Abstract
Genomes are vulnerable to selfish genetic elements that enhance their own transmission often at the expense of host fitness. Examples are cytoplasmic elements such as maternally inherited bacteria that cause feminization, male-killing, parthenogenesis and cytoplasmic incompatibility. We demonstrate, for the first time, that segregation distortion, a phenomenon so far seen only for nuclear genetic elements, can also be caused by a cytoplasmic element, the ubiquitous endosymbiotic bacterium Wolbachia. For Eurema mandarina butterfly lineages with a Z0 sex chromosome constitution, we provide direct and conclusive evidence that Wolbachia induces production of all-female progeny by a dual role: the compensation for the female-determining function that is absent in Z0 lineages (feminization) and the prevention of maternal sex chromosome inheritance to offspring as a specific type of segregation distortion. Therefore, our findings highlight that both sex determination and chromosome inheritance — crucially important developmental processes of higher eukaryotes — can be manipulated by cytoplasmic parasites.
Introduction
Genomes of sexually reproducing organisms are exposed to genetic conflicts. For example, some genes bias reproduction towards male offspring while other genes within the same genome may favor reproduction of more daughters. Selfish genetic elements (SGEs), such as meiotic drivers, cytoplasmic sex ratio distorters and transposons, are extreme examples, which enhance their own transmission often at the expense of their hosts’ fitness [1,2]. There is growing evidence that SGEs, and their genetic conflict with host genomes, trigger important evolutionary change and innovation in eukaryotes [2].
Segregation distortion (SD), also referred to as meiotic drive, is a violation of Mendelian law as it leads to the more frequent inheritance of one copy of a gene than the expected 50% [3,4]. A segregation distorter that sits on a sex chromosome biases the sex ratio. For example, X-linked segregation distorter (X drive) and Y-linked segregation distorter (Y drive) in flies (Diptera), result in female-biased and male-biased sex ratios, respectively [4]. In male-heterogametic species, X and Y segregation distorters are expected to be encoded in the nuclear genome. In female-heterogametic species, however, W chromosome and cytoplasm behave as a single linkage group and thus distortion of sex chromosome inheritance in female-heterogametic species can theoretically also be caused by cytoplasmic elements. Although this possibility has previously been proposed [5,6], lack of empirical evidence questions whether it is mechanistically possible for cytoplasmic elements to cause SD.
Wolbachia pipientis (Alphaproteobacteria), simply referred to as Wolbachia, attracts significant interest in evolutionary and developmental biology but also in applied fields such as pest management because it can manipulate reproduction of arthropods in various ways such as cytoplasmic incompatibility, parthenogenesis induction, feminization and male-killing [7]. Here we demonstrate for the first time that Wolbachia is responsible for the disruption of sex chromosome inheritance, which can also be seen as a form of segregation distortion, in any host species. We do this by providing multifaceted and conclusive evidence that in the butterfly Eurema mandarina Wolbachia-induced SD is the underlying mechanism for the production of all-female progeny. In most populations, E. mandarina is infected with the cytoplasmic-incompatibility (CI)-inducing Wolbachia strain wCI at a high prevalence of close to 100% [8,9]. Hiroki et al. [10,11] first reported all-female offspring production in E. mandarina (then known as Eurema hecabe yellow type), which was considered to be due to the feminization of genetic males (ZZ) by co-infections with the Wolbachia strain wFem (hereafter referred to as double infection CF while single infection with wCI is referred to as C). Three observations about CF lineages supported this view, i.e., (a) antibiotic treatment of adult females led to the production of all-male offspring [10], (b) antibiotic treatment of larvae resulted in intersex adults [12] and (c) females did not have the W chromatin body [10,12]. This has recently been challenged, because it was demonstrated that CF females have only one Z chromosome and that this Z chromosome always derived from their fathers implying that a SD mechanism may be in place albeit it was not clear whether Wolbachia induced this SD [13]. As a consequence two novel (yet untested) hypotheses were formed, namely, that CF females have either a Z0 or a W’Z sex chromosome set (whereby W’ cannot be visualized in W chromatin assays and does not have a female-determining function), and that the disruption of Z chromosome inheritance occurs in CF lineages due to Wolbachia or another factor, such as those encoded by the host nucleus.
