GreenshellTM Mussel Research Articles

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0°C for 5min, then cooling at 1°Cmin−1 to −10°C, holding for a further 5min, then cooling at 0.5°Cmin−1 to −35°C followed by a 5min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.

Determination of Brevetoxins in Shellfish by LC/MS/MS: Single-Laboratory Validation

A single-laboratory validation is reported for an LC/MS/MS quantification of six brevetoxins in four matrixes (Greenshell mussel, eastern oyster, hard clam, and Pacific oyster). Recovery and precision data were collected from seven analytical batches using shellfish flesh at 0.05 mg/kg. Method recoveries and within-laboratory reproducibility ranged from 73 to 112%, with an RSD between 14 and 18% for brevetoxin-3, brevetoxin B5, brevetoxin B2, and S-desoxy brevetoxin B2. The recovery and within-laboratory reproducibility for brevetoxin-2 was 61%, with an RSD of 27%. Brevetoxin B1 gave an RSD of 12%, but no reference material was available and this toxin was only recorded in a hard clam sample naturally contaminated with brevetoxins. One naturally contaminated sample of each shellfish matrix, with brevetoxin levels ranging from 0.012 to 9.9 mg/kg, was tested in multiple batches, and the RSDs were similar to those for fortified samples at 0.05 mg/kg. Comparisons with limited data for the neurotoxic shellfish poisoning mouse bioassay for four naturally contaminated shellfish samples showed that the regulatory action limit of 0.8 mg/kg is conservative with respect to the bioassay regulatory limit of 20 mouse units/100 g.

The toxic effects of three dinoflagellate species from the genus Karenia on invertebrate larvae and finfish

Three species of Kareniaceae, Karenia brevis, Karenia brevisulcata and Karenia mikimotoi were assessed for toxicity to juvenile Chinook salmon (Oncorhynchus tshawytscha), snapper (Pagrus auratus) and six species of marine invertebrate larvae (GreenshellTM mussel, pacific oyster, paua [New Zealand abalone], sea slug, sea urchin and brine shrimp). Karenia brevis was ichthyotoxic to juvenile salmon with a LT50 of 0.75 h at 0.18 × 106 cells L–1 but juvenile snapper were less sensitive (LT50 of 4.5 h at 5.9 × 106 cells L–1). Karenia brevisulcata also showed differential toxicity to juvenile salmon and snapper with LT50 of 0.48 h at 11 × 106 cells L–1 and 7.5 h at 16 × 106 cells L–1 respectively. However, no acute symptoms were observed in the fish following 48 h exposure to K. mikimotoi at 3.4 × 106 cells L–1. Karenia brevis at the highest level tested (8.7 × 106 cells L–1) did not cause observable symptoms in any of the invertebrate larvae over 24 h. Karenia mikimotoi only affected paua larvae (LT50 10 h at 12 × 106 cells L–1). Karenia brevisulcata was highly toxic to mussel, sea urchin and paua larvae (LT50 1–6.1 h at 7.5–9.6 × 106 cells L–1), toxic to sea slug and oyster larvae (LT50 6 h at 80 × 106 cells L–1 and 9.2 h at 34 × 106 cells L–1 respectively) but did not affect brine shrimp. Analysis of test cultures by liquid chromatography-mass spectrometry confirmed that K. brevis produced brevetoxins (52 pg cell–1; mainly brevetoxin-2). Karenia brevisulcata produced brevisulcatic acids (BSXs) and the more toxic Karenia brevisulcata toxins (KBTs). Karenia mikimotoi did not produce brevetoxin, BSXs or KBTs. The present study confirms the broad threats that blooms of K. brevisulcata pose to marine ecosystems and aquaculture, and the hazards of K. brevis to finfish and K. mikimotoi to abalone respectively.

