Zebrafish Receptors Research Articles

Biodistribution of synthesized polyethylene terephthalate fibers in adult zebrafish, their sex hormone disruption effect, and mitigation using natural organic matter

Although polyethylene terephthalate (PET) fibers are a representative form of plastic pollutants, studies on their toxicity are currently limited compared to other plastic types. Moreover, the effect of natural organic matter (NOM) on their toxicity has not been investigated. In this study, female and male adult zebrafish were exposed to synthesized PET fibers at concentrations of 0.1, 1, 10, and 100 mg/L in the presence and absence of 10 mg/L of NOM for 10 d. Bioaccumulation of PET fibers in zebrafish intestine, liver, and gills was identified and expression levels of reactive oxygen species (ROS) generation, sex hormones, and oxidative stress and sex hormone-related genes were measured. In addition, the developmental stages of gonadal cells were examined through histological analysis. We found that PET fibers bioaccumulated in the intestine and liver of zebrafish. ROS generation significantly increased at 100 mg/L of PET fibers, the expression of oxidative stress-related genes decreased in female and increased in male zebrafish. Exposure to 100 mg/L of PET fibers did not affect 17-beta estradiol, but significantly decreased the testosterone levels in male zebrafish. Sex hormone-related genes significantly decreased in both female and male zebrafish, except for androgen receptor in female zebrafish. However, these changes were exacerbated by the removal of NOM, suggesting a protective effect of NOM against PET fibers toxicity. We demonstrated that the accumulated PET fibers may lead to oxidative stress and sex hormone alteration, and disrupt the development of gonadal cells. Additionally, the NOM coating did not alter bioaccumulation considerably, but mitigated the adverse effects at the hormone level in PET fiber-exposed zebrafish. Thus, this study provides a basis for further research on the toxicity assessment of PET fibers and interactions between NOM and PET fiber-related toxicity.

Exploring estrogen antagonism using CRISPR/Cas9 to generate specific mutants for each of the receptors

Endocrine disruptors are chemicals that have been in the spotlight for some time now. Their modulating action on endocrine signaling pathways made them a particularly interesting topic of research within the field of ecotoxicology. Traditionally, endocrine disrupting properties are studied using exposure to suspected chemicals. In recent years, a major breakthrough in biology has been the advent of targeted gene editing tools to directly assess the function of specific genes. Among these, the CRISPR/Cas9 method has accelerated progress across many disciplines in biology. This versatile tool allows to address antagonism differently, by directly inactivating the receptors targeted by endocrine disruptors. Here, we used the CRISPR/Cas9 method to knock out the different estrogen receptors in zebrafish and we assessed the potential effects this generates during development. We used a panel of biological tests generally used in zebrafish larvae to investigate exposure to compounds deemed as endocrine disrupting chemicals. We demonstrate that the absence of individual functional estrogen receptors (Esr1, Esr2b, or Gper1) does affect behavior, heart rate and overall development. Each mutant line was viable and could be grown to adulthood, the larvae tended to be morphologically grossly normal. A substantial fraction (70%) of the esr1 mutants presented severe craniofacial deformations, while the remaining 30% of esr1 mutants also had changes in behavior. esr2b mutants had significantly increased heart rate and significant impacts on craniofacial morphometrics. Finally, mutation of gper1 affected behavior, decreased standard length, and decreased bone mineralization as assessed in the opercle. Although the exact molecular mechanisms underlying these effects will require further investigations in the future, we added a new concept and new tools to explore and better understand the actions of the large group of endocrine disrupting chemicals found in our environment.

Developmental Delay and Male-Biased Sex Ratio in esr2b Knockout Zebrafish.

The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Angpt1 binding to Tie1 regulates the signaling required for lymphatic vessel development in zebrafish.

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish.

(-)-Epicatechin gallate ameliorates cyprodinil-induced cardiac developmental defects through inhibiting aryl hydrocarbon receptor in zebrafish.

