DEVELOPMENT AND VALIDATION OF PROCEDURES FOR MONITORING ENDOCRINE AND REPRODUCTIVE FUNCTION IN FRESHWATER MUSSELS

T.S. Gross^1,2, C.M. Wieser¹, N.J. Kernaghan², and D.S. Ruessler¹

 ¹USGS - Florida Integrated Science Center, Gainesville, Florida
²College of Veterinary Medicine, Department of Physiological Sciences
University of Florida, Gainesville, Florida

 

ABSTRACT

Considerable evidence has indicated that a wide variety of environmental stressors such as habitat degradation and environmental contaminants can adversely alter or disrupt reproductive and endocrine function in wildlife populations. Most of these data have focused on effects in fish and or other macro-faunal vertebrates. The development and validation of procedures for monitoring endocrine and reproductive function in freshwater invertebrates, such as freshwater mussels, would be critical to evaluations of reproductive health. The current study included the development and validation of procedures for monitoring endocrine function in the freshwater mussel, Elliptio buckleyi, as well as an evaluation of seasonal reproductive cycles. Body tissues were collected from adult mussels and extracted for endocrine analyses. Standard RIA procedures were utilized for androgen, progestin, and estrogen analyses. Sex steroid concentrations were compared to histological assessments of sex and status to validate and develop these procedures. These efforts have detected significant concentrations of estradiol, progesterone and testosterone as well as gender differences and seasonal cycles. The development of these procedures for use with freshwater mussel species will be critical to the elucidation of potential habitat and contaminant effects on reproductive function, as well as evaluations of reproductive status and function for critical populations and species.

INTRODUCTION

         Considerable evidence has indicated that a wide variety of environmental stressors such as habitat degradation and environmental contaminants can adversely alter or disrupt reproductive and endocrine function in wildlife populations.  Much of the attention has focused on a variety of xenobiotic agents and other environmental stressors/contaminants introduced into the environment which exert endocrine disrupting effects within biological systems. Much of the data indicating endocrine-disrupting effects of environmental contaminants in wildlife, has focused on effects in fish and/or other macro-faunal vertebrates. Indeed, as with many ecosystems, the macro fauna (i.e., the vertebrates) have been studied extensively, while invertebrates have largely been ignored. While much attention has been focused recently on declining populations of vertebrates, such as amphibians, similar or greater declines may be occurring for many aquatic invertebrates as well. Aquatic invertebrates would be expected to be exposed to both sediment and water partitioning of stressors/contaminants. Little is known about the effects of environmental stressors/contaminants on invertebrates. However, invertebrate species have been recognized as important environmental models and serve as models for a wide variety of toxicity tests that utilize mortality and lethality as the endpoints of significance. Indeed, invertebrates are a major, important, trophic level within ecosystems and a taxa with high exposure potentials to a wide variety of environmental contaminants/chemicals. Selected invertebrate species should be developed as models for general toxicity as well as for the first trophic level of potential effects for stressors/contaminants in the environment. Invertebrates could, indeed, serve as important environmental sentinels, early signals, of environmental effects for stressors/contaminants such as endocrine-disruptors.
         Techniques need to be developed and validated for monitoring reproductive-endocrine function in invertebrates. This study reports the development and validation of endocrine monitoring procedures for a freshwater mussel species: Elliptio buckleyi. Additional development and validation of these techniques may be useful for the detection of potential effects of stressors/contaminants, as well as enable valuable studies of reproductive function in freshwater mussels.

OBJECTIVES:

The objectives of these preliminary studies  were :

1. Develop and validate procedures for the determination of reproductive-endocrine biomarkers: total estrogen, estradiol, progesterone and testosterone.
2. Document seasonal patterns for these reproductive-endocrine biomarkers and correlations to reproductive events.

MATERIALS AND METHODS

Experiment 1:
         Freshwater mussels (Elliptio buckleyi) were collected from an experimental pond located at the Fisheries Department, University of Florida, Gainesville. All mussels collected were in the size range of 32-56mm in shell length. Mussels were sacrificed and the following tissues and/or samples collected from each mussel: body fluids, mantle, foot and gonadal biopsy. Tissue weights and fluid volumes were recorded and all samples stored at -80°C. Gonadal tissues were examined to identify sex. Samples from 3 females and 5 males were utilized for the validation and development of endocrine analyses.

