Function In Teleost Fish Research Articles

Functional comparison of Rab3aa and Rab3ab in grass carp (Ctenopharyngodon idella) immune response and GCRV replication

Rab GTPases not only play a crucial role in the exocytosis and membrane transport of all eukaryotic cells, but are also gradually being found to be involved in innate immunity in mammals. However, the immunological function of Rab3a in fish remains unclear. In this study, two members of Rab3a, CiRab3aa and CiRab3ab, were cloned from grass carp (Ctenopharyngodon idella) for the first time. It was observed that CiRab3aa exhibited a prompt response to grass carp reovirus (GCRV) infection, and suppressing the expression of the CiRab3aa effectively inhibited GCRV replication, whereas the expression level of the CiRab3ab gene did not affect GCRV replication. Furthermore, overexpression of CiRab3aa significantly suppressed the mRNA levels of TLR9, TNF-α, IL-1β, IL-6, IL-10, ERK, and IFN-I following stimulation with CpG ODN 1670 A, suggesting that CiRab3aa is a negative regulator of TLR9 signaling pathway. In conclusion, this study not only revealed functional differences between CiRab3aa and CiRab3ab in grass carp immunity, providing new sights into understanding their function in teleost fish, but also provided a new target for the treatment strategy of grass carp hemorrhagic disease. These results will help address the harm and economic losses caused by GCRV infection in grass carp breeding industry.

Non-invasive dsRNA delivery via feeding for effective gene silencing in teleost fish: A novel approach in the study of gene function analysis

RNA interference (RNAi) is a powerful technique for gene silencing, involving post-transcriptional regulation of gene expression. Successful application of RNAi has been demonstrated in various organisms like nematodes, insects, and oysters by ingesting double-stranded RNA (dsRNA)-expressing bacteria. In this study, we attempted a non-invasive feeding method in the marine economy teleost fish, Sebastes schlegelii. To validate the feasibility of this approach in S. schlegelii, we utilized rotifers, Artemia nauplii, and commercial pellet feed as vectors to deliver Escherichia coli strain HT115 engineered to express dsRNA, targeting dead end (dnd) gene, known to be important for primordial germ cells (PGCs) migration and proliferation in fish. Our experimental results consistently showed that knockdown of the dnd gene effectively inhibited the development of PGCs in S. schlegelii, resulting in gonadal dysplasia and even sex reversal. This study marks a significant advancement in understanding gene function in teleost fish, laying the groundwork for future large-scale research in this field.

Characterization of ccl20a.3 and ccl20l as gene markers for Th17 cell in turbot

T-helper 17 lymphocytes (Th17) are the most common inflammatory cells identified in mammals. However, the identification of Th17 cells and the clarification of their function in teleost fish remain largely unknown. In this study, we took advantage of the single-cell RNA sequencing-based immune cell atlas that was identified in turbot (Scophthalmus maximus), and revealed two chemokine-related genes, ccl20a.3 and ccl20l, that were specifically expressed in Th17 cells. Moreover, through immuno-fluorescence analysis, we found that CCL20a.3 or CCL20l was co-expressed with the classical makers in Th17 cells, including IL17a/f1 and IL22. Furthermore, through a Th17 lineage-specific transcription factor RORc inhibitor GSK805 treatment, we found that the expression of ccl20a.3 and ccl20l was significantly impaired, compared with other T cell markers. Besides, we also found that ccl20a.3 and ccl20l exhibited the same dynamic response with the classical markers that were identified in Th17 cells during bacterial infection. Taken together, these results provide potential gene markers for better understanding of the dynamic immune responses of Th17 cells in teleost fish.

Functional characterization of melanocortin-3 receptor in rainbow trout (Oncorhynchus mykiss).

