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applied soil ecology 36 (2007) 199–204
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Evaluation of the ‘‘bait-lamina test’’ to assess soil microfauna
feeding activity in mixed grassland
Chantal Hamel a,b,*, Michael P. Schellenberg a, Keith Hanson a, Hong Wang a
a
b
Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, SK S9H 3X2 Canada
Department of Soil Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8 Canada
abstract
Article history:
The soil fauna plays an important role in regulating nutrient cycling through predation and
Accepted 27 February 2007
comminution of organic residues. The bait-lamina test was proposed as a practical mean to
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article info
assess soil faunal feeding activity. The test consists of vertically inserting 16-hole-bearing
plastic strips filled with a plant material preparation into the soil. We assessed the value of the
Soil faunal feeding activity
bait-lamina test in the mixed grassland ecoregion of Canada, using 5-year-old seeded grass
assessment
field plots. There were five treatments of grass communities, i.e. monocultures of (1) Russian
Methodology
wild rye, (2) switchgrass, (3) green needlegrass, or (4) western wheatgrass, and (5) a grass
Soil biological activity
mixture of western wheatgrass + green needlegrass + switchgrass + little blue stem, which
Soil animals
were distributed in four complete blocks, and six bait mix treatments, i.e. bait made with
Field experiment
ground tissue of (1) Russian wild rye, (2) switchgrass, (3) green needlegrass, (4) western
Bait-lamina test
wheatgrass, (5) alfalfa, or with (6) wheat bran, which were randomized in each plot. Bait-
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rs
Keywords:
lamina strips were used in groups of five strips inserted in the soil; they were equally spaced
across the inter-row. The analysis revealed a marginally significant effect (P = 0.065) of plant
community on soil faunal feeding activity with more activity in mixed grass species compared
to green needlegrass plots. Bait flavour had no significant effect (P = 0.22) on feeding. More
(P < 0.0005) feeding activity was detected close to the soil surface (0–5 mm deep) than at 20 mm
r's
below the surface and at deeper depths. Feeding activity was relatively low over the 65-day
period of the test (13 June to 17 August 2005), with 8.3% and 2.0% of the lamina (holes filled)
showing signs of feeding in the 0–5 and 5–15 mm soil layer. We conclude that the bait-lamina
1.
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test is appropriate to assess the activity of surface litter processing fauna in mixed grassland,
but it is not a good indicator of the decomposition occurring in the soil matrix, where most
litter is produced. We recommend the use of a large number of replicated bait-lamina strips
and a whole growing season incubation period in soils of the semiarid prairie ecoregion where
the surface litter layer is thin and faunal saprobes are relatively scarce.
Introduction
The soil is a complex environment where plants, microorganisms and small animals interact in the recycling of live and
dead organic matter, which impacts soil chemistry. In
terrestrial ecosystems, above- and belowground plant inputs
# 2007 Published by Elsevier B.V.
constitute the main sources of carbon and energy for a large
and extraordinarily diverse community of living organisms
(Hättenschwiler et al., 2005).
Soil bacteria and fungi have the largest biomass and
are responsible for most of the mineralization of nutrients
from soil organic matter. Although bacteria and fungi can
* Corresponding author at: Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, SK S9H 3X2
Canada. Tel.: +1 306 778 7264; fax: +1 306 778 3188.
E-mail address: [email protected] (C. Hamel).
0929-1393/$ – see front matter # 2007 Published by Elsevier B.V.
doi:10.1016/j.apsoil.2007.02.004
200
applied soil ecology 36 (2007) 199–204
Materials and methods
2.1.
Site description
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The experiment was conducted on a Brown Chernozem
(Haverhill clay loam) located at the South Farm of the Semiarid
Prairie Agricultural Research Centre in Swift Current Saskatchewan, Canada (latitude: 508170 N; longitude: 1078410 W;
elevation 825 m). The region receives 361 mm of annual
precipitation and has a yearly mean temperature of 3.6 8C
with mean monthly temperatures ranging from 13.2 8C in
January to 18.6 8C in June (54-year averages). Soil temperature
at 5 cm depth under a sod and rainfall received during the
period of the study were measured at a weather station
located approximately 300 m from the study sites.
