Developmental plasticity in the thermal tolerance of zebrafish Danio rerio

To evaluate developmental plasticity in thermal tolerance of zebrafish Danio rerio , common-stock zebrafish were reared from fertilization to adult in the five thermal regimes (two stable, two with constant diel cycles and one stochastic diel cycle) and their thermal tolerance at three acclimation temperatures compared. The energetic cost of developing in the five regimes was assessed by measuring body size over time. While acclimation accounted for most of the variability in thermal tolerance, there were also significant differences among fish reared in the different regimes, regardless of acclimation. Fish reared in more variable environments (as much as ±6° C diel cycle) had a greater tolerance than those from non-variable environments at the same mean temperature. Fish from the more variable environments were also significantly smaller than those from non-variable environments. These results indicate that the thermal history of individual zebrafish induces irreversible changes to the thermal tolerance of adults.     

Thermally induced plasticity of body shape in adult zebrafish Danio rerio (Hamilton, 1822)

We examined the effect of temperature during the early development on the phenotypic plasticity of Danio rerio. The effect of temperature was examined during two different early developmental periods of 280°d (the product of days × temperature) each, 28‐308°d or 280‐560°d, by subjecting the experimental populations to three different water temperatures (22°C, 28°C, and 32°C). Before and after the end of the 280°d period of the different thermal exposure, all populations were cultured in standard temperature (28°C). Five to 10 months after exposure to the different thermal regimes, the body shape of the adults was analyzed by geometric morphometrics. In both ontogenetic windows and experimental repetitions, the results showed that developmental temperature and sex significantly affected the body shape of adult zebrafish. Thermally induced shape variation discriminated the fish that developed at 22°C from those developed at 28°C–32°C. In the early developmental period (DP1, 28–308°d postfertilization), dorsal, anal, and caudal fin structures differed between the animals that developed at 22°C and 28°C–32°C. In the later developmental period (DP2, 280–560°d postfertilization), caudal, anal, pectoral, and pelvic fins, as well as the gill cover and lower jaw, were affected when animals developed at different temperatures. These results show that thermal history during a short period of embryonic and larval life affects the body form of adult zebrafish with potentially functional consequences. Based on previous data on the effects of temperature on fish development, we suggest thermally induced muscle and bone remodelling as possible mechanism underlying the observed plasticity. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

The effects of chronic developmental hypoxia on swimming performance in zebrafish

Relative to normoxia-reared fish, hypoxia-reared zebrafish Danio rerio achieved 24·9 and 21·4% slower maximal swim velocities in both normoxic and hypoxic waters, respectively. Hypoxia-reared fish also produced 26·1 and 63·9% less lactate during resting conditions across both normoxic and hypoxic waters, respectively. During exercise, this trend continued as hypoxia-reared fish produced 68·2 and 55·1% less lactate across both normoxic and hypoxic waters, respectively. This reduction in performance, rather than representing a purely pathological (maladaptive) response to hypoxia, appears to represent a fundamental shift in the metabolic response to hypoxia.     

Timing and plasticity of shoaling behaviour in the zebrafish, Danio rerio

The zebrafish has become a major model system for biomedical research and is an emerging model for the study of behaviour. While adult zebrafish express a visually mediated shoaling preference, the onset of shoaling behaviour and of this preference is unknown. To assess the onset of these behaviours, we first manipulated the early social environment of larval zebrafish subjects, giving them three model shoaling partners of the same pigment phenotype. We then assayed the subjects' preferences using binary preference tests in which we presented subjects with two shoals, one shoal of fish exhibiting the same pigment pattern phenotype as their models and another shoal with a radically different pigment pattern. To determine whether or not the visually mediated preference could be altered once it was established, we further manipulated the social environment of a number of subjects, rearing them with one model shoal and testing them, then changing their social consorts and retesting them. Our results demonstrate that larval zebrafish shoal early in their development, but do not exhibit a shoaling preference until they are juveniles. Moreover, we find that the shoaling preference is stable, as changing the social environment of fish after they had acquired a preference did not change their preference. These data will facilitate investigations into the mechanisms underlying social behaviour in this vertebrate model system.     

