Assessing differences in the bioaccessibility of phenolics present in two wine by-products using an in-vitro model of fish digestion.

Increasing attention is currently being paid to the protective role of polyphenols in health and oxidative status in fish. For this reason, the potential use of different natural sources of such compounds, like wine by products, is under study. One key step required to gain a better understanding on the biological roles of polyphenols for a given species is to assess the different factors affecting their digestive bioaccessibility, and a great number of such studies is based in the use of in vitro digestion models. In the present study the potential digestive bioavailability of the phenolic compounds present in wine bagasse and lees was evaluated for two fish species showing great differences in their digestive phisyiology: the omnivorous gilthead sea bream (Sparus aurata) and the herbivorous flathead grey mullet (Mugil cephalus). The study was developed using in vitro models adapted to simulate their digestion and a factorial experimental design that simultaneously evaluated the effects of the ingredient used as source of polyphenols, presence or absence of feed matrix, fish species and digestion time. The release of the phenolic compounds was evaluated using ultra-high performance liquid chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS) detection. Both the presence of feed matrix and the type of wine by-product showed a significant effect on the digestive release of both total and specific types of polyphenols while fish species showed to be significant only for some specific compounds, like eriodyctiol or syringic acid. The time of digestion was not identified as a statistically significant factor in the release of phenolic compounds due to the great variability in the patterns observed that were classified as early, sustained and late. The observed great variations in the patterns of release of different types of phenolic compounds with time suggest an important effect of gut transit rates on the net bioavailability of a given phenolic compound in the live fish. The present study is, to our knowledge, the first one on which an in vitro approach was applied to assess to what extent the possible complexation of wine polyphenols present in wine by-products with either digestive enzymes or components of the feed matrix could limit their bioaccessibility if included in diets of two different fish species.

Dietary Effects of a Short-Term Administration of Microalgae Blend on Growth Performance, Tissue Fatty Acids, and Predominant Intestinal Microbiota in Sparus aurata.

Given the potential of microalgae as new aquafeed ingredients, this study focuses on using a blend of microalgae, Tisochrysis lutea, Nannochloropsis gaditana, and Scenedesmus almeriensis, as a dietary ingredient for feeding Sparus aurata juveniles. The growth performance, carcass composition, tissue fatty acid profile, and intestinal microbiota were evaluated after a 30 day-feeding period. A microalgae-free diet was used as control, and three experimental diets were formulated containing 5%, 15%, and 25% of the microalgae blend (MB-5%, MB-15%, and MB-25%, respectively). After 7, 15, and 30 days of feeding experimental diets, biological samples were taken. Growth performance and nutrient utilization were not significantly modified at the end of the experiment. Microalgae inclusion tended to decrease body lipids and affected the fatty acid profile, especially MB-25 diet increased DHA levels. Diet MB-25 promoted appropriate microbial diversity, favoring the presence of probiotic bacteria, such as Lactobacillus, and significantly influencing the fatty acid composition and lipid metabolism in fish. In conclusion, using a short pulse of dietary administration of 25% microalgal blend in S. aurata modulates the intestinal microbiota and lipid composition while maintaining growth performance.

Stress response of Rhamdia quelen to the interaction stocking density - Feeding regimen.

This study aimed to verify the effect of different feeding and stocking conditions during 14 days on the gene expression of several hormones and enzymes related to the stress cascade and metabolic parameters in silver catfish Rhamdia quelen under the following experimental conditions: 1) fed at low stocking density (2.5 kg m-3, LSD-F); 2) fed at high stocking density (32 kg m-3, HSD-F); 3) food-deprived at LSD (LSD-FD); and 4) food-deprived at HSD (HSD-FD). Fish from LSD-F and HSD-F groups were fed daily (1 % of their body mass), while fish from food-deprived groups (LSD-FD and HSD-FD) were not fed during the experimental time. Plasma metabolic parameters (glucose, lactate, triglycerides, and proteins) and hepatosomatic index (HSI) were evaluated. In addition, mRNA expression of genes related to the stress axis (crh, pomca, pomcb, nr3c2, star, hsd11b2 and hsd20b), heat shock protein family (hsp90 and hspa12a), sodium-dependent noradrenaline transporter (slc6a2), and growth axis (gh and igf1) were also assessed. Specific growth rate and HSI decreased in food-deprived fish regardless of stocking density. The HSD-FD group showed weight loss compared to the HSD-F, LSD-F, and LSD-FD groups. Plasma glucose and triglycerides were reduced in food-deprived groups, while lactate and protein levels did not change. The expression of key players of the stress response (crh, pomca, pomcb, hsd11b2, nr3c2, and hsp90b) and growth (gh and igf1) pathways were differently regulated depending on the experimental condition, whereas no statistical difference between treatments was found for hsd20b, scl6a2, hspa12a, and star mRNAs expression. This study suggests that LSD acts as a stressor affecting negatively the physiological status of fed fish, as demonstrated by the reduction in growth rates, altered metabolic orchestration, and a higher crh mRNA expression. In addition, food deprivation also increased mRNA expression of other assessed genes (nr3c2, hsp90b, pomca, and pomcb) in fish from the HSD group, indicating higher responsiveness to stress in this stocking density when combined with food deprivation.

