Summary
In contrast to the important role of hormones in the development of sexual dimorphic traits in vertebrates [1], the differentiation of these traits in insects is attributed exclusively to variation in cell-autonomous mechanisms controlled by members of the sex determination pathway [2], such as doublesex (dsx). Although hormones can shape the development of sexual traits in insects, and interact with dsx to create dimorphisms, variation in hormone levels are not known to cause dimorphism in these traits [3]. Here we show that butterflies use sex-specific differences in 20-hydroxyecdysone (20E) hormone titers to create sexually dimorphic wing ornaments, without the local involvement of dsx. Females of the dry season (DS) form of Bicyclus anynana display a larger sexual ornament on their wings than males, whereas in the wet season (WS) form both sexes have similarly sized ornaments [4]. High levels of circulating 20E during larval development in DS females and WS forms cause proliferation of the cells fated to give rise to this wing ornament, and results in sexual dimorphism in the DS forms. This study advances our understanding of how the environment regulates sex-specific patterns of plasticity of sexual ornaments and conclusively shows that sex-specific variation in hormone titers can play a role in the development of secondary sexual traits in insects, just like they do in vertebrates.
Highlights
Sex-specific levels of 20E, an insect molting hormone, regulate secondary sexual trait dimorphism and plasticity in butterflies.
20E levels above a threshold promote local patterns of cell division in one sex, but not in the other sex, to create sexually dimorphic eyespots.
eTOC Sexual selection drives the evolution of ornaments for individuals to display to the opposite sex. Yet, the mechanisms by which sexual selection operates are still not well understood. Here Bhardwaj et al. provide conclusive evidence, for the first time, that male and female insects use variation in levels of hormones to create dimorphism in their sexual ornaments. Authors show that 20-hydroxyecdyone, the insect molting hormone, also functions as a sex hormone in a butterfly. They also show how the environment shapes the development of sexual ornaments at a proximate level.
Introduction
Recent studies have shown that sexual traits are neither under constant, or even similar direction of selection over time and space [5-7]. This is because organisms do not live in stable biotic and abiotic environments. One consequence of predictable and recurrent environmental changes, such as seasons, is the evolution of plasticity in sexual traits [8,9] Understanding the mechanisms behind the development of such plastic traits can help in developing better models of phenotypic evolution by focusing research on the actual genetic loci of evolution [10].
Bicyclus anynana butterflies evolved in a seasonal environment in Africa, experiencing predictable and recurrent dry and wet seasons (DS and WS) [11]. As a consequence of this heterogeneity this species evolved a complex pattern of plasticity in its sexual behavior as well as in the size of its sexual ornaments, the bright, UV-reflective dorsal eyespot centers (Fig. 1) [4]. Essentially, DS individuals display sexual dimorphism in the size of the ornaments, with the courting DS females avidly displaying their unusually large sexual ornaments to the choosy cryptic males which have overall smaller eyespots (Fig. 1) [4]. In the WS, both sexes develop large eyespots characteristic of the season and males avidly court choosy females. This leads to a pattern of sexual dimorphism in the DS and plasticity in the sexual ornament that is male-limited (Fig. 1) [4].
While the ultimate selective factors behind the patterns of sexual dimorphism and plasticity in ornament size in B. anynana are becoming increasingly clear [4], the proximate factors behind these patterns are not understood. Here we set out to examine the developmental mechanisms that regulate sexual ornament size dimorphism in DS individuals and male-limited plasticity in this butterfly species.
Results
Because ornament size in males is controlled by rearing temperature [4], we began by identifying the developmental window that is critical for eyespot size regulation using temperature shift experiments. Low rearing temperature typical of the DS (17°C) leads to DS butterflies, whereas high temperature typical of the WS (27°C) leads to WS butterflies [12]. We experimentally manipulated rearing temperature for brief windows of 48h at different stages of development by moving animals from one temperature to the alternate temperature, and then returning them back to the original temperature (Fig. 2). WS animals reared at 27°C, which were moved to 17°C during the wandering (Wr) stage of larval development showed the strongest decrease in eyespot size (Fig 2A). The opposite pattern, an increase in eyespot size, was seen in animals reared throughout at 17°C, and moved briefly to 27°C for a 48h interval during the same Wr stage (Fig 2B). These experiments show that the Wr stage is critical for the determination of dorsal eyespot center size in males. Therefore, we focused our subsequent investigations of eyespot center size around this developmental stage.
