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Testing soil nematode extraction efficiency using different variations of the Baermann funnel method

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Nematodes are increasingly used as powerful bioindicators of soil food web composition and functioning in ecological studies. Todays' ecological research aims to investigate not only local relationships but global patterns, which requires consistent methodology across locales. Thus, a common and easy extraction protocol of soil nematodes is needed. In this study, we present a detailed protocol of the Baermann funnel method and highlight how different soil pre-treatments and equipment (soil type, amount of soil, sieving, filter type) can affect extraction efficiency and community composition by using natural nematode communities. We found that highest nematode extraction efficiency was achieved using lowest soil weight (25 g instead of 50 g or 100 g) in combination with soil sieving, and by using milk filters (instead of paper towels). PCA at the family level revealed that different pre-treatments significantly affected nematode community composition. Increasing the amount of soil increased the proportion of larger-sized nematodes being able to overcome long distances. Sieving is suggested to break up soil aggregates and, therefore, facilitate moving in general. Interestingly, sieving did not negatively affect larger nematodes that are supposed to have a higher probability of getting bruised during sieving. The present study shows that variations in the extraction protocol can alter the total density and community composition of extracted nematodes and provides recommendations for an efficient and standardized approach in future studies. Having a simple, cheap, and standardized extraction protocol can facilitate the assessment of soil biodiversity in global contexts.
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1
1
2
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4Testing soil nematode extraction efficiency using different
5variations of the Baermann funnel method
6
7Annika Eva Schulz1, Nico Eisenhauer1,2,3, Simone Cesarz1,2,3*
8
91Institute of Ecology, Friedrich Schiller University of Jena, Dornburger Str. 159, 07743 Jena,
10 Germany
11 2 German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher
12 Platz 5e, 04103 Leipzig, Germany
13 3 Institute of Biology, University of Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany
14
15 *Corresponding author
16 Email: simone.cesarz@idiv.de
.CC-BY 4.0 International licensenot peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was. http://dx.doi.org/10.1101/318691doi: bioRxiv preprint first posted online May. 9, 2018;
2
17 Abstract
18 Nematodes are increasingly used as powerful bioindicators of soil food web composition and
19 functioning in ecological studies. Todays’ ecological research aims to investigate not only
20 local relationships but global patterns, which requires consistent methodology across locales.
21 Thus, a common and easy extraction protocol of soil nematodes is needed. In this study, we
22 present a detailed protocol of the Baermann funnel method and highlight how different soil
23 pre-treatments and equipment (soil type, amount of soil, sieving, filter type) can affect
24 extraction efficiency and community composition by using natural nematode communities.
25 We found that highest nematode extraction efficiency was achieved using lowest soil weight
26 (25 g instead of 50 g or 100 g) in combination with soil sieving, and by using milk filters
27 (instead of paper towels). PCA at the family level revealed that different pre-treatments
28 significantly affected nematode community composition. Increasing the amount of soil
29 increased the proportion of larger-sized nematodes being able to overcome long distances.
30 Sieving is suggested to break up soil aggregates and, therefore, facilitate moving in general.
31 Interestingly, sieving did not negatively affect larger nematodes that are supposed to have a
32 higher probability of getting bruised during sieving. The present study shows that variations
33 in the extraction protocol can alter the total density and community composition of extracted
34 nematodes and provides recommendations for an efficient and standardized approach in future
35 studies. Having a simple, cheap, and standardized extraction protocol can facilitate the
36 assessment of soil biodiversity in global contexts.
37
38
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3
39 Introduction
40 Nematodes appear in nearly any kind of soil from dry desert sand to the tundra [1]. Their
41 ubiquitousness, high species richness, and characteristic responses to environmental stressors
42 make them unique biological indicators. Next to information about soil conditions, soil health,
43 and soil processes [2], nematodes and indices based on their community composition can be
44 used to describe soil food web complexity, nutrient enrichment, and decomposition channels
45 (Ferris 2001). These variables can provide important information in ecological research, e.g.,
46 studying the consequences of environmental change and biodiversity loss [3–5].
47 To study general patterns and ecological principles, global networks of ecological
48 experiments have been set up, such as Nutrient Network (https://www.nutnet.umn.edu/),
49 investigating the consequences of multiple nutrient additions in grasslands [6], or TreeDivNet
50 (http://www.treedivnet.ugent.be/) comprising different tree diversity experiments across the
51 globe [7]. Thus far, soil nematodes have mostly been studied in single and local experiments
52 [e.g., 5,8–10], but global assessments of the responses of soil nematodes as bioindicators to
53 environmental changes are scarce. Generally, the assessment of soil biodiversity is largely
54 neglected, leading to a strong under-representation of soil biodiversity in databases, especially
55 at the global scale [11]. Reasons may be a bias towards charismatic species (e.g. vertebrates)
56 and difficulties in sampling procedures.
