Nematode Extraction Techniques

Nematodes may be extracted by a variety of techniques, either active or passive in nature. The principal advantage of the oldest, active method, namely the Baermann funnel method, is that it is simple, requiring no sophisticated equipment or electricity. It is based on the animal's movement and gravity. Samples are placed on coarse tissue paper, on a coarse mesh screen, and then placed in the cone of a funnel and immersed in water. After crawling through the moist soil and filter paper, the nema-todes fall down into the neck of the funnel and fall to the bottom of the funnel stem, which is closed off with a screw clamp on a rubber hose. At the conclusion of the extraction (typically 48 h), the nematodes in solution are drawn off into a vial and kept preserved for examination later. Drawbacks to the technique are that only active nematodes are extracted. It also allows dormant nematodes to become active and eggs to hatch into juveniles and be extracted, yielding a slightly inflated estimate of the true, "active" population at a given time. For more accuracy in determination of populations, the passive, or flotation, techniques are generally preferred. Passive methods include filtration, or decanting and sieving, and flotation/centrifugation (Coleman et al., 1999) to remove the nematodes from the soil suspension. Elutriation methods can be employed for handling larger quantities of soil, usually greater than 500 g, or to recover large amounts and a greater diversity of nematodes. Elutriation methods rely on fast mixing of soil and water in funnels. Semiautomatic elutriators, which enhance the number of soil samples to be extracted, are available (Byrd et al., 1976). There are many references comparing methods, including McSorley and Frederick (2004), Schouten and Arp (1991), and Whitehead and Hemming (1965). Anhydrobiotic nematodes can be extracted in a high-molarity solution such as sucrose, which prevents the nematodes from rehydrating (Freckman et al., 1977).

microarthropods

Large numbers of the microarthropod group (mainly mites and collembolans) are found in most types of soils. A square meter of forest floor may contain hundreds of thousands of individuals representing thousands of species. Microarthropods have a significant impact on the decomposition processes in the forest floor and are important reservoirs of biodiversity in forest ecosystems. Many microarthropods feed on fungi and nematodes, thereby linking the microfauna and microbes with the meso-fauna. Microarthropods in turn are prey for macroarthropods, such as spiders, beetles, ants, and centipedes, thus bridging a connection to the macrofauna.

In the size spectrum of soil fauna, the mites and collembolans are mesofauna. Members of the microarthropod group are unique, not so much because of their body size as because of the methods used for sampling them. Small pieces of habitat (soil, leaf litter, or similar materials) are collected and the microarthropods extracted from them in the laboratory. Most of the methods used for microarthropod extraction are either variations of the Tullgren funnel, which uses heat to desiccate the sample and force the arthropods into a collection fluid, or flotation in solvents or saturated sugar solutions followed by filtration (Edwards, 1991). Generally, flotation methods work well in low organic, sandy soils, while Tullgren funnels perform best in soils with high organic matter content. Flotation procedures are more laborious than the Tullgren extraction. Better estimates of species number may be achieved using fewer, larger samples. However, valid comparisons of microarthro-pod abundance in different habitats may be obtained even if extraction efficiencies, though unknown, are similar.

Microarthropod densities vary during seasons within and between different ecosystems. Generally, temperate forest floors with large organic matter content support high numbers (33-88 X 103m~2) and coniferous forests may have in excess of 130 X 103m~2. Tropical forests, where the organic layer is thin, contain fewer microarthropods (Seastedt, 1984; Coleman et al., 2004). Tillage, fire, and pesticide applications typically reduce populations, but recovery may be rapid and microarthropod groups respond differently. Soil mites usually outnumber collem-bolans but the latter become more abundant in some situations. In the springtime, forest leaf litter may develop large populations of "snow fleas" (Hypogastrura nivicola and related species). Among the mites themselves, the oribatids usually dominate but the delicate Prostigmata may develop large populations in cultivated soils with a surface crust of algae. Estimation of species richness is a difficult problem for most soil fauna, including ants.

enchytraeids

In addition to earthworms (discussed under Macrofauna), another important family of terrestrial Oligochaeta is the Enchytraeidae. This group of small, unpig-mented worms, also known as "potworms," is classified within the "microdrile" oligochaetes and consists of some 600 species in 28 genera. Species from 19 of these genera are found in soil, the remainder occur primarily in marine and freshwater habitats (Dash, 1990; van Vliet, 2000). The Enchytraeidae are thought to have arisen in cool temperate climates where they are commonly found in moist forest soils rich in organic matter. Various species of enchytraeids are now distributed globally from subarctic to tropical regions.

Keys to the common genera were presented by Dash (1990). Identification of enchytraeid species is difficult, but genera may be identified by observing internal structures through the transparent body wall of specimens mounted on slides.

The Enchytraeidae are typically 10-20 mm in length and are anatomically similar to the earthworms, except for the miniaturization and rearrangement of features overall. They possess setae (with the exception of one genus), and a clitellum in

Clitellum Female Worm
FIGURE 7.6 Two enchytraeid worms tunneling through a pine needle, indicated by arrows (fecal pellets have been deposited on the outside). F1 layer (modified from Ponge, 1991).

segments XII and XIII, which contains both male and female pores. Sexual reproduction in enchytraeids is hermaphroditic and functions similarly to that in earthworms. Cocoons may contain one or more eggs, and maturation of newly hatched individuals ranges from 65 to 120 days depending on species and environmental temperature (van Vliet, 2000). Enchytraeids also display asexual strategies of parthenogenesis and fragmentation, which enhance their probability of colonization of new habitats (Dosza-Farkas, 1996).

