Sex-specific expression profiles of ecdysteroid biosynthesis and ecdysone response genes in extreme sexual dimorphism of the mealybug Planococcus kraunhiae (Kuwana).

Insect molting hormone (ecdysteroids) and juvenile hormone regulate molting and metamorphic events in a variety of insect species. Mealybugs undergo sexually dimorphic metamorphosis: males develop into winged adults through non-feeding, pupa-like stages called prepupa and pupa, while females emerge as neotenic wingless adults. We previously demonstrated, in the Japanese mealybug Planococcus kraunhiae (Kuwana), that the juvenile hormone titer is higher in males than in females at the end of the juvenile stage, which suggests that juvenile hormone may regulate male-specific adult morphogenesis. Here, we examined the involvement of ecdysteroids in sexually dimorphic metamorphosis. To estimate ecdysteroid titers, quantitative RT-PCR analyses of four Halloween genes encoding for cytochrome P450 monooxygenases in ecdysteroid biosynthesis, i.e., spook, disembodied, shadow and shade, were performed. Overall, their expression levels peaked before each nymphal molt. Transcript levels of spook, disembodied and shadow, genes that catalyze the steps in ecdysteroid biosynthesis in the prothoracic gland, were higher in males from the middle of the second nymphal instar to adult emergence. In contrast, the expression of shade, which was reported to be involved in the conversion of ecdysone into 20-hydroxyecdysone in peripheral tissues, was similar between males and females. These results suggest that ecdysteroid biosynthesis in the prothoracic gland is more active in males than in females, although the final conversion into 20-hydroxyecdysone occurs at similar levels in both sexes. Moreover, expression profiles of ecdysone response genes, ecdysone receptor and ecdysone-induced protein 75B, were also analyzed. Based on these expression profiles, we propose that the changes in ecdysteroid titer differ between males and females, and that high ecdysteroid titer is essential for directing male adult development.

Introduction

Arthropods (insects, arachnids, and crustaceans) and nematodes belong to Ecdysozoa, a superphylum characterized by molting events–developing an exoskeleton that sheds during their development or lifetime. Among them, insects later acquired the ability to undergo metamorphosis which, combined to molting, this allowed insects to evolve highly diverse phenotypes and enabled them to conquer a wide range of habitats, contributing to their extraordinary diversity today [1]. Insect molting and metamorphosis are strictly regulated by two major hormones, juvenile hormone (JH) and ecdysteroids [2-4]. The titers of these hormones fluctuate throughout development, and work in concert to both determine the next state of developmental stage and molting event timing. As such, ecdysteroid pulses accompanied with high JH titer usually induce status quo molts, whereas ecdysteroid pulses with low JH titer induce metamorphic molts [5]. Thus, ecdysteroids are essential to trigger the successive molts throughout insect post-embryonic development [6].

The ecdysteroid biosynthetic pathway consists of a chain of enzymatic reactions. First, cholesterol from dietary sources is converted to 7-dehydrocholesterol (7dC) by a Rieske oxygenase Neverland [7, 8]. 7dC is then converted into 3β,14α-dihydroxy-5β-cholest-7-en-6-one (5β-ketodiol) through several steps. The detailed pathway from 7dC to 5β-ketodiol has not been clarified yet, hence named the “Black Box” [9-12]. To date, Spook (Spo, CYP307A1), Spookier (Spok, CYP307A2), CYP6T3, and Non-molting glossy/Shroud (Nm-g/Sro) were shown to be involved in the “Black Box” reactions [13-17]. Among the ecdysteroid biosynthetic genes, sro encodes a short-chain dehydrogenase/reductase, whereas spo, spok, and cyp6T3 encode cytochrome P450 monooxygenases. spo and spok are paralogous gene copies, and have different functions in the fruit fly Drosophila melanogaster [13, 16]. Another paralogous gene, spookiest (spot, Cyp307b1), was identified in Coleoptera, Hymenoptera, and Diptera species other than Drosophilidae [16, 18]. The reaction from 5β-ketodiol to 2,22-dideoxyecdysone (5β-ketotriol) is catalyzed by Phantom (Phm, CYP306A1) [19, 20]. 5β-Ketotriol is converted into 2-deoxyecdysone by Disembodied (Dib, CYP302A1) [21, 22], and finally Shadow (Sad, CYP315A1) converts it into ecdysone [22]. Ecdysone is released from the prothoracic gland into the hemolymph and metabolized to 20-hydroxyecdysone (20E) by the ecdysone 20-monooxygenase Shade (Shd, CYP314A1) in peripheral tissues such as fat body, midgut, and Malpighian tubules [23].

The function of these ecdysteroidogenic enzymes was first studied in D. melanogaster: loss-of-function mutations of spo, sro, phm, dib, sad, or shd resulted in embryonic lethality, and a phenotype with naked and polished cuticles was observed [21]. Thus, these genes were named “Halloween genes” [24, 25]. Expression analysis of some of the Halloween genes in the silkworm Bombyx mori revealed that their developmental expression profiles were positively correlated with hemolymph ecdysteroid titer [15, 19, 26].

Mealybugs (Hemiptera: Pseudococcidae) are characterized by remarkable sexual dimorphism, as a result of unusual diverging post-embryonic development. In Japanese mealybug, Planococcus kraunhiae (Kuwana), males and females are phenotypically undistinguishable until the middle of the second nymphal instar (N2). After this stage, males metamorphose through non-feeding quiescent stages called prepupa (Pre) and pupa (Pu) into winged adults, whereas N2 females develop through successive molting events to the adult stage, retaining the wingless, juvenile features of nymphal instars. Hormonal regulation has been reported to be involved in sex-specific morphogenesis in some insects. For example, horn length in the dung beetle Onthophagus taurus is regulated by JH [27, 28], while the sex-specific mandible growth in the stag beetle Cyclommatus metallifer is affected by JH [29]. Moreover, sex-specific wing patterns in the butterfly Bicyclus anynana are controlled by diverging 20E titers [30]. Finally, our previous study suggests that the JH titer in P. kraunhiae is lower in females than in males, so JH is likely to be involved in establishing sexual dimorphism in mealybugs [31]. We further showed that the adult specifying transcription factor E93, which is involved in in hormonal signaling pathways, is only expressed at the end of male adult development [32]. Since their titers have not been measured in P. kraunhiae, the involvement of ecdysteroids in sexual dimorphism remains unknown. In order to better understand the role of ecdysteroids in mealybug sex-specific post-embryonic development, measuring their titer is a critical step.