The melanocortin-3 receptor (MC3R) is an important regulator of energy homeostasis and inflammation in mammals. However, its function in teleost fish needs to be further explored. In this study, we characterized rainbow trout MC3R (rtMC3R), which encoded a putative protein of 331 amino acids. Phylogenetic and chromosomal synteny analyses showed that rtMC3R was closely related to bony fishes. Quantitative PCR (qPCR) revealed that the transcripts of rtMC3R were highly expressed in the brain and muscle. The cellular function of rtMC3R was further verified by the signal-pathway-specific luciferase reporter assays. Four agonists such as α-MSH, β-MSH, ACTH (1-24), and NDP-MSH can active rtMC3R, increasing the production of intracellular cAMP and upregulating MAPK/ERK signals. Moreover, we found that rtMC3R stimulated with α-MSH and NDP-MSH can significantly inhibit the NF-κB signaling pathway. This research will be helpful for further studies on the function of MC3R in rainbow trout, especially the role of energy metabolism and immune regulation.

Hormonal and neuroendocrine control of reproductive function in teleost fish.

Reproduction is one of the important physiological events for the maintenance of the species. Hormonal and neuroendocrine regulation of teleost requires multiple and complex interactions along the hypothalamic-pituitary-gonad (HPG) axis. Within this axis, gonadotropin-releasing hormone (GnRH) regulates the synthesis and release of gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Steroidogenesis drives reproduction function in which the development and differentiation of gonads. In recent years, new neuropeptides have become the focus of reproductive physiology research as they are involved in the different regulatory mechanisms of these species' growth, metabolism, and reproduction. However, especially in fish, the role of these neuropeptides in the control of reproductive function is not well studied. The study of hormonal and neuroendocrine events that regulate reproduction is crucial for the development and success of aquaculture.

Immune responses, subcellular localization, and antiviral activity of interferon-induced protein 35 (IFP35) in rock bream (Oplegnathus fasciatus)

Interferon-induced protein 35 kDa (IFP35) has been demonstrated to play important roles in antiviral defense, inflammatory response and cancer progression. However, its precise function in teleost fish remains to be elucidated. Herein, we functionally characterized the rock bream (Oplegnathus fasciatus) IFP35 (OfIFP35) to understand its expression pattern, subcellular localization, antiviral activity, and regulation of downstream genes. OfIFP35 consists of an 1107 bp open reading frame encoding 368 amino acids, including two N-myc-interactor (Nmi)/IFP35 domains (NIDs). The predicted molecular weight of OfIFP35 was 42 kDa, with a theoretical isoelectric point (pI) of 5.10. Evolutionary conservation of IFP35 was analyzed using multiple, pairwise alignments and phylogenetic tree analysis. OfIFP35 in rock bream was found to be highest expressed in the gills. Immune challenges with iridovirus, polyinosinic:polycytidylic acid, lipopolysaccharide, and live bacteria (Streptococcus iniae and Edwardsiella tarda) significantly upregulated its mRNA expression in gill and liver tissues of the rock bream. GFP-tagged OfIFP35 was localized in the cytoplasm of FHM cells, and its overexpression significantly suppressed VHSV transcription in vitro. Moreover, the analysis of downstream gene expression revealed that OfIFP35 could activate the type I interferon pathway. Collectively, these findings indicate that OfIFP35 is important for the immune system of rock bream as it promotes defense responses during viral infections.

Revisiting the Teleost Thymus: Current Knowledge and Future Perspectives.

Simple SummaryThe thymus is the immune organ producing T lymphocytes that are essential to create immunity after encountering pathogens or vaccination. This review summarizes the thymus localization and histological studies, cell composition, and function in teleost fishes. We also describe how seasonal changes, photoperiod, water temperature fluctuations, and hormones can affect thymus development in fish species. Overall, the information helps identify future studies needed to understand thymus function in fish species and the immune system’s evolutionary origins. Since fish are exposed to pathogens, especially under aquaculture conditions, knowledge about the fish thymus and T lymphocyte can also help improve fish farming protocols, considering intrinsic and environmental conditions that can contribute to achieving the best vaccine responsiveness for disease resistance.The thymus in vertebrates plays a critical role in producing functionally competent T-lymphocytes. Phylogenetically, the thymus emerges early during evolution in jawed cartilaginous fish, and it is usually a bilateral organ placed subcutaneously at the dorsal commissure of the operculum. In this review, we summarize the current understanding of the thymus localization, histology studies, cell composition, and function in teleost fishes. Furthermore, we consider environmental factors that affect thymus development, such as seasonal changes, photoperiod, water temperature fluctuations and hormones. Further analysis of the thymus cell distribution and function will help us understand how key stages for developing functional T cells occur in fish, and how thymus dynamics can be modulated by external factors like photoperiod. Overall, the information presented here helps identify the knowledge gaps and future steps needed for a better understanding of the immunobiology of fish thymus.

