Electronic Supplementary Material
ESM 1 GenBank accession numbers of representative DNA sequences of target and non-target taxa. Columns
show the taxonomic affiliation of the invertebrate taxa, the gene for which sequences are provided and an
indication of whether the sequences have been generated for this study (the identity of all taxa was assured by
qualified taxonomists) or retrieved from GenBank
taxa
Carabidae, Acupalpus parvulus
Carabidae, Anchomenus dorsale
Carabidae, Agonum muelleri
Carabidae, Amara aenea
Carabidae, Amara bifrons
Carabidae, Amara similata
Carabidae, Asaphidion flavipes
Carabidae, Bembidion lampros
Carabidae, Bembidion quadrimaculatum
Carabidae, Bembidion tetracolum
Carabidae, Bembidion guttula
Carabidae, Calathus fuscipes
Carabidae, Calathus melanocephalus
Carabidae, Carabus cancellatus
Carabidae, Carabus granulatus
Carabidae, Clivina fossor
Carabidae, Harpalus affinis
Carabidae, Harpalus rufipes
Carabidae, Loricera pilicornis
Carabidae, Nebria brevicollis
Carabidae, Patrobus longicornis
Carabidae, Poecilus cupreus
Carabidae, Poecilus versicolor
Carabidae, Pterostichus melanarius
Carabidae, Pterostichus niger
Carabidae, Synuchus vivalis
Carabidae, Trechus quadristriatus
Carabidae, Trechus secalis
Staphylinidae, Atheta gregaria
Staphylinidae, Philonthus fimetarius
Staphylinidae, Stenus providus
Staphylinidae, Stenus clavicornis
Staphylinidae, Tachinus rufipes
Staphylinidae, Tachyporus chrysomelinus
Staphylinidae, Tachyporus formosus
Staphylinidae, Tachyporus hypnorum
Staphylinidae, Tachyporus obtusus
Staphylinidae, Xantholinus tricolor
Coccinellidae, Adalia bipunctata
Coccinellidae, Adalia decempunctata
GenBank accession nos
JN619253
KT204374
KT204317
KT204375
KT204318
KT204376
FJ173123
FN868610
KT204377
KT204319
KT204378
KT204320
KT204379
KT204321
KT204380
AF201402
KT204381
KT204322
KT204382
FJ173118
KJ962712
JX279744
KT204383
KT204323
KT204384
KT204324
KT204385
KT204325
KT204386
AF201396
KT204387
AF201395
KT204388
AF002786
KT204326
KT204389
KT204327
KT204390
KT204328
KT204391
KT204329
KT204392
FJ173120
KT204393
KT204330
KT204394
KT204331
KT204395
KT204332
KT204396
JN619017
AY745615
KT204397
KT204333
KT204398
KT204334
KT204399
AJ293031
KT204336
KT204400
KT204335
KT204337
KT204401
KT204338
KT204402
EF512323
JQ757053
gene
18S
COI
18S
COI
18S
COI
18S
COI
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
18S
COI
18S
COI
18S
COI
COI
18S
COI
18S
18S
COI
18S
COI
18S
COI
present study



















































1
Coccinellidae, Anatis ocellata
Coccinellidae, Anisosticta novedecimpunctata
Coccinellidae, Coccinella septempunctata
Coccinellidae, Exochomus quadripustulatus
Coccinellidae, Harmonia axyridis
Coccinellidae, Propylea quatuordecimpunctata
Chrysomelidae, Phyllotreta striolata
Chrysomelidae, Phyllotreta undulata
Chrysomelidae, Phyllotreta vittula
Chrysomelidae, Psylliodes brettinghami
Elateridae, Agriotes obscurus
Lycosidae, Alopecosa cuneata
Lycosidae, Alopecosa trabalis
Lycosidae, Alopecosa virgata
Lycosidae, Pardosa agrestis
Lycosidae, Pardosa amenata
Lycosidae, Pardosa palustris
Lycosidae, Pardosa prativaga
Lycosidae, Pardosa nigra
Lycosidae, Pirata piraticus
Lycosidae, Pirata procurvus
Lycosidae, Trochosa ruricola
Lycosidae, Trochosa spinipalpis
Lycosidae, Trochosa terricola
Linyphiidae, Araeoncus humilis
Linyphiidae, Bathyphanthes gracilis
Linyphiidae, Diplocephalus christatus
Linyphiidae, Erigone atra
Linyphiidae, Erigone dentipalpis
Linyphiidae, Agyneta rurestris
Linyphiidae, Oedothorax apicatus
Linyphiidae, Oedothorax retusus
Linyphiidae, Oedothorax fuscus
Linyphiidae, Porrhomma microphthalmum
Linyphiidae, Tenuiphanthes sp.
