Sole Juveniles Research Articles

Water-in-oil adjuvant challenges in fish vaccination: An experimental inactivated adjuvanted vaccine against betanodavirus infection in Senegalese sole.

The extensive growth of intensive fish farming has led to a massive spread of infectious diseases. Nervous necrosis virus (NNV) is the causative agent of the viral encephalo- and retinopathy disease which has become a major threat for fish farming all over the globe. The devastating mortality rates recorded in disease outbreaks, especially when infected specimens are at early stages of development, have a high economic impact on the sector. Currently, vaccines are the most cost-effective preventing tool in the fight against viruses. Inactivated vaccines have the advantage of simplicity in their development at the same time as present the antigen in a similar manner than the natural infection in the host. Nevertheless, they usually trigger weaker immune responses needing adjuvants to boost their effectiveness. In this work, we have intraperitoneally vaccinated Senegalese sole juveniles (Solea senegalensis) with a previously designed inactivated vaccine against NNV based on binary ethylenimine (BEI), mixed or not with an oil-adjuvant. Our results demonstrated the potential activation of different immune pathways when the vaccine was administered alone compared to the oil-adjuvanted vaccine, both resulting in an equivalent partial improvement in survival following a NNV challenge. However, whilst the vaccine alone led to a significant increase in specific antibodies, in the adjuvanted version those antibodies were kept basal although with a slight improvement in their neutralization capacity. At transcriptional level, neither vaccine (adjuvanted or not) triggered the immune system activation during the vaccination period. However, after NNV infection, the BEI-inactivated vaccines alone and oil-adjuvanted both elicited the stimulation of antiviral responsive genes (rtp3, herc4), antigen presentation molecules (mhcii) and T-cell markers (cd8a) in the head-kidney. Additionally, the oil-adjuvanted vaccine appears to stimulate mediator cytokines (il6) and B-cell markers (ight and ighm). Surprisingly, when the adjuvant was administered alone, fish showed the highest survival rates concomitantly with a lack of NNV-IgM production, pointing to the possible induction of different immune pathways than the B-cell responses via antibodies by the adjuvant. Since this combined vaccine did not succeed in the full extension of protection against the pathogen, further studies should be performed focusing on unravelling the molecular mechanisms through which adjuvants trigger the immune response, both independently and when added to a vaccine antigen.

Toxicopathic effects of waterborne Cd in sole juveniles, a prospective pollution monitoring sentinel for estuarine ecosystems

IntroductionEstuarine ecosystems are under high anthropogenic pressure and receive a wide Q7 variety of contaminants, including metals. They can remain trapped in estuarine sediments at high concentrations for long periods but eventually they can be released to the overlying water and become toxic for the resident benthic biota. As a consequence, biomonitoring programs surveying the health status of estuaries and coastal areas count with benthic species for the assessment of health disturbances in these specific ecosystems. The present investigation aims at recognizing toxicopathic effects elicited in estuarine life stages of the benthic fish Solea senegalensis on exposure to waterborne Cd, a model compound for metal contamination.MethodsBiological responses to metal exposure were assessed based on the application of a “biomarker + histopathology” approach. Two-year old sole juveniles were exposed to various concentrations of waterborne Cd for 3 and 7 days (Control; Low Cd: 1 µg/l; Mid Cd: 10 µg/l and High Cd: 1000 µg/l). Liver samples were collected for chemical analysis at day 7. Biological samples were collected at days 3 and 7 for biochemical (brain and liver), histochemical (liver) and histopathological analysis (liver, gills and gonad).ResultsBrain acetyl cholinesterase was inhibited after 7 days exposure, indicating neurotoxic effects on exposure to 1000 µg Cd/l. Hepatic catalase and glutathione-Stransferase were induced at day 3 and inhibited at day 7, which suggests a bell-shaped response. A dose-dependent lysosomal membrane destabilization in hepatocytes was recorded at day 7. In parallel, histopathological lesions in gills, liver and gonad were more frequent at day 7 in soles exposed to high Cd concentrations.DiscussionOverall, the “biomarker + histopathology” approach revealed that waterborne Cd causes toxicopathic effects in sole juveniles upon exposure to environmentally relevant concentrations (10 µg Cd/l). The effects were clearly time dependent, and thus it is conceivable that more severe biological effects would be elicited by these low concentrations of Cd at longer exposure periods. Therefore, biomarkers and histopathology in combination provide early-warning indications of altered health status after waterborne Cd exposure in sole juveniles, a prospective sentinel for biological effects assessment of metal contamination in pollution monitoring programs in estuaries and coastal areas.

