The aim of this study was to thoroughly characterize the impact of high temperature exposure of female rainbow trout ( Oncorhynchus mykiss ) during the reproductive season on offspring emotional and cognitive phenotypes, using specific behavioural tests previously validated in the laboratory ( Colson et al., 2015a ; Poisson et al., 2017 ; Sadoul et al., 2016 ). We also aimed at deciphering the molecular mechanisms mediating such intergenerational effects by analysing genome-wide gene expression in eggs and developing embryos following maternal exposure to high temperature.

Several studies have, however, shown that maternal history can impact offspring behaviour and phenotypic plasticity (i.e., ability of an organism to change its morphology, physiology, or behaviour according to stressful environmental conditions ( Bijlsma & Loeschcke, 2005 ). This intergenerational effect on offspring behaviour was observed in salmonid fish in which stress during reproductive season, or at least artificial exposure to stress hormones, has a significant intergenerational impact on offspring phenotypic plasticity, including modifications of cognitive abilities ( Sloman, 2010 ) and emotional reactivity ( Colson et al., 2015b ; Eriksen et al., 2011 ; Espmark et al., 2008 ). In contrast, the underlying mechanisms mediating these effects remain poorly documented. In mammals, profound long lasting behavioural deficits have been observed in mice originating from stressed mothers, possibly due to epigenetic modifications occurring in the mother and transmitted to offspring ( Weiss et al., 2011 ). In fish, a recent study has demonstrated the existence of the programming of stress axis function in zebrafish ( Danio rerio ) offspring by maternal social status ( Jeffrey & Gilmour, 2016 ). Another study showed that three-spined stickleback ( Gasterosteus aculeatus ) embryos respond to maternal exposure to predation risk via changes in gene expression ( Mommer & Bell, 2014 ). A better understanding of underlying mechanisms is, however, necessary to fill the gap between maternal history and offspring phenotypic plasticity. Identifying molecular players mediating intergenerational effects will contribute to identifying the mechanisms underlying the neural changes leading to a change in fear response and learning capacity.

Material and Methods

Ethics statement Fish were reared in INRA LPGP facilities, which hold full approval for animal experimentation (C35-238-6). All fish were reared and handled in strict accordance with French and European policies and guidelines of the INRA LPGP Institutional Animal Care and Use Committee, which specifically approved this study (no. T-2016-55-VC-CV).

Maternal treatment and fertilization Two-year old female rainbow trout were exposed to either 12 °C (12 °C group, standard reproduction conditions) or 17 °C (17 ° C group, high suboptimal temperature) for six weeks before ovulation (Experimental schedule is given in Fig. 1). The temperature of 17 °C was selected because it is known to induce a dramatic decrease in embryonic survival (Aegerter & Jalabert, 2004) and to correspond to actual aquaculture situations, especially in photoperiod-induced out of season spawning during summer. For each group, 30 marked (external tag placed on the dorsal fin) females were kept in 2.5 m3 tanks (2 × 2 × 0.62 m, length × width × water height). In the 17 °C group, females initially reared at 12 °C were acclimated for five days to an increase of 1 °C/day until 17 °C. For three weeks before ovulation, females were checked every two-three days to detect ovulation, by applying gentle pressure to the abdomen under anaesthesia (females transferred to a 100-l tank containing water circuit + 100 ml tricaïne + 100 ml bicarbonate of sodium). In both experimental group, eggs originating from four simultaneously ovulating females of each group were collected and fertilized using a pool of sperm collected from four males held at 12 °C. The use of males held under optimal conditions, including normal temperature is standardly used in gamete biology studies in fish and other vertebrates (Cabrita, Robles & Herráez, 2009). Fertilization was performed immediately in both groups in order to avoid any bias on subsequent behavioural phenotypes that would have been induced by differences during embryo development. For each female, fertilization of 800 eggs was performed at 10 °C using sperm extender ActiFish medium (IMV, L’Aigle, France; 100 ml ActiFish + 400 ml water) and fertilized eggs were distributed within a tray (20 × 50 cm) in two incubators (10 ×10 cm) (approximately 400 eggs/incubator and two incubators/tray) supplied with 10 °C flow-though recycled water. Each tray was covered with a lid to avoid exposure to light. Figure 1: Experimental schedule.

Monitoring of developmental success Developmental success was monitored at eyeing stage, i.e., 19 days post-fertilization (dpf), hatching (32–33 dpf), and completion of yolk-sac resorption (YSR, 55 dpf) by counting dead embryos that were removed from incubators (Fig. 1). The occurrence of malformations was obtained by taking a picture of euthanized malformed fry in each incubator at YSR. The types of malformed fry observed in this study were: torsion (T), yolk sac resorption defects (YSD) and other malformations (O) as described in (Bonnet, Fostier & Bobe, 2007a). For each female, the occurrence of each type of malformations was calculated in comparison to the total number of malformed fry. Percentages of mortalities and malformations per incubator were obtained by counting the final number of live fry at swim-up stage, before transfer into rearing tanks.

