Electronic Supplementary Material(ESM) S1: Material and methods
EightEurema mandarina females were captured on Tanegashima Island, Japan, from populations previously reported to be infected with both Wolbachia strains wCI and wFem [1]. These females were kept inisolation for egg-laying.Five females showed a female-only offspring ratio, whereas three had unbiased offspring ratios(ESM S1 Table 1). For the qPCR experiment we used 11 daughters of all-female broods; for unbiased broods we usedeight males and eight females. The unbiased broods were either infected with wCI (a total of five females and five males from mothers 6-8) or cured from infection after tetracycline treatment (three females and three males from mother 6).
ESM 1 Table 1.Eurema mandarina from Tanegashima and their offspring included in this study
Mother# / Daughters
n / Daughters
used in qPCR / Sons
n / Sons
used in qPCR
1 / 8 / 3 / - / -
2 / 8 / 3 / - / -
3 / 2 / 2 / - / -
4 / 3 / 3 / - / -
5 / 6 / 0 / - / -
6 / 7 / 5 / 6 / 5
7 / 1 / 1 / 1 / 1
8 / 3 / 2 / 4 / 2
As control we included three female and three male E. mandarina from Hachijō-jima Island, Japan, from a population previously found to be infected only by wCI. We also included fivefemales and five males of each wCI infectedE. hecabe anduninfected E. smilax from various locations in Australia(ESM S2).
DNA isolation
Genomic DNA was extracted from single legs (the standard tissue used for Wolbachia detection in E. hecabe [1])using the GenElute Mammalian Genomic Miniprep kit (Sigma) according to the manufacturer’s protocol. DNA was eluted in 100µL elution solution and then stored at 4°C.
Wolbachiascreening PCR
PCR-based screening of Eurema individuals was performed using strain specific wspprimers for wCI andwFem[1].Cycling conditions were 3 min at 94 °C, then 35 cycles of 94 °C for 30 s, 60 °C for 45 s, 72 °C for 60 s, and a final step at 72 °C for 10 min, and the master mix was as listed in ESM 1 Table 2.
PCR of theTpigene
The Tpi gene is located on the Z chromosome of lepidopteran species. We amplified a highly variable intron using primers that were positioned in the conserved exon region of Tpi and previously used in [2]. The PCR cycling conditions were 3 min at 94 °C, then 40 cycles of 94 °C for 30 s, 50 °C for 45 s, 72 °C for 1 min 20 s, and a final step at 72 °C for 10 min. PCR products were cleaned for direct sequencing by treatmentwith a combination of 0.5 U Exonuclease I (New England Biolabs, Ipswich, MA) and 0.25 U Shrimp AlkalinePhosphatase (Promega), with incubation at 37°C for 30min, then 95°C for 5min, prior tocapillary sequencing by Macrogen (Seoul, Korea).
ESM 1 Table 2. PCR conditions for Wolbachia detection and the Tpi gene
PCR protocol / wsp (wCI, wFem) / TpiTotal volume / 10 µL / 20 µL
5x MyTaq Red Reaction Buffer / 1x / 1 x
Primer 1 / 0.4 µM / 0.8 µM
Primer 2 / 0.4 µM / 0.8 µM
MyTaq™ Red DNA Polymerase (Bioline) / 0.5 U / 1 U
DNA Template / 1 µL / 1 µL
Sequence data analysis
In total we direct sequenced Tpi from 57 specimens of 8 mothers and their offspring (11 males and 38 females, see ESM S3). No indels or SNPs were observed in sequence chromatograms of females; some males where heterozygous due to detected double peaks and shifts of sequence reads. For this reason we sequenced males form both sides and assigned the ambiguous positions using the IUPAC code. We used PHASE [3] algorithm as implement in DnaSP v5.10.01 [4]to infer putative Tpi alleles (coded as Allele 1 and Allele 2 in ESM S3).The sequences were then
trimmed and edited in Geneious version 8.0.4 [5]. The 485 bp alignment consisted of 13 parsimony informative sites, representing 17 haplotypes.The overall haplotype
diversity was 0.914. We conducted a network analysis using the median-joining method[6] implemented in PopART[7]. The network (ESM S4) shows the high haplotype diversity. Of the 17 haplotypes only 5 haplotypes were shared between families. There was also no clear divergence of alleles found inwCI and wFem co-infected individuals.We also confirmed the presence of the qPCR primer sequences
within individual sequences and thus excluded the presence of any pseudogenes in
our sequence alignment.
qPCR primer design
Tpi primers were designed in an exon region ofpreviously published sequences of E. mandarina (GenBank accession numbers: AB231163-AB231194) [8].Primers amplifying an exon region of the kettin gene were designed based on an RNAseq sequence of E. mandarina (D Kageyama, personal communication) and sequencedfrom two females each ofE. mandarina, E. hecabe and E. smilax(GenBankaccession numbers KM502545-KM502550).Primers for EF-1α were designed from previously published E. mandarina sequences (GenBank accession numbers: AB194749-AB194754). All primers are shown in ESM 1 Table 3 and were designed using the software PrimerQuest (
ESM 1 Table 3.qPCR primers used for the amplification of Tpi, kettin and Ef-1α
Name / Primer sequence (5’ 3’) / Amplicon length (bp) / Tm (℃)TPI-F / GGC CTC AAG GTC ATT GCC TGT / 60
TPI-R
Ket-F
Ket-R
EF-1F
EF-1R / ACA CGA CCT CCT CGG TTT TAC C
TCA GTT AAG GCT ATT AAC GCT CTG
ATA CTA CCT TTT GCG GTT ACT GTC
AAA TCG GTG GTA TCG GTA CAG TGC
ACA ACA ATG GTA CCA GGC TTG AGG / 73
73
73 / 60
60
60
60
60
Tm, primer melting temperature
qPCR conditions
qPCRreactions were performed on a Rotor-GeneQ PCR system (Qiagen). Using 20 µL reactions, each qPCR reaction contained 1xSensiFAST™ SYBR No-ROX (Bioline), 0.8 µL of each primer and 1 µL of template DNA. Each DNA template was diluted 1:10 prior to reaction set up. Cycling conditions for all qPCR reactions were 2 min at 95 °C, then 40 cycles of 5 s at 95 °C, 10 s at 60 °C and 10 s at 72 °C. A melt curve for each primer pair was conducted to clarify that there was no primer dimer formation. Every run was performed analysing each specimen in three technical replicates with all three primer sets including a no-template control. One E. mandarina male was selected as a run calibrator in all runs in order to compare runs.
Quantitative Analysis of Gene Dose Ratio
PCR amplification efficiencies weremeasured for each individual reaction using LinRegPCR[9]and tested that they were approximately equal [9]. The relative copy number of the Z-linked genes was obtained as gene dose ratio (GDR) using the2-ΔΔCt method, ΔCt = Cttarget- Ctreference[10]. The target and the reference PCR efficiencies where approximately equal with less than 10 % difference and the ratios were calculated without efficiency correction [10].
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