Boldness and physiological variation in round goby populations along their Baltic Sea invasion front.

The round goby (Neogobius melanostomus) is a fish native to the Ponto-Caspian region that is highly invasive through freshwater and brackish habitats in northern Europe and North America. Individual behavioural variation appears to be an important factor in their spread, for example a round goby's personality traits can influence their dispersal tendency, which may also produce variation in the behavioral composition of populations at different points along their invasion fronts. To further analyze the drivers of behavioral variation within invasive round goby populations, we focused on two populations along the Baltic Sea invasion front with closely comparable physical and community characteristics. Specifically, this study measured personality within a novel environment and predator response context (i.e., boldness), and directly analyzed links between individuals' personality traits and their physiological characteristics and stress responses (i.e., blood cortisol and lactate, brain neurotransmitters). In contrast to previous findings, the more recently established population had similar activity levels but were less bold in response to a predator cue than the older population, which suggests that behavioral compositions within our study populations may be more driven by local environmental conditions rather than being a result of personality-biased dispersal. Furthermore, we found that both populations showed similar physiological stress responses, and there also appeared to be no detectable relationship between physiological parameters and behavioral responses to predator cues. Instead, body size and body condition were important factors influencing individual behavioral responses. Overall, our results reinforce the importance of boldness traits as a form of phenotypic variation in round goby populations in the Baltic Sea. We also highlight the importance of these traits for future studies specifically testing for effects of invasion processes on phenotypic variation in the species. Nonetheless, our results also highlight that the physiological mechanisms underpinning behavioural variation in these populations remain unclear.

Environmental Enrichment for Rainbow Trout Fingerlings: A Case Study Using Shelters in an Organic Trout Farm.

Physical enrichment can improve the welfare of captive fish. Previous research has shown that fish often show preference for enriched environments, which can also result in improvements in growth performance. However, effects of enrichment are not always positive and the design and extent of the enrichment needs to be carefully considered. In this regard, information in real aquaculture scenarios is limited. The aim of this study was to serve as a proof of concept to test the feasibility of using simple PVC immersed shelters as a tool for better welfare in an organic rainbow trout farm. Our shelters induced little extra work in farm routines and had no negative effects on fish performance, health or mortality. The behavioral assessment pointed to a preference for sheltered areas in undisturbed conditions. However, no benefits were observed in terms of stress responses during standardized stress tests, and fish showed no obvious shelter-seeking behavior after disturbance. The results in terms of shelter-seeking behavior were probably limited by the short duration of the experiment, which was due to the farm's routines and needs. It is recommended that strategies for enrichment in real scenarios should be tested covering a relevant part of the life cycle of the fish in captivity, to fully account for their potential to improve welfare in aquaculture.

Cohabitation With Atlantic Salmon (Salmo salar) Affects Brain Neuromodulators But Not Welfare Indicators in Lumpfish (Cyclopterus lumpus).

Lumpfish are utilized to combat ectoparasitic epidemics in salmon farming. Research gaps on both cleaning behavior and client preferences in a natural environment, emphasizes the need to investigate the physiological impacts on lumpfish during cohabitation with piscivorous Atlantic salmon. Lumpfish (39.9 g, S.D ± 8.98) were arranged in duplicate tanks (n = 40 per treatment) and exposed to Live Atlantic salmon (245.7 g, S.D ± 25.05), salmon Olfaction or lifelike salmon Models for 6 weeks. Growth and health scores were measured every second week. In addition, the final sampling included measurements of neuromodulators, body color, and plasma cortisol. A stimulation and suppression test of the hypothalamic-pituitary-interrenal (HPI) axis was used for chronic stress assessment. Results showed that growth, health scores, and body color remained unaffected by treatments. Significant reductions in levels of brain dopamine and norepinephrine were observed in Live compared to Control. Plasma cortisol was low in all treatments, while the stimulation and suppression test of the HPI axis revealed no indications of chronic stress. This study presents novel findings on the impact on neuromodulators from Atlantic salmon interaction in the lumpfish brain. We argue that the downregulation of dopamine and norepinephrine indicate plastic adjustments to cohabitation with no negative effect on the species. This is in accordance with no observed deviations in welfare measurements, including growth, health scores, body color, and stress. We conclude that exposure to salmon or salmon cues did not impact the welfare of the species in our laboratory setup, and that neuromodulators are affected by heterospecific interaction.

