Exploring the Hawaiian Ala Wai Watershed with Zebrafish.

The Ala Wai Canal is an artificial waterway in the tourist district of Waikiki in Honolulu, HI. Originally built to collect runoff from industrial, residential, and green spaces dedicated to recreation, the Ala Wai Canal has since experienced potent levels of toxicity due to this runoff entering the watershed and making it hazardous for both marine life and humans at current concentration, including Danio rerio (zebrafish). A community of learners at educations levels from high school to postbaccalaureate from Oahu, HI was connected through the Consortium for Increasing Research and Collaborative Learning Experiences (CIRCLE) distance research program. This team conducted research with an Investigator and team from Mayo Clinic in Rochester, MN, with the Ala Wai Canal as its primary subject. Through CIRCLE, research trainees sent two 32 oz bottles of Ala Wai- acquired water to a partnered laboratory at the Mayo Clinic in which zebrafish embryos were observed at differing concentrations of the sampled water against a variety of developmental and behavioral assays. Research trainees also created atlases of developmental outcomes in zebrafish following exposure to environmental toxins and tables of potential pesticide contaminants to enable the identification of the substances linked to structural defects and enhanced stress during Ala Wai water exposure experiments.

DNA mimic foldamers affect chromatin composition and disturb cell cycle progression.

The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. We therefore systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. Our studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.

Relationships between Isomeric Metabolism and Regioselective Toxicity of Hydroxychrysenes in Embryos of Japanese Medaka (Oryzias latipes).

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are ubiquitous contaminants that can be formed through oxidation of parent PAHs. Our previous studies found 2-hydroxychrysene (2-OHCHR) to be significantly more toxic to Japanese medaka embryos than 6-hydroxychrysene (6-OHCHR), an example of regioselective toxicity. We have also previously identified a sensitive developmental window to 2-OHCHR toxicity that closely coincided with liver development, leading us to hypothesize that differences in metabolism may play a role in the regioselective toxicity. To test this hypothesis, Japanese medaka embryos were treated with each isomer for 24 h during liver development (52-76 hpf). Although 6-OHCHR was absorbed 97.2 ± 0.18% faster than 2-OHCHR, it was eliminated 57.7 ± 0.36% faster as a glucuronide conjugate. Pretreatment with cytochrome P450 inhibitor, ketoconazole, reduced anemia by 96.8 ± 3.19% and mortality by 95.2 ± 4.76% in 2-OHCHR treatments. Formation of chrysene-1,2-diol (1,2-CAT) was also reduced by 64.4 ± 2.14% by ketoconazole pretreatment. While pretreatment with UDP-glucuronosyltransferase inhibitor, nilotinib, reduced glucuronidation of 2-OHCHR by 52.4 ± 2.55% and of 6-OHCHR by 63.7 ± 3.19%, it did not alter toxicity for either compound. These results indicate that CYP-mediated activation, potentially to 1,2-CAT, may explain the isomeric differences in developmental toxicity of 2-OHCHR.

Polyhalogenated carbazoles (PHCZs) induce cardiotoxicity and behavioral changes in zebrafish at early developmental stages.

Polyhalogenated carbazoles (PHCZs) are widely present in the environment, and their health risks are of increasing concern. Available studies primarily confirm their dioxin-like toxicity mechanism based on biomarkers, such as aryl hydrocarbon receptor (AHR) and CYP1A1, while few studies have investigated their actual toxic effects at the level of individual organisms. In the present study, the developmental toxicity of two typical PHCZs with a high detection rate and high concentration in the environment (3,6-dichlorocarbazol (3,6-DCCZ) and 3,6-dibromocarbazole (3,6-DBCZ)) was investigated based on a fish embryo acute toxicity test (FET, zebrafish) and transcriptomics analysis. The 96 h LC50 values of 3,6-DCCZ and 3,6-DBCZ were 0.636 mg/L and 1.167 mg/L, respectively. Both tested PHCZs reduced the zebrafish heart rate and blocked heart looping at concentrations of 0.5 mg/L or higher. The swimming/escaping behavior of zebrafish larvae was more vulnerable to 3,6-DBCZ than 3,6-DCCZ. Transcriptomics assays showed that multiple pathways linked to organ development, immunization, metabolism and protein synthesis were disturbed in PHCZ-exposed fish, which might be the internal mechanism of the adverse effects. The present study provides evidence that PHCZs cause cardiac developmental toxicity and behavioral changes and improves our understanding of their health risks.

