Role of melanocortin system in the locomotor activity rhythms and melatonin secretion as revealed by agouti-signalling protein (asip1) overexpression in zebrafish.

Temporal signals such as light and temperature cycles profoundly modulate animal physiology and behaviour. Via endogenous timing mechanisms which are regulated by these signals, organisms can anticipate cyclic environmental changes and thereby enhance their fitness. The pineal gland in fish, through the secretion of melatonin, appears to play a critical role in the circadian system, most likely acting as an element of the circadian clock system. An important output of this circadian clock is the locomotor activity circadian rhythm which is adapted to the photoperiod and thus determines whether animals are diurnal or nocturnal. By using a genetically modified zebrafish strain known as Tg (Xla.Eef1a1:Cau.asip1)iim04, which expresses a higher level of the agouti signalling protein 1 (Asip1), an endogenous antagonist of the melanocortin system, we observed a complete disruption of locomotor activity patterns, which correlates with the ablation of the melatonin daily rhythm. Consistent with this, in vitro experiments also demonstrated that Asip1 inhibits melatonin secretion from the zebrafish pineal gland, most likely through the melanocortin receptors expressed in this gland. Asip1 overexpression also disrupted the expression of core clock genes, including per1a and clock1a, thus blunting circadian oscillation. Collectively, these results implicate the melanocortin system as playing an important role in modulating pineal physiology and, therefore, circadian organisation in zebrafish.

Exploring the potential for an evolutionarily conserved role of the taste 1 receptor gene family in gut sensing mechanisms of fish.

In this study, we investigated the transcriptional spatio-temporal dynamics of the taste 1 receptor (T1R) gene family repertoire in seabream (Sparus aurata [sa]), during larval ontogeny and in adult tissues. In early larval development, saT1R expression arises heterochronously, i.e. the extraoral taste-related perception in the gastrointestinal tract (GIT) anticipates first exogenous feeding (at 9 days post hatching [dph]), followed by the buccal/intraoral perception from 14 dph onwards, supporting the hypothesis that the early onset of the molecular machinery underlying saT1R expression in the GIT is not induced by food but rather genetically hardwired. During adulthood, we characterized the expression patterns of sa T1R within specific tissues (n = 4) distributed in oropharingeal, GIT and brain regions substantiating their functional versatility as chemosensory signaling players to a variety of biological functions beyond oral taste sensation. Further, we provided for the first time direct evidences in fish for mRNA co-expression of a subset of saT1R genes (mostly sa T1R3, i.e. the common subunit of the heterodimeric T1R complexes for the detection of "sweet" and "umami" substances), with the selected gut peptides ghrelin (ghr), cholecystokinin (cck), hormone peptide yy (pyy) and proglucagon (pg). Each peptide defines the enteroendocrine cells (ECCs) identity, and establishes on morphological basis, a direct link for T1R chemosensing in the regulation of fish digestive processes. Finally, we analyzed the spatial gene expression patterns of 2 taste signaling components functionally homologous to the mammalian G(i)α subunit gustducin, namely sa G( i )α1 and sa G( i )α2, and demonstrated their co-localization with the saT1R3 in EECs, thus validating their direct involvement in taste-like transduction mechanisms of the fish GIT. In conclusion, data provide new insights in the evolutionary conservation of gut sensing in fish suggesting a conserved role for nutrient sensors modulating entero-endocrine secretion.

Distribution of two isoforms of tryptophan hydroxylase in the brain of rainbow trout (Oncorhynchus mykiss). An in situ hybridization study.

Serotonin (5-HT) is one of the principal neurotransmitters in the nervous system of vertebrates. It is initially synthesized by hydroxylation of tryptophan (Trp) by means of tryptophan hydroxylase or TPH which is the rate-limiting enzyme in the production of 5-HT. In most vertebrates, there are two isoforms of TPH present, TPH1 and TPH2, which exhibit different catalytic or substrate specificity as well as different expression domains. Studies carried out in mammals show that only tph2 is expressed in the brain whereas tph1-mRNA is primarily localized in the enterochromaffin cells and pineal gland. A large number of neurons are also considered to be serotonergic or "pseudo-serotonergic" as they accumulate and release 5-HT yet do not produce it as no amine-synthetic enzymes are expressed, yet a combination of 5-HT transporters is observed. Therefore, tph expression is considered to be the only specific marker of 5-HT-producing neurons that can discriminate true 5-HT from pseudo-serotonergic neurons. This work examined in situ hybridization to study the mRNA distribution of one paralogue for tph1 and tph2 in the central nervous system of rainbow trout. Results show a segregated expression for both paralogues that predominantly match previous immunocytochemical studies. This study thus adds valuable information to the scarce analyses focusing on the central distribution of the expression of serotonergic markers, particularly tphs, in the vertebrate brain thus characterizing the true serotonergic brain territories.