Isolation and characterization of an enzyme from the Greenshell™ mussel Perna canaliculus that hydrolyses pectenotoxins and esters of okadaic acid

An enzyme capable of hydrolysing pectenotoxins (PTXs) and okadaic acid (OA) esters within the hepatopancreas of the Greenshell™ mussel Perna canaliculus was isolated and characterized. The enzyme was purified by sequential polyethylene glycol fractionation, anion exchange, hydrophobic interaction, gel filtration and hydroxyapatite chromatography. The enzyme was an acidic (pI ∼ 4.8), monomeric, 67 kDa, serine esterase with optimum activity at pH 8.0 and 25 °C. PTX2 and PTX1 were hydrolysed but the enzyme was inactive against PTX11, PTX6 and acid isomerised PTX2 and PTX11. PTX11 and PTX2b competitively inhibited PTX2 hydrolysis. The enzyme also hydrolysed short and medium chain length (C2–C10) 4-nitrophenyl-esters, okadaic acid C8–C10 diol esters and DTX1 7-O-palmitoyl ester (DTX3). MALDI-Tof MS/MS analysis showed that the enzyme had some homology with a juvenile hormone esterase from the Red Flour Beetle Tribolium castaneum, although BLAST searches of several data bases using de novo amino acid sequences failed to identify any homology with known proteins.

Performance of single and multi-strain probiotics during hatchery production of Greenshell™ mussel larvae, Perna canaliculus

Earlier work identified two novel strains of probiotic bacteria, Alteromonas macleodii 0444 and Neptunomonas sp. 0536, for Greenshell™ mussel (Perna canaliculus) larvae. Herein, we investigated whether the combination of the two probiotics in a multi-strain mix provided a) enhanced larval production during routine rearing and b) improved larval protection during two separate pathogen-challenge tests (Vibrio sp. DO1 and Vibrio splendidus). The response of larvae to multi-strain or single-strain probiotic administration was compared to that of larvae without probiotics. Two concentrations of each probiotic were tested (107 and 108CFUml−1). Addition of 108CFUml−1 multi-strain mix in the routine rearing of larvae yielded smaller-sized larvae and a lower feeding rate when compared with the 107CFUml−1 and control groups. During the challenge test against Vibrio sp. DO1 and V. splendidus, protection by single-strain probiotic administration was observed at both levels of probiotic administration (107 and 108CFUml−1), with no apparent added protection from multi-strain probiotics. Although 108CFUml−1 levels provided protection against pathogen attack, they were also potentially detrimental to normal larval rearing when administered in combination and, as such, where administration of A. macleodii 0444 and Neptunomonas sp. 0536 would be applied together, a multi-strain mix of probiotics at 107CFUml−1, is recommended as the best concentration of each probiotic.

Cryopreservation of Greenshell™ mussel (Perna canaliculus) sperm. I. Establishment of freezing protocol

A series of eight experiments was conducted to determine the most suitable cryoprotectants (CPAs), dilution rates and methods of CPA addition as well as freezing method for the cryopreservation of Greenshell™ mussel (Perna canaliculus) sperm. The experiments utilized a fertilization assay to determine the treatments that gave the highest post-thaw fertility. The assays revealed that dimethyl sulphoxide (DMSO) gave the best results of the seven CPAs tested. A concentration of DMSO of approximately 12% [volume: volume] in Milli-Q water provided the best results. A dilution rate of 1:1 (sperm: CPA solution) was superior to 1:3 indicating that more concentrated sperm had higher post-thaw fertility. The time that sperm were exposed to the CPA solution prior to freezing had no effect over the range tested which gives some flexibility in the amount of time sperm can be exposed to CPA prior to freezing. The addition of the CPA in either one step or in 4 equal volume steps over two min had no significant effect on fertility and the two different freezing methods (programmable freezer or rack floating above liquid nitrogen) produced similar results. The post-thaw fertility of mussel sperm using the final protocol developed (12% DMSO plus 0.2M trehalose, sperm diluted 1:1, loaded into 0.5mL straws and cooled on a rack 3cm above liquid nitrogen followed by immersion in liquid nitrogen) was significantly reduced in comparison to fresh sperm. On average, approximately 363 x more sperm were required to achieve an equivalent level of fertilization as that of fresh sperm. The method is being used in mussel selective breeding, however, there is considerable between-animal variability. Further refinement to the method may help to improve post-thaw fertility.

Biofouling on Greenshell™ mussel (Perna canaliculus) farms: a preliminary assessment and potential implications for sustainable aquaculture practices

