1 SCIENTIFIC REPORTS | (2018) 8:2430 | DOI:10.1038/s41598-018-20835-x www.nature.com/scientificreports CPAP3 proteins in the mineralized cuticle of a decapod crustacean Shai Abehsera 1,2 , Shir Zaccai 1 , Binyamin Mittelman 1,2 , Lilah Glazer 4 , Simy Weil 1,2 , Isam Khalaila 2,3 , Geula Davidov 1,2 , Ronit Bitton 1,2 , Raz Zarivach 1,2 , Shihao Li 5 , Fuhua Li 5 , Jianhai Xiang 5 , Rivka Manor 1,2 , Eliahu D. Afalo 1,2 & Amir Sagi 1,2 The pancrustacean theory groups crustaceans and hexapods (once thought to comprise separate clades within the Arthropoda) into a single clade. A key feature common to all pancrustaceans is their chitinous exoskeleton, with a major contribution by cuticular proteins. Among these, are the CPAP3’s, a family of cuticular proteins, frst identifed in the hexapod Drosophila melanogaster and characterized by an N-terminal signaling peptide and three chitin-binding domains. In this study, CPAP3 proteins were mined from a transcriptomic library of a decapod crustacean, the crayfsh Cherax quadricarinatus. Phylogenetic analysis of other CPAP3 proteins from hexapods and other crustaceans showed a high degree of conservation. Characterization of the crayfsh proteins, designated CqCPAP3’s, suggested a major role for CPAP3’sin cuticle formation. Loss-of-function experiments using RNAi supported such a notion by demonstrating crucial roles for several CqCPAP3 proteins during molting. A putative mode of action for the CqCPAP3 proteins –theoretically binding three chitin strands– was suggested by the structural data obtained from a representative recombinant CqCPAP3. The similarities between the CqCPAP3 proteins and their hexapod homologues further demonstrated common genetic and proteinaceous features of cuticle formation in pancrustaceans, thereby reinforcing the linkage between these two highly important phylogenetic groups. Te Pancrustacean theory groups together into a single clade the Crustacea and the Hexapoda, two taxa that were once thought to be separate clades within the Arthropoda 1–3 . One of the defning characteristics common to all pancrustaceans and arthropods in general, is the presence of a rigid exoskeleton whose principal organic components are chitin and proteins. Te rigidity of the exoskeleton dictates that all arthropods must undergo a molt cycle if they are to metamorphose and grow. Tus, many of the features of the exoskeleton composition and of the molt cycle are common across the ‘modern’ pancrustacean taxa and presumably predate the divergence of the Crustacea and the Hexapoda 4 . In the Crustacea – the taxon of interest to us here – the molt cycle is composed of four main consecutive stages: inter-molt, the stage between two molting events during which the animal has a fully developed exoskel- eton; pre-molt, during which the old exoskeleton is partly reabsorbed in parallel to the initial formation of the new exoskeleton; ecdysis, the actual act of shedding the old exoskeleton; and post-molt, during which the cuticle continues to be deposited and is hardened through sclerotization and mineralization 5 . Our study animal, the decapod crustacean Cherax quadricarinatus (Cq), is characterized by two cuticular structures – the gastrolith and the molar tooth – in addition to the cuticular exoskeleton, common to all crusta- ceans. Te two gastroliths, which are located one on each side of the stomach wall, are transient calcium storage organs having a chitinous scafold which is mineralized with stabilized amorphous calcium carbonate during the pre-molt stage (as opposed to most of the cuticle, which is mineralized with calcium carbonate in the post-molt stage). During post-molt, the gastroliths collapse into the stomach where they are fully digested, thereby provid- ing the calcium for cuticular mineralization 6–8 . Te two molar teeth are part of the mandibles of Cherax quadri- carinatus. Te mandibles form the anterior mouthparts and are located directly in front of the oral opening. Te molar teeth serve as a grinding surface for mastication and processing of food. Te molar teeth are cuticular structures that are mineralized with a calcium carbonate (innermost) to calcium phosphate (outermost) gradient. 1 Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel. 2 The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel. 3 Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel. 4 Department of Psychiatry and Behavioral Science, Duke University Medical Center, Durham, USA. 5 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China. Correspondence and requests for materials should be addressed to A.S. (email: sagia@bgu.ac.il) Received: 16 August 2017 Accepted: 24 January 2018 Published: xx xx xxxx OPEN