The wing melanization with 20-hydroxyecdysone modulation in Idea leuconoe clara (Lepidoptera: Nymphalidae)

Abstract

Temperature-induced changes in the physiological signals altered the wing coloration in Bicyclus anynana (Butler, 1879). The morphogenetic hormone 20-hydroxyecdysone (20E) increases with temperature and regulates the seasonal morph formation of the species. Although melanization in Idea leuconoe clara Butler (Lepidoptera: Nymphalidae) can be induced by temperature shock at 10, 35, and 40 °C, the relationship between this phenomenon and 20E remains unexplored. This study hypothesizes that the melanic phenotype of I. leuconoe clara is affected by distinct hormonal responses under extreme temperatures. The 20E levels were measured at the 3% and 20% pupal development stages of I. leuconoe clara under temperature shock. The relationship between 20E and melanization was investigated through hormone injection. The results demonstrated that the relationship between melanization under cold shock and 20E was negative and that 20E regulated the degree of melanin deposition rather than the patterning of melanized areas. This study investigates the effects 20E on wing melanization under extreme temperatures in I. leuconoe clara. Future studies could further determine the melanic changes in pigment and structural coloration through transcriptomic analysis and scanning electron microscopy based on the temperature shock procedure.

Introduction

Temperature and other environmental factors influence the development of many lepidopteran species, leading to different phenotypes from the same genotype (Beldade et al. 2011). Dynamic temperature regulation, in particular, can result in distinct butterfly phenotypes (Roskam and Brakefield 1999). Wing coloration develops during the late final instar larval stage and early pupal stage, with pigment genes activating at pupation and becoming visible as the pupal cuticle hardens (Koch et al. 2000, Iwata et al. 2018). Mateus et al. (2014) showed that different eyespot patterns in Bicyclus anynana (Butler, 1879) wings could be induced by simulating dry (19 °C) or wet (27 °C) conditions during the prepupation wandering phase.

The 20-hydroxyecdysone (20E) morphogenetic hormone primarily guides environmentally induced wing coloration changes (Mateus et al. 2014, Bear et al. 2017). In B. anynana, the eyespot in the CuA1 region of the hindwing enlarges with increasing temperature, corresponding to elevated 20E levels and distinct ecdysone receptor (EcR) expression (Bear et al. 2017). However, Bhardwaj et al. (2020) found that despite increased 20E levels with temperature in Idea leuconoe (Danainae), wing patterns show minimal changes at 30 °C. EcR expression remains unresponsive, suggesting alternative pathways for 20E-induced pigment deposition. In hormone injection experiments with the larvae of Papilio xuthus, high 20E concentrations suppressed multiple melanin genes while inducing the melanin gene yellow (Futahashi and Fujiwara 2007).

I. leuconoe clara Butler (Lepidoptera: Nymphalidae) is widely distributed across tropical and subtropical island regions of Asia (Morishita 1985) and is commonly bred in butterfly houses throughout Taiwan and many other countries. While typically displaying black patterns on white wings, several museums have preserved melanic specimens. However, the specific causes of melanism in I. leuconoe clara remain unclear. Chen et al. (2003) reported only 18% larval survival at 30 °C and no egg hatching at 35 °C, while Chu et al. (2020) demonstrated melanization induced by temperature shocks of 10, 35, and 40 °C.

Given the varied temperature sensitivity among butterfly species and the incomplete understanding of melanin synthesis mechanisms, it is hypothesized that extreme temperatures could trigger distinct hormonal responses in I. leuconoe, resulting in melanic phenotypes. Elevated 20E levels may indicate temperature stress effects on wing coloration. The current study aims to investigate (i) variations in 20E hormone levels under extreme temperature conditions and (ii) the relationship between temperature-induced melanism and 20E secretion, discussing the role 20E in the overall coloration system through hormone injection experiments under controlled temperature conditions.

Materials and Methods

Idea leuconoe clara Rearing Conditions and Colony Maintenance

Five female butterflies of I. leuconoe clara obtained from Muh Sheng Museum of Entomology (Puli township, Nantou county, Taiwan) were used to establish a colony in a greenhouse at National Pingtung University of Science and Technology (Neipu township, Pingtung county, Taiwan). Larvae were fed Parsonsia laevigata, while adults were offered Asclepias curassavica and Kalimeris indica. The greenhouse was maintained at an average temperature of 31 °C during the day and 26 °C at night.

