Indole-producing bacteria from the biosynthetic organs of a muricid mollusc could contribute to Tyrian purple production.
KEY WORDS: Dicathais orbita, muricid, hypobranchial gland, 6,6'-dibromoindigo, microbial symbionts, biosynthesis, marine natural products
Molluscs are an important source of potentially useful secondary metabolites, including biologically active compounds and dyes (Benkendorff 2010). Marine gastropods of the family Muricidae produce Tyrian purple (6,6'-dibromoindigo; Cooksey 2001), a dye of significant historical importance that can be obtained from no other natural source (Westley & Benkendorff 2008). The precursors of Tyrian purple have interesting pharmacological properties (Benkendorff 2013), including the specific inhibition of a range of cancer cell lines (Benkendorff et al. 2011, Edwards et al. 2012, Esmaeelian et al. 2013) and prevention of early stage colon cancer formation in rodent models (Westley et al. 2010b, Esmaeelian et al. 2014). These brominated indole precursors are stored as choline ester salts in the hypobranchial glands of Muricidae (Baker & Sutherland 1968, Benkendorff 2013). The main choline ester is murexine, which has a muscle relaxing activity and is an effective pain killer (Roseghini et al. 1996). These pharmacological properties of Muricidae extracts and secondary metabolites have raised interest in their potential for development as new nutraceutical products (Benkendorff 2009, Benkendorff 2013).
Sustainable supply is a major impediment to the development of new marine nutraceuticals and pharmaceuticals (Benkendorff 2009, Molinski et al. 2009, Berrue et al. 2011). Many marine natural products are complex and difficult or expensive to chemically synthesize (Sipkema et al. 2005, Berrue et al. 2011), as is the case for some of the bioactive brominated indole precursors of Tyrian purple (Benkendorff 2013). In such cases, further insight into the biosynthetic origin, including potential microbial symbionts and, the specific genes involved in the biosynthesis of these secondary metabolites may provide options for supplying sufficient amounts of marine compounds for clinical testing and commercialization (Sipkema et al. 2005, Berrue et al. 2011, Lane & Moore 2011).
Secondary metabolites associated with marine invertebrates are often produced by symbiotic bacteria (Berrue et al. 2011) and comparative genetic studies of 16S rRNA highlight the coevolution between symbiotic microbes and their invertebrate hosts (Radjasa et al. 2011), particularly in the Porifera (sponges; Thacker & Starnes 2003). Secondary metabolites, such as the polyketide onnamides with antitumor properties, arise from bacterial symbionts of the host sponge Theonella swinhoei (Piel et al. 2004), whereas bryostatins, anticancer agents first isolated from the bryozoan Bugula neritina, require the microbial symbiont Endobugula sertula for their synthesis (Davidson et al. 2001, Lopanik et al. 2004, Sudek et al. 2007). Several other secondary metabolites are due to microbial symbionts associated with marine sponges (e.g., Unson et al. 1994, Sacristan-Soriano et al. 2011), although microbial populations associated with other marine invertebrates are less well studied.
Nevertheless, studies in the last decade have begun to investigate the microbial origin of some secondary metabolites found in molluscs. For example, dolastatin 10, an antitumor agent (Kindler et al. 2005) originally isolated from the marine gastropod Dolabella auricularia (Pettit et al. 1987) was recently found to be associated with the marine cyanobacterium Symploca sp. (Luesch et al. 2001). Another antitumor depsipeptide, kahalalide F (Cruz et al. 2009), was originally isolated from Elysia rufescens, but is now thought to be of bacterial (Davis et al. 2013) or algal (Bryopsis pennata) origin (Henriquez et al. 2005). Recently, Streptomyces sp. cultivated from the gastropod Lienardia totopotens, was reported to be the source of the peptide compound totopotensamide A and totopotensamide B (Lin et al. 2012). Lin et al. (2013) also reported that pyrone polyketides found in the cone snail Conus rolani are synthesized by bacteria. These examples highlight the potential for shelled molluscs to be a source of microbially derived secondary metabolites.
Structural similarity between marine invertebrate secondary metabolites and microbial compounds has been used to infer a microbial origin. For example, Rath et al. (2011), hypothesized that Trabectedin (ET-743; D'Incalci & Galmarini 2010) is the product of a marine bacterial symbiont Ecteinascidia turbinata because of its structural similarities to secondary metabolites of the bacterial species Myxococcus xanthus, Streptomyces lavendulae, and Pseudomonasfluorescens (Arai et al. 1980, Ikeda et al. 1983, Irschik et al. 1988, Rath et al. 2011). A large number of natural products from marine invertebrates, including nonribosomal peptides, polyketides, and hybrid molecules are consistent with bacterial metabolites (Faulkner 2000). These structural similarities suggest many marine invertebrate natural products could be partially or wholly derived from microbial symbionts rather than being synthesized by the host alone (Lane & Moore 2011).
The biosynthetic origin of Tyrian purple which has been exclusively isolated from Muricidae molluscs is not currently known. Indigo (which has the same structure as Tyrian purple but without the two attached bromines), is produced by a diverse range of bacterial species residing in soil (Lim et al. 2005), marine (Mercadal et al. 2010) and sludge environments (Qu et al. 2010, 2012). The structural similarities between indigo and 6,6'-dibromoindigo suggest the possible involvement of symbiotic bacteria in the biosynthesis of this compound in muricid molluscs.
Tyrian purple is generated from indoxyl sulfate precursors in the hypobranchial glands of Muricidae (Baker & Sutherland 1968). The essential amino acid tryptophan has been hypothesized to be the ultimate precursor for indole production, in which tryptophanase enzymes act on tryptophan to produce indoles (Verhecken 1989, Naegel & Alvarez 2005, Westley et al. 2006, Benkendorff 2013, Laffy et al. 2013). Indeed, tryptophan appears to be stored in the hypobranchial glands of a range of muricids (Srilakshmi 1991, Naegel & Aguilar-Cruz 2006). Westley and Benkendorff (2009), have identified the presence of tryptophan in the hypobranchial gland, and female reproductive organs, of the Australian muricid Dicathais orbita based on histochemical analysis. This is consistent with reports of Tyrian purple and indole precursors occurring in the hypobranchial glands, reproductive organs (Westley & Benkendorff 2008) and egg capsules of Muricidae (Palma et al. 1991, Benkendorff et al. 2000, 2001). Like all animals, molluscs cannot biosynthesize tryptophan (Verhecken 1989, Rouhbakhsh et al. 1997, Westley et al. 2006) so this essential amino acid must be derived from either the diet or symbiotic bacteria. Previous researchers have assumed a dietary origin for tryptophan (Verhecken 1989, Westley et al. 2006, 2010a), but here we propose a bacterial role in Tyrian purple precursor synthesis.