Cyprodinil is a widely used fungicide with broad-spectrum activity, but it has been associated with cardiac abnormalities. (-)-Epicatechin gallate (ECG), a natural polyphenolic compound, has been shown to possess protective properties in cardiac development. In this study, we investigated whether ECG could mitigate cyprodinil-induced heart defects using zebrafish embryos as a model. Zebrafish embryos were exposed to cyprodinil with or without ECG. Our results demonstrated that ECG significantly improved the survival rate, embryo movement, and hatching delay induced by cyprodinil. Furthermore, ECG effectively ameliorated cyprodinil-induced cardiac developmental toxicity, including pericardial anomaly and impairment of cardiac function. Mechanistically, ECG attenuated the cyprodinil-induced alterations in mRNA expression related to cardiac development, such as amhc, vmhc, tbx5, and gata4, as well as calcium ion channels, such as ncx1h, atp2a2a, and cdh2. Additionally, ECG was found to inhibit the activity of the aryl hydrocarbon receptor (AhR) signaling pathways induced by cyprodinil. In conclusion, our findings provide evidence for the protective effects of ECG against cyprodinil-induced cardiac developmental toxicity, mediated through the inhibition of AhR activity. These findings contribute to a better understanding of the regulatory mechanisms and safe utilization of pesticide, such as cyprodinil.

Lrp13a and Lrp13b serve as vitellogenin receptors in the ovary of zebrafish†.

In oviparous animals, egg yolk is largely derived from vitellogenin, which is taken up from the maternal circulation by the growing oocytes via the vitellogenin receptor. Recently, a novel member of the lipoprotein receptor superfamily termed low-density lipoprotein receptor-related protein 13 was identified and proposed as a candidate of vitellogenin receptor in oviparous animals. However, the roles of low-density lipoprotein receptor-related protein 13 in vitellogenesis are still poorly defined. Here, we investigated the expression, vitellogenin-binding properties, and function of low-density lipoprotein receptor-related protein 13 in zebrafish. Two different lrp13 genes termed lrp13a and lrp13b were found in zebrafish. Reverse transcription polymerase chain reaction and quantitative polymerase chain reaction revealed both lrp13s to be predominantly expressed in zebrafish ovary, and in situ hybridization detected both lrp13s transcripts in the ooplasm of early stage oocytes. Two yeast hybrid studies showed that among eight vitellogenins of zebrafish, Vtg1, 2, and 3 bind to Lrp13a, while Vtg1, 2, and 5 bind to Lrp13b. We created zebrafish lrp13a and lrp13b mutant lines using CRISPR/Cas9. Knockout of lrp13a leads to a male-biased sex ratio and decreased diameter of embryo yolk, while knockout of lrp13b and double knockout of lrp13a and lrp13b leads to the delay of vitellogenesis, followed by follicular atresia. These phenotypes of mutants can be explained by the disruption of vitellogenesis in the absence of Lrp13s. Taken together, our results indicate that both Lrp13a and Lrp13b can serve as vitellogenin receptors in zebrafish among other vitellogenin receptors that are not yet described.

Characterizing the SREB G protein-coupled receptor family in fish: Brain gene expression and genomic differences in upstream transcription factor binding sites