Parental selection for growth and early-life low stocking density increase the female-to-male ratio in European sea bass.

In European sea bass (Dicentrarchus labrax), as in many other fish species, temperature is known to influence the sex of individuals, with more males produced at relatively high temperatures. It is however unclear to what extent growth or stress are involved in such a process, since temperature is known to influence both growth rate and cortisol production. Here, we designed an experiment aiming at reducing stress and affecting early growth rate. We exposed larvae and juveniles originating from both captive and wild parents to three different treatments: low stocking density, food supplemented with tryptophan and a control. Low stocking density and tryptophan treatment respectively increased and decreased early growth rate. Each treatment influenced the stress response depending on the developmental stage, although no clear pattern regarding the whole-body cortisol concentration was found. During sex differentiation, fish in the low-density treatment exhibited lower expression of gr1, gr2, mr, and crf in the hypothalamus when compared to the control group. Fish fed tryptophan displayed lower crf in the hypothalamus and higher level of serotonin in the telencephalon compared to controls. Overall, fish kept at low density produced significantly more females than both control and fish fed tryptophan. Parents that have been selected for growth for three generations also produced significantly more females than parents of wild origin. Our findings did not allow to detect a clear effect of stress at the group level and rather point out a key role of early sexually dimorphic growth rate in sex determination.

Interactions of temperature and dietary composition on juvenile European lobster (Homarus gammarus, L.) energy metabolism and performance.

Optimal rearing temperatures for European lobster Homarus gammarus in aquaculture differ from those prevalent in their aquatic ecosystems and acclimating juveniles to the prevailing temperatures before release may aid in the success of re-stocking programs. As the dietary nutritional composition is important for optimal performance of H. gammarus, in this study we aimed to investigate whether juvenile growth and energy metabolism responses to temperature variation could be modulated by the diet. Prior to the trial start, the juveniles were divided into two groups. One was maintained at 19 °C and the other gradually adapted to 13 °C. From this point and for a 24-day period, juveniles (~ 100 mg) within each temperature group were assigned one of two experimental diets: a carbohydrate-rich (HC) or a protein-rich (HP) extruded feed. Antarctic krill (AK) was used as a control diet within each temperature group. Feed intake, growth, glycogen, glucose, lactate, and protein concentrations of H. gammarus in each group were evaluated. Regardless the dietary treatment, feed intake, cephalothorax protein and glucose, and abdominal glycogen and glucose levels decreased at colder temperature. The effect of lower temperature on growth (SGR and moulting rate declines) and energy metabolism (reduction on cephalothorax glycogen and protein) was more severe in HC-fed lobsters. Results showed that the impact of lower temperature on juvenile H. gammarus can be modulated by diet highlighting the importance of designing optimized diets not only for growth and feed efficiency but also for resilience to environmental variation.

Replacement of Antarctic krill (Euphausia superba) by extruded feeds with different proximate compositions: effects on growth, nutritional condition and digestive capacity of juvenile European lobsters (Homarus gammarus, L.).

Extruded feeds are widely used for major aquatic animal production, particularly for finfish. However, the transition from fresh/frozen to extruded/pelleted feeds remains a major obstacle to progressing sustainable farming of European lobster (Homarus gammarus). The aim of the present study was to investigate the effects of using extruded feeds with different protein levels and lipid/carbohydrate ratios on growth, feed utilisation, nucleic acid derived indices (sRD) and digestive enzymatic activity of H. gammarus juveniles. Six extruded feeds were formulated to contain two protein levels (400 and 500 g/kg), with three lipid/carbohydrate ratios (LOW - 1:3; MEDium - 1:2; HIGH - 1:1). The extruded feeds were tested against Antarctic krill (Euphausia superba) used as control (CTRL). Overall, the CTRL and 500MED feed supported the highest growth and nutritional condition estimated by means of sRD, while the poorest results were observed for the 400HIGH and 400MED groups. The FCR was significantly lower in the CTRL than all extruded feeds, among which the most efficient, i.e., lower FCR, was the 500MED. The highest activity of trypsin and amylase in lobsters fed the 400MED and 400HIGH feeds points to the activation of a mechanism to maximise nutrients assimilation. The highest lipase activity observed for the 500LOW and 500MED groups indicates a higher capacity to metabolise and store lipids. Overall, the results suggest that the 500MED feed (500 g/kg protein, 237 g/kg carbohydrates and 119 g/kg lipids) is a suitable extruded feed candidate to replace Antarctic krill, commonly used to grow lobster juveniles.

