raise stickleback

Basic stickleback husbandry

This page describes the basic procedures we use to make crosses and raise stickleback. Do not use water obtained from copper pipes, as the copper ions kill stickleback. Set up the aquarium in a cool room. 17 degrees C is perfect for our local populations. Above 20 degrees is dangerous.

Standalone aquariums – McPhail Room

We use 100 L aquaria to keep and raise stickleback. The fish grow well under these conditions provided densities are kept low. Standalone tanks are stacked on shelves in the McPhail room of the InSEAS aquatic facility. The greatest challenge is getting the nitrogen cycle going in a new standalone tank.

All wild fish go into the McPhail Room — do not bring wild fish into the Flow-through Room.

Initial setup

  • Dechlorinated tap water is fine. Add extra conditioning, such as Prime or Amquel, as backup dechlorination in case of a spike in chlorine in the water supply. Add baking soda if pH is below 7 (Vancouver water is sometimes acidic).
  • To aid fish health, add 100-200 g of synthetic sea salt (e.g., Deep Ocean Synthetic Sea Salts) to each 100 L aquarium. Add even more, 500 g, if you are raising crosses between freshwater and Little Campbell River marines (LCM), which tend to develop poorly at low salinity. This will bring salt concentration up to about 5 ppt.
  • Add bicarbonate of soda to raise the pH to desired levels (see next section for information on adjusting pH).
  • Add crushed coral or limestone to the tank. This keeps the pH high and the water hard, which is best for minimizing disease. Many but not all of our study populations live in hard water lakes.
  • One airstone per 100 L aquarium is fine (you don’t need to add one if you are also using a sponge filter). Keep the flow light when the fish have just hatched, because they are not strong swimmers.
  • Use sponge filters for babies and add power filters for juveniles and adults. For small juveniles, put a sponge on the end of the intake pipe to prevent sucking up fish. Use angel hair or double up the sponges in the filter box for extra surface area and filtration of fine particulates. The foam inserts of the power filters last a long time but need periodic cleaning. Squeeze the sponge filters every two weeks or so to keep the sponges from plugging up.

Start the nitrogen cycle

This is the greatest challenge when starting up a tank for fish. The filters provide a surface for the bacteria that break down ammonia to nitrite and then to nitrate. Ammonia and nitrite are toxic, whereas nitrate is relatively harmless. However, it takes at least a month for the bacteria to reach sufficient numbers to handle all the waste produced by the fish.

To get the nitrogen cycle up and running:

  • Set up new tanks at least one month before introducing your valuable fish.
  • While you wait, keep a goldfish, sculpin, or a very small number of non-essential stickleback in the tank to help get the nitrogen cycle going before you replace them with your valuable fish one month later.
  • Another technique, while you wait, is to add ammonia daily directly to a fish-less tank and keep testing the water until nitrites drop to zero. Your tank is ready to receive important fish.
  • If you don’t have the luxury of a one-month advance, but must throw your fish straight into a new, sterile tank, then you will need to change the water frequently to keep waste levels down for weeks while the nitrogen cycle gets going. Add a filter sponge taken from a older tank whose nitrogen cycle is functioning (beware of communicating disease). Try replacing 1/3 to 1/2 of the water every two days for the first two weeks at least. Add Amquel or other conditioner to help remove nitrite as well as ammonia (not all products will do both). Make arrangements for water changes on weekends if you are not around to do it yourself, especially long weekends.

Manage the nitrogen cycle

  • Keep fish densities low, even after the nitrogen cycle has started to work. A 100 L tank can handle about 15 adult fish when everything is going well (maybe even 20 if you are not dealing with large benthic or marine fish). If you go higher, be prepared for the higher risk. Go lower if the fish are irreplaceable.
  • Remove dead fish immediately. Take special care on weekends, if you are alone in the lab. You might be able to avert disaster this way.
  • Do not overfeed fish. If the fish food that drops to the bottom of the tank isn’t eaten within a few minutes, siphon the excess away.
  • Test ammonia and nitrites regularly. See the next section below.
  • Apply regular water changes (see below).
  • If you are raising fish from eggs, you might end up with as many as 200 juveniles from one clutch in a single aquarium. This is unsustainable. Families should be divided among multiple tanks as soon as the young fish are large enough to tolerate a move.
  • Make sure your filters are working properly. Restart them immediately if there has been a power outage.