Ultraviolet avoidance by embryonic buoyancy control in three species of marine fish.

Pelagic fish embryos are thought to float in or near surface waters for the majority of their development and are presumed to have little to no control over their mobility, rendering these embryos at high risk for damages associated with surface stressors such as ultraviolet radiation (UVR). We recently challenged these long-standing paradigms by characterizing a potential mechanism of stressor avoidance in early-life stage mahi-mahi (Coryphaena hippurus) in which embryos sense external cues, such as UVR, and modify their buoyancy to reduce further exposure. It is unknown whether embryos of other marine fish with pelagic spawning strategies have similar capabilities. To fill this knowledge gap, we investigated buoyancy change in response to UVR in three additional species of marine fish that utilize a pelagic spawning strategy: yellowfin tuna (Thunnus albacares), red snapper (Lutjanus campechanus), and cobia (Rachycentron canadum). Embryos of all three species displayed increased specific gravity and loss of buoyancy after exposures to environmentally relevant doses of UVR, a response that may be ubiquitous to fish with pelagic embryos. To gain further insight into this response, we investigated recovery of buoyancy, oxygen consumption, energy depletion, and photolyase induction in response to UVR exposures in at least one of the three species listed above.

Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo.

Significant regulation of gene expression is mediated at the translation level. Here, we describe protocols for imaging and analysis of translation at single mRNA resolution in both fixed and living Drosophila embryos. These protocols use the SunTag system, in which the protein of interest is visualized by inserting a peptide array that is recognized by a single chain antibody. Simultaneous detection of individual mRNAs using the MS2/MCP system or by smFISH allows translation sites to be identified and quantified. For complete information on the generation and use of this protocol, please refer to Vinter et al. (2021).

Cardiac dysfunction affects eye development and vision by reducing supply of lipids in fish.

Developing organisms are especially vulnerable to environmental stressors. Crude oil exposure in early life stages of fish result in multiple functional and developmental defects, including cardiac dysfunction and abnormal and smaller eyes. Phenanthrene (Phe) has a reversible impact on cardiac function, and under exposure Phe reduces cardiac contractility. Exposure to a known L-type channel blocker, nicardipine hydrochloride (Nic) also disrupts cardiac function and creates eye deformities. We aimed to investigate whether cardiac dysfunction was the major underlying mechanism of crude oil-, Phe- and Nic-induced eye malformations. We exposed Atlantic haddock (Melanogrammus aeglefinus) early embryos to Nic and crude oil (Oil) and late embryos/early larvae to Phe exposure. All three exposures resulted in cardiac abnormalities and lead to severe, eye, jaw and spinal deformities at early larval stages. At 3 days post hatching, larvae from the exposures and corresponding controls were dissected. Eyes, trunk, head and yolk sac were subjected to lipid profiling, and eyes were also subjected to transcriptomic profiling. Among most enriched pathways in the eye transcriptomes were fatty acid metabolism, calcium signaling and phototransduction. Changes in lipid profiles and the transcriptome suggested that the dysfunctional and abnormal eyes in our exposures were due to both disruption of signaling pathways and insufficient supply of essential fatty acids and other nutrients form the yolk.

Immunotoxicity of F53B, an alternative to PFOS, on zebrafish (Danio rerio) at different early life stages.

As an alternative to perfluorooctane sulfonate (PFOS), 6:2 chlorinated polyfluorinated ether sulfonate (F53B) has emerged in the Chinese market in recent years and has been frequently detected in the aquatic environment, but its ecological risk assessment is limited. In this study, zebrafish embryos and larvae were separately exposed to F53B, and their 96-h LC50 values were estimated to be 15.1 mg/L and 2.4 mg/L, respectively, suggesting that embryos were more resistant to F53B than larvae. The bioconcentration factor in larvae was basically higher than that of embryos, and the body growth of larvae was significantly affected by F53B rather than embryos, indicating that F53B may cause more severe toxicity to larvae. In addition to the excessive production of ROS and NO, the expression of many immune-related genes was increased in both embryos and larvae, but the number of dysregulated genes in larvae was more than that in embryos. Finally, the results of Point of Departure (PoD) indicated that the immunotoxicity of F53B was more sensitive to larvae than embryos at the molecular level. Our findings revealed the ecological risk of F53B by exploring the adverse effects of immunoregulation at different early life stages of zebrafish and indicated that the zebrafish larvae were more sensitive than embryos.

Using RNA-binding proteins for immunoprecipitation of mRNAs from Xenopus laevis embryos.

