Expanding non-invasive approaches for fish-health monitoring: A survey of the epidermal mucous metabolomes of phylogenetically diverse freshwater fish species.

There is a pressing need for more-holistic approaches to fisheries assessments along with growing demand to reduce the health impacts of sample collections. Metabolomic tools enable the use of sample matrices that can be collected with minimal impact on the organism (e.g., blood, urine, and mucus) and provide high-throughput, untargeted biochemical information without the requirement of a sequenced genome. These qualities make metabolomics ideal for monitoring a wide range of fish species, particularly those under protected status. In the current study, we surveyed the relative abundances of 120 endogenous metabolites in epidermal mucus across eight freshwater fish species belonging to seven phylogenetic orders. Principal component analysis was used to provide an overview of the data set, revealing strong interspecies relationships in the epidermal mucous metabolome. Normalized relative abundances of individual endogenous metabolites were then used to identify commonalities across multiple species, as well as those metabolites that showed notable species specificity. For example, taurine was measured in high relative abundance in the epidermal mucus of common carp (Cyprinus carpio), northern pike (Esox lucius), golden shiner (Notemigonus crysoleucas), rainbow trout (Oncorhynchus mykiss), and rainbow smelt (Osmerus mordax), whereas γ-amino butyric acid (GABA) exhibited a uniquely high relative abundance in flathead catfish (Pylodictis olivaris). Finally, hierarchical cluster analysis was used to evaluate species relatedness as characterized by both the epidermal mucous metabolome (phenotype) and genetic phylogeny (genotype). This comparison revealed species for which relatedness in the epidermal mucous metabolome composition closely aligns with phylogenetic relatedness (e.g., N. crysoleucas and C. carpio), as well as species for which these two measures are not well aligned (e.g., P. olivaris and Polyodon spathula). These, and other findings reported here, highlight novel areas for future research with fish, including development of epidermal mucous-based markers for non-invasive health monitoring, sex determination, and hypoxia tolerance.

Safety assessment of subchronic feeding of insect-resistant and herbicide-resistant transgenic soybeans to juvenile channel catfish (Ictalurus punctatus).

Transgenic soybean is one of the most planted crops for human food and animal feed. The channel catfish (Ictalurus punctatus) is an important aquatic organism cultured worldwide. In this study, the effect of six different soybean diets containing: two transgenic soybeans expressing different types of cp4-epsps, Vip3Aa and pat genes (DBN9004 and DBN8002), their non-transgenic parent JACK, and three conventional soybean varieties (Dongsheng3, Dongsheng7, and Dongsheng9) was investigated in juvenile channel catfish for eight weeks, and a safety assessment was performed. During the experiment, no difference in survival rate was observed in six groups. The hepatosomatic index (HSI) and condition factor (CF) showed no significant difference. Moreover, comparable feed conversion (FC), feeding rate (FR), and feed conversion ratio (FCR) were found between transgenic soybean and JACK groups. Assessment of growth performance showed that the weight gain rate (WGR) and specific growth rate (SGR) of channel catfish were consistent. In addition, there were no changes in enzyme activity indexes (lactate dehydrogenase (LDH), total antioxidant capacity (T-AOC), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) in channel catfish among treatments. The research provided an experimental basis for the aquaculture feed industry to employ transgenic soybean DBN9004 and DBN8002 for commercial purposes.

Assessment of the Live Attenuated and Wild-Type Edwardsiella ictaluri-Induced Immune Gene Expression and Langerhans-Like Cell Profiles in the Immune-Related Organs of Catfish.

