Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

All animal experiments were conducted under approval of IACUC protocols, numbered 14-05-05-1018-3-01, 13-04-29-1018-3-01, and 14-04-17-1018-3-01.

1. Animal Collection, Handling, and Ethical Care

1. Collect Gambusia affinis from the field site (identification guide at http://www.sms.si.edu/irlspec/Gambusia_affinis.htm) using a small dip net and place into 19 L buckets. Use visual inspection to identify species.
2. Rest fish for 1 – 2 d in a bucket with pond water. Afterwards, transfer fish into 76 or 189 L aquarium tanks filled with half pond water/half tap water at 25 °C, aerated with a bubbling airstone. Use a small dip net when handling fish as to minimally disturb their skin microbiome.
   NOTE: Fish density should be no higher than 20 fish/38 L of aquarium water. Fish are acclimated to the aquarium for at least 5 d before experimentation.
3. Feed fish on a daily regimen with 5 mg of flake food per fish. Hold fish with a 12 h light/ 12 h dark cycle.

2. Initial Antibiotic Exposure for All Experiments

1. Drawing all fish from each experiment from the same aquarium, place into two separate groups (treated: exposed to antibiotic & control: unexposed) in 19 L buckets. Previous data collected shows that fish in the same aquarium have homogenized skin microbiomes that have little variation in community composition.¹²
2. During experiments, keep fish in 2 L of artificial pond water (APW; 0.33 g/L CaCl₂, 0.33 g/L MgSO₄, 0.19 g/L NaHCO₃) that is sterilized by autoclaving before usage.
3. Prepare the rifampicin antibiotic stock solution by adding 50 mg/mL into dimethyl sulfoxide (DMSO). Rifampicin is a representative broad-spectrum antibiotic that is non-toxic to the fish. In humans and mice, it distributes well in the tissues.
4. From the rifampicin stock administer a final concentration of 25 µg/mL in 2 L of APW for antibiotic exposure of the treated fish group for 3 days. Note that after 3 d, culturable skin bacterial numbers return similar to that of pretreated fish skin.
5. In parallel, keep a group of untreated fish in APW and give an equivalent amount of DMSO (0.5 mL per L of APW) into the water column to act as a control group.
6. As experiments are intended to measure effects of an antibiotic-altered microbiome on fish phenotypes, in order to avoid effects directly from the antibiotic itself, following the three-day antibiotic exposure (or control) period, transfer fish into a bucket with 2 L of fresh APW.
   1. Use a 10 h rest period so the antibiotic is removed by the bodies of the fish. Base this elimination time on experiments where fish were exposed to 25 μg/mL of antibiotic for three days. Euthanize the fish by snipping the spinal cord followed by pithing, then homogenize the entire body using a tissue grinder.
   2. Determine antibiotic presence by placing 50 μL of this suspension after 0.2 µm filtering into the center of an agar petri dish. Make a hole for this suspension by pressing an upside down sterile 200 μL pipette tip into the agar, which removes a small plug.
   3. Use agar plates with 20 mL measured volume of nutrient agar, and spread evenly using a cotton swab with an indicator organism, Bacillus subtilis, which is sensitive to rifampicin. Obtain the B. subtilis from a nutrient agar incubated at 25 °C O/N and using the cotton swab to obtain a colony. Observing a zone of inhibition after overnight incubation at 25 °C indicates presence of antibiotic in the fish tissues.

