Embryonic exposure to produced water can cause cardiac toxicity and deformations in Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) larvae.

Regular discharges of produced water from the oil and gas industry represents the largest direct discharge of effluent into the marine environment worldwide. Organic compound classes typically reported in produced water include saturated hydrocarbons, monoaromatic and polyaromatic hydrocarbons (MAHs, PAHs) as well as oxygenated compounds, such as phenols, acids and ketones. This forms a cocktail of known and suspect toxicants, but limited knowledge is yet available on the sub-lethal toxicity of produced water to cold-water marine fish species. In the present work, we conducted a 4-day exposure of embryos of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) to produced water extracts equivalent to 1:50, 1:500 and 1:5000 times dilutions of raw effluent. No significant reduction in survival or hatching success was observed, however, for cod, hatching was initiated earlier for exposed embryos in a concentration-dependent manner. During recovery, significantly reduced embryonic heart rate was observed for both species. After hatch, larvae subjected to embryonic exposure to produced water extracts were smaller, and displayed signs of cardiotoxicity, jaw and craniofacial deformations. In order to improve risk assessment and regulation of produced water discharges, it is important to identify which produced water components contribute to these effects.

Automatic determination of heart rates from microscopy videos of early life stages of fish.

Toxic effects of organic hydrophobic contaminants include impacts on fish heart rate (HR) and cardiac functioning. Thus, in ecotoxicology as well as aquaculture and even medicine, fish heart functioning plays an important role in application areas. The aim of this study was to assemble a pipeline of image processing and statistical techniques to extract HR information from microscopy videos of the embryo and larval stages of three species of fish (Atlantic cod, haddock, and Atlantic bluefin tuna). The method enables automatic processing for a large number of individuals, saving a significant amount of time compared with manual processing, while simultaneously eliminating the type of errors such a manual process might incur.

Novel adverse outcome pathways revealed by chemical genetics in a developing marine fish.

Crude oil spills are a worldwide ocean conservation threat. Fish are particularly vulnerable to the oiling of spawning habitats, and crude oil causes severe abnormalities in embryos and larvae. However, the underlying mechanisms for these developmental defects are not well understood. Here, we explore the transcriptional basis for four discrete crude oil injury phenotypes in the early life stages of the commercially important Atlantic haddock (Melanogrammus aeglefinus). These include defects in (1) cardiac form and function, (2) craniofacial development, (3) ionoregulation and fluid balance, and (4) cholesterol synthesis and homeostasis. Our findings suggest a key role for intracellular calcium cycling and excitation-transcription coupling in the dysregulation of heart and jaw morphogenesis. Moreover, the disruption of ionoregulatory pathways sheds new light on buoyancy control in marine fish embryos. Overall, our chemical-genetic approach identifies initiating events for distinct adverse outcome pathways and novel roles for individual genes in fundamental developmental processes.

Histological Aspects of the Early Development of the Digestive System of Burbot Lota lota L. (Lotidae, Gadiformes).

The ontogeny of the digestive tract was studied histologically in burbot, Lota lota L., from hatching to 42 days post-hatch (dph). At hatching, the digestive tract consisted of a straight tube with discernible digestive accessory glands (the liver and the pancreas) dorsally attached to the yolk sac. Most of the yolk sac reserves were consumed during the first 12 days and were completely depleted by 17 dph. The first PAS-positive goblet cells appeared at 6 dph, dispersed within the epithelium of the oesophagus and increasing substantially in number and distribution as development progressed. At 12 dph, the first vacuoles (neutral lipids) appeared in the intestine, indicating the functional absorption of nutrients from food. Differentiation of gastric glands was first noticed at 17 dph and was extensive by 27 dph. L. lota larvae have a morphologically complete digestive tract by 32 dph. These findings on the development of the digestive system in L. lota may contribute to a better understanding of its ontogeny and can be useful for improvement of the larval rearing techniques of this promising species for freshwater aquaculture diversification.

The effect of temperature on embryonic and yolk-sac larval development in the burbot Lota lota.

The present study investigated the effect of various temperature regimes on embryonic and yolk-sac larval development of the burbot Lota lota. At constant temperature regimes a high percentage of ready-to-hatch embryos, hatched larvae and normally shaped larvae was observed at the lowest temperature (2° C), which significantly decreased with increasing temperatures (4 and 6° C). No larvae hatched at 9° C. The stream temperature regime had no effect on the percentage of ready-to-hatch embryos, but significantly decreased the percentage of hatched larvae and of normally shaped larvae. The lake temperature regime did not affect the viability variables. Also an abrupt temperature increase from 2 to 4° C after 8 days and from 2 to 9° C after 48 days had no effect on the evaluated viability variables. For yolk-sac larvae no temperature related mortalities or abnormalities were observed between 2 and 9° C.

Establishment and partial characterization of a cell line from burbot Lota lota maculosa: susceptibility to IHNV, IPNV and VHSV.