Edwardsiella ictaluri is a Gram-negative intracellular pathogen that causes enteric septicemia of catfish (ESC). Successful vaccination against intracellular pathogens requires T cell priming by antigen presenting cells (APCs) that bridge innate and adaptive immunity. However, the evidence on immunological mechanisms that underscore E. ictaluri pathogenesis and the protective role of live attenuated vaccines (LAVs) is scarce. We assessed the expression of immune genes related to antigen presentation by real-time PCR and the distribution patterns of Langerhans-like (L/CD207+) cells by immunohistochemistry in the immune-related tissues of channel catfish challenged with two novel E. ictaluri LAVs, EiΔevpB, and ESC-NDKL1 and wild type (WT) strain. Our results indicated significantly elevated expression of IFN-γ gene in the anterior kidney (AK) and spleen of vaccinated catfish at the early stages of exposure, which correlated with increased numbers of L/CD207+ cells. In general, the ESC-NDKL1-induced IFN-γ gene expression patterns in the AK resembled that of the patterns induced by EiΔevpB. However the MHCII gene expression patterns differed between the strains with significant increases at 6 h post-challenge (pc) with the EiΔevpB and at 7 d pc with the ESC-NDKL1 strains, respectively. Significant increases in activity of T helper type polarization genes such as IFN-γ and T cell co-receptors after exposure to ESC-NDKL1, in combination with elevated numbers of L/CD207+ cells at 7 d pc with both LAVs compared to uninfected and the WT-exposed counterparts, were documented in the spleen. The dominant pro-inflammatory environment with dramatically overexpressed inflammatory genes in the AK and 7 d pc in the spleen in response to E. ictaluri was found in exposed catfish. In general, the pro-inflammatory gene expression profiles in the ESC-NDKL1 pc showed more similarities to the WT strain-induced gene profiles compared to the EiΔevpB counterpart. In addition, E. ictaluri WT significantly decreased the numbers of Langerhans-like L/CD207+ cells in the AK and spleen at 3 and 7 days pc. In conclusion, we report the differential framework of initiation of innate and adaptive immune responses between E. ictaluri strains with both LAVs having a potential of satisfying the stringent requirements for successful vaccines.

Mercury concentrations in fish from three major lakes in north Mississippi: Spatial and temporal differences and human health risk assessment.

The goal of this study was to compare total mercury (Hg) concentrations in fish muscle tissue and assess consumption health risks of fish collected from three north Mississippi lakes (Sardis, Enid, and Grenada) that are extensively used for fishing and recreation. Largemouth bass (LMB; n = 64), channel catfish (CC; n = 72), and white crappie (WC; n = 100), which represent a range of trophic levels, were collected during spring 2013 and 2014. Creel data estimated that anglers harvested approximately 370,000 kg of WC, 27,000 kg of CC, and 15,000 kg of LMB from the lakes annually. Median Hg wet weight concentrations were highest in LMB (443 ng/g), followed by CC (211 ng/g) and WC (192 ng/g). Fish-Hg concentrations were lower than those reported in fish >10 years ago. There were significant differences between lakes consistent across species. Grenada length-normalized fish-Hg concentrations were higher than those from Enid and Sardis. Because existing consumption advisories for CC are length based, the lack of relationship between length and Hg concentration indicated that the recommendations may not be sufficiently protective. Further, five different risk assessment paradigms yielded hazard quotient (HQ) values suggesting that existing fish consumption advisories may be insufficient to protect adults and especially children from exposure to Hg.

Toxic airborne S, PAH, and trace element legacy of the superhigh-organic-sulphur Rasa coal combustion: Cytotoxicity and genotoxicity assessment of soil and ash.

This paper presents the levels of sulphur, polycyclic aromatic hydrocarbons (PAHs), and potentially toxic trace elements in soils surrounding the Plomin coal-fired power plant (Croatia). It used domestic superhigh-organic-sulphur Rasa coal from 1970 until 2000. Rasa coal was characterised by exceptionally high values of S, up to 14%, making the downwind southwest (SW) area surrounding the power plant a significant hotspot. The analytical results show that the SW soil locations are severely polluted with S (up to 4%), and PAHs (up to 13,535ng/g), while moderately with Se (up to 6.8mg/kg), and Cd (up to 4.7mg/kg). The composition and distribution pattern of PAHs in the polluted soils indicate that their main source could be airborne unburnt coal particles. The atmospheric dispersion processes of SO2 and ash particles have influenced the composition and distribution patterns of sulphur and potentially toxic trace elements in studied soils, respectively. A possible adverse impact of analysed soil on the local karstic environment was evaluated by cytotoxic and genotoxic methods. The cytotoxicity effects of soil and ash water extracts on the channel catfish ovary (CCO) cell line were found to be statistically significant in the case of the most polluted soil and ash samples. However, the primary DNA-damaging potential of the most polluted soil samples on the CCO cells was found to be within acceptable boundaries.