Simultaneous Measurement of Sex Steroid Hormones in Largemouth Bass (Micropterus salmoides) Plasma by Application of Liquid Chromatography-Tandem Mass Spectrometry.

Sex steroid hormones are potential biomarkers of reproductive function in teleost fish, but their measurement continues to rely on antibody-based assays. The objective of this study was to optimize a robust and simultaneous liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measurement of eight steroid hormones (cortisol, 11-ketotestosterone, estradiol, 17α-ethynyl estradiol, estrone, estriol, progesterone and testosterone) in fish plasma. The extraction was followed by liquid-liquid extraction with tert-butyl methyl ether and time scheduled multi-reaction monitoring (sMRM) was used for quantitation of steroids. Validation of method performance using charcoal-stripped human plasma showed extraction recoveries for eight steroids ranged from 85.5 to 108.2% with matrix effects>80%. The limits of quantitation were 0.01pg/μL for testosterone, 0.05pg/μL for cortisol and progesterone, 0.1pg/μL for 11-ketotestosterone, estradiol and estrone, 0.125pg/μL for estriol and 0.25pg/μL for 17α-ethynyl estradiol. The proposed method was applied to plasma samples of largemouth bass (Micropterus salmoides) collected from contaminated (Lake Apopka) and reference sites (Wildcate Lake) in Florida. Concentrations of testosterone, cortisol, estradiol and estrone were significantly different in female fish, but plasma concentration of cortisol was only statistically different in male fish between two sites (P<0.05). This study demonstrates the application of a robust LC-MS/MS analysis for a range of sex steroid hormones representative of endocrine function in a top predator, largemouth bass.

Fish TRAF2 promotes innate immune response to RGNNV infection

Tumour necrosis factor receptor-associated factors (TRAFs) are key regulatory proteins in the NF-κB signaling pathways. TRAF2 participates in the activation of both canonical and non-canonical NF-κB pathways, which are crucial for cell inflammation and cell survival. To elucidate its function in teleost fish, TRAF2 homologues of yellow grouper (Epinephelus awoara) and golden pompano (Trachinotus ovatus) have been cloned and characterized in this study. The open reading frame (ORF) of grouper TRAF2 (EaTRAF2) consists of 1563 nucleotides encoding a 521 amino acid protein with a predicted molecular mass of 58.70 kDa. The ORF of golden pompano TRAF2 (ToTRAF2) consists of 1563 nucleotides encoding a 521 amino acid protein with a predicted molecular mass of 58.66 kDa EaTRAF2 and ToTRAF2 share 99.23% and 99.42% identity with orange-spotted grouper (Epinephelus coioides) TRAF2 (EcTRAF2), respectively. Quantitative real-time PCR analysis indicated that the expression of EaTRAF2 was increased in grouper spleen (GS) cells after Red-spotted grouper nervous necrosis virus (RGNNV) infection; while the expression of ToTRAF2 was decreased in golden pompano brain (TOGB) cells after RGNNV infection. Both EaTRAF2 and ToTRAF2 were identified as a cytosolic protein and suggested to be associated with vesicles scattering in the cytoplasm. Both EaTRAF2 and ToTRAF2 enhanced RGNNV replication during viral infection in vitro. Further studies showed that EaTRAF2 and ToTRAF2 overexpression decreased the expression levels of interferon associated cytokines and pro-inflammatory factors. Taken together, these results are important for better understanding of the function of TRAF2 in fish and reveal its involvement in host response to immune challenges in RGNNV.

