Irradiation of rainbow trout at early life stages results in a proteomic legacy in adult gills. Part B; the effect of a second radiation dose, after one year, on the proteomic responses in the irradiated and non-irradiated bystander fish.

This study extends the investigation of the legacy effects of exposure to a single radiation dose at one of four early life stages, in adult rainbow trout (Part A), by examining the effects of a second identical dose after one year; i.e. egg 48 h after fertilisation (48 h egg) + 1 year, eyed egg + 1 year, yolk sac larvae (YSL) + 1 year and first feeder + 1 year. This included the induction of a bystander effect in non-irradiated trout which had swam with the irradiated fish. The second radiation dose negated any beneficial proteomic responses following early life stage irradiation only, particularly irradiation of 48 h eggs and eyed eggs (Part A). Instead the responses after early life stage + 1 year irradiation are consistently associated with tumorigenesis, cancer progression, or are otherwise damaging: upregulation of alpha-globin 1 (YSL + 1 year and first feeders + 1 year) and downregulation of histone H1, type II keratin, malate dehydrogenase 2-2, Na/K ATPase alpha subunit isoform 1b, nucleoside diphosphate kinase (48 h egg + 1 year), electron transfer flavoprotein subunit alpha (eyed egg + 1 year), 60 S ribosomal protein L30 (YSL + 1 year) and haemoglobin subunit beta-4 (first feeder + 1 year). Most significantly the second radiation dose also negated the overwhelmingly beneficial bystander effect proteomic responses induced by trout irradiated at an early life stage only (Part A). Instead the bystander effect proteomic changes induced by trout irradiated at an early life stage and again at 1 year have been associated with uncertain, with respect to tumorigenesis, or detrimental effects; upregulation of alpha-globin 1 (YSL + 1 year and first feeder + 1 year) and downregulation of malate dehydrogenase 2-2, nucleoside diphosphate kinase (48 h egg + 1 year), transferrin precursor (eyed egg + 1 year), 60 S ribosomal protein L30 (YSL + 1 year) and serine / threonine-protein phosphatase 2 A 65 kDa (first feeder + 1 year). This difference between the bystander effect induced proteomic changes following early life stage irradiation only and early life stage + 1 year irradiation may indicate a fundamental change in the non-targeted effects of radiation following multiple exposure to radiation.

Irradiation of rainbow trout at early life stages results in a proteomic legacy in adult gills. Part A; proteomic responses in the irradiated fish and in non-irradiated bystander fish.

Exposure to a single 0.5 Gy X-ray dose of eggs at 48 h after fertilisation (48 h egg), eyed eggs, yolk sac larvae (YSL) and first feeders induces a legacy effect in adult rainbow trout. This includes the transmission of a bystander effect to non-irradiated adult trout which had swam with the irradiated fish. The aim of this study was to investigate this legacy by analysing the gill proteome of these irradiated and bystander fish. Irradiation at all of the early life stages resulted in changes to proteins which play a key role in development but are also known to be anti-tumorigenic and anti-oxidant: upregulation of haemoglobin subunit beta (48 h egg), haemoglobin, serum albumin 1 precursor (eyed eggs), clathrin heavy chain 1 isoform X10 (eyed eggs and first feeders), and actin-related protein 2/3 complex subunit 4 (first feeders), downregulation of pyruvate dehydrogenase, histone 1 (48 h egg), triosephosphate isomerase (TPI), collagen alpha-1(1) chain like proteins (YSL), pyruvate kinase PKM-like protein (YSL and first feeders), ubiquitin-40S ribosomal proteins S27 and eukaryotic translation initiation factor 4 A isoform 1B (first feeders). However irradiation of YSL and first feeders (post hatching early life stages) also induced proteomic changes which have a complex relationship with tumorigenesis or cancer progression; downregulation of alpha-1-antiprotease-like protein precursor, vigilin isoform X2 and nucleoside diphosphate kinase (YSL) and upregulation of hyperosmotic glycine rich protein (first feeders). In bystander fish some proteomic changes were similar to those induced by irradiation: upregulation of haemoglobin subunit beta (48 h egg), haemoglobin (eyed eggs), actin-related protein 2/3 complex subunit 4, hyperosmotic glycine rich protein (first feeders), and downregulation of alpha-1-antiprotease-like protein, vigilin isoform X2, nucleoside diphosphate kinase (YSL), pyruvate kinase PKM-like protein and ubiquitin-40S ribosomal protein S27a-like (first feeders). Other proteomic changes were unique to bystander fish; downregulation of TPI, ubiquitin-40S ribosomal protein S2 (eyed egg), cofilin-2, cold-inducible RNA-binding protein B-like isoform X3 (YSL) and superoxide dismutase (first feeder), and upregulation of haemoglobin subunit alpha, collagen 1a1 precursor, apolipoprotein A-1-1 and A-1-2 precursor (first feeders). These bystander effect proteomic changes have been shown to be overwhelmingly anti-tumorigenic or protective of the fish gill.