The SREB (Super-conserved Receptors Expressed in Brain) family of orphan G protein-coupled receptors is highly conserved in vertebrates and consists of three members: SREB1 (orphan designation GPR27), SREB2 (GPR85), and SREB3 (GPR173). SREBs are associated with processes ranging from neuronal plasticity to reproductive control. Relatively little is known about similarities across the entire family, or how mammalian gene expression patterns compare to non-mammalian vertebrates. In fish, this system may be particularly complex, as some species have gained a fourth member (SREB3B) while others have lost genes. To better understand the system, the present study aimed to: 1) use qPCR to characterize sreb and related gene expression patterns in the brains of three fish species with different systems, and 2) identify possible differences in transcriptional regulation among the receptors, using upstream transcription factor binding sites across 70 ray-finned fish genomes. Overall, regional patterns of sreb expression were abundant in forebrain-related areas. However, some species-specific patterns were detected, such as abundant expression of receptors in zebrafish (Danio rerio) hypothalamic-containing sections, and divergence between sreb3a and sreb3b in pufferfish (Dichotomyctere nigroviridis). In addition, a gene possibly related to the system (dkk3a) was spatially correlated with the receptors in all three species. Genomic regions upstream of sreb2 and sreb3b, but largely not sreb1 or sreb3a, contained many highly conserved transcription factor binding sites. These results provide novel information about expression differences and transcriptional regulation across fish that may inform future research to better understand these receptors.

A ligand-receptor interactome atlas of the zebrafish

Studies in zebrafish can unravel the functions of cellular communication and thus identify novel bench-to-bedside drugs targeting cellular communication signaling molecules. Due to the incomplete annotation of zebrafish proteome, the knowledge of zebrafish receptors, ligands, and tools to explore their interactome is limited. To address this gap, we de novo predicted the cellular localization of zebrafish reference proteome using deep learning algorithm. We combined the predicted and existing annotations on cellular localization of zebrafish proteins and created repositories of zebrafish ligands, membrane receptome, and interactome as well as associated diseases and targeting drugs. Unlike other tools, our interactome atlas is based on both the physical interaction data of zebrafish proteome and existing human ligand-receptor pair databases. The resources are available as R and Python scripts. DanioTalk provides a novel resource for researchers interested in targeting cellular communication in zebrafish, as we demonstrate in applications studying synapse and axo-glial interactome. DanioTalk methodology can be applied to build andexplore the ligand-receptor atlas of other non-mammalian model organisms.

A comprehensive pharmacological analysis of fenoterol and its derivatives to unravel the role of β2-adrenergic receptor in zebrafish

β-adrenergic receptors (βARs) belong to a key molecular targets that regulate the most important processes occurring in the human organism. Although over the last decades a zebrafish model has been developed as a model complementary to rodents in biomedical research, the role of β2AR in regulation of pathological and toxicological effects remains to elucidate. Therefore, the study aimed to clarify the role of β2AR with a particular emphasis on the distinct role of subtypes A and B of zebrafish β2AR. As model compounds selective β2AR agonists – (R,R)-fenoterol ((R,R)-Fen) and its new derivatives: (R,R)-4’-methoxyfenoterol ((R,R)-MFen) and (R,R)-4'-methoxy-1-naphtylfenoterol ((R,R)-MNFen) – were tested. We described dose-dependent changes observed after fenoterols exposure in terms of general toxicity, cardiotoxicity and neurobehavioural responses. Subsequently, to better characterise the role of β2-adrenergic stimulation in zebrafish, we have performed a series of molecular docking simulations. Our results indicate that (R,R)-Fen displays the highest affinity for subtype A of zebrafish β2AR and β2AAR might be involved in pigment depletion. (R,R)-MFen shows the lowest affinity for zebrafish β2ARs out of the tested fenoterols and this might be associated with its cardiotoxic and anxiogenic effects. (R,R)-MNFen displays the highest affinity for subtype B of zebrafish β2AR and modulation of this receptor might be associated with the development of malformations, increases locomotor activity and induces a negative chronotropic effect. Taken together, the presented data offer insights into the functional responses of the zebrafish β2ARs confirming their intraspecies conservation, and support the translation of the zebrafish model in pharmacological and toxicological research.

Fishing for a balance: Toward promyelinating drug discovery

In this issue, Häberlein etal. demonstrate a role for GPR17 in regulating zebrafish oligodendrocyte differentiation. Zebrafish expressing a humanized GPR17 respond to modulators, which are inactive against the endogenous zebrafish receptor. These findings highlight the potential for humanized zebrafish as an invivo platform for targeted remyelination drug screens.

