Comparative Biochemistry and Physiology Part D: Genomics and Proteomics
Suppressive subtractive hybridisation transcriptomics provides a novel insight into the functional role of the hypobranchial gland in a marine mollusc
Introduction
The function of the hypobranchial gland, present within the mantle cavity of gastropod molluscs, has been the subject of investigation and speculation for many years. It is known that the hypobranchial gland is responsible for the amalgamation of particulate matter within the mantle cavity and it has been characterised as a predominant tissue involved in mucus production (Fretter and Graham, 1994). Further studies into the gland suggest varied roles for this pallial organ, including possible roles in aestivation and antiseptic production (Andrews and Little, 1972), olfactory response during feeding (Roller et al., 1995), the production of pheromones involved in aggregation and spawning behavior (Kuanpradit et al., 2010) and in the production of the embedding medium into which egg capsules are deposited onto benthic substrata (Westley, 2008). Several secondary metabolites, including pharmacologically active choline esters (Whittaker, 1960, Baker and Duke, 1976, Roseghini et al., 1995), brominated indole derivatives (Naegel and Cooksey, 2002, Kelley et al., 2003, Westley and Benkendorff, 2008) and an attractin-like protein (Kuanpradit et al., 2010) have been extracted from this gland. Furthermore, arylsulphatase and bromoperoxidase enzyme activity has been identified within the gland in the Muricidae (Erspamer, 1946, Jannun and Coe, 1987, Westley, 2008, Westley and Benkendorff, 2009).
In the Muricidae family of neogastropods, the hypobranchial gland is well known as a source of the ancient dye Tyrian purple (Baker, 1974, Cooksey, 2001). This pigment is a brominated indole that was first identified in 1909 by Friedlander (1909). The dye is produced from tryptophan-derived indoxyl sulphate prochromagens, which are hydrolysed by arylsulphatase enzymes (Baker and Duke, 1976, Cooksey, 2001, Naegel and Cooksey, 2002) to yield intermediate precursors that exhibit antibacterial and anticancer activities (Benkendorff et al., 2000, Benkendorff et al., 2001b, Benkendorff et al., 2011, Westley et al., 2006, Edwards et al., 2012). Dicathais orbita (Fig. 1A) is an ideal model species in which to investigate gene expression of the hypobranchial gland, due to its well-documented production of bioactive compounds. These compounds are also found in the egg masses (Fig. 1A) and their associated biological activity has led to suggestions that hypobranchial gland secretions play a role in maternal defence of the egg masses (Benkendorff et al., 2000, Benkendorff et al., 2001a), as well as an immune role within adult snails (Westley et al., 2006). Bromination of secondary metabolites has also been associated with the hypobranchial gland in other families of molluscs, including at least one species in the distantly related Vetigastropoda. 6-Bromo-2-mercaptoptrytamine, isolated from the hypobranchial gland of the vetigastropod Calliostoma canaliculatum, has been characterised as a potent neurotoxin, targeting potassium channels (Kelley et al., 2003, Wolters et al., 2005). These secondary metabolites within the hypobranchial gland of distinct gastropod lineages suggests that one of the roles of the gland is the production of bioactive secondary metabolites, which may have key roles in the chemical defence of molluscs.
A modern approach for investigating biosynthesis and the functional role of the hypobranchial gland is to examine gene expression. Molluscs have been used historically as model organisms to study the central nervous system and several transcriptome studies investigating neuronal interactions, chronic pain and neurological disorders have been performed (Moroz et al., 2004, Moroz et al., 2006, Feng et al., 2009, Walters and Moroz, 2009). More recently, the transcriptome of several gastropods has been investigated, at the embryonic, larval and metamorphic developmental stages, in order to better understand cellular differentiation and embryogenesis (Jackson and Degnan, 2006, Henry et al., 2010, Heyland et al., 2011). Transcriptomics has also been applied to the investigation of biomineralisation, shell development and deposition in molluscs (Jackson and Degnan, 2006, Bai et al., 2010, Clark et al., 2010, Joubert et al., 2010, Berland et al., 2011, Fang et al., 2011). Most recently an extensive transcriptome analysis of Thais clavigera using pyrosequencing has been used to identify selected genes involved in the innate immune system of this mollusc (Rhee et al., 2012). In all of these studies, transcriptomics has been used as a tool to better understand biological processes and has helped resolve how global gene expression influences physiological changes.