Dose-dependent effects of developmental mercury exposure on C-start escape responses of larval zebrafish Danio rerio

Zebrafish Danio rerio embryos were exposed to 0, 25, 50 or 75 ppb Hg²⁺ from 0 to 24 h post‐fertilization (hpf) then placed into Hg²⁺‐free water. Inductively coupled plasma‐mass spectrophotometer analysis of whole embryo Hg²⁺ content at 24 hpf showed a positive correlation with exposure regime (Pearson's one‐tailed, r²= 0·698, P < 0·01); at 5 days post‐hatch (dph), whole larval Hg²⁺ content was not detectable. Hg²⁺‐induced behavioural deficits in larvae were, therefore, due to changes during embryogenesis and not to residual Hg²⁺ in the larvae. At 5 dph, larvae were tested for responses to different frequencies but equal intensities of vibrational stimuli generated by a remotely controlled plastic hammer. Data were recorded by high‐speed videography and computer‐analysed for latency of response (ms), amplitude of the response as measured by maximum initial velocity [normalized as body (standard) lengths s^?1; V_max] and duration of behaviour from initial head movement to cessation of caudal tail movement (ms). A single mechanical stimulus resulted in behavioural outcomes that were related to embryonic Hg²⁺ uptake. Response latency increased with exposure level and displayed an increase of ×1·5–2·5 over control values (ANOVA, P < 0·01). The V_max decreased with exposure level to a low of 71% of control at the highest Hg²⁺ concentration (ANOVA, P < 0·01). Duration of behaviour displayed a biphasic response pattern in which exposure to 0, 50 or 75 ppb Hg²⁺ did not result in a significantly different response yet exposure to 25 ppb Hg²⁺ caused a significantly longer time of active response (ANOVA, P < 0·01). Repeated stimulation (1, 2 or 4 hits s^?1) resulted in a concentration‐dependent increase in response failures. Regardless of stimulation frequency, larvae exposed to 0 or 25 ppb Hg²⁺ as embryos maintained higher V_max levels for longer intervals during the testing period than those exposed as embryos to either 50 or 75 ppb Hg²⁺.     

Developmental toxicity of triclocarban in zebrafish (Danio rerio) embryos

Triclocarban (TCC), which is used as an antimicrobial agent in personal care products, has been widely detected in aquatic ecosystems. However, the consequence of TCC exposure on embryo development is still elusive. Here, by using zebrafish embryos, we aimed to understand the developmental defects caused by TCC exposure. After exposure to 0.3, 30, and 300 μg/L TCC from 4‐hour postfertilization (hpf) to 120 hpf, we observed that TCC exposure significantly increased the mortality and malformation, delayed hatching, and reduced body length. Exposure to TCC also affected the heart rate and expressions of cardiac development–related genes in zebrafish embryos. In addition, TCC exposure altered the expressions of the genes involved in hormonal pathways, indicating its endocrine disrupting effects. In sum, our data highlight the impact of TCC on embryo development and its interference with the hormone system of zebrafish.     

Plasticity of the skeleton and skeletal deformities in zebrafish (Danio rerio) linked to rearing density

The teleost zebrafish (Danio rerio), an established model for human skeletal diseases, is reared under controlled conditions with defined parameters for temperature and photoperiod. Studies aimed at defining the proper rearing density have been performed with regard to behavioural and physiological stress response, sex ratio and reproduction. Studies concerning the effect of rearing density on the skeletal phenotype are lacking. This study analyses the response of the skeleton to different rearing densities and describes the skeletal deformities. Wild-type zebrafish were reared up to 30 dpf (days post-fertilization) in a common environment. From 30 to 90 dpf, animals were reared at three different densities: high density (HD), 32 fish l^–1; medium density (MD), 8 fish l^–1 and low density (LD), 2 fish l^–1. Animals at 30 and 90 dpf were collected and whole-mount stained with Alizarin red S to visualize mineralized tissues. The entire skeleton was analysed for meristic counts and 172 types of deformities. The results showed that the rearing density significantly influenced the specimens’ average standard length, which decreased with the increase in the rearing density. Differences in meristic counts among the three groups were not observed. Rearing density–independent malformations affected the ribs, neural arches and the spines of the abdominal region, as well as vertebrae of the caudal complex. The HD group showed the highest number of deformities per specimen, the highest number of observed types of deformities and, together with the MD group, the highest frequency of specimens affected by severe deformities. In particular, the HD group showed deformities affecting arches, spines and vertebral centra in the caudal region of the vertebral column. This study provides evidence of an effect of the rearing density on the development of different skeletal phenotypes.     

