Marie-Laure Bégout

Systematic Screening of Behavioral Responses in Two Zebrafish Strains

Wild-type (WT) zebrafish are commonly used in behavioral tests, but the term WT is not a precise description, and corresponds to many different strains (e.g., AB, TU, WIK, and others). Previous studies compared the physiological, behavioral, or metabolic characteristics of different zebrafish strains (indigenous WT populations versus laboratory WT strains). AB and TU are widely used, but at least one study has demonstrated behavioral differences between them. To choose the most appropriate strain for our experiments, we systematically screened behavioral responses of AB and TU fish in several assays. We analyzed the locomotion activity and responses to a light/dark challenge in adults and larvae, and exploratory behavior and color conditioning in adults. Differences were observed for all tests, the strains displaying particular behavior depending on the tests. As larvae, TU displayed a wider activity range than AB larvae at the onset of locomotor behavior; as adults, TU were more reactive to sudden light transitions and recovered the swimming activity faster in T-maze or homebase release in novel tank tests, whereas AB fish had more contrasted circadian rhythms and performed better in color learning. Strain-specific behavior should be considered when designing experiments using behavior.

De novo assembly, characterization and functional annotation of Senegalese sole (Solea senegalensis) and common sole (Solea solea) transcriptomes: integration in a database and design of a microarray

BackgroundSenegalese sole (Solea senegalensis) and common sole (S. solea) are two economically and evolutionary important flatfish species both in fisheries and aquaculture. Although some genomic resources and tools were recently described in these species, further sequencing efforts are required to establish a complete transcriptome, and to identify new molecular markers. Moreover, the comparative analysis of transcriptomes will be useful to understand flatfish evolution.ResultsA comprehensive characterization of the transcriptome for each species was carried out using a large set of Illumina data (more than 1,800 millions reads for each sole species) and 454 reads (more than 5 millions reads only in S. senegalensis), providing coverages ranging from 1,384x to 2,543x. After a de novo assembly, 45,063 and 38,402 different transcripts were obtained, comprising 18,738 and 22,683 full-length cDNAs in S. senegalensis and S. solea, respectively. A reference transcriptome with the longest unique transcripts and putative non-redundant new transcripts was established for each species. A subset of 11,953 reference transcripts was qualified as highly reliable orthologs (>97% identity) between both species. A small subset of putative species-specific, lineage-specific and flatfish-specific transcripts were also identified. Furthermore, transcriptome data permitted the identification of single nucleotide polymorphisms and simple-sequence repeats confirmed by FISH to be used in further genetic and expression studies. Moreover, evidences on the retention of crystallins crybb1, crybb1-like and crybb3 in the two species of soles are also presented. Transcriptome information was applied to the design of a microarray tool in S. senegalensis that was successfully tested and validated by qPCR. Finally, transcriptomic data were hosted and structured at SoleaDB.ConclusionsTranscriptomes and molecular markers identified in this study represent a valuable source for future genomic studies in these economically important species. Orthology analysis provided new clues regarding sole genome evolution indicating a divergent evolution of crystallins in flatfish. The design of a microarray and establishment of a reference transcriptome will be useful for large-scale gene expression studies. Moreover, the integration of transcriptomic data in the SoleaDB will facilitate the management of genomic information in these important species.

Risk-taking behaviour variation over time in sea bass Dicentrarchus labrax: effects of day-night alternation, fish phenotypic characteristics and selection for growth.

Differences in bold and shy personality on sea bass Dicentrarchus labrax were investigated between a population (wild) produced from wild-brood fish and a population (selected) produced from selected-brood fish. During the experiment (112 days), fish were reared under self-feeding condition to characterize the feeding behaviour of each individual fish. Three risk-taking tests (T1, T2 and T3 of 24 h with day-night alternation) were carried out at >1 month intervals on 180 fish of each strain in order to monitor D. labrax behaviour over time and in relation to the light:dark period. A risk-taking score was evaluated via a preference choice between a safe zone (without food) and a risky zone (potentially with food) by recording the number and the duration of individual passages through an opening in an opaque divider. Results showed that fish performed passages preferentially during the night period and that wild fish were generally bolder than selected fish during T1 and T2 but showed a decrease in risk taking during T3, contrary to selected fish which showed a constant increase in their risk-taking behaviour. The phenotypic characteristics of the bold fish were different in the two strains: wild bold fish were the smallest within the wild strain and selected bold fish presented the higher growth rate within the selected strain. For both strains, these bold fish were also generally characterized by a high feed-demand activity. Fish hunger state thus seemed to be the highest motivation for risk-taking behaviour under the present conditions. Furthermore, behavioural variations over tests such as higher risk taking (number of passages) and faster exploratory responses (higher score emergence) could be interpreted as relevant indicators of the learning process and habituation. According to the results, however, no real difference in coping strategy between strains could be observed at this first stage of domestication and selection.

