Functional analysis of a tyrosinase gene involved in early larval shell biogenesis in Crassostrea angulata and its response to ocean acidification

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Abstract

The formation of the primary shell is a vital process in marine bivalves. Ocean acidification largely influences shell formation. It has been reported that enzymes involved in phenol oxidation, such as tyrosinase and phenoloxidases, participate in the formation of the periostracum. In the present study, we cloned a tyrosinase gene from Crassostrea angulata named Ca-tyrA1, and its potential function in early larval shell biogenesis was investigated. The Ca-tyrA1 gene has a full-length cDNA of 2430 bp in size, with an open reading frame of 1896 bp in size, which encodes a 631-amino acid protein that includes a 24-amino acid putative signal peptide. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that Ca-tyrA1 transcription mainly occurs at the trochophore stage, and the Ca-tyrA1 mRNA levels in the 3000 ppm treatment group were significantly upregulated in the early D-veliger larvae. WMISH and electron scanning microscopy analyses showed that the expression of Ca-tyrA1 occurs at the gastrula stage, thereby sustaining the early D-veliger larvae, and the shape of its signal is saddle-like, similar to that observed under an electron scanning microscope. Furthermore, the RNA interference has shown that the treatment group has a higher deformity rate than that of the control, thereby indicating that Ca-tyrA1 participates in the biogenesis of the primary shell. In conclusion, and our results indicate that Ca-tyrA1 plays a vital role in the formation of the larval shell and participates in the response to larval shell damages in Crassostrea angulata that were induced by ocean acidification.

Introduction

Anthropogenic CO2 emissions are currently acidifying the world's oceans. Ocean acidification has significantly reduced the pH of seawater in the past century, which in turn has largely impacted marine organisms, particularly calcifying marine invertebrates. Numerous studies on calcifying marine invertebrates have demonstrated that ocean acidification can impact survival, growth, development, and physiology (Talmage and Gobler, 2010, Hofmann et al., 2010, Valerio et al., 2012). Marine calcifiers are particularly vulnerable to ocean acidification because it is more difficult for them to produce calcium carbonate in acidified water (Orr et al., 2005, Fabry et al., 2008). Research on the early development of Haliotis discus hannai has shown that ocean acidification results in decreased fertilization and hatching rates, an increase in malformation rate, and a decrease in settlement (Li et al., 2013). Besides, ocean acidification makes it difficult for pteropods to survive because of instability in their biological skeleton, coupled with shell thinning in some foraminifera (Li et al., 2013). It has been reported that Amphiura filiformis adapts to an increase in calcification and metabolic rates during ocean acidification; however, as pH decreases, the function of wrist muscles is impaired (Wood et al., 2008). Similar findings have also been reported in Hemicentrotus pulcherrimus and Echinometra mathaei (Shirayama and Thornton, 2005). These findings indicate that the effect of ocean acidification is widespread in marine animals, and induces marine calcifiers to utilize less calcium carbonate (Watson et al., 2012).

Several mollusks are recognized as economically significant calcifiers. However, shells are vital for most mollusk species. It is reported that the typical adult shells of mollusks are composed of several layers, including the external cuticle layer (periostracum) covering the outer surface of the mollusk shell and inner layers containing the CaCO3 polymorph (calcite and aragonite) (Marin and Luquet, 2004). The shell can be primarily discriminated in gastrulation, and the periostracum is the first observable part of the shell. When larvae develop from gastrulae into early trochophores, an initial shell is formed (Mouëza et al., 2006). To distinguish this from the calcified shell that grows later, Huan et al. (2013) defined this shell as the initial non-calcified shell (InCaS). The InCaS is the first shell during molluscan ontogenesis and thus it is very important to understand molecular mechanisms underlying its biogenesis

It has previously been reported that enzymes involved in phenol oxidation such as tyrosinase and phenoloxidases probably participate in the formation of the periostracum (Checa, 2000). Moreover, tyrosinases have been distinguished in molluscan shells (Marin and Luquet, 2004). Thus, it is important to determine whether tyrosinases take part in InCaS biogenesis. Tyrosinase (EC 1.14.18.1) contains two conserved copper-binding domains, which include conserved amino acid residues. It can catalyze the oxidation of both monophenols and o-diphenols into reactive o-quinones (Chang, 2009) and is involved in mammalian melanogenesis, enzymatic browning reactions in damaged fruits, and sclerotization of insect cuticle (Andersen, 2010, Chang, 2009). Tyrosinase cloned from Pinctada fucata has also been shown to be involved in periostracum formation (Zhang et al., 2006). In addition, an earlier research study on InCaS biogenesis in Crassostrea gigas reported the successful cloning of a kind of tyrosinase (cgi-tyr1) that was expressed in the domain of InCaS by whole mount in situ hybridization (WISH), thereby suggesting that is was involved in InCaS biogenesis (Huan et al., 2013). On the other hand, considering that there are multiple tyrosinases and the shell structure of larvae and adults differs, it is essential to understand the relationship between tyrosinases and InCaS formation.

Our current understanding of the impact of ocean acidification on the Fujian oyster Crassostrea angulata larvae is limited. In laboratory experiments designed to mimic seawater chemistry in oceans, we tested the impact of short-term exposure to elevated pCO2 (1500 ppm and 3000 ppm, compared to a control, 400 ppm) on C. angulata, which is one of the most important economic aquaculture varieties. In the present study, we cloned a tyrosinase (Ca-tyrA1 of the oyster Crassostrea angulata) and determined by quantitative RT-PCR, whole mount in situ hybridization (WISH), and RNA interference (RNAi) that it might be involved in the biogenesis of larval InCaS.

Section snippets

Animal and samplings

Adult oysters of C. angulata were collected from the coast of Xiamen and dissected to obtain different tissues, including gills, visceral mass, gonads, mantle, muscle and palps. There are six parallel samples for each tissue. The oyster larvae were obtained from the adult oysters. Sexually mature adults (three males and three females) were used in the collection of sperm and oocytes. After separately standing for 30 min, the sperm and oocytes were respectively mixed and incubated at different CO2

Sequence analysis

The Ca-tyrA1 cDNA sequence and deduced amino acid sequence are shown in Fig. 1. The open reading frame was 1896 bp in length and encoded a protein of 631 amino acid residues. The full-length of the cDNA was 2430 bp, which included a 26-bp 5″ untranslated region that was located at upstream of the putative start codon (ATG) and a 432-bp 3″ untranslated region that ended with a poly(A) tail sequence. A putative polyadenylation signal (AATAAA) was located at position 2399, which is upstream of the

Discussion

The tyrosinase gene family has been reported to be classified into three types: secreted form (Type A), cytosolic form (Type B) and membrane-bound form (Type C) based on the arrangement of histidine, (Aguilera et al., 2014). in the Pacific oyster all the tyrosinases belong to Type A just by the arrangement of histidine, but in the Pacific oyster some tyrosinase including signal peptide belong to secreted form (Type A) and other tyrosinase locate in the plasma membrane (Type B) or has the

Conflict of interest

All the authors in this manuscript have no conflict of interest.

Acknowledgements

This study was funded by NSFC (No. 41176113), Marine nonprofit industry research special funds (No. 201305016), the Earmarked Fund for Modern Agro-industry Technology Research System (No. nycytx-47), the National Natural Science Foundation of China under Grant (41276127), the Regional Demonstration Projects for Innovation and Development of Marine Economy in Xiamen under Grant (12PZB001SF09),Young and middle-aged teachers education scientific research project of Fujian (No. JA15811) and Science

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