Introduction.
Surveys for aquatic oligochaetes and other macroinvertebrates occurring in the Great Smoky Mountains National Park were begun in September 1999. Perennial funding via mini-grants from DLIA (1999-2004, 2006) have supported our continuing surveys in the Park, in September 2000, April and September/October 2001, April and August 2002, April/May and September 2003, May and October 2004, August 2005, and April 2006 (one collection). No additional collections were made in 2006 because of other project commitments; however, one field collecting trip is tentatively scheduled for August 2007. On each visit to the Park, qualitative macroinvertebrate samples have been collected from a variety of aquatic and semi-aquatic habitatas using D-ring dip nets and fine-mesh aquarium hand nets, and by hand-picking of specimens from various natural substrates (e.g., gravel, cobble, boulders, sticks, branches, logs, leaf packets, and root mats) and incidental (but extremely rare) non-indigenous substrates (e.g., bottles, cans, trash, and various forms of aggregate substrates such as cement, asphalt, bricks, and tiles).
As noted on this website's homepage, under 'Permits', all research associated with this project in the GSMNP is conducted according to the guidelines set forth in a Scientific Research and Collecting Permit issued by the USDI-NPS.
General Collecting Techniques.
Aquatic oligochaetes and other macroinvertebrates:
During the collecting process, riffle, run, pool, backwater, cascade, and torrent habitats are agitated by hand and foot, with dislodged material drifting into nets; material captured in the nets is placed in plastic buckets with habitat water, then gently agitated and swirled around in the bucket using our hands - basically, a simple elutriation process. Substrates (primarily sticks, stones, and leaves) are rinsed off by hand in the bucket, then placed back into the stream. The contents of the bucket are again elutriated by hand in habitat water (bucket about 1/3 full), strained through a fine-meshed aquarium net, then placed into whirl pacs or Nalgene jars in a small amount of habitat water. Habitat materials remaining in the bucket (e.g., mud, sand, gravel sediments, small detritus) are rinsed in similar fashion 2-3 more times to maximize retention of specimens collected (especially snails, and the case-building caddisflies that use sand, small stones, a variety of plant materials, and even snail shells in the construction of their cases). The sample (= the elutriated material that has been strained through the small aquarium nets) is then placed in the whirl pacs or Nalgene jars; samples are first relaxed in a 5-10% ethanol solution for 15-20 minutes [vis, the walk from the sampling site to the vehicle]; the use of a weak ethanol solution has two purposes - serving as both an anesthetic and as relaxant to reduce muscular contraction. After this short period of anesthetization/relaxation, the sample is fixed with 10% buffered formalin. [I strongly discourage the presence or use of formalin and other tissue fixative fluids at your aquatic sites -- an accidental spill could have serious consequences, affecting both the non-target flora, fauna, and water quality at and downstream of your site.] Many of the larger aquatic macroinvertebrates captured in the nets (e.g., crayfishes, aquatic Gerromorpha, tipulid larvae, large beetles, some larger annelids) are hand-picked directly from the nets prior to sample elutriation, relaxed in weak ethanol solution, then preserved in 80+% ethanol (or rarely, in buffered formalin) in small sample containers separate from other collected material.
Branchiobdellidans and Crayfishes:
Crayfishes are hand-picked from hand or dip-nets (and minnow seines when we use them), placed into individual whirl pacs or jars with habitat water, relaxed for 15-20 minutes by adding small, incremental amounts of 5-10% ethanol to their container, then preserved in 80+% ethanol; in this manner, specific associations of the host crayfish by ecto- and endo-commensal branchiobdellidans can be maintained. Collections of crayfishes formerly were fixed in buffered formalin; however, the crayfish and branchiobdellidan systematists we work with now prefer ethanol preservation rather than formalin fixation of specimens, especially to assure that tissue will be useful for DNA sequencing. It is recommended that the ethanol used for initial preservation be changed within a short period of time after collection so that the tissue of both the crayfish and branchiobdellidans remains suitable for DNA analyses; be sure to use a very fine-meshed net to filter rinse water (when rinsing off the crayfishes) and the fluid in your field sample containers....to retain all branchiobdellidan species that may still be loosely attached to the crayfish, or had dropped off the host during preservation, residing as residue at the bottom of the initial sample preservation jar or whirl pac. Be sure to sort through this fluid using a petri dish and dissecting microscope, and be sure to include collection and host association information on labels you then place in the vials and jars in which the branchiobdellidan and host crayfish specimens are stored. Please include a simple but unique letter or numbering system on labels placed in the vials and jars, so that future identifications of each group can be associated with one another. Host specificity of branchiobdellidans with crayfishes has not been widely documented; however, we believe that unique associations can only be substantiated if relationships are clearly established at the time of collection, and maintained during initial sample processing and curation.
