Whitehead Lab: EXPERIMENTAL DESIGN
Fields are in bold, examples are in red.
Experiment title:
Freshwater Challenge, Range-finding (Part of Osmotic Regulation study)
People Involved:
Andrew Whitehead (PI), Fernando Galvez (PI), Shujun Zhang (Ph.D. student)
Date this document was prepared:
June 18, 2008
Who prepared this document?
Shujun Zhang
Experiment goal:
Overall Study - Assess the variation in physiological and gene regulation responses between populations
adapted to a variety of salinities to a strong decrease in salinity. Use this variation in response to determine
the regulatory basis for adaptation along a salinity cline.
Freshwater Challenge – Assess the core processes and genomic underpinnings to acclimation to freshwater
from a saltwater environment. Correlate consistent and divergent patterns of gene expression/regulation
with physiological response, habitat of origin, and evolutionary relatedness. These correlations allow for
the potential to infer in areas such as fundamental processes for acclimation and adaptation, variation in
patterns of phenotypic plasticity, variability in mechanisms controlling stress response pathways.
Specific – Attempt to detect the most physiologically informative points for the definitive Freshwater
Challenge experiment using the two most likely constrained populations in rapid acclimation to low
salinity. {Galvez} how?criteria?
Experiment hypotheses:
(need more work)
 Populations with strong saltwater fidelity, low phenotypic plasticity in salinity variation, will
exhibit decreased plasticity in gene expression in osmotic regulation, cellular repair, and stress
response pathways.
 Independent pathways contribute to salinity tolerance plasticity. These patterns are more
conserved between species of similar tolerance range than through evolutionary history.
 Populations adapted to high salinity environments will have a much stronger and varied response
during acclimation to freshwater than populations adapted to freshwater environments and more
phenotypically plastic populations.
Location of experiment:
Acclimation will occur in LSB 627 “Brackish” system for no less than 21 days. Fish will be transferred and
held in freshwater10g aquaria in LSB 687. The 10g aquaria in LSB 687 will be replicate systems with 10
fish per tank. These 3 tanks per treatment per population or 18 total aquaria.
Nature of treatments:
All fish will be acclimated for no less then 21 days at 31-33ppt salinity. Two treatments and a control
population of both Fhet NH and Fhet GA will be used. The two treatments are at 0-0.2ppt salinity and 5ppt
salinity and the control will remain 31-33 ppt salinity. 6 fish per population will be dissected prior to the
beginning of the treatments. At each of the 5 chosen time points 6 fish from each treatment and 6 from the
control will be sampled, dissected, and stored for gene expression and physiological study. The timeframes
and order of sampling are as follows:
Population
Fhet NH
Fmaj GA
Treatment
Start 11/9
6hr 11/9
24hr 11/10
72hr 11/12
7d 11/16
14d 11/23
0ppt
5ppt
32ppt
0ppt
5ppt
32ppt
Add 30/pre
11am
11am
11am
2:00p
2:00p
2:00p
Sample 6
5:00p
5:00p
5:00p
8:00p
8:00p
8:00p
Sample 6
11am
11am
11am
2:00p
2:00p
2:00p
Sample 6
11am
11am
11am
2:00p
2:00p
2:00p
Sample 6
11am
11am
11am
2:00p
2:00p
2:00p
Sample 6
11am
11am
11am
2:00p
2:00p
2:00p
Fish will follow a normal twice daily feeding regime.
Species:
Fundulus heteroclitus sampled in New Hampshire (Fhet NH)
Fundulus majalis sampled in coastal Georgia (Fmaj GA)
Fish acclimation and holding:
All fish will be placed into tanks in LSB 627 “brackish” rack system with a shared water system at 32ppt
for no less then 21 days prior to treatment.
All fish will be moved to 10g aquariums in LSB 687 on Nov 9. These tanks will be stand alone aquaria
with side hanging power filters. Each tank will be conditioned with 3 Fhet GA during the acclimation
process. During the experiment, 10 fish will be added to each tank per treatment per species. All Fhet GA
fish used for the conditioning will be returned to the Fhet GA population in the large sump in LSB687.
There will be 3 of each tank salinity population combination: Fhet NH (32ppt, 5ppt, 0ppt) and Fmaj (32ppt,
5ppt, 0ppt).
Age/lifestage of organisms:
Fish from identical age classes are preferred if possible. This will not be the case for some populations;
especially in the case of Fmaj GA. Effects from this will be minimized by randomizing the age breakdown
of each tank of fish throughout the acclimation and treatment process.