Growth Performance After Agouti-Signaling Protein 1 (Asip1) Overexpression in Transgenic Zebrafish.

The melanocortin system is a key structure in the regulation of energy balance. Overexpression of inverse agonists, agouti-signaling protein (ASIP), and agouti-related protein (AGRP) results in increased food intake, linear growth, and body weight. ASIP regulates dorsal-ventral pigment polarity through melanocortin 1 receptor (MC1R) and overexpression induces obesity in mice by binding to central MC4R. Asip1 overexpression in transgenic zebrafish (asip1-Tg) enhances growth, yet experiments show fish overexpressing Asip1 do not develop obesity even under severe feeding regimes. Asip1-Tg fish do not need to eat more to grow larger and faster; thus, increased food efficiency can be observed. In addition, asip1-Tg fish reared at high density are able to grow far more than wild-type (WT) fish reared at low density, although asip1-Tg fish seem to be more sensitive to crowding stress than WT fish, thus making the melanocortin system a target for sustainable aquaculture, especially as the U.S. Food and Drug Association has recently approved transgenic fish trading.

Functional nutrition modulates the early immune response against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout.

Although viruses represent a major threat for cultured fish worldwide, the commercialization of vaccines capable of providing effective and long-lasting protection is still lacking for most of these viral diseases. In this situation, the use of supplemented diets could be a suitable strategy to increase the immune status of the fish and reduce the impact of viral pathogens. Among possible immunostimulants that could be included in these functional feeds, some studies have previously shown that certain ß-glucans can significantly increase certain immune parameters of fish and reduce the impact of viral diseases. However, the mechanisms through which ß-glucans exert their activity have not been fully elucidated yet. In the current study, we have studied the immune response of different tissues to viral haemorrhagic septicaemia virus (VHSV) in rainbow trout fed with a non-supplemented control diet as well as in fish fed a commercial functional aquafeed (Protec™, Skretting) containing ß-glucans, vitamin C, vitamin E and zinc. For this, after 30 days of feeding the fish with one of the two diets, they were subsequently infected with VHSV by bath or mock-infected. After 2 or 6 days post-infection, fish were sacrificed and the levels of transcription of different immune genes such as IgM, IgT, IgD, Mx, interferon γ (IFN γ) and perforin studied in different tissues (kidney, gut and gills). Additionally, the levels of natural IgMs in serum were also determined. Our results demonstrate that fish fed the functional diet were capable of mounting an increased IgM, IgT, IgD and Mx transcriptional response to the virus. Additionally, these fish also showed increased levels of natural IgMs in serum. These results reveal a previously undescribed effect of functional diets on fish Ig production and point to Protec™ as an adequate diet to be incorporated in holistic programs aimed at mitigating the effect of viral diseases.

Effect of vitamin C on innate immune responses of rainbow trout (Oncorhynchus mykiss) leukocytes.

Vitamin C, also known as ascorbic acid, is an essential micronutrient that influences a wide variety of physiological processes, including immunological functions. Although the positive effects of vitamin C supplementation on the immunological status of fish has been established in different species, the bases for these positive effects are still unknown. Hence, the aim of our study was to evaluate the in vitro effect of vitamin C on several innate immune functions of rainbow trout (Oncorhynchus mykiss) leukocyte populations. For this, we assessed the effects exerted on the established rainbow trout monocyte-macrophage cell line RTS11, and compared them to those observed in trout head kidney leukocytes. Our results demonstrate that vitamin C increases the production of reactive oxygen species and the percentage of phagocytic cells in both cell populations. On the other hand, vitamin C had no effect on the surface MHC II levels and only in the case of RTS11 cells increased the capacity of these cells to migrate towards the CK9 chemokine. Finally, vitamin C also increased the transcription of several pro-inflammatory and antimicrobial genes elicited by Escherichia coli, with some differences depending on the cell population studied. Our results contribute to further understand how vitamin C supplementation regulates the fish immune system.

