Introduction

Predation exerts significant selective pressure on the evolution of various species, driving the development of anti-predator strategies across the animal kingdom(Galloway et al., 2020; Stevens & Ruxton, 2018). Camouflage is a widely employed strategy, allowing animals to blend into their surroundings by adjusting their coloration and patterns to reduce detection by visual predators(Hughes et al., 2019; Skelhorn & Rowe, 2016). Nevertheless, the presence of predators with multispectral vision, capable of perceiving non-visible light spectra like infrared and ultraviolet, can compromise traditional color and pattern-based camouflage(Galloway et al., 2020; Jones et al., 2007; Stevens, 2007; Thery & Gomez, 2010), paralleling the challenges of military camouflage under non-visible light(Xi et al., 2023). Consequently, achieving alignment between an animal’s surface optical characteristics and its surroundings becomes a crucial requirement in visual camouflage strategies.

Insects, one of the most diverse animal groups on earth, contend with intense predation pressure(Cinel et al., 2020; Sheikh et al., 2017). Many of their predators, including birds, reptiles, and predatory arthropods, rely on visual cues, with some possessing highly developed ultraviolet (UV) vision, where UV light plays a critical role in locating insect prey(Lim & Ben-Yakir, 2020; Stobbe et al., 2008). Furthermore, the natural backgrounds where insects reside, such as leaves, tree bark, and soil, exhibit minimal light reflection(Endler, 1997). Consequently, light reflections originating from an insect’s body significantly elevate its risk of exposure to predators(Tovee, 1995). To counter this challenge, the various nano- and microstructural features of the insect cuticle have excellent antireflective properties that affect insect body coloration, reduce surface light reflection and aid camouflage(Vukusic & Sambles, 2003; Watson et al., 2017). The first instances of anti-reflective structures were observed in the compound eyes of moths, characterized their corneas are covered by hexagonally arranged protrusions measuring approximately 200 nanometers apart (Bernhard, 1965; Blagodatski et al., 2015). Subsequent research has revealed that anti-reflective structures are widespread on insect body surfaces, primarily found on compound eyes and wings(Blagodatski et al., 2015; Chotard et al., 2022; Ho et al., 2016; Monteiro et al., 2019; Stavenga et al., 2005). These nano- and micro-structures on the cuticular layer are believed to possess anti-reflective properties, effectively minimizing light reflections from their bodies and thus preventing detection by predators(Blagodatski et al., 2015; Chotard et al., 2022; Ho et al., 2016; Monteiro et al., 2019; Stavenga et al., 2005).

Leafhoppers (Cicadellidae), one of the largest insect families with over 22,000 species, possess unique extra-cuticular coating known as brochosome. Brochosomes, typically hollow, honeycomb-like spheres with a diameter ranging from 0.2 to 0.6 micrometers, are synthesized in the Malpighian tubules, secreted through the hindgut, and applied as a coating on the fresh cuticle following molting(Rakitov, 2000; Rakitov, 2009). Brochosomes primarily comprise lipids and proteins, with protein content ranging from 45% to 75%, and the exact proteins responsible for brochosome formation remain unknown(Li et al., 2022; Rakitov et al., 2018; Yuan et al., 2023). Some have proposed that brochosomes may serve as a protective layer on the leafhopper’s cuticle surface, with a potential role in enhancing the hydrophobicity of their cuticle surface, providing defense against pathogens and predators(Rakitov & Gorb, 2013a; Rakitov, 2009). Nevertheless, despite the known function of hydrophobicity and the shedding of brochosomes in helping leafhopper escape spider webs, there is currently no definitive biological evidence supporting other hypothesized functions of brochosomes(Lin et al., 2021; Rakitov & Gorb, 2013a, 2013b). Recent studies have explored bio-inspired materials mimicking the structure of brochosomes have shown outstanding antireflection properties in recent studies(Lei et al., 2020; Yang et al., 2017). While these artificial materials do not perfectly replicate the intricate structure of brochosomes, they offer valuable insights, indicating that leafhoppers may use a unique strategy, setting them apart from other insects, to achieve anti-reflection properties on their surfaces. Therefore, we hypothesize that brochosomes could potentially serve as a camouflage coating on the leafhopper’s cuticle surface, helping it reduce surface optical reflections and stay hidden to avoid detection by visual predators, ultimately escaping from predation.

In this study, we combined spectrophotometry, electron microscopy, transcriptome analysis, proteome analysis, gene function validation, and leafhopper and jumping spider bioassays to characterize the protein composition and the function of brochosome in leafhopper camouflage. We focused our research on the rice green leafhopper, Nephotettix cincticeps, a well-known agricultural pest with an extensive research history(Hibino, 1996; Wei et al., 2018; Yan et al., 2021). Our results revealed the pronounced effectiveness of brochosome coverage in reducing light reflection on leafhoppers N. cincticeps, especially within the scope of the ultraviolet spectrum. By conducting RNAi experiments, we successfully identified four structural proteins of brochosome for the first time. The suppression of their synthesis induced changes in brochosome morphology, influencing the optical characteristics of the leafhopper’s cuticle surface. Furthermore, our analysis of the evolutionary origin of brochosomes indicates that they likely originated through a process involving duplication–divergence. Our findings support brochosomes as a unique camouflage coating that enables leafhoppers to evade visual predators.

