Examining the effects of elevated carbon dioxide on Japanese medaka (Oryzias latipes) behaviour and the use of alarm cue responses for ecologically relevant behavioural studies

Elevations in atmospheric carbon dioxide (CO2) have been measured by experts for decades. These atmospheric elevations cause changes in aquatic environments as increased CO2 reduces pH. Acidification has been shown to cause changes to the behaviour of many fish species including reducing homing behaviour, foraging behaviour, and predator avoidance. Many studies have analyzed the effects of CO2 on fish behaviour, however, wide variation in responses between species is known. Moreover, not many studies have analyzed potential acclimation to CO2. The aim of chapter 2 was to identify whether elevated levels of CO2 affect behaviours of Japanese medaka (Oryzias latipes) and whether they acclimate over time to these elevated levels. To test this, I exposed a total of 300 fish to varying levels of CO2 (2500 µatm, 5500 µatm, 8500 µatm) with different exposure times (0, 5, 10, 15, 20 d). After each exposure period, I performed two behavioural tests, the first, where I placed individual fish into circular arenas, measured cumulative distance moved, immobility duration, and percent of time spent in the outer zone. For the second test, I placed 6 fish at a time into a small tank where half of the tank was marked with a black line. I then measured the amount of time spent over the line and the number of crosses above the line for individual fish. I also performed another behavioural test only at the highest CO2 treatment where I placed 3 fish into one circular arena and measured the same parameters to identify whether solitude influenced behaviour. My results showed that CO2 at 8500 µatm affects behaviour of Japanese medaka only in a group setting. There was also evidence of acclimation around day 10 to 20; however, increased individual variation was seen. My study suggests that CO2 changes behaviours of Japanese medaka under certain conditions, and thus Japanese medaka may be affected by rising atmospheric CO2. Although it is important to analyze basic behaviours of fish to determine baselines, it is also important to study ecologically relevant behaviours to understand what is going on in the environment. Alarm cue behaviours act as a good way to analyze predator avoidance behaviours of prey fish. Although a lot of work has been done in this area, not a lot of studies have analyzed these behaviours in Japanese medaka and new methods for using synthetic cues are still being studied. The aim of chapter 3 was to investigate whether Japanese medaka respond behaviourally and neurophysiologically to both synthetic alarm cues and skin extracts. To quantify behavioural responses, I exposed lab-reared Japanese medaka to three different concentrations of both a synthetic cue (hypoxanthine-3-N oxide) and a prepared natural skin extract and then monitored fish for cumulative distance moved, immobility duration, and the percent of time spent in the outer zone of the arena. To quantify neurophysiological responses, I exposed Japanese medaka to three concentrations of the synthetic cue or natural skin extracts for 5 d and then removed the brains and measured acetylcholinesterase (AChE) activity. There were no changes in behaviour or AChE activity for fish exposed to either cue, which suggest two things: one, Japanese medaka lack the ability to sense the cues; or two, the cues were not appropriate for Japanese medaka. Identifying differences in the structure and function of olfactory systems of Japanese medaka and their putative alarm cue chemical structure may help to explain my results.

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