Previous studies on the developmental basis of sexual traits in insects have pointed exclusively to variation in cell-autonomous mechanisms involving the activation of members of the sex-determination pathway, such as the gene doublesex (dsx), in the cells that develop the trait [3, 13-15]. Therefore, we asked whether dsx was being expressed in the eyespot centers at the wandering stage of development. Insituhybridizations with a probe generated against a common region of dsx, (i.e., made to identify both male and female isoforms of this gene) identified dsx expression in the developing androconial organs, a sex-pheromone producing organ [16-18] in the wings of males (Fig. 3A). However, no dsx expression could be detected in the developing eyespot centers of Wr larvae (Fig. 3A, Suppl. Fig. 3).
This led us to ask whether the sexual ornaments could be under the control of sex-specific hormone titers. Previous studies have implicated insect hormones in the evelopment and maintenance of sexual traits in insects [3], but to date no study to our knowledge has ever shown sexual dimorphism in hormone titers leading to the development of sexual traits in insects. Furthermore, previous research in this species showed that levels of the molting hormone, 20-hydoxyecdysone (20E), were involved in regulating ventral eyespot center size in females during the Wr stages of development. We, therefore, asked whether levels of this hormone could be different between males and females at the Wr stage.
We collected hemolymph from developing male and female larvae at finely spaced intervals during the Wr stage, and observed a rise in 20E titers in all WS and DS forms towards the end of this stage, just before the Wr larvae turned into prepupae. Furthermore, male and female 20E titers were different within each seasonal form, with females having higher titers than males (F1,41=55.78, P<0.001) (Fig 3B). In addition, WS titers were higher than DS titers, as previously reported for females [19,20] (F1,41=52.11, P<0.001), with no interaction between season and sex (F1,41=0.001, P=0.977).
Steroid hormones such as 20E exert effects on cells only if such cells express correspondent hormone receptors [21]. We looked for the presence of the Ecdysone Receptor (EcR) at two different stages during the Wr stage, an early stage (∼40% development) and a later stage (∼90% development), flanking the period before and after the rise in 20E tiers. At the early Wr stage, EcR was expressed in the dorsal eyespot centers in a similar extent in each sex and seasonal form(Fig 3C i-iv: panel 1), confirming the ability of these cells to respond to the subsequent rising titers of 20E, and the potential for this hormone to impact the developmental fate of these cells. At the later Wr stage, however, we observed a difference in the extent of EcR staining. DS males still expressed EcR in a small group of cells, whereas DS females and both WS sexes expressed EcR in a larger cluster of cells (Fig. 3C i-iv: panel 3). This suggests that the size control of the sexual ornament appears to be taking place in between these two time points, primarily via an increase in cell number.
20E levels above certain thresholds are known to promote cell division in larval wing imaginal discs [22,23]. Therefore, to visualize whether such localized cell divisions were taking place in the region of the future sexual ornaments, we studied the localization of a mitotic marker, phospho-histone H3 (pH3) [24], using fluorescently labeled anti-pH3 antibodies in the wing discs. At 40% of the Wr stage, when the 20E titers are low, we observed no pH3 staining (green, Fig 3C i-iv: panel 2). However, at the later stage (90% Wr), when 20E titers are surging, cell division was taking place in all groups, except DS males (Fig. 3C i-iv: panel 4). We hypothesized that cell division is initiated only once a critical threshold of 20E is attained. The cells making up the sexual ornament of DS males, having the lowest 20E titers, may never reach this threshold, and hence do not experience 20E signaling at similar levels as the other groups, and do not divide.
To test this hypothesis, we manipulated 20E signaling in the four butterfly groups. We elevated 20E signaling in DS males by injecting them with 20E at approximately 60% of the Wr stage; and lowered 20E signaling in the other three groups by injecting individuals with a EcR antagonist, Cucurbitacin B (CucB) [25] (Fig. 4A). Injections of 20E caused an increase in eyespot center size in DS males, relative to injections with vehicle (Fig 4B i; DS M-F1,37=18.38, P<0.01), while injections of CucB significantly reduced the eyespot center size in the other three groups relative to injections with vehicle (Fig 4B ii-iv; DS Fem: F1,46=6.43, P=0.015, WS Mal: F1,44=13.75, P=0.001, WS Fem: F1,37=4.617, P=0.038), indicating a functional role of 20E signaling in dictating the size of these sexual ornaments.
Discussion
Here we have shown that sex differences in levels of a steroid hormone, during a brief period of development, controls a very localized pattern of division in cells that express the hormone receptor, which later develop into the bright UV-reflective scale cells that make up a sexual ornament in adult butterflies. Females produce more of this hormone than males, and WS forms more than DS forms. However, all groups, except DS males, produce sufficient hormone to trigger a process of local cell division at the center of the dorsal eyespots. This creates sexual dimorphism in ornament size in DS animals, and plasticity in ornament size in males.