Temperature increase and its effects on fish stress physiology in the context of global warming.

The capacity of fishes to cope with environmental variation is considered to be a main determinant of their fitness and is partly determined by their stress physiology. By 2100, global ocean temperature is expected to rise by 1-4°C, with potential consequences for stress physiology. Global warming is affecting animal populations worldwide through chronic temperature increases and an increase in the frequency of extreme heatwave events. As ectotherms, fishes are expected to be particularly vulnerable to global warming. Although little information is available about the effects of global warming on stress physiology in nature, multiple studies describe the consequences of temperature increases on stress physiology in controlled laboratory conditions, providing insight into what can be expected in the wild. Chronic temperature increase constitutes a physiological load that can alter the ability of fishes to cope with additional stressors, which might compromise their fitness. In addition, rapid temperature increases are known to induce acute stress responses in fishes and might be of ecological relevance in particular situations. This review summarizes knowledge about effects of temperature increases on the stress physiology of fishes and discusses these in the context of global warming.

Consistent individual competitive ability in rainbow trout as a proxy for coping style and its lack of correlation with cortisol responsiveness upon acute stress.

For a given fish species, individuals are different in their ability to cope with stressors; each individual has its own set of physiological and behavioral responses to stress (stress-coping style). This individual diversity is of importance when considering the welfare of fish reared in aquaculture facilities. In this study with rainbow trout (Oncorhynchus mykiss) we investigated the link between the ability to compete for food of each individual (used as a proxy of dominance behavior/proactive stress-coping style) and its ability to cope with stress; we hypothesized that fish that are better competitors would be more robust against common aquaculture stressors. We screened 680 rainbow trout individuals for competition ability. This was done by submitting groups of 20 individuals to a 1-week competition trial where they were kept at low stocking density and were provided a restricted amount of food. A 15% of the screened fish were selected as "winners" and another 15% were selected as "losers", based on growth rates during the competition trials. Fish were re-tested in a second competition trial after several weeks, to assess for consistency of competitive ability. Winner and loser fish were individually exposed to confinement and their neuroendocrine stress response was evaluated (serotonergic activity in telencephalon and brain stem, plasma levels of cortisol, glucose and lactate). Furthermore, behavioral responses to confinement and net restraining tests were also investigated. The results showed good temporal consistency of competitive ability in the lapse of time of the experiments. Besides, competitive ability showed a positive association to fish activity during the net restraining tests. However, plasma stress marker data showed a lack of relevant differences between the acute stress responses of winner and loser fish, adding up to the body of evidence suggesting that stress responsiveness might not be consistently linked to SCS in vertebrates. This, together with the inability of winner fish to outperform loser fish in usual stocking density conditions, suggests that there is no clear welfare or performance benefits in selecting fish of a specific coping style for fish farming, at least in the domesticated trout population used in the current study.

Coping styles in European sea bass: The link between boldness, stress response and neurogenesis.

Coping styles consist of a coherent set of individual physiological and behavioral differences in stress responses that are consistent across time and context. Such consistent inter-individual differences in behavior have already been shown in European sea bass (Dicentrarchus labrax), but the associated mechanisms are still poorly understood. Here, we combine physiological measurements with individual behavioral responses in order to characterize coping styles in fish. Fish were tagged and placed in a tank for group risk-taking tests (GRT) at 8 months of age to evaluate boldness using the proxy latency of leaving a sheltered area towards an open area. A subsample of these fish were individually challenged 16 months later using an open field test (OFT), in which the boldness was assessed after being placed in a shelter within an open arena. Latency to exit the shelter, time spent in the shelter, and distance travelled were recorded for this purpose. The blood and brain were then collected to evaluate plasma cortisol concentration and neurotransmitter levels (dopamine, norepinephrine, serotonin, and related metabolites), as well as brain transcription of key genes involved in stress axis regulation (gr1, gr2, mr, crf), neurogenesis (neurod1, neurod2, pcna), and neuronal development (egr1). Fish acting bolder in the GRT were not necessarily those acting bolder in the OFT, highlighting the relatively low consistency across different types of tests performed with a 16-months interval. There was, however, a significant correlation between stress markers and boldness. Indeed, mRNA levels of mr, crf, gr2, egr1, and neurod2, as well as norepinephrine levels were higher in shy than bold fish, whereas brain serotonergic activity was lower in shy fish. Overall, our study highlights the fact that boldness was not consistent over time when testing context differed (group vs. alone). This is in agreement with previous literature suggesting that social context play a key role in boldness measurement and that the particular life history of each individual may account in shaping the personality fate of a fish.

