The molluscan mantle is a thin tissue from which proteins are secreted into the extrapallial fluid; these proteins dictate the animals shell construction and microstructure. As a conserved organ involved in shell formation throughout mollusks, the mantle is an excellent foundation from which to study biomineralization . In this study a P. maxima mantle tissue-specific cDNA microarray has been generated termed PmaxArray 1.0, comprising 4992 cDNA clones derived from the mantle tissue of several P. maxima individuals. This tool has provided significant power to interrogate the role of proteins in shell formation.
Microarray analysis has spatially mapped the expression of a number of known and unknown ESTs with reference to specific mantle zones. 2012 ESTs present on PmaxArray 1.0 were expressed as significantly different to the control condition and approximately one third of those were sequenced and aligned resolving a total of 184 unique ESTs. The majority of those sequences could not be annotated via the Genbank database as no molluscan genome has yet been sequenced, let alone functionally annotated. Other non-model organisms also report a high proportion of unannotated genes [Crustaceans, 60% ; Scallop, 73% . As such, where sequence homologies are absent, functional significance of ESTs identified in this study are interpreted with reference to their pattern of expression (microarray EST differential expression and in situ hybridization) and the relevance this bears to mantle associated responsibilities. Five major expression profiles were observed among the mantle zones indicative of specialized molecular functions and ESTs clustering in each of these profiles will be discussed within these groupings.
The spatial expression profile in cluster A suggests a role associated with the nacreous shell formation of P. maxima. Sudo et al.  along with others [26, 27] support this supposition noting a close spatial link between transcript expression in mantle zones and shell microstructure inclusion. Of particular interest within this cluster are PM077 and PM044, as both ESTs possess two tandem KUNTIZ/Bovine pancreatic trypsin domains (KUNTIZ BPT1). PM077 is a significant match to papilin; an extracellular matrix glycoprotein occurring widely from nematodes to humans and known to contain several KUNTIZ domains . Likewise the presence of KUNTIZ domains is expected for PM044 which shares sequence similarity with a pancreatic trypsin inhibitor domain protein. KUNTIZ BPT1 domains are generally regarded as serine protease inhibitors involved in clotting and tissue remodeling . Similarly shell formation is known to involve a number of inhibitory components limiting mineralization. Proteoglycans are one such component, essential to shell formation yet intrinsically inhibit biomineralization [30, 31]. The protease inhibiting domains of PM077 and PM044 may act to maintain the viscous silk gel detailed by Adaddi et al.  as necessary for nacre formation. PM077 is expressed in the DM epithelial cells overlying the nacre microstructure in conjunction with the immediate cessation of expression toward the VM zone and prismatic microstructure. Taken together, tissue localization and sequence homologies suggest that PM077 and possibly PM044 are glycoproteins with inhibitory protease activity specific for nacre formation.
ESTs PM037 and PM041 are unannotated however in situ hybridization demonstrated a very specific localization to the epithelium of the DM zone, as already described for PM077. This same distribution of expression is also demonstrated for N14 gene [11, 26] and MSI60 gene [10, 26] both of which code for nacre matrix proteins. The exclusive expression of these two novel ESTs, PM037 and PM041, suggest a role in nacre formation which along with PM077, are the only reported cases of in situ hybridization localizing ESTs to the DM zone since Sudo et al.  reported MSI60 gene expression.
This cluster is the largest and most ubiquitous of all the expression profiles identified in this study. ESTs in cluster B display similar expression values across a number of seemingly unrelated mantle tissues. The anatomy and function of the mantle organ is generally considered as follows: OF is secretory (periostracum and shell), MF is sensory, IF is muscular, VM and DM are secretory (shell) . Therefore in the perceived absence of a specialized function uniting these tissues, cluster B most likely represents ESTs involved in general cellular maintenance and regulation rather than shell formation. This proposition is supported by the identification of a number of 'housekeeping' genes (HKGs) not seen in any of the other clusters including cytochrome c oxidase, glutathione peroxidase, ezrin/radixin/moesin binding proteins and ribosomal proteins.