In this study, we examined the ecdysteroid titers in P. kraunhiae life cycle, with a focus on post-embryonic development. We initially attempted to measure the direct titers of ecdysteroids in pooled nymphs (approximately 200 individuals; ca. 10 mg in total) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Detection of ecdysteroids was unsuccessful probably because of their small body size (Muramatsu et al., unpublished). We therefore estimated ecdysteroid titers by analyzing the expression profiles of the Halloween genes using quantitative RT-PCR. To further validate the estimated ecdysteroid titers, the expression profiles of ecdysone response genes, ecdysone receptor (EcR), and ecdysone-induced protein 75B (E75), were also measured. Our results suggest that ecdysteroid titer fluctuates in a sex-specific manner.

Materials and methods

Insect rearing conditions

The P. kraunhiae mealybugs were reared at 23°C (16L8D) on sprouted broad beans (Kokusai Pet Food, Kobe, Japan) as described in a previous study [31]. In these conditions, from egg oviposition to adult emergence, development times were approximately as follows: 9–11 days for the embryonic stage (E) after oviposition, 11 days for the first-instar nymphs (N1), 4 days for the phenotypically undifferentiated second-instar nymphs (N2), 3–4 days for the differentiated female second-instar nymphs (N2♀), 5 days for differentiated second-instar nymphs (N2♂), 9 days for the female third-instar nymphs (N3), 4 days for the male prepupae (Pre), and 5–6 days for the male pupae (Pu).

Sex ratio estimation and collection strategy of staged individuals

Males and females of P. kraunhiae from E to N2D3 are not distinguishable from their external morphology. However, because the sex ratio of the eggs that a female lays depends on oviposition time, sex-biased eggs can be collected at different oviposition days, as previously reported [31, 33]. Samples from E to N2D3 for quantitative RT-PCR were therefore collected using the sex-biased strategy as follows: eggs laid on day 1 of oviposition were collected as male-biased samples, and eggs laid on day 5 of oviposition were collected as female-biased samples. In order to obtain staged nymphs, mated adult females were separated in glass dishes containing a sprouted broad bean, and the eggs were collected every 24 hours and monitored for development in separate glass dishes until they attained the desired stage. Staged pooled individuals were then homogenized in TRIzol reagent (Thermo Fisher Scientific Inc., MA, USA) for RNA extraction. In order to confirm the sex ratio, some individuals were left to develop until N3 or Pre stages for observation.

cDNA cloning of Halloween genes and ecdysone response genes in P. kraunhiae

To identify homologous sequences of spo, phm, dib, sad and shd in P. kraunhiae, tblastn searches were performed using the unpublished RNA-seq database of P. kraunhiae from our laboratory (Vea et al., unpublished) with the amino acid sequences of other insects, as listed in S1 Table. Similarly, tblastn searches were performed in the RNA-seq database (DDBJ/EMBL-Bank/GenBank accession number DRA004114) [34] using EcR and E75 sequences from other insects.

Total RNA was extracted from pooled individuals of different stages and sexes using TRIzol reagent as reported previously [31]. Oligo-dT-primed reverse transcription was performed with PrimeScript II 1st strand cDNA synthesis kit (Takara Bio Inc., Shiga, Japan). Primers for RT-PCR were designed based on putative nucleotide sequences identified in RNA-seq databases. PCR products were purified and subcloned into pGEM-T Easy Vector (Promega Corp., WI, USA) and sequenced. To obtain the complete nucleotide sequences of E75 variants at the 5’ end, 5’ RACE PCR was performed with a SMARTer RACE cDNA Amplification Kit (Takara Bio) as previously reported [31, 32].

Primer sequences are listed in S2 Table. Obtained nucleotide sequences were deposited in the DDBJ/EMBL-Bank/GenBank International Nucleotide Sequence Database with the following accession numbers: spo, LC508221; dib, LC508220; sad, LC508223–LC508225; shd, LC508222; EcR, LC508219; E75 isoforms, LC508214–LC508218.

To confirm the homology of the candidate Halloween genes (cytochrome P450 gene family), we aligned the translated amino acid sequences of all P. kraunhiae Halloween genes with known sequences of other insects, using MAFFT v.7 via the online service [35] and using the L-INS-i method [36]. The phylogeny was then inferred using the Bayesian method, and were carried out with MrBayes v3.2.6 [37] using the mixed amino acid model, through the Cipres Science Gateway Portal [38]. Four independent runs were carried out for 1 million generations each, and trees were sampled every 100 generations. After the analysis, the phylogeny was estimated based on the majority consensus of sampled trees, after removing the first 25% trees (burn-in). The nexus file containing the sequence alignment and analysis script can be found in S1 File.

Gene expression

Absolute quantitative RT-PCR was performed as described previously [31]. Briefly, samples were collected every 24 h after oviposition up to adult emergence. From E to N2D3 were collected using a sex-biased strategy. Total RNA was extracted from pooled individuals as described above. These RNA samples were reverse transcribed using a Prime Script RT reagent Kit with gDNA Eraser (Takara Bio). Quantitative RT-PCR was carried out in a 14 μl reaction volume containing SYBR Ex Taq (Takara Bio), 0.2 μM of each primer (see S2 Table) and 1 μl of template cDNA or standard plasmids. PCR conditions were 95°C for 30 s, followed by 40–45 cycles at 95°C for 5 s and 60°C for 30 s. After thermal cycling, the absence of unwanted byproducts was confirmed using melting curve analysis. Serial dilutions of a plasmid containing a part of the ORF of each gene were used as standards. Transcript levels of the target genes were normalized to that of ribosomal protein L32 (rpL32) levels in the same samples.