Attenuated Infectious Hematopoietic Necrosis Virus with Rearranged Gene Order as Potential Vaccine.

The genome of infectious hematopoietic necrosis virus (IHNV), a salmonid novirhabdovirus, has been engineered to modify the gene order and to evaluate the impact on a possible attenuation of the virus in vitro and in vivo By reverse genetics, eight recombinant IHNVs (rIHNVs), termed NxGy according to the respective positions of the nucleoprotein (N) and glycoprotein (G) genes along the genome, have been recovered. All rIHNVs have been fully characterized in vitro for their cytopathic effects, kinetics of replication, and profiles of viral gene transcription. These rIHNVs are stable through up to 10 passages in cell culture. Following bath immersion administration of the various rIHNVs to juvenile trout, some of the rIHNVs were clearly attenuated (N2G3, N2G4, N3G4, and N4G1). The position of the N gene seems to be one of the most critical features correlated to the level of viral attenuation. The induced immune response potential in fish was evaluated by enzyme-linked immunosorbent spot assay (ELISPOT) and seroneutralization assays. The recombinant virus N2G3 induced a strong antibody response in immunized fish and conferred 86% of protection against wild-type IHNV challenge in trout, thus representing a promising starting point for the development of a live attenuated vaccine candidate. IMPORTANCE: In Europe, no vaccines are available against infectious hematopoietic necrosis virus (IHNV), one of the major economic threats in fish aquaculture. Live attenuated vaccines are conditioned by a sensible balance between attenuation and pathogenicity. Moreover, nonsegmented negative-strain RNA viruses (NNSV) are subject to a transcription gradient dictated by the order of the genes in their genomes. With the perspective of developing a vaccine against IHNV, we engineered various recombinant IHNVs with reordered genomes in order to artificially attenuate the virus. Our results validate the gene rearrangement approach as a potent and stable attenuation strategy for fish novirhabdovirus and open a new perspective for design of vaccines against other NNSV.

Distinct Differentiation Programs Triggered by IL-6 and LPS in Teleost IgM(+) B Cells in The Absence of Germinal Centers.

Although originally identified as a B cell differentiation factor, it is now known that mammalian interleukin-6 (IL-6) only regulates B cells committed to plasma cells in response to T-dependent (TD) antigens within germinal centers (GCs). Even though adaptive immunity is present in teleost fish, these species lack lymph nodes and GCs. Thus, the aim of the present study was to establish the role of trout IL-6 on B cells, comparing its effects to those induced by bacterial lipopolysaccharide (LPS). We demonstrate that the effects of teleost IL-6 on naïve spleen B cells include proliferation, activation of NF-κB, increased IgM secretion, up-regulation of Blimp1 transcription and decreased MHC-II surface expression that point to trout IL-6 as a differentiation factor for IgM antibody-secreting cells (ASCs). However, LPS induced the secretion of IgM without up-regulating Blimp1, driving the cells towards an intermediate activation state in which antigen presenting mechanisms are elicited together with antibody secretion and expression of pro-inflammatory genes. Our results reveal that, in trout, IL-6 is a differentiation factor for B cells, stimulating IgM responses in the absence of follicular structures, and suggest that it was after follicular structures appeared that this cytokine evolved to modulate TD responses within the GC.

T cell immunity in the teleost digestive tract.

Fish (along with cyclostomes) constitute the most ancient animal group in which an acquired immune system is present. As in higher vertebrates, both B and T lymphocytes cooperate in implementing an adequate response. Although there is still a debate on whether fish possess a true gut associated lymphoid tissue (GALT), the presence of diffuse B and T lymphocytes throughout all mucosal surfaces has been demonstrated in a wide variety of fish species. The lack of antibodies against T lymphocyte markers has hampered the performance of functional assays in both systemic and mucosal compartments. However, most components associated with T lymphocyte function have been identified in fish through extensive genomic research, suggesting similar functionalities for fish and mammalian T lymphocytes. Thus, the aim of this review is to briefly summarize what is known in teleost concerning the characteristics and functionalities of the different T cell subsets, to then focus on what is known to date regarding their presence and role in the gastrointestinal tract, through either direct functional assays or indirectly by conclusions drawn from transcriptomic analysis.

