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The early stages of mussel farming are often inefficient with a large proportion of seed (spat) typically lost early in the production cycle. An effective approach for decreasing such losses is to grow spat to larger sizes in nursery systems prior to seeding. However, for such an approach to be commercially viable, spat must first be separated from their settlement substrates into a single seed format, that is, singulated. This study investigated the efficacy of three commercial protease enzymes (Enzidase PAP, PXT and BAP) and chlorine (NaOCl) in singulating Greenshell mussel (Perna canaliculus) spat from spat‐collector ropes. Two distinct experiments involved immersing segments of spat collector rope in seawater solutions with varying concentrations (2.5% or 5%) of each enzyme or chlorine (NaOCl) for different exposure times (10, 30 and 60 min for enzymes; 2 and 5 min for chlorine). After the singulation experiments, all spat were recovered with sieves and reintroduced into the tanks with renewed seawater. After 24 h of rearing, the recovery of alive spat was measured. Results indicate that in Experiment 1, involving spat ∼5 mm in size, the singulation rate was up to 65% for protease enzymes (i.e., in the case of 5% PAP at 30 min) and 61% for chlorine (i.e., at 5% for 2 min). In Experiment 2, involving spat ∼1.5 mm in size, the singulation rate was up to 85% for protease enzymes (i.e., in the case of 5% PXT for 60 min) and 78% for chlorine (i.e., of 2.5% for 5 min). However, these treatments generally resulted in elevated spat mortality, which was exacerbated by increasing concentrations and exposure times, resulting in recovery rates of up to 35% (i.e., for 2.5% PAP for 30 min) in Experiment 1 and 26.0% (i.e., for 5% chlorine for 2 min) in Experiment 2. These findings emphasise the necessity for consideration of chemical concentrations and exposure times to optimise the singulation process while highlighting challenges in achieving high post‐singulation survival rates. These insights contribute to ongoing efforts aimed at improving the efficiency and sustainability of mussel farming practices, demonstrating the potential utility of protease enzymes and chlorination in singulating spat from settlement substrate prior to their transfer to nursery systems for further ongrowing. For mussel spat to be grown in nursery culture systems they must first be separated from their associated settlement substrate (i.e., singualted). This work demonstrated that protease enzymes and chlorination is an effective approach for separating Greenshell mussel spat from spat‐catching ropes. [ABSTRACT FROM AUTHOR]

Aquaculture has maintained a sustained growth trend over the last 50 years, worldwide and in Aotearoa New Zealand.This industry is known to have environmental consequences, such as impacts on benthos, water column and higher trophic levels. Some of these impacts have been thoroughly studied, while others, such as the effects on cetaceans, are not well understood.This study used passive acoustic monitoring (PAM) to assess, for the first time, the overlap between kūtai—New Zealand greenlipped mussel (Perna canaliculus)—farms and habitat use by endemic endangered Hector's dolphins (Cephalorhynchus hectori hectori) within the Banks Peninsula Marine Mammal Sanctuary.T‐PODs were deployed in six bays, three with mussel farms and three without, between 2018 and 2019. Occurrence of dolphins, expressed as 'dolphin positive minutes per day' (DPM/day), was modelled using generalized linear mixed models.Results showed a strong interaction between farm (presence/absence) and season (summer/winter), with fewer acoustic detections of dolphins in winter if farms were present. Furthermore, in bays with mussel farms, there were significantly more click train detections on the opposite side of the bay, compared to the side by the mussel farm.Considering that results from this study suggest potential displacement of the dolphins due to the mussel farms, the recommendation is a precautionary approach. Further expansion of aquaculture in Hector's dolphin habitat should be done carefully, taking into account their habitat use, and conducting intensive monitoring including a robust 'before and after' design prior to the establishment of new farms. [ABSTRACT FROM AUTHOR]

