BEHAVIORAL COMPARISON AMONG WILD AND DOMESTICATED ZEBRAFISH STRAINS: EFFECTS OF DOMESTICATION

Emmanouil Diakos*, Alain Pasquet, Antoine Hardy, Céline Chevalier, Pascal Fontaine, Thomas Lecocq

University of Lorraine, INRAE, UR AFPA, Nancy, France

Email: Thomas.lecocq@univ-lorraine.fr

 

 

Introduction

Domestication is the process by which a species bred in a captive environment, and modified across successive generations from its wild ancestors, becomes more useful to humans who control increasingly its reproduction and food supply. Domestication has been described in five levels, as the acclimatization of wild fish to human facilities (level 1), the completion of the life cycle in captivity partially (level 2), then completely with wild outputs (level 3) or without wild outputs (level 4), and, finally, to selection programs to meet specific breeding goals (level 5) (Teletchea and Fontaine, 2012). Among traits that change under a domestication process, behaviors as phenotypes are the most directly influenced (Pasquet, 2018). However, few studies have investigated the behavioural differences between wild fish at early domestication (i.e., levels 1 & 2) and fish bred for several generations in captivity (levels 3 and above), despite the fact that such changes can occur very rapidly in the domestication process (Huntingford, 2004). Using the mirror-biting test, we compared behavioral traits involved in activity, aggression and stress, between wild caught zebrafish acclimatized to captivity (F0), their offspring (F1) reared in captivity, and three laboratory wild-type strains (AB, TU, and WIK).

 

Materials and methods

We tested 174 zebrafish in the mirror biting test under 5 different groups considering the different levels of their domestication: F0 (level 1), F1 (level 4), AB, TU, and WIK (all laboratory strains were considered at level 5). The F0 were wild fish collected from Bangladesh and acclimatized to captive conditions from June 2022. The F1 was the first generation produced by the F0 in captive condition in March 2023. The individuals were housed in 3.5 L tanks part of a recirculated water standalone rack (Tecniplast) under conditions that met the physico-chemical requirements of the species.

An individual 18h acclimatization in the experimental tank with all sides covered occurred before the beginning of each test. All tracking was performed with the R- package trackR (Garnier 2022) in RStudio (R Core Team 2022; version 4.2.1). The tracking was conducted for each individual, when the mirror cover was removed and lasted one hour generating 90,000 frames. Behavioural analysis was done using RStudio (R Core Team 2022; version 4.2.1), by coding an optimized script to obtain behavioural results from the data table produced by trackR after tracking. The most relevant behaviors for the mirror-biting test were based on definitions used in the literature (see (Audira et al., 2018; Kalueff et al., 2013)). All statistical analyses and formatting of results were carried out using RStudio. First, correlation between traits were assessed (i.e. Pearson’s correlation) and for highly correlated traits, one of the traits was randomly chosen. Second, a principal component analysis (PCA) was done to assess divergences between the groups tested. Third, the potential divergences between the five groups of zebrafish were further assessed by a global multi-response permutation procedures (MRPP) using R-package vegan (Oksanen et al., 2020) and 10000 permutations. In parallel, we also tested a potential divergence between sexes and the two AB age classes with a MRPP. As these results were not statistically significant (p-value = 0.56 and 0.62, respectively), we no longer distinguished between the sexes and ages in the analyses that followed. Fourth, when the global MRPP was significant (e.g. p-value < 0.05), we performed a pairwise MRPP between each pair of groups and applied a Bonferroni correction. Fifth, an indicator value (IndVal) (Dufrêne and Legendre, 1997) R-package labdsv (Roberts, 2023) was performed to determine which behaviors were most expressed by each of the five groups of fish. Finally, we investigated the intragroup variability by calculating the distance between individuals and the centroid of each group in PCA space. These distances were then compared between groups using a global Kruskal-Wallis test and followed, if the test result was statistically significant, i.e. p-value <0.05) by a Dunn's test using the R-package rstatix (Kassambara, 2023).

 

Results

The three first axes of the PCA were taken into consideration and they represented 27.8% of the variance (axis 1), 21.3% (axis 2) and 14.8% (axis 3) (Figure). The behaviors contributing to the maximum of variance on the three axes differed. The first axis represented activity, opposing immobility to travelled distance. The second axis represented aggressiveness opposing presence in the contact zone, and aggression to the mirror and presence in zones farther from the mirror. The third axis was more linked to stress opposing thigmotaxis and acceleration to the presence in zones near the mirror. The different strains and generation differed relatively to the PCA analysis. The overall MRPP was significant (A=0.0963, p<0.01).  AB and ABw differed from all the other strains and F1. Wild (F0) differed from F1. According to IndVal, the F0 were characterized by periods of immobility and aggressiveness to the mirror. The F1 displayed more erratic behaviors to the mirror alternating aggressiveness (U-turns, aggressive acceleration) and stress (thigmotaxis). AB and ABw were more characterized by activity, and exploration of the different zones. The Dunn’s test showed intra-strains variance with AB showing less variability than Wild and F1 (Dunn test: z=4.81 p<0.0005, z=6.2, p=0.0006 respectively).

 

Discussion

The results showed behavioural differences between the two generations of wild zebrafish (F0 and F1), while AB differed from TU and WIK, although they are supposed to be at the same level of domestication (i.e., 5). Lower aggressiveness in F1 agrees with the domestication increasing docility scenario (Wilkins et al., 2014). Activity, stress and aggressiveness have been linked to the domestication of organisms as modifications from their wild counterparts (Milla et al., 2021). This divergence from the wild during the domestication process is due to the increasing control of humans over a species life cycle, along with the decreasing gene flow from the wild in successive generations (Teletchea and Fontaine, 2012). Behavioral traits have been described as the first to be affected by this process in fish (Huntingford, 2004; Pasquet, 2018) as well as in mammals (Sánchez-Villagra et al., 2016) with main changes involving increased docility (Lu et al., 2022), reduction in avoidance behavior (Álvarez and Nicieza, 2003), changes in risk-taking behavior and in general decreased stress response in the rearing environment (Milla et al., 2021; Pasquet, 2018).

 

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