The Use of Sand Substrate Modulates Dominance Behaviour and Brain Gene Expression in a Flatfish Species

Physical complexity adds physical enrichment to rearing conditions. This enrichment promotes fish welfare and reduces detrimental characteristics that fish develop in captivity. Senegalese sole (Solea senegalensis) is an important species for European aquaculture, where it is reared in intensive conditions using fibreglass tanks. However, reproductive dysfunctions present in this species do not allow it to complete its life cycle in captivity. Recently, dominance behaviour has been studied to try to solve this problem. The present study aimed to assess the effect of sand as environmental enrichment in the dominance behaviour and brain mRNA abundance of Senegalese sole juveniles. Four tanks of sole (n = 48 fish in total) were established in two different environments (with and without sand). Juveniles were subjected to dominance tests of feeding and territoriality. Behaviours analysed by video recordings related to the distance from the food delivered and harassment behaviour towards other individuals (e.g., resting of the head on another individual). In both environments, dominant sole were the first to feed, displayed more head-resting behaviour and dominated the area close to the feeding point, where the events were reduced in fish maintained in the sand. mRNA expression related to differentiation of dopamine neurons (nr4a2) and regulation of maturation (fshra) were significantly upregulated in dominant fish in the sand environment compared to dominants maintained without sand. The use of an enriched environment may affect Senegalese sole dominance, enhance welfare and possibly advance future maturation.

Fish Microbiome Modulation and Convenient Storage of Aquafeeds When Supplemented with Vitamin K1.

Vitamin K (VK) is a fat-soluble vitamin necessary for fish metabolism and health. VK stability as dietary component during aquafeed storage and its potential effect on intestinal microbiome in fish have not yet been completely elucidated. The convenient storage conditions of aquafeeds when supplemented with phylloquinone (VK1), as well as its potential effects on the gut microbiota of Senegalese sole (Solea senegalensis) juveniles, have been explored. Experimental feeds were formulated to contain 0, 250 and 1250 mg kg-1 of VK1 and were stored at different temperatures (4, -20 or -80 °C). VK stability was superior at -20 °C for short-term (7 days) storage, while storing at -80 °C was best suited for long-term storage (up to 3 months). A comparison of bacterial communities from Senegalese sole fed diets containing 0 or 1250 mg kg-1 of VK1 showed that VK1 supplementation decreased the abundance of the Vibrio, Pseudoalteromonas, and Rhodobacterace families. All these microorganisms were previously associated with poor health status in aquatic organisms. These results contribute not only to a greater understanding of the physiological effects of vitamin K, particularly through fish intestinal microbiome, but also establish practical guidelines in the industry for proper aquafeed storage when supplemented with VK1.

First releases of hatchery-produced Senegal sole (Solea senegalensis), brill (Scophthalmus rhombus), and wedge sole (Dicologoglossa cuneata) juveniles in the South-western Spanish coast

The regression of fisheries in the Gulf of Cadiz is evident since current fish catches are 33% of that 30 years before. Consequently, some initiatives for the replenishment of exhausted wild stocks are welcome. The objective of the present work is to describe and analyse the results coming from the first flatfish stock enhancements in Andalusia. A total of 3189 fish from three flatfish species: Senegal sole (Solea senegalensis Kaup), wedge sole (Dicologlossa cuneata Moreau), and brill (Scophthalmus rhombus Linnaeus) were tagged and released. Several variables were calculated through the data analysis of recovered fish. Some variables were calculated only for Senegal soles since wedge sole and brill recaptures were not significant. The Senegal sole recapture rate was 2.71 ± 0.72, similar to other published data, the recapture rates for bigger fish being higher though not significant. No significant differences were detected for distance, time, growth, or recapture rate amongst initial Senegal sole sizes. Around 80% of recaptures were registered within 15 weeks after release. The results show that it is possible for the release and recovery of tagged Senegal soles in the Gulf of Cadiz. Future long-term programmes on stock enhancement could help to determine the effects on fisheries and recover stocks.