Results

Brochosomes are a distinctive coating on the cuticle surface of leafhopper N. cincticeps.

The cuticle surface of rice green leafhopper N. cincticeps is coated with brochosomes (Figure 1A and B), which are approximately 350nm in diameter and featuring a hollow sphere with a honeycomb-like structure (Figure 1C and D). The leafhopper N. cincticeps possesses two pairs of Malpighian tubules, each consisting of proximal segment, distal segment, and terminal segment (Figure 1E). Brochosomes are synthesized in the distal segment of the Malpighian tubule, which exhibits a swollen and rod-shaped appearance (Figure 1E). The epithelial cells in this segment contain large spherical nuclei, an extensive rough endoplasmic reticulum, and multiple Golgi regions (Figure 1E and F). Within the Golgi-derived vacuole, the brochosomes undergo progressive development, transforming their initially spherical granules into a honeycomb-like surface with closely spaced invaginations (Figure 1G-I). Mature brochosomes are stored in secretory vacuoles at the boundaries of the cells and are subsequently secreted into the lumen of the Malpighian tubules (Figure 1F).

Figure 1.

The optical properties of the leafhopper’s cuticle surface are intricately connected to the brochosome coating

To investigate the interplay between brochosome coverage and the optical features of N. cincticeps cuticle surface, we observed the relationship for male and female adults at 5, 10, 15, 20, and 25 days post-eclosion. N. cincticeps exhibited a gradual shift in body color with increasing eclosion time, manifesting clear distinctions between males and females (Figure 2A-figure supplement 1A and B). Males transformed from light green to dark green, while females progressively transitioned from light green to translucent, displaying an iridescence (Figure 2A-figure supplement 1A and B). Furthermore, the brightness of the leafhopper cuticle surface under UV light intensified with the extension of post-eclosion time, and the UV reflectance of female cuticle surface was significantly higher than that of males after 15 days post-eclosion (Figure 2B-figure supplement 1A and B).

To validate the correlation between brochosome coverage, body color, and UV light reflectance, we examined the distribution of brochosomes on the cuticle surface of male and female N. cincticeps at various post-eclosion intervals using scanning electron microscopy (SEM). SEM results unveiled a gradual reduction in brochosome coverage on the cuticle surface of both genders as post-eclosion time progressed. Specifically, male coverage decreased from 90% to approximately 40%, whereas female coverage descended from 90% to around 10% (Figure 2C-figure supplement 1A-C). Microscopic analyses of the Malpighian tubule revealed that, with prolonged post-eclosion time, the distal segment of the tubule underwent gradual atrophy, particularly pronounced at 15 days post-eclosion. Notably, females exhibited a more marked atrophy in the distal segment compared to males. By 25 days post-eclosion, the distal segment of the Malpighian tubule in females became indistinguishable from other segments (Figure 2D-figure supplement 1A and B). This denotes a progressive reduction in post-eclosion brochosome synthesis, resulting in a corresponding decrease in brochosome coverage on the cuticle surface. Furthermore, the distribution of brochosomes on the leafhopper cuticle surface may be correlated with its optical characteristics. To substantiate this hypothesis, we analyzed the light reflection characteristics of leafhopper forewings at various post-eclosion time points. The findings revealed a positive correlation between brochosome coverage on the forewings and light reflection values, underscoring the remarkable anti-reflective attributes of the brochosome coating on leafhopper cuticle surface (Figure 2E-figure supplement 1D and E). The brochosome coating efficiently diminishes the reflectance of UV and visible light on the cuticle surface of N. cincticeps.

Preferential capture of leafhoppers with fewer brochosomes by jumping spiders

Given that UV light serves as a crucial visual cue for arthropod predators, especially common visual hunters like jumping spiders(Cronin & Bok, 2016; Li & Lim, 2005; Morehouse et al., 2017; Silberglied, 1979; Zou et al., 2011), the brochosome coating on leafhoppers’ cuticle surface significantly reduces UV light reflectance. This prompted us to investigate the effect of brochosome coatings on reducing predation risk in leafhoppers. Predation experiments were conducted using Plexippus paykulli (Figure 2F-figure supplement 2), a common jumping spider in rice fields known to prey on leafhoppers(Yang et al., 2018). Predation rates and times were scrutinized as parameters to assess predation efficiency(Taylor et al., 2014; Walker & Rypstra, 2002). In the experimental group comprising females and males at 5 days post-eclosion, P. paykulli did not exhibit a clear predation preference (Figure 2G). However, in the experimental group involving females and males at 25 days post-eclosion, there was a higher probability of predation on female N. cincticeps compare to male ones (Figure 2H). For male N. cincticeps in both the 5-day and 25-day post-eclosion experimental groups, P. paykulli showed a stronger inclination towards preying on individuals that were eclosed for 25 days, resulting in significantly shorter predation times compared to those at 5 days post-eclosion (Figure 2I). These patterns were consistently observed for female leafhoppers in both the 5-days and 25-days post-eclosion (Figure 2J). The cumulative findings strongly suggest a distinct preference of the jumping spider P. paykulli for individuals with lower brochosome coverage on the cuticle surface. Based on these experiments, we proposed a hypothesis that the brochosome coating on the leafhopper cuticle surface may mitigate predation risk by reducing surface light reflection, particularly in the UV spectrum.

Figure 2.

