Exposure to silver and titanium dioxide nanoparticles at supra-environmental concentrations decreased sperm motility and affected spermatozoa subpopulations in gilthead seabream, Sparus aurata.

Marine pollution by nanoparticles (NPs) can be reprotoxic for fish and disturb successful reproduction of wild populations. In gilthead seabream (Sparus aurata), a mild effect on sperm motility was observed after exposure to high concentrations of silver NPs. Considering the great heterogeneity traits within a sperm sample, it is possible that NPs affect spermatozoa accordingly, modulating subpopulation profile. Thus, this work aimed to analyse NP effects in sperm motility in general and considering spermatozoa population structure, using a subpopulation approach. Seabream sperm samples from mature males were exposed for 1 h to increasing concentrations of titanium dioxide (1, 10, 100, 1000 and 10,000 µg L-1) and silver (0.25, 25 and 250 µg L-1) NPs, including Ag NP and Ag+, dissolved in a non-activating medium (0.9 % NaCl). Concentrations chosen include realistic (10-100 and 0.25 µg L-1, respectively, for TiO2 and Ag) and supra-environmental values. The mean particle diameter was determined as 19.34 ± 6.72 and 21.50 ± 8.27 nm in the stock suspension, respectively, for titanium dioxide and silver. After the ex vivo exposure, sperm motility parameters were determined using computer-assisted sperm analysis, and sperm subpopulations were later identified using a two-step cluster analysis. Results revealed a significant reduction in total motility after exposure to the 2 highest concentrations of titanium dioxide NPs, while curvilinear and straight-line velocities were not altered. Exposure to silver NPs (Ag NP and Ag+) lowered significantly total and progressive motilities at all concentrations, while curvilinear and straight-line velocities were significantly lower only at the highest concentration. Sperm subpopulations were also affected by the exposure to both titanium dioxide and silver NPs. In both cases, the highest levels of NPs triggered a decrease in the percentage of fast sperm subpopulations (38.2% in TiO2 1000 µg L-1, 34.8.% in Ag NP 250 µg L-1, and 45.0% in Ag+ 250 µg L-1 vs 53.4% in the control), while an increase on slow sperm subpopulations. A reprotoxic effect was proven for both NPs, but only at supra-environmental concentrations.

Stressor controllability modulates the stress response in fish.

BACKGROUND: In humans the stress response is known to be modulated to a great extent by psychological factors, particularly by the predictability and the perceived control that the subject has of the stressor. This psychological dimension of the stress response has also been demonstrated in animals phylogenetically closer to humans (i.e. mammals). However, its occurrence in fish, which represent a divergent vertebrate evolutionary lineage from that of mammals, has not been established yet, and, if present, would indicate a deep evolutionary origin of these mechanisms across vertebrates. Moreover, the fact that psychological modulation of stress is implemented in mammals by a brain cortical top-down inhibitory control over subcortical stress-responsive structures, and the absence of a brain cortex in fish, has been used as an argument against the possibility of psychological stress in fish, with implications for the assessment of fish sentience and welfare. Here, we have investigated the occurrence of psychological stress in fish by assessing how stressor controllability modulates the stress response in European seabass (Dicentrarchus labrax). RESULTS: Fish were exposed to either a controllable or an uncontrollable stressor (i.e. possibility or impossibility to escape a signaled stressor). The effect of loss of control (possibility to escape followed by impossibility to escape) was also assessed. Both behavioral and circulating cortisol data indicates that the perception of control reduces the response to the stressor, when compared to the uncontrollable situation. Losing control had the most detrimental effect. The brain activity of the teleost homologues to the sensory cortex (Dld) and hippocampus (Dlv) parallels the uncontrolled and loss of control stressors, respectively, whereas the activity of the lateral septum (Vv) homologue responds in different ways depending on the gene marker of brain activity used. CONCLUSIONS: These results suggest the psychological modulation of the stress response to be evolutionary conserved across vertebrates, despite being implemented by different brain circuits in mammals (pre-frontal cortex) and fish (Dld-Dlv).

Investigating the kisspeptin system in the hermaphrodite teleost gilthead seabream (Sparus aurata).

