Fish Brain Research Articles

Raymond Michael Gaze. 22 June 1927—11 September 2012

Mike Gaze devoted his career to understanding how nerves form precise connections. His experiments focused on the nerves connecting the eye to the brain in frogs and fish. Mike pioneered the use of electrophysiological recording in investigating how the nervous system develops. This technique enabled him a greater degree of anatomical precision than had previously been possible. The dominant theory on how nerve connections are formed was Sperry's chemoaffinity theory (Roger Wolcott Sperry (ForMemRS 1976)), based on the results of regeneration experiments. Mike repeated Sperry's experiments using his better technique. He then extended Sperry's work: first, to the development of retinotectal connections following various surgical operations on the embryonic eye; second, to regeneration following surgical reductions in the size of the tectum or retina; and third, to normal development. In each case he showed, for the first time, that connections are plastic . Even during normal development, retinal fibres are continually changing their connections. This was a radical finding. It implied, contrary to Sperry's theory, that retinal fibres interact with each other in forming tectal connections. Mike called this dynamic process systems matching . Mike then investigated how retinal fibres behave before they reach the brain. In further original work, Mike, with former student Mike Keating, showed, contrary to the prevailing view, that functional value can affect the development of specific nerve connections. In the 30 years since Mike retired there has been much work on the molecular bases underlying nerve connections. These have confirmed Mike's original findings and ideas.

Evolutionary implications of size-selective mortality on the ontogenetic development of shoal cohesion: a neurochemical approach using a zebrafish, Danio rerio, harvest selection experiment

Size-selective mortality may evolutionarily alter life-history as well as individual behavioral and physiological traits. Moreover, size-selective mortality can affect group behavioral traits, such as shoaling and collective properties (e.g., shoal cohesion), which are relevant for finding food and reducing risk of predation. Here, we present experimental evidence using selection lines of zebrafish (Danio rerio) that were exposed to positive (large-harvested), negative (small-harvested), and random (control) size-selective mortality for five generations, followed by eight generations during which harvesting was halted to remove maternal effects and to study evolutionarily fixed outcomes. We investigated changes in shoal cohesion and turnover in monoamines in zebrafish through ontogeny. To that end, we repeatedly measured inter-individual distance in groups of eight fish and the turnovers of dopamine and serotonin in brains of fish from juvenile to the adult stage at 40-day intervals. We, firstly, found that shoal cohesion was overall consistent through ontogeny at group levels suggesting the presence of collective personality. Secondly, we found a decrease in shoal cohesion through ontogeny in the small-harvested and control lines, while the large-harvested line did not show any ontogenetic change. Thirdly, the selection lines did not differ among each other in shoal cohesion at any ontogenetic stage. Fourthly, dopamine turnover increased through ontogeny in a similar way for all lines while the serotonin turnover decreased in the large-harvested and control lines, but not in the small-harvested line. The large-harvested line also had higher serotonin turnover than controls at specific time periods. In conclusion, intensive size-selective mortality left an evolutionary legacy of asymmetric selection responses in the ontogeny of shoal cohesion and the underlying physiological mechanisms in experimentally harvested zebrafish in the laboratory.Significant statementThe evolution of animal behavior can be affected by human activities both at behavioral and physiological levels, but causal evidence is scarce and mostly focusing on single life-stages. We studied whether and to what extent size-selective harvesting, a common selection pattern in fisheries, can be an evolutionary driver of the development of shoal cohesion during ontogeny. We used a multi-generation experiment with zebrafish to study cause-and-effects of opposing size-selection patterns. We quantified shoal cohesion, and serotonin and dopamine turnover in the brain. We found that shoal cohesion emerged as a collective personality trait and that behavioral and physiological responses were asymmetrical with respect to the opposing selection patterns.

Temperature dependent cellular, and epigenetic regulatory mechanisms underlying the antiviral immunity in sevenband grouper to nervous necrosis virus infection

Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFβ. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.