Comparison of extracellular DNase- and protease-producing spoilage bacteria isolated from Delaware pond-sourced and retail channel catfish (Ictalurus punctatus).

BACKGROUND: Spoilage of fishery products begins immediately following filleting due to microbial growth that degrades fish tissue quality prior to consumption. Extensive research has been conducted to identify such bacterial populations. A better understanding of the mechanisms involved in fish spoilage is necessary as a novel remedy for microbial spoilage inhibition has yet to be established for fish tissue. The present study identified, for the first time, bacterial populations that produce extracellular DNase and protease from Delaware and local retail distributed channel catfish (Ictalurus punctatus) fillets. RESULTS: A clear trend was identified between bacteria derived from catfish filleted under aseptic conditions where Pseudomonas was the dominant genus. Bacteria isolated from retail catfish contained high quantities of DNase-producing isolates, in contrast to aseptic-filleted catfish tissue which had none. Both types of catfish sample maintained high populations of protease-producing bacterial colonies throughout the duration of the study. Most bacteria isolated from catfish intestines exhibited DNase production with no protease production. CONCLUSION: Specific spoilage organism populations were significantly higher on retail-derived catfish in comparison to lab-filleted Delaware cultured catfish tissue. It is suggested that DNase production and protease production contribute to the spoilage of fish tissue as a result of mishandling and septic filleting being the major cause of rapid catfish tissue spoilage.

In vitro cytotoxicity assessment of imidazolium ionic liquids: biological effects in fish Channel Catfish Ovary (CCO) cell line.

Increasing interest in the application of ionic liquids as green replacement for volatile organic solvents emphasized the need for the evaluation of their toxic effects at different biological systems in order to reduce the risk for human health and environment. To our knowledge, effects of imidazolium ionic liquids on cellular level of fish cell lines have not been studied yet. The cytotoxicity of imidazolium ionic liquids containing different anions and alkyl chain lengths as the substituent at the cation ring towards the fish CCO cell line was determined by WST-1 proliferation assay. Morphological alterations were examined by fluorescent microscopy using acridine orange/ethidium bromide staining and flow cytometry analysis was also performed. The results showed concentration-dependent cytotoxicity of ionic liquids in CCO cells, related to the type of anion and alkyl chain length, while EC50 values showed moderate to high cytotoxicity of tested imidazolium ionic liquids. Distinct morphological changes observed under fluorescence microscope and data obtained by flow cytometry suggest that the toxicity of imidazolium ionic liquids with longer alkyl chains could be related to necrosis. Results presented in here may be helpful for filling existing gaps of knowledge about ionic liquids toxicity and their impact on aquatic environment.

Assessment of the health status of wild fish from the Wheatley Harbour Area of Concern, Ontario, Canada.

The overall health and endocrine function of wild brown bullhead (Ameiurus nebulosus) and goldfish (Carassius auratus) from the Wheatley Harbour Area of Concern (Lake Erie, Ontario, Canada) was assessed using a suite of physiological and biochemical endpoints. Smaller gonads were detected in female brown bullhead and goldfish from Wheatley Harbour compared with Hillman Marsh (Ontario, Canada) reference fish. Female brown bullhead exhibited decreased in vitro synthesis of 17ß-estradiol. Female goldfish had decreased plasma vitellogenin concentrations. Plasma testosterone and 11-ketotestosterone were significantly depressed in males of both species. Perturbations in the thyroid status were detected, but varied between sexes and species. Observed differences included lower plasma concentrations of thyroid hormones and/or elevated liver deiodinase activity. Histological evaluation of the thyroid tissue indicated that in the case of female goldfish, those perturbations stimulated the thyroid (as indicated by increased thyroid epithelial cell height) and partially depleted the thyroxine reserves, as indicated by decreased colloid and elevated thyroid activation index. Increased mixed-function oxygenase activity in brown bullhead from Wheatley Harbour was consistent with exposure to planar aromatic contaminants. A principal component analysis of selected variables showed the separation of fish by collection site. The endpoints most strongly associated with the separation were generally those exhibiting significant differences between sites. The results of the present study indicate that the health of fish populations within Wheatley Harbour warrants continued attention.