This study describes the development and partial characterization of a continuous fibroblastic-like cell line (BEF-1) developed from late stage embryos of North American burbot Lota lota maculosa. This cell line has been maintained for over 5 yr and 100 passages in vitro. Cells were cultured using Eagle's minimum essential medium with Earle's salts (MEM) supplemented with GlutaMAX, and 10% fetal bovine serum (FBS), pH 7.4. The addition of penicillin-streptomycin-neomycin (PSN) antibiotic mixture (0.05, 0.05, 0.1 mg m(-1), respectively) did not negatively influence cell replication; however, the antimycotic FungizoneTM (2.5 microg m(-1), amphotericin B) caused cell rounding and resulted in a severe decrease in cell proliferation. Optimal incubation temperature has been observed between 15 and 23 degrees C, and at these temperatures cultures are routinely passed using standard trypsinization methods every 5 to 7 d at a split ratio of 1:3 or 1:4. The cell line was susceptible to isolates of the M and U North American genotypes of infectious hematopoietic necrosis virus (IHNV), and to isolates of genotypes I, IVa, and IVb of viral hemorrhagic septicemia virus (VHSV). In contrast, the cell line was refractory to infection by 2 North American isolates of infectious pancreatic necrosis virus (IPNV) from serotypes A1 and A9. This cell line provides a new laboratory tool, will allow further investigation into viral diseases of burbot and possibly other species, and is the first immortalized cell line reported from a species in the Gadidae (cod) family.

Microarray analysis of polychlorinated biphenyl mixture-induced changes in gene expression among Atlantic tomcod populations displaying differential sensitivity to halogenated aromatic hydrocarbons.

Several populations of fishes inhabiting contaminated Atlantic Coast estuaries exhibit resistance to early life-stage (ELS) toxicities induced by halogenated aromatic hydrocarbons such as coplanar polychlorinated biphenyls (PCBs). These toxicities include mortality, circulatory failure, edema, and craniofacial malformations. The mechanisms behind resistance to halogenated aromatic hydrocarbon toxicity in these populations are unknown. First and second generation Atlantic tomcod Microgadus tomcod embryos derived from the Hudson River ([HR]; New York, USA) population are highly resistant to PCB-induced cytochrome P4501A (CYP1A) expression and ELS toxicity when compared to embryos of Miramichi River ([MR]; New Brunswick, Canada) and Shinnecock Bay ([SB]; New York, USA) origin. The present study sought to identify novel genes involved in population differences in response to PCB exposure using custom microarrays. Microarray probes consisted of unsequenced inserts of randomly picked clones from a tomcod cardiac cDNA library. Tomcod embryos from three populations (HR, MR, and SB) were exposed to two doses of an environmentally relevant mixture of coplanar PCBs and screened for dose- and population-specific patterns of gene expression. Clones displaying significant differences between populations exposed to the high dose of PCBs were identified by DNA sequencing. Of the 28 identified nonribosomal protein clones, none displayed expression patterns highly similar to CYP1A (altered in MR and SB, but not in HR). However, several transcripts representing biomarkers of cardiomyopathy in mammals (cardiac troponin T2, cathepsin L, and atrial natriuretic peptide) were differentially altered among the three tomcod populations by PCBs. Although the present study did not identify any novel genes associated with PCB resistance in tomcod, several potential molecular biomarkers of PCB exposure were revealed.

Larval development of Argentine hake Merluccius hubbsi.

The ontogeny of the developmental stages of the hake Merluccius hubbsi is described. Fish larvae and post-transitional juveniles were collected in the Nor-Patagonian area from 1989 to 2004. The opening of the mouth and the pigmentation of the eyes are coincident with yolk resorption, finishing the yolk-sac stage. This species presents pigmentation on the head, trunk and tail typical of gadiform larvae. Pectoral fin development is completed during the transformation stage. The post-transitional juvenile stage begins when the fin-ray complements are complete and squamation begins. The fins become fully formed in the following sequence: pelvic fins, first dorsal fin, second dorsal and anal fins together, caudal fin and pectoral fins. The caudal complex is totally developed in larvae of 22.0-23.0 mm standard lengths (L(S)) and all vertebral elements are first observed in larvae of 8.5 mm L(S). The rate of development of M. hubbsi observed in this study could be faster than the rates reported for other species of Merluccius by different authors.

A cell line (HEW) from embryos of haddock (Melanogrammus aeglefinius) and its capacity to tolerate environmental extremes.

Cell lines can be useful experimental tools for studying marine fish, which are often difficult to routinely obtain and maintain in the laboratory. As few cell lines are available from coldwater marine fish, cultures were initiated from late gastrula embryos of haddock (Melanogrammus aeglefinus) in Leibovitz's L-15 with fetal bovine serum (FBS). From one culture, a cell line (HEW) emerged that has been grown for close to 100 population doublings, was heteroploid, and expressed telomerase activity, all of which suggest HEW is immortal. Growth occurred only if FBS was present and was optimal at 12 to 18 degrees C. Usually most cells had an epithelial-like morphology, but under some conditions, cells drew up into round central bodies from which radiated cytoplasmic extensions with multiple branches. These neural-like cells appeared within a few hours of cultures being placed at 28 degrees C or being switch to a simple salt solution (SSS). At 28 degrees C, cells died within 24 h. In SSS, HEW cells survived as a monolayer for at least 7 days. The sensitivity of HEW cells to morphological change and their capacity to withstand starvation should make them useful for investigating cellular responses to environmental stresses.