Results

P. kraunhiae Halloween genes cloning and expression profiles

The search for Halloween gene homologs in the P. kraunhiae transcriptome retrieved different candidate transcripts. Using designed primers, we performed RT-PCR on cDNA synthesized from total RNA extracted from pooled individuals of different stages and obtained the partial sequences of the following genes: a 953-bp long Pkspo transcript, a 1286-bp Pkdib transcript, and a 1640-bp Pkshd transcript. We retrieved three variants of Pksad (here designated as variants A, B and C): variant-A (1535 bp), variant-B (with a 20-bp deletion compared with variant-A), and variant-C (which had an additional 146-bp deletion). Pksad transcripts are likely a result of alternative splicing and a blastp search of the predicted protein sequences retrieved variant-B with the highest homology to other insect functional sad proteins (data not shown).

To further confirm the homology of the identified Halloween genes, the predicted amino acid sequences were aligned with Halloween genes from different insect species and a phylogenetic tree was inferred using the Bayesian method. Our phylogenetic tree retrieved the different Halloween genes of P. kraunhiae into their respective groups (Fig 1). Moreover, the amino acid sequences were aligned for each Halloween gene with the sequences of D. melanogaster, Tribolium castaneum and B. mori and showed that these genes are highly conserved (S1 Fig). For instance, in PkSpo, the alignment highlighted the conserved signature sequences of a cytochrome P450 protein, such as “PERF” domain (PxxFxPxRF) and heme-binding domain (PFxxGxRxCxG) (S1 Fig). Among the Halloween genes involved in ecdysteroid biosynthesis, we were not able to identify a candidate sequence for phm in P. kraunhiae.

Fig 1

Cytochrome P450 gene family phylogeny of representatives from different insect orders.

The phylogenetic reconstruction was inferred using MrBayes based on the majority consensus obtained from 4 runs at 1 million generations, with trees sampled every 100 generations, using the mixed amino acid model. Posterior probability values are indicated at each node. Genes from Planococcus kraunhiae are highlighted in red in the phylogeny.

To compare ecdysteroid titers between male and female developments, we indirectly measured the titers by quantifying transcript levels of Pkspo, Pkdib, Pksad, and Pkshd every 24 h from oviposition to adult emergence in both sexes (Fig 2).

Fig 2

Developmental expression profiles of Planococcus kraunhiae spook (A), disembodied (B), shadow (C), and shade (D). Transcript levels were determined using absolute quantitative RT-PCR, and the values were normalized to those of PkrpL32. RNA was isolated from pooled individuals. E: Egg, N1: first-instar nymph, N2: second-instar nymph, N3: female third-instar nymph, Pre: male prepupa, Pu: male pupa, Ad: adult. Numbers on x-axis indicate the ages in days within each developmental stage. Solid circles represent males, while open circles represent females.

Pkspo, Pkdib and Pksad were highly expressed during embryonic development after oviposition and sex-specific expression was found in Pkspo and Pksad, where females had higher levels of transcripts (Fig 2). The N1 stage did not show notable expression differences between Halloween genes or sexes, except for very low expression of Pkspo and Pkshd in the first few days after hatching. Most of the sex-specific expression differences were found starting at the end of N2. Generally, all Halloween genes had higher expression levels during male development, with peaks preceding molting events. Although in a lesser extent, the same pattern of expression timed to molting events also occurred in females, with notably two peaks of Pkspo and Pkshd at the end of N2 and N3. Finally, Pkshd expression profile presented a distinct expression pattern compared to the other Halloween genes: expression levels remained low from E to the middle of N1, increasing during the last four days of N1, before molting to N2, in both sexes. Interestingly, Pkshd mRNA highest peaks coincided with the onset of metamorphosis for males (before prepupal stage), and the onset of adult molting event, at the end of N3 for females (Fig 2D).

Expression of ecdysone response genes in P. kraunhiae

To further assess how ecdysone is involved in the establishment of extreme sexual dimorphism, we cloned and measured the expression of ecdysone receptor (EcR) and one of the early response genes in the ecdysone signaling pathway E75. Using RT-PCR, a 1565-bp fragment for PkEcR was amplified and sequenced, which revealed that the amino acid sequence of PkEcR is most similar to EcR-A isoforms in other insect species (S2 Fig). Regarding E75, cDNA sequences of five variants, generated from different transcription initiation sites and alternative splicing, were obtained by RT-PCR and 5’ RACE PCR (Fig 3). These variants were named E75A, E75B, E75C, E75D, and E75E, based on the homology with other insect counterparts. An alignment of the E75A amino acid sequences (S3 Fig) showed that these sequences were conserved especially within the putative DNA-binding and ligand-binding domains.

Fig 3

Structure of Planococcus kraunhiae E75 isoforms.

Open reading frames are shown as boxes. The putative DNA-binding domain and ligand binding domain are shown in dark grey. Numbers in boxes indicate amino acids for each domain. E75D lacks 28 amino acids in putative DBD, whereas E75E lacks 74 amino acids.

PkEcR expression coincides with hatching and molting events throughout both male and female development (Fig 4A). Between the embryonic stage and the end of N2, PkEcR expression was progressive and happened as small peaks at hatching and N1–N2 transition, with female transcripts being slightly higher. From N2, PkEcR levels in males increased progressively at each molting event, reaching the highest expression at the Pu–Ad transition. In females, however, the highest peak of expression occurred at the end of female N2, but remained very low during the N3–Ad transition.

Fig 4

Developmental expression profiles of Planococcus kraunhiae E75 and EcR.

Transcript levels of EcR (panel A), E75A (panel B), E75B (panel C), E75C (panel D), E75D (panel E), E75E (panel F), and E75 common region (panel G) were determined by quantitative RT-PCR, and the values were normalized to those of PkrpL32. RNA was isolated from pooled individuals. E: Egg, N1: first-instar nymph, N2: second-instar nymph, N3: female third-instar nymph, Pre: male prepupa, Pu: male pupa, Ad: adult. Numbers on the x-axis indicate the ages in days within each developmental stage. Solid circles represent males, while open circles represent females.

