Does a ghrelin stimulus during zebrafish embryonic stage modulate its performance on the long-term?

Metabolic programming refers to the induction, deletion, or impaired development of a somatic structure or "setting" of a physiological system by an early life stimulus operated at a critical period during development. Ghrelin is the only known orexigenic gut hormone, is an acylated peptide that acts as an endogenous ligand specific for growth-hormone secretagogue-receptor. The aim of the present work was to evaluate if an in ovo ghrelin administration could positively influence the zebrafish performance in the long-term and to gain insight on the mechanisms associated to ghrelin regulation of food intake during the larval phase. Food intake, growth potential, protein metabolism, expression of target genes involved in ghrelin, feeding behaviour regulation and locomotor activity were assessed in zebrafish (Danio rerio) larvae at 25 days post-fertilization. Elevated levels of acylated ghrelin in zebrafish eggs did not result in increased growth or food intake. Differences in mRNA expression between larvae fasted for 16 h before and 1 h after feeding were found for igf1ra, gh1 and pomca. Moreover, ghrelin treated larvae showed higher swimming activity, indicating that the peptide may have an important role on foraging activity. The present study addressed for the first time the effects of an early stimulus of ghrelin during the embryonic stage of zebrafish, however, further studies are needed to clarify the metabolic pathways affected by the early stimulus as well as focus on the effects on metabolic regulation of energy balance through lipid and carbohydrate metabolism.

Supervised machine learning outperforms taxonomy-based environmental DNA metabarcoding applied to biomonitoring.

Biodiversity monitoring is the standard for environmental impact assessment of anthropogenic activities. Several recent studies showed that high-throughput amplicon sequencing of environmental DNA (eDNA metabarcoding) could overcome many limitations of the traditional morphotaxonomy-based bioassessment. Recently, we demonstrated that supervised machine learning (SML) can be used to predict accurate biotic indices values from eDNA metabarcoding data, regardless of the taxonomic affiliation of the sequences. However, it is unknown to which extent the accuracy of such models depends on taxonomic resolution of molecular markers or how SML compares with metabarcoding approaches targeting well-established bioindicator species. In this study, we address these issues by training predictive models upon five different ribosomal bacterial and eukaryotic markers and measuring their performance to assess the environmental impact of marine aquaculture on independent data sets. Our results show that all tested markers are yielding accurate predictive models and that they all outperform the assessment relying solely on taxonomically assigned sequences. Remarkably, we did not find any significant difference in the performance of the models built using universal eukaryotic or prokaryotic markers. Using any molecular marker with a taxonomic range broad enough to comprise different potential bioindicator taxa, SML approach can overcome the limits of taxonomy-based eDNA bioassessment.

Environmental DNA metabarcoding of benthic bacterial communities indicates the benthic footprint of salmon aquaculture.

We evaluated benthic bacterial communities as bioindicators in environmental impact assessments of salmon aquaculture, a rapidly growing sector of seafood industry. Sediment samples (n=72) were collected from below salmon cages towards distant reference sites. Bacterial community profiles inferred from DNA metabarcodes were compared to reference data from standard macrofauna biomonitoring surveys of the same samples. Deltaproteobacteria were predominant in immediate vicinity of the salmon cages. Along the transect, significant shifts in bacterial community structures were observed with Gammaproteobacteria dominating the less-impacted sites. Alpha- and beta-diversity measures of bacterial communities correlated significantly with macrofauna diversity metrics and with five ecological status indices. Benthic bacterial communities mirror the reaction of macrofauna bioindicators to environmental disturbances caused by salmon farming. The implementation of bacterial eDNA metabarcoding in future Strategic Framework Directives is an alternative cost-effective high-throughput biomonitoring solution, providing a basis for management strategies in a matter of days rather than months.

Are coping styles consistent in the teleost fish Sparus aurata through sexual maturation and sex reversal?