Adjust ammonia and pH

These instructions are from the standard operating procedures, SOP for adjusting ammonia and the SOP for adjusting pH.

Ammonia. Test ammonia (NH3) using manufacturer’s instruction. Ammonia levels should be at 0 ppm (mg/L) any level above zero can harm fish. Ammonia builds up due to the breakdown of organic waste and the nitrogen cycle and can be toxic. The presence of ammonia indicates possible overfeeding, too many fish, or inadequate biological filtration. If the ammonia test reveals ammonia levels above 0 ppm, follow the instructions below:

  1. Scrub off excess algae with a brush and perform a 25% water change.
  2. Ensure tank is seeded/developed with nitrogenous bacteria to break down ammonia.
  3. Ensure that the filters are circulating water at a sufficiently high rate.
  4. If the tank has only a foam filter, add a higher-flow (box) filter.
  5. Lower the density of fish in the tank to decrease the amount of ammonia produced.
  6. Test the water again after 24 hours.

pH. Test pH using manufacturer’s instruction. Stickleback from most populations prefer pH between 7.4 – 7.6 (stickleback from limestone lakes, such as Paxton, Priest, and Cranby on Texada Island, can tolerate pH up to 8.3). If pH falls below the preferred range, gradually increase pH using one or more of the following methods.

  1. Add limestone or crushed coral to the aquarium. The carbonate in these compounds will react with and remove excess H+ ions in the water.
    • Crushed coral and limestone are advantageous as the release of carbonate is governed by the surrounding water chemistry. As a result, crushed coral and limestone will raise the pH to a maximum of 7.8 regardless of the amount added. Crushed coral and limestone will develop a hard surface layer and should be scrubbed once every 3 months to ensure reactivity.
  2. Add baking soda to the aquarium (gradually).
    • The direct addition of baking soda can raise the pH very quickly and it is possible to overshoot the target pH. This can cause pH shock in the fish. It is best to gradually add baking soda by dissolving 1 teaspoon of baking soda in 1 cup of water for every 5 gallons of water in the tank. Most of our tanks are 30 gallon. Add ¼ of the baking soda-water mixture to the tank, wait 30 minutes or longer and add another ¼. Continue this procedure until the mixture is used up. Wait 24 hours and test the pH again.
  3. Aeration can raise the pH slightly.
    • Aerate the water by adding an air stone to the fish tank or increasing the flow of air. Excess CO2 dissolved in the water decreases pH (makes it more acidic). As the tank water is exposed to atmospheric air through aeration, the tendency of the dissolved gases to approach equilibrium will transfer oxygen into the water and carbon dioxide out of the water, resulting in a higher pH. This method will result in only a slight change in pH. Use an alternate method if you require a larger magnitude change.
  4. Use a commercial alkaline buffer and follow manufacturer’s instructions.

Regular water changes

A functioning nitrogen cycle in an aquarium will convert toxic ammonia and nitrites to nitrates, which are less harmful to fish. The way to remove nitrates is via regular water changes. The following is from the standard operating procedure SOP for recommended frequencies and amounts of water changes to standalone tanks.

  • Once your tank is fully cycled (tests reveal zero nitrites and zero ammonia), tank water needs to be changed on a routine basis.
  • The frequency and volume of water change depends primarily on the amount of ammonia being produced; this varies with fish density and feeding schedule (over feeding raises ammonia).
  • A 25% water change should be carried out every 2-3 weeks on healthy tanks (low fish density, no detectable ammonia). If 25% water changes cause too much stress, decrease the volume of water being replaced to 10% and increase water change frequency.
  • To minimize the stress caused by water changes, use aged water (same temperature, adjusted pH levels).
  • Increase the volume or frequency of water changes when tanks show spikes in ammonia and continue to test ammonia levels daily until the tank stabilizes at zero.
  • To achieve targets, it may be necessary to lower fish density and the amount of feed.

Flow-through system

The Flow Room is for lab-raised fish only, to minimize disease transmission. Do not bring fish from the wild into this room, or dip nets and other equipment from the McPhail room to minimize the chances of contamination.