Replicates per treatment:
6 replicate fish per treatment; 3 treatments per sample point; 5 sample points per population; 2 populations
used.
Dose range:
The acclimation range is 31-33ppt beginning >21days prior to the experiment. The two treatments will be
0-0.3ppt and 4-6ppt for “0ppt” and “5ppt” treatments respectively.
Preparation of treatments:
The 18 tanks and filters will be washed with filtered water and set up on racks in LSB 687. Three 60 gallon
batches of water, 32ppt, 5ppt, and 0ppt will be mixed the day prior to setup of the tanks using filtered water
from the filtration system in the room in aerated plastic bins on the floor. Six evenly distributed by vertical
and horizontal arrangement of tanks will be filled with 32ppt followed by six more with 5ppt. Remaining
tanks will be filled with 0 ppt.
Exposure time:
The exposure times are chosen based on estimated based on a spectrum of time points relevant to gene
expression, protein replacement, and cell recruitment timeframes. These times are initially set for: 6h, 24h,
72h, 7d, and 14d. Based on preliminary data a following experiment to increase resolution at particular
points can be done. Due to the sensitivity of these time points all sampling points are staggered to attempt
to limit dissection time to less then 90mins.
Container size and volume:
Independent 10g aquaria with tank side filters with biofilter activated by 7 days submerged in the
freshwater system in 627.
Container preparation/cleaning:
All tanks and power filters will be rinsed and scrubbed using filtered water and aquaria scrubbing pads. All
biobags will be soaked in the freshwater sump for 11day prior to treatment. {are biofilters necessary with
33% daily water changes over a 2 week period}
Feeding:
Twice daily, flake food in the morning and thawed brine shrimp in the afternoon.
Lighting:
Light will be ambient fluorescent room lighting set on a timer to turn on at 6am and off at 6pm every day.
This is consistent throughout the acclimation and experimental process.
Temperature:
Ambient room temperature will be used.
Water source:
Water used for this experiment will be from the filtration system in the room. Any salt needed for the 5ppt
and 32ppt tanks will come from pre-purchased Instant Ocean salt mixed as needed in common batches for
consistency. 30gallon batches of water will be mixed every day and aerated over night for the next days
water changes.
Randomization of container assignment:
The process of catching and transporting the fish between rooms in common tanks should serve to
randomize them very effectively.
Water chemistry endpoints:
Salinity will be corrected for any deviation from these ranges (5-8%):
 “32ppt” – 31-33ppt
 “5ppt” – 4.8-5.2ppt
 “1ppt” – 0.1-0.3ppt
Dissolved oxygen should be no lower then 5mg/L and temperature should be between 18-25°C. Treatments
for deviations from these ranges as well as any noticeable buildup of NH4+ or NO2 will be developed on a
case by case basis.
Frequency of water chemistry:
NH4+, NO2, Salinity, DO, and Temp will be recorded daily in all tanks @ ?? time for consistency. 3-4
gallon water changes will be performed every day using water mixed and aerated the day before on all
systems.
Test termination procedure:
At predetermined sampling time points, a sample of 6 from each population and from each treatment will
be taken. These sampled fish will be dissected within 90minutes of sampling time in an assembly line
fashion.
1. Euthanize and draw blood from the fish. {prepare and store blood}
2.
3.
4.
Take liver and gill basket. Preserve left side of gill and liver in mRNA fixing solution in 1.5mL
centrifuge tube in chilled RNAlater for future mRNA extraction.
Remove intestines and flush with DI water. Place {which portion} in 1.5mL centrifuge tube in
chilled RNAlater for future mRNA extraction. Place remainder of intestines in {where} for
physiological study.
Remove {remaining tissues for Galvez lab} for future study. {store how} Remainder of fish must
be bagged and moved to the animal disposal freezer in the DLAM facility according to protocol.
Daily plan:
NH4+, NO2, Salinity, DO, Temp, and overall population/treatment health will be observed daily. Fish will
be fed twice daily. After water quality is done, 3-4 gallon water changes will be done on all tanks.
Experiment termination plan:
The experiment will be terminated upon sampling the final fish.
All unused portions of the dissected fish will be placed in the animal discard freezer in the DLAM. No
other action will be taken since the same setup will be necessary for the following definitive experiment.
Tissue storage:
The tissues for mRNA study, intestine, liver, and gill, will be stored in cold RNAlater -80 in the Whitehead
lab deep freezer. {remaining tissues for Galvez lab}
Questions:
Are biofilters necessary?
Does RNAlater need -80,-20, refrigeration?
How should we try and control for chemical or spatial density effects from sampling?