Quantification of 20E Concentration Under Temperature Stress

The prepupae at the wandering stage of I. leuconoe clara were subjected to temperature shocks of 10, 35, and 40 °C based on Chu et al. (2020). Hemolymph samples were collected from 24 larvae for 20E concentration measurement, with each treatment performed in 3 biological replicates. Six individuals from each temperature treatment group, including the 25 °C control, were sampled at 3% and 20% of pupal development following Mateus et al. (2014). The timing of pupal development at 3% and 20% under each temperature was determined through Y-shaped epicranial suture molting (Chu et al. 2020). At 10 °C, larvae reached 3% and 20% of pupal development in 1.35 and 9 d, respectively. At 25 °C, these milestones were reached in 0.48 and 3.22 d; at 35 °C in 0.34 and 2.26 d; and at 40 °C in 0.39 and 2.6 d. We extracted 50 μl of hemolymph from the head region using a 10 μl microsyringe (HAMILTON; 80330) with a 0.3 mm needle. Samples were stored at −80 °C until analysis.

The 20E concentrations were quantified using a 20E ELISA Kit (Cayman Chemical Co., Ann Arbor, MI, United States). Hemolymph samples were prepared by adding 150 μl of 75% MetOH to 50 μl of hemolymph, followed by centrifugation at 10,000 rpm for 10 min at 4 °C. The supernatant was vacuum-concentrated and reconstituted in 125 μl Assay Buffer for analysis following the manufacturer’s protocol. Three technical replicates were performed for standards and samples, and the standard curve correlation coefficient (R²) was confirmed to be 0.99.

20E Injection Assay

To verify the role 20E in melanization, hormone injection experiments were conducted using 25 prepupal larvae at 25 °C during the wandering stage. We calculated the maximum natural 20E level to be 1,237,100 pg following Bhardwaj et al. (2020) based on the mean body weight of wandering larvae and the total hemolymph volume. This approach effectively guided our selection of 20E concentrations that induce wing melanization without causing mortality or disrupting development. Four concentrations of 20E (H5142, Sigma Aldrich Co. LLC, United States) (5,000, 10,000, 20,000, and 30,000 pg/μl) were prepared and dissolved in 10% EtOH. Each individual received 4 μl injection, corresponding to total doses of 20,000, 40,000, 80,000, and 120,000 pg of 20E per individual. Five individuals per concentration received 4 μl injections, with 90% saline containing 10% EtOH as a control. All specimens were reared at 25 °C until eclosion and then analyzed for melanization.

Melanization Analysis

Wing melanization was quantified using ImageJ software (version 1.53a) following Chu et al. (2020). Specimens were photographed using a Nikon D5600 camera with a SIGMA 18–200 mm lens under standardized conditions: automatic mode with aperture F4.5–5.0, shutter speed 1/100–1/125 s, ISO 100, auto white balance, and maximum resolution of 24.2 megapixels. Images were captured in JPG format against a standard background. Adobe Photoshop removed the black background, and the head, thorax, and abdomen were excluded from the analysis. The processed images were saved in JPG format and analyzed in ImageJ to calculate the ratio of black area to total wing area and measure the gray value intensity of melanized regions (Chu et al. 2020).

Statistical Analysis

Before statistical analysis, hormone concentration data were tested for normality using the Shapiro–Wilk test. After confirming normal distribution, differences in 20E concentrations among temperature treatments were analyzed using one-way analysis of variance (ANOVA). Post hoc comparisons between treatment groups were conducted using Tukey’s Honestly Significant Difference (HSD) (P < 0.05). Effect sizes for ANOVA were calculated using eta squared (η²). All statistical analyses were performed using R software version 4.1.0 (R Core Team 2023).

