USE OF ANALYTICAL METHODS TO DETERMINE HEAVY METAL CONCENTRATION OR LOCATION IN FRUITING STRUCTURES OF SLIME MOLDS (MYXOMYCETES)

Carolyn J. McQuattie, USDA Forest Service, 359 Main Road, Delaware, Ohio 43015, and Steven L. Stephenson, Fairmont State College, Department of Biology, Fairmont, West Virginia 26554. cmcquattie@fs.fed.us

ABSTRACT

Fruiting bodies of slime molds collected from decaying wood or bark in the Great Smoky Mountains National Park were analyzed by atomic absorption (Pb) or inductively coupled plasma emission spectrometry (Fe, Mn, Cu, Zn, Al) to determine heavy metal concentration. Additional specimens were prepared for electron microscopy (TEM) and X-ray microanalysis (EDX) to determine location or accumulation of heavy metals in various regions of the fruiting body. Of the species examined, Fuligo septica (Physarales) had the highest concentrations of Pb, Mn, and Zn. By EDX, metals (Fe, Cr, Mn, Si, Al) were detected as crystalline precipitates in the stalks of fruiting bodies. Viewed by TEM, clusters of bacteria were observed in stalk and hypothallus regions; bacteria and individual spores in sporangia contained polyphosphate bodies (containing P, Ca, K) in their cell cytoplasm. The analytical methods used demonstrate bioaccumulation of heavy metals in myxomycetes and suggest that metal precipitation in the stalk region may provide tolerance to high metal levels.

INTRODUCTION

Heavy metals in forest soils and litter are derived from both anthropogenic (pollutant) and natural (soil weathering) sources. The metal load may vary by elevation (Reiners et al. 1975).

Plasmodia of slime molds (myxomycetes) obtain nutrients by ingesting bacteria, fungal hyphae and spores, and particles of organic matter from their immediate environment (Stephenson and Stempen 1994). Some myxomycetes appear to be tolerant of high levels of heavy metals and apparently accumulate them vigorously (Setala and Nuorteva 1989). Although myxomycetes may bioaccumulate metals, it is not known if heavy metals are dispersed throughout plasmodia or sequestered in distinct regions of the fruiting bodies that develop from plasmodia. Because myxomycetes are ubiquitous in terrestrial ecosystems and accumulate material only from their immediate environment, they may be useful in biomonitoring heavy metal contamination at different locations/elevations across the landscape. The objectives of this preliminary study were to 1) analyze myxomycete fruiting bodies collected from different elevations in the Great Smoky Mountains National Park for element concentration, and 2) determine potential sites of element accumulation in various regions of the fruiting body.

METHODS

During the 1999 field season, fruiting bodies of myxomycetes were collected from decaying wood or bark at high (H, >1700 m), mid (M, ca. 1200 m), or low (L, <700m) elevation sites in the Great Smoky Mountains National Park (in western North Carolina and eastern Tennessee). Individual specimens from H and L elevations (species listed in Table 1) were air-dried and sent to Brookside Laboratories (New Knoxville, OH) for analysis of element concentration. Sample material was treated by standard nitric acid-perchloric acid digestion methods. Analysis of Pb was carried out on a Varian 220 Zeeman graphite furnace atomic absorption spectrophotometer in order to achieve low detection levels. All other trace metals were analyzed on a Thermo Jarell ash inductively coupled plasma spectrometer (ICP). Collection numbers are those of the second author.

Individual mature fruiting bodies (sporangium plus stalk and/or hypothallus) collected from H elevation (Trichia decipiens 11545 and Physarum viride 11575), from M elevation (Hemitrichia calyculata 11604 and P. viride 11614) or from L elevation (H. calyculata 11620, T. decipiens 11621, and T. favoginea 11622) were prepared for light microscopy (LM), transmission electron microscopy (TEM), and energy-dispersive X-ray microanalysis (EDX). All fruiting bodies were collected from 7/16/99-7/18/99. Specimens were fixed in 3% glutaraldehyde, dehydrated in an ethanol series (30%-100%), and embedded in PolyBed-Araldite epoxy resin. Semi-thin (2 μm) cross sections of stalks and sporangia were examined with an Olympus BH2 light microscope. Ultrathin (90-110 nm) sections were cut with a diamond knife using an ultramicrotome, transferred to Cu grids, and examined in a JEOL JEM-1010 TEM. EDX analysis of sections was performed in the same TEM, which was equipped with a Link ISIS microanalysis system (Oxford Instruments) utilizing a Link Pentafet Si(Li) detector. TEM conditions for EDX included: 80 kV accelerating voltage, current density 2.0 pA/cm² , goniometer tilt 30^o, magnification at 50,000x. All spectra were collected for 100 seconds. Significant element peaks from each cellular component were identified by the Link Systems AUTO-Identification program (Link ISIS Microanalysis Group, Buckinghamshire, England).

RESULTS

Overall, concentrations of Pb, Fe, Mn, and Zn were highest in Fuligo septica fruiting bodies (Table 1). No consistent differences between specimens from H vs. L elevation sites were apparent, although a collection of Trichia favoginea from H elevation displayed higher concentrations of several metals than a collection of the same species from L elevation.