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and the usefulness of this test in a semiarid environment is
only speculative. We therefore evaluated the bait-lamina test
in mixed grassland soils. The trial was conducted in an
agricultural soil growing 5-year-old stands of different forage
grasses. We wanted to assess the value of the bait-lamina test
for mixed grassland soil environments and, in particular, we
wanted to determine if the plant material used to prepare the
bait strips would be differentially utilized by the soil faunal
population present under differing plant species.
2.2.
rs
accomplish the complete degradation of organic matter, they
rarely act alone (Whalen and Hamel, 2004). Some protozoa
and nematodes are saprotrophic and directly contribute to
decomposition, whereas others are predatory and regulate
the activity of fungal and bacterial populations. The mesofauna mainly represented by springtails (Collembola) and
mites (Acarina) are involved in the regulation of soil
microorganisms but also consume and process considerable
amounts of litter. Millipedes (Diplopoda), isopods (Isopoda),
enchytraeids (Tubificida) and earthworms (Haplotaxida) displace and fragment litter producing large amounts of feces,
which are easily utilized by soil microorganisms. They move
surface litter into the soil where the environment is more
favourable for microbial decomposers. Understanding soil
processes requires the consideration of the soil fauna.
Clearly, litter-feeding invertebrates play an important role
in the decomposition and recycling of dead organic matter,
and their contribution to nutrient cycling needs to be
considered together with that of soil fungi and bacteria.
Different methods have been developed to study soil
animals’ contribution to organic matter decomposition. Among
these, the bait-lamina test, proposed by Von Törne (1990), was
found to best reflects the feeding activity of soil animals (Helling
et al., 1998; Von Gestel et al., 2003). The test consists of vertically
inserting 16-hole-bearing plastic strips filled with a plant
material preparation into the soil. After a period of time, the
bait-lamina strips are removed and examined for evidence of
feeding. Helling et al. (1998) have shown high feeding activity of
Collembola and Enchytraeidae using bait-lamina strips in
microcosm experiments, and a reduced impact of microorganisms on the disappearance of the bait. They also demonstrated
the preference of the organisms for bait-lamina made with
nettle tissues over those made with wheat bran, as initially
proposed by Von Törne (1990).
Soil organisms preferentially feed on certain litter types
(Hättenschwiler et al., 2005). Consequently, bait materials
more closely resembling substrates found in different plant
communities may be preferred by soil fauna occurring within.
The bait-lamina test has been repeatedly used to assess litterfeeders’ activity (Paulus et al., 1999; Filzek et al., 2004), but it
remains unclear how different plant stands can influence the
results of the test. The bait-lamina test was successfully used
in mesic environments, in micro- (Helling et al., 1998) and
mesocosms (Von Gestel et al., 2003), in forest ecosystems (Von
Törne, 1990; Paulus et al., 1999), and in grassland sites (Filzek
et al., 2004), to assess the activity of the soil fauna. Moisture
deficit characterizes the mixed grassland ecoregion, which
offer soil environments less conducive to biological activity
than more mesic soils, but soil invertebrates are present. A 5year survey conducted in the Northern part of the North
American mixed grassland region at Matador Saskatchewan,
approximately 60 km north of our site, found averages of
490 m2 Elatridae; 74,823 m2 Collembola; 31,191 m2 Acarina;
2.7 106 Nematoda; 34,083 m2 Enchytraeidae in the top 0–
30 cm soil layer (Willard, 1974). These numbers are similar to
those reported by others from prairie ecosystems (Stanton,
1988), but generally lower than those reported for European
grasslands (Paul and Clark, 1996).
The bait-lamina test has never been used to assess the
feeding activity of soil fauna in the mixed grassland ecoregion,
Experimental design
The bait-lamina method was evaluated in 5-year-old field
plots planted with monocultures of Russian wild rye (Elymus
junceus Fisch.), switchgrass (Panicum virgatum L.), green
needlegrass (Stipa viridula Trin.), or western wheatgrass
(Agropyron smithii Rydb.), or with a mixture of western
wheatgrass, green needlegrass, little bluestem (Schizachyrium
scoparium Michx.) and switchgrass. Each plant species treatment was grown in 4 m 8 m plots arranged in four
randomized complete blocks. Six bait flavour treatments were
factorially arranged and randomized within each plot. Baitlamina strips are 1-mm thick 6 mm 150 mm plastic strips
bearing a series of 16 holes (2 mm diameter) pierced at 5 mm
interval in their lower end, which is inserted into the soil.