Initial setup of tanks

Water quality is managed centrally, so there’s little setup or maintenance needed at individual tanks.

  1. Choose empty aquaria and add your fish.
  2. Look over each tank and ensure that water is flowing out of the tap and the water level is not too high.
  3. If the water level is high, lower the flow from the tap and rinse out the sponge over the outflow pipe as it may be clogged.

Manage water quality

  1. Check room temperature and record it in the daily monitoring spreadsheet.
  2. Every 2 weeks, clean the end of the pH probe on the pH doser in the back corner of the room. Use a soft toothbrush.
  3. Record the pH reading on the pH doser in the back corner of the room. pH should be 7.4. If pH is below 7.2, clean the pH probe to ensure an accurate reading (see above step).
  4. The doser is pre-set to adjust the system pH to 7.4 by adding soda ash from the barrel. When the pH reading stabilizes, top up the barrel by adding dense soda ash and dechlorinated water. The amount of soda ash added does not need to be precise.
  5. To change the dosing parameters on the system, refer to the Pinpoint pH Controller User’s Guide.
  6. Check the water temperature and O2 saturation using the PointFour monitoring system in Room 1208A. Press the “home” button (house icon). Our recirculation system is identified as SYSTEM B3. Measures of water temperature and oxygen saturation should appear on the screen. Record these values in the record book.
  7. Water temperature is currently set to 17 C. The system will allow the water temperature to fluctuate ±l degree before setting off a temperature alarm.
  8. If the temperature is outside the desired range, check that the heat exchange pumps are functioning (you should be able to feel the machines vibrating if you touch them). They are the two machines against the right wall as you enter the room. They are labeled “Heat exchange pumps for system B3”. The screen should read “17” degrees. If it the reading is above or below 17, contact Patrick Tamkee and ask him to adjust the “top up” water to the system in the Biosciences basement. Turning the top up water ON will lower the water temperature in the system, as cool water from the UBC well source will be introduced into the system. Turning OFF the top up will allow the heat exchange pumps to maintain a water temperature of 17 degrees. If there are any other problems with water temperature, contact Patrick Tamkee right away.

Adjust water flow to the recirculating system

  1. The main valve that controls the water pressure is located in the back right corner of the control room (see below).

    This valve determines how much water gets diverted back to the filter pump and into the pipes connected to the fish tanks. In an emergency (i.e water overflowing), adjust this valve first.

  2. There are additional ball valves located above each rack in the main Flow Room that control how much water feeds each row of tanks.

    BE CAREFUL when adjusting these valves. Turning off all the taps at the tank level will cause a build up of water pressure that could lead to cracked pipes or leaks between each joint. Always adjust the main valve in the support room before any major adjustments at the tank level.

Make crosses

Obtaining eggs from females:

A female ready to spawn can be identified by her abdomen shape at the cloaca. If ready to lay eggs, one can almost see the first egg; at this point her abdomen at the cloaca is sharply angled, almost like the corner of a box. The eggs should come after gently squeezing her body above and forward of the egg mass and, while maintaining pressure, sliding your fingers posteriorly. The eggs, when they appear, will stick to one another in a clump. If they dis-aggregate then the female was not ready and you should throw the eggs out. Keep the eggs covered with water, but keep them near the surface for oxygen.

Testes:

We haven’t developed a method to extract sperm without killing the male. Extract testes from the male after giving him an overdose of anaesthetic. If you do not actually require separate crosses, you can fertilize several clutches with the testes from one male. Male sperm will keep in Hanks solution for a few days (see Storing sperm, below). Shred the testes with tweezers and stir around the egg mass .

Remove the testes after a few minutes otherwise they might decay and attract fungus. It is worthwhile to check that the eggs are fertilized and begin development (the easiest way to tell is under a low-power microscope—look for the separation of outer and inner membranes).

Preserve sperm in the fridge for up to 2 weeks

Materials needed

  • 49.5mL of Ginsburg’s ringer solution (a buffer solution – recipe is below)
  • 0.25mL of Gibco antibiotic/antimycotic (Invitrogen cat# 15240-096, 100x concentration),
  • 0.25mL of Gentamycin sulfate hydrate, (Invitrogen cat# 15750-060, 10ml, 50mg/ml liquid)
  • 1 falcon tube.