RNAseq Analysis of Brain Aging in Wild Specimens of Short-Lived Turquoise Killifish: Commonalities and Differences With Aging Under Laboratory Conditions.

A vast body of studies is available that describe age-dependent gene expression in relation to aging in a number of different model species. These data were obtained from animals kept in conditions with reduced environmental challenges, abundant food, and deprivation of natural sensory stimulation. Here, we compared wild- and captive aging in the short-lived turquoise killifish (Nothobranchius furzeri). These fish inhabit temporary ponds in the African savannah. When the ponds are flooded, eggs hatch synchronously, enabling a precise timing of their individual and population age. We collected the brains of wild fish of different ages and quantified the global age-dependent regulation of transcripts using RNAseq. A major difference between captive and wild populations is that wild populations had unlimited access to food and hence grew to larger sizes and reached asymptotic size more rapidly, enabling the analysis of age-dependent gene expression without the confounding effect of adult brain growth. We found that the majority of differentially expressed genes show the same direction of regulation in wild and captive populations. However, a number of genes were regulated in opposite direction. Genes downregulated in the wild and upregulated in captivity were enriched for terms related to neuronal communication. Genes upregulated in the wild and downregulated in captive conditions were enriched in terms related to DNA replication. Finally, the rate of age-dependent gene regulation was higher in wild animals, suggesting a phenomenon of accelerated aging.

Effects of selected light wavelengths on the transcript levels of photoreceptors and growth-related hormones and peptides in the Malabar grouper Epinephelus malabaricus

Fish growth is influenced by environmental cues, including light conditions. The duration of exposure (photoperiod), the intensity of exposure (illumination), and quality (light spectrum) play crucial roles in maximizing growth. This study aimed to clarify how selected light wavelengths are perceived and transduced as an internal signal to stimulate growth of the Malabar grouper Epinephelus malabaricus, an important aquaculture species in Asian countries. Four opsins (RH1, RH2, SWS2, and LWS) were selected from the database of photoreceptors of groupers, and reverse-transcription polymerase chain reaction revealed that these opsins were expressed in the eyes and brain. When fish were reared under conditions of long-day (light:dark = 14:10) with red (peak at 632 nm), green (519 nm), and blue (465 nm) LED and natural light, the specific growth rate was higher (P < 0.05) in fish exposed to blue LED light than to red LED light. Rearing fish under shorter light wavelengths (blue or green) resulted in high transcript levels (P < 0.05) of RH1 and RH2 in the eyes (retina) and of RH1 and SWS2 in the brain (diencephalon), suggesting that certain light wavelengths activate photoreceptors in light perceptive organs. The transcript levels of growth hormone (GH) and insulin-like growth factor (IGF-I) increased significantly (P < 0.05) in the pituitary and liver of fish reared under blue LED light, respectively. Moreover, similar increases (P < 0.05) in the transcripts of neuropeptide Y (NPY) and pro-opiomelanocortin were observed in the brain of fish reared under blue LED light. We concluded that blue light perceived by visual opsins in the eyes and brain activates the growth of the endocrine axis and its interaction with NPY. Consequently, rearing the Malabar grouper under specific light wavelengths helps maximize their growth.

Neuroactive pharmaceuticals in estuaries: Occurrence and tissue-specific bioaccumulation in multiple fish species

