Freshwater mussels in an impacted watershed: Influences of pollution from point and non-point sources.

The Grand River watershed in a densely populated region of Ontario supports one of the richest assemblages of freshwater mussels in Canada. However, water quality in this watershed is influenced by urban development, agriculture, and industry. Mussel populations and water chemistry in the lower Grand River and the Boston Creek tributary were evaluated to determine whether point sources of pollution such as discharges of domestic wastewater and industrial effluent, and non-point sources of pollution are affecting mussel distribution and population structure. Semi-quantitative population surveys conducted at 9 study sites identified 20 mussel species, including 3 Species at Risk. Mussel abundance (34-160 mussels/search hour) and species richness indicated that mussel populations in the lower Grand River watershed are continuing to recover from historical lows reported in the 1970s. However, changes in populations at some sites were consistent with altered water chemistry. Most notable was that the three most abundant mussel species in the Boston Creek tributary downstream of a gypsum plant discharge were significantly smaller in length than those upstream of this site. The water chemistry in this habitat was characterized by elevated conductivity (∼2000 µS/cm) and calcium (∼500 mg/L), as well as concentrations of sulfate (∼1000 mg/L) that can be toxic to freshwater mussels. In the Grand River downstream of the confluence with Boston Creek, there tended to be (p > 0.05) fewer mussels (mean 34 ± 20/search h) compared to upstream (mean 67 ± 15/search h) and this corresponded to altered water chemistry, including elevated sulfate (239 mg/L) downstream of the confluence relative to upstream (58 mg/L). These data indicate that chronic exposures to high levels of major ions is likely driving changes to mussel population structure. In addition, the discharges of wash water from a gypsum plant may be impacting sensitive biota in the main stem Grand River well beyond the immediate tributary receiving environment.

Effects of municipal wastewater effluents on the digestive gland microbiome of wild freshwater mussels (Lasmigona costata).

Gut microbial communities are vital for maintaining host health, and are sensitive to diet, environment, and chemical exposures. Wastewater treatment plants (WWTPs) release effluents containing antimicrobials, pharmaceuticals, and other contaminants that may negatively affect the gut microbiome of downstream organisms. This study investigated changes in the diversity and composition of the digestive gland microbiome of flutedshell mussels (Lasmigona costata) from upstream and downstream of two large (service >100,000) WWTPs. Mussel digestive gland microbiome was analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16 S rRNA gene. Bacterial alpha diversity decreased at sites downstream of the second WWTP and these sites were dissimilar in beta diversity from sites upstream and downstream of the first upstream WWTP. The microbiomes of mussels collected downstream of the first WWTP had increased relative abundances of Actinobacteria, Bacteroidetes, and Firmicutes, with a decrease in Cyanobacteria, compared to upstream mussels. Meanwhile, those collected downstream of the second WWTP increased in Proteobacteria and decreased in Actinobacteria, Bacteroidetes, and Tenericutes. Increased Proteobacteria has been linked to adverse effects in mammals, but their functions in mussels is currently unknown. Finally, effluent-derived bacteria were found in the microbiome of mussels downstream of both WWTPs but not in those from upstream. Overall, results show that the digestive gland microbiome of mussels collected upstream and downstream of WWTPs differed, which has implications for altered host health and the transport of WWTP-derived bacteria through aquatic ecosystems.

Salt-Laden Winter Runoff and Freshwater Mussels; Assessing the Effect on Early Life Stages in the Laboratory and Wild Mussel Populations in Receiving Waters.

The widespread use of road salt for winter road maintenance has led to an increase in the salinity of surface water in many seasonally cold areas. Freshwater mussels have a heightened sensitivity to salt, which is a concern, because many Canadian mussel species at risk have ranges limited to southern Ontario, Canada's most road-dense region. This study examined the effect of winter road runoff on freshwater mussels. The impact of two bridges that span mussel habitat in the Thames River watershed (Ontario, Canada), the second most species-rich watershed for mussels in Canada, were studied. During a winter melt event, bridge runoff, as well as creek surface water surrounding the bridges were collected. Chloride concentrations in samples from bridge deck and tile drains varied (99-8250 mg/L). In general, survival of Lampsilis fasciola glochidia exposed to those samples reflected chloride levels (e.g. 84% at 99 mg/L; 0% at 8250 mg/L), although potassium (60 mg/L) may have at least contributed to toxicity in one sample. Serial dilution exposures with the two most toxic runoff samples revealed 48-h glochidia EC50s of 44% (McGregor Creek Tile Drain) and 26% (Baptiste Creek Deck Drain). During the melt event, the chloride concentrations in creek surface waters downstream of the bridges ranged from 69 to 179 mg Cl-/L; effects on glochidia (viability 77-91%) exposed to those waters was minimal. There were no live mussels surrounding one bridge (Baptiste Creek), likely due to poor habitat. At the other targeted bridge (McGregor Creek), fewer mussels were found close (< 100 m up- or downstream) to the bridge than further (> 200 m) away. However, other contributing factors, including agriculture, were present at both study areas.