Individual differences in behaviour and physiological responses to stress are associated with evolutionary adaptive variation and thus raw material for evolution. In farmed animals, the interest in consistent trait associations, i.e. coping styles, has increased dramatically over the last years. However, one of limitations of the available knowledge, regarding the temporal consistency, is that it refers always to short-term consistency (usually few weeks). The present study used an escape response during a net restraining test, previously shown to be an indicative of coping styles in seabream, to investigate long-term consistency of coping styles both over time and during different life history stages. Results showed both short-term (14 days) consistency and long-term (8 months) consistency of escape response. However, we did not found consistency in the same behaviour after sexual maturation when the restraining test was repeated 16, 22 and 23 months after the first test was performed. In conclusion, this study showed consistent behaviour traits in seabream when juveniles, and a loss of this behavioural traits when adults. Therefore, these results underline that adding a life story approach to data interpretation as an essential step forward towards coping styles foreground. Furthermore, a fine-tuning of aquaculture rearing strategies to adapt to different coping strategies may need to be adjusted differently at early stages of development and adults to improve the welfare of farmed fish.

Stress and fear responses in the teleost pallium.

Evolution has resulted in behavioural responses to threat which show extensive similarities between different animal species. The reaction to predator cues is one example of such prevailing responses, and functional homologies to mammalian limbic regions involved in threat-sensitive behaviour have been found in the teleost telencephalon. The dorsolateral (Dl) and dorsomedial (Dm) regions of the pallium are thought to perform hippocampus and amygdala-like functions respectively. To what degree these regions are involved in the neuroendocrine responses to stress and predator cues however remains largely unknown. In the present study the involvement of Dl and Dm in such responses was investigated by exposing Nile tilapia (Oreochromis niloticus) to a standardized confinement stress and to skin extract from conspecifics. Nile tilapia develops a characteristic anticipatory behaviour to hand feeding, and effects of skin extract on this behaviour and locomotor activity were studied to characterise threat sensitive behaviour. Nile tilapia responded behaviourally to conspecific alarm cues by reducing feeding anticipatory behaviour. This may reflect a general elevation of alertness, and further studies combining skin extract with other challenges are needed to reveal neuroendocrine effects associated with this predator cue. Confinement stress resulted in an elevation of cortisol and serotonin (5-hydroxytryptamine, 5-HT) metabolism in both Dl and Dm. A similar tendency was observed in fish exposed to chemical alarm cues, but this effect did not reach the level of statistical significance. Hence, limbic responses to stress and fear, akin to those seen in extant mammals, are also present in the teleost lineage.

Behavioural stress responses predict environmental perception in European sea bass (Dicentrarchus labrax).

Individual variation in the response to environmental challenges depends partly on innate reaction norms, partly on experience-based cognitive/emotional evaluations that individuals make of the situation. The goal of this study was to investigate whether pre-existing differences in behaviour predict the outcome of such assessment of environmental cues, using a conditioned place preference/avoidance (CPP/CPA) paradigm. A comparative vertebrate model (European sea bass, Dicentrarchus labrax) was used, and ninety juvenile individuals were initially screened for behavioural reactivity using a net restraining test. Thereafter each individual was tested in a choice tank using net chasing as aversive stimulus or exposure to familiar conspecifics as appetitive stimulus in the preferred or non preferred side respectively (called hereafter stimulation side). Locomotor behaviour (i.e. time spent, distance travelled and swimming speed in each tank side) of each individual was recorded and analysed with video software. The results showed that fish which were previously exposed to appetitive stimulus increased significantly the time spent on the stimulation side, while aversive stimulus led to a strong decrease in time spent on the stimulation side. Moreover, this study showed clearly that proactive fish were characterised by a stronger preference for the social stimulus and when placed in a putative aversive environment showed a lower physiological stress responses than reactive fish. In conclusion, this study showed for the first time in sea bass, that the CPP/CPA paradigm can be used to assess the valence (positive vs. negative) that fish attribute to different stimuli and that individual behavioural traits is predictive of how stimuli are perceived and thus of the magnitude of preference or avoidance behaviour.

