In Vivo Toxicity Evaluation of Sugar Adulterated Heterotrigona itama Honey Using Zebrafish Model.

Honey is prone to be adulterated through mixing with sugars, cheap and low-quality honey, and other adulterants. Consumption of adulterated honey may cause several health issues such as weight gain, diabetes, and liver and kidney dysfunction. Therefore, studying the impact of consumption of adulterated honey on consumers is critical since there is a lack of study in this field. Hence, the aims of this paper were: (1) to determine the lethal concentration (LC50) of adulterated honey using zebrafish embryo, (2) to elucidate toxicology of selected adulterated honey based on lethal dose (LD50) using adult zebrafish, (3) to determine the effects of adulterated honey on histological changes of zebrafish, and (4) to screen the metabolites profile of adulterated honey by using zebrafish blood serum. The LC50 of Heterotrigona itama honey (acacia honey) and its sugar adulterants (light corn sugar, cane sugar, inverted sugar, and palm sugar in the proportion of 1-3% (w/w) from the total volume) was determined by the toxicological assessment of honey samples on zebrafish embryos (different exposure concentrations in 24, 48, 72, and 96 h postfertilization (hpf)). Pure H. itama honey represents the LC50 of 34.40 ± 1.84 (mg/mL) at 96 hpf, while the inverted sugar represents the lowest LC50 (5.03 ± 0.92 mg/mL) among sugar adulterants. The highest concentration (3%) of sugar adulterants were used to study the toxicology of adulterated honey using adult zebrafish in terms of acute, prolong-acute, and sub-acute tests. The results of the LD50 from the sub-acute toxicity test of pure H. itama honey was 2.33 ± 0.24 (mg/mL). The histological studies of internal organs showed a lesion in the liver, kidney, and spleen of adulterated treated-honey groups compared to the control group. Furthermore, the LC-MS/MS results revealed three endogenous metabolites in both the pure and adulterated honey treated groups, as follows: (1) S-Cysteinosuccinic acid, (2) 2,3-Diphosphoglyceric acid, and (3) Cysteinyl-Tyrosine. The results of this study demonstrated that adulterated honey caused mortality, which contributes to higher toxicity, and also suggested that the zebrafish toxicity test could be a standard method for assessing the potential toxicity of other hazardous food additives. The information gained from this research will permit an evaluation of the potential risk associated with the consumption of adulterated compared to pure honey.

Meis1, Hi1α, and GATA1 are integrated into a hierarchical regulatory network to mediate primitive erythropoiesis.

During development, erythroid cells are generated by two waves of hematopoiesis. In zebrafish, primitive erythropoiesis takes place in the intermediate cell mass region, and definitive erythropoiesis arises from the aorta-gonad mesonephros. TALE-homeoproteins Meis1 and Pbx1 function upstream of GATA1 to specify the erythroid lineage. Embryos lacking Meis1 or Pbx1 have weak gata1 expression and fail to produce primitive erythrocytes. Nevertheless, the underlying mechanism of how Meis1 and Pbx1 mediate gata1 transcription in erythrocytes remains unclear. Here we show that Hif1α acts downstream of Meis1 to mediate gata1 expression in zebrafish embryos. Inhibition of Meis1 expression resulted in suppression of hif1a expression and abrogated primitive erythropoiesis, while injection with in vitro-synthesized hif1α mRNA rescued gata1 transcription in Meis1 morphants and recovered their erythropoiesis. Ablation of Hif1α expression either by morpholino knockdown or Crispr-Cas9 knockout suppressed gata1 transcription and abrogated primitive erythropoiesis. Results of chromatin immunoprecipitation assays showed that Hif1α associates with hypoxia-response elements located in the 3'-flanking region of gata1 during development, suggesting that Hif1α regulates gata1 expression in vivo. Together, our results indicate that Meis1, Hif1α, and GATA1 indeed comprise a hierarchical regulatory network in which Hif1α acts downstream of Meis1 to activate gata1 transcription through direct interactions with its cis-acting elements in primitive erythrocytes.

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis.

AIMS/HYPOTHESIS: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. METHODS: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. RESULTS: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. CONCLUSIONS/INTERPRETATION: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. DATA AVAILABILITY: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.

Exposure of zebrafish to environmentally relevant concentrations of mercury during early life stages impairs subsequent reproduction in adults but can be recovered in offspring.