Holes are filled with a ground plant-based mix and, thus,
called ‘‘bait-lamina’’. Five bait-lamina strip replicates constituted one experimental unit. Thus, 600 bait-lamina strips
were used in this experiment. These strips were inserted in the
soil on 13 June 2005. A wooden template containing holes was
laid down between two plant rows and the tool used to prepare
holes in the soil. The strips were then placed into the holes in
the soil using a machined steel insertion tool, which supported
and protected the strips during insertion. Bait-lamina strips
were placed just deep enough in the soil for the edge of the
uppermost bait-lamina to be 3 mm below soil surface (Fig. 1).
Four supplementary bait-lamina strips of each flavour were
also inserted in the soil of each plot. They were harvested at
different time intervals to monitor feeding and to determine
the incubation time most appropriate for the test, under mixed
grassland field conditions. Experimental bait-lamina strips
were removed from the soil on 17 August 2005, prior to forage
harvest. The number of lamina showing feeding activity were
201
applied soil ecology 36 (2007) 199–204
Fig. 1 – Arrangement of one experimental subplot, each of
which received one bait flavour treatment.
2.3.
Preparation of the bait-lamina
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Bait materials were prepared by mixing ground dry plant
tissues with agar-agar (Anachemia Canada Inc., Montreal,
The significance of treatment effects could not be assessed by
ANOVA due to lack of normality, according to the Shapiro
Wilks’ test calculated using JMP1 v. 3.2.6 (SAS Institute, Cary,
USA). The nonparametric Kruskal–Wallis test (Mcbean and
Rovers, 1998) was conducted by PROC NPAR1WAY and
treatments were compared by PROC MULTTEST using Bonferroni–Holms correction (Westfall and Wolfinger, 1997). The
data were averaged over the 16 depths of each bait-lamina
strip to assess plant species treatment effect. The bait flavour
effects on soil faunal feeding activity within each plant
community treatments as well as the overall bait flavour effect
were also computed on data averaged over the 16 bait-lamina
of each strip. Percentage of bait-lamina position with evidence
of feeding was calculated over each plot, and these data were
used to assess the impact of soil depth on feeding across all
on
counted and their location on the strips, i.e. their depth in soil,
recorded. Some bait-lamina had dislodged during manipulation, and some had cracked. They were recorded as such but
were not considered as indicators of feeding. Feeding activity
was expressed as percentage of individual bait-lamina
showing evidence of feeding.
Statistical analysis
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Quebec), cellulose powder and bentonite (Sigma–Aldrich Inc.,
St. Louis, MO) in a 1/6.5/1.5/1 proportion calculated on a weight
basis. Several applications of the bait preparation were
required to load the lamina, because of shrinkage. The recipe
was modified by excluding bentonite in later applications.
Plant tissue from the 2004 harvest and commercial wheat bran
was used to prepare the bait. This material was analysed for: N
by the Kjeldahl method (McGill and Figueiredo, 1993), P
according to Murphy and Riley (1962), and crude protein by
multiplying Kjeldahl N by 6.25. The percentages of N, P and
crude protein in the plant materials are presented in Table 1.
Table 1 – Analysis of the plant materials used to make the bait-lamina filling
Plant tissues used as bait-lamina material
r's
Russian wild rye
Western wheatgrass
Green needlegrass
Switchgrass
Alfalfa
Wheat bran
Analyses (mg g1 dry weight basis)
Nitrogen
Phosphorus
9.5
8.8
7.2
4.7
22.2
11.5
0.78
0.88
0.95
0.90
4.20
7.90
Crude protein
59
55
45
29
144
75
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All the material was harvested from the field experiment the previous year except alfalfa, which was used as a nitrogen rich control, and
wheat bran, which was initially proposed as the bait ingredient.
