Maternal effect and dietary supplementation of estradiol-17ß on female zebrafish (Danio rerio) affects the swimming behavior and stress-coping styles of its offspring.

Extrinsic estradiol-17ß (E2) is an environmental hormone. Female fish exposure to waterborne E2 might affect the development of craniofacial cartilage of its offspring. The present study investigates the effects of maternal E2 on larval craniofacial cartilage development by administering oral feed containing E2 (F-E2) to female zebrafish, and examines whether the swimming behavior and their stress coping style are influenced by maternal E2. The results showed that E2 contents responded to dosage and time in male fish after being fed with a diet containing E2. In addition, the E2 contents in female ovaries showed a significant increase after 250 mg of E2/kg treatment for 14 d. On the other hand, the fecundity rate of F-E2 group was lower around 2 folds than FC (female fed 0 mg of E2/kg) group. Craniofacial chondrogenesis on 72 hpf (hours of post fertilization) of F-E2 larvae were abnormalities, and a recovery to a normal developmental pattern was observed at the 96 hpf stage. The swimming speed was slower for F-E2 larvae compared to the FC larvae; and the F-E2 juvenile seems to be less responsive to cortisol (LRC) after cold stress. According to the results, we suggested that F-E2 larvae might have worse environmental adaptability than FC larvae.

Effects of bisphenol A or diethyl phthalate on cartilage development and the swimming behavior of zebrafish (Danio rerio) through maternal exposure.

Waterborne bisphenol A (BPA) and diethyl phthalate (DEP) are endocrine disruptive chemicals that impact the reproductive system of fish. The present study checks the effectiveness of the reproductive capacity on zebrafish after BPA and DEP exposure, and consequently investigates its effect on their development and the swimming behavior of its offspring. The exposure of BPA and DEP to zebrafish reveals that the levels of ovarian 17ß-estradiol (E2) and relative mRNA expression (RRE) ratios (Treatment/Control) of hepatic vitellogenin (vtg1) could be induced and decreased. Liver RRE levels in estrogen receptors (ERs) are also affected. Among the ERs, esr2a significantly increased upon BPA exposure, and esr1 and esr2b decreased upon DEP exposure. In addition, the ceratohyal cartilage (CH) angle of larvae whose mothers were exposed to BPA (F-BPA) was significantly bigger, but the CH angle of larvae whose mothers were exposed to DEP (F-DEP) was significantly smaller than the control. The swimming performance of larvae from F-DEP was more compromised than the control, but the situation did not appear in the larvae from the F-BPA group. The success rate of larvae hatching from F-BPA and F-DEP was lower than control group. Moreover, the successful rate of female spawns was higher in the control group compared to the treatment groups exposed to BPA and DEP. We suggested that both maternal BPA and DEP disrupt E2 levels, and influence the CH development of larvae, resulting in a decrease in successful hatching. Only the swimming behavior of larvae from maternal DEP was disrupted.

Spatial and seasonal distribution of microplastics on sandy beaches along the coast of the Hengchun Peninsula, Taiwan.

Here we present the first report on microplastic pollution on the beaches along the coast of the Hengchun Peninsula, which is one of the major tourist attractions in Taiwan. By using a standard operating procedure, sand samples were collected from eight beaches in June and November in 2017, and the microplastics in the sand samples were quantified and characterized in the laboratory. The average density of microplastics ranged from 80 to 480 particles/kg dry weight sand. There was no apparent seasonal difference but there were significant spatial differences among sampling sites. No significant difference in microplastic levels was observed among the west, south, and east coasts, but microplastic density was higher on beaches with higher tourism activity levels. The most abundant type of microplastics was fiber (>97%) and the most common color was white/transparent (57%). In addition, using a Fourier Transform Infrared (FTIR) spectrophotometer we identified microplastics as polyethylene (PE) and polypropylene (PP). Our results show that microplastics are ubiquitous along the coast of the Hengchun Peninsula, and the major factor associated with the abundance of microplastics is tourism activity.

Intra-Colonial Functional Differentiation-Related Modulation of the Cellular Membrane in a Pocilloporid Coral Seriatopora caliendrum.