Indole biosynthesis can be detected in microbial communities using biochemical tests such as the indole (tryptophan degradation) test and tryptophanase activity (Holding & Collee 1971, Lammert 2007). More generally, biochemical kits can be used to identify bacteria and elucidate differences in bacterial communities based on the substrates that they can metabolize. Biochemical kits, such as API 20E, are typically used for identification of Gram negative rods and Enterobactericeae, but have been successfully applied to the preliminary characterization of microbial communities in marine samples (Alcaide et al. 1999, Xie et al. 2007, Peters et al. 2012) and this approach enables functional profiling of heterotrophic bacterial communities in different tissues.
The ability to culture bacteria using traditional techniques is an advantage for subsequent large scale production of marine natural products and so it is useful to investigate the diversity of bacteria that can be cultured from biosynthetic organs using traditional culture techniques. The main aim of this study was to compare the biochemical profiles of microbial communities derived from different tissues of Dicathais orbita, to establish whether any unique bacteria or distinct bacterial communities are associated with the biosynthetic organs compared with tissues that are known not to be involved in Tyrian purple production. A secondary aim was to culture aerobic heterotrophic bacteria from these tissues and then identifiy any indole-producing bacteria using 16S rRNA sequencing. Using this approach, three indole-producing bacteria were isolated and cultured from the biosynthetic organs of D. orbita, thus confirming symbiotic bacteria could contribute to Tyrian purple production.
MATERIALS AND METHODS
Sample Collection and Preparation
The muricid mollusc Dicathais orbita was collected under permit number P10/0069-1.0 issued by Primary Industries, NSW Government, Australia. The snails were collected from the subtidal and intertidal rocky reefs near Ballina (28[degrees] 84' S and 153[degrees] 60' E) on the north coast of NSW, Australia, during low tides in August and November, 2013. Snails were transported live to Southern Cross University in Lismore (~40 min) and held in aerated seawater tanks until processing (within 24 h). Twelve snails were collected in August for standardized comparison of the predominant culturable heterotrophic bacteria from different tissues; three male and three female D. orbita were used for biochemical characterization and a further three independent males and three females were used for culturing the predominant heterotrophic symbiotic bacteria. From each sample, swabs were taken from the outer surface of the tissues before dissection and homogenates were subsequently prepared from the inner tissue surfaces which were untouched during swabbing.
Supplementary sampling during November 2013 involved seven snails (swabs and homogenates) and egg capsules of D. orbita (Fig. 1A). These were used to obtain additional cultures and further characterize the bacterial colonies by Gram staining, motility, oxidase, and indole tests (see below).
The shell of D. orbita was removed according to Westley and Benkendorff (2008), using a bench vice with pressure applied at the point between the primary body whorl and spire. Tissues from the foot, hypobranchial, rectal, prostate (males, Fig. 1B) and capsule glands (females, Fig. 1C) were dissected under sterile conditions (Westley & Benkendorff 2008). An incision was made into each tissue and swabbed across a 0.3 [cm.sup.2] surface area with a sterile cotton bud, then placed in 2 mL micro centrifuge tubes containing 0.5 mL sterile seawater. Swabs were taken from three male and three female snails to provide independent replicates for each tissue type (Fig. 1). Additionally, tissue homogenates weighing 0.1 g were taken from a separate region of each sample of foot muscle, hypobranchial, rectal, prostate, and capsule glands. These were prepared in 0.5 mL sterile seawater by homogenizing the tissues with a UV-treated mortar and pestle.
Biochemical characterization of the bacterial communities from each of the triplicate tissue swabs and homogenate samples was undertaken using a biochemical characterization kit, (API 20E, BioMerieux, Marcy I'Etoile, France) according to the manufacturers' instructions. Triplicate samples of fresh seawater from the same location where the snails were collected were used as background controls. The swab and homogenate suspensions were diluted in 5 mL sterile seawater. After vortexing, 200 [micro]L of the suspension was pipetted into each well of the biochemical test strip. Anaerobic conditions were maintained by overlaying the wells with mineral oil for the tests of arginine dihydrolase (ADH), ornithine decarboxylase (ODC), lysine decarboxylase (LDC), hydrogen sulfide, and urease. The mineral oil does not create an anoxic environment itself but effectively provides a barrier against the continual diffusion of oxygen into the media and thus prevents potential false positives. Tests for citrate (CIT), acetoin production (VP) and gelatinase utilization (GEL) were performed by filling both the well and cupule of the test strips. All other tests were performed with only the well filled, but not the cupule. Test strips were incubated at 25[degrees]C for 48 h.
Heterotrophic Bacterial Culture and Biochemical Identification of Indole-Producing Bacteria
Two types of agar, marine agar (Difco Marine Broth 2,216 and Bacto Agar) and nutrient agar (Peptone, 5 g; Yeast extract, 3 g; NaCl, 5 g; Agar, 15 g; Milli-Q water, 1,000 mL) were used for the recovery of aerobic bacteria. A 3-fold dilution series was prepared from each of the triplicate swab and homogenate samples and then aliquots (100 pL) were plated in triplicate and spread evenly onto agar and incubated at 25[degrees]C for 48-72 h. Seawater controls were prepared in triplicate. Morphologically distinct colonies were described and counted at the lowest dilution possible then converted to colony forming units per mL.
Supplementary swabs and homogenates from the tissues of seven additional snails were undertaken by streaking onto fresh marine agar to obtain pure cultures. Gram staining and oxidase tests were performed for each of the pure cultures and each isolate was analyzed for indole production using API 20E test strips. The indole test (BioMerieux, Marcy I'Etoile, France) was performed using 5 mL of sterile seawater containing a loop of an individual colony from each pure culture. The test strips were inoculated with the bacterial suspension and incubated for 24 h at 25[degrees]C. After 24 h of incubation, one drop of JAMES reagent (BioMerieux, Marcy FEtoile, France) was added to the test strips. The formation of a pink color in the whole cupule was scored as a positive reaction. The indole positive cultures were preserved by diluting 1:1 in sterile marine broth containing 30% glycerol and stored at -80[degrees]C pending further analysis.
