Dose addition in chemical mixtures inducing craniofacial malformations in zebrafish (Danio rerio) embryos.

A challenge in cumulative risk assessment is to model hazard of mixtures. EFSA proposed to only combine chemicals linked to a defined endpoint, in so-called cumulative assessment groups, and use the dose-addition model as a default to predict combined effects. We investigated the effect of binary mixtures of compounds known to cause craniofacial malformations, by assessing the effect in the head skeleton (M-PQ angle) in 120hpf zebrafish embryos. We combined chemicals with similar mode of action (MOA), i.e. the triazoles cyproconazole, triadimefon and flusilazole; next, reference compounds cyproconazole or triadimefon were combined with dissimilar acting compounds, TCDD, thiram, VPA, prochloraz, fenpropimorph, PFOS, or endosulfan. These mixtures were designed as (near) equipotent combinations of the contributing compounds, in a range of cumulative concentrations. Dose-addition was assessed by evaluation of the overlap of responses of each of the 14 tested binary mixtures with those of the single compounds. All 10 test compounds induced an increase of the M-PQ angle, with varying potency and specificity. Mixture responses as predicted by dose-addition did not deviate from the observed responses, supporting dose-addition as a valid assumption for mixture risk assessment. Importantly, dose-addition was found irrespective of MOA of contributing chemicals.

Developmental Toxicity of a Neonicotinoid Insecticide, Acetamiprid to Zebrafish Embryos.

Agricultural use of neonicotinoid insecticides is increasing worldwide, posing a risk to nontarget organisms. The present study investigated developmental toxicity of a widely used neonicotinoid, acetamiprid, to zebrafish embryos. Sublethal (malformations, hatchability, heart rate, body length, alteration of spontaneous movement and touch responses) and lethal effects were monitored during exposure period from 6 h post fertilization (hpf) to 120 hpf. Zebrafish embryos exhibited significant mortality and teratogenic effects at acetamiprid concentration greater than 263 mg/L, with bent spine being the main malformation. Toxicity spectra were constructed to rank the sensitivity of individual end points to acetamiprid exposure and impaired spontaneous movement was the most sensitive end point of those tested. The present study provides the basis for understanding developmental toxicity of acetamiprid exposure to zebrafish embryos. This information is critical for future studies evaluating aquatic risk from neonicotinoids as little is known regarding adverse effects of neonicotinoids to aquatic vertebrate species.

Thermal experience during embryogenesis contributes to the induction of dwarfism in whitefish Coregonus lavaretus.

Ecotype pairs provide well-suited model systems for study of intraspecific phenotypical diversification of animals. However, little is still known about the processes that account for the development of different forms and sizes within a species, particularly in teleosts. Here, embryos of a normal-growing 'large' form and a dwarf form of whitefish Coregonus lavaretus were incubated at two temperatures that are usually experienced at their own spawning sites (2°C for the normal and 6°C for the dwarf form). All fish were subjected to similar thermal treatment after hatching. The present data demonstrate for the first time that different thermal experience in embryonic life has lasting effects on body and muscle growth of this ecotype pair and contributes to the development of the dwarf form. Thus, juvenile fish of the regular form are much smaller and have less muscle mass when pre-hatching thermal conditions were similar to those typical for the spawning sites of the dwarf form (6°C) than when subjected to conditions of their own spawning sites (2°C). Surprisingly, fish of the dwarf form exhibit a similar pattern of response to thermal history (2°-fish much larger than 6°-fish), indicating that in their case, normal spawning site temperature (6°C) is indeed likely to act as a growth limiting factor. Results also demonstrate that the hypertrophic and hyperplastic muscle growth modes are similarly affected by thermal history. Immunolabelling experiments for Pax7, H3P and Mef2 provide evidence that the cellular mechanisms behind the increased growth rates after cold incubation in both ecotypes are increased proliferation and reduced differentiation rates of muscle precursor cells. This is of major significance to aspects of ecological and developmental biology and from the evolutionary perspective.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Inhibitory effect of cadmium on estrogen signaling in zebrafish brain and protection by zinc.

The present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERß1 and ERß2) was also used. Embryos were exposed either to estradiol (E2 ), Cd, E2 +Cd or E2 +Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2 -stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2 -treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement. Copyright © 2016 John Wiley & Sons, Ltd.

Cichlid fishes as a model to understand normal and clinical craniofacial variation.

