Precise staging of beetle horn formation in Trypoxylus dichotomus reveals the pleiotropic roles of doublesex depending on the spatiotemporal developmental contexts

Many scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex differentiation gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.


Introduction
Beetle horns are used as weapons for intraspecific combats between males.Beetle horns display sexual dimorphism in many Scarab species, and their shapes, numbers, sizes and forming regions are highly diverged even among closely related species [1][2][3].Interestingly, the diversified horn forms are associated with the fighting styles employed by the beetles, such as scooping up, piercing and throwing [4].Furthermore, beetle horns are thought to be an evolutionary novelty.Horns are an outgrowth structure derived not from an appendage but from a dorsal epidermal sheet.Elucidating how these novel traits were acquired in Scarab species will lead to better understanding the mechanisms of morphological diversification during evolution.Therefore, beetle horns are an attractive model for studying not only the association of trait novelty with sexually dimorphic development but also the evolution of novel traits.
Beetle horn development has been investigated in several horned beetles including the Japanese rhinoceros beetle, Trypoxylus dichotomus (Coleoptera, Scarabaeidae, Dynastinae).In T. dichotomus, male adults have sexually dimorphic exaggerated horns on the head and prothorax, which are used in combat among conspecific males as weapons [5,6].The head horn is shaped like a plow with a long stalk, and bifurcated twice at the distal tip, while the prothoracic horn is shorter than the head horn, and bifurcated once at the distal tip.During development, the horns are first formed as thickened epidermal primordia at the prepupal stage.Male adults have exaggerated horns at the head and prothoracic regions, whereas females do not have these structures.However, females have a small head horn at the pupal stage (Fig 1B,S1 Fig).
Sexual dimorphism of horns can be first observed in horn primordia formed during the prepupal stage, and the horn primordia grow larger through cell growth before pupation [7,8].In males, the length of the pupal horn primordia after pupation is almost the same as that of an adult horn [9].However, the shape of a pupal horn is rounded, and slightly larger than an adult horn (Fig 1B,S1 Fig).During the pupal period, the horn primodium is transformed to become an sophisticated adult horn shape through a process known as "horn remodeling" during which programmed cell death sculpts specific regions of the horn primordium into the adult morphology [7,10,11].
In insects, orthologs of a transcription factor gene doublesex (dsx) play a pivotal role in sexual differentiation.These orthologs are involved in the formation of sexually dimorphic traits through the expression of sex-specific isoforms (dsxM and dsxF) [8,[12][13][14][15][16][17].In addition, tissue-specific expression of dsx is elevated in regions where sexually dimorphic structures are formed to induce their development [17][18][19][20][21].In horned beetles including T. dichotomus, the dsx orthologs also regulate sexually dimorphic horn formation during larval-pupal development.RNA interference (RNAi) targeting dsx results in intersexual phenotypes both in males and females [8,14].(c) The sagittal section of head.Head horn primordia were formed above the clypeus in the clypeolabrum (S1 Movie).(B) Morphological changes of head and thoracic horn primordia during larval-pupal development revealed by the time-lapse photography system.Both in males (the upper panels in the upper half) and in females (the upper panels in the lower half), horn primordia were formed in the clypeolabrum.In males, the frontal views are presented concerning the head horn primordia from 12 h APF to 36 h APF, and the anterodorsal views are presented concerning the head horn primordia from 48 h APF to 120 h APF.In females, the frontal views are presented demonstrated the head horn primordia from 12 h APF to 48 h APF, and the anterodorsal views are presented demonstrating the head horn primordia from 60 h APF to 120 h APF.The dorsal views are presented demonstrating thoracic horn primordia.APF, after pupal-chamber formation; HH, head horn primordia; TH, thoracic horn primordia; Orange arrowheads, the firstly bifurcated distal tips of a head horn; Green arrowheads, the secondly bifurcated distal tips of a head horn; Blue arrowheads, the long column shaped stalk of a head horn; Purple arrowheads, the bifurcated distal tips of a thoracic horn.Scale bars are 5 mm.(C) Snapshot images from time-lapse photography (S2 Movie).The panel on the left is a single raw image of time-lapse photography observing 64 larvae bottled in 100 ml The molecular mechanisms of sex-specific splicing of dsx has been intensely studied in Drosophila melanogaster [12,22].In D. melanogaster, Sex-lethal (Sxl), the master sex determination gene, initiates the sex determination cascade.Sxl encodes an RNA-binding protein that directly binds to target RNAs.Functional Sxl protein is translated only in females [23][24][25], and controls sex-specific splicing of transformer (tra) to produce functional Tra protein in females [26].This TRA molecule then forms a heterodimer with a ubiquitously expressed RNA-binding protein, Transformer2 (Tra2), to regulate the sex-specific alternative splicing of dsx by directly binding to dsx transcripts in females [27,28].The resultant dsxF isoform in females and dsxM isoform in males regulate a battery of downstream genes to form sexually dimorphic traits by binding to the target DNA sequences with the Dsx binding motif [29,30], and by activating or repressing their transcription [31].intersex (ix) functions as a female-specific co-activator by directly binding to female-specific isoforms of the DsxF protein, and regulates development of female-specific traits [32].
The Sxl-[tra/tra2]-[dsxF/ix] pathway described above is only activated in females.In males, default mRNA splicing results in expression of the male-specific splicing variant of dsx, dsxM [12,22].Loss of function of the sex determination genes in females, and gain of function in males result in sex transformation [12,22].
Orthologous genes corresponding to the D. melanogaster sex determination genes described above are conserved among many holometabolous insects.In addition, the function of splicing regulatory factors (Tra/Tra2) and a transcription co-factor (Ix) that are supposed to directly interact with dsx transcripts or Dsx protein are conserved in many holometabolous insects (e.g.Diptera: Drosophila melanogaster [33], the housefly Musca domestica [34], the Mediterranean fruit fly Ceratitis capitata [35], the Australian sheep blowfly Lucilia cuprina [36], the oriental fruit fly Bactrocera dorsalis [37], Hymenoptera: the honeybee Apis mellifera [38], the parasitic wasp Nasonia vitripennis [39], Coleoptera: the red flour beetle, Tribolium castaneum [40,41]).However, whether these genes are involved in the sex determination pathways to form sexually dimorphic horns in T. dichotomus remains elusive.In addition, when and how dsx interacts with the gene regulatory network (GRN) for horn formation to drive cellular activities such as cell growth, cell death and cell movement is also unknown.
To understand the developmental and genetic mechanisms underlying sexually dimorphic horn formation in T. dichotomus, we first describe a precise time course of the morphogenetic changes of male and female horn primordia during larval-pupal development using time-lapse photography.Next, we examined the function of putative sex determination genes in T. dichotomus using larval RNAi by focusing on orthologs of known D. melanogaster sex determination genes.Moreover, we investigated the initiation timing of the GRN for horn formation by knocking down Tdic-tra at multiple developmental timepoints, and by evaluating the extent of sex transformation phenotypes.Based on these experiments, we concluded that the GRN for horn formation, which is supposed to be modified by dsxM and dsxF functions in males and females, is driven at a very early stage of larval-pupal development before clear morphological changes in horn primordia can be detected.Furthermore, we show that dsxM has different functions in both the prepupal and pupal stages during the formation of appropriate morphologies in pupal and adult horns in males.Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic horn formation in horned beetles.
plastic test tubes at the same time.The three panels on the right side are the magnified images of T. dichotomus at different developmental stages.(D) The durations of the prepupal periods in males and females estimated using the time-lapse photography system.Median, the upper/lower quantile, and minimum/ maximum values were presented by a box-and whisker-plot.The median duration of prepupal periods in males and females were 131 hours and 129 hours, respectively.The mean duration of prepupal period in Tdic-tra RNAi females was 127 hours, which was slightly shorter (4 hours) than that of normally developed females.https://doi.org/10.1371/journal.pgen.1008063.g001

