Bivalve shell formation through development and evolution

Abstract

Bivalves are not as charismatic as whales, sharks, or even corals. Yet, bivalves are among the most evolutionarily successful taxa; they fuel global aquaculture and coastal ecosystems and forge durable, functional, complex, and fascinating shells that are considered a primary reason for the success of this class. However, despite the long, ongoing research effort, the biologically controlled process of bivalve shell formation is still elusive. To broaden the current knowledge on bivalve shell formation, I focused primarily on bivalve shell proteins and their regulation through developmental and evolutionary scales.

Microscopic biominerals are ubiquitous in the ocean and as complex as larger ones. However, challenges in sample preparation have rendered this group underrepresented. Therefore, I developed a biomineral protein extraction method for microscopic samples and applied it to bivalve larval shells, revealing the need for sample-specific treatments. Bivalve larvae are a bottleneck in their development and are often negatively impacted by environmental stress, such as adverse carbonate chemistry. To resolve potential compensatory mechanisms in resilient populations, I investigated the plasticity of larval shell formation under control and adverse carbonate chemistry using shell proteomics for the first time. Hong Kong oyster larvae can maintain shell formation aided by a diverse shell proteome and through upregulation of calcium-binding sequences and downregulation of calcification inhibitors and proton-generating processes. Further, a novel larval biologically induced mechanism similar to bacterial biomineralization is discussed. The larval shell is hypothesized to represent an ancestral stage in bivalve development. However, evidence shows that the downstream regulation is species-specific, yet quantitative shell protein expression analyses are lacking. Therefore, I compared the expression of 33 shell protein orthologs among larvae and adults from two sister branches, the Mytilidae and the Ostreidae. Fewer shell proteins are differentially expressed between larvae than adults, possibly indicating a more conserved larval shell-forming apparatus. Only four shell proteins are relatively more abundant in larvae than adults, and mussel larvae display heightened biological control over oyster larval and adult mussel shell formation. In adult stages, mussels upregulate organic matrix pathways, while oysters focus on biomineral growth. Finally, shell protein evolutionary studies have relied only on a few species from the same clade. Therefore, I investigated adult shell formation across the Bivalvia phylogenetic tree from a shell protein and structural viewpoint. Eleven previously undescribed species from the major clades were investigated. The shell structure and shell protein diversity show that bivalve shells are more strongly influenced by shell shape, behavior, and shell microstructure than phylogeny. A biomineralization toolkit of six protein domains was identified; however, nacreous shells have the largest shell proteomes, and their toolkit spans 55 domains, suggesting that complex, conserved proteomes are needed for nacre assembly but not other for microstructures. Finally, all species shared a common ancestry of two shell protein orthogroups with chitin-binding abilities, supporting the emergence of bivalve shells from a chitinous ancestor.

This thesis does not resolve all the unknowns of bivalve shell formation; however, novel developmental and evolutionary mechanisms are characterized, and new directions and hypotheses are set for future research.