Spatial organization of olfactory receptor gene choice in the complete V1R-related ORA family of zebrafish

The vertebrate sense of smell employs four main receptor families for detection of odors, among them the V1R/ORA family, which is unusually small and highly conserved in teleost fish. Zebrafish possess just seven ORA receptors, enabling a comprehensive analysis of the expression patterns of the entire family. The olfactory organ of zebrafish is representative for teleosts, cup-shaped, with lamella covered with sensory epithelium protruding into the cup from a median raphe. We have performed quantitative in situ hybridization on complete series of horizontal cryostat sections of adult zebrafish olfactory organ, and have analysed the location of ora-expressing cells in three dimensions, radial diameter, laminar height, and height-within-the-organ. We report broadly overlapping, but distinctly different distributions for all ora genes, even for ora3a and ora3b, the most recent gene duplication. Preferred positions in different dimensions are independent of each other. This spatial logic is very similar to previous reports for the much larger families of odorant receptor (or) and V2R-related olfC genes in zebrafish. Preferred positions for ora genes tend to be more central and more apical than those we observed for these other two families, consistent with expression in non-canonical sensory neuron types.

Regulation of heart rate following genetic deletion of the ß1 adrenergic receptor in larval zebrafish.

AimAlthough zebrafish are gaining popularity as biomedical models of cardiovascular disease, our understanding of their cardiac control mechanisms is fragmentary. Our goal was to clarify the controversial role of the ß1‐adrenergic receptor (AR) in the regulation of heart rate in zebrafish.MethodsCRISPR‐Cas9 was used to delete the adrb1 gene in zebrafish allowing us to generate a stable adrb1 −/− line. Larval heart rates were measured during pharmacological protocols and with exposure to hypercapnia. Expression of the five zebrafish adrb genes were measured in larval zebrafish hearts using qPCR.ResultsCompared with genetically matched wild‐types (adrb1 +/+ ), adrb1 −/− larvae exhibited ~20 beats min−1 lower heart rate, measured from 2 to 21 days post‐fertilization (dpf). Nevertheless, adrb1 −/− larvae exhibited preserved positive chronotropic responses to pharmacological treatment with AR agonists (adrenaline, noradrenaline, isoproterenol), which were blocked by propranolol (general ß‐AR antagonist). Regardless of genotype, larvae exhibited similar increases in heart rate in response to hypercapnia (1% CO2) at 5 dpf, but tachycardia was blunted in adrb1 −/− larvae at 6 dpf. adrb1 gene expression was abolished in the hearts of adrb1 −/− larvae, confirming successful knockout. While gene expression of adrb2a and adrb3a was unchanged, adrb2b and adrb3b mRNA levels increased in adrb1 −/− larval hearts.ConclusionDespite adrb1 contributing to the setting of resting heart rate in larvae, it is not strictly essential for zebrafish, as we generated a viable and breeding adrb1 −/− line. The chronotropic effects of adrenergic stimulation persist in adrb1 −/− zebrafish, likely due to the upregulation of other ß‐AR subtypes.

Kisspeptin stimulates oocyte maturation, and food deprivation modulates the abundance of kisspeptin system in zebrafish gonads