Feeding motivation as a personality trait in Nile tilapia (Oreochromis niloticus): role of serotonergic neurotransmission.

Consistent individual variation in behaviour and physiology (i.e. animal personality or coping style) has emerged as a central topic in many biological disciplines. Yet, underlying mechanisms of crucial personality traits like feeding behaviour in novel environments remain unclear. Comparative studies, however, reveal a strong degree of evolutionary conservation of neural mechanisms controlling such behaviours throughout the vertebrate lineage. Previous studies have indicated duration of stress-induced anorexia as a consistent individual characteristic in teleost fishes. This study aims to determine to what degree brain 5-hydroxytryptamine (5-HT, serotonin) activity pertains to this aspect of animal personality, as a correlate to feed anticipatory behaviour and recovery of feed intake after transfer to a novel environment. Crucial to the definition of animal personality, a strong degree of individual consistency in different measures of feeding behaviour (feeding latency and feeding score), was demonstrated. Furthermore, low serotonergic activity in the hypothalamus was highly correlated with a personality characterized by high feeding motivation, with feeding motivation represented as an overall measure incorporating several behavioural parameters in a Principle Component Analyses (PCA). This study thus confirms individual variation in brain 5-HT neurotransmission as a correlate to complex behavioural syndromes related to feeding motivation.

Can we predict personality in fish? Searching for consistency over time and across contexts.

The interest in animal personality, broadly defined as consistency of individual behavioural traits over time and across contexts, has increased dramatically over the last years. Individual differences in behaviour are no longer recognised as noise around a mean but rather as adaptive variation and thus, essentially, raw material for evolution. Animal personality has been considered evolutionary conserved and has been shown to be present in all vertebrates including fish. Despite the importance of evolutionary and comparative aspects in this field, few studies have actually documented consistency across situations in fish. In addition, most studies are done with individually housed fish which may pose additional challenges when interpreting data from social species. Here, we investigate, for the first time in fish, whether individual differences in behavioural responses to a variety of challenges are consistent over time and across contexts using both individual and grouped-based tests. Twenty-four juveniles of Gilthead seabream Sparus aurata were subjected to three individual-based tests: feed intake recovery in a novel environment, novel object and restraining and to two group-based tests: risk-taking and hypoxia. Each test was repeated twice to assess consistency of behavioural responses over time. Risk taking and escape behaviours during restraining were shown to be significantly consistent over time. In addition, consistency across contexts was also observed: individuals that took longer to recover feed intake after transfer into a novel environment exhibited higher escape attempts during a restraining test and escaped faster from hypoxia conditions. These results highlight the possibility to predict behaviour in groups from individual personality traits.

Cortisol and finfish welfare.

Previous reviews of stress, and the stress hormone cortisol, in fish have focussed on physiology, due to interest in impacts on aquaculture production. Here, we discuss cortisol in relation to fish welfare. Cortisol is a readily measured component of the primary (neuroendocrine) stress response and is relevant to fish welfare as it affects physiological and brain functions and modifies behaviour. However, we argue that cortisol has little value if welfare is viewed purely from a functional (or behavioural) perspective-the cortisol response itself is a natural, adaptive response and is not predictive of coping as downstream impacts on function and behaviour are dose-, time- and context-dependent and not predictable. Nevertheless, we argue that welfare should be considered in terms of mental health and feelings, and that stress in relation to welfare should be viewed as psychological, rather than physiological. We contend that cortisol can be used (with caution) as a tractable indicator of how fish perceive (and feel about) their environment, psychological stress and feelings in fish. Cortisol responses are directly triggered by the brain and fish studies do indicate cortisol responses to psychological stressors, i.e., those with no direct physicochemical action. We discuss the practicalities of using cortisol to ask the fish themselves how they feel about husbandry practices and the culture environment. Single time point measurements of cortisol are of little value in assessing the stress level of fish as studies need to account for diurnal and seasonal variations, and environmental and genetic factors. Areas in need of greater clarity for the use of cortisol as an indicator of fish feelings are the separation of (physiological) stress from (psychological) distress, the separation of chronic stress from acclimation, and the interactions between feelings, cortisol, mood and behaviour.

