Quantification of Photobacterium swingsii and characterisation of disease progression in the New Zealand Greenshell™ mussel, Perna canaliculus.

Greenshell™ mussels (Perna canaliculus) are endemic to New Zealand and support the largest aquaculture industry in the country. Photobacterium swingsii was isolated and identified from moribund P. canaliculus mussels following a mass mortality event. In this study, a challenge experiment was used to characterise, detect, and quantify P. swingsii in adult P. canaliculus following pathogen exposure via injection into the adductor muscle. A positive control (heat-killed P. swingsii injection) was included to account for the effects of injection and inactive bacterial exposure. Survival of control and infected mussels remained 100% during 72-hour monitoring period. Haemolymph was sampled for bacterial colony counts and haemocyte flow cytometry analyses; histology sections were obtained and processed for histopathological assessments; and adductor muscle, gill, digestive gland were sampled for quantitative polymerase chain reaction (PCR) analyses, all conducted at 12, 24, 48 h post-challenge (hpc). The most profound effects of bacterial injection on mussels were seen at 48 hpc, where mussel mortality, haemocyte counts and haemolymph bacterial colony forming were the highest. The quantification of P. swingsii via qPCR showed highest levels of bacterial DNA at 12 hpc in the adductor muscle, gill, and digestive gland. Histopathological observations suggested a non-specific inflammatory response in all mussels associated with a general stress response. This study highlights the physiological effects of P. swingsii infection in P. canaliculus mussels and provides histopathological insight into the tissue injury caused by the action of injection into the adductor muscle. The multi-technique methods used in this study can be applied for use in early surveillance programs of bacterial infection on mussel farms.

The first use of LC-MS/MS proteomic approach in the brown mussel Perna perna after bacterial challenge: Searching for key proteins on immune response.

The brown mussel Perna perna is a valuable fishing resource, primarily in tropical and subtropical coastal regions. Because of their filter-feeding habits, mussels are directly exposed to bacteria in the water column. Escherichia coli (EC) and Salmonella enterica (SE) inhabit human guts and reach the marine environment through anthropogenic sources, such as sewage. Vibrio parahaemolyticus (VP) is indigenous to coastal ecosystems but can be harmful to shellfish. In this study, we aimed to assess the protein profile of the hepatopancreas of P. perna mussel challenged by introduced - E. coli and S. enterica - and indigenous marine bacteria - V. parahaemolyticus. Bacterial-challenge groups were compared with non-injected (NC) and injected control (IC) - that consisted in mussels not challenged and mussels injected with sterile PBS-NaCl, respectively. Through LC-MS/MS proteomic analysis, 3805 proteins were found in the hepatopancreas of P. perna. From the total, 597 were significantly different among conditions. Mussels injected with VP presented 343 proteins downregulated compared with all the other conditions, suggesting that VP suppresses their immune response. Particularly, 31 altered proteins - upregulated or downregulated - for one or more challenge groups (EC, SE, and VP) compared with controls (NC and IC) are discussed in detail in the paper. For the three tested bacteria, significantly different proteins were found to perform critical roles in immune response at all levels, namely: recognition and signal transduction; transcription; RNA processing; translation and protein processing; secretion; and humoral effectors. This is the first shotgun proteomic study in P. perna mussel, therefore providing an overview of the protein profile of the mussel hepatopancreas, focused on the immune response against bacteria. Hence, it is possible to understand the immune-bacteria relationship at molecular levels better. This knowledge can support the development of strategies and tools to be applied to coastal marine resource management and contribute to the sustainability of coastal systems.

Perkinsus olseni in green-lipped mussels Perna canaliculus: diagnostic evaluation, prevalence, and distribution.

Perkinsus olseni (Perkinsidae) is a molluscan parasite notifiable to the World Organisation for Animal Health that is reported in several shellfish hosts in New Zealand, including the native green-lipped mussel Perna canaliculus. Green-lipped mussels comprise over half of New Zealand's aquaculture export value and have historically been considered free of serious diseases based on extensive histology-based surveillance. The discovery of P. olseni in green-lipped mussels has raised questions about future disease threats to green-lipped mussels, particularly under changing ocean climatic conditions. Using mussels collected from farmed (n = 358) and wild (n = 236) populations, we aimed to determine the distribution and prevalence of P. olseni in green-lipped mussels around New Zealand, and assess the performance of diagnostic tests, including real-time PCR, conventional PCR, and culture using Ray's fluid thioglycolate medium (RFTM). Prevalence and diagnostic test performance was evaluated using Bayesian latent class analysis with informative priors. The prevalence of P. olseni was 0-3%, except for 1 wild population from a harbour where prevalence was 22%. Real-time PCR had the highest diagnostic sensitivity (87%) compared to 62 and 21% for conventional PCR and RFTM, respectively. Diagnostic specificity was similar among all methods (96-98%). No mortality was observed during the study. Our results suggest that real-time PCR is the diagnostic test best suited for surveillance of P. olseni in subclinically infected green-lipped mussels under New Zealand conditions.