Kisspeptin is a hormone involved in the neuroendocrine control of reproduction in fish. We hypothesized that kisspeptin stimulates oocyte maturation and modulates other reproductive hormones in zebrafish; and the gonadal kisspeptin system in zebrafish is affected during energy unavailability. The main goals of this research were to test in vitro effects of kisspeptin on oocyte maturation and mRNA abundance in zebrafish ovarian follicles and determine how short-term feed restriction affects kisspeptin and its receptors in zebrafish testis and ovary. This study demonstrates the presence of kisspeptin and its receptors in zebrafish (Danio rerio) gonads and its direct action on ovarian follicles in vitro. Kisspeptin (10 ng/mL) induced oocyte maturation, as indicated by germinal vesicle break down at 18- and 24- hours post-treatment. Kisspeptin significantly increased the abundance of mRNAs encoding reproductive hormones and its receptors in zebrafish oocytes. This suggests that kisspeptin-10 affects ovarian functions by modulating other hormones. Reproduction is a process that requires energy. Therefore, whether energy availability affects the kisspeptin system in zebrafish gonads was determined. Food deprivation modulated kisspeptin expression differently in zebrafish testis and ovary. Kiss2 and kiss1ra were upregulated while kiss1rb was downregulated in the testis post-food deprivation. Meanwhile, no changes in kiss in the ovary were found after food deprivation. However, kiss1rb was downregulated in unfed fish at 3- and 7-days post-food deprivation. Overall, our results suggest sex- and tissue-specific changes in the gonadal abundance of the kisspeptin system in zebrafish. The fine tuning of reproduction during energy fluctuations in fish is likely mediated via changes in hormones, including kisspeptin as shown in this research.

Botulinum toxin promotes orofacial antinociception by modulating TRPV1 and NMDA receptors in adult zebrafish

The aim of the study was to evaluate the possible involvement of transient receptor potential (TRP) channels, Acid-sensing ion channels (ASIC) and N-Methyl-D-aspartate receptor (NMDAR) in the orofacial antinociceptive behaviour effect of botulinum toxin type A (BoNT/A) in adult zebrafish. Initially, the open field test was performed to evaluate the effect of BoNT/A on the locomotor activity of zebrafish. Subsequently, the animals were pretreated with BoNT/A (0.05U, 0.1U or 0.5U/masseter) and acute orofacial nociception was induced by cinnamaldehyde, capsaicin, menthol, acid saline or glutamate applied to the lip or masseter muscle. In another group of experiments, animals were pre-treated with capsazepine (TRPV1 antagonist) or ketamine (NMDAR antagonist) to investigate the mechanism of antinociception. The possible involvement of central C-fibre afferents was also investigated using capsaicin desensitized animals. A molecular docking study was performed to observe the in silico interaction of BoNT/A with TRPV1 and NMDA channels. Pretreatment with BoNT/A reduced the nociceptive behaviour induced by capsaicin and glutamate. Antinociception was effectively inhibited by capsazepine and ketamine, as well as by capsaicin-induced desensitization. Consistent with these in vivo findings, the molecular docking study indicated that BoNT/A can interact with TRPV1 and NMDAR. The results indicate the involvement of TRP and NMDAR mechanisms in the orofacial antinociceptive behaviour effect of BoNT/A. The results also confirm the pharmacological relevance of BoNT/A as an inhibitor of orofacial nociception behaviour.

An Ancient Adenosine Receptor Gains Olfactory Function in Bony Vertebrates.

Nucleotides are an important class of odorants for aquatic vertebrates such as frogs and fishes, but also have manifold signaling roles in other cellular processes. Recently, an adenosine receptor believed to belong to the adora2 clade has been identified as an olfactory receptor in zebrafish. Here, we set out to elucidate the evolutionary history of both this gene and its olfactory function. We have performed a thorough phylogenetic study in vertebrates, chordates and their sister group, ambulacraria, and show that the origin of the zebrafish olfactory receptor gene can be traced back to the most recent common ancestor of all three groups as a segregate sister clade (adorb) to the adora gene family. Eel, carp, and clawed frog all express adorb in a sparse and distributed pattern within their olfactory epithelium very similar to the pattern observed for zebrafish that is, consistent with a function as olfactory receptor. In sharp contrast, lamprey adorb-expressing cells are absent from the sensory region of the lamprey nose, but form a contiguous domain directly adjacent to the sensory region. Double-labeling experiments confirmed the expression of lamprey adorb in nonneuronal cells and are consistent with an expression in neuronal progenitor cells. Thus, adorb may have undergone a switch of function in the jawed lineage of vertebrates towards a role as olfactory receptor.