Contamination of surface waters by pharmaceuticals is an emerging problem globally. This is because the increased access and use of pharmaceuticals by a growing world population lead to environmental contamination, threatening non-target species in their natural environment. Of particular concern are neuroactive pharmaceuticals, which are known to bioaccumulate in fish and impact a variety of individual processes such as fish reproduction or behaviour, which can have ecological impacts and compromise fish populations. In this work, we investigate the occurrence and bioaccumulation of 33 neuroactive pharmaceuticals in brain, muscle and liver tissues of multiple fish species collected in four different estuaries (Douro, Tejo, Sado and Mira). In total, 28 neuroactive pharmaceuticals were detected in water and 13 in fish tissues, with individual pharmaceuticals reaching maximum concentrations of 1590 ng/L and 207 ng/g ww, respectively. The neuroactive pharmaceuticals with the highest levels and highest frequency of detection in the water samples were psychostimulants, antidepressants, opioids and anxiolytics, whereas in fish tissues, antiepileptics, psychostimulants, anxiolytics and antidepressants showed highest concentrations. Bioaccumulation was ubiquitous, occurring in all seven estuarine and marine fish species. Notably, neuroactive compounds were detected in every water and fish brain samples, and in 95% of fish liver and muscle tissues. Despite variations in pharmaceutical occurrence among estuaries, bioaccumulation patterns were consistent among estuarine systems, with generally higher bioaccumulation in fish brain followed by liver and muscle. Moreover, no link between bioaccumulation and compounds’ lipophilicity, species habitat use patterns or trophic levels was observed. Overall, this work highlights the occurrence of a highly diverse suite of neuroactive pharmaceuticals and their pervasiveness in waters and fish from estuarine systems with contrasting hydromorphology and urban development and emphasizes the urgent need for toxicity assessment of these compounds in natural ecosystems, linked to internalized body concentration in non-target species.

Tissue-specific differences and temperature-dependent changes in Na,K-ATPase of the roach (Rutilus rutilus)

Na,K-ATPase activity is vital for virtually all cells of the animal body and the major consumer of cellular energy. However, Na,K-ATPase activity, the number of Na+ pump units, the catalytic rate of the pump, and their responses to thermal acclimation in different tissues of the same fish species have been seldom measured. Therefore, roach (Rutilus rutilus), eurythermal freshwater fish, was acclimated at 4 °C (cold-acclimated, CA) and 18 °C (warm-acclimated, WA), and the Na+ pump density and the temperature-dependence of Na,K-ATPase activity were determined in the heart, gills, kidney and brain. There were prominent tissues specific differences in Na,K-ATPase activity. The activity was highest in the brain (100%), whereas the activity of renal, branchial and cardiac enzymes was 55%, 9%, and 4% of the brain activity in the WA roach at 25 °C. The corresponding values for brain, kidney, gills and heart of the CA fish at 25 °C were 100%, 56%, 5% and 4%, respectively. The number of Na,K-ATPase α-subunits, determined by [3H]ouabain binding, strongly correlated with the Na,K-ATPase activity at R2 values of 0.99 and 0.85 for WA and CA fish, respectively. Acclimation to cold, increased the number of Na,K-ATPase α-subunits in the heart, kidney and brain (P < 0.05), but this did not appear as an increase in Na,K-ATPase activity. In the gills, Na,K-ATPase α-subunits did not change in cold-acclimation, but Na,K-ATPase activity was depressed. Although tissue-specific differences in Na,K-ATPase activity are largely determined by the density of α-subunits, differences in Na,K-ATPase activity within the same tissue due to temperature acclimation involve also other factors in addition to the number of Na+ pump units.

A COMPARATIVE STUDY ON BRAIN-TROPISM OF BETANODAVIRUSES IN YOUNG MARINE FISH: A WAY TO PERFORM HOST SPECIFICITY

Investigation on the brain-tropism was carried out through direct injection of the virus into the vitreous body of the eye using five marine fish species: kelp grouper, striped jack, Japanese flounder, threeline grunt and yellow tail. When the brains of intravitreously-injected fish (106 TCID50 Fish-1) were examined at 72 h post infection, striped jack nervous necrosis virus (SJNNV; the type species of the genus Betanodavirus) was frequently detected from striped jack and flounder while kelp grouper nervous necrosis virus (KGNNV) were detected from kelp grouper, striped jack, and flounder. No constant growth of the virus was noticed in the brains of threeline grunt and yellow tail. Considering that natural outbreaks of VNN (viral nervous necrosis) have been frequently reported in the former three species but not in the later two. The present result supports that the brain cell-tropism might be one of the tools to perform the host-specificity of betanodavirus.