Molecular cloning

Transcription factor c-Jun is a member of AP-1 transcription complex that can be induced by various pathogens and plays a broad regulatory role in vertebrate immune response. In this study, the complete c-Jun cDNA of large yellow croaker Larimichthys crocea (Lcc-Jun) was cloned, whose open reading frame (ORF) is 984 bp long and encodes a protein of 327 amino acids (aa). The deduced Lcc-Jun protein contains three highly conserved domains, a transactivation domain (TAD, Met1-His118), a DNA binding domain (DBD, Lys218-Arg243), and a Leucine zipper domain (LZD, Leu271-Leu299), as found in other specie c-Jun. Lcc-Jun was constitutively expressed in all examined tissues, with the higher levels in blood, heart, and head kidney. Its transcripts were not only induced in spleen and head kidney by poly (I: C) or LPS, but also up-regulated in primary head kidney leukocytes (PKL), macrophages (PKM), and granulocytes (PKG), suggesting that Lcc-Jun may be involved in immune responses induced by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial component. Overexpression of Lcc-Jun in PKL increased the expression of cytokines and transcription factors involved in T helper 1 (Th1: TNF-α, IFN-γ, and T-bet) and Th2 (IL-4/13 A/B, IL-6, and GATA3) cell development and differentiation, suggesting that Lcc-Jun may play a role in regulation of Th1/Th2 cell response. These results therefore led us to suggest that the c-Jun-mediated signaling pathways may have an important immune-modulatory function in teleost fish.

Molecular cloning, expression analysis, and the immune-related role of a thymosin β in the goldfish, Carassius auratus.

β-Thymosins play critical roles in the regulation of many important physiological processes, but their function in teleost fishes remains poorly understood. In this study, the full-length cDNA coding for a thymosin β (Tβ) was cloned and identified in goldfish, Carassius auratus (gfTβ). The gfTβ cDNA consisted of 653bp with an open reading frame of 135bp that encodes a 44 amino acid polypeptide. Sequence analysis revealed one thymosin domain and a highly conserved actin-binding motif (18LKKTET23). Expression of gfTβ transcript was detected ubiquitously in all tissues examined, with relatively higher levels in the brain, intestine, spleen, gill, skin, kidney, and testis. Cadmium and H2O2 exposure induced increases in gfTβ transcript levels in the liver and spleen. Moreover, gfTβ transcription was upregulated in response to LPS challenge in the spleen while Poly I:C treatment did not affect gfTβ expression. In vivo injection of recombinant gfTβ generated from an Escherichia coli system induced expression of T lymphocyte-related genes (RAG1 and CD8α). These results suggest that gfTβ may be involved in the immune response of teleost fishes via modulation of T lymphocyte development.

TRAF3 enhances STING-mediated antiviral signaling during the innate immune activation of black carp

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a main regulator of antiviral and anti-inflammatory pathways in mammals, which is considered to induce type I interferon (IFN) activation and negatively regulate the activation of the canonical and non-canonical NF-κB pathways. To elucidate its function in teleost fish, TRAF3 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. The open reading frame (ORF) of black carp TRAF3 (bcTRAF3) consists of 1722 nucleotides and bcTRAF3 contains 574 amino acids. bcTRAF3 protein migrated around 65 KDa in immunoblot analysis of both EPC and HEK293T cells. bcTRAF3 was identified as a cytosolic protein and suggested to form aggregates or be associated with vesicles scattering in the cytoplasm. It was interesting that both NF-κB and IFN transcription was activated by bcTRAF3 in reporter assay. When co-expressed with black carp STING (bcSTING), bcTRAF3 was redistributed in the cytoplasm and its subcellular location overlapped with that of bcSTING no matter what the cells was infected with GCRV or not, which suggested the association between these two molecules. bcSTING-mediated IFN production was up-regulated by bcTRAF3 in a dose dependent manner in reporter assay. Accordingly, EPC cells transfected with both bcSTING and bcTRAF3 showed enhanced antiviral activity comparing EPC cells expressing bcSTING alone. Taken together, the data generated in this paper supported the conclusion that bcTRAF3 was recruited into host innate immune activation and positively regulated bcSTING-mediated antiviral signaling.