Researchers collecting crayfishes in the Park (and elsewhere) are encouraged to follow the above collecting and preservation procedures, taking care not to discard the 'sediment' in the bottom of preservation containers used when you first collect the crayfishes (e.g., via the common practice of rinsing samples after the initial collection). In this way, branchiobdellidan specimens can be sorted from the sediment, and associated with the host, prior to transferring the crayfish during routine curation of those specimens. Although branchiobdellidan taxonomists and systematists often look in crayfish collections for branchiobdellidans on the bottom of curated specimens, this usually is less than rewarding, as most crayfish specimens in both institutional and personal collections have been 'rinsed' at least once after collection and initial preservation or fixation; more often, they have been rinsed two or more times prior to 'final' deposition in a collection. Obviously, this common procedure reduces or eliminates the possibility that some, if any, of the branchiobdellidans originally associated with that crayfish....are still in the jar with that original host.
Several branchiobdellidans may remain attached to the exterior of the host crayfish after initial preservation, but most seem to drop off of the host and settle to the bottom of the the jar. A few genera and species of branchiobdellidans occur in the gill chambers of host crayfishes; these are more difficult to obtain, as they do not commonly 'fall' out of, and off, of the host crayfish; these must be dissected out of the gill chambers.
Mollusks:
Mollusks (Gastropoda, and representatives of the families Ancylidae and Sphaeriidae), when collected, are first relaxed in a weak (5-10%) ethanol solution, then preserved in 80+% ethanol. To date, neither live specimens nor shell material of the pelecypod families Unionidae, Corbiculidae, or Dreissenidae have been observed at any of the sites that we have surveyed in the Park.
Tissue analysis for DNA:
We have not yet collected any material for DNA analyses, but intend to do so in 2007, specifically, for crayfishes and associated branchiobdellidans. Tissue preservation techniques we will use will be posted here soon. Those of you wishing to preserve invertebrate tissue using near-absolute [80-95%, non-denatured] ethanol so that future DNA studies can be conducted....should first verify the proper procedures for taking and preserving animal tissue by contacting one or more professionals who focus on DNA sequencing. In a pinch, grain alcohol (everclear or gemclear) from a local liquor store could be used.
Sensitivity to Uniqueness of Habitats.
A priority during our surveys for aquatic macroinvertebrates has been to minimize physical disturbance of the habitat present in those sites we choose to survey. In particular, we are extremely careful to abbreviate collecting efforts for aquatic macroinvertebrates in the finite habitats associated with any springs, seep, and rock-face areas; only a small area of the substrate is agitated at the springhead, regardless of its size; additionally, only one finite area in the springrun (usually between 0.5 and 25m downstream of the springhead) is surveyed; collections in seeps and rock-face areas are also completed with minimal disturbance. Because formalin is a poisonous fluid and known carcinogen, we never take it near any aquatic habitat. The fixation and preservation of all samples is always completed some distance from the surveyed habitat and its drainage, usually at the field vehicle. As biologists, we can often be the most significant, negative impact on a habitat, even though our interests and the objectives of our research are to contribute to the understanding of communities occurring there, and the long-term protection of that habitat. To paraphrase Pogo, 'We have met the enemy, and he is us.'
Sensitivity To / Incidental Take of Non-targeted Fauna.
Fishes and amphibians are occasionally collected in our dipnets, an inevitable expectation because of our collecting techniques; when representatives of these groups are observed (within seconds after removal of net from the aquatic habitat), they are carefully removed from the net using a wet hand and immediately returned live to the specific habitat from which they were obtained. Occasionally, however, a few small specimens of these two groups are collected incidentally. After samples are returned to the lab, these incidental specimens are processed, properly labeled, identified if possible, and forwarded to systematic experts associated with the ATBI project who are studying those groups.
Other Studies Supported by this Fieldwork.
1. Aquatic fungi: During the course of our surveys at stream, spring, and standing water habitats, a variety of partially/fully denuded sticks (dia.: ~0.2 to 1 cm; length: ~2 to 4 cm) are collected. These short sticks are placed in zip-lock bags, drained of excess water, then stored on ice while in the field and during transport back to Champaign. These sticks are then given to Dr. Carol Shearer, University of Illinois Department of Plant Biology, Champaign. Dr. Shearer's research focuses on the ecology of freshwater saprophytic fungal communities, the systematics and biogeography of freshwater Ascomycetes and their anamorphs, and Fungi Imperfecti; she also has been working for several years with graduate students in their study of these groups fungi and their occurrence in the Great Smoky Mountains National Park. To date, samples of denuded sticks collected from over 90 sites in the Park (1999-2004) have been transferred to Dr. Shearer's lab.