Development of sexual dimorphism in T. dichotomus horn primordia
To identify the developmental timepoint when sexual dimorphism of horns first appears in T. dichotomus, we described morphological changes of head and thoracic horn primordia during the prepupal period.Micro-CT analysis of a head horn primordium at an early stage (24 hours after pupal-chamber formation; 24 h APF) in the prepupa revealed that a head horn primordium was formed in the clypeolabral region during the prepupal stage as investigated in Onthophagus taurus, Onthophagus sagittarius and Onthophagus gazella ( Until recently, developmental staging of T. dichotomus prepupae had been difficult because they form pupation chambers and pupate underground.Using our time-lapse photography system, we found that the head-rocking behavior at the end of pupal-chamber formation can be an unambiguous marker for the initiation of the prepupal stage (S2 Movie).We could minimize the developmental deviation between individuals within 10 hours using this precise developmental marker (Fig 1C, S2 Movie).The average prepupal period was 5.5 ± 0.19 days (131 ± 4.7 hours) in males and 5.4 ± 0.17 days (129 ± 4.3 hours) in females (Fig 1D).Based on this staging paradigm, we manually dissected out horn primordia every 12 h after pupal-chamber formation (APF).We found that sexual dimorphism of horn primordia appeared at 36 h APF (Fig 1B).Therefore, we concluded that the GRN driving the formation of horn sexual dimorphism would be activated before 36 h APF in T. dichotomus.In addition, we also found that apolysis occurring at 36 h APF can be another unambiguous developmental marker.Larval mandibular tendons that tightly connect mandibular muscles and apodemes (S3 Movie) [44][45][46] were completely detached at 36 h APF.This feature, along with the apolysis occurring at every body part including the neighboring ocular region also allowed us to know the timing of the onset of sexual dimorphism in these beetles.