The expression profiles of PkE75 isoforms markedly differed among each other (Fig 4B–4F). For instance, PkE75A and PkE75C showed a distinct male-specific peak of expression at the N2–Pre transition (Fig 4B and 4D), although there was a transient peak in females at Day4 of the embryonic stage. Alternatively, the peak of PkE75B coincided to molting events in both males and females, although males peaks were generally higher (Fig 4C). PkE75D was highly expressed at the male Pu–Ad transition, while there was no obvious peak in other developmental stages as well as during female development (Fig 4E). Finally, the transcript levels of PkE75E increased during the latter half of each instar, and was prominent at the end of E, as well as during male Pre and Pu stages (Fig 4F). The expression profile with primers for PkE75 common region reflected those of the five isoforms: it was high in the latter half of each instar, and prominent in male Pre to Ad (Fig 4G).

Taken together, the transcript levels of PkE75 isoforms, especially PkE75C and PkE75E, were higher at the onset of male metamorphosis (Pre and Pu stages) compared to females, while PkE75D was only high at the male Pu–A transition.

Discussion

The role of ecdysteroids in insect metamorphosis is already extensively investigated in selected insect models that undergo complete metamorphosis, such as D. melanogaster and B. mori. However, little is known of ecdysteroid involvement in hemimetabolous insects, and more specifically how it establishes sex-specific metamorphosis in mealybugs. The goal of this study was to first provide evidence of how ecdysteroid titer is linked to sexually dimorphic development in the mealybug P. kraunhiae. Because our attempts to directly measure ecdysteroids using LC-MS/MS methods were unsuccessful, we estimated indirectly ecdysteroid titers using the expression profiles of Halloween genes, as well as those of EcR and E75.

Expression of ecdysteroid biosynthetic genes and ecdysone response genes were previously assessed in another mealybug, Phenacoccus solenopsis, by a transcriptome analysis but was limited to a few developmental stages [39]. Here we present, for the first time, a detailed developmental expression pattern of ecdysteroid biosynthesis and ecdysone response genes, highlighting the major differences in gene expression between male and female development in the mealybug Planococcus kraunhiae. We first examined the developmental expression profiles of spo, dib, sad, and shd, Halloween genes that are highly conserved in arthropod groups [10]. In the silkworm B. mori, expression profiles of Halloween genes are positively correlated with the hemolymph ecdysteroid titer throughout development [15, 19, 26]. This suggested that the transcript levels of Halloween genes can be used as a good indicator of the ecdysteroid titer. We found that the transcript levels of Pkspo, Pkdib, Pksad, and Pkshd usually start increasing during the second half of N1 and N2 stages (Fig 2). This indicates that ecdysteroid biosynthesis in the prothoracic gland becomes active before each nymphal molt in P. kraunhiae. Importantly, from the middle of the N2 stage, when sexual dimorphism becomes visible, the expression profiles of Pkspo, Pkdib, Pksad, and Pkshd start differing between males and females. In particular, the transcript levels of Pkspo, Pkdib, and Pksad remain higher in males compared with females from mid-N2 to the adult. We conclude that ecdysteroid biosynthesis in the prothoracic gland is more active in males than in females.

It is worth mentioning that the developmental expression profile of Pkshd was somewhat different from those of the other three Halloween genes tested: only Pkshd expression peaks in females at N3–Ad molting, which was not observed in Pkspo, Pkdib, or Pksad (Fig 2). In other insect species such as D. melanogaster and Schistocerca gregaria, Spo, Dib and Sad are generally located in the prothoracic gland, whereas Shd converts ecdysone into 20E by hydroxylation in peripheral tissues such as the fat body, midgut, and Malpighian tubules [23, 40]. Therefore, we propose that the conversion of ecdysone to 20E occurs before adult metamorphosis in both sexes, i.e., at the end of N2 for males and at the end of N3 for females. Nevertheless, since the transcript levels of Pkspo, Pkdib, and Pksad from mid-N2 to the adult were higher in males compared with those of females, the total amount of ecdysteroids in males might be higher than in females. In contrast, a high expression of Pkshd in females at N3–adult molting would be necessary for transient peak of 20E, which might be essential to induce female adult differentiation such as ovarian development. In addition, our results suggested that the transcription of Pkspo, Pkdib, and Pksad, the ecdysteroidogenic enzymes involved in the biosynthetic pathway from 7dC to ecdysone, is regulated in a similar manner, whereas the transcriptional regulation of Pkshd is distinct from the others. This might be because the tissue localization is different between shd and others.

We also examined the expression profiles of E75 and EcR, both of which are known as ecdysone response genes in several insect species and are well understood in D. melanogaster [41] and B. mori [42, 43]. The transcript levels of the E75 common region and EcR were high in male Pu stage (Fig 4A and 4G), suggesting that ecdysteroid titer is high during male adult development. Using 5’ RACE PCR, we identified five isoforms of E75 in P. kraunhiae as shown in Fig 3. Although the developmental expression profiles of these E75 isoforms were generally similar, peaks of expression shifted slightly among isoforms (Fig 4). Similar observations have been reported in other species such as Manduca sexta [44] and B. germanica [45]. These isoforms must have distinct roles in insect development, and the transcriptional regulation mechanism differs among isoforms. For instance, involvement of JH in regulating E75 transcription has already been reported, where JH suppresses 20E-induced transcription of E75C in adult development of M. sexta [44]. We reported previously that JH levels were higher in males during metamorphosis in P. kraunhiae [31]. Therefore, we suggest that the sex-specific expression of PkE75 isoforms could be regulated by both ecdysteroids and JH.

