Involvement of Ecdysone Signaling in the Expression of the doublesex Gene during Embryonic Development in the Silkworm, Bombyx mori

In mammals, sexual differentiation is induced by sex hormones produced in the gonad. The gender is determined by the action of the SRY(sex-determining region Y) gene located in the Y chromosome, and the gonad primordium begins to differentiate into the ovary or the testis [Berta et al., 1990; Jäger et al., 1990; Koopman et al., 1991]. The ovaries secrete estrogen, and the testes secrete testosterone, which act on follicle maturation and testicular development, respectively, resulting in the sexual development of the body. Estrogen and testosterone are both steroid hormones. In fish, testis-ova are formed when adult males are treated with estrogen [Egami, 1955]. In reptiles whose sex is determined by the temperature of the environment, treatment with estrogen at male induction temperature induces gonadal feminization, and treatment with estrogen inhibitors at female induction temperature induces gonadal masculinization [Chardard et al., 1995; Richard-Mercier et al., 1995]. Similarly, in birds, it has been revealed that female-to-male sex reversal in gonads occurs when estrogen synthesis is inhibited [Wartenberg et al., 1992]. Thus, sexual differentiation is induced by sex hormones in many vertebrates including mammals.

Many “vertebrate-type” steroids, such as estradiol, testosterone, dihydrotestosterone, androsterone, and progesterone, have been chemically identified in insects [De Loof, 1987; Swevers et al., 1991; Darvas et al., 1997]. However, there is no evidence that they play a role in sexual differentiation or reproduction. Typical steroid hormones in insects are ecdysteroids, including ecdysone, ecdysterone, turkesterone, and 20-hydroxyecdysone (20E), which are essential for development, molting, and metamorphosis [Krishnakumaran and Schneiderman, 1970; De Loof, 2006; Margam et al., 2006]. In the silkworm, Bombyx mori, ecdysteroids are synthesized from cholesterol in the prothoracic gland by stimulation of prothoracic stimulating hormone secreted by the brain [Huang et al., 2008]. Ecdysone is converted into its active form, 20E, in the target tissue, and 20E binds to the ecdysone receptor (EcR), which forms a dimer with Ultraspiracle [Yao et al., 1993]. This complex then binds to the ecdysone response element (EcRE) to regulate the transcription of downstream target genes [Thummel et al., 1996].

In the silkworm, the continuous supply of 20E is required for embryonic development [Makka et al., 2002]. During the embryonic stage, 20E is provided not only by de novo synthesis, but also by dephosphorylation of maternally derived ecdysteroid conjugates (ecdysteroid-phosphates) [Horike and Sonobe, 1999; Sonobe et al., 1999; Makka and Sonobe, 2000]. In the de novo synthesis of 20E, hydroxylation at the C-20 position of ecdysone, which is catalyzed by ecdysone 20-hydroxylase (E20OHase), is a rate-limiting step [Makka and Sonobe, 2000]. The conversion of ecdysteroid-phosphates is catalyzed explicitly by ecdysteroid-phosphate phosphatase (EPPase) [Yamada and Sonobe, 2003].

A growing body of evidence supports that there is a close link between ecdysteroids and sexual differentiation in insects. The 20E titer in the hemolymph in several fly species (Sarcophaga, Calliphora, Lucilia, Phormia) is much higher in females than in males [Briers and De Loof, 1980; Briers et al., 1983a, b]. 20E is the only factor that can induce vitellogenin synthesis in adult Sarcophagamales [Huybrechts and De Loof, 1977]. Moreover, the feeding of 20E to males directs their behavior in a female direction [De Clerck and De Loof, 1983]. In Drosophila melanogaster, it is known that EcRand E74, a small set of genes induced directly by ecdysone, are required to maintain germline stem cells in the ovary [Morris and Spradling, 2012]. Similarly, EcRtogether with E75, a gene also induced directly by ecdysone [Segraves and Hogness, 1990], promotes stem cell maintenance in the Drosophilatestis [Li et al., 2014]. Furthermore, ecdysone also affects sexual behavior in Drosophila[Ganter et al., 2007, 2011, 2012]. However, it remains obscure whether ecdysteroids or ecdysone signaling play a crucial role in sex determination, which usually occurs during embryonic development.