Sexual dimorphism in some vertebrate traits, such as the length of digits in mice, is controlled by two hormones, androgen and estrogen steroids, present in different relative amounts in each sex during a small window of development [26]. Our study indicates that sexual dimorphism in insects can be achieved via the use of a single hormone, 20-hydroxyecdysone, present in each sex at different levels.
It is likely that this butterfly species, which has evolved a complex mechanism for the regulation of plasticity in the size of its ventral eyespots [11,19], which function in predator-prey interactions [27,28], simply co-opted this mechanism to also regulate the size of its dorsal eyespots. The selection pressures working on the dorsal eyespots, however, are different from those on the ventral eyespots; so, the mechanism of plasticity had to be tweaked to allow eyespots on different surfaces to display different reaction norms for size in response to environmental temperature. Part of the tweaking appears to have been the rise in 20E hormone titers in DS females relative to DS males, allowing females to develop large dorsal eyespots in the DS. Why DS females are able to maintain small ventral hindwing eyespots requires further experiments. Additional work will also be necessary for a better understanding of how and when the sexual dimorphism in the hormone titers actually evolved.
An important advance of this work is the demonstration that different levels of a steroid hormone in an insect control sexually dimorphic traits. Differentiation of male and female traits in insects has, so far, been attributed exclusively to cell-autonomous mechanisms involving the expression of sex-specific splice variants and factors from the sex determination pathway, such as Feminizer (Fem), Transformer (Tra), Fruitless (Fru), and Doublesex (Dsx), in cells that build the sexually dimorphic trait [3, 29, 30]. For instance, previous work showed that the sexually dimorphic mandibles of a stag beetle, Cyclommatus metallifer,were regulated by Juvenile Hormone (JH) interacting with sex-specific isoforms of doublesex expressed in the mandibles, but levels in JH titers across the sexes were found to be similar [31,32].Previous reports also implicated hormones in the maintenance of sexual dimorphism of adult insects [33], but no study conclusively reported sexual differences in levels of insect hormones as developmental regulators of sexual traits [34]. Interestingly, higher levels of 20E were found to be present in the hemolymph of females and horness males Ontophagus taurusdung beetles, relative to horned males, during a feeding stage of the last larval instar [35]. These sex-differences in titers, however, were not tested for function. Here, we show conclusively, that sexual differences in hormone titers can regulate dimorphism in sexual traits, without the need of cell-autonomous factors also being expressed in the trait.
Sexual trait development in insects has, thus, been considered distinct from sexual trait development in vertebrates, where sex-specific variation in steroid hormones, such as testosterone and estrogen, are important regulators of sexual dimorphism [26,36]. Until recently, hormones were considered the exclusive means by which vertebrates regulate their sexual traits [37], but the appearance of gynandromorphic finches [38], displaying half male and half female plumage patterns, finally led researchers to consider the presence of cell-autonomous mechanisms of sexual trait development in vertebrates. The striking appearance of gynandromorphic insects [39], in turn, led most biologists to assume insects used cell-autonomous processes exclusively to differentiate sexual traits. Our work now conclusively shows that both mechanisms are playing a role in vertebrates and insects and calls for additional comparative work to understand how these two convergent mechanisms of sexual trait development may have diversified and evolved.
Materials and Methods
Butterfly husbandry
B. anynana butterflies, originally from Malawi, were reared in two climate rooms at 17°C and 27°C, at 70% relative humidity, 12:12 hours light: dark cycle, to produce the dry and wet season forms, respectively. Larvae were fed young corn, whereas adults were fed ripe mashed banana.
Eyespot and eyespot center size measurements
B. anynana adults from each season and sex were dissected and imaged using a Leica Stereo Microscope. Area measurements for dorsal forewings, individual posterior Cu1 eyespot, and white centers were calculated using ImageJ (NIH, v1.45s), as described previously [19].
Wandering stage sampling
Late 5th instar larvae were kept with ample food in transparent containers and imaged at 5 min intervals using the time-lapse feature of a RICOH Pentax WG-3 Camera, using method described previously [19]. Initiation of wandering stage happened when the larvae left the food and started wandering up. End of wandering stage happened when the animal begun hanging from the container, upside down.
doublesex in-situ hybridization
A fragment of doublesex mRNA from B. anynana was amplified from the cDNA using the primers AM0016 (5’-GGTGTCCGTGGGCCCGTG-3’-forward) and AM0017 (5’-CCGGTCCAGCTCCAGGCG-3’-reverse) and cloned into the pGEMT-Easy vector (Promega). See Supp. Fig. 1 for the position of the probe and primers. The insert was amplified using universal M13 primers and the amplicon was used as atemplate to synthesize DIG-labeled RNA probes. Wing discs were collected from the Wr stage larvae and used for RNA in-situ hybridization as described previously [40]. A Leica stereo microscope was used for imaging the stained tissues.