In a multifaceted approach, by combining fluorescence in situ hybridization (FISH), genome sequencing, quantitative PCR, reverse transcription PCR and antibiotic treatment, we have tested these two hypotheses and revealed that CF females are Z0, and that Wolbachia is the cause for both the disruption of Z chromosome inheritance and the feminization of Z0 individuals. Our results demonstrate, for the first time, Wolbachia as the agent that is responsible for distorted sex chromosome inheritance, and thereby highlight that cytoplasmic elements can have profound effects on oogenesis, sex chromosome inheritance and sex determination – fundamental biological processes of eukaryotes.
Results
All-female-producing CF females have a Z0 sex chromosome constitution
We performed FISH on E. mandarina chromosomes prepared from CF females, C females, and C males collected on Tanegashima Island (Figure 1; Figure 1—figure supplement 1). In the mitotic complement of C females, which harbor a 2n = 62 karyotype, genomic probes highlighted the W chromosome, with scattered signals on the other chromosomes (Figure 2A; see Materials and Methods for technical details). A probe for the Z-linked gene Kettin (Ket) identified the single Z chromosome in C females (Figure 2A), and also hybridized to the Z chromosome paired with the W chromosome in pachytene bivalents (Figure 2J). The Ket probe identified two Z chromosomes in the mitotic complement of C males (Figure 2B; 2n = 62). No painted W chromosome was observed in interphase nuclei (Figure 2H, I), the mitotic complement (Figure 2C) and pachytene complement (Figure 2L) of CF females, but the Ket signal appeared on the single Z chromosome in the mitotic complement (Figure 2C) and Z univalent in the pachytene complement (Figure 2L). Based on the relative read counts homologous to Bombyx mori Z-linked and autosomal genes in females and males, our genome sequencing data support the notion that CF and C females have one Z chromosome (Figures 2M–O; Figure 2—figure supplement 1), which is consistent with genomic qPCR data based on two loci, Triosephosphate isomerase (Tpi) and Ket, relative to the autosomal gene EF-1α[13]. Thus, our results directly reveal the sex chromosome constitution of C females, C males, and CF females as WZ, ZZ, and Z0, respectively. This confirms one of two previously suggested sex chromosome constitution of CF females [13] while it disproves another previous interpretation based on W-body diagnosis that CF females are ZZ [10,12].
All embryos oviposited by CF females are Z0
We performed real-time genomic qPCR (to detect Z-linked Tpi or Ket relative to autosomal EF-1α) on individual fertilized eggs, and found that C females oviposited embryos with either one or two Z chromosomes at nearly equal frequencies (Figure 3A left; Figure 3—figure supplement 1). In contrast, all embryos oviposited by CF females were single Z carriers (Figure 3A middle; Figure 3—figure supplement 1). These findings indicate that the progeny of CF females are exclusively Z0 individuals, supporting the view that the maternal Z chromosomes are not inherited in CF lineages.
Wolbachia causes the exclusive production of Z0 embryos by CF females
To abolish the effects of Wolbachia, tetracycline (tet) was administered to adult CF females previously inseminated by antibiotic-treated male offspring of C females. The Z-linked gene dose of embryos laid by these tet-treated females ranged from approximately 0.5–1.0, indicating that some embryos are Z0 and others are ZZ (Figure 3A right; Figure 3—figure supplement 1). This suggests that the Wolbachia strain wFem in CF females causes the exclusive production of gametes without sex chromosomes that then develop as Z0 embryos after fertilization. Therefore, our finding is the first empirical evidence that in a female-heterogametic species the sex-specific linkage disequilibrium can be caused by cytoplasmic elements [5,6]. Furthermore, Wolbachia-like structures were observed near the chromosomes in CF females while less apparent in C females and C males, and this may represent different tropism and function of wFem when contrasted with wCI (Figure 2C). Sixty-nine adults (15 females and 54 males) were obtained from offspring produced by five tet-treated adult CF females (Figure 3B). Three of these tet-treated females produced only male offspring. Exclusive production of males was previously observed in tet-treated E. mandarina females derived from a different population on Okinawa-jima Island, Okinawa Prefecture, Japan [10]. In this study, we obtained 15 female offspring from two broods in the first days after tet treatment; however, the mothers produced more males as the duration of tet treatment increased, and eventually produced only males. Examination of the Z-linked gene dose of these offspring by genomic qPCR showed that the females had one Z chromosome, whereas almost all of the males had two Z chromosomes (Figure 3C). The nucleotide sequences of the introns of the Tpi gene demonstrate that, in brood 19-1, all females (n = 12) were hemizygous and nine out of 10 males were heterozygous (Figure 3C; Figure 3—figure supplement 2). Curiously, one male (21m) that exhibited the lowest gene dose of Ket (0.588) appeared to be hemizygous (Figure 3C). These results suggest that the emerged females had a Z0 sex chromosome constitution, whereas most males had a ZZ sex chromosome constitution, with one exception (21m) of either Z0 or ZZ’ (Z’ represents partial deletion/mutation in Z). These results also demonstrate that, in principle, tet-treated adult CF females can oviposit embryos with either a Z0 or ZZ sex chromosome constitutions (Figure 3A right). However, Z0 individuals appear to have zero or very low survival rates because few emerge as adults.