57 The high trophic and functional diversity of nematodes comes along with a large
58 number of extraction methods available (reviewed in [12]), highlighting that there may not be
59 one ideal technique for all taxa, and different research questions can ask for different
60 approaches [13]. In addition, the diversity of extraction methods is usually accompanied with
61 complex equipment like the Oostenbrink elutriator [14]. However, labs agreeing to extract
62 nematodes in the frame of global ecological networks need one extraction technique, which
63 can be easily implemented and is low cost.
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4
64 In this study, we focus on the very common Baermann funnel method [15] as a simple,
65 fast, and cheap approach for nematode extraction in global assessments (Fig 1). By the use of
66 the Baermann funnel method, many samples (hundreds) can be extracted in parallel, and it
67 can be rebuild easily by laboratories without having experience in nematode extraction. The
68 amount of soil needed is relatively small and only a low amount of water is required. Another
69 advantage to other methods is the cleanliness of the final solution (less soil particles) making
70 microscope work easier and faster. However, this method only selects active nematodes,
71 thereby excluding cysts and inactive forms.
72
73 Fig 1. Schematic Baermann funnel apparatus for nematode extraction from soil. A
74 silicone hose is fixed with hot glue to a funnel with an inner diameter of 11 cm. The end of
75 the funnel is closed with a clip to prevent leaking of the nematode solution. A PVC tube of an
76 inner diameter of 7 cm with a 250 µm mesh at the bottom is covered with a milk filter to
77 prevent soil particles to enter the soil solution, which is than filled with soil. The apparatus is
78 filled with tap water until it touches the soil (no submerging) to moisten the soil and increase
79 nematode movement. Nematodes will accumulate at the bottom of the closed silicone hose.
80
81 Building on many previous studies testing nematode extraction efficiencies [13,16,17],
82 we provide a detailed protocol to extract soil nematodes, e.g., for global assessments, focusing
83 on a consistent methodology and high extraction efficiency, which can be used by many
84 laboratories worldwide. By doing so, we highlight how common soil pre-treatments and
85 equipment (sieving, amount of soil, filter type) can affect extraction efficiency and
86 community composition and should therefore be considered in future studies.
87 Generally, soil samples are homogenized before extraction by sieving [18]. Mesh sizes
88 from 1 mm up to 5 mm are commonly used, whereby small mesh sizes require gentle sieving
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5
89 to not bruise nematodes [12]. Mortality can occur when samples are handled roughly, and loss
90 of species after sieving was shown to be species-specific [13,19]. By contrast,
91 homogenization breaks soil aggregates and is assumed to facilitate the movement of
92 nematodes through the soil, likely resulting in higher nematode extraction efficiency.
93 However, despite the inconsistency of mesh sizes used in different studies, the consequences
94 for extraction efficiency and comparability of results have not been tested before.
95 In ecological long-term experiments or in pristine habitats, destructive samplings like
96 taking soil cores often are strongly limited to prevent destruction of the plots, and only small
97 amounts of soil may be available for nematode extraction. Using large amounts of soil, on the
98 other hand, may also reduce extraction efficiency as less mobile nematodes are discriminated
99 [12,17]. Thus, exploring the role of the amount of extracted soil for nematode extraction
100 efficiency is required to provide general recommendations.
101 Different permeable filters are used to separate nematodes from soil. Most often
102 cotton-wool milk filters are used, but also cheesecloth, filter paper, or paper tissue are
103 suggested. However, knowledge of the influence of different filters on extraction efficiency is
104 missing. Regarding the availability of materials and costs, we test milk filters and common
105 paper towels in this study.
106 A well-chosen combination of the settings described above may help to increase
107 nematode extraction efficiency and to avoid potential biases of different extraction protocols.
108 In this study, we evaluated different settings of the Baermann-funnel method by varying 1)
109 different sieving mesh sizes, 2) different amounts of soil, and 3) two different filter types to
110 investigate the consequences for the total amount of extracted nematodes and for nematode
111 community composition. In addition, two very different soil types, i.e., loamy and sandy soil,
112 were used to enable us to make general recommendations.
113
114
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115 Methods
116
117 We tested the effects of four variables in soil nematode extraction in a full-factorial
118 design: two soil types (loamy and sandy soil), three soil sieving treatments (2 mm mesh size,
119 5 mm mesh size, no sieving), three amounts of extracted soil (25 g, 50 g, and 100 g fresh
120 weight), and two permeable filters (milk filters and paper towels). All treatments were
121 replicated five times resulting in 180 samples.
122 The loamy soil was taken from the Jena Experiment, a grassland biodiversity experiment in
123 Jena, Germany [20]. Adjacent to the experimental plots, soil samples were taken from a soil
124 depth of 0 to 20 cm with pH 8.1, carbon concentration 4.6%, nitrogen concentration 0.3%,
125 and C-to-N ratio 15.7. Clay content was 14%, silt content 41%, and sand content 45% [21].
