Sex recognition in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

We investigated sex recognition in female zebrafish (Danio rerio) to better understand the underlying sensory mechanisms and identify male secondary sexual traits. Females were simultaneously presented with two fish, a male and a female, in a flow-chamber apparatus, and females’ relative attraction towards males was observed under different conditions. With domesticated fish, females were more attracted to males when presented with both visual and chemosensory cues from stimulus fish. They still discriminated the sexes when only visual cues were provided, but not when white ambient light was changed to yellow, indicating that colour plays a role. Sex discrimination under yellow light was improved when chemosensory cues were also provided. Surprisingly, females’ attraction to males was not more pronounced in the morning when mating occurs. Domesticated females discriminated the sexes when presented with wild-derived, as well as domesticated fish, whereas wild-derived females did not show any biases for domesticated or wild-derived males. Behavioural observations indicated that the wild-derived females were distressed, which explains their lack of attraction to males. In summary, domesticated female zebrafish discriminated the sexes using both visual (body colour) and olfactory cues; however, wild-derived zebrafish were too distressed for behavioural experiments under these laboratory conditions.     

Expression of peroxisome proliferator-activated receptors in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

Peroxisomes increase in size and number in responsive animals ranging from mammals to marine mussels and fish species when treated with certain compounds named peroxisome proliferators. This phenomenon, known as peroxisome proliferation, is mediated by nuclear receptors termed peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described (alpha, beta, and gamma) and in mammals PPARalpha is mainly expressed in tissues that catabolize fatty acids, PPARbeta is ubiquitously distributed, and PPARgamma is mainly expressed in the adipose tissue and immune system. The aim of this study was to analyze the tissue distribution of different PPAR subtypes in zebrafish Danio rerio using commercially available antibodies against PPARalpha, PPARbeta, and PPARgamma. In western blots, specific bands were detected at about 58 kDa for PPARalpha and PPARbeta. For PPARgamma the band was detected at 56 kDa. Similar results were obtained in mouse liver homogenates used as positive control, indicating the specificity of the antibodies. Immunohistochemistry was performed in paraformaldehyde-fixed tissue using either microwave or microwave plus trypsin pretreatment for antigen retrieval. In zebrafish, PPARalpha was expressed mainly in liver parenchymal cells, proximal tubules of kidney, enterocytes, and pancreas. PPARbeta showed a widespread distribution and was expressed in the liver, proximal and distal tubules and glomeruli of the kidney, pancreas, enterocytes and smooth muscle of the intestine, skin epithelium, lymphocytes, and male and female gonads. PPARgamma expression was weak in pancreatic cells, intestine, and gonads for both pretreatments. Most of the signal detected was cytoplasmic; only in the cases of PPARalpha and PPARbeta was some nuclear labeling detected in the liver. In mouse tissues, the distribution of PPAR subtypes was similar to that described previously for rats. Our results demonstrate that all three distinct PPAR subtypes are present in zebrafish. The tissue and cellular distribution of PPAR subtypes in zebrafish resembled partly that described before in mammals. Further studies are needed to decipher the functions of PPAR subtypes in zebrafish and other aquatic organisms and particularly their role in regulation of metabolic responses to xenobiotic exposure.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Cortactin mediated morphogenic cell movements during zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.     

Expression of peroxisome proliferator-activated receptors in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) depending on gender and developmental stage

Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors involved in embryo development and differentiation of several tissues in mammals. The aim of the present study was to investigate the possible differential expression of the three PPAR subtypes (PPAR, PPAR, and PPAR) in relation to gender and developmental stage in zebrafish. For this purpose PPAR expression was assessed by immunohistochemistry in 7-day-old larvae, 1-month-old juveniles, and 1-year-old adults. Additionally, the activity of peroxisomal acyl-CoA oxidase (AOX), a gene regulated by PPARs, and the volume density of catalase-immunolabeled liver peroxisomes (V_VP) was examined. No significant gender-related differences were detected in the tissue distribution of the three PPAR subtypes or in peroxisomal AOX activity and V_VP. The percentage of PPAR-positive hepatocytes was significantly higher in females than in males suggesting a specific regulatory role of this subtype in female zebrafish. The three PPAR subtypes were already expressed at the larval stage, with a similar tissue distribution pattern to that found in adults. For all stages, PPAR and PPAR were expressed at higher levels than PPAR, and PPAR immunolabeling was stronger in juveniles than in larval or adult stages. The percentages of hepatocyte nuclei immunolabeled for PPARs was higher in early developmental stages than in adults, similarly to AOX activity and V_VP. In conclusion, our results indicate that PPAR expression, the activity of its target gene AOX, and peroxisomal biogenesis are developmentally modulated in zebrafish.     

Molecular analysis shows differential expression of <Emphasis Type="Italic">R</Emphasis>-<Emphasis Type="Italic">spondin1</Emphasis> in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) gonads

R-spondin1 (RSPO1) is a potential female-determining gene in human (Homo sapiens) and mouse (Mus musculus). Its differential expression in these mammals is correlated with signaling for sex determination. As a way of studying sex determination in fish we cloned and analyzed a RSPO1 gene in zebrafish (Danio rerio). Using real-time PCR, we observed that RSPO1 is expressed more strongly in ovaries than in testes, suggesting that RSPO1 may have a role in gonad differentiation. High RSPO1 expression was detected in some non-gonadal organs like muscle and kidneys. In situ hybridization results demonstrate that RSPO1 is expressed in premature germ cells, in oogonia and primary oocytes in ovaries and in spermatogonia and spermatocytes in testes. It is also expressed in gonad somatic cells during gonadal development: in granulosa cells and theca cells of early and late cortical-alveolar stage follicles in ovaries, and in Leydig cells in testes. This differential expression may indicate that RSPO1 has a role(s) in zebrafish gonad development and differentiation. By fusing zebrafish RSPO1 with a green fluorescent protein gene, we found that RSPO1 is located in the cytosol and Golgi apparatus but not the nucleus of fish epithelioma papulosum cyprinid (EPC) cells. These preliminary findings suggest some aspects of RSPO1 like differential expression linked to sex determination may be conserved in fish while other aspects like subcellular localization differ from the mammalian RSPO1.     

A shifted repertoire of endocannabinoid genes in the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.     

Rapid growth and out-crossing promote female development in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

Sex determination in fishes is often enigmatic, a situation that is often made even more complex by the fact that the process of sexual differentiation in many species may be influenced by environmental conditions. This situation is typified in zebrafish, a popular model organism. Despite the vast array of information available for the species, the genetic controls of sex are unknown. Further, environmental parameters, such as rearing densities, seem to exert an influence on the sex ratios of captive stocks. In an effort to dissect the genetic and environmental controls underlying the expression of sex in this species, we manipulated growth of pure-bred and out-crossed zebrafish by varying their food supply during development. Faster-growing zebrafish were more likely to be female than siblings that were fed less, and out-crossed broods had higher proportions of females than broods from pure-bred crosses. The dependence of sex ratio on feeding rate is readily understood in terms of adaptive sex allocation: zebrafish life history seems to confer the greater pay-off for large size on females. A similar male/female difference in the pay-off for hybrid vigor could similarly account for the female bias of out-crossed broods—and it could be a manifestation of Haldane’s rule.     

The effects of habitat complexity on aggression and fecundity in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

Female zebrafish housed in aquaria with spatial complexity (plastic plants) over a 13–16-week period showed reduced levels of aggressive behavior compared to females in bare tanks. In tanks with plants, there was no relationship between levels of aggression and fecundity but, in bare tanks, females experiencing the highest levels of aggression showed reduced fecundity. Our results suggest that it may be beneficial, when maintaining zebrafish at moderate to high densities or working with especially aggressive strains, to house them in spatially complex conditions.     