Site selection is one of the biggest challenges affecting coastal restoration success, and can be particularly difficult in the intertidal zone, where species are often living close to their physiological thermal limits. Climate change is causing atmospheric and marine heat waves, with the summer of 2023 having the highest ever recorded global average temperatures. This changing climate, along with other intertidal stressors, increases the difficulty for managers to choose restoration locations in the intertidal zone. In this study, green‐lipped mussels (Perna canaliculus) were transplanted into the intertidal zone in a series of 12 plots (9 m2) to examine the in situ influence of temperature on mussel survival over a 6‐month period. The plots were deployed at varying intertidal heights with differing periods of aerial exposure and temperature. Six months post‐deployment the plots had highly variable mussel survival with a range of 0–95%. Results showed that temperature, specifically hours exceeding 25°C, had a negative relationship with mussel survival, and reducing aerial exposure reduces the number of hours exposed to temperatures above 25°C in the summer months. This study demonstrates the utility of deploying temperature loggers by restoration practitioners prior to restoration trials to determine localized variances in temperature and exposure that may aid in site selection in the intertidal zone. In a warming world, our findings illustrate the importance of understanding the potentially critical fine‐scale interplay between temperature and exposure time in intertidal shellfish restoration. [ABSTRACT FROM AUTHOR]

Abstract The early stages of mussel farming are often inefficient with a large proportion of seed (spat) typically lost early in the production cycle. An effective approach for decreasing such losses is to grow spat to larger sizes in nursery systems prior to seeding. However, for such an approach to be commercially viable, spat must first be separated from their settlement substrates into a single seed format, that is, singulated. This study investigated the efficacy of three commercial protease enzymes (Enzidase PAP, PXT and BAP) and chlorine (NaOCl) in singulating Greenshell mussel (Perna canaliculus) spat from spat‐collector ropes. Two distinct experiments involved immersing segments of spat collector rope in seawater solutions with varying concentrations (2.5% or 5%) of each enzyme or chlorine (NaOCl) for different exposure times (10, 30 and 60 min for enzymes; 2 and 5 min for chlorine). After the singulation experiments, all spat were recovered with sieves and reintroduced into the tanks with renewed seawater. After 24 h of rearing, the recovery of alive spat was measured. Results indicate that in Experiment 1, involving spat ∼5 mm in size, the singulation rate was up to 65% for protease enzymes (i.e., in the case of 5% PAP at 30 min) and 61% for chlorine (i.e., at 5% for 2 min). In Experiment 2, involving spat ∼1.5 mm in size, the singulation rate was up to 85% for protease enzymes (i.e., in the case of 5% PXT for 60 min) and 78% for chlorine (i.e., of 2.5% for 5 min). However, these treatments generally resulted in elevated spat mortality, which was exacerbated by increasing concentrations and exposure times, resulting in recovery rates of up to 35% (i.e., for 2.5% PAP for 30 min) in Experiment 1 and 26.0% (i.e., for 5% chlorine for 2 min) in Experiment 2. These findings emphasise the necessity for consideration of chemical concentrations and exposure times to optimise the singulation process while highlighting challenges in achieving high post‐singulation survival rates. These insights contribute to ongoing efforts aimed at improving the efficiency and sustainability of mussel farming practices, demonstrating the potential utility of protease enzymes and chlorination in singulating spat from settlement substrate prior to their transfer to nursery systems for further ongrowing.

The nutritional condition of seed mussels (spat) at the time of seeding is thought to play a major role in influencing spat losses on mussel farms, with nutritionally compromised spat thought to be more likely to be lost from aquaculture substrata than those in good condition. New Zealand's Greenshell™ mussel (Perna canaliculus) aquaculture industry relies almost exclusively (∼85%) on wild caught spat, with the vast majority, typically > 90%, lost shortly after being seeded onto coastal mussel farms. This study sought to quantify the extent of the variability in the nutritional condition (i.e., calorific, protein, lipid and carbohydrate content) of samples of spat used by the Greenshell™ industry between 2014 and 2021 (i.e., wild caught and hatchery produced) as a first step towards determining whether variability in nutritional condition may be a significant contributor to the marked variation observed in spat performance after it is seeded onto coastal farms. All parameters measured were highly variable among wild spat samples, with all but lipid content varying, including for samples of wild spat harvested at different times within the same month. Furthermore, there were no clear patterns in the nutritional condition of spat across months or years, suggesting environmental factors that limit feeding during the harvest and transport of spat may be contributing to marked differences in the quality of spat being used by the industry to seed farms. These results provide a baseline for assessing the nutritional condition of Greenshell™ spat and point towards a need to develop interventions to improve the quality of wild spat supply for this industry to help and prevent the ongoing massive losses of spat from farms. These measures could include, testing nutritional condition of spat at harvest or prior to seeding, development of nursery culture methods and development of artificial spat feeds that have the potential to replace live phytoplankton during nursery culture. [ABSTRACT FROM AUTHOR]