126 The sandy soil was taken from the Kreinitz Experiment, a tree biodiversity experiment in
127 Zeithain, Saxony, Germany [22]. Soil samples were taken at a distance of around 10 m to the
128 experimental plots from 0 to 20 cm depth. Soil pH was 5.5, carbon concentration 1.1 %,
129 nitrogen concentration 0.1%, and C-to-N ratio was 11.4. Clay content was 2%, silt content
130 5%, and sand 94%.
131 Before any treatments were applied, soil was gently mixed. Afterwards, a fraction of the soil
132 was sieved with a mesh of 2 mm or 5 mm. One fraction of the soil was not sieved, but roots
133 and stones were removed by hand to correctly evaluate soil weight [12]. Three different
134 amounts of fresh soil were used for extraction: 25 g, 50 g, and 100 g, representing a thickness
135 of the soil during extraction of about 1, 2, and 4 cm, respectively. Finally, two different filter
136 types were used: commonly used milk filters (Sana, type FT 25) and paper towels (ZVG
137 Zellstoff-Vertriebs-GmbH & Co. KG, EAN: 4026899028532).
138
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139 Baermann funnel apparatus
140 The Baermann funnel apparatus consisted of a funnel with an inner diameter of 11 cm.
141 Using a different diameter is possible but should be constant among and within studies. A 12
142 cm piece of silicone hose was attached to the funnel ending and fixed with hot glue to prevent
143 water leakage. The tube was closed with a squeezer clip at the end of the silicon hose. The
144 Baermann funnel apparatus was installed in a horizontal position without buckling of the
145 silicon hose. Soil of a certain amount was filled into circular PVC tubes (7 cm in diameter)
146 with a mesh of 250 µm at the bottom, allowing nematodes to traverse the mesh. The mesh
147 was covered with a filter (milk filter or paper towel) to prevent soil particles to enter the
148 nematode solution (Fig 1). To obtain clean samples, we used a large piece of the filter
149 material to prevent soil particles to enter the nematode solution from the side. This, however,
150 increases evaporation and water has to be added if necessary to prevent that the soil falls dry.
151
152 Procedure
153 The first step was to check if the apparatus is tight by filling fresh tap water (room
154 temperature or below) into the funnel with the closed silicone hose at the bottom until it
155 reached the lower end of the funnel. The silicone hose had to be squeezed several times to
156 remove air from the silicone hose.
157 The weight of the empty PVC tube including the filter and a label to identify the
158 sample was noted. Fresh soil of 25 g, 50 g, or 100 g was filled into the PVC tubes. The exact
159 weight has to be noted to get soil water content and relate nematodes to g dry soil. The height
160 of the soil volume was 0.9 cm, 1.9 cm, and 3.8 cm for 25 g, 50 g, and 100 g of fresh soil
161 weight, respectively. Afterwards, the PVC tube with soil was inserted in the funnel.
162 Fresh tap water was added from the side until the bottom of the mesh of the PVC tube
163 touched the water to saturate the sample with water to increase nematode mobility. Samples
164 were not submerged with water to prevent oxygen limitation. After 72 h [12], nematodes
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165 accumulating at the bottom of the tube were transferred into vials and fixed in 4% hot
166 formalin. Therefore, the silicone hose was opened and the water containing nematodes rinsed
167 through a sieve with a 15 µm mesh to separate nematodes from water. Nematodes
168 accumulating on the mesh were transferred into a vial by rinsing the mesh with hot formalin
169 (4%). After extraction, the soil with the PVC tube were dried and weighed to obtain nematode
170 densities per g soil dry weight.
171
172 Nematode counting and identification
173 All nematodes within one sample were counted with a microscope (Leica, DMI 3000
174 B) using 50× magnification. To detect changes in nematode community composition due to
175 the different treatments, a subset of treatments showing the strongest differences in the total
176 number of extracted nematodes (see below) were identified to family level after Bongers
177 (1994) and Andrássy (2005) using 1000× magnification. We randomly identified 100
178 individuals per sample. The proportional value of each family was extrapolated to the total
179 number of nematodes in the sample.
180
181 Statistical analysis
182 The full-factorial experiment was analyzed using four-way Analysis of Variance
183 (ANOVA) with the factors soil sieving (three levels: 5 mm mesh size, 2 mm mesh size, no
184 sieving), amount of extracted soil (three levels: 25 g , 50 g, and 100 g fresh soil), filter type
185 (two levels: milk filters and paper towels), and all possible interactions. Each treatment was
186 replicated five times resulting in 180 samples. As four-way interactions are complex, we
187 simplified the analysis by separating the dataset by soil type as main differences arouse due to
188 strong differences in densities (mean ± sd of nematodes extracted from 1 g of sandy soil was
189 1.4 ±0.9 compared to samples from the loamy soil with 17.5 ± 12.4, respectively.) Analyses
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190 were performed using R (R i386 3.3.1; [25]). Model residuals were checked and fulfilled
191 assumptions of the performed tests.