Short-term exposure to pharmaceuticals negatively impacts marine flatfish species: Histological, biochemical and molecular clues for an integrated ecosystem risk assessment

The marine habitat and its biodiversity can be impacted by released pharmaceuticals. The short-term (7 days) effect of 3 commonly used drugs – warfarin, dexamethasone and imidazole – on Senegalese sole (Solea senegalensis) juveniles was investigated. Occurrence of hemorrhages, histopathological alterations, antioxidant status, activity of antioxidant enzymes and expression of genes involved in the xenobiotic response (pxr, abcb1 and cyp1a), were evaluated. The results showed a time and drug-dependent effect. Warfarin exposure induced hemorrhages, hepatocyte vacuolar degeneration, and altered the activity of glutathione peroxidase (GPx) and the expression of all the studied genes. Dexamethasone exposure increased liver glycogen content, altered antioxidant status, GPx and superoxide dismutase activities, as well as abcb1 and cyp1a expression. Imidazole induced hepatocyte vacuolar degeneration and ballooning, and altered the antioxidant status and expression of the tested genes. The present work anticipates a deeper impact of pharmaceuticals on the aquatic environment than previously reported, thus underlining the urgent need for an integrated risk assessment.

Evaluation of an oral DNA nanovaccine against photobacteriosis in Solea senegalensis

Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for aquaculture in southern Europe, whose production is affected by the appearance of bacterial diseases such as photobacteriosis, a septicemia caused by Photobacterium damselae subsp. piscicida (Phdp). The aim of this study was to obtain an oral DNA nanovaccine and to evaluate its efficacy against Phdp in S. senegalensis juveniles. For this purpose, the amplified product corresponding to the protein inosine-5′-monophophate dehydrogenase (IMPDH) from Phdp, was cloned into the expression vector pcDNA™6.2/C-EmGFP-GW obtaining the DNA vaccine named as pPDPimpdh. The correct transcription and protein expression was verified at 48 h post tansfection in HEK293 cells. Chitosan nanoparticles (CS-TPP NPs) were prepared by ionotropic gelation and their features were appropriate for use as oral delivery system. Therefore, pPDPimpdh was protected with chitosan CS-TPP NPs throughout complex coacervation method giving as a result a DNA nanovaccine referred as CS-TPP+pPDPimpdh NPs. Sole juveniles were vaccinated orally with CS-TPP NPs, pPDPimpdh and CS-TPP+pPDPimpdh NPs followed by a challenge with Phdp at 30 days post vaccination (dpv). The relative percentage survival (RPS) for pPDPimpdh vaccinated groups was 6.25%, probably due to its degradation in the digestive tract. RPS value obtained for CS-TPP NPs and CS-TPP+pPDPimpdh NPs was 40% and antibodies were observed in both cases. However, a delay in mortality was observed in sole juveniles vaccinated orally with CS-TPP+pPDPimpdh NPs. In fact, an upregulation of tf, mhcII, cd8a and igm in the posterior gut and c3, hamp1, tf and cd4 in spleen was observed in juveniles vaccinated with CS-TPP+pPDPimpdh NPs. After challenge, a modulation of cd8a and cd4 expression levels in the posterior gut and c3, tf, lyg, cd4, igm and igt expression levels in spleen was observed. Moreover, the concentration of lysozyme in skin mucus significantly increased in fish vaccinated orally with CS-TPP+pPDPimpdh NPs at 11 dpc. These data indicate that oral vaccination with CS-TPP+pPDPimpdh NPs could be acting through the non-specific immune responses as well as the specific humoral and cell mediated immunity and provide the first step toward a development of an oral DNA nanovaccine against Phdp in sole.