Sex determination in the silkworm occurs between 29 and 32 h after oviposition [Sakai et al., 2014]. The femaleness of the silkworm is predominantly determined by the presence of the W chromosome [Hashimoto, 1933]. During the sex determination stage, Feminizer(Fem), the master regulatory gene for femaleness, is transcribed from the W chromosome in females [Kiuchi et al., 2014]. Femtranscripts yield PIWI-interacting RNAs (piRNAs), causing degradation of transcripts from the Masculinizer (Masc) gene, which is an essential regulatory gene for maleness [Sakai et al., 2016]. In the absence of Mascexpression, Bombyx doublesex(Bmdsx) pre-mRNA undergoes female-specific splicing to produce the female-type isoform of Bmdsx(BmdsxF), enhancing the female development of somatic cells [Suzuki et al., 2003; Kiuchi et al., 2014; Sakai et al., 2015]. The male stably expresses the Mascgene due to the lack of the W chromosome, inducing the expression of the male-specific isoform of the Bombyx homolog of the insulin-like growth factor II mRNA-binding protein gene (Imp^M) [Kiuchi et al., 2014; Sakai et al., 2015]. Imp^M is involved in the male-specific splicing of Bmdsxpre-mRNA, resulting in the production of the male-type isoform of Bmdsx (BmdsxM) [Suzuki et al., 2010]. BmdsxMpromotes male development of the body [Suzuki et al., 2005]. Bmdsxstarts sexual dimorphic expression within 48 h after oviposition [Sakai et al., 2014].

In the silkworm, by using the sex-limited strain, female embryos can be easily discriminated from male ones based on the pigmentation of the eggshell [Sakai et al., 2014]. Taking advantage of this useful feature, in this study, we investigated whether ecdysteroids or ecdysone signals are involved in sexual differentiation during the embryonic development of the silkworm. For this purpose, ecdysone and 20E titers in male and female eggs were measured, and a comparison of the expression levels of ecdysone biosynthesis-related genes was made between the male and female embryos. This study provides several lines of evidence that there is a strong link between ecdysone signaling and Bmdsxexpression by knockdown (KD) experiments of several ecdysone synthesis-related genes.

To quickly and precisely discriminate female eggs from male eggs, the sex-limited black egg strain, S-2, was used as described previously [Sakai et al., 2015]. The developing eggs were incubated at 25°C. Larvae were reared on an artificial diet (Nihon Nosan) at approximately 25°C.

Eggs of the S-2 strain were collected at determined points in time after oviposition and divided into males and females based on eggshell color. Twenty eggs of each sex were homogenized using a pestle in 350 μL sonic buffer (150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EGTA pH 8.0). The resulting suspension was mixed with 350 μL of 1-butanol for 15 min at room temperature. The supernatant obtained by centrifugation (1,500 rpm, 4°C, 10 min) was collected, and the solvent was distilled off for 2 h at room temperature using an evaporator. The solvent-distilled sample was dissolved in 500 μL of 70% methanol and was shaken with 500 μL of hexane for 15 min to extract hydrophobic components such as cholesterol. The lower layer obtained by centrifugation (1,500 rpm, 4°C, 10 min) was collected, and the solvent was distilled off for 2 h at room temperature using an evaporator. The solvent-distilled sample was stored at -20°C until analysis.

Prominence gradient HPLC (Shimadzu) and QTRAP 5000 (AB SCIEX) were used for LC-MS/MS analysis. The sample stored at -20°C as described above was redissolved in 100 μL of 100% methanol. The supernatant obtained by centrifugation (14,000 rpm, 4°C, 10 min) was used for the analysis. The conditions of LC-MS/MS were as follows. Column: PEGASIL ODS (2.0 × 100 mm), flow rate: 0.25 mL/min, gradient curve: 10-80% acetonitrile. The concentration of the component corresponding to 20E was calculated from the peak area value obtained from LC-MS/MS analysis.