192 We used principal component analysis (PCA) to detect if specific nematode families
193 were selected by the different extraction treatments. Based on the strongest treatments effects
194 on the total number of extracted nematodes, three treatments with n = 3 were chosen for more
195 detailed identification and PCA analysis: i) 100 g soil fresh weight sieved at 5 mm, ii) 25 g
196 soil fresh weight sieved at 5 mm, and iii) 25 g soil fresh weight without sieving. All samples
197 used for the multivariate analysis were extracted with milk filter. As we assumed nematode
198 body size to reflect different levels of mobility, three size classes were considered, i.e., small
199 (up to 0.5 mm), intermediate (0.5 to 1.0 mm), and large (>1.0 mm). Therefore, the mean size
200 of all species/genera per family was calculated (Table 1) using values listed in Bongers
201 (1994), and in Andrássy (2005) for the family Microlaimidae. In addition, nematode families
202 were assigned to the five c-p classes according to [26] and [27] reflecting life strategie
203 histories with cp 1 and cp 2 indicate r-strategists and cp 3 to cp 5 indicate K-strategists.
204 Furthermore, nematode families were classified according their occurrence to abundant (up to
205 5%), medium (5-1%), and rare (below 1%) families using mean relative occurrence of
206 nematode families in loamy and sandy soils. PCA was performed with R i386 3.3.1 [25].
207
208
209
210
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211 Table 1. Nematode families extracted from loamy and sandy soil. List of nematode families extracted from loamy (L) and sandy (S) soil using
212 the Baermann funnel method with assigned c-p classes after Bongers (1990) and Bongers and Bongers (1998) and trophic groups after Yeates et al.,
213 (1993). In addition, assignment to r- and K strategists. R-strategsits were nematodes from c-p class 1 and 2, whereas c-p classes 3 to 5 are classified
214 as K-strategists [36]. Size classes (small, intermediate, and large) of all nematode families were calculated as the mean size of the minimum (min)
215 and maximum (max) of all genera and species belonging to one family being listed in Bongers (1994), and in Andrássy (2005) for the family
216 Microlaimidae. Occurrence describes in which soil types nematodes occurred. Using mean relative occurrence of nematode families in loamy and
217 sandy soils were used to assign nematodes to abundant (up to 5%), medium (5-1%), and rare (below 1%) families. Means ± SD of nematode
218 families are given for samples displayed in the PCA using different extraction treatments, i.e., using i) 25 g of fresh soil sieved at 5 mm and ii) no
219 sieving, respectively, as well as using 100 g of fresh soil sieved at 5 mm (n = 3). Taxa were sorted by overall mean of extracted nematodes.
Familiy
cp
class
Trophic group
r/K
strat
egy
Mean
size
Size
Mean %
occurence loam
Mean % occurence
sand
Occurence in
loamy soil
Occurence in
sandy soil
Cephalobidae
2
Bacterial feeder
r
0.65
medium
6.67
±
2.00
25.57
±
8.59
abundant
abundant
Tylenchidae
2
Plant feeder + Fungal feeder
r
0.6
medium
13.33
±
4.66
9.18
±
3.67
abundant
abundant
Dolichodoridae
3
Plant feeder
K
0.85
medium
18.44
±
7.04
3.18
±
3.12
abundant
medium
Plectidae
2
Bacterial feeder
r
0.8
medium
8.78
±
4.24
8.86
±
4.66
abundant
abundant
Rhabditidae
1
Bacterial feeder
r
1.35
large
5.22
±
2.17
11.95
±
5.24
abundant
abundant
Hoplolaimidae
3
Plant feeder
K
0.9
medium
12.56
±
3.94
0.00
±
0.00
abundant
not occuring
Qudsianematidae
4
Omnivore
K
1.45
large
4.22
±
2.33
6.29
±
4.24
medium
abundant
Aphelenchoididae
2
Fungal feeder
r
0.65
medium
1.44
±
1.59
6.35
±
3.31
medium
abundant
Paratylenchidae
2
Plant feeder
r
0.35
small
6.22
±
2.68
1.21
±
3.62
abundant
medium
Alaimidae
4
Bacterial feeder
K
1.55
large
2.33
±
1.94
4.48
±
3.87
medium
medium
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Monhysteridae
1
Bacterial feeder + Substrate
ingestion
r
0.75
medium
1.44
±
1.01
5.02
±
4.86
medium
medium
Aporcelaimidae
5
Omnivore
K
4.3
large
0.11
±
0.33
5.23
±
3.95
rare
abundant
Diphtherophorida
e
3
Fungal feeder
K
0.7
medium
4.44
±
1.94
0.54
±
1.13
medium
rare
Mononchidae
4
Predator
K
1.5
large
2.11
±
1.76