TRAF2 of black carp upregulates MAVS-mediated antiviral signaling during innate immune response

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of the tumor necrosis factor (TNF) mediated signaling of higher vertebrates. To elucidate its function in teleost fish, TRAF2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. The open reading frame (ORF) of black carp TRAF2 (bcTRAF2) consists of 1611 nucleotides and bcTRAF2 contains 536 amino acids. bcTRAF2 protein migrated around 65 KDa in immunoblot analysis of both EPC and HEK293T cells. bcTRAF2 was identified as a cytosolic protein and suggested to be associated with vesicles scattering in the cytoplasm. NF-κB transcription instead of IFN transcription was activated by bcTRAF2 in reporter assay. It was interesting that bcMAVS-mediated IFN production was up-regulated by bcTRAF2 in a dose dependent manner in reporter assay. Accordingly, EPC cells transfected with both bcMAVS and bcTRAF2 showed enhanced antiviral activity comparing EPC cells only expressing bcMAVS. When co-expressed with bcMAVS, bcTRAF2 was redistributed in the cytoplasm and its subcellular location overlapped with the subcellular location of bcMAVS, which suggested the association between these two molecules. Taken together, the data generated in this paper supported the conclusion that bcTRAF2 was recruited into host innate immune response and positively regulated bcMAVS-mediated antiviral signaling.

Characterization of the black carp TRAF6 signaling molecule in innate immune defense

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a vital role in the innate immune response of higher vertebrates. To elucidate its function in teleost fish, TRAF6 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Black carp TRAF6 (bcTRAF6) transcription in Mylopharyngodon piceus fin (MPF) cells was up-regulated in response to both poly (I:C) treatment and viral infection, but was suppressed by LPS stimulation. bcTRAF6 migrated around 72 KDa in immunoblot analysis and was identified as a cytosolic protein suggested to be associated with vesicles scattering in the cytoplasm. Reporter assay demonstrated that NF-κB instead of IFN was activated by bcTRAF6; and EPC cells expressing bcTRAF6 presented the same cytopathic effect (CPE) ratio to that of control cells. When co-expressed with bcMAVS, bcTRAF6 was redistributed and overlapped with the subcellular location of bcMAVS. It was interesting that bcMAVS mediated the IFN induction was up-regulated by low input of bcTRAF6 but down-regulated by high input of bcTRAF6. Taken together, the data generated in this paper supported the conclusion that bcTRAF6 associated with bcMAVS and was recruited into bcMAVS mediated signaling during host innate immune response.

Effects of exposure to Streptococcus iniae on microRNA expression in the head kidney of genetically improved farmed tilapia (Oreochromis niloticus)