3. Microbiome Extraction

1. Extract a skin microbiome sample from the outer mucosal epidermis by placing the fish into a sterile 15 mL conical tube filled with 2 mL sterile PBST (137 mM NaCl, 10 mM phosphate, 0.1% Tween-20, pH 7.4) solution and vortexing at a setting of 10 for 1 min with 10 s pausing increments. Detergent and salt in the buffer promotes even dispersion of bacteria in solution. Comparable results were found with 0.85% saline but lowered bacterial counts with pure water.
2. Transfer the fish from the conical tube to a recovery bucket. Lethality of skin extraction is typically lower than 10%. Either, analyze the bacterial suspension in the tube immediately upon extraction or pellet the bacteria by a 2 min spin in a centrifuge at room temperature at 15,000 x g and store at -80 °C for future analysis.
   NOTE: All culture and biochemical analyses are immediate; DNA or protein extraction can be from frozen samples.
3. To obtain a gut microbiome sample, first place the fish in a sterile petri dish. Euthanize the fish by severing the cervical spinal cord directly behind the head with surgical scissors, followed by pithing, and then externally sanitize the body with 70% ethanol wipes.
4. After dissection, remove the entire gut by cutting after the esophagus and before the anus.
5. Cut the gut into small sections 1 – 2 mm in length and place all sections into two mL PBST solution in a conical tube. After vortexing for 1 min, remove supernatant into another clean tube (take caution to not draw up the gut sections).
   NOTE: The supernatant contains extracted gut bacteria that can either be used for direct analyses or pelleted by the previous method mentioned for skin samples.

4. Infection Model Preparation and Bath of a Specific Pathogen

1. Obtain an infectious strain of Edwardsiella ictaluri (E. ictaluri) and keep the culture stored at -80 °C. Strain 93 – 146 used in this study, isolated from an infected catfish, was obtained from Mark Lawrence, College of Veterinary Medicine, Mississippi State University.
2. Streak E. ictaluri stock onto a selective & differential media called E. ictaluri media (EIM) agar¹⁹ to culture the bacteria and incubate for 2 d at 27 °C.
3. Transfer a pure isolated colony from the culture and inoculate a 150 mL flask containing 40 mL of nutrient broth (NB; 5 g/L peptone, 4 g/L beef extract). Place flask in a shaker set at 150 rpm for 2 d at 27 °C.
4. After incubation, dilute a sample of the culture in PBST to a spectrometer reading of 0.2 OD at 650 nm to calibrate E. ictaluri culture for desired infectious dose volumes.
5. Next transfer both treated (after 3 d of antibiotic exposure) and untreated fish groups into individual plastic cups containing 130 mL of fresh artificial pond water, or APW for approximately 10 h for drug clearance from fish tissues.
6. Then from both groups, transfer again each fish into 8-ounce plastic cups containing 130 mL of clean APW.
7. Give each fish a lethal dose containing 2.8 x 10 ⁶ CFU/mL of E. ictaluri culture into the APW (bath infection model) and keep in a 27 °C incubator for 24 h.
8. After the E. ictaluri bath, transfer fish individually into new cups with 130 mL of APW.
9. Following water replacement, record fish mortality over the duration of a week. Fish are not fed over this period. In contrast to catfish, Gambusia show no external signs of infection, therefore it is essential to use lethality as an experimental endpoint.

5. Polymicrobial Challenge with Feces & Soil

1. Feces Treatment
   1. Obtain fresh human feces from a healthy volunteer subject who has not received antibiotics in the previous two weeks, using sterile gloves and a sterile 50 mL conical tube.
   2. Upon collection, gather fecal matter in a sterile plastic bag and then transfer into a sterile 15 mL conical tube. Create a stock concentration by suspending feces in PBST to give a stock solution of 500 – 800 mg/mL. This thick mixture is best to transfer using a 1 mL or larger plastic pipette tip that has been cut at the bottom with sterile scissors so that the opening is larger.
   3. After initial antibiotic exposure of treated and control untreated fish groups, separate into individual plastic cups containing fecal suspension at final concentrations of either 15 mg/mL or 10 mg/mL into APW with a total volume of 130 mL. Hold cups in an incubator at 25 °C.
   4. Record fish mortality during fecal exposure by close observation over 2 d.
2. Soil Treatment
   1. Collect 1 – 2 kg of rich organic topsoil (should contain the highest density and diversity of microbes) approximately 7 – 15 cm down in depth and place into a clean plastic container. Note that the soil should be used within two days following collection.
   2. Directly after initial exposure period, separate both groups of antibiotic treated and untreated fish into individual plastic cups containing 18.2 g of soil in 130 mL of APW. Mix the soil in the water well by hand before adding the fish. Most of the particulates do not suspend and stay at the bottom of the cup.
   3. Expose fish for a duration of 3 d at 25 °C and record any mortality.

