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Ares(2018)3472032 - 29/06/2018

Work package number 9 WP3 Lead beneficiary 10 4 - RRes

Work package title Linking crop yields with off-crop functional biodiversity
Start month 1 End month 54

Objectives

Main objective of WP3 is to quantify the relationship between ESP, pests and crop yield by combining experimental
provisioning of resources for ESP with yield maps from GPS-enabled harvesters.
- Link off-crop habitat provision with in-crop yield variation, by using farmers’ yield map data from GPS-enabled
harvesters.
- Establish experimental framework to optimize off-crop habitats to support ESP populations and movement for service
provision at field/landscape scale
- Track shifts in abundance of ESP between in-crop and off-crop habitats through the growing season using time lapse
imagery and machine learning tools
- Quantify the direct and indirect interactions of pests, natural enemies and pollinators between crops and off-crop
habitats by combining DNA-barcoding and ecological network analysis

Description of work and role of partners

WP3 - Linking crop yields with off-crop functional biodiversity [Months: 1-54]
RRes, SLU, AU, UNE, ISARA, JKI, UKA, AUPL, FBUB, UBL , UJA, UC, UB, ProAg, FOR, AT, LLH, UTU
Coordinator: Samantha Cook (RRes), deputies: Milan Plećaš (FBUB), Darren Evans (UNE)
Spatio-temporal dynamics of various ESP functional groups of arthropods within agricultural landscapes is influenced
by a variety of in-crop and off-crop interactions (both mutualistic and antagonistic), hence the effectiveness of ESP on
yield level is context dependent (Tscharntke et al. 2012). Numerous approaches have been designed to improve yield
by manipulating the quality and availability of semi-natural habitats around the crops on a varied landscape scales, with
mixed success across crops, landscape types, and (bio)geographical regions, and even between similar studies across
years (Plećaš et al. 2014). However, the complex interactions between agricultural plants and insects across trophic levels
is poorly understood. We will experimentally manipulate spatio-temporal availability of off-crop flowering resources in
varied landscape contexts across a biogeographical gradient, and quantify the changes in yield and population levels of
key insect pests, natural enemies and pollinators, as well as their movements between respective habitats (crop-fields,
flower strips, semi-natural habitats, etc), i.e. spill-over effects of ESP both to and from mass-flowering crops. Advances
in Next Generation Sequencing will be developed to create highly-resolved species interaction networks, the structures
of which will be compared between experimental treatments. We will develop a spatially- explicit sampling design for
accurate sampling of ESP to assess and monitor population dynamics, interactions and movements at the landscape
scale and in a phenological context, to identify the key actors and evaluate their relative functional importance to the
ecosystem services they provide, and to model these factors in relation to crop yield.
Task 3.1. Yield-data -based assessment of biodiversity benefits on crop yields
Leads: Samantha Cook & Alice Milne (RRes)
By linking yield monitor data (which is recorded by most GPS enabled combine harvesters) with spatially explicit data
on the locations, quality and quantity of landscape features known to support ESP (primarily pollinators and natural
enemies of crop pests), we will quantify their effects on crop yield and the distance into the crop over which any effects
operate. This will give growers information on the real value of such features and will ultimately be able to optimize
positioning of interventions to improve yield via effective provisioning for pollination and biocontrol services.
Yield monitor data generated over the course of field experiments within this Project (WP3; WP4) as well as historical
data from Partners and farms within our Network (WP2) will be collated (RRes & UH). Code will be developed to extract
information including these raw data from the Farm management systems (e.g. Gatekeeper) used to create yield maps
(RRes). Detailed landscape maps will be created using high-resolution satellite imagery encompassing the 1km area
surrounding each crop with monitor yield data and other field data such as soil type, EC and meteorological data relating
to the fields will be collated (RRes). Statistical and modelling exercises will then be done to relate yield variations
within the crop to mapped landscape features known to support ESP (RRes). We recognize that there will be signals
other than ESP affecting yield, such as soil moisture, soil type, pest, weed and disease pressure etc., and these will be
accounted for in as many sites as possible from sampling undertaken in WP3 and WP4. Clear relationships between
habitat quality, quantity and location with yield effects will be communicated to farmers and policy makers via WP2 and