Linyphiidae, Tenuiphanthes tenuis
Linyphiidae, Walckenaeria clavicornis
Linyphiidae, Walckenaeria palustris
Tetragnathidae, Pachygnatha clercki
Tetragnathidae, Pachygnatha degeeri
Tetragnathidae, Tetragnatha extensa
Tetragnathidae, Tetragnatha maxillosa
Theridiidae, Theridion impressum
Thomisidae, Xysticus obscurus
Thomisidae, Xysticus sicus
Aphididae, Metopolophium dirhodum
Aphididae, Rhopalosiphum padi
Aphididae, Sitobion avenae
Aphididae, Chaitophorus capreae
Lumbricidae, Allolobophora chlorotica
Lumbricidae, Aporrectodea caliginosa
Lumbricidae, Aporrectodea trapezoides
Lumbricidae, Dendrobaena clujensis
GU073676
GU073920
AY748146
KT204339
KT204403
GU073721
AJ429493
GU073689
HQ978630
KT204340
KT204404
FJ973971
KT204341
KT204405
KT204342
KT204406
FJ973976
HQ333805
HM542030
KT204343
KT204407
KT204408
JN816762
KT204344
KT204409
KT204345
KT204410
KT204346
KT204411
KT204347
KT204412
JQ746513
HQ924465
JN816771
KT204348
KT204413
KT204349
KT204414
KT204350
KT204415
KT204351
KT204352
KT204416
GU338490
HQ924452
KT204353
KT204417
KT204354
KT204418
KT204355
KT204419
KT204356
KT204420
KT204357
KT204421
KT204358
KT204359
KT204422
GU338514
KT204423
GU338483
GU683830
KT204360
KT204424
KT204361
FJ899819
GU684028
AY425723
KT204425
KF369067
JN816831
KT204362
KT204426
KT204363
KT204427
KT204364
KT204428
HM988752
HM417954
JQ908896
HQ621897
AJ272527
18S
COI
18S
18S
COI
18S
COI
18S
COI
18S
COI
18S
18S
COI
18S
COI
18S
18S
COI
18S
COI
COI
18S
18S
COI
18S
COI
18S
COI
18S
COI
COI
COI
18S
18S
COI
18S
COI
18S
COI
18S
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
18S
18S
COI
18S
COI
18S
COI
18S
COI
18S
COI
COI
18S
COI
COI
18S
18S
COI
18S
COI
18S
COI
18S
COI
COI
18S
18S




















































2
Lumbricidae, Dendrobaena octaedra
Lumbricidae, Dendrodrilus rubidus
Lumbricidae, Eisenia andrei
Lumbricidae, Eisenia fetida
Lumbricidae, Lumbricus terrestris
Lumbricidae, Octolasium lacteum
Collembola, Cryptopygus caecus
Collembola, Entomobrya dorsosignata
Collembola, Entomobrya nivalis
Collembola, Folsomia candida
Collembola, Folsomia sp.
Collembola, Isotomiella minor
Collembola, Isotoma riparia
Collembola, Isotoma viridis
Collembola, Isotomurus palustris
Collembola, Onychiurus yodai
Collembola, Parisotoma notabilis
Collembola, Sminthurus viridis
Collembola, Sminthurinus elegans
Syrphidae, Episyrphus balteatus
Syrphidae, Eristalis arbustorum
Syrphidae, Eristalis tenax
Syrphidae, Helophilus hybridus
Syrphidae, Scaeva pyrastri
Syrphidae, Sericomyia silentis
Syrphidae, Sericomyia chrysotoxoides
Syrphidae, Sphaerophoria scripta
Syrphidae, Syrphus vitripennis
Anthomyiidae, Anthomyiidae sp.