Proactive coping style in early emerging rainbow trout carries a metabolic cost with no apparent return.

The timing with which salmonid larvae emerge from their gravel nests is thought to be correlated with a particular suite of behavioural and physiological traits that correspond to the stress coping style of the individual. Among these traits, aggressiveness, dominance and resilience to stress, are potentially interesting to exploit in aquaculture production. In the present study a series of experiments were performed, with the purpose of characterising behavioural, metabolic and production related traits in rainbow trout juveniles from different emergence fractions. Newly hatched rainbow trout were sorted according to their emergence time from an artificial redd. The early, middle, and late fractions were retained and assessed for their physiological response to stress, growth performance, metabolism, fasting tolerance, and potential for compensatory growth. The early emerging fraction showed proactive behavioural traits; they were faster to reappear following startling, showed a reduced cortisol response following stress, and a reduced metabolic cost of recovery. Emergence time was not correlated with any differences in standard or maximum metabolic rates, but was however, correlated with higher routine metabolic rates, as demonstrated by significantly bigger weight losses during fasting in the early emerging group. Growth rates and feed conversion efficiencies were not significantly different when fish were co-habitated under a restrictive feeding regime, suggesting that early emerging fish are not able to monopolise food resources. The intermediate emerging group, which makes up the bulk of a population and is often ignored, appears to possess the best growth performance traits, possibly because they do not expend excessive energy on dominance behaviour such as the early emerging group, while they are also not overly timid or stress prone such as the late emerging group.

Differences in stress and disease resilience related to emergence time for first feeding in farmed rainbow trout (Oncorhynchus mykiss).

Salmonid individuals show a relatively high variability in the time required to abandon the gravel nest where they hatch, the so-called 'emergence time'. Different behavioral and physiological traits have been shown to be associated with emergence time in wild salmonids. In general, early- and late-emerging fish have traits resembling those of proactive and reactive stress coping styles, respectively. Proactive fish are considered to be more resilient to stress and probably to disease, so it was hypothesized that fish with different emergence times have different abilities to resist repeated episodes of stress without suffering deleterious effects on their welfare or health status. In this study, rainbow trout eyed eggs were hatched and larvae were fractionated according to their emergence time (early fraction: first 20% of fish to emerge; intermediate fraction: mid 20%; late fraction: last 20%). When the fish were 4 months old, they were exposed to a daily repeated stress protocol for 15 days. The next day, both naïve and repeatedly stressed fish were exposed to an acute stress challenge. Different plasma (cortisol, glucose, lactate) as well as CNS (serotonergic activity) stress markers were assessed to evaluate the stress resilience of the different groups. Furthermore, an intraperitoneal infection challenge with Flavobacterium psychrophilum was carried out to assess disease resilience. Altogether, the results showed that fish from different fractions displayed differences in activation of the hypothalamus-pituitary-inter-renal axis, indicating a higher stress resilience in the fish with early emergence times. However, those differences were not reflected in the ability of the different fractions to grow and perform well in terms of growth, or in the ability to overcome infection with bacteria, which was similar for all the emergence fractions. This suggests that discriminating fish according to emergence time would probably have little effect in improving the performance and the welfare of farmed fish.

Influence of vegetable diets on physiological and immune responses to thermal stress in Senegalese sole (Solea senegalensis).