HDL biodistribution and brain receptors in zebrafish, using HDLs as vectors for targeting endothelial cells and neural progenitors

High density lipoproteins (HDLs) display pleiotropic functions such as anti-inflammatory, antioxidant, anti-protease, and anti-apoptotic properties. These effects are mediated by four main receptors: SCARB1 (SR-BI), ABCA1, ABCG1, and CD36. Recently, HDLs have emerged for their potential involvement in brain functions, considering their epidemiological links with cognition, depression, and brain plasticity. However, their role in the brain is not well understood. Given that the zebrafish is a well-recognized model for studying brain plasticity, metabolic disorders, and apolipoproteins, it could represent a good model for investigating the role of HDLs in brain homeostasis. By analyzing RNA sequencing data sets and performing in situ hybridization, we demonstrated the wide expression of scarb1, abca1a, abca1b, abcg1, and cd36 in the brain of adult zebrafish. Scarb1 gene expression was detected in neural stem cells (NSCs), suggesting a possible role of HDLs in NSC activity. Accordingly, intracerebroventricular injection of HDLs leads to their uptake by NSCs without modulating their proliferation. Next, we studied the biodistribution of HDLs in the zebrafish body. In homeostatic conditions, intraperitoneal injection of HDLs led to their accumulation in the liver, kidneys, and cerebral endothelial cells in zebrafish, similar to that observed in mice. After telencephalic injury, HDLs were diffused within the damaged parenchyma and were taken up by ventricular cells, including NSCs. However, they failed to modulate the recruitment of microglia cells at the injury site and the injury-induced proliferation of NSCs. In conclusion, our results clearly show a functional HDL uptake process involving several receptors that may impact brain homeostasis and suggest the use of HDLs as delivery vectors to target NSCs for drug delivery to boost their neurogenic activity.

At the Root of T2R Gene Evolution: Recognition Profiles of Coelacanth and Zebrafish Bitter Receptors.

The careful evaluation of food is important for survival throughout the animal kingdom, and specialized chemoreceptors have evolved to recognize nutrients, minerals, acids, and many toxins. Vertebrate bitter taste, mediated by the taste receptor type 2 (T2R) family, warns against potentially toxic compounds. During evolution T2R receptors appear first in bony fish, but the functional properties of bony fish T2R receptors are mostly unknown. We performed a phylogenetic analysis showing the “living fossil” coelacanth (Latimeria chalumnae) and zebrafish (Danio rerio) to possess T2R repertoires typical for early-diverged species in the lobe-finned and the ray-finned clade, respectively. Receptors from these two species were selected for heterologous expression assays using a diverse panel of bitter substances. Remarkably, the ligand profile of the most basal coelacanth receptor, T2R01, is identical to that of its ortholog in zebrafish, consistent with functional conservation across >400 Myr of separate evolution. The second coelacanth receptor deorphaned, T2R02, is activated by steroid hormones and bile acids, evolutionary old molecules that are potentially endogenously synthesized agonists for extraoral T2Rs. For zebrafish, we report the presence of both specialized and promiscuous T2R receptors. Moreover, we identified an antagonist for one of the zebrafish receptors suggesting that bitter antagonism contributed to shape this receptor family throughout evolution.

Taurine-mediated aggression is abolished via 5-HT1A antagonism and serotonin depletion in zebrafish

