Brochosomes as an antireflective camouflage coating for leafhoppers

Reviewer #1 (Public review):

Summary:

Evading predation is of utmost importance for most animals and camouflage is one of the predominant mechanisms. Wu et al. set out to test the hypothesis of a unique camouflage system in leafhoppers. These animals coat themselves with brochosomes, which are spherical nanostructures that are produced in the Malpighian tubules and are distributed on the cuticle after eclosion. Based on previous findings on the reflectivity properties of brochosomes, the authors provide very good evidence that these nanostructures indeed reduce the reflectivity of the animals thereby reducing predation by jumping spiders. Further, they identify four proteins, which are essential for the proper development and function of brochosomes. In RNAi experiments, the regular brochosome structure is lost, the reflectivity reduced and the respective animals are prone to increased predation. Finally, the authors provide some phylogenetic sequence analyses and speculate about the evolution of these essential genes.

Strengths:

The study is very comprehensive including careful optical measurements, EM and TM analysis of the nanoparticles and their production line in the malphigian tubules, in vivo predation tests, and knock-down experiments to identify essential proteins. Indeed, the results are very convincingly in line with the starting hypothesis such that the study robustly assigns a new biological function to the brochosome coating system.

A key strength of the study is that the biological relevance of the brochosome coating is convincingly shown by an in vivo predation test using a known predator from the same habitat.

Another major step forward is an RNAi screen, which identified four proteins, which are essential for the brochosome structure (BSMs). After respective RNAi knock-downs, the brochosomes show curious malformations that are interesting in terms of the self-assembly of these nanostructures. The optical and in vivo predation tests provide excellent support for the model that the RNAi knock-down leads to a change of brochosomes structure, which reduces reflectivity, which in turn leads to a decrease of the antipredatory effect.

Weaknesses:

The reduction of reflectivity by aberrant brochosomes or after ageing is only around 10%. This may seem little to have an effect in real life. On the other hand, the in vivo predation tests confirm an influence. Hence, this is not a real weakness of the study - just a note to reconsider the wording for describing the degree of reflectivity.
The single gene knockdowns seemed to lead to a very low penetrance of malformed brochosomes (Figure Supplement 3). Judging from the overview slides, less than 1% of brochosomes may have been affected. A quantification of regular versus abnormal particles in both, wildtype and RNAi treatments would have helped to exclude that the shown aberrant brochosomes did not just reflect a putative level of "normal" background defects. Of note, the quadruple knock-down of all BSMs seemed to lead to a high penetrance (Figure 4), which was already reflected in the microtubule production line. While the data shown are convincing, a quantification might strengthen the argument.

While the RNAi effects seemed to be very specific to brochosomes and therefore very likely specific, an off-target control for RNAi was still missing. Finding the same/similar phenotype with a non-overlapping dsRNA fragment in one off-target experiment is usually considered required and sufficient. Further, the details of the targeted sequence will help future workers on the topic.

The main weakness in the current manuscript may be the phylogenetic analysis and the model of how the genes evolved. Several aspects were not clearly or consistently stated such that I felt unsure about what the authors actually think. For instance: Are all the 4 BSMs related to each other or only BSM2 and 3? If so, not only BSM2 and 3 would be called "paralogs" but also the other BSMs. If they were all related, then a phylogenetic tree including all BSMs should be shown to visualize the relatedness (including the putative ancestral gene if that is the model of the authors). Actually, I was not sure about how the authors think about the emergence of the BSMs. Are they real orphan genes (i.e. not present outside the respective clade) or was there an ancestral gene that was duplicated and diverged to form the BSMs? Where in the phylogeny does the first of the BSMs or ancestral proteins emerge (is the gene found in Clastoptera arizonana the most ancestral one?)? Maybe, the evolution of the BSMs would have to be discussed individually for each gene as they show somewhat different patterns of emergence and loss (BSM4 present in all species, the others with different degrees of phylogenetic restriction). Related to these questions I remained unsure about some details in Figure 5. On what kind of analysis is the phylogeny based? Why are some species not colored, although they are located on the same branch as colored ones? What is the measure for homology values - % identity/similarity? The homology labels for Nephotetix cincticeps and N. virescens seem to be flipped: the latter is displayed with 100% identity for all genes with all proteins while the former should actually show this. As a consequence of these uncertainties, I could not fully follow the respective discussion and model for gene evolution.