Demand feeding and welfare in farmed fish

Following the development of demand-feeding systems, many experiments have been conducted to explore feeding motivation and feed intake in farmed fish. This work aims to review a selection of studies in the field, focusing on three key factors, related to demand feeding and fish welfare. Firstly, we outline how demand feeders should be considered when developing feed management strategies for improving welfare in production conditions. Secondly, via laboratory demand-feeding experiments, we show self-feeding activities depend not only on feeding motivation and social organisation, but also on individual learning capacity and risk-taking behaviour. Thirdly, we report encouraging results demonstrating that when presented with two or more self-feeders containing complementary foods, fish select a diet according to their specific nutritional requirements, suggesting that demand feeders could be used to improve welfare by allowing fish to meet their nutritional needs.

Long-term dietary-exposure to non-coplanar PCBs induces behavioral disruptions in adult zebrafish and their offspring

The use of polychlorinated biphenyls (PCBs) has been banned for several decades. PCBs have a long biological half-life and high liposolubility which leads to their bioaccumulation and biomagnification through food chains over a wide range of trophic levels. Exposure can lead to changes in animal physiology and behavior and has been demonstrated in both experimental and field analyses. There are also potential risks to high trophic level predators, including humans. A maternal transfer has been demonstrated in fish as PCBs bind to lipids in eggs. In this study, behavioral traits (exploration and free swimming, with or without challenges) of contaminated zebrafish (Danio rerio) adults and their offspring (both as five-day-old larvae and as two-month-old fish reared under standard conditions) were measured using video-tracking. Long-term dietary exposure to a mixture of non-coplanar PCBs was used to mimic known environmental contamination levels and congener composition. Eight-week-old fish were exposed for eight months at 26-28 °C. Those exposed to an intermediate dose (equivalent to that found in the Loire Estuary, ∑(CB)=515 ng g⁻¹ dry weight in food) displayed behavioral disruption in exploration capacities. Fish exposed to the highest dose (equivalent to that found in the Seine Estuary, ∑(CB)=2302 ng g⁻¹ dry weight in food) displayed an increased swimming activity at the end of the night. In offspring, larval activity was increased and two-month-old fish occupied the bottom section of the tank less often. These findings call for more long-term experiments using the zebrafish model; the mechanisms underlying behavioral disruptions need to be understood due to their implications for both human health and their ecological relevance in terms of individual fitness and survival.

Electronic individual identification of zebrafish using radio frequency identification (RFID) microtags.

SUMMARY Although individual electronic tagging using passive integrated acoustic (PIT) tags is established, it is mainly for fish >60 mm in length and is unsuitable for fish of <30 mm, like zebrafish. We used radio frequency identification (RFID) microtags (1 mm in diameter and 6 mm in length, with a mass of ~10 mg) to individually identify juvenile zebrafish (length 16–42 mm, mass 138–776 mg) for the first time, and studied the effects of intracoelomic implantation on fish survival and microtag loss, growth, spawning and exploratory behaviour. After 5.5 months, both high survival (82%) and low microtag loss (11%) were achieved. The smallest surviving fish weighed 178 mg, and success in microtag reading was 73% for the size class 350–450 mg (26 mm). Greater success was achieved when fish were larger at the time of tagging but no negative effects on growth were observed for any size class and some tagged fish spawned. No significant differences in behavioural responses could be detected between tagged fish and untagged controls after 2 months. Overall, the results suggest that the tagging method is highly suitable for fish as small as zebrafish juveniles. We think this method will provide significant advances for researchers of the ever-growing fish model community and more generally for all small-fish users. Tagging is essential when one needs to identify fish (e.g. particular genotypes with no external cue), to run longitudinal monitoring of individual biological traits (e.g. growth) or to repeat assays with the same individual at discrete points in time (e.g. behaviour studies). Such a method will find applications in physiology, genetics, behaviour and (eco)toxicology fields.