Table 2 – Feeding activity, revealed by the bait-lamina test, in plots with different grass species and pair-wise
comparisons of treatment meansa
Grass community
Holes fed upon
Comparison
x2
P
3.2
2.6
1.9
2.4
3.2
GNG vs. RWR
GNG vs. SG
GNG vs. WGLS
GNG vs. WWG
RWR vs. SG
5.62
0.29
7.39
1.11
2.99
0.017
0.592
0.007
0.291
0.083
Average
2.7
RWR vs. WGLS
RWR vs. WWG
SG vs. WGLS
SG vs. WWG
WGLS vs. WWG
0.21
1.63
4.11
0.21
2.28
0.648
0.202
0.043
0.649
0.131
a
Au
Russian wild rye (RWR)
Western wheatgrass (WWG)
Green needlegrass (GNG)
Switchgrass (SG)
Mix community (WGLS)
Significant
ns
ns
b
ns
ns
ns
ns
ns
ns
ns
Whole model P value = 0.065.
Differences between treatment means are considered significant only after applying Bonferroni–Holms correction for multiple comparisons.
Each comparison represent a Kruskal–Wallis chi-square approximation with 1 d.f.
b
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applied soil ecology 36 (2007) 199–204
treatments. Analyses were conducted using the SAS/STAT1
software (SAS Institute Inc., Cary, NC, USA).
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Fig. 3 – Daily precipitation, and minimum (~) and
maximum (^) soil temperature at 5 cm below soil surface
from 1 June to 31 August 2005.
normal. The soil temperature and the distribution of the
precipitation received during the study period are presented in
Fig. 3.
4.
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Overall soil faunal feeding activity was low over the 65-day
period of the trial, with an average rate of 2.7% of the baitlamina positions showing evidence of feeding (Table 2). Under
the conditions of the test, bait-lamina strips prepared with
different plant materials were not differentially consumed
(P = 0.22; mean range: 2.0–3.6% of lamina with evidence of
feeding). Furthermore, the plant species did not influence soil
faunal feeding preference, as indicated by a lack of significant
bait-lamina flavour effect when tested within all five plant
species treatments used in this trial (data not shown).
Marginally larger feeding activity (P = 0.065) was found in
the grass mix than in green needlegrass plots, and the
difference between treatment means was small with feeding
activity values spanning from 1.9% for green needlegrass, to
3.2% for the grass species mix (Table 2). Feeding activity
decreased (P < 0.0005) from 8.3% of lamina showing evidence
of feeding at 5 mm below soil surface to 2.0%, on average, in
the 20–80 mm soil layer (Fig. 2).
Air temperature and precipitation during the study period
were close to normal (120-year average). June was wetter
(123.2 mm, normal: 73.2 mm) and July drier (21.4 mm, normal:
52.0 mm) than normal, resulting in a slightly drier 1 June to 14
August period (134.2 mm, normal: 146.7 mm). Air temperature
during the same period averaged 16.8 8C, i.e. 0.5 8C below
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Results
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3.
Fig. 2 – Variation in soil animal feeding activity with depth
across all treatments, as revealed by the bait-lamina test
(P = 0.0005; N = 20). Multiple mean comparisons are
considered significant (P = 0.05) only after applying
Bonferroni–Holms correction for multiple comparisons to
the test. Each pair-wise comparison represent a Kruskal–
Wallis chi-square approximation with 1 d.f.
Discussion
The soil faunal feeding activity measured over a period of 65
days under summer mixed grassland conditions was relatively low (2.7% on average). As a comparison, mean feeding
activities measured with the bait-lamina test ranged from
about 50 to 1% over 6 days in grasslands sites in UK (Filzek
et al., 2004). Values between 15.8 and 4.1% over 44 h were
found in German forest soils (Von Törne, 1990), and Helling
et al. (1998) reported mean feeding activities ranging from 13%
to less than 4% with incubation times of 15–43 days, in
microcosms containing various combinations of Enchytraeidae, Collembola and microorganisms. Differentiating evidence of feeding from cracked or lost bait is largely
subjective. Our determinations were conservative and this
may have also contributed to the low percentage of feeding we
found in comparison with similar studies.