Evaluation of the Combined Administration of Chlorella fusca and Vibrio proteolyticus in Diets for Chelon labrosus: Effects on Growth, Metabolism, and Digestive Functionality.

This study aimed to evaluate the combined effect of dietary Chlorella fusca and ethanol-inactivated Vibrio proteolyticus DCF12.2 (C + V diet) in Chelon labrosus juveniles, highlighting their nutritional, physiological, and morphological effects. The results showed that the combined dietary inclusion of C. fusca and V. proteolyticus significantly enhanced growth performance and feed utilization compared to the control group. The C + V diet increased the fish lipid quality index (FLQ), n-3 polyunsaturated fatty acids, and n-3/n-6 ratio, which might be beneficial in terms of human nutrition. The C + V diet considerably increased carbohydrate metabolic activity by statistically boosting plasma glucose. The dietary inclusion of C. fusca in conjunction with V. proteolyticus increased metabolic enzyme activity as well as intestinal absorption capacity compared to that found in the control group. In conclusion, the experimental diet was suitable for feeding C. labrosus, increasing their growth and the nutritional characteristics of the muscle and intestine, without causing tissue damage.

The antibacterials ciprofloxacin, trimethoprim and sulfadiazine modulate gene expression, biomarkers and metabolites associated with stress and growth in gilthead sea bream (Sparus aurata).

The high consumption and subsequent input of antibacterial compounds in marine ecosystems has become a worldwide problem. Their continuous presence in these ecosystems allows a direct interaction with aquatic organisms and can cause negative effects over time. The objective of the present study was to evaluate the effects of exposure to three antibacterial compounds of high consumption and presence in marine ecosystems (Ciprofloxacin CIP, Sulfadiazine SULF and Trimethoprim TRIM) on the physiology of the gilthead sea bream, Sparus aurata. Plasma parameters, enzymatic biomarkers of oxidative stress and damage and expression of genes related to stress and growth were assessed in exposed S. aurata specimens. For this purpose, sea bream specimens were exposed to individual compounds at concentrations of 5.2 ± 2.1 µg L-1 for CIP, 3.8 ± 2.7 µg L-1 for SULF and 25.7 ± 10.8 µg L-1 for TRIM during 21 days. Exposure to CIP up-regulated transcription of genes associated with the hypothalamic-pituitary-thyroid (HPT) (thyrotropin-releasing hormone, trh) and hypothalamic-pituitary-interrenal (HPI) axes (corticotropin-releasing hormone-binding protein, crhbp) in the brain, as well as altering several hepatic stress biomarkers (catalase, CAT; glutathione reductase, GR; and lipid peroxidation, LPO). Similar alterations at the hepatic level were observed after exposure to TRIM. Overall, our study indicates that S. aurata is vulnerable to environmentally relevant concentrations of CIP and TRIM and that their exposure could lead to a stress situation, altering the activity of antioxidant defense mechanisms as well as the activity of HPT and HPI axes.

Acute Inflammation Induces Neuroendocrine and Opioid Receptor Genes Responses in the Seabass Dicentrarchus labrax Brain.