Total RNA was extracted from either single eggs or 10 pooled eggs using Isogen (Nippon Gene), as described previously [Suzuki et al., 2012]. cDNA synthesis and quantitative real-time RT-PCR (qRT-PCR) were performed according to the protocol of Suzuki et al. [2012]. The primer sequences used for qRT-PCR are shown in Table 1.

DNA templates for double-stranded RNA (dsRNA) synthesis were amplified by RT-PCR using primers described in Table 2. Each primer contained a T7 promoter site. dsRNA synthesis and dsRNA injection into eggs were performed according to the protocol of Suzuki et al. [2012]. Briefly, fertilized eggs of B. moriS-2 stain were collected within 2 h after oviposition and were glued onto the surface of glass slides. dsRNAs were injected into the eggs at the preblastoderm stage (between 6-8 h after oviposition at 25°C) at a final concentration of 1 μg/μL.

Testes were dissected out from day-0 fifth instar larvae of the w1pnd strain provided by Dr. Megumi Sumitani, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO). The dissected testes were washed 3 times with 1× PBS (TaKaRa). The testes were separately cultured using a 96-well cell culture plate (Iwaki). Each well contained 200 μL of TC-100 insect medium (Thermo Fisher). Ponasterone A (kindly provided by Dr. Shinji Nagata, University of Tokyo) was dissolved in 99.5% ethanol and added to the medium to give the desired concentrations prior to the culture. After 6 h of culture at 25°C, total RNA was extracted from each testis, and the expression level of Bmdsxwas quantified by qRT-PCR according to the protocol described above.

Quantitative analysis using mass spectrometer LC-MS/MS was attempted to compare ecdysone and 20E titers between sexes at the embryonic stage. For this purpose, male and female eggs at 2, 3, 4, 5, 6, 7, 8, 9, and 10 days post-oviposition (dpo) were subjected to LC-MS/MS analysis. Although we could not quantitate ecdysone, the 20E titer in male and female eggs was quantified as shown in Figure 1A. The 20E titer in both sexes increased as embryonic development progressed, peaked at 6 dpo, and then gradually decreased (Fig. 1A). However, there was no significant difference in the 20E titer between sexes at all the stages examined in this study.

In order to verify the above results from another angle, we decided to investigate the expression level of 2 genes encoding EPPase and E20OHase, both of which are important for the biosynthesis of ecdysone and 20E in eggs. As described above, EPPase dephosphorylates ecdysone 22-phosphate accumulated in eggs, yielding free ecdysone [Yamada and Sonobe, 2003]. E20OHase catalyzes the hydroxylation at the C-20 position of ecdysone and is essential for the de novo synthesis of 20E in eggs [Makka and Sonobe, 2000]. Quantification of EPPase and E20OHasemRNAs by qRT-PCR demonstrated that the expression levels of both genes reached the highest peak at 4 dpo, and then gradually decreased with embryo development (Fig. 2A, B). However, no significant differences were seen in the expression levels of the 2 genes between males and females. These results were consistent with the fact that EPPase and E20OHase are involved in the biosynthesis of 20E in the egg since the expression level of EPPaseand E20OHasepeaked before the 20E titer became highest as shown in Figure 1A.