BackgroundGenetically improved farmed tilapia (GIFT, Oreochromis niloticus) are susceptible to infection by Streptococcus iniae when maintained in modern intensive culture systems. GIFT are commercially important fishes that are cultured widely in southern China. The role of microRNAs (miRNAs) in the regulatory response of GIFT to S. iniae infection has been underestimated and has not yet been well studied. Head kidney has an important immune function in teleost fishes. The main aim of this study was to determine the possible function of miRNAs in head kidney of S. iniae-infected GIFT. MiRNAs are small, non-coding RNAs that regulate gene expression by binding to the 3’-untranslated regions of their target mRNAs. MiRNAs are known to regulate immune-regulated signaling and inflammatory response pathways.ResultsHigh-throughput deep sequencing of two libraries (control group [CO] and infected group [IN]) of RNA extracted from GIFT head kidney tissues generated 12,089,630 (CO) and 12,624,975 (IN) clean reads. Bioinformatics analysis identified 1736 and 1729 conserved miRNAs and 164 and 165 novel miRNAs in the CO and IN libraries, respectively. Three miRNAs (miR-310-3p, miR-92, and miR-127) were found to be up-regulated and four miRNAs (miR-92d-3p, miR-375-5p, miR-146-3p, and miR-694) were found to be down-regulated in the S. iniae-infected GIFT. The expressions of these miRNAs were verified by quantitative real-time PCR. RNAhybrid and TargetScan were used to identify complementary miRNA and mRNA target sites, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to annotate and predict potential downstream regulation of biological pathways. Seven target genes, which encode immune-related proteins (complement C3, cytidine deaminase, regulator of G-protein Rgs22, mitogen-activated protein kinase Mapk1, metabotropic glutamate receptorm GluR8, calcium-sensing receptor CaSR, and microtubule-associated protein Map1S) were predicted to play crucial roles in the GIFT response to S. iniae infection.ConclusionsS. iniae outbreaks have hindered the development of the tilapia industry in China. Understanding the miRNA transcriptome of S. iniae-infected GIFT is important for exploring the immune responses regulated by miRNAs as well as for studying novel regulated networks to prevent and treat S. iniae infections in the future.

Neutrophil Development, Migration, and Function in Teleost Fish.

It is now widely recognized that neutrophils are sophisticated cells that are critical to host defense and the maintenance of homeostasis. In addition, concepts such as neutrophil plasticity are helping to define the range of phenotypic profiles available to cells in this group and the physiological conditions that contribute to their differentiation. Herein, we discuss key features of the life of a teleost neutrophil including their development, migration to an inflammatory site, and contributions to pathogen killing and the control of acute inflammation. The potent anti-microbial mechanisms elicited by these cells in bony fish are a testament to their long-standing evolutionary contributions in host defense. In addition, recent insights into their active roles in the control of inflammation prior to induction of apoptosis highlight their importance to the maintenance of host integrity in these early vertebrates. Overall, our goal is to summarize recent progress in our understanding of this cell type in teleost fish, and to provide evolutionary context for the contributions of this hematopoietic lineage in host defense and an efficient return to homeostasis following injury or infection.

Independently evolved upper jaw protrusion mechanisms show convergent hydrodynamic function in teleost fishes

A protrusible upper jaw has independently evolved multiple times within teleosts and has been implicated in the success of two groups in particular: Acanthomorpha and Cypriniformes. We use digital particle image velocimetry (DPIV) to compare suction feeding flow dynamics in a representative of each of these clades: goldfish and bluegill. Using DPIV, we contrast the spatial pattern of flow, the temporal relationship between flow and head kinematics, and the contribution of jaw protrusion to the forces exerted on prey. As expected, the spatial patterns of flow were similar in the two species. However, goldfish were slower to reach maximal kinematic excursions, and were more flexible in the relative timing of jaw protrusion, other jaw movements and suction flows. Goldfish were also able to sustain flow speeds for a prolonged period of time as compared with bluegill, in part because goldfish generate lower peak flow speeds. In both species, jaw protrusion increased the force exerted on the prey. However, slower jaw protrusion in goldfish resulted in less augmentation of suction forces. This difference in force exerted on prey corresponds with differences in trophic niches and feeding behavior of the two species. The bluegill uses powerful suction to capture insect larvae whereas the goldfish uses winnowing to sort through detritus and sediment. The kinethmoid of goldfish may permit jaw protrusion that is independent of lower jaw movement, which could explain the ability of goldfish to decouple suction flows (due to buccal expansion) from upper jaw protrusion. Nevertheless, our results show that jaw protrusion allows both species to augment the force exerted on prey, suggesting that this is a fundamental benefit of jaw protrusion to suction feeders.