Ontogeny of odorant receptor gene expression in zebrafish,Danio rerio

We cloned three putative odorant receptor (OR) genes from the zebrafish to use as in situ hybridization probes to follow the temporal patterns of neurons expressing OR genes through a developmental progression from embryo (12 h postfertilization) to adult. The identification of these genes is supported by sequence homology to previously reported ORs and by the morphology and location of labeled cells in in situ hybridization experiments. Cells expressing OR mRNA were first observed in the olfactory placodes between 31 and 38 h after fertilization (fish reared at 26°C). Initially, only single cells were observed to hybridize the probe; the number of labeled cells increased throughout the remainder of embryogenesis and through postembryonic growth and morphogenesis of the olfactory organ. At all ages, the positively hybridizing cells were scattered throughout the olfactory epithelium but not in the nonsensory epithelium of the olfactory organ. © 1996 John Wiley & Sons, Inc.     

Allozyme and microsatellite genetic variation in natural samples of zebrafish,Danio rerio

In this paper, we report the results from allozyme and microsatellite markers in natural populations of the zebrafish, a species of great significance in biological studies. Four zebrafish wild samples from West Bengal, India, were analysed in a preliminary survey of levels and patterns of genetic variation. Results indicate high levels of genetic variability and weak genetic structure, although the latter is consistent with the geographical features of the area under study, sampling sites being located in the Ganges and Brahmaputra delta region, which is characterized by high waterways connectivity.     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

Expression patterns of the creatine metabolism‐related molecules AGAT,GAMT and CT1 in adult zebrafish Danio rerio

AGAT, GAMT and CT1, three creatine synthesis and transport‐related molecules, have been widely studied in mammals. To explore their homologous genes in adult zebrafish Danio rerio, the gene expression patterns of these three genes in D. rerio were investigated. The results reveal that AGAT, GAMT and CT1 are expressed widely in diverse tissues of D. rerio where the homologous genes in mammals are also expressed.     

Parent-of-origin specific effects on the methylation of a transgene in the zebrafish,Danio rerio

We have examined the inheritance of a transgene locus in the zebrafish, Daniorerio and demonstrated that its methylation is af fected by the sex of the parent contributing the allele. This parent-of-origin effect on the zebrafish transgene appears to be identical to imprinting as seen in mammals except that in zebrafish, passage of the locus through a female tended to decreased its methylation, whereas passage through a male increased it. Methylation of the transgene in gametic tissues differed from somatic tissue with the locus being hypomethylated in sperm and hypermethylated in the unfertilized egg. The potential identification of imprinting in the zebrafish has important ramifications with respect to the evolution of the process as well as for understanding the role of imprinting in mammals. © 1995 Wiley-Liss, Inc.     

Effects of environmental enrichment on survivorship,growth, sex ratio and behaviour in laboratory maintained zebrafish Danio rerio

Environmental enrichment involves increasing the complexity of a fish's environment in order to improve welfare. Researchers are legally obliged to consider the welfare of laboratory animals and poor welfare may result in less robust data in experimental science. Laboratory zebrafish Danio rerio are usually kept in bare aquaria for ease of husbandry and, despite being a well-studied species, little is known about how laboratory housing affects their welfare. This study shows that environmental enrichment, in the form of the addition of gravel substratum and plants into the tank, affects survivorship, growth and behaviour in laboratory-maintained D. rerio. Larvae reared in enriched tanks had significantly higher survivorship compared with larvae reared in bare tanks. Effects of the tank conditions on growth were more variable. Females from enriched tanks had a higher body condition than females maintained in bare tanks, but intriguingly this was not the case for males, where the only difference was a more variable body condition in males maintained in bare tanks. Sex ratio in the rearing tanks did not differ between treatments. Resource monopolisation was higher for fish in enriched tanks than for those in bare tanks. Fish from enriched tanks displayed lower levels of behaviours associated with anxiety compared with fish from bare tanks when placed into a novel environment. Thus, this study demonstrates differences in welfare for D. rerio maintained under different environmental conditions with enhancements in welfare more commonly associated with tank enrichment.     