Identification of genes regulating the sex-specific splicing of Tdic-dsx during T. dichotomus horn formation
In T. dichotomus, the regulatory factors associated with sex-specific splicing of dsx had not been identified.We searched for such regulatory factors focusing on T. dichotomus orthologs of known D. melanogaster sex determination genes (Sxl, tra, tra2 and ix) [12,22].
First, we investigated whether these genes produce sex-specific splicing variants in T. dichotomus by RT-PCR.All of these genes were expressed in male and female prepupal head and thoracic horns (  [47,48].Among these genes, sex-specific splicing variants were detected only in Tdic-tra (Fig 2B).Next, to test whether these genes function as sex determination genes, we performed larval RNAi experiments using dsRNA targeting the common regions between sexes (Fig 2A, black bars, S1 Table, S2 Table ).
In females, morphological changes was observed in the RNAi treatments targeting Tdic-tra and Tdic-ix, whereas no morphological changes were observed in males (Fig 3A,S2 Fig).Such female-specific phenotypes were comparable with the mutant phenotypes of tra, tra2 and ix in D. melanogaster [12,22].Concerning Tdic-tra2, we could not observe adult phenotypes due to the prepupal lethality of RNAi injection in both sexes.The phenotypes in Tdic-tra RNAi females and Tdic-ix RNAi females were different and the Tdic-ix RNAi phenotype was similar to the effects of the Tdic-dsx RNAi phenotype (Fig 3A).In the Tdic-tra RNAi females, ectopic horn formation was observed in both the head and prothorax (Fig 3A ), whereas in Tdic-ix RNAi females, ectopic horns were formed only in the head, and these horns were significantly shorter than the ectopic head horns in Tdic-tra RNAi females (Fig 3A and 3B).Such a difference in morphology was also observed in another sexually dimorphic structure, the intercoxal process of the prosternum (IPP).Male IPPs are generally larger than female IPPs (Fig 3A).Although both the Tdic-tra and Tdic-ix RNAi females have larger IPPs than female controls (EGFP RNAi), Tdic-tra RNAi females have much larger IPPs than Tdic-ix RNAi females (Fig 3A).These results and the intermolecular interactions reported in D. melanogaster led us to predict that Tdic-tra may also regulate the sex-specific splicing of Tdic-dsx in T. dichotomus.
To test this, we investigated the splicing patterns of Tdic-dsx in the above RNAi-treated males and females.We designed PCR primer sets to amplify the region including the whole female specific exon, which is spliced out in males (Fig 2A, light green bars, S1 Table ).We found that the sex-specific splicing pattern observed in wild type (Fig 2B) was switched in Tdic-tra and Tdic-tra2 RNAi treatments, whereas splicing patterns were not changed by Tdic-Sxl and Tdicix RNAi treatments (Fig 3C).These data indicate that Tdic-tra and Tdic-tra2 regulate femalespecific splicing of Tdic-dsx in T. dichotomus.The switching of sex-specific splicing of Tdic-dsx and the resultant morphological sex transformation in Tdic-tra RNAi females implies that the sex-specific splicing regulation of Tdic-dsx by Tdic-tra is also conserved in T. dichotomus as in other holometabolous insects investigated so far [33][34][35][36][37][38][39][40][41].Taken together, we concluded that Tdic-tra functions as a sex determination gene during horn formation in T. dichotomus.

Estimation of the onset of the developmental program for sexually dimorphic horn formation using Tdic-tra RNAi
As sexual dimorphism of horn primordia first appeared at 36 h APF (Fig 1B ), we speculated that the onset of the developmental program for sexually dimorphic horn formation was initiated before 36 h APF.To estimate this timepoint more accurately, we performed Tdic-tra RNAi in females at multiple developmental timepoints during pupal chamber formation periods and prepupal periods, and evaluated the extent of sexual transformation in horns.
If the timing of Tdic-tra RNAi in females is early enough, the ectopic Tdic-dsxM would be expressed in female horn primordium from the onset of developmental program for sexual dimorphism formation, and full sexual transformation can be achieved.On the other hand, the later the timing of Tdic-tra RNAi treatment becomes, the more the initial phases of the male-specific horn formation program driven by ectopically expressed Tdic-dsxM are trimmed, and at the same time repressed by normally expressed dsxF.Therefore, by determining the latest RNAi injection timing when a full sexual transformation phenotype is observed, we can estimate the onset of the sexually dimorphic horn formation program mediated by the ectopic Tdic-dsxM expression.
In this experiment, fully matured female last instar larvae and unstaged female prepupae were injected with EGFP or Tdic-tra dsRNA (S3 Table ).We performed time-lapse photography until pupation using these larvae, and retrospectively estimated the exact timing of injection before pupation.In addition, we estimated how many hours after pupal chamber formation (APF) each injection had been performed by subtracting the duration between each injection timepoint and pupation timepoint from the mean duration of the prepupal period in Tdic-tra RNAi females (127 hours) (Fig 1D).Our data indicated that Tdic-tra RNAi females treated earlier than the timepoint of -7 h APF formed fully developed male horns (Fig 4A(i), magenta dot).In contrast, Tdic-tra RNAi females treated later than the timepoint at -3 h APF showed either no morphological changes or only modest sex transformation of head or horn, if any (Fig 4A (ii) and (iii), green dot).These data suggested that the estimated timepoint of the onset of the developmental program for sexually dimorphic horn formation is around -7 h APF.
Since this timepoint was estimated by means of RNAi, we speculated that there should be time lag between the timing of injection and the timing of the decrease in functional protein levels followed by the mRNA degradation.Thus, we also quantified expression dynamics of Tdic-tra mRNA after RNAi treatment by qRT-PCR.It was technically impossible to monitor the expression dynamics of Tdic-dsx at -7 h APF, which is estimated to be before pupal chamber formation (Fig 1D

The spatiotemporal expression pattern of Tdic-dsx in the head horn primordium
The expression level of Dsx protein is frequently upregulated region-specifically during development of sexually dimorphic traits in insects (e.g.Sex comb formation in Drosophila, mimetic wing morph formation in Papilio, and wing pheromone gland formation in Bicyclus), presumably to facilitate sexual dimorphism formation [18][19][20][21].To test whether Tdic-Dsx protein also exhibits region-specific upregulation in the horn primordium, we raised anti-Tdic-Dsx polyclonal antibodies, and performed immunohistochemistry at the onset of sexually dimorphic horn formation (36 h APF).As a result, Tdic-Dsx protein showed higher expression in the head primordial epidermis than in the surrounding head epidermis and was mainly localized in nuclei (Fig 5D-5F and 5D'-5F', S3 Fig) .In accordance with this result, mRNA expression level of Tdic-dsxM were also higher in the head horn primordial epidermis than in the surrounding head epidermis (Fig 5G and 5H).In contrast, Tdic-dsxF did not show significantly higher expression in the horn primordium at this stage (Fig 5G and 5H).Therefore, we concluded that expression level of Dsx protein is upregulated region-specifically to form sexually dimorphic horns during development.
Interestingly, we found that Tdic-Dsx showed higher expression in the head horn primordial epidermis even before the onset of sexually dimorphic horn formation (12 h APF) (Fig 5A-5C and 5A'-5C', S3 Fig), but the localization of expression was cytoplasmic.This finding suggests that the region-specific expression of Tdic-Dsx has been already initiated before it activates downstream genes, but its transcription factor activity is triggered only after it is translocated to nuclei.