Suppressive subtractive hybridisation (SSH) is a powerful technique that allows the rapid and cost effective investigation of gene expression between different tissue types or at different stages of development and produces a library of differentially expressed genes. It was first developed by Diatchenko et al. (1999) and has been used for a variety of different investigations. In particular it has been used to examine the role of the cerebral ganglia in the growth, feeding behavior and reproduction of the tropical abalone Haliotis asinina (York et al., 2010) and the hibernating brain of the greater horseshoe bat Rhinolophus ferrumequinum (Yuan et al., 2008). In these two examples, SSH has been a very useful transcriptomics tool that has allowed further understanding of the biology of these species. So far the only known genes that have been identified from D. orbita are highly conserved sequences used in phylogenetic studies (Colgan et al., 2000, Colgan et al., 2003, Colgan et al., 2007, Laffy et al., 2009). Thus the use of SSH in this study should allow us to identify functional genes within the hypobranchial gland, to identify genes that may be involved in Tyrian purple synthesis and to expand on our general knowledge of this gland in gastropod molluscs.
The mantle tissue, which surrounds the mantle cavity and protects the visceral mass, including the hypobranchial gland, was used as the subtractor (driver) in this SSH study. The epithelium of the mantle tissue in gastropod molluscs is responsible for shell biomineralisation and thus the mantle has been the focus of several gene expression studies in gastropods (Jackson and Degnan, 2006, Nagai et al., 2007, McDougall et al., 2011). More importantly, histochemistry and chemical extractions demonstrate the mantle does not produce Tyrian purple (Westley and Benkendorff, 2008, Westley et al., 2010). We hypothesise that a number of different genes will be identified from a SSH cDNA library containing expressed sequence tags (EST) that are differentially or uniquely expressed within the hypobranchial gland, relative to the mantle tissue of D. orbita, corresponding to the roles this gland plays in organismal cell biology. Quantitative real-time PCR was applied to a subset of the identified genes to confirm differential expression.
Section snippets
Tissue collection and RNA isolation
A study investigating the chemical composition of Tyrian purple precursors within D. orbita identified that there are distinct differences in precursor composition between the hypobranchial glands of male and female individuals (Westley and Benkendorff, 2008). In order to minimise the potential gene expression differences that may be involved in this sexual dimorphism, our study focussed only on the gene expression within female snails. Two female D. orbita individuals (e.g. Fig. 1A) were
EST manual analysis
A total of 554 randomly picked EST clones were sequenced from our hypobranchial gland cDNA library, and after performing quality control on sequence chromatographs we were left with 437 usable sequences (Table 2, Table 3). When assembly of sequences was performed eliminating overlap, as well as the removal of all clones with inserts less than one hundred base pairs, we were left with 311 singletons, and 37 contigs, comprising in total 348 non-redundant sequences (Table 2).
From these 437 EST
Discussion
This study resulted in a total of 437 high quality EST sequences that are up-regulated or uniquely expressed in the hypobranchial gland, relative to mantle tissue, in D. orbita. When sequences underwent preliminary manual sequence analysis, the putative functions of 96 D. orbita protein coding sequences were identified, as well as 28 ribosomal RNA sequences and 256 novel sequences that were not assigned a function. A further 57 sequences were identified as having significant similarities to
Acknowledgements
Patrick Laffy was supported firstly by a Flinders University Faculty of Science and Engineering Research scholarship, followed by a Flinders University Postgraduate Research scholarship. This research was funded by a grant from a philanthropic foundation to KB and CAA. We would also like to thank the South Australian Partnership for Advanced Computing, for the use of their BLAST portal and Dr Chantel Westley for her assistance in specimen dissection.
References (68)
- et al.
Isolation of choline and choline ester salts of tyrindoxyl sulphate from the marine molluscs Dicathais orbita and Mancinella keineri
Tetrahedron Lett.
(1976) - et al.
