GILES: Data collection manual

(Deliverable 2.1)
Version 02.02.2017

1. General considerations

The existing data on plant losses to insects were collected for different purposes and using
different methodologies, and the data on the diversity of the key ecosystem components may
not be easily available to complement the published data on herbivory. To overcome this
problem, we establish a new network to monitor, with standardized methods, selected
ecosystem components in four participating countries (Argentina, Peru, Spain and Finland)
which considerably differ in climatic conditions, biodiversity and land use.

It is critically important that all partners follow this protocol precisely. If the partner foresee
problems with implementation of any part of this protocol, this partner should inform all other
partners well in advance in order to find a mutually acceptable solution. Any modification of
this protocol should be implemented simultaneously by all partners.

This protocol will be discussed at the kick-off meeting (Lima, 21-22 February 2017), and the
training session will be arranged to demonstrate some practical aspects of sampling and
processing of samples. Whenever possible, the key aspects will be video recorded.

2. Study sites and study plants

Each partner will select study sites in (i) pristine/semi-natural forests, (ii) heavily managed or
plantation forests, and (iii) urban forests or urban plantings. The latter plots should be selected
in large cities (urban population >200,000), as close to city centre as possible.

Each managed/urban forest site should be paired to a pristine forest site to calculate the effect
of land use as the relative weighed difference between values recorded in ‘treatment’ and
‘control’ sites (as it is commonly done for calculation of effect sizes in meta-analysis).

Each pairs of study sites (managed/pristine and urban/pristine) should be replicated three
times within each participating country. Thus, each partner will monitor 12 study sites. The
sites of each type should be at least 10 km apart. It is not desirable to have two urban sites in
the same city, even in a megapolis. Whenever possible, study sites should be selected among
those sites for which the partner has earlier monitoring data.

At each site, we will measure intensity of land use (as percent of non-forest habitats within
500 m and 5000 m), diversity of trees, herbaceous vegetation (whenever possible), plant-
feeding insects and insect-feeding birds, stand productivity, and record selected soil
characteristics (thickness of humus layer, pH, nutritional quality) using standard methods of
data collection. Climate data will be obtained from the weather stations nearest to our study
sites. This manual does not specify the methods that are to be used for collecting this
background information; the partners will discuss some of these methods at the kick-off
meeting, and the manual will be extended after that. The third part of the manual (data
analysis) will be prepared by October 2017.

The stability of plant-herbivore-predator interactions at each study site will be monitored

using three species of trees or large shrubs. Plant species and study sites should be selected in

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such a way, that foliage of the selected plant species is easily accessible from the ground. In
other words, we will collect samples at the height not exceeding 2 m.

One angiosperm tree species (Betula pubescens in Finland; Quercus ilex in Spain; Alnus
acuminata in Peru; Nothofagus pumilio in Argentina) will be present in all plots within each
participating country and serve as reference, whereas two other species will be randomly
selected from a list of most common angiosperm species available at each pair of study sites.
Thus, two sites within each pair should have the same set of study plants, but sites belonging
to different pairs must share only one species. By using this design, we will assure the
generality of our conclusions and separate the effects of land use from the effects of species
composition.

The partners are expected to present their tentative sampling design, including selections of
study sites and plant species, by the time of kick-off meeting.

3. Field data collection and processing

3.1. Selecting the sampling time

The losses of foliar biomass accumulate during the entire lifetime of a leaf, although in many
plants the larger part of these losses occurs when leaves are young. The researchers working
with deciduous plants in temperate regions often measure standing herbivory near the end of
the growing season, assuming that this sampling strategy yields lifetime foliar damage.
However, this sampling may underestimate the level of herbivory due to premature abscission
of leaves that were damaged early in the season. On the other hand, densities of sap-feeding
insects are likely to peak in the middle of the growth season. Therefore we will measure
insect herbivory twice in the season.

We agreed that the sampling will be conducted in the following months:

 Argentina: in February and December;
 Finland: in June and August;
 Peru: in May and November;
 Spain: in April and June;

3.2. Sampling of tree branches

For the current project, we will sample the branches that could be assessed from the ground
without disturbing insects feeding with plant foliage. This may preclude generalization of the
obtained absolute estimates of herbivory to the entire tree canopy, but is unlikely to distort our
conclusions on temporal variation in herbivory.

Selection of plant individuals for sampling should assure absence of a bias. We will select
trees within a site following a ‘first found, first sampled’ approach. The sampled trees will not
be tagged and, therefore, repeated samples will generally be collected from different trees.
However, the use of the same trees is allowed, especially when the number of suitable plant
individuals is low (e.g., in urban environment).

To avoid unconscious selection of a branch within the tree crown, resulting from the
collector’s opinion on the ‘typical’ level of herbivory, the collector will choose a branch
from a distance that does not allow visual evaluation of foliar damage, e.g. 5–10 m.