We have made great strides towards understanding the etiology of craniofacial disorders, especially for 'simple' Mendelian traits. However, the facial skeleton is a complex trait, and the full spectrum of genetic, developmental, and environmental factors that contribute to its final geometry remain unresolved. Forward genetic screens are constrained with respect to complex traits due to the types of genes and alleles commonly identified, developmental pleiotropy, and limited information about the impact of environmental interactions. Here, we discuss how studies in an evolutionary model - African cichlid fishes - can complement traditional approaches to understand the genetic and developmental origins of complex shape. Cichlids exhibit an unparalleled range of natural craniofacial morphologies that model normal human variation, and in certain instances mimic human facial dysmorphologies. Moreover, the evolutionary history and genomic architecture of cichlids make them an ideal system to identify the genetic basis of these phenotypes via quantitative trait loci (QTL) mapping and population genomics. Given the molecular conservation of developmental genes and pathways, insights from cichlids are applicable to human facial variation and disease. We review recent work in this system, which has identified lbh as a novel regulator of neural crest cell migration, determined the Wnt and Hedgehog pathways mediate species-specific bone morphologies, and examined how plastic responses to diet modulate adult facial shapes. These studies have not only revealed new roles for existing pathways in craniofacial development, but have identified new genes and mechanisms involved in shaping the craniofacial skeleton. In all, we suggest that combining work in traditional laboratory and evolutionary models offers significant potential to provide a more complete and comprehensive picture of the myriad factors that are involved in the development of complex traits.

Molecular cloning, characterization and expression of cathepsin D from grass carp (Ctenopharyngodon idella).

Cathepsin D is a lysosomal aspartic proteinase which participates in various degradation functions within the cell. In this current study, we cloned and characterized the complete cDNA of grass carp cathepsin D through 5'- and 3'-RACE. The cathepsin D contained a 56 bp 5' terminal untranslated region (5'-UTR), a 1197 bp open reading frame encoding 398 amino acids, and a 394 bp 3'-UTR. Grass carp cathepsin D shared high similarity with those from other species, and showed the highest amino acid identity of 91% to Danio rerio. Unlike many other organisms, the grass carp cathepsin D contains only one N-glycosylation site closest to the N-terminal. Real-time quantitative RT-PCR demonstrated that Cathepsin D expressed in all twelve tissues (bladder, brain, liver, heart, gill, muscle, fin, eye, intestines, spleen, gonad and head kidney). The relative expression levels of Cathepsin D in gonad and liver were 26.58 and 24.95 times as much as those in fin, respectively. The expression level of Cathepsin D in muscle approximately 16-fold higher, in intestines and spleen were 12-fold higher. The cathepsin D expression showed an upward trend during embryonic development. After challenged with Aeromonas hydrophil, the expression of grass carp cathepsin D gene showed significant changes in the four test tissues (liver, head kidney, spleen and intestines). The fact that the bacterial infection can obviously improve the cathepsin D expression in immune-related organs, may suggest that cathepsin D plays an important role in the innate immune response of grass carp.

Cloning, characterization and expression analysis of a cold shock domain family member YB-1 in turbot Scophthalmus maximus.

The Y-box proteins are a family of highly conserved nucleic acid binding proteins. In this report we have identified a new member, YB-1 from turbot (Scophthalmus maximus) spleen cDNA library. The full-length cDNA sequence of turbot YB-1 was obtained and then the expression at transcriptional level was researched by qRT-PCR. In normal organs, the expression of YB-1 was higher in liver, brain, gill and heart, respectively. YB-1 had the highest expression level at gastrula stage during the early stages of embryo development. In the liver, kidney and spleen, the turbot YB-1 expression level was the highest at 72 h after challenge with lymphocystis disease virus (LCDV) and the highest at 12 h after challenge with Vibrio anguillarum (V. anguillarum). Furthermore, the expression of turbot YB-1 also distinctly increased in turbot kidney cells (TK) at 24 h after challenge with V. anguillarum and LCDV. These results indicated that the turbot YB-1 protein may play a significant role in the immune response of turbot.

Ontogeny of anti-viral hemorrhagic septicemia virus (VHSV) immunity in developing Japanese flounder.

We examined the ability of developing Japanese flounder (Paralichthys olivaceus) to acquire protective immunity after exposure to viral hemorrhagic septicemia virus (VHSV). Juveniles measuring 9.8 cm average body length were not susceptible to infection with VHSV at 20 °C, while the smaller fish were susceptible. Mortality was not observed after secondary infection at 15 °C in the 9.8 cm cohort that had previously been exposed to the virus at 20 °C, while the smaller fish were susceptible to secondary infection. The expression of interferon (IFN)-related genes was shown to be better developed in larger fish upon virus infection and basal expression levels of the virus recognition proteins were higher in larger fish. Virus-specific antibody was detected in the larger fish, but not in smaller fish. These data indicate that the largest juvenile (9.8 cm) acquired immunity against VHSV infection at the first virus challenge, but smaller fish did not. The anti-viral immune system in the Japanese flounder matures when juveniles reach approximately 10 cm.

The inflammatory bowel disease (IBD) susceptibility genes NOD1 and NOD2 have conserved anti-bacterial roles in zebrafish.