Endocrine Analyses:
         Samples were analyzed for the following sex steroids: estradiol, testosterone, total estrogen, and progesterone, using standard radioimmunoassay (RIA) procedures. Tissues were diluted with 3x volume of 30% KOH and incubated for 20 minutes in a 60°C water bath to facilitate tissue digestion. Fluid samples (200µl) and digested tissues (mantle: 150µl; foot: 50µl; gonad: 100µl) were extracted twice with 4 ml diethyl ether prior to RIA analysis. Each sample was analyzed in duplicate for each of the sex steroids. Standard curves were prepared in buffer with known amounts of radioinert sex steroid (1, 5, 10, 25, 50, 100, 250, 500 and 1000 pg). Cross-reactivities of the estradiol-17ß antiserum with other steroids were: 11.2 % for estrone; 1.7% for estriol; <1.0 % for estradiol-17alpha and androstenedione; and <0.1% for all other steroids examined. Cross-reactivities of the total estrogen antiserum with other steroids were: 100% for estrone, 63% for estriol, 0.65 % for testosterone, <1.0 % for androstenedione, and <0.1% for all other steroids examined. Cross-reactivities of the testosterone antiserum with other steroids were: 18.5% for dihydrotestosterone; 3% for androstenediol; <1.0% for androstenedione, androstanedion, estradiol and progesterone; and <0.01% for all other steroids examined. Cross-reactivities of the progesterone antiserum with other steroids were: 5.4% for 20-dihydroprogesterone; 3.8% for desoxycorticosterone; <1.0% for corticosterone, 17-hydroxyprogesterone, pregnenolone, androstenedione, and testosterone; and <0.01% for all other steroids examined.
         Validations included the analysis of pooled samples to generate inhibition curves which were parallel to the respective standard curve, with the tests for homogeneity of regression indicating that the curves did not differ. Further characterization of the assays involved measurement of known amounts (1, 2, 5, 10, 25, 50, 100, 250 and 500 pg) of sex steroids in pooled samples.
         Results were calculated as concentrations: pg/g tissue or pg/ml fluid. A potential sex specific index: estradiol:testosterone ratio was also calculated as an index of sex specific sex steroid pattern.

Experiment 2:  Seasonal Anaylses
         Freshwater mussels (Elliptio buckleyi) were collected at approximate biweekly intervals from January 31, 2000 through May 4, 2000 to document seasonal reproductive cycles.  Mussels were sacrificed (n=15 to 20 per collection/date) and the following tissues and/or samples collected from each mussel: mantle, foot and gonadal biopsy. Tissue weights were recorded and all samples stored at -80°C. Gonadal tissues were examined histologically to identify sex and status. Mantle tissues were utilized for the evaluation of endocrine parameters: estradiol and teststerone.

Figure 4:  Experiment 2 Results
Concentrations of sex steroids for mantle tissues from the freshwater mussel: Elliptio buckleyi collected biweekly: January 31, 2000 through May 4, 2000. Sex steroids are reported as pg/g mantle tissue. Note the seasonal patterns for both sexes and correlations to reproductive status. These results demonstrate gender specific patterns and clear seasonal reproductive cycles for the freshwater mussel: Elliptio buckleyi.

DISCUSSION

         Few procedures have been developed or validated for the detection of reproductive endocrine factors in invertebrates. The results from these efforts are among the first to demonstrate sex specific patterns of sex steroids in freshwater invertebrates. Future efforts will include a thorough examination of seasonal reproductive trends and correlations to specific gonadal stages, additional species, an evaluation of mantle biopsy methods, and the evaluation of the effects of environmental contaminants/chemicals on freshwater mussels.
         Considerable evidence has accumulated over the past several years that a wide variety of chemical compounds present in the environment can disrupt reproductive endocrine function in exposed wildlife populations. To understand just how endocrine-disrupting chemicals (EDCs), such as mercury and organochlorine pesticides, harm invertebrates, it is important to study the non-lethal, physiological and endocrine effects. At the biochemical level, the exact mechanisms by which EDCs affect freshwater mussels is unclear. The development and validation of procedures to quantitate and monitor endocrine parameters in freshwater mussels and other aquatic invertebrates could enable a documentation of mechanisms of action for EDC's on invertebrates. The additional development and validation of these procedures will be critical to the elucidation of EDC effects in freshwater mussels.