In fish, as observed in mammals, any stressful event affects the immune system to a larger or shorter extent. The neuroendocrine-immune axis is a bi-directional network of mobile compounds and their receptors that are shared between both systems (neuroendocrine and immune) and that regulate their respective responses. However, how and to what extent immunity modulates the neuroendocrine system is not yet fully elucidated. This study was carried out to understand better central gene expression response patterns in a high-valued farmed fish species to an acute peripheral inflammation, focusing on genes related to the hypothalamus-pituitary-interrenal axis and the opioid system. European seabass, Dicentrarchus labrax, were intra-peritoneally injected with either Freund's Incomplete Adjuvant to induce a local inflammatory response or Hanks Balances Salt Solution to serve as the control. An undisturbed group was also included to take into account the effects due to handling procedures. To evaluate the outcomes of an acute immune response, fish were sampled at 4, 24, 48, and 72 h post-injection. The brain was sampled and dissected for isolation of different regions: telencephalon, optic tectum, hypothalamus, and pituitary gland. The expression of several genes related to the neuroendocrine response was measured by real-time PCR. Data were statistically analyzed by ANOVA and discriminant analyses to obtain these genes' responsiveness for the different brain regions. Serotonergic receptors were upregulated in the telencephalon, whereas the optic tectum inhibited these transcription genes. The hypothalamus showed a somewhat delayed response in which serotonin and glucocorticoid receptors were concerned. Still, the hypothalamic corticotropin-releasing hormone played an important role in differentiating fish undergoing an inflammatory response from those not under such conditions. Opioid receptors gene expression increased in both the hypothalamus and the telencephalon, while in the optic tectum, most were downregulated. However, no changes in the pituitary gland were observed. The different brain regions under immune stimulation demonstrated clear, distinct responses regarding gene transcription rates as well as the time period needed for the effect to occur. Further, more integrative studies are required to associate functions to the evaluated genes more safely and better understand the triggering mechanisms.

Differential Metabolic and Transcriptional Responses of Gilthead Seabream (Sparus aurata) Administered with Cortisol or Cortisol-BSA.

Cortisol is the main glucocorticoid hormone promoting compensatory metabolic responses of stress in teleosts. This hormone acts through genomic and membrane-initiated actions to exert its functions inside the cell. Experimental approaches, using exogenous cortisol administration, confirm the role of this hormone during short (minutes to hours)- and long-term (days to weeks) responses to stress. The role of membrane-initiated cortisol signaling during long-term responses has been recently explored. In this study, Sparus aurata were intraperitoneally injected with coconut oil alone or coconut oil containing cortisol, cortisol-BSA, or BSA. After 3 days of treatment, plasma, liver, and skeletal muscle were extracted. Plasma cortisol, as well as metabolic indicators in the plasma and tissues collected, and metabolism-related gene expression, were measured. Our results showed that artificially increased plasma cortisol levels in S. aurata enhanced plasma glucose and triacylglycerols values as well as hepatic substrate energy mobilization. Additionally, cortisol stimulated hepatic carbohydrates metabolism, as seen by the increased expression of metabolism-related genes. All of these responses, observed in cortisol-administered fish, were not detected by replicating the same protocol and instead using cortisol-BSA, which exclusively induces membrane-initiated effects. Therefore, we suggest that after three days of cortisol administration, only genomic actions are involved in the metabolic responses in S. aurata.

Toward a More Comprehensive View of α-Amylase across Decapods Crustaceans.

Decapod crustaceans are a very diverse group and have evolved to suit a wide variety of diets. Alpha-amylases enzymes, responsible for starch and glycogen digestion, have been more thoroughly studied in herbivore and omnivore than in carnivorous species. We used information on the α-amylase of a carnivorous lobster as a connecting thread to provide a more comprehensive view of α-amylases across decapods crustaceans. Omnivorous crustaceans such as shrimps, crabs, and crayfish present relatively high amylase activity with respect to carnivorous crustaceans. Yet, contradictory results have been obtained and relatively high activity in some carnivores has been suggested to be a remnant trait from ancestor species. Here, we provided information sustaining that high enzyme sequence and overall architecture conservation do not allow high changes in activity, and that differences among species may be more related to number of genes and isoforms, as well as transcriptional and secretion regulation. However, recent evolutionary analyses revealed that positive selection might have also occurred among distant lineages with feeding habits as a selection force. Some biochemical features of decapod α-amylases can be related with habitat or gut conditions, while less clear patterns are observed for other enzyme properties. Likewise, while molt cycle variations in α-amylase activity are rather similar among species, clear relationships between activity and diet shifts through development cannot be always observed. Regarding the adaptation of α-amylase to diet, juveniles seem to exhibit more flexibility than larvae, and it has been described variation in α-amylase activity or number of isoforms due to the source of carbohydrate and its level in diets, especially in omnivore species. In the carnivorous lobster, however, no influence of the type of carbohydrate could be observed. Moreover, lobsters were not able to fine-regulate α-amylase gene expression in spite of large changes in carbohydrate content of diet, while retaining some capacity to adapt α-amylase activity to very low carbohydrate content in the diets. In this review, we raised arguments for the need of more studies on the α-amylases of less studied decapods groups, including carnivorous species which rely more on dietary protein and lipids, to broaden our view of α-amylase in decapods crustaceans.