Based on our qRT-PCR results, we estimated the ecdysteroid titers throughout post-embryonic development of P. kraunhiae. Among the genes that we examined in this study, we selected Halloween genes and EcR as indicators to measure ecdysteroid titer indirectly. As stated above, the transcription of E75 isoforms seems to be regulated by both ecdysteroids and JH in isoform-specific manners, which makes it difficult to estimate ecdysteroid titer from the expression profiles of E75 isoforms alone. As shown in Fig 5, in male development, there are peaks of ecdysteroids, which are likely to induce metamorphic molts to Pre, Pu and Ad. We propose that high ecdysteroid titer in males is essential to activate transcription factors such as br and E93. br is a pupal specifier in holometabolous insects, whereas it is involved in progressive wing formation in hemimetabolous species [46-51]. E93 is a transcription factor that induces adult morphogenesis in both hemimetabolous and holometabolous species [52, 53]. It has been reported that the transcription of both br and E93 is regulated by ecdysteroids and JH [51-54]. In P. kraunhiae, br expression is higher in males than in females, while E93 is exclusively expressed during male adult metamorphosis [31, 32]. Higher ecdysteroids during male adult development would have a significant role in promoting adult morphogenesis through br and E93 (Fig 5, upper panel). In females, by contrast, ecdysteroid titer remains relatively low compared with males, although a transient peak is observed at N2-N3 transition (Fig 5, lower panel). The overall low ecdysteroid titer in females might account for their neotenic development and wingless adult stages.

Fig 5

Diagram of estimated ecdysteroid titer in Planococcus kraunhiae based on our study.

Ecdysteroid titer is shown in green. The expression profiles of br (blue line) and E93 (magenta line) are based on our previous studies [31, 32]. N2: second-instar nymph, Pre: male prepupa, Pu: male pupa, N3: female third-instar nymph, Ad: adult.

The reason why our attempts to measure ecdysteroid titers using LC-MS/MS were not successful is not clear. One possibility is that due to their small body size, especially during the juvenile stages, it is not possible to collect hemolymph from the mealybugs, which might decrease the purity of extracted ecdysteroids for the analysis. Another possibility is the involvement of metabolism of ecdysteroids in the hemolymph of mealybugs: in the insect body, a part of the ecdysteroids isgenerally metabolized into polar metabolites such as esters and conjugates [55]. It is possible that most of the ecdysteroids in mealybugs are rapidly metabolized, which makes it difficult to identify ecdysteroids by LC-MS/MS. In order to extract enough amount of ecdysteroids, it will be necessary to collect a higher number of individuals in which ecdysteroids biosynthesis is active: collecting individuals prior to ecdysis might help for this purpose.

In summary, our results suggest that the changes in ecdysteroid titer are diverse between females and males, and that higher ecdysteroids in males may play a significant role in promoting male-specific adult morphogenesis. Taken together with our previous studies [31, 32], we conclude that both ecdysteroids and JH play an essential role in establishing sexually dimorphic metamorphosis of mealybugs. Further studies such as promoter analysis of br and E93 should provide insights into any crosstalk between ecdysteroids and JH.

Alignment of protein sequences of spook, disembodied, shadow and shade.

Protein sequences of Spook (A), Disembodied (B), Shadow (C) and Shade (D) were aligned among Planococcus kraunhiae (Pk), Bombyx mori (Bm), Drosophila melanogaster (Dm) and Tribolium castaneum (Tc). Putative “PERF” domains as well as heme-binding domains were indicated with lines. Accession numbers follow gene names.

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Alignment of EcR protein sequences.

BgEcR, Blattella germanica EcR (accession number, CAJ01677.1); TcEcR, Tribolium castaneum EcR-A (NP_001107650.1); NvEcR, Nezara viridula EcR-A (ADQ43370.1); PkEcR, Planococcus kraunhiae EcR (this study). Asterisks indicate fully-conserved amino acid residues, while colons and periods represent conservation with strong and weak similarity, respectively. The DNA binding domain (C region) and ligand binding domain (E region) are boxed. The putative junction between EcR-A and EcR-B isoforms is shown by an arrow.

(PDF)

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Alignment of E75A protein sequences.

PkE75A, Planococcus kraunhiae E75 isoform A (this study); BgE75A, Blattella germanica E75A (accession number, CAJ87513.1); BmE75A, Bombyx mori E75A (NP_00106079.1). Asterisks indicate fully-conserved amino acid residues, while colons and periods represent conservation with strong and weak similarity, respectively. The DNA-binding domain and ligand-binding domain are boxed.

(PDF)

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Accession numbers of the query amino acid sequences of Halloween genes.

(PDF)

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Primer sequences for RT-PCR, RACE PCR and qRT-PCR.

(PDF)

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Nexus file of aligned Cytochrome P450 genes of different insect species.

Nexus format file including aligned amino acid sequences of cytochrome P450 gene regions and command lines used for the MrBayes phylogenetic inference.

(NEX)

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4 Feb 2020

PONE-D-20-01381

Sex-specific expression profiles of ecdysteroid biosynthesis and ecdysone response genes in extreme sexual dimorphism of the mealybug Planococcus kraunhiae (Kuwana)

PLOS ONE

Dear Dr. Minakuchi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewer 1 suggests developing an analytical method to directly detect and quantify ecdysteroids from mealybug by increasing the number of individuals analyzed. As both reviewer 2 and 3 are fully accepting of the use of expression profiles as proxies for titer and reviewer 3 clearly highlights the challenges that conjugation of ecdysteroids present in quantification, I believe reviewer 1’s request is beyond the scope of the current manuscript.  Please address the remaining concerns of all three reviewers, including comment 2 from reviewer 3 and pay particular attention to indicating biological replicate data in your qPCR results.

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Reviewer #3: Yes

**********

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Reviewer #1: Manuscript #: PONE-D-20-01381

Title: "Sex-specific expression profiles of ecdysteroid biosynthesis and ecdysone response genes in extreme sexual dimorphism of the mealybug Planococcus kraunhiae (Kuwana)"

Authors: Miyuki Muramatsu et al.

Comments

Mealybugs are characterized by remarkable sexual dimorphic traits as a result of unusual diverging post-embryonic development. In some insect species, such sexual dimorphic traits were regulated by hormones such as JH and ecdysteroids. However, ecdysteroid titers have not been measured in the Japanese mealybug, Planococcus kraunhiae, and thus the involvement of ecdysteroids in sexual dimorphism in this species remains unknown.

