Identification and characterization of sexually dimorphic neurons that express the sex-determining gene doublesex in the brain of silkmoth Bombyx mori

https://doi.org/10.1016/j.ibmb.2021.103518Get rights and content

Highlights

  • Doublesex expression is sexually dimorphic in the brains of developing silkworms.

  • Whole brain neural activity-mapping methods were established in silkmoths.

  • Doublesex-positive neurons were not activated by sexual behavior in silkmoths.

Abstract

Sexual differences in behavior are generated by sexually dimorphic neural circuits in animals. In insects, a highly conserved sex-determining gene doublesex (dsx) plays essential roles in the development of sexual dimorphisms. In the present study, to elucidate the neural basis of sexual differences in behaviors of silkmoth Bombyx mori, we investigated Bombyx mori dsx (Bmdsx) expression in the brains through development. In the brain, Bmdsx was differentially expressed in sex- and developmental stage-dependent manners. BmDSX protein-expressing cells were located in the dorsomedial region of the pupal and adult brains, and constituted two and one neural clusters in males and females, respectively. The number of BmDSX-positive cells was developmentally regulated and peaked at the early to middle pupal stages, suggesting that the sexually dimorphic neural circuits are established during this period. The detection of a neural activity marker protein BmHR38 suggested that the BmDSX-positive cells are not active during sexual behavior in both male and female moths, even though the cells in the vicinity of the BmDSX-positive cell clusters are active. These results imply that Bmdsx plays roles in the development of sexually dimorphic neural circuits, but the neural circuits are not related to sexual behavior in silkmoths.

Introduction

Many animals exhibit sexual differences in their external morphologies and behaviors. Recent studies have revealed the molecular mechanisms of sex determination, which are the basis of sexual dimorphisms, in various organisms (Bachtrog et al., 2014; Gempe and Beye, 2011). Silkmoth Bombyx mori, a model Lepidopteran insect, exhibits clear sexual differences in both morphology and behavior. B. mori has a ZW-type sex determination system, where Feminizer (Fem) PIWI-interacting RNA (piRNA) produced from the W chromosome epistatically determines the sex (Kiuchi et al., 2014). The Fem piRNAs target and silence Masculinizer (Masc) mRNA. Since Masc located on the Z chromosome encodes a male-determining transcription factor, the silencing of the gene leads to feminization (Kiuchi et al., 2014). In the male embryos, Masc protein induces the expression of the male-specific isoform of insulin-like growth factor II mRNA binding protein (IMPM) (Sakai et al., 2015; Suzuki et al., 2010, 2014). IMPM functions as an RNA-binding protein by interacting with its partner protein B. mori P-element somatic inhibitor (BmPSI), and induces the male-type splicing of B. mori doublesex (Bmdsx) mRNA (Suzuki et al., 2008). In contrast, in the female embryos, the lack of Masc leads to the default (female-type) Bmdsx mRNA splicing (Fig. 1A). Bmdsx belongs to the Doublesex Mab-3 Related Transcription factor (DMRT) family, an evolutionarily conserved sex-determining gene in vertebrates and invertebrates (Smith et al., 1999). The sex-differential splicing of Bmdsx mRNAs generates distinct C-terminal domains, which are important for the oligomerization with other transcription factors, and leads to the sex-differential expression of downstream genes (Ohbayashi et al., 2001; Suzuki et al., 2003, 2005). Through the ectopic expression or mutating of male- or female-type Bmdsx genes, it had been shown that Bmdsx is essential for the sex-determination of somatic cells in silkmoths (Suzuki et al., 2003, 2005; Xu et al., 2014, 2017; Yuzawa et al., 2020).

B. mori has a unique trait in that the adult moths are specialized in reproduction and do not exhibit any behavior other than courtship behavior (Sakurai et al., 2014). In addition, the courtship behavior is clearly sex-differential and simple, where a single sex pheromone component synthesized by females regulates the male courtship behavior (Butenandt et al., 1959; Sakurai et al., 2015). Males exhibit a series of stereotypic courtship behavior, which consists of zig-zag turns, round walks, and abdominal bending, all upon detection of bombykol, the major component of female sex pheromones (Obara, 1979). BmOR1, the bombykol receptor, is expressed exclusively in the male antennae, and only males exhibit the courtship behavior in response to bombykol (Sakurai et al., 2004). Meanwhile, mature females extend their pheromone glands and expose their gland surface to the air, leading to male attraction. After copulation, the females become unreceptive to males and start to lay eggs. These unique traits and clear sexual differences of silkmoth behavior prompted us to investigate the sexual difference of neural circuits in silkmoth brains.