Fish community responses to multiple municipal wastewater inputs in a watershed.

Municipalities utilize aquatic environments to assimilate their domestic effluent resulting in eutrophication, anoxia, toxicity and endocrine disruption of aquatic biota. The objective of this study was to assess the potential cumulative impacts of municipal wastewater effluent (MWWE) discharges in the Grand River on the health status of a sentinel species and the fish community downstream of 2 MWWE discharges. The fish communities downstream of the MWWE outfalls demonstrated differences in the abundance and diversity, species and family richness, % tolerance and % vulnerability when compared to the fish community upstream or further downstream of these points of effluent discharge. In both years studied, the fish community exposed to MWWE in the riffle-run habitats demonstrated reductions in the proportion of the most prominent fish (Rainbow Darter, Ethoestoma caeruleum) downstream of the outfalls, and a significant increase in the proportion of large mobile, tolerant-omnivorous fish species such as suckers and sunfish. There was less variability in the responses of the fish community to MWWE in the same season between years than between seasons within the same year. An examination of how impaired health of a sentinel species exposed to MWWE discharges parallels changes in the fish community is also conducted. This study successfully demonstrates the cumulative impact of urban development, including multiple outfalls of treated wastewater effluents on fish populations and communities. Municipalities are the major source of nutrients and pharmaceuticals and personal care products to aquatic systems, and they need to consider their impacts carefully with increasing urban population growth and ageing demographics.

Reproductive and histopathological effects in wild fish inhabiting an effluent-dominated stream, Wascana Creek, SK, Canada.

During the winter low flow periods, Wascana Creek, Saskatchewan, Canada, can be 100% treated municipal wastewater downstream of the City of Regina's Wastewater Treatment Plant. The objective of this study was to determine if exposure to municipal effluent affects the health and reproductive development of fish in an effluent-dominated stream. Field studies were conducted on post-spawning (August 2006), spawning (June 2007), recrudescent (October 2007) and pre-spawning (May 2008) sentinel fish [Fathead Minnow Pimephales promelas and Brook Stickleback Culaea inconstans] to assess responses in terms of growth (condition factor), reproduction (in vitro sex steroid biosynthetic capacity, and gonadosomatic indices, histology) and survival associated with the effluent outfall. Sentinel species demonstrated varying responses depending on the season of field collections. While Stickleback collected downstream of the sewage discharge were often longer, heavier and had greater condition, Fatheads from the same site were shorter and lighter. Exposed fish of both species exhibited delayed spawning and altered gonadal development depending on the season. Exposed male Fathead Minnows also had significantly lower scores of secondary sexual characteristics (fewer nuptial tubercles, little or no development of the dorsal pad, and the lack of presence of a dorsal fin dot). Histopathology of exposed Fathead Minnows revealed thickening of the gill lamellae and alterations in structure of the kidneys (inflammation of the proximal tubules and Bowman's capsule). It is not known if the effluents are affecting natural reproduction and recruitment into this population or if these impacted populations rely on immigration from upstream reaches to sustain the populations. Climate change and human population growth will further challenge this effluent-dominated stream's ability to assimilate nutrients and contaminants which may further impair the performance of fish in this arid environment.

Genetic and phylogenetic characterization of the type II cyclobutane pyrimidine dimer photolyases encoded by Leporipoxviruses.

Shope fibroma virus and myxoma virus encode proteins predicted to be Type II photolyases. These are enzymes that catalyze light-dependent repair of cyclobutane pyrimidine dimers (CPDs). When the Shope fibroma virus S127L gene was expressed in an Escherichia coli strain lacking functional CPD repair pathways, the expressed gene protected the bacteria from 70-75% of the ultraviolet (UV) light-induced cytotoxic DNA damage. This proportion suggests that Leporipoxvirus photolyases can only repair CPDs, which typically comprise approximately 70% of the damage caused by short wavelength UV light. To test whether these enzymes can protect virus genomes from UV, we exposed virus suspensions to UV-C light followed by graded exposure to filtered visible light. Viruses encoding a deletion of the putative photolyase gene were unable to photoreactivate UV damage while this treatment again eliminated 70-90% of the lethal photoproducts in wild-type viruses. Western blotting detected photolyase protein in extracts prepared from purified virions and it can be deduced that the poxvirion interior must be fluid enough to permit diffusion of this approximately 50-kDa DNA-binding protein to the sites where it catalyzes photoreactivation. Photolyase promoters are difficult to categorize using bioinformatics methods, as they do not obviously resemble any of the known poxvirus promoter motifs. By fusing the SFV promoter to DNA encoding a luciferase open reading frame, the photolyase promoter was found to exhibit very weak late promoter activity. These data show that the genomes of Leporipoxviruses, similar to that of fowlpox virus, encode catalytically active photolyases. Phylogenetic studies also confirmed the monophyletic origin of poxviruses and suggest an ancient origin for these genes and perhaps poxviruses.