The same analysis was performed to investigate expression levels of the 20E primary response genes EcRand E75. The expression level of EcR-A, one of the isoforms of EcR, peaked at 7 dpo (Fig. 2C) when the 20E titer still had a high level (Fig. 1A). E75B, which is one of the E75isoforms, showed an expression pattern similar to that of EcR-A(Fig. 2G). These results were consistent with the findings that expressions of EcRand E75are both induced directly by ecdysone [Segraves and Hogness, 1990; Morris and Spradling, 2012]. On the other hand, the expression levels of other isoforms of EcR(EcR-B1and EcR-B2) reached the highest peak already at 4 dpo (Fig. 2D, E). Differing from the expression patterns of the genes examined above, E75A, which is another isoform of E75, showed higher expression levels during 2 and 4 dpo (Fig. 2F). There was no significant difference in the expression levels of all analyzed genes between male and female eggs. Taken together, it seemed unlikely that ecdysone titer, ecdysone biosynthesis, and ecdysone signaling showed a difference between sexes in the embryonic stage.

In order to examine the possibility that sex differentiation of the silkworm embryo is under the control of ecdysone signaling, the expression level of Bmdsxat various embryogenic stages was quantified by qRT-PCR. As shown in Figure 1B, the expression level of Bmdsxin male and female embryos gradually increased with the progress of embryogenesis and reached the highest peak at 6 dpo. The expression pattern was almost similar to the developmental change in the 20E titer (Fig. 1A). These results suggest that Bmdsxexpression in the embryo may be under the control of ecdysone signaling.

Although the above results indicate that ecdysone signaling is not likely involved in sexual development, the expression level of the master regulatory gene for sexual differentiation, Bmdsx, seemed to correlate with the developmental changes in the 20E titer in embryos. To examine whether the expression level of Bmdsxis indeed under the control of 20E, we investigated the effect of the RNAi-mediated KD of EPPase, which dephosphorylates ecdysone 22-phosphate accumulated in eggs, supplying a lot of free ecdysones [Yamada and Sonobe, 2003]. The dsRNA injection targeting EPPasesignificantly repressed the expression of the target gene (Fig. 3A). However, efficiency for EPPase KD was slightly lower in male embryos. To evaluate whether EPPaseKD caused a reduction in ecdysone and 20E titers, we quantified expression levels of several ecdysone responsive genes examined above. Expression levels of all the examined genes in the EPPaseKD embryos significantly decreased compared with those in negative control embryos (Fig. 3B). Especially, the extent of reduction in the expression level of EcR-Aand E75Bin the EPPaseKD eggs was greater than in other ecdysone responsive genes. Based on these results, it would be expected that EPPaseKD might cause a reduction in ecdysone and 20E titers in eggs.

The same analysis was performed to quantify the expression level of Bmdsx. A reduced expression level of Bmdsxwas observed in both female and male EPPase KD embryos (Fig. 3C). These results suggest that the expression level of Bmdsxin male and female embryos is under the control of ecdysone signaling. Either EcR-Aor E75Bmay be involved in the regulation of Bmdsxexpression in embryos since a relatively greater extent of reduction in the expression levels of EcR-Aand E75Bwas observed in the EPPase KD embryos (Fig. 3B).

To further verify whether the expression of Bmdsxin embryos is under the control of ecdysone signaling, we next investigated the effect of KD of EcRand E75, whose expression levels were decreased in the EPPaseKD embryos. As shown in Figure 4A, dsRNA injection targeting EcR-Asuccessfully reduced the expression level to less than 10% of that in the negative control embryos (Fig. 4A). KD of EcR-Acaused a significantly reduced expression level of Bmdsxboth in male and female embryos (Fig. 4B). Although we successfully knocked down 2 other EcRisoforms, EcR-B1and EcR-B2(Fig. 4C), the expression level of Bmdsxwas not affected by the KD of EcR-Bisoforms (Fig. 4D). Similarly, KDs of E75Aand E75Bcaused no significant change in the Bmdsx expression level (Fig. 4E, F). These results demonstrated that ecdysone signaling mediated by EcR-A directly or indirectly regulates the proper expression of Bmdsx during the embryonic stage.