Abstract Growing seed mussels (spat) to larger sizes in single‐seed nursery culture systems prior to seeding onto coastal mussel farms is an effective approach for greatly reducing costly spat losses during this stage of mussel aquaculture. However, the production of single‐seed mussel spat in nursery culture systems relies on first separating spat from the settlement substrata to which they are attached, whether spat are settled in a hatchery or gathered from the wild. This study sought to determine whether chlorination could be an effective approach for separating wild Greenshell mussel (Perna canaliculus) spat from beachcast macroalgal material (Kaitaia spat material) harvested from a beach in northern New Zealand. The results demonstrate that chlorination (i.e. in solutions of up to 0.625% sodium hypochlorite) is a highly effective approach for separating spat, resulting in the detachment of up to 94.6% in one treatment without increasing mortality relative to controls. The effectiveness of chlorination was modulated by spat density, with increasing spat densities, lowering rates of detachment. While chlorination is effective at separating small spat (i.e. successfully separating 72.4% of spat 750 μm). The high survival (relative to the control treatments) also suggests that chlorine concentrations and exposure durations could be increased from those used in this study. These results demonstrate that chlorination can be used to separate spat from substrate prior to their transfer to nursery systems for ongrowing, paving the way for such approaches for reducing spat losses to be integrated into the aquaculture production cycle.

Abstract The nutritional condition of seed mussels (spat) at the time of seeding is thought to play a major role in influencing spat losses on mussel farms, with nutritionally compromised spat thought to be more likely to be lost from aquaculture substrata than those in good condition. New Zealand's Greenshell™ mussel (Perna canaliculus) aquaculture industry relies almost exclusively (∼85%) on wild caught spat, with the vast majority, typically > 90%, lost shortly after being seeded onto coastal mussel farms. This study sought to quantify the extent of the variability in the nutritional condition (i.e., calorific, protein, lipid and carbohydrate content) of samples of spat used by the Greenshell™ industry between 2014 and 2021 (i.e., wild caught and hatchery produced) as a first step towards determining whether variability in nutritional condition may be a significant contributor to the marked variation observed in spat performance after it is seeded onto coastal farms. All parameters measured were highly variable among wild spat samples, with all but lipid content varying, including for samples of wild spat harvested at different times within the same month. Furthermore, there were no clear patterns in the nutritional condition of spat across months or years, suggesting environmental factors that limit feeding during the harvest and transport of spat may be contributing to marked differences in the quality of spat being used by the industry to seed farms. These results provide a baseline for assessing the nutritional condition of Greenshell™ spat and point towards a need to develop interventions to improve the quality of wild spat supply for this industry to help and prevent the ongoing massive losses of spat from farms. These measures could include, testing nutritional condition of spat at harvest or prior to seeding, development of nursery culture methods and development of artificial spat feeds that have the potential to replace live phytoplankton during nursery culture.