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The selected branch should have 50-150 leaves to allow accurate assessment of herbivory,
and branch position on a tree should allow collecting without disturbing the invertebrates
inhabiting the foliage.

The branch is collected as follows: one of two collectors places a mesh bag attached to a ring
(60 cm in diameter) under the selected branch, while the second collector cuts the branch in
such a way that it fell into the bag together with the arthropods that dropped from the branch
when disturbed.

If the selected plant species has a resin or sticky sap, then branch section should be closed by
e.g. polyethylene to prevent insects from damage by plant sap, and to prevent contamination
of a bag used for sampling.

The bag should be immediately closed, labelled and transported to the laboratory for
processing. Each bag should contain one branch. The care should be taken to keep leaves
fresh and invertebrates alive until processing of a sample. In practice, this means that samples
should not be pressed, dried, or overheated - the use of cool boxes is desirable.

Two trained persons need 1 hour or less to collect 15 branches at a site. To perform this task,
they need 15 bags, pruning shears (hand pruners), small pieces of polyethylene and small
rubber rings (if the selected plants produce sticky sap), and pre-printed labels.

3.3. Processing of tree branches

In the laboratory, use large desk with white surface, or cover your desk with white paper, to
sort the samples. You will need excellent illumination not to miss any animal. Open the bag
carefully; be prepared that some invertebrates, especially spiders, will immediately try to
escape. Collect all invertebrates from a bag and from a branch into individual vial containing
70% ethanol. It is useful to shake a branch within a bag, then inspect the foliage for insects
and, finally, beat a table surface with a branch to assure that all invertebrates are removed

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from foliage. Use pre-printed labels for samples preserved in alcohol: one branch=one or
more vials; do not combine invertebrates collected from different branches!

After all invertebrates are collected, separate all leaves from the woody parts of a branch. Cut
woody parts into pieces and pack these into a paper envelope (use pre-printed labels) for
drying (105ºC for 48 h) and weighing (to the nearest 1 mg). After weighing, branches can be
trashed.

Count the leaves, record their number. Separate intact leaves from leaves damaged by insects.
Even the smallest damage imposed by an insect, i.e. loss of 1 mm2 of leaf area, makes the
leaf damaged. The damage might include loss of leaf margins, holes, skeletonization, mines
and galls.

Count undamaged leaves, record their number. Randomly select five undamaged leaves (e.g.
mesh these leaves on a desk and take the uppermost ones); pack these into a paper envelope
(use pre-printed labels) for drying (105ºC for 24 h) and weighing (to the nearest 1 mg). After
weighing, these leaves can be trashed. If you do not have five undamaged leaves in your
sample, then proceed to the next step.

Select leaves which have mines or galls. The search for these types of damage requires
looking at both leaf surfaces; in particular, epidermal mines can be seen only from one side of
a leaf. Examination of a leaf against strong light source helps to recognize minor mines.

Attribute each leaf that has a mine or a gall to one of the six damage classes, according to the
visual estimate of the percentage of the leaf area covered by mine(s) or gall(s). The damage
classes are: 0.01-1, 1-5, 5-25, 25-50, 50-75 and 75-100%. At this stage, do not pay any
attention to damage imposed by leaf-chewing insects, which may also occur on these leaves.

Two methods could facilitate visual estimation of the percentage of leaf damage. First, you
can prepare a figure, which is approximately of a size and shape of a leaf of your study plant,
and show the size of damage corresponding to first three damage classes on this figure (see
below). Leaves belonging to high-damage classes are usually infrequent, and their visual
classification does not cause problems.

The second method is to measure (using a palette, or image processing software) the
percentage of the consumed or otherwise damaged leaf area in several dozens of leaves, and
then use the images of these leaves (see below) to classify further damaged leaves.

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Of course it is possible to measure damage of each leaf with a software, but several
intercalibration projects concluded that image processing, which is significantly more
laborious than the visual assessment (in particular, due to the need to manually reconstruct the
edge of each damaged leaf, and difficulties in tracing minor damage on leaf images), has no
advantages over visual estimation of the missed leaf area in terms of the accuracy of
measurements (Wint 1983; Schaffer et al. 1997). The relative difference between the mean
values obtained by these two methods, instrumental and visual, ranged from 2−5% (Basset
1991; Blundell and Peart 2000). Moreover, the results of visual estimation made by different
evaluators were quite similar to each other (Schaffer et al. 1997).

Record the numbers of leaves with mines and galls, belonging to each damage class, in a pre-
printed form (see an example below: damage records for foliage of five trees of Salix caprea).

After that, examine each of these leaves for damage imposed by other groups of herbivores,
e.g. search for galls and bite marks or holes eaten away by defoliating insects in mined leaves.
Record the results to the same form (line «Defoliators»).