Conclusion:

The authors successfully tested their hypothesis in a multidisciplinary approach and convincingly assigned a new biological function to the brochosomes system. The results fully support their claims - only the quantification of the penetrance in the RNAi experiments would be helpful to strengthen the point. The author's analysis of the evolution of BSM genes remained a bit vague and I remained unsure about their respective conclusions.

The work is a very interesting study case of the evolutionary emergence of a new system to evade predators. Based on this study, the function of the BSM genes could now be studied in other species to provide insights into putative ancestral functions. Further, studying the self-assembly of such highly regular complex nano-structures will be strongly fostered by the identification of the four key structural genes.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors investigate the optical properties of brochosomes produced by leafhoppers. They hypothesize that brochosomes reduce light reflection on the leafhopper's body surface, aiding in predator avoidance. Their hypothesis is supported by experiments involving jumping spiders. Additionally, the authors employ a variety of techniques including micro-UV-Vis spectroscopy, electron microscopy, transcriptome and proteome analysis, and bioassays. This study is highly interesting, and the experimental data is well-organized and logically presented.

Strengths:

The use of brochosomes as a camouflage coating has been hypothesized since 1936 (R.B. Swain, Entomol. News 47, 264-266, 1936) with evidence demonstrated by similar synthetic brochosome systems in a number of recent studies (S. Yang, et al. Nat. Commun. 8:1285, 2017; L. Wang, et al., PNAS. 121: e2312700121, 2024). However, direct biological evidence or relevant field studies have been lacking to directly support the hypothesis that brochosomes are used for camouflage. This work provides the first biological evidence demonstrating that natural brochosomes can be used as a camouflage coating to reduce the leafhoppers' observability of their predators. The design of the experiments is novel.

Weaknesses:

(1) The observation that brochosome coatings become sparse after 25 days in both male and female leafhoppers, resulting in increased predation by jumping spiders, is intriguing. However, since leafhoppers consistently secrete and groom brochosomes, it would be beneficial to explore why brochosomes become significantly less dense after 25 days.

(2) The authors demonstrate that brochosome coatings reduce UV (specular) reflection compared to surfaces without brochosomes, which can be attributed to the rough geometry of brochosomes as discussed in the literature. However, it would be valuable to investigate whether the proteins forming the brochosomes are also UV absorbing.

(3) The experiments with jumping spiders show that brochosomes help leafhoppers avoid predators to some extent. It would be beneficial for the authors to elaborate on the exact mechanism behind this camouflage effect. Specifically, why does reduced UV reflection aid in predator avoidance? If predators are sensitive to UV light, how does the reduced UV reflectance specifically contribute to evasion?

(4) An important reference regarding the moth-eye effect is missing. Please consider including the following paper: Clapham, P. B., and M. C. Hutley. "Reduction of lens reflection by the 'Moth Eye' principle." Nature 244: 281-282 (1973).

(5) The introduction should be revised to accurately reflect the related contributions in literature. Specifically, the novelty of this work lies in the demonstration of the camouflage effect of brochosomes using jumping spiders, which is verified for the first time in leafhoppers. However, the proposed use of brochosome powder for camouflage was first described by R.B. Swain (R.B. Swain, Notes on the oviposition and life history of the leafhopper Oncometopta undata Fabr. (Homoptera: Cicadellidae), Entomol. News. 47: 264-266 (1936)). Recently, the antireflective and potential camouflage functions of brochosomes were further studied by Yang et al. based on synthetic brochosomes and simulated vision techniques (S. Yang, et al. "Ultra-antireflective synthetic brochosomes." Nature Communications 8: 1285 (2017)). Later, Lei et al. demonstrated the antireflective properties of natural brochosomes in 2020 (C.-W. Lei, et al., "Leafhopper wing-inspired broadband omnidirectional antireflective embroidered ball-like structure arrays using a nonlithography-based methodology." Langmuir 36: 5296-5302 (2020)). Very recently, Wang et al. successfully fabricated synthetic brochosomes with precise geometry akin to those natural ones, and further elucidated the antireflective mechanisms based on the brochosome geometry and their role in reducing the observability of leafhoppers to their predators (L. Wang et al. "Geometric design of antireflective leafhopper brochosomes." Proceedings of the National Academy of Sciences 121: e2312700121 (2024))