Distribution of T Cells in Rainbow Trout (Oncorhynchus mykiss) Skin and Responsiveness to Viral Infection.

Although the skin constitutes the first line of defense against waterborne pathogens, there is a great lack of information regarding the skin associated lymphoid tissue (SALT) and whether immune components of the skin are homogeneously distributed through the surface of the fish is still unknown. In the current work, we have analyzed the transcription of several immune genes throughout different rainbow trout (Oncorhynchus mykiss) skin areas. We found that immunoglobulin and chemokine gene transcription levels were higher in a skin area close to the gills. Furthermore, this skin area as well as other anterior sections also transcribed significantly higher levels of many different immune genes related to T cell immunity such as T cell receptor α (TCRα), TCRγ, CD3, CD4, CD8, perforin, GATA3, Tbet, FoxP3, interferon γ (IFNγ), CD40L and Eomes in comparison to posterior skin sections. In agreement with these results, immunohistochemical analysis revealed that anterior skin areas had a higher concentration of CD3(+) T cells and flow cytometry analysis confirmed that the percentage of CD8(+) T lymphocytes was also higher in anterior skin sections. These results demonstrate for the first time that T cells are not homogeneously distributed throughout the teleost skin. Additionally, we studied the transcriptional regulation of these and additional T cell markers in response to a bath infection with viral hemorrhagic septicemia virus (VHSV). We found that VHSV regulated the transcription of several of these T cell markers in both the skin and the spleen; with some differences between anterior and posterior skin sections. Altogether, our results point to skin T cells as major players of teleost skin immunity in response to waterborne viral infections.

Identification of Teleost Skin CD8α+ Dendritic-like Cells, Representing a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells.

Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleosts has been addressed only briefly, and the identification of a specific DC subset in teleosts remained elusive because of the lack of specific Abs. In mice, DCs expressing CD8α(+) in lymphoid tissues have the capacity to cross-present extracellular Ags to T cells through MHC I, similarly to tissue-derived CD103(+) DCs and the human CD141(+) DC population. In the current study, we identified a large and highly complex subpopulation of leukocytes coexpressing MHC class II and CD8α. This CD8α(+) MHC II(+) DC-like subpopulation constituted ∼1.2% of the total leukocyte population in the skin, showing phenotypical and functional characteristics of semimature DCs that seem to locally regulate mucosal immunity and tolerance in a species lacking lymph nodes. Furthermore, we identified trout homologs for CD141 and CD103 and demonstrated that, in trout, this skin CD8(+) DC-like subpopulation expresses both markers. To our knowledge, these results provide the first evidence of a specific DC-like subtype in nonimmune tissue in teleosts and support the hypothesis of a common origin for all mammalian cross-presenting DCs.

Characterization, tissue distribution and regulation by fasting of the agouti family of peptides in the sea bass (Dicentrarchus labrax).

The melanocortin system is one of the most complex hormonal systems in vertebrates. Atypically, the signaling of melanocortin receptors is regulated by the binding of endogenous antagonists, named agouti-signaling protein (ASIP) and agouti-related protein (AGRP). Teleost specific genome duplication (TSGD) rendered new gene copies in teleost fish and up to four different genes of the agouti family of peptides have been characterized. In this paper, molecular cloning was used to characterize mRNA of the agouti family of peptides in sea bass. Four different genes were identified: AGRP1, ASIP1, AGRP2 and ASIP2. The AGRP1 gene is mainly expressed in the brain whereas ASIP1 is mainly expressed in the ventral skin. Both ASIP2 and AGRP2 are expressed in the brain and the pineal gland but also in some peripheral tissues. Immunocytochemical studies demonstrated that AGRP1 is exclusively expressed within the lateral tuberal nucleus, the homologue of the mammalian arcuate nucleus in fish. Long-term fasting (8-29 days) increased the hypothalamic expression of AGRP1 but depressed AGRP2 expression (15-29 days). In contrast, the hypothalamic expression of ASIP2 was upregulated during short-term fasting suggesting that this peptide could be involved in the short term regulation of food intake in the sea bass.

Effects of dopaminergic system activation on feeding behavior and growth performance of the sea bass (Dicentrarchus labrax): a self-feeding approach.