Osmoregulatory Plasticity of Juvenile Greater Amberjack (Seriola dumerili) to Environmental Salinity.

Osmotic costs in teleosts are highly variable, reaching up to 50% of energy expenditure in some. In several species, environmental salinities close to the isosmotic point (~15 psu) minimize energy demand for osmoregulation while enhancing growth. The present study aimed to characterize the physiological status related to osmoregulation in early juveniles of the greater amberjack, Seriola dumerili, acclimated to three salinities (15, 22, and 36 psu). Our results indicate that plasma metabolic substrates were enhanced at the lower salinities, whereas hepatic carbohydrate and energetic lipid substrates decreased. Moreover, osmoregulatory parameters, such as osmolality, muscle water content, gill and intestine Na+-K+-ATPase activities, suggested a great osmoregulatory capacity in this species. Remarkably, electrophysiological parameters, such as short-circuit current (Isc) and transepithelial electric resistance (TER), were enhanced significantly at the posterior intestine. Concomitantly, Isc and TER anterior-to-posterior intestine differences were intensified with increasing environmental salinity. Furthermore, the expression of several adeno-hypophyseal genes was assessed. Expression of prl showed an inverse linear relationship with increasing environmental salinity, while gh mRNA enhanced significantly in the 22 psu-acclimated groups. Overall, these results could explain the better growth observed in S. dumerili juveniles kept at salinities close to isosmotic rather than in seawater.

High Stocking Density and Food Deprivation Increase Brain Monoaminergic Activity in Gilthead Sea Bream (Sparus aurata).

In teleosts, brain monoamines (dopamine and serotonin) participate in the early response to different acute stressors. However, little is known regarding their role during chronic stress. In a 2 × 2 factorial design, the influence of a high stocking density (HSD) and/or food deprivation (FD) on the brain monoaminergic activity in gilthead sea bream (Sparus aurata) was evaluated. Following a 21-day experimental design, samples from the plasma and brain regions (telencephalon, hypothalamus, and optic tectum) were collected. The dopamine (DA), serotonin (5HT), and their main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5 hydroxyindoleacetic acid (5HIAA), contents were HPLC-assessed in brain tissues, and the ratios DOPAC/DA and 5HIAA/5HT were calculated as indicators of enhanced monoaminergic activity. The plasma levels of cortisol and catecholamine were also evaluated. The cortisol levels increased in fish exposed to HSD and normally fed but, also, in all FD groups, whereas the NA levels decreased in LSD-FD animals. Within the brain, the dopaminergic and serotonergic activities in telencephalon and hypothalamus increased in fish subjected to HSD and in the telencephalon of LSD-FD fish. While DA (hypothalamus) and 5HT (telencephalon) increased in the animals submitted to a HSD, food-deprived fish did not show such an increase. Taken together, our results supported the hypothesis of brain monoaminergic activity participating in maintaining and orchestrating the endocrine response to chronic stress in fish.

Aflatoxicosis Dysregulates the Physiological Responses to Crowding Densities in the Marine Teleost Gilthead Seabream (Sparus aurata).