Growing seed mussels (spat) to larger sizes in single‐seed nursery culture systems prior to seeding onto coastal mussel farms is an effective approach for greatly reducing costly spat losses during this stage of mussel aquaculture. However, the production of single‐seed mussel spat in nursery culture systems relies on first separating spat from the settlement substrata to which they are attached, whether spat are settled in a hatchery or gathered from the wild. This study sought to determine whether chlorination could be an effective approach for separating wild Greenshell mussel (Perna canaliculus) spat from beachcast macroalgal material (Kaitaia spat material) harvested from a beach in northern New Zealand. The results demonstrate that chlorination (i.e. in solutions of up to 0.625% sodium hypochlorite) is a highly effective approach for separating spat, resulting in the detachment of up to 94.6% in one treatment without increasing mortality relative to controls. The effectiveness of chlorination was modulated by spat density, with increasing spat densities, lowering rates of detachment. While chlorination is effective at separating small spat (i.e. successfully separating 72.4% of spat <750 μm in shell length) overall spat densities, further investigation is required to determine how to effectively separate larger spat (i.e. with chlorination only successfully separating 21.5% of spat >750 μm). The high survival (relative to the control treatments) also suggests that chlorine concentrations and exposure durations could be increased from those used in this study. These results demonstrate that chlorination can be used to separate spat from substrate prior to their transfer to nursery systems for ongrowing, paving the way for such approaches for reducing spat losses to be integrated into the aquaculture production cycle. [ABSTRACT FROM AUTHOR]

Conservation and restoration projects have emerged globally to address extensive declines in mussel reefs and the ecosystem services they provide. The success of these efforts relies on the re‐establishment of self‐sustaining populations, which requires mussels to effectively undergo every stage in their complex development cycle from larvae to adult mussels. In particular, the settlement of early life stages presents a potential bottleneck for recruitment in species like the green‐lipped mussel (Perna canaliculus) that rely on juvenile settlement onto macroalgae before they transition to a reef. However, it is largely unknown whether mussels can undergo settlement in areas after overharvesting where only remnant populations remain and natural recovery is vital.In this study, wild and transplanted macroalgae were sampled in an area with historically near‐extirpated mussel reefs (Kenepuru Sound, New Zealand) to examine the patterns of abundance of plantigrades, an early mussel life stage, in a degraded environment. The results suggest that plantigrades can settle to and survive on natural substrates following the removal of mussel reefs, but not into ecologically relevant areas. Specifically, almost no plantigrades were recorded on substrates near the sea bed, the area where almost all settlement to macroalgae would naturally occur. Instead, plantigrades were primarily recorded on macroalgae on docks suspended above the sea bed.A transplantation experiment verified this trend, demonstrating that substrates transferred to surface waters recorded a 36‐fold increase in plantigrade abundance compared with substrates near the sea bed. Turbidity was significantly higher near the sea bed than near the water surface, suggesting that high turbidity following the loss of wild mussels may be hindering natural plantigrade settlement or survival.This study identifies a potential bottleneck to natural mussel recovery and demonstrates the importance of a life cycle‐informed approach to restoration that prioritizes the removal of bottlenecks at multiple life stages. [ABSTRACT FROM AUTHOR]

Temperature is considered to be a major abiotic factor influencing aquatic life. Marine heatwaves are emerging as threats to sustainable shellfish aquaculture, affecting the farming of New Zealand's green-lipped mussel [Perna canaliculus (Gmelin, 1791)]. In this study, P. canaliculus were gradually exposed to high-temperature stress, mimicking a five-day marine heatwave event, to better understand the effects of heat stress on the metabolome of mussels. Following liquid chromatography-tandem mass spectrometry analyses of haemolymph samples, key sugar-based metabolites supported energy production via the glycolysis pathway and TCA cycle by 24 h and 48 h of heat stress. Anaerobic metabolism also fulfilled the role of energy production. Antioxidant molecules acted within thermally stressed mussels to mitigate oxidative stress. Purine metabolism supported tissue protection and energy replenishment. Pyrimidine metabolism supported the protection of nucleic acids and protein synthesis. Amino acids ensured balanced intracellular osmolality at 24 h and ammonia detoxification at 48 h. Altogether, this work provides evidence that P. canaliculus has the potential to adapt to heat stress up to 24 °C by regulating its energy metabolism, balancing nucleotide production, and implementing oxidative stress mechanisms over time. The data reported herein can also be used to evaluate the risks of heatwaves and improve mitigation strategies for aquaculture. [ABSTRACT FROM AUTHOR]