16S rRNA Analysis
Indole positive colonies isolated from biosynthetic tissues were identified by 16S rRNA sequencing. DNA of the indole positive isolates was extracted (QIAamp DNA Mini Kit, Qiagen) and 16S rRNA amplified using primer pair 27F-5'-GAGAGTTTGATCCTGGCTCAG-3' and 1.492R 5'-CTACGGCTACCTTGTTACGA-3' (Li & Liu 2006, Chen et al. 2012). The polymerase chain reaction (PCR) mix included 2.5 [micro]L of 10X PCR buffer; 2.5 [micro]L of dNTPs (2 mM), 1.25 [micro]L of 50 mM Mg[Cl.sub.2]; 1 [micro]L genomic DNA (35-80 ng); 0.4 [micro]L Taq polymerase and 1 [micro]L forward primer (FP) (10 pM), 1 [micro]L reverse primer (RP) (10 [micro]M), 15.35 [micro]L Milli-Q water in a final volume of 25 [micro]L. PCR cycle conditions comprised an initial denaturation at 94[degrees]C for 5 min followed by 30 cycles of 45 sec at 95[degrees]C, 1 min at 58[degrees]C and 1 min at 72[degrees]C. The PCR amplicons were separated by agarose gel electrophoresis (1.5%) followed by visualization with GelRed staining under UV irradiation, purified in accordance with the manufacturer's instructions (QIAquick PCR Purification Kit, Qiagen) and sequenced by the Australian Genome Research Facility (AGRF), Brisbane, using Applied Biosystems 3,730 and 3,730x1 capillary sequencers. DNA sequences were analyzed using Sequence scanner software vl.O and compared with sequences in the NCBI GenBank database by BLASTN. All nucleotide sequences were submitted to GenBank with the following GenBank accession numbers: KM242644, KM242645, KM242646, KM242647, KM242648, and KM242649.
Multivariate analyses of the biochemical activity (presence/ absence) were analyzed using PRIMER 6 and PERMANOVA. Owing to a lack of independence in the source snails, separate analyses were run for the swab and homogenate samples. Similarity matrices were generated using Euclidean distance for the biochemical profiles. All analyses were run using 9,999 permutations of the data. For the biochemical data, two factor nested PERMANOVAs were run with gender (male, female) and tissue (foot, hypobranchial gland, rectal gland, prostate, capsule gland) nested in gender. Pairwise tests were then undertaken on the significant factors. Nonmetric multidimensional scaling (nMDS) plots were used to graphically represent the data. Seawater was included as an additional variable in the plots for homogenates.
To assess the richness (number of morphologically distinct colony types), total abundance (CFU/mL from 0.3 [cm.sup.2] swabs or 0.1 g homogenates) and diversity (Shannon's H index) from the standardized sampling of seawater and each Dicathais orbita tissue, the DIVERSE function in PRIMER 6 was used. Three factor nested univariate analyses were then performed on the Bray-Curtis similarity matrices, with a dummy variable of one. Separate analyses were run on the swabs and homogenates. The factors used in each analysis were gender, tissue (nested in gender), and agar (marine or nutrient) with pairwise tests undertaken to establish significant differences between tissues, split for agar type in cases where the interaction term was significant. Multivariate analyses were not undertaken on the cultured bacterial communities because of uncertainty regarding whether morphologically similar bacteria were actually the same across all the different tissue samples.
Biochemical Comparison of the Bacterial Communities Associated With Different Tissues of Dicathais orbita
API 20E biochemical analysis of swabs and homogenates revealed the
same biochemical substrates were utilized by bacteria in the foot (non-Tyrian purple producing tissue) of males and females and seawater (Table 1). There was urea metabolism in foot tissue swabs, which was absent in foot homogenates and all other Dicathais orbita tissues (Table 1). No biochemical reactions were observed in homogenates of the hypobranchial and rectal gland from male and female snails (Table 1). Positive biochemical reactions were recorded in swabs taken from the hypobranchial and rectal glands, although there was some inconsistency between replicate samples for many of the tests, with only one or two out of three positive reactions for many of the biochemical tests (Table 1). All other tissues showed a high degree of consistency in the biochemical profiles between replicates (Table 1). Indole positive reactions were observed in swab samples from all tissues, as well the seawater and homogenates from all tissues except the hypobranchial and rectal gland. Arginine dihydrolase (L-arginine) metabolism was absent in swabs from the hypobranchial gland of both sexes, but was present in some samples from all other tissues. The metabolism of ODC (L-ornithine) and LDC (L-lysine) were less frequent in swabs from the hypobranchial gland, but these amino acids were consistently metabolized in all other tissues. The metabolism of sodium thiosulfate, which leads to hydrogen sulfide (EPS) production, was less apparent in swabs from male tissues compared with females (Table 1). The metabolism of sugars such as RHA (L-rhamnose) and MEL (D-melibiose) was only detected in swabs from one replicate of the female biosynthetic organs (hypobranchial and rectal glands) and generally not in these tissues from males, except one replicate of the male prostate gland (Table 1). L-Arabinose (ARA) was only associated with the female internal organs, but was not detected in homogenates or swabs from the male tissues except for the foot (Table 1).
Multivariate analyses for biochemical activity profiles from bacterial communities associated with swabs from Dicathais orbita showed that there was no significant difference between males and females (Pseudo F = 0.702, P = 0.626). There was, however, a significant difference between tissue samples within the sexes (Pseudo F = 3.781, P < 0.01). Multivariate analyses for the biochemical activity profiles of bacterial communities from the homogenates from D. orbita showed similar results to the swabs (Fig. 2). There was no significant difference between the homogenates sampled from males and females (Pseudo F= 0.377, P = 0.945), but there was a significant difference between the tissue samples within the sexes (Pseudo F= 157.5, P < 0.01).
Nonmetric multidimensional scaling of swab samples revealed some variation among samples, with the ordination showing separation of hypobranchial and prostate gland samples from foot samples (Fig. 2A). The nMDS analysis and pairwise tests between tissues from swab samples of male and female showed that the hypobranchial gland was significantly different to the foot in females (P = 0.026) and males (P = 0.009). The female capsule glands and male prostate and rectal glands were also significantly different to the foot (P < 0.05). The rectal gland was not significantly different from the hypobranchial gland (P = 0.302), capsule (P = 0.489), or prostate gland (P = 0.358).