This protocol is developed for identifying mRNAs that form complexes with mRNA-binding proteins (mRBPs) in Xenopus laevis embryos at different developmental stages. Here, we describe the use of the Ybx1 mRBP for immunoprecipitation-based mRNA isolation. This protocol features the translation of the mRBP of interest directly in living embryos following injection of synthetic mRNA templates encoding a hybrid of this protein with a specific tag. This approach allows precipitation of mRNA-protein complexes from embryonic lysates using commercially available anti-tag antibodies. For complete details on the use and execution of this protocol, please refer to Parshina et al. (2020).

Microsporidia infection upregulates host energy metabolism but maintains ATP homeostasis.

Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.

The role of gut microbial community and metabolomic shifts in adaptive resistance of Atlantic killifish (Fundulus heteroclitus) to polycyclic aromatic hydrocarbons.

Altered gut microbiomes may play a role in rapid evolution to anthropogenic change but remain poorly understood. Atlantic killifish (Fundulus heteroclitus) in the Elizabeth River, VA have evolved resistance to polycyclic aromatic hydrocarbons (PAHs) and provide a unique opportunity to examine the links between shifts in the commensal microbiome and organismal physiology associated with evolved resistance. Here, 16S rRNA sequence libraries derived from fish guts and sediments sampled from a highly PAH contaminated site revealed significant differences collected at similar samples from an uncontaminated site. Phylogenetic groups enriched in the libraries derived from PAH-resistant fish were dissimilar to their associated sediment libraries, suggesting the specific environment within the PAH-resistant fish intestine influence the gut microbiome composition. Gut metabolite analysis revealed shifts between PAH-resistant and non-resistant subpopulations. Notably, PAH-resistant fish exhibited reduced levels of tryptophan and increased levels of sphingolipids. Exposure to PAHs appears to impact several bacterial in the gut microbiome, particularly sphingolipid containing bacteria. Bacterial phylotypes known to include species containing sphingolipids were generally lower in the intestines of fish subpopulations exposed to high concentrations of PAHs, inferring a complex host-microbiome relationship. Overall, killifish microbial community shifts appear to be related to a suppression of overall metabolite level, indicating a potential role of the gut in organismal response to anthropogenic environmental change. These results on microbial and metabolomics shifts are potentially linked to altered bioenergetic phenotype observed in the same PAH-resistant killifish populations in other studies.

Isotopic enrichment in Rhizoprionodon porosus (Poey, 1861) and Carcharhinus porosus (Ranzani, 1840) embryos.

Isotopic values of two Caribbean sharpnose shark Rhizoprionodon porosus litters (Poey, 1861) with two and three embryos and one litter of 11 smalltail shark Carcharhinus porosus embryos showed enriched 15 N and 13 C compared to their mothers. In R. porosus, embryonic isotope values were 3.06 ± 0.07‰ and 0.69 ± 0.15‰ greater than their mothers' for δ15 N and δ13 C, respectively, whereas in C. porosus, δ15 N and δ13 C were 1.79 ± 0.09‰ and 1.31 ± 0.17‰ greater in embryos than their mothers.

Bioorthogonal Activation of Dual Catalytic and Anti-Cancer Activities of Organogold(I) Complexes in Living Systems.

Controllably activating the bio-reactivity of metal complexes in living systems is challenging but highly desirable because it can minimize off-target bindings and improve spatiotemporal specificity. Herein, we report a new bioorthogonal activation approach by employing Pd(II)-triggered transmetallation reactions to conditionally activate the bio-reactivity of NHC-Au(I)-phenylacetylide complexes (1 a) in vitro and in vivo. A combination of 1 H NMR, LC-MS, DFT calculation and fluorescence screening assays reveals that 1 a displays a reasonable stability against biological thiols, but its phenylacetylide ligand can be efficiently transferred to Pd(II), leading to in situ formation of labile NHC-Au(I) species that is catalytically active inside living cells and zebrafish, and can meanwhile effectively suppress the activity of thioredoxin reductase, potently inhibit the proliferation of cancer cells and efficiently suppress angiogenesis in zebrafish models.

A turn-on red-emitting fluorescent probe for determination of copper(II) ions in food samples and living zebrafish.