Greenshell™ mussel (Perna canaliculus) culture is the primary aquaculture industry in New Zealand. However, our knowledge of biofouling on Greenshell™ mussel farms, and its contribution to farm ecotrophic effects, is poor. We conducted a preliminary study of biofouling accumulation at two Greenshell™ mussel farms during Intermediate and Final seed on-growing stages (each of 6 months duration) with sampling of mussel ropes at 0, 3, 5 and 6 months during each on-growing stage. A diverse range of biofouling organisms (71 distinct taxa) accumulated on mussel ropes, with biofouling biomass dominated by suspension-feeding organisms (~88% of biofouling biomass) such as other bivalves, ascidians and, to a lesser degree, bryozoans. Biofouling biomass increased with culture time, varied between farms and was generally greater at 2 m than at 8 m depth. After 6 months, biofouling organisms on average comprised 54% of the total rope biomass. The reseeding of ropes between Intermediate and Final seed crops reduced the amount of non-Greenshell™ mussel biofouling. However, after 6 months, non-Greenshell™ mussel biofouling on average still comprised 15% of the total rope biomass. To evaluate potential ecotrophic effects of biofouling on Greenshell™ mussel farms, we compare the clearance rates of Greenshell™ mussel longlines based on Greenshell™ mussels alone and when combined with the two dominant biofouling species observed in our study (the mussel Mytilus galloprovincialis and the ascidian Ciona intestinalis). Our study shows that accumulated biofouling biomass on Greenshell™ mussel ropes can be significant and recommends further investigation as to actual ecotrophic effects of biofouling to ensure sustainable mussel farm practices.

Variation in nutrient loading from the Motueka River into Tasman Bay, New Zealand, 2005–2009: implications for the river plume ecosystem

The monthly mass transport of nitrate nitrogen (NO3-N), ammonium nitrogen (NH4-N), total nitrogen (TN), dissolved reactive phosphorus (DRP), total phosphorus (TP) and dissolved reactive silica (DRSi) from the Motueka River catchment into Tasman Bay (South Island, New Zealand) was estimated for the period 2005–2009 based on observed historical relationships between recorded river flows and the associated measured concentrations of each nutrient. The resulting average annual loading estimates for TN, TP and DRSi ranged from 397–829, 25–72 and 4053–6576 tonnes, respectively, over the 5-year period. Large seasonal variations in nutrient loadings were also estimated with the highest rates generally, but not always, occurring during winter. The observed patterns suggest that phytoplankton biomass within the river outwelling plume may also vary in response to seasonal and inter-annual variations in catchment nutrient loading rates with potential follow-on food web effects including the growth of cultured GreenshellTM mussels. The estimated average annual TN loading rate (2005–2009) from the Motueka River catchment (613 tonnes) is only c. 20% of the annual loss of nitrogen based on extrapolated literature values for denitrification rates in Tasman Bay. We therefore suggest that, under the present land use scenario in the Motueka catchment, there is little potential for resulting dysfunctional river plume ecosystem enrichment effects to occur.

Prevalence and biofilm-forming ability of Listeria monocytogenes in New Zealand mussel (Perna canaliculus) processing plants

Greenshell™ mussels are New Zealand’s largest seafood export species. Some export markets require compliance with ‘zero’ tolerance legislation for Listeria monocytogenes in 25 g of product. Even though individually quick frozen (IQF) mussel products are labeled ‘to be cooked’, and are not classified as ready-to-eat, some markets still require them to comply with the strict policy. Three mussel processing plants were assessed for the pattern of L. monocytogenes contamination on raw material, environment, food contact surfaces, and in the final product. Cultures (n = 101) obtained from an industrial Listeria monitoring program from August 2007 to June 2009 were characterized by serotyping and pulsed field gel electrophoresis. Using the crystal violet method, isolates were assessed for their ability to form biofilms. This work confirmed the presence of L. monocytogenes in raw and processed product, and the importance of cross-contamination from external and internal environments. Processing plants had L. monocytogenes pulsotypes that were detected more than once over 6 months. No correlation was found between biofilm-forming ability and persistent isolates. Two pulsotypes (including a persistent one), were previously isolated in human cases of listeriosis in New Zealand, but none of the pulsotypes matched those involved in international outbreaks.

Relative importance of natural cues and substrate morphology for settlement of the New Zealand Greenshell™ mussel, Perna canaliculus