2.83
±
2.64
medium
medium
Criconematidae
3
Plant feeder
K
0.45
small
3.33
±
2.00
0.62
±
1.85
medium
rare
Pratylenchidae
3
Plant feeder
K
0.75
medium
3.44
±
2.51
0.48
±
0.73
medium
rare
Tripylidae
3
Predator
K
1.4
large
3.11
±
2.57
0.23
±
0.46
medium
rare
Trichodoridae
4
Plant feeder
K
0.8
medium
0.00
±
0.00
3.04
±
3.44
not occuring
medium
Prismatolaimidae
3
Bacterial feeder
K
1.05
large
0.11
±
0.33
1.57
±
1.83
rare
medium
Thornenematidae
5
Omnivore
K
1.95
large
1.00
±
1.32
0.11
±
0.33
medium
rare
Microlaimidae
3
Bacterial feeder
r
0.55
medium
0.89
±
1.05
0.00
±
0.00
rare
not occuring
Osstellidae
2
Bacterial feeder
r
0.45
small
0.00
±
0.00
0.87
±
2.60
not occuring
rare
Aulolaimidae
3
Bacterial feeder
K
0.95
medium
0.00
±
0.00
0.72
±
0.88
not occuring
rare
Anguinidae
2
Fungal feeder
r
1.65
large
0.00
±
0.00
0.67
±
1.07
not occuring
rare
Leptonchidae
4
Fungal feeder +
K
1
medium
0.00
±
0.00
0.46
±
0.92
not occuring
rare
Panagrolaimidae
1
Bacterial feeder
r
1.1
large
0.44
±
0.53
0.00
±
0.00
rare
not occuring
Discolaimidae
5
Predator
K
1.4
large
0.22
±
0.44
0.19
±
0.57
rare
rare
Diplopeltidae
3
Bacterial feeder
K
0.95
medium
0.00
±
0.00
0.17
±
0.52
not occuring
rare
Bastianidae
3
Bacterial feeder
K
1.5
large
0.00
±
0.00
0.17
±
0.52
not occuring
rare
Anatonchidae
4
Predator
K
2.5
large
0.11
±
0.33
0.00
±
0.00
rare
not occuring
220
221
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222 Results
223
224 Nematode extraction efficiency was affected by a significant four-way interaction of
225 soil type, sieving, amount of soil, and filter (Table S1). To better identify specific treatment
226 effects, datasets were divided by soil type as nematode densities were mainly affected by soil
227 type. Mean ± sd nematode density in loamy soil was 17.5 ± 12.4 nematodes g-1 dry soil
228 compared to 1.4 ± 0.9 nematodes g-1 dry soil in sandy soil. The combination of treatments was
229 of importance for nematode extraction efficiency in the loamy soil as indicated by the three-
230 way interaction, but this was not the case in the sandy soil (Table 2).
231
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232 Table 2. Treatment effects on nematode extraction. ANOVA table of F and P values of the
233 effect of soil weight (25 g, 50 g, and 100 g fresh soil), type of filter (milk filter and paper
234 towel), and sieving (2 mm, 5mm, no sieving), and all possible interactions on nematode
235 extraction efficiency (total nematode densities expressed as individuals g-1 dry soil) in two
236 different soil types (loamy and sandy soil) using the Baermann funnel technique. df: degrees
237 of freedom. Significant results are marked in bold.
Factors
df
F
P
Loamy soil
Soil weight
2,72
94.92
<.0001
Filter
1,72
340.21
<.0001
Sieving
2,72
15.85
<.0001
Soil weight:Filter
2,72
27.22
<.0001
Soil weight:Sieving
4,72
4.13
0.005
Filter:Sieving
2,72
4.85
0.011
Soil weight:Filter:Sieving
4,72
2.97
0.025
Sandy soil
Soil weight
2,71
33.34
<.0001
Filter
1,71
20.80
<.0001
Sieving
2,71
0.24
0.789
Soil weight:Filter
2,71
0.94
0.397
Soil weight:Sieving
4,71
0.32
0.866
Filter:Sieving
2,71
1.93
0.152
Soil weight:Filter:Sieving
4,71
1.18
0.325
238
239 In loamy soil, highest nematode extraction efficiency was achieved using 25 g of fresh
240 soil sieved with a mesh size of 5 mm and using milk filters (42.2 ± 7.7 nematodes g-1 dry soil
241 weight), with milk filters generally increasing nematode extraction efficiency (Fig 2a).
242 Sieving 25 g of soil with a smaller mesh resulted in only slightly fewer nematodes (40.4 ± 5.6
243 nematodes g-1 dry soil weight; -4% in comparison to sieving at 5 mm) and did not differ
244 significantly from sieving with 5 mm. In contrast, nematode extraction efficiency was
245 significantly lower when the soil was not sieved. No sieving of 25 g fresh soil resulted in 31.8
246 nematodes g dry soil-1, that is, 25% fewer nematodes compared to highest number of
247 extracted nematodes. In sandy soil, sieving was not of significant importance (Table 2, Fig
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248 2b). Similar as for the loamy soil though, increasing the amount of soil decreased nematode
249 extraction efficiency significantly (Table 2; Fig 2c).