Finally, evaluate the TOTAL damage imposed by all feeding guilds of insects on these leaves,

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and record the results in the «Total» line. Do not be confused: if a leaf has a gall, a mine and a
hole, it will then be recorded in all four lines.

Note that we cannot calculate the sum of damage from three first lines to get the «Total»
result. Imagine you have a leaf with a gall (6%), a mine (10%), and a hole (7%). This leaf will
be recorded in the column «5-25%» in lines «Defoliators», «Miners» and «Galls», but also in
the same column in the «Total» line (6+10+7=23%). But another leaf recorded in the same
column in lines «Defoliators», «Miners» and «Galls», may easily go to the column «50-75%»
in the line «Total», if each type of damage covers 20% of leaf area (20+20+20=60%).

Mines and galls can often be identified to the species' level, and therefore it is desirable to
preserve mined and galled leaves as ordinary herbarium specimens for future processing. Of
course, if some type of gall or mine is common, there is no need to preserve all mined and
galled leaves.

Processing of the remaining leaves, which are damaged by externally feeding insects only, is
simpler: you classify these leaves by damage classes and record the results twice: in the lines
«Defoliators» and «Total».

If your sample did not contain five undamaged leaves (see above), then select five leaves with
the lowest level of damage and preserve these leaves in a paper envelope for drying and
weighing.

After you complete processing of five branches of the same tree species from a site, then
combine the leaves damaged by externally feeding defoliators, which were collected from
these branches, and randomly select 100 of these leaves. If you have less than 100 damaged
leaves, then preserve all of them. Press-dry these leaves as ordinary herbarium specimens for
measurements of the diversity of damage (the protocol will be provided later on). The
remaining leaves (if any) can be trashed.

A team of three trained persons during a working day can process 20-40 branches, depending
on the number of leaves on these branches and the intensity of damage. The equipment and
consumables needed for this work: vials, 70% ethanol, soft forceps, a brush (wet brush is used
to take minor invertebrates from the desk and place these into a vial with ethanol), pre-printed
labels for different types of samples, pre-printed data recording forms and paper envelopes for
different types of samples.

3.4. Sampling of soil-dwelling insects and plant roots

The data on root losses to insects generally refer to the entire plant community, because it is
rather difficult to sort roots by plant species and to learn the diet of each collected specimen of
root-feeding insects. We will calculate root losses to insects from insect load, i.e. the insect
biomass per unit of dry weight of roots. To obtain these data, insects and roots will be
quantitatively collected from a known amount of soil.

Collect seven soil samples of 25×25×30 (depth) cm at each study site at each sampling date.
The site selection bias is unlikely to occur in studies of root herbivores, because their density,
as well as root density, can not be estimated before the sample is processed. Still some kind of
random choice of sampling sites, e.g. by throwing a stone at random over the study area, is
desirable.

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Before sampling, plants should be cut and removed from the selected area. Use a shovel to dig
out the topsoil (A horizon), and pack it separately from the soli from more deep layers. A
shovel which is 25 cm wide is most suitable for this sampling.

If the sampled soil contains many big stones, then visually estimate and record the percentage
of their volume in the collected soil sample.

Packing of samples should prevent soil from drying, and do not allow soil animals to escape.
We use 10-L plastic bags to pack topsoil, and collect deeper soil layers into 14-L plastic
containers (see below).

Next to these seven samples, please collect seven soil cores (approx. to 30 cm depth) using a
sampler 30-50 mm diameter (see below). We use a sampler with internal diameter 36 mm.

Place a core into a plastic bag in such a way, that it remains intact or nearly intact, so that its
total length and the thickness of individual layers can be measured later on. Alternatively, you
can measure cores and split them into individual samples (as described below, sect. 3.5)
immediately after their extraction.

A team of two trained persons needs 30-45 min to sample one study site. The collecting time
increases greatly when the soil contains numerous stones: it may take 5-10 min to locate a
point where soil corer reached the desired depth of 30 cm. Instruments: a ruler (if the width of
your shovel is less than 25 cm), shovel, soil corer, 10-L plastic bags for topsoil, plastic
containers for deeper soil horizons (can be replaced by 20-L plastic bags in combination with
a bucket: place a bag into a bucket for collecting a soil), 3-L bags (for soil cores), tape (for
packing soil cores), waterproof pre-printed labels, form for recording the percentage of stones
in soil samples.

3.5. Extraction of invertebrates from soil cores

Soil samples collected for extraction of invertebrates could be preserved 3-5 days at +10-
12ºC, and 1-2 days at +16-22ºC. Avoid overheating or longer storage. Alive (moving)

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invertebrates are easily recognised, but it is extremely difficult to collect dead bodies of
invertebrates from soil samples.