Identifying the major structural proteins of brochosomes

Despite the initial discovery of brochosomes in the early 1950s and the confirmation in the 1960s that they are protein-lipid particles(Gouranton & Maillet, 1967), the specific protein composition of brochosomes remains unknown. Recent suggested that they are primarily composed of brochosomins (BSM) and brochosome-associated proteins (BSAP)(Li et al., 2022; Rakitov et al., 2018). BSM, a novel class of secreted proteins with molecular weights ranging from 21 to 40 kDa, is considered as the major structural component of brochosomes(Rakitov et al., 2018). However, there remains uncertainty regarding the specific proteins implicated in brochosome formation.

To further elucidate the essential protein component of brochosome and the relationship between the brochosome coating and optical properties of leafhopper’s cuticle surface, transcriptomic and proteomic data derived from brochosome of N. cincticeps were comprehensively analyzed. Based on our prior integrated analysis of the brochosome transcriptome and proteome(Wu et al., 2023), in conjunction with literature search results, we selected 50 candidate genes for functional analysis using RNAi-mediated gene silencing. Through RNAi experiments, we successfully identified four genes encoding brochosome structural proteins that led to significant morphological changes in brochosomes (Figure 3A-figure supplement 3). According to the coding sequence (CDS) length of these genes, the four proteins were names as BSM-1 to BSM-4 (GenBank accession number PP273097, PP273098, PP273099, PP273100) (Figure 3B). Comparative analysis with previous reported brochosome proteins indicated that BSM2 and BSM3 may form a pair of paralogous genes based on their highly conserved protein sequences (figure supplement 4A and C); although BSM2 and BSM3 show lower sequence homology, their structural similarities is notably high (figure supplement 4B). In contrast, BSM-1 and BSM-4 lack homologous genes within the other reported leafhopper brochosome protein repertoire. Results of RT-qPCR showed that microinjection of dsRNA effectively suppressed the expression of these four BSM-coding genes (Figure 3C-F). SEM observations also indicated that the inhibition of these four BSM-coding genes individually led to a reduction in brochosome coverage on the cuticle surface of N. cincticeps (figure supplement 3). Specifically, knockdown of these genes resulted in significant morphological changes in the brochosome, characterized by an increase in the diameter of certain brochosomes and deformation of the honeycomb-like structures on the surface (Figure 3A-figure supplement 3). In addition, investigation of temporal and spatial expression patterns showed that all the four BSM-coding genes were specifically expressed in the Malpighian tubules (Figure 3G-J) and exhibited relatively stable expression during the early post-eclosion period, followed by a gradual decrease after 10 days (Figure 3K-N). By 25 days post-eclosion, the expression of BSM-coding genes declined to around 10% in females and 30-40% in males, respectively (Figure 3K-N). This phenomenon is consistent with our initial microscopic observations, suggesting that the gradual reduction in brochosome synthesis contributing primarily to the decrease of brochosome coverage on the cuticle surface of N. cincticeps after adulthood.

Figure 3.

Brochosome coating diminishes light reflection and facilitates predator avoidance

To further investigate the function of brochosomes in leafhopper cuticle surface associated with light reflection and predator avoidance, we implemented RNAi to simultaneously suppress the expression of all four BSM-coding genes with dsBSM mixture injection. RT-qPCR results demonstrated a substantial downregulation in the expression of these genes compared to the dsGFP control (Figure 4F). Notably, under ultraviolet light, both male and female leafhoppers exhibited a significant increase in UV reflection on their cuticle surface following dsBSM treatment (Figure 4A). Spectral data corroborated these findings, showing elevated emission values in the ultraviolet spectrum on the leafhopper’s cuticle surface (Figure 4G). Electron microscopy observations revealed pronounced alterations in brochosome morphology and distribution after dsBSM treatment. Reduced distribution area and the accumulation of deformed brochosomes were observed, differing from the uniform distribution in the dsGFP treatment (Figure 4B and C-figure supplement 5A and B). Transmission electron microscopy of the Malpighian tubules indicated numerous Golgi-derived vacuoles without brochosome distribution in epithelial cells after dsBSM treatment (Figure 4D and E-figure supplement 5C and D). Predation experiments with jumping spiders revealed an increased predation preference for leafhoppers treated with dsBSM. For male leafhoppers, the proportion attacked of the predator was 70%, with a significantly shorter predation time (71.3 s) compared to the dsGFP control (100.4 s) (Figure 4H). Similarly, for females, there was a higher predation rate (68.3%) and a significantly shorter predation time (79.6 s) for dsBSM treated groups compared to the dsGFP ones (30% and 109.9 s, respectively) (Figure 4I).

Figure 4.

Normal structure of the brochosome correlates with its antireflective properties

To further elucidate the correlation between brochosome morphology and its optical performance, we collected brochosomes from the wings of leafhoppers treated with dsGFP and dsBSM, respectively. Subsequently, these brochosomes were applied to the wings of brown planthoppers and quartz slides. SEM observations revealed that the coverage of brochosomes derived from both dsGFP and dsBSM treatments on the wings of brown planthoppers and quartz slides were nearly identical (figure supplement 6). Spectral measurements indicated that the application of brochosomes from both dsGFP and dsBSM treatments effectively reduced the reflectance of the brown planthopper wings and quartz slides. It should be noted that brochosomes from the dsGFP treatment exhibited significantly higher anti-reflective performance compared to those from the dsBSM treatment (Figure 4J and K). This indicates a close correlation between brochosome morphology and its optical performance.