Sex-specific metabolic dysregulation in digestive glands of green mussels following exposure to triazophos.

As a ubiquitous environmental pollutant in China, triazophos (TP) is known to have neurotoxicity, oxidative stress, and reproductive toxicity to mussels. To investigate the molecular mechanisms of TP toxicity, metabolic changes in the digestive glands of Perna viridis in different sexes were examined after treated with 35 µg/L TP. Notably, 158 significant different metabolites (SDMs) were detected in TP-treated mussels and more than half of the SDMs were lipids and lipid-like molecules, which suggested that TP disturbed the lipid metabolism of P. viridis. In addition, metabolites associated with neurotoxicity and reproductive disturbance were also detected in female and male mussels. Moreover, a larger number of SDMs were found in male mussels (120 SDMs) than females (99 SDMs), and 60 common metabolites exhibited consistent variation tendency and similar magnitude in both sexes. The metabolic alternations in female and male mussels displayed similar protective mechanisms and also sex-specific responses, male mussels were more sensitive to TP exposure. This research provided new data about the molecular mechanisms of TP toxicity and the gender specific changes in mussels after treated by chemicals.

The effect of simulated marine heatwaves on green-lipped mussels, Perna canaliculus: A near-natural experimental approach.

Marine heatwaves (MHW) are projected for the foreseeable future, affecting aquaculture species, such as the New Zealand green-lipped mussel (Perna canaliculus). Thermal stress alters mussel physiology highlighting the adaptive capacity that allows survival in the face of heatwaves. Within this study, adult mussels were subjected to three different seawater temperature regimes: 1) low (sustained 18 °C), 2) medium MHW (18-24 °C, using a +1 °C per week ramp) and 3) high MHW (18-24 °C, using a +2 °C per week ramp). Sampling was performed over 11 weeks to establish the effects of temperature on P. canaliculus survival, condition, specific immune response parameters, and the haemolymph metabolome. A transient 25.5-26.5 °C exposure resulted in 61 % mortality, with surviving animals showing a metabolic adjustment within aerobic energy production, enabling the activation of molecular defence mechanisms. Utilisation of immune functions were seen within the cytology results where temperature stress affected the percentage of superoxide-positive haemocytes and haemocyte counts. From the metabolomics results an increase in antioxidant metabolites were seen in the high MHW survivors, possibly to counteract molecular damage. In the high MHW exposure group, mussels utilised anaerobic metabolism in conjunction with aerobic metabolism to produce energy, to uphold biological functions and survival. The effect of exposure time was mainly seen on very long-, and long chain fatty acids, with increases observed at weeks seven and eight. These changes were likely due to the membrane storage functions of fatty acids, with decreases at week eleven attributed to energy metabolism functions. This study supports the use of integrated analytical tools to investigate the response of marine organisms to heatwaves. Indeed, specific metabolic pathways and cellular markers are now highlighted for future investigations aimed at targeted measures. This research contributes to a larger program aimed to identify resilient mussel traits and support aquaculture management.

Microplastics in Perna viridis and Venerupis species: assessment and impacts of plastic pollution.

This study is divided into two parts. The first part aims to verify the presence of microplastics in bivalves, namely Perna viridis and Venerupis spp. using microscopy and Fourier transform infrared spectroscopy. The second part explores the knowledge, attitude and perception (KAP) of bivalve gleaners on microplastics and plastics. Results of the study confirmed the presence of microplastics in both bivalves, with polyamide fibers being the most common polymer found in the bivalves. The mean size of microplastics found in Perna viridis and Venerupis spp. was 0.25 ± 0.05 mm and 0.33 ± 0.03 mm, respectively. Varying colors and shapes were also observed in both bivalves. Further, results of the KAP showed the lack of knowledge of the gleaners in terms of the basic information about microplastics. Nevertheless, they showed a positive attitude in terms of reducing plastic pollution and perceived coastal waters as important to them. The data on the two parts were used to compute for the estimate of the amount of microplastics that can be transferred to humans through consumption of bivalves, which was found to be 0.003 mg/day. Supplementary Information: The online version contains supplementary material available at 10.1007/s13762-023-04982-x.

