Methods

Our study lake, South Sparkling Bog (SSB) (46.003°N, 89.705°W), is a bog lake
located in Vilas County in Northern Wisconsin. South Sparkling Bog is a dystrophic,
dimictic lake with a maximum depth of 8 m, a mean depth of 3.6 m, and a surface area of
0.44 ha. South Sparkling Bog is surrounded by a sphagnum bog mat and has no shoreline
development. During the winters of 2019-2020 and 2020-2021, snow was removed from the
surface of South Sparkling Bog following any snow accumulation event. Removal was
conducted via a snowplow attached to the front of an ARGO all-terrain vehicle and a
snowblower. The winter of 2018-2019 served as a reference year, and snow was not removed
from South Sparkling Bog’s surface. While ice cover persisted, plankton samples were
collected at the deep spot for each lake during on a biweekly-to-monthly basis each
winter. On each sampling date, one integrated zooplankton tow was taken at a depth of 0-7
m using a 56 µm mesh Wisconsin net. All zooplankton samples were collected into glass
sample jars, preserved in 90% ethanol, and saved for laboratory analysis. In the lab,
zooplankton samples were filtered through 53 µm mesh and diluted to a known volume, and
three sub-sample replicates were taken using a 1 mL Hensen-Stempel pipette. Sub-sample
replicates were counted to at least 100 individuals, otherwise the entire sample was
quantified. Sub-sample data was then converted to the known diluted volume and finally
converted to total filtered volume (from the integrated tow sample) to estimate density
(individuals L-1). Zooplankton samples were processed using a Leica M8Z dissecting scope
and Leica imaging software. Replicate subsamples were averaged to estimate total abundance
and density, and average lengths (mm) for each sample taxa were calculated from measures
of the first 30 taxa found within a sample date.<br/>

Methods

In the laboratory, three measurements of body size and seven measurements of biomass were captured. First, any foreign material found adhering to the external surface of specimens was completely removed. Body size directional measurements of shell length (L), width (W), and height (H) were recorded for every specimen with the aid of callipers (0.01 mm). Following this, any excess water was removed from surfaces by drying the external shell with tissue paper. Further, using a scalpel blade and tweezers, excess water was removed from the mantle cavity by gently forcing bivalves to gape, taking care not to cut the adductor muscle or damage tissues. Using high-resolution scales, living-weight (LW) was obtained for each specimen. Then each specimen was fully opened, which in most cases involved cutting of the adductor muscles. To remove additional fluid from the mantle and other cavities, each specimen was then placed with the valve gape (flesh) facing downwards onto absorbent tissue, for ~5-10 minutes. A wet-weight (WW) was obtained for each specimen. Following this, the soft tissue was dissected from the shell, then both soft tissue and shell were dried together within an oven (60-72 degreeC) for ~48 hrs, or until they reached a constant weight. Specimens were cooled to room temperature in a desiccator before final weighing. A combined dry-weight (DW) was recorded, as were weights for the soft tissue and shell separately, i.e. shell free dry-weight (SFDW) and dry shell-weight (SW), respectively. Following the establishment of SW, SFDW was calculated subtracting SW from the total DW (i.e. SFDW = DW–SW). To obtain an ash-weight (AW), the soft and hard tissue structures of specimens were incinerated (500–550 degreeC) together within a muffle furnace for 4–6 hrs. In all cases, the ash free dry-weight (AFDW) was then calculated for the entire specimen (soft tissue and shell) by subtracting the AW from DW, i.e. AFDW = DW–AW.<br/>

Methods

We sampled larval zebra mussels (veligers) using a 64 microm mesh, 0.5 m diameter
plankton net and stored them in 80% ethanol in 200 mL containers at 25degree C for 0-12
weeks until processing. At each site we performed triplicate 8 m depth plankton tows by
pulling a net from 2 m above the lake bottom at the 10 m depth sites of transects A-C
developed for adult zebra mussel collection. We collected samples approximately every 14
days from June to August in 2016, and June to November in 2017-2019. During fall
sampling, poor weather conditions occasionally limited the number of sites or replicates
collected. We also sampled veligers biweekly in 2019 but reduced sampling to one
replicate per site and only sampled at one site after September. Because veligers are
small and difficult to see, enumeration was time consuming. <br/>We sampled larval zebra mussels (veligers) using a 64 microm mesh, 0.5 m diameter
plankton net and stored them in 80% ethanol in 200 mL containers at 25degree C for 0-12
weeks until processing. At each site we performed triplicate 8 m depth plankton tows by
pulling a net from 2 m above the lake bottom at the 10 m depth sites of transects A-C
developed for adult zebra mussel collection. We collected samples approximately every 14
days from June to August in 2016, and June to November in 2017-2019. During fall
sampling, poor weather conditions occasionally limited the number of sites or replicates
collected. We also sampled veligers biweekly in 2019 but reduced sampling to one
replicate per site and only sampled at one site after September. Because veligers are
small and difficult to see, enumeration was time consuming. <br/>We sampled larval zebra mussels (veligers) using a 64 microm mesh, 0.5 m diameter
plankton net and stored them in 80% ethanol in 200 mL containers at 25degree C for 0-12
weeks until processing. At each site we performed triplicate 8 m depth plankton tows by
pulling a net from 2 m above the lake bottom at the 10 m depth sites of transects A-C
developed for adult zebra mussel collection. We collected samples approximately every 14
days from June to August in 2016, and June to November in 2017-2019. During fall
sampling, poor weather conditions occasionally limited the number of sites or replicates
collected. We also sampled veligers biweekly in 2019 but reduced sampling to one
replicate per site and only sampled at one site after September. Because veligers are
small and difficult to see, enumeration was time consuming.

