Analysis across diverse fish species highlights no conserved transcriptome signature for proactive behaviour.

BACKGROUND: Consistent individual differences in behaviour, known as animal personalities, have been demonstrated within and across species. In fish, studies applying an animal personality approach have been used to resolve variation in physiological and molecular data suggesting a linkage, genotype-phenotype, between behaviour and transcriptome regulation. In this study, using three fish species (zebrafish; Danio rerio, Atlantic salmon; Salmo salar and European sea bass; Dicentrarchus labrax), we firstly address whether personality-specific mRNA transcript abundances are transferrable across distantly-related fish species and secondly whether a proactive transcriptome signature is conserved across all three species. RESULTS: Previous zebrafish transcriptome data was used as a foundation to produce a curated list of mRNA transcripts related to animal personality across all three species. mRNA transcript copy numbers for selected gene targets show that differential mRNA transcript abundance in the brain appears to be partially conserved across species relative to personality type. Secondly, we performed RNA-Seq using whole brains from S. salar and D. labrax scoring positively for both behavioural and molecular assays for proactive behaviour. We further enriched this dataset by incorporating a zebrafish brain transcriptome dataset specific to the proactive phenotype. Our results indicate that cross-species molecular signatures related to proactive behaviour are functionally conserved where shared functional pathways suggest that evolutionary convergence may be more important than individual mRNAs. CONCLUSIONS: Our data supports the proposition that highly polygenic clusters of genes, with small additive effects, likely support the underpinning molecular variation related to the animal personalities in the fish used in this study. The polygenic nature of the proactive brain transcriptome across all three species questions the existence of specific molecular signatures for proactive behaviour, at least at the granularity of specific regulatory gene modules, level of genes, gene networks and molecular functions.

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).

Using model selection to choose a size-based condition index that is consistent with operational welfare indicators.

Quantitative and qualitative measures of fish health and welfare are essential for management of both wild capture and aquaculture species. These measures include morphometric body condition indices, energetic condition and aquaculture operational welfare indicators (OWIs). Measures vary in ease of measurement (and may require destructive sampling), and it is critical to know how well they correlate with fish health and welfare so appropriate management decisions can be based on them. Lumpfish (Cyclopterus lumpus) is a new farming species that needs nondestructive OWIs to be developed and validated. In this study, we developed a C. lumpus fin damage score. Four different body condition indexes based on individual weight relative to either length-weight relationships or relative to other fish in its local environment were tested (using model selection) as predictors of individual fin damage. Results showed severity of fin damage was predicted by small size relative to the other individuals in the tank or cage. Body condition based on length-weight relationship was not found to predict fin damage, indicating that using established indices from fisheries or from other species would not predict welfare risks from fin damage. Implications are that especially in hatchery conditions grading will improve the condition index, and is expected to mitigate fin damage, but that low weight at length was not of use in predicting fin damage. Model selection to choose between a suite of possible indices proved powerful and should be considered in other applications where an easily measured index is needed to correlate with other health measures.

Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients.

Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.

The expression of TRPV channels, prostaglandin E2 and pro-inflammatory cytokines during behavioural fever in fish.

A fever, or increased body temperature, is a symptom of inflammation, which is a complex defence reaction of the organism to pathogenic infections. After pathogens enter the body, immune cells secrete a number of agents, the functions of which stimulate the body to develop a functional immune and fever response. In mammals it is known that PGE2 is the principal mediator of fever. The extent to which PGE2 and other pro-inflammatory cytokines such as TNF-α, IL-6, or IL-1ß could be involved in the induction of behavioural fever in fish remains to be clarified. Several members of the transient receptor potential (TRP) family of ion channels have been implicated as transducers of thermal stimuli, including TRPV1 and TRPV2, which are activated by heat. Here we show that members of the TRP family, TRPV1 and TRPV4, may participate in the coordination of temperature sensing during the behavioural fever. To examine the behavioral fever mechanism in Salmo salar an infection with IPNV, infectious pancreatic necrosis virus, was carried out by an immersion challenge with 10 × 105 PFU/mL-1 of IPNV. Behavioural fever impacted upon the expression levels of both TRPV1 and TRPV4 mRNAs after the viral challenge and revealed a juxtaposed regulation of TRPV channels. Our results suggest that an increase in the mRNA abundance of TRPV1 is tightly correlated with a significant elevation in the expression of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and PGE2) in the Pre-Optic Area (POA) and cytokine release in plasma. Together, these data indicate that the reduction of TRPV4 expression during behavioural fever may contribute to the onset of behavioural fever influencing movement toward higher water temperatures. Our data also suggest an effect of TRPV channels in the regulation of behavioural fever through activation of EP3 receptors in the central nervous system by PGE2 induced by plasma-borne cytokines. These results highlight for first time in mobile ectotherms the key role of pro-inflammatory cytokines and TRPV channels in behavioural fever that likely involves a complex integration of prostaglandin induction, cytokine recognition and temperature sensing.