Each sample should be hand-sorted. The use of soil sieves (4-6 mm grid) facilitates the
procedure: small portions of soils are sieved above a white tray, so that invertebrates became
visible. All invertebrates should be collected and placed into a vial with 70% ethanol: one
sample = one or more vials. Keep animals from topsoil separately from animals extracted
from deeper soil horizons.
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02A trained person can sort 5-10 samples (including both topsoil and deeper soil layers)
during one working day. This work requires soil sieves, white trays, vials, rubber gloves, 70%
ethanol, soft forceps, a brush (wet brush is used to take minor insects from the soil and place
these into a vial with ethanol), and pre-printed labels for invertebrate samples.

3.6. Processing of soil cores

Soil cores, if properly packed to prevent them from drying, can be preserved in refrigerator
for 1-3 weeks without obvious problems with their subsequent processing. They can be even
frozen for indefinitely long time; however, thawed samples are more difficult to process: fine
roots more easily break in parts, and it is more difficult to distinguish between dead and alive
roots.

Start core processing from measuring the depth of topsoil; then separate topsoil (with a knife)
from deeper soil layers. Record the length of the remaining part of a core, starting from the
lower border of topsoil, and split this remaining part into pieces as shown on a picture
(below). The measurements of the length are needed because the cores will be of different
length, and therefore their deepest piece will be of variable length. We will use the length of
each part of a core to express root biomass per unit of soil volume.

Process each part of a core separately. Extract roots by hand-sorting and hand-washing over a
sieve (2-3 mm grid). The step-by-step description of the procedure:
 carefully break up the sample, separating plant roots from other things;
 green plant tissues do NOT belong to roots, whereas dark or white underground stems
of plants (rhizomes) do belong to roots;
 place all discovered roots into the white plate filled with water;
 wash the roots (mycorrhyza is a part of the root) out from soil and other substances
(use brush whenever necessary) and move roots to the next white plate filled with
water; repeat this procedure until the roots are clean from soil particles;
 take the roots out from a plate and place them on filter paper;
 sort the roots into four categories (use callipers whenever necessary):

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 - thick (>2 mm diam) alive roots;
- thin (<2 mm diam) alive roots;
- thick (>2 mm diam) dead roots;
- thin (<2 mm diam) dead roots;
 If a piece of a root contains both thick and thin parts, then cut it into pieces to have
thick and thin roots separated;
 pack each of these subsamples (note that some samples not contain some categories of
roots) into individual envelopes with corresponding labels.

Notes:
 Dead roots are easily broken, whereas alive roots are flexible.
 Do not waste your time trying to collect small (<5 mm length) thin pieces of roots –
keep in mind that they contribute little to the total biomass; the loss of 1-2 % of root
biomass is unavoidable and acceptable.

Processing of a topsoil layer from a core may easily take several hours; in Finland, it took ca.
1 hour on average. The deeper soil horizons are processed much faster (5-15 min per 10-cm
part of a core) by washing over soil sieve with 2-3 mm grid. Thus, one person can sort 4-6
cores during one working day. This work requires white plates, strong forceps, a brush,
scissors, callipers, paper envelopes (10-15 per each soil core), and pre-printed labels for root
samples.

The roots should be oven-dried (105ºC at 48 h) and weighed to the nearest 1 mg. After
weighing, roots can be trashed.

3.7. Intensity of bird predation

Bird predation on free-living defoliators will be measured using artificial plasticine larvae;
this method is increasingly used to study the avian predation pressure on insect herbivores in
different environments (Sam et al., 2015; Muiruri et al., 2016). Three larvae (approx. 30 mm
length and 4 mm diameter) of different colours (we used green, yellow and brown) will be
attached to branches of each of five trees of Betula pubescens in Finland, Quercus ilex in
Spain, Alnus acuminata in Peru, and Nothofagus pumilio in Argentina (i.e., 15 dummy larvae
per site).

The timing of this experiment should correspond to the timing of herbivore sampling. The
duration of exposure, i.e. time period between the introduction of dummy larvae and
evaluation of damage imposed by predators, primarily bird’s beak marks, should be
established experimentally for each country to allow sufficient damage of these dummy larvae
but avoid the situation when nearly all of them are damaged. In Finland, we successfully used
one-month interval.

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We observed only one type of damage on our plasticine larvae, but the diversity could be
higher in tropics. If you observe different types of damage, then please count them separately.
We counted the numbers of attacks on dummy larvae as long as individual damages could be
counted. After that, we evaluated the percentage of plasticine that was removed by predators
(25, 50, 75 or 99%).

Preparation of a dummy larva requires, on average, 3 min.; thus, 180 larvae needed for a team
(15 larvae × 12 sites) could be prepared in 6 hours. Introduction of larvae and data recording
each take 20-30 min per site.

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