From the Field to the Lab: Best Practices for Field Preservation of Bat Specimens for Molecular Analyses

Studies in molecular ecology depend on field-collected samples for genetic information, and the tissue sampled and preservation conditions strongly affect the quality of the DNA obtained. DNA yields from different tissue types have seldom been compared, and the relative performance of storage media has never been directly tested, even though these media may influence DNA degradation under field conditions. We analyzed DNA yield from buccal swabs and wing punches harvested from live bats using nucleic acid quantification as well as quantitative PCR for a single-copy nuclear locus. We also compared DNA yields from wing tissue preserved in three media: ethanol, NaCl-saturated dimethyl sulfoxide (DMSO), and silica desiccant. Wing punches yielded more total DNA than did buccal swabs, and wing tissues preserved in silica beads yielded significantly more total and nuclear DNA than those preserved in DMSO or ethanol. These results show that tissue type and preservation media strongly influence the quantity of DNA obtained from non-lethal genetic samples, and based on these effects we provide recommendations for field collection of tissues for genetic analyses

Zebra Mussel (Dreissena Polymorpha) Promotion of Cyanobacteria in Low-Nutrient Lakes and the Subsequent Production and Fate of Microcystin

The ability of established populations of the non-native zebra mussel (Dreissena polymorpha) to influence phytoplankton communities and promote Microcystis aeruginosa, a potentially toxic cyanobacterium, has been reported by Fahnenstiel et al. (1995), Vanderploeg et al. (2002) and others, in the Great Lakes region. This study documents changes following zebra mussel establishment in six low-nutrient inland lake basins in northwest lower Michigan (Leelanau County). Shifts in phytoplankton communities that occurred only in basins with zebra mussels included declines in spring diatoms and chrysophytes prior to blooms of cyanobacteria. Decreases in these taxa support a competitive release hypothesis for M. aeruginosa dominance. It should also be noted that study basins did not experience increases in phosphorus or summer temperatures. M. aeruginosa proliferation may be related to zebra mussel filtering behavior, a combination of total zebra mussels and lake morphology. Zebra mussel populations were estimated using underwater video, M. aeruginosa densities were quantified from surface water and lake bathymetry and basin-wide zebra mussel densities were estimated using ArcGIS. Underwater video ground-truthed using SCUBA was an effective, yet labor intensive method to estimate zebra mussel populations, and kernel interpolation provided acceptable zebra mussel density estimates basin-wide. The relationship between M. aeruginosa density and zebra mussel filtering capacity was not significant, however the sample size may have been an issue. Microcystin (MC), the hepatotoxin produced by M. aeruginosa and other cyanobacteria, was measured before and after blooms of M. aeruginosa over depths and across seasons in whole water, sediment, macroinvertebrates, bivalves and fishes, using enzyme linked immunosorbent assay. A subset of sediment and Hexagenia spp. samples were analyzed using high performance liquid chromatography and mass spectrometry for microcystin-LR. MC was present in all components, including spring samples prior to M. aeruginosa blooms and exceeded the recreational and total daily intake guidelines of the World Health Organization in whole water (on at least one occasion) and in fish white muscle, for consumption by men, women and children. Hexagenia spp. and walleye contained the highest concentrations of MC. MC should be monitored in these lake basins because of the potential risk of chronic sub-lethal exposure to humans

The Effectiveness of Constructed Wetlands

Wetland construction represents a vital tool to increase the number and extent of wetlands in the United States. However, there is uncertainty as to how effective constructed wetlands actually are and if they continue to function efficiently as they age. This study’s objective was to evaluate the constructed wetlands on Grand Valley State University’s Allendale campus. The wetlands studied were constructed in both 2009 (n=3) and 2011 (n=5), not specifically to mitigate for wetland loss; rather they are a proactive attempt to reduce erosion from excessive stormwater runoff in the GVSU ravines. We compared these to wetlands constructed in the mid 1980’s (n=3) located at the near-by Bass River Recreation Area. Specifically, aquatic macroinvertebrates were sampled throughout May 2012, following rapid bioassessment protocols used by the Michigan DNR, while water chemistry parameters (specific conductivity, pH, dissolved oxygen, temperature, turbidity, riparian coverage, chloride, and total dissolved solids) were measured bi-weekly throughout the summer. The macroinvertebrate Family richness and diversity were significantly different (p\u3c0.05, ANOVA) and values for each metric ranged from 21.3, 20.67, and 6.6 and 2.31, 2.13, and 1.01 between 1980’s, 2009, and 2011 sites, respectively. These differences in the insect community assemblages were evident in a multivariate test as well (NMDS). Thus, at a community level there was a rapid improvement in the aquatic insects in just three years suggesting these constructed wetlands will rapidly develop into healthier communities

