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Extracting Invertebrates from Bryophytes

Article  in  Journal of Insect Conservation · March 1999

DOI: 10.1023/A:1009682523054

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Journal of Insect Conservation, 3,
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 commu-
nities use techniques to sample and extract inverte-
brates 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 sub-
strates (Geurs et al., 1991; Walter et al., 1987). Determin-
ing 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, hep-
tane flotation) have been devised to extract inverte-
brates 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 microinverte-
brates, extraction techniques are required that are effec-
tive in extracting a high proportion of these taxa as
well as maintaining the samples in an adequately pre-
served state. In view of all these limitations, it is essen-
tial 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
53–55 (1999)
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 terres-
trial invertebrate samples. In this paper we outline the
technique, and compare its effectiveness to sugar flota-
tion, which is one of the most commonly used extrac-
tion 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). Bryo-
phyte samples were placed in 95% ethanol upon collec-
tion 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 kero-
sene 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 col-
lected. The test tube sides were washed with 95% etha-
nol 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 prelimi-
nary study, the higher taxonomic level used was
deemed appropriate due to time, money and resource Figure 1. Mean number ( 6 SD) of individuals (asterisks) and
constraints (Cranston and Hillman, 1992). invertebrate orders (solid bars) extracted from bryophyte samples
Total abundance and number of orders recovered (n 5 12) using kerosene and sugar extraction treatments.
from different extraction treatments were analysed
using a one-way ANOVA. The count data were square invertebrates from bryophyte substrates. Many studies
root transformed to improve the normality of the
are restricted to using preserved samples, and under-
underlying distribution (Zar, 1984).
standing the efficiency of the chosen extraction tech-
nique is critical to any study which investigates the
Results
ecology and conservation of invertebrate communities
The kerosene extraction recovered significantly more (Edwards, 1991; McSorley and Walter, 1991; Edwards
invertebrate individuals than the sugar extraction (F 5 and Fletcher, 1971). The majority of available techni-
10; p 5 0.004; d.f 5 1; Fig. 1) and similar numbers of ques are designed for the extraction of animals from
orders (F 5 3.03; p 5 0.1; d.f 5 1; Fig. 1). soil and leaf litter. Some of these techniques have been
Acari, Collembola and Diptera were extracted by used to extract invertebrates from bryophytes, how-
each extraction treatment. Only one individual of each ever, few studies have tested the efficiency of extraction
of Hymenoptera, Protura and Psocoptera were found, of invertebrates from this substrate. This study found
all from one mossbed in the Kerosene treatment. How- that kerosene-phase separation is at least as effective as
ever, because of the low numbers of these taxa, no sugar flotation for collecting invertebrates from bryo-
meaningful comparison can be made. phyte samples.
Preliminary investigations suggest that for samples Comparing extraction techniques can be difficult
that are complex, for example, densely tufted bryo- since they may extract different taxa at different den-
phytes, pre-washing samples in 95% ethanol may be sities (Block, 1967; Freckman and Virginia, 1993). This
useful. This involves prising the bryophyte apart and study found that the number of individuals extracted
gently shaking within the ethanol to help dislodge by the two techniques differed considerably (Fig. 1),
invertebrates caught within the tuft. but the number of taxa recovered was relatively con-
sistent (Fig. 1). Since there were six taxa extracted over-
Discussion all, with only three taxa being extracted collectively by
The present study has demonstrated that for samples kerosene-phase separation and sugar flotation, a sig-
which have been preserved in ethanol, kerosene-phase nificant difference in number of taxa extracted would
separation maybe a useful technique for extracting be unlikely. A higher taxonomic resolution may be nee-

54
Extracting invertebrates from bryophytes
ded in order for a more detailed analysis of the effi-
ciency of particular extraction techniques to recover
different species.
Acknowledgements
We would like to thank Graham Osler for initially sug-
gesting that kerosene-phase separation may be useful
for extracting microinvertebrates from bryophyte sam-
ples. Alan York, Kris French and Ian Oliver commented
on an earlier draft of this manuscript and their com-
ments were much appreciated.
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