Molecular cloning and expression analysis of mytilin-like antimicrobial peptides from Asian green mussel Perna viridis.

Mytilin is one of the most important CS-αß peptides involved in innate immune response in Mytilidae. In this study, we successfully identified four mytilin-like antimicrobial peptides (pernalins) from Asian green mussel Perna viridis by aligning the P. viridis transcriptome with 186 mytilins and myticins related sequences collected from the transcriptome data of six Mytilus species. Analysis on gene structure showed that pernalin genes had high conservation with mytilin B of Mediterranean mussel Mytilus galloprovincialis. Interestingly, all pernalin genes have a similar tissue expression feature, evidenced by the highest transcription level observed in the hemocytes and followed by the mantle. The lowest transcription level was observed in the foot and gills. qRT-PCR analysis showed that all pernalin genes were significantly down-regulated at each time points from 3 h to 48 h after Vibrio parahaemolyticus infection, suggesting their timely immune responses after bacterial infection.

Effects of seawater temperature and acute Vibriosp. challenge on the haemolymph immune and metabolic responses of adult mussels (Perna canaliculus).

The New Zealand Greenshell™ mussel (Perna canaliculus) is an endemic bivalve species with cultural importance, that is harvested recreationally and commercially. However, production is currently hampered by increasing incidences of summer mortality in farmed and wild populations. While the causative factors for these mortality events are still unknown, it is believed that increasing seawater temperatures and pathogen loads are potentially at play. To improve our understanding of these processes, challenge experiments were conducted to investigate the combined effects of increased seawater temperature and Vibrio infection on the immune and metabolic responses of adult mussels. Biomarkers that measure the physiological response of mussels to multiple-stressors can be utilised to study resilience in a changing environment, and support efforts to strengthen biosecurity management. Mussels acclimated to two temperatures (16 °C and 24 °C) were injected with either autoclaved, filtered seawater (control) or Vibriosp. DO1 (infected). Then, haemolymph was sampled 24 h post-injection and analysed to quantify haemocyte immune responses (via flow-cytometry), antioxidant capacity (measured electrochemically) and metabolic responses (via gas chromatography-mass spectrometry) to bacterial infection. Both seawater temperature and injection type significantly influenced the immune and metabolite status of mussels. A lack of interaction effects between temperature and injection type indicated that the effects of Vibrio sp. 24 h post-infection were similar between seawater temperatures. Infected mussels had a higher proportion of dead haemocytes and lower overall haemocyte counts than uninfected controls. The proportion of haemocytes showing evidence of apoptosis was higher in mussels held at 24 °C compared with those held at 16 °C. The proportion of haemocytes producing reactive oxygen species did not differ between temperatures or injection treatments. Mussels held at 24 °C exhibited elevated levels of metabolites linked to the glycolysis pathway to support energy production. The saccharopin-lysine pathway metabolites were also increased in these mussels, indicating the role of lysine metabolism. A decrease in metabolic activity (decreases in BCAAs, GABA, urea cycle metabolites, oxidative stress metabolites) was largely seen in mussels injected with Vibrio sp. Itaconate increased as seen in previous studies, suggesting that antimicrobial activity may have been activated in infected mussels. This study highlights the complex nature of immune and metabolic responses in mussels exposed to multiple stressors and gives an insight into Vibrio sp. infection mechanisms at different seawater temperatures.

Histopathological and immunological changes in green mussel, Perna viridis, challenged with Vibrio alginolyticus.