Methods

Two 30-meter vertical tows (0.5m diameter, 400um mesh net) are collected at the deepest part of Trout Lake each time the lake is visited for routine LTER sampling during open water. On occasion, tows are collected on additional dates. Samples are visually scanned in their entirety for number of Bythotrephes present. The samples are not preserved or archived.

Methods

One zooplankton sample was collected in June 2015 at the deepest part of each lake, via vertical tow with a Wisconsin net (20cm diameter, 80um mesh). Contents of the net were preserved in the field with cold 95% ethanol. Subsamples of each vertical tow sample were counted for zooplankton species, using enough volume to count at least 300 individuals. A larger volume was then visually scanned to look for presence of additional species not seen in the count volume, until at least 2000 individuals had been seen.

Methods

formulas are based on data in literature or were determined in samples from research lakes:

Culver D.A. et.al. 1985. Can. J. Fish. Aquat. Sci. Vol 42, 1380-1390.
Biomass of freshwater crustacean zooplankton from length-weight regressions.

Downing, John A. and Frank H. rigler. 1984.
A manual on methods for the assessment of secondary productivity in fresh waters. Second edition.

Dumont, H.J., I. Van de Velde and S. Dumont. Ref??
The dry weight estimate of biomass in a selection of cladocera, copepoda and rotifera from the plankton, periphyton and benthos of continental waters.

Hawkins, Bethany E. and M.S. Evans. 1979. J.Great Lakes Res. 5(3-4):256-263
Seasonal cycles of zooplankton biomass in southeastern Lake Michigan

Lawrence, S.G., D.F. Malley, W.J. Findlay, M.A. MacIver and I.L. Delbaere. 1987. Can J. Fish. Aquat. Sci. 44: 264-274.
Methods for estimating dry weight of freshwater planktonic crustaceans from measures of length and shape.

Pace M.L. and J.D. Orcutt. 1981. Limnol. Oceanogr. 26(5), 822-830.
The relative importance of protozoans, rotifers, and crustaceans in a freshwater zooplankton community.

Yan N.D. and G.L. Mackie. 1987. Can. J. Fish. Aquat. Sci. Vol 44, 382-389.
Improved estimation of the dry weight of Holopedium gibberum using clutch size, a body fat index, and lake water total phosphorus concentration.

Ruttner-Kolisko A. 1977. Arch. Hydrobiol. Beih. Ergebn. Limnol. 8, 71-76.
Suggestions for biomass calculations of plankton rotifers.

Methods

We collect zooplankton samples at the deepest part of the lake using two different gear types. We take one vertical tow with a Wisconsin Net (80um mesh), and a series of Schindler Patalas (53um mesh) samples spanning the water column. All samples are preserved in cold 95percent EtOH.
After collection we combine subsamples of the individual Schindler Patalas trap samples to create one hypsometrically pooled sample for each lakeordate. The individual depth samples are discarded after pooling except from one August sampling date per year. The Hypsometrically Pooled sample and the Wisconsin Net sample are archived in the UW Zoology museum.
We count zooplankton in one or two subsamples, each representing 1.8L of lake water, of the hypsometrically pooled samples to calculate zooplankton abundance. We count one sample date per month from the open water season, and the February ice cover sample. We identify individuals to genus or species, take length measurements, and count eggs and embryos.
Protocol log: 1981-May1984 -- a 0.5m high, 31L Schindler Patalas trap with 80um mesh net was used. Two Wisconsin Net tows were collected. Preservative was 12percent buffered formalin.
June1984 -- changed to 53um mesh net on Schindler trap.
July1986 -- began using the 2m high, 45L Schindler Patalas trap. Changed WI Net collection to take only one tow.
2001 -- changed zooplankton preservative from 12percent buffered formalin to 95percent EtOH.
The number of sample dates per year counted varies with lake and year, from 5 datesoryear to 17 datesoryear.
1981-1983 -- pooled samples are of several types: Total Pooled (TP) were created using equal volume subsamples of the Schindler samples. Epi, Meta, Hypo pooled used equal volume subsamples from the Schindler samples collected from each of the thermal strata. Strata Pooled used equal volume subsamples from the Epi, Meta, Hypo pooled samples to create an entire lake sample. Hypsometrically Pooled (HP) is our standard, which uses subsample volumes weighted to represent the hypsometry of the lake.

