Transcriptomics provides insight into Mytilus galloprovincialis (Mollusca: Bivalvia) mantle function and its role in biomineralisation
Introduction
Biomineralisation is a biologically controlled process through which living organisms generate mineralised structures (Wilbur and Saleuddin, 1983). The minerals are naturally occurring, of inorganic origin and possess a crystalline structure. Biomineralisation is a widespread and ancient process, occurring in both prokaryotes and eukaryotes, in plants and in animals (Yoshida et al., 2010, Knoll, 2003). The structures produced can have multiple functions, including tissue support, protection from the external environment, feeding and sensing (Miyamoto et al., 2013, Wilbur and Saleuddin, 1983). According to the pioneers of the field of biomineralisation, the process of shell mineralisation occurs in two main steps. First, ions are transported across an epithelium, proteins are then synthesized and secreted. Second, calcium carbonate (CaCO3) crystals are deposited and grown concomitantly with an organic matrix which envelops the crystals (Wilbur and Saleuddin, 1983).
When one thinks of shells, in general one immediately thinks of sea shells; the shells of molluscs. Phylum Mollusca is a highly successful and species-rich group of organisms, yet much of their biology remains poorly understood despite their importance from an ecological and socio-economic perspective. Shellfish account for 22.8% of global aquaculture and mussels are the third most produced family of bivalves (The State of World Fisheries and Aquaculture, Food and Agriculture Organization of the United Nations, 2014). In addition, due to their sessile and filter-feeding qualities, mussels are also used in environmental monitoring programmes as biological indicators of pollution and other changes to the marine environment, such as ocean acidification (De Zwaan and Eertman, 1996, Fitzer et al., 2014, Dupont and Pörtner, 2013, Huning et al., 2013). Therefore, understanding the underlying mechanisms of biomineralisation and the effects of external stress are vital for the maintenance of food security (Cheung et al., 2010), ecological stability (Bijma et al., 2013) and a sustainable shellfish industry (Narita et al., 2012).
The Mytilus shell is composed of an organic matrix, two forms (aragonite and calcite) of CaCO3 and an uncalcified periostracum that covers the whole structure (De Paula and Silveira, 2009). The organic matrix, which surrounds the calcite and aragonite crystals, is made up largely of polysaccharides (i.e. chitin) and glycoproteins (De Paula and Silveira, 2009). The organic matrix is synthesized in the mantle and secreted into the extrapallial cavity of the animal, where it becomes a template for the nucleation, orientation and growth of CaCO3 crystals (Nudelman et al., 2006, Simkiss and Wilbur, 1989). This controlled biological process, whereby a hard structure is formed from the synthesis of organic molecules, is known as biomineralisation (Yoshida et al., 2010).
Bivalves generally uptake calcium (Ca2 +) and bicarbonate (HCO−3) from their environment and metabolize them to form the CaCO3 that is found in the shell, however the exact mechanism and regulation of this process is still ambiguous. The mantle tissue appears to be important for shell formation; it is where proteins such as chitin and silk fibroin are produced and secreted to form the organic matrix (Freer et al., 2014, Marin et al., 2000, Marin et al., 2008, Miyamoto et al., 2013, Suzuki et al., 2009). This tissue has several known functions including sensory activities, secretory activities, shell growth and shell repair (Watabe, 1983), however the expression of genes associated with these functions has never been mapped to specific regions along the mantle edge. Gardner et al. (2011) used microarrays to identify clusters of transcripts in different regions of Pinctada maxima's mantle and Jolly et al. (2004) localized zones where shell matrix proteins were expressed in the Haliotis tuberculata mantle. Nonetheless, despite the existence of several large mantle transcriptome datasets generated by next generation sequencing (NGS) (Artigaud et al., 2014, Clark et al., 2010, Craft et al., 2010, Freer et al., 2014, Philipp et al., 2012), none have contemplated the reported functional variations and consider the mantle edge as a homogeneous tissue.
The present study was designed to test the hypothesis, the mantle edge of the Mediterranean mussel, M. galloprovincialis, is a functionally heterogeneous tissue from the posterior tip to the umbo. M. galloprovincialis was chosen as the model for the study as it has two symmetrical valves of the multi-layered CaCO3 shell and is readily available in Southern Europe. We present, for the first time, RNA sequencing of three distinct regions of the mantle edge (posterior, middle region, and umbo) of M. galloprovincialis. The data from the present study will be a resource for future studies in which the goal is to characterize regulatory mechanisms involved in the control of shell growth/turnover. Functional heterogeneity was inferred from 1) presence/absence of candidate biomineralisation genes in the mantle regions and 2) histological analysis of the mantle edge.
Section snippets
Sample preparation
M. galloprovincialis specimens (n = 4) were sampled from the Ria Formosa in Faro, Portugal on July 23, 2014. The samples were removed by hand from bridge pillars at low tide (37°0′22″N 7°58′3″W). The mussels were transported live to the Centre of Marine Sciences (CCMAR), University of the Algarve, anaesthetized using magnesium chloride (10%) and the mantle tissue dissected out. Tissue samples were taken within one hour of mussel collection. Individuals chosen for RNA extraction or histology were
Histological analysis
The histology of the mussel mantle was reminiscent of that reported in other species and it is composed of a thin membrane with a simple epidermis covering the inner (facing the mantle cavity) and outer (facing the shell) surface of the centrally located connective tissue (Audino et al., 2015, Chagot and Cirizcl, 1992). The histological sections of the M. galloprovincialis mantle revealed differences in morphology across the mantle edge in the three regions analysed (Fig. 2). The PAS stain was
Acknowledgements
This study was financed by the European Regional Development Fund (ERDF) COMPETE and Portuguese funds through Foundation for Science and Technology (FCT, project UID/Multi/04326/2013) and CACHE a Marie Curie ITN (PITN-GA-2013-605051). RCF is in receipt of a FCT Postdoctoral grant SFRH/BDP/8911/2012 and AMC is in receipt of the FCT fellowship (SFRH/BPD/85408/2012). The authors would also like to thank Joao Cardoso for help with sampling, Rute Martins and Anna-Patricia Mateus for help with
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