Christopher Thome

A Self-Contained, Controlled Hatchery System for Rearing Lake Whitefish Embryos for Experimental Aquaculture

AbstractA self-contained, small-scale research hatchery was constructed in a modified chromatography refrigerator equipped with a filtered and UV-sterilized water recirculation system. Lake Whitefish Coregonus clupeaformis embryos were raised in conventional upwelling hatching jars, in dishes with a continuous slow “drip feed,” and in a variety of static water incubation systems in petri dishes and multiwell plates. The optimal rearing density for petri dishes was found to be 50 embryos per dish, with weekly water changes. The highest survival in multiwell plates was seen in the 6- and 24-well sizes. Survival rates in most multiwell plates and petri dishes, as well as in the hatching jar incubators, were between 40% and 60%, which is in line with survival rates seen in commercial large-scale rearing. Overall, these techniques permitted the rearing of large numbers of whitefish in separate batches and under controlled conditions, while greatly reducing space requirements and material costs. Our system is w...

Embryonic development of lake whitefish Coregonus clupeaformis: a staging series, analysis of growth and effects of fixation

A reference staging series of 18 morphological stages of laboratory reared lake whitefish Coregonus clupeaformis is provided. The developmental processes of blastulation, gastrulation, neurulation as well as development of the eye, circulatory system, chromatophores and mouth are included and accompanied by detailed descriptions and live imaging. Quantitative measurements of embryo size and mass were taken at each developmental stage. Eggs were 3·19 ± 0·16 mm (mean ± s.d.) in diameter at fertilization and embryos reached a total length (LT ) of 14·25 ± 0·41 mm at hatch. Separated yolk and embryo dry mass were 0·25 ± 0·08 mg and 1·39 ± 0·17 mg, respectively, at hatch. The effects of two common preservatives (formalin and ethanol) were examined throughout development and post hatch. Embryo LT significantly decreased following fixation at all points in development. A correction factor to estimate live LT from corresponding fixed LT was determined as live LT = (fixed LT )(1·025) . Eye diameter and yolk area measurements significantly increased in fixed compared with live embryos up to 85-90% development for both measurements. The described developmental stages can be generalized to teleost species, and is particularly relevant for the study of coregonid development due to additionally shared developmental characteristics. The results of this study and staging series are therefore applicable across various research streams encompassing numerous species that require accurate staging of embryos and descriptions of morphological development.

Initial Characterization of the Growth Stimulation and Heat-Shock-Induced Adaptive Response in Developing Lake Whitefish Embryos after Ionizing Radiation Exposure

Ionizing radiation is known to effect development during early life stages. Lake whitefish (Coregonus clupeaformis) represent a unique model organism for examining such effects. The purpose of this study was to examine how ionizing radiation affects development in lake whitefish embryos and to investigate the presence of an adaptive response induced by heat shock. Acute exposure to 137Cs gamma rays was administered at five time points corresponding to major developmental stages, with doses ranging from 0.008 to 15.5 Gy. Chronic gamma-ray exposures were delivered throughout embryogenesis within a custom-built irradiator at dose rates between 0.06 and 4.4 mGy/day. Additionally, embryos were given a heat shock of 3, 6 or 9°C prior to a single acute exposure. Radiation effects were assessed based on survival, development rate, morphometric measurements and growth efficiency. Embryos showed high resistance to acute exposures with an LD50/hatch of 5.0 ± 0.7 Gy immediately after fertilization, increasing to 14.2 ± 0.1 Gy later in development. Chronic irradiation at all dose rates stimulated growth, with treated embryos up to 60% larger in body mass during development compared to unirradiated controls. Chronic irradiation also accelerated the time-to-hatch. A heat shock administered 6 h prior to irradiation reduced mortality by up to 25%. Overall, low-dose chronic irradiation caused growth stimulation in developing lake whitefish embryos and acute radiation mortality was reduced by a heat-shock-induced adaptive response.