Feeding Protocol Modulates the Digestive Process in Senegalese Sole (Solea senegalensis) Juveniles

Understanding the daily digestive function could lead to more efficient feeding practices in the farmed fish species. In this work, the effect of several daily feeding protocols was assessed on the daily expression of the genes related to the circadian system (arntl1, clock, per1, and cry1), appetite (cck), nutrients hydrolysis (try1, ctrb1, pga, atp4a, cel, and amy2a), peptides transport (slc15a1), pH, and activity of key digestive (trypsin, chymotrypsin, amylase, 4C- and 7C-like lipases) and absorptive (alkaline phosphatase) enzymes in the gastrointestinal tract (GIT) of Senegalese sole (Solea senegalensis) juveniles. Some of the tested feeding protocols demonstrated a significant influence on the entrainment of the peripheral oscillator. The synchrony of slc15a1 and some digestive proenzymes with per1 in some of the experimental treatments could underline the role of clock genes in the regulation of these biological functions. Unlike gene expression of the digestive proenzymes wherein feeding protocol occasionally affected the temporal organization, the influence of this factor was determinant on the daily average production and/or circadian pattern of their activities. Daily pH was maintained at approximately neutral levels in all the GIT segments, and no pepsin activity was detected. Fish fed a single diurnal meal showed the lowest activity for all the studied enzymes, except for amylase, suggesting an insufficient utilization of the ingested nutrients. Higher amylase activity in this group, on the other hand, implies the possible activation of alternative compensatory metabolic pathways. Overall, our results pointed to the suitability of higher feeding frequencies, especially during dark hours, for the aquaculture production of this species.

Biological responses and toxicopathic effects elicited in Solea senegalensis juveniles by waterborne exposure to benzo[a]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are priority contaminants in coastal and estuarine ecosystems under anthropogenic pressure. Although PAHs tend to accumulate in the sediment, toxicity for benthic flat fish such as soles may be caused by PAHs released from the sediment to the water column. Within this context, the present investigation aims at recognizing toxicopathic effects elicited after waterborne exposure to benzo[a]pyrene B[a]P, a model individual PAH compound, in juvenile Solea senegalensis. Sole juveniles were exposed to various concentrations of waterborne B[a]P for 3 and 7 days. Brain, liver, gills and gonad were the target tissues selected to determine biochemical and lysosomal biomarkers, and histopathology. Biological responses and toxicopathic effects were consistent with B[a]P concentration and exposure time. From day 3, hepatic catalase inhibition indicated potential oxidative effects of B[a]P. At day 7, contaminant exposure produced hepatic glutathione-S-transferase induction at low concentrations and inhibition at higher levels, evidencing a bell-shaped response. A clear gradient in lysosomal membrane destabilisation was observed in relation with B[a]P concentrations. Histopathological lesions were more frequent at day 7 and at higher contaminant levels. It seems that environmentally relevant waterborne concentrations of B[a]P (1000 ng/l) would suffice to cause toxicopathic effects on sole juveniles in relatively short exposure times. In agreement, the Integrative Biological Response index (IBR/n) indicated a dose-dependent decline in health condition upon exposure to B[a]P (IBR/nHighB[a]P > IBR/nMidB[a]P > IBR/nLowB[a]P > IBR/nDMSO > IBR/nControl). Overall, changes in antioxidant enzymes activity, lysosomal biomarkers and gill and liver histopathology are responsive early-warning signs of health disturbance in sole juveniles exposed to waterborne PAHs.

Daily rhythms of intestinal cholecystokinin and pancreatic proteases activity in Senegalese sole juveniles with diurnal and nocturnal feeding

The influence of diurnal and nocturnal feeding on daily rhythms of gut levels of cholecystokinin (CCK) and the activity of two key pancreatic proteases, trypsin and chymotrypsin, were examined in juveniles of Senegalese sole (Solea senegalensis), a species with nocturnal habits. Four feeding protocols were performed: P1) One morning meal; P2) Six meals during the light period; P3) Six meals during the dark period; and P4) 12 meals during 24 h. Daily activity patterns of both proteases were remarkably similar and showed a high correlation in all the experimental protocols. In P1, daily patterns of CCK and digestive enzymes showed a single maximum. In P2, CCK levels exhibited two peaks. Digestive enzymes activities showed slightly delayed peaks compared to CCK, although their daily fluctuations were not significant. In P3, intestinal CCK concentration exhibited two peaks at the end of light and dark periods, but only the second one was significant. The first maximum level of chymotrypsin activity occurred 4 h after the first CCK peak, while the second one coincided with the second CCK peak. Fluctuations of trypsin activity were not significant. In P4, CCK concentration showed three small peaks. Digestive enzymes daily fluctuations were not significant, although they showed an inverted trend with respect to CCK. The daily pattern of the gut CCK content in our study is in agreement with the anorexigenic function of this hormone. Our results support the existence of a negative feedback regulatory loop between CCK and pancreatic proteolytic enzymes in Senegalese sole juveniles.