Several studies in fish have shown that aflatoxin B1 (AFB1) causes a disparity of species-dependent physiological disorders without compromising survival. We studied the effect of dietary administration of AFB1 (2 mg AFB1 kg-1 diet) in gilthead seabream (Sparus aurata) juveniles in combination with a challenge by stocking density (4 vs. 40 g L-1). The experimental period duration was ten days, and the diet with AFB1 was administered to the fish for 85 days prior to the stocking density challenge. Our results indicated an alteration in the carbohydrate and lipid metabolites mobilization in the AFB1 fed group, which was intensified at high stocking density (HSD). The CT group at HSD increased plasma cortisol levels, as expected, whereas the AFB1-HSD group did not. The star mRNA expression, an enzyme involved in cortisol synthesis in the head kidney, presented a ninefold increase in the AFB1 group at low stocking density (LSD) compared to the CT-LSD group. Adenohypophyseal gh mRNA expression increased in the AFB1-HSD but not in the CT-HSD group. Overall, these results confirmed that chronic AFB1 dietary exposure alters the adequate endocrinological physiological cascade response in S. aurata, compromising the expected stress response to an additional stressor, such as overcrowding.

Dysregulation of Intestinal Physiology by Aflatoxicosis in the Gilthead Seabream (Sparus aurata).

Aflatoxin B1 (AFB1) is a mycotoxin often present in food. This study aimed to understand the physiological effects of AFB1 on the seabream (Sparus aurata) gastrointestinal system. In a first in vitro approach, we investigated ion transport using the short-circuit current (Isc) technique in Ussing chambers in the anterior intestine (AI). Application of apical/luminal AFB1 concentrations of 8 and 16 µM to healthy tissues was without effect on tissue transepithelial electrical resistance (TER), and apparent tissue permeability (Papp) was measured using fluorescein FITC (4 kD). However, it resulted in dose-related effects on Isc. In a second approach, seabream juveniles fed with different AFB1 concentrations (1 and 2 mg AFB1 kg-1 fish feed) for 85 days showed significantly reduced gill Na+/K+-ATPase (NKA) and H+-ATPase (HA) activities in the posterior intestine (PI). Moreover, dietary AFB1 modified Isc in the AI and PI, significantly affecting TER in the AI. To understand this effect on TER, we analyzed the expression of nine claudins and three occludins as markers of intestinal architecture and permeability using qPCR. Around 80% of the genes presented significantly different relative mRNA expression between AI and PI and had concomitant sensitivity to dietary AFB1. Based on the results of our in vitro, in vivo, and molecular approaches, we conclude that the effects of dietary AFB1 in the gastrointestinal system are at the base of the previously reported growth impairment caused by AFB1 in fish.

Low dietary inclusion of nutraceuticals from microalgae improves feed efficiency and modifies intermediary metabolisms in gilthead sea bream (Sparus aurata).

The aim of this work was to evaluate two functional feeds for the gilthead seabream, Sparus aurata, containing low inclusion of two microalgae-based products (LB-GREENboost, LBGb; and LB-GUThealth, LBGh). Fish (12-13 g) were fed for 13 weeks a control diet or one of the four diets supplemented with both products at 0.5% or 1%. LBGb and LBGh did not affect specific growth rate or survival, but increased feed efficiency by decreasing feed intake and enlarging the intestines. LBGb increased hepatosomatic index and reduced cortisol levels in plasma, while both products lowered plasma lactate. Extensive metabolite and metabolic enzyme profiling revealed that microalgae supplementations, especially 1% LBGh: (i) decrease plasma lactate and increase hepatic glycogen, (ii) reduce hepatic gluconeogenesis, (iii) enhance hepatic lipogenic activity and lipid secretion, (iv) led fish to double triglyceride content in muscle and to stimulate its lipid oxidative capacity, and (v) increase the content of monounsaturated fatty acids and the omega-3 alpha-linolenic acid in muscle. This study demonstrates that both microalgae-based products are suited to improve feed efficiency and orchestrate significant changes in the intermediary metabolism in gilthead seabream juveniles.

Quercetin attenuates endocrine and metabolic responses to oxytetracycline in silver catfish (Rhamdia quelen).