Mercury (Hg) is a global pollutant that poses potential threats to health of fishes. Although effects of Hg on reproduction of fishes have been documented, little is known about effects of exposure to Hg2+ during early life stages on subsequent reproductive fitness of adults or whether these effects can be transferred to offspring. In this study, zebrafish embryos were exposed to environmentally relevant concentrations of Hg2+ (0.6, 3 or 15 µg/L) for 5 days and then depurated in clean water for another 115 days. Exposure to Hg2+ during early life stages disturbed the balance of sex hormones and gametogenesis by altering expression of mRNA for genes involved in the hypothalamic-pituitary-gonadal axis, which resulted in delayed gonadal development and lesser gonado-somatic index, thereby resulting in lesser fecundity. A similar, but less pronounced effect was observed in F1 females that were not exposed directly to Hg, whereas such damage was neither observed in F1 males nor either sex during the F2 generation. Exposure to Hg2+ during early life can impair subsequent reproduction in adults and has intergenerational effects on F1 females, but this reproductive damage can be recovered in F1 males and in F2 females.

Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS.

Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17ß-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17ß-estradiol were highly correlated with R2 of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17ß-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.

Advanced blood flow assessment in Zebrafish via experimental digital particle image velocimetry and computational fluid dynamics modeling.

Over the past few decades, Zebrafish has become a widely used vertebrate model for cardiovascular research. Easy genetic manipulation, low cost, high fecundity, embryonic transparency, and ability to survive in the early stages of development without active circulation are among the advantages of Zebrafish. Cardiac malformations can be induced through genetic manipulations for elucidating the influence of mechanobiological stimuli on the development and progress of the cardiovascular diseases. For this purpose, a reliable in vivo assessment of cardiac function and disturbed hemodynamics is required. Therefore, it is necessary to accurately determine the complex blood flow patterns and associated hemodynamic shear stresses within the developing heart and cardiovascular system. In the traditional approach, brightfield microscopy is used to track the motion of cells in two-dimensions (2D). However, with the development of advanced modalities such as light-sheet fluorescent microscopy, it is now possible to perform 4D (three-dimensional space + time) imaging of Zebrafish embryo and larvae. The integration of digital particle image velocimetry (DPIV) and computational fluid dynamics (CFD) provide an opportunity for detailed investigations using in vivo images. In this review, DPIV and CFD methods are explained for blood flow assessment, and recent relevant research findings from Zebrafish studies are summarized.

Effect of 3 Euthanasia Methods on Serum Yield and Serum Cortisol Concentration in Zebrafish (Danio rerio).

Zebrafish are an important model in neuroscience and developmental biology and are also an emerging model in hematology and immunology. Little information is available for zebrafish regarding the physiologic impact of different euthanasia methods and whether a chosen method of euthanasia can impact serum yield. These parameters could impact the choice of euthanasia method for a study. To that end, the current study compared 3 methods of adult zebrafish euthanasia and their effects on 3 distinct criteria; time to loss of opercular movement, volume of serum obtained, and serum cortisol concentration. Blood was collected using a postmortem tail amputation and centrifugation blood collection technique. Time to loss of opercular movement differed significantly among euthanasia methods, with animals undergoing rapid chilling displaying the shortest time (mean Rapid Chilling: 40 s; Benzocaine: 86 s; MS222: 96 s). All methods of euthanasia resulted in a comparable average serum yield (Rapid Chilling = 7.5 µL; Benzocaine = 8.5 µL; MS222 = 7.5 µL per fish). None of the euthanasia methods tested resulted in average cortisol concentrations above the reported physiologic range. Although no significant differences were observed in serum yield or serum cortisol concentration, rapid chilling remains the preferred method for painless, humane euthanasia.

Transcriptomic analysis reveals potential mechanisms of toxicity in a combined exposure to dibutyl phthalate and diisobutyl phthalate in zebrafish (Danio rerio) ovary.