Involvement of Wolbachia in the sex determination of Eurema mandarina
Next, we fed CF larvae a tet-containing diet. As previously observed [12], all individuals treated in this way developed an intersex phenotype at the adult stage, typically represented with male-like wing color and an incomplete male-specific structure on the wing surface (Figure 4E and H; Figure 4—figure supplement 2). The qPCR assay to assess the Z-linked gene dose revealed that these intersexes (n = 23) had just one Z chromosome (Figure 4I), and therefore a Z0 genotype. Because these Z0 individuals were destined to develop as females without tet treatment, wFem is likely to be responsible for female sex determination. Further evidence in support of this idea was obtained by examining the sex-specific splicing products of dsx (Figure 4—figure supplement 3), a widely conserved gene responsible for sexual differentiation [14]. Similar to B. mori [15], C females exhibited female-specific splicing products of E. mandarina dsx (EmdsxF), whereas C males had a male-specific splicing product of E. mandarina dsx (EmdsxM; Lanes 1 and 2 in Figure 4A, respectively; Figure 4B). Similarly to C females, CF females exhibited exclusive expression of EmdsxF (Lanes 3 and 4 in Figure 4A; Figure 4B). Intersexual butterflies, generated by feeding the larval offspring of CF females a tet-containing larval diet, expressed both EmdsxF and EmdsxM (Lanes 5 and 6 in Figure 4A; Figure 4—figure supplement 1).
Discussion
We provide comprehensive and conclusive indirect (qPCR of Z gene dosage) and direct (W chromosome painting; genomic analyses) evidence for the loss of the W chromosome from CF individuals. Furthermore, we demonstrate that the Wolbachia strain wFem is directly responsible for chromosomal segregation distortion (SD) by causing the disruption of sex chromosome inheritance in CF females of E. mandarina. This is the first empirical proof for previous theoretical predictions that cytoplasmic SGEs, such as Wolbachia, can cause SD. In E. mandarina, wFem has a dual role in both causing segregation distortion and feminization in Z0 lineages that have lost W chromosome and its feminizing function.
Wolbachia disrupts Z chromosome inheritance in Z0 females
Our data provides evidence that the exclusive production of Z0 embryos by CF females is due to a yet unidentified developmental process that leads to the disruption of sex chromosome inheritance in CF females prior to oviposition, thereby the absence of maternal Z chromosome in CF offspring. This process can be referred to as SD, according to established conceptual frameworks [5,6]. We believe that two mutually exclusive hypotheses can account for the SD observed in CF individuals (Figure 5A). The first assumes that a gamete without the maternal Z chromosome (or without any sex chromosome overall), is always selected to become an egg pronucleus (meiotic drive sensu stricto) (Figure 5A left) [16]. The second assumes that meiosis itself is normal, and that maternal Z chromosomes (or sex chromosomes in general), are selectively eliminated from Z-bearing gametes during, or possibly after, meiosis (Figure 5A right). At present, it is unclear which of the two scenarios (meiotic drive sensu stricto or elimination of the maternal Z at a later stage) is more plausible. However, it is noteworthy that, in the moth Abraxas grossulariata, a matriline consisting of putative Z0 females was observed to produce only females or a great excess of females, and the underlying mechanism was considered to be the selective elimination of Z chromosomes [17–20]. However, the presence of cytoplasmic bacteria such as Wolbachia has not yet been examined for this moth species. If we assume that the elimination of the maternal Z chromosome is the mechanism of the SD in E. mandarina, the exceptional individual 21m (Figure 3C) could be viewed as ZZ’ rather than Z0, wherein Z’ is a maternal Z chromosome that was only partially deleted in the position including Tpi and Ket by the incomplete action of wFem. It is possible to further speculate that the presence of wFem results in the elimination of sex chromosomes in general (Z or W chromosomes) and, therefore, the absence of W chromosomes in CF females may also be a direct effect of wFem.
