In preparation
Sébastien Alfonso1,2, Manon Peyrafort1, Xavier Cousin2,3, Marie-Laure Bégout1
1
Ifremer, Laboratoire Ressources Halieutiques, Place Gaby Coll, F-17137 L'Houmeau, France.
2
Ifremer, UMR MARBEC, Laboratoire Adaptation et Adaptabilités des Animaux et des Systèmes, Route de Maguelone, F-34250 Palavas- les-Flots, France.
3
INRA, UMR GABI, INRA, AgroParisTech, Domaine de Vilvert, Bâtiment 231, F-78350 Jouy-en-Josas, France.
Abstract
Coping style is defined as a set of individual physiological and behavioural differences consistent across time and context. In zebrafish, as well as in many other animals, several covariations were established between behavioural, physiological and molecular responses. There is, however, a lack of study addressing consistency across time starting at larval stage onwards. The aim of our study was to bring a better understanding of behavioural consistency across context and time in zebrafish from larval to juvenile stages. Fish boldness was determined at three ages (8, 21 and 60 days post fertilization, dpf) using risk taking test to identify bold (proactive) and shy (reactive) individuals. A second test, either open field test or novel tank challenge, was then perfor med to evaluate consistency across context and to define a behavioural syndrome. Thereafter, groups of fish (either bo ld or shy) were bathed in alizarin red S solution for later identification. Fish were challenged again at the following age to evaluate behavioural consistency across time. Our results showed behavioural consistency across context with bold fish being more active and expressing higher thigmotaxis whatever the age. A lack of behavioural consistency was however observed across time between larval (8 dpf) and juvenile stage (60 dpf) suggesting behavioural plasticity.
1. Introduction
Coping style is defined as a coherent set of individual physiological and behavioural traits consistent across time and context, i.e. in different times and situations (Koolhaas et al., 1999). The existence of coping styles was demonstrated in many animal species, including fish, and is described as a continuum between two extremes phenotypes which can be called proactive and reactive (Castanheira et al., 2017; Ferrari et al., 2015; Øverli et al., 2007; Tudorache et al., 2013). Temperament (Réale et al., 2007), personality (Gostling, 2001; Réale et al., 2010) or behavioural syndrome (Sih et al., 2004) are terminology related to coping s tyle as defined Koolhaas et al. (1999). In fish, proactive individuals are usually bolder (Huntingford et al., 2010; Øverli et al., 2006), more aggressive (Castanheira et al., 2013; Øverli et al., 2004), more active (Øverli et al., 2002) and e xplore their environment faster than reactive ones (Ferrari et al., 2015; Millot et al., 2009).
Behavioural consistency across time and context was shown in adult fish including zebrafish (Rey et al., 2013; Toms and Echevarria, 2014). As examples, boldness, aggression, fear and exploration, measured in 5 distinct contexts, were consistent across contexts and within a period of 7 days (Toms and Echevarria, 2014). Rey et al. (2013) also showed that boldness, measured in risk-taking test, was pos itively correlated with aggressive behaviour and strongly consistent acros s 10 months in adults. In most cases, however, includ ing those ment ioned above, coping styles were studied in juveniles or adults and consistency across time evaluated through repeated assays, generally after a short time interval but only within a single stage. Nevertheless, coping styles were also monitored at other stages but only occasionally at early stages, i.e. larval stages in fish. In zebrafish 8 days post fertilization (dpf) larvae, Tudorache et al. (2015) showed that swimming behaviour was different depe nding on emergence order from a sheltered compartment. Early emerging larvae expressed lower velocity and travelled less distance than later emerging ones. Moreover, early emerging larvae expressed higher cortisol level in response to netting stress but reached baseline level faster than late emerging ones which is consistent with a proactive coping style. Pasquet et al. (2016) de monstrated that Northern pike (Esox lucius) larvae exhibited consistency in be havioural responses across context in maze and novel object tests. More active larvae visited a higher number of zones in the maze and spe nt more time near the novel ob ject than others which indicated that coping styles were present and consistent across context. Such cross context cons istency is
Ontogenic studies evaluating coping style consistency across stages are even scarcer. Coping style consistency between early (likely larval) and later stages was evaluated in mangrove killifish and nine-spine sticklebacks and authors concluded to a lack of consistency (Edenbrow and Croft, 2013; Herczeg et al., 2013). In both studies, however, individuals were kept isolated to allow for individual ide ntification. This rearing method can introd uce several biases such as divergence in phys ico-chemical variables, unintentional differences in feeding (especially with such small individuals). In addition, in some fish species, social isolation was shown to ind uce stress (e.g. in zebrafish, Pagnussat et al. (2013)).
An option to circumvent these limitations is to use batch marking of larvae. Indeed, this method allows a common garden rearing of fish characterized for their coping style. Such marking can be accomplished using vital staining. Alizarin red S (C14H7NaO7S), is a fluorescent dye that can label calcium-containing tissue and therefore has been used to monitor skeletal mineralization in vivo in zebrafish (Bensimon-Brito et al., 2016; DeLaurier et al., 2010; Tu and Johnson, 2011). Recently, the use of alizarin red S was developed for mass marking at early stage in several fish species showing no particular mortality or growth delay (Caraguel et al., 2015; Lu et al., 2015; Stanczak et al., 2015). Larvae mass marking procedure can be performed by ingestion of Artemia salina previously bathed into alizarin red S solution (Stanczak et al., 2015) or directly by bathing larvae into alizarin red S solution (Caraguel et al., 2015; Lu et al., 2015). Therefore, alizarin red S seems to be a promising non- invasive tool to mark larvae and monitor behavioural consistency in zebrafish across life stages. Such information is indeed essential to understand how development and/or environment may shape behaviour and coping style.