Methods

Primary publications that provide more information about taxa, methods, and data are:
Carpenter, S.R., J.J. Cole, M.L. Pace, R.D. Batt, W.A. Brock, T. Cline, J. Coloso, J.R. Hodgson, J.F. Kitchell, D.A. Seekell, L. Smith and B. Weidel. 2011. Early warnings of regime shifts: A whole-ecosystem experiment. Science 332: 1079-1082.
Cline, T.J., D. A. Seekell, S. R. Carpenter, M. L. Pace, J. R. Hodgson, J. F. Kitchell, and B. C. Weidel 2014. Early warnings of regime shifts: evaluation of spatial indicators from a whole-ecosystem experiment. Ecosphere 5:art102. http://dx.doi.org/10.1890/ES13-00398.1
Pace, M.L., S.R. Carpenter, R.A. Johnson and J. T. Kurzweil. 2013. Zooplankton provide early warnings of a regime shift in a whole-lake manipulation. Limnology and Oceanography 58: 525-532.
For an explanation of our rationale and expected results see:
Carpenter, S. R., Brock, W. A., Cole, J. J., Kitchell, J. F., & Pace, M. L. 2008. Leading indicators of trophic cascades. Ecology Letters, 11(2), 128-138. doi:DOI 10.1111/j.1461-0248.2007.01131.x

Methods

Sampling:
Zooplankton were sampled approximately weekly with two net hauls through the water column (30 cm diameter net, 80 um mesh). Tows were taken at standard depths for almost all years. The standard depths are as follows: Peter, East Long, West Long, Crampton and Tuesday Lakes: 12m, Paul Lake: 8m, Ward Lake: 6m; exceptions are: for 2012 and beyond Tuesday Lake was sampled at 10m, Peter was sampled at 10m from 1984-1986, Paul was sampled at 7.5m in 1995. Samples were preserved with cold sugared formalin or Lugol's solution.

Methods

general monitoring for spiny water flea:
The dataset contains collected Bythotrephes longimanus monitoring efforts from 8 invaded lakes in Wisconsin that took place over the course of 2009 through 2014 using a zooplankton net. Monitoring efforts were conducted to 1) obtain more accurate estimates of Bythotrephes densities using a more appropriately sized net (50-cm diameter over 30-cm diameter) and 2) obtain detailed demographic measurements of Bythotrephes morphology and reproduction in each lake. Here only Bythotrephes densities are included.
The majority of samples occurred at a lakes deep hole with a 50-cm diameter and 150-micron mesh zooplankton net. Nets are lowered to 2 m off of the lake bottom before being towed to the surface. Samples are processed in their entirety
Exceptions to this are those at sites containing “LTER” (e.g., site IDs LTER-DH and LTER-MB) in their ID which were samples taken according to the Southern Lakes LTER zooplankton collection protocol with a 30-cm and 83-micron mesh. Other exceptions include sites outside the deep hole of the lake (site ID 5m = 5m lake depth north of the Center for Limnology on Lake Mendota; CFL = 15m lake depth north of the Center for Limnology; DH = deep hole but specific to Lake Mendota; MB = 15m lake depth southwest of Maple Bluffs in Madison on Lake Mendota; MO.5m = a 5m lake depth site in Lake Monona; MO.Y = 5m lake depth site at the mouth of the Yahara River on Lake Monona; TL = 15m lake depth west of Tenney Locks in Madison on Lake Mendota; WS = 15m site in northwestern basin of Lake Mendota, east of Picnic Point; WP = 5m site south of Warner Park on Lake Mendota). Several tows were taken using a 200m oblique (i.e., horizontal) net tow with the 50-cm diameter net (DH-ObliqueTow). Efforts in Southern Wisconsin were led by Jake Walsh while efforts in Northern Wisconsin were led by Carol Warden (site ID = CW), Pam Montz (site ID = PM), Sam Christel (site ID = SC), Sam Oliver (site ID = SM), as well as a researcher with initials (site ID) “EM”.