Calliphoridae, Calliphora nigribarbis
Calliphoridae, Calliphoridae sp.
Chironomidae, Chironomidae sp.
Dolichopodidae, Dolichopodidae sp.
Drosophilidae, Drosophila melanogaster
Empididae, Empididae sp.
Muscidae, Muscidae sp.
Sciaridae, Sciaridae sp.
Tipulidae, Dolichopeza subalbipes
Tipulidae, Tipulidae sp.
Chrysopidae, Chrysoperla carnea
Chrysopidae, Chrysoperla plorabunda
Thysanoptera, Frankliniella intonsa
Thysanoptera, Frankliniella occidentalis
Thysanoptera, Limothrips denticornis
Thysanoptera, Aeolothrips fasciatus
Thysanoptera, Anaphothrips incertus
Thysanoptera, Anaphothrips obscurus
Thysanoptera, Haplothrips graminis
Thysanoptera, Haplothrips aculeatus
Braconidae, Aphidius rhopalosiphi
Braconidae, Ephedrus persicae
JQ909051
GU901868
JQ909082
AY874511
AB558505
HQ691211
JQ909131
AJ272312
HQ592688
AY596360
HG422599
AY555515
HG422608
HG422636
HG422621
AY596361
DQ016560
AY037171
JQ935202
AY859604
JQ909238
KT204365
KT204429
KT204366
KT204430
JN991985
KT204367
EU431553
JN992029
KT204368
JF442710
EU241860
HQ845768
HQ979118
AB466039
KC135914
HQ979200
GU013594
HM102299
HQ939431
JF870659
HQ979047
AY521834
KC136018
KT204369
KT204431
L10183
KT204370
HM246175
KC512959
JX235929
KT204371
KT204372
KT204432
KC512926
HM246168
KC512965
KC513158
HQ605967
KT204373
KT204433
AJ009329
COI
18S
COI
COI
18S
18S
COI
18S
COI
18S
COI
18S
COI
COI
COI
18S
18S
18S
COI
18S
COI
18S
COI
18S
COI
COI
18S
18S
COI
18S
COI
18S
18S
COI
18S
COI
COI
COI
COI
COI
COI
COI
18S
COI
18S
COI
18S
18S
COI
18S
COI
18S
18S
COI
18S
COI
18S
COI
COI
18S
COI
18S














3
ESM 2 Further details on the molecular approach, prey-specific primers, and customised multiplex PCR assays
Standard singleplex PCR protocol for amplification of 18S and COI DNA
These PCRs were performed in 10 µl reactions containing 1.5 µl of DNA extract, 0.25 U OneTaq® DNA
polymerase (NEB, Ipswich, USA), 1× reaction buffer (NEB) and additional MgCl 2 to a final concentration of 3
mM, 0.2 mM dNTPs (Genecraft, Köln, Germany), 5 µg bovine serum albumin (BSA), 1 µM of each primer, and
PCR-grade water to adjust the volume. Amplifications were carried out under the following thermocycling
conditions: initial denaturation of 2 min at 94°C, 35 cycles of 20 s at 94°C, 30 s at 50°C, and 1 min at 68°C
followed by a final elongation of 3 min at 68°C. Note that this protocol was used for amplification of the 18S
and COI gene for subsequent DNA sequencing, for DNA template generation (sensitivity tests) and also to check
extraction negative controls as well as ‘screening-negatives’ (two carabid DNA extracts) with the universal
primers.
Evaluation of newly developed primers in singleplex PCR
The specificity and sensitivity/diagnostic efficacy of all primer pairs were evaluated in singleplex PCRs based on
the optimized multiplex PCR protocols (Qiagen, see Results). Those employed in one of the three multiplex PCR
assays were tested using the respective conditions of the multiplex PCR protocol, with the exception of a
modification in primer concentration of 0.5 µM and annealing temperature of 62°C. The primers not included in
multiplex PCR assays were tested as follows: the ladybeetle primer pair and the second primer versions for
aphids, springtails, and dipterans were tested with the MPI protocol, the second version for Pachygnatha spp.
with the MPII spiders, and the primer pair for Trechus spp. and second versions for Harpalus spp. and C.
septempunctata with the MPII beetles/thrips protocol.
