Research Questions
- What levels of poultry litter-based estrogenicity can induce TO development in largemouth bass?
- At what age do largemouth bass respond most profoundly to estrogenic compounds through increasing Vtg levels and development TOs?
- How does poultry litter contamination of water correlate with intersex occurrence in bass in a natural setting?
The Phases of our research
Our project has two main phases:
- A laboratory study of the effects of poultry litter exposure on intersex in largemouth bass.
- A study to validate the use of non-lethal surgical techniques to determine intersex.
MEthodology
General Experimental Design:
We have obtained 300 fish from a Maryland Department of Natural Resources hatchery where they have been raised since hatch in May 2012. The fish have been held in large tanks at the Crane Aquaculture Facility near campus since October 2012. In May of 2013, we will obtain an additional 200 to 500 fish. We will then have two groups of fish: an ‘old’ group spawned in the spring of 2012 and a ‘young’ group spawned in the spring of 2013.
We plan to expose these healthy largemouth bass to varying concentrations of poultry litter-derived EDCs as done in previous experiments with fathead minnows. We will expose a subsample from both the old and young groups to three different levels of contamination: high, low and no poultry litter. Exposures will take place in separate tanks for up to three month periods, after which the fish will be returned to their holding tanks. Exposures will begin in the spring of 2013.
We will then repeat this exposure protocol on another two subsamples of fish from the old and young groups, which are now three months older at the start of exposure as compared to the fish from the first exposure. We will repeat this protocol every three months on increasingly older samples of fish for up to a year. Our final experimental group will sample fish that are one year older at the start of their exposure than the initially exposed fish. By monitoring fish throughout the exposure cycles, we hope to find a period of time, or ‘window of sensitivity,’ in which the fish are most affected by EDCs. In addition, we hope to simultaneously determine whether both high and low levels of poultry litter can induce intersex.
YES Assay
We will be using a Yeast Estrogen Screen Assay (YES Assay) to measure the estrogenicity, or potential estrogenic activity, of water samples. The YES-assay is a biological test system for the detection of estrogens in water that is based on genetically modified yeast. We have chosen to use a Bioluminescent Yeast Estrogen Screen Assay (BLYES-assay) as opposed to other methods of measuring estrogenicity because it is a straightforward procedure for which we already have access to the needed equipment.
The BLYES assay uses the Saccharomyces cerevisiae strain of yeast, which is sensitive to estrogens. When an estrogenic compound comes in contact with the yeast, it will bind with the human estrogen receptor (hER-α) that acts as a promoter for lacZ, which signals the transcription of lacZ. If the promoter is activated, lacZ transforms to a red colored product that can be measured by absorbance at 540 nm.
ELISA Assay for Vtg Levels
A common indicator of exposure to environmental estrogen in fish is elevated vitellogenin levels. Vitellogenin is a protein that acts as the energy source for developing eggs in mature female fish. The expression of the Vtg gene in male fish has been used as a biomarker for the presence of endocrine disrupting compounds in the environment.
In order to use vitellogenin as a biomarker in our research, it is necessary to determine the levels of vitellogenin protein in the blood plasma of fish, most commonly obtained through an Enzyme-Linked Immunosorbant Assay (ELISA). ELISA is used to detect the presence and quantity of a certain antigen in a sample that binds with a specific enzyme linked antigen. After an enzyme substrate is added to the antibody, a colored product is formed. Its intensity correlates with the amount of antigen present.
Novel Methodology:
Electronarcosis
In order to extract plasma samples from the bass for analysis, the subject fish must first be anesthetized. For studies of non-releasable laboratory fish, we will use the anesthetic tricaine mesylate or MS-222. MS-222 has been used in a variety of fish studies. Due to current FDA restrictions, it is difficult to release MS-222 anesthetized fish back into the environment during field studies. FDA law requires a 21-day quarantine period between treatment with MS-222 and the release of any fish that could potentially be harvested for food.
Thus, for any fish we plan to release, we want to implement and validate a novel anesthetic procedure known as electronarcosis. Electronarcosis uses an electric current instead of a chemical agent to induce unconsciousness. This technique leads to reduced recovery times and a reduced threat to cohabitant piscivores. Though electronarcosis has been studied episodically for decades, the design specifications of a portable and cost-efficient electronarcosis apparatus have been recently published and tested in largemouth bass.
With this method of anesthesia, we hope to cause as little pain to the fish as possible. We also hope to achieve sufficient immobilization for surgery and to have the ability to release the tested fish into the wild once the procedure is over.
Laparoscopy
Laparoscopy is a minimally invasive procedure involving visual imaging, and biopsy (when applicable), of internal tissues. This procedure is used routinely in medical and veterinary settings for examining and biopsying tumors and cysts. This method involves either a small incision or entry through a natural orifice, which decreases the likelihood of infections and the time necessary to recover. Current sampling methods to determine intersex in largemouth bass involve sacrificing fish; generally, intersex is determined post mortem through histological sampling. We aim to validate the use of an established method of non-lethal laparoscopy for the purpose of oocyte detection.