Thermal preference predicts animal personality in Nile tilapia Oreochromis niloticus.

Environmental temperature gradients provide habitat structure in which fish orientate and individual thermal choice may reflect an essential integrated response to the environment. The use of subtle thermal gradients likely impacts upon specific physiological and behavioural processes reflected as a suite of traits described by animal personality. In this study, we examine the relationship between thermal choice, animal personality and the impact of infection upon this interaction. We predicted that thermal choice in Nile tilapia Oreochromis niloticus reflects distinct personality traits and that under a challenge individuals exhibit differential thermal distribution. Nile tilapia were screened following two different protocols: 1) a suite of individual behavioural tests to screen for personality and 2) thermal choice in a custom-built tank with a thermal gradient (TCH tank) ranging from 21 to 33 °C. A first set of fish were screened for behaviour and then thermal preference, and a second set were tested in the opposite fashion: thermal then behaviour. The final thermal distribution of the fish after 48 h was assessed reflecting final thermal preferendum. Additionally, fish were then challenged using a bacterial Streptococcus iniae model infection to assess the behavioural fever response of proactive and reactive fish. Results showed that individuals with preference for higher temperatures were also classified as proactive with behavioural tests and reactive contemporaries chose significantly lower water temperatures. All groups exhibited behavioural fever recovering personality-specific thermal preferences after 5 days. Our results show that thermal preference can be used as a proxy to assess personality traits in Nile tilapia and it is a central factor to understand the adaptive meaning of animal personality within a population. Importantly, response to infection by expressing behavioural fever overrides personality-related thermal choice.

Fish can show emotional fever: stress-induced hyperthermia in zebrafish.

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as 'emotional fever'. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2-4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes.

Draft genome sequence of Klebsiella pneumoniae KLEB-33: a convergent biofilm hyperforming multiresistant strain belonging to the emerging ST16 lineage harboring multiple hypervirulence genes.

The emergence of convergent Klebsiella pneumoniae strains showing multiresistance, characteristic of nosocomial pathotypes and hypervirulent traits typical of community-acquired isolates, makes them important models for studying K. pneumoniae pathogenesis. Here, we describe the convergent, multidrug-resistant KLEB-33 strain harboring several hypervirulence genes and make its genome available to the scientific community.

Behavioural fever is a synergic signal amplifying the innate immune response.

Behavioural fever, defined as an acute change in thermal preference driven by pathogen recognition, has been reported in a variety of invertebrates and ectothermic vertebrates. It has been suggested, but so far not confirmed, that such changes in thermal regime favour the immune response and thus promote survival. Here, we show that zebrafish display behavioural fever that acts to promote extensive and highly specific temperature-dependent changes in the brain transcriptome. The observed coupling of the immune response to fever acts at the gene-environment level to promote a robust, highly specific time-dependent anti-viral response that, under viral infection, increases survival. Fish that are not offered a choice of temperatures and that therefore cannot express behavioural fever show decreased survival under viral challenge. This phenomenon provides an underlying explanation for the varied functional responses observed during systemic fever. Given the effects of behavioural fever on survival and the fact that it exists across considerable phylogenetic space, such immunity-environment interactions are likely to be under strong positive selection.

Imported malaria in an area in southern Madrid, 2005-2008.