Sexually dimorphic morphogenesis in male and female horn primordia
T. dichotomus adult males have exaggerated horns at the head and prothoracic regions whereas adult females only have three small protrusions at the clypeolabral region (Fig 6A, magenta arrowhead).We focused on whether the distal tips of the male head horn and three female head protrusions were formed in the same region or whether they originated from distinct regions in the head (Fig 6A ,  However, due to the lack of clear morphological landmarks indicating the formation region of male head horns and female head protrusions, within the head, the formation regions of these traits remained elusive.Unexpectedly, however, we obtained an intermediate sexual transformation phenotype of Tdic-tra RNAi to solve this problem.When we injected Tdic-tra dsRNA in small amounts (2.5 μg), we obtained several adults possessing both the three small protrusions similar to those in females, and a small ectopic anterior protrusion, seemingly analogous to a male head horn (Fig 6B and 6C).This result suggests that at least the anterior region of a male head horn is not formed from the same region as female head protrusions.
Next, we asked whether Tdic-dsx regulates the entire cellular activity during sexually dimorphic horn formation.We especially focused on the two distinct developmental processes, "horn growth" before pupation, and "horn remodeling" after pupation (see details in the Introduction section).The morphological changes before and after pupation are qualitatively comparable between males and females, but the extent of the morphological changes during development is different between them.During the prepupal stage, males form longer head and thoracic pupal horns, whereas females only form smaller pupal head horns.During the pupal stage, both male head and thoracic horns become slimmer, whereas a substantial portion of the female pupal head horn disappears to form small three protrusions (S1 Fig) .We tested whether Tdic-dsx regulates the above sexually dimorphic morphogenetic processes by injecting dsRNA targeting Tdic-dsx into male larvae at several developmental stages (S3 Table ).As previously reported, male head horns became shorter, and thoracic horns were not formed in adults when Tdic-dsx dsRNA was injected at sufficiently early timepoints [8].In these conditions, the sizes of the pupal head horns and the pupal thoracic horns were also smaller proportionally to that of shortened adult horns (-85 h APF) (Fig 7B ,

Discussion
Here, we have described the development of sexually dimorphic horns in a Japanese rhinoceros beetle, T. dichotomus, from both morphological and genetic points of view.We focused on Tdic-dsx as the major regulatory gene for sexually dimorphic horn formation and investigated its time of action through expression analysis and loss-of-function analysis of sex determination genes in multiple developmental timepoints during the horn morphogenesis period.To manipulate sex-specific alternative splicing of Tdic-dsx, we identified Tdic-tra as a key regulatory factor for the sex-specific splicing of Tdic-dsx.We discuss below the genetic regulatory framework of sex determination in T. dichotomus, the detailed actions of Tdic-dsx in males and females to form sexually dimorphic horns, and evolutionary background realizing the exaggerated horn formation in horned beetles.

Genetic regulatory mechanisms of sex determination in T. dichotomus
As mentioned in the Introduction section, the function of regulatory factors that are supposed to bind directly to dsx transcripts (Tra, Tra2) or Dsx protein (Ix) are conserved in many holometabolous insects.Our loss-of-function analysis data suggest that the genetic regulatory mechanisms of sex determination in T. dichotomus follow this framework.
The conserved function of Tdic-Tra and Tdic-Tra2 as splicing factors targeting Tdic-dsx seems to be conserved (Fig 3C).In addition, the biological functions of these genes are at least similar to those in other beetles because the RNAi phenotypes of tra and tra2 orthologs in T. dichotomus (i.e. the viable masculinized phenotype in Tdic-tra RNAi females, and the lethal phenotype in Tdic-tra2 RNAi females) were comparable with those reported in other beetles (T.castaneum; [40,41], the stag beetle Cyclommatus metallifer; [49]).
In D. melanogaster, Ix directly binds to DsxF in females but not to DsxM in males, and functions as a co-activator to facilitate transcription activity of target genes of DsxF [32].The intersexual phenotype observed only in Tdic-ix RNAi-treated females (Fig 3A ) implies that Tdic-Ix might also interact with Tdic-DsxF to regulate female-specific sex differentiation as in D. melanogaster.A female-specific intersexual transformation phenotype reported in the stag beetle C. metallifer [49] suggests that the female-specific function of ix may be conserved among Polyphaga as well.
In contrast, our RNAi experiments implied that Tdic-Sxl did not regulate the sex-specific alternative splicing of Tdic-tra in females (Fig 3), which is a direct regulatory target of Sxl in D. melanogaster females.Because the splicing of tra orthologs are not regulated by Sxl orthologs even in other Dipteran species [50,51], the sex-specific alternative splicing of tra may be regulated by unknown species-specific factors other than Tdic-Sxl.To summarize the regulatory mechanisms of T. dichotomus sex determination discussed above, functional Tdic-Tra is first expressed female-specifically through species-specific unknown mechanisms.Then, as in many other holometabolous insects, a Tdic-Tra/Tdic-Tra2 heterodimer is formed only in females and produces Tdic-dsxF mRNA and Tdic-DsxF to promote female differentiation by interacting with Tdic-Ix (Fig 8).On the other hand, in males, functional Tdic-Tra is not expressed, and default splicing of Tdic-dsx would result in Tdic-dsxM mRNA and Tdic-DsxM production to promote male differentiation (Fig 8).

