Methods
Summary
Field study
I conducted a pilot field study in August 2015, where I attempted to collect 20-30 individual Mottled Sculpin (60 – 80 mm) and Mountain Sucker (80 – 100 mm) from seven headwater streams in the Wyoming Range. Sampling was conducted in August to ensure responses were not influenced by reproductive status. Well density within each stream’s drainage will be used as a measure of development intensity and will represent our main stressor of interest for the field study. Changes to in-situ water quality (i.e., conductivity and temperature) will be used as measures of disturbance to assess fish physiological responses.
Immediately after being captured, individuals were placed in a solution of MS-222 and anesthetized for safe extraction of blood. Within 3 minutes of capture, a baseline blood sample was collected from the caudal vessel for each fish. After blood samples were collected, length (mm) and weight (0.01 g) was recorded for each individual before being placed into a recovery flow through container in the stream. Fishes were retained in the flow through container under natural stream conditions, monitored for mortality for 48 hours, and released back to the site of original capture. This was repeated again in August 2016 with the addition of stress responses (e.g., stress-induced and stress-reactivity cortisol). Three additional reference streams were also sampled in August 2016.
Laboratory experiment
Because many exogenous factors could be influencing our responses from fishes in the field and we cannot tease apart which factors are the true culprits, the laboratory experiment will focus on two stressors that are commonly associated with ONG development (increased temperature and salts) and their interactive effects on fish physiology.
The laboratory experiment will begin in summer 2017, where 300 individuals of each Mountain Suckers and Mottled Sculpin will be collected from headwater streams in the Upper Green River basin via backpack electrofishing. In the field, fishes will be placed in aerated containers and transported back to the University of Wyoming’s Red Buttes facility. Fishes will be housed in aerated aquaria and allowed to acclimate to laboratory conditions for approximately one month. Once fishes have acclimated, I will randomly assign them among 60 10-gallon aquaria. Before being randomly assigned to treatment aquaria, I will measure length and weight and mark all individuals to monitor fish growth over the study period.
To assess the acute and chronic, as well as the interactive, effects of two stressors associated with ONG development, I will manipulate temperature and salt (NaHCO3) in aquaria using 2 temperature treatments X 7 salt concentrations in a factorial design (5 replicates per treatment combination). Temperature and salt treatments were chosen based on levels measured at our field sites near Big Piney, Wyoming and those used in recent toxicity studies related to ONG development. Temperature treatments will include a control treatment of 16-17°C (this represents the optimal temperature of these fishes) and a stressful temperature of 20-23°C. Salt treatments will include specific conductivity level of 400, 500, 600, 800 900, 1000, 1200, and 1300 µS/cm.
To measure acute physiological responses (e.g., cortisol, stress-induced cortisol, glucose, and innate immunity), baseline blood samples will be collected from all anesthetized individuals after 24 hours of treatment exposure. Immediately after a baseline blood sample has been collected, fish will be suspended in a dipnet at the water’s surface for 30 minutes in their respective aquaria to allow for recovery and to induce a secondary stressor (net confinement). This will allow me to measure physiological stress-reactivity among individuals across treatment combinations. At the end of exposure to the secondary stressor, a stress-induced blood sample will be collected in the exact same manner as the baseline blood sample. Fish will be returned to their original aquaria to continue the experiment for an additional 30 days to measure the response of chronic exposure.
At the end of the 30 day chronic exposure, individual fish will be removed from aquaria, anesthetized, measured for final length and weight, and bleed for a baseline sample. I will then place individuals in a suspended dipnet in their respective aquaria to induce a secondary stressor for 30 min. After a chronic stress-induced blood sample is extracted, fish will be placed back in aquaria to recovery and monitored for mortality for 48 hours. All blood samples will be processed within 4.5 hours for preservation for future laboratory assays to be conducted.
Blood processing
Directly following blood extraction, blood glucose will be measured using an ACCU-CHECK Aviva glucose meter and 0.6µl of whole blood. Blood samples will then be placed on ice until further processing can take place (<5 hours), at which time plasma will be separated from red blood cells via centrifuging. Plasma samples will then be frozen and stored at -20°C until further laboratory assays can be conducted.
Bactericidal assay
Bacteria killing assays (BKAs) are a relevant measure of an animal’s innate immune system. The immune components in an animal’s plasma (e.g., monocytes, neutrophils, etc.) are exposed to controlled amounts of bacteria (in this case, Escherichia coli) at a controlled temperature to allow bacterial killing to take place, then the bacteria are given a nutrient broth and allowed to grow for a given amount of time. Using spectrophotometry and 96-well plates, absorbance of sample wells are compared with positive (with bacteria but no plasma) and negative (no bacteria or plasma) wells to determine the percent killing ability of the plasma samples. BKAs will be performed in Dr. Susannah French’s laboratory at Utah State University.
Radioimmunoassay
Circulating cortisol concentrations will be determined using a protocol modified from Moore (1986). Briefly, extractions will be performed using a solution of 30 % ethyl acetate: isooctane. Samples will then be resuspend using phosphate-buffered solution and assayed in duplicate for cortisol. Individual recoveries will be determined using a separate aliquot of the resuspended fractions. The recoveries account for any potential loss during the assay and will allow us to adjust the final sample concentration.
Pre Analysis Plan
Hypothesis Testing
With this research, I plan to test the following three competing physiological hypotheses: the acclimation/habituation hypothesis, the facilitation hypothesis, and the inhibition hypothesis. Acclimation or habituation occurs after repeated or chronic exposure to a stressor and the animal reduces its cortisol response because it no longer considers the stressor to be a threat. Facilitation is the idea that acclimation to one stressor heightens the response to a novel stressor. Inhibition occurs when chronic exposure to a stressor diminishes the functioning of the hypathalamus-pituitary-adrenal axis and renders the animal incapable of mounting an appropriate physiological response.
Protocols
This project has not yet shared any protocols.