Nonlinear relationship between Silver Carp density and their eDNA concentration in a large river.

Although environmental DNA (eDNA) is increasingly being used to survey for the presence of rare and/or invasive fishes in aquatic systems, the utility of this technique has been limited by a poor understanding of whether and how eDNA concentrations relate to fish density, especially in rivers. We conducted a field study to systematically test whether the eDNA released by a model invasive fish, Silver Carp (Hypophthalmichthys molitrix), was related to the density of this species in a large river. We quantified fish density throughout the 460 km long Illinois River using hydroacoustic surveys at 23 sites while concurrently collecting 192 surface water samples for eDNA analysis. We found that Silver Carp numerical density and biomass density were positively and non-linearly related to eDNA concentration and detection rate. Both eDNA concentration (copy number) and detection rate increased rapidly as Silver Carp density increased but plateaued at moderate densities. These relationships could prove useful for estimating Silver Carp relative abundance in newly invaded locations where population numbers are low to moderate. Future studies should explore the causes of this nonlinear relationship as it would ultimately benefit aquatic species monitoring and management programs.

Attracting Common Carp to a bait site with food reveals strong positive relationships between fish density, feeding activity, environmental DNA, and sex pheromone release that could be used in invasive fish management.

Measurement of environmental DNA (eDNA) is becoming a common technique to survey for rare and invasive fish due to its sensitivity and specificity. However, its utility is limited by an incomplete understanding of factors governing its sources and fates. Failure to detect eDNA is especially difficult to interpret so surveillance techniques often collect large numbers of samples across broad regions. If, however, fish could be reliably attracted to a single location where their eDNA could be easily measured that would be useful. We conducted a proof-of-concept study of this idea using invasive Common Carp. We monitored the distribution of radio-tagged Carp and their eDNA across a 67 ha lake focusing at the bait site while a pheromone (Prostaglandin F2α; PGF 2α) was also measured to determine their reproductive condition. Prior to baiting, Carp were patchily distributed and while eDNA was occasionally detectable, it was patchy and only loosely associated with moderately dense groups of fish. Further, neither Carp, nor their eDNA were consistently measurable at the bait site and surrounding region, and the pheromone was not measurable at all. However, once baiting commenced, Carp started visiting the bait site and feeding, especially at night, where eDNA levels increased 500-fold as fish densities doubled and PGF 2α became detectable. Fish presence, eDNA and pheromone concentrations peaked at night after 6 days, strongly suggesting feeding activity was the main driver. While the presence of eDNA precisely coincided with this aggregation, levels had dropped dramatically within 5 m. PGF 2α levels dropped less rapidly and demonstrated the presence of live mature fish. We suggest that food could be used to train fish to come to locations where they otherwise are too scarce to be reliably measured, increasing their eDNA release, making them measurable, and their reproductive condition also discernable by measuring pheromones.

Correlations between pathogen concentration and fecal indicator marker genes in beach environments.

The concentrations of several potential pathogens were measured using a microfluidic quantitative PCR (MFQPCR) platform in beach water, sand, and sediment samples collected in Duluth, MN. Among the 19 pathogen marker genes examined, eaeA from Escherichia coli and plc from Clostridium perfringens were most frequently detected in all samples. In beach water and wastewater samples, positive correlations were observed between quantities of potential pathogens and most of the fecal indicator genetic markers. Such correlations, however, were not frequently observed in sand and sediment samples. Our results suggest that the behavior of potential pathogens and FIB may vary by sample type and source of contamination. Consequently, appropriate FIB marker genes need to be chosen for reliable water/sand quality monitoring.

Environment shapes the fecal microbiome of invasive carp species.

BACKGROUND: Although the common, silver, and bighead carps are native and sparsely distributed in Eurasia, these fish have become abundant and invasive in North America. An understanding of the biology of these species may provide insights into sustainable control methods. The animal-associated microbiome plays an important role in host health. Characterization of the carp microbiome and the factors that affect its composition is an important step toward understanding the biology and interrelationships between these species and their environments. RESULTS: We compared the fecal microbiomes of common, silver, and bighead carps from wild and laboratory environments using Illumina sequencing of bacterial 16S ribosomal RNA (rRNA). The fecal bacterial communities of fish were diverse, with Shannon indices ranging from 2.3 to 4.5. The phyla Proteobacteria, Firmicutes, and Fusobacteria dominated carp guts, comprising 76.7 % of total reads. Environment played a large role in shaping fecal microbial community composition, and microbiomes among captive fishes were more similar than among wild fishes. Although differences among wild fishes could be attributed to feeding preferences, diet did not strongly affect microbial community structure in laboratory-housed fishes. Comparison of wild- and lab-invasive carps revealed five shared OTUs that comprised approximately 40 % of the core fecal microbiome. CONCLUSIONS: The environment is a dominant factor shaping the fecal bacterial communities of invasive carps. Captivity alters the microbiome community structure relative to wild fish, while species differences are pronounced within habitats. Despite the absence of a true stomach, invasive carp species exhibited a core microbiota that warrants future study.