250
251 Fig 2. Treatment effects on nematode extraction efficiency. Nematode extraction
252 efficiency of the Baermann funnel method in a) loamy and b-d) sandy soil as affected by
253 sieving soil with different mesh sizes (2 mm, 5mm, no sieving), using different filters (milk
254 filter and paper towels) to obtain clean samples, and by using different amounts of fresh soil
255 (25 g, 50 g, and 100 g of fresh soil weight). Significant single factor effects of the sandy soil
256 are given in c) for soil weight and in d) for filter type as indicated by asterisks. Asterisk in
257 panel a) shows the significant three way interaction. Different letters indicate significant
258 difference (Tukey's HSD; α=0.05). *P < 0.05, ***P < 0.001.
259
260 Generally, paper towels significantly reduced overall nematode efficiency by 65% in
261 loamy soil and by 34% in sandy soil. Increasing the amount of soil decreased nematode
262 extraction efficiency from 25 g to 50 g by 30% and from 25 g to 100 by 61% in loamy soil,
263 whereas in sandy soil the reduction was 42% and 60%, respectively (Table 3).
264
265 Table 3. Comparison of nematode extraction efficiency and total amount of extracted
266 nematodes. Extracted nematodes from three different amounts of fresh soil (25 g, 50 g, and
267 100 g) related to fresh and dry soil weight and the proportional difference between soil
268 amounts.
Fresh
weight
Soil dry
weight
Nematodes
(g)
(g)
Total nematodes
extracted from
fresh soil sample
(individuals)
(individuals g-1 dry
soil)
n
Difference to
25 g soil fresh
weight (%)
loamy soil
25
20.77
±
0.59
525.83
±
306.97
25.25
±
14.61
30
---
50
41.03
±
0.80
720.47
±
427.10
17.57
±
10.38
30
-30
100
80.85
±
0.80
792.17
±
426.83
9.80
±
5.28
30
-61
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sandy soil
25
22.60
0.42
49.60
21.27
2.19
0.96
30
---
50
45.01
0.94
57.57
25.49
1.28
0.58
30
-42
100
89.25
1.49
79.24
31.41
0.88
0.36
29
-60
269
270
271 To assess the consequences of sieving and soil weight on nematode community
272 composition, we used a selection of three specific treatment combinations (all using milk
273 filters as densities were highest here), i.e., i) sieving with 5 mm and using 100 g fresh soil
274 (lowest yield in loamy soil), ii) sieving with 5 mm and using 25 g of fresh soil (highest yield
275 in loamy soil), and iii) no sieving and using 25 g of fresh soil (medium yield in loamy soil).
276 Generally, nematode family composition differed strongly between soil types/sites (explained
277 58.6% of the variation, first axis; Fig 3). In the loamy soil, extraction treatments had a
278 stronger effect of the family composition, whereas in sandy soil family composition was more
279 homogenous and sieving and soil weight were of lower importance. In loamy soil, family
280 composition in samples extracted from 100 g fresh soil was more different from samples
281 using 25 g of fresh soil, whereas the family composition of samples without sieved soil and
282 using 25 g fresh soil was intermediate. In loamy soil, more K-strategists were extracted from
283 100 g fresh soil compared to 25 g fresh soil (Fig 4a). No sieving did not increase the amount
284 of K-strategists, i.e., larger organisms that are supposed to be more likely to be damaged by
285 sieving (Fig S1). However, in samples using 100 g loamy soil, more large (Fig S1) and rare
286 (Fig S2) nematodes were extracted. Generally, nematode families from different c-p classes
287 distributed more homogenously among the treatments in sandy soils, indicating a less strong
288 effect of sieving and soil weight in sandy soils.
289
290 Fig 3. Effect of soil type and soil pre-treatments on nematode community composition.
291 Principal component analysis (PCA) of the nematode community (family level) as
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292 affected by soil type and different treatments (sieving with 5 mm mesh size and no sieving,
293 and different amounts of soil [25 g and 100g fresh soil]) prior to extraction reflecting
294 treatment combinations with highest (sieving with 5 mm and 25 g soil), medium (no sieving
295 and 25 g soil), and lowest (5 mm sieving and 100 g soil) nematode extraction efficiency in
296 loamy soil. Symbols represent the specific treatment combinations with larger symbols
297 display centroids. Numbers in brackets are variation explained by the first (Dim1) and second
298 (Dim2) PCA axis, respectively.
299
300 Fig 4. Distribution of nematode c-p classes after extracting nematode with
301 different pre-treatments. Principal component analysis (PCA) of the nematode community
302 (family level) as affected by different treatments (sieving with 5 mm mesh size and no
303 sieving, and different amounts of soil [25 g and 100g fresh soil]) prior to extraction reflecting
304 treatment combinations with highest (sieving with 5 mm and 25 g soil), medium (no sieving
305 and 25 g soil), and lowest (5 mm sieving and 100 g soil) nematode extraction efficiency in a
306 a) loamy and b) sandy soil. Nematode families were assigned to the five c-p classes according
307 to Bongers (1990) and Bongers & Bongers (1998) with blueish colors indicate r-strategists
308 and reddish colors K-strategists. Numbers in brackets are variation explained by the first
309 (Dim1) and second (Dim2) PCA axis, respectively.