The kisspeptin system, a known regulator of reproduction in fish, was investigated during two key phases within the gilthead seabream (Sparus aurata) life cycle: protandrous sex change and larval ontogeny. Seabream specific partial cDNA sequences were identified for two key targets, kissr4 and kiss2, which were subsequently cloned and qPCR assays developed. Thereafter, to examine association in expression with sex change, a group of adult seabream (2+ years old) undergoing sex change were sampled for gene expression at two different periods of the annual cycle. To study the kisspeptin system ontogeny during early life stages, transcript levels were monitored in larvae (till 30 days-post-hatch, DPH) and post-larvae (from 30 till 140 DPH). During sex change, higher expression of kissr4 and kiss2 was observed in males when compared to females or individual undergoing sex change, this is suggestive of differential actions of the kisspeptin system during protandrous sex change. Equally, variable expression of the kisspeptin system during early ontogenic development was observed. The higher expression of kissr4 and kiss2 observed from 5 DPH, with elevations at 5-20 and 90 DPH for kissr4 and at 5, 10, 20, and 60 DPH for kiss2, is coincident with the early ontogeny of gnrh genes previously reported for seabream, and possibly related with early development of the reproductive axis in this species.

Kisspeptin Influences the Reproductive Axis and Circulating Levels of microRNAs in Senegalese Sole.

Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain-pituitary-gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis.

Meagre's melatonin profiles under captivity: circadian rhythmicity and light sensitiveness.

The present study reveals the first characterization of the plasma melatonin rhythms of the meagre (Argyrosomus regius) under aquaculture conditions. Melatonin levels were monitored during a 24 h cycle under a photoperiod of 16 L:8D and under constant darkness (DD), respectively to characterize the daily rhythm of this indoleamine and to test its endogenous origin. Besides, to identify which light intensities are perceived as night or day by this species, the degree of inhibition of nocturnal melatonin production caused by increasing intensities of light was tested (3.3, 5.3, 10.5, and 120 µW/cm2), applying 1 h light pulses at Mid-Dark. The result for melatonin daily rhythm in plasma showed a typical profile: concentration remained low during all daytime points, increasing greatly during dark points, with maximum values at 16:00 and 22:00 h, zeitgeber time. Under DD conditions, the plasma melatonin profile persisted, with a similar acrophase but with a lower amplitude between subjective day and night periods, indicating this rhythm as being endogenously driven. Moreover, meagre seemed to be very sensitive to dim levels of illumination during the night, since an intensity of just 3.3 µW/cm2 inhibited melatonin production. However, only the pulse of 5.3 µW/cm2 caused a melatonin drop till daytime concentrations. Thus, the threshold of light detection by the pineal organ was suggested as being located between 3.3 and 5.3 µW/cm2. Such results are an added value for this species biology knowledge, and in consequence to its adaptation to aquaculture conditions, allowing the improvement of culture husbandry protocols.

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.

Endocrine (plasma cortisol and glucose) and behavioral (locomotor and self-feeding activity) circadian rhythms in Senegalese sole (Solea senegalensis Kaup 1858) exposed to light/dark cycles or constant light.

The existence of daily rhythms under light/dark (LD) cycles in plasma cortisol, blood glucose and locomotor and self-feeding activities, as well as their persistence (circadian nature) under constant light (LL), was investigated in Senegalese sole (Solea senegalensis). For the cortisol and glucose rhythms study, 48 soles were equally distributed in 8 tanks and exposed to a 12:12 LD cycle and natural water temperature (experiment 1). After an acclimation period, blood was sampled every 3 h until a 24-h cycle was completed. Blood glucose levels were measured immediately after sampling, while plasma cortisol was measured later by ELISA. In experiment 2, the fish were exposed to LL for 11 days, and after this period, the same sampling procedure was repeated. For the study of locomotor and self-feeding rhythms (experiment 3), two groups of sole were used: one exposed to LD and the other to LL. Each group was distributed within 3 tanks equipped with infrared photocells for the record of locomotor activity, and self-feeders for feeding behavior characterization. The results revealed a marked oscillation in cortisol concentrations during the daily cycle under LD, with a peak (35.65 ± 3.14 ng/ml) in the afternoon (15:00 h) and very low levels during the night (5.30 ± 1.09 ng/ml). This cortisol rhythm persisted under LL conditions, with lower values (mean cortisol concentration = 7.12 ± 1.11 ng/ml) and with the peak shifted by 3 h. Both rhythms were confirmed by COSINOR analysis (p < 0.05). The synchronizing role of temperature and feeding schedule, in addition to light, is also discussed. Diel rhythms of glucose were not evident in LD or LL. As to locomotor and self-feeding activity, a very marked rhythm was observed under LD, with higher activity observed during the night, with acrophases located at 2:14 and 3:37 h, respectively. The statistical significance of daily rhythms was confirmed by COSINOR analysis. Under LL, both feeding and locomotor rhythms persisted, with an endogenous period (τ) around 22.5 h. In short, our findings described for the first time the existence of circadian cortisol and behavioral circadian rhythms in flat fish. Such results revealed the importance of taking into account the time of day when assessing stress responses and evaluating physiological indicators of stress in fish.