Phthalate esters (PAEs), which are notable plasticizers, can be prolific contaminants in aquatic environments, and have been shown to induce reproductive toxicity. However, the studies concerning their toxicity towards aquatic species are based on individual chemicals, and the combined toxicity of PAEs to aquatic organisms remains unclear. The aim of this study was to explore the potential toxicity mechanisms associated with combined exposure to dibutyl phthalate (DBP) and diisobutyl phthalate (DiBP) in adult female zebrafish ovaries. Zebrafish were exposed to DBP, DiBP and their mixtures for 30 days, and their effects on ovarian histology, plasma sex hormones and ovarian transcriptomics were investigated. Plasma estradiol (E2) levels were significantly decreased by 38.9% in the DBP-1133 exposure group and 41.0% in the DiBP-1038 exposure group. The percentage of late/mature oocytes was also significantly decreased by 17.3% under DBP-1133 exposure and 16.2% under DiBP-1038 exposure, while that under combined exposure was not significantly affected. Nevertheless, transcriptome sequencing revealed 2564 differentially expressed genes (DEGs) in zebrafish ovaries after exposure to the mixtures. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were involved in the neuroactive ligand-receptor interaction, GnRH, progesterone-mediated oocyte maturation, oocyte meiosis and steroid hormone biosynthesis signaling pathways. These results revealed that combined exposure exerts potential reproductive toxicity at the molecular level.

Artery-vein specification in the zebrafish trunk is pre-patterned by heterogeneous Notch activity and balanced by flow-mediated fine-tuning.

How developing vascular networks acquire the right balance of arteries, veins and lymphatic vessels to efficiently supply and drain tissues is poorly understood. In zebrafish embryos, the robust and regular 50:50 global balance of intersegmental veins and arteries that form along the trunk prompts the intriguing question of how does the organism keep 'count'? Previous studies have suggested that the ultimate fate of an intersegmental vessel (ISV) is determined by the identity of the approaching secondary sprout emerging from the posterior cardinal vein. Here, we show that the formation of a balanced trunk vasculature involves an early heterogeneity in endothelial cell behaviour and Notch signalling activity in the seemingly identical primary ISVs that is independent of secondary sprouting and flow. We show that Notch signalling mediates the local patterning of ISVs, and an adaptive flow-mediated mechanism subsequently fine-tunes the global balance of arteries and veins along the trunk. We propose that this dual mechanism provides the adaptability required to establish a balanced network of arteries, veins and lymphatic vessels.

Recovery of reproductive function of female zebrafish from the toxic effects of microcystin-LR exposure.

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 µg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17ß-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17ßhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.

Palmitic Acid-Enriched Diet Induces Hepatic Steatosis and Injury in Adult Zebrafish.

Palmitic acid (PA) is the most abundant saturated fatty acid in fast foods and is known to induce inflammation and cellular injury in various tissues. In this study, we investigated whether a PA-enriched diet can induce hepatic steatosis and injury in adult zebrafish. The adult zebrafish exhibited increased body weight, hyperlipidemia, hyperglycemia, and steatosis and a hepatic injury phenotype after being fed with a PA-enriched diet for 6 weeks. The quantitative polymerase chain reaction analysis demonstrated that genes associated with hepatic injury were all significantly increased in the liver. Furthermore, livers from the PA-fed group showed an increased messenger RNA (mRNA) expression associated with oxidative stress and endoplasmic reticulum (ER) stress responses. We also found significant upregulation of genes involved in lipid metabolism and triacylglyceride accumulation. Ultrastructural analysis revealed mitochondrial cristae injury and a dilated ER phenotype in the PA-fed hepatocytes, which can be causes of hepatic injury. PA-enriched diet induced hepatic steatosis and injury in adult zebrafish that recapitulated typical metabolic changes and pathophysiological changes as well as increased oxidative stress and ER stress observed in patients with nonalcoholic fatty liver disease.

Chronic exposure to environmental concentrations of phenanthrene impairs zebrafish reproduction.

Phenanthrene (PHE) is a tricyclic polycyclic aromatic hydrocarbon which distributed extensively in the aquatic environment. However, the knowledge about its impact on fish reproduction is still limited, particularly under a chronic exposure regime. In this study, we exposed zebrafish (Danio rerio) embryos to environmentally relevant concentrations (0.2, 1.0, and 5.0 µg/L) of PHE for 4 months and assessed the impact on reproduction. The results demonstrated that egg production was decreased in fish exposed to PHE, with a significant reduction at 5.0 µg/L. The exposure significantly decreased the circulating concentrations of estradiol (E2) and testosterone (T) in female fish or E2 in male fish. In addition, plasma vitellogenin levels were significantly inhibited after PHE exposure in female fish. The transcription of hypothalamic-pituitary-gonadal (HPG) axis related genes (GnRH2, FSHß, LHß, 17ß-HSD, CYP11A1, and CYP19a) were significantly altered in a sex-specific manner. In addition, embryos derived from exposed parents exhibited increased malformation and decreased hatching success in the F1 generation. Taken together, these results demonstrate that chronic exposure to environmentally relevant concentration of PHE could cause adverse effects on reproduction and impair the development of offspring, ultimately leading to fish population decline in aquatic environment.