Beetle/thrips-primers
A second version of the beetles/thrips-forward primer which perfectly matches ladybeetles (S405.1) was
designed and we suggest using 1:1 mixes of the two primers A405 and A405.1 if an inclusion of ladybeetles is
desired. Note that a 10–15 bp shorter fragment was amplified in PCR with DNA of rove-, lady-, leafbeetles, and
thrips compared to DNA of carabid beetles which is due to gaps in the respective region of the 18S gene. DNA
of the carabid beetle Nebria sp. could not be amplified because of mismatches at the 3’ end of the forward
primer. This is also true for the two tested elaterids, Agriotes obscurus and Hemicrepidius niger. Note that DNA
of Cantharidae, Silphidae, and the ladybeetle Exochomus sp. could not always be amplified – most likely due to
similar deficiencies of these group-specific primers.
4
Genus-specific primers for Carabidae
The genus-specific primers for Poecilus spp. were designed based on the COI gene and are thus most likely P.
cupreus/versicolor-specific; DNA of Poecilus sericeus (the only other species tested in PCR) could not be
amplified. On the contrary, the 18S-based primers for Pterostichus spp. and Bembidion spp. might also work for
congeners as primers perfectly fit P. illigeri when tested in silico and DNA of B. guttula and B. properans was
successfully amplified in PCR. The primers for Harpalus spp. (version 1) should also work for H. aenaeus
(tested in silico only). The second primer pair for Harpalus spp., however, might only be used for the species H.
rufipes – DNA of the closely-related H. affinis was not always amplified in PCR due to mismatches of the
reverse primer (A476) for Harpalus spp. (version 2).
It was not always possible to ‘exclude’ closely-related taxa when designing these genus-specific primers: the
COI-based forward (S475) and reverse primer (A486) for Poecilus spp. also fit on Pterostichus spp. and
Bembidion spp., respectively. No cross-reactions could be observed, though, when using these primers in
singleplex PCR as the respective other primer is specific in each case. Moreover, in the multiplex PCR assay
MPII beetles/thrips both Pterostichus spp. and Bembidion spp. are targeted on the 18S gene making undesired
amplifications impossible. Likewise, the use of the reverse primer (A467.1) for Pterostichus spp., which
perfectly fits on Harpalus spp. and Poecilus spp., doesn’t constitute a problem in MPII beetles/thrips, as both of
the latter were based on the COI gene.
We suggest mixing the two versions of the forward primer for Pterostichus spp. to achieve amplification of both
species P. melanarius (S467) and P. niger (S467.1). The two closely-related genera Bembidion and Trechus
(both subfamily Trechinae) share the same forward primer (S468); note also that the combination for Trechus
spp. amplifies a shorter amplicon length for T. quadristriatus (142 bp) compared to T. secalis (152 bp) due to a
gap in the respective region on 18S in the former (Table 2).
Thrips-primers
Note that the so-called group-specific primer pair for thrips most probably amplifies DNA of Frankliniella spp.
and Limothrips denticornis only (both species are commonly found in spring-sown cereals). Other genera within
this highly diverse group (e.g. Aeolothrips, Haplothrips, Anaphothrips) could not be included.
Spider- and lycosid-primers
DNA of tetragnathid spiders produced a slightly longer (~10 bp) fragment compared to other spider families in
PCR with the group-specific spiders-primers. DNA of the spider family Theridiidae did not always amplify –
5
however, a longer fragment of ~430 bp was detected when using the MPI assay (not observed in singleplex
PCR).
Note
that
the
so-called
family-specific
primers
for
lycosids
(primarily
designed
for
Pardosa/Trochosa/Alopecosa) might also amplify DNA of Rabidosa and Lycosa as seen in silico; DNA of
Pirata sp., for which DNA extracts were available, did not produce amplicons in PCR, though.