The nMDS analyses of homogenates produced an ordination where tight clustering of hypobranchial and rectal gland samples occurred and both showed clear separation from all the other tissues (Fig. 2B). In addition, the ordination showed foot and female capsule gland samples grouping close to seawater samples (Fig. 2B). Pairwise tests between tissues for the homogenates in PERMANOVA confirmed that the hypobranchial and rectal glands were different to all other tissues (P < 0.05). Furthermore, the rectal gland was different to the prostate gland (P < 0.01). The prostate gland was also different from the hypobranchial gland (P < 0.01), but not to the foot (P = 0.054).
Bacterial Diversity Cultured From Different Tissues of Dicathais orbita and Identification of Indole-Producing Bacteria
There was substantial variation in the types of bacterial colonies isolated from the different tissue and seawater samples, with on average less than four colony types per sample (Table 2), compared with a collective total of 16 distinct colonies (Table 3). The maximum number of morphologically distinct bacteria cultured from individual samples from seawater and some prostate samples was five (data not shown). The highest abundance and diversity of bacterial colonies was found in the seawater and the foot samples, whereas the lowest bacterial richness, abundance, and diversity was recovered from the hypobranchial and rectal glands of Dicathais orbita (Table 2). The reduced bacterial diversity in these biosynthetic tissues is likely to account for the fewer biochemical reactions (Table 1) and consequently the separate clustering based on biochemical profiles (Fig. 2), in comparison with other tissues.
Univariate PERMANOVA for the richness of bacterial colony types from the homogenate samples revealed that on average, significantly more morphologically distinct types of bacteria were cultured on marine agar (mean 1.9 [+ or -] 1.3) compared with nutrient agar (mean 1.1 [+ or -] 1.1) (Pseudo F = 6.791, P = 0.026). Similarly for the swab samples, there was a greater richness of bacterial species cultured on marine (mean 2.3 [+ or -] 1.3) than nutrient agar (mean 1.4[+ or -]1) (Pseudo F= 11.448, P = 0.008). For both homogenate and swab samples, there was no significant interaction between agar and gender or tissue (P > 0.05) and no significant difference between males and females (P > 0.4). There was, however, a significant difference in the richness of bacterial species cultured from the different tissues (Homogenates: Pseudo F = 4.072, P = 0.001; Swabs; Pseudo F = 2.967, P = 0.012). Pairwise tests on the homogenates revealed fewer types of distinct bacteria in the hypobranchial gland of females compared with all other tissues (Table 2). In males, the hypobranchial glands and the rectal glands had fewer types of bacteria than the foot, but the rectal glands were not different from any other tissue (Table 2). Very similar results were found for bacterial richness in the swabs, with significantly fewer morphological types isolated from the hypobranchial glands in comparison with the foot tissue, as well as the capsule gland in females (Table 2).
Analysis of the total abundance of bacteria from homogenate samples revealed no significant differences or interactions between agar and gender (P > 0.05). There was, however, significantly higher abundances of colony-forming units (CFUs) on marine agar (mean 41.5 [+ or -] 43) than nutrient agar (15.5 [+ or -] 19) (Pseudo F = 4.787, P = 0.033) from swab samples. For both the homogenate and swab samples, there was a significant difference between tissues (homogenates Pseudo F = 9.95, P < 0.01; swabs Pseudo F = 3.689, P < 0.01). The hypobranchial gland homogenates had significantly fewer CFUs than all other tissue homogenates, except the male rectal glands (Table 2). There were also fewer bacteria cultured from female rectal glands compared with female foot tissues (Table 2). Similarly in female swab samples, there were significantly fewer bacteria cultured from the hypobranchial glands than all other tissues, whereas in the males, significantly fewer CFUs were cultured from both the rectal and hypobranchial gland compared with the foot (Table 2). Very similar statistical outcomes were found using the Shannon's diversity index (Table 2), with tissue type being the main significant factor in both homogenates (Pseudo F= 3.957, P = 0.003) and swabs (Pseudo F = 4.152, P = 0.002).
In total, 16 morphologically distinct bacterial colonies were isolated from the supplementary sampling of Dicathais orbita adults and egg capsules (Table 3). Only two of the most abundant colony types (LC1 and SC2) were ubiquitous across all tissues and seawater (Table 3). Gram staining, however, revealed at least two distinct bacteria with similar colony morphologies for LC1, as well as LC6. Most of the bacterial isolates were Gram negative, oxidase positive and motile, except isolates LCl-a, SB4 and LC61-a which were Gram positive (Table 3). Three Gram negative isolates, namely LCl-b isolated from rectal gland, LR9 from prostate gland and SC2 from hypobranchial gland homogenates and egg capsules, were found to be indole positive based on API 20E biochemical tests (Table 3). These isolates were also oxidase positive and motile.
Molecular Identification of Indole-Producing Bacteria
Analysis of 16S rRNA gene sequences using BLASTN revealed isolate LCl-b had 100% (FP) sequence similarity to Vibrio sp. P1S6 (GenBank accession no. JX477117.1) and 98% (RP) similarity to Vibrio pomeroyi strain VSG520, (GenBank accession no. KC534198.1; Table 4). Isolate SC2 had 97% (FP) and 99% (RP) sequence similarity to Vibrio sp. (GenBank accession no. KF577048.1) and Vibrio sp. V140 (GenBank accession no. DQ146978.1), respectively. Similarly, isolate LR9 showed 99% (FP and RP) sequence similarity to Vibrio gigantis strain PJ-21, (GenBank accession no. KC261280.1) and Vibrio sp. V140, (GenBank accession no. DQ146978.1). Overall, partial sequencing of 16S rRNA revealed that all three indole-producing bacteria from Dicathais orbita are likely to be Vibrio spp.
This study establishes the potential for ubiquitous indole-producing marine bacteria to contribute to Tyrian purple precursor synthesis in Muricidae molluscs. Biochemical analysis revealed microbial communities have the capacity to produce indole in all tissues sampled from Dicathais orbita, as well as in seawater controls. Three indole positive bacterial species were isolated from various D. orbita tissues and these all closely matched to Vibrio sp. The abundance, diversity, and richness of morphologically distinct bacteria from foot tissue were similar to seawater, but significantly fewer bacteria were isolated from the hypobranchial and rectal glands. No bacteria were identified that were unique to the hypobranchial and rectal glands, which are organs that store tryptophan (Westley & Benkendorff 2009) and are the main site for Tyrian purple production (Westley & Benkendorff 2008). The reduced microbial biochemical substrate utilization in these purple producing glands appears to be due to a relatively low diversity and abundance of culturable bacteria.