BACKGROUND: In Spain, malaria cases are mostly due to migrants and travellers returning from endemic areas. The objective of this work was to describe the malaria cases diagnosed at the Severo Ochoa University Hospital (HUSO) in Leganés in the south of the Madrid Region from 2005 to 2008. METHODS: Descriptive retrospective study performed at HUSO. Data sources are registries from the Microbiology Department and malaria cases notified to the Preventive Medicine Department. Analysed parameters were: administrative, demographical, related to the stay at the endemic country, clinical, microbiological diagnosis method, pregnancy, treatment and prophylaxis, co-infections, and days of hospital stay. RESULTS: Fifty-seven patients diagnosed with malaria were studied. Case distribution per year was 13 in 2005, 15 in 2006, 15 in 2007 and 14 in 2008. Thirty-three patients were female (57.9%) and 24 male (42.1%). Mean age was 27.8 years. Most of the malaria cases were acquired in Nigeria (49.1%) and Equatorial Guinea (32.7%). 29.1% of the patients were immigrants who had arrived recently, and 61.8% acquired malaria when travelling to their countries of origin to visit friends and relatives (VFR). Majority of cases were diagnosed between June and September. Microscopy was positive in 39 cases (68.4%) immunochromatography in 42 (73.7%) and PCR in the 55 cases where performed. Plasmodium falciparum was responsible for 94.7% of the cases. The more frequent symptoms were fever (77.2%), followed by headache and gastrointestinal symptoms (33.3%). Nine cases needed hospital admittance, a pregnant woman, three children, four VFR and an African tourist, but all evolved favourably. Chemoprophylaxis data was known from 55 patients. It was taken correctly in one case (1.8%), in five (9.1%) the prophylaxis was improper while the others 49 (89.1%) cases had not followed any anti-malarial prophylaxis. CONCLUSIONS: Children, pregnant women and the VFR have the highest risk to present severe malaria and to need hospital admittance. Another important risk factor for acquiring malaria is incorrect prophylaxis. The first place for malaria acquisition was Nigeria and the main species causing malaria was P. falciparum.

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.

Liver colour scoring index, carotenoids and lipid content assessment as a proxy for lumpfish (Cyclopterus lumpus L.) health and welfare condition.

Ensuring lumpfish health and welfare in salmon farms is vital to reduce the high mortality rates reported and to guarantee a high delousing efficiency. Recent observations of farmed lumpfish livers have shown colours ranging from pale (colours 1 and 2), through bright orange (colours 3 and 4), to dark reddish-brown (colours 5 and 6), some of which may be related to welfare condition. To characterize the status of lumpfish deployed in four Faroese salmon farms, several welfare indicators were assessed: a weight-length relationship, scoring of external physical damage, and after dissection, stomach content and liver colour scoring. Liver samples were weighed, stored and analysed for lipid content, lipid classes, total pigments, fatty acid profile and histopathology to explain the differences between the mentioned liver colours. Bright orange livers, liver colours 3 and 4, were related to increased levels of carotenoid pigments rather than levels of lipids and appear to reflect good fish welfare. However, dark reddish-brown colours, liver colours 5 and 6, were associated with very low levels of triacyl glycerides in the liver, indicating use of lipid reserves and poor welfare condition. Histopathology confirmed that the dark reddish-brown livers, liver colours 5 and 6, formed a distinct group. Thus, liver colour was shown to be a good welfare indicator and should be monitored in farms.

Physiological responses during acute stress recovery depend on stress coping style in European sea bass, Dicentrarchus labrax.

Individual stress coping style (reactive, intermediate and proactive) was determined in 3 groups of 120 pit tagged European seabass using the hypoxia avoidance test. The same three groups (no change in social composition) were then reared according to the standards recommended for this species. Then, 127 days later, individuals initially characterized as reactive, intermediate or proactive were submitted to an acute confinement stress for 30 min. Blood samples were taken to measure plasma cortisol levels 30 min (Stress30) or 150 min (Stress150) after the end of the confinement stress. Individuals were then sacrificed to sample the telencephalon in order to measure the main monoamines and their catabolites (at Stress30 only). Individuals from Stress150 were sampled for whole brain for a transcriptomic analysis. The main results showed that reactive individuals had a lower body mass than intermediate individuals which did not differ from proactive individuals. The physiological cortisol response did not differ between coping style at Stress30 but at Stress150 when intermediate and proactive individuals had recovered pre stress levels, reactive individuals showed a significant higher level illustrating a modulation of stress recovery by coping style. Serotonin turnover ratio was higher in proactive and reactive individuals compared to intermediate individuals and a significant positive correlation was observed with cortisol levels whatever the coping style. Further, the confinement stress led to a general increase in the serotonin turnover comparable between coping styles. Stress150 had a significant effect on target mRNA copy number (Gapdh mRNA copy number decreased while ifrd1 mRNA copy number increased) and such changes tended to depend upon coping style.

Proactive avoidance behaviour and pace-of-life syndrome in Atlantic salmon.