Production from aquaculture of the New Zealand Greenshell™ mussel, Perna canaliculus, is worth more than US$190 M annually. However, expansion of the industry is currently limited by the number of naturally occurring seed available for growout. Previous work suggests that natural chemical cues and settlement surface topography may be important factors in the settlement and retention of P. canaliculus larvae. This study investigated the effects both of these factors separately and in conjunction on the settlement of P. canaliculus larvae under laboratory conditions. Eight species of marine algae (5 red and 3 brown) and one species of hydrozoan that have high numbers of mussels attached in the field were targeted as sources of chemical cues. In laboratory assays, non-polar extracts from four of the eight species ( Osmundaria colensoi, Ballia callitricha, Melanthalia abscissa and Scytothamnus australis) increased settlement relative to controls, although differences were non- or marginally significant. Polar extracts generally decreased settlement relative to controls. Further assays using a concentration gradient of non-polar extracts of these same four species showed weak positive effects on larval settlement. The four non-polar extracts were further fractionated and subsequently tested on both rough and smooth surfaces. Settlement was significantly higher on rough compared to smooth surfaces either with, or without extracts. Although the application of extracts did not significantly enhance settlement, the hexane and 10% EtOAc:hexane fractions of S. australis had a positive effect on settlement when added to rough surfaces. The strong effect of surface structure, and the weak effect of natural chemical cues, supports future studies concentrating on the manipulation material surface properties and surface structure to enhance the settlement of larvae and retain Greenshell™ mussel spat.

Effect of water velocity on growth and retention of cultured Greenshell™ mussel spat, Perna canaliculus (Gmelin, 1791)

The GreenshellTM mussel, Perna canaliculus, is a commercially important species forming New Zealand’s largest aquaculture export product. Losses of P. canaliculus spat from culture ropes between larval settlement and the time mussels reach initial reseeding size (c. 10 mm) are common. To test whether water velocity affects growth and retention of post-settlement P. canaliculus spat, and whether there is a threshold effect of increasing velocities on spat migration, a laboratory-based experiment was conducted. Spat were grown for 8 weeks in experimental tanks on culture ropes at four velocities typical of velocities within mussel farms (40, 10, 4, and 1 cm s−1). Spat migration was observed at all the velocities tested, but the number of spat migrating decreased as water velocity increased. Spat retention was highest at the 40 cm s−1 velocity. Mean spat size increased significantly with increasing water velocity. At the highest velocity tested (40 cm s−1), migration increased as the spat grew suggesting that migration was density-driven. The results of this experiment indicate the potential for mussel farmers to reduce seeded spat loss and to increase the growth rate of spat by modification of the water flow within their farms, or placement of seeded ropes in locations of certain existing water velocities, and indicate that velocities in the range 15–40 cm s−1 promote higher spat growth and spat retention for P. canaliculus.

75. Potential biomarkers of oxidative stress during cryopreservation of Greenshell™ mussel oocytes

75. Potential biomarkers of oxidative stress during cryopreservation of Greenshell™ mussel oocytes

Regiospecific Analyses of Triacylglycerols of Hoki (Macruronus novaezelandiae) and Greenshell™ Mussel (Perna canaliculus)

AbstractThe lipid profiles of the two most important New Zealand marine oil sources were investigated, with particular attention to the regioisomeric compositions of triacylglycerides (TAG), using 13C‐nuclear magnetic resonance analysis. Oils from hoki (Macruronus novaezelandiae) and Greenshell™ mussel (Perna canaliculus) (GSM) were analyzed for their lipid content, lipid class and fatty acid profile. The regiospecific distribution of long chain (C ≥ 20) polyunsaturated fatty acids (LC‐PUFA) between the sn‐1,3 and sn‐2 glycerol positions was calculated from 13C responses in the carbonyl region in the triacylglycerol fraction. Rendered hoki oil (RHO) produced from the viscera and filleting discards, had a similar lipid profile to that of hoki liver oil (HLO) confirming that the liver is the major source of oil in RHO. The regioisomeric distribution of fatty acids showed differences between the two oil sources. Docosahexaenoic acid (DHA) had a regioisomeric distributional preference to the sn‐2 position in TAG from all the oils (59.2% HLO, 54.3% RHO and 63.4% GSM). Eicosapentaenoic acid (EPA) had a more even distribution along the triacylglycerol backbone in hoki TAG (29.1% HLO, 33.6% RHO) while there was a slight sn‐2 positional preference in the GSM TAG (37.6%). This regioisomeric information is vital to distinguish LC‐PUFA‐rich marine oils from other marine sources for authentication purposes.