Molecular markers of cytotoxicity induced with copper-contained pesticides

The applying copper sulphate is a cheap, however it leads to global the environment contamination. Especially, the contamination of the water sources is crucial for fish harvesting and farming. The copper contamination in some areas are caused by decades. The impact of copper ions is dependent on the inhibition of invasive aquatic plants, many microorganisms and protozoa. The family of copper-contained chemicals is frequently used as algaecides in swimming pools. Despite the copper ions are well known as a high toxic for fish in freshwater ponds, the copper sulphate remains one of the prevalent pesticides in fish farming everywhere. High cytotoxicity and accumulation of the copper ions in the sediments require studying and calculation of the optimal dosage to use it as an antiseptic agent that should prevent the detrimental effect in various tissue types of aquatic organisms. The main depicted mechanism, which is associated with cytotoxicity is the generation of oxidative stress.&#x0D; Neural tissue cells are extremely susceptible to oxidative damages. Neural damaging is important cause of the CNS disfunction as well as is the critic to the organism vitality. Glial cells maintain the structure and many vital functions of neuronal cells. Glial fibrillary acidic protein (GFAP), transcriptional nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and poly(ADP-ribose) polymerase (PARP) are critic regulators in a cell response to the environment toxicity. Based on this fact, it could be applied as the boimarkers of heavy metal toxicity. In presented study, we investigated the effects of copper ions on PARP, NF-kB, and GFAP expression in the rainbow trout (Oncorhynchus mykiss) brain tissue. For 96 hours the fishes were exposed to the copper sulphate sublethal concentrations 380 µg/L, estimated as 1/4 of the LC50 value. Western blot analysis of GFAP, NF-kB and PARP was used to assess further effects in the brain tissue.&#x0D; The modulatory effect of copper ions on the expression of GFAP was detected in fish brain. Reciprocal changes were observed in respect with 24 hours exposure in compare to both 48 hours and 96 hours treatment. Obtained results demonstrated meaningful disturbance in the expression of transcriptional factor NF-kB in the brain of fish group exposed to copper in compare to control group. Furthermore, the time-depended effect in a range 24 – 96 hours was detected. However, the short time exposure to copper ions initiated no effect in fish group treated 24 hours while in compare to both 48 hours and 96 hours treatment. The similar changes of PARP content in the brain of fish were determined as well. It deserves to be mentioned that all of aforementioned changes of GFAP, NF-kB and PARP expression were accompanied by increasing in reactive oxygen species content. Thus, copper ions cytotoxic effect could be initiated with oxidative injury in a brain tissue.&#x0D; On the other hand, the observed effect of copper in the fish brain evidences the unspecific toxicity of this ion in brain tissue cells.&#x0D; The results showed that copper ions could induce astroglial response accompanied by modulations of NF-kB and PARP-1 expression. Obtained in presented study data suggest that copper sulphate has significant effect on astrogliosis and DNA damage in the fish brain.

Relaxin ligand/receptor systems in the developing teleost fish brain: Conserved features with mammals and a platform to address neuropeptide system functions.

The relaxins (RLNs) are a group of peptide hormone/neuromodulators that can regulate a wide range of physiological processes ranging from reproduction to brain function. All the family members have originated from a RLN3-like ancestor via different rounds of whole genome and gene specific duplications during vertebrate evolution. In mammals, including human, the divergence of the different family members and the emergence of new members led to the acquisition of specific functions for the various relaxin family peptide and associated receptor genes. In particular, in mammals, it was shown, that the role of RLN3 is correlated to the modulation of arousal, stress responses, emotion, social recognition, and other brain functions, positioning this gene/peptide as a potential therapeutic target for neuropsychiatric disorders. This review highlights the evolutionary conservation of relaxin family peptide and receptor gene expression and their associated brain neural circuits. In the zebrafish, the expression pattern of the different relaxin family members has specific features that are conserved in higher species, including a likely similar functional role for the ancestral RLN3-like gene. The use of different model organisms, particularly the zebrafish, to explore the diversification and conservation of relaxin family ligands and receptor systems, provides a relatively high-throughput platform to identify their specific conserved or differential neuromodulatory roles in higher species including human.