We have obtained 300 fish from a Maryland Department of Natural Resources hatchery where they have been raised since hatch in May 2012. The fish have been held in large tanks at the Crane Aquaculture Facility near campus since October 2012. In May of 2013, we will obtain an additional 200 to 500 fish. We will then have two groups of fish: an ‘old’ group spawned in the spring of 2012 and a ‘young’ group spawned in the spring of 2013.
We plan to expose these healthy largemouth bass to varying concentrations of poultry litter-derived EDCs as done in previous experiments with fathead minnows. We will expose a subsample from both the old and young groups to three different levels of contamination: high, low and no poultry litter. Exposures will take place in separate tanks for up to three month periods, after which the fish will be returned to their holding tanks. Exposures will begin in the spring of 2013.
We will then repeat this exposure protocol on another two subsamples of fish from the old and young groups, which are now three months older at the start of exposure as compared to the fish from the first exposure. We will repeat this protocol every three months on increasingly older samples of fish for up to a year. Our final experimental group will sample fish that are one year older at the start of their exposure than the initially exposed fish. By monitoring fish throughout the exposure cycles, we hope to find a period of time, or ‘window of sensitivity,’ in which the fish are most affected by EDCs. In addition, we hope to simultaneously determine whether both high and low levels of poultry litter can induce intersex.
YES Assay
We will be using a Yeast Estrogen Screen Assay (YES Assay) to measure the estrogenicity, or potential estrogenic activity, of water samples. The YES-assay is a biological test system for the detection of estrogens in water that is based on genetically modified yeast. We have chosen to use a Bioluminescent Yeast Estrogen Screen Assay (BLYES-assay) as opposed to other methods of measuring estrogenicity because it is a straightforward procedure for which we already have access to the needed equipment.
The BLYES assay uses the Saccharomyces cerevisiae strain of yeast, which is sensitive to estrogens. When an estrogenic compound comes in contact with the yeast, it will bind with the human estrogen receptor (hER-α) that acts as a promoter for lacZ, which signals the transcription of lacZ. If the promoter is activated, lacZ transforms to a red colored product that can be measured by absorbance at 540 nm.
ELISA Assay for Vtg Levels
A common indicator of exposure to environmental estrogen in fish is elevated vitellogenin levels. Vitellogenin is a protein that acts as the energy source for developing eggs in mature female fish. The expression of the Vtg gene in male fish has been used as a biomarker for the presence of endocrine disrupting compounds in the environment.
In order to use vitellogenin as a biomarker in our research, it is necessary to determine the levels of vitellogenin protein in the blood plasma of fish, most commonly obtained through an Enzyme-Linked Immunosorbant Assay (ELISA). ELISA is used to detect the presence and quantity of a certain antigen in a sample that binds with a specific enzyme linked antigen. After an enzyme substrate is added to the antibody, a colored product is formed. Its intensity correlates with the amount of antigen present.
Novel Methodology:
Electronarcosis
In order to extract plasma samples from the bass for analysis, the subject fish must first be anesthetized. For studies of non-releasable laboratory fish, we will use the anesthetic tricaine mesylate or MS-222. MS-222 has been used in a variety of fish studies. Due to current FDA restrictions, it is difficult to release MS-222 anesthetized fish back into the environment during field studies. FDA law requires a 21-day quarantine period between treatment with MS-222 and the release of any fish that could potentially be harvested for food.
Thus, for any fish we plan to release, we want to implement and validate a novel anesthetic procedure known as electronarcosis. Electronarcosis uses an electric current instead of a chemical agent to induce unconsciousness. This technique leads to reduced recovery times and a reduced threat to cohabitant piscivores. Though electronarcosis has been studied episodically for decades, the design specifications of a portable and cost-efficient electronarcosis apparatus have been recently published and tested in largemouth bass.
With this method of anesthesia, we hope to cause as little pain to the fish as possible. We also hope to achieve sufficient immobilization for surgery and to have the ability to release the tested fish into the wild once the procedure is over.
Laparoscopy
Laparoscopy is a minimally invasive procedure involving visual imaging, and biopsy (when applicable), of internal tissues. This procedure is used routinely in medical and veterinary settings for examining and biopsying tumors and cysts. This method involves either a small incision or entry through a natural orifice, which decreases the likelihood of infections and the time necessary to recover. Current sampling methods to determine intersex in largemouth bass involve sacrificing fish; generally, intersex is determined post mortem through histological sampling. We aim to validate the use of an established method of non-lethal laparoscopy for the purpose of oocyte detection.