PRACTICAL CONSERVATION. Extracting invertebrates from bryophytes. Nigel Andrew* and Louise Rodgerson. Australian Flora and Fauna Research Centre, ...
Journal
of
Insect
Conservation,
3,
53–55
(1999)
PRACTICAL CONSERVATION
Extracting invertebrates from bryophytes Nigel Andrew* and Louise Rodgerson Australian Flora and Fauna Research Centre, Department of Biological Sciences, University of Wollongong, 2522, Australia Received: 8 June 1998; accepted: 16 July 1998
Keywords: Kerosene-phase separation; sugar flotation; Acari; Collembola; Diptera
Introduction Many studies that are concerned with the ecology and conservation of invertebrate populations and communities use techniques to sample and extract invertebrates from their habitat. The effectiveness of different techniques to extract invertebrates from substrates has been the source of much debate amongst ecological entomologists. There is no single method which is 100% effective in extracting all invertebrates from substrates (Geurs et al., 1991; Walter et al., 1987). Determining the most appropriate method for a given study involves a compromise between cost and efficiency, the type of substrate sampled and the aims of the study (Edwards, 1991; McSorley and Walter, 1991; Walter et al., 1987). In addition, it is often impossible to extract live invertebrates from samples when working in remote locations, consequently methods are needed for preserved samples. Within these constraints several methods (e.g. sugar flotation, centrifugal flotation, heptane flotation) have been devised to extract invertebrates from substrate in preserved samples (Edwards, 1991; Geurs et al., 1991; McSorley and Walter, 1991; Upton, 1991; Kethley, 1990; Norton, 1990; van Gundy, 1982). When working with soft-bodied microinvertebrates, extraction techniques are required that are effective in extracting a high proportion of these taxa as well as maintaining the samples in an adequately preserved state. In view of all these limitations, it is essential when choosing an extraction technique for a given study that several techniques be trialed, preferably experimentally, however, it appears this is rarely done. Keeping these criteria in mind we devised an extrac* To whom correspondence should be addressed.
1366–638X © 1999 Kluwer Academic Publishers
tion technique, kerosene-phase separation, to extract invertebrates from preserved samples of bryophytes. The technique relies on the binding of kerosene to the waxy cuticle of invertebrates (Walter et al., 1987). It has been used for freshwater samples (Barmuta, 1984), however, it has not previously been reported for terrestrial invertebrate samples. In this paper we outline the technique, and compare its effectiveness to sugar flotation, which is one of the most commonly used extraction techniques for soil and litter invertebrate samples.
Materials and methods Bryophyte samples were collected on the 12th June 1997 from Bald Hill, Stanwell Tops, Australia (34°13'30"S 150°58'30"E). Twenty-four haphazardly chosen, 5 cm 3 2.5 cm, samples were collected down to the rock substrate. Twelve samples were randomly allocated to each of the two extraction treatments (kerosene-phase separation and sugar flotation). Bryophyte samples were placed in 95% ethanol upon collection in the field and left for two weeks before extractions proceeded. For sugar flotation treatment the protocol of Pask and Costa (1971) was followed. Kerosene-phase separation involved placing each sample into two large test tubes (2 cm wide 3 17 cm long) and adding 95% ethanol so that each test tube was approximately 3⁄4 full. Kerosene was added to each test tube to within 1 cm of the top. The tubes were then shaken vigorously to ensure that the ethanol and kerosene were fully mixed. After 10–15 minutes of settling, each test tube was rolled to allow any trapped bubbles of kerosene to rise from the bottom and sides. A dis-
N. Andrew and L. Rodgerson
tinct interface formed between the ethanol (below) and kerosene (on top) after the ethanol and kerosene had separated. The invertebrates settled onto this interface layer. The kerosene layer was pipetted off to within 5 mm of the interface and discarded. The remaining kerosene together with the interface were pipetted off and collected. The test tube sides were washed with 95% ethanol to dislodge any kerosene that had stuck to the sides of the tube and the new interface was re-pipetted and collected. The whole procedure was repeated to increase the effectiveness of the extraction. The second extraction increased the total number of invertebrates collected by 16%. The interface mixture was transferred to petri dishes and examined under a binocular microscope in a fume hood. Invertebrates in the kerosene layer were pushed into the ethanol, using a fine brush, to dislodge the kerosene from the cuticle. The invertebrates were then collected and sorted to order. Since this was a preliminary study, the higher taxonomic level used was deemed appropriate due to time, money and resource constraints (Cranston and Hillman, 1992). Total abundance and number of orders recovered from different extraction treatments were analysed using a one-way ANOVA. The count data were square root transformed to improve the normality of the underlying distribution (Zar, 1984).