In order to cultivate a better understanding for the role of ecdystderoids in sexual-dimorphic development in the mealybug, the authors first attempted to directly measure the ecdysteroid titers using LC-MS/MS. But they failed to detect ecdysteroids by their method. Therefore, the authors tried to estimate ecdysteroid titers by quantifying mRNA levels of the Halloween genes together with ecdysone response genes such as EcR and E75 by qRT-PCR. Finally, they propose that the changes in ecdysteroid titer are different between males and females, and that high ecdysteroid titer is essential for directing male adult development.

Overall, this is a nicely done, and several interesting observations are represented and discussed. But I think that there are several unignorable problems especially in their qRT-PCR data. Moreover, English used in this manuscript has a lot of problems. Resolution of the figures is so poor that I cannot precisely recognize characters described on them. Consequently, this manuscript is not suitable for publication in this current form.

Major requirements for revision

1. page 4, line 109. The authors said that they were not able to detect any ecdysteroid, probably due to the small size of the mealybug. If so, then the authors should increase the number of individuals subjected to the LC-MS/MS analysis.

2. page 9, lines 242-244 and Fig. S2. The authors identified an EcR ortholog of the mealybug, which showed highest similarity to EcR-A isoforms in other insect species. Were there any other EcR isoforms such as EcR-B1 and EcR-B2? If not, then the authors should show several lines of evidence that the mealybug examined in this study has only one isoform. And also, they should perform phylogenetic analysis as described in Fig. 1 to evaluate whether the PkEcR is indeed an ortholog of EcR in the mealybug.

3. Fig 2 and 3. How many trials did the authors perform qRT-PCR analyses? In general, researchers perform at least triplicate trials of qRT-PCRs and give SE to statistically assess the quantified data. The authors should provide such kind of information about statistical analysis on their qRT-PCR data.

4. page 10, lines 272-273. The authors said that expression levels of PkE75C and PkE75E were higher in males than females at the onset of male metamorphosis. But the expression level of PkE75C was specifically higher in females than males at day-4 embryo. The level was as same as that in male at pre-pupal stage. The authors should give some explanations about this female-specific increment of PkE75C expression level.

5. page 12, lines 343-345. The authors said that the sex-specific expression of PkE75 isoforms could be regulated by both ecdysteroids and JH. If so, then expression levels of PkE75 isoforms do not simply reflect ecdysteroids titers. This means that people cannot estimate ecdysteroid titers based on the expression levels of PkE75 isoforms. Therefore, the authors should directly quantify the ecdysteroid titers.

Minor requirements for revision

1. Overall. All text describing experimental results should be written in the past tense. For example, in the line 9, the author described that "PkEcR expression is progressive and happens as small peaks at hatching". But they should change this phrase to " PkEcR expression was progressive and happened as small peaks at hatching.

2. page 4, line 88. I think "a result of" should be better to change to "as a result of".

3. page 4, line 97. Add period immediately after "[29]".

4. page 4, line 101. I think "which is" should be better to insert prior to "involved in".

5. page 4, line 107. "directs" should be replaced with "direct".

6. page 6, line 166. "(" before [37] should be removed.

Reviewer #2: Sexual dimorphism on metamorphosis is known in meal bug. Generally, juvenile hormone (JH) and ecdysteroid play crucial roles on regulation of polypenism. Authors has already showed the JH titer of Planococus kraunhiae. In this manuscript, authors estimate the ecdysteroid profiles of male and female in the meal bug, P. kraunhiae. It is impossible to measure the ecdysteroid titer directly because the bug is too small to get enough volume of hemolymph. The ecdysteroid titer was estimated from the expression profiles of 4 ecdysterodgenic genes and 2 ecdysone responsible genes. According to data, they discussed ecdysteroid titer in both sex and speculates the role of ecdysteorid on sexual dimophism. The manuscript is written well and statement is clear.

In general, I do not see serious problems on the scientific aspects of the manuscript. Thus, I’d add only some comments.

1. In figure 5, ecdysteroid titer was estimated. The ecdysteroid level of female seems too low levels in N3 nymph. The expression levels of spo, dib and sad in N3 were similar to these in N2. What did you calculated the level is based on?

2. Line 97. Followed by [97], please add full stop.

3. Lin 100. What “the transcription factor” does indicate?

4. Line 107. I think it is mistype, please correct “the directi titers”.

5. Line 166. Please remove “(“.

6. Line 176. About what amount total RNA did you use for RT? Please mention.

7. In discussion section, from line 312 to 318 is very similar to line 319-330. Please rewrite these paragraphs.

8. In figure 1, “a” is present at upper of the figure. Please remove this.

Reviewer #3: The authors presented a nice piece of research on very interesting theme with sex-dependent effects. Here in the agriculture important insect, the mealybug Planococcus kraunhiae. The paper starts from a very interesting observation, a fascination in nature of the insects. The paper is well written and the research questions well formulated. I like that the discussion is balanced and critical. The research is done with high precision and quality.

I have two suggestions to the authors to improve their manuscript.

1. On the qPCR data, please write the number of biological and technical repeats done; although the work is done using a protocol as published before. Also the authors should confirm the stability in expression of the rpL32 as reference gene over the different stages tested. Why did the authors not sued 2 reference genes?

2. I appreciate that the authors did their upmost to measure the titer of ecdysteroids and even identify the different forms, however it did not work out successfully. It is not evident as I know from my own research. But the authors should also reflect in the discussion that the amount of free ecdysteroids can also depend on the process of conjugation, to bind or liberate ecdysteroid hormone and so reducing or increasing the titer concentration in the insect body.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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18 Mar 2020

We would like to thank the Editor and the Reviewers for reviewing our manuscript and for their comments. All the comments are very valuable and will improve our manuscript. Please find the responses to the comments below. In the revised manuscript, changes are highlighted with yellow.

Through careful proofreading, we noticed additional points that need to be revised, most of which are grammatical errors. These changes are highlighted with cyan.