Pharmaceuticals as Neuroendocrine Disruptors: Lessons Learned from Fish on Prozac

Pharmaceuticals are increasingly detected in a variety of aquatic systems. One of the most prevalent environmental pharmaceuticals in North America and Europe is the antidepressant fluoxetine, a selective serotonin reuptake inhibitor (SSRI) and the active ingredient of Prozac. Usually detected in the range below 1 μg/L, fluoxetine and its active metabolite norfluoxetine are found to bioaccumulate in wild-caught fish, particularly in the brain. This has raised concerns over potential disruptive effects of neuroendocrine function in teleost fish, because of the known role of serotonin (5-HT) in the modulation of diverse physiological processes such as reproduction, food intake and growth, stress and multiple behaviors. This review describes the evolutionary conservation of the 5-HT transporter (the therapeutic target of SSRIs) and reviews the disruptive effects of fluoxetine on several physiological endpoints, including involvement of neuroendocrine mechanisms. Studies on the goldfish, Carassius auratus, whose neuroendocrine regulation of reproduction and food intake are well characterized, are described and represent a reliable model to study neuroendocrine disruption. In addition, fish studies investigating the effects of fluoxetine, not only on reproduction and food intake, but also on stress and behavior, are discussed to complement the emerging picture of neuroendocrine disruption of physiological systems in fish exposed to fluoxetine. Environmental relevance and key lessons learned from the effects of the antidepressant fluoxetine on fish are highlighted and may be helpful in designing targeted approaches for future risk assessments of pharmaceuticals disrupting the neuroendocrine system in general.

Sublethal Concentrations of Ammonia Impair Performance of the Teleost Fast‐Start Escape Response

The fast-start escape response in fish is essential for predator avoidance, but almost nothing is known about whether sublethal concentrations of pollutants can impair this reflex. Ammonia, a pervasive pollutant of aquatic habitats, is known to have toxic effects on nervous and muscle function in teleost fish. Golden gray mullet (Liza aurata L.) were exposed for 24 h to sublethal ammonia concentrations in seawater (control, 400 micromol L(-1), or 1,600 micromol L(-1) NH(4)Cl), and then their response to startling with a mechanical stimulus was measured with high-speed video. Initiation of the escape response was significantly slowed by ammonia exposure: response latency rose proportionally from <50 ms in controls to >300 ms at a concentration of 1,600 micromol L(-1 ) NH(4)Cl. This indicates toxic effects on nervous function within the reflex arc. Impaired escape performance was also observed: maximum turning rate, distance covered, velocity, and acceleration were significantly reduced by >45% at a concentration of 1,600 micromol L(-1) NH(4)Cl. This indicates toxic effects on fast-twitch glycolytic white muscle function, the muscle type that powers the fast-start response. These neuromotor impairments were associated with significant ammonia accumulations in venous plasma and white muscle and brain tissue. These results indicate that anthropogenic ammonia pollution in aquatic habitats may increase the vulnerability of fish to predation, especially by birds and mammals that are not affected by water ammonia concentrations.

Generation and use of monoclonal antibodies as leucocyte-markers to study the immune function in teleost fish (B219)

Abstract Fish possess the first full immune system in the vertebrate evolution. To date, leucocyte abundance, distribution and function has attempted to elucidate how similar or different are the fish and mammalian immune system. Fish possess lymphocytes, monocyte-macrophages and granulocytes with main differences in the tissue origin, distribution and functioning. Lack of proper molecular markers, such as commercial specific antibodies, for fish leucocytes is still hampering the success in this field. Considering this, we immunized mice with gilthead seabream (Sparus aurata L.) leucocytes and generated monoclonal antibodies following standard protocols. We chose 3 clones (H4, D2 and C1) and characterised their specificity for seabream leucocytes by means of flow cytometry, immunocytochemistry, magnetic-activated cell sorting (MACS) and gene expression by RT-PCR. Functional studies with the isolated leucocyte populations were also conducted to ascertain their implication in phagocytosis, cell-mediated cytotoxicity or oxygen-free radical production. Generation of monoclonal antibodies is a promising field in order to get good markers for further characterisation of the fish immune system. Work partially funded by European (QLK2-CT-2002-00722), national (AGL2005-07454-C02-01) and regional (02958/PI/05) projects.