ELECTRONIC SUPPLEMENTARY MATERIAL (ESM)
This pdf provides supplementary material describing studies sites, primer testing (specificity and
sensitivity) methods and results for:
Potential for exploitative competition, not intraguild predation, between invasive harlequin
ladybirds and flowerbugs in urban parks
Journal: Biological Invasions
Andy G. Howe1, Hans Peter Ravn1, Christian Bressen Pipper2, Alexandre Aebi3
1
Department of Geosciences and Natural Resource Management, University of Copenhagen,
Rolighedsvej 23, DK-1958 Frederiksberg C, Denmark
2
Department of Public Health, Section of Biostatistics, University of Copenhagen, Øster
Farimagsgade 5, DK-1014 København K, Denmark
3
Institut de biologie, Laboratoire de Biologie du Sol, Université de Neuchâtel
Emile-Argand 11, CH-2000 Neuchâtel, Switzerland
Corresponding author: Andy G. Howe, andy@ign.ku.dk
Study sites
We studied the dominant generalist predators in the crowns of mature (>15 m high) common lime
trees, Tilia x europaea L. (Malvaceae) in managed urban green spaces (public parks) in inner city
Copenhagen, Denmark, a small city with a human density of 1.749.405 and area of 2.553 km2
(Statistikbanken 2013).
The greatest densities of H. axyridis in Denmark occur in Copenhagen where Tilia spp. incidentally
comprise 59.1% of the tree species used in inner city public green spaces (Municipality of
Copenhagen 2012).
In 2009, seven urban green spaces (including parks and other spaces, <16.7 km from the city centre)
were sampled on six dates in October (2nd, 7th, 14th, 15th, 19th and 22nd). Green spaces are situated
between 55°47'16.6"N 12°29'39.1"E and 55°40'08.5"N 12°25'32.8"E.
In 2011, five trees in five inner city parks were sampled. Distances between trees (i.e. parks) were
0.68 to 4.95 km, and were assumed to be spatially independent, while parks were between 0.53 and
5.46 km from the city centre (distance from town hall). Inner city park names are Churchillparken,
Ørstedsparken, Genforeningspladsen, Bispebjerg Parkallé, Østre Anlæg.
Specificity and sensitivity of primers
Primers were tested for cross-reactivity against two or more individuals from 26 insect species
found in the crowns of Tilia spp. in city parks. The majority of these specimens originated from
Denmark although insects collected in Switzerland, Belgium and England were added where
possible in order test primer specificity over a larger geographic area (Table ESM 1). Positive
controls in specificity PCRs were either A. nemoralis or E. tiliae.
Furthermore, the sensitivity of primers to detect target prey in predator DNA was assessed
following the method by Thomas et al. (2013), using target prey DNA (E. tiliae or A. nemoralis)
diluted in a 1:1 to 1: 106 (prey : predator) dilution series in predator DNA (H. axyridis or A.
nemoralis) as PCR templates.
Post-feeding detection period of prey
Laboratory cultures of H. axyridis collected from Copenhagen parks were reared on a diet of E.
kuehniella eggs and kept in Petri dishes at 20 °C ± 1 °C, 16:8 L:D and RH 50% ± 20% , with access
to a moistened cotton pad. Cultured A. nemoralis were supplied by EWH BioProduction
(Tappernøje, Denmark); these predators originated from Danish and German field populations
collected in 2010.
When in the laboratory they were kept in insect cages (20 °C ± 1 °C, 16:8 L:D and RH 50% ±
20%). Within 12 hours of moulting to 4th instars (H. axyridis) or adults (H. axyridis and A.
nemoralis) predators were starved for 24 or 48 hours in individual 30 ml plastic containers with
ventilation and a moistened cotton pad. Starvation times of forty-eight hours were chosen for the 4th
instar H. axyrids larvae offered 2nd instar A. nemoralis nymphs and for A. nemoralis offered E. tiliae
based on pilot-studies which revealed predator reluctance to reliably ingest prey within an hour of
prey being offered.
The following four combinations of prey detection times were investigated: 4th instar H. axyridis
larvae ingesting one 2nd instar A. nemoralis nymph; 4th instar H. axyridis larvae ingesting one E.
tiliae adult; H. axyridis adults ingesting one E. tiliae adult and; A. nemoralis adults ingesting one E.
tiliae adult.
Predators were offered prey in 1.5 ml Eppendorf tubes and observed for an hour until ingestion
which was noted to the nearest quarter hour. Six to eight H. axyridis were frozen as soon as
ingestion was complete (time = 0), whereas for A. nemoralis ingestion lasted up to an hour (time =
1). Thereafter all predators were transferred to Petri dishes kept in climate chambers (20 °C ± 1 °C,
16:8 L:D and RH 50% ± 20%). Initially, H. axyridis adults offered E. tiliae were given up to 24 h
digestion time, which was not long enough to calculate a prey detectability half-life; we were
unable to extend digestion times beyond 24 h for this combination. However, remaining
combinations were allowed 2, 4, 6, 8, 24 and 36 h to digest prey (see ESM Table 2). At each time
interval 6 live individuals were transferred to a freezer (- 20°C), until PCR of six H. axyridis from
each digestion time tested for prey DNA digestion. Six A. nemoralis were tested, except at times t =
1 (n = 5), t = 6 (n = 5), t = 8 (n = 5) and t = 36 (n = 4). Despite starving A. nemoralis for 48 h, we
were unable to achieve greater sample sizes due to their reluctance to reliably ingest prey and the
constraints this places on available resources. Longer starvation times were not practised due to
increased chance of mortality of flowerbugs. Future studies investigating anthocorids are
recommended to use increased sample sizes.