Reviewer #1:

Comments

Mealybugs are characterized by remarkable sexual dimorphic traits as a result of unusual diverging post-embryonic development. In some insect species, such sexual dimorphic traits were regulated by hormones such as JH and ecdysteroids. However, ecdysteroid titers have not been measured in the Japanese mealybug, Planococcus kraunhiae, and thus the involvement of ecdysteroids in sexual dimorphism in this species remains unknown.

In order to cultivate a better understanding for the role of ecdystderoids in sexual-dimorphic development in the mealybug, the authors first attempted to directly measure the ecdysteroid titers using LC-MS/MS. But they failed to detect ecdysteroids by their method. Therefore, the authors tried to estimate ecdysteroid titers by quantifying mRNA levels of the Halloween genes together with ecdysone response genes such as EcR and E75 by qRT-PCR. Finally, they propose that the changes in ecdysteroid titer are different between males and females, and that high ecdysteroid titer is essential for directing male adult development.

Overall, this is a nicely done, and several interesting observations are represented and discussed. But I think that there are several unignorable problems especially in their qRT-PCR data.

Moreover, English used in this manuscript has a lot of problems.

Response: Thank you for reviewing our paper. We carefully proofread the manuscript, and corrected the Result description.

Resolution of the figures is so poor that I cannot precisely recognize characters described on them. Consequently, this manuscript is not suitable for publication in this current form.

Response: Thank you for the comments. We checked the PDF file and found that the figures were in low resolution, but the high resolution original figures can be downloaded on the link at the top right of the figure pages.

Major requirements for revision

1. page 4, line 109. The authors said that they were not able to detect any ecdysteroid, probably due to the small size of the mealybug. If so, then the authors should increase the number of individuals subjected to the LC-MS/MS analysis.

Response: We agree with the Reviewer #1 that LC-MS/MS analysis of ecdysteroids with increased number of individuals will be desirable. However, in our trials to analyze ecdysteroids with LC-MS/MS, we used enough amount of pooled mealybugs (approximately 200 individuals, equivalent to ca. 10 mg) which was comparable to those used in other insect species such as the fruit fly Drosophila melanogaster. At present, it is unknown whether this is due to low titer of ecdysteroids in the mealybugs, or there is some unidentified humoral factors that inhibit the detection of ecdysteroids by LC-MS/MS. In any case, we believe that detection and quantification of ecdysteroids will be possible if the number of the individuals is dramatically increased. However, we are afraid that this is beyond the scope of the current manuscript, and will be performed in our future study.

2. page 9, lines 242-244 and Fig. S2. The authors identified an EcR ortholog of the mealybug, which showed highest similarity to EcR-A isoforms in other insect species.

Were there any other EcR isoforms such as EcR-B1 and EcR-B2?

If not, then the authors should show several lines of evidence that the mealybug examined in this study has only one isoform.

And also, they should perform phylogenetic analysis as described in Fig. 1 to evaluate whether the PkEcR is indeed an ortholog of EcR in the mealybug.

Response: Thank you for the comment. We have only performed sequencing of a partial fragment of EcR and therefore did not identify all the isoforms. However, based on the alignment of the fragment that we sequenced (S2 Fig.), we are confident that the covered region matches EcR-A isoform.

EcR is a highly conserved gene with ligand-binding and DNA-binding domains (S2 Fig.). As opposed to the Halloween genes which are close paralogous copies and where a phylogenetic tree is interesting to perform to separate each of the copies, we do not think that providing a phylogenetic tree of the EcR sequence we identified is necessary.

3. Fig 2 and 3. How many trials did the authors perform qRT-PCR analyses? In general, researchers perform at least triplicate trials of qRT-PCRs and give SE to statistically assess the quantified data. The authors should provide such kind of information about statistical analysis on their qRT-PCR data.

Response: Thank you for the comment and we understand the concern. We used pooled individuals for each point, and did not do biological replicates for this experiment. In fact, we have another set of samples for analyzing developmental expression profiles, from the embryonic stage to the adults, that was used in our previous study (Vea et al., PLOS One 2016). For some of the genes including E93, we have confirmed that the profiles are consistent between two set of cDNA samples. However, it was not possible for us to combine the results from two sets of cDNA samples for calculating the average or SE because there was slight difference of the duration of each developmental stage: for example, the length of the the embryonic stage after oviposition and that of the first-instar nymphs are 10 days and 14–15 days respectively in the previous study, while they are 9–11 days and 11 days respectively in the present study. For these reasons, we showed the results from only one replicate in the manuscript.

4. page 10, lines 272-273. The authors said that expression levels of PkE75C and PkE75E were higher in males than females at the onset of male metamorphosis.

But the expression level of PkE75C was specifically higher in females than males at day-4 embryo.

The level was as same as that in male at pre-pupal stage. The authors should give some explanations about this female-specific increment of PkE75C expression level.

Response: Thank you for the comment. We agree with the Reviewer #1 that there is a female-specific increment of PkE75C at Day 4_Embryo. But we will not discuss on it in details because this study is focused on post-embryonic development. We revised the sentence to mention that there is a female-specific increment of PkE75C in the embryonic stage (L261–263 in the revised manuscript).

5. page 12, lines 343-345. The authors said that the sex-specific expression of PkE75 isoforms could be regulated by both ecdysteroids and JH. If so, then expression levels of PkE75 isoforms do not simply reflect ecdysteroids titers. This means that people cannot estimate ecdysteroid titers based on the expression levels of PkE75 isoforms. Therefore, the authors should directly quantify the ecdysteroid titers.

Response: As we pointed out in the Discussion, we used several markers to assess the titers of ecdysteroids, including PkE75. We agree that the potential interaction with other pathways may influence the transcriptional regulation of PkE75. We totally agree with the Reviewer #1 that direct quantification of ecdysteroids will be the best way to identify ecdysteroid titer. However, as we have stated above, this will be beyond beyond the current manuscript. In Discussion section (L370–380), we added a statement on quantifying ecdysteroid titers directly with increased number of individuals.