310
311
312 Discussion
313
314 In the present study, we found that the combination of different extraction treatments
315 significantly affected nematode extraction efficiency. Although treatment combinations were
316 of different importance in loamy and sandy soil, overall highest numbers of extracted
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317 nematodes were observed when using milk filters and the lowest amount of soil, i.e., 25 g
318 fresh soil. Sieving was important in the loamy soil, while it did not matter if soil was sieved
319 with 2 or 5 mm mesh size. Although not sieving soil yielded highest nematode numbers in the
320 sandy soil, no significant difference was found between sieving and not sieving. Therefore, to
321 achieve high nematode extraction efficiency in different types of soil, it is recommended to
322 use small amounts of soil in combination with sieving and using milk filters.
323 Sieving was of higher importance in the loamy soil than in the sandy soil. Loamy soil
324 has more stable soil aggregates than sandy soil, which is why we suggest that breaking up soil
325 aggregates by sieving increases nematode mobility in loamy soil as nematodes are no longer
326 limited by soil structure and pore space [28]. Using 5 mm compared to 2 mm mesh size
327 resulted in slightly higher (4%) extraction efficiency and may reflect losing some rare families
328 when using a smaller mesh size. These rare families were larger in body size in the present
329 study and may have had a higher probability to be injured by sieving with 2 mm. As we did
330 not analyze the community composition of samples sieved with 2 mm, we cannot provide
331 specific information about the consequences for nematode communities. However, as the
332 observed effect was small (4%) and non-significant, we believe that both mesh sizes can be
333 recommended.
334 Extraction efficiency decreased with an increased amount of soil. The higher
335 proportion of large nematodes in 100 g loamy soil suggests that small nematodes may not
336 have been able to pass and or exit thick soil volumes during the common extraction time of 72
337 h. [29] observed an entomopathogenic nematode to overcome maximally 80 mm in 14 days,
338 indicating that nematodes may be rather slow, which is why the standard [12] even suggests
339 to use a soil volume of only a few millimeters in height. In addition, a thick soil layer can
340 reduce oxygen supply [30], which may decrease nematode survival in the soil sample. PCA
341 revealed that using 100 g of soil increased the number of extracted nematodes of rare families.
342 This may be the result of a higher probability that 100 g soil contains more rare species.
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343 Moreover, rare species often are large in body size, which is why they may have a higher
344 chance to be extracted from larger amounts of soil, as mentioned above. However, the soil
345 volume of 100 g samples also selected against the majority of other nematode families. In
346 summary, using the combination of treatments that resulted in highest nematode extraction
347 efficiency may select against some rare species but may better reflect total densities. To
348 overcome this tradeoff, reducing the thickness of the soil layer by increasing the diameter of
349 the funnel/PVC tube-system may help to increase the amount of soil used and the surface
350 area, allowing nematodes to exit the soil for improved qualitative and quantitative nematode
351 community assessments.
352 Using milk filters resulted in a significantly higher number of extracted nematodes
353 than using paper towels. Paper towels are supposed to adsorb water, whereas milk filters are
354 supposed to filter a solution. The fabric of paper towels is probably chosen such that the fibers
355 will take up water, and this paper structure may hamper nematodes to pass the paper towel.
356 Instead of using paper towels as an alternative for milk filters for biodiversity assessments,
357 they may be used to artificially reduce nematode densities according to morphological traits
358 and alter community composition for targeted experiments.
359 Although we analyzed only a small fraction of possible treatment combinations on the
360 family level, we were able to show that pretreating the soil can change the community
361 composition of extracted nematodes. These results highlight the need to standardize nematode
362 extraction protocols and to account for potential differences when comparing data from
363 multiple sites and studies in syntheses and meta-analyses. The present study may guide the
364 implementation of common nematode extraction protocols for future research.
365 Nematodes are a powerful indicator taxon, and global assessments of soil nematode
366 communities could increase our understanding of global distribution patterns. Generally, only
367 few datasets of global belowground biodiversity exist [31–34], but these still have insufficient
368 data coverage. In this study, we present a rather simple method, i.e. the Baermann funnel
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369 method, to extract nematodes from different soils. The simplicity of the method and the
370 suggested standardized approach allows also non-experts to extract nematodes to participate
371 in global soil biodiversity assessments.
372
373 Acknowledgements
374 We thank UFZ to access the Kreinitz tree diversity platform and two anonymous reviewers
375 for previous comments, which strongly improved the manuscript. In addition, we thank Anja
376 Zeuner for extracting nematodes from the sandy soil.
377
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477
478 Supporting information
479
480 S1 Table. Treatment effects on nematode extraction. ANOVA table of F and P values of
481 the effect of soil type (loamy and sand soil), sieving (2 mm mesh size, 5mm mesh size, no
482 sieving), type of filter (milk filter and paper towel), soil weight (25 g, 50 g, and 100 g fresh
483 soil) and all possible interactions on nematode extraction efficiency (total nematode densities
484 expressed as individuals g-1 dry soil) using the Baermann funnel technique. df: degrees of
485 freedom.