Experimental assessment of the effects of a Neotropical nocturnal piscivore on juvenile native and invasive fishes

We experimentally examined the predator-prey relationships between juvenile spotted sorubim Pseudoplastystoma corruscans and young-of-the-year invasive and native fish species of the Paraná River basin, Brazil. Three invasive (peacock bass Cichla piquiti, Nile tilapia Oreochromis niloticus, and channel catfish Ictalurus punctatus) and two native (yellowtail tetra Astyanax altiparanae and streaked prochilod Prochilodus lineatus) fish species were offered as prey to P. corruscans in 300 L aquaria with three habitat complexity treatments (0%, 50% and 100% structure-covered). Prey survival was variable through time and among species (C. piquiti < O. niloticus < A. altiparanae < P. lineatus < I. punctatus), depending largely on species-specific prey behavior but also on prey size and morphological defenses. Habitat complexity did not directly affect P. corruscans piscivory but some prey species changed their microhabitat use and shoaling behavior among habitat treatments in predator's presence. Pseudoplatystoma corruscans preyed preferentially on smaller individuals of those invasive species with weak morphological defensive features that persisted in a non-shoaling behavior. Overall, our results contrast with those in a companion experiment using a diurnal predator, suggesting that nocturnal piscivores preferentially prey on different (rather diurnal) fish species and are less affected by habitat complexity. Our findings suggest that recovering the native populations of P. corruscans might help controling some fish species introduced to the Paraná River basin, particularly C. piquiti and O. niloticus, whose parental care is expected to be weak or null at night.

A relação predador-presa entre juvenis de pintado Pseudoplatystoma corruscans (piscívoro nativo) e jovens do ano de espécies nativas e invasoras de peixes da bacia do rio Paraná, Brasil, foi testada experimentalmente. Três espécies de peixe invasoras (o tucunaré Cichla piquiti, a tilápia do Nilo Oreochromis niloticus e o bagre do canal Ictalurus punctatus) e duas nativas (o lambari do rabo amarelo Astyanax altiparanae e o curimbatá Prochilodus lineatus) foram oferecidas como presa para P. corruscans em microcosmos, com três tratamentos de complexidade de habitat (0%, 50% e 100% de cobertura por estruturas submersas). A sobrevivência de presas variou ao longo do tempo e entre espécies (C. piquiti < O. niloticus < A. altiparanae < P. lineatus < I. punctatus), sendo governada, em grande parte, por diferenças espécies-específicas no comportamento das presas, mas também pelo tamanho e defesas morfológicas das mesmas. A complexidade de habitat não afetou diretamente a piscivoria de P. corruscans, mas, na presença do predador, algumas espécies-presa alteraram seu comportamento quanto ao uso de micro-habitat e grau de agregação entre os níveis de complexidade de habitat. Pseudoplatystoma corruscans predou preferencialmente sobre os indivíduos menores das espécies invasoras que apresentaram estruturas morfológicas defensivas pouco desenvolvidas e que não formavam cardume. Em geral, os resultados obtidos contrastam com os de um experimento análogo, no qual foi usado um piscívoro diurno, sugerindo que piscívoros noturnos predam preferencialmente sobre espécies de peixes diferentes (de hábitos mais diurnos) e que são menos afetados pela complexidade estrutural de habitats. Os resultados obtidos sugerem que a recuperação de populações nativas de P. corruscans poderia contribuir para o controle de algumas espécies de peixes introduzidas no rio Paraná, especialmente C. piquiti e O. niloticus, dos quais se espera que o cuidado parental seja reduzido ou nulo à noite.

Channel catfish (Ictalurus punctatus) muscle protein isolate performance processed under different acid and alkali pH values.

