Red and black: A β-carotene-binding protein carrying a red pigment regulates body-color transition in locusts

Changes of body color have important effects for animals in adapting to variable environments. The migratory locust exhibits body color polyphenism between solitary and gregarious individuals, with the former displaying a uniform green coloration and the latter having a prominent pattern of black dorsal and brown ventral surface. However, the molecular mechanism underlying the density-dependent body color changes of conspecific locusts remain largely unknown. Here, we found that up regulation of β-carotene-binding protein promotes the accumulation of red pigment, which added to the green color palette present in solitary locusts changes it from green to black, and that down regulation of this protein led to the reverse, changing the color of gregarious locusts from black to green. Our results provide insight that color changes of locusts are dependent on variation in the red β-carotene pigment binding to βCBP. This finding of animal coloration corresponds with trichromatic theory of color vision.

showed the expected uniform green coloration ( Figure 1A). 105 The body coloration of gregarious locusts is characterized as a black 106 tergum of the thorax and abdomen, in particular, a very black pronotum. To 107 delineate gene activity changes potentially associated with the regulation of 108 6 phase-dependent body color traits, we performed transcriptome sequencing on 109 pronotum integuments of gregarious and solitary locusts. We identified a total 110 of 1653 DEGs between the gregarious and solitary locusts (Figure 1-figure   111 supplementary 1A), and 26% (430) of these DEGs were protein-coding genes 112 (Figure 1-figure supplementary 1B). Gene ontology analysis of these 430 113 protein-coding DEGs indicated that many of them encode cuticle metabolism-114 related proteins involved in chitin metabolic, melatonin metabolic, transport, 115 and catecholamine catabolic activities (Figure 1-figure supplementary 1C). 116 Sixty-eight of the 430 protein-coding DEGs are involved in pathways 117 associated with animal coloration ( Figure 1B). The top 17 DEGs (Log FC > 2.8, 118 FDR < 1e-5) of these 68 genes were used to validate the differential expression 119 patterns between the gregarious and solitary locusts via qPCR ( Figure 1C).  To explore whether these 8 genes regulate body color plasticity during 129 locust phase transition, we carried out transcript knockdown analyses of these showed a significant effect on the regulation of body color transition when it was 132 silenced. After molting, the dark pattern in the pronotum decreased markedly in 133 60% of the gregarious locusts after βCBP knockdown (Figure 1figure   134 supplementary 2). These data imply that CBP serves as a key gene involved in 135 body color change.

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Because the expression pattern of βCBP in the pronotum was consistent 168 with that in the tergum of the thorax and abdomen, the pronota were considered 169 representative of all terga of locusts in the following analyses. We also found 170 that the level of CBP protein in the locust pronotum integuments was 171 significantly higher in the gregarious locusts than in the solitary locusts 172 ( Figure 2D). Moreover, the protein levels of βCBP declined when the locusts 173 were isolated and increased under the crowding conditions ( Figure 2D). This   The β-carotene content in the gregarious locusts was 2.8-fold higher than that      To confirm the role of the βCBP-pigment complex in black color pigmentation, 261 we performed a pigment immunoprecipitation assay using an antibody against 262 13 the βCBP protein in vitro and in vivo to examine the binding capacity and 263 complex coloration of βCBP with β-carotene ( Figure 5A and B). We incubated 264 recombinant βCBP (rβCBP) with β-carotene under immunoprecipitation by 265 using βCBP antibody conjugated with protein A-Sepharose ( Figure 5A). 266 Immunoprecipitation of β-carotene-rβCBP formed a red complex, whereas no 267 red precipitate formed when β-carotene was added to bovine serum albumin 268 (BSA) or when β-carotene in the absence of rβCBP was incubated in the 269 binding buffer followed by immunoprecipitation ( Figure 5A). It could be inferred    Therefore, the mechanisms of color variation are highly conserved and can 395 offer insights into the adaptive evolution of gene regulation. Our study shows a 396 new "palette effect" mechanism by which the red βCBP-β-carotene pigment 397 complex can act as a switch to coordinate between black and green coloration.

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This mechanism of color pattern change may be evolutionarily conserved 399 among multiple species, although additional functional work is needed to 400 assess this hypothesis.      insects were fed a synthetic diet without β-carotene and exposed to crowding.

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Rearing jars were cleaned and fresh diets were provided every day. A total of 464 29 nymphs were fed a synthetic diet in each of the treatment and control groups.     The same sample was re-extracted twice according to the same protocol as  The following figure supplement is available for figure 1: 785 Source data 1. Numerical data that are represented as graphs in Figure 1C.  *p < 0.05; **p < 0.01; ***p < 0.001.

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The following figure supplement is available for figure 3: 866 Source data 1. Numerical data that are represented as graphs in Figure 3A,