Christopher M. Somers

Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location

Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.

Air pollution induces heritable DNA mutations

Hundreds of thousands of people worldwide live or work in close proximity to steel mills. Integrated steel production generates chemical pollution containing compounds that can induce genetic damage (1, 2). Previous investigations of herring gulls in the Great Lakes demonstrated elevated DNA mutation rates near steel mills (3, 4) but could not determine the importance of airborne or aquatic routes of contaminant exposure, or eliminate possible confounding factors such as nutritional status and disease burden. To address these issues experimentally, we exposed laboratory mice in situ to ambient air in a polluted industrial area near steel mills. Heritable mutation frequency at tandem-repeat DNA loci in mice exposed 1 km downwind from two integrated steel mills was 1.5- to 2.0-fold elevated compared with those at a reference site 30 km away. This statistically significant elevation was due primarily to an increase in mutations inherited through the paternal germline. Our results indicate that human and wildlife populations in proximity to integrated steel mills may be at risk of developing germline mutations more frequently because of the inhalation of airborne chemical mutagens.

The Invasive Round Goby (Neogobius melanostomus) in the Diet of Nestling Double-crested Cormorants (Phalacrocorax auritus) in Hamilton Harbour, Lake Ontario

Populations of the invasive round goby (Neogobius melanostomus) have expanded dramatically since their discovery in the Laurentian Great Lakes in 1990. The abundance of these fish and possible competitive displacement of native species from aquatic food webs suggest that they will become an important prey item for predatory birds and sport fish. To date, there is very little information on the predation of round gobies by piscivorous birds in the Great Lakes. We used an abdominal palpation technique to stimulate regurgitation by double-crested cormorant (Phalacrocorax auritus) chicks in ground nests from colonies in Hamilton Harbour, western Lake Ontario, during the 2002 nesting season. We collected and identified all fish species present in regurgitated boluses. For tree-nesting birds, we collected fish that were regurgitated and fell to the ground as a result of targeted disturbance of particular nest trees. At all locations and times, alewife (Alosa pseudoharengus) was the most abundant fish species present by a large margin. The second most abundant species was the round goby, which was present in the regurgitated stomach contents of chicks in a total of 18 percent of nests surveyed, and made up 1.8 to 11 percent of all individual fish specimens identified. Our results show that the round goby is already an important food item for breeding cormorants in Hamilton Harbour, despite relatively recent establishment of goby populations in western Lake Ontario. Fish species of sport or commercial interest were detected in our samples with extremely low frequency (< 0.1%).

Ambient air pollution exposure and damage to male gametes: human studies and in situ 'sentinel' animal experiments

Globally there is concern that adverse reproductive outcomes and fertility impairment in humans may be caused by exposure to environmental contaminants. Air pollution in particular has been linked to DNA damage, abnormal sperm morphology, and reduced sperm performance in men. Experimental studies using model species (mice and rats) exposed in situ provide evidence that ambient air pollution can cause damage to the respiratory system and other tissues or organs. This can take the form of DNA damage and other genetic changes throughout the body, including induced mutations, DNA strand breaks, and altered methylation patterns in male germ cells. Human and animal studies together provide strong evidence that air pollution, especially airborne particulate matter, at commonly occurring ambient levels is genotoxic to male germ cells. The mechanistic link between air pollution exposure and induced genetic changes in male germ cells is currently unclear. ‘Sentinel’ animal experiments explicitly examining air pollution affects on sperm quality in laboratory rodents have not been conducted and would provide a critical link to observations in humans. The importance of air pollution compared to other factors affecting fertility and reproductive outcomes in humans is not clear and warrants further investigation.

Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq)

Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next‐generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double‐digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0‐, 12‐, 48‐ and 96‐h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12–48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96‐h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2‐ to 16‐fold), and additional read‐length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.

Cormorant- fisheries conflicts: Stable isotopes reveal a consistent niche for avian piscivores in diverse food webs

Global conflict between piscivorous cormorants ( Phalacrocorax spp.) and fish harvesters is one of the most widespread wildlife management issues in history. Despite the persistent belief that these birds adversely affect economically important fish populations, relatively little is known about cormorant trophic ecology and habitat use. We examined the diet and trophic position of breeding populations of Double-crested Cormorants ( Phalacro- corax auritus; hereafter cormorants) from three different lakes using stable carbon and nitrogen isotopes (5l3C and 615N). The 815N values revealed that cormorants generally occupied top-predator positions in all food webs examined; their trophic position (5.0 ± 0.5) was consistent within and between years in all lakes studied. The 513C values showed that cormorants occupied very similar dietary niches in three different freshwater food webs. Mean centroid distance, a measure of diversity, varied significantly among food webs (range 1.5- 2.9), but not among cormorants from different lakes (range 0.8-1.2). In all three lakes, cormorants relied heavily on non-benthic or pelagic prey. Stable isotopes mixing models demonstrated that cisco ( Coregonus artedii ), yellow perch ( Perca flavescens), and ninespine stickleback ( Pungitius pungitius) were most important for cormorant diet independent of the local food web structure. The isotopie values of cormorants and pelagic predatory fish were sometimes similar, suggesting that dietary overlap is possible. Based on our study, we suggest that cormorants may have more specific and uniform dietary niche requirements than previously considered. Consequently, the potential for adverse effects on food webs will heavily depend on whether economically valuable species fill the prey niches consumed by cormorants. For example, cormorants should have little economic impact where food webs are diverse in abundant prey species and niches. Alternately, food webs with less diversity of prey species and niches may be more affected by cormorant predation. Managers should avoid simply assuming that cormorants will have negative impacts on fisheries and should instead consider the structure of the food web as well as the niches occupied by cormorants and fish species of economic interest.