Channel catfish (Ictalurus punctatus) muscle was subjected to 6 protein extraction and precipitation techniques using acid solubilization (pH 2.0, 2.5, and 3.0) or alkaline solubilization (pH 10.5, 11.0, 11.5) followed by precipitation at pH 5.5. The catfish protein isolate was compared with ground defatted white muscle. Alkali-processed catfish showed increased gel rigidity, gel strength, and gel flexibility compared to acid-processed catfish, which exhibited inconsistent functional performance, increasing and decreasing gel rigidity, gel strength, and gel flexibility. The gel rigidity (G') at pH 3.0 in the absence of salt had the highest G' of the acid treatments and was not significantly different from the alkaline-treated catfish muscle (P>0.05). However in the presence of added salt pH treatment it had the lowest G' and was different from alkaline treatments (P<0.05) during break force testing. These results show that pH-shift processing of channel catfish muscle provides highly functional isolates with a potentially broad range of applications. This range of applications is possible due to the modification of the textural properties of catfish muscle protein produced using different acidic or alkaline pH solubility treatments.

Leukocyte and thrombocyte reference values for channel catfish (Ictalurus punctatus Raf), with an assessment of morphologic, cytochemical, and ultrastructural features.

BACKGROUND: Hematology tests are useful to evaluate physiologic disturbances in fish and can provide important information for the diagnosis and prognosis of disease. OBJECTIVES: The primary purpose of this study was to define reference intervals for thrombocytes and leukocytes in healthy channel catfish (Ictalurus punctatus). In addition, the morphologic, cytochemical, and ultrastructural features of blood cells were assessed. METHODS: Blood samples (0.5 mL) were collected into EDTA from 40 clinically healthy catfish on a commercial fish farm in Jaboticabal, Brazil. Thrombocyte, total WBC, and differential WBC counts were determined and reference intervals were calculated as the 25-95th percentiles of data. Thrombocyte and leukocyte morphology was assessed in blood smears stained with May Grünwald-Giemsa-Wright and ultrastructurally by transmission electron microscopy. Cytochemical staining patterns were described using periodic acid-Schiff (PAS), peroxidase, nonspecific esterase, alkaline phosphatase, and toluidine blue. RESULTS: Reference intervals were as follows: thrombocytes 58,802-99,569/microL; total WBCs 27,460-41,523/microL; lymphocytes 5380-11,581/microL; monocytes 2949-7459/microL; neutrophils 12,529-22,748/microL, and basophils 736-2003/microL. Neutrophils were positive for peroxidase and PAS; monocytes were positive for nonspecific esterase; and basophils were positive with toluidine blue. CONCLUSION: The morphologic and staining features of neutrophils and monocytes of channel catfish are similar to those of mammals, and the presence of basophils in this species was verified. These reference intervals and morphologic findings provide a foundation for future investigations on the functions and alterations of blood cells in channel catfish.

Repeat structure of the catfish genome: a genomic and transcriptomic assessment of Tc1-like transposon elements in channel catfish (Ictalurus punctatus).

We have assessed the distribution and diversity of members of the Tc1/mariner superfamily of transposable elements in the channel catfish (Ictalurus punctatus) genome as well as evaluating the extent of transcription of Tc1 transposases in the species. Through use of PCR amplification and sequencing, assessment of random BAC end sequences (BES) equivalent to 1.2% genome coverage, and screening of over 45,000 catfish ESTs, a significant proportion of Tc1-like elements and their associated transcripts were captured. Up to 4.2% of the catfish genome in base pairs appears to be composed of Tc1-like transposon-related sequences and a significant fraction of the catfish cellular mRNA, approximately 0.6%, was transcribed from transposon-related sequences in both sense and antisense orientations. Based on results of repeat-masking, as much as 10% of BAC end sequences from catfish, which is a random survey of the genome, contain some remnant of Tc1 elements, suggesting that these elements are present in the catfish genome as numerous, small remnants of the transposons. Phylogenetic analysis allowed comparison of catfish Tc1 transposase types with those found in other vertebrate and invertebrate species. In spite of the existence of many types of Tc1-like sequences that are not yet able to be placed in clades with strong statistical support, it is clear that multiple families of Tc1-like elements exist in channel catfish.