Results
Specificity/sensitivity of primers and post-feeding detection times
Our primers were specific for target species when screened for cross-reactivity against 26 insect
species found in Tilia spp., producing amplicons of A. nemoralis (bands at 79 bp) and E. tiliae (134
bp). Slightly weaker PCR amplicons were observed for A. nemoralis of UK origin, which could
reflect sub-optimal DNA extraction or slight genotypic differences between populations.
The primer pair for E. tiliae was moderately sensitive detecting target DNA at a presence of 0.01%,
i.e. a mixture of 1 part prey to 104 part predator, and was independent of predator type(ESM Table
2).
In contrast, the A. nemoralis primer pair displayed low sensitivity, not detecting target DNA below
1% (i.e. in a 1 part prey to 102 part H. axyridis DNA mix). Prey detectability half-lives are reported
and discussed in the main article.
References
Municipality of Copenhagen (2012) Bytræerfordeling i Københavns Kommune. Teknik- og Miljøforvaltning,
Københavns Kommune.
Statistikbanken (2013) Population and area of Copenhagen, Denmark 2013. Danmarks Statistik.
Thomas,A.P., Trotman,J., Wheatley,A., Aebi,A., Zindel,R. & Brown,P.M.J. (2013) Predation of native
coccinellids by the invasive alien Harmonia axyridis (Coleoptera: Coccinellidae): detection in Britain
by PCR-based gut analysis. Insect Conservation and Diversity, 6, 20-27.
ESM Table 1 Insect species found in Tilia x europaea canopies tested for cross-reactivity against primer pairs AnsF2/AnsR2 and Eti-F5.1/EtiR2. DK: Denmark, GB: England, BE: Belgium, CH: Switzerland – denoting the origin of
samples primers were tested against. *not collected on Tilia spp.
Order
Family
Species
Origin of specimens
Specificity tested with primer
pair
Ans-F2/R2
Coleoptera
Coccinellidae
Eti-F5/R2.1
Harmonia axyridis (Pallas, 1773)
DK, GB, BE, CH
DK, GB
Adalia bipunctata (Linnaeus, 1758), A.
DK
DK
decempunctata*(L., 1758), Exochomus
quadripustulatus (L., 1758), Calvia
quattuordecimguttata (L., 1758), Anatis
ocellata (L., 1758), Coccinella
septempunctata (L., 1758)
Halyzia sedecimguttata (L., 1758)
Hemiptera
DK
Propylea quattuordecimpunctata (L., 1758)
DK
Aphididae
Eucallipterus tiliae (L, 1758.)
DK
DK
Anthocoridae
Anthocoris nemoralis (Fabricius, 1794)
DK, GB
DK, GB
A. confusus (Reuter, 1884)
DK
DK
A. nemorum (L. 1761)
DK, GB
DK
A. limbatus (Fieber, 1836), Orius minutus (L.
GB
GB
1758)
Miridae
O. vicinus (Ribaut, 1923)
BE
BE
Neolygus viridis (Fallén, 1807), Deraeocoris
DK
DK
DK
DK, CH
lutescens (Schilling, 1837), Phytocoris tiliae
(Fallén, 1776), Blepharidopterus angulatus
(F., 1807)
D. flavilinea (Costa, 1862)
P. populi (L., 1758)
DK
Acanthosomatidae
Elasmostethus interstinctus (L., 1758)
DK
DK
Lygaeidae
Scolopostethus sp.
GB
GB
Diptera
Syrphidae
Episyrphus balteatus (De Geer, 1776)
CH
CH
Neuroptera
Chrysopidae
Chrysoperla carnea*(Stephens, 1836)
CH
CH
ESM Table 2 Number of positives detected (prey DNA within a predator) for each predator-prey combination at
different post-ingestion times and the detectability half-life (DS50). DS50 values followed by a different letter are
significantly different (Pearson’s chi-square statistic with Yates continuity correction).
Time since feeding (h)
Predator
Prey
0h
1h
2h
4h
(life stage)
(life stage)
Anthocoris
Eucallipterus
5 (5)* 6 (6)
6 (6)
nemoralis
tiliae
(adult)
(adult)
Harmonia
A. nemoralis
6 (6)
6 (6)
4 (6)
axyridis
(nymph)
(larva)
H. axyridis
E. tiliae
5 (6)
6 (6)
6 (6)
(larva)
(adult)
H. axyridis
E. tiliae
6 (6)
5 (6)
6 (6)
(adult)
(adult)
* Numbers in parentheses denote number of samples (n).
†
Post-digestion times did not extend long enough to evaluate DS50.
6h
8h
24 h
36 h
DS50 ± SE
5 (5)
5 (5)
2 (6)
1 (4)
24.8 ± 3.4a
4 (6)
0 (6)
0 (6)
0 (6)
5.6 ± 0.6b
6 (6)
6 (6)
3 (6)
3 (6)
32.4 ± 6.8a
6 (6)
6 (6)
6 (6)
-
Not
calculated†