The Adaptive Response and Protection against Heritable Mutations and Fetal Malformation

There are a number of studies that show radiation can cause heritable mutations in the offspring of irradiated organisms. These “germ-line mutations” have been shown to occur in unique sequences of DNA called “minisatellite loci”. The high frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be measured at low doses of exposure in a small population, making minisatellite mutation a powerful tool to investigate radiation-induced heritable mutations. However, the biological significance of these mutations is uncertain, and their relationship to health risk or population fitness is unknown. We have adopted this mutation assay to study the role of adaptive response in protecting mice against radiation-induced heritable defects. We have shown that male mice, adapted to radiation with a low dose priming exposure, do not pass on mutations to their offspring caused by a subsequent large radiation exposure to the adapted males. This presentation and paper provide a general overview of radiation-induced mutations in offspring and explain the effect of low dose exposures and the adaptive response on these mutations. It is also known that exposure of pregnant females to high doses of radiation can cause death or malformation (teratogenesis) in developing fetuses. Malformation can only occur during a specialized stage of organ formation known as organogenesis. Studies in rodents show that radiation-induced fetal death and malformation can be significantly reduced when a pregnant female is exposed to a prior low dose of ionizing radiation. The mechanism of this protective effect, through an adaptive response, depends on the stage of organogenesis when the low dose exposures are delivered. To better understand this process, we have investigated the role of an important gene known as p53. Therefore, this report will also discuss fetal effects of ionizing radiation and explain the critical stages of development when fetuses are at risk. Research will be explained that investigates the biological and genetic systems (p53) that protect the developing fetus and discuss the role of low dose radiation adaptive response in these processes.

Non-destructive sampling of maternal DNA from the external shell of bird eggs

The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.

Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings

Critical windows are periods of developmental susceptibility when the phenotype of an embryonic, juvenile or adult animal may be vulnerable to environmental fluctuations. Temperature has pervasive effects on poikilotherm physiology, and embryos are especially vulnerable to temperature shifts. To identify critical windows, we incubated whitefish embryos at control temperatures of 2°C, 5°C, or 8°C, and shifted treatments among temperatures at the end of gastrulation or organogenesis. Heart rate (fH) and oxygen consumption ( [Formula: see text] ) were measured across embryonic development, and [Formula: see text] was measured in 1-day old hatchlings. Thermal shifts, up or down, from initial incubation temperatures caused persistent changes in fH and [Formula: see text] compared to control embryos measured at the same temperature (2°C, 5°C, or 8°C). Most prominently, when embryos were measured at organogenesis, shifting incubation temperature after gastrulation significantly lowered [Formula: see text] or fH. Incubation at 2°C or 5°C through gastrulation significantly lowered [Formula: see text] (42% decrease) and fH (20% decrease) at 8°C, incubation at 2°C significantly lowered [Formula: see text] (40% decrease) and fH (30% decrease) at 5°C, and incubation at 5°C and 8°C significantly lowered [Formula: see text] at 2°C (27% decrease). Through the latter half of development, [Formula: see text] and fH in embryos were not different from control values for thermally shifted treatments. However, in hatchlings measured at 2°C, [Formula: see text] was higher in groups incubated at 5°C or 8°C through organogenesis, compared to 2°C controls (43 or 65% increase, respectively). Collectively, these data suggest that embryonic development through organogenesis represents a critical window of embryonic and hatchling phenotypic plasticity. This study presents an experimental design that identified thermally sensitive periods for fish embryos.

Mercury concentrations in surface water and harvested waterfowl from the prairie pothole region of Saskatchewan.

Mercury cycling in prairie ecosystems is poorly understood. We examined methylmercury (MeHg) concentrations in whole water from 49 diverse prairie wetlands and lakes in Saskatchewan. We also determined total Hg (THg) concentrations in waterfowl harvested by hunters for consumption. Average whole water MeHg concentrations ranged from 0.02 to over 4 ng L(-1) and were higher in water from wetland ponds compared to those in lakes. High MeHg concentrations in prairie wetlands present the possibility of increased Hg concentrations in biota inhabiting these and other similar systems. We therefore measured THg in 72 birds representing 13 species of waterfowl that commonly use prairie aquatic habitats. A large range in THg concentrations was observed among individual birds, with values ranging from below the detection limit to over 435 ng g(-1). When waterfowl were classified according to diet, we observed clear evidence of THg biomagnification with increasing proportion of animal prey consumed. THg concentrations in waterfowl collected by hunters did not exceed consumption guidelines of 0.5 mg kg(-1) developed for fish. This is the first study that has reported MeHg concentrations in water from the prairie pothole region of southern Saskatchewan.