This cluster is the smallest and contains ESTs which showed no significant similarities with any reported protein or nucleotide sequences. The in situ hybridization results for ESTs PM317 and PM316 showed association with the periostracal groove in which the outer epithelium of the MF is included. The main function of the periostracal groove is to secrete a glycocalyx coating forming the periostracum. A glycocalyx is a network of polysaccharides that project from cellular surfaces usually secreted by epithelial cells for a range of adhesion functions. The distil expression of PM316 in the MF outer epithelium indicates this EST may code for a glycoprotein incorporated in the mature stages of the outer glycocalyx coating . Similarly, expression of PM317 in the proximal epithelial cells of the periostracal groove may also code a protein involved in glycocalytic coatings and the stepwise construction of the periostracum.
EST PM315 has a peculiar in situ expression pattern in that the transcript is found below the epidermal layer, interspersed throughout the inner region of the MF and the outer region of the IF. Bivalves expose these mantle folds to the external environment . Chemoreceptors, photoreceptors and mechanoreceptors are all usually present in the epidermal layer of these folds in order to elicit closure of the shell valves in response to negative stimulus . Considering PM315 is expressed sub-dermally, it is less likely that this EST has a direct sensory role but rather associated with what appears to be nerve fibres , possibly involved in a signal transduction cascade .
The expression profile of cluster D ESTs suggests an exclusive role of these genes in the OF tissue, specifically concerned with the inner epithelia. This epithelium forms the bottom half of the periostracal groove, which is a highly dynamic tissue responsible for formation, maturation and extrusion of the complex periostracum layer. The proteinaceous layer functions to seal the extrapallial space and protect the shell from dissolution as well as serve as an initial matrix for mineralization . In situ localization to the inner epithelium of the OF tissue signify a periostracum-related function.
Neuromacin  and theromacin  are a family of antimicrobial peptides known to occur in a number of invertebrates. These peptides are part of a immediate immune response characterized predominantly by cationic and hydrophobic amino acids . EST PM238 shows a significant sequence similarity to the gene encoding these antimicrobial peptides and its in situ expression profile maps it to where the internal periostracum is formed. Cationic and hydrophobic properties of these peptides  are synonymous with the characteristics of the periostracum and water insoluble matrix (WISM) of shells [14, 39]. Specifically, a scenario for PM238 may be that poly-anionic glycoproteins (shell precursors) bind to cationic peptides in the periostracum, effectively anchoring the hydrophilic macromolecules to the hydrophobic WISM. This in turn facilitates active nucleation sites by which microstructure mineralization occurs.
Lysine-rich matrix protein (KRMP) is a family of proteins seemingly specific to mollusks and shell formation of the prismatic design. Cluster D includes seven ESTs significantly similar to the KRMP gene class. Zhang et al.  first described these proteins noting predominate expression in the inner epithelial cells of the OF and outer epithelium of the mantle edge region. The deduced amino acid sequence includes an N-terminal signal peptide, a lysine-rich basic region potentially interacting with acidic proteins or CO3
2-, and a glycine/tyrosine-rich region considered involved in protein cross-linking via the quinone-tanning process. The expression in the mantle edge region and similarities among the signal peptide of other prismatic shell matrix proteins lead Zhang et al. (2006a) to assign a putative prismatic microstructural function to the KRMP family in P. fucata. However unlike Zhang's et al.  observations of dual expression in the periostracal groove and the prismatic mantle region, these P. maxima ESTs are exclusively expressed in the OF, a number of which are localized by in situ hybridization to the inner epithelium of the fold (PM233, PM234, PM235, PM239), representing the lower half of the periostracal groove. This deviation from Zhang's et al.  original characterization is potentially explained by sequence analysis. The newly identified KRMP members appear to be concatenated versions of P. fucata KRMP possessing only the signal peptide and lysine rich region typical of the class. In many of the ESTs the C-terminal region is significantly reduced and/or replaced with serine and aspartic acid residues. The absence of the glycine/tyrosine-rich region suggests that the predicted proteins coded by these ESTs are not quinone-tanned. PM239 is the most divergent of the KRMP members and displays a different local expression being present along the middle region of the OF inner epithelium, suggesting a different function, specific to periostracum formation. Unclear however, is whether these ESTs are a novel sub-family of KRMP or a species specific evolutionary adaption of KRMP in P. maxima. The conservation of the lysine-rich region confers the positive charge required to attract and bind acidic glycoproteins necessary for nucleation  while expression in the periostracal groove suggests they are incorporated in the periostracum. In summary, the seven KRMP homologs in cluster D are considered specifically adapted for periostracal formation in P. maxima.