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WP10 respectively, to improve uptake and development of successful agri-environment schemes to optimise provision
of resources in the landscape for ESP for maximum yield benefits.
Task 3.2 Optimising spatio-temporal availability of off-crop habitats for improved ESP effectiveness,
Leads: Milan Plećaš & Željko Tomanović (FBUB); Samantha Cook (RRes)
The task aims to improve ecosystem service provision in-crop by optimizing provision of off-crop resources, which
provide habitat, food, and alternative hosts for crop pollinators and natural enemies of crop pests. Providing early-
flowering resources in the vicinity of target crops should attract and maintain pollinators to the crop vicinity before
focal crop flowering period enabling better pollination while late-flowering resources should support pollinators after
crop flowering ends enabling pollinators to finish the season cycle. Likewise, early flowering resources support natural
enemies of crops pests when resources are scarce, helping to build-up natural enemy populations in the crop early enough
to prevent pest outbreaks, and late-flowering resources provide food, habitat and alternative prey species after mass-
flowering crops have finished blooming, therefore promoting better survival of natural enemies for the next season).
We will undertake a manipulative field experiment, replicated in each country and repeated across at least 3 different
European pedo-climatic zones, comprising paired oilseed rape (OSR) crops supplemented with biogeographically
optimised flowering field margins (early- and late-flowering) and controls without any additional sown habitat resources
(FBUB, RRes, AU). Flower mixes will be developed to cover the entire phenology of all important ESP functional
groups (beyond the flowering period of OSR), in order to attract, build-up and maintain ESP populations throughout
their life-cycles. We expect extended provision of complementary resources to change the abundance and/or diversity
of beneficial insects. We will identify the functionally most important members of each ESP guild, and monitor
relevant ESP and pest community parameters (structure, interactions, stability, spatial dynamics), by use of standardized
sampling techniques for each group (Williams et al. 2007; Westphal et al. 2008; Vaissière et al. 2011; Popic et al. 2013
as well as by estimation by digital imagery (Task 3.4). The surrounding landscape (1km-radius) of each experimental
site will be visited and crops and landscape features recorded to assess the quality and quantity of habitat provision
for ESP and landscape complexity, and to relate to maps produced of these sites as part of Task 3.1. Sampling will be
conducted at spatially-explicit sampling points throughout the crop and field margins. At each sample point, selected
plants will be intensively studied, measuring pest infestation, parasitisation, predation and pollination. Samples of plant
and arthropod materials will be taken and related to ESP dynamics assessed via molecular methods in Task 3.3. At the
end of the season, yield will be estimated for each sample point and related to yield maps (if available) produced by GPS
enabled combines (Task 3.1). ESP samples collected in this task will also be used also in WP7 to construct a database
of the most important ESP in study areas and to determine in-field status of ESP sensitivity to agrochemicals.
Task 3.3. Tracking ESP dynamics using molecular tools
Lead: Darren Evans (UNE)
Considerable effort is needed to quantify the direct and indirect interactions between plants and animals, especially insect
pests and their predators, before ESP can be exploited commercially. Complete DNA barcodes are necessary for the
correct placement of species in phylogenetically-structured networks, which can then be used for trait-based analyses.
Presently, DNA barcodes for key ESP are either woefully incomplete (e.g. hymenopteran parasitoids, Evans et al. 2016)
or erroneous in open-resource genetic sequence databases (e.g. GenBank). This Task will develop and combine DNA
metabarcoding approaches with ecological network analysis to determine the interactions between ESP and understand
their movements between in-crop and off-crop plants. First, an up-to-date and complete DNA-barcode library of the
focal organisms will be created. Second, novel ‘nested-tagging’ DNA metabarcoding protocols will be developed and
validated for the construction of species-interaction networks. This will be used to quantify ESP interactions within
and between crop and non-crop habitats. Third, a PCR-diagnostic tool for rapidly determining parasitism rates of insect
pests will be developed in partnership with growers (WP2) to assist in pesticide application decision-making.
UNE have developed a novel ‘nested tagging’ metabarcoding approach (which allows for individual level sample
tracking through the use of hierarchically organised molecular identification tags) for the rapid construction of large,
phylogenetically structured species-interaction networks (Evans et al. 2016). The following ecological networks will be
constructed at the study sites described in Task 3.2 using standardized sampling methods (Pocock et al. 2012): a) flower-
visitor networks based on (i) direct observation of insects caught on flowers and later identified; ii) pollen identified
on caught insect mouth-parts; and iii) the presence of insect DNA in plant nectaries); b) host-parasitoid networks
(based on i) screening collected hosts for parasitoid DNA (i.e. egg, larvae) and ii) screening collected adult parasitoids
for host DNA on the body or in the gut) and c) intraguild interactions based on presence of predated insect DNA
within the gut. Second, by systematically sampling insects within and between crop and non-crop habitats, quantitative,
phylogenetically-structured ecological networks will be constructed following Pocock et al. (2012) to determine: a)
the extent of plant and habitat sharing by antagonists and mutualists; b) the degree of parasitoid sharing; c) variation
in network structures and d) the ‘robustness’ (a measure of the tolerance of the network) of the networks to species