Sublethal ammonia induces alterations of emotions, cognition, and social behaviors in zebrafish (Danio rerio).

Ammonia pollutants were usually found in aquatic environments is due to urban sewage, industrial wastewater discharge, and agricultural runoff and concentrations as high as 180mg/L (NH4+) have been reported in rivers. High ammonia levels are known to impair multiple tissue and cell functions and cause fish death. Although ammonia is a potent neurotoxin, how sublethal concentrations of ammonia influence the central nervous system (CNS) and the complex behaviors of fish is still unclear. In the present study, we demonstrated that acute sublethal ammonia exposure can change social behavior of adult zebrafish. The exposure to 90mg /L of (NH4+) for 4h induced a strong fear response and lower shoaling cohesion; exposure to 180mg /L of (NH4+) for 4h reduced the aggressiveness, and social recognition, while the anxiety, social preference, learning, and short-term memory were not affected. Messenger RNA expressions of glutaminase and glutamate dehydrogenase in the brain were induced, suggesting that ammonia exposure altered glutamate neurotransmitters in the CNS. Our findings in zebrafish provided delicate information of ammonia neurotoxicity in complex higher-order social behaviors, which has not been revealed previously. In conclusion, sublethal and acute ammonia exposure can change specific behaviors of fish, which might lead to reductions in individual and population fitness levels.

Social buffering of the stress response: insights from fishes.

Social buffering of stress refers to the effect of a social partner in reducing the cortisol or corticosterone response to a stressor. It has been well studied in mammals, particularly those that form pair bonds. Recent studies on fishes suggest that social buffering of stress also occurs in solitary species, gregarious species that form loose aggregations and species with well-defined social structures and bonds. The diversity of social contexts in which stress buffering has been observed in fishes holds promise to shed light on the evolution of this phenomenon among vertebrates. Equally, the relative simplicity of the fish brain is advantageous for identifying the neural mechanisms responsible for social buffering. In particular, fishes have a relatively small and simple forebrain but the brain regions that are key to social buffering, including the social behaviour network, the amygdala and the hypothalamic–pituitary–adrenal/interrenal axis, are functionally conserved across vertebrates. Thus, we suggest that insight into the mechanistic and evolutionary underpinnings of stress buffering in vertebrates can be gained from the study of social buffering of stress in fishes.

In Vivo Diagnostic Real-time Wireless Sensing of Glucose in Human Urine and Live Fish Deep Brain Cells

Background: A method for the diagnosis of glucose was developed using square-wave anodic stripping voltammetry (SWASV). Objective: With mercury immobilized onto the carbon nanotube using a handmade paste electrode (HgPE). In this study, the method of modification probe was first tried directly on live cells and untreated human urine. Methods: The optimized results indicated a sensitive peak signal of glucose on the HgPE. Curves were obtained within a concentration range of 10ngL-1~14mgL-1 with a preconcentration time of 100 sec. Results: The observed relative standard deviation (RSD) was 0.279 (n=15), with a glucose concentration of 1 ugL-1 under optimum conditions and a 50-sec accumulation. A low detection limit (S/N) of 0.6 ngL-1 (2.99×10-12 M) was also attained, which is better sensitive than other methods of 58 M, 7 uM, 2×10-6M, 0.13 mg/ml, 8 mM, 40 mM. Conclusion: The final results indicate that the method could be applied to the diagnosis of glucose in human urine and deep live fish brain cells in real-time using wireless, and the method can be used in assays, in vivo and in real-time, without requiring any pretreatment and other pharmaceutical or medicinal analysis, as well as other materials requiring diagnostic analyses.