The gene regulatory network driving horn dimorphism formation in T. dichotomus
A previous study revealed that Tdic-dsx RNAi in both males and females does not result in a total loss of horns, but results in intermediate-lengthened head horn formation and loss of a thoracic horn (Fig 3) [8].The head horn phenotype suggests that there exists a GRN for head horn formation that is operated independently of Tdic-dsx.Tdic-dsxM seems to enhance this GRN whereas Tdic-dsxF seems to suppress it.On the other hand, the thoracic horn phenotypes suggest that thoracic horn formation seems to be totally dependent on Tdic-dsxM function, and that action of Tdic-dsxM on horn formation might be different between head and thoracic horns during prepupa and/or pupa.However, in the course of our Tdic-dsx RNAi experiments, we noticed that thoraces of severely intersexually transformed individuals are always slightly bulged at the thoracic horn formation region (Fig 3A).This implies that Tdic-dsxF expression in the female thorax still has suppressive activity against the GRN for thoracic horn formation.If this is the case, the regulatory relation between Tdic-dsx and GRN for horn formation has partial similarity between the head and the thorax.The phenotypic difference previously described in head and thoracic horns would be due to difference in length of horn formed in each body region.In the sections below, we mainly discuss the GRN for horn formation based on our head horn data in which interpretation of phenotypes is less ambiguous.
Our knockdown experiment of Tdic-tra revealed that the onset of Tdic-dsx modulating GRN for head horn formation is as early as 29 h APF ( Therefore, Tdic-dsx seems to modulate head horn primordium formation just before the onset of tissue growth, and following tissue morphogenesis during prepupal stages.Several genes involved in the GRN for horn formation have been identified in horned beetle species, primarily by focusing on Drosophila appendage patterning genes [52][53][54].These studies propose an attractive evolutionary model in which large portions of the GRN for proximodistal appendage patterning were recruited to acquire beetle horns in the head and thoracic regions.Still, the overall framework of GRN for horn formation including the key regulatory gene sets involved, spatiotemporal expression dynamics, and regulatory relation among those genes, has not been unveiled so far.Therefore, investigating regulatory relations between appendage patterning genes and other head/thoracic patterning genes expressed in horn primordia focusing on this developmental timepoint will lead to verification of this model in the future studies.In addition, because candidates of the dsx target genes involved in GRN for horn formation were identified recently in a horned beetle, O. taurus [55], functional analyses focusing on orthologs of those genes expressed in Trypoxylus horn primordia at this timepoint will also lead to understanding of conserved and divergent aspects of sexually dimorphic horn formation in horned beetles. Region-specific upregulation of dsx is another essential feature to drive GRN for sexual dimorphism formation in insects [17][18][19][20][21]. Intense studies focusing on regulatory mechanisms of sex comb formation in D. melanogaster revealed that such region-specific upregulation of dsx is mediated by a positive feedback loop between dsx and a Hox gene, Sex combs reduced [18].Therefore, region-specific upregulation of Tdic-dsx observed in a head horn primordium might reflect an analogous positive feedback loop mechanism (Fig 5).Future research focusing on the patterning mechanisms of beetle horns and its interaction with dsx will be an important issue to discuss conserved genetic framework for sexual dimorphism formation.Moreover, we found nuclear translocation of Tdic-Dsx at the onset of male head horn formation ( Fig 5,S3 Fig).As far as we know, such a mode of regulation against Dsx during development is not reported in other insects.Understanding the molecular mechanisms underlying this phenomenon will be another direction to take in the future studies.