Semi Quantitative RT-PCR
To complement our findings from the in-situ hybridization, we performed semi-quantitative RT-PCR in two different sectors of the wings of Wr larvae. Late Wr stage larval wing discs were extracted and dissected into a proximal and a distal sector (Fig. S2). Proximal sectors contain the male androconial organ and hair pencils (only in hindwings), whereas distal sectors contain the sexually dimorphic eyespots. Wings were stored in TRIzol reagent (Life Technologies, Cat #15596-018) at −80°C immediately after dissection. Extracted wing tissues were homogenized in TRIzol using a bullet blender, followed by a chloroform-isopropanol precipitation and ethanol wash. Subsequently, we treated extracted RNA with DNAse, and incubated at 37°C for 15 min, followed by 3M NaoAC treatment and incubation at −80°C for precipitation. Extracted RNA was followed through one round of phenol-chloroform RNA extraction. We then used 500ng of RNA from each tissue sample to do a reverse transcription by adding dNTPs, Reverse transcriptase and RNAse inhibitor at 42°C for one hour to generate cDNA. A fragment of doublesex was amplified from this cDNA using the primers AM0462 (5’-AGTACCGCTTGTGGCCCTTC-3’-forward) and AM0463 (5’-GTCCGCGTGCGAAATACATC-3’-reverse). We used a housekeeping gene, EF-1 a, as an internal control, which was amplified using primers AM0110 (5’-TGGGCGTCAACAAAATGGA-3’-forward) and AM0111 (5’-GCAAAAACAACGAT-3’-reverse).
Male proximal forewing sectors, containing the androconial organ, expressed doublesex, whereas distal forewing sectors containing eyespots, completely lacked doublesex expression at this stage in development. Females, which lack the androconial organ, lacked dsx expression in both proximal and distal sectors. In addition, we observed similar expression patterns of dsx in hindwing anterior and posterior sectors. Anterior sectors, which contain androconial organs and hair pencils in males, show presence of dsx, which is absent in posterior sectors with eyespots. These results reinforce the idea that doublesex is not involved in regulating sexual dimorphism in eyespots.
Hemolymph collection
A small puncture was made to the first abdominal proleg of individual wanderers, and pre-pupae, and 20 μl of hemolymph were collected using a pipet. Hemolymph collections were taken from WS and DS male and female wonderers at five time points following the onset of wandering (20, 40, 60, 80 and 100%), and from pre-pupae (at 2 μm after the onset of pre-pupae). N = 4 per time point per seasonal form, but N>12 for Wr 80 and Wr 100%. Sample preparation followed an established protocol [41].
Hormone extraction
We added 800 μl of HPLC grade water to the 200 μl sample of 20 μl of hemolymph + 45 μl methanol + 45 μl iso-octane and then vortexed the solution. We used a previously described protocol [19].
Hormone titer measurements using UPLC/MS
20 μL of sample was transferred into sample vial and 5 μL of 250 μg/mL deuterated-2,2,4,4-chenodeoxycholic acid (Catalogue #DLM-6780-PK, Cambridge Isotopes Laboratories, Andover, MA, USA) (additional internal control against loss of MS sensitivity upon repeated exposure) was spiked into the sample (to make a final concentration of 50 μg/mL d4-chenodeoxycholic acid as internal standard). A series concentration of 20-hydroxyecdysone commercial hormone (Sigma-Aldrich, Catalogue#H5142, Lot#060M1390V) (1, 2, 5, 8 and 10 μg/mL) were all spiked with a constant amount of d4-chenodeoxycholic acid (50μg/mL) and analyzed via LC-MS on an Agilent 1100 LC system coupled with an ABSciex 4000 QTrap mass spectrometer. Liquid chromatography was performed on an Eclipse XDB-C18, 5μm, 4.6 × 150 mm column (Agilent Technologies Corp, Santa Clara CA). HPLC conditions: injection volume 10μL; mobile phase A and B consisted of reverse osmotic water and methanol, both containing 0.1% of formic acid; flow rate 0.5 ml/min, 30% B for 0.1min, and linearly changed to 80% B in 0.2 min; then linearly switched to100% B in 1.2 min and maintained for 1.3 min, and then linearly changed to 30% B in 2.6 min and maintained for 7.4 min. Then, the flow rate and the mobile phase were returned to the original ratio. Mass spectrometry was recorded under the positive ESI mode. A blank injection of 100% MeOH was run after each sample injection to ensure no carry over. Response factor (F) of commercial hormone to the internal standard, d4-chenodeoxycholic acid was determined. The linear range of detection for each standard was determined via the LC-MRM parameters. The result of a standard titration at 1, 2, 5, 8 and 10 μg/mL were subjected to linear regression analysis, and relation coefficient (R2). Lipids of hormone samples were measured using the validated LC-MRM parameters. Approximate concentration of butterfly hormone was calculated using the peak area under the curve. Intensity of individual hormone species was quantified by normalizing against the respective calibration curve of standards and labeled steroid.