Next, to examine whether an increased 20E titer promotes the expression of Bmdsxin embryos, we injected ponasterone A (Fig. 5A), which was shown to induce molting in the silkworm and to act efficiently as a ligand for EcR [Kobayashi et al., 1967; Saez et al., 2000], into fertilized eggs at the preblastoderm stage. However, most injected eggs died within 4 days after injection either due to technical problems in the injection technique or the presumably toxic effect of ponasterone A. The expression level of EcRquantified by qRT-PCR using cDNAs prepared from barely survived eggs showed no significant change between control eggs and ponasterone A-injected eggs (Fig. 5B). We used an in vitro system to overcome this problem. Kambysellis and Williams [1972] succeeded in the in vitro production of sperms in cultured testis from silkworm pupae by adding ecdysone to the medium. According to their method, testes of fifth instar larvae were excised and cultured in medium with or without ponasterone A. After 6 h of culture at 25°C, total RNA was extracted from the testis, and the expression level of Bmdsxwas quantified by qRT-PCR. The expression level of Bmdsx in the cultured testis increased in a dose-dependent manner of ponasterone A (Fig. 5C). However, if ponasterone A treatment causes some changes in the cell type composition in the testis (i.e., more germ cells/less somatic cells), then such changes may lead to an apparent increase in Bmdsxexpression levels. To investigate whether the ponasterone A treatment affects the number of somatic cells versus germ cells in the testis, we quantified the expression level of a Bombyxortholog of the vasagene (Bombyx morivasa like protein gene, Bmvlg), a well-known germ cell marker gene in various animals including the silkworm [Raz, 2000; Nakao et al., 2006; Cao et al., 2012]. In contrast to the Bmdsxexpression, no apparent changes were observed in the expression level of Bmvlg(Fig. 5D), suggesting that ponasterone A treatment did not affect the number of somatic cells versus germ cells under our experimental conditions. These results strongly suggest that stimulation by 20E increases the expression level of Bmdsx.

In order to examine the possibility that EcR-A directly regulates Bmdsxexpression, we searched for a sequence identical to EcRE or EcRE-like sequences in the region near the transcription start site of Bmdsxor within the Bmdsxgene body, but could not find any (data not shown). Therefore, it is conceivable that EcR indirectly regulates Bmdsx expression. In Drosophila, transcription of let-7microRNA is known to be directly regulated by EcR [Chawla and Sokol, 2012]. Interestingly, ecdysone stimulates let-7 miRNA expression through EcR, resulting in changes of expression levels of sex-determining genes such as Sex-lethal, transformer, and dsx[Fagegaltier et al., 2014]. To test whether the miRNA pathway is also involved in the regulation of Bmdsxexpression, we investigated the effect of KD of Argonaute-1 (Ago1), an essential component for the formation of miRNA-induced silencing complexes [Baumberger and Baulcombe 2005; Mallory and Vaucheret, 2010], on Bmdsxexpression in embryos. As shown in Figure 6A and D, KD of Ago1 caused significant decrement in the expression level of Bmdsxin male and female embryos. The same KD experiments did not affect expression levels of Femand Imp^M (Fig. 6B, C). As is the case in Drosophila, it is highly probable that EcR may also regulate the expression of dsxin the silkworm via miRNAs such as let-7miRNA.

In order to investigate whether ecdysteroids or ecdysone signals are involved in sexual differentiation at the sex determination stage of the silkworm, in this study, we first measured ecdysone and 20E titers in male and female eggs by LC-MS/MS. However, we could not detect a compound whose retention time of the LC precisely matched the ecdysone standard. It is known that most ecdysone in the silkworm egg is accumulated in the phosphorylated state [Yamada and Sonobe, 2003] and that ecdysone is rapidly epimerized in the egg [Sonobe et al., 1999; Makka and Sonobe, 2000]. Therefore, the amount of free ecdysone extracted from eggs in this study (20 eggs per sample) may be insufficient for the analysis.