The substitution of fish resources as ingredients for aquafeeds by those based on vegetable sources is needed to ensure aquaculture sustainability in the future. It is known that Senegalese sole (Solea senegalensis) accepts high dietary content of plant ingredients without altering growth or flesh quality parameters. However, scarce information is available regarding the long-term impact of vegetable diets (combining the inclusion of both vegetable protein and oils) on the stress response and immunity of this fish species. This study aims to evaluate the concomitant effect of the extended use of vegetable protein-based diets with fish oil (FO) replacement (0, 50 or 100%) by vegetable oils (VO), on the response to acute (10 min) or prolonged (4 days) stress, induced by thermal shock. Plasma levels of cortisol, glucose and lactate as well as hepatic levels of glucose, glycogen and lactate were evaluated as primary and secondary responses to stress, 6 and 18 months after feeding the experimental diets (6 and 18 MAF). The brain monoaminergic activity in telencephalon and hypothalamus, and non-specific immune parameters were also evaluated. As expected, thermal shock induced an increase in values of plasma parameters related to stress, which was more evident in acute than in prolonged stress. Stress also affected lactate levels in the liver and the values of the alternative complement pathway-ACH50 in the plasma. Dietary substitution of FO induced an effect per se on some parameters such as decreased hepatic glucose and glycogen levels and peroxidase activity in plasma as well enhanced serotonergic activity in brain of non-stressed fish. The results obtained in some parameters indicate that there is an interaction between the use of vegetable diets with the physiological response to thermal stress, as is the case of the hepatic lactate, serotonergic neurotransmission in brain, and the activity of ACH50 in plasma. These results suggest that the inclusion of VO in plant protein based diets point to a slightly inhibited stress response, more evident for an acute than a prolonged stress.

Behavior Analysis of Novel Wearable Indoor Mapping System Based on 3D-SLAM.

This paper presents a Wearable Prototype for indoor mapping developed by the University of Vigo. The system is based on a Velodyne LiDAR, acquiring points with 16 rays for a simplistic or low-density 3D representation of reality. With this, a Simultaneous Localization and Mapping (3D-SLAM) method is developed for the mapping and generation of 3D point clouds of scenarios deprived from GNSS signal. The quality of the system presented is validated through the comparison with a commercial indoor mapping system, Zeb-Revo, from the company GeoSLAM and with a terrestrial LiDAR, Faro Focus3D X330. The first is considered as a relative reference with other mobile systems and is chosen due to its use of the same principle for mapping: SLAM techniques based on Robot Operating System (ROS), while the second is taken as ground-truth for the determination of the final accuracy of the system regarding reality. Results show that the accuracy of the system is mainly determined by the accuracy of the sensor, with little increment in the error introduced by the mapping algorithm.

Interplay between daily rhythmic serum-mediated bacterial killing activity and immune defence factors in rainbow trout (Oncorhynchus mykiss).

Circadian rhythm is emerging as an important regulator of immune functions. However, there is a paucity of information on the influence of this biological phenomenon in the antimicrobial factors in teleost fish. This study investigated the dynamics and interplay of serum-mediated bacterial killing activity and immune defence factors throughout the light:dark (LD) cycle in rainbow trout (Oncorhynchus mykiss). The juvenile fish came from two different emergence time fractions (i.e., late and early) that were believed to exhibit behavioural and physiological differences. Serum collected during the day from fish (mean ± SD: 39.8 ± 6.3 g) reared under 14L:10D photoperiod demonstrated bactericidal activity against Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida of varying magnitude, but no significant differences between the emergence fractions were observed. A day-night comparison in the same batch of fish revealed time-of-day dependence in the bactericidal activity against F. psychrophilum and Y. ruckeri amongst emergence fractions. A group of fish (63.3 ± 4.7 g) from each fraction was entrained to 12L:12D photoperiod for 21 days to investigate whether serum bactericidal activity exhibited daily rhythm. Serum-mediated bacterial killing activity against F. psychrophilum and Y. ruckeri displayed significant daily rhythm in both emergence fractions, where the peak of activity was identified during the light phase. Moreover, several serum defence factors manifested variations during the LD cycle, where anti-protease (ANTI) and myeloperoxidase (MPO) activities exhibited significant daily oscillation. However, there were no remarkable differences in the daily changes of serum factors amongst emergence fractions. Acrophase analysis revealed that the peaks of activity of alkaline phosphatase (only in late fraction), ANTI, lysozyme (only in early fraction) and MPO were identified during the light phase and corresponded with the period when serum-mediated bacterial killing activity was also at its highest. The daily dynamics of bactericidal activity and immune defence factors displayed positive correlation, particularly between MPO and, the two pathogens (i.e., F. pyschrophilum and Y. ruckeri). Taken together, the study revealed that serum-mediated bacterial killing activity and immune defence factors remarkably varied during the LD cycle in rainbow trout. In addition, the two emergence fractions displayed nearly comparable immunological profiles.