The feminizing effect of Wolbachia compensates for the loss of the W chromosome in Z0 individuals
In general, lepidopterans species with Z0/ZZ sex chromosome constitution are considered to determine their sexes by Z-counting mechanisms, wherein ZZ is male and Z0 is female [21,22]. However, the appearance of the male phenotype in Z0 individuals of E. mandarina after antibiotic treatment suggests that wFem in Z0 individuals compensates for the loss of W and its feminizing function (Figure 5B). We speculate that the W chromosome of E. mandarina acts as an epistatic feminizer. In B. mori, the W chromosome – more specifically, a piRNA located on the W chromosome – acts as an epistatic feminizer by silencing Masculinizer on the Z chromosome [23].
Reduced survival of Z0 individuals or their offspring after antibiotic treatment of larvae or adults, respectively, may suggest improper dosage compensation in Z0 males. Improper dosage compensation was also proposed to be the cause of male- and female-specific lethality in Wolbachia-infected and cured lines of Ostrinia moths [24–27].
How did the coordinated dual effects of Wolbachia evolve?
We demonstrated that wFem causes SD and feminization in E. mandarina in two steps (Figure 5B). This is similar to the dual role of Wolbachia and Cardinium in haplodiploid parasitoid wasps where they induce thelytokous parthenogenesis in a two-step mechanism, comprising diploidization of the unfertilized egg followed by feminization [28,29]. Here, we develop the potential evolutionary scenario that led to the appearance of both effects in E. mandarina (Figure 6). A WZ female Eurema butterfly may have acquired wFem that exerted a feminizing effect on ZZ males. The feminizing effect was lethal to ZZ individuals because of improper dosage compensation, as evident in Wolbachia-infected Ostrinia moths (Figure 6A) [26,27]. This could be viewed as a manipulation similar to a male-killing phenotype [30,31]. However, the feminizing effect of wFem was redundant in WZ females where the W chromosome acted as a female determiner [23]. Conversely, the function of W had also become redundant in CF individuals and this could have led to the loss of the W chromosome and the rise of a Z0 lineage (Figure 6B). Similarly, in Ostrinia moths, a female-determining function is thought to have been lost from the W chromosome in Wolbachia-infected matrilines [25]. Spontaneous loss of a nonfunctional W chromosome may be easier than expected: in a wild silkmoth Samia cynthia, the W chromosome does not have a sex-determining function, and Z0 females are frequently obtained in experimental crosses between subspecies [32]. Wolbachia has previously been found to be involved in the loss and birth of W chromosomes in the woodlouse Armadillidium vulgare [33,34]. However, in A. vulgare it has not yet been tested whether Wolbachia interferes with chromosome segregation and inheritance as we have mechanistically demonstrated it for E. mandarina; i.e., after the loss of the W chromosome in CF lineages, Wolbachia then acquired a novel function that affected female oogenesis and resulted in SD (Figure 6C). It is unlikely that SD arose prior to the feminization function of Wolbachia: if the appearance of SD were to precede the loss of the W chromosome, the feminizing or female-determining function would become unnecessary for Wolbachia because there would be no males. In the short term, disruption of Z chromosome inheritance in females in a female-heterogametic species represents a great advantage to cytoplasmic symbionts because all vertically transmitted symbionts gain the opportunity to survive. However, males are still required for fertilization, and fixation of the symbionts in the host population will inevitably lead to the extinction of both the symbionts and the hosts [35]. In the long term, suppressors against sex ratio distortion, as has been observed for the male-killing phenotypes in the butterfly Hypolimnas bolina or a ladybird beetle [36,37], can be expected to evolve in the host. However, the evolutionary outcomes of the suppression of a combined SD and feminization would be different from that of male-killing suppression, because it would lead to all-male progeny, resulting in the loss of the matriline that inherits the feminizing and sex-distorting Wolbachia. This process thereby selects for an increased frequency of WZ females.