Channel catfish BAC-end sequences for marker development and assessment of syntenic conservation with other fish species.

In the present study, 25 195 BAC ends for channel catfish (Ictalurus punctatus) were sequenced, generating 20 366 clean BAC-end sequences (BES), with an average read length of 557 bp after trimming. A total of 11 414 601 bp were generated, representing approximately 1.2% of the catfish genome. Based on this survey, the catfish genome was found to be highly AT-rich, with 60.7% A+T and 39.3% G+C. Approximately 12% of the catfish genome consisted of dispersed repetitive elements, with the Tc1/mariner transposons making up the largest percentage by base pair (4.57%). Microsatellites were detected in 17.5% of BES. Catfish BACs were anchored to the zebrafish and Tetraodon genome sequences by BLASTN, generating 16% and 8.2% significant hits (E < e(-5)) respectively. A total of 1074 and 773 significant hits were unique to the zebrafish and Tetraodon genomes, respectively, of which 417 and 406, respectively, were identified as known genes in other species, providing a major genome resource for comparative genomic mapping.

A screening-level assessment of lead, cadmium, and zinc in fish and crayfish from Northeastern Oklahoma, USA.

The objective of this study was to evaluate potential human and ecological risks associated with metals in fish and crayfish from mining in the Tri-States Mining District (TSMD). Crayfish (Orconectes spp.) and fish of six frequently consumed species (common carp, Cyprinus carpio; channel catfish, Ictalurus punctatus; flathead catfish, Pylodictis olivaris; largemouth bass, Micropterus salmoides; spotted bass, M. punctulatus; and white crappie, Pomoxis annularis) were collected in 2001--2002 from the Oklahoma waters of the Spring River (SR) and Neosho River (NR), which drain the TSMD. Samples from a mining-contaminated site in eastern Missouri and from reference sites were also analyzed. Individual fish were prepared for human consumption in the manner used locally by Native Americans (headed, eviscerated, and scaled) and analyzed for lead, cadmium, and zinc. Whole crayfish were analyzed as composite samples of 5--60 animals. Metals concentrations were typically higher in samples from sites most heavily affected by mining and lowest in reference samples. Within the TSMD, most metals concentrations were higher at sites on the SR than on the NR and were typically highest in common carp and crayfish than in other taxa. Higher concentrations and greater risk were associated with fish and crayfish from heavily contaminated SR tributaries than the SR or NR mainstems. Based on the results of this and previous studies, the human consumption of carp and crayfish could be restricted based on current criteria for lead, cadmium, and zinc, and the consumption of channel catfish could be restricted due to lead. Metals concentrations were uniformly low in Micropterus spp. and crappie and would not warrant restriction, however. Some risk to carnivorous avian wildlife from lead and zinc in TSMD fish and invertebrates was also indicated, as was risk to the fish themselves. Overall, the wildlife assessment is consistent with previously reported biological effects attributed to metals from the TSMD. The results demonstrate the potential for adverse effects in fish, wildlife, and humans and indicate that further investigation of human health and ecological risks, to include additional exposure pathways and endpoints, is warranted.

Hazard assessment of a simulated oil spill on intertidal areas of the St. Lawrence River with SPMD-TOX.