Brochosomes as a camouflage coating for the leafhoppers in the family Cicadellidae

Based on the forementioned findings, brochosomes of N. cincticeps can be considered as an anti-reflective stealth coating against visual recognition by the predator P. paykulli. However, it remains unknown whether brochosomes also serve as a stealth coating in other leafhopper species. Therefore, we extended our investigation to include additional leafhopper species in the family Cicadellidae, including Recilia dorsalis, Empoasca onukii, and Psammotettix alienus. We carefully removed the brochosomes from the forewings of these leafhopper species using acetone, and found a significant enhancement in UV reflectance on the forewings of the leafhoppers (Figure 5A and B). In addition to N. cincticeps, we observed a considerably higher brightness of acetone-treated forewings compared to the untreated ones under UV light for R. dorsalis, E. onukii, and P. alienus (Figure 5A). This result suggests that brochosomes as an anti-reflective stealth coating may be commonly existed in various species of the family Cicadellidae.

Figure 5.

Previous research indicated that brochosome as a unique secretion in Cicadellidae (Rakitov & Gorb, 2013b; Rakitov et al., 2018). To investigate the conservation of genes linked to brochosome synthesis in Cicadellidae, we systematically screened these four BSM coding genes across 116 other hemipteran insect species (Supplementary Table 2). Results of homology alignment reveal that BSM coding genes exhibit marked orphan gene characteristics, with homologous genes exclusively present in Membracoidea. However, there are variations in the species distribution of different BSM coding genes. In Membracoidea species, homologous genes for BSM3 and 4 are observed, while BSM2 is identified in Cicadellidae insects, and BSM1 exclusively in Deltocephalinae insects (Figure 5C). Furthermore, the degree of homology of all BSM genes is positively correlated with taxonomic classification. An intriguing observation is that in the majority of Cicadellidae species with confirmed brochosome distribution, homologous gene distribution for BSM2 is detectable. In Cicadellidae species without brochosome distribution and in the majority of Membracidae, BSM2 genes are largely absent (Figure 5C). Since Membracidae evolved from Cicadellidae, we hypothesize that BSM homologous gene duplication/loss may be a key factor in brochosome formation/loss. In conclusion, our data suggest that in Cicadellidae, brochosome is synthesized by a conserved group of BSM coding genes, making it a widely distributed anti-reflective camouflage coating in Cicadellidae.

Discussion

Leafhoppers exhibit a distinctive grooming behavior, resulting in the deposition of a unique extra-cuticular coating known as brochosome(Rakitov, 2009). However, the precise characteristics and exact adaptive significance of this specialized coating remain largely unknown. Although they are widely believed to confer hydrophobicity, protecting leafhoppers from water and their own excreta(Rakitov & Gorb, 2013a), similar functionalities are attributed to cuticular waxes on the external surfaces of various insects, including leafhoppers(Andersen, 1979; Bello et al., 2022). Consequently, the functionalities of brochosomes may extend beyond this conventional understanding. In this study, we demonstrated that brochosomes exhibit robust anti-reflective properties, markedly decreasing the reflectance of the leafhopper cuticle surface, particularly in the ultraviolet spectral range. This substantial reduction is visibility to visual predators, contributing to the evasion and predation avoidance(Vukusic & Sambles, 2003; Watson et al., 2017). Thus, brochosomes function as a natural camouflage coating on the leafhopper cuticle surface, playing a pivotal role in reducing leafhoppers’ visibility to visual predators and offering a distinct advantage for their survival in predation scenarios as illustrated in Fig. 6.

Fig. 6.

The present work has elucidated the function of brochosome as a natural anti-reflective camouflage coating on leafhoppers’ extra-cuticular, with its coverage directly influencing the anti-reflective properties of the cuticle surface (Figure 2A-E and Figure 4A-G). Previous studies suggested that arthropods employ various nanostructures formed by proteins, lipids, wax, or adhesive substances on their cuticle surfaces to achieve anti-reflectivity(Silberglied, 1979). These nanostructures, initially identified in the compound eyes of moths, have been subsequently found to be widely distributed on the cuticle surfaces of insects(Bernhard, 1965; Blagodatski et al., 2015; Chotard et al., 2022; Ho et al., 2016; Monteiro et al., 2019; Stavenga et al., 2005). The nano- and microstructures on insect cuticle surfaces generate a gradual change in refractive index near the surface, leading to antireflectivity(Raut et al., 2011). The degree of reflection reduction depends on the shape of these nanostructures, studies in Drosophila melanogaster have confirmed that modifying the morphology of nanostructures in compound eyes directly impacts their optical performance(Kryuchkov et al., 2020). In this study, a thorough examination of the structural protein composition of brochosome, coupled with RNAi-mediated suppression of BSM coding gene expression, yielded brochosomes with regular structure (dsGFP treatment) and structurally abnormal brochosomes (dsBSM treatment) (Figure 4B-E). Subsequent in vitro experiments provided further evidence of the correlation between the normal structure of brochosome and its anti-reflective performance (Figure 4J and K). This correlation is likely attributed to the fact that the diameter of the hollow pits in brochosome structure (approximately 100nm) is considerably smaller than most spectral wavelengths(Burks et al., 2022; Rakitov, 2009). As light passes through these small pits, it undergoes diffraction, while light passing through the ridges of brochosome induces scattering. The interference of diffracted and scattered light between different pits and ridges contributes to the observed extinction features in brochosome(Raut et al., 2011). Biomimetic material and mathematical modeling analyses have clarified the relationship between the extinction features of brochosome and the spacing between hollow pits(Banerjee et al., 2023; Yang et al., 2017). Therefore, the regular morphology of brochosome is a robust guarantee for its excellent anti-reflective performance.

