Evolutionary origin of sex differentiation system in insects

The evolution of the functionality of genes and genetic systems is a major source of animal diversity. Its best example is insect sex differentiation systems: promoting male and female differentiation (dual-functionality) or only male differentiation (single-functionality). However, the evolutionary origin of such functional diversity is largely unknown. Here, we investigate the ancestral functions of doublesex, a key factor of insect sex differentiation system, using the apterygote insect, Thermobia domestica, and reveal that its doublesex is essential for only males at the phenotypic level, but contributes to promoting female-specific vitellogenin expression in females. This functional discordance between the phenotypic and transcription-regulatory levels in T. domestica shows a new type of functionality of animal sex differentiation systems. Then, we examine how the sex differentiation system transited from the single-functionality to the dual-functionality in phenotypes and uncover that a conserved female-specific motif of doublesex is detected in taxa with the dual-functional doublesex. It is estimated that the role of the sex differentiation system for female phenotypes may have evolved through accumulating mutations in the protein motif structures that led to the enhancement of its transcription-regulatory function.


Introduction
. In T. domestica female, the ovipositor consists of two pairs of appendices 178 (gonapophysis) and is derived from the retracted vesicles on the abdomen VIII and IX 179 (Emeljanov, 2014; Matsuda, 1976). This ovipositor is an autapomorphy of Ectognatha 180 (Beutel, 2017;Kristensen, 1975). The gonapophyses on the abdomen VIII (valvula I) 181 were the ventral part of the ovipositor and a paired structure. The gonapophyses on In females that were treated with RNAi for dsx, dsx isoforms, dsx-like, or both 205 genes, the external genital organ was the same as the ovipositor of the control females 206 that described in the above section ( Figure 2D Table 4). However, the seminal vesicle in males became round in shape in the dsx 277 RNAi males in contrast to the bean pod-like shape observed in the control males    domestica were more than 10000-fold higher in females than in males ( Figure 4A-C). 314 We found that the vtg mRNAs were expressed at 40-100-fold higher levels in dsx 315 RNAi males than in the control males ( Figure 4D and Table 2). Notably, the dsx 316 RNAi females produced around half the amount of vtg mRNA as the controls. The 317 dsx-like RNAi and a double-knockdown of dsx and dsx-like also significantly reduced 318 the expression of vtg in females ( Figure 4E and Table 2).

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This result is the first case that dsx can promote female-specific vitellogenin 320 expression in non-holometabolan species, even though it does not affect female   Table 2 (C and 333 D). Results of Brunner-Munzel tests are indicated by asterisks: *P < 0.05; **P < 334 0.01; ***P < 0.001 and is also described in Table 2. P > 0.05 is not shown.

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Differences of doublesex sequences between single-and dual-functional species 337 Genes can gain new functions due to gene duplication, co-factor function, we explored the remaining possibilities: novel exons or trans-regions. 347 We found that there are alternative splice types in T. domestica, which has an 348 alternative 5ʹ splice site, and in Pterygote insects, which have a mutually exclusive 349 exon, but did not find any differences of exon structure between species with single-350 functionality of dsx, and those with dual-functionality ( Figure 5A). 351 We finally discovered amino acid sequences in the female-specific region that

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A novel type of the output in the sex differentiation system 368 We show that the dsx of Thermobia domestica is essential for producing male 369 phenotypes, but does not contribute to female phenotypes. In contrast to the 370 phenotypes, this study showed that the female-specific vitellogenin genes are slightly 371 promoted by dsx in T. domestica females. These facts indicate that dsx in T. domestica 372 has an opposite transcription-regulatory function in males and females. Therefore, in 373 T. domestica, dsx contributes only to male differentiation at the phenotypic level, but 374 affects both sexes at the transcription-regulatory level (seemingly useless nature).

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There have been two known types of outputs of the insect sex differentiation is also responsible for producing traits of both sexes in other tissues. Therefore, it is 383 likely that the former type is the primary capability of the sex differentiation systems 384 in these species and that the function for promoting female differentiation may tissue-385 specifically become silent. In contrast, the seemingly useless nature of dsx for females 386 in T. domestica is a third type of the insect sex differentiation system ( Figure 6A).  On the origin of the role of the insect sex differentiation system for females 411 There are discrepancies in the output of the sex differentiation system in T.

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The question is how the function of dsx for the female phenotype evolved. We

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On the other hand, the female isoform of dsx shows no function in female phenotypes.
Our hypotheses are models that a non-functional isoform gains functionality at the 481 phenotypic level. The heterotypic evolution may need to be divided into modification 482 from existing functions and innovation from non-functionality at phenotypic level.

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In this study, we succeeded in detecting the transcription-promoting ability of    Wilmington, USA). fs-cDNA was synthesized from 350 ng of the total RNA using 623 SuperScript III Reverse Transcriptase (Thermo Fisher Scientific K.K., Tokyo, Japan). 624 We diluted the fs-cDNA to 1:2 with MilliQ water and preserved it at −30°C until it 625 was used in RT-qPCR assay. The RT-qPCR assays were performed using a  Table 6. Each RT-qPCR was technically replicated three times. Some samples were 639 excluded before analyzing the data when the Ct value of any genes was not detected 640 (ND) in one or more replicates or when the Ct value of the reference gene deviated 641 from that of other samples. In these removed data, a technical error was suspected.  Table 2. Also, its source data can be found in Table2-Source Data 1. In the dsx 649 expression of the RNAi male, we performed the Smirnov-Grubbs (SG) test for ΔCt 650 value using the grubbs.test function of the "outliers" package in R (https://cran.r-651 project.org/web/packages/outliers/index.html) ( Table 3). An outlier was detected in 652 the dsx RNAi male. We repeatedly performed the SG test using the data excluding the outlier. No further outliers were detected. Lastly, we re-analyzed the data, excluding 654 the outlier, using the BM test (Table 2).  (Figures 1-figure supplement 3, Figure2-figure supplement 1A). Therefore, at least in repeatedly into the adults every three days. The dsRNA was initially injected into 690 adults 12 hours after molting. We sampled the adults at 720±20 minutes after 691 subsequently molts, to analyze the vtg mRNA levels.

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Phenotype observation 693 We dissected thirteenth instar individuals in PBS using tweezers and removed 694 the thoraxes, reproductive systems, and external genital organs. We took images using  Also, the source data are reported in Table 4-source data 1 and Table 5-source data 1.       Smirnov-Grubbs' test are shown in Table 3. Results of the Brunner-Munzel test are 1137 indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001 and is also described in 1138   Table 2. P > 0.05 is not shown. Each plot is an individual. White plot is the outlier.

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Sample size are listed in Table 2.