Seven days before the experiment, I familiarized the fish with shelters (which were used in later parts of my experiment) by putting one into the fish‟s maintenance container. In the field the fish are often found hiding in a shelter (Davis 1990).
Laboratory-hatched individuals placed in a container with a shelter often utilized the shelter within one day and stayed in it for most of the time thereafter. I therefore concluded that the shelter-hiding may be an innate tendency, and used this tendency as the basis of my study.
Between 9 and 10am on the first day of the experiment, a water sample was collected from the focal individual for baseline hormone analyses. (See “2.3.1 Hormone collection and extraction” for a description.) Afterwards, the fish received its pre-assigned winning, losing or no-contest experience. (See “2.3.2 Experience training” for details.) After training the fish was replaced in its maintenance container and fed newly hatched brine shrimp.
Between 9 and 10 am on the second day of the experiment, another water sample was collected from the fish for post-experience hormone analyses. The fish was placed back in its maintenance container for 15 minutes and fed a small amount (0.5 ml) of brine shrimp. The fish was then exposed to three tests (aggressiveness, boldness, exploratory tendency) consecutively in a pre-determined order. Each test lasted for 30 minutes, but because the fish was allowed to acclimate in the test tank for an hour before the test started, each test took an hour and a half to complete. When the three tests were completed, the fish was transferred to a T-maze for the spatial learning test. Procedures for conducting these tests are described below in sections
(2.3.3 - 2.3.6). After completing the learning test, the fish was returned to its maintenance container.
2.3.1 Hormone collection and extraction
At 9 am on the first and the second days of the experiment, I removed the focal individual from its maintenance container for an hour to analyze its baseline and post-experience hormone levels, respectively, as mentioned above. The fish was placed in a 400 ml glass beaker filled with 200 ml 25 ppt clean synthetic sea water and housed inside an individual translucent plastic container, visually isolating it in order to reduce the risk of disturbance. The fish stayed in the beaker for an hour. At 10 am, after removing the fish from the beaker, hormones were extracted from the water sample using a C18 solid phase extraction column (Lichrolut RP-18, 500 mg, 3.0 ml; Merck) fitted to a 12-port manifold. Before use, the columns were primed by 2 consecutive washes with 2 ml methanol (MeOH) followed by 2 consecutive washes with 2 ml pure water. Tubing was then fastened to the top of each column and placed into the water sample collected from the focal fish. A vacuum pump was connected to the tubing and the water sample was extracted through the tubing into the column.
After processing all the water from the sample, I used 2 consecutive 2 ml washes with pure water to purge the seawater salt from the column and then stored it in a freezer at -80ºc until future processing. Freeze storage of water samples and columns has been determined not to influence steroid concentrations (Ellis et al. 2004). Hormones were eluted from the columns into 6 ml vials by 2 consecutive 2 ml washes with EA (Ethyl Acetate). The 4 ml of eluted solvent was evaporated at 37ºc with nitrogen gas to facilitate efficient evaporation. The resulting hormone powder was then stored at -20ºc until being assayed.
Both hormones (testosterone and cortisol) were assayed using Cayman Chemicals Inc. EIA kits and following the procedures recommended by the manufacturer. Plates were read at 405 nm on a BioMek microplate reader.
2.3.2 Experience training
On day one, using random-selection procedures, the fish were given a pre-designated randomly assigned experience (W, L or N). The relative standard length (the distance from the tip of the snout to the beginning of the tail fin) is an important factor in determining the likelihood that individuals of K. marmoratus will win a contest (Hsu et al. 2008). To ensure that the focal individuals received their pre-designated losing or wining experiences, I fought them against much larger/
smaller (SL difference >2 mm) standard winners/ losers, which were strong/ weak and had won/ lost several fights with similar-sized opponents. For experience training, a fish was placed in each of two symmetrical compartments (randomly selected) of a standard aquarium (12 × 8 × 20 cm3, containing water 16 cm deep and 2 cm of gravel) divided by an opaque partition, which was removed to start the training. Individuals received their pre-designated experience quickly due to the large size differences and were again separated by the partition soon after the contest was decided. Fish assigned to receive no (N) experience were treated exactly as above except with no opponent in the standard aquarium. After training, the fish were replaced in their maintenance containers and fed newly hatched brine shrimp.
2.3.3 Aggressiveness test
I used a mirror test to quantify the fishes‟ aggressiveness (Earley et al. 2000).
The test was carried out in a 16 10 20 cm3 experimental aquarium (Fig. 1),
containing water 12 cm deep with 2 cm of gravel. Each aquarium had a mirror on one of the narrow sides and, 10 cm away from the mirror, a black opaque removable partition. The focal individual was placed in the part of the aquarium behind the partition, separated from its mirror image, for an hour‟s acclimation. After the acclimation, I removed the partition and recorded the fish‟s behavior for 30 minutes.