Scleractinian corals have displayed phenotypic gradients of polyps within a single genotypic colony, and this has profound implications for their biology. The intrinsic polymorphism of membrane lipids and the molecular interactions involved allow cells to dynamically organize their membranes to have physicochemical properties appropriate for their physiological requirements. To gain insight into the accommodation of the cellular membrane during ontogenetic shifts, intra-colony differences in the glycerophosphocholine profiling of a pocilloporid coral, Seriatopora caliendrum, were characterized using a previously validated method. Specifically, several major polyunsaturated phosphatidylcholines showed higher levels in the distal tissue of coral branches. In contrast, the corresponding molecules with 1-2-degree less unsaturation and plasmanylcholines were expressed more highly in the proximal tissue. The lipid profiles of these two colonial positions also contrasted sharply with regard to the saturated, monounsaturated, and lyso-glycerophosphocholine ratios. Based on the biochemical and biophysical properties of these lipids, the associated modulation of cellular membrane properties could be related to the physiological requirements, including coral growth and aging, of the functionally differentiated polyps. In this study, the metabolic regulation of membrane lipids involved in the functional differentiation of polyps within a S. caliendrum colony was identified.

Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.

BACKGROUND: Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene. METHODOLOGY/PRINCIPAL FINDINGS: Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1)(cy18) displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1)(cy18), the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/ß, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1)(cy18) caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1)(cy18). In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1)(cy18), suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues. CONCLUSION/SIGNIFICANCE: Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity.

Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo.

BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

Chronic exposure of 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) alters locomotion behavior in juvenile zebrafish (Danio rerio).

In the present study, we used zebrafish (Danio rerio) as a model to address possible effects of chronic exposure of polybrominated diphenyl ether (PBDE) flame retardants on locomotion behavior, body size, and gonad development in fish. Zebrafish were fed food dosed with PBDE-47 (control, solvent control, low, medium, and high dose groups) from 21 days post hatch (dph) to 90 dph. Fish locomotion parameters, including maximum swimming speed, total distance moved, and percent time active, were assessed using a video-based animal movement analysis system. At the end of the exposure, all fish were euthanized for length and weight measurement, and then subjected to either whole fish histological analysis or tissue PBDE-47 measurement. Survival, body size, and gonad histology were similar between the five groups. However, both total swimming distance and percent time active were negatively correlated with tissue PBDE-47 concentration and were significantly lower in the high dose group. Tissue levels of PBDE-47 in the exposed fish were comparable to that reported in previous field studies. In summary, this study showed that developmental exposure of PBDE-47 at an ecologically relevant level altered locomotion behavior without affecting body size or gonad development of zebrafish.

Growth and transcriptional effect of dietary 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) exposure in developing zebrafish (Danio rerio).

In this study, zebrafish (Danio rerio) were fed food dosed with pure PBDE-47 (2,2',4,4'-tetrabromodiphenyl ether) congener or a blank from 20 to 60 day post-hatch (dph). At 38 dph, half of the fish were sampled for body size measurements and gene expression analyzes (CYP1A1, VTG, TTR, D1, and TSH-beta). At 60 dph, body size was measured again for all fish remaining. Whole-fish histology was performed and the PBDE levels in fish were determined. PBDE treated fish was significantly smaller at 38 dph but not at 60 dph. No apparent histopathological effect was observed. In the PBDE treated fish, there was a weak induction of CYP1A1 mRNA transcription, but not of the other genes. The tissue levels of PBDE-47 were comparable to that found in other wild fish reported in the literature, indicating that our exposure level was ecologically relevant.

Lethal and sublethal effects of chronic cadmium exposure on northern leopard frog (Rana pipiens) tadpoles.

Data on the chronic effects of cadmium on amphibians are lacking in spite of widespread anthropogenic contamination of terrestrial and aquatic systems. We exposed embryos and tadpoles of northern leopard frogs (Rana pipiens) to Cd (control, 0.25, 5.0, and 20.0 microg/L as CdCl2, nominal concentrations) in a static renewal system from embryonic stages to complete tail resorption. Survival of embryos (Gosner stage [GS] 17-25) was greater than 97% in all treatments. Tadpole survival was negatively correlated with dose and was significantly reduced in the 5.0 and 20.0 microg/L treatments compared with controls. Tadpole survival was greater than 80% through GS 42, forelimb emergence, for all other treatments. Tadpoles exposed to 0.25 and 5.0 microg/L exhibited increased growth by week 11; tadpoles exposed to 5.0 microg/L were significantly younger at forelimb emergence. Whole-tadpole body burdens of Cd were positively correlated with increasing Cd treatments. Cadmium was shown to alter growth and development in a native amphibian species at ecologically relevant concentrations. The existing chronic water quality criterion for Cd appears to be protective of amphibians. However, additional studies with other chemicals are needed to further explore the potential for adverse effects of contaminants on the complex life cycle of amphibians.