In spite of the relatively low activity found in our study
plots, our analysis was precise enough to detect a significant
effect of soil depth. The difference in feeding activity in the
grass mix and green needlegrass plots was marginally
significant (P = 0.065) and small, with feeding activity values
of 1.9% in green needlegrass and 3.2% for the grass species mix
(Table 2). Greater feeding in mixed grasses stands may reflect
lower invertebrate abundance under monoculture, as it was
reported by others (St. John et al., 2006). We conclude from
these observations that, technically, the bait-lamina test can
be used to assess soil faunal feeding over a normal North
American mixed grassland summer period and that using
wheat bran in the bait is as appropriate as using other plant
materials.
Feeding activity revealed by the bait-lamina test was
reduced below 15 mm of the soil surface. This feeding
reduction may be surprising considering that plant residues
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applied soil ecology 36 (2007) 199–204
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Acknowledgements
Thanks to D. Wiebe for his perseverance in loading the baitlamina strips and in data acquisition and to D. Judiesch for
providing the weather data.
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Von Gestel, C.A.M., Kruidenier, M., Berg, M.P., 2003. Suitability
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Conclusion
The bait-lamina test is appropriate to assess the activity of
soil animals involved in the comminution of soil litter of
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plant origin, a process that seems largely restricted to the
few uppermost millimetres of the soil, in mixed grassland
soils. We measured low feeding activity in our mixed
grassland plots indicating that the number of bait-lamina
strips per plot must be high for the test to achieve good
precision. We used 30 bait-lamina strips per plot. This level
of duplication should be maintained or increased in further
work. We also recommend to incubate the bait-lamina
strips from early spring to the first killing frost, in Canadian
mixed grassland soils.
rs
are abundant in soil. Sixty to 80% of total net primary
productivity (NPP) occurs belowground in mixed grassland
(Stanton, 1988). Mean root:shoot ratio vary from 2:1 to 13:1
across North American prairie regions, with higher values
associated with cooler regions like the Swift Current area. The
bait-lamina test detected lower activity belowground where
most NPP occurs suggesting that this test does not assess
belowground decomposition processes, at least in our mixed
grassland. While the general principle of the technique seems
appropriate for the assessment of epigeic animal feeding
associated with surface litter decomposition, its value to
assess belowground nutrient cycling-related activity appears
questionable.
The bait-lamina test has yielded results different from that
of other methods of biological activity assessment suggesting
that different methods reflect the activity of different groups
(Kula and Rombke, 1998; Paulus et al., 1999). The activity of
Collembola and Enchytraeidae may be revealed by the test, but
the activity of microorganisms is hard to detect (Helling et al.,
1998) probably due to the low surface to volume ratio of the baitlamina. Von Gestel et al. (2003) found increasing bait-lamina
consumption rate with increasing earthworm density, and no
significant consumption in mesocosms containing only Collembola and Acarina, over a period of 60 days. Earthworms
appear as very effective feeders on bait-lamina strips and the
bait-lamina test seems to largely reflect their activity.
Dry soils are not a favourable habitat for earthworms, which
are rarely or never found in arid and semiarid grasslands
(Stanton, 1988). No earthworms were ever found at the field
research station where our study was located and no earthworms were reported in a 5-year survey conducted 60 km away
from our study site (Willard, 1974). The absence of earthworms
in our study site may explain the low feeding activity revealed
by the bait-strip test, in particular at lower depths (15–80 mm).
Standing microbial biomass is about equivalent to half that of
net primary productivity (NPP) and in grassland, mineralization
was explained by the activity of bacterivores and fungivores,
mostly protozoa and nematodes (Stanton, 1988), which are
quite evenly distributed throughout the top 300 mm of the soil
in mixed grass prairie (Willard, 1974). Such activities are not
revealed by the bait-lamina test. Our results support the
conclusion that animal feeding on plant materials in mixed
grassland soils is significant only in the surface litter layer,
which is well colonized by epigeic saprobes such as the Acarina.
Belowground mineralization processes should be better
described through enumeration of bacterivorous and fungivorous invertebrates extracted from soil.
Another limitation of the bait-lamina test comes from the
fact that the data produced with this test has a non-normal
distribution (Von Törne, 1990), which can rarely be corrected
with data transformation. This prevents the use of ANOVA,
the most powerful statistical analysis for experimental data
(Scherrer, 1984) and restricts statistical treatment to one-way
analyses.
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applied soil ecology 36 (2007) 199–204
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