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extinctions. This latter approach allows for the disproportionately important species and habitats for network integrity
to be identified, which can then be used for targeted management (Evans et al. 2013).
UNE have aided in the development of a universal method for the detection and identification of Aphidiinae
parasitoids within their aphid hosts (following Derocles et al. 2012b). Briefly, DNA amplification (based on 16S
rRNA mitochondrial gene and LWRh nuclear gene) with primers common to aphid parasitoids and sequencing of the
amplified fragment is used to rapidly detect and identify parasitoids in their hosts, without prior knowledge on the
species potentially present. By focusing on aphid-parasitoid interactions in wheat crops determined using the PCR-
diagnostics approach (with RRes), this Task will: a) quantify parasitism rates of aphids throughout 3 growing seasons;
b) determine the rate at which aphid infestation and/or parasitism crop yield is affected (using controlled glass-house
experiments at UNE) and c) develop and test a lab-to-farm diagnostic facility for rapid deployment (in collaboration
with The Institute for Agri-Food Research and Innovation (IAFRI) at Newcastle University - a joint venture between
Newcastle University and Fera Science Ltd (Fera).
Task 3.4. Tracking ESP dynamics using image-based tools
Lead: Toke Høye (AU)
Since the functional importance of pollinators and natural enemies in agricultural systems is closely linked to their
abundance, a key challenge is to track temporal and spatial variation in abundance of ESP. Traditional methods (manual
observation and trapping) are either very labour intensive or confound activity and abundance. Using image-based
methods, we can control for variation in activity by operating cameras at high temporal frequency to offer a cost-effective
solution for quantifying abundance without the need to manually sort insect samples (Steen 2017, Gilpin et al. 2017).
So far, image-based techniques have been used to demonstrate this potential. This task will advance this technique for
large-scale application across multiple sites. This entails the development of field protocols and hardware solutions and
the construction of image reference libraries to train neural networks for arthropod detection and identification.
We will develop a standardized field setup for image-based recording of ESP adapted to track abundant species of pests,
pollinators and natural enemies across experimental study sites. Time lapse cameras will be used to record occurrence
rates of pest species, pollinators, and natural enemies of crop pests directly from images of flowers, plant parts and
patches of ground. The development and testing of field protocol will be carried out in year 1 and implemented in years
2-4 across sites. The field experimentation carried out in Task 3.2 will provide the basis for assessing the fine scale
temporal dynamics of ESP and will be used to infer ESP use of in-crop vs. off-crop resources. In this way, this task
supplements the detailed interaction networks resolved in Task 3.3.
The detection of animals will rely on object-oriented image analysis and the automatic identification of dominant ESP
will be based on machine learning, which is a specific expertise of partner AU. By integration with Task 3.2 and Task 3.3,
an image database of the dominant species in each functional group at each site will be constructed. The image database
of species of known identity will be used to train neural network algorithms for the automatic identification of abundant
taxa using machine learning. Machine learning is used by companies like Facebook and Google to automatically
extract information from images and has already been exploited in the automatic fine-grained classification of aquatic
macroinvertebrates (Raitohariju et al. 2016, Arje et al. 2017). We will adapt and validate existing algorithms to infer
abundance, identity, and biomass from reference data. With the neural network algorithms in place, we will generate
co-occurrence patterns and compare these to the molecular ecological networks constructed in Task 3.3. In years 2-4,
we will implement camera traps in connection with field trials in treatment and control plots to quantify spatial and
temporal dynamics of ESP and pest species as well as co-occurrence patterns across sites and countries. These data will
be used to assess the importance resource provisioning across the season for ESP and thus yield (in Task 3.1.) while
controlling for climatically related bias in activity as well as landscape context.