BACKGROUND: Marine‐derived omega‐3 polyunsaturated fatty acids (PUFA) are used globally as food supplements for their anti‐inflammatory properties and to reduce the risk of obesity, cardiovascular diseases, and cancer. New Zealand green‐lipped mussels (Perna canaliculus) contain diverse valuable compounds, including omega‐3 PUFA. The most abundant omega‐3 PUFA in Perna canaliculus are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both of which account for many of the health benefits accredited to these mussels. This research investigates the feasibility of using deep eutectic solvents (DESs) to isolate EPA and DHA from Perna canaliculus. RESULTS: Twenty‐two hydrophobic DESs were screened using COSMO‐RS software to predict their affinity for EPA and DHA. Nine top‐ranked DESs were used for experimental extraction validation. The results exposed that the extraction capacity of the menthol: lidocaine DES (1:1 M ratio) for EPA and DHA was superior (172.04 μg EPA g−1 dry weight (DW) of biomass and 602.79 μg DHA g−1 DW) amongst other DESs. This was a considerably higher extraction yield than that observed using ethanol (95.65 μg EPA g−1 DW and 311.49 μg DHA g−1 DW). The optimisation of the menthol: lidocaine ratio identified a 1:2 M ratio as the optimal composition for this DES. The highest yields of EPA (267.59 μg g−1 DW) and DHA (1014.84 μg g−1 DW) were obtained by this optimised DES ratio at optimum extraction conditions (extraction temperature 65 °C, extraction time 2.12 h, and biomass loading 5 w/v%). The EPA and DHA showed good stability in the menthol: lidocaine DES over 7 days. This DES also showed good reusability, with a comparable extraction efficiency being observed after five extraction cycles. CONCLUSION: This research revealed that menthol‐based DES can be a potential green solvent for the extraction of EPA and DHA from New Zealand green‐lipped mussels and it may be applicable for extracting other bioactive compounds with similar characteristics. © 2023 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

In this study, Reactive Black 5 (RB5) removal potential was investigated by Spirulina and Perna canaliculus biocomposite (SPC) in batch and continuous systems. FTIR, Point of Zero Charge, and SEM analyzes were performed to elucidate the structure of the biocomposite. In the batch system, the effects of pH, biosorbent dose, contact time, temperature, and initial RB5 concentration on the biosorption process were investigated. The flow rate and bed height studies were carried out in the continuous system. Langmuir, Freundlich, and Scatchard isotherm models were used to describe the biosorption process. The experimental data fitted best to the Langmuir isotherm model, and the maximum monolayer biosorption capacity was determined as 277.6 mg/g. In kinetic studies, pseudo-first-order, pseudo-second-order, and intraparticle diffusion models were applied, and it was determined that the process followed the pseudo-second-order kinetic model. Equilibrium was established in 30 min for RB5 removal with SPC biocomposite. As a result of the studies carried out in the continuous system, the biosorption efficiency was higher at the low flow rate and increased with the rising bed height. In biosorption-desorption cycle studies, RB5 removal by SPC was successful for up to 10 cycles. Also, the biosorption process was applied to simulated wastewater and the results indicated that the biosorption capacity was not affected significantly by the existence of many other metallic and organic pollutants. As a result of the findings, it was concluded that SPC could be an effective and alternative biosorbent in RB5 removal. [ABSTRACT FROM AUTHOR]

Ecosystem restoration has proliferated across the globe to combat widespread ecosystem decline. Translocations of viable individuals into degraded habitats form a core component of restoration efforts, but the selection of source populations poses challenges because phenotypic differences between source populations and depleted populations result in low post‐translocation survival. Intertidal mussel restoration is typically reliant on subtidal source populations despite their weaker shells, lighter weights, and poorer respiratory abilities compared to intertidal mussels. Intertidal mussel restoration using subtidal mussels has recorded very low survival, leading to suggestions that subtidal mussels are unfit for the intertidal zone, effectively eliminating the possibility of translocation‐based intertidal mussel restoration. In this study, small‐scale intertidal mussel restoration was tested using subtidal cultured mussels transplanted to paired plots, half in the lower intertidal and half in the upper subtidal. The results demonstrate the first scientific evidence of the successful restoration of intertidal mussel reefs using subtidal mussels at this scale. Specifically, one location recorded very high survival after 12 months (95.1 ± 0.6%; mean ± SE), comparable to the survival recorded in the subtidal zone (97.6 ± 0.3%). Translocations to the intertidal at the other two locations experienced mortality during the Austral summer (final survival: 81.3 ± 1.6% and 56.6 ± 7.4%), which directly correlated with exposure to extreme temperatures (>40°C). These results reveal that despite phenotypic differences, subtidal cultured mussels are potential sources for successful intertidal restoration. However, care should also be taken to avoid locations prone to high temperatures, particularly in light of a warming global environment. Ultimately, to achieve the massive upscaling in restoration called for by the UN Decade on Ecosystem Restoration, we encourage restoration managers to think broadly about potential source populations, avoid rejections based solely on phenotypic differences, and explore every potential avenue for restoration success. [ABSTRACT FROM AUTHOR]