No biochemical reactions were observed in the homogenates of hypobranchial and rectal glands, indicating that something within these glands may be inhibiting the habitation, growth, viability, or metabolism of heterotrophic aerobic bacteria. This is likely to be due to the presence of brominated indole precursors of Tyrian purple in homogenates from these glands, which show strong inhibitory activity against a range of Gram positive and Gram negative bacteria (Benkendorff et al. 2000, Benkendorff et al. 2001). It could be assumed that any specialized endosymbionts involved in Tyrian purple precursor synthesis would be naturally resistant to the bioactive properties. Indeed in previous studies on sponges, chemical extracts from the sponges have been used to effectively "simulate" the sponge environment. Using this approach, Li and Liu (2006), successfully isolated bacteria belonging to Actinobacterium and Bacteroidetes from the sponge Craniella austrialiensis. This approach is, however, not likely to be successful if the microbial symbionts produce inactive precursors that are stored by the host invertebrate with a controlled release mechanism. The hypobranchial gland of Dicathais orbita has an highly compartmentalized structure with nine distinct cell types storing a range of vesicles with different staining reactions (Westley et al. 2010a). Two distinct types of secretory cells are believed to be associated with the separate storage of tyrindoxyl sulfate and an aryl sulfatase enzyme (Westley et al. 2010a). Homogenization of the hypobranchial gland would break down the cell structure, thus initiating the hydrolysis of tyrindoxyl sulfate by aryl sulfatase (Westley & Benkendorff 2008, Benkendorff 2013). Any bacteria associated with the gland would then be exposed to the antimicrobial brominated indole precursors of Tyrian purple, resulting in potential cell death or the inhibition of normal metabolic activity. This would then lead to no or few viable bacteria remaining in hypobranchial gland homogenates, thus explaining the lack of biochemical reactions in these samples.
Swabs taken from incisions of hypobranchial and rectal glands also resulted in relatively few biochemical reactions and significantly fewer bacteria were cultured from these glands in comparison with other tissues. Low bacterial diversity and abundance in the biosynthetic glands might be due to the presence of "unculturable bacteria." Many bacterial symbionts are difficult to culture, with only an estimated 0.001 %-0.1 % of marine microbes being successfully cultured (Ferguson et al. 1984, Amann et al. 1995). Furthermore, tissues associated with particular secondary metabolites, such as Tyrian purple precursors, are likely to support distinct chemical environments. This may lead to selection of well-adapted highly specialized symbiotic bacteria that are particularly difficult to culture using standard culture conditions. For example, preliminary data indicates that the hypobranchical glands of Dicathais orbita have a pH of less than 5 and a different oxidation-reduction potential in comparison with other tissues, such as the foot (unpublished data). Recent studies on the effects of low pH on marine bacterial communities have demonstrated significant effects on community composition and metabolism (Krause et al. 2012, Siu et al. 2014). Similarly, multivariate analyses of the biochemical profiles of swabs from D. orbita confirmed that the hypobranchial and rectal glands were statistically different to other tissue samples. This implies that the bacteria associated with these glands have distinct metabolic requirements, likely to result from adaptations to the specific chemical environment within these glands.
The hypobranchial gland is a reducing environment with high production of mercaptans, including dimethyl disulfide (Benkendorff et al. 2001). Many bacterial species can metabolize compounds such as thiosulfate to produce [H.sub.2]S (Clarke 1953). Biochemical tests revealed thiosulfate metabolism, leading to hydrogen sulfide production was more common in bacterial swabs from female hypobranchial glands and reproductive organs than the equivalent male tissues. This is consistent with previous findings of higher concentrations of methane thiol containing intermediate precursors of Tyrian purple in female glands compared with oxidized products in the males (Westley & Benkendorff 2008). Consequently, bacteria involved in thiosulfate metabolism may facilitate the synthesis of reduced Tyrian purple precursors in females, which are then transfered to the egg capsules for maximal antimicrobial protection of the encapsulated embryos (Benkendorff et al. 2000).
Biochemical tests can provide a simple culture independent method for assessing some properties of the resident bacteria. The absence of urea metabolism in most of the Dicathais orbita tissue samples, with the exception of swabs from the foot, suggest that urea is not an important source of nitrogen for these symbiotic bacteria and the source of nitrogen for the microbial communities might be amino acids (Table 1). The preferred source of nitrogen for most bacteria is ammonium (Warner 1956, Masepohl et al. 2001), amino acids, purines, and polyamines (Masepohl et al. 2001). The absence of L-arginine metabolism and reduced levels of L-ornithine and L-lysine metabolism from the hypobranchial gland swabs may indicate the presence of uncommon enteric bacterial communities that are not easily cultured in nutrient rich broth, as is often the case with marine bacteria. The use of dilute nutrient media can facilitate the culturing of previously unculturable bacteria from aquatic environments (Connon & Giovannoni 2002, Rappe et al. 2002). Enzyme activity associated with the metabolism of sugar substrates, such as L-rhamnose, D-melibiose, and L-arabinose was also less common in the Tyrian purple producing biosynthetic organs, particularly in male D. orbita. This biochemical profile is in common with Roseivirga echinicomitans sp. nov., a novel marine bacterium isolated from sea urchins (Strongylocentrolus intermedins) which also does not use these sugars (Nedashkovskaya et al. 2005). These biochemical profiles further support the presence of specific bacterial communities that are using alternative carbon sources in the biosynthetic tissues of D. orbita.
Ubiquitous bacteria were present in all the non-Tyrian purple-associated organs of Dicathais orbita. The similar biochemical profiles and bacterial communities present in the seawater and foot of D. orbita suggests the foot is mostly occupied by opportunistic bacteria from the external environment. The foot of marine gastropods is directly exposed to seawater, whereas the internal organs may be exposed to reduced bacterial loads within the mantle cavity. Anatomical studies, have suggested that the production of mucus from the hypobranchial gland in gastropods facilitates the binding and removal of particulate matter introduced in the respiratory current (Fretter & Graham 1994, Fretter et al. 1998). The hypobranchial glands of D. orbita secrete highly sulfated mucopolysaccharides (Westley et al. 2010a, Laffy et al. 2013), which are commonly associated with antimicrobial defense (Westley et al. 2010a) and could therefore effectively trap and kill bacteria as seawater passes into the mantle cavity. Although some seawater-associated bacteria were found in all of the reproductive organs, particularly in the male prostate, mucus secretions may reduce the number of opportunistic bacteria reaching other internal organs.
