A significant potential for genetic improvement of resistances to acute hyperthermia and acute hypoxia was demonstrated in rainbow trout (Oncorhynchus mykiss) through selection
. However, the physiological mechanisms underlying these resistances are still under debate. It is therefore necessary to understand what makes a genotype resistant by identifying the mechanisms underlying these resistances. In previous experiments
, we observed expression of a large behavioral repertoire during acute hyperthermia and hypoxia stress suggesting that behavior may be linked to resistances .
In the present study , we seek to understand whether resistance to acute hyperthermia and hypoxia could be linked to behavior and discuss the possibility of using some behavior traits as a novel way of phenotyping these resistances. For this purpose , we used the rainbow trout isogenic lines, previously shown to exhibit different levels of resistance to either acute hyperthermia or hypoxia
Materials and methods
Six heterozygous rainbow trout isogenic lines were produced at the INRAE PEIMA experimental fish farm (doi : 10.15454/1.5572329612068406E12, Sizun , France). At 177 days post fecundation (dpf), 6 fish per isogenic line were randomly chosen and subjected to an individual challenge and then gathered in a group challenge under moderate heat stress. In individual challenge, fish were placed in 12-l aquariums backlighted by an infrared device. The aquarium was divided into two zones: on one side, a gated zone covered by an opaque plate (safe zone) and on the other side, an uncovered zone (risk zone). After a 5-min acclimation in the safe the zone, the gate was open and behavior recorded over 25 minutes. Immediately after the end of the individual challenge, the six fish were transferred in a 75 x 75 cm tank for group challenge. Temperature was gradually increased, from 12°C to 23°C in two hours and kept at 23°C for 0.5 hour. At the end of the challenge, fish were anesthetized, weighed and euthanized. Both tests were repeated three times for each line. Fish activity was video recorded and later analyzed with EthoVision XT15.0 software. A total of seven traits were measured in individual challenge: the maximum acceleration (ACC_MAX, cm.s-2 ), the distance travelled (DIST_TRAV, cm), the zone-change frequency (FRQ_CHAN, #.min-1), the absolute meander (MEANDER, deg.cm-1 ), the moving duration (MOV%, % of the time), the risk-zone duration (RISK%, % of the time) and the maximum velocity (VEL_MAX, cm.s-1). In group challenge, 7 traits were measured : ACC_MAX, the average inter-fish distance (DISPERSION, cm), DIST_TRAV, the body contact frequency (FRQ_CONTACT, #.min-1), MEANDER, MOV, and VEL_MAX. A linear mixed model was fitted for each variable, with replica as random effect , time bins and line as fixed effects, body weight as covariate, and the interactions between time bins, line and body weight.
At 182 dpf, isogenic lines were phenotyped for acute hyperthermia and hypoxia resistances with a robust experimental design constituted of 150 fish per line and type of stress. The effect of isogenic line on acute hyperthermia and hypoxia resistances were analyzed by fitting a linear mixed model with replica as a random effect, line as a fixed effect, body weight as a covariate, and the interaction between line and body weight as explained in Lagarde et al. (2023).
Different individuals were phenotyped for resistance to acute hyperthermia and hypoxia and behavioral traits. Nevertheless, within each isogenic line, fish had the same genotype and could be considered as repetitions of the same genotype. Behavior traits for which least squared means of isogenic lines on behavioral traits were highly correlated with least squared means of isogenic lines on resistance phenotypes were considered as potentially genetically linked with resistance phenotypes and therefore discussed.
Results and discussion
There were significant differences between isogenic lines for most behavior variables. Some behaviors were found to be highly correlated with acute hyperthermia and hypoxia resistances (Figure 1). In general, lines with a higher activity level were found to be more sensitive to acute hyperthermia and more resistant to acute hypoxia than lines with a lower activity level . It suggests that some behavior variables could be used as proxies for acute hyperthermia and hypoxia resistances in fish , leading to more ethical phenotyping methods t han classical resistances phenotyping ones.
These results must be confirmed in additional studies as only 6 isogenic lines were used.
This study was supported by the European Maritime and Fisheries Fund and FranceAgrimer (Hypotemp project, n° P FEA470019FA1000016).
Lagarde, H., et al., 2022. Genetic architecture of acute hyperthermia resistance in juvenile rainbow trout and genetic correlations with production traits. bioRxiv .
Lagarde, H., et al., 2023. Are resistances to acute hyperthermia or hypoxia stress similar and consistent between early and late ages in rainbow trout using isogenic lines? Aquaculture 562, 738800.
Perry, G.M.L., et al., 2005. Genetic parameters for upper thermal tolerance and growth-related traits in rainbow trout (Oncorhynchus mykiss). Aquaculture 250, 120–128.
Prchal, M., D’Ambrosio , J., et al., 2023. Genome-wide association study and genomic prediction of tolerance to acute hypoxia in rainbow trout. Aquaculture 565, 739068.