An acoustic telemetry study of seabream (Sparus aurata L.): first results on activity rhythm, effects of environmental variables and space utilization

The movements of seabream (Sparus aurata L) were recorded in two earthen ponds (250 m2 and 400 m2) by tracking fish tagged with miniature acoustic transmitters. Five seabreams, 500 g in weight, were tracked for periods of four or five days. Fish positions were recorded continuously using an acoustic telemetry system. Each of the five tagged seabreams were tracked individually. Two fishes were released schooled with numerous other individuals, two others were released in isolation and one was grouped with three other individuals.

Exposures of zebrafish through diet to three environmentally relevant mixtures of PAHs produce behavioral disruptions in unexposed F1 and F2 descendant

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment has increased very substantially over the last decades. PAHs are hydrophobic molecules which can accumulate in high concentrations in sediments acting then as major secondary sources. Fish contamination can occur through contact or residence nearby sediments or though dietary exposure. In this study, we analyzed certain physiological traits in unexposed fish (F1) issued from parents (F0) exposed through diet to three PAH mixtures at similar and environmentally relevant concentrations but differing in their compositions. For each mixture, no morphological differences were observed between concentrations. An increase in locomotor activity was observed in larvae issued from fish exposed to the highest concentration of a pyrolytic (PY) mixture. On the contrary, a decrease in locomotor activity was observed in larvae issued from heavy oil mixture (HO). In the case of the third mixture, light oil (LO), a reduction of the diurnal activity was observed during the setup of larval activity. Behavioral disruptions persisted in F1-PY juveniles and in their offspring (F2). Endocrine disruption was analyzed using cyp19a1b:GFP transgenic line and revealed disruptions in PY and LO offspring. Since no PAH metabolites were dosed in larvae, these findings suggest possible underlying mechanisms such as altered parental signaling molecule and/or hormone transferred in the gametes, eventually leading to early imprinting. Taken together, these results indicate that physiological disruptions are observed in offspring of fish exposed to PAH mixtures through diet.

Assessment of Genetic Variability of Fish Personality Traits using Rainbow Trout Isogenic Lines

The study of inter-individual variability of personality in fish is a growing field of interest but the genetic basis of this complex trait is still poorly investigated due to the difficulty in controlling fish genetic origin and life history. When available, isogenic lines that allow performing independent tests on different individuals having identical genotype constitute a very relevant experimental material to disentangle the genetic and environmental components of behavioural individuality. We took advantage of heterozygous isogenic lines to investigate the personality in rainbow trout through the analysis of their reactions to different experimental situations. To this end, seven to ten rainbow trout isogenic lines were screened for their spatial exploratory behaviour, their flight response toward a stressor and their risk taking behaviour. Results showed that some lines seemed less sensitive to new events or environmental changes and could be defined as low responsive, while others were very sensitive and defined as high responsive. The use of isogenic lines highlighted the importance of genetic factors, in combination with life history, in the expression of personality in domesticated fish.

First Insight into Exploration and Cognition in Wild Caught and Domesticated Sea Bass (Dicentrarchus labrax) in a Maze

European sea bass aquaculture is so recent that very little is known on the effects of the early steps of its domestication. Behavioural parameters are sensitive indicators of the domestication process since they are generally impacted as soon as the first generation. The present work compared wild-caught and domesticated sea bass juvenile swimming activity, exploration and ability to learn to discriminate between two 2-D objects associated to a simple spatial task that enabled the tested individual to visually interact with an unfamiliar congener (the reward) located behind a transparent wall at the end of one of the two arms of a maze. Ten fish from each origin were individually tested 3 times in a row during 3 days (9 trials in total). Fish were placed in a start box closed by a transparent wall located in front of two 2-D objects. Fish were filmed during 10 min after the removal of the start box wall. Different swimming variables including angular velocity, total distance travelled and velocity mean, were analyzed from videos as well as the time spent in each of 6 virtual zones including the reward zone near the congener (Cong) and the zone opposite to the reward zone (OpCong). Two learning criteria were chosen: the number of successful turns and time to reach Cong. Behavioural differences were found between domesticated and wild fish. Angular velocity was higher in wild fish while the distance travelled and the velocity mean were higher in domesticated ones. Wild and domesticated fish spent most of the time in Cong and in OpCong. No differences were seen in learning ability between wild and domesticated fish. However, our findings for learning require confirmation by further studies with larger numbers of learning sessions and experiments designed to minimise stress. This study therefore demonstrated an impact of domestication on swimming behaviour but not on spatial learning.