As my index of aggressiveness, I recorded the time elapsed until the fish first took part in gill display (erected its operculum during display), the most aggressive fish having the shortest latency to gill display during the mirror test. I used this index for aggressiveness because gill display was usually the first aggressive behavior displayed and was therefore probably less influenced by the mirror image than other agonistic behaviors such as the latency to the attack or attack frequency. The mirror test was terminated after 30 minutes even if the focal fish did not display its gills towards its mirror image. After the mirror test, I moved the fish to the next experiment aquarium, to acclimatize for an hour.
2.3.4 Boldness test
Boldness is defined as the tendency to take risk (Wilson 1994). Following Brown et al. (2005), I used the latency to emerge from a small dark plastic shelter as my index of boldness, bolder individuals having a shorter latency to emerge from the shelter. The testing aquarium (Fig. 2) was the same size as that used for the mirror test, containing water 12 cm deep with 2 cm of gravel. The aquarium was fitted with a (6 6 8 cm3) black plastic shelter with a removable sliding door, through which the fish could freely swim when open. I placed the fish to be tested in the black plastic shelter for an hour‟s acclimation, pulled up the door at the beginning of the boldness test and recorded the time elapsed until the fish first emerged. I terminated the test after 30
minutes even if the fish had not come out.
2.3.5 Test of tendency to explore
I used the time elapsed before the fish started exploring a new environment (with a shelter) as my index to quantify the tendency to explore (Gerlai et al. 1990;
Dingemanse and Goede 2004). Fish with a greater tendency to explore novel environments should arrive at the shelter sooner. The experimental aquarium (Fig. 3) was 34 12 26 cm3, containing water 16 cm deep with 2 cm of gravel. I put a new shelter at the end of the wide side and a black opaque partition 6 cm from the opposite end. I marked a small dot 4 cm from the shelter on the surface of the aquarium as the
„well explored region‟. The focal individual was placed in the smaller compartment, behind the partition, for an hour to acclimatize to the new experimental settings. At the start of the experiment, I pulled the partition up to let the focal fish freely explore the surroundings and recorded the time elapsed until the fish went into the „well explored region‟ (< 4 cm distance from the shelter). I terminated the experiment after 30 minutes even if the fish did not get close to the shelter.
2.3.6 Test of spatial learning performance
I used a T-maze test to test the fishes‟ spatial learning performance. Fish which found their way into the target end of the T-maze during the test were deemed (on average) to have better spatial learning performance than those that did not. The T-maze (Fig. 4) was a slightly modified version of that used in a previous study by Darland and Dowling (2001), and consisted of a cross piece (70 cm long), with a down piece (35 cm long) joining the mid-point of the cross piece. Both sections were 10 cm wide, 12 cm deep and contained water 5 cm deep. At each end of the
cross-piece, there was a 22.5 22.5 22.5 cm3 reservoir, with 1 cm of gravel, 3 shelters and water 13 cm deep. As the fish tend to stay in shelter when it is available (see “experimental procedures” above) the reservoir area with its gravel and shelters should be desirable to them. The reservoirs and the shelters inside them were arranged symmetrically, to avoid any possible visual clues to the fish. There was an opaque removable partition at the entrance to each reservoir, controlling whether the fish could see and get into the reservoir area or not and a further removable partition 10 cm away from the end of the down piece of the T-maze (start point). I placed the focal fish at the end of the down piece of the maze, behind the removable partition, to acclimatize for an hour before the test.
Before the test, one of the two reservoirs was chosen at random as the target. The fish was then given two training sessions (I and II), in which to learn to go into the target reservoir, and a test to see whether it had learnt from its training, all as described below.
a) Training phase I: I pulled up the partition of the pre-assigned reservoir
before the test so that the fish could enter it, but not the reservoir opposite. After acclimatization for an hour, I pulled up the partition of the start point so that the focal fish could freely explore the t-maze. After the fish entered the target reservoir, I closed the door (removable partition) so that the fish was restricted to the shelter-rich environment. Five minutes later, I terminated the first training period. If, after 30 minutes, the fish had not moved out of the down piece of the T-maze, I forced it to move to the junction of the T-maze by using a white opaque partition, leaving it to make its own turning decision there. If, after a further 30 minutes, the fish still had not arrived at the target reservoir, I forced it in into the reservoir using an opaque partition as above. After the first training period I placed the fish back at the start point, with
the start partition closed, to acclimatize for 10 minutes in preparation for the next
Fish that did enter the reservoir were placed at the start point, with the start partition closed, to acclimatize for 10 minutes in preparation for testing phase.
c) Testing phase: I pulled up the partitions at the entrances to both the reservoirs,
allowing the fish to choose for itself whether to enter the correct or the incorrect reservoir. At the start of the test phase, I pulled up the partition at the start point allowing the fish to swim freely in the T-maze. If the fish found its own way to the correct reservoir it was deemed to have passed the T-maze test. If the fish did not move out of the start point, I forced it out of using an opaque partition as above. If the fish moved out of the start point but failed to get into the target reservoir in 30 minutes, I defined that the fish had failed the T-maze test.