Moreover, we noticed a distinct contrast in the anti-reflective capabilities when applying brochosomes with regular and irregular structures onto different substrates (quartz glass and brown planthopper wings), especially in the UV region (Figure 4J and K). This indicates that the reduction in reflected light is not only related to the structure of brochosome but also to other factors. SEM observations revealed that some brochosome is embedded in the grooves of the leafhopper cuticle surface or brown planthopper wings, which is not observed on the smooth surface of quartz glass (Figure 4B-figure supplement 6). When the wavelength of light is higher than the size of the structure and the surface has a gradient refractive index, light interacts entirely with the rough surface, causing the light to gradually bend(Raut et al., 2011). Combined with the complex structure formed by brochosomes and nanostructures on the cuticle surface, this result in multiple internal reflections of light within these structures, increasing light absorption and significantly enhancing anti-reflective performance. Furthermore, the aromatic ring structures, conjugated double bonds, and peptide bonds in the proteins of both the cuticle and brochosomes may contribute to the absorption of ultraviolet light, providing another potential reason for their excellent anti-reflective capabilities in the ultraviolet spectral region. Therefore, the outstanding anti-reflective performance of brochosome on the insect cuticle surface might be a result of the combined effect of brochosome and cuticular nanostructures.

UV light serves as a crucial visual cue for various insect predators, enhancing foraging, navigation, mating behavior, and prey identification(Cronin & Bok, 2016; Morehouse et al., 2017; Silberglied, 1979), while the reflection of ultraviolet light from the insect cuticle surface increases the risk of predation by disrupting camouflage (Tovee, 1995) . In this study, we utilized the jumping spider P. paykulli to explore the impact of the brochosome coating on the leafhopper’s camouflage against predation. Hunting spiders, such as jumping spiders and wolf spiders, are primary predators of leafhoppers(Liu et al., 2015; Oraze & Grigarick, 1989) and the wavelength of UV light plays a crucial role as a visual cue for jumping spiders in identifying and locating their prey compare to the other wavelengths(Li & Lim, 2005; Zou et al., 2011). Our results revealed that P. paykulli displayed a preference for preying on N. cincticeps with a lower brochosome coverage on their cuticle surface (Figure 2G-J and Figure 4H-I). Thus, we hypothesize that the combination of brochosome and leafhopper cuticular microstructures exhibits superior anti-reflective performance in the UV region, effectively reducing UV reflection from leafhopper bodies and consequently diminishing the predator’s ability to identify them. Additionally, spectral measurement data indicated that the wing reflection spectrum of the brown planthopper (Figure 4J) closely resembled the body spectrum of 25-day-old female N. cincticeps (Figure 2E). It is possible that, in the same living environment, other Hemipteran insects would be more conspicuous to visual predators than leafhoppers. Brochosome coating could provide an advantage for leafhoppers to evade visual predators, thereby enhancing their survival rates in interspecific competition. This adaptation could potentially favor the survival of Cicadellidae in environments with high predation pressure, allowing them to thrive in a wider range of environments. Additionally, within the leafhopper population, the brochosome coverage on the cuticle surface of older individuals was notably lower than that of younger individuals, making elderly leafhoppers more susceptible to be detected and captured by predators (Figure 2F-I-figure supplement 1). This may contribute to eliminating elderly and weaker individuals in the leafhopper population, conserving food resources, and protecting the entire population. Therefore, we consider brochosome, as a form of anti-reflective camouflage coating on the leafhopper’s cuticle surface, represents a more advanced evolutionary strategy against visual predation. It not only provides a significant advantage in terms of camouflage and evasion from visual predators but also plays a crucial role in inter-species competition and stably maintenance of the insect population, highlighting its importance in the survival and evolution of leafhoppers.