Irradiation of rainbow trout at early life stages results in trans-generational effects including the induction of a bystander effect in non-irradiated fish.

The bystander effect, a non-targeted effect (NTE) of radiation, which describes the response by non-irradiated organisms to signals emitted by irradiated organisms, has been documented in a number of fish species. However transgenerational effects of radiation (including NTE) have yet to be studied in fish. Therefore rainbow trout, which were irradiated as eggs at 48h after fertilisation, eyed eggs, yolk sac larvae or first feeders, were bred to generate a F1 generation and these F1 fish were bred to generate a F2 generation. F1 and F2 fish were swam with non-irradiated bystander fish. Media from explants of F1 eyed eggs, F1 one year old fish gill and F1 two year old fish gill and spleen samples, and F2 two year old gill and spleen samples, as well as from bystander eggs/fish, was used to treat a reporter cell line, which was then assayed for changes in cellular survival/growth. The results were complex and dependent on irradiation history, age (in the case of the F1 generation), and were tissue specific. For example, irradiation of one parent often resulted in effects not seen with irradiation of both parents. This suggests that, unlike mammals, in certain circumstances maternal and paternal irradiation may be equally important. This study also showed that trout can induce a bystander effect 2 generations after irradiation, which further emphasises the importance of the bystander effect in aquatic radiobiology. Given the complex community structure in aquatic ecosystems, these results may have significant implications for environmental radiological protection.

Exposure to acute levels of waterborne aluminium modifies the legacy of early life stage irradiation, including the communication of radiation-induced bystander signals, in adult rainbow trout.

PURPOSE: To determine if the legacy of early life stage irradiation seen in rainbow trout is modified in adult fish by acute exposure to waterborne aluminium (Al). METHODS: Two-year-old trout which had been irradiated as either eggs 48 h after fertilization, eyed eggs, yolk sac larvae or as first feeders, were exposed to 100 or 200 µg l(-1) Al. These fish were then paired with untreated fish. Gill and skin samples from both the irradiated and Al-exposed, and the untreated bystander fish, were then assayed for the production of toxic or pro-death signals, using the HPV-G reporter cell line. RESULTS: Depending on the life stage, irradiated and the tissue assayed acute Al exposure modified both the radiation legacy, and the radiation-induced bystander effect in untreated fish, either by reducing reporter cell survival or increasing reporter cell growth. CONCLUSIONS: The effects of early irradiation and adult Al exposure were complex but clearly demonstrated that both the direct radiation effects and the communication of a radiation-induced bystander effect can be modified by a second environmental stressor.

Tissue-specific effects of acute aluminium exposure on the radiation-induced bystander effect in rainbow trout (Oncorhynchus mykiss, Walbaum).

