SUPPLEMENTAL MATERIAL FOR:
Candidate Genes for Individual Recognition in Polistes fuscatus Paper Wasps
Journal of Comparative Physiology A
AJ Berens1,2, EA Tibbetts3, and AL Toth1,2,4
1. Program in Bioinformatics and Computational Biology, Iowa State University, Ames, IA 50011,
USA
2. Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, IA
50011, USA
3. Department of Ecology and Evolutionary Biology, University of Michigan Ann Arbor, MI
48109, USA
4. Department of Entomology, Iowa State University, Ames, IA 50011, USA
Corresponding author: AJ Berens, berens.ali@gmail.com
SUPPLEMENTAL METHODS
Selection of control gene
Quantitative reverse transcription polymerase chain reaction data are routinely normalized to control or
“housekeeping” genes in order to account for difference between samples such as variation in RNA
levels and reverse-transcription efficiency. Mounting evidence in model organisms suggest that there are
no true “housekeeping” genes with expression that remains constant under all condition in a given
organism (Savonet et al., 1997). Thus, determining such appropriate control genes in emerging model
organisms, like Polistes paper wasp, can be challenging.
Current approaches typically select control genes based on previous studies of the same or
somewhat closely-related organism, despite differences in tissue type and experimental conditions, and
are not guaranteed to yield viable controls (Daugherty et al., 2011). Based on previous control genes
from honey bees (Ament et al., 2008), we tested two ribosomal protein genes (RP49 and RPS8) as
internal control genes. RPS8 was up-regulated during the second meeting of wasp pairs for both Polistes
fuscatus and metricus, whereas RP49 initially showed no expression differences between social
interaction groups for both species (A.J. Berens, unpublished data). Thus, we selected RP49 for
normalization across samples.
For three candidate genes (IP3K, IP3R, and mGluR), we performed qPCR for the social
interaction gene expression experiment at a later date than all other candidate genes, so we re-ran the
RP49 control gene at that time. In these later runs, RP49 gene expression was significantly different
between groups in P. fuscatus (ANOVA, p-value = 7.08e-6). To investigate whether changes in RP49
gene expression were caused by cDNA degradation, we performed a correlation test of the RP49
expression from the initial and later qPCR runs. We identified high correlation between the two dates
(Pearson correlation coefficient = 0.86, p-value = 7.154e-8), which suggests that there was not
degradation of the samples. Although we observed some change of the level of RP49 expression (fold
changes are 1.3, 1.8, and 2.5 for pairwise comparisons between social interaction groups), but these fold
changes fall within an acceptable range of variability for control genes (Derisi et al., 1996).
Therefore, RP49 was still used for normalization of IP3K, IP3R, and mGluR for the social
interaction gene expression experiment so that these results would be comparable to the other 15
candidate genes and comparable across species.
SUPPLEMENTAL FIGURES
Figure 1: Mean a) non-aggressive time, b) number of aggressive contacts, and c) aggressive index with
standard error between the first meeting (light grey) and second meeting (dark grey) during
manipulation of social interactions behavioral assay (n = 34 wasp pairs per meeting). Test statistics and
p-values provided for each pairwise t-test.
SUPPLEMENTAL TABLES
Table 1: Primers for candidate and control genes were developed from previously published Polistes metricus sequences (Toth et al.,
2007).
Gene Name
Acetylcholinesterase
Brahma associated
protein 60kD