PM241 is a novel transcript expressed in the inner epithelia cells of the OF at the base of the periostracal groove. Periostracum development begins with the formation of the pellicle providing a framework on which coatings of the glycocalyx thicken and develop the periostracum . In bivalves, the pellicle typically originates from a row of basal cells at the bottom of the periostracal groove . The spatial expression of PM241 closely matches the area described for pellicle formation and its deduced sequence is dominated by tyrosine and glycine, typical of a quinone-tanned protein [43, 44]. As the pellicle provides the structural backbone on which ensuing glycocalyx coatings mature the periostracum, its formation would be largely concerned with the hardening of the structure.
ESTs in this cluster are characterised by expression primarily in the OF and VM tissues. The outer epithelia of both these tissues are considered homogenous in function, attributed to prismatic shell formation [10, 26, 27]. In situ hybridization of several cluster E ESTs confirms dual expression in the outer epithelia of the OF and VM, consistent with involvement in prismatic shell formation.
ESTs PM264, PM273, PM274, PM262, PM246, PM255 and PM245 represent the shematrin protein family. While P. maxima isoforms for shematrin have already been reported (accession: B1Q4VA) all the ESTs presented here, except PM274, are novel isoforms. Shematrin is a family of glycine-rich shell matrix proteins known to be present in the prismatic microstructure of several pearl oyster species. Yano et al.  suggests shematrins are framework proteins facilitating calcification of the prismatic microstructure. This investigation maps shematrin isoform PM273 via in situ hybridization to the outer epithelium from the tip of the OF to the VM/DM mantle border, parallel with the prismatic/nacreous shell border, adding to the characterization of the shematrin family in relation to the prismatic microstructure.
ESTs PM265 and PM269 show significant sequence homologies with mantle protein 10 and alveolin3 respectively, and both appear to be related to cytoskeletal protein family articulin. Articulins are part of the membrane skeleton of eukaryotic cells stabilizing plasma membranes [45, 46]. It is suggested that ESTs PM265 and PM269, function as plateins, a new family of articulins described by Kloetzel et al. . Plateins contain modified articulin core domains typical of secreted structural proteins as well as a novel predicted signal peptides detected in intra-alveolar sacs, an extracellular environment. Likewise, PM265 and PM269 also contain predicted signal peptides indicating a secretory pathway and EST PM265 has been detected by in situ hybridization specifically to the epithelial cells of both the lower periostracal groove and mantle outer epithelium, in contact with the prismatic shell. These tissues are noted for their secretions reinforcing the secretory pathway of PM265 and PM269. In summary, gene sequence homology of PM265 and PM269 with membrane skeleton proteins, coupled with their differential expression to secretory tissues and detection of signal peptides suggest these ESTs are putative members of a new articulin family, differentiated by extracellular function. These ESTs may encode framework proteins involved in the formation of the prismatic microstructure in P. maxima shell.
A functional link between the periostracal groove secretions and prismatic shell formation has previously been suspected based on a structural continuity between the outer periostracum and interprismatic matrices . Zhang et al.  demonstrated shell matrix protein KRMP as expressed in both secretory tissues. However, the presence of ten KRMP related ESTs found to be expressed specifically in the outer epithelia of the ventral mantle zone three of which were confirmed with in situ hybridisation (PM268, PM280, PM281) represent a break from that observed by Zhang et al. . The KRMP family has already been discussed in reference to seven EST homologs found to be specific to the periostracal groove of P. maxima. The observation of these two separate expression patterns for KRMP related ESTs in the periostrcum and prismatic shell formation mantle regions differs from reports in the related pearl oyster P. fucata . In contrast, where KRMP homologues appear to perform dual periostracum/prismatic microstructure roles; P. maxima appear to use additional KRMP homologs to accomplish the periostracum related task. This corroborates Jackson's et al.  supposition that the 'secretome' is a rapidly evolving collection of proteins capable of significant molecular differences in building molluscan shells. In summary, cluster E contains specific KRMP isoforms potentially involved in the prismatic microstructure formation of the P. maxima shell. A functional linkage between the periostracum and prismatic shell formation is probable, however the mode by which this occurs is highly adaptable, and unlikely to be conserved among species.