Publications: Research reports and publications

Biochemistry of digestion and algal-biotoxin metabolism in bivalves

1 January, 2013
A Thesis submitted to the University of Otago in fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry. University of Otago.


This study was primarily focussed on the identification and isolation of enzymes involved in the digestion of phytoplankton in the bivalve gut and the assimilation and biotransformation of secondary metabolites released by these digestive processes within the digestive gland (hepatopancreas). The main hypothesis was that these enzymes play important roles in algal biotoxin sequestration and elimination and an improved understanding of the nature of these enzymes may lead to solutions to the harmful effect of algal toxin contamination on shellfish aquaculture productivity. The function of the crystalline style in these processes was an essential component of this objective incorporating the hypothesis that major matrix proteins play an important role in the gelling properties of the styles. A second objective was to identify digestive enzymes that might be useful as biochemical indicators for the discrimination of superior family traits within a bivalve selective breeding programme, and test the performance of these markers against mussel families selected for differing growth capacities.

An endo β-1,4 glucanase (cellulase) that mediates the digestion of dinoflagellate thecal plates in the bivalve gut was tentatively identified and partially purified from the crystalline style of the GreenshellTM mussel Perna canaliculus. This enzyme appears to play a key role in the lysis of the toxic dinoflagellate Dinophysis (spp.) thereby liberating okadaic acid and pectenotoxins for absorption within the hepatopancreas. The enzyme was isolated using dissociated dinoflagellate thecal plates as substrate and assayed by the release of reducing sugars. After selective adsorption/desorption using a thecal plate suspension the enzyme was further purified by gel filtration and an active fraction of 43-44 kDa isolated. One major band of carboxymethyl cellulose (CMC) hydrolysis activity was observed on CMC-zymogram gels corresponding to a Coomassie stained protein of 46kDa on non-reducing SDS-PAGE gels. Under reducing conditions the activity on zymogram gels was maintained (at 46kDa) but the Coomassie stained band was shifted to 30kDa. However de novo amino acid sequencing of bands from heated aliquots on an SDS-PAGE gel revealed the presence of a 63 kDa protein which contained tryptic peptides with close sequence similarity to glycoside hydrolase family 9 (GHF9) cellulases found in a variety of invertebrate taxa including the blue mussel Mytilus galloprovincialis. This demonstrated that the 46 kDa activity bands on the zymograms were possibly artifacts created by complexing of the enzyme with probably at least two other proteins. The enzyme is almost certainly endogenous although this has yet to be confirmed by sequence analysis of cDNA and genomic DNA.

An enzyme capable of hydrolyzing pectenotoxins (PTXs) and okadaic acid (OA) esters within the hepatopancreas of the GreenshellTM mussel Perna canaliculus was isolated and characterized. It is believed this is the first report of the purification of any esterase or lipase from the digestive gland of a bivalve. PTX2 and PTX1 were hydrolyzed by the enzyme but it was inactive against PTX11, PTX6 and acid isomerised PTX2 and PTX11. PTX11 and PTX2b competitively inhibited PTX2 hydrolysis. The enzyme also hydrolyzed short and medium chain length (C2-C10) 4-nitrophenyl-esters, okadaic acid C8-C10 diol esters and DTX1 7-O-palmitoyl ester (DTX3). The ability of the enzyme to hydrolyze the diol and acyl esters of okadaic acid, with which PTXs are invariably associated in natural shellfish contamination events, raises the possibility that it may participate in other associated process such as the synthesis of OA and PTX seco acid esters in vivo. MALDI-Tof MS/MS determination of de novo amino acid sequence and BLAST searches of several data bases failed to identify any similarity to known proteins.

Partial characterization of the crystalline style matrix proteins of Perna canaliculus and Mytilus galloprovincialis was carried out to attempt to identify the biochemical basis of differences in the physical structure of the styles. P. canaliculus has a crystalline style that is typical of those bivalves with permanent hard-type styles while the style of M. galloprovincialis is typical of bivalves with transient soft-type styles. The consistency of the styles is clearly related to their respective moisture content and both style types contain a suite of high molecular weight (>800 kDa and ~95-170 kDa) heavily glycosylated proteins and medium molecular weight (40-50 kDa) more lightly glycosylated protein duplexes. These protein pairs appear to play a role in gel formation, and differences in the nature of these proteins may determine the difference between hard and soft styles. One and two dimensional SDS-PAGE, MALDI-TOF MS/MS peptide analysis and de novo amino acid sequencing, showed there was a high degree of similarity between the sequences of several peptide fragments of the prominent40-50 kDa protein duplexes and 'Myosinases' I and II annotated within a M. galloprovincialis EST data base ("Mytibase"). 'Myosinases' belong to a large family of astacin-like metalloproteinases that are widely distributed in nature, although in many cases their function is unknown. This research establishes a new role for this important group of proteins. Analysis of the electrophoretic behaviour of the style proteins from a number of New Zealand endemic bivalve species showed that medium MW lightly glycosylated proteins were a major component of them all. It was hypothesised that astacin-like proteins determine the character of the crystalline styles of all bivalve species.

The activities of amylase, laminarinase, cellulase and chitinase enzymes in the crystalline styles of several New Zealand bivalve species were visualised, quantified and partially characterised. Alpha amylase and laminarinase constituted the major activities in Perna canaliculus and Mytilus galloprovincialis. Protein-specific N-acetyl glucosaminidase (NAGase) activity was highest in the style of C. gigas followed by the gastric fluids of P. galloprovincialis and the style of P. canaliculus. NAGase activity was very low in the style and gastric fluids of the surf clam Paphies subtriangulata and the gastric fluids of P. canaliculus. Chitobiase and chitotriase activities also varied in different ways between styles and gastric fluids in the various species. Whether the observed activities represented true chitinase activity was questionable because assays using chitin azure did not relate to the relative activities observed using fluorescently labelled proxy substrates in oyster and mussel style extracts. It was speculated that the NAGase activities observed may more realistically represent lysozymes than chitinases.

Assays of chitinase, cellulase and amylase activities in the crystalline styles of selected fast and slow growing families of the mussel Perna canaliculus were carried out on two occasions over six months, during which time the shellfish were growing in a normal mussel cultivation situation. The data did not support the hypothesis that differences in growth rate by these families could be related to the different expression of digestive enzymes leading to improved nutritional efficiency by faster growing families. On the contrary faster growing families in general exhibited lower rates of enzyme activity and these differences between families appeared to be related to the size of the individuals rather than their genetic heritage.

Key words Perna canaliculus; Mytilus galloprovincialis; bivalves; selective breeding; digestion; crystalline style; hepatopancreas; enzymology; cellulase; esterase; myosinase; dinoflagellates; Protoceratium reticulatum; Dinophysis acuta; pectenotoxins; okadaic acid; okadaic acid esters; MALDI TOF-MS