Minor requirements for revision

1. Overall. All text describing experimental results should be written in the past tense. For example, in the line 9, the author described that "PkEcR expression is progressive and happens as small peaks at hatching". But they should change this phrase to "PkEcR expression was progressive and happened as small peaks at hatching.

Response: We corrected the description of the results into the past tense, as highlighted in yellow in the revised manuscript.

2. page 4, line 88. I think "a result of" should be better to change to "as a result of".

Response: Revised as suggested (L85).

3. page 4, line 97. Add period immediately after "[29]".

Response: Revised as suggested (L94).

4. page 4, line 101. I think "which is" should be better to insert prior to "involved in".

Response: Revised as suggested (L98).

5. page 4, line 107. "directs" should be replaced with "direct".

Response: Revised as suggested (L104).

6. page 6, line 166. "(" before [37] should be removed.

Response: Revised as suggested (L164).

Reviewer #2: Sexual dimorphism on metamorphosis is known in meal bug. Generally, juvenile hormone (JH) and ecdysteroid play crucial roles on regulation of polypenism. Authors has already showed the JH titer of Planococus kraunhiae. In this manuscript, authors estimate the ecdysteroid profiles of male and female in the meal bug, P. kraunhiae. It is impossible to measure the ecdysteroid titer directly because the bug is too small to get enough volume of hemolymph. The ecdysteroid titer was estimated from the expression profiles of 4 ecdysterodgenic genes and 2 ecdysone responsible genes. According to data, they discussed ecdysteroid titer in both sex and speculates the role of ecdysteorid on sexual dimophism. The manuscript is written well and statement is clear.

In general, I do not see serious problems on the scientific aspects of the manuscript. Thus, I’d add only some comments.

Response: Thank you for reviewing our paper.

1. In figure 5, ecdysteroid titer was estimated. The ecdysteroid level of female seems too low levels in N3 nymph. The expression levels of spo, dib and sad in N3 were similar to these in N2. What did you calculated the level is based on?

Response: We estimated the ecdysteroid titer in Figure 5 based on the expression profiles of Halloween genes and EcR, as described in L347–349.

2. Line 97. Followed by [97], please add full stop.

Response: A full stop was added (L94). Thank you for the comment.

3. Lin 100. What “the transcription factor” does indicate?

Response: We revised the sentence as “We further showed that the adult-specifying transcription factor E93” (L97–98), so that it is clear to the readers.

4. Line 107. I think it is mistype, please correct “the directi titers”.

Response: This was corrected as suggested (L104).

5. Line 166. Please remove “(“.

Response: This was corrected as suggested (L164).

6. Line 176. About what amount total RNA did you use for RT? Please mention.

Response: In our RNA isolation, we used glycogen as coprecipitant to increase the yield of RNA, as we described in our previous study (Vea et al., 2016). Since glycogen shows a UV absorbance and interfere RNA quantification with spectrophotometer, we were not able to quantify total RNA. Equal volume of total RNA were used for RT.

7. In discussion section, from line 312 to 318 is very similar to line 319-330. Please rewrite these paragraphs.

Response: This part was rewritten and organized as suggested (L312–328).

8. In figure 1, “a” is present at upper of the figure. Please remove this.

Response: This was corrected as suggested.

Reviewer #3: The authors presented a nice piece of research on very interesting theme with sex-dependent effects. Here in the agriculture important insect, the mealybug Planococcus kraunhiae. The paper starts from a very interesting observation, a fascination in nature of the insects. The paper is well written and the research questions well formulated. I like that the discussion is balanced and critical. The research is done with high precision and quality.

I have two suggestions to the authors to improve their manuscript.

Response: Thank you for reviewing our paper. We appreciate valuable comments.

1. On the qPCR data, please write the number of biological and technical repeats done; although the work is done using a protocol as published before. Also the authors should confirm the stability in expression of the rpL32 as reference gene over the different stages tested. Why did the authors not sued 2 reference genes?

Response: As we described in Response to Reviewer #1’s comments, we used pooled individuals for each point, and did not do biological replicates for this experiment. We have another set of samples for analyzing developmental expression profiles, from the embryonic stage to the adults, that was used in our previous study (Vea et al., PLOS One 2016). For some of the genes such as E93, we have confirmed that the profiles are consistent between two set of cDNA samples (data not shown in the manuscript).

Regarding adding another reference gene, we agree with the Reviewer #3 that using two reference genes would be desirable. This will enable us to quantify the transcript levels more precisely. In the present study we used rpL32 as the reference gene because this is one of the most frequently utilized one for qRT-PCR and had been routinely used for scale insects in particular. Since other reference genes have not been established in this species yet, if we consider adding a second reference gene, we would need to properly examine which candidate housekeeping gene would be the most appropriate as the reference gene: developmental expression profiles of many candidate genes would need to be examined and compared. Since it will take quite a long time to determine the second reference gene, we would rather not do it in the present study, but will consider this point in future studies.

2. I appreciate that the authors did their upmost to measure the titer of ecdysteroids and even identify the different forms, however it did not work out successfully. It is not evident as I know from my own research. But the authors should also reflect in the discussion that the amount of free ecdysteroids can also depend on the process of conjugation, to bind or liberate ecdysteroid hormone and so reducing or increasing the titer concentration in the insect body.

Response: Thank you for the suggestion. We added a paragraph regarding the possible conjugation of ecdysteroids in the hemolymph (L370–380) in Discussion section.

Submitted filename: Response to reviewers comments_200317.docx

Click here for additional data file.

25 Mar 2020

Sex-specific expression profiles of ecdysteroid biosynthesis and ecdysone response genes in extreme sexual dimorphism of the mealybug Planococcus kraunhiae (Kuwana)

PONE-D-20-01381R1

Dear Dr. Minakuchi,

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

30 Mar 2020

PONE-D-20-01381R1

Sex-specific expression profiles of ecdysteroid biosynthesis and ecdysone response genes in extreme sexual dimorphism of the mealybug Planococcus kraunhiae (Kuwana)

Dear Dr. Minakuchi:

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