Negative Effect of Lambda-cyhalothrin (Insecticide) on Alazani River Fish, Luciobarbus Mursa

Pesticides are complex organic substances that are an important source of water pollution. Using pesticides is one of the important actions for harvest protection. This, however, does not negate the fact that its usage can result to negative consequences as well. The study of toxic action of lambda-cigalotrin (the piretroid of the insecticides which is included in icons, karate, commodore, samurai) on quantitative contents of fatty acids and structural lipids in muscle, gills, liver, and in the brain of fishes in river Alazani was carried out. Based on the actual material, one can conclude that intoxication of the fish tissues with lambda-cyhalotrin processes appear, which stimulates oxidation of free radicals of structural lipids. The peroxides of the initiators of free radicals are formed, which stimulate lipid per oxidation. Exposure to insecticides caused an induction of antioxidant enzyme activities indicating the activation of the insecticides detoxification pathways and the antioxidant defenses. The non-protein sulfhydryl groups increased to 1,75  0,109 compared to the control (1,04  0,123), while the total sulfhydryl groups statistically increased to 39,16  1,44 compared to the control (34,00  1,22 ).This indicates an increase in the concentration of protein and total sulfhydryl groups compared to controls. The study also identified that excess amounts of insecticides in the tissues disrupts the biochemical parameters. These changes may be potentially disruptive for the survivability of fish in natural resources. This fact should thus be taken into consideration when this insecticide is used for pest control in agriculture fields surrounding surface water and ground water resources.

MERCURY CHLORIDE (HgCl2) EXPOSURE CHANGES THE HISTOPATHOLOGICAL FIGURE OF EYE AND BRAIN OF TILAPIA FISH (Oreochromis mossambicus)

Mercury pollution brings harmful effects to aquatic animals, the environment and eventually to human health. Mercury accumulates in the liver, kidney, eye lens and brain of fish, resulting in organ damage. This study aimed to determine the effect of HgCl2 exposure on anatomical pathology and histopathology of tilapia fish eye and brain. A total of 36 male tilapia fish were allotted into 4 treatment groups with 3 replications. Fish were exposed to 0.00, 0.25, 0.50, and 0.75 ppm of HgCl2 for 10, 20, and 30 days. Subsequently, the anatomical pathology was observed followed by histopathological examination. Anatomical pathology examination of fish eye on day 30 showed white membrane on the eye lens surface, pupil diminution, and sunken eyes. The brain demonstrated hemorrhage, necrosis, discolorations, and granulated area. The retina showed necrosis, retina pigmentation flexiform layer widened, and cone cell atrophy. Brain depicted structural and cellular damage such as degeneration necrosis and vacuolation. HgCl2 exposure changes the anatomical pathology and histopathology of tilapia fish eye and brain.

Characterization of three novel cell lines derived from the brain of spotted sea bass: Focusing on cell markers and susceptibility toward iridoviruses

Despite tens of cell lines originating from fish brain tissue have been constructed, little is known about the definite cell types they belong to. Whether fish cell lines derived from the brain shares similar characteristics is not well-answered yet. Here, we constructed three cell lines designated as LMB-S, LMB-M, LMB-L using brain tissue of spotted sea bass (Lateolabrax maculatus). Among them, LMB-L was identified as astroglia-like cells considering the high expression of GFAP, DCX, PTX, S100b, which are regarded as astrocyte-specific or astrocyte-associated cell markers. LMB-M exhibited smooth muscle-like features showing strong expression of LMOD1, SLAMP, M-cadherin, MGP, which are confirmed as muscle-restricted or myogenesis-involved cell markers. Although LMB-S was not definitely identified, it appeared an activation of WNT/β-catenin pathway. Besides the distinct expression profiles of cell markers, the three cell lines also presented differences in transfection efficiency and susceptibility to iridovirus infection. Relying on the established cell lines, a novel megalocytivirus, named LMIV (Lateolabrax maculatus iridovirus), was first isolated from diseased spotted sea bass. Genetic analysis of major capsid protein (MCP) and adenosine triphosphatase (ATPase) manifested that LMIV was clearly distinguishable from other representative teleost iridoviruses. Further investigations revealed that LMIV could replicate most efficiently in LMB-L cells obtaining the highest viral load (2.16 × 1010 copy/mL). By contrast, LMB-S cells gave rise to the highest viral load up to 3.86 × 108 copy/mL, when the three cell lines were infected with MRV, a newly emerged ranavirus. Moreover, LMIV infection caused lots of cells to be detached from monolayers, generating adherent and non-adherent cells. An opposite expression profiling of type I IFN pathway-related genes (JAK1, STAT1, STAT2, IRF9, Mx1) was found between adherent and non-adherent cells. Combined with the analysis of MCP gene expression, it is speculated that inhibiting type I IFN pathway in non-adherent cells allowed the facilitation of virus duplication. Taken together, the present study broadens our understanding about the diversity of cell lines derived from fish brain tissue and screening cells more susceptible to virus is not only meaningful for the development of vaccine, but also provide clues for further clarification of cell-iridovirus interactions.