Mucosal immune responses in Senegalese sole (Solea senegalensis) juveniles after Tenacibaculum maritimum challenge: A comparative study between ocular and blind sides

Most pathogens start the process of infection at the mucosal surfaces and therefore the mucosal immune response plays an essential role in the course of the infection. Due to the Senegalese sole (Solea senegalensis Kaup) condition of flatfish, the present comparative study aimed to analyse several immune-related enzymes as well as the bactericidal activity in the skin mucus from ocular and blind sides. For this purpose, Senegalese sole juveniles were bath challenged with a sub-lethal dose of Tenacibaculum maritimum for 24 h and sampled at 1, 2 and 3 weeks. The haematological profile and immune-related parameters were also measured in plasma in order to evaluate the systemic immune response after T. maritimum challenge. Results from this study showed that most parameters tested increased in skin mucus of bath challenged fish compared to unchallenged ones. In contrast, the sub-lethal dose tested did not influence the haematological profile including peripheral numbers the different leucocyte types. No variations were observed in plasma lysozyme, peroxidase, protease and haemolytic complement activities between unchallenged and bath challenged fish. This study suggests that the studied innate immune-related molecules are constitutively present in both skin mucus sides but at different levels. Interestingly, the levels of most parameters measured were higher on the ocular side than on the blind side, possibly due to the higher exposure to invasion by waterborne microorganisms on this side. Therefore, the present study brings some insights regarding local immune responses after bacterial challenge in skin mucus from the ocular and blind sides in one of the most valuable flatfish species in southern Europe.

Effects of the sulfated polysaccharide ulvan from Ulva ohnoi on the modulation of the immune response in Senegalese sole (Solea senegalensis)

Sulfated polysaccharides derived from green seaweeds exhibit many beneficial biological activities and have great potential to be used as nutraceutical in aquaculture. In this work, we evaluated the effects of the sulfated polysaccharide ulvan from Ulva ohnoi on Senegalese sole (Solea senegalensis) juveniles at the transcriptomic level. Cytotoxicity assay performed in liver primary cell cultures from sole determined that the different ulvan concentrations assayed did not impair cell viability. Juveniles were intraperitoneally (IP) injected with ulvan (0.5 mg/fish) followed by a challenge with Photobacterium damselae subsp. piscicida (Phdp) at 7 days. RNASeq analyses at 2 days post injection (dpi) revealed that 402 transcripts were differentially expressed in liver between ulvan IP injected and control groups before the challenge. Genes related to bacterial and antiviral defence, complement system, chemokines, proteasomes and antigen presentation were upregulated in ulvan treated groups. A detailed expression analysis of sixteen genes related to innate and adaptive immune system was performed in two systemic tissues: liver and spleen. Ulvan injection provoked the upregulation of tlr22 and a transient inflammatory response was initiated in both liver and spleen at 2 dpi. As consequence, expression of acute phase proteins, antimicrobial peptides and complement genes was induced. Moreover, expression of mhcI, mhcII, psmb10 and bcl6 was also induced 2 dpi. At 2 dpi with Phdp, inflammatory cytokines and genes related to bacterial and antiviral defense, iron metabolism, complement system and antigen presentation were differentially modulated in survival juveniles previously IP injected with ulvan. Moreover, mortality was retarded in ulvan treated juveniles. These results provide new evidence about the role of ulvan as a bioactive compound with immunomodulatory activity in Senegalese sole as well as its possible use as vaccine adjuvant against Phdp. This is the first published study that evaluates the transcriptomic response of Senegalese sole IP injected with ulvan.

