Supplementary Methods
RNA isolation and cDNA synthesis
5
For each of the eight sampling times for both males and females, total RNA of wasp
samples were extracted using EasyPureTM RNA kits (TransGen Biotech, Beijing, China)
and dissolved in Rnase free water. Because pollinating fig wasps are very small, we used
30 individuals for each RNA sample. Genomic DNA was removed by treating with
DNase
10
I
according
to
the
manufacturer’s
instruction.
A
NanoDrop
2000
Spectrophotometer (Thermo, USA) was used to measure RNA purity and concentration.
In total, 48 RNA samples (three replicates per group) with values of A260/A280 ranging
from 1.9 to 2.2 and an A260/A230 ratio of more than 2.0 were selected for further
analysis. The integrity of the RNA samples was evaluated by electrophoresis on 1.0%
agarose gels stained with ethidium bromide. Singlestrand cDNA was synthesized from 1
15
μg total RNA with oligodT per 20μl reaction, using TransScript II FirstStrand cDNA
Synthesis SuperMix (TransGen Biotech, Beijing, China).
PCR amplification and sequencing of the five circadian genes
20
For each PCR reaction, a volume of 25μl contained 0.2 mM of each dNTP, 0.2 μM of
each primer, and 5 U of TranstaqTM DNA Polymerase High Fidelity (TransGenBiotech,
Beijing, China). The PCR cycle started with 5 min at 95°C followed by 35 cycles of 30 s
at 95°C, 30 s at 55 °C, and 1 min at 72°C, finishing with 10 min at 72°C. All PCR
reactions were carried out on an Applied BiosystemsVeritiTM 96-Well Thermal Cycler
25
(ABI, USA). The primers used for PCR were designed using Primer Premier 5.0 (Table
S1). Purified PCR products were cloned with pEASY-T3 cloning kits (TransGenBiotech,
Beijing, China), and at least three positive clones were sequenced by Biosune Sequencing
Center, Beijing, China.
30
Real time qPCR expression analysis
RT-qPCR was used to analyze daily transcript levels of five circadian genes. RPL13a and
UBC were selected as the reference genes for normalizing the RT-qPCR data [1]. Based
on the full coding sequences of five circadian genes, five gene-specific primer pairs were
35
designed using Primer Premier 5.0 (Table S2) [2]. Amplification efficiencies and R2
coefficients of the primer pairs were determined from the slopes of the standard curves
generated from plasmid standards. Products obtained via gene-specific primers were
cloned with the PEASY-T3 cloning kit (TransGenBiotech, Beijing, China). Clones with
appropriate insert size were verified by PCR and sequencing. Plasmids were prepared
40
with EasyPure Plasmid MiniPrep Kits (TransGenBiotech, Beijing, China). We
determined the amount of plasmids using a NanoDrop-2000 Spectrophotometer (Thermo,
USA), which enabled us to calculate the copy numbers of the plasmids. Ten-fold serial
dilutions to 108, 107, 106, 105, and 104 copies per 20μl RT-qPCR reaction, were made for
each plasmid with two technical replicates to generate standard curves. The formula E=10
45
(-1/slope) was used to calculate amplification efficiencies (Es), which reflected the
efficacy of each primer pair.
The Stratagene Mx3000p qPCR system (Stratagene, La Jolla, CA) was used to conduct
the RT-qPCR experiments. A 20μl PCR mixture containing 1μl of template, 10μ
50
lTransStart Green qPCRSuperMix UDG(2x) (TransGenBiotech, Beijing, China), 0.4 μl
Passive Reference Dye II (50x) (TransGenBiotech, Beijing, China), 0.8μl primer mix
(0.2μM), and 7.8μl sterile water was prepared. Melting curves were constructed for all
runs to confirm amplification specificity. RT-qPCR reactions of all genes for each sample
were duplicated (technical replicates) to account for variation between runs. The same
55
suite of thermal conditions were used for all RT-qPCR reactions: 50°C for 2 min, 95°C
for 10 min, and then the following: 95°C for 10 s, 54°C for 15 s and 72°C for 15 s for 40
cycles. A template for no reverse transcription (no-RT) control was prepared for each
sample. All cDNA templates were stored at -20 °C. No-reverse transcription controls for
all 48 samples were performed to check for genomic DNA contamination, and a
60
no-template control was also conducted for each run to preclude reagent contamination.