Alteromonas macleodii 0444 and Neptunomonas sp. 0536, two novel probiotics for hatchery-reared Greenshell™ mussel larvae, Perna canaliculus

Antibiotic management of aquacultured animals, such as Greenshell™ mussel (GSM) larvae, Perna canaliculus, is undesirable because of health concerns and political pressures; hence, alternatives are needed. Herein, two novel probiotic bacteria were identified and trialled in a GSM larval rearing hatchery. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately at the Glenhaven Aquaculture Centre hatchery facility during routine larval rearing and when the larvae were challenged with both a high (10 7 and 10 6 CFU ml −1) and low (10 6 and 10 5 CFU ml −1) pathogenic dose of Vibrio sp. DO1 and V. splendidus respectively. In all experiments, probiotic application improved larval survival significantly when administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. The administration of probiotics prior to pathogen challenge resulted in larval survival of 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This is the first report of A. macleodii and Neptunomonas sp. as probiotic bacteria in a large scale production facility.

Optimising the delivery of the key dietary diatom Chaetoceros calcitrans to intensively cultured Greenshell™ mussel larvae, Perna canaliculus

The diatom Chaetoceros calcitrans forma pumilum is an important dietary component for cultured Greenshell™ mussel larvae, Perna canaliculus. However concerns surrounding potentially deleterious side effects have motivated a series of experimental trials to optimise the safe usage of this alga. Bioassays involved raising veliger larvae in the purpose-built Cawthron Ultra-Density Larval rearing (CUDL) system; an array of 2.5-L tanks were stocked with 2-day-old veligers (200 larvae mL − 1) supplied with inflowing water dosed with sufficient microalgae to maintain a fixed concentration of cells, after compensating for ingestion. The nutritional role of C. ‘ calcitrans’ was examined by adjusting its cellular fraction in the feed environment. Diets of 0, 5, 66, 95 and 100% C. ‘ calcitrans’ were each offered to six replicate rearing tanks, using Isochrysis aff . galbana (T-Iso clone) to maintain a total of 40 cells μL − 1 in the larval cultures. The 66% C. ‘ calcitrans’ diet was also offered at 3, 20, 60 and 120 cells µL − 1 ( n = 6). Higher C. ‘ calcitrans’ fractions sustained faster growth, with 95% and 100% treatments producing 23-day-old pediveligers of 240 ± 15 μm and 228 ± 5 μm mean shell length, respectively; compared to 212 ± 7 μm in the 66% treatment (40 cells μL − 1). However, high C. ‘ calcitrans’ treatments were more volatile, had higher mortality and greater predisposition towards population crashes. Similarly, reduced survival was observed in the high feed treatment of 120 cells µL − 1, reflected in a final pediveliger yield of 29 ± 6%, compared to 47 ± 3% at 60 cells µL − 1 and 36 ± 7% at 40 cells µL − 1. The 5% C. ‘ calcitrans’ and 20 cells µL − 1 treatments showed signs of nutrient limitation, while larvae fed 0% C. ‘ calcitrans’ or a total of 3 cells µL − 1 starved, failing to reach metamorphosis. A standard diet consisting of 66% C. ‘ calcitrans’ and 34% I. aff. galbana maintained at 40 cells μL − 1 was used to test the hypothesis that culture age increased deleterious side effects associated with feeding C. ‘ calcitrans’. The final stage of batch culture, using 20-L nylon carboys inoculated with 10 10 C. ‘ calcitrans’ cells was allowed to age for 2, 3, 4, 5 or 6 days before being fed to larvae. After 21 days eating 2-day-old C. ‘ calcitrans’ larvae reached a mean shell length of 236 μm, were eating 35,000 cells larva − 1 day − 1, and had a survival rate of 59 ± 3%; in contrast, larvae eating 6-day-old cells only reached 214 µm, eating 17,000 cells larva − 1 day − 1, with an overall survival of 46 ± 7%. While C. ‘ calcitrans’ is valuable in the larval culture of P. canaliculus and many other bivalves, appropriate feeding protocols are needed when using this diatom.

Screening for probiotics of Greenshell™ mussel larvae, Perna canaliculus, using a larval challenge bioassay

A bioassay was developed to screen and select bacterial strains as probiotics for an alternative to antibiotic usage in the management of bacterial pathogens of Greenshell™ mussel (GSM) larvae, Perna canaliculus. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate, Vibrio sp. DO1, were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls ( p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 h revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics.