Concluding remarks
In summary, we demonstrate for the first time that the manipulation of sex chromosome inheritance and cytoplasmically induced SD can be added to the repertoire of host manipulations induced by Wolbachia. Therefore, the host effects of this bacterium are far more diverse and profound than previously appreciated. Disentangling these complex interactions between insects and Wolbachia may provide further exciting discoveries in the areas of host–parasite interactions, endosymbiosis as well as cell and chromosome biology in years to come, and perhaps also provide new avenues for pest population control.
Materials and methods
Collection and rearing of E. mandarina
Female adults of E. mandarina (Lepidoptera: Pieridae) were collected on Tanegashima Island, Kagoshima, Japan (Figure 1—figure supplement 1). In the laboratory, each female was allowed to lay embryos on fresh leaves of Lespedeza cuneata (Fabales: Fabaceae) in a plastic cup with absorbent cotton immersed with 5% honey solution. The artificial diet for larvae was prepared by mixing leaf powder of Albizia julibrissin (Fabales: Fabaceae) in the custom-made Silkmate (Nihon-Nosa, Yokohama, Japan) devoid of mulberry leaves. Insects were reared under the 16 h/8 h light /dark photoperiod at 25°C.
Antibiotic treatment
We performed antibiotic treatment of two different stages (larval stage and adult stage) of E. mandarina. For larval antibiotic treatment, larvae were fed with the artificial diet (shown above) containing 0.05% tetracycline hydrochloride (tet). For adult antibiotic treatment, female adults were fed with 5% honey solution containing 0.1% tet. Specifically, CF females were mated to antibiotic-treated male offspring of C females. Antibiotic treatment of these males was performed in the larval stage and prevented CI in the crossing. After mating, each CF female was allowed to lay embryos on fresh leaves of L. cuneata in a plastic cup with absorbent cotton immersed with 5% honey solution containing 0.1% tet. Fresh leaves of L. cuneata and cotton with tet-containing honey solution were exchanged daily.
Diagnosis of Wobachia strains
To diagnose Wolbachia strains in E. mandarina, several legs of each adult were homogenized in STE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 150 mM NaCl) and incubated at 56°C for 30 min followed by 92°C for 5 min. After centrifugation at 15,000 rpm for 2 min, the supernatant was used for polymerase chain reaction (PCR) using different primer pairs. The primer pair wsp81F (5′–TGGTCCAATAAGTGATGAAGAAAC–3′) and wsp691R (5′–AAAAATTAAACGCTACTCCA–3′) amplifies a ca. 610-bp fragment of the Wolbachia wsp gene [38]. The primer pair wsp81F and HecCIR (5′–ACTAACGTCGTTTTTGTTTAG–3′) amplifies a 232-bp fragment of the wsp gene of wCI, while the primer pair HecFemF (5′–TTACTCACAATTGGCTAAAGAT–3′) and the wsp691R amplifies a 398-bp fragment of wsp gene of wFem [11,39].
Whole genome sequencing and de novo assembly
We performed whole genome sequencing for three types of E. mandarina individuals (CF females, C females and C males) that were collected on Tanegashima Island, Japan (Figure 1—figure supplement 1). Six genomic DNA libraries (two libraries for each sample type derived from two individuals) were constructed following manufacturer’s instructions (http://www.illumina.com). The average insert size of the libraries was approximately 350 bp and each library was multiplexed using a single indexing protocol. The genomic DNA libraries were sequenced by Illumina MiSeq using MiSeq Reagent Kit v3 (600-cycle) (Illumina, San Diego, CA). Generated raw reads (8.31 Gb, 5.34 Gb, and 6.94 Gb for CF females, C females and C males, respectively) were filtered by Trimmomatic [40] and then mapped to the complete genome of Wolbachia strain wPip (GenBank: NC_010981.1) by Bowtie2 [41]. Mapped reads were discarded and then remaining reads of the three samples were merged and de novo assembled by SGA assembler [42]. Generated genome contig sequences were used for further analysis.