Shipping can be achieved but cargo arrives with damage - titanium dioxide nanoparticles affect the DNA of Pacific oyster sperm.

Titanium dioxide nanoparticles (TiO2 NP) were reported to be reprotoxic in humans and fish. However, the effects of these NP on the reproduction of marine bivalves, namely oysters, remain unknown. Thus, a short-term (1 h) direct exposure of sperm of the Pacific oyster (Crassostrea gigas) to two TiO2 NP concentrations (1 and 10 mg.L-1) was performed, and sperm motility, antioxidant responses, and DNA integrity were evaluated. Although no changes occurred in sperm motility and the activities of the antioxidants, the genetic damage indicator increased at both concentrations, demonstrating that TiO2 NP affects the DNA integrity of oyster sperm. Although DNA transfer can happen, it does not fulfill its biological mission since the transferred DNA is not intact and may compromise the reproduction and recruitment of the oysters. This vulnerability of C. gigas sperm towards TiO2 NP highlights the importance of studying the effects of NPs exposure to broadcast spawners.

Silver nanoparticles and silver ions indistinguishably decrease sperm motility in Pacific oysters (Magallana gigas) after short-term direct exposure.

The present study aimed to evaluate the reprotoxicity of environmental (0.25 µg.L-1) and supra-environmental (25 µg.L-1 and 250 µg.L-1) levels of silver nanoparticles (Ag NP) on the Pacific oyster (Magallana gigas), by determining sperm quality. For that, we evaluated sperm motility, mitochondrial function and oxidative stress. To determine whether the Ag toxicity was related to the NP or its dissociation into Ag ions (Ag+), we tested the same concentrations of Ag+. We observed no dose-dependent responses for Ag NP and Ag+, and both impaired sperm motility indistinctly without affecting mitochondrial function or inducing membrane damage. We hypothesize that the toxicity of Ag NP is mainly due to adhesion to the sperm membrane. Blockade of membrane ion channels may also be a mechanism by which Ag NP and Ag+ induce toxicity. The presence of Ag in the marine ecosystem is of environmental concern as it may affect reproduction in oysters.

Antioxidants in Fish Sperm and the Potential Role of Melatonin.

In recent years, the effects of novel antioxidants have played an important role in the research focusing on fish cell protection. As food demand grows, aquaculture production becomes more intensive, and fish are more exposed to oxidative stress conditions, like high densities, temperature shifting, frequent fish handling and samplings, and prophylactic or disease treatments, which expose fish to a different environment. Particularly in reproduction, germ cells lose antioxidant capacity with spermatogenesis, as spermatozoa are more prone to oxidative stress. Antioxidants have been used in a variety of fish physiological problems including in reproduction and in the establishment of cryopreservation protocols. From the most used antioxidants to natural plant food and herbs, and endogenously produced antioxidants, like melatonin, a review of the literature available in terms of their effects on the protection of fish spermatozoa is presented here in a classified structure. Several direct and indirect approaches to improve gamete quality using antioxidants administration are mentioned (through feed supplementation or by adding in cryopreservation media), as well as factors affecting the efficiency of these molecules and their mechanisms of action. Special attention is given to the unclear melatonin pathway and its potential scavenger activity to prevent and counteract oxidative stress damage on fish spermatozoa.