Preparation of the storage solution

  1. measure out 49.5mL of Ginsburg’s ringer solution and pour into a falcon tube.
  2. pipette 0.25mL of both the Gibco and Gentamycin solutions into the Ginsburg’s solution in the falcon tube.
  3. close cap and shake well in order to thoroughly mix solution

Sperm storage

  1. after removing the testes from the male stickleback place each testis (or portion of testis) in it’s own Eppendorf tube.
  2. pipette in enough Ginsburg’s solution to fill half the tube.
  3. make sure that each testis (or portion of testis) is completely submerged in the solution (it should sink to the bottom).
  4. store in fridge
  5. replace with fresh Ginsburg’s solution every 7-10 days for storage up to 6 weeks.

Recipe for Ginsburg’s ringer solution

  1. To 900 mL of ddH2O, Add: 6.6 g NaCl 0.25g KCl 0.3 g CaCl2
  2. Add 0.2g NaHCO3 last
  3. Mix well, and bring up to 1 L
  4. Autoclave

Cryopreserve sperm

The following is derived from the Medaka book, which is based on Aoki et al (1997)

Contributed by Tom Howes:
“The main modifications I made are that I use 200 ul of medium to mince the testes (instead of 60) and store aliquots in 5 ul microcapillary tubes (Drummond microcaps) instead of 10 ul. That gives
plenty of aliquots that can be stored inside several 1.8 mL cryotubes (Nunc #377267).”

Freezing Stickleback Sperm

  1. Remove the lids from eight 14 mL conical tubes and embed them in dry ice in a styrofoam container (Can use a hammer to tamp them down into the dry ice bed).
  2. In a regular ice bucket, start chilling eight labeled 1.8 mL cryotubes, and a 1.5 mL tube with the lid open and resting on the ice. Start thawing an aliquot of Fetal Bovine Serum (heat inactivated and cleared).
  3. When ready to dissect out the testes, prepare the freezing medium fresh (180 ul FBS + 20 ul
    dimethylformamide) and place it in the cap of the 1.5 mL tube on ice. Place the dissected testes in the medium and mince thoroughly. Remove any large tissue fragments.
  4. Use some of the fresh sperm prep if needed (5 ul is plenty). Draw the rest into individual 5 ul
    microcapillary tubes (the Drummond microcaps come with their own squeeze bulb that you can use to
    create a small amount of negative pressure). Distribute the microcap tubes to the 1.8 mL cryotubes on
    ice. Put the lids on the cryotubes, making sure the capillary tubes go inside the threading of the lid so they don’t break.
  5. Put the cryotubes inside the 14 mL conical tubes embedded in dry ice and leave them there for 20
    min. Then dump the cryotubes into liquid nitrogen and move them to storage in a liquid nitrogen-
    cooled freezer.

Thawing an aliquot

Warm ~100 ul of Hank’s buffered saline solution to 30 C. Take out a cryotube and keep it in liquid
nitrogen until ready. Move the cryotube onto dry ice and remove one microcap tube without thawing
the others (use forceps chilled on dry ice). Hold the microcap tube between the fingers, and it will
quickly thaw enough that you can use the squeezebulb to eject the contents into the Hank’s solution.
Move the cryotube back to liquid nitrogen. Mix the thawed sperm solution briefly and use for
fertilization.

Raise crosses

Raise eggs

We use either of two approaches to raise the eggs to hatching. The egg-tank method involves raising eggs for the first 7 days in small 5 gallon fish tanks and then transferring the eggs to standard 100 L tanks just before they hatch. The 7 day period assumes that the rooms are at 17 degrees. If the room is warmer they may hatch sooner. Egg-tanks should be cleaned out and made fresh every 2 weeks. The big-tank method involves raising eggs entirely in the 100 L tanks.