2. Other aquatic macroinvertebrates: A broad diversity of non-annelid aquatic macroinvertebrates are collected during our surveys; these are clean-sorted to order and family level, placed in 70-80% ethanol in glass vials and jars with archival-quality locality labels that include complete georeference information, then transferred to other ATBI researchers for assimilation into their science programs. At the annual DLIA meeting convened in Gatlinburg, TN in early December 2002, over 300 vials and jars (>5,000 specimens) were transferred to ATBI personnel. Subsequent transfers of aquatic insects (especially Trichoptera - the caddisflies) have been made to Dr. Chuck Parker at the Twin Creeks Research Center, and to other ATBI systematists at the annual DLIA meeting in December 2004. Numerous other specimens from our 1999-2005 collections await time and funding for proper curation and transfer to ATBI specialists.
Field Water Quality Measurements:
Several physical and chemical water quality parameters are measured prior to biological collections at each site. These include ambient and water temperature (hand-held thermometer or meter thermistor); dissolved oxygen (YSI Model 57, or YSI Model 95D dissolved oxygen/temperature meter); conductivity and salinity (YSI Model 33 S-C-T conductivity and salinity meter); turbidity (Hanna Instruments Model HI 93703 microprocessor turbidity meter); hydrogen ion concentration (as pH) and total dissolved solids (TDS) (Hanna Watercheck 1 meter). The above parameters were measured (using the stated equipment) during the surveys conducted in 1999 and 2000.
A Yellow Springs Instrument Company, Inc. (YSI) Model 556 multimeter [water temperature, dissolved oxygen, % saturation DO, pH, specific conductivity, salinity, total dissolved solids, ORP, resistivity, and barometric pressure] was used during the April and September/October 2001, April and August 2002, April/May and September 2003, May and October 2004, and August 2005 collecting trips. This meter was kindly loaned to us by YSI (Tim Grooms, YSI Research and Development team) for the collecting trips in 2001. Subsequent to my field evaluation of this new meter, YSI facilitated a corporate research contribution (reduction in retail price of the 556 multi-meter) to this and other research programs at the INHS. [I cannot provide specific endorsements of commercially available products; however, I have used numerous meters in the YSI product line for over 30 years, and they have been extremely reliable. The Model 556 meter has performed flawlessly during our investigations in the Park, and also on two 9-10 day, 230 mi rafting /field collecting expedition on the Colorado River and its tributaries in the Grand Canyon National Park in 2001 and 2006. In addition, this meter also served flawlessly in an 'emergency field situation' after a comparable meter (manufactured by a competing company) failed to 'meet expectations'.]
Habitat characterization:
Habitat characterization of each site, including the width and depth of the major habitats present, and substrate composition, and the presence of submerged or floating vegetation (e.g., roots, vascular plants, algae, mosses) are recorded while the water quality meter is stabilizing for the parameters being measured. Each site is geo-referenced (Garman eTrex Vista GPS unit) and noted in pencil on 7.5' USGS topographic quadrangle maps. A map of is drawn on the back of the data sheet for each site. Photographs (SLR-color slide, digital p&s, and/or digital SLR) are also taken at each site. Interesting observations are reported to NPS and DLIA/ATBI personnel, when appropriate.
Data Associated with Collections:
We complete a field data sheet for each site we survey in the Park. This information includes specific locality information (e.g., distance from known landmarks, georeference [lat/long, UTM] coordinates obtained from a field GPS unit, and information obtained from U.S. Geological Survey topographic quadrangle maps we have with us in the field), time, elevation, values for measured field water quality parameters, general habitat conditions, site drainage hierarchy, hand-drawn map of the site, information related to digital and film photographs of the site, and other observations. All geo-reference information recorded and obtained during field studies is verified against topographic quadrangle maps and the resources provided by TopoZone.com. Odd or other interesting observations (e.g., evidence of poachers, hogs and other wildlife, damage to Park property, and unsafe road conditions) are also recorded, and reported to Park personnel, as appropriate. Specific locality information for all sites we have visited during this study, and procedure for verification of data associated with each site, are available via the link "Site Map, Locality Information" in the site navigation bar at the bottom of this page.
All data recorded on our field data sheets are then entered into a project database (FileMakerPro, v. 6) upon return to the INHS in Champaign. We are in the process of transferring all data associated with our collections to the ATBI database being maintained by Chuck Parker, Michael Kunze, and Chuck Cooper at the Twin Creeks Research Center.
Permits:
All research for this project complies with and has been conducted under the following Scientific Research and Collecting Permits, issued by the USDI-NPS to Mark J. Wetzel: GRSM-99-124; GRSM-00-133; GRSM-2001-SCI-0094, and GRSM-2002-SCI-0015 [2002-2004], and GRSM-2005-SCI-0020 [valid 1 April 2005 - 31 March 2008]. Investigator Annual Reports (IAR's) summarizing the objectives, findings, and results of our research have been submitted to the NPS, in partial fulfillment of permit requirements.
I encourage you to use the site navigation bar, below, to familiarize yourself with the other aspects of this specific project, other research projects and educational opportunities associated with Discover LIfe in America and the All Taxa Biodiversity program, and the Great Smoky Mountains National Park.
page update: 18 January 2008