Inflammatory bowel disease (IBD), in the form of Crohn's disease (CD) or ulcerative colitis (UC), is a debilitating chronic immune disorder of the intestine. A complex etiology resulting from dysfunctional interactions between the intestinal immune system and its microflora, influenced by host genetic susceptibility, makes disease modeling challenging. Mutations in NOD2 have the highest disease-specific risk association for CD, and a related gene, NOD1, is associated with UC. NOD1 and NOD2 encode intracellular bacterial sensor proteins acting as innate immune triggers, and represent promising therapeutic targets. The zebrafish has the potential to aid in modeling genetic and environmental aspects of IBD pathogenesis. Here, we report the characterization of the Nod signaling components in the zebrafish larval intestine. The nod1 and nod2 genes are expressed in intestinal epithelial cells and neutrophils together with the Nod signaling pathway genes ripk2, a20, aamp, cd147, centaurin b1, erbin and grim-19. Using a zebrafish embryo Salmonella infection model, morpholino-mediated depletion of Nod1 or Nod2 reduced the ability of embryos to control systemic infection. Depletion of Nod1 or Nod2 decreased expression of dual oxidase in the intestinal epithelium and impaired the ability of larvae to reduce intracellular bacterial burden. This work highlights the potential use of zebrafish larvae in the study of components of IBD pathogenesis.

Effects of vitamin B1 (thiamine) deficiency in lake trout (Salvelinus namaycush) alevins at hatching stage.

The objectives of this study were to examine the relationship between thiamine concentrations in unfertilized eggs and yolksac individuals of lake trout (Salvelinus namaycush), along with any associated histopathological changes in the tissues of alevins at the hatching stage. We address these questions in a lake trout population from different spawning grounds of Lake Michigan (North and South), known for compromised survival due to early mortality syndrome (EMS). However, a dichotomous forage base of lake trout spawning stocks, with a dietary thiaminase-rich alewife in the North, and dietary low-thiaminase round goby in the South, provides the basis for the assumption that different diets may lead to differences in severity of EMS between different stocks. Lake trout eggs of 18 females were collected and fertilized individually with the sperm of several males. The eggs, eyed embryos and newly-hatched alevins were sampled to examine thiamine utilization during embryogenesis. Progenies of females with low (< 0.73 nmol/g) and high (> 0.85 nmol/g) levels of thiamine were chosen for histological studies. The obtained results showed that total thiamine levels in the body and yolk of eyed embryos and alevins at hatching were influenced by thiamine levels of unfertilized eggs and it decreased during embryogenesis (to 51% in eyed embryos and 28% in newly-hatched alevins in comparison to unfertilized eggs). The survival of lake trout until hatching stage does not correlate with the thiamine level, however it was affected by collection site and was significantly higher in fish from the South site (Julian's Reef). At the hatching stage, no pathological changes were observed in the brain, olfactory lobe, retina or liver in embryos regardless of thiamine concentrations in unfertilized eggs. It has been concluded that an enhanced thiamine requirement for the fast muscle mass growth near the swim-up stage is responsible for overt and histopathological signs of EMS. Current study confirms earlier findings that lake trout suffering from EMS can be successfully treated by immersion in thiamine solution as late as at the swim-up stage.

[Analysis of parasitic communities in fishes from Lake Baikal].

Analysis of infracommunities and component communities of fish parasites in Lake Baikal has been conducted for the first time. It has been revealed that parasite infracommunities for the majority of Baikal fishes are weakly balanced and impoverished (the Berger-Parker Index is > 0.5; Evension is < 0.5; the Brillouin Index is < 1). The highest diversity and balance of the communities are characteristic for carnivorous fishes (Brachymystax lenok, Hucho taimen, Thymallus arcticus, Esox lucius, and Percafluviatilis). The component parasitic communities of Leuciscus leuciscus baicalensis, Rutilus rutilus, and Leocottus kesslerii are the most diverse in Lake Baikal since the Shennon index for L. leuciscus baicalensis, R. rutilus, and L. kesslerii is 2.4, for Paracotlus knerii--2.2, Limnocoitus godlewskii--2.3, Phoxinus phoxinus--2.1, Lota lota and Limnocuttus pallidus--1.9, P. fluviatilis--1.8, Leuciscus idus--1.8. The component parasitic communities of other fishes in Lake Baikal have low indices of biological diversity (H = 0.5-1.05, Smp is close to 1). A classification of mature and immature components of parasitic communities based on the ratio of specialist species and generalist species has been proposed. It is established that the component parasitic communities in sublitoral, profundal, and pseudoabyssal zones are mature, while in the littoral zone they are immature (impoverished and weakly balanced). The component parasitic communities in benthophagous fishes and predators are mature, in planktivorous fishes they are immature. The component parasitic communities are mature in the family Cyprinidae and immature in the families Coregonidae and Cottidae. The component parasitic communities of the Boreal Plain and Boreal Submountain faunal complexes are mature, but they are immature in Lake Baikal and Arctic freshwater complexes.

Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish development.

Polycyclic aromatic hydrocarbons (PAHs), derived largely from fossil fuels and their combustion, are pervasive contaminants in rivers, lakes, and nearshore marine habitats. Studies after the Exxon Valdez oil spill demonstrated that fish embryos exposed to low levels of PAHs in weathered crude oil develop a syndrome of edema and craniofacial and body axis defects. Although mechanisms leading to these defects are poorly understood, it is widely held that PAH toxicity is linked to aryl hydrocarbon receptor (AhR) binding and cytochrome P450 1A (CYP1A) induction. Using zebrafish embryos, we show that the weathered crude oil syndrome is distinct from the well-characterized AhR-dependent effects of dioxin toxicity. Blockade of AhR pathway components with antisense morpholino oligonucleotides demonstrated that the key developmental defects induced by weathered crude oil exposure are mediated by low-molecular-weight tricyclic PAHs through AhR-independent disruption of cardiovascular function and morphogenesis. These findings have multiple implications for the assessment of PAH impacts on coastal habitats.

The effects of dimethylated and alkylated polycyclic aromatic hydrocarbons on the embryonic development of the Japanese medaka.

The Japanese medaka (Oryzias latipes) early-life stage assay was used to investigate the effects of a number of commercially available dimethylated polycyclic aromatic hydrocarbons (PAHs) (3,6-dimethylphenanthrene, 7,12-dimethylbenz[a]anthracene, and 4,6-dimethyldibenzothiophene) and their unsubstituted congeners, dimethylated and unsubstituted tertiary mixtures, and a complex environmental mixture (with elevated C2-substituted dibenzothiophene) on embryo larval development. Unsubstituted PAHs showed trends of increased blue sac disease (BSD) relative to dimethylated PAHs, although the severity of BSD induction varied. Results demonstrated that the dibenzothiophene congeners were the strongest inducers of BSD of the commercial PAHs tested. These compounds reduced the hatching success of embryonic medaka, an effect that was enhanced in the mixture. The base neutral extract significantly increased the frequency and severity of BSD abnormalities, while significantly reducing larval hatch length. Based on these results, a sublethal maximum allowable toxicant concentration (MATC) of 13.91 microg PAHs/L was calculated.

Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin.

The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue.

Trebius shiinoi n. sp. (Trebiidae: Siphonostomatoida: Copepoda) from uteri and embryos of the Japanese angelshark (Squatina japonica) and the clouded angelshark (Squatina nebulosa), and redescription of Trebius longicaudatus.

Trebius shiinoi n. sp. is described from females and males collected from the uterine linings and on embryos within the uteri of 2 near-term Japanese angelsharks (Squatina japonica Bleeker, 1858) captured in Suruga Bay, off central Japan, and from female specimens reported by Shiino in 1963 that were found on embryos of the clouded angelshark (Squatina nebulosa Regan, 1906) captured off Shirahama, central Japan. Shiino identified his specimens as Trebius longicaudatus Shiino, 1954. However, our comparisons between Shiino's specimens and those newly collected revealed both to represent the same species, and comparisons of these specimens to 5 syntypes of T. longicaudatus and to published information detailing other Trebius species revealed them to be a new species that differs most notably from its congeners by the enormous length of its transformed adult female's abdomen and by the presence of a distinctive nublike seta on her caudal ramus. Trebius shiinoi n. sp. is an unusual copepod because it is an endoparasite of adult female angelsharks as well as an ectoparasite of embryo angelsharks, and it is proposed that flushing of the uterine-cloacal chambers of clouded angelsharks and Japanese angelsharks may facilitate T. shiinoi infections. A redescription of T. longicaudatus is also provided.

Effects of Beauveria bassiana on embryos of the inland silverside fish (Menidia beryllina).

A chemical toxicity and teratogenicity test was adapted to assess potential adverse effects of a microbial pest control agent on a nontarget fish. Developing embryos of the inland silverside, Menidia beryllina, were exposed to conidiospores of the insect-pathogenic fungus Beauveria bassiana. Embryo rupture and death were observed. Embryo rupture did not always result in death, nor was death always associated with embryo rupture. Adherence of spores to the chorion, followed by germination and penetration by the germ tube, probably caused the embryos to rupture. Statistically significant (P less than or equal to 0.05) responses were observed in tests in which conidiospore concentrations were greater than or equal to 8.3 x 10(4) or less than or equal to 1.5 x 10(6)/ml. Conidiospores treated with a dispersant (biological detergent) showed significantly less binding (P less than or equal to 0.01) to embryos than did untreated spores. Both detergent-treated and heat-killed spores failed to cause significant adverse effects.