In either case, place the eggs in a yoghurt cup having mesh screen on the bottom. Suspend the cup from the side of the aquarium so that the eggs are well submerged. Door screen is best, because the fry drop through after hatching (another reason to make sure to transfer eggs from egg-tanks to 100 L tanks before hatching begins). Put an airstone nearby to provide oxygen and maintain a current around the eggs. Avoid fine streams of bubbles directly underneath the eggs because air may get trapped under in the egg mass, bringing it to the surface, where the eggs may dry out.

The water in the tank should be dechlorinated. Adjust the pH to 7 (using baking soda or pH adjuster). Add 100-200 g of synthetic sea salt (e.g., Deep Ocean Synthetic Sea Salts) per 100 L water. Add at least 500 g/100 L if you are raising crosses between freshwater and Little Campbell River marines (LCM), which tend to develop poorly at low salinity. Also add Methylene Blue solution to the tanks to help reduce fungus attack. Mix the powder thoroughly with water in a falcon tube and then add a few drops to the tank so that the water color is a pale blue (don’t add the powder directly to the tank). The 100 L tanks should be set up at least a day in advance of the egg transfer, so the filters have time to clean the water and help dissolve/mix the salt and baking soda. Use only a sponge filter in the tank. Make sure power filters are shut off. Use only sponge filters until young are large enough to handle the current and suction of a power filter.

Tend the eggs daily and remove clots of fungus that appear. Be careful not to tear nearby eggs. You will never manage to be free of fungus, but plenty of aeration and the methylene blue will help to avoid the worst. Our experience is that if fungus is out of control in a clutch of eggs only a couple of days after fertilization, then the clutch probably wasn’t fertilized after all.

Raise young fry

When the young hatch they will sink to the bottom and stay there for a couple of days. Then they will swim up and gulp some air from the surface to establish neutral buoyancy. Make sure that the sponge filters and airstones are not bubbling too vigorously at this time (keep the power filters off until the babies are older). After a few days you will start to see the tiny babies hanging out together off the bottom, especially in the corners of the aquarium.

Optional: Start adding a squirt of infusoria to tanks the day they hatch. You may also add a few drops of pet-shop liquid food for egg-layers. They don’t eat the stuff; rather, they eat the paramecium and possibly some bacteria. Continue adding infusoria for about 5 days after hatch, by which time all the fry will be free-swimming.

One day after hatching, start feeding them microworms, if available, and small quantities of first-instar brine-shrimp nauplii. Add enough brine shrimp so that after they have fed their bellies are orange and swelled. It is best to feed twice daily until they are a few weeks old, but once daily will also work if you don’t mind the slower growth.

When the young reach about 2 cm, start feeding them frozen bloodworms. Continue to feed them a small amount of brine shrimp nauplii as well, even as adults.

Troubleshooting

Nitrites are high

Usually a problem in recently cleaned tanks, or overstocked tanks. Monitor N levels monthly and note level on tank. When a tank has detectable nitrites:

  • change 1/2 of the water immediately
  • add 5-15 ml of Prime (more directions on bottle of Prime)
  • add another filter
  • move some fish to another tank
  • recheck N next day, continue above steps until problem solved

Continue to replace 1/3 of the water in the tank daily until nitrites are undetectable .

Filters not working/water not flowing properly

Check whether it’s a single filter or a bunch in one area. If single:

  • check if it’s plugged in (make sure it’s not a new baby tank and therefore supposed to be unplugged)
  • check power bar is working
  • jiggle motor fan with finger to get it working again (sometimes they jam)
  • check u-tube and downspout are attached properly near the motor and are sucking up water (sometimes the filter is on, but no water is cycling – especially important to check after cleaning filters)
  • check that the downspout isn’t blocked (plant material/dead fish/mesh clogged with dirt)
  • check for broken parts and discard broken bits, re-assemble filter with new part (there’s boxes of filter parts in one of the rooms, including motors, bodies, fans etc)
  • follow the power to where it doesn’t work (the power bar has probably gotten wet or there’s a short somewhere)
  • Jump start all filters that don’t automatically restart themselves, but do it after you’ve sorted the problem out or you’ll be restarting things all day.

Rooms Are Warm

This needs to be fixed immediately or fish will die.

  • If Patrick is around, tell him about the problem. If he’s not around, phone plant ops ‘trouble calls (ext 2-2173)‘ immediately, and tell them it is an animal care emergency.