Results

20E Concentration Under Temperature Stress

Analysis of 20E concentrations during early pupal development revealed significant differences across temperature treatments (F_3,8 = 181.4, P = 1.08 × 10⁻⁷, η² = 0.99 at 3% pupal development; F_3,8 = 74.4, P = 3.49 × 10⁻⁶, η² = 0.97 at 20% pupal development). At 3% pupal development, the highest 20E concentration was observed at 40 °C (8,747.00 ± 454.10 pg/μl, 95% confidence interval [CI] [7,618.96, 9,875.04]), followed by 35 °C (5,345.00 ± 758.45 pg/μl, 95% CI [3,460.92, 7,229.08]) and 25 °C (3,133.33 ± 297.27 pg/μl, 95% CI [2,394.87, 3,871.80]). At the same time, 10 °C treatment showed the lowest concentration (147.57 ± 17.92 pg/μl, 95% CI [103.04, 192.09]) (Fig. 1). Similarly, at 20% pupal development, 40 °C treatment maintained the highest 20E concentration (9,260.67 ± 103.32 pg/μl, 95% CI [9,004.01, 9,517.32]), with 35 °C showing comparable levels (8,642.33 ± 1,185.38 pg/μl, 95% CI [5,697.70, 11,586.97]). The control temperature (25 °C) exhibited intermediate levels (4,778.33 ± 816.73 pg/μl, 95% CI [2,749.46, 6,807.21]), while the 10 °C treatment consistently showed the lowest concentration (1,550.00 ± 98.32 pg/μl, 95% CI [1,305.76, 1,794.24]) (Fig. 1).

Fig. 1.

The mean 20E titers ± standard deviation in the early pupal stage of Idea leuconoe clara at 4 temperatures. Three extreme temperatures (40, 35, and 10 °C) with 25 °C as control. The vertical axis represents the concentration of 20E titers (pg/μl), the white bar represents the 3% development of the pupal stage, and the gray bar represents the 20% development of the pupal stage. Different capital letters represent 3%, and lowercase letters represent 20%, according to Tukey’s HSD post hoc tests when significant differences were detected (P < 0.05).

Relationship Between Temperature-Induced Melanization and 20E Secretion

The relationship between 20E concentration and wing melanization was further investigated through hormone manipulation experiments. When different concentrations of 20E (20,000–120,000 pg) were injected into prepupae, the overall proportion of black wing patterns showed no significant differences across treatments (F_4,20 = 1.7, P = 0.18, η² = 0.25). The control group exhibited 36.2% black wing area, while specimens treated with increasing 20E concentrations showed similar proportions, decreasing from 35.8% to 33.8% as hormone concentration increases (Fig. 2A).

Fig. 2.

The effect of 20E hormone manipulations on the wing melanization of Idea leuconoe clara. A) Schematic diagram of specimens and blackening of the injection of 5 different titers of 20E. The treatment group has 4 different 20E titers (20,000, 40,000, 80,000, 120,000 pg), and the control group is 10% EtOH mixed saline. The percentages indicate the area of the wings’ black markings. There is no significant difference in the concentration of 5 titers of 20E (F_4,20 = 1.746, P = 0.18, η² = 0.25). B) The effect of 20E manipulations on the gray value of the wings of Idea leuconoe clara. The higher the gray value, the brighter the black markings on the wings. Tukey’s HSD revealed a significant difference among the 5 titers 20E (F_4,20 = 228.6, P = 2 × 10⁻¹⁶, η² = 0.98).

However, the wing pattern’s gray value intensity analysis revealed significant differences among treatment groups (F_4,20 = 228.6, P = 2 × 10⁻¹⁶, η² = 0.98). The gray value indicated the brightness of black markings and showed a positive correlation with 20E concentration. The control group and specimens treated with lower concentrations (20,000 pg) showed significantly darker patterns (mean gray value ≈ 4.4). Specimens treated with 40,000 and 80,000 pg displayed slightly lighter coloration and intermediate brightness (mean gray value ≈ 4.7 and ≈ 4.9, respectively). In contrast, those treated with the highest concentration (120,000 pg) exhibited the brightest wing patterns (mean gray value ≈ 5.0) (Fig. 2B). Post hoc analysis using Tukey’s HSD test indicated distinct groupings, with specimens receiving higher 20E concentrations (80,000 and 120,000 pg) forming significantly different groups from those receiving lower concentrations or control treatment.

