Fathead Minnow Research
During the academic year
The research conducted in my laboratory during the academic year focuses on the cumulative impacts of habitat-related and contaminant-related stress on the reproductive success of freshwater fish. We are investigating the reproductive success in terms of fertile egg productivity, growth and survival of offspring, and reproductive hormone profiles (using Enzyme Linked Immunosorbent Assay techniques, or ELISA) of adults in breeding pairs of fathead minnows (Pimephales promelas).
We chose to use fathead minnows because they are important in aquatic toxicology research. They are easily reared in a lab, their spawning behavior is well known, and they are an important forage fish in the food chain. We exposed breeding pairs of adult fathead minnows at different quantitative levels of habitat to investigate whether habitat availability either directly or indirectly affects reproductive success. The direct effects could be through available spawning substrate, whereas the indirect mechanism is postulated to be through a physiological stress response mechanism.
Experimental Design
The pilot project of this experiment had a setup with 20 five gallon aquaria, each of which was randomly assigned a breeding pair of fathead minnows and was also randomly assigned an amount of physical habitat (none, 1x, 5x, or 10x). Therefore, each of the four habitat levels had five replicate tanks. The "physical habitat" was created to try to mimic the three dimensional pattern of instream vegetation (both in terms of materials and construction) that these fish would actually encounter in nature. They were made of wooden dowels and approximately 3.5" x 4" wooden boards. Each of the wooden boards was considered to be one of the x's from 1x, 5x, and 10x. When the pairs begin to reproduce, they lay their eggs on the underside of the wooden boards. So, with different amounts of boards (habitat) available to lay eggs on, we assume that this would also impose different amounts of stress on the breeding pairs.
Our fish are kept in a controllable growth room with a temperature of between
~23-25°C and a 16 hour light, 8 hour dark photoperiod, to mimic the summer breeding months for these
fish. In addition to normal tank maintenance of feeding and changing the water twice a week, we
also check water chemistry (dissolved oxygen concentration, conductivity, salinity, alkalinity, pH and
temperature) twice a week. Habitats are checked morning and night for eggs, which are counted,
recorded, and relocated to incubation chambers. Once the eggs hatch, the larvae are kept in
separate 1 L glass jars with an air stone. The survival and growth of offspring are tracked and
recorded at day 0, 2, 4, 7, 14, 21, and 28 for each clutch of eggs. Adult fish are also weighed
and measured once a week. At the end of the pilot experimental period, whole adult fish were
homogenized and hormones were extracted from the whole body samples using diethyl ether. 17-b
estradiol and testosterone levels were analyzed using ELISA kits purchased
from Cayman Chemical Company, Ann Arbor, MI.
The pilot project of this experiment has helped us to identify many areas that need improvement. Some improvements we are working on include trying to prevent the adult fish from dying prematurely, preventing eggs and larvae from dying from fungal infection, and modifying our methods of extracting hormones so we can get more pure samples and more accurate concentration readings. Many changes have been made to the setup, and the experiment was conducted again with only 1/2x habitat (half of a wooden board) and 5x habitat levels. This new setup increased the amount of replicates at each habitat level (10 tanks instead of 5), as well as increased the difference between amounts of habitat. However, we still continued to experience problems with adult fish dying and eggs/larvae dying. We are currently continuing to modify our techniques to improve survival of adult fish as well as offspring until closer to the end of the 6 week experimental period. Once we have optimized this experimental regime, we intend to repeat the experiment at different levels of exposure to an environmental endocrine disrupting compound to determine whether these two very different types of stressors (habitat-related and contaminant-related) are antagonistic, additive, or synergistic.