6. Osmotic Stress Challenge

1. After treatment followed by a 10 h rest period as described above, individualize fish into separate cups containing NaCl at 17.5 mg/mL (300 mM) in 150 mL of APW. This is half of the typical salinity of seawater, which is not fully lethal (<50%) to control fish but is strongly stressful, requiring effective osmoregulation.
2. Observe for fish mortality over a duration of 36 h.

7. Nitrate Toxicity Challenge

1. Rest fish for a 10 h period after antibiotic exposure, and then separate fish into individual cups containing a sodium nitrate concentration of either 10 mg/mL (118 mM) or 17.5 mg/mL (206 mM) in 130 mL of APW.
2. Record for mortality over 4 d for low dose and 1 d for high dose.

8. Growth Analysis of Individualized or Grouped Fish

1. Complete 3 d antibiotic exposure or control period as groups in buckets.
2. For Individual, fill 8-oz Styrofoam cups with 150 mL APW each, transfer an individual fish into the cup and record total body weight for initial assessment.
   1. Repeat for all fish in both treated and untreated groups. Use white Styrofoam cups instead of clear plastic cups because fish will not eat when in the clear cups.
3. Feed fish daily with the standard diet of two pellets per fish. Pellets were used rather than flakes because pellets have low variance in individual mass (3.53 ±0.42 mg each, or 8% variation), resulting in fish receiving similar amounts of food. Monitor consumption by visually recording uneaten pellets. Consumption rates were typically above 80%.
4. At the end of each week over 4 weeks, determine fish weight. Prepare a new cup with fresh APW, place on a balance, and then record the weight. Then, pour a fish into a dip net from the original cup and transfer into the new cup, which is then weighed again. Fish are not handled to avoid stress.
5. For Groups, place 2 L of APW into a 19 L bucket and tare the scale. Keep fish together in both groups when transferring into new APW buckets then record total group weight. Record fish group weight each week over a month time span by transferring to a new bucket.

9. Gut Transit Time

1. Use fluorescein-labeled 70 kDa anionic dextran. Dextran is not significantly digested, and is too large to be absorbed across the gut layer, thus passage will measure transit time through the gut. Labeled dextran was incorporated into fish food.
2. Into a sterile 1.7 mL tube, add 360 μL of sterile deionized water and 52 mg of gelatin (13% solution), and place the tube on a 60 °C heat block until the gelatin melts and is fully dissolved.
   1. Grind goldfish food flakes to a fine powder using a mortar and pestle, and add 40 mg of this powder to the tube, along with 20 μL of fish oil. After mixing, add 1.6 mg of FITC-dextran.
3. Dispense the hot liquid mixture into 20 μL drops onto Parafilm on the lab bench, and let them quickly cool and solidify. Drops can be stored for up to 4 d before they become too hard for fish to eat. Store drops in the refrigerator by placing the Parafilm onto half of a petri dish, which is then floated on sterile water inside a sealed plastic container, which keeps the drops humidified.
4. Just before feeding, quarter drops into sections using a razor blade. Acclimate the fish to the Styrofoam cups individually for 2 days without feeding (Note: Only unexposed control fish were used). Place four quarters of the food into the cup with the fish, and observe the fish for 30 minutes for how many pieces of the food they each eat.
5. To begin the monitoring period, transfer fish individually into cups with 80 mL of APW using a dip net. Every 2 h, gently swirl the APW by hand, and then take a 1 mL sample and store in a labeled 1.7 mL tube at 4 °C. Take samples for 36 h.
6. Record fluorescence, with a fluorescence spectrophotometer, of all samples at the same time after completion of the assay. Place the entire 1 mL sample into a cuvette, and record the fluorescence measured using excitation at 495 nm and emission at 520 nm.