Phytoremediation in a simulated crude oil spill was studied with a "minimalistic" approach. The SPMD-TOX paradigm-a miniature passive sorptive device to collect and concentrate chemicals and microscale tests to detect toxicity-was used to monitor over time the bioavailability and potential toxicity of an oil spill. A simulated crude oil spill was initiated on an intertidal freshwater grass-wetland along the St. Lawrence River southwest of Quebec City, Quebec, Canada. Several phytoremediation treatments were investigated; to dissipate and ameliorate the spill, treatments included nutrient amendments with inorganic nitrogen sources (ammonium nitrate and sodium nitrate) and phosphate (super triple phosphate) with and without cut plants, with natural attenuation (no phytoremedial treatment) as a control. Sequestered oil residues were bioavailable in all oil-treated plots in Weeks 1 and 2. Interestingly, the samples were colored and fluoresced under ultraviolet light. In addition, microscale tests showed that sequestered residues were acutely toxic and genotoxic, as well as that they induced hepatic P(450) enzymes. Analysis of these data suggested that polycyclic aromatic hydrocarbons were among the bioavailable residues sequestered. In addition, these findings suggested that the toxic bioavailable fractions of the oil spill and degradation products dissipated rapidly over time because after the second week the water column contained no oil or detectable degradation products in this riverine intertidal wetland. SPMD-TOX revealed no evidence of bioavailable oil products in Weeks 4, 6, 8, and 12. All phytoremediation efforts appeared to be ineffective in changing either the dissipation rate or the ability to ameliorate the oil toxicity. SPMD-TOX analysis of the water columns from these riverine experimental plots profiled the occurrence, dissipation, and influence of phytoremediation on the bioavailability and toxicity of oil products (parent or degradation products).

Development of an enzyme-linked immunosorbent assay for the measurement of plasma growth hormone (GH) levels in channel catfish (Ictalurus punctatus): assessment of environmental salinity and GH secretogogues on plasma GH levels.

We report the development of a sensitive, and specific, competitive, antigen-capture enzyme-linked immunosorbent assay for the measurement of channel catfish (Ictalurus punctatus) growth hormone (cfGH). The detection limit of the assay (90% binding) was 2.0ng/ml and the ED(50) value (standard curve range 150-0.59 ng/ml) was 67.3 ng/ml. Recovery of cfGH-spiked plasma samples was determined to be 102%. Dose-response inhibition curves using serially diluted pituitary homogenates and plasma samples consistently showed parallelism with the standard curves using purified cfGH. The GH antibody (rabbit anti-catfish GH) specificity was demonstrated in competitive binding curves employing heterologous hormones and purified channel catfish prolactin (cfPRL). These studies show that there was no significant (0.006%) binding of cfPRL (competitive inhibition of cfGH binding), or heterologous hormones, within the working range of the assay. To physiologically validate the assay, catfish were injected (100 microg/g body weight, 3 injections every 5 days) with either bovine GHRH(1-29)-amide or the synthetic hexapeptide GHRP-2 (KP-102: D-Ala-D-beta-Nal-Ala-Trp-D-Phe-Lys-NH(2)) suspended in corn oil. Following the last injection, half of the animals were sampled for plasma and the remaining transferred from fresh water (FW) to 12 ppt seawater (BW: brackish water). Twenty-four hours after transfer to BW, animals were again sampled for plasma. Plasma GH levels were significantly (p<0.001) elevated in all the BW groups (control, KP-102, and bGHRH), compared with the FW (fresh water) groups. In addition, plasma GH levels were significantly (p<0.001) elevated by treatment with either of the GH secretogogues, KP-102 or bGHRH. Our findings demonstrate that two regulatory mechanisms of GH elevation, one which is seen in euryhaline teleosts (salinity-induced GH levels) and another, which has been recently described in teleosts (GHRP-induced GH levels), are present in the stenohaline channel catfish.

Toxicity of boron to rainbow trout: a weight-of-the-evidence assessment.

From the large data set available on the toxicity of boron to aquatic organisms, the toxicity of boron to the early life stages of rainbow trout (Oncorhyncus mykiss) is the seminal issue relative to setting water quality criteria and effluent standards. Issues associated with the early life stage studies are the flat concentration-response curve, the low threshold of toxicity, and teratogenic effects observed. Recent laboratory and field studies offer new experimental data that make a weight-of-the-evidence assessment timely. In a re-examination of the effect of boron on the embryo-larval stage in rainbow trout and zebrafish, adverse effects due to boron deficiency are observed which decrease with increasing dose. It was found that low concentrations of boron stimulate embryonic growth in rainbow trout and increase the viability and survival of embryonic zebrafish. As boron concentration is further increased, the dose-response curve becomes flat as homeostatic processes are active; this is followed at higher doses by a new adverse response that increases with increasing dose. As a result, the dose-response relationship is U shaped, consistent with the characteristic shape of an essential micronutrient. Thus, effects originally reported to be toxicity at low exposures rather may be due to boron deficiency. Water analyses in trout hatcheries and field studies in wild trout streams add additional information on the toxicity of boron to trout. Of particular note is a controlled field study carried out in the Firehole River in Yellowstone Park (WY, USA), where trout populations survive and reproduction successfully occurs in natural water containing boron concentrations up to and in some cases greater than 1.0 mg B/L. Teratogenic effects due to boron exposure were not observed in any of these more recent studies.