The 20E titer in both sexes increased as embryonic development progressed, peaked at 6 dpo, and then gradually decreased (Fig. 1A). This result was almost similar to the changes in the level of 20E during embryonic development reported previously [Yamada and Sonobe, 2003], supporting the high reliability of our quantification of 20E titer in the egg. Our results demonstrated that there was no significant difference in the 20E titer between the sexes during embryonic development. However, we still cannot completely rule out the possibility that the ecdysone titer, which we failed to quantify in this study, may differ between male and female eggs. Further research is needed to clarify whether a sexual difference in ecdysone titer is observed during the embryonic stage.

Not only the 20E titer, but also the expression levels of genes related to the biosynthesis of ecdysone and 20E in the egg did not show a significant difference between males and females during the embryonic stage (Fig. 2A, B). Moreover, no significant difference in the expression levels of 20E primary response genes was observed between male and female embryos (Fig. 2C-G). These results suggest that ecdysteroids such as ecdysone and 20E are unlikely to act as a sex hormone that is important for sexual differentiation during embryogenesis in the silkworm. Nevertheless, KD of EPPaseand EcR-Aresulted in decreased expression of Bmdsxin male and female embryos (Fig. 3A, C, 4A, B). From these findings, it could be possible that ecdysone signaling plays a role in indirectly controlling the expression of some genes involved in sexual differentiation through inducing expression of Bmdsx, which is a master regulator of sexual differentiation in the silkworm.

Since EcRE or EcRE-like sequences were not found in the region near the transcription start site of Bmdsxor within the Bmdsxgene body, it is conceivable that EcR indirectly regulates Bmdsxexpression. In Drosophila, ecdysone stimulates let-7 miRNA expression through EcR, resulting in changes in the expression level of dsx[Fagegaltier et al., 2014]. Our results, where Ago1KD decreased the expression level of Bmdsx, support that there is a close link between the miRNA pathway and Bmdsxexpression. In B. mori, the expression level of let-7miRNA is higher at the end of each instar and the beginning of each molt than at other periods, coinciding with the pulse of ecdysone as a whole [Liu et al., 2007]. Consistent with this finding, analysis using transgenic silkworms carrying a miRNA sponge targeting the let-7seed region demonstrates that let-7miRNA regulates molting and metamorphosis in B. mori[Ling et al., 2014]. As is the case in Drosophila, it is highly probable that EcR may also regulate the expression of dsxin the silkworm via miRNAs such as let-7miRNA.

In Drosophila, the expression of dsxis not maintained at a constant level throughout life and also varies in each tissue. For example, expression of dsxin the genital primordium is kept at a low level until the third instar larval stage but increases rapidly from the late third instar larval stage to the pupal stage when differentiation of genital primordia starts [Chatterjee et al., 2011]. On the other hand, dsxis scarcely expressed in the fat bodies from the larval stage to the early pupal stage, but its expression level increases explosively from the late pupal stage to adults [Chatterjee et al., 2011]. Gonads and genital primordia in the silkworm show the most intensive growing during the pupal stage [Tazima, 1978]. It would be reasonable to consider that the expression of Bmdsxalso changes dramatically depending on the tissue and developmental stage and that some of such changes are controlled via ecdysone signaling. There is still room to investigate whether ecdysone signaling also affects Bmdsxexpression in developmental stages other than the embryonic stage.

Injection of ponasterone A, a well-known 20E analog, into fertilized eggs did not increase the expression level of EcR (Fig. 5B). The expression levels of all the EcRisoforms tended to decrease rather than increase depending on the concentration of ponasterone A. This may be due to the deleterious effect of higher concentrations of ponasterone A. Another plausible explanation is that ponasterone A might be inactivated immediately after injection because of the rapid epimerization of ecdysone in eggs as reported previously [Sonobe et al., 1999; Makka and Sonobe, 2000]. In a previous study, Makka et al. [2002] injected 20E into diapause eggs (18-21 h after oviposition) in order to examine the effect of 20E on embryonic development. Only approximately 7% of eggs were able to develop beyond the gastrula stage without entering diapause. The authors speculated that the low efficiency of injected 20E to induce embryonic development might be due to the immediate inactivation of 20E, since exogenous ecdysone and 20E are indeed promptly inactivated due to epimerization and phosphorylation [Makka and Sonobe, 1998, 2000; Sonobe et al., 1999]. Similarly, it is highly plausible that injected ponasterone A was also promptly inactivated and, therefore, had no effect on EcR and E75 expressions as observed in this study.