Tdic-dsx-mediated sexually dimorphic morphogenesis in head and thoracic horn formation during larval-pupal development
As previously reported, Tdic-dsx RNAi from early stages resulted in short head horn formation in males and females (Fig 3A) [8].These data indicate that during the prepupal stage Tdic-dsxM promotes the growth of head and thoracic horn primordium, whereas Tdic-dsxF suppresses growth of head and thoracic horn primordium (Fig 8, prepupal stage) [8].On the other hand, RNAi treatments in later stages revealed distinct functions of Tdic-dsx in sexually dimorphic horn formation.During pupal-adult development, Tdic-dsxM regulates remodeling of a thoracic horn primordium from a rounded shape to a slender hooked shape ( These results indicate that Tdic-dsxM and Tdic-dsxF regulate different aspects of morphogenesis at both prepupal and pupal stages.Furthermore, to what extent Tdic-dsx is required during larval-pupal development is also finely regulated during male head horn formation, male thoracic horn formation, female head protrusion formation, and female flat prothorax formation.Importantly, in contrast to the previous study that suggested that functions of Tdic-dsxM and Tdic-dsxF in T. dichotomus head and thoracic horn formation are to some extent analogous as described in the previous section (that is, in both of the head and the thoracic horn formation, Tdic-dsxM functions as a positive regulator whereas Tdic-dsxF functions as a negative regulator) [8], our experiments clarified that both spatial cues (i.e.different developmental contexts in head and prothorax) and temporal cues (i.e.different developmental contexts in prepupa and pupa) modulate the actions of Tdic-dsxM and Tdic-dsxF to drive appropriate morphogenetic activity in each horn primordium at each developmental stage.Since spatiotemporal modularity of gene function is often coded in modular regulatory elements in the genome (reviewed in [56]; e.g.[57][58][59][60]), the regulatory elements of dsx that integrates the above distinct spatiotemporal cues could be coded at the Tdic-dsx locus in the genome.Identification of such regulatory elements will be an important issue to elucidate evolution of sexually dimorphic horn formation in the future studies.
A notable feature of Tdic-dsx during sexually dimorphic development is that the onset of action during horn development seems to be categorized earlier (i.e. during the prepupal period) than that of many other sexually dimorphic adult traits in holometabolous insects reported so far (i.e. the pupal period).In D. melanogaster sex comb formation, Papilio mimetic wing morph formation, and Bicyclus wing pheromone gland formation, tissue-specific high expression of dsx orthologs are detected after pupation [18][19][20][21].Such a difference in dsx's onset of action seems to be due to the requirement of drastic growth during sexually dimorphic structure formation.In either case mentioned above, the finally formed structure accompanies little to no tissue-level drastic growth during development.On the other hand, as in T. dichotomus horn formation, D. melanogaster genital organ formation, and C. metallifer stag beetle mandible formation, whose sexually dimorphic morphogenesis is regulated by dsx, accompanies drastic sexually dimorphic growth during larval-pupal development [17,[61][62][63], and its onset of action is as early as the prepupal stage.Association between dsx's earlier onset of action and requirement of drastic growth during sexual dimorphism formation suggest that recruitment of the dsx function in the earlier stage may be one of the prerequisites to form structurally drastically different sexual dimorphism in insects.
The intermediate head horn formation via Tdic-tra RNAi in females (Fig 6A -6C) revealed that the anterior region of male head horn and female small protrusions are formed from different regions in the clypeolabrum.Such a distinct developmental origin of head horn within the clypeolabrum is also clearly demonstrated in Onthophagus species of dung beetles [3], in which the dsx orthologs regulate the formation of distinct sexually dimorphic horns in either the anterior or the posterior region of the head.Analogy of multiple horn formation regions within the clypeolabrum in distinct horned beetle species implies that clypeolabrum is a hotspot of morphological innovations in horned beetles.A comprehensive understanding of the GRN for horn formation in both Trypoxylus and Onthophagus and comparative developmental studies in the future will lead to understanding of molecular mechanisms underlying the evolutionary origin and evolvability of exaggerated horns in beetles.
Here we described the accurate developmental time course of horn primordial morphogenesis during the prepupal stage in the horned beetle T. dichotomus using a time-lapse photography system.In addition, we functionally characterized both Tdic-tra and Tdic-tra2, genes that regulate the sex-specific splicing of Tdic-dsx.By manipulating expression levels of Tdic-tra and Tdic-dsx during different developmental time points, and by quantifying the extent of sex transformation, we revealed the following three crucial features of Tdic-dsx function during the development of sexually dimorphic horn formation: (1) Tdic-dsx modulates the GRN for horn formation as early as 29 h APF, a timepoint which corresponds to 7 h before sexual dimorphisms of horn primordia first appears; (2) Tdic-dsx regulates different aspects of the tissue growth, tissue death and tissue movement of horn primordia depending on both spatial (head/prothorax) and temporal (prepupa/pupa) contexts; (3) Tdic-dsxM and Tdic-dsxF promotes the formation of outgrowth structure in distinct regions within the clypeolabrum.These findings inform our understanding of the patterning mechanisms at play during T. dichotomus horn formation, as well as provide information regarding regulatory shifts in these mechanisms during the evolution of sexually dimorphic traits in horned beetles.The present study provides a good starting point to elucidate such issues.

Insects
We purchased T. dichotomus larvae from Loiinne (Japan), and Urakiso Tennen Kabuto no Sato (Japan).The last instar larvae were sexed as described previously [8], individually fed on humus in plastic containers, and kept at 10˚C until use.Larvae were moved to room temperature at least 10 days, and reared at 28˚C.

Micro-CT analysis
A male head tissue at 24 hours after pupal-chamber formation (24 h APF) was fixed in Carnoy solution at room temperature overnight, washed in 70% ethanol and stored in 70% ethanol.The sample was rehydrated through a graded ethanol series, and stained with 25% Lugol solution [64][65][66] for 5 days.The stained sample was scanned using an X-ray micro-CT device (ScanXmate-E090S105, Comscantechno Co., Ltd., Japan) at a tube voltage peak of 60 kVp and a tube current of 100 μA.The sample was rotated 360 degrees in steps of 0.24 degrees, generating 1500 projection images of 992 × 992 pixels.The micro-CT data were reconstructed at an isotropic resolution of 13.3 × 13.3 × 13.3 μm, and converted into an 8-bit tiff image dataset using coneC-Texpress software (Comscantechno Co., Ltd., Japan).Three-dimensional tomographic images were obtained using the OsiriX MD software (version 9.0, Pixmeo, SARL, Switzerland) and Imaris software (version 9.1, Carl Zeiss Microscopy Co., Ltd., Japan).Supplemental videos were edited using Adobe Premiere Pro CC (Adobe Systems Co., Ltd., Japan).sections.Tiled array images were obtained using a confocal microscopes equipped with a motorized stage (Olympus 3D mosaic imaging system).apodeme at 24 h APF.Apolysis was incomplete in the ocular and the mandibular apodeme at 24 h APF [44][45][46].After this stage, prepupal horn primordia can be readily dissected out due to apolysis at the ocular and the mandibular apodeme (Fig 1B).This morphological character is unambiguous developmental marker to know the onset of sexual dimorphism formation (36 h APF).(MP4)