This study aimed to verify whether dietary quercetin protects against the detrimental effects induced by oxytetracycline (OTC) administration in silver catfish (Rhamdia quelen). Fish were divided into different experimental groups that received OTC and/or quercetin, either during 14 or 21 days. To determine the endocrine system stress response, we have measured the brain mRNA expression levels of corticotropin-releasing hormone (crh), proopiomelanocortins (pomca and pomcb) and some of the pituitary hormones (growth hormone [gh], somatolactin [sl], and prolactin [prl]). We have also quantified the levels of cortisol as well as some metabolites (glucose, glycogen, lactate, and triglycerides) in the plasma. Moreover, the enzymatic activity of hexokinase, phosphorylase (active GPase), fructose-biphosphatase (FBP), glycerol-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate dehydrogenase (GDH) and gill Na+/K+-ATPase were measured. The results demonstrated that OTC activates the silver catfish stress response by increasing the plasma cortisol and decreasing the glucose levels at 14 and 21 days. Additionally, OTC also altered the fish hepatic metabolic status as demonstrated by an increase in triglycerides levels and the enzymatic activity of both FBP and GDH after 14 days. OTC also stimulated Na+/K+-ATPase activity in the gill after 14 days and altered the hypophyseal expression of gh (at 14 and 21 days) and prl (at 14 days). The co-treatment with 1.5 g of quercetin could prevent most of the alterations caused by OTC, strongly suggesting quercetin as a beneficial compound when added to the fish diet.

Daily rhythms in endocrine factors of the somatotropic axis and their receptors in gilthead sea bream (Sparus aurata) larvae.

Living organisms have adapted to environmental oscillations in light and temperature through evolving biological clocks. Biological rhythms are pervasive at all levels of the endocrine system, including the somatotropic (growth) axis. The objective of the present research was to study the existence of daily rhythms on the somatotropic axis of a marine teleost species, specifically, the gilthead sea bream (Sparus aurata). Larvae of S. aurata at 30 dph (days post hatching), kept under a 9 L:15D (light-dark) photoperiod, were collected every 3 h throughout a 36 h cycle. The expression of the following somatotropic axis genes was analyzed by quantitative PCR: pituitary adenylate cyclase-activating polypeptide 1 (adcyap1), prepro-somatostatin-1 (pss1), growth hormone (gh), growth hormone receptor types 1 and 2 (ghr1 and ghr2, respectively), insulin-like growth factor 1 (igf1) and igf1 receptor a (igf1ra). All genes displayed significant differences among time points and, with the exception of adcyap1, all showed statistically significant daily rhythms. The acrophases of gh, ghr1, ghr2, igf1 and igf1ra were located around the end of the dark phase, between ZT19:44 and ZT0:48 h, whereas the highest expression levels of adcyap1 occurred at ZT18 h. On the other hand, the acrophase of pss1, an inhibitor of Gh secretion, was located at ZT10:16 h, hence it was shifted by several hours with respect to the other genes. The present results provide the first thorough description of somatotropic axis rhythms in gilthead sea bream. Such knowledge provides insights into the role of rhythmic regulation of the Gh/Igf1 axis system in larval growth and metabolism, and it can also improve the implementation of more species-specific feeding regimes.

New Perspectives Related to the Bioluminescent System in Dinoflagellates: Pyrocystis lunula, a Case Study.

Pyrocystis lunula is considered a model organism due to its bioluminescence capacity linked to circadian rhythms. The mechanisms underlying the bioluminescent phenomenon have been well characterized in dinoflagellates; however, there are still some aspects that remain an enigma. Such is the case of the presence and diversity of the luciferin-binding protein (LBP), as well as the synthesis process of luciferin. Here we carry out a review of the literature in relation to the molecular players responsible for bioluminescence in dinoflagellates, with particular interest in P. lunula. We also carried out a phylogenetic analysis of the conservation of protein sequence, structure and evolutionary pattern of these key players. The basic structure of the luciferase (LCF) is quite conserved among the sequences reported to date for dinoflagellate species, but not in the case of the LBP, which has proven to be more variable in terms of sequence and structure. In the case of luciferin, its synthesis has been shown to be complex process with more than one metabolic pathway involved. The glutathione S-transferase (GST) and the P630 or blue compound, seem to be involved in this process. In the same way, various hypotheses regarding the role of bioluminescence in dinoflagellates are exposed.

An "omic" approach to Pyrocystis lunula: New insights related with this bioluminescent dinoflagellate.