Valorisation and enhanced sustainability of Senegalese sole (Solea senegalensis) aquaculture by dietary use of ditch shrimp (Palaemonetes varians) meal

The European Blue Growth Strategy requires innovative proposals contributing to sustainable development of the aquaculture activity. Within this context, ditch shrimp (Palaemonetes varians) meal obtained from extensively managed aquaculture wetlands was assessed as a potential source to substitute dietary fishmeal (FM) in compound diets for Senegalese sole (Solea senegalensis) juveniles. Estuarine ditch shrimp biomass from an integrated aquaculture system was converted into a meal that contained 61.9% protein and 9.2% lipid on a dry matter (DM) basis. The n-3 highly unsaturated fatty acids (HUFA) constituted the main fraction of the polyunsaturated fatty acids (PUFA). Three isonitrogenous (55% DM), isolipidic (12% DM) and isoenergetic (17 kJ g−1 DM) diets containing three substitution levels of FM by ditch shrimp meal (SM) were elaborated: 100% FM + 0% SM (SM0); 50% FM + 50% SM (SM50) and 0% FM + 100% SM (SM100). The experimental diets were tested on duplicate groups of sole juveniles (~4 g body weight) in an experiment that lasted 63 days. The substitution of dietary FM with SM did not influence growth and feed intake of the reared fish. Juveniles fed SM100 significantly decreased their muscle and skin lipid depositions in comparison with soles fed SM0. Moreover, muscle lipid analyses showed arachidonic acid (ARA) enrichment caused by the SM dietary use and a major presence of the n-3 over n-6 PUFA. Overall results demonstrate that dietary incorporation of P. varians meal in compound diets for Senegalese sole would add nutritional value to the harvested product with no impact on growth performance of reared fish. The dietary use of a low trophic level marine resource is an innovative practice that may contribute to a further sustainable development of aquaculture of carnivorous fish species.

Senegalese sole (Solea senegalensis) coping styles are consistent over time: behavioural and physiological responses during ontogenesis

Individuals differ in how they cope with stressful situations along a behavioural continuum, being proactive and reactive at the extremes of this continuum. Proactive individuals are usually bold, highly active and take risks, while reactive organisms are generally shy, exhibit low activity and avoid risky situations. Definitions of stress coping styles state that proactive and reactive traits are consistent over time and across contexts. The present study evaluated the individual differences in stress coping style, physiological changes and reproductive status in Senegalese sole juveniles and breeders over three and two-years, respectively. To determine stress coping style, the fish were subjected to three individual (restraining, new environment, confinement) and one group screening test (risk taking). Both groups were tested on three occasions, juveniles were tested each year and adults were tested in the first year and twice (spring and autumn) in the second year. On the third year, a proportion of the juveniles initiated puberty and the reproductive status of all individuals was assessed and compared with their behavioural responses. Results demonstrated individual differences that were consistent with proactive and reactive traits in juveniles and breeders. Significant intra-individual repeatability and consistency of juveniles and breeder's behavioural responses were observed over time and across situations. In addition, glucocorticoid levels (cortisol) were consistent for individuals. Another result to highlight was that juveniles that past puberty and initiated gametogenesis had significant higher activity, risk predisposition and lower plasma cortisol levels compared to fish that remained immature (did not initiate puberty).

Linking stress coping styles with brain mRNA abundance of selected transcripts for Senegalese sole (Solea senegalensis) juveniles

In fish, proactive and reactive individual stress copying styles (SCS) have been used to resolve variation in molecular expression data. Stress coping styles have been previously described in several stages of Solea senegalensis by validating for the species the use of standard behavioural screening tests. The present study aimed to link behavioural SCS tests with brain transcript abundance in early Senegalese sole juveniles in order to observe the natural variation in a molecular pathway in this species. A total of 50 juveniles were subjected to three individual behavioural (Restraining, New environment and Confinement) and one group (Risk-taking) screening tests. The fish were classified in SCS categories by applying a hierarchical cluster to the variable “Total activity” (the total activity time that the fish was moving in each individual test). Three categories were defined, proactive, intermediate and reactive sole. Six transcripts were chosen and tested, one related to basic metabolism (gapdh-2), three to feeding behaviour (per1, igf-Ia, pparß) and two to the stress response (crh-BP and hsp90aa) in 30 juveniles (10 individuals per SCS category) using rt-qPCR to observe differences in the abundance of those transcripts among SCS. Four transcripts were differentially expressed (DETs) among them. The transcript gapdh-2 showed up-regulation for proactive and intermediate SCS sole while reactive individuals showed down-regulation. Target mRNAs per1, igf-Ia and pparß, showed different levels of up-regulation for proactive and reactive fish while intermediates were highly down-regulated. Surprisingly no differences in stress related transcripts were observed. Correlations were found between variation in coping styles and variation in the abundance of mRNAs involved in important biological functions in Senegalese sole. These results are the first evidence of the relationship between the behavioural individual variation and the fluctuation in brain transcripts abundance in Senegalese sole.