Herein, we developed a turn-on red-emitting fluorescent probe for the sensitive and selective detection of copper ions (Cu2+) in food samples and living zebrafish. The probe employs a hemicyanine scaffold as the fluorophore and a 2-pyridinecarbonyl group as the recognition receptor and quenching moiety. The 2-pyridinecarbonyl moiety can be specifically cleaved by Cu2+ and results in an approximately 18-fold fluorescence enhancement of the probe, thereby providing a fluorescence turn-on assay for Cu2+. Additionally, the probe exhibited excellent selectivity, high sensitivity, a broad linear relationship (0.020 to 8.0 µM), and a low limit of detection (4.0 nM, S/N = 3) for Cu2+. Concomitantly, the probe exhibited satisfactory analytical performance when used with actual food samples. Moreover, the probe could be used for in situ determination of Cu2+ in both living plant tissues and in living zebrafish.

The Use of Stem Cell Differentiation Stage Factors (SCDSFs) Taken from Zebrafish Embryos during Organogenesis and Their Role in Regulating the Gene Expression of Normal and Pathological (Stem) Cells.

Studies conducted on Zebrafish embryos in our laboratory have allowed for the identification of precise moments of organogenesis in which a lot of genes are switched on and off, a sign that the genome is undergoing substantial changes in gene expression. Stem cell growth and differentiation stage-factors present in different moments of organogenesis have proven to have different specific functions in gene regulation. The substances present in the first stages of cell differentiation in Zebrafish embryos have demonstrated an ability to counteract the senescence of stem cells, reducing the expression of the beta-galactosidase marker, enhancing the genes Oct-4, Sox-2, c-Myc, TERT, and the transcription of Bmi-1, which act as key telomerase-independent repressors of cell aging. The molecules present in the intermediate to late stages of cell differentiation have proven to be able to reprogram pathological human cells, such as cancer cells and those of the basal layer of the epidermis in psoriasis, which present a higher multiplication rate than normal cells. The factors present in all the stages of cell differentiation are able to counteract neurodegeneration, and to regenerate tissues: It has been possible to regenerate hair follicles in many patients with androgenetic alopecia through transdermal administration of stem cell differentiation stage factors (SCDSFs) by means of cryopass-laser.

Embryonic substances induce alarm response in adult zebrafish (Danio rerio).

Many aquatic animals rely on chemicals released by injured individuals of the same species to assess predation risk. Among these chemical cues, alarm substances released from the injured skin of ostariophysan fishes have been extensively examined. In most fish species examined, these cues appear to be released by all injured individuals (including larvae, juveniles and adults) and elicit alarm responses in conspecifics. Adult alarm cues also affect development and physiology of embryos. Nonetheless, whether embryos produce alarm cues that affect adults is not known. This study reports that extracts of zebrafish (Danio rerio) embryos at 36 h post-fertilization or later induce antipredator behaviours reminiscent of those induced by skin alarm substances. At an equivalent of 10-6 g embryo per millilitre, the extract induced bottom-dwelling and freezing in adults. These behaviours are consistent with those induced by adult alarm substances. This study concludes that zebrafish embryos produce alarm substances.

Embryonic geometry underlies phenotypic variation in decanalized conditions.

During development, many mutations cause increased variation in phenotypic outcomes, a phenomenon termed decanalization. Phenotypic discordance is often observed in the absence of genetic and environmental variations, but the mechanisms underlying such inter-individual phenotypic discordance remain elusive. Here, using the anterior-posterior (AP) patterning of the Drosophila embryo, we identified embryonic geometry as a key factor predetermining patterning outcomes under decanalizing mutations. With the wild-type AP patterning network, we found that AP patterning is robust to variations in embryonic geometry; segmentation gene expression remains reproducible even when the embryo aspect ratio is artificially reduced by more than twofold. In contrast, embryonic geometry is highly predictive of individual patterning defects under decanalized conditions of either increased bicoid (bcd) dosage or bcd knockout. We showed that the phenotypic discordance can be traced back to variations in the gap gene expression, which is rendered sensitive to the geometry of the embryo under mutations.

Differences in δ15 N and δ13 C between embryonic and maternal tissues of the ovoviviparous bluntnose sixgill shark Hexanchus griseus.

Stable nitrogen (δ15 N) and carbon (δ13 C) isotope ratios from muscle, liver and yolk were analysed from the mother and embryos of an ovoviviparous shark, Hexanchus griseus. Embryonic liver and muscle had similar δ15 N and δ13 C ratios or were depleted in heavy isotopes, compared to the same maternal somatic and reproductive yolk tissues, but no relationship existed between δ15 N or δ13 C and embryo length, as expected, because a switch to placental nourishment is lacking in this species. This study expands the understanding of maternal nourishment and embryonic stable isotope differences in ovoviviparous sharks.

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT7 genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.