Hypoxia enhances blood O2 affinity and depresses skeletal muscle O2 consumption in zebrafish (Danio rerio).

Zebrafish (Danio rerio) are widely used animal models. Nevertheless, the mechanisms underlying hypoxia tolerance in this species have remained poorly understood. In the present study, we have determined the effects of hypoxia on blood-O2 transport properties and mitochondrial respiration rate in permeabilized muscle fibres of adult zebrafish exposed to either 1) a gradual decrease in O2 levels until fish lost equilibrium (~1 h, acute hypoxia), or 2) severe hypoxia (PO2 ∼ 15 Torr) for 48 h (prolonged hypoxia). Acute, short-term hypoxia caused an increase in hemoglobin (Hb) O2 affinity (decrease in P50), due to a decrease in erythrocyte ATP after erythrocyte swelling. No changes in isoHb expression patterns were observed between hypoxic and normoxic treatments. Prolonged hypoxia elicited additional reponses on O2 consumption: lactate accumulated in the blood, indicating that zebrafish relied on glycolysis for ATP production, and mitochondrial respiration of skeletal muscle was overall significantly inhibited. In addition, male zebrafish had higher hypoxia tolerance (measured as time to loss of equilibrium) than females. The present study contributes to our understanding of the adaptive mechanisms that allow zebrafish, and by inference other fish species, to cope with low O2 levels.

The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

Medroxyprogesterone acetate affects sex differentiation and spermatogenesis in zebrafish.

Medroxyprogesterone acetate (MPA) is a widely used synthetic progestin and it has been frequently detected in aquatic environments. However, its effects on aquatic organisms remain largely unknown. Here we investigated the chronic effects of MPA on sex differentiation and gonad development in zebrafish. Zebrafish larvae at 20 days post fertilization (dpf) were exposed to 4.32, 42.0, and 424 ng L-1 of MPA until they reached 140 dpf. The results showed that chronic exposure to 42.0 ng L-1 of MPA caused 60% proportion of males as well as significant up-regulation of dmrt1 (˜1.79 fold) and hsd17b3 (˜1.92 fold). Histological analysis showed MPA significantly increased the frequency of immature spermatocytes accompanied with the increased transcription of dmrt1 (˜2.06 fold) and ar (˜1.73 fold) in the testes. Meanwhile, MPA exposure significantly increased the transcription of lhb at all exposure concentrations in the males. In contrast, it significantly suppressed the transcription of lhb (˜-8.06-fold) and fshb (˜-6.35-fold) at 42.0 ng L-1 in the females. Collectively our results demonstrated that MPA had androgenic activity, and could affect sex differentiation and spermatogenesis in zebrafish at environmentally relevant concentrations. The findings from this study suggest that MPA in the aquatic environment may pose potential androgenic risks to fish populations.

Optimization of Hyperglycemic Induction in Zebrafish and Evaluation of Its Blood Glucose Level and Metabolite Fingerprint Treated with Psychotria malayana Jack Leaf Extract.

A standard protocol to develop type 1 diabetes in zebrafish is still uncertain due to unpredictable factors. In this study, an optimized protocol was developed and used to evaluate the anti-diabetic activity of Psychotria malayana leaf. The aims of this study were to develop a type 1 diabetic adult zebrafish model and to evaluate the anti-diabetic activity of the plant extract on the developed model. The ability of streptozotocin and alloxan at a different dose to elevate the blood glucose levels in zebrafish was evaluated. While the anti-diabetic activity of P. malayana aqueous extract was evaluated through analysis of blood glucose and LC-MS analysis fingerprinting. The results indicated that a single intraperitoneal injection of 300 mg/kg alloxan was the optimal dose to elevate the fasting blood glucose in zebrafish. Furthermore, the plant extract at 1, 2, and 3 g/kg significantly reduced blood glucose levels in the diabetic zebrafish. In addition, LC-MS-based fingerprinting indicated that 3 g/kg plant extract more effective than other doses. Phytosterols, sugar alcohols, sugar acid, free fatty acids, cyclitols, phenolics, and alkaloid were detected in the extract using GC-MS. In conclusion, P. malayana leaf aqueous extract showed anti-diabetic activity on the developed type 1 diabetic zebrafish model.

Effect of MS222 on Hemostasis in Zebrafish.