With the aim of bringing new knowledge which could benefit zebrafish welfare in hus ba ndr y, characterise strains or explain variability observed within a strain (Castanheira et al., 2017; Réale et al., 2007; van den Bos et al., 2017), objectives of this study were first to monitor behavioural consistency o f coping styles across context in zebrafish at larval (8 or 21 dpf) and juve nile stage (60 dpf). For this purpose, fish were screened as proactive or reactive using risk-taking test and thereafter challenged in open field test or novel tank diving test at these three ages. Second, behavioural consistency of coping styles across time was monitored by comparing fish behavioural responses between ages (i.e., between 8 and 21, and between 21 and 60 dpf) using alizarin red S bath marking for group ide nt ification.
2. Material and methods
This study was conducted under the approval of the Animal Care Committee of Poitou-138 Charentes # 84 COMETHEA (France) under project authorization number CE2012-23 and followed the recommendations of Directive 2010 /63/EU.
2.1. Fish rearing
The study was conducted o n zebrafish TU strain (ZFIN ID: ZDBGENO-990623-3) kept at the Fish Ecophysiology Platform (PEP - http://wwz.ifremer.fr/pep_eng) and originated from the European Zebrafish Resources Center (EZRC, Karlsruhe, Germany).
To provide larvae for behavioural experiments, eggs were obtained by random pairwise mating using adults from our stock at around 180 dpf. Briefly, one male and one female were placed together in the evening before eggs were required in spawning boxes (AquaSchwarz, Germany). Eggs were collected in the following morning and the fertilization rate was assessed within 2 h of collection. Spawns above 80% fertilization rate were kept for experiments and placed in Petri dishes filled with 30 mL of the isotonic medium E3 (1 L: 17.2 g NaCl, 0.76 g HCl, 2.9 g CaCl2. 2 H2O, 4.9 MgSO4.7 H2O) and methylene blue (250 µL.L-1) and incubated at 28 °C under a 14h/10h dark/light cycle. The day after, 10 eggs from 6 different spawns were mixed, in order to avoid spawn-effects, to provide 60 embryos for one Petri dish. Multiple Petri dishes were prepared depending on the number of replicates needed. At 72 hours post fertilization (hpf), after hatching, chorions were removed from the Petri dishes, and larvae were returned to the incubator until 5 dpf. They were then transferred from Petri dishes to 1 L tanks. Initial behavioural tests and alizarin red S marking were performed at 8 and 21 dpf. After these first characterization and marking steps, larvae from both coping styles were kept in common garden (see below for details). At 8 dpf, larvae were reared in 1L tank (24 larvae per tank, n=12 per coping style), at 21 dpf, larvae were reared in 10 L tanks in the rearing system (20 larvae per tank, n=10 per coping style), in both case under a 14h/10h dark/light cycle. Physical water conditions were maintained constant during the experiment: temperature at 27±1°C, conductivity 300±50 ȝ6.cm-1
and pH 7.5±0.5 using a mixture of reverse osmosis water and tap water, both being initially treated with charcoal and sediments filters. Ammonia, nitrites, and nitrates were monitored weekly and remained within recommended ranges (Lawrence, 2007). Fish were fed three times a day, i.e. morning and
sequentially with SDS 100 µm, 200 µm, and 300 µm (Special Diet Service; Dietex international).
2.2. Experime ntal procedure to monitor behavioural consistency across context and time
Two experiments were performed covering larval stage (Larval experiment) and a juvenile stage (Juvenile experiment) and the succession of steps is described Figure 1. In both cases, the first step was a risk-taking test performed in groups to measure bo ldness and thus determine coping style after which individuals were kept isolated overnight. This was performed at 8 dpf (Larval experiment) and at 21 dpf (Juvenile experiment) after which individuals were kept isolated overnight. The day after, a second test was performed to evaluate coping style consistency across context. Thereafter fish were stained using alizarin red S and fish of both coping styles further reared in common garden to avoid possible rearing bias. Later, at 21 dp f for larval experiment and 60 dp f for juvenile experiment, fish were tested again to evaluate coping style consistency across time at population level and alizarin red S was read to obtain initial coping style. The whole expe riment was repeated (see details below) with marking of either bold or shy individuals in order to take possible effect of marking into account.
Figure 1. Graphical protocol used to monitor behavioural consistency across context and time
in fish larval (Larval experiment) and juvenile (Juvenile experiment) stages.
c
Group risk-taking test for coping style screening;d
Individual behavioural characterization using either open field test (at 8 and 21 dpf) or novel tank diving test (at 60 dpf);e
Marking using alizarin red S;f
Lecture of alizarin red S staining.2.3. Behavioural experime nts
For larval behavioural experiments (8 or 21 dpf), videos were recorded using DanioVision (Noldus, The Netherlands). For juvenile behavioural experiments, videos were recorded with an analogue camera ICD-48E (Ikegami) and a 2.1-13.5 lens (Fujinon) linked to a computer
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