Individuals in a fish population differ in key life-history traits such as growth rate and body size. This raises the question of whether such traits cluster along a fast-slow growth continuum according to a pace-of-life syndrome (POLS). Fish species like salmonids may develop a bimodal size distribution, providing an opportunity to study the relationships between individual growth and behavioural responsiveness. Here we test whether proactive characteristics (bold behaviour coupled with low post-stress cortisol production) are related to fast growth and developmental rate in Atlantic salmon, Salmo salar. Boldness was tested in a highly controlled two-tank hypoxia test were oxygen levels were gradually decreased in one of the tanks. All fish became inactive close to the bottom at 70% oxygen saturation. At 40% oxygen saturation level a fraction of the fish actively sought to avoid hypoxia. A proactive stress coping style was verified by lower cortisol response to a standardized stressor. Two distinct clusters of bimodal growth trajectories were identified, with fast growth and early smoltification in 80% of the total population. There was a higher frequency of proactive than reactive individuals in this fast-developing fraction of fish. The smolts were associated with higher post-stress plasma cortisol than parr, and the proactive smolts leaving hypoxia had significant lower post-stress cortisol than the stayers. The study demonstrated a link between a proactive coping and fast growth and developmental ratio and suggests that selection for domestic production traits promotes this trait cluster.

'Love at first sight': The effect of personality and colouration patterns in the reproductive success of zebrafish (Danio rerio).

Individual differences in animal personality and external appearance such as colouration patterns have both been extensively studied separately. A significant body of research has explored many of pertinent ecological and biological aspects that can be affected by them and their impact upon fitness. Currently little is known about how both factors interact and their effect on reproductive success. In this study, we evaluated two major parameters contributing to the fitness of the species: reproduction and offspring survival. We selected two different phenotypes of the zebrafish (Danio rerio) selected by their colouration patterns: phenotype 1) named Wild type, UAB line (WT-UAB) with a homogeneous colouration pattern (clear and defined lateral stripes) and phenotype 2) Wild type indefinite (WT-I) had a heterogeneous colouration pattern and different degrees of lateral stripe definition. All animals were also screened for personality. We then compared their reproductive success (spawning rate) and offspring survival at different stages, from egg to larvae, and for 2 successive generations (parental generation was G0; First and second generations were G1 and G2 respectively). Our results show that personality traits were the main source of variability between the fitness components measured: both personalities had similar total numbers of eggs spawned but proactive animals, for both colour phenotypes, had higher reproductive success. This was reflected in a higher percentage of spawning viability at 1day post fertilization (dpf), higher total survival and growth rates at larval stages. Proactive phenotypes from WT-UAB population had a higher overall fitness in comparison to the other phenotypes studied. Our findings imply that fitness of this species when kept under similar husbandry conditions is significantly influenced by parental personality and not by their external appearance. Under these conditions the reproductive success is enhanced. The implications of this study are important for zebrafish breeding and husbandry in captivity and are relevant toward understanding the underlying drivers of trait selection in natural environments.

Sustainable production and use of cleaner fish for the biological control of sea lice: recent advances and current challenges.

Currently, cleaner fish are one of the most widely used sea lice control strategies in Atlantic salmon aquaculture. Two species are currently being farmed in North Atlantic countries, ballan wrasse (Labrus bergylta) and lumpfish (Cyclopterus lumpus), and the sector in most countries is rapidly expanding towards self-sufficiency. The species are very different both in terms of their biology and life histories and, consequently, production and husbandry methods must be tailored to each species. There are numerous health challenges currently experienced in both species, with bacterial and parasitic diseases being the most prevalent, and cohabitation with salmon may increase the risk of disease. Good husbandry and routine health monitoring are essential, although treatment is often required when disease outbreaks occur. Ballan wrasse and lumpfish are both proven to be effective salmon delousers, although delousing efficacy can be variable in farmed fish; the provision of suitable habitat and acclimation to net-pen conditions may encourage natural behaviours, including delousing, and the use of operational welfare indicators can highlight potential welfare issues. Cleaner fish research is progressing rapidly, although much of the basic knowledge regarding the species' biology remains unknown. The simultaneous domestication of two new marine aquaculture species is a significant challenge demanding sustained effort and funding over a prolonged period of time. Research must focus on enhancing the robustness of the farmed stocks and increasing hatchery outputs to meet the urgent demands from the salmon sector and protect wild stocks from overfishing.

Behavioural fever in zebrafish larvae.