MS222 is a compound used in anesthetizing vertebrates, including fish and frogs. Several side effects of this anesthetic have been reported, but its effect on hemostasis has not been studied. In our laboratory, we have used zebrafish for more than 2 decades as a model system to study hemostasis. During this period, we have had trouble in collecting blood from anesthetized zebrafish and observed more rapid blood clotting than in nonanesthetized counterparts. However, no systematic studies regarding the effect of MS222 on zebrafish hemostasis are available. In this study, we performed various assays such as gill bleeding, measurement of Hct, total blood cell counts, thrombocyte counts, thrombocyte aggregation, and coagula- tion and measured the amount of blood collected. We found that Hct values, the amount of blood collected, bleeding, and coagulation differed significantly between anesthetized and nonanesthetized fish. Our results suggest that blood collected after MS222 anesthesia of zebrafish has altered hemostasis.

Analysis of O-Acetylated Sialic Acids in Dried Blood Spots.

Sialic acid is a family of N- and O-substitutions of neuraminic acid. Plasma or serum sialic acid has been established as a potential disease marker. For example, the presence of 9- O-acetyl on the sialic acid of some glycans and glycoconjugates (e.g., 9- O-acetyl GD3 ganglioside) could be related to cancer occurrence. A variety of assays are available to measure serum or plasma sialic acid; however, sample preparation and storage can alter the O-acetylation profile due to the loss of O-acetyl groups and/or the migration of O-acetyl groups. Herein, we report dried blood spot (DBS) sampling, in combination with diamino-4,5-methylenedioxybenzene derivatization, for profiling sialic acids in blood samples with minimal alteration in O-acetylation patterns. The feasibility of the method was first evaluated by analyzing sialic acids in crucian carp blood and comparing with traditional blood/plasma sample preparation procedures. A total of 19 different sialic acids were identified by using liquid chromatography-Orbitrap mass spectrometry, including four mono-O-acetylated N-acetylneuraminic acids, four mono-O-acetylated N-glycolylneuraminic acids, six di-O-acetylated N-acetylneuraminic acids, and three tri-O-acetylated N-acetylneuraminic acids. The long-term storage study indicated that DBS sampling could effectively preserve the O-acetylation information for at least 6 weeks. Thus, it is demonstrated that this method is a valuable tool for the study of sialic acid diversity, especially for the characterization of isomeric structures.

Plasma proteomic analysis of zebrafish following spring viremia of carp virus infection.

To better understand spring viremia of carp virus (SVCV) pathogenesis in zebrafish proteomic analysis was used to examine the plasma protein profile in SVCV-infected zebrafish. A total of 3062 proteins were identified. Of those 137, 63 and 31 proteins were enriched in blood samples harvested at 1, 2 and 5 days post SVCV infection, respectively. These altered host proteins were classified based on their biological function: 23 proteins under the response to stimulus term were identified. Interestingly, at the top of the up-regulated proteins during SVCV infection were the proteins of the vitellogenin family (Vtg) and the grass carp reovirus-induced gene (Gig) proteins. Real-time RT-PCR evaluation of samples from internal organs verified that SVCV infection induced vtg and gig2 gene expression already at day 1 post-infection. Western blot analysis revealed the presence of Vtg protein only in blood of SVCV-infected fish. This is the first proteomic study to reveal the involvement of Vtg proteins in adult fish response to viral challenge. It also highlights the role of Gig proteins as important factors in antiviral response in fish. This work provides valuable relevant insight into virus-host interaction and the identification of molecular markers of fish response to virus.

High-speed quantitative 3D imaging by dual-illumination holographic microscopy.

A new blood flow imaging (BFI) technique using digital holography with double illumination of the sample is proposed. We imaged the moving red blood cells (RBCs) using a two microscope objective lenses setup. The setup consists in a larger angle of separation (90 °) between the two illumination beams, allowing a wider angular rotation at good z resolution. Moreover, the setup geometry allows an easier displacement of the sample in all directions. Results show that this technique is able to perform phase-shifting reconstruction for the two beams at the same time which is more suitable for the future implementation of live 3D holography. Experimental results are carried out for the verification of the effectiveness of the proposed technique on a zebrafish larvae sample. RESEARCH HIGHLIGHTS: Blood flow imaging techniques are often invasive and image analysis is time consuming. To alleviate this issue an imaging technique based on dual illumination in holographic domain is proposed. This method has been validated on zebrafish embryos.