Understanding of pathogenicity and immunity is crucial in producing disease-resistant cultured mollusk varieties. This study aimed to isolate pathogenic Vibrio alginolyticus from naturally infected Perna viridis, and to determine histopathological and immunological changes after challenge test with the same bacteria. Biochemical tests and 16S rDNA identified the pathogen as V. alginolyticus (99%). Antibiotic susceptibility test showed ampicillin resistance of the pathogen. Pathogenicity assay was conducted by immersing P. viridis in 1.5 × 106 CFU mL-1V. alginolyticus for 60 min and observed for 5 days. Clinical signs, histopathological and immunological alterations were observed and monitored. Infected groups showed 60% mortality and decreased immunity factors, including total hemocyte count and lysozymes activity. Histopathological examination revealed pathological lesions in the hepatopancreas at 24 h post-challenge and hemocyte proliferation as part of a severe inflammatory reaction. Karyomegaly in the hepatopancreas tissue, concomitant with necrosis demolition of tubules cells, was also observed. V. alginolyticus was determined to be pathogenic to P. viridis, causing mortality as a result of multiple organ lesions and dysfunction in digestive gland and immune organs. This study demonstrated the role of histopathological and immunological parameters as potential biomarkers in assessing vibriosis caused by Vibrio species in green mussel, P. viridis.

Cinnamaldehyde Could Reduce the Accumulation of Diarrhetic Shellfish Toxins in the Digestive Gland of the Mussel Perna viridis under Laboratory Conditions.

Diarrhetic shellfish toxins (DSTs), some of the most important phycotoxins, are distributed almost all over the world, posing a great threat to human health through the food chain. Therefore, it is of great significance to find effective methods to reduce toxin accumulation in shellfish. In this paper, we observed the effects of four phytochemicals including cinnamaldehyde (CA), quercetin, oridonin and allicin on the accumulation of DSTs in the digestive gland of Perna viridis after exposure to the DSTs-producing Prorocentrum lima. We found that, among the four phytochemicals, CA could effectively decrease the accumulation of DSTs (okadaic acid-eq) in the digestive gland of P. viridis. Further evidence demonstrated that CA could reduce the histological alterations of the digestive gland of a mussel caused by DSTs. RT-qPCR showed that CA could suppress the CYP3A4 induction by DSTs, suggesting that the DSTs' decrease induced by CA might be related to the inhibition of CYP3A4 transcription induction. However, further studies on the underlying mechanism, optimal treatment time, ecological safety and cost should be addressed before cinnamaldehyde is used to decrease the accumulation of DSTs in field.

Lactic fermentation of cooked, comminuted mussel, Perna canaliculus.

The endogenous microflora of mussels, filter feeders, can include pathogens with resulting food safety concerns. The aim was to develop a cook-then-ferment technology to extend shelf life and safety of a ready-to-eat mussels. Only after cooking to destroy the mussel's endogenous microflora could an edible product be made as determined by pH decline after fermentation and the fate of common pathogens. Perna canaliculus was bought live at retail on many dates. Fermentation was with commercial lactic cultures incubated under vacuum at 30 °C for four days. Using one culture containing Pediococcus pentosaceus and Staphylococcus carnosus as a model, pH typically declined to 4.5 to 4.7, and common pathogens, Staphylococcus aureus, Salmonella and Vibrio parahaemolyticus were absent or reduced to acceptable levels. The fate of Listeria monocytogenes was studied with five cultures. These were variably effective at inhibition with one clear success, Chr Hansen's T-SC-150 containing a specific strain of Lactobacillus sakei, and flavour-generating Staphylococcus carnosus. This culture's efficacy was confirmed with sterile extracts of LAB challenging L. monocytogenes in vitro. This culture was also the most rapid fermenter by pH fall. Cook-then-ferment technology may be applied to other novel foods to minimise a disruptive endogenous microflora.

Accumulation and ecotoxicological risk of weathered polyethylene (wPE) microplastics on green mussel (Perna viridis).

Recent studies have shown that organisms including humans are exposed to microplastics directly or indirectly. The present study aims to examine the ingestion of these microplastics and the consequences of the same by studying the accumulation behavior of weathered Polyethylene (wPE) microplastics. The Perna viridis were exposed chronically to three different environmentally relevant concentrations of wPE for 30 days, followed by a one-week depuration phase. There was no mortality observed in the control and exposed groups, but the feeding rate was observed to have substantially decreased in the group exposed to higher concentration (3 µgL-1) of wPE. It was also observed that a higher number of wPE particles accumulated in the intestine of exposed organisms. Interestingly, the present study revealed the presence of the substantial number of wPE particles in exposed organisms, which may adversely affect the internal organs as well as growth and reproduction. This study perceived that accumulation is marginally influenced by size of wPE. Similarly, biomarker analysis showed that wPE exposure significantly altered both the metabolism and histology of the internal organs of the exposed organisms. Overall, the study confirmed that the intestine was the most sensitive organ followed by gills, adductor muscles, and foot tissue adding new insights into the adverse effects of wPE in the marine ecosystem.