In addition, we successfully identified four proteins essential for the structure of brochosomes for the first time (Figure 3). These BSM-encoded genes exhibit typical orphan gene characteristics, consisting with previous findings(Li et al., 2022; Rakitov et al., 2018; Yuan et al., 2023). Orphan genes originating from processes like de novo evolution, horizontal gene transfer (HGT), and duplication–divergence(Light et al., 2014; Tautz & Domazet-Lošo, 2011). To elucidate the evolutionary origin of the four identified BSM genes, we aligned them with all prokaryotic genomes in NCBI and approximately 1000 arthropod genomes. Interestingly, the homologous genes of these BSM genes were exclusively found in leafhopper genomes in the family Cicadellidae, suggesting that the origin of these BSM genes might not because of the de novo evolution or horizontal gene transfer. Combining the species distribution of the four identified BSM genes (as well as their homology) (Figure 5C), we hypothesize that the BSMs were potentially originated from a process involving duplication– divergence. In this scenario, a new gene would be created through gene duplication or transposition, undergoing fast adaptive evolution and losing similarity to the original gene(Light et al., 2014; Tautz & Domazet-Lošo, 2011). For Membracoidea insects, the majority insects in the family Cicadellidae (leafhoppers) can secrete brochosomes, while many Membracidae (treehoppers) insects exhibit grooming behavior without observable distinct setae on the cuticle surface(Rakitov, 1996). Considering that Membracidae are derived from Cicadellidae (Dietrich et al., 2001), the formation of the symbiotic relationship between treehoppers and ants might due to the loss of brochosome synthesis capability. Ants create a protective environment around treehoppers, deterring threats and reducing predation pressure, providing a relatively secure living environment, leading to lower predation pressure compared to leafhoppers(Del-Claro & Oliveira, 2000; Nelson & Mooney, 2022). Consequently, the need for the protective disguise represented by brochosomes is likely diminished. We speculate that environmental changes serve as one of the major driving forces in the evolution of brochosomes. Moreover, the distribution of paralogous genes BSM2 and BSM3 in Membracoidea (Figure 5C-figure supplement 4C) suggests that gene duplication/loss may be a primary factor causing the difference in brochosome synthesis capability between the insects of the families Cicadellidae and Membracidae. In eukaryotes, gene duplication/loss is a common mechanism for generating/losing gene functions, and this process promotes protein diversity and functional redundancy through the production of multiple homologous genes (Wong & Belov, 2012). Therefore, we propose that gene duplication may be a significant factor contributing to the formation and morphological/functional diversity of brochosomes in leafhoppers.

In summary, our exploration has underscored the indispensable role of brochosomes as a vital anti-reflective camouflage coating in leafhoppers, granting them a significant edge in eluding visual predators. The identification of the four BSM encoding genes, a groundbreaking revelation in brochosome research, points towards the likely involvement of gene duplication in the formation of these adaptive structures. Our study not only deepens our understanding of leafhoppers’ anti-predator tactics but also illuminates the distinct evolutionary trajectories utilized by these insects within their ecological niche, thus contributing to the development of biomimicry and the advancement of state-of-the-art camouflage technologies.

Materials and Methods

Insect

The leafhopper N. cincticeps and R. dorsalis adults were collected from a rice field in Jiaxing, Zhejiang Province, China in September 2020. P. alienus adults were collected from a wheat field in Shenyang, Liaoning Province, China in May 2020. E. onukii adult were collected from a tea-garden in Fuzhou, Fijian province, China in May 2020. The identity of the leafhopper species was confirmed using stereomicroscopy and mitochondrial cytochrome oxidase subunit 1 (CO1) sequence analysis (Supplementary Table 1). Collected leafhopper N. cincticeps and R. dorsalis are maintained in insect-proof greenhouses at 26 ± 1°C under a 16:8 h light:dark photoperiod and 50 ± 5% relative humidity on rice variety TaiChung Native 1(TN1).

The jumping spider P. paykulli were collected from a rice field in Ningbo, Zhejiang Province, China. Species identification was confirmed by assessing its morphological characteristics using stereomicroscopy and CO1 sequence analysis (Supplementary Table 1). Jumping spiders were fed about their own mass of leafhoppers N. cincticeps three times each week and examined 14-52 days following collection, as previously reported (Taylor et al., 2014).

Electron microscopy

The Malpighian tubules of N. cincticeps were dissected using an astereomicroscope, serially fixed overnight at 4°C with 2.5% glutaraldehyde in 0.1 mol/L PBS, and post-fixed for 2 hours at room temperature with 1% OsO4. Following that, samples were dehydrated in a series of ethanol solutions (50%, 70%, 80%, 90%, and 95%), then permeabilized with 100% ethanol and 100% acetone, followed by a series of epoxies in acetone (50%, 75%, and 100%). The pierced tissue was then immersed in epoxy resin and baked for more than 24 hours at 70°C. Ultrathin slices were cut and stained with uranyl acetate for 15 minutes and lead citrate for 5 minutes, and examined by Hitachi H7800 microscope (Hitachi, Japan).

Scanning electron microscopy was used to observe the morphology of leafhopper brochosomes. Samples were prepared as previously described(Rakitov & Gorb, 2013a). Leafhopper forewings were obtained by dissection under the stereomicroscope and glued onto SEM metal stubs with a double-sided carbon tape. The dried samples were coated with gold for 1 min in a MC1000 Ion Sputter Coater (Hitachi, Japan) and viewed by a HITACHI Regulus 8100 SEM (Hitachi, Japan).

Spider predation experiment

Jumping spiders were starved for three days prior to the predation experiment to ensure that participating spiders were hungry enough to attack leafhoppers in our predation preference tests, but not so hungry that they would attack the first prey they encountered (without being choosy)(Taylor et al., 2014). For the jumping spiders to prey on, we placed two leafhoppers in a 9 cm diameter test arena (the bottom of the dish was covered with filter paper). Before starting the test, the spiders were placed in the arena for 15 minutes to adapt, and then they were released and permitted to prey on the leafhoppers. We directly observed the feeding process of the jumping spiders and recorded when the spiders attacked the first leafhopper and which leafhopper they preyed on. The predation finishes when the first leafhopper is attacked or when the jumping spider does not attack any leafhopper within 5 minutes. Each pair of jumping spider predation tests were independently replicated more than 60 times.