The mean individual PCR efficiency (Em) gives more reliable results than a standard
curve-derived efficiency[3-5]. Thus, we determined the baseline and calculated an Em of
individual reactions for each primer pair from the raw RT-qPCR data using LinRegPCR[6,
7]. Subsequently, Cq and Em values were used to calculate the relative expression of all
65
five genes with respect to the reference genes RPL13a and UBC according to the
following equation:
(1/ (1+ Em _ i )Cq _ i )
Ri
=
R ref
(1 / (1+ Em _ RPL13a )Cq _ RPL13a )2 + (1 / (1+ Em _UBC )Cq _UBC )2
Ri/Rref was the expression of each circadian gene normalized to the reference genes
70
RPL13a and UBC.
Data analysis
Statistical analyses were performed using SPSS v19 (SPSS Inc., Chicago, Ill). Two
75
repeated measures ANOVAs, one for males and another for females, were used to assess
rhythmic expression of mRNA levels for each of the five genes investigated. Correlations
between gene expression and time since first male eclosion in each sex were analysed
with cosinus models. This enable us to determine how closely the oscillations fitted a
generalized circadian model. For these analyses we used R [8].
80
For differences in mean overall gene expression between the sexes, we used a series of
ANOVAs, with sex (male or female) as the fixed factor.
The expression ratios between males and females
85
After normalizing to reference genes RPL13a and UBC, we obtained three relative
expression values for each gene at each time point. First, we calculated the expression
ratios between males and females at each time point by using the mean value of the three
relative expression values, thus producing a total of eight ratio values. We obtained the
90
expression ratios between males and females for each gene by averaging the eight ratios.
MIQE guidelines
We followed the Minimum Information for publication of Quantitative real-time PCR
experiments (MIQE) guidelines to increase the reliability and integrity of our results, and
95
to promote experimental consistency and transparency between research laboratories[9].
A MIQE checklist is provided in Table S3.
References
1. Wang B, Xiao JH, Bian SN, Gu HF, Huang DW. 2013 Adaptive evolution of
vertebrate-type cryptochrome in the ancestors of Hymenoptera. Biol. Lett. 9, 20120958.
100
(doi:10.1098/rsbl.2012.0958).
2. Lalitha S. 2000 Primer premier 5. Biotech Software & Internet Report1,
270-272.(doi:10.1089/152791600459894).
3. Karlen Y, McNair A, Perseguers Sb, Mazza C, Mermod N. 2007 Statistical
significance of quantitative PCR. BMC Bioinformatics8, 131.
105
(doi:10.1186/1471-2105-8-131).
4. Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H. 2006
Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene
expression's CT difference" formula. J. Mol. Med. 84, 901-910.
(doi:10.1007/s00109-006-0097-6).
110
5. Peirson SN, Butler JN, Foster RG. 2003 Experimental validation of novel and
conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids
Res.31, e73-e73. (doi:10.1093/nar/gng072).
6. Ruijter J, Ramakers C, Hoogaars W, Karlen Y, Bakker O, Van den Hoff M, Moorman
A. 2009 Amplification efficiency: linking baseline and bias in the analysis of quantitative
115
PCR data. Nucleic Acids Res.37, e45-e45. (doi: 10.1093/nar/gkp045).
7. Ramakers C, Ruijter JM, Deprez RHL, Moorman AF. 2003 Assumption-free analysis
of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett.339, 62-66.
(doi:10.1016/S0304-3940(02)01423-4).
8. Shemesh Y, Eban-Rothschild A, Cohen M, Bloch G. 2010 Molecular dynamics and
120
social regulation of context-dependent plasticity in the circadian clockwork of the honey
bee. J. Neurosci.30, 12517-12525. (doi:10.1523/JNEUROSCI.1490-10.2010).
9. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R,
Nolan T, Pfaffl MW, Shipley GL. 2009 The MIQE guidelines: minimum information for
publication of quantitative real-time PCR experiments. Clin.Chem.55, 611-622.
125
130
(doi:10.1373/clinchem.2008.112797).
Table S1. Primers used for amplifying coding sequences of the five circadian genes
(including sequence accession numbers).
Gene
Forward primer (5’-3’)
Reverse primer (5’-3’)
name
Amplicon
Sequence accession
length
No.
(bp)
135
clk
AGAAAGTCTAGGAATTTGAGTGAG
TCACGTTGGCCTTTGCTGTTGG
1392
KF042886
cyc
ATGATTCCAGGGACCGGCAAC
TTAATTCTTGATCCTGAAAC
1560
KF042888
per
TCAAATCGAAGCCTTACAGATTC
ACTGCCGCTATTGCCGCTTTGCT
700
KF042889
pdf
ATGAAATTTTGGATGAATCG
CTTTCCAGCATTATTCATAT
249
KF042890
cwo
ATGGTTACGCACAGCATGGAGA
CTACTGTATGTGATGGGTCGGAC
1194
KF042887
Table S2. Five primer pairs used for Real Time qPCR.
Gene
Forward primer (5’-3’)
Reverse primer (5’-3’)
Amplicon
PCR
length (bp)
Efficiency(
R2
%)
140
pdf
TCTACTTCCGCAACGATTA
TTCCAGCATTATTCATATTCTTTG
91
101.5
0.997
cyc
GTAGGTGGTGTTCAAGGT
TGGCAACAAGGCAATAATG
95
96.2
0.994
clk
CATCAGAGTGTATCGGGTGTCAG
TGTTGCTGTGAGTTGGGATTGG
92
99.5
0.999
cwo
TCGCACAGAATCATAGAA
TCTAAGACCTTGAAGATGTT
129
91.2
0.996
per
ACGACTTGCTGGCGAATA
CGAGGAGCGACACAATCT
80
100.6
0.998
UBC
GAAGCGGATCAACAAGGAACT
GGACTGTCAGGTGGACCCATAAT
97
104.4
0.997
RPL13a
CTGCTCGTGGTCCTTTCCATTTTC
GCATCCTTGCCTCTTTGTGTCTTG
123
96.6
0.992
Table S3. MIQE checklist.
Item to check
Importance
Details
Sample
Description
E
Pollinators (Ceratosolensolmsimarchali) of Ficushispida
Processing procedure
E
Female and male fig pollinators were darked treated and collected
If frozen, how and how quickly
E
from fig fruits
Insect samples were immediately frozen in liquid nitrogen after
they were collected
If fixed, with what and how quickly?
E
Stored in sample Protector (TAKARA, China) immediately after
frozen
Sample storage conditions and
E
Samples were held at -20 oC for less than a week before RNA
isolation
duration
Experimental design
Definition of experimental and control
E
No relative quantification were involved in this work, thus no
control groups were defined
groups
Number within each group
E
3
E
For each RNA sample, total RNA of 40 individuals was extracted
Nucleic acid extraction
Procedure and/or instrumentation
by using an EasyPureTM RNA kit (TransGen Biotech, China)
Name of kit and details of any
E
EasyPureTM RNA kit (TransGen Biotech, China). We exactly
followed the protocols of the kit
modifications
Details of DNase or RNase treatment
E
Genomic DNA was removed by treating with DNase I according
to the standard protocols
Contamination assessment (DNA or
E
RNA sample to assess the absence of DNA.
RNA)
Nucleic acid quantification
No reverse transcription control (NRC) was performed for each
E
RNA concentration was determined by measuring the
abosorbance at 260nm UV light
Instrument and method
E
NanoDrop-2000 Spectrophotometer (Thermo, USA)
RNA integrity: method/instrument
E
RNA integrity was assessed by electrophoresis on 1.0% agarose
gels stained with ethidium bromide
RIN/RQI or Cq OF 3’ and 5’
E
N/A
E
Standard curve analyses were sufficient to test inhibition
E
TransScript II First-Strand cDNA Synthesis SuperMix (TransGen
transcripts
Inhibition testing (Cq dilutions, spike,
or other)
Reverse transcription
Complete reaction conditions
Biotech, China) was used to generate single-stranded cDNA total
RNA with oligo-dT. For each sample, a template for no reverse
transcription-control was prepared.