PURPOSE: To investigate if aluminium (Al) modifies the rainbow trout response to radiation exposure and/or the induction of a radiation-induced bystander effect. METHODS: Rainbow trout were exposed to 100 or 200 µg l(-1) Al (for 3 h), a 0.5 Gy X-ray dose or Al followed immediately by irradiation. The exposed fish were then swum with completely untreated bystander fish. A human reporter cell clonogenic assay was used to determine whether Al exposure modified the effects of irradiation on the skin and gills from directly exposed fish and also the radiation-induced bystander effect in untreated fish. RESULTS: Al exposure did not modify the response to direct irradiation by the skin, or the gill. Al did not modify the bystander effect in the skin. However Al did modify the bystander effect in the gill. Gills of bystander fish swum with fish exposed to 200 µg l(-1) Al, followed by irradiation, caused a greater reduction in HPV-G cell survival than was caused by irradiation only. Interestingly Al exposure only also caused a bystander effect (reduced HPV-G survival) in the gill. CONCLUSION: This study shows that, in a multiple stressor scenario, the communication of radiation-induced stress signals is modified on a tissue-specific basis by acute Al exposure. Aside from the implications this has for radiological protection this response may also have potential for environmental monitoring where detection of the bystander effect could act as an indicator of radiation exposure when direct exposure responses are not evident.

The induction of a radiation-induced bystander effect in fish transcends taxonomic group and trophic level.

PURPOSE: To extend the investigations of bystander effect induction in fish of the same species as the irradiated fish, to bystander effect induction between fish species and between trophic levels. MATERIALS AND METHODS: To investigate interspecies bystander effect induction, zebrafish and medaka were irradiated with a 0.5 Gy X-ray dose and then swum with non-irradiated fish of the same and opposite species. To investigate trophic level bystander effect induction, California blackworms were irradiated with the same X-ray dose and then fed to non-irradiated rainbow trout. RESULTS: Reductions in clonogenic survival of the HPV-G (non-transformed human keratinocytes, immortalized with the human papilloma virus) reporter cell line, treated with tissue explant media, revealed that zebrafish and medaka induced a pro-apoptotic bystander effect in the other species and that, in trout, the normally anti-apoptotic effect caused by the consumption of non-irradiated blackworms was significantly reduced or lost if the blackworms had been irradiated. CONCLUSIONS: These results are the first to show that a radiation- induced bystander effect can transcend taxonomic group and trophic level in fish. This provides further evidence that bystander signals are widespread and conserved and may be transmitted through an ecosystem, as well as between individuals of the same species.

Investigating fear in rainbow trout (Oncorhynchus mykiss) using the conditioned-suppression paradigm.

Trout learned the operant task of pendulum-pressing for a food-reward in a mean of 4.3 sessions lasting 1 hr. In a separate phase, fish also learned--through classical conditioning--to associate a neutral light cue with an aversive stimulus. When again allowed to pendulum-press for food, after aversive classical conditioning, there was a drop in the rate of responding. The mean rate dropped from 3.6-2.9 responses per min. Most important, when the light-stimulus was superimposed on a steady bout of pendulum-pressing, trout ceased to press the pendulum and did not resume activity until termination of the light-stimulus (mean number of responses during a 3-min interval immediately prior to light-stimulus = 14.3 vs. during 3-min light-stimulus = 0.1). Psychologists have used this decrease in operant responding, or "conditioned emotional response," as a tool to examine the psychological nature of this type of aversive conditioning. In this study, the fish demonstrated various results under this paradigm similar to those shown by "higher" nonhuman animals, therefore challenging the view of fish as unconscious, nonsentient animals.

Estimating aquatic phosphorus concentrations 30 metres down-current from a rainbow trout cage array.

Aquatic total phosphorus (Tot-P) is measured 30 m distant from rainbow trout (Oncorhynchus mykiss) cages, for regulatory compliance on the Canadian side of Lake Huron. The ability to predict changes in near-field Tot-P concentrations from changes in production, or to explain near-field concentrations in the event of compromised reference sites, would be very useful for managers and farmers. A logical first step in the development of a near-field model is the estimation of down-current Tot-P concentrations. A nutritional mass balance approach was used to calculate the amount of non-settleable Tot-P which was divided by the corresponding flushing volume over four separate production periods. Despite a number of uncertainties, such as cage effects on flushing volume, the contribution of particulate phosphorus, fish distribution, feed conversion ratio, and ingestion-excretion time; accuracy (slope = 1.06) and precision (r(2) = 0.75) of modelled estimates fit well with empirical results. It was concluded that 30 m down-current Tot-P concentrations could be modelled under a diverse set of culture and environmental conditions, suggesting typical 'near-field' concentrations of Tot-P can be explained on the basis of farm activities.