Evaluation of Ulva ohnoi as functional dietary ingredient in juvenile Senegalese sole (Solea senegalensis): Effects on the structure and functionality of the intestinal mucosa

This study evaluates the effects of the macroalgae Ulva ohnoi on growth and intestinal functionality and integrity of Solea senegalensis juveniles in a 90-day feeding trial. Ulva diet (U-5) included 5% dry U. ohnoi biomass, whereas an algae-free diet was used as control. After being fed for 45 and 90 days, fish body weight was recorded for growth evaluation, and biological samples were taken. The inclusion of U. ohnoi (5%) significantly affected growth performance of sole juveniles, given that after being fed for 45 and 90 days fish showed lower body weight, specific growth and weight gain rates than fish fed with the control diet. Compared to fish fed with control diet, protease activities measured in the pancreatic secretion was also reduced when Ulva was included in the diet, but these fish also showed increased alkaline phosphatase activity in their intestinal mucosa. Quantitative PCR revealed changes in the relative expression of a set of genes coding for enzymes involved in lipid absorption and metabolism, several digestive enzymes, as well as for some stress response indicators. Electron microscopy revealed a lack of damage in the intestinal mucosa, as well as significantly increased mucosal absorptive surface in those fish fed with Ulva-supplemented diet for 45 days, compared to control fish. The results suggest that U. ohnoi at low inclusion level could protect the intestinal epithelium of fish, but a relatively short administration period is recommended with the aim of avoiding negative effects on Senegalese sole growth and proteolytic digestive enzyme activities.

Effects of dietary vegetable oils on the expression of eicosanoid receptors genes in Senegalese sole (Solea senegalensis) intestine

Vegetable oils (VO) are being extensively used as lipid sources in marine fish diets, due to the decreased availability of fish oil. The use of VO could induce some imbalances on essential fatty acids (EFAs) with the subsequent well described detrimental effects on fish immunity. EFAs are directly associated with several immune functions, acting as important elements for the fluidity of cytoplasmic membranes and as precursors of eicosanoids. At intestinal level, eicosanoids produced via cyclooxygenase (COX-2) enzymes are involved in gastrointestinal cyto-protection by influencing the homeostasis regulation and the onset of gut inflammation process through the action on eicosanoid receptors (EPs), which modulate gastrointestinal mucosal integrity amongst other functions. Senegalese sole is a flatfish with high susceptibility to deleterious effects due to stress and diseases and highly sensitive to changes in dietary fatty acids. In this study, Senegalese sole juveniles were fed diets (56% crude protein, 12% crude lipid) containing linseed (100LO), soybean (100SO) or fish (100FO) oils as unique oil source. Intestinal fatty acid composition plus relative expression of intestinal EPs and other genes related with eicosanoid cascade were determined after 90 days. Moreover, at the end of the experiment a stress test (5 min of net chasing) was carried out. The use of VO altered the fatty acid profile of intestine and the expression of EP genes. The use of SO (rich in n-6 fatty acids) induced an overexpression of cox-2 in basal (non-stress) situation, plus a down-regulation of intestinal ep4 gene expression. Besides, fish fed soybean oil in the diet seems to be unable to respond properly to chasing stress.

Characterization of bacterioplankton communities from a hatchery recirculating aquaculture system (RAS) for juvenile sole (Solea senegalensis) production.

There is a growing consensus that future technological developments of aquaculture systems should account for the structure and function of microbial communities in the whole system and not only in fish guts. In this study, we aimed to investigate the composition of bacterioplankton communities of a hatchery recirculating aquaculture system (RAS) used for the production of Senegalese sole (Solea senegalensis) juveniles. To this end, we used a 16S rRNA gene based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing analyses to characterize the bacterioplankton communities of the RAS and its water supply. Overall, the most abundant orders were Alteromonadales, Rhodobacterales, Oceanospirillales, Vibrionales, Flavobacteriales, Lactobacillales, Thiotrichales, Burkholderiales and Bdellovibrionales. Although we found a clear distinction between the RAS and the water supply bacterioplankton communities, most of the abundant OTUs (≥50 sequences) in the hatchery RAS were also present in the water supply. These included OTUs related to Pseudoalteromonas genus and the Roseobacter clade, which are known to comprise bacterial members with activity against Vibrio fish pathogens. Overall, in contrast to previous findings for sole grow-out RAS, our results suggest that the water supply may influence the bacterioplankton community structure of sole hatchery RAS. Further studies are needed to investigate the effect of aquaculture practices on RAS bacterioplankton communities and identification of the key drivers of their structure and diversity.