Figure 1. Mean number ( 6 SD) of individuals (asterisks) and invertebrate orders (solid bars) extracted from bryophyte samples (n 5 12) using kerosene and sugar extraction treatments.
invertebrates from bryophyte substrates. Many studies are restricted to using preserved samples, and understanding the efficiency of the chosen extraction technique is critical to any study which investigates the ecology and conservation of invertebrate communities (Edwards, 1991; McSorley and Walter, 1991; Edwards and Fletcher, 1971). The majority of available techniques are designed for the extraction of animals from soil and leaf litter. Some of these techniques have been used to extract invertebrates from bryophytes, however, few studies have tested the efficiency of extraction of invertebrates from this substrate. This study found that kerosene-phase separation is at least as effective as sugar flotation for collecting invertebrates from bryophyte samples. Comparing extraction techniques can be difficult since they may extract different taxa at different densities (Block, 1967; Freckman and Virginia, 1993). This study found that the number of individuals extracted by the two techniques differed considerably (Fig. 1), but the number of taxa recovered was relatively consistent (Fig. 1). Since there were six taxa extracted overall, with only three taxa being extracted collectively by kerosene-phase separation and sugar flotation, a significant difference in number of taxa extracted would be unlikely. A higher taxonomic resolution may be nee-
Results The kerosene extraction recovered significantly more invertebrate individuals than the sugar extraction (F 5 10; p 5 0.004; d.f 5 1; Fig. 1) and similar numbers of orders (F 5 3.03; p 5 0.1; d.f 5 1; Fig. 1). Acari, Collembola and Diptera were extracted by each extraction treatment. Only one individual of each of Hymenoptera, Protura and Psocoptera were found, all from one mossbed in the Kerosene treatment. However, because of the low numbers of these taxa, no meaningful comparison can be made. Preliminary investigations suggest that for samples that are complex, for example, densely tufted bryophytes, pre-washing samples in 95% ethanol may be useful. This involves prising the bryophyte apart and gently shaking within the ethanol to help dislodge invertebrates caught within the tuft.
Discussion The present study has demonstrated that for samples which have been preserved in ethanol, kerosene-phase separation maybe a useful technique for extracting 54
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ded in order for a more detailed analysis of the efficiency of particular extraction techniques to recover different species.
todes from Dry Valley Antarctic soils. Polar Biol. 13, 483–487. Geurs, M., Bongers, J. and Brussaard, L. (1991) Improvements to the heptane flotation method for collecting microarthropods from silt loam soil. Agric. Ecosystems Environ. 34, 213–221. Kethley, J. (1990) Acarina: Prostigmata (Actinedida). In Soil Biology Guide (D.L. Dindall, ed.) pp. 667–755. John Wiley and Sons, Brisbane. McSorley, R. and Walter, D.E. (1991) Comparison of soil extraction methods for nematodes and microarthropods. Agric. Ecosystems Environ. 34, 201–207. Norton, R.A. (1990) Acarina: Oribatida. In Soil Biology Guide (D.L. Dindall, ed.) pp. 779–803. John Wiley and Sons, Brisbane. Pask, W.M. and Costa, R. (1971) Efficiency of sucrose flotation in recovering insect larvae from benthic stream samples. Can. Entomol. 103, 1649–1652. Upton, M.S. (1991) Methods for collecting, preserving, and studying insects and allied forms. The Australian Entomological Society, Brisbane. van Gundy, S.D. (1982) Nematodes. In Methods of Soil Analysis Part 2: Chemical and Microbiological Properties (A.L. Page, R.H. Miller and D.R. Keeney, eds) pp. 1121–1130. American Society of Agronomy, Inc., Wisconsin. Walter, D.E., Kethley, J. and Moore, J.C. (1987) A heptane flotation method for recovering microarthropods from semiarid soils, with comparison to the Merchant-Crossley high-gradient extraction method and estimates of microarthropod biomass. Pedobiologia 30, 221–232. Zar, J.H. (1984) Biostatistical Analysis. 2nd edition. PrenticeHall International, New Jersey.
Acknowledgements We would like to thank Graham Osler for initially suggesting that kerosene-phase separation may be useful for extracting microinvertebrates from bryophyte samples. Alan York, Kris French and Ian Oliver commented on an earlier draft of this manuscript and their comments were much appreciated.
References Barmuta, L.A. (1984) A method for separating benthic arthropods from detritus. Hydrobiologia 112, 105–107. Block, W. (1967) Recovery of mites from peat and mineral soils using a new flotation method. J. Anim. Ecol. 36, 323–327. Cranston, P. and Hillman, T. (1992) Rapid assessment of biodiversity using ‘Biological Diversity Technicians’. Aust. Biol. 5, 144–154. Edwards, C.A. (1991) The assessment of populations of soilinhabiting invertebrates. Agric. Ecosystems Environ. 34, 145–176. Edwards, C.A. and Fletcher, K.E. (1971) A Comparison of extraction methods for terrestrial arthropods. In: Methods of Study in Quantitative Soil Ecology: population, production and energy flow (J. Phillipson, ed.) pp. 150–185. Blackwell Scientific Publications, Oxford. Freckman, D.W. and Virginia, R.A. (1993) Extraction of nema-
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