The slide show below shows the main steps taken over the course of the study.
Open field test and measured behavior
-Why the open field test?-
Recording behaviour requires tests that are theoretically and empirically validated (Reale et al. 2007). Tests need to be reliable and valid to produce reasonable data. However, reliability and validity are often weighed against aspects of feasibility and practicality. It is safe to say that in most cases the optimal test does not exist (Hanell & Marklund 2014). An open field test (Figure 9) seemed to be the best choice to measure these behavioural traits, since they all can be assessed when individuals are exposed to a novel environment. Open field tests have been used for a long time to measure boldness and exploratory behaviour reliably and moreover depict natural scenarios most fish species can experience when facing a novel environment (Burns 2008; Crusio 2001).
Figure 9. The two stages of each behavioural trial phase. Inner and outer areas in picture a) were defined post recording but pre-tracking with EthoVision. a) acclimatisation phase for two minutes in a small retractable refuge. b) refuge is removed and fish allowed to move freely in the novel environment.
Behavioral proxys
-Swimming distance-
An often-considered measured variable in an open field test is Movement. Movement is often captured by the total distance a test-subject moves during the test or by the movement rate when not freezing (Burns 2008). Swimming distance is suggested to be regarded as a measure for exploratory behaviour, either by curiosity or by means of returning to a familiar environment (Suarez & Gallup 1985). Therefore, measured total swimming distance in a given time will be used as an indicator for exploratory behaviour in Heterandria formosa (Burns 2008).
-Time spent moving-
The time an individual spends moving compared to time not moving (Moving time, Table 2) can also be regarded as exploratory behaviour. It has been argued to be a boldness-shyness indicator but for instance Mikheev and Andreev (1993) concluded it to be mostly exploratory behaviour, since most fish increased their time spent moving when given new environments to explore compared to making smaller changes to the environment (Mikheev & Andreev 1993). Factorial analysis by Budaev attributed swimming rate to both boldness and exploration but he also included predator interactions which were not part of this experiment (Budaev 1997). Therefore, the proportional time each fish spent moving compared to being inactive will be used as an indicator for exploratory behaviour.
-Actual velocity-
As the third behavioural variable measured I chose actual velocity. Actual velocity is the swimming speed of an individual when it is actually moving, and not just the mean swimming speed over the complete time interval studied. Actual velocity can be seen as a general descriptor for activity since it indicates higher ground coverage and energy expenditure, (Yanase et al. 2007) and will be used as such here.
-Time spent in different areas-
The fourth behavioural variable, time spent in the outer area compared to the time spent in the inner area (Time spent in outer area), should be interpreted as a boldness-shyness measure as traditionally assumed (Brown et al. 2005; Dahlbom et al. 2011). Spending more time in the outer area and closer to the “safe” walls (Thigmotaxis) would be attributed to the shyer individuals whereas bolder specimen would frequent the potentially “unsafe” open centre for longer periods of time (Walsh & Cummins 1976). Consequently, I will use the time spent in the outer area as a shyness indicator.
Table 1. The four measured attributes during the experiment and their corresponding behavior.
Assessment frequeny - How often was behavior measured -
The goal was to identify potential behavioural types and their development over ontogeny under low and high-food conditions. Therefore, is important to measure repeatability, since repeatability of individual behaviour and the differences between specimen defines a behavioural type (Réale et al. 2007; Sih et al. 2004b). To calculate the repeatability of different behavioural traits, like boldness or exploration, a basic framework should be considered. Two repeated measurements for each trait and individual are the absolute minimum to be able to detect among and within-individual variances (Dingemanse & Dochtermann 2013). Dingemanse and Dochtermann also suggest an increase in individual repeated measurements (Table 2) rather than an increase in test subjects (Dingemanse & Dochtermann 2013). I therefore chose to measure each fish 4 times on 4 consecutive days for each life history stage. For sample size, Dingemanse & Dochtermann recommend an ample sample size of 40 individuals to increase measurement power, hence our decision to choose 80 juveniles (40 for each treatment) (Dingemanse & Dochtermann 2013).
Table 2. Behavioral data collection scheme for each of the 80-individual fish across life-stages and the variances estimable from this design. The design allows to estimate both variance components and differences between measured behavioural attributes.