Ecdysone Receptor and Phospho-histone H3 (pH3) immunostainings
Wing discs were dissected from wanderers at different stages. Monoclonal (mouse) antibodies raised against a Manduca sextaEcR peptide shared across all EcR isoforms (Developmental Studies Hybridoma Bank, #10F1)[42] were used at a concentration of 1:5. Goat anti-mouse (Molecular Probes, #A-11001) was used as secondary antibody at a concentration of 1:800. Polyclonal antibodies raised against rabbit mitosis marker anti-Phospho-histone H3 (Ser 10) was used at a concentration 1:150 (Merck Milipore, #06-570). Goat anti-rabbit (Molecular probes, #A-11034) was used as a secondary antibody at the concentration of 1:800. Wings were dissected, fixed in PFA, dehydrated in MeOH at −20°C, rehydrated using a gradient of MeOH and water, and then treated with primary and secondary antibodies. All wings were double immunostained with pH3 and EcR, and mounted with ProLong Gold (Invitrogen, Carlsbad, CA, USA). Images were captured on a LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). Serial Z-optic sections were done in order to distinguish dorsal from ventral EcR expression. At least three biological replicates were obtained for each immunostaining.
Hormone injections
Male DS wanderers (60% Wr) were injected with 4 μΙ of 2000 pg/μΙ of 20E (8000 μg total) (Sigma-Aldrich, Catalogue#H5142, Lot#060M1390V) or 4 μl of vehicle (1 ethanol: 9 saline solution). Female DS and male WS wanderers (60% Wr) were injected with 3 μl of 5600 μg/μl of cucurbitacin B (16,800 pg total) (Sigma-Aldrich, Catalogue#C8499, Lot#035M47104V) or 3 μl of vehicle (1 ethanol: 9 saline solution). Female WS wanderers (60% Wr) were injected with 4 μl of 5600 μg/μl of cucurbitacin B (22,400 pg total) or 4 μl of vehicle (1 ethanol: 9 saline solution). All solutions were stored at −20°C. The injections were done using a Hamilton syringe (10 μl 700 series hand fitted microliter syringe with a 33 gauge, 0.5-inch needle). The injection site was on the dorsal surface in between the integument of the second and third thoracic leg after the larvae had been chilled for 30 min on ice.
Statistical analyses
Eyespot center size was compared across seasonal forms or treatments using analyses of covariance (ANCOVA), where wing area was used as a covariate. Fixed factors appearing in the model were evaluated at a wing area of 175.265mm2 for WS and 193.021mm2 for DS wings. Hemolymph titers were compared using 2-way ANOVAs with seasonal form and sex as fixed factors. All analyses used the GLM procedure in SPSS Statistics (version 19). Data was log-transformed to meet homogeneity of variance criteria (as determined by a Levene’s test). Pair-wise comparisons, using a Bonferroni correction for multiple comparisons, were used to detect which developmental time switch points produced significant differences in eyespot traits in the temperature-shift analyses. Graphs were made inMicrosoft Excel (version 14.6.5 for the Mac) and Adobe Illustrator CC2015 using reverse transformed data (when applicable).
Author contributions
Conceived and designed the experiments: SB, KLP and AM. Performed the experiments: SB, KLP, AB, MDG, BRW, XT, WFC. Analyzed the data: SB, WFC, MRW, AM. Wrote the paper SB, AM.
Acknowledgments
Work was supported by NSF award IOS 1146933 to AM and KLP, Singapore Ministry of Education award MOE2014-T2-1-146 to AM. Work at SLING (MRW) is supported by grants from the National University of Singapore via the Life Sciences Institute (LSI), the National Research Foundation (NRFI2015-05) and a BMRC-SERC joint grant (BMRC-SERC 112 148 0006) from the Agency for Science, Technology and Research (A*Star). We acknowledge Anne K Bendt for excellent SLING scientific program management and operations support.
Footnotes
1 Lead Author: Antónia Monteiro (antonia.monteiro{at}nus.edu.sg)
↵§ Equal contributions