MICROCYSTIN IN LITTLE BROWN BATS (MYOTIS LUCIFUGUS) FOLLOWING CONSUMPTION OF ADULT MAYFLIES OF THE GENUS HEXAGENIA

Microcystin (MC) is a hepatotoxin, implicated globally in the poisoning of humans, livestock and other organisms (Sivonen and Jones 1999). Substantial research documents potential accumulation and transfer of MC from one ecosystem component to another within aquatic ecosystems (Mohamed 2001; Vasconcelos 1995; Kotak et al. 1996; Falconer et al. 1992), and between aquatic and terrestrial ecosystems (Miller et al. 2010, Stewart et al. 2008). Since bat consumption of mayflies was determined by Clare et al. (2011), and mayfly nymphs of the genus Hexagenia were demonstrated to contain MC (Smith et al. 2008), it is possible that if bats consume adult Hexagenia spp., they will be exposed to MC. We analyzed MC equivalents from adult Hexagenia spp. and fecal material from a maternal colony of little brown bats (Myotis lucifugus) collected from Leelanau County, Michigan, USA in June 2013, using enzyme linked immuno-sorbent assay. All Hexagenia spp. (mean = 325.40 ng/g ± 51.72, n = 25) and M. lucifugus (mean = 262.10 n/g ± 31.08, n = 20) samples contained MC. Hexagenia consumption by bats was verified in fecal material using polymerase chain reaction. Greater understanding of the fate of MC and the potential impact of MC in terrestrial ecosystems is imperative for management of both aquatic and terrestrial ecosystems

Changing Water Levels in Lake Superior, MI (USA) Impact Periphytic Diatom Assemblages in the Keweenaw Peninsula

Predicted climate-induced changes in the Great Lakes include increased variability in water levels, which may shift periphyton habitat. Our goal was to determine the impacts of water level changes in Lake Superior on the periphyton community assemblages in the Keweenaw Peninsula with different surface geology. At three sites, we identified periphyton assemblages as a function of depth, determined surface area of periphyton habitat using high resolution bathymetry, and estimated the impact of water level changes in Lake Superior on periphyton habitat. Our results suggest that substrate geology influences periphyton community assemblages in the Keweenaw Peninsula. Using predicted changes in water levels, we found that a decrease in levels of 0.63 m resulted in a loss of available surface area for periphyton habitat by 600 to 3000 m2 per 100 m of shoreline with slopes ranging 2 to 9°. If water levels rise, the surface area of substrate will increase by 150 to 370 m2 per 100 m of shoreline, as the slopes above the lake levels are steeper (8–20°). Since periphyton communities vary per site, changes in the surface area of the substrate will likely result in a shift in species composition, which could alter the structure of aquatic food webs and ecological processes

Changing Water Levels in Lake Superior, MI (USA) Impact Periphytic Diatom Assemblages in the Keweenaw Peninsula

Predicted climate-induced changes in the Great Lakes include increased variability in water levels, which may shift periphyton habitat. Our goal was to determine the impacts of water level changes in Lake Superior on the periphyton community assemblages in the Keweenaw Peninsula with different surface geology. At three sites, we identified periphyton assemblages as a function of depth, determined surface area of periphyton habitat using high resolution bathymetry, and estimated the impact of water level changes in Lake Superior on periphyton habitat. Our results suggest that substrate geology influences periphyton community assemblages in the Keweenaw Peninsula. Using predicted changes in water levels, we found that a decrease in levels of 0.63 m resulted in a loss of available surface area for periphyton habitat by 600 to 3000 m2 per 100 m of shoreline with slopes ranging 2 to 9°. If water levels rise, the surface area of substrate will increase by 150 to 370 m2 per 100 m of shoreline, as the slopes above the lake levels are steeper (8–20°). Since periphyton communities vary per site, changes in the surface area of the substrate will likely result in a shift in species composition, which could alter the structure of aquatic food webs and ecological processes

Data from: From the field to the lab: best practices for field preservation of bat specimens for molecular analyses

Studies in molecular ecology depend on field-collected samples for genetic information, and the tissue sampled and preservation conditions strongly affect the quality of the DNA obtained. DNA yields from different tissue types have seldom been compared, and the relative performance of storage media has never been directly tested, even though these media may influence DNA degradation under field conditions. We analyzed DNA yield from buccal swabs and wing punches harvested from live bats using nucleic acid quantification as well as quantitative PCR for a single-copy nuclear locus. We also compared DNA yields from wing tissue preserved in three media: ethanol, NaCl-saturated dimethyl sulfoxide (DMSO), and silica desiccant. Wing punches yielded more total DNA than did buccal swabs, and wing tissues preserved in silica beads yielded significantly more total and nuclear DNA than those preserved in DMSO or ethanol. These results show that tissue type and preservation media strongly influence the quantity of DNA obtained from non-lethal genetic samples, and based on these effects we provide recommendations for field collection of tissues for genetic analyses

DNA quantification by Nanodrop Spectrophotometer

Analysis data of DNA yield from buccal swabs and wing punches harvested from live bats using nucleic acid quantification as well as DNA yields from wing tissue preserved in three media: ethanol, NaCl-saturated dimethyl sulfoxide (DMSO), and silica desiccant

Estimated coefficients of the effect of wing punch preservation methods on the natural logarithm of gene copies detected after accounting for differences between qPCR runs and among individual bats.

<p>Estimated coefficients of the effect of wing punch preservation methods on the natural logarithm of gene copies detected after accounting for differences between qPCR runs and among individual bats.</p

Relationship between the median number of copies of the single-copy nuclear gene obtained from repeated quantitative PCR reactions and concentration of whole DNA (wing punches in three preservation treatments).

<p>The correlation was significant whether including (<i>r</i> = 0.32, <i>p</i> = 0.018), or excluding the outlier measurements from sample individual ALR096 (<i>r</i> = 0.47, <i>p</i> = 0.00034).</p