Behavioural fever has been reported in different species of mobile ectotherms including the zebrafish, Danio rerio, in response to exogenous pyrogens. In this study we report, to our knowledge for the first time, upon the ontogenic onset of behavioural fever in zebrafish (Danio rerio) larvae. For this, zebrafish larvae (from first feeding to juveniles) were placed in a continuous thermal gradient providing the opportunity to select their preferred temperature. The novel thermal preference aquarium was based upon a continuous vertical column system and allows for non-invasive observation of larvae vertical distribution under isothermal (TR at 28 °C) and thermal gradient conditions (TCH: 28-32 °C). Larval thermal preference was assessed under both conditions with or without an immersion challenge, in order to detect the onset of the behavioural fever response. Our results defined the onset of the dsRNA induced behavioural fever at 18-20 days post fertilization (dpf). Significant differences were observed in dsRNA challenged larvae, which prefer higher temperatures (1-4 °C increase) throughout the experimental period as compared to non-challenged larvae. In parallel we measured the abundance of antiviral transcripts; viperin, gig2, irf7, trim25 and Mxb mRNAs in dsRNA challenged larvae under both thermal regimes: TR and TCh. Significant increases in the abundance of all measured transcripts were recorded under thermal choice conditions signifying that thermo-coupling and the resultant enhancement of the immune response to dsRNA challenge occurs from 18 dpf onwards in the zebrafish. The results are of importance as they identify a key developmental stage where the neuro-immune interface matures in the zebrafish likely providing increased resistance to viral infection.

Dominance behaviour in a non-aggressive flatfish, Senegalese sole (Solea senegalensis) and brain mRNA abundance of selected transcripts.

Dominance is defined as the preferential access to limited resources. The present study aimed to characterise dominance in a non-aggressive flatfish species, the Senegalese sole (Solea senegalensis) by 1) identifying dominance categories and associated behaviours and 2) linking dominance categories (dominant and subordinate) with the abundance of selected mRNA transcripts in the brain. Early juveniles (n = 74, 37 pairs) were subjected to a dyadic dominance test, related to feeding, and once behavioural phenotypes had been described the abundance of ten selected mRNAs related to dominance and aggressiveness was measured in the brain. Late juveniles were subjected to two dyadic dominance tests (n = 34, 17 pairs), related to feeding and territoriality and one group test (n = 24, 4 groups of 6 fish). Sole feeding first were categorized as dominant and sole feeding second or not feeding as subordinate. Three social behaviours (i. "Resting the head" on another fish, ii. "Approaching" another fish, iii. "Swimming above another" fish) were associated with dominance of feeding. Two other variables (i. Total time occupying the preferred area during the last 2 hours of the 24 h test, ii. Organisms occupying the preferred area when the test ended) were representative of dominance in the place preference test. In all tests, dominant fish compared to subordinate fish displayed a significantly higher number of the behaviours "Rest the head" and "Approaches". Moreover, dominant sole dominated the sand at the end of the test, and in the group test dominated the area close to the feed delivery point before feed was delivered. The mRNA abundance of the selected mRNAs related to neurogenesis (nrd2) and neuroplasticity (c-fos) in dominant sole compared to subordinate were significantly different. This is the first study to characterise dominance categories with associated behaviours and mRNA abundance in Senegalese sole and provides tools to study dominance related problems in feeding and reproduction in aquaculture.

The Lower Danube River-Danube Delta-North West Black Sea: A pivotal area of major interest for the past, present and future of its fish fauna--A short review.

The complex Danube-Black Sea geoecosystem, created by a unique combination of integrated biotopes and biocoenoses related forces and counter-forces in time and space, forms a rich "ichthyosystem". The equilibrium among the fish species captured in the Danube Delta reveals its structural and functional roles in the connectivity of the Danube and Black Sea. The key role of the delta is evidenced by the fact that 57.26% of the Lower Danube-Danube Delta-North West Black Sea fish species use two or three of the subsystems in terms of habitats. Therefore, this convergence area can be considered to be a dynamic and rich "ichthyosystem", with three subsystems. All three evolved interdependently, which permits their flexibility and adaptation in an interdependent way. The habitat heterogeneity, native economic and conservation priority fish species of the Lower Danube-Danube Delta-North Western Black Sea have decreased significantly, and there are no indications that this trend will be halted soon. The Danube "sub-ichtyosystem" seems to be more directly affected than the others. The Lower Danube-Danube Delta-North Western Black Sea "ichthyosystem" exhibits a significant level of flexibility, resilience and adaptation over geological time, but has become much more sensitive to environmental perturbations due to the last century of human impact. This "ichthyosystem" is affected by non-native fish species. The study area represents an interdependent ecological net, without which the specific "ichthyosystem" formed over geological time will disappear. The studied ecological net fish fauna is an accurate indicator of various human pressures. The Lower Danube-Danube Delta-North West Black Sea geoecosystem, in which the Danube Delta provides the pivotal habitat element, is the matrix for a unique "ichthyosystem." However, human impacts decrease its resilience and can induce its extinction.