Publisher Summary
This chapter focuses on the techniques used for identifying, isolating, propagating, assaying, and preserving nematodes that are parasitic in or pathogenic to insects. Nematodes are nonsegmented animals with excretory, nervous, digestive, reproductive, and muscular systems but lacking circulatory and respiratory systems. The stage of entomogenous and entomopathogenic nematodes that is infective varies depending on the group. A good stereomicroscope is essential for nematode identification and should have a range of magnification between 10 and 100X, a fairly fiat field, and good resolution. The gonads and other structures of fixed nematodes may be obscured by the granular appearance of the intestine. Specimens can be cleared by processing to lactophenol or glycerin. The cephalic structures and the number of longitudinal chords are diagnostic characters for genetic or specific determination of certain groups of nematodes. Extraction methods for insect nematodes are derived from techniques developed with plant-parasitic nematodes. It is found that the most common methods are the Baermann funnel, sieving, elutriation, and centrifugal flotation.

Techniques in insect nematology

A technique based on progressive fumigation was used to reduce soil microbial biodiversity, and the effects of such reductions upon the stability of key soil processes were measured. Mineral soil samples from a grassland were fumigated with chloroform for up to 24 h and then incubated for 5 months to allow recolonisation by surviving organisms. The diversity of cultivable and non-cultivable bacteria, protozoa and nematodes was progressively reduced by increasing fumigation times, as was the number of trophic groups, phyla within trophic groups, and taxa within phyla. Total microbial biomass was similar within fumigated soils, but lower than for unfumigated soil. There was no direct relationship between biodiversity and function. Some broad-scale functional parameters increased as biodiversity decreased, e.g. thymidine incorporation, growth on added nutrients, and the decomposition rate of plant residues. Other more specific parameters decreased as biodiversity decreased, e.g. nitrification, denitrification and methane oxidation. Thus specific functional parameters may be a more sensitive indicator of environmental change than general parameters. Although fumigation reduced soil microbial biodiversity, there was evidence to suggest that it selected for organisms with particular physiological characteristics. The consequences of this for interpreting biodiversity – function relationships are discussed. The stability of the resulting communities to perturbation was further examined by imposing a transient (brief heating to 40°C) or a persistent (addition of CuSO4) stress. Decomposition of grass residues was determined on three occasions after such perturbations. The soils clearly demonstrated resilience to the transient stress; decomposition rates were initially depressed by the stress and recovered over time. Resilience was reduced in the soils with decreasing biodiversity. Soils were not resilient to the persistent stress, there was no recovery in decomposition rate over time, but the soils with the highest biodiversity were more resistant to the stress than soils with impaired biodiversity. The study of functional stability under applied perturbation is a powerful means of examining the effects of biodiversity.

Ecosystem response of pasture soil communities to fumigation-induced microbial diversity reductions: an examination of the biodiversity-ecosystem function relationship

Nematode diversity in agroecosystems

Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well-acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)-based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-294X.2009.04473.x

Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises.

This microcosm study is concerned with understanding those factors which regulate ecosystem processes of nutrient cycling and plant productivity in a montane grassland ecosystem. We examined the effects of different groups of soil fauna, namely bacterial-feeding nematodes and Collembola, on nutrient mineralization (N and P) in an acid, organic soil taken from a montane grassland in the Peak District National Park, United Kingdom. We also examined whether faunal influences on nutrient release, a measure of nutrient mineralization, resulted in changes in nutrient uptake and biomass production of an indigenous montane grass species (Nardus stricta (L.)). We found that in the presence of Collembola, and when nematodes and Collembola were combined, N mineralization, nutrient leaching and shoot N contents of N. stricta was significantly increased relative to a defaunated control. We also found that net P mineralization and leaching increased (although not significantly) in the presence of both nematodes and Collembola, resulting in a significant increase in shoot P content of N. stricta. The presence of nematodes alone, which were largely bacterial-feeders, had no effect on the mineralization of N or P, or shoot nutrient content. We suggest that differences in the effect of faunal treatments on nutrient mineralization are related to the feeding strategies of the added fauna, and to their consequent effect on the size of the soil microbial biomass. The treatments that increased N mineralization and plant nutrient content (N and P) also significantly reduced plant growth (shoot and root). We suggest that high NH4+–N concentrations in the soil solution of Collembola treatments inhibited the growth of N. stricta and that the growth of other grassland species may benefit from this improvement in nutrient availability.

Experimental evidence that soil fauna enhance nutrient mineralization and plant nutrient uptake in montane grassland ecosystems

SUMMARY
When 200 ml. dispersed soil is sedimented from an obliquely rising water current in a simple compartmented tank about three-quarters of the nematodes are extracted. About 95% of the nematodes in the concentrated suspension can be separated finally from mineral and heavy organic particles by centrifugal notation. When mobile nematodes were finally separated from soil particles by paper tissue, this sedimentation method extracted as many nematodes from sand and loam as Seinhorst's two-flask and elutriation methods, but in one test extracted fewer Tylenchorhynchus from clay and in another fewer Paratylenchus from clay than the elutriation method. The method is quicker (4 or 6 instead of 30–45 min.) and easier.

Mobile nematodes can be extracted from 300 ml. soil spread out on paper tissue in 23. 33 cm. trays of 8 mesh/cm. phosphor-bronze gauze, just resting on shallow water. The suspension obtained after 24 hr. at 16–18°C. was concentrated to 10–15 ml. without loss by sedimentation in two tapered cylinders, one of 8 cm. bore, the other of 2.6 cm. bore. This method usually extracted significantly more nematodes than the sedimentation, two-flask and elutriation methods.

J. R. Hemming

Papers

A comparison of some quantitative methods of extracting small vermiform nematodes from soil