Abstract Ecosystem restoration has proliferated across the globe to combat widespread ecosystem decline. Translocations of viable individuals into degraded habitats form a core component of restoration efforts, but the selection of source populations poses challenges because phenotypic differences between source populations and depleted populations result in low post‐translocation survival. Intertidal mussel restoration is typically reliant on subtidal source populations despite their weaker shells, lighter weights, and poorer respiratory abilities compared to intertidal mussels. Intertidal mussel restoration using subtidal mussels has recorded very low survival, leading to suggestions that subtidal mussels are unfit for the intertidal zone, effectively eliminating the possibility of translocation‐based intertidal mussel restoration. In this study, small‐scale intertidal mussel restoration was tested using subtidal cultured mussels transplanted to paired plots, half in the lower intertidal and half in the upper subtidal. The results demonstrate the first scientific evidence of the successful restoration of intertidal mussel reefs using subtidal mussels at this scale. Specifically, one location recorded very high survival after 12 months (95.1 ± 0.6%; mean ± SE), comparable to the survival recorded in the subtidal zone (97.6 ± 0.3%). Translocations to the intertidal at the other two locations experienced mortality during the Austral summer (final survival: 81.3 ± 1.6% and 56.6 ± 7.4%), which directly correlated with exposure to extreme temperatures (>40°C). These results reveal that despite phenotypic differences, subtidal cultured mussels are potential sources for successful intertidal restoration. However, care should also be taken to avoid locations prone to high temperatures, particularly in light of a warming global environment. Ultimately, to achieve the massive upscaling in restoration called for by the UN Decade on Ecosystem Restoration, we encourage restoration managers to think broadly about potential source populations, avoid rejections based solely on phenotypic differences, and explore every potential avenue for restoration success.

The onset of the Anthropocene has coincided with enormous global declines in natural ecosystems, leading to losses in the valuable goods and ecosystem services they provide. This global decline, in conjunction with growing recognition of the ecological importance of natural ecosystems, has generated a pressing need for restoration. Effective ecosystem restoration relies on accurate identification of the cause of decline and clear metrics of success, which are only possible with baseline data of both the pre‐degradation and pre‐restoration ecosystems. However, the establishment of these baselines can be difficult as different potential information sources each have benefits and drawbacks. Determining an efficient method to balance these diverse information sources and generate robust baselines is vital to achieving the United Nations' goal of massively scaled‐up ecosystem restoration. Here we expand on the concept of multidisciplinary baselines, or the combined use of sources and methods across a wide disciplinary spectrum to establish comprehensive and reliable ecosystem baselines, and use mussel reefs in the South Island of New Zealand as a test case. Using a combination of comprehensive historical review, extensive shoreline surveys, and local ecological knowledge, we demonstrate that local mussel abundances decreased by 97% since the mid‐1960s as a result of overharvesting, leaving the extant populations scattered, small, and without recovery. This study demonstrates that harnessing multidisciplinary baselines allows for the consolidation of qualitative and quantitative estimates of ecosystem change over hundreds of years, as well as confirmation of causes of ecosystem degradation, and clear documentation of current ecosystem state beyond what is possible from any individual source. This approach to establishing ecosystem baselines also provides valuable avenues for the advancement of restoration by quantifying the temporal and geographic scales of ecosystem decline, identifying areas for intervention, and establishing clear metrics of success. [ABSTRACT FROM AUTHOR]