The REPAIR Project: Examining the Biological Impacts of Sub-Background Radiation Exposure within SNOLAB, a Deep Underground Laboratory

Considerable attention has been given to understanding the biological effects of low-dose ionizing radiation exposure at levels slightly above background. However, relatively few studies have been performed to examine the inverse, where natural background radiation is removed. The limited available data suggest that organisms exposed to sub-background radiation environments undergo reduced growth and an impaired capacity to repair genetic damage. Shielding from background radiation is inherently difficult due to high-energy cosmic radiation. SNOLAB, located in Sudbury, Ontario, Canada, is a unique facility for examining the effects of sub-background radiation exposure. Originally constructed for astroparticle physics research, the laboratory is located within an active nickel mine at a depth of over 2,000 m. The rock overburden provides shielding equivalent to 6,000 m of water, thereby almost completely eliminating cosmic radiation. Additional features of the facility help to reduce radiological contamination from the surrounding rock. We are currently establishing a biological research program within SNOLAB: Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR project). We hypothesize that natural background radiation is essential for life and maintains genomic stability, and that prolonged exposure to sub-background radiation environments will be detrimental to biological systems. Using a combination of whole organism and cell culture model systems, the effects of exposure to a sub-background environment will be examined on growth and development, as well as markers of genomic damage, DNA repair capacity and oxidative stress. The results of this research will provide further insight into the biological effects of low-dose radiation exposure as well as elucidate some of the processes that may drive evolution and selection in living systems. This Radiation Research focus issue contains reviews and original articles, which relate to the presence or absence of low-dose ionizing radiation exposure.

Is There a Trade-Off between Radiation-Stimulated Growth and Metabolic Efficiency?

Beneficial protective effects may result from an adaptive respose to low dose radiation exposure. However, such benefits must be accompanied by some form of cost because the responsible biological mechanisms are not normally maintained in an upregulated state. It has been suggested that stimulation of adaptive response mechanisms could be metabolically costly, or that the adaptive response could come at a sacrifice to other physiological processes. We exposed developing lake whitefish embryos to a fractionated regime of gamma radiation (662 keV; 0.3 Gy min–1) to determine whether radiation-stimulated growth was accompanied by a trade-off in metabolic efficiency. Developing embryos were exposed at the eyed stage to different radiation doses delivered in four fractions, ranging from 15 mGy to 8 Gy per fraction, with a 14 day separation between dose fractions. Dry weight and standard length measurements were taken 2–5 weeks after delivery of the final radiation exposure and yolk conversion efficiency was estimated by comparing the unpreserved dry weight of the yolk to the unpreserved yolk-free dry weight of the embryos and normalizing for size-related differences in somatic maintenance. Our results show that the irradiated embryos were 8–10% heavier than the controls but yolk conversion efficiency was slightly improved. This finding demonstrates that stimulated growth in developing lake whitefish embryos is not “paid for” by a trade-off in the efficiency of yolk conversion.

Developmental effects of the industrial cooling water additives morpholine and sodium hypochlorite on lake whitefish (Coregonus clupeaformis)

Chemicals used in the prevention of corrosion and biofouling may be released into the environment via industrial cooling water discharges. The authors assessed the impacts of 2 commonly used chemicals, morpholine and sodium hypochlorite, on development in lake whitefish (Coregonus clupeaformis). Embryos were exposed chronically, beginning at fertilization or at the eyed stage. Acute 96-h exposures were also examined at 4 development stages. Chronic morpholine resulted in median lethal concentrations (LC50s) of 219 ± 54 mg/L when exposure began at fertilization and 674 ± 12 mg/L when exposure began at the eyed stage, suggesting that embryos are more sensitive earlier in development. Chronic morpholine exposure advanced hatching by up to 30%, and the early hatching embryos were up to 10% smaller in body length. A decrease in yolk conversion efficiency was also observed in embryos exposed to chronic morpholine concentrations of 1000 mg/L. The majority of effects from morpholine exposure manifested near hatch, possibly reflecting changes in chorion permeability at the end of embryonic development. Sodium hypochlorite only impacted survival with chronic exposure from fertilization, where the total residual chlorine LC50 was 0.52 ± 0.11 mg/L. Acute exposures to both chemicals had minimal effects up to the highest tested concentrations. Overall, the results suggest that the risk during development from exposure to morpholine and sodium hypochlorite is low under normal operating conditions. Environ Toxicol Chem 2017;36:1955-1965.

