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Supplementary Materials
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Title: Pollen contaminated with field-relevant levels of cyhalothrin affects honey bee survival,
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nutritional physiology, and pollen consumption behavior
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Authors: Adam G Dolezal (1), Jimena Carrillo-Tripp (2), W. Allen Miller (2), Bryony C.
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Bonning (3), Amy L. Toth (1,3)
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Affiliations: (1) Department of Ecology, Evolution, and Organismal Biology, (2) Department of
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Plant Pathology and Microbiology, (3) Department of Entomology, Iowa State University,
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Ames, IA USA
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Supplementary Methods
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Viral infection screening
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Honey bee pathogens have been detected in bee collected pollen (Singh et al., 2010). Therefore,
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in order to rule out the effects of viral pathogens on bee mortality, physiology, and behavior, we
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screened for several honey bee viruses commonly found in our apiary: deformed wing virus
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(DWV), black queen cell virus (BQCV), and Israeli acute paralysis virus (IAPV). Live bees were
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collected from each cage at day 14 of the respective experiments and screened via one step
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reverse transcription-qPCR using iScript – SYBR Green kit (BioRad; see Supplementary Table 1
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for primers). A melting curve step was included at the end of the amplification program to
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corroborate the identity of amplicons. For each treatment, we screened RNA from two pools of
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bees, each containing one bee from each of 5 different cages. It is difficult to find standard
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reference genes for use in quantifying virus titers (Boncristiani et al. 2013); here, we follow
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similar procedure and use quantification cycle (Cq) to estimate levels of virus infection. Cq
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values of bees sampled from hives positive for viruses were included as reference.
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Supplementary Results
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Screening for virus infection
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Analysis of Cq values showed that IAPV and BQCV were at very low levels, close to the lower
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detection limit of the assay (Cq=40) for all samples (Summary data shown in Supplementary
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Table 2). DWV was detected in some samples but at levels substantially lower than those
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observed in local hives assayed at the same time (Summary data shown in Supplementary Table
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2).
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Supplementary Table 1. List of primers used for virus screening
Gene
Black queen
cell virus
Deformed
wing virus
Israeli acute
paralysis
virus
Primer
Forward
Reverse
Forward
Reverse
Forward
Reverse
Sequence 5’ – 3’
TTTAGAGCGAATTCGGAA
ACA
GGCGTACCGATAAAGATG
GA
GAGATTGAAGCGCATGAA
CA
TGAATTCAGTGTCGCCCAT
A
GCACAGTCTTCTGGTGATT
GC
GTTAGCACACGATTGGTTA
TCAGC
Product
size (bp)
Product
Tm (°C)
140
79.0
Reference
(VanEngelsdorp
, Evans et al.
2009)
130
80
200
80.5
This work
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Supplementary Table 2: Summary of Cq values observed during virus screening. Average, low,
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and high Cq values detected via RT-qPCR for 40 samples taken from cages fed unmanipulated
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field pollen (10/treatment group). Compared to the Cq values found for similar samples taken
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from local hives (one seemingly healthy hive and two weak hives).
BQCV
DWV
IAPV
Average
Cq
36.11
32.55
38.26
Low
Cq
31.67
26.22
33.12
High Representative Representative Representative
Cq
healthy hive
weak hive 1
weak hive 2
14.55
18.03
40
24.02
29.61
25.34
38.08
14.96
21.54
19.84
40
27.41
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Supplemental Fig. 1: Proportion of original 30 bees surviving per cage averaged across all
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cages in a treatment (Control, 140 ppb, 280 ppb, 560 ppb cyhalothrin-contaminated pollen). No
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significant differences in mortality were observed over 14 days (Cox proportional hazards
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model, alpha adjusted p<0.033).
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References
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Boncristiani HF, Evans JD, et al. (2013). In Vitro Infection of Pupae with Israeli Acute Paralysis
Virus Suggests Disturbance of Transcriptional Homeostasis in Honey Bees (Apis
mellifera). Plos One 8(9). doi ARTN e73429DOI 10.1371/journal.pone.0073429
Singh R, Levitt AL, Rajotte EG, Holmes EC, Ostiguy N, Vanengelsdorp D, Lipkin WI,
Depamphilis CW, Toth AL, Cox-Foster DL (2010) RNA Viruses in Hymenopteran
Pollinators: Evidence of Inter-Taxa Virus Transmission via Pollen and Potential Impact
on Non-Apis Hymenopteran Species. Plos One 5.
VanEngelsdorp D, Evans JD, et al. (2009). Colony collapse disorder: a descriptive study. PLoS
One 4(8): e6481. doi 10.1371/journal.pone.0006481
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