Pigments of marine animals VIII. Precursors of 6,6′-dibromoindigotin (Tyrian Purple) from the mollusc Dicathais orbita Gmelin
Tetrahedron Lett.
(1968) - et al.
Chemical defense in the egg masses of benthic invertebrates: an assessment of antibacterial activity in 39 mollusks and 4 polychaetes
J. Invertebr. Pathol.
(2001) - et al.
Transcriptome analysis in Concholepas concholepas (Gastropoda, Muricidae): mining and characterization of new genomic and molecular markers
Mar. Genomics
(2011) - et al.
Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing
BMC Genomics
(2010) - et al.
Molecular phylogenetics of Caenogastropoda (Gastropoda: Mollusca)
Mol. Phylogenet. Evol.
(2007) - et al.
Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes
Methods Enzymol.
(1999) - et al.
A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis
Mol. Biochem. Parasitol.
(2002) - et al.
Crystal structure of dodecameric vanadium-dependent bromoperoxidase from the red algae Corallina officinalis
J. Mol. Biol.
(2000) - et al.
Bromoperoxidase from the marine snail Murex trunculus
Comp. Biochem. Physiol. B
(1987)
Characterization of a novel gastropod toxin (6-bromo-2-mercaptotryptamine) that inhibits shaker K channel activity
J. Biol. Chem.
Neuronal transcriptome of Aplysia: neuronal compartments and circuitry
Cell
Tyrosinase localization in mollusc shells
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
Towards a phylogeny of gastropod molluscs: an analysis using morphological characters
Zool. J. Linn. Soc.
Immune gene mining by pyrosequencing in the rockshell, Thais clavigera
Fish Shellfish Immunol.
The angiotensin system elements in invertebrates
Brain Res. Rev.
Regulation of elongation factor G GTPase activity by the ribosomal state. The effects of initiation factors and differentially bound tRNA, aminoacyl-tRNA, and peptidyl-tRNA
J. Biol. Chem.
X-ray structure determination of a vanadium-dependent haloperoxidase from Ascophyllum nodosum at 2.0 angstrom resolution
J. Mol. Biol.
Immunocytochemical localisation of glycosaminoglycan-like activity in the mucus and associated tissues of terrestrial slug species (Gastropoda: Pulmonata)
Comp. Biochem. Physiol. B
Differential expression analysis of Liprin-alpha 2 in hibernating bat (Rhinolophus ferrumequinum)
Prog. Nat. Sci.
Structure and function in the excretory system of some terrestrial prosobranch snails (Cyclophoridae)
J. Zool.
Identification of genes potentially involved in pearl formation by expressed sequence tag analysis of mantle from freshwater pearl mussel (Hyriopsis Cumingii Lea)
J. Shellfish. Res.
Tyrian purple: an ancient dye, a modern problem
Endeavour (Oxford)
Tyrian purple precursors in the egg masses of the Australian muricid, Dicathais orbita: a possible defensive role
J. Chem. Ecol.
Indole derivatives from the egg masses of Muricid molluscs
Molecules
Bioactivity of the Murex homeopathic remedy and of extracts from an Australian muricid mollusc against human cancer cells
Evid. Based Complement. Alternat. Med.
Coupling proteomics and transcriptomics for the identification of novel and variant forms of mollusk shell proteins: a study with P. margaritifera
ChemBioChem
Gastropod evolutionary rates and phylogenetic relationships assessed using partial 28S rDNA and histone H3 sequences
Zool. Scr.
Gastropod phylogeny based on six segments from four genes representing coding or non-coding and mitochondrial or nuclear DNA
Molluscan Res.
Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research
Bioinformatics
Tyrian purple: 6,6′-dibromoindigo and related compounds
Molecules
Marine compounds selectively induce apoptosis in female reproductive cancer cells but not in primary-derived human reproductive granulosa cells
Mar. Drugs
Ricerche chimiche e farmacologiche sugli estratti di ghiandola ipobranchiale di Murex (Truncularia) trunculus (L.), Murex (Biolinus) brandaris (L.) e Tritonalia erinacei (L.)
Pub. Stat. Zool. Napoli
Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata
PLoS One
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