Fig 1 .
Fig 1. Morphological change of horn primordia and the prepupal period in Trypoxylus dichotomus.(A) (a) Schematic of the frontal view of larval head from T. dichotomus.Green, ocular (OC); Magenta, clypeolabrum; CL, clypeus; LB, labrum.(b), (c) Micro-CT images of prepupal head at 24 h APF from T. dichotomus.(b) The frontal view.Green arrowheads indicate the position of a sagittal section in (c).(c) The sagittal section of head.Head horn primordia were formed above the clypeus in the clypeolabrum (S1 Movie).(B) Morphological changes of head and thoracic horn primordia during larval-pupal development revealed by the time-lapse photography system.Both in males (the upper panels in the upper half) and in females (the upper panels in the lower half), horn primordia were formed in the clypeolabrum.In males, the frontal views are presented concerning the head horn primordia from 12 h APF to 36 h APF, and the anterodorsal views are presented concerning the head horn primordia from 48 h APF to 120 h APF.In females, the frontal views are presented demonstrated the head horn primordia from 12 h APF to 48 h APF, and the anterodorsal views are presented demonstrating the head horn primordia from 60 h APF to 120 h APF.The dorsal views are presented demonstrating thoracic horn primordia.APF, after pupal-chamber formation; HH, head horn primordia; TH, thoracic horn primordia; Orange arrowheads, the firstly bifurcated distal tips of a head horn; Green arrowheads, the secondly bifurcated distal tips of a head horn; Blue arrowheads, the long column shaped stalk of a head horn; Purple arrowheads, the bifurcated distal tips of a thoracic horn.Scale bars are 5 mm.(C) Snapshot images from time-lapse photography (S2 Movie).The panel on the left is a single raw image of time-lapse photography observing 64 larvae bottled in 100 ml Fig 1A and 1B, S1 Movie) [3, 42-44].In addition, head horn primordia were formed above the clypeus in the clypeolabrum (Fig 1A, S1 Movie).To determine the exact timepoint when protrusion of the primordium is initiated, we established a time-lapse photography system (Fig 1C, S2 Movie).

Fig 2 .
Fig 2. Schematic representation of putative exon-intron structures and sex-specific splicing patterns of the sex determination genes.(A) Schematics of putative exon-intron structures of the sex determination genes in T. dichotomus.Orange box, RRM (RNA binding motif); Green box, CAM (C, Ceratitis; A, Apis; M, Musca) domain [34, 47], which is conserved in all tra orthologs identified in other insects except for those in Drosophila species and the sandfly (Phlebotomus papatasi) [48]; Light blue box, Med29 (mediator complex subunit 29) domain; Brown box, DM (Doublesex/Mab-3 DNA-binding) domain; Yellow box, OD2 (Oligomerization dmain 2); Black bars, template sequences for dsRNA synthesis; Green bars, the amplified regions in qRT-PCR analysis; Light green bars, the amplified region to test sex-specific splicing variants; Magenta lines, translation start sites; Purple lines, stop codons.(B) Evaluation of expression and sex-specific splicing patterns in the sex determination genes in male and female prepupal head horn primordia.Template cDNAs were derived from prepupal horn primordia at 72 h APF.Tdic-RpL32 was used as an internal control for RT-PCR.Blue arrows, PCR products with male specific splicing variants; Magenta arrows, PCR products with female specific splicing variants; Black arrows, PCR products amplified in both sexes.https://doi.org/10.1371/journal.pgen.1008063.g002

Fig 3 .
Fig 3. RNAi-mediated loss-of-function phenotypes of the sex determination genes.(A) Representative individuals in each RNAi treatment in males (the left half) and females (the right half).Each dsRNA was injected into last-instar larvae.The negative control RNAi treatment (EGFP dsRNA) showed no morphological defects.The upper row, the dorsal views of adults; the second row, the lateral views of a head and a prothorax in adults; the third row, the ventral views of adults; the forth row, magnified views of orange squares (IPP, intercoxal process of prosternum) in the third row.Scale bars are 1 cm in the upper three rows and 1 mm in the fourth row.(B) Quantification of the relative head and thoracic horn length in RNAi-treated individuals.Relative head and thoracic horn length in RNAi-treated males and females are plotted in the blue dots and the magenta dots, respectively.The relative horn lengths were standardized by dividing the horn length by the body size in each RNAi treated individual and by the mean horn length of the EGFP RNAi-treated males.Differences in horn length among individual RNAi treatments were compared using Brunner-Munzel test.These p-values were adjusted by the Bonferroni correction.Asterisks denote significance: n.s., p > 0.05; � , p < 0.05.(C) Sex-specific splicing of Tdic-dsx in RNAi treatments targeting Tdic-Sxl, Tdic-tra, Tdic-tra2 and Tdic-ix.Blue arrowheads, male specific splicing patterns.Magenta arrowheads, female specific splicing patterns.Tdic-RpL32 was used as an internal control for RT-PCR.https://doi.org/10.1371/journal.pgen.1008063.g003 Fig 4A).Then, we monitored the expression dynamics as early as possible (dsRNA injection at 24 h APF), instead.The expression levels of Tdic-tra and Tdic-dsxF were quantified every 12 hours up to 36 hours after injection (Fig 2A, green bars, Fig 4B).As a result, the expression levels of Tdic-tra and Tdic-dsxF were decreased to less than half of that of the negative control at 36 hours after injection (Fig 4B).Since mRNA started to be degraded 36 hours after RNAi treatments (Fig 4B), we estimated that this timepoint corresponded to 29 h APF (−7 plus 36) (Fig 4C).Taking morphological data into account, this timepoint