Evaluation of bioavailability of trace metals through bioindicators in a urbanized estuarine system in southeast Brazil.

The mussel Perna perna is one of the most used bioindicators of coastal areas and the most economically exploited species in Brazil through mariculture. In the present study, P. perna was used to investigate metal pollution in the estuarine area of Vitória Bay. Four sampling sites were located along an estuarine branch of Vitória Bay and stations were sampled during three campaigns. Trace metals in the tissues of P. perna were evaluated as well as dissolved trace metals and other ancillary variables in the water column. Dissolved Cd, Pb, Cu, Ni, and Fe concentrations surpassed the tolerance limits stablished by legislation in all the sampling campaigns. P. perna exhibited concentrations in disagreement with the Brazilian legislation for Cr and As. A general trend of higher concentrations in outer stations was observed for most metals, what suggested the occurrence of flocculation process in the lower estuary, reducing the concentrations of dissolved elements and increasing their bioavailability for the biota through the particulate form. Cd was highlighted with elevated concentrations in dissolved fraction but not detected in P. perna, probably due to chlor-complex formation under influence of more saline waters. Al, Ba, Mn, Fe, Cu, Zn, Ni, Cr, Pb, and As were considered bioavailable, once they were accumulated in the mussels' tissues. Hazard index (HI) and target cancer risk (TCR) showed that the consumption of mussels from the study area offers health risk issues, being iron and arsenic the main contributors for the high indexes.

Response of green mussels (Perna viridis) subjected to chlorination: investigations by valve movement monitoring.

Perna viridis Linnaeus (1758) is a major foulant in the cooling water systems of electric power stations located on the East coast of India. Though chlorination is considered an effective fouling control measure, the strategy may fail in the case of bivalve mussels, due to the ability of the mussels to close their shells and still survive for extended periods of time. In a given power station, continuous low dose (exomotive) chlorination (0.2 ± 0.1 mg l-1) is practiced to control biofouling. Laboratory experiments were carried out to assess the mortality and valve movement response of Perna viridis exposed to chlorine, using a Mosselmonitor®. All size groups tested showed progressive reduction in valve opening upon chlorination. However, continuous dosing of chlorine concentration as high as 1.0 mg l-1 was required for sustained and complete valve closure response in this mussel. At lower concentration (0.7 mg l-1), the mussels were able to open their shells and feed. Sustained valve closure resulted in physiological stress to the mussels due to reduced feeding, subsequently leading to death. Time to 100% mortality was dependent on the size of the mussels. At 1.0 mg l-1 chlorine residual, smaller size group (30-50 mm) mussels showed 100% mortality in 79.3 h, while larger groups (50-70 mm and 70-90 mm) took 152 h and 243 h, respectively. Frequency of valve opening was high in smaller size group mussels (30-50 mm), compared with larger groups (70-90 mm). Even though the time taken for killing was size-dependent, frequency of valve opening and time period between successive openings were found to be characteristic of individual mussels. The observations provide new insight into the response of bivalve mussels to continuous chlorination in the context of biofouling control and point to the need to adopt pragmatic strategies to prevent mussel spat settlement rather than killing of adult mussels, thereby reducing environmental burden due to chlorine residuals. Usage of target-specific biocidal strategies (intermittent/pulse dosing) or alternative biocides (chlorine dioxide) may help mitigate green mussel fouling in tropical cooling water systems.

Metabolomics investigation of summer mortality in New Zealand Greenshell™ mussels (Perna canaliculus).

Increasing water temperatures due to climate change have resulted in more frequent high mortality events of New Zealand Greenshell™ mussels (Perna canaliculus Gmelin 1791). These events have significant impacts within mussel farms which support a major shellfish industry for New Zealand. The present study investigates metabolic responses of farmed mussels during a summer mortality event in order to identify health impacts and elucidate mechanistic effects of external stressors on mussels. A gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach was used to identify metabolic perturbations and flow cytometry assays were used to assess viability, oxidative stress and apoptosis of haemocytes from healthy and unhealthy mussels during a summer mortality event. The results showed significantly higher mortality and apoptosis of haemocytes in unhealthy mussels compared to healthy mussels. Reactive oxygen species (ROS) production, which is an indicator of oxidative stress was very high in both mussel groups, but no differences were observed between the two mussel groups. Metabolomics revealed alterations of many metabolites in both haemolymph and hepatopancreas (digestive gland) of unhealthy mussels compared to healthy mussels, reflecting perturbations in several molecular pathways, including energy metabolism, amino acid metabolism, protein degradation/tissue damage and oxidative stress. An increased level of itaconic acid which is an antimicrobial metabolite and biomarker of pathogen infection was observed in haemolymph, but not in hepatopancreas samples. This investigation provides the first detailed metabolic characterization of mussel immune responses to a summer mortality event and illustrates the benefits of using an integrated metabolomics and flow cytometry workflow for mussel health assessment and biomarker identification for summer mortality early detection.