Dopamine is synthesized from l-dopa and subsequently processed into norepinephrine and epinephrine. Any excess neurotransmitter can be taken up again by the neurons to be broken down enzymatically into DOPAC. The effect of dopamine on mammalian food intake is controversial. Mice unable to synthesize central dopamine die of starvation. However, studies have also shown that central injection of dopamine inhibits food intake. The effect of dopaminergic system in the fish feeding behavior has been scarcely explored. We report that the inclusion of l-dopa in the diets results in the activation of sea bass central dopaminergic system but also in the significant increase of the hypothalamic serotonin levels. Dietary l-dopa induces a decrease of food intake and feed conversion efficiency that drives a decline of all growth parameters tested. No behavioral effects were observed after l-dopa treatment. l-dopa treatment stimulated central expression of NPY and CRF. It suggests that CRF might mediate l-dopa effects on food intake but also that CRF neurons lie downstream of NPY neurons in the hierarchical forebrain system, thus controlling energy balance. Unexpectedly, dietary administration of haloperidol, a D2-receptor antagonist, cannot block dopamine effects but also induces a decline of the food intake. This decrease seems to be a side effect of haloperidol treatment since fish exhibited a decreased locomotor activity. We conclude that oral l-dopa inhibits sea bass food intake and growth. Mechanism could also involve an increase of hypothalamic serotoninergic tone.

Molecular characterization and functional regulation of melanocortin 2 receptor (MC2R) in the sea bass. A putative role in the adaptation to stress.

The activation of melanocortin 2 receptor (MC2R) by ACTH mediates the signaling cascade leading to steroid synthesis in the interrenal tissue (analogous to the adrenal cortex in mammals) of fish. However, little is known about the functional regulation of this receptor in fish. In this work described, we cloned sea bass MC2R from a liver cDNA. SbMC2R requires the melanocortin 2 receptor accessory protein (MRAP) for its functional expression. Dietary cortisol but not long-term stress protocols downregulated interrenal sbMC2R expression. Data suggest the existence of a negative feedback on interrenal sbMC2R expression imposed by local or systemic glucocorticoids. This feedback could be involved in long-term stress adaptation by regulating interrenal sensitivity to ACTH. ACTH-induced MC2R activation stimulates hepatic lipolysis, suggesting that ACTH may mediate stress-induced effects upstream of cortisol release.

Stress-induced effects on feeding behavior and growth performance of the sea bass (Dicentrarchus labrax): a self-feeding approach.

Repetitive aquaculture-related protocols may act as cyclic stressors that induce chronic stress in cultured fish. The sea bass is particularly sensitive to stressful conditions and the mere presence of humans will disturb feeding behavior. In this paper, we study whether chronic stress induced by repetition of acute stress protocols affects long-term feeding behavior and growth performance in sea bass and whether exogenous cortisol may induce stress-like changes in these parameters. We demonstrate that both chronic stress and dietary cortisol decrease food intake and have a negative effect on feed conversion efficiency, severely impairing sea bass performance. Both experimental approaches induced changes in the daily feeding activity by lengthening the active feeding periods. Fish subjected to a cyclic stressor modify their daily feeding pattern in an attempt to avoid interference with the time of the stressor. The delay in feeding when fish are acutely and repeatedly stressed could be of substantial adaptive importance.

Sex steroid-induced inhibition of food intake in sea bass (Dicentrarchus labrax).

This study was conducted to test the sensitivity to gonadal steroids of the systems regulating food intake in sea bass. Animals were treated with silastic implants containing 17-beta-estradiol or testosterone. Self-feeding was recorded for 31 days using computerized demand feeders and unfed-pellet recovery systems. Both steroids strongly decreased self-feeding levels, feed efficiency and specific growth rates. The linear growth of fish treated with testosterone was higher than in 17-beta-estradiol treated fish. In the second experiment, fish were treated with lower 17-beta-estradiol doses and 11-keto-androstenedione, a precursor of the main fish androgen (11-keto-testosterone). The results demonstrated a dose-response effect of estrogen and no effect of non-aromatizable androgens on food intake or growth performance. The inhibitory effect of testosterone on food intake seems to be mediated by its aromatization to estradiol, while linear growth promotion is mediated by the androgen per se. Data suggest that gonadal steroids may be involved in the seasonal feeding pattern of sea bass. The results demonstrate the sensitivity of the mechanisms regulating food intake to estrogenic compounds and point to the risk of including feed containing estrogenic substances in fish diets as well as the risk involved in exposure to "estrogenic environments".