Chronic exposure to realistic concentrations of metformin prompts a neurotoxic response in Danio rerio adults

Metformin (MET) is among the most consumed drugs around the world, and thus, it is considered the uppermost drug in mass discharged into water settings. Nonetheless, data about the deleterious consequences of MET on water organisms are still scarce and require further investigation. Herein, we aimed to establish whether or not chronic exposure to MET (1, 20, and 40 μg/L) may alter the swimming behavior and induce neurotoxicity in Danio rerio adults. After 4 months of exposure, MET-exposed fish exhibited less swimming activity when compared to control fish. Moreover, compared with the control group, MET significantly inhibited the activity of AChE and induced oxidative damage in the brain of fish. Concerning gene expression, MET significantly upregulated the expression of Nrf1, Nrf2, BAX, p53, BACE1, APP, PSEN1, and downregulated CASP3 and CASP9. Although MET did not overexpress the CASP3 gene, we saw a meaningful rise in the activity of this enzyme in the blood of fish exposed to MET compared to the control group, which we then confirmed by a high number of apoptotic cells in the TUNEL assay. Our findings demonstrate that chronic exposure to MET may impair fish swimming behavior, making them more vulnerable to predators.

Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research.

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Development of cell lines from brain, spleen and heart of ornamental blood parrot cichlid and their susceptibility to Tilapia tilapinevirus

Blood parrot cichlid, a hybrid of Amphilophus citrinellus (♂) and Paraneetroplus synspilus (♀), is among the most popular ornamental fishes globally. In this study, three cell lines were established from brain, spleen, and heart tissues of the blood parrot cichlid using explant method and these were designated as parrot fish brain (PFB), parrot fish spleen (PFS) and parrot fish heart (PFH), respectively. All the cell lines have been subcultured for over 120 passages and these grow optimally at 28–32 °C in Leibovitz's L-15 medium containing 20% fetal bovine serum. PFB and PFH cells depict epithelial morphology whereas PFS cells consist of fibroblast-like cells. Although the chromosome number of blood parrot cichlid was 2n = 48, the cell lines exhibited heteroploidy and aneuploidy, suggesting transformation. The sequencing of amplified fragments of two mitochondrial genes, namely COI and 16S rRNA confirmed that the cell lines originated from blood parrot cichlid. The three cell lines were free of Mycoplasma contamination. Besides, their successful transfection with a GFP reporter gene suggested potential application in transient gene expression studies. All the three cell lines were susceptible to Tilapia tilapinevirus, also known as Tilapia lake virus (TiLV), which was indicated by appearance of cytopathic effects and RT-PCR. Of the three cell lines, PFS was found to be the most permissive with maximum TiLV copy number of 2.5 × 108 per 100 ng of total RNA, as determined by qRT-PCR. Importantly, the TiLV disease was successfully reproduced in naive tilapia after injection of cell culture supernatant from TiLV-infected PFS cell line, and this was evidenced by clinical signs, histopathological alterations, reisolation of virus and specific amplification in RT-PCR. The three newly established cell lines were submitted to National Repository on Fish Cell Lines at ICAR-NBFGR. This forms the first report of development of cell lines from blood parrot cichlid, and these would be useful for surveillance of diseases suspected to be of viral origin in this candidate species and also other ornamental cichlids.