Effects of Temperature and Trophic State on Degradation of Environmental DNA in Lake Water.

Degradation of environmental DNA (eDNA) in aquatic habitats can affect the interpretation of eDNA data and the ability to detect aquatic organisms. The effect of temperature and trophic state on the decay of Common Carp (Cyprinus carpio) eDNA was evaluated using lake water microcosms and quantitative PCR for a Common Carp-specific genetic marker in two experiments. The first experiment tested the effect of temperature on Common Carp eDNA decay. Common Carp eDNA exhibited exponential decay that increased with temperature. The slowest decay rate was observed at 5 °C, with a T90 value (time to 90% reduction from initial concentration) of 6.6 days, as opposed to ∼1 day at higher temperatures. In a second experiment, decay was compared across waters from lakes of different trophic states. In this experiment, Common Carp eDNA exhibited biphasic exponential decay, characterized by rapid decay for 3-8 days followed by slow decay. Decay rate was slowest in dystrophic water and fastest in oligotrophic water, and decay rate was negatively correlated to dissolved organic carbon concentration. The overall rapid decay of eDNA and the effects of temperature and water quality should be considered in protocols for water sample storage and field sampling design.

Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.

Few studies have examined capture and extraction methods for environmental DNA (eDNA) to identify techniques optimal for detection and quantification. In this study, precipitation, centrifugation and filtration eDNA capture methods and six commercially available DNA extraction kits were evaluated for their ability to detect and quantify common carp (Cyprinus carpio) mitochondrial DNA using quantitative PCR in a series of laboratory experiments. Filtration methods yielded the most carp eDNA, and a glass fibre (GF) filter performed better than a similar pore size polycarbonate (PC) filter. Smaller pore sized filters had higher regression slopes of biomass to eDNA, indicating that they were potentially more sensitive to changes in biomass. Comparison of DNA extraction kits showed that the MP Biomedicals FastDNA SPIN Kit yielded the most carp eDNA and was the most sensitive for detection purposes, despite minor inhibition. The MoBio PowerSoil DNA Isolation Kit had the lowest coefficient of variation in extraction efficiency between lake and well water and had no detectable inhibition, making it most suitable for comparisons across aquatic environments. Of the methods tested, we recommend using a 1.5 µm GF filter, followed by extraction with the MP Biomedicals FastDNA SPIN Kit for detection. For quantification of eDNA, filtration through a 0.2-0.6 µm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal. These results are broadly applicable for laboratory studies on carps and potentially other cyprinids. The recommendations can also be used to inform choice of methodology for field studies.

The relationship between the distribution of common carp and their environmental DNA in a small lake.

Although environmental DNA (eDNA) has been used to infer the presence of rare aquatic species, many facets of this technique remain unresolved. In particular, the relationship between eDNA and fish distribution is not known. We examined the relationship between the distribution of fish and their eDNA (detection rate and concentration) in a lake. A quantitative PCR (qPCR) assay for a region within the cytochrome b gene of the common carp (Cyprinus carpio or 'carp'), an ubiquitous invasive fish, was developed and used to measure eDNA in Lake Staring (MN, USA), in which both the density of carp and their distribution have been closely monitored for several years. Surface water, sub-surface water, and sediment were sampled from 22 locations in the lake, including areas frequently used by carp. In water, areas of high carp use had a higher rate of detection and concentration of eDNA, but there was no effect of fish use on sediment eDNA. The detection rate and concentration of eDNA in surface and sub-surface water were not significantly different (p≥0.5), indicating that eDNA did not accumulate in surface water. The detection rate followed the trend: high-use water > low-use water > sediment. The concentration of eDNA in sediment samples that were above the limit of detection were several orders of magnitude greater than water on a per mass basis, but a poor limit of detection led to low detection rates. The patchy distribution of eDNA in the water of our study lake suggests that the mechanisms that remove eDNA from the water column, such as decay and sedimentation, are rapid. Taken together, these results indicate that effective eDNA sampling methods should be informed by fish distribution, as eDNA concentration was shown to vary dramatically between samples taken less than 100 m apart.