The global loss in ecosystem engineers has initiated calls for restoration, which includes the UN declaration of 2021–2030 as the decade of ecosystem restoration. As researchers dive into this decade it is important to consider the current state of an ecosystem to ensure restoration success. Pilot‐scale restoration has been recommended by global guidelines and standards as an effective starting point for restoration to provide valuable initial information to increase efficiency and success of subsequent larger‐scale restoration. To test habitat suitability, 4 t of green‐lipped mussels were placed onto three 2.25 m2 plots at five locations with differing benthic environments and assessed over 2 years. This study had a mean of 85% mussel survival at four of the five locations, while the restored shellfish in one location were completely extirpated, most likely by sea star predation. This pilot‐scale restoration revealed location‐specific differences that have implications for ensuring larger‐scale restoration success, including mussel survival, density, and health, and recommends a timeframe of at least 18 months to properly assess habitat suitability. Overall, the results validated the efficacy of using small‐scale pilot experiments to test habitat suitability and optimize location selection to maximize success and efficiency of larger‐scale restoration efforts. [ABSTRACT FROM AUTHOR]

Growth of the Greenshell™ mussel (Perna canaliculus) industry, New Zealand's most significant aquaculture sector, is limited by spat abundance in the early stages of the aquaculture cycle. However, there are few management techniques to improve spat production. Here, we tested the effects of seeding density (low: 257, mid: 515, and high: 1030 mussels per 10 cm of culture rope) – a putative method to manage spat seeding efficiency and yield – on the abundance and size of P. canaliculus spat at four depths at two sites in the Marlborough Sounds. We also recorded the abundance and size of blue mussels, Mytilus galloprovincialis , which are a problematic biofouling species, as well as the biomass and composition of the biofouling accumulating on mussel culture ropes under the same experimental treatments. Although the abundance of P. canaliculus was greatest at the highest seeding density (∼200 mussels per 10 cm of rope at 87 days), this seeding density also experienced the greatest losses. However, while fewer spat were lost from the low-density seeding treatment, the overall number of spat per 10 cm was significantly reduced, resulting in few operational gains in terms of biofouling reduction or spat size. Overall, the seeding density of suggesting that seeding density of ∼1030 spat per 10 cm of rope was the optimal density tested in this study and could be a useful target density for mussel farmers. • Effects of seeding density (SD) on abundance and growth of mussel spat was tested. • Abundance remained highest at the highest SD over time. • Biofouling accumulation and biomass was generally lowest at the highest SD. • Mussel size and abundance was impacted by SD and additional factors together • Highest SD tested was preferred to optimise mussel abundance and reduce biofouling. [ABSTRACT FROM AUTHOR]

Abstract The onset of the Anthropocene has coincided with enormous global declines in natural ecosystems, leading to losses in the valuable goods and ecosystem services they provide. This global decline, in conjunction with growing recognition of the ecological importance of natural ecosystems, has generated a pressing need for restoration. Effective ecosystem restoration relies on accurate identification of the cause of decline and clear metrics of success, which are only possible with baseline data of both the pre‐degradation and pre‐restoration ecosystems. However, the establishment of these baselines can be difficult as different potential information sources each have benefits and drawbacks. Determining an efficient method to balance these diverse information sources and generate robust baselines is vital to achieving the United Nations' goal of massively scaled‐up ecosystem restoration. Here we expand on the concept of multidisciplinary baselines, or the combined use of sources and methods across a wide disciplinary spectrum to establish comprehensive and reliable ecosystem baselines, and use mussel reefs in the South Island of New Zealand as a test case. Using a combination of comprehensive historical review, extensive shoreline surveys, and local ecological knowledge, we demonstrate that local mussel abundances decreased by 97% since the mid‐1960s as a result of overharvesting, leaving the extant populations scattered, small, and without recovery. This study demonstrates that harnessing multidisciplinary baselines allows for the consolidation of qualitative and quantitative estimates of ecosystem change over hundreds of years, as well as confirmation of causes of ecosystem degradation, and clear documentation of current ecosystem state beyond what is possible from any individual source. This approach to establishing ecosystem baselines also provides valuable avenues for the advancement of restoration by quantifying