Fig 4 .
Fig 4. Estimation of the onset of the Tdic-dsx dependent horn developmental program by Tdic-tra RNAi.(A) Relationship between the timepoints of Tdic-tra dsRNA injection and the extent of sex transformation.The extent of the sex transformation (masculinization) phenotypes of Tdic-tra RNAi females were categorized into three classes: (i) prominent HH and TH were formed (< -7 h APF); (ii) only small HH was formed (5 h APF ~73 h APF); (iii) neither the HH nor TH was formed as in normal females (-3 h APF ~127h APF).HH, head horn primordia; TH, thoracic horn primordia.The dot plot panel was timepoints of Tdic-tra dsRNA injection before pupation in each class.The green dot, the magenta dot, and the blue dot indicate -3 h APF, -7 h APF and 73 h APF, respectively.Scale bars are 5 mm.(B) Time course of relative Tdic-tra and Tdic-dsxF mRNA expression level after Tdic-tra dsRNA injection at 24 h APF. mRNA expression levels at each timepoint were quantified by qRT-PCR.The expression levels of Tdic-tra were significantly decreased at 36 hours after injection (p<0.015).The expression level of Tdic-dsxF tended to be decreasing as time went on, but there were no statistically significant difference (p<0.056).(C) Summary of sex transformation phenotypes and the estimated onset of the Tdic-dsx dependent horn developmental program.The magenta line is the boundary timepoints of phenotype (i).Red arrowheads indicate the timepoints corrected by the mRNA degradation delay (36 hours), which estimated by qRT-PCR.APF, after puapal-chamber formation; Red arrowhead, the estimated onset of the developmental program for sexually dimorphic horn formation driven by Tdic-dsx.https://doi.org/10.1371/journal.pgen.1008063.g004 S1 Fig).During prepupal stages, a head horn primordium in both males and females seemed to be formed in the same region (the almost entire clypeolabral region in the head) (Fig 1, S1 Fig).
S4 Fig).On the other hand, males treated with Tdic-dsx dsRNA at later stages (13 h APF) formed thickened adult thoracic horns similar to a pupal thoracic horn before remodeling (Fig 7C)and the head horn was comparable with the wild type head horn (S4 Fig).These data indicate that DsxM is required for horn remodeling only in the thorax, and is dispensable in the head horn remodeling.

Fig 5 .
Fig 5. Spatial and temporal expression of Tdic-Dsx protein in the head horn primordium during larval-pupal development.(A-F) The head epidermis including the head horn primordium was stained with DAPI (magenta) to label nuclei and with anti-Tdic-Dsx antibody (green) to label Tdic-Dsx protein.Tdic-Dsx protein showed higher expression in the head primordial epidermis than in the surrounding head epidermis.Between Orange-Orange arrowheads is the head horn primordium.Between Orange-Blue arrowheads is the surrounding head epidermis.(A-C) Tdic-Dsx expression pattern in 12 h APF, Scale bars are 500 μm.(A'-C') A higher magnification of the head horn primordium, respectively.Tdic-Dsx protein localization was cytoplasmic.Scale bars are 50 μm.(D-F) Tdic-Dsx expression pattern in 36 h APF, Scale bars are 500 μm.(D'-F') A higher magnification of the head horn primordium, respectively.Tdic-Dsx protein localization were nuclei.Scale bars are 50 μm.(G) The epidermal regions used for qRT-PCR.The 3D volume image was reconstructed from sequential micro CT images.Blue, head horn primordium (HH); Purple, the head epidermis (non-HH).(H) The relative Tdic-dsx expression level in head horn primordia and head epidermis at 36 h APF in both sexes.Asterisks denote significance: n.s., p > 0.05; � , p < 0.05.HH, head horn primordium epidermis; non-HH, the head epidermis.https://doi.org/10.1371/journal.pgen.1008063.g005

Fig 6 .
Fig 6.Horn formation phenotypes induced by Tdic-tra RNAi at late prepupal stages.(A-C) Comparison of wild type female head and ectopic intermediate sexual transformation head horn in female heads induced by Tdic-tra RNAi treatments.(A) A wild type female head.(B) Small ectopic head horn formation in a female head.(C) Middle-sized ectopic head horn formation in a female head.Three small protrusions are formed in clypeolabrum on the wild type female head (A-C, magenta arrowheads), whereas ectopic small-or middle-sized ectopic head horns were formed in the region anterior to the three small protrusions (B, C, green arrowheads) in Tdic-tra RNAi treatments.Scale bars are 5 mm.https://doi.org/10.1371/journal.pgen.1008063.g006

Fig 8 .
Fig 8.A regulatory model for the formation of sexually dimorphic horns in T. dichotomus.In males, default splicing generates Tdic-dsxM, whereas in females, Tdic-tra (a female isoform, traF) and Tdic-tra2, generates Tdic-dsxF by female-specific splicing of Tdic-dsx pre-mRNA.Prepupal Tdic-dsxM expression in males positively regulates the growth of head and thoracic horn primordia, whereas prepupal Tdic-dsxF in females suppresses the horn growth activity.In pupa, Tdic-dsxM regulates thoracic horn remodeling, but is dispensable for remodeling of head horn.GRN, gene regulatory network.https://doi.org/10.1371/journal.pgen.1008063.g008 Fig 4).This is approximately 7 hour before the appearance of sexual dimorphism of head horn primordium (Fig 1B).Our qRT-PCR and immunohistochemistry analysis focusing on Tdic-Dsx were consistent with this estimation: transcription and translation of dsx was initiated before 0 h APF, but nuclear translocation of Dsx was initiated betweeen 12 h and 36 h APF (Fig 5, S3 Fig, S5 Fig).