gainesville, FL 32653
³USGS - Appalachian Research Laboratory, Wellsboro, PA 16901

Abstract

 Thiamine deficiency has been linked to early mortality syndrome in salmonids in the Great Lakes. The present study was conducted to compare thiamine concentrations in alligator eggs from sites with high embryo mortality and high exposure to organochlorine pesticides (OCPs) (Apopka, Griffin, and Emeralda Marsh) to that from a site that has historically exhibited low embryo mortality and low OCPs (Orange/Lochloosa).  During 2000 and 2001, a total of 56 clutches were collected from these sites, and artificially incubated. Clutches were monitored for embryo mortality and hatch rates, and thiamine measured in one egg/clutch. Eggs from the reference site had two times the amount of total thiamine compared to the impacted sites (1603 pmol/g vs. 847 pmol/g), and clutches with > 65% hatch rates had twice the amount of total thiamine compared to clutches with < 64% hatch rates (990 pmol/g vs. 485 pmol/g). These results suggest that thiamine deficiency might be playing an important role in alligator embryo survival and development. Causes for this deficiency are unknown at this time, but might be related to differences in the nutritional value of prey items across the sites studied. In addition, it is remains unknown what the role of OCPs are in the overall differences observed in egg nutritional quality across the sites studied (Funded by NIEHS-SFBRP).
 

Introduction

 Researchers from the Great Lakes and the Baltic Sea have linked mortalities of early-life stages of trout and salmon with lower than normal concentrations of a critical vitamin (vitamin B₁ or thiamine). Clinical signs of the disease include loss of equilibrium, anorexia, reduced yolk-sac utilization, and death. The ability of thiamine to reverse and prevent this syndrome has led to the speculation of a cause-effect relationship between low egg thiamine levels and early mortality syndrome in salmonids. Recent studies, however, raise the potential for a multifactor etiology because not all progeny of salmonids having low thiamine exhibit this syndrome.  This suggests the involvement of other factors. In this respect, it is known that contaminants such as PCBs and organochlorine pesticides (DDT and dieldrin), can reduce thiamine storage in laboratory animals (rodents).
 

Objective

The overall objective of this study was to:

v To compare thiamine concentrations in alligator eggs from sites with high embryo mortality and high exposure to organochlorine pesticides (OCPs) (Apopka, Griffin, and Emeralda Marsh) to that from a site that has historically exhibited low embryo mortality and low OCPs (Orange/Lochloosa).
 

Materials and Methods

 During the summers of 2000 and 2001, alligator egg clutches (13 to 15 per site, total of 56 clutches) were collected from Lakes Apopka, Griffin, and from the reclaimed/flooded agricultural properties adjacent to Lake Griffin (Emeralda Marsh). Lakes Orange and Lochloosa served as the reference sites for these studies (Fig. 1). In the laboratory, eggs were candled to determine whether they were fertile or "banded", and if so, nested in moist sphagnum moss and incubated at 30.5^oC and 91-95% humidity. One egg/clutch was opened to determine stage of development.  The following parameters were recorded from all clutches: percentage of "unbanded" eggs [(number of unbanded eggs)/(fecundity) x 100]; percent hatchability [(number of eggs that hatched)/(fecundity) x 100], and neonatal mortality.  Neonatal mortality was classified as either pre-hatch early (1-35 days of development) or late (36 days - hatching).  One representative egg/clutch was analyzed for [OCPs] via GC-MS and for thiamine concentrations as explained below.  In addition, thiamine changes over embryo development were examined in two clutches (3 eggs/clutch were opened at 5 day intervals, from day 5 to day 40 of embryo development).

 Thiamine concentrations were determined by HPLC separation of thiamine pyrophosphate (TPP), thiamine monophosphate (TP) and free thiamine (T) as described by Brown et al. (1998).  Briefly, thiamine is extracted into a trichloroacetic (TCA) solution. The extract is then washed with ethyl acetate:hexane (3/2, vol/vol) to remove excess TCA.  An aliquot of the washed solution is reacted with potassium ferricyanide to produce thiochrome derivatives.  The resulting derivatives are separated on a Hamilton PRP-1 column and detected with a spectrofluorometer (excitation 375 nm, emission 433 nm). Authentic standards of TPP, TP and thiamine-HCL (Sigma Chemical Co.) were used to quantify the amount of thiamine in each sample.
 

Conclusions

v Alligators from sites with moderate to high [OCPs] produced clutches that hatched at lower rates when compared to sites with low to no [OCPs] (Figs. 2 and 3A). The primary pesticides observed were DDE (a DDT metabolite) and toxaphene (Fig. 3B).

v Concentrations of total and free thiamine were significantly higher in eggs from the reference site (Lochloosa/Orange) when compared to Griffin, Apopka, and Emeralda. (Fig. 4). However, there were no differences in the concentrations of mono and pyrophosphate thiamine across sites. Concentrations of total thiamine in the impacted sites fell below the threshold known to induce embryo mortality syndrome in salmonids.

v Thiamine monophosphate and triphosphate (the active form of thiamine), are not produced until after day 25 of embryo development (Fig. 5).

v Free thiamine and thiamine monophosphate were decreased in clutches with high early and late embryo mortalities (Figs. 6A & B). Likewise, there was a significant positive relationship between free thiamine in yolk and hatch rates (all clutches combined) (Fig. 6E). However, there were no differences in thiamine concentrations among clutches with low, moderate, and high percentage of unbanded eggs (Figs. 6A, B & C).

v There was not a clear pattern in total yolk OCP concentrations among clutches with different viabilities and thiamine concentrations (Fig. 6D).

v All combined these results would suggest that thiamine deficiency might be playing a role in the alligator embryo mortalities observed in some Florida Lakes. Causes for the decline in the concentration of this thiamine are unknown at this time, but might be related to differences in the nutritional value of prey items across the sites studied.

v An additional mechanism that could explain the alligator embryo mortalities, relates to the exacerbation of the oxidative damage caused by exposure of embryos to environmental contaminants.  Since thiamine is a cofactor required in many essential enzymatic processes (including repair and prevention of oxidative damage), its deficiency coupled with the presence of contaminants, could act synergistically to increase the incidence of alligator mortalities.

v These hypotheses should be further tested through the conduction of laboratory controlled studies.
 

Reference

 v Brown, S., D. C. Honeyfield, and L. Vandenbyllaardt. 1998.  Thiamine analysis in fish egg and tissues by high pressure liquid chromatography. In: G. McDonald, J. Fitzsimons, and D.C. Honeyfield (Eds). Early life stage mortality syndrome in fishes of the Great Lakes and Baltic Sea. Symposium 21, p 73-81. American Fisheries Society, Bethesda, MD 20814.

Acknowledgements

v NIEHS, Superfund Project: "Chlorinated pesticides and developmental mortality in wildlife".

v University of Florida, Department of Physiological Sciences, College of Veterinary Medicine.

v USGS - Florida Integrated Science Center.

v Florida Fish and Wildlife Conservation Commission.