Comments on 'Significance of developmental expression of amphioxus Branchiostoma belcheri and zebrafish Danio rerio Hsd17b10 in biological and medical research'

The reported data on the developmental expression of Hsd17b10 gene in Danio rerio is crucial to the utilization of the D. rerio embryo as an animal model for human developmental disorders caused either by mutations on HSD17B10 (formerly HADH2 ) or by defective expression of the gene. Related diseases were summarized, and it was noticed that hyperinsulinaemic hypoglycaemia is not linked to HSD17B10 . This inherited disease is actually caused by a deletion in the HADH gene on chromosome 4. Moreover, it was found by a revision of the reported phylogenetic tree that hydroxyacyl-CoA dehydrogenase II or rather hydroxysteroid (17beta) dehydrogenase 10 (HSD10) of amphioxus Branchiostoma belcheri —occupies a transition position from HSD10 orthologs of invertebrates to those of vertebrates.     

Hesperidin inhibits expression of hypoxia inducible factor-1 alpha and inflammatory cytokine production from mast cells

The citrus unshiu peel has been used traditionally as a medicine to improve bronchial and asthmatic conditions or cardiac and blood circulation in Korea, China, and Japan. Here, we report the effects of citrus unshiu peel water extract (CPWE) on the phorbol myristate acetate (PMA) + calcium ionophore A23187-induced hypoxia-inducible factor-1α (HIF-1α) activation and inflammatory cytokine production from the human mast cell line, HMC-1 cells. We compared CPWE with hesperidin, a common constituent of citrus unshiu. CPWE and hesperidin inhibited the PMA + A23187-induced HIF-1α expression and the subsequent production of vascular endothelial growth factor (VEGF). In addition, CPWE suppressed PMA + A23187-induced phosphorylation of the extracellular signal-regulated kinase (ERK). We also show that the increased cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α level was significantly inhibited by treatment of CPWE or hesperidin. In the present study, we report that CPWE and hesperidin are inhibitors of HIF-1α and cytokines on the mast cell-mediated inflammatory responses.     

Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1α expression by HIF-1α specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1α expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.     

缺氧诱导因子1α、血管内皮生长因子和成纤维细胞生长因子在胰腺癌中的表达及意义

目的：研究胰腺癌组织中缺氧诱导因子1alpha（Hypoxia-inducible factor-1alpha,HIF-1α）、血管内皮生长因子（vascular endothelial growth factor,VEGF）和成纤维细胞生长因子（fibroblast growth factor,FGF）的表达并探讨其意义。方法：Western blot法检测22例胰腺癌及癌旁组织中HIF-1α、VEGF和FGF蛋白的表达,分析HIF-1α与VEGF、FGF之间的相关性以及与性别、年龄、肿瘤大小、淋巴结转移和TNM分期之间的关系。结果：HIF-1α、VEGF和FGF在胰腺癌组织中的蛋白表达水平明显高于胰腺癌周组织（P〈0.01）,HIF-1α与VEGF、FGF之间的表达具有显著相关性（P〈0.01）。HIF-1α的表达与胰腺癌的TNM分期、肿瘤大小和淋巴结转移有关（P〈0.01）,VEGF和FGF的表达与胰腺癌的肿瘤大小和淋巴结转移有关（P〈0.05）。结论：HIF-1α可以上调VEGF和FGF的表达,在胰腺癌的发生、发展中起着重要作用。     

Hypoxia-inducible factor-1 mediates the expression of DNA polymerase iota in human tumor cells

Hypoxia generated in tumors has been shown to contribute to mutations and genetic instability. However, the molecular mechanisms remain incompletely defined. Since reactive oxygen species (ROS) are overproduced immediately after reoxygenation of hypoxic cells and generate oxidized guanine, we assumed that the mechanisms might involve translesion DNA polymerases that can bypass oxidized guanine. We report here that hypoxia as well as hypoxia mimetics, desferrioxamine, and CoCl(2), enhanced the expression of DNA polymerase iota (pol iota) in human tumor cell lines. Searching the consensus sequence of hypoxia response element to which HIF-1 binds revealed that it locates in the intron 1 of the pol iota gene. These results suggest that HIF-1-mediated pol iota gene expression may be involved in the generation of translesion mutations during DNA replication after hypoxia followed by reoxygenation, thereby contributing to the accumulation of genetic changes in tumor cells.