Mollusca - Oyster genome

Key findings:

 Oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively

shaping variation.  

The

expansion of genes coding for heat shock protein  and inhibitors of apoptosis is probably central to the oyster’s

adaptation to sessile life in the highly stressful intertidal zone. 

Our analyses also show that shell formation in molluscs

is more complex than currently understood and involves extensive participation of cells and their exosomes. 

Devlopmental biology

One notable finding of developmental interest is that the oyster

Hox gene cluster is broken into four sections with flanking

non-Hox genes. 

We did not find a clear

Antennapedia gene, which is present in other bivalves such as Pecten

we find that Hox genes

in the oyster are not activated in an order matching their identity or

genomic position, with, for example, HOX4 and HOX1 peaking before

gastrulation, LOX5 and POST2 during the trochophore stage and

HOX5 during the pediveliger stage.

Adaptation to environmental stress

. The oyster genome contains

88 heat shock protein 70 (HSP70) genes, which have crucial roles

in protecting cells against heat and other stresses, compared with,17

in humans and 39 in sea urchins. 

a) Phylogenetic analysis finds clustering

of 71 oyster HSP70 genes to themselves, suggesting that the expansion

is specific to the oyster (Supplementary Fig. 19). 

b) Also expanded

are cytochrome P450 (Supplementary Fig. 20) and multi-copper

oxidase gene families, which are important in the biotransformation

of endobiotic and xenobiotic chemicals26, and extracellular superoxide

dismutases, which are important in defence against oxidative stress.

c) The oyster genome has 48 genes coding for inhibitor of apoptosis

proteins (IAPs), compared with 8 in humans and 7 in sea urchins,

indicating a powerful anti-apoptosis system in oysters. 

d) Genes encoding

lectin-like proteins, including C-type lectin, fibrinogen-related

proteins and C1q domain-containing proteins (C1QDCs), are highly

over-represented in the oyster genome (P,0.0001; Supplementary

Table 18); these genes have important roles in the innate immune

response in invertebrates27–29. 

e) Interestingly, many immune-related

genes, including genes coding for Gram-negative bacteria-binding

proteins, peptidoglycan-recognition proteins, defensin, C-typelectin-

domain-containing proteins and C1QDCs, are highly expressed

in the digestive gland (Supplementary Fig. 21), indicating that the

digestive system of this filter feeder is an important first-line defence

organ against pathogens.

 f) The finding of key genes belonging to both

intrinsic (BAX, BAK, BAG, BCL2, BI1 and procaspase) and extrinsic

(TNFR and caspase 8) apoptosis pathways indicates that oysters have

advanced apoptosis systems. Powerful inhibition of apoptosis as shown

by genomic and transcriptomic analyses may be central to the ability of

oysters to tolerate prolonged air exposure and other stresses.

g) Heat stress induced a ,2,000-fold increase in expression of five

highly inducible HSP70 genes or a 13.9-fold increase in average

expression of all HSP70 genes, amounting to 4.2% of all transcripts

(Supplementary Figs 24c and 25). The genomic expansion and massive

upregulation of HSP genes help to explain why C. gigas can

tolerate temperatures as high as 49 uC when exposed to summer

sun at lowtide33. 

h) Genes involved in signal transduction, including genes coding for

G-protein-coupled receptors and Ras GTPase, were also activated by

stressors  and over-represented in the oyster

genome.

Shell formation

 Two models have

been advanced for molluscan shell formation. The matrix model

posits that mineralization occurs in a mantle-secreted matrix of

chitin, silk fibroin and acidic proteins35,36. Chitin and silk proteins

are proposed to provide matrix structure, whereas acidic proteins

control the nucleation and growth of CaCO3 crystals. 

The cellular

model suggests that biomineralization is cell-mediated; that is,

crystals are formed in haemocytes and then deposited at the mineralization

front37.

identified 259 shell proteins (Supplementary Text H3 and

Supplementary Table 24). Although our search found evidence for

the involvement of chitin, we did not find any silk-like proteins

encoded in the oyster genome (Supplementary Text H2) but found,

instead, many diverse proteins that may have roles in matrix construction

and modification.

 Notably, a gene coding for a fibronectin like

protein was highly expressed at the early developmental stage,

when larval shells are formed, in unison with chitin synthase (Fig. 4a)

and was mostly expressed in the adult mantle (403 other organ

average; Fig. 4b); the fibronectin-like protein was among the most

abundant proteins found in oyster shells. 

Genes coding for laminin

and some collagen proteins were also highly expressed in the mantle

(Supplementary Fig. 27a) and found in shells. These are typical

extracellular matrix (ECM) proteins, and their presence in shells

suggests that the shell matrix has similarities to the ECM of animal

connective tissues and basal lamina. 

Unlike silk fibroins that can self

assemble38, the formation of fibronectin fibrils in the ECM is cell

mediated39. Oyster fibronectin-like proteins have five type-III

domains for integrin binding and cell adhesion. 

Genes coding for

integrins were highly expressed in haemocytes (43 other organ

average, Supplementary Fig. 27b). Thus, haemocytes may organize

fibronectin-like fibril formation in the shell matrix as they do in ECM.

l. Furthermore, 84% of the 259 shell proteins identified

are not classical secreted proteins (Supplementary Text H3.4 and

Supplementary Table 24); they may be part of cells or deposited by

exosomes. Supporting the presence of exosomes, 61 of the 259 shell

proteins matched proteins in the exosome database41. Cells and

exosome-like vesicles containing calcite crystals have been observed

at the mineralization front37,42, although their significance in shell

formation is debated. This study provides molecular evidence for

their presence inside shells and their probable participation in shell

formation.

Many shell proteins are enzymes that may be involved in matrix

construction or modification. A homologue of penicillin-binding

protein is exclusively expressed in mantle (723 other organ average)

and highly abundant in shells (Supplementary Fig. 27d). Penicillinbinding

protein is a transpeptidase that crosslinks glycopeptides in

bacterial cell walls43 and may have similar functions in the shell

matrix. 

Another notable enzyme found is tyrosinase. The oystergenome has an expanded set of 26 genes coding for tyrosinase,

compared with one in Caenorhabditis elegans and two in humans;

most genes coding for tyrosinase are mantle specific (Fig. 4d) and

highly enriched among shell proteins (P5831026). 

Althoughtyrosinase is a key enzyme in melanogenesis44,45, it is most highly

expressed in the non-pigmented pallial mantle (Fig. 4d), indicating

that it has other functions in the oyster. 

The mantle secretes tyrosinerich

proteins46, and oxidation of tyrosine may be essential for shell

matrix maturation. Several proteinases and proteinase inhibitors are

highly mantle specific and abundant in shells, and may be involved

in matrix formation, modification and protection (Supplementary

Table 24). 

Together, these results indicate that oyster shell matrix is

not formed simply by self-assembling silk-like proteins but by diverse

proteins through complex assembly and modification processes that

may involve haemocytes and exosomes.

Concluding remarks

The draft assembly provided insight

into a molluscan genome characterized by high polymorphism,

abundant repetitive sequences and active transposable elements.

Genomic, transcriptomic and proteomic analyses show unique

adaptations of oysters to sessile life in a highly stressful intertidal

environment and the complexity of shell formation.