Analysis of mapped read counts on chromosomes
To verify that CF and C females have one Z chromosome, we compared normalized mapped read counts of the three samples on Z chromosomes and remaining chromosomes. The filtered reads of each sample were mapped to the genome contigs by Bowtie2 (only concordantly and uniquely mapped reads were counted) and then normalized mapped read count of each sample on each contig was calculated based on the ratio of the number of total mapped reads between the three samples. Nucleotide sequences of relatively long genome contigs (length is 2 kb or more) with enough coverage (20 or more mapped reads) were extracted and compared with the gene set A of B. mori [43] by blastx search (cutoff e-value is 1e-50). Genome contigs with blastx hits were extracted and classified into 28 chromosomes based on the location of the homologous B. mori genes. For each chromosome, the average number of relative normalized mapped read counts was calculated for each sample (the number of C males was normalized to 1) using the normalized mapped read counts in the classified genome contigs, respectively.
Sanger sequencing
To genotype Z chromosomes, a highly variable intron of Z-linked triosephosphate isomerase (Tpi) gene was PCR amplified using the primers, 5′–GGTCACTCTGAAAGGAGAACCACTTT–3’ and 5′–CACAACATTTGCCCAGTTGTTGCAA–3′, located in coding regions [44]. The PCR products were treated with ExoSAP-IT® (Affymetrix Inc., Santa Clara, CA) and subjected to direct sequencing at Eurofins Genomics K.K. (Tokyo, Japan). 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. By sequencing from both sides, it was possible to obtain the genotypes of males and females (Figure 3—figure supplement 2).
FISH analysis
In most lepidopteran species a conspicuous heterochromatic body is exclusively found in female polyploid nuclei. Since W derived-BAC as well as genomic probes have highlighted the W chromosomes and heterochromatin bodies in B. mori [45,46], there is no doubt that the bodies consist of the W chromosomes. The diagnosis however retains unreliable if a species of interest carries a W–autosomal translocation and/or partial deletion of the W [47,48]. Hiroki et al. [10] as well as Narita et al. [12] relied on the W-body diagnosis for C and CF females and concluded that they have WZ and ZZ sex chromosome constitutions, respectively. However, Kern et al. [13] has recently found that, on the basis of genomic qPCR designed to amplify Z-linked gene sequences (Tpi and Ket) relative to an autosomal gene (EF-1α), both CF and C females have only one Z chromosome while males have two Z chromosomes. This finding rejected the previous conclusion that the sex chromosome constitution of CF females is ZZ [10,12] but was inconclusive about whether CF females have a Z0 or W’Z system (with W’ as a modified W that has lost the feminization function and cannot be detected by the W-body assay). Hence we carried out more extensive chromosome analysis (other than just the W-body) to directly prove whether CF females carry the W or not.
In Lepidoptera, the W chromosome can be highlighted by FISH using probes prepared from whole genomic DNA of males or females. The capablity of FISH probes in detecting the W chromosome is due to the numerous repetitive short sequences occupying the W chromosome, which is then prone to be hybridized by random sequences. Genomic probes also paint repetitive regions scattered across other chromosomes, albeit at a lower density (autosomes and Z chromosome). Here we made mitotic and pachytene chromosome preparations from wing discs and gonads, respectively, in the last instar larvae of C and CF individuals of E. mandarina (see [49] for details). Genomic DNA was extracted from tet-treated C female larvae. Insect telomeric repeats were amplified by non-template PCR [50]. Kettin (Ket) gene fragments were amplified from adult cDNA synthesized by PrimeScript™ RT reagent Kit (TaKaRa, Otsu, Japan) and cloned by TOPO® TA Cloning® Kit (Thermo Fisher Scientific, Waltham, MA). We used 4 pairs of primers, Em_kettin_F1:5′–AGGTAATCCAACGCCAGTCG–3′ and Em_kettin_R1: 5′–TGCTTGCCCTAAGGCATTGT–3′, Em_kettin_F2: 5′–ACAATGCCTTAGGGCAAGCA–3′ and Em_kettin_R2: 5′–TGGGCAAAGCCTCTTCATGT–3′, Em_kettin_F3: 5′–AGATTCCGCACTACGCATGA–3′ and Em_kettin_R3: 5′–TAAATTGTGGTGGGACGGCA–3′, Em_kettin_F5: 5′–ACATGAAGAGGCTTTGCCCA–3’ and Em_kettin_R5: 5′–TCATGCGTAGTGCGGAATCT–3′, for PCR amplification with 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 3 min finalized by 72°C for 10 min. Probe labeling was done by using the Nick Translation Kit (Abbott Molecular, Des Plaines, IL). We selected Green-dUTP, Orange-dUTP (Abbott Molecular Inc.) and Cy5-dUTP (GE Healthcare Japan, Tokyo) fluorochromes for genomic DNA, Ket and insect telomeric repeat (TTAGG)n probes respectively. Hybridizations were carried out according to protocols described elsewhere [49]. Signal and chromosome images were captured with a DFC350FX CCD camera mounted on a DM 6000B microscope (Leica Microsystems Japan, Tokyo) and processed with Adobe Photoshop CS2. We applied green, red and yellow pseudocolors to signals from Green, Orange and Cy5 respectively.