The distribution of cuticle surface brochosomes and the optical properties of male and female insects at various periods post-eclosion

To investigate the distribution of brochosomes on the cuticle surface of adult N. cincticeps at various post-eclosion periods, we first collected female and male adult leafhoppers at 5-, 10-, 15-, 20-, and 25-days post-eclosion and dissected the forewings under a Nikon SMZ25 microscope (Nikon, Japan). The forewings were gold-sprayed and examined using HITACHI Regulus 8100 SEM (Hitachi, Japan). At each time point, 30 samples were randomly selected for SEM imaging. A 10 μm × 10 μm area within the SEM images was used to assess brochosome coverage, and ImageJ was employed for analysis(Schneider et al., 2012). Since brochosomes are synthesized at the distal section of the Malpighian tubule and the morphology of the Malpighian tubule in leafhoppers can also reflect brochosomes synthesis, we used Nikon SMZ25 microscope (Nikon, Japan) to examine the morphology of the Malpighian tubule in female and male adult leafhoppers at 5-, 10-, 15-, 20-, and 25-days post-eclosion.

To examine the optical properties of male and female insects at various periods post-eclosion, we first collected female and male adult leafhoppers at 5-, 10-, 15-, 20-, and 25-days post-eclosion, freeze-killed them and placed them under white or UV light for observation through a Nikon SMZ25 microscope (Nikon, Japan). Following that, the forewings of the leafhoppers were meticulously dissected, and the specular reflectance spectra of the forewings (25 × 25 μm) were precisely measured utilizing a 20/30 PV UV-Vis-NIR microspectrophotometer (CRAIC Technologies Inc., USA). UV-Vis-NIR reflectance spectra (250 to 1000 nm) were obtained using a 10× UV-absorbing glass objective lens, with a minimum of five replicates per sample.

Identification of brochosome structural protein-coding genes

Candidate brochosome structural protein-coding genes were identified through a combined analysis of transcriptomic and proteomic data. To knock down the expression of these candidates, RNAi was conducted by microinjecting double-stranded RNA (dsRNA) into the abdomens of fifth instar nymphs. The dsRNAs targeting 500-1000 bp regions of Malpighian tubule-specific expression genes or GFP were synthesized in vitro using the T7 RiboMAX Express RNAi System (Promega, USA). The 50 fifth instar nymphs of N. cincticeps were microinjected with 30 nl dsRNA (0.5 μg μl^-1) using the Nanoject II Auto-Nanoliter Injector (Drummond, USA). Thereafter, they were transferred to healthy rice seedlings for recovery. Forewings of treated leafhoppers were collected 7 days after microinjection (about 4-5 days post-eclosion), sprayed with gold, and analyzed by HITACHI Regulus 8100 SEM (Hitachi, Japan). Screening identified four brochosome structural protein-coding genes, and inhibiting their expression resulted in significant changes in brochosome morphology. The full-length sequence of these genes was obtained using by 5’-rapid amplification of cDNA ends (RACE) and 3’-RACE using SMARTer RACE 5’/3’ Kit (Clontech, USA).

To analyze the temporal expression profiles of four brochosome structural protein-coding genes (GenBank accession number PP273097, PP273098, PP273099, PP273100), the whole body, salivary gland, midgut, ovary, and testis were dissected from 200 adult insects, total RNA in different tissue were extracted using TRIzol Reagent (Invitrogen, USA). The relative expression of these genes in different tissues of N. cincticeps adults was detected by RT-qPCR assay using a QuantStudio^TM 5 Real-Time PCR system (Thermo Fisher Scientific, USA). The detected transcript levels were normalized to the transcript level of the housekeeping gene elongation factor 1 alpha (EF1α) (GenBank accession number AB836665) and estimated by the 2−△△Ct (cycle threshold) method. Using the same procedure, the expression levels of these brochosome structural protein-coding genes were evaluated in female and male adult leafhoppers at 5-, 10-, 15-, 20-, and 25-days post-eclosion.

Knocking down in vivo expression of BSM-coding genes in N. cincticeps

Following the prior procedure, dsRNA produced from four BSM-coding genes was injected into the abdomen of fifth instar nymphs of N. cincticeps by microinjection, and a control group injected with dsGFP was established. Seven days after injection, the forewings and Malpighian tubules of the dsBSM and dsGFP treated leafhoppers were dissected, and the morphology of brochosomes on the cuticle surface of leafhoppers and the synthesis of brochosomes in Malpighian tubules were examined by SEM and TEM, respectively. To examine the optical properties, the dsBSM and dsGFP treated leafhoppers, freeze-killed them and placed them under white or UV light for observation through a Nikon SMZ25 microscope (Nikon, Japan). Subsequently, the forewings of the leafhoppers were meticulously dissected, and the specular reflectance spectra of the forewings (25 × 25 μm) were precisely measured utilizing a 20/30 PV UV-Vis-NIR microspectrophotometer (CRAIC Technologies Inc., USA). UV-Vis-NIR reflectance spectra (250 to 1000 nm) were obtained using a 10× UV-absorbing glass objective lens, with a minimum of five replicates per sample. Finally, the predation preference of the jumping spiders on the dsBSM and dsGFP treated leafhoppers was examined by a predation experiment.