Green-lipped mussel (Perna canaliculus) hemocytes: A flow cytometric study of sampling effects, sub-populations and immune-related functions.

Green-lipped mussels (Perna canaliculus) are a commercially and culturally important bivalve species in New Zealand (NZ). As the highest value export aquaculture product in NZ, understanding and safeguarding the health of this species is imperative. The identification and characterization of hemocytes can provide useful information regarding the health of this species. Using flow cytometry (FCM), the present study assessed for the first time the use of different antiaggregant solutions and storage times on the immune-related parameters of hemocytes from cultured adult P. canaliculus. In addition, characterization of the immune-related functions of hemocyte sub-populations within the hemolymph were assessed. The two antiaggregant solutions tested (Modified Alserver's, MAS, A and B) maintained similar numbers of hemocytes in circulation over a 60 min period but, reduced the viability (MAS A) and increased the ROS production (MAS B) of the hemocytes compared to hemocytes diluted in cold filtered seawater (FSW). Hemocytes diluted in FSW and kept on ice showed significant aggregation after 2 h and a reduction in viability from 4 h. Three different hemocyte sub-populations were identified, discernible by their relative size and internal complexity: blast-like cells, hyalinocytes and granulocytes, which accounted for approximately 4, 67 and 29% of the total hemolymph population respectively. Granulocytes showed significantly higher reactive oxygen species production, phagocytic capabilities and neutral lipid content compared to hyalinocytes and blast-like cells. Results indicate that maintaining extracted hemolymph in cold FSW, completing analysis of fresh samples within 2 h of extraction and FCM assay incubation times of no longer than 30 min are best to obtain accurate results. Formalin fixation can also be used for future determination of hemocyte sub-populations and internal structures. Results from this study will allow effective future study of the effects of various stressors on P. canaliculus health and lead to improved management and production strategies in this species.

Population connectivity and genetic structure of Asian green mussel, Perna viridis along Indian waters assessed using mitochondrial markers.

Perna viridis (Linnaeus, 1758), the Asian green mussel, belonging to the family Mytilidae is widely distributed along the Indian coast. The species is majorly found in southeastern countries and is considered an ideal candidate for aquaculture due to its high nutritional value and growth rate. Obtaining their genetic information is essential for their sustainable capture-based production. In the present study, genetic variation, population structure, and demographic processes of the populations across the distribution of this species were assessed using the mitochondrial DNA ATPase6 and cytb gene. In total, we selected 170 samples from five localities across the Indian subcontinent including Andaman Sea. Sequence analysis of partial cytb (885 bp) and ATPase6 (714 bp) genes revealed 45 and 58 haplotypes, respectively. The significant coefficient of genetic differentiation (FST: 0.255 for cytb and 0.252 for ATPase6) and analyses of molecular variance indicated three varieties of stocks, namely Arabian Sea, Bay of Bengal, and Andaman Sea. All the populations showed low nucleotide diversity, suggesting severe historical bottleneck events and high haplotype diversity, indicating population expansion. The genetic variation and demographic process reported in this study will form the baseline information for framing policies, which can be adopted while planning stock specific ranching and relaying programmes in the Indian subcontinent with view to enhance and manage the fishery.

Development of a method to cryopreserve Greenshell mussel™ (Perna canaliculus) veliger larvae.