Decay of genetic markers for fecal bacterial indicators and pathogens in sand from Lake Superior.

Beach sands impact water quality and pathogen loads, however, the comparative decay of the fecal indicator bacteria (FIB) Enterococcus spp. and Escherichia coli, and pathogens in freshwater sand have not been examined. In this study, freshwater sand microcosms were inoculated with sewage and pure cultures of bacterial pathogens to compare relative decay rates. The abundance of culturable Enterococcus spp. and E. coli, genetic markers for Enterococcus spp. (Entero1), total Bacteroides (AllBac), and human-specific Bacteroides (HF183), and genetic markers for the pathogens Campylobacter jejuni, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella enterica subsp. enterica serovar Typhimurium, and Shigella flexneri were monitored over the course of two weeks using conventional culture methods and quantitative PCR (qPCR). The effect of moisture on the persistence of culturable FIB and all genetic markers was also determined. In addition, propidium monoazide (PMA) treatment was used to examine differences in the persistence of total genetic markers and those from live cells. Decay rates were statistically compared using Tukey's test. Moisture had a significant (p ≤ 0.05) effect on the decay rates of culturable indicator bacteria, total AllBac markers, and genetic markers for FIB, Salmonella, and MRSA from live cells. At 14% sand moisture, the decay rate of total markers was slower than that of live cells for all qPCR assays, but at 28% moisture, there was no difference in the decay rates of total and live markers for any assay. AllBac and MRSA markers increased in sand at 28% moisture, probably indicating cellular growth. Overall, culturable FIB and HF183 had decay rates that were most comparable to the bacterial pathogen markers examined in this study, whereas Entero1 and AllBac rarely exhibited decay rates similar to the bacterial pathogens in this study. The choice of FIB for assessment of fecal contamination in freshwater sand should take into account the pathogen of concern and sand moisture conditions.

Distribution of genetic markers of fecal pollution on a freshwater sandy shoreline in proximity to wastewater effluent.

Water, sand, and sediment from a Lake Superior harbor site continuously receiving wastewater effluent was sampled monthly for June to October 2010 and from May to September 2011. Understanding the dynamics of genetic markers of fecal bacteria in these matrices is essential to accurately characterizing health risks. Genetic markers for enterococci, total Bacteroides, and human-associated Bacteroides were measured in site-water, sand, and sediment and in final effluent by quantitative PCR. The similarity between the quantity of molecular markers in the water column and effluent indicated that the abundance of genetic markers in the water column was likely controlled by effluent inputs. Effluent turbidity was positively correlated (p ≤ 0.05) with AllBac and HF183 in final effluent and AllBac in the water column. In sand and sediment, Entero1 and AllBac were most abundant in the upper 1-3 cm depths, whereas HF183 was most abundant in the upper 1 cm of sand and at 7 cm in sediment. The AllBac and Entero1 markers were 1- and 2-orders of magnitude more abundant in sand and sediment relative to the water column per unit mass. These results indicate that sand and sediment may act as reservoirs for genetic markers of fecal pollution at some freshwater sites.

Tertiary-treated municipal wastewater is a significant point source of antibiotic resistance genes into Duluth-Superior Harbor.

In this study, the impact of tertiary-treated municipal wastewater on the quantity of several antibiotic resistance determinants in Duluth-Superior Harbor was investigated by collecting surface water and sediment samples from 13 locations in Duluth-Superior Harbor, the St. Louis River, and Lake Superior. Quantitative PCR (qPCR) was used to target three different genes encoding resistance to tetracycline (tet(A), tet(X), and tet(W)), the gene encoding the integrase of class 1 integrons (intI1), and total bacterial abundance (16S rRNA genes) as well as total and human fecal contamination levels (16S rRNA genes specific to the genus Bacteroides ). The quantities of tet(A), tet(X), tet(W), intI1, total Bacteroides , and human-specific Bacteroides were typically 20-fold higher in the tertiary-treated wastewater than in nearby surface water samples. In contrast, the quantities of these genes in the St. Louis River and Lake Superior were typically below detection. Analysis of sequences of tet(W) gene fragments from four different samples collected throughout the study site supported the conclusion that tertiary-treated municipal wastewater is a point source of resistance genes into Duluth-Superior Harbor. This study demonstrates that the discharge of exceptionally treated municipal wastewater can have a statistically significant effect on the quantities of antibiotic resistance genes in otherwise pristine surface waters.