Growth performance, body composition, and digestive functionality of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles fed diets including microalgae freeze-dried biomass.

Senegalese sole is one of the most promising fish species cultivated in the Southern European countries. This study was aimed at assessing the effects of microalgae biomass added to diets for Senegalese sole juveniles on fish growing and condition status. Three isoproteic (52%) and isolipidic (10%) were formulated containing 15% Tisochrysis lutea (TISO), Nannochloropsis gaditana (NAN), or Scenedesmus almeriensis (SCE) biomass, respectively. An experimental microalgae-free diet (CT) and a commercial diet (COM) were used as controls. Fish were fed at 3% of their body weight for 85days. Final body weight of fish fed microalgae-supplemented diets did not differ from group fed CT diet. Fish-fed CT, TISO, NAN, and SCE showed higher growth performance and nutrient utilization figures than specimen-fed COM diet. The highest carcass lipid content was found in COM group (141gkg-1), and no differences were observed in body protein content. Ash was significantly higher in TISO, NAN, and SCE groups compared to fish-fed CT. Muscle EPA and DHA contents were not modified owing to the different dietary treatments. The n3/n6 and EPA/DHA ratios in muscle were similar in all the experimental groups. The quantification of digestive proteolytic activities did not differ among experimental groups, although differences in the protease pattern in digestive extracts by zymography were revealed in those fish fed on COM diet. Both α-amylase activity in the intestinal lumen and leucine aminopeptidase in the intestinal tissue were significantly lower in COM fish. Specimens fed on SCE diet showed a higher leucine aminopeptidase activity associated to the intestinal tissue compared to NAN-fed fish (0.40 and 0.25Ugtissue-1, respectively). The ultrastructural study revealed that the dietary inclusion of algal biomass, especially T. lutea and N. gaditana, had a positive impact on the absorptive capacity of the intestinal mucosa. The highest values for the parameters microvilli length and microvilli absorption surface were observed in fish fed on NAN diet (1.99μm and 45.93μm2, respectively). Even though further studies aimed at optimizing commercial formulas for Senegalese sole are required prior to any large-scale practical utilization, the results obtained clearly suggest the potential of microalgae as dietary ingredients for this fish species.

Acute hyperoxia induces systemic responses with no major changes in peripheral tissues in the Senegalese sole (Solea senegalensis Kaup, 1858)

Senegalese sole Solea senegalensis is currently farmed in recirculation aquaculture systems that often involve water re-oxygenation, which in turn may cause acute or prolonged hyperoxia exposures. In order to understand the impact of acute hyperoxia on the fish immune system and peripheral tissues such as gills and gut, Senegalese sole juveniles (30.05 ± 1.72 g) were exposed to normoxia (100% O2sat) as control and two hyperoxic conditions (150 and 200% O2sat) and sampled at 4 and 24 h. Fish haematological profile, total and differential blood cell counts and plasma immune parameters were analysed. Histomorphology and immunofluorescence analyses of gills and intestine were performed, respectively, whereas head-kidney samples were used for assessing the expression of immune-related genes. Results indicate that acute hyperoxia exposure may reduce fish erythrocyte and haemoglobin levels. Moreover, decreases in total leucocytes numbers, circulating lymphocytes, monocytes, alternative complement pathway activity and expression of cyclooxygenase-2 were observed in fish exposed to hyperoxia. In contrast, hyperoxia did not induce major effects on gill histomorphology nor in the protein content of ion and glucose cotransporters as well as a macrophage marker (V-ATPase) in the intestine. Although the activation of humoral mechanisms and immune-related genes were not dramatically affected by acute hyperoxia, the compromised immune cell status and the reduction of some inflammatory indicators are issues to consider under acute hyperoxia conditions.