In vitro measurements were conducted to assess the relationship between brochosome morphology and their optical performance

To collect approximately 500 N. cincticeps treated with dsGFP or dsBSM, dissect the forewings of the leafhoppers and immerse them in acetone in a 50 mL centrifuge tube. Place the centrifuge tubes on a room temperature orbital shaker at 50 rpm for 12 hours to separate the forewings from the leafhoppers due to friction. Following this, centrifuge at 1000g for 10 minutes to separate the brochosomes. Resuspend the particles in fresh acetone, briefly sonicate, then centrifuge at 1000g for 10 minutes. Repeat this process three times. Resuspend the collected brochosomes from dsGFP and dsBSM-treated leafhopper forewings in acetone and adjust the two suspension solutions to the same OD280 reading. Next, using a pipette, carefully drop the brochosome solution onto quartz slides and brown planthopper wings. Allow the acetone to completely evaporate at room temperature. At the same time, we also set up quartz slides and brown planthopper wings that were treated with acetone alone as negative controls. Prepared quartz slides and brown planthopper wings can be observed under SEM to examine the distribution of brochosomes. Additionally, UV-Vis-NIR reflectance spectra (250 to 1000 nm) were obtained using a 20/30 PV UV-Vis-NIR microspectrophotometer (CRAIC Technologies Inc., USA), with a minimum of five replicates per sample.

Removal of leafhopper forewing brochosome by acetone

Previous research has shown that acetone may efficiently remove brochosomes from the cuticle surface of leafhoppers(Rakitov et al., 2018). We manually removed the brochosomes from the forewings of leafhoppers using acetone and examined the imaging under white or UV light for observation through a Nikon SMZ25 microscope (Nikon, Japan). UV-Vis-NIR reflectance spectra (250 to 1000 nm) were obtained using a 20/30 PV UV-Vis-NIR microspectrophotometer (CRAIC Technologies Inc., USA), with a minimum of five replicates per sample.

Bioinformatics analysis and phylogenetic tree

The brochosome is a unique secretion produced by leafhoppers (Cicadellidae). In order to determine the distribution of brochosome structural protein-coding genes in Hemiptera. The brochosome structural protein-coding genes were used to queries to search for homologous sequences in Hemiptera transcriptomes. We examined 116 Hemiptera species, encompassing major families (Acanaloniidae, Achilidae, Aetalionidae, Aphrophoridae, Caliscelidae, Cercopidae, Cicadellidae, Cicadidae, Cixiidae, Clastopteridae, Delphacidae, Derbidae, Dictyopharidae, Epipygidae, Eurybrachidae, Flatidae, Fulgoridae, Issidae, Machaerotidae, Melizoderidae, Membracidae, Myerslopiidae, Nogodinidae, Peloridiidae, Ricaniidae, Tettigarctidae, Tettigometridae, Theaceae, Tropiduchidae). Transcriptome data of 116 Hemiptera species were downloaded from the NCBI Sequence Read Archive (Supplementary Table 2), and assembled using SOAPdenovo-Trans (version 1.01)(Xie et al., 2014). The resulting transcripts were filtered to remove potential contaminants. We compared four identified BSM-encoded genes with these transcriptomes using the tblastn (E-value < 1.0e–5). Heatmaps were generated using TBtools software(Chen et al., 2023). At least one homologous gene for the four BSM genes was identified in 66 species. A species phylogenetic tree for these 66 Hemiptera species was constructed following the methods outlined in previous studies(Johnson et al., 2018).

The amino acid sequences of BSM2 and BSM3 were aligned using the Clustal W multiple sequence alignment program(Larkin et al., 2007). AlphaFold2 was employed to predict the three-dimensional structures of BSM2 and BSM3, and structural alignments were conducted using PyMOL(DeLano, 2002; Jumper et al., 2021). In Membracoidea, the maximum likelihood (ML) phylogenetic tree for BSM2 and BSM3 was constructed using IQtree in PhyloSuite(Minh et al., 2020; Xiang et al., 2023).

Statistical analyses

All experiments were performed at least three independent replicates and statistical analyses were performed with GraphPad Prism8.0 software. Statistical significance was calculated by a two-tailed Student’s t-test, one-way ANOVA, two-way ANOVA and/or unpaired Student’s t-test. P-values < 0.05 were considered statistically significant.

Acknowledgements

This work received support from the National Natural Science Foundation of China (U23A6006, U20A2036, 32270150) and the Natural Science Foundation of Ningbo Municipality (2023J112). The author acknowledges Dr. Yuan Cheng from the Instrumentation and Service Center for Molecular Science (ISCMS) at Westlake University for assistance with optical measurement and data interpretation. We are grateful for the support provided by the Bioimaging Center of the Bioimaging Center, State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Virology, Ningbo University, in TEM and SEM measurement and data interpretation. Additionally, we thank Professor Shou-Wei Ding (University of California, Riverside, CA, United States) for his valuable and constructive suggestions for manuscript improvement.

Author contributions

W.W. J.-M.L., C.-X.Z., and J.-P.C. designed research; W.W. Q.M. Z.Y. Z.L. performed research; W.W. and Z.Y. analyzed data; and W.W. H.-W.S., J.-M.L., C.-X.Z., and J.-P.C wrote the paper. All authors gave final approval for publication. All authors have read and agreed to the published version of the manuscript.

Data availability

All the data needed to understand and assess the conclusions of this research are available in the article, Supplementary Materials, and GenBank (accession number PP273097, PP273098, PP273099, PP273100). Any additional information required to reanalyse the data reported in this paper is available from the lead contact upon request.

Declaration of interests

The authors declare no competing interests.