Quantitative polymerase chain reaction (qPCR)
Embryos of mated females were sampled 48 h after the oviposition and stored at –80°C until DNA extraction. Embryos were individually subjected to DNA extraction using DNeasy® Blood & Tissue Kit (Qiagen, Tokyo, Japan). Real-time fluorescence detection quantitative PCR (qPCR) was performed using SYBR Green and a LightCycler® 480 System (Roche Diagnostics K.K., Tokyo, Japan). Z-linked Tpi was amplified using TPI-F (5′–GGCCTCAAGGTCATTGCCTGT–3′) and TPI-R (5′–ACACGACCTCCTCGGTTTTACC–3′), Z-linked Ket was amplified using Ket-F (5′–TCAGTTAAGGCTATTAACGCTCTG–3′) and Ket-R (5′–ATACTACCTTTTGCGGTTACTGTC–3′), and autosomal EF-1α was amplified using EF-1F (5′–AAATCGGTGGTATCGGTACAGTGC–3′) and EF-1R (5′–ACAACAATGGTACCAGGCTTGAGG–3′) [13]. For each qPCR, a standard dilution series of PCR products (108, 107, 106, 105, 104 and 103 copies per microliter) was included in order to estimate the absolute copy numbers of the target sequence in the samples. To prepare standard samples, PCR products were gel-excised and purified by Wizard® SV (Promega). Copy numbers of the standard samples were estimated by the concentration measured by a spectrophotometer, considering that the molecular weight of a nucleotide is 309 g/mol. For each qPCR, two replicates were performed that delivered similar results. All qPCRs were performed using a temperature profile of 40 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 10 s. The qPCR data were analyzed by the Absolute Quantification analysis using the Second Derivative Maximum method implemented in the LightCycler® 480 Instrument Operator Software Version 1.5 (Roche).
RT-PCR
RNA was extracted from adult abdomens that were stored at -80°C using RNeasy® Mini Kit (Qiagen, Tokyo, Japan). The cDNA synthesized by using Superscript™ III (Invitrogen) and Oligo(dT) was used as a template for RT-PCR. A partial sequence of dsx which contains alternative splicing sites was amplified using a primer pair, E520F (5′–GCAACGACCTCGACGAGGCTTCGCGGA–3′) and EhdsxR4 (5′–AGGGGCAGCCAGTGCGACGCGTACTCC–3′) and a temperature profile of 94°C for 2 min, 30 cycles of 94°C for 1 min, 57°C for 1 min and 72°C for 1 min 30 s, followed by 72°C for 7 min. The sequences of seven dsxF isoforms and a dsxM isoform were deposited in DDBJ/EMBL/Genbank (LC215389-LC215396).
Additional information
Competing interests
The authors declare no conflict of interest.
Funding
Author contributions
DK, KS, designed the research; DK, MO, TS, AY, TK, SK, HK, YK, SN, MM, MR, KS, performed the research; DK, AJ, KS, analyzed the data; DK, MR, KS, wrote the paper with input from AY.
Acknowledgements
We thank Isao Kobayashi for help in collecting butterflies and Ranjit Kumar Sahoo for stimulating discussion. A part of this study is supported by NIAS technical support system at NARO.