Springtail-primers
When testing the specificity of the group-specific primers for springtails it was not known which species were
used in PCR. We have, however, included all of the important taxa within Arthropleona (e.g. Folsomia sp.,
Entomobrya sp., Onychiurus sp., Isotoma sp., Isotomurus sp., Cryptopygus sp., Parisotoma sp., and others) and
Sminthurus sp. (Symphypleona) in the 18S sequence alignment for in silico evaluation.
Dipteran-primers
Note that the aphidophagous hoverflies (Diptera: Syrphidae) are also covered by the group-specific primers for
dipterans. If disentanglement is desired, family-specific primers as developed by Gomez-Polo et al. (2014) and
Sint et al. (2014) might be used subsequently. Our in silico evaluations showed that all of the three primers for
dipterans (S413, S414, A416) have several deficiencies regarding the family of Chironomidae. Only the
dipterans2-primers did sometimes amplify Chironomidae DNA in PCR.
6
ESM 3 Carabid beetles collected in two barley fields in Southern Sweden in spring 2012 and subjected to molecular gut content analysis. Columns show the species and body
size allocation of the carabids (L, large, i.e., >10 mm; S, small), the sampling date (May: aphid colonisation and/or June: peak aphid density/population crash) and field (A and/or
B) where a specific carabid species was found and number of specimens collected (sampling dates and fields pooled). The prey DNA detection per species is provided as number
of individuals testing positive for each prey taxon targeted in the three multiplex PCR assays. IGP refers to intraguild prey; note that the values for intraguild predation of spiders
and carabids are pooled detections of MPII spiders (2 families, 1 genus) and MPII beetles/thrips (4 carabid genera), respectively
species
size
sampling dates
fields
Acupalpus dorsalis
Acupalpus exiguus
Anchomenus dorsale
Agonum muelleri
Amara aenea
Amara apricaria
Amara curta
Amara plebeja
Amara tibialis
Asaphidion flavipes
Bembidion lampros
Bembidion obtusum
Bembidion tetracolum
Calathus melanocephalus
Clivina fossor
Harpalus affinis
Harpalus distinguendus
Harpalus rufipes
Loricera pilicornis
Nebria brevicollis
Poecilus cupreus
Poecilus lepidus
Poecilus versicolor
Pterostichus diligens
Pterostichus melanarius
Pterostichus niger
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
L
L
L
S
L
L
L
L
L
L
L
May
May
May + June
May + June
May
June
June
May+June
May+June
May
May+June
May
May+June
May+June
May+June
May+June
May+June
May+June
May+June
May
May
June
May+June
May
May+June
June
B
B
A+B
A+B
B
B
B
A+B
B
B
A+B
B
A+B
A+B
A+B
A+B
B
A+B
B
B
A+B
B
A+B
B
A+B
B
number of
specimens
collected
2
1
29
11
6
1
1
13
2
5
171
1
129
6
23
12
3
13
5
8
26
1
30
2
58
1
aphid DNA
detection
springtail
DNA
detection
earthworm
DNA detection
1
1
3
2
thrips DNA
detection
spider DNA
detection
(IGP)
carabid DNA
detection (IGP)
Coccinella
septempunctata DNA
detection (IGP)
2
12
6
2
1
1
9
1
1
91
41
5
18
5
2
5
1
7
21
1
53
2
2
2
3
1
2
1
1
1
1
1
33
1
9
1
8
2
4
1
23
1
2
2
1
1
1
2
2
2
3
9
2
1
1
1
1
1
3
3
6
1
2
7
ESM 4 Pooled prey DNA detection rates for aphids, alternative prey groups, and intraguild prey (IGP) in carabid
beetles collected in Southern Sweden in late June 2012 in two barley fields: a, field A, where aphid population
had reached its peak density of approx. 30 aphids per tiller (large, N=55 and small, N=49 carabid beetles) and b,
field B, where aphid population had already crashed leaving less than one aphid per tiller (large, N=20 and
small, N=47 carabid beetles). Asterisk indicates significantly different DNA detection rates in large and small
carabid beetles (P<0.05, as tilting confidence intervals [TCI] are not overlapping). Note that non-detected prey
taxa are not shown and that the values for intraguild predation of spiders and carabids are pooled detections of
MPII spiders and MPII beetles/thrips, respectively
8