Biomarker assessment of the effects of coal strip-mine contamination on channel catfish.

A suite of biomarkers was used to evaluate acute (1-day) to semichronic (3-month) heavy metal-induced toxicity in channel catfish, lctalurus punctatus, caged at an abandoned strip mine and a noncontaminated reference site. Assays performed include indicators of metabolic, hematological, osmoregulatory, and genotoxic stress. Two cage designs were used to evaluate the importance of exposure routes: one excluding contact with the sediments and the other allowing contact with water and sediments. Significant DNA strand breakage was observed in catfish exposed to both exposure regimens, but evidence of DNA repair was observed only in water-exposed catfish. Transient increases in hemoglobin, delta-aminolevulinic acid dehydratase, and hematocrit levels were observed at 1 month's exposure for both exposure regimens, followed by a return to control levels for the duration of the study. Environmental conditions (i.e., weather-related changes in water quality) may have contributed to the variable plasma chloride and glucose levels observed in all catfish exposed to strip-mine wastes. The transient changes in biomarkers followed by a return to reference values represent an initial stress and an acclimation to normal levels.

Immunochemical characterization of hepatic cytochrome P450 isozymes in the channel catfish: assessment of sexual, developmental and treatment-related effects.

The profiles of immunoreactive proteins recognized by antibodies raised against purified trout P-450 isoforms (CYP1A1, CYP2M1 and CYP2K1) were examined in channel catfish liver by Western blot analysis. Gender differences in basal expression of these isoforms, as well as responses to known inducers of mammalian isoforms (ethanol, beta-naphthoflavone and clofibric acid) and early life stage (3 and 6 months) profiles are described. Two similar protein bands were detected by Western blotting in mature untreated catfish with CYP2K1 and CYP2M1 antibodies. A third band is detected by anti-2K1 in fish treated with beta-naphthoflavone; this band was verified as CYP1A, with about twice the level of expression in males versus females. No difference between sexes was seen in the expression of the 51-kDa CYP2-reactive bands; however, a significant difference (female > male) was seen in the lower molecular weight CYP2 band (47-kDa). Ethanol treatment caused a dose-dependent decrease in the 47-kDa CYP2-reactive isoforms but no change in the 51-kDa band. Clofibric acid treatment caused an increase in both the 51-kDa CYP2 protein as well as in liver somatic index. Age-dependent changes in isoform expression were also detected in CYP2-reactive forms, with a novel protein (53-kDa) detected in 3-month-old fish. The results from this study provide insight into the regulation of constitutive catfish CYP isoforms and prepares a foundation for further examination of the biotransformation capabilities of an important aquatic species.

Biomarker assessment of the effects of petroleum refinery contamination on channel catfish.

A suite of biomarkers was used to evaluate acute (1- and 7-day) heavy metal-induced toxicity in channel catfish, Ictalurus punctatus, caged at an abandoned petroleum refinery and a non-contaminated reference site. Assays performed include indicators of metabolic, hematological, osmoregulatory, and genotoxic stress. Two cage designs were utilized to evaluate the importance of exposure routes: one allowing exclusive contact with the water column and the other allowing contact with water and sediments. Data collected at 1 and 7 days postexposure indicated that the experimental fish were stressed, having significant increases in blood glucose. After 7 days of exposure, two hemodynamic parameters, hemoglobin and delta-aminolevulinic acid dehydratase, were significantly increased. There were no significant differences observed in other parameters measured.