Only three indole-producing bacterial species were cultured from Dicathais orbita tissues and these were all found to have greater than 97% 16S rRNA sequence similarity to Vibrio sp. Greater than 97% 16S rRNA sequence similarity is the benchmark for bacterial species differentiation (Stackebrandt & Goebel 1994). Species of the highly diverse Vibrionaceae family are commonly associated with marine invertebrates (Cheng et al. 1995, Sawabe et al. 2003, Thompson et al. 2004, Chimetto et al. 2011, Lasa et al. 2013) and marine sediments (Baross & Liston 1970, Williams & Larock 1985, Urakawa et al. 2000). Furthermore, many marine Vibrios produce indole, including V. gigantis, V. pomeroyi (Beleneva & Kukhlevskii 2010), and V. parahaemolyticus (Alcaide et al. 1999) which were most similar to the indole-producing bacteria from D. orbita. Consequently, these Vibrio spp. could be providing some of the basic building blocks for Tyrian purple precursor synthesis, which may be acquired opportunistically and stored within the biosynthetic organs. Furthermore, some Vibrio sp. produce tryptophanase (Klug & DeMoss 1971) and may also produce haloperoxidase (Small & McFall-Ngai 1999, Nishiguchi et al. 2004). Bromination of tryptophan or indole precursors is essential for Tyrian purple biosynthesis and bromoperoxidase activity has been reported in the hypobranchial glands of Muricidae (Jannun et al. 1981), including D. orbita (Westley & Benkendorff 2009). More broadly, marine Vibrionaceae have thiol peroxidase (Cha et al. 2004) and produce a range of bioactive compounds including the indole derivative indazole-3-carbaldehyde (Indazole) (Fotso Fondja Yao et al. 2010). Consequently, it is plausible that endosymbiotic Vibrio spp. are involved in the biosynthesis of Tyrian purple precursors. Nevertheless, further studies, similar to those used for identifying halogenases in sponge microbiota (Ozturk et al. 2013), will be required to confirm this.
In conclusion, this study is the first report on the diversity of bacterial communities associated with Dicathais orbita. The presence of indole-producing bacteria in the tissues of D. orbita that are associated with Tyrian purple synthesis suggest a possible role for ubiquitous symbiotic bacteria in the production of early precursors to Tyrian purple in Muricidae molluscs. Nevertheless, to further investigate any unique symbionts and their capacity to brominate indole precursors, other bacteria associated with the hypobranchial gland may need to be identified through culture independent methods. Our preliminary metagenome data reveals higher diversity of bacterial taxa in the foot than the hypobranchial gland of D. orbita and also indicates higher proportion of Vibrio spp. in the hypobranchial glands (unpublished data), thus supporting the conclusions drawn from heterotrophic cultural bacterial in this study.
We would like to thank Mr. Paul Kelly, Southern Cross University (SCU), for his assistance in collecting the snail samples. We appreciate feedback on the draft manuscript from Dr. Joshua Smith, Mrs. Roselyn Regino, and Ms. Nongmaithem Bijayalakshmi Devi (SCU). This study was supported by the funding from the Marine Ecology Research Centre, Southern Cross University, a philanthropic grant to K.B. and an International Postgraduate Research Scholarship (IPRS), and Australian Postgraduate Award (APA) for A.K.N's PhD research, Southern Cross University.
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AJIT KUMAR NGANGBAM, (1) DANIEL L. E. WATERS, (2) STEVE WHALAN, (1) ABDUL BATEN (2) AND KIRSTEN BENKENDORFF (1) *
(1) Marine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University, P.O. Box 157, Lismore, NSW2480, Australia; (2) Southern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, Australia
* Corresponding author. E-mail: email@example.com
TABLE 1. API 20E biochemical test of Dicathais orbita tissue homogenates and swabs. Homogenates RG HBG Foot CG PG SW Test F M F M F M F M SW ONPG +++ +++ - - - - +++ +++ +++ ADH +++ +++ - - - - +++ +++ +++ LDC +++ +++ - - - - +++ +++ +++ ODC +++ +++ - - - - +++ +++ +++ CIT +++ +++ - - - - +++ +++ +++ [H.sub.2]S +++ +++ - - - - +++ +++ +++ URE - - - - - - - - - TDA +++ +++ - - - - +++ +++ +++ IND +++ +++ - - - - +++ +++ +++ VP +++ +++ - - - - +++ +++ +++ GEL +++ +++ - - - - +++ +++ +++ GLU +++ +++ - - - - +++ +++ +++ MAN +++ +++ - - - - +++ +++ +++ INO - - - - - - - - - SOR +++ +++ - - - - +++ +++ +++ RHA +++ ++ - - - - +++ +++ +++ SAC +++ +++ - - - - +++ +++ +++ MEL +++ ++ - - - - +++ +++ +++ AMY +++ +++ - - - - +++ +++ +++ ARA +++ - - - - - +++ +++ +++ Swabs RG HBG Foot CG PG Test F M F M F M F M ONPG +++ +++ +++ +++ +++ ++ +++ +++ ADH +++ +++ +++ + - - +++ +++ LDC +++ +++ ++ +++ + - +++ +++ ODC +++ +++ +++ +++ + + +++ +++ CIT +++ +++ +++ +++ +++ + +++ +++ [H.sub.2]S ++ - + - + - +++ +++ URE - + + - - - ++ +++ TDA +++ +++ +++ +++ +++ ++ +++ +++ IND +++ +++ +++ +++ +++ ++ +++ +++ VP +++ +++ +++ +++ +++ ++ +++ +++ GEL +++ +++ +++ +++ ++ ++ +++ +++ GLU +++ +++ +++ +++ +++ ++ +++ +++ MAN +++ +++ +++ +++ +++ ++ +++ +++ INO - - - - - - - - SOR +++ ++ ++ +++ +++ - +++ +++ RHA ++ + + - + - +++ +++ SAC +++ +++ +++ +++ +++ +++ +++ +++ MEL - + + + + - +++ +++ AMY +++ +++ +++ +++ +++ +++ +++ +++ ARA +++ - ++ - + - +++ +++ "+," positive biochemical test in a single replicate; "++" and "+++" indicate positive tests in two or three replicate samples, respectively; negative biochemical test in all three replicates. CG, capsule gland; PG, prostate gland; RG, rectal gland; HBG, hypobranchial gland; SW, seawater; F, female; M, male; Biochemical test: ONPG, ortho-NitroPhenyl-[beta]-D-galactopyranoside; ADH, arginine dihydrolase; LDC, lysine decarboxylase; ODC, ornithine decarboxylase; CIT, citrate utilization; [H.sub.2]S, [H.sub.2]S production; URE, urease; TDA, tryptophan deaminase; IND, indole production; VP, acetoin production; GEL, gelatinase; GLU, glucose; MAN, mannitol; INO, inositol; SOR, sorbitol; RHA, rhamnose; SAC, saccharose; MEL, melibiose; AMY, amygdalin; ARA, arabinose. TABLE 2. Diversity of bacteria cultured from different tissues of Dicathais orbita. Richness Preparation Agar Sex Tissue (mean [+ or -] SD) Homogenates Marine NA Seawater 3.33 [+ or -] 2.08 agar Female Foot 3.0 [+ or -] 0.0 (a) Hypobranchial 0.33 [+ or -] 0.57 (b) gland Rectal gland 1.66 [+ or -] 0.57 (a) Capsule gland 2.33 [+ or -] 0.57 (a) Male Foot 3.0 [+ or -] 1.0 (a) Hypobranchial 1.0 [+ or -] 0.0 (b) gland Prostate 1.33 [+ or -] 0.57 (ab) gland Rectal gland 1.0 [+ or -] 0.0 (b) Nutrient NA Seawater 3.0 [+ or -] 2.0 agar Female Foot 1.0 [+ or -] 1.73 (a) Hypobranchial 0.0 [+ or -] 0.0 (b) gland Rectal gland 0.33 [+ or -] 0.57 (a) Capsule gland 1.0 [+ or -] 1.0 (a) Male Foot 2.0 [+ or -] 1.0 (a) Hypobranchial 1.0 [+ or -] 1.0 (b) gland Prostate 1.33 [+ or -] 0.57 (ab) gland Rectal gland 0.66 [+ or -] 0.57 (b) Swabs Marine Female Foot 3.0 [+ or -] 0.0 (a) agar Hypobranchial 1.0 [+ or -] 0.0 (b) gland Rectal gland 2.0 [+ or -] 1.0 (ab) Capsule gland 2.0 [+ or -] 0.0 (a) Male Foot 3.0 [+ or -] 0.0 (a) Hypobranchial 2.0 [+ or -] 1.0 (b) gland Prostate 3.0 [+ or -] 2.64 (ab) gland Rectal gland 1.33 [+ or -] 0.57 (b) Nutrient Female Foot 1.0 [+ or -] 1.0 (ab) agar Hypobranchial 0.66 [+ or -] 0.57 (a) gland Rectal gland 1.66 [+ or -] 1.52 (ab) Capsule gland 1.33 [+ or -] 0.57 (b) Male Foot 1.0 [+ or -] 1.0 (a) Hypobranchial 0.66 [+ or -] 0.57 (b) gland Prostate 1.66 [+ or -] 1.52 (ab) gland Rectal gland 1.0 [+ or -] 0.0 (ab) Total abundance Preparation Agar Sex Tissue (mean [+ or -] SD) Homogenates Marine NA Seawater 18.66 [+ or -] 10.40 agar Female Foot 14.0 [+ or -] 5.29 (a) Hypobranchial 0.33 [+ or -] 0.57 (c) gland Rectal gland 2.33 [+ or -] 0.57 (b) Capsule gland 7.33 [+ or -] 6.11 (ab) Male Foot 14.66 [+ or -] 4.50 (a) Hypobranchial 1.0 [+ or -] 0.0 (b) gland Prostate 9.66 [+ or -] 2.51 (a) gland Rectal gland 1.33 [+ or -] 0.57 (b) Nutrient NA Seawater 13.0 [+ or -] 6.24 agar Female Foot 3.66 [+ or -] 6.35 (a) Hypobranchial 0.0 [+ or -] 0.0 (c) gland Rectal gland 0.33 [+ or -] 0.57 (b) Capsule gland 5.33 [+ or -] 6.80 (bc) Male Foot 7.66 [+ or -] 4.04 (a) Hypobranchial 1.66 [+ or -] 2.08 (b) gland Prostate 8.66 [+ or -] 2.51 (a) gland Rectal gland 0.66 [+ or -] 0.57 (b) Swabs Marine Female Foot 55.33 [+ or -] 32.47 (a) agar Hypobranchial 2.66 [+ or -] 1.52 (b) gland Rectal gland 24.66 [+ or -] 10.40 (a) Capsule gland 46.66 [+ or -] 51.38 (a) Male Foot 78.67 [+ or -] 28.59 (a) Hypobranchial 11.0 [+ or -] 5.29 (b) gland Prostate 90.33 [+ or -] 78.42 (ab) gland Rectal gland 22.66 [+ or -] 29.14 (b) Nutrient Female Foot 22.33 [+ or -] 32.80 (a) agar Hypobranchial 0.66 [+ or -] 0.57 (b) gland Rectal gland 20.66 [+ or -] 21.54 (a) Capsule gland 22.0 [+ or -] 33.80 (a) Male Foot 25.67 [+ or -] 9.02 (a) Hypobranchial 1.33 [+ or -] 1.52 (b) gland Prostate 27.66 [+ or -] 24.37 (ab) gland Rectal gland 3.33 [+ or -] 1.15 (b) H index Preparation Agar Sex Tissue (mean [+ or -] SD) Homogenates Marine NA Seawater 0.92 [+ or -] 0.80 agar Female Foot 0.95 [+ or -] 0.07 (a) Hypobranchial 0.0 [+ or -] 0.0 (b) gland Rectal gland 0.45 [+ or -] 0.39 (ab) Capsule gland 0.68 [+ or -] 0.21 (a) Male Foot 0.86 [+ or -] 0.31 (a) Hypobranchial 0.0 [+ or -] 0.0 (b) gland Prostate 0.15 [+ or -] 0.27 (b) gland Rectal gland 0.0 [+ or -] 0.0 (b) Nutrient NA Seawater 0.83 [+ or -] 0.75 agar Female Foot 0.27 [+ or -] 0.48 (a) Hypobranchial 0.0 [+ or -] 0.0 (b) gland Rectal gland 0.0 [+ or -] 0.0 (b) Capsule gland 0.23 [+ or -] 0.40 (a) Male Foot 0.51 [+ or -] 0.52 (a) Hypobranchial 0.22 [+ or -] 0.38 (b) gland Prostate 0.16 [+ or -] 0.28 (b) gland Rectal gland 0.0 [+ or -] 0.0 (b) Swabs Marine Female Foot 0.78 [+ or -] 0.27 (a) agar Hypobranchial 0.0 [+ or -] 0.0 (b) gland Rectal gland 0.49 [+ or -] 0.43 (a) Capsule gland 0.50 [+ or -] 0.16 (a) Male Foot 0.63 [+ or -] 0.29 (a) Hypobranchial 0.51 [+ or -] 0.45 (b) gland Prostate 0.64 [+ or -] 0.56 (ab) gland Rectal gland 0.15 [+ or -] 0.27 (b) Nutrient Female Foot 0.22 [+ or -] 0.39 (a) agar Hypobranchial 0.0 [+ or -] 0.0 (b) gland Rectal gland 0.43 [+ or -] 0.39 (a) Capsule gland 0.13 [+ or -] 0.24 (a) Male Foot 0.79 [+ or -] 0.23 (a) Hypobranchial 0.0 [+ or -] 0.0 (b) gland Prostate 0.44 [+ or -] 0.41 (ab) gland Rectal gland 0.0 [+ or -] 0.0 (b) Richness, number of morphologically distinct bacteria; Total abundance, number of colony forming units per mL from a 0.3 [cm.sup.2] swab or 0.1 g tissue homogenate; H, Shannon's diversity index. Different small letters indicate statistically difference between tissues nested in sex in PERMANOVA pairwise tests (P < 0.05). TABLE 3. Summary of the morphologically distinct bacteria isolated from seawater and different tissues of Dicathais orbita. Bacterial Gram Motility Oxidase isolates Description stain test test LC1 Large colony, circular, creamy color LCl-a (Gram +) Gram + Motile + LCl-b (Gram -) Gram - Motile - SC2 Small colony, circular, Gram - Motile + creamy color ST3 Small colony, circular, Gram - Motile + transparent SB4 Small colony, circular, Gram + Motile + dark brown in color ST5 Small colony, watery Gram - Motile + appearance, transparent LC6 Large colony, irregular edge, creamy color LC6-a (Gram +) Gram + Motile + LC6-b (Gram -) Gram - Motile + SG7 Small colony, circular, NT NT NT light green color LY8 Large colony, circular, NT NT NT yellow color LR9 Large colony, irregular, Gram - Motile + rhizoid LT10 Large colony, circular, Gram - Motile + transparent SP11 Small colony, circular, Gram - Motile + pink color LP12 Large colony, circular, Gram - Motile + pink color SCY13 Small colony, circular, Gram - Motile + yellow color RC14 Round colony, dull creamy Gram - Motile + color LI515 Large colony, irregular Gram - Motile + shape, transparent STS16 Small colony, transparent, Gram - Motile + shiny appearance Bacterial Indole isolates Description test SW F HBG LC1 Large colony, circular, X X X creamy color LCl-a (Gram +) - X LCl-b (Gram -) + SC2 Small colony, circular, + X X X creamy color ST3 Small colony, circular, - X X transparent SB4 Small colony, circular, - X dark brown in color ST5 Small colony, watery - appearance, transparent LC6 Large colony, irregular X X edge, creamy color LC6-a (Gram +) - LC6-b (Gram -) - SG7 Small colony, circular, NT light green color LY8 Large colony, circular, NT X yellow color LR9 Large colony, irregular, + rhizoid LT10 Large colony, circular, - X transparent SP11 Small colony, circular, X X pink color LP12 Large colony, circular, - X pink color SCY13 Small colony, circular, - X yellow color RC14 Round colony, dull creamy - color LI515 Large colony, irregular - shape, transparent STS16 Small colony, transparent, X shiny appearance Bacterial isolates Description RG PG CG EC LC1 Large colony, circular, X X X creamy color LCl-a (Gram +) LCl-b (Gram -) X SC2 Small colony, circular, X X X X creamy color ST3 Small colony, circular, X X transparent SB4 Small colony, circular, X dark brown in color ST5 Small colony, watery X X appearance, transparent LC6 Large colony, irregular X edge, creamy color LC6-a (Gram +) X X X X LC6-b (Gram -) X X X SG7 Small colony, circular, X light green color LY8 Large colony, circular, X yellow color LR9 Large colony, irregular, X rhizoid LT10 Large colony, circular, transparent SP11 Small colony, circular, X X pink color LP12 Large colony, circular, X X pink color SCY13 Small colony, circular, X yellow color RC14 Round colony, dull creamy X color LI515 Large colony, irregular X shape, transparent STS16 Small colony, transparent, shiny appearance SW, seawater; F, foot; HBG, hypobranchial gland; RG, rectal gland; PG, prostate gland; CG, capsule gland; EC, egg capsules. X indicates presence in the respective tissue and those in capital letters were isolated and cultured for Gram staining, motility, oxidase, and indole testing. NT, not tested due to insufficient growth in culture. TABLE 4. Results of BLASTN analysis showing the closest match to other 16S rRNA gene partial sequences in GenBank for each indole-producing bacterial isolate cultured from Dicathais orbita tissues. Bacterial GenBank isolates accession nos. Length (base pair) Identity (%) LC1-b FP KM242647 427 100 RP KM242644 904 98 SC2 FP KM242648 648 97 RP KM242645 1,115 99 LR9 FP KM242646 435 99 RP KM242649 605 99 Bacterial isolates Closest match and accession number LC1-b FP Vibrio sp. P1S6, (JX477117.1) RP Vibrio pomeroyi strain VSG520, (KC534198.1) SC2 FP Vibrio sp. (KF577048.1) RP Vibrio sp. V140, (DQ146978.1) LR9 FP Vibrio gigantis strain PJ-21, (KC261280.1) RP Vibrio sp. V140, (DQ146978.1) FP, forward primer; RP, reverse primer.
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|Author:||Ngangbam, Ajit Kumar; Waters, Daniel L.E.; Whalan, Steve; Baten, Abdul; Benkendorff, Kirsten|
|Publication:||Journal of Shellfish Research|
|Date:||Aug 1, 2015|
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