Viewed by LM, stalks of Hemitrichia and Physarum were filled with cytoplasmic material (Fig. 1), whereas stalks of Trichia had an outer ring of cytoplasmic material surrounding an inner empty cavity (lumen). By TEM, the stalk of Hemitrichia had clusters of bacteria encased as discrete inclusions that were interspersed with similar regions containing deteriorated (possibly plasmodial) material (Fig. 2). Physarum stalks contained crystalline compounds or dense precipitates (Fig. 3) adjacent to amorphous cytoplasmic material and bacteria containing electron-dense polyphosphate (polyP) bodies (Fig. 3). Sporangia from each species contained individual spores enclosed by either a thick (Trichia) or thin (Hemitrichia, Physarum) peridium. Individual spores contained cytoplasm, numerous cellular organelles, and polyphosphate bodies (not shown).

In this study, heavy metals were detected only in the stalk region. EDX analysis showed the electron-dense precipitate in Physarum (Fig. 3) contained Fe, Si, Al, Mg (labelled M on spectrum), Cl, Cr, and Mn (Fig. 4). By contrast, regions of the Hemitrichia stalk with deteriorated plasmodial material (Fig. 2) contained only P and Cl. The most common metals detected in stalk precipitates were Fe, Al, and Cr. Precipitates of Al, Si, K, and Cl on outer stalk or peridium (Fig. 5) walls were seen frequently in all species examined and possibly represent minute soil particles. In all species, the polyP bodies seen in spores (within sporangia) and in bacteria from stalk regions contained P, Ca, and occasionally K (spectrum not shown).

DISCUSSION

Trace metals were detected in myxomycete stalk or hypothallus regions (near the base of the fruiting structure) as electron-dense precipitates among bacteria and areas of cytoplasmic material. These metals appeared to be ‘complexed’ and thus may not affect myxomycete growth or reproduction. Setala and Nuorteva (1989) reported a higher concentration of Zn in the hypothallus of Fuligo septica than in other areas of the fruiting body. Tolerance to high levels of heavy metals may be related to the ability to sequester them in regions where reproduction is unaffected.

Members of Physarales (Fuligo and Physarum) had higher metal content than other species analyzed in this study (i.e., Fuligo from element concentration analyses and Physarum from EDX analysis). Lead concentration was highest in Fuligo. This species may be useful as an indicator of pollution, and further examination of Fuligo (to determine the location of Pb in the fruiting body) should be pursued. Moreover, the considerable variation in metal concentrations between the two Fuligo collections from L elevation indicates that additional collections of all myxomycete species are needed to determine whether a relationship exists between metal concentration and site elevation in the Great Smoky Mountains National Park.

In myxomycete spores, elements (P, Ca, K) were detected only in polyphosphate bodies. Whallon et al. (1989) reported that approximately 0.1% concentration (by weight) is needed for significant element detection by EDX. It is possible that some trace metals in sporangia (or stalks) may be too dispersed to be detected by EDX. Therefore, the use of atomic absorption and ICP (to analyze whole fruiting structures and detect elements at very low concentrations) and TEM/EDX (to determine where metals are concentrated) represents a good combination of analytical methods to determine heavy metal uptake and localization.

REFERENCES

1) Reiners, W.A., Marks, R.H., Vitousek, P.M. 1975, Oikos 26:264-275.

2) Setala, A., Nuorteva, P. 1989, Karstenia 29:37-44.

3) Stephenson, S.L., Stempen, H. 1994, Myxomycetes: A Handbook of Slime Molds. Timber Press, Portland, Oregon.

4) Whallon, J.H., Flegler, S.L., Klomparens, K.L., 1989, Bioscience 39:256-259.

Table 1. Metal concentrations (ppm) in individual slime molds collected from decaying wood or bark at two elevations in the Great Smoky Mountains National Park.

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Species Elevation Pb Fe Mn Cu Zn Al

Fuligo septica 2401 H 7.65 116 1049 9 533 135

Fuligo septica 11698 L 3.48 143 2173 27 3477 195

Fuligo septica 15706 L 6.02 40 1015 12 386 57

Trichia favoginea 15689 H 2.05 146 128 10 94 193

Trichia favoginea 15671 L 0.20 41 145 10 69 37

Stemonitis splendens 15714 L 0.37 44 109 6 68 63

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Fig. 1. Cross sections of two Hemitrichia calyculata stalks, LM. Bar = 200 mm.

Fig. 2. H. calyculata stalk, TEM. Clusters of bacteria (B) and areas of deteriorated plasmodial material (P) appeared to be separated by distinct wall material (D). W, outer stalk wall.

Bar = 2 μm.

Fig. 3. Physarum viride stalk, TEM. Electron-dense precipitates (E) accumulated between cytoplasmic material (P) and bacteria (B) containing polyP bodies (arrowhead). Bar = 1 μm.

Fig. 4. X-ray spectrum (0-10 KeV) of elements from crystalline precipitate in Fig. 3. Cu peaks are from the Cu support grid used; peaks between 0-1 KeV are C and O. Vertical axis = total counts.

Fig. 5. Spectrum from precipitate on peridium wall. Total counts, Cu, C, and O peaks, as above.