Taurine is one of the most abundant amino acids in vertebrates involved in important physiological functions, including osmoregulation, membrane stability, and neuronal activity. The pleiotropic effects of taurine support the existence of different mechanisms of action (e.g., modulation of GABAA, strychnine-sensitive glycine, and NMDA receptors), which can play a role in aggressive-related responses. However, the mechanisms underlying the effects of taurine on aggression are still poorly understood. Because aggression has been associated with diverse central mechanisms, especially serotonergic activity, we aimed to investigate the involvement of this system in taurine-induced aggression in zebrafish. We treated adult zebrafish with ρ-chlorophenylalanine (ρCPA), an inhibitor of the serotonin synthesis, as well as 5-HT1A receptor antagonist and agonist (WAY100135 and buspirone, respectively). Taurine effects were tested individually at three concentrations (42, 150, and 400 mg/L) for 60 min. We further analyzed the effects on aggression and locomotion using the mirror-induced aggression test. Taurine concentration that changed behavioral responses was selected to the succeeding pharmacological experiments using ρCPA, WAY100135, and buspirone. We found that buspirone did not alter the aggression. Yet, 42 mg/L taurine increased aggression, which was abolished by ρCPA and WAY100135, indicating the involvement of 5-HT1A receptors in taurine-mediated aggression. These set of data support an indirect mechanism mediating taurine-induced aggression via serotonin release and activation of 5-HT1A receptors in zebrafish. While the exact mechanisms underlying aggression are still unclear, our novel findings reveal a key role of the serotonergic system in the effects of taurine, supporting the use of zebrafish models to understand the neural basis of aggression in vertebrates.

Genome-wide analysis of Toll-like receptors in zebrafish and the effect of rearing temperature on the receptors in response to stimulated pathogen infection.

Water temperature has a major influence on the host innate immune defence and the infectivity of pathogens in ectothermic teleosts. Toll-like receptors (TLRs) are the first and well-characterized innate immune receptors that are conserved in vertebrates. However, little is known about the effect of temperature variation on TLRs in fish species. In this study, we used adult zebrafish as a research model to investigate the effect of water temperature on TLRs. Whole genome searches identified 20 TLR homologue genes in zebrafish. Multiple sequence alignment and protein structure analysis revealed the conserved domains for these TLR proteins. To identify TLR genes related to temperature variation, TLR family genes from 12 species with different body temperatures were assigned to conduct phylogenetic analyses. Based on the phylogenetic relationships, TLR3, TLR4, TLR5 and TLR20~21 were selected as candidate genes. Immunostimulation data indicated that TLR3, TLR5, and TLR21 were more sensitive to temperature variation and their expression levels were affected in response to pathogen stimulation. Taken together, our results provide a new opportunity to understand the roles of temperature on host innate immune response in fishes and have broader implications for disease prevention in aquaculture.

Two highly related odorant receptors specifically detect α-bile acid pheromones in sea lamprey (Petromyzon marinus)

Pheromones play critical roles in habitat identification and reproductive behavior synchronization in the sea lamprey (Petromyzon marinus). The bile acid 3-keto petromyzonol sulfate (3kPZS) is a major component of the sex pheromone mixture from male sea lamprey that induces specific olfactory and behavioral responses in conspecific individuals. Olfactory receptors interact directly with pheromones, which is the first step in their detection, but identifying the cognate receptors of specific pheromones is often challenging. Here, we deorphanized two highly related odorant receptors (ORs), OR320a and OR320b, of P. marinus that respond to 3kPZS. In a heterologous expression system coupled to a cAMP-responsive CRE-luciferase, OR320a and OR320b specifically responded to C24 5α-bile acids, and both receptors were activated by the same set of 3kPZS analogs. OR320a displayed larger responses to all 3kPZS analogs than did OR320b. This difference appeared to be largely determined by a single amino acid residue, Cys-792.56, the C-terminal sixth residue relative to the most conserved residue in the second transmembrane domain (2.56) of OR320a. This region of TM2 residues 2.56-2.60 apparently is critical for the detection of steroid compounds by odorant receptors in lamprey, zebrafish, and humans. Finally, we identified OR320 orthologs in Japanese lamprey (Lethenteron camtschaticum), suggesting that the OR320 family may be widely present in lamprey species and that OR320 may be under purifying selection. Our results provide a system to examine the origin of olfactory steroid detection in vertebrates and to define a highly conserved molecular mechanism for steroid-ligand detection by G protein-coupled receptors.