The effects of early and adult social environment on zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) behavior

Because early social experience can have a profound effect on later mate and social choices, the availability of options and decisions made early in development can have major effects on adult behavior. Herein, we use strain differences among zebrafish, Danio rerio, as an experimental tool to test the effects of social experience on behavior. By manipulating the strain composition of groups in which the subject fish are housed at different ages, we tested (1) whether mixing with dissimilar individuals influenced subsequent behavior, (2) whether prolonged mixing during the juvenile stage had a more pronounced effect than a shorter period of mixing during adulthood and (3) whether mixing had a lasting effect after animals were resorted into groups of same strain animals. We found that social experience had a profound impact on social behavior. Both Nadia and TM1 individuals engaged in more frequent biting after having been in mixed strain groups compared to pure strain groups. This was true of groups mixed as juveniles, as well as adults, indicating that this response was not dependent on exposure during a critical developmental period. Also, TM1 fish (but not Nadia) having recently been housed in mixed-strain groups were more willing to leave the immediate vicinity of a shoal than were TM1 fish raised in pure strain groups. This change was more pronounced in groups mixed as juveniles than as adults. In addition, the observed changes persisted after mixed groups were separated into pure strain groups for a month. Other behavioral measures including Activity Level, Predator Response and Stress Recovery were unaffected by previous social experience.     

Completion of meiosis in male zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) despite lack of DNA mismatch repair gene <Emphasis Type="Italic">mlh1</Emphasis>

Mlh1 is a member of DNA mismatch repair (MMR) machinery and is also essential for the stabilization of crossovers during the first meiotic division. Recently, we have shown that zebrafish mlh1 mutant males are completely infertile because of a block in metaphase I, whereas females are fertile but have aneuploid progeny. When studying fertility in males in a two-fold more inbred background, we have however observed low numbers of fertilized eggs (approximately 0.4%). Histological examination of the testis has revealed that all spermatogenic stages prior to spermatids (spermatogonia, primary spermatocytes, and secondary spermatocytes) are significantly increased in the mutant, whereas the total weight of spermatids and spermatozoa is highly decreased (1.8 mg in wild-type vs. 0.1 mg in mutants), a result clearly different from our previous study in which outbred males lack secondary spermatocytes or postmeiotic cells. Thus, a delay of both meiotic divisions occurs rather than complete arrest during meiosis I in these males. Eggs fertilized with mutant sperm develop as malformed embryos and are aneuploid making this male phenotype much more similar to that previously described in the mutant females. Therefore, crossovers are still essential for proper meiosis, but meiotic cell divisions can progress without it, suggesting that this mutant is a suitable model for studying the cellular mechanisms of completing meiosis without crossover stabilization. Marcelo C. Leal and Harma Feitsma contributed equally to this work. This work was supported by the Brazilian Foundation CAPES, the Cancer Genomics Center (Nationaal Regie Orgaan Genomics), the European Union-funded FP6 Integrated Project ZF-MODELS, and Utrecht University.     

Influence of magnetic field on the spatial orientation in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) (Cyprinidae) and Roach (<Emphasis Type="Italic">Rutilus rutilus</Emphasis>) (Cyprinidae)

Preferred direction of motion under influence of geomagnetic field and its modifications was registered in zebrafish (Danio rerio) raised in laboratory culture and in roach (Rutilus rutilus) from the Rybinsk Reservoir. In the geomagnetic field, specimens of zebrafish prefer two opposite directions oriented towards the north and south, while they prefer towards east and west at 90° turning of the horizontal component of geomagnetic field. The specimens of roach in the geomagnetic field prefer only the direction oriented towards east–northeast. This direction coincides with the direction along the canal where roach was sampled to the main river channel part of the Rybinsk Reservoir. At 90° rotation of the horizontal component of geomagnetic field, the direction turns to the south–southeast. The reasons for selection of certain directions in the geomagnetic field are discussed.     

Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

Preparation and regeneration of spheroplasts from <Emphasis Type="BoldItalic">Arthrospira</Emphasis><Emphasis Type="BoldItalic">platensis</Emphasis> (Spirulina)

Ultrasonic pretreatment, lysozyme, inorganic osmotics and bovine albumin were used to prepare the spheroplasts of Arthrospira platensis (Spirulina platensis). The average cell number of the fragments from the filaments of strain A9 was about 2.2 cells after 80-s ultrasonic pretreatment. These fragments could regenerate and were suitable material for isolating spheroplasts, so the optimum conditions for doing this were investigated. The best enzymolysis parameters were designed. During the isolation process, gentle shaking of the enzymolysis sample for several times greatly enhanced the proportion of spheroplasts. However, no spheroplasts were obtained when organic compounds were used as osmotics. The spheroplasts could form typical colonies on plate of inorganic medium, with a regeneration rate of about 3%. These spheroplasts might be used as competence cells to carry on the research of genetic transformation.     

The effect of rearing temperature on body shape and meristic characters in zebrafish (<Emphasis Type=Italic>Danio rerio</Emphasis>) juveniles

Considering its immediate costs of producing dispensable males, the maintenance of sexual reproduction is a major paradox in evolutionary biology. Asexual lineages that do not face such costs theoretically should replace sexuals over time. Nonetheless, several systems are known in which closely related sexual and asexual lineages stably coexist. In the present study, we studied a sexual/asexual mating complex of a sperm-dependent parthenogenetic fish (amazon molly, Poecilia formosa) and its sexual congeners, the sailfin molly P. latipinna and the Atlantic molly P. mexicana. We asked whether differences in feeding behavior could contribute to their stable coexistence. We conducted a laboratory experiment to compare feeding efficiencies and also measured the competitive abilities between the two reproductive forms. Additionally, we measured gut fullness of fishes caught in natural habitats. Contrary to our predictions, we could not find P. formosa to be less efficient in feeding. We argue that food competition in mollies plays a minor role in mediating coexistence between closely related asexual and sexual mollies.     

Isolation and characterization of the major histocompatibility complex <Emphasis Type="BoldItalic">DQA1</Emphasis> and <Emphasis Type="BoldItalic">DQA2</Emphasis> genes in gayal (<Emphasis Type="BoldItalic">Bos frontalis</Emphasis>)

The species origin of Yunnan gayal has been controversial since many years. However, few recent genetic studies have suggested that it has perhaps originated from the hybridization between male Bos frontalis and female B. taurus or B. indicus. Being an important semi-wild bovid species, this has also been listed under the red list of International Union of Conservation of Nature and Natural Resources. However, there is limited information available about the immunogenicity of this precarious species of Bos. Major histocompatibility complex (MHC) plays a pivotal role in immune response to infectious diseases in vertebrates. In the present study, we have investigated the structural and functional characteristics and possible duplication of the MHC-DQA genes in gayal (B. frontalis). Two full-length cDNA clones of the MHC-DQA genes were amplified and designated as Bofr-DQA1 (DQA*0101) and Bofr-DQA2 (DQA*2001) with GenBank accession numbers KT318732 and KT318733, respectively. A comparison between Bofr-DQA1, Bofr-DQA2 and to other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of these two identified MHC-DQA genes have more similarity to alleles of specific DQA1 and DQA2 molecules from other Ruminantia species than to each other. The phylogenic investigation also demonstrated a large genetic distance between these two genes than to homologous from the other species. The large genetic distance between Bofr-DQA1 and Bofr-DQA2, and the presence of different bovine DQA putative motifs clarify that these sequences are nonallelic type. These results could suggest that duplication of the DQA genes has also occurred in gayal. The findings of the present study have strengthened our understanding to MHC diversity in rare ruminants and mutation of immunological functions, selective and evolutionary forces that affect MHC variation within and between species.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.