Neuroendocrine and Immune Responses Undertake Different Fates following Tryptophan or Methionine Dietary Treatment: Tales from a Teleost Model.

Methionine and tryptophan appear to be fundamental in specific cellular pathways involved in the immune response mechanisms, including stimulation of T-regulatory cells by tryptophan metabolites or pro-inflammatory effects upon methionine supplementation. Thus, the aim of this study was to evaluate the immunomodulatory effect of these amino acids on the inflammatory and neuroendocrine responses in juveniles of European seabass, Dicentrarchus labrax. To achieve this, goal fish were fed for 14 days methionine and tryptophan-supplemented diets (MET and TRP, respectively, 2× dietary requirement level) or a control diet meeting the amino acids requirement levels (CTRL). Fish were sampled for immune status assessment and the remaining fish were challenged with intraperitoneally injected inactivated Photobacterium damselae subsp. piscicida and sampled either 4 or 24 h post-injection. Respiratory burst activity, brain monoamines, plasma cortisol, and immune-related gene expression showed distinct and sometimes opposite patterns regarding the effects of dietary amino acids. While neuroendocrine intermediates were not affected by any dietary treatment at the end of the feeding trial, both supplemented diets led to increased levels of plasma cortisol after the inflammatory insult, while brain monoamine content was higher in TRP-fed fish. Peripheral blood respiratory burst was higher in TRP-fed fish injected with the bacteria inoculum but only compared to those fed MET. However, no changes were detected in total antioxidant capacity. Complement factor 3 was upregulated in MET-fed fish but methionine seemed to poorly affect other genes expression patterns. In contrast, fish fed MET showed increased immune cells numbers both before and after immune challenge, suggesting a strong enhancing effect of methionine on immune cells proliferation. Differently, tryptophan effects on inflammatory transcripts suggested an inhibitory mode of action. This, together with a high production of brain monoamine and cortisol levels, suggests that tryptophan might mediate regulatory mechanisms of neuroendocrine and immune systems cooperation. Overall, more studies are needed to ascertain the role of methionine and tryptophan in modulating (stimulate or regulate) fish immune and neuroendocrine responses.

Emergence Time and Skin Melanin Spot Patterns Do Not Correlate with Growth Performance, Social Competitive Ability or Stress Response in Farmed Rainbow Trout.

In wild salmonid fish, specific individual behavioral traits have been correlated with the timing of fry emergence from their gravel spawning nests; Early emerging fish display more aggressive behavior and have a higher probability of becoming socially dominant, compared to fish that emerge at a later stage. Apart from aggression and dominance, other behavioral and metabolic traits, such as boldness, metabolic rate, or growth, have also been linked to emergence time. Altogether, the traits of early- and late-emerging fish resemble those of the proactive and reactive stress-coping style, respectively. As proactive fish are considered more resilient to stress, it may be desirable to select these for aquaculture production. However, it is currently unclear to what extent the link between emergence time and stress-coping styles is maintained in the selective breeding of farmed fish. In the present study, eyed eggs from a commercial supplier were hatched, and larvae fractionated according to their emergence time. Later on, juvenile fish from different emergence fractions were subjected to a stress challenge and also tested to evaluate their competitive ability for food. Beyond some slight dissimilarities in the acute stress responses, emergence fraction displayed no correlation with growth rates, or the ability to compete for feed. Within the whole group of fish utilized in the experiments, no relationship between skin melanin spot pattern and growth performance, stress response intensity, or competitive ability was found. Altogether, the differences in physiological traits related to emergence time were not as strong as those found in earlier studies. It is hypothesized, that the origin and degree of domestication of the fish might be partly responsible for this. The predictive value of skin spots or emergence time to infer the fish stress coping style in farmed fish is also discussed.

Characterization of melatonin synthesis in the gastrointestinal tract of rainbow trout (Oncorhynchus mykiss): distribution, relation with serotonin, daily rhythms and photoperiod regulation.