Discussion

At 20% of the total pupal development time, I. leuconoe clara had a higher 20E concentration than the 3% time point, and the concentration of 20E increased with temperature. The 3% and 20% of the total pupal development time corresponded to the period before ecdysone secretion started and the ascending phase of ecdysone levels, respectively (Oostra et al. 2011, Mateus et al. 2014). Ecdysteroid plays a crucial role in molting and metamorphosis during insect development (Nijhout 2003, Mateus et al. 2014). The 20E, as a hormone mediating development, worked on the pupal stage of B. anynana and was detected in high concentrations at higher temperatures (Mateus et al. 2014, Bear et al. 2017, Bhardwaj et al. 2020). A high 20E titer was also detected at the 3% and 20% time points during pupal development at 40 °C in I. leuconoe clara. The peak of 20E dynamics at higher temperatures occurred earlier during the pupal stage, with a shorter development time than at lower temperatures in B. anynana (Oostra et al. 2011).

Chu et al. (2020) provided insight into the melanization process of I. leuconoe clara, indicating that this phenomenon is induced by both heat shocks at temperatures of 35 and 40 °C and cool shocks at 10 °C. Our results demonstrated a notable distinction: a higher concentration of 20E was observed under heat shock conditions, whereas a lower concentration was recorded under cool shock conditions.

Heat shock increased ecdysteroid levels and activated the stress response. The cold-shock hormone (CSH) may also play a role, as Mahdi et al. (2011) suggested, given that heat conditions can produce phenotypes similar to those observed under cold conditions. The CSH, a humoral factor induced by cold exposure, functions as a phenotypic plasticity mechanism that affects color pattern change. Under cold shock conditions, CSH expression is upregulated, triggering adaptive changes in wing pigmentation (Otaki 2008, Mahdi et al. 2011). It is plausible that the melanization of I. leuconoe clara during exposure to heat is primarily regulated by 20E, while a distinct mechanism may govern melanization under cool shock conditions. For example, Polyommatus icarus is known to alter its scale structure and coloration in response to cold stress (Kertész et al. 2017).

In the 20E injection experiment, the wing pattern of I. leuconoe clara was also brighter under high-concentration 20E injection. Genes manipulate pigment production, with the yellow gene up-regulating melanin synthesis and the aaNAT gene regulating the formation of colorless scales. (Matsuoka and Monteiro 2018, Kuwalekar et al. 2020). B. anynana individuals with 20E injection inhibited yellow gene expression and simultaneously promoted aaNAT gene expression (Connahs et al. 2022). Furthermore, the wing of Vanessa indica with 20E injection exhibited a paler orange coloration compared to the noninjected wing, consistent with the established hypothesis that ecdysteroid (20E) secretion alters wing coloration and reveals its role in the evolution of color patterns and speciation within the genus (Otaki 2008). The formation of the bright wing in the current study might be manipulated by 20E, which decreased yellow expression and increased aaNAT expression.

Overall, temperature and ecdysteroid secretion influence pigment gene expression and scale structure, leading to melanization in individuals (Kertész et al. 2017, Connahs et al. 2022). It is speculated that melanization in I. leuconoe clara is associated with changes in pigment gene expression and scale arrangement induced by 20E and extreme temperature. It could be further confirmed through transcriptomic analysis and scanning electron microscopy in future studies based on the above temperature shock experiment.

In conclusion, these results suggest that 20E manipulation influences the melanin intensity but not the pattern distribution. While extreme temperatures affect 20E secretion and melanization, their relationship appears nonlinear, suggesting multiple regulatory pathways in temperature-induced melanization in I. leuconoe clara.

Author Contributions

C.L. Chu (Conceptualization [equal], Formal Analysis [lead], Investigation [equal], Methodology [equal], Project Administration [equal], Visualization [equal], Writing Original Draft [equal]), Y.C. Chen (Data Curation [equal] Visualization [equal], Writing Original Draft [supporting]), T. Hua (Supervision [supporting], Validation [supporting], Writing Review Editing [supporting]), C.T. Lai (Conceptualization [supporting], Methodology [supporting], Project Administration [supporting], Resources [supporting]), and Li Hsin Wu (Funding Acquisition [equal], Supervision [equal], Writing Review Editing [equal])

Funding

This research was funded by the Ministry of Science and Technology, Taiwan, MOST-106-2313-B-020-014-MY2, and by National Pingtung University of Science and Technology, Taiwan.

Conflicts of Interest

None declared.

Data Availability

The data underlying this article are available in the article.