Towards cryopreservation of Greenshell™ mussel (Perna canaliculus) oocytes

Cryopreservation is a powerful tool for selective breeding in aquaculture as it enables genetic material from selected stock to be stored and crossed at will. The aim of this study was to develop a method for cryopreserving oocytes of the Greenshell™ mussel (Perna canaliculus), New Zealand’s main aquaculture species. The ability of oocytes to be fertilized post-thawing was used as the criterion for success in initial experiments and then subsequently, the ability of frozen oocytes to develop further to D-stage larvae was assessed. Ethylene glycol, propylene glycol, dimethyl sulphoxide and glycerol were evaluated at a range of concentrations with and without the addition of 0.2M trehalose using post-thaw fertilization as the endpoint. Ethylene glycol was most effective, particularly when used in combination with trehalose. A more detailed investigation revealed that ethylene glycol at 9% or 10% in the presence of 0.2–0.4M trehalose afforded the best protection. In experiments varying sperm to egg ratio and egg density in post-thaw fertilization procedures, D-larval yield averaged less than 1%. Following these results, a detailed experiment was conducted to determine the damaging steps in the cryopreservation process. Fertilization losses occurred at each step whereas D-larval yield approximately halved following CPA addition and was almost zero following cooling to −10°C. Cryomicroscopy studies and fertilization results suggest that the inability of oocytes to develop to D-larvae stage after cooling to −10°C and beyond are most likely related to some form of chilling injury rather than extracellular ice triggering intracellular ice formation. Further research is needed to determine the causes of this injury and to reduce CPA toxicity and/or osmotic effects.

Uptake of paralytic shellfish poisoning and spirolide toxins by paddle crabs ( Ovalipes catharus) via a bivalve vector

The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab ( Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell™ mussels ( Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3 ± 91.6 fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5 ± 1.6 pg STXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5 μg STXequiv./100 g, with 3/4 of the crabs exceeding the regulatory limit of 80 μg STXequiv./100 g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0 ± 19.5 fmol/cell; 1.4 ± 0.3 pg STXequiv./cell) and spirolides (1.8 pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8 μg STXequiv./100 g and spirolides from 6 to 7 μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption.

Development and evaluation of microsatellite markers for identification of individual Greenshell™ mussels (Perna canaliculus) in a selective breeding programme

A set of 49 microsatellite loci isolated from the endemic New Zealand Greenshell™ mussel, Perna canaliculus, were evaluated for inclusion in a parentage assignment marker suite by assessing their ease of PCR amplification, allele scoring and conformity to Mendelian inheritance in hatchery-produced families. Ten polymorphic loci (mean He=0.78 and polymorphic information content (PIC)=0.72) were identified as being suitable for parentage assignment. These 10 microsatellite loci gave a combined non-exclusion probability of <0.001 (probability that an unrelated parent pair will not be excluded from parentage of an arbitrary offspring), based on allele frequencies from 16 broodstock mussels. Simulations predicted an assignment success rate of 99.9% with all 10 loci and >95% with the best 5 or more loci (mean PIC=0.84). In actual parentage assignments, 124 offspring from 8 full-sib families were assigned to the correct parent pair with 4 or more loci. We found evidence for null alleles and extensive size homoplasy in many loci, highlighting the importance of thoroughly characterizing and evaluating microsatellite markers prior to parentage assignment and other applications.

The Effects of Food Concentration and Quality on the Feeding Rates of Three Size Classes of the GreenshellTM Mussel, Perna canaliculus

In order to calibrate carrying capacity models, investigations were conducted into the effects of food concentration and food quality on the feeding rates of small (25–50 mm), medium (60–85 mm) and large (90–115 mm) Greenshell mussels (Perna canaliculus). Experimental diets varying from 3.3 to 6.0 μg l−1 chlorophyll a concentration and 12–25% organic content were fed to mussels housed in individual flow through chambers. Not surprisingly, this study found that the main factor affecting feeding rates is mussel size. Small mussels were observed to maintain a constant filtration rate of approximately 20 mg h−1 irrespective of food concentration or quality, whereas mussels of greater than 60 mm length had more variable filtration rates between 30 and 80 mg h−1. The filtration rates of these large mussels were also observed to increase positively with organic content, and showed no sign of levelling out, even at the highest organic content tested (25%). Highest rejection rates (50–70 mg h−1) were observed when the organic content of the available seston was low, suggesting that P. canaliculus are able to selectively reject organic material, thereby organically enriching their diet. It appears that the organic content of the seston is the primary determinant of the net efficiency with which food is selected from the available seston by the mussel. The present study shows that P. canaliculus of all sizes are capable of adapting their feeding behaviour to compensate for changes in the food supply, which may occur over relatively short time periods, in the culture environment.