Sample collection during embryo development In both experimental groups, and in all egg clutches, biological samples were collected at four different stages (Fig. 1): unfertilized eggs (0 dpf), following zygotic genome activation (Nagler, 2000) (5 dpf), hatching after removing yolk-sac (32–33 dpf), and YSR (55 dpf), which also corresponded to the stage of behavioural phenotyping. Entire (i.e., whole body) embryos were sampled. All samples were frozen in liquid nitrogen and held at −80 °C until further processing.

Fry rearing After yolk-sac resorption, at 55 dpf (Fig. 1), swim-up fry originating from the two incubators of each female were combined and transferred to seven distinct tanks (50 × 60 × 28 cm) (approximately 200 fry/84 L), corresponding to seven different females (four from the 12 °C group and three from the 17 °C group). The mortality rate of one of the 17 °C female was 98.4% and we did not obtain enough offspring to perform behavioural phenotyping. For this female, we however sampled remaining fry to perform transcriptome analyses. Water temperature was maintained at 12 °C. Fish were fed manually four times a day on a commercial diet (Biomar, 48% protein and 22% lipid, 0.5 mm diameter pellets). Tanks were automatically illuminated from 8:00 to 20:00. Before each behavioural test, fish were starved for 24 h. At the end of each test, fish were netted and transferred into individual bowls containing 250 ml of the tank water to which a lethal dose of anaesthetic (tricaïne: 4.5 ml + bicarbonate of sodium: 5 ml) had been added.

Phenotyping of offspring behaviour For each female (three 17 °C females and four 12 °C females), different fry were subjected to the following behavioural tests thoroughly described in Poisson et al. (2017).

Assessment of offspring emotional reactivity Fish propensity to express fear-related behaviour (e.g., emotional reactivity) was evaluated individually in a novel-tank test (social isolation in a novel tank) at 75–76 dpf (Fig. 1). The novel tank (30 × 19 × 16 cm) was supplied with 12 °C flow-though recycled water. Fifteen fish per female, randomly netted either at the top or the bottom of the water column, were observed. The treatment order was randomly chosen. Behavioural responses were video-recorded for 30 min. We analysed the first (0–5 min) and the last (25–30 min) 5-min intervals with EthovisionXT software (Noldus, Netherland). The following behavioural parameters were calculated for each individual: total distance travelled (cm), maximum swimming velocity (cm/s), angular velocity (° /s) (i.e., erratic swim), and time spent (%) in the border zone (i.e., thigmotaxis) corresponding to the mean length of all fish tested (3.51 ± 0.03 cm). At the end of the test, the body weight (W) and length (L) were measured. For each fish, the condition-factor was calculated as followed: K-factor = 100 (W/L33).

Assessment of offspring spatial learning abilities Between 111 and 135 dpf (Fig. 1), offspring propensity to locate a food-rewarded arm was assessed in a T-maze supplied with 12 °C flow-though recycled water (see Poisson et al., 2017) for a complete setup description). Five randomly netted fish per female were tested. After 24 h of food deprivation and 30 min of acclimation in the start-box of the T-maze, a remote-controlled guillotine door was pulled-up and fish position in the T-maze was video-recorded. A visual cue (black cross) was located on the wall of the T-maze at the entrance of the reward arm. When the fish crossed an invisible line separating the rewarded arm from the rest of the maze, a mechanic ridge, remotely-controlled by an experimenter observing live videos in an adjacent control room, released pellets. Then the fish was left to eat the pellets for at least 5 min before being gently netted and introduced in its individual holding tank until the next trial. Eleven successive trials were run for four consecutive days (two trials on the first day and three on the other days). The treatment order was randomly chosen on the first day. We measured the latency to leave the start-box (Latency SB), the latency to reach the reward arm after the fish had left the start-box (e.g., right choice, Latency RC) and the ability of the fish to make the right choice (i.e., to choose the rewarded arm first after leaving the start-box). We counted the number of fish making the right choice first in less than 900 s in at least four out of the last seven trials (57% correct choices).

Gene expression profiling Transcriptome analysis was conducted using four egg batches originating from females held at 12 °C and four egg batches originating from females held at 17 °C, with the exception of YSR/12 °C for which only three RNA samples of sufficient quality could be obtained. RNA was extracted from 20 eggs sampled at fertilization, 20 eggs at 5 dpf, six embryos at hatching and six fry sampled at YSR. Each sample was collected randomly (either at the border or the center of the incubator) or randomly when hatched fish were swimming. Frozen tissues were lysed with a Precellys Evolution Homogenizer (Ozyme, Bertin Technologies) in TRI Reagent (TR118; Euromedex) and total RNA was extracted according to the manufacturer procedure and followed by Nucleospin RNA isolation kit (740955; Macherey Nagel). Gene expression profiling was conducted using an Agilent 8 × 60 K microarray (GPL24910) as previously described (Zarski et al., 2017). Samples were randomly distributed on the microarray for hybridization. The data were processed with GeneSpring software (Agilent v.14.5) using gMedianSignal values. After data processing, one sample from the hatching/17 °C group, which behaved differently from other samples, even after normalization, was removed from subsequent analysis. Corresponding data were deposited in Gene Expression Omnibus (GEO) database under the reference GSE113377.