Amount of RNA and reaction volume
E
Amount of RNA: 1μg; Reaction volume: 20μl
Priming oligonucleotide and
E
oligo-dT: 2μM
E
50 oC for 1 hour
E
PCR reactions were performed in a Mx3000P Real Time
concentration
Temperature and time
qPCR protocol
Complete reaction conditions
Thermocycler (Stratagene, USA). A 20 μl PCR mixture was
prepared containing 1 μl of template, 10μl TransStart Green
qPCRSuperMixUDG(2x) (TransGen Biotech, China), 0.4 μ l
Passive Reference Dye II(50x) (TransGen, China), 0.8μl primer
mix(0.2μM), and 7.8 μl sterile water. The following thermal
conditions for RT-qPCR were used: 50oC for 2 min, 95oC for 10
min, and then the follwing: 95oC for 10 s, 54oC for 15 s and 72oC
for 15 s for 40 cycles
Reaction volume and amount of
E
volume
cDNA/DNA
Primer, (probe), Mg2, and dNTP
Reaction volume: 20μl; amount of cDNA: 1μl per reaction
E
500nM primers; 3mM MgCl2 ; 0.2 mMdNTP
E
TransStart Green qPCRSuperMix UDG (2x) (TransGen Biotech,,
concentrations
Polymerase identity and concentration
China)
Buffer/kit identity and manufacturer
E
TransStart Green qPCRSuperMix UDG (2x) (TransGen Biotech,,
China)
Additives (SYBR Green I, DMSO, and
E
Passive Reference Dye II(50x) (TransGen Biotech,, China)
E
50oC for 2 min, 95oC for 10 min, and then the follwing: 95oC for
so forth)
Complete thermocycling parameters
10 s, 54oC for 15 s and 72oC for 15 s for 40 cycles
Specificity (gel, sequence, melt, or
E
Melting curve analysis, gel electrophoresis and sequencing
E
The signal of the amplification plot was late (Cq>30), difference
digest)
For SYBR Green I, Cq of the NTC
of Cq between NTC controls and cDNA samples was large
E
-3.58~-3.10
E
Table S2 of Paper
R of calibration curve
E
Table S2 of paper
Linear dynamic range
E
Cqvariation at LOD
E
Evidence for LOD
E
Cq<35 for all samples
If multiplex efficiency and LOD OF
E
N/A
E
MxPro QPCR Software
E
Cq values were determined using threshold, which is determined
Calibration curves with slope and y
intercept
PCR efficency calculated from slope
2
each assay
qPCR analysis analysis program
(sourse, version)
Method of Cq determination
using the Amplification-based Threshold method
Outlier identification and disposition
E
None of Cq values was discarded
Results for NTCs
E
The signal of the amplification plot was very late (C q>35)
Justification of number and choice of
E
UBC; RPL13a
Description of normalization method
E
N/A
Number and stage (RT or qPCR)v OF
E
Duplicate
E
Triplicate
Repeatability (intraassay variation)
E
∆Cq< 0.5 for all duplicates
Statistical methods for results
E
One way ANOVA
E
SPSS v19
Gene symbol
E
Text
Sequence accession number
E
Amplicon length
E
In silico specificity screen (BLAST,
E
reference genes
technical replicates
Number and concordance of biological
replicates
significnce
Software (source, version)
qPCR target information
Table S2 of paper
and so on)
Location of each primer by exon or
E
Primers were designed spanning the splicing sites
E
N/A
intron (if applicable)
What splice variants are targeted
145
150
Fig.S1. The expression ratios between males and females among five circadian genes
155
160
Fig.S2. The daily expression of relative mRNA of clk, cyc pdf and cwo