Choline
acetyltransferase
Deep orange
Ebony
Golden goal
Grunge
Inositol 1,4,5triphosphate kinase 1
Inositol 1,4,5trisphosphate receptor
Metabotropic
Glutamate Receptor
Na+/Ca2+-K+
exchanger 30C
N-methyl-D-aspartate
receptor 1
Notch
Protostome-specific
guanine nucleotide
exchange factor
Ribosomal protein 49
(control)
Scabrous
Suppressor of
variegation 2-10
Staufen
Synaptotagmin 7
Gene
Abbreviation
Ace
Bap60
P. metricus
contig
cn49499
cn7690
Forward Primer Sequence (5’ to 3’)
Reverse Primer Sequence (5’ to 3’)
CCGTGCCAAGGTTCAATGGGTAAT
CACAATGGGATCAGGTGGATGCAA
GTCTCGTACGTGGATTCAGTCGTA
GGAACAAATCTTTGCTTGTCCCAG
Cha
cn32427
GTCAACGTTTATACCAGGTCGTCG
GGACAGGTCCGAATTGGAAATCGATAAGG
dor
e
gogo
Gug
IP3K
cn40356
cn11524
cn45874
cn13769
cn34626
CCTATGTAAATATAATAATTCTGGTGGAGG
TTCCGGTTCGTCCCGTAGATCAAA
AATGAAGGGTGGAAGGTGGTGCAA
CTCTCTCTGCTTGTTTCCTTGATCT
CTGATCTCGCCATGGTCTTT
AGATCCTTTAGGCTATCATTTGTT
AGTTGTGGGAAGCGTTTATTGAAA
ACTTCAAGGTGATCGTGTGCGAGT
TACGACATTGGAATCGAGGCGA
GAGACACGTTCATCGAGTTACA
IP3R
cn43881
CGCATCAGCTCATTCATTCTAAC
GATACCGGGTGGACAAGTAAA
mGluR
cn30490
GTGGTCGGCCTTACCAATAATA
AGTGGCGTGTCTTCTCTTTC
Nckx 30C
cn44473
AAGTAGATCAGGGTGAGTAGGCTTGC
TCCCGTACCGTATTATCCCTCACA
Nmdar1
cn54652
TGATGAGAATATGGCGGGACGGTT
GTATCTCTTCGCGTGACTCAGCAT
N
PsGEF
cn15158
cn20219
ACGAGCGTTAAGATTCGTTGCCCT
TGCCGTTGCAACAAGTTATGCCAG
AGAACACCTCTCCATTCAGCCGT
CGTCCTCGGAGACGAGTCATCATATT
RP49
cn13098
TTCTTGTTGCTTCCGTAACCAA
TGATAACAGAGTTCGTAGGCGTTT
sca
Su(var)2-10
cn38025
cn21226
CCTGCTGTGTAATCGTTCCAAGTTCG
CTTGAAAGCTTCCTTACAAGATATACAGC
GCACCGTTACTAGCCTCGTGTCATAA
GCCTCTGGTAGGCAATCAGATTCA
stau
Syt7
cn8504
cn11653
ATATGAACGAGCCGTGAAACCGGA
CATCGAGAGAACACTCTCTGATCTT
TGTCGAACGTGAAACGTAAGCCTC
GTGCACTCTCAATCCTGTATTCAACG
Table 2: Statistical results of P. fuscatus normalized gene expression (to RP49) across social interaction groups (single, first meeting,
and second meeting) using a one-way ANOVA for each gene and post-hoc pairwise t-tests for genes with significant social interaction
effects (ANOVA, p-value < 0.05).
Gene
Abbreviation
Ace
Bap60
Cha
dor
e
gogo
Gug
IP3K
IP3R
mGluR
N
Nckx30C
Nmdar1
PsGEF
Sca
Stau
Su(var)2-10
Syt7
ANOVA
F(2,21)
0.86
5.48
1.53
2.08
2.40
0.66
3.97
11.75
3.39
4.32
0.68
7.94
4.06
4.31
5.33
1.49
14.34
3.20
p-value
0.44
0.012
0.24
0.15
0.12
0.53
0.035
< 0.001
0.05
0.027
0.52
0.002
0.032
0.027
0.01
0.25
<0.001
0.06
Single vs. First Meeting
t(21)
-2.10
-1.56
-1.51
1.56
-1.95
0.21
-1.70
-1.60
-2.30
1.29
-
FDR
0.024
0.067
0.073
0.933
0.032
0.583
0.052
0.062
0.016
0.895
-
Post-hoc pairwise t-tests
Single vs. Second Meeting
t(21)
0.43
0.27
-4.06
-1.65
1.44
-2.65
0.58
0.11
-2.30
-4.11
-
FDR
0.664
0.605
<0.001
0.057
0.918
0.007
0.715
0.542
0.016
<0.001
-
First vs. Second Meeting
t(21)
-2.24
-1.79
-2.43
-1.65
-1.95
-2.93
-1.75
-1.96
2.04
-4.11
-
FDR
0.018
0.044
0.012
0.057
0.032
0.004
0.047
0.032
0.973
<0.001
-
Table 3: Statistical results of P. metricus normalized gene expression (to RP49) across social interaction groups (single, first meeting,
and second meeting) using a one-way ANOVA for each gene and post-hoc pairwise t-tests for genes with significant social interaction
effects (ANOVA, p-value < 0.05).
Gene
Abbreviation
Ace
Bap60
Cha
dor
e
gogo
Gug
IP3K
IP3R
mGluR
N
Nckx30C
Nmdar1
PsGEF
Sca
Stau
Su(var)2-10
Syt7
ANOVA
F(2,21)
1.06
0.27
1.77
1.74
0.89
0.11
0.22
3.48
4.06
1.41
1.30
0.08
1.59
0.36
0.77
0.28
0.15
1.33
p-value
0.37
0.77
0.19
0.20
0.42
0.89
0.81
0.05
0.03
0.27
0.29
0.93
0.23
0.70
0.48
0.76
0.87
0.28
Single vs. First Meeting
t(21)
0.23
1.03
-
FDR
0.588
0.843
-
Post-hoc pairwise t-tests
Single vs. Second Meeting
t(21)
-1.35
-1.81
-
FDR
0.095
0.042
-
First vs. Second Meeting
t(21)
-1.61
-1.81
-
FDR
0.061
0.042
-
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