Toxicity of Clostridium botulinum type E neurotoxin to Great Lakes fish: implications for avian botulism.

Since 1999, large-scale mortalities of fish-eating birds have been observed on the Great Lakes, and more specifically on Lake Erie. Type E botulism has been established as the primary cause of death. The mechanism of type E botulism exposure in fish-eating birds is unclear. Given that these birds are thought to eat live fish exclusively, it seems likely that their prey play a key role in the process, but the role of fish as potential transport vectors of botulinum neurotoxin type E (BoNT/E) to birds has not been adequately investigated. Between June 2003 and April 2004 a methodological model for exposing fish to Clostridium botulinum was developed and used to compare the sensitivity of rainbow trout (Oncorhynchus mykiss), round goby (Neogobius melanostomas), walleye (Stizostedion vitreum), and yellow perch (Perca flavescens) to four doses (0, 800, 1,500, and 4,000 Mouse Lethal Doses) of Clostridium botulinum type E neurotoxin. Each fish species expressed unique changes in both behavior and skin pigmentation prior to death. Yellow perch survived significantly longer (P < 0.05) than the three other species at all toxin treatments. Results of this study suggest that live fish can represent a significant vector for transfer of BoNT/E to birds.

Repeated low-level exposure of the round goby (Neogobius melanostomas) to Clostridium botulinum type E neurotoxin.

In a 4-mo study (June 2004-September 2004), round gobies (Neogobius melanostomas) were dosed orally every 72 hr for up to 21 days with Clostridium botulinum neurotoxin type E (BoNT/E) at one of four doses: 0, 50, 250, and 500 mouse lethal doses (MLD). Fish were observed for changes in pigmentation and behavior for the duration of the experiment. Mortality was observed with all treatments, with the exception of the 0 MLD control. Clinical signs observed were consistent with prior research and appeared to occur in a threshold manner. The mean times to death and percent mortalities were dose dependent. Hazard ratios were determined to have a significant positive (parameter estimate = 0.03) linear relationship with dose. The hazard ratio showed that per one unit dose increase, the instantaneous probability of a fish dying increased 1.02%. Postmortem analysis of experimental fish demonstrated that 11% (3/27) of fish contained detectable BoNT/E in their visceral fraction. The other 89% tested negative for BoNT/E, despite the fact that all fish died as a result of BoNT/E exposure. Therefore, botulism should not necessarily be ruled out as the cause of a fish kill, even if the fish test negative for BoNT/E.

Near-field loading dynamics of total phosphorus and short-term water quality variations at a rainbow trout cage farm in Lake Huron.

Aquatic total phosphorus (Tot-P) is measured at fish-cages in Lake Huron for environmental regulatory compliance. An improved understanding of how Tot-P is manifested in the near-field (

Persistence of Clostridium botulinum neurotoxin type E in tissues from selected freshwater fish species: implications to public health.

Rainbow trout (Oncorhynchus mykiss), round gobies (Neogobius melanostomas), yellow walleye (Stizostedion vitreum), and yellow perch (Perca flavescens) were given Clostridium botulinum neurotoxin type E (BoNT/E) at four doses (0, 800, 1500, and 4000 mouse lethal doses). BoNT/E was sought in the fish tissues at death or at the conclusion of the experiment (10 days after treatment). Fish were divided into a "fillet" (axial musculature) and a "nonfillet" sample before testing for BoNT/E toxicity with a mouse bioassay. BoNT/E was detected in all species. The percentage of positive BoNT samples ranged across the species and doses from 0 (trout, perch, and walleye) to 17% (round goby) in fillet tissues and from 0 (perch) to 92% (round goby) in nonfillet tissues. The lack of positive fillet samples in three key commercial fish species suggests that the public health implications of eating these fish are minimal. However, the presence of toxin in the nonfillet compartment of a high proportion of fish supports the hypothesis that live intoxicated fish are a vehicle for the transfer of BoNT/E to fish-eating birds, which are then in turn, intoxicated.