Cryopreservation of larvae of Greenshell™ mussel Perna canaliculus, the most cultivated species in New Zealand, can provide flexibility for selective breeding programmes and enhance its global production. In this study, we set out to develop a reliable protocol for freezing D-stage larvae of Greenshell™ mussels that ensured long-term survival for successful rearing of thawed larvae in the hatchery. The effects of different combinations of cryoprotecting agents (CPA), varying CPA equilibration times, larval concentrations per straw as well as different larval development stages (48 h vs 72 h old) were evaluated by assessing the behavioural response (swimming activity, algal consumption), shell size and survival of larvae, up to 4 days post-thawing. The protocol yielding the best larval performances was a combination of the following CPA (final concentrations): 14% ethylene-glycol (EG) + 0.6 M trehalose (TRE) + 1% polyvinyl-pyrrolidone (PVP), prepared with Milli-Q water. Stocking densities ranging from 50,000 to 150,000 larvae per straw (0.25 mL) and a 20 min equilibration time gave the best results, while no significant differences in fitness were found between larvae cryopreserved at 48 h nor 72 h-old. Using the improved cryopreservation protocol, over 50% of previously cryopreserved D-larvae were able to survive after 4 days of rearing, compared with 65% in the unfrozen control. More importantly, about one third of thawed larvae were able to swim and feed, and to potentially develop further. These findings contribute to enhance the selective breeding programmes for this species.

Development stage of cryopreserved mussel (Perna canaliculus) larvae influences post-thaw impact on shell formation, organogenesis, neurogenesis, feeding ability and survival.

Cryopreservation of genetic material from farmed aquatic species is a valuable technique to advance selective breeding programs for stock improvement. In this study, effects of cryopreservation on development of trochophore and D-stage larvae of Greenshell™ mussel (Perna canaliculus) were evaluated through histology, light microscopy, scanning electron microscopy, and confocal microscopy. Larvae of both life stages were motile immediately post-thawing, but survival declined rapidly from 4 days post-fertilisation (dpf). At 18 dpf, ~23% of non-cryopreserved control larvae had progressed to the pediveliger stage, while <1% of cryopreserved larvae had survived. Control larvae grew faster and larger, and consumed more food than larvae cryopreserved at either life stage (trochophore or D-stage). Settlement competency was achieved in the control larvae at 21 days post-fertilization, with most remaining individuals developing eye spots. Organogenesis was delayed in all cryopreserved larvae, and eyespots did not appear at all. Neurogenesis was stunted in cryopreserved trochophore larvae but seemed to progress almost normally in their cryopreserved D-stage counterparts. Developing abnormalities in shell morphology rapidly became apparent in all mussels post-thaw, with trochophore larvae being most highly afflicted. These delays in organogenesis and overall development are indicative of cryo-injuries sustained at a cellular level. Our results show that D-stage larvae are somewhat more resilient to cryopreservation than trochophore larvae. D-larvae are good life-stage candidates for cryobanking genetic resources in this species because there is generally an excess of larvae from selective breeding family crosses and these can be banked and stored for later use. Further on-going research aims to improve the long-term viability of cryopreserved D-larvae for successful rearing.

Bioavailability of Orally Administered Active Lipid Compounds from four Different Greenshell™ Mussel Formats.

Greenshell™ mussel (GSM, Perna canaliculus) is New Zealand's most important aquaculture species. They are a good source of long chain-polyunsaturated fatty acids (n-3 LC PUFA). Beyond a traditional food product, GSMs are also sold as mussel powders and oil extract formats in the nutraceutical markets. In this study, a four-sequence, single dose, randomized crossover human trial with eight evaluable healthy male participants was undertaken to determine the bioavailability of the n-3 LC PUFA in four different GSM formats (oil, powder, food ingredient and half-shell unprocessed whole mussel) by measuring area under the curve (AUC) and maximal concentration (CMax). Blood samples were collected at baseline and up to 48 h after initiation of product consumption in each administration period. There were minor differences between the bioavailability of FA (fatty acid) between the different GSM formats. Eicosapentaenoic acid (EPA) peak concentrations and plasma exposures were significantly lower with GSM oil compared to GSM half-shell and GSM powder formats, which resulted in AUC0-48 for the intake of GSM half-shell mussel and GSM powder being significantly higher than that for GSM oil (p = 0.013, f= 4.84). This equated to a 20.6% and 24.3% increase in the amount of EPA present in the plasma after consumption of half-shell mussels and mussel powder respectively compared to GSM oil. GSM oil produced the shortest median time to maximal plasma n-3 LC PUFA concentration of all evaluated products demonstrated by a shorter maximum measured plasma concentration (TMax = 5 h). Docosahexaenoic acid (DHA) and n-3 LC PUFA plasma exposure parameters were statistically comparable across the four GSM products evaluated.