Melatonin is synthesized in peripheral locations of vertebrates, including the gastrointestinal tract (GIT). In teleost, information regarding this topic is scarce. Here we studied the presence and synthesis of melatonin at the rainbow trout GIT. Different sections of trout GIT (from esophagus to hindgut) were dissected out and assayed for contents of melatonin, serotonin (5-HT) and its metabolite, 5-hydroxyindole acetic acid, as well as for aanat1, aanat2 and hiomt mRNA abundance. A trout group was pinealectomized to evaluate changes in plasma and gut melatonin content. Finally, the daily profile of melatonin and 5-HT content, and aanat1, aanat2 and hiomt mRNA abundance were analyzed in gut of trout kept under normal lighting, and then under constant darkness. Melatonin was detected in all GIT regions with higher concentrations in the muscular wall than in the mucosa, a similar trend to that of 5-HT. In contrast, transcripts of melatonin synthesis enzymes were more abundant in the mucosa. Pinealectomy did not affect melatonin levels in midgut and hindgut either at day or at night. Additionally, no daily rhythms could be defined for melatonin content in gut tissues but increases during late light phase and at midnight occurred. However, aanat1, aanat2 and hiomt mRNA abundance showed clear daily rhythms with peaks at night. These rhythms remained with a 3-h phase advanced peak in fish exposed to constant darkness. Our results provide clear evidence for a local synthesis of melatonin in trout GIT that might be influenced by the content of 5-HT in the tissue. The process is affected by environmental light cycle and is likely to be under circadian regulation.

Effects of Tributyltin and Other Retinoid Receptor Agonists in Reproductive-Related Endpoints in the Zebrafish (Danio rerio).

Both field and experimental data examined the influence of exposure to environmental contaminant tributyltin (TBT) on marine organisms. Although most attention focused on the imposex phenomenon in gastropods, adverse effects were also observed in other taxonomic groups. It has been shown that imposex induction involves modulation of retinoid signaling in gastropods. Whether TBT influences similar pathways in fish is yet to be addressed. In this study, larvae of the model teleost Danio rerio were exposed to natural retinoids, all-trans-retinoic acid, 9-cis-retinoic acid, and all-trans-retinol, as well as to the RXR synthetic pan-agonist methoprene acid (MA) and to TBT. Larvae were exposed to TBT from 5 days post fertilization (dpf) to adulthood, and reproductive capacity was assessed and correlated with mode of action. TBT significantly decreased fecundity at environmentally relevant levels at 1 µg TBT Sn/g in diet. Interestingly, in contrast to previous reports, TBT altered zebrafish sex ratio toward females, whereas MA exposure biased sex toward males. Since fecundity was significantly altered in the TBT-exposed group with up to 62% decrease, the potentially affected pathways were investigated. Significant downregulation was observed in brain mRNA levels of aromatase b (CYP19a1b) in females and peroxisome proliferator activated receptor gamma (PPARg) in both males and females, suggesting an involvement of these pathways in reproductive impairment associated with TBT.

Is gill cortisol concentration a good acute stress indicator in fish? A study in rainbow trout and zebrafish.

Cortisol is the main biomarker of physiological stress in fish. It is usually measured in plasma, which requires blood collection. Though cortisol is produced in the anterior kidney, it can diffuse easily through cell membranes due to its lipophilic nature. Taking advantage of that, some non-invasive techniques have been developed to measure cortisol directly in the water from fish-holding tanks, in skin mucus or in scales. In this study, we explored the possibility to analyze fish cortisol from gill filaments as a reliable acute stress marker. Our results show that gill cortisol levels correlate well with plasma cortisol levels in both rainbow trout and zebrafish exposed or not to an acute stress protocol. Measuring cortisol in gill filaments increases the available possibilities for stress assessment in fish. Although this approach should yet be tested for its use with other stressors, it has several advantages: In relatively large fish (i.e. above 30 g) gill cortisol levels could be measured in vivo. Sampling of gill biopsies is very fast and easy, and the procedure does not induce stress if properly performed, making it an ideal option for in vivo stress assessment. In small fish, the use of gill tissue to measure cortisol has important technical advantages with respect to the current methods using whole-body homogenates. Gill homogenates could be used directly for ELISA cortisol analysis, avoiding the need of tedious and expensive cortisol extraction protocols, and, since no organic solvent is required, contributing for a more environmentally friendly analysis.