486 S1 Fig. Distribution of nematode size classes after extracting nematode with different
487 pre-treatments. Principal component analysis (PCA) of the nematode community (family
488 level) as affected by different treatments (sieving with 5 mm mesh size and no sieving, and
489 different amounts of soil [25 g and 100g fresh soil]) prior to extraction reflecting treatment
490 combinations with highest (sieving with 5 mm and 25 g soil), medium (no sieving and 25 g
491 soil), and lowest (5 mm sieving and 100 g soil) nematode extraction efficiency in a a) loamy
492 and b) sandy soil. Nematode families were assigned to different size classes according to
493 overall mean values of individuals calculated from sizes given in Bongers (1994) and
494 Andrássy (2005). Numbers in brackets are variation explained by the first (Dim1) and second
495 (Dim2) PCA axis, respectively.
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496 S2 Fig. Distribution of nematode occurrence after extracting nematode with different
497 pre-treatments. Principal component analysis (PCA) of the nematode community (family
498 level) as affected by different treatments (sieving with 5 mm mesh size and no sieving, and
499 different amounts of soil [25 g and 100g fresh soil]) prior to extraction reflecting treatment
500 combinations with highest (sieving with 5 mm and 25 g soil), medium (no sieving and 25 g
501 soil), and lowest (5 mm sieving and 100 g soil) nematode extraction efficiency in a a) loamy
502 and b) sandy soil. Nematode families were assigned according their mean relative occurrence
503 to abundant (up to 5%), medium (5-1%), and rare (below 1%). Numbers in brackets are
504 variation explained by the first (Dim1) and second (Dim2) PCA axis, respectively.
505 S1 File. Raw data of total numbers of nematodes extracted. 180 samples were taken in a
506 full four-way factorial design with the factor soil type (loam and sand), sieving (2 and 5 mm
507 mesh size, no sieving), filter (milk filter and paper towels), and soil weight (25 g, 50 g and
508 100 g fresh soil weight). Total numbers of extracted nematodes are provided as well as
509 nematodes related to g dry soil.
510 S2 File. Raw data of nematode family composition. Identified nematode families from a
511 subset (n=15) of samples for estimating nematode extraction efficiency. Selected treatments
512 had highest, lowest and medium nematode extraction efficiency in loamy soil.
513
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... From each soil sample, a sub-sample of 25 g of fresh soil was taken to extract nematodes using a modified Baermann method (Ruess 1995; Appendix S1). This amount of soil was shown to have high nematode extraction efficiency (Schulz et al. 2018). Nematodes were then grouped into different feeding groups (plant-feeders, bacterialfeeders, fungal-feeders, omnivores, and predators), and their abundances were assessed. ...
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Four of every five multicellular animals on the planet are nematodes. They occupy any niche that provides an available source of organic carbon in marine, freshwater and terrestrial environments. Nematodes vary in sensitivity to pollutants and environmental disturbance. Recent development of indices that integrate the responses of different taxa and trophic groups to perturbation provides a powerful basis for analysis of faunal assemblages in soil as in situ environmental assessment systems.
Article
Plant species identity and diversity may greatly influence the composition of the nematode fauna. The abundance of various nematode populations was investigated in a field experiment on plant diversity. 58 plots in an arable field planted with plant species growing alone or together up to a richness of 12 species were sampled after seven years for analysis of composition of the nematode fauna. Two additional control plots without vegetation were also sampled. Plant species identity was generally more important than plant diversity for the composition of the nematode fauna. Only the omnivorous Aporcelaimidae was positively related to plant species richness, and the fungal-feeding Aphelenchus and the bacterial-feeding Prismatolaimus were affected by functional diversity. Some nematode populations were strongly influenced by plant species composition, e.g. the plant-feeder Tylenchorhynchus maximus was clearly coupled to the grass Phleum pratense. Nematode species within a feeding group sometimes had a rather specific abundance patterns under various plant species and plant species combinations. This was especially the case with the plant-feeding nematodes some of which obviously were directly influenced by the host suitability of specific plant species. Other nematode species were probably more influenced by indirect effects of plants on edaphic and nutrient conditions including biotic interactions from other components in the soil organism community. Yet other nematode species were little influenced by the kind of vegetation in the different plots. Our results show that to fully understand plant community effects on the nematode fauna there is a need to go further than just a division into nematode feeding groups.
Stuttgart: Franckh'sche Verlagshandlung
  • A Meyl
Meyl A. Fadenwürmer (Nematoden). Stuttgart: Franckh'sche Verlagshandlung; 1961. 380 2.
Assessing the Response of Nematode 386
  • R Gingold
  • T Moens
  • A Rocha-Olivares
Gingold R, Moens T, Rocha-Olivares A. Assessing the Response of Nematode 386