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

(A) Male adult of the green rice leafhopper N. cincticeps. Bar, 1 mm. (B) Brochosomes on the surface of the forewing of N. cincticeps. Bar, 1 μm. (C, D) The morphologies of brochosomes by SEM (C) and TEM (D). Bar, 200 nm. (E) Alimentary tract and the Malpighian tubules of N. cincticeps. The leafhopper N. cincticeps have two pairs of Malpighian tubules, each divided into proximal segment, distal segment, and terminal segment. The brochosomes are synthesized in the distal segment, which is dilated and rod-shaped. Bar, 1 mm. (F) The distal segment epithelial cell displays an extensive rough endoplasmic reticulum and multiple Golgi regions (red arrow) containing developing brochosomes in its basal portion, as well as a number of secretory vacuoles with mature brochosomes near the cell border. Bar, 5 μm. (G) The initial stage of the development of brochosome. Bar, 500 mm. (H) The two brochosomes that are developing inside the primary vesicles are shown in close-up. Regular invaginations appear on the surface of the growing brochosome at the same time that its matrix separates into a looser core and a denser wall. Bar, 500 nm. (I) Larger vesicles containing numerous BS, formed by the fusion of multiple primary vesicles. The surface of the brochosomes has regular cell-like invaginations, and it is surrounded by amorphous flocculent material with a moderate electron density. Bar, 1 μm. (J) A vesicle filled with mature brochosomes, each mature brochosome has a spherical inner cavity and a well-swollen outer margin of the septa. Bar, 1 μm. E, F, G, H are the enlargement of boxed area in D. Fc, filter chamber; Mg, midgut; Mt, Malpighian tubules; mtd, distal segment of the Malpighian tubule; mtp, proximal segment of the Malpighian tubule; mtt, terminal segment of the Malpighian tubule. All images are representative of at least three replicates.

The distribution of brochosomes on the cuticle surface of leafhopper N. cincticeps are associated with predation by jumping spiders.

(A, B) Images of leafhopper N. cincticeps males and females in white (A) and ultraviolet light (B) at 5 and 25 days post-eclosion, respectively. Bar, 1 mm. (C, D) The distribution of brochosomes on the surface of a forewing (C) and morphological changes of the Malpighian tubules (D) of N. cincticeps males and females at 5 and 25 days post-eclosion. Bar, 5 μm in C; Bar, 1 mm in D. (E) Reflectance spectra of female and male forewing of N. cincticeps at 5 and 25 days post-eclosion. (F) Images of the jumping spider P. paykulli. Bar, 2 mm. (G-J) Jumping spiders prefer leafhoppers with little brochosome covering as food. In predation experiment, jumping spiders offered N. cincticeps male and female at 5 days post-eclosion (G), male and female at 25 days post-eclosion (H), males at 5 and 25 days post-eclosion (I), and females at 5 and 25 days post-eclosion (J). Data on predation times are displayed using the traditional box and whisker shapes. All box plots with whiskers represent the data distribution based on five number summary statistics (maximum, third quartile, median, first quartile, minimum), each dot in box plot represents an independent experiment. **P < 0.01, ****P < 0.0001, ns no significance, Statistical significance was determined by unpaired t test with Welch’s correction method. Predation preference is shown in the pie chart. All images are representative of at least three replicates.

Identification of brochosome structural proteins.

(A) Morphological of brochosomes on the forewing of leafhopper N. cincticeps at 7 days post-microinjection of dsRNA. Bar, 500 nm. (B) Gene structures of BSM-1, BSM-2, BSM-3 and BSM-4. (C-F) Transcript levels of BSM-1 (C), BSM-2 (D), BSM-3 (E) and BSM-4 (F) at 7 days post-microinjection of dsRNA. Each data point represents the result of one independent experiment. (G-J) The abundance of BSM transcripts in different tissues and whole bodies of N. cincticeps was determined by RT-qPCR. Notably, BSM-1 (G), BSM-2 (H), BSM-3 (I), and BSM-4 (J) exhibited specific expression in the Malpighian tubules. Each data point represents the result of one independent experiment. (K-N) The expression patterns of BSM-1 (K), BSM-2 (L), BSM-3 (M) and BSM-4 (N) transcripts were examined in male and female leafhopper at 5, 15, and 25 days post-eclosion. Results were obtained from 3 independent experiments. For C-N, data shown are mean ± SD values. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significance (C-F, two tailed Student’s t-test; G-J, one-way ANOVA; K-N, two-way ANOVA). All images are representative of at least three replicates.

RNAi inhibits brochosome synthesis, alters brochosome morphology, and influences predation in jumping spiders.

(A) Images of leafhopper N. cincticeps males and females in white and ultraviolet light after dsGFP or dsBSM treatment, respectively. Bar, 1 mm. (B, C) Morphology of the brochosome on the forewings of leafhoppers after dsGFP (B) and dsBSM (C) treatment. Bar, 2 μm. (D, E) Morphology of the brochosome in the distal segment epithelial cells of Malpighian tubules after dsGFP (D) and dsBSM (E) treatment. Bar, 2 μm. (F) The transcript levels of BSM-1, BSM-2, BSM-3 and BSM-4 at 7 days after dsGFP and dsBSM treatment. Each data point represents the outcome of an individual independent experiment. The presented data are expressed as mean ± SD values. Statistical significance is denoted as *P < 0.05 and **P < 0.01, determined by two-way ANOVA. (G) Reflectance spectra of female and male forewing of N. cincticeps at 7 days after dsGFP and dsBSM treatment. (H and I) Jumping spiders prefer to prey on dsBSM-treated leafhoppers. Predation efficiency and preference of jumping spiders on males (H) and females (I) N. cincticeps after dsGFP and dsBSM treatment in the predation experiment. Data on predation times are displayed using the traditional box and whisker shapes. All box plots with whiskers represent the data distribution based on five number summary statistics (maximum, third quartile, median, first quartile, minimum), each dot in box plot represents an independent experiment. **P < 0.01, ***P < 0.001, Statistical significance was determined by unpaired t test with Welch’s correction method. Predation preference is shown in the pie chart. (J, K) The morphology of brochosomes is related to their optical performance. Collect brochosomes treated with dsGFP and dsBSM separately, apply them to brown planthopper wings (J) and quartz slides (K), and set up a control group with acetone application only. All images are representative of at least three replicates.

Brochosome coating on Cicadellidae cuticle surface is essential for their optical qualities.

(A) Images of forewings of four leafhopper species before and after acetone treatment in white and ultraviolet light. Bar, 1mm. (B) Reflection spectra of the forewings of the N. cincticeps before and after acetone treatment. (C) Phylogenetic associations between the BSM and phylogeny of Hemipteran lineages. The left column describes the phylogeny of 66 representative species of Hemiptera with BSM proteins. In the right column, the presence of BSM-encoded genes in different Hemiptera species is illustrated along with their homology analysis with N. cincticeps. White indicates the absence of related genes; the color gradient represents the degree of sequence homology. All images are representative of at least three replicates.

Brochosomes serve as an anti-reflective camouflage coating on the cuticle surface of leafhoppers.

Brochosomes effectively reduce the reflection of various wavelengths of light, particularly in the UV region. UV light is a crucial visual cue for numerous visual predators to identify and locate prey. Brochosomes efficiently decrease the UV light reflection on the surface of leafhoppers, thereby reducing their exposure risk to visual predators and facilitating evasion of visual predation.