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Caption : figure s1. schematic representation of crusticul protein functional domains, as derived by smart (see materials and methods) and signal peptide prediction. a) all crusticuls contain a chitin-binding domain. red boxes denote signal peptides, purple boxes denote low complexity regions. b) signal peptide prediction for all crusticul proteins by signalp-5.0, with calculated probabilities of signal peptide presence. all crusticuls are predicted to contain a signal peptide. 

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Caption : figure s2. crusticul proteins sequence alignment and similarity. a) protein sequence alignment of all nine crusticul proteins expressed in the carapace cuticle-forming epithelium. conserved regions and domains are indicated below the alignment. b) crusticul protein sequence similarities. c) crusticul protein conserved domain sequence similarities.    

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Caption : figure s3. cdna and protein gels showing crusticul expression in the c. quadricarinatus exoskeleton and its forming epithelia during the molt cycle.  a) spatial-temporal expression of crusticul genes expressed mainly during late premolt. for each gene, spatial-temporal expression is shown in five tissues sampled in animals at four different molt stages. nc – negative control. b) crustacyanin and crusticul proteins separated by sds-page after elution from a homemade ms-ms column with egta or urea buffer (see materials and methods). mw = molecular weight. 

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Caption : figure s4. the effects of crusticul silencing on c. quadricarinatus cuticle protein profiles. a) sds-page of rcqcrusticuls1 at known concentrations and the resulted calibration curve (protein peak intensity measured in arbitrary units), which generated a standard curve for determining crusticul protein concentrations upon transcript silencing. r-squared coefficient and calibration curve linear equations are shown. b) ms-ms analysis of a non-crusticul proteins from control and silenced cuticle protein extracts.

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Caption : figure s5. cuticle cross-section reveals crusticul silencing-mediated effects on chitin fibers. a) sem image of a control cuticle cross-section. b) sem image of a crusticul-silenced cuticle cross-section. the three cuticular layers are identified by red arrows in both samples. scale bars, 10 µm.

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Caption : figure s6. protein chitin-binding and protein activity assays performed in ionic liquid. a) chitin-binding assay of bsa and recombinant crusticul 1 protein (rcrus). positive control – molecular weight standard protein ladder, pure bsa and recombinant crusticul 1 protein samples. wash – excess protein wash fractions. nb – non-bound fractions. chitin - chitin-bound protein fractions. b) absorption measured with a lysozyme detection kit after lysis of the micrococcus lysodeikticus substrate (measured as absorbance at 450 nm).

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Caption : figure s7. crusticuls phylogeny across decapod crustaceans. maximum likelihood phylogenetic tree of crusticuls from different decapod crustaceans. phylogeny of c. quadricarinatus, p. clarkii, l. vannamei, m. rosenbergii and p. platyceros crusticul proteins is portrayed. bootstrap test (n=1000) results are shown at each node junction. crusticul proteins from c. quadricarinatus appear in blue text color against a light orange background.

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Caption : video s1. reconstructed block of control cuticle, revealing the wild type organization of the c. quadricarinatus exoskeleton chitinous scaffold. ortho-slices along both the x and y axes are shown, revealing the different cuticular layers.   

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Caption : video s2. reconstructed block of treated cuticle, revealing impairment of wild type organization of the c. quadricarinatus exoskeleton chitinous scaffold following crusticul knock-down. ortho-slices along both the x and y axes are shown, revealing the different cuticular layers.