On the other hand, an increased expression level of Bmdsxin the cultured testis was observed in a dose-dependent manner of ponasterone A (Fig. 5). Spermatogenesis occurred in the cultured testis of Cynthia silkworms only when ecdysone was added to the medium [Kambysellis and Williams, 1972]. In Drosophila, spermatocyte meiosis and spermatogenesis can be induced in cultured spermatocytes under the presence of 20E [Ogata et al., 2012]. It is also known that EcR is required for promoting germline stem cell maintenance in the Drosophilatestis [Li et al., 2014]. dsxcontrols the sexually dimorphic development of somatic gonadal cells via nonautonomous cell-cell signaling [DeFalco et al., 2008] and is responsible for how the somatic gonad influences sex determination in the germline [Steinmann-Zwicky, 1994]. If we consider these findings together, it could be speculated that ecdysone signaling may promote spermatogenesis by inducing the expression of dsxat the proper level with proper timing. Among the EcR isoforms, EcR-A shows the highest expression in spermatocytes and is thought to play an important role in germline cells in Drosophila[Ogata et al., 2012]. This is reminiscent of our results where KD of EcR-Aseverely reduced the expression level of Bmdsx(Fig. 4B). EcR-A may have a conserved function to transmit ecdysone signals to dsx.

Another example where ecdysone triggers the differentiation of sexually dimorphic traits is reported in the tussock moth Orgyia recens. Females of this species have vestigial wings, whereas males have normal wings [Yamada, 1982]. During the early pupal stage, female wing discs degenerate drastically, while male wing discs develop normally. In vitro culture experiments demonstrate that only female wing discs start degradation after 20E stimulation [Lobbia et al., 2003]. This finding suggests that the same 20E stimulus is converted to a signal that results in a different sexual development. Such signaling conversion may be achieved by sex-specifically expressed factors such as dsx. Further studies are needed to uncover more detailed molecular mechanisms underlying the close link between ecdysteroids and sexual differentiation.

In this study, we focused only on ecdysone signaling via EcR. However, several lines of evidence suggest that the activity of some isoforms of the Ca²⁺-ATPases in the different membrane systems such as plasma membrane, rough endoplasmic reticulum, and smooth endoplasmic reticulum are subject to regulation by lipophilic signaling substances such as endogenous sesquiterpenoids (e.g., farnesol [De Loof et al., 2014]) and hormones (e.g., steroid hormones [Bhargava et al., 2000; Strehler and Zacharias, 2001]). Based on these findings, De Loof [2015] proposed a novel paradigm named “Calcigender,” where differential Ca²⁺ homeostasis enabled by the interplay between farnesol-like endogenous sesquiterpenoids (juvenile hormones in insects) and steroid hormones (ecdysteroids in insects) governs the female-male physiological dimorphism. Depending on the species, the plasma membrane Ca²⁺ pumps can occur in more than 1 allele (4 alleles in human [Strehler and Zacharias, 2001], only 1 coding gene in Drosophilabut 11 isoforms [De Loof et al., 2014]).

If sex-specific allele or splice variants of Ca²⁺-ATPases exist, it can be easily imagined that they will produce the sexual difference in Ca²⁺ homeostasis in response to ecdysone and 20E stimuli. Further studies are needed to verify the validity of this hypothesis.

We are grateful to Dr. Mari Ogiwara and Dr. Masatoshi Iga for their LC-MS/MS analysis. This work was supported by Grant-in-Aid for Scientific Research (B), 17H03940, 2017.

The authors have no ethical conflicts to disclose.

The authors have no conflicts of interest to declare.