Enchytraeids ingest both mineral and organic particles in the soil, although typically of smaller size ranges than those of earthworms. Numerous investigators have noted that finely divided plant materials, often enriched with fungal hyphae and bacteria, are a principal portion of the diet of enchytraeids. Microbial tissues are probably the fraction most readily assimilated because enchytraeids lack the gut enzymes to digest more recalcitrant soil organic matter (van Vliet, 2000). Didden (1990, 1993) suggested that enchytraeids feed predominantly upon fungi, at least in arable soils, and classified a community as 80% microbivorous and 20% saprovorous. As with several other members of the soil mesofauna, the mixed microbiota that occur on decaying organic matter, either litter or roots, are probably an important part of the diet of these creatures. The remaining portions of organic matter, after the processes of ingestion, digestion, and assimilation, become part of the slow-turnover pool of soil organic matter. Zachariae (1964) suggested that so-called "collembolan soil," said to be dominated by collembolan feces (particularly low-pH mor soils), were actually formed by Enchytraeidae. Mycorrhizal hyphae have been found in the fecal pellets of enchytraeids from pine litter (Fig. 7.6,

Ponge, 1991). Enchytraeids probably consume and further process larger fecal pellets and castings of soil macrofauna, such as collembolans and earthworms (Zachariae, 1964; Rusek, 1985). Thus it is clear that fecal contributions to soil by soil-dwelling invertebrates provide feedback mechanisms affecting the abundance and diversity of other soil-dwelling animals.

Enchytraeid densities range from <1000 individuals m~2, in intensively cultivated agricultural soil in Japan, to > 140,000 individuals m~2 in a peat moor in the United Kingdom. In a subtropical climate, enchytraeid densities of 4000 to 14,000 individuals m~2 occur in agricultural plots in the Piedmont of Georgia, USA, whereas higher densities (20,000 to 30,000 individuals m~2) are found in surface layers of deciduous forest soils in the southern Appalachian mountains of North Carolina (van Vliet et al., 1995). Although enchytraeid densities are typically highest in acid soils with high organic content, Didden (1995) found no statistical relationship over a broad range of data between average enchytraeid density and several environmental variables such as annual precipitation, annual temperature, or soil pH. It appears that local variability may be at least as great as variation on a wider scale, as enchytraeid densities show both spatial and seasonal variations. Vertical distributions of enchytraeids in soil are related to organic matter horizons. Up to 90% of populations may occur in the upper layers in forest and no-tillage agricultural soils, but densities may be higher in the Ah horizon of grasslands (Davidson et al., 2002). Seasonal trends in enchytraeid population densities appear to be associated with moisture and temperature regimes (van Vliet, 2000).

Enchytraeids have been shown to have significant effects on soil organic matter dynamics and on soil physical structure. Litter decomposition and nutrient mineralization are influenced primarily by interactions with soil microbial communities. Enchytraeid feeding on fungi and bacteria can increase microbial metabolic activity and turnover, accelerate the release of nutrients from microbial biomass, and change species composition of the microbial community through selective grazing. However, Wolters (1988) found that enchytraeids decreased mineralization rates by reducing microbial populations and possibly by occluding organic substrates in their feces. The influence of enchytraeids on soil organic matter dynamics is therefore the net result of both enhancement and inhibition of microbial activity depending on soil texture and population densities of the animals (Wolters, 1988; van Vliet, 2000).

Enchytraeids affect soil structure by producing fecal pellets, which, depending on the animal size distribution, may enhance aggregate stability in the 600- to 1000-^m aggregate size fraction. In forest floors, these pellets are composed mainly of fine humus particles, but in mineral soils, organic matter and mineral particles may be mixed into fecal pellets with a loamy texture. Davidson et al. (2002) estimated that enchytraeid fecal pellets constituted nearly 30% of the volume of the Ah horizon in a Scottish grassland soil (Fig. 7.7). Encapsulation or occlusion of organic matter into these structures may reduce decomposition rates. Burrowing activities of enchytraeids have not been well studied, but there is evidence that soil porosity and pore continuity can increase in proportion to enchytraeid body size (Rusek, 1985; Didden, 1990).

Soil Fecal Pellets Fauna
FIGURE 7.7 Thin-section micrographs of fecal pellets in a grassland soil. (a) Derived from enchytraeids (scale bar, 0.5 mm). (b) Derived from earthworms (scale bar, 1.0 mm) (from Davidson et al., 2002).

Enchytraeids are typically sampled in the field using cylindrical soil cores of 5- to 7.5-cm diameter. Large numbers of replicates may be needed for a sufficient sampling due to the clustered distribution of enchytraeid populations (van Vliet, 2000). Extractions are often done with a wet-funnel technique, similar to the Baermann funnel extraction used for nematodes. In this case, soil cores are submerged in water on the funnel and exposed for several hours to a heat and light source from above; enchytraeids move downward and are collected in the water below. Van Vliet (2000) provides a comparison of modifications of this technique.

macrofauna

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Responses

  • Yohannes
    What is the active method of nematode extraction from soil?
    1 year ago
  • delma
    Does nematodes extraction involves microbiology?
    1 year ago
  • Gualtiero
    What filter paper to use with nematode extraction?
    3 months ago

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