Accumulating evidence from the studies in the vinegar fly Drosophila melanogaster revealed that the sexual differences of neural circuits and behaviors are governed by the actions of sex-determining genes (Yamamoto and Koganezawa, 2013). In D. melanogaster, two sex-determining genes, fruitless and doublesex, cooperatively regulate the development of sexually dimorphic neural circuits essential for courtship behavior in the male brains (Demir and Dickson, 2005; Kimura et al., 2008; Pan and Baker, 2014; Pan et al., 2012; Pan et al., 2011; Rideout et al., 2007; Rideout et al., 2010; von Philipsborn et al., 2011). In contrast, the development of neural circuits that control the courtship behavior of females is mainly regulated by doublesex, whose expression pattern is sexually dimorphic in the fly brains (Kimura et al., 2015; Lee et al., 2002; Rezával et al., 2016; Rezaval et al., 2012; Rideout et al., 2010; Zhou et al., 2014). Based on these lines of evidence, we hypothesized that Bmdsx has sexually dimorphic expression patterns and regulates the development of neural circuits that govern sexually different courtship behaviors in the silkmoth brains, similar to D. melanogaster. In this study, we revealed, for the first time, that Bmdsx is expressed in the pupal and adult brains in a sexually dimorphic manner. In contrast to our expectations, activity mapping by a neural activity marker gene demonstrated that the BmDSX-positive neurons are not activated by sexual behavior in both male and female moths. These results suggest that, neurons expressing Bmdsx in the adult stage are not involved in regulation of reproductive behavior, although Bmdsx may play roles in the development of sexually dimorphic neural circuits at earlier developmental stages.

Section snippets

Insects

Eggs of non-diapausing or diapausing white strain (w1-pnd and w/C, respectively) and a racial hybrid of B. mori, Kinshu × Showa, were obtained from the National Bio-Resource Project (NBRP) and a local dealer (Ueda Sanshu, Nagano, Japan), respectively. Larvae were reared on an artificial diet (Nihon Nosan Kogyo, Yokohama, Japan) at 25 °C under a 12-h light/12-h dark photoperiodic cycle. Adult moths within 1–3 days after eclosion were used for experiments.

Quantitative RT-polymerase chain reaction (QRT-PCR)

QRT-PCR was conducted as previously

Bmdsx is differentially expressed in sex- and developmental stage-dependent manners in the brain

To investigate the timing and relative levels of Bmdsx expression in the brain of B. mori, we conducted qRT-PCR analysis using various tissues, in addition to the brain, of males and females from different developmental stages (Fig. 1C–E). In the present study, we used primers designed to amplify a common region between male- and female-type Bmdsx mRNAs (Fig. 1A) to compare expression levels between sexes. Consistent with a previous report (Ohbayashi et al., 2001), Bmdsx was highly expressed in

Discussion

In this study, we revealed that Bmdsx is expressed in the brain of B. mori from the pupal to adult stages and the number and location of BmDSX-positive cells are sexually dimorphic. Interestingly, neural activity mapping using the neural activity marker BmHR38 suggested that cells expressing BmDSX at an adult stage are not activated by sexual behavior, implying that these neurons are not involved in sexual behavior. These results raise a possibility that in B. mori, the transient Bmdsx

Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgement

This work was supported by JSPS KAKENHI (Grant Number 16H05053 and 19H02968) to T.K. We thank late Prof. Bruce S. Baker and the Developmental Studies Hybridoma Bank for anti-DSX and anti-Synapsin mouse monoclonal antibodies, respectively, Mr. Jorge Colque Pedraza for English editing and the National Bio-Resource Project (NBRP): Silkworms from Japan Agency for Medical Research and Development (AMED) for white strain.

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