Behavioural indicators of welfare in farmed fish.

Behaviour represents a reaction to the environment as fish perceive it and is therefore a key element of fish welfare. This review summarises the main findings on how behavioural changes have been used to assess welfare in farmed fish, using both functional and feeling-based approaches. Changes in foraging behaviour, ventilatory activity, aggression, individual and group swimming behaviour, stereotypic and abnormal behaviour have been linked with acute and chronic stressors in aquaculture and can therefore be regarded as likely indicators of poor welfare. On the contrary, measurements of exploratory behaviour, feed anticipatory activity and reward-related operant behaviour are beginning to be considered as indicators of positive emotions and welfare in fish. Despite the lack of scientific agreement about the existence of sentience in fish, the possibility that they are capable of both positive and negative emotions may contribute to the development of new strategies (e.g. environmental enrichment) to promote good welfare. Numerous studies that use behavioural indicators of welfare show that behavioural changes can be interpreted as either good or poor welfare depending on the fish species. It is therefore essential to understand the species-specific biology before drawing any conclusions in relation to welfare. In addition, different individuals within the same species may exhibit divergent coping strategies towards stressors, and what is tolerated by some individuals may be detrimental to others. Therefore, the assessment of welfare in a few individuals may not represent the average welfare of a group and vice versa. This underlines the need to develop on-farm, operational behavioural welfare indicators that can be easily used to assess not only the individual welfare but also the welfare of the whole group (e.g. spatial distribution). With the ongoing development of video technology and image processing, the on-farm surveillance of behaviour may in the near future represent a low-cost, noninvasive tool to assess the welfare of farmed fish.

Exploring novelty: a component trait of behavioural syndromes in a colonial fish.

Individual differences in exploratory behaviour have been shown to be consistent across contexts and suggested to be part of behavioural syndromes in a diversity of species, including fish. Exploration has also been shown to be a key factor in understanding complex ecological processes such as sexual selection and cooperation. Another important question in ecology is why animals breed in colonies. Exploration syndromes, by affecting prospecting behaviour, dispersal and public information use may also contribute to our understanding of coloniality. This study aims at investigating whether an exploration syndrome exists in a colonial fish species, Neolamprologus caudopunctatus. Individuals of this species were subjected to two consecutive tests, a novel environment and a novel object test. Results show that more explorative individuals in a novel environment are also less neophobic in the presence of a novel object, suggesting that the tendency to engage with novelty per se is a consistent trait and part of an exploration syndrome. These results are discussed in light of the contribution of an exploration syndrome to explain colony formation in animals.

Linking fearfulness and coping styles in fish.

Consistent individual differences in cognitive appraisal and emotional reactivity, including fearfulness, are important personality traits in humans, non-human mammals, and birds. Comparative studies on teleost fishes support the existence of coping styles and behavioral syndromes also in poikilothermic animals. The functionalist approach to emotions hold that emotions have evolved to ensure appropriate behavioral responses to dangerous or rewarding stimuli. Little information is however available on how evolutionary widespread these putative links between personality and the expression of emotional or affective states such as fear are. Here we disclose that individual variation in coping style predicts fear responses in Nile tilapia Oreochromis niloticus, using the principle of avoidance learning. Fish previously screened for coping style were given the possibility to escape a signalled aversive stimulus. Fearful individuals showed a range of typically reactive traits such as slow recovery of feed intake in a novel environment, neophobia, and high post-stress cortisol levels. Hence, emotional reactivity and appraisal would appear to be an essential component of animal personality in species distributed throughout the vertebrate subphylum.