Pyrocystis lunula (Schutt) is a photoautotrophic dinoflagellate without armored form, frequently found in marine environments. Today, there are several biotechnological applications derived from the bioluminescent system of this species. From a post-genomic perspective, in order to have a starting point for studying the proteome of P. lunula, an "omics" approach (transcriptomics-proteomics) was assessed using fresh microalgae samples. A total of 80,874,825 raw reads were generated (11,292,087,505 bp; 55.82% GC) by mRNA sequencing. Very high-quality sequences were assembled into 414,295 contigs (219,203,407 bp; 55.38% GC) using Trinity software, generating a comprehensive reference transcriptome for this species. Then, a P. lunula proteome was inferred and further employed for its analysis on this species. A total of 17,461 peptides were identified, yielding 3182 protein identification hits, including 175 novel proteins. The identified proteins were further categorized according to functional description and gene ontology classification. SIGNIFICANCE: The major contribution of the present work is making available a reference transcriptome and proteome of P. lunula, that is now accessible for the research community, and a functional description of the 3182 proteins inferred from the transcriptome, including 175 novel proteins, which have already been deposited in the ProteomeXchange and NCBI SRA databases, respectively. In addition to this, a series of important factors related to the bioluminescent system and the regulation of gene expression, were identified and described.

Acute-Stress Biomarkers in Three Octopodidae Species After Bottom Trawling.

Several Octopodidae species have a great potential for the diversification of worldwide aquaculture. Unfortunately, the lack of stress-related biomarkers in this taxon results an obstacle for its maintenance in conditions where animal welfare is of paramount relevance. In this study, we made a first approach to uncover physiological responses related to fishing capture in Eledone moschata, Eledone cirrhosa, and Octopus vulgaris. Captured octopus from all three species were individually maintained in an aquaculture system onboard of oceanographic vessel in south-western waters of Europe. Haemolymph plasma and muscle were collected in animals at the moment of capture, and recovery was evaluated along a time-course of 48 h in Eledone spp., and 24 h for O. vulgaris. Survival rates of these species captured in spring and autumn were evaluated. Physiological parameters such as plasma pH, total CO2, peroxidase activity, lysozyme, hemocyanin, proteases, pro-phenoloxidase, anti-proteases, free amino acids, lactate and glucose levels, as well as muscle water percentage, free amino acids, lactate, glycogen and glucose values were analyzed. The immune system appears to be compromised in these species due to capture processes, while energy metabolites were mobilized to face the acute-stress situation, but recovery of all described parameters occurs within the first 24 h after capture. Moreover, this situation exerts hydric balance changes, as observed in the muscle water, being these responses depending on the species assessed. In conclusion, three Octopodidae species from south-western waters of Europe have been evaluated for stress-related biomarkers resulting in differentiated mechanisms between species. This study may pave the way to further study the physiology of stress in adult octopuses and develop new methodologies for their growth in aquaculture conditions.

Essential Oils as Stress-Reducing Agents for Fish Aquaculture: A Review.

In fish, stressful events initiate a hormone cascade along the hypothalamus-pituitary-interrenal and hypothalamus-sympathetic-chromaffin (HSC) axis to evoke several physiological reactions in order to orchestrate and maintain homeostasis. Several biotic and abiotic factors, as well as aquaculture procedures (handling, transport, or stocking density), activated stress system inducing negative effects on different physiological processes in fish (growth, reproduction, and immunity). In order to reduce these consequences, the use of essential oils (EOs) derived from plants has been the focus of aquaculture studies due to their diverse properties (e.g., anesthetic, antioxidant, and antimicrobial), which have been shown to reduce biochemical and endocrine alterations and, consequently, to improve the welfare status. Recently, several studies have shown that biogenic compounds isolated from different EOs present excellent biological activities, as well as the nanoencapsulated form of these EOs may potentiate their effects. Overall, EOs presented less side effects than synthetic compounds, but their stress-reducing efficacy is related to their chemical composition, concentration or chemotype used. In addition, their species-specific actions must be clearly established since they can act as stressors by themselves if their concentrations and chemotypes used are not suitable. For this reason, it is necessary to assess the effect of these natural compound mixtures in different fish species, from marine to freshwater, in order to find the ideal concentration range and the way for their administration to obtain the desired biological activity, without any undesired side effects. In this review, the main findings regarding the use of different EOs as stress reducers will be presented to highlight the most important issues related to their use to improve fish welfare in aquaculture.