Molecular Analysis of Atypical Family 18 Chitinase from Fujian Oyster Crassostrea angulata and Its Physiological Role in the Digestive System

RESEARCH ARTICLE Molecular Analysis of Atypical Family 18 Chitinase from Fujian Oyster Crassostrea angulata and Its Physiological Role in the Digestive System Bingye Yang1,2,3, Mingming Zhang4, Lingling Li4, Fei Pu2,3, Weiwei You3, Caihuan Ke2,3* 1 Xiamen Medical College, Xiamen, 361008, PR China, 2 State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, 361005, PR China, 3 College of Ocean and Earth Science, Xiamen University, Xiamen, 361005, PR China, 4 College of Life Science, Xiamen University, Xiamen, 361005, PR China * Abstract OPEN ACCESS Citation: Yang B, Zhang M, Li L, Pu F, You W, Ke C (2015) Molecular Analysis of Atypical Family 18 Chitinase from Fujian Oyster Crassostrea angulata and Its Physiological Role in the Digestive System. PLoS ONE 10(6): e0129261. doi:10.1371/journal. pone.0129261 Academic Editor: Wan-Xi Yang, Zhejiang University, CHINA Received: August 13, 2014 Accepted: May 6, 2015 Published: June 5, 2015 Copyright: © 2015 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chitinolytic enzymes have an important physiological significance in immune and digestive systems in plants and animals, but chitinase has not been identified as having a role in the digestive system in molluscan. In our study, a novel chitinase homologue, named Ca-Chit, has been cloned and characterized as the oyster Crassostrea angulate. The 3998bp fulllength cDNA of Ca-Chit consisted of 23bp 5-UTR, 3288 ORF and 688bp 3-UTR. The deduced amino acids sequence shares homologue with the chitinase of family 18. The molecular weight of the protein was predicted to be 119.389 kDa, with a pI of 6.74. The Ca-Chit protein was a modular enzyme composed of a glycosyl hydrolase family 18 domain, threonine-rich region profile and a putative membrane anchor domain. Gene expression profiles monitored by quantitative RT-PCR in different adult tissues showed that the mRNA of CaChit expressed markedly higher visceral mass than any other tissues. The results of the whole mount in-situ hybridization displayed that Ca-Chit starts to express the visceral mass of D-veliger larvae and then the digestive gland forms a crystalline structure during larval development. Furthermore, the adult oysters challenged by starvation indicated that the CaChit expression would be regulated by feed. All the observations made suggest that Ca-Chit plays an important role in the digestive system of the oyster, Crassostrea angulate. Data Availability Statement: All relevant data are within the paper. Funding: This study was funded by NSFC (41176113), The National Basic Research Program of China (No. 2010CB126403), the Special Fund for Marine Scientific Research in the Public Interest (201305016) and the Earmarked Fund for Modern Agro-industry Technology Research System (CARS48). Competing Interests: The authors have declared that no competing interests exist. Introduction Chitin belongs to a nitrogen-containing polysaccharide bio-polymer and is found widely spread across the earth. The Chitin polymer has been found as a structural component in the shell of crustaceans, as seen in shrimp, crabs, insects, the organs of invertebrate, the cell wall of fungi and some algae. Chitin is one of the most abundant carbohydrates present in the marine environment and the second most abundant bio-polymer on earth, next to cellulose [1]. PLOS ONE | DOI:10.1371/journal.pone.0129261 June 5, 2015 1 / 13 Gene of Atypical Family 18 Chitinase Involved in the Digestive System Annual biosynthesis estimates range from 1010 to 1011 metric tons [2]. Chitin plays an important role in the ocean’s food chain and carbon cycle because of its ubiquitous and extensive presence in marine ecosystems. The Fujian oyster crassostrea angulata is an important edible marine bivalve, which has been harvested from the wild and cultivated for centuries. The growth and development of oysters is closely related to its digestive system. Oyster belong to bivalves take algae which contain chitin as main food, degradation of chitin-containing structures requires chitinases, so which suggested that the digestive gland of organism in bivalves probably secrete chitinases when the algae as food enter into their digestive system. The bivalve digestive system is comprised of a complex stomach and associated structures, but an otherwise simple intestine, actually in the bivalve Scrobicularia plana, Chitinase had been found in the gastric shield of digestive gland by histochemiscal technique [3], and also some chitinases are detected in the digestive tract of mammalians [4,5] and mollusks [6,7], But until now, there is no gene expression analysis of chitinase reported in bivalves. The crystalline style is also an important part of digestive gland, which is present in nearly all bivalve mollusks and some plant-eating Prosobranchia [8,9]. The crystalline style is known to undergo cyclical changes in size and/or occurrence in many species [10–13], which is a distinct formation with a cylindrical shape, concentric layered structure, jelly consistency, and complex chemical composition [14,15]. The crystalline style has many functions, but the main digestive function which enhances the digestion of food particles was beyond a doubt. The crystalline structure is the principal organ of extracellular digestion, which is rotated in its sac by cilia, which head projects into the stomach and grinds against part of the chitinous gastric shield lined stomach wall. Chitinase activity has been detected in the crystalline structure of the American oyster Crassostrea virginica [16]. In other respects, two genes encoding the chitinase protein and chitinase-like protein had been characterized with their cDNAs from the oyster Crassostrea gigas, but both of them functioned on the immunity [17,18]. While the chitinase activities were measured by the crystalline structure and the digestive tract of several molluscs [19], no chitinase gene of family 18 has been identified at the molecular level in the Bivalves. In this study, a chitinase gene was characterized and the full cDNA sequence was identified in Crassostrea angulata. Tissue distribution and the temporal spatial pattern of expression during larval development were established by real time PCR and in situ hybridization. We also examined the gene chitinase expression of the adult oyster after starvation and food treatment. Our results provided insight into the study of chitinase and its effects on the digestive system of oysters at the molecular level. Materials and Methods Sample collection and larva culture The oyster Crassostrea angulata is not a protected species, and collections were only made from public access areas, no specific permits were required to collect this species in Wuguan aquaculture farm of xiam (...truncated)