Participation per Partner

Partner number and short name WP3 effort

2 - SLU 3.00
3 - AU 30.00
4 - RRes 76.00
5 - UNE 48.00
6 - ISARA 2.00
7 - JKI 1.00

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Partner number and short name WP3 effort

8 - UKA 2.00
10 - AUPL 15.00
11 - FBUB 30.00
12 - UBL 12.00
13 - UJA 4.00
14 - UC 4.00
15 - UB 2.00
16 - ProAg 3.00
19 - FOR 2.00
20 - AT 1.00
21 - LLH 2.00
23 - UTU 2.00
Total 239.00

List of deliverables

Due
Deliverable
Deliverable Title Lead beneficiary Type 15
Dissemination level 16 Date (in
Number14
months)17
DNA diagnostic tool for
D3.1 5 - UNE Demonstrator Public 24
pesticide application
Impact of floral resources
D3.2 11 - FBUB Report Public 36
on ESP dynamics
DNA-barcode inventories
D3.3 of ESP and interacting 5 - UNE Demonstrator Public 36
organisms
Guidance materials
D3.4 for best use of floral 11 - FBUB Report Public 48
resources
Mathematical model
D3.5 predicting the ESP 4 - RRes Other Public 48
impact of yield
DNA-metabarcoding
D3.6 protocols to quantify ESP 5 - UNE Report Public 48
interactions
ESP abundance across
D3.7 3 - AU Demonstrator Public 48
pedo-climatic zones

Description of deliverables

3.1 A rapid DNA diagnostic tool for pesticide application decision-making based on parasitism rates of key
agricultural insect pests (24)
3.2 Documented impact of how temporal availability of floral resources affects dynamics and movement of ESP, and
crop yield for mass flowering crops (36)

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3.3 DNA-barcodes and accompanying inventory for a complete range of ESP and the organisms they interact with
(36)
3.4 Guidance materials for farmers on how to best use floral resources for improving yields (48)
3.5 Mathematical model to relate quality, quantity and location of habitats for ESP with yield effects from yield maps
(48).
3.6 New DNA-metabarcoding protocols to quantify ESP interactions, with quantitative ecological network
information of in-crop and off-crop usage for targeted management (48)
3.7 A generic, low maintenance image-based tool to track ESP abundance across pedo-climatic zones (48)
D3.1 : DNA diagnostic tool for pesticide application [24]
A rapid DNA diagnostic tool for pesticide application decision-making based on parasitism rates of key agricultural
insect pests
D3.2 : Impact of floral resources on ESP dynamics [36]
Documented impact of how temporal availability of floral resources affects dynamics and movement of ESP, and crop
yield for mass flowering crops
D3.3 : DNA-barcode inventories of ESP and interacting organisms [36]
Development of DNA-barcodes and accompanying inventory for a complete range of ESP and the organisms they
interact with
D3.4 : Guidance materials for best use of floral resources [48]
Guidance materials for farmers on how to best use floral resources for improving yields
D3.5 : Mathematical model predicting the ESP impact of yield [48]
Mathematical model to relate quality, quantity and location of habitats for ESP with yield effects from yield maps
D3.6 : DNA-metabarcoding protocols to quantify ESP interactions [48]
New DNA-metabarcoding protocols to quantify ESP interactions, with quantitative ecological network information of
in-crop and off-crop usage for targeted management
D3.7 : ESP abundance across pedo-climatic zones [48]
A generic, low maintenance image-based tool to track ESP abundance across pedo-climatic zones

Schedule of relevant Milestones

Due
Milestone
Milestone title Lead beneficiary Date (in Means of verification
number18
months)
Selection, sowing of floral
MS8 Experimental site network 4 - RRes 12 strips and landscape analyses
carried out
Protocols for sampling of
Protocols for sampling of ESP ESP and pest populations and
MS9 4 - RRes 14
and pest populations assessment of their effects
made available
Collection of yield maps and
MS10 Yield maps 4 - RRes 40
related ecological data
Mathematical model to relate
Mathematical model
MS11 off-crop ESP with in-crop 4 - RRes 48
developed
yield effects
Availability of DNA-barcodes
MS12 DNA-barcodes 5 - UNE 36 and inventory for ESP and
organisms they interact with

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Schedule of relevant Milestones

Due
Milestone
Milestone title Lead beneficiary Date (in Means of verification
number18
months)
Availability of DNA-
New DNA-metabarcoding
MS13 5 - UNE 48 metabarcoding protocols to
protocols
quantify ESP interactions
Rapid DNA diagnostic tool Prototype of tool developed
MS14 5 - UNE 24
for pesticide decision-making and tested
A generic, low maintenance
Iimage-based tool to track
MS15 3 - AU 48 image-based tool to track ESP
ESP
abundance

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