The relative biological effectiveness of low-dose mammography quality X rays in the human breast MCF-10A cell line.

Mammography is used to screen a large fraction of the population for breast cancer, and mammography quality X rays are speculated to be more damaging than the higher energy X rays used for other diagnostic procedures. The radiation dose delivered to breast cells as a result of these screening exposures may be a concern. The purpose of this current study was to determine the relative biological effectiveness (RBE) of low-energy mammography X rays for radiation-induced DNA double-strand breaks evaluated using a highly sensitive automated 53BP1 assay. Automation of the 53BP1 assay enabled the quantification and analysis of meaningful image-based features, including foci counting, within the cell nuclei. Nontumorigenic, human breast epithelial MCF-10A cells were irradiated in the low-dose range with approximately 3–30 mGy of 29 kVp mammography X rays or 137Cs (662 keV) gamma rays. The induction and resolution of the 53BP1 foci did not differ significantly between exposures to 137Cs gamma rays and 29 kVp X rays. The RBE was calculated to be 1.1 with a standard deviation of 0.2 for the initial number of radiation-induced double-strand breaks. The radiation dose from a single mammogram did not yield a significant change in the number of detectable foci. However, analysis of additional features revealed subtle differences in the distribution of 53BP1 throughout the nuclei after exposure to the different radiation qualities. A single mammogram was sufficient to alter the distribution of 53BP1 within the nuclear area, but not into discrete foci, while a dose-matched gamma exposure was not sufficient to alter the distribution of 53BP1. Our results indicate that exposure to clinically relevant doses of low-energy mammography quality X rays does not induce more DNA double-strand breaks than exposure to higher energy photons.

Micronuclei formation in rainbow trout cells exposed to multiple stressors: Morpholine, heat shock, and ionizing radiation

Discharges from industrial cooling water systems can include low levels of morpholine (a chemical pH regulator and corrosion inhibitor), as well as transiently higher temperature effluent water which present a potential source of environmental impact to aquatic biota. The effects of environmental levels of morpholine or heat shock (HS) treatment alone and in combination with a challenge high-dose of 137Cs ionizing radiation were studied using the cytokinesis block micronucleus assay in a rainbow trout cell line (RTG-2). Morpholine treatment of 10 or 100mgL-1 alone produced no significant effects, and no interaction was observed in combination with 7.75Gy radiation. A 9°C magnitude HS treatment alone significantly increased micronuclei formation. A synergistic response was observed when 9°C HS was combined with 7.75Gy radiation, with 15% more cells containing 3 or more micronuclei than the sum of each individual stressor. A synergistic increase in the average number of micronuclei was observed when morpholine and a 9°C HS were co-treated. These results indicate that morpholine at environmentally-relevant levels does not impact micronuclei formation or cell cycle progression however 9°C HS may be of potential concern both alone and in combination with other stressor treatments.

A method to transform a variable thermal regime to a physiologically equivalent effective temperature

We present a method to characterize variable thermal regimes in terms of an equivalent or effective temperature. Our method is based on a first order exponential transformation of a time series of temperatures to yield an exponentially-weighted mean temperature characteristic of the regime and independent of any particular species or end point. The resulting effective temperature or exponential mean, Te¯, offers an improved method for summarizing mean temperature where biological response scales exponentially to temperature. The exponential mean allows growth under varying thermal regimes to be predicted using constant temperature models and offers a compact descriptor communicating the growth capacity of variable thermal regimes. The method combines mathematical simplicity with translatability to different Q10 values without recourse to the underlining time series data. It also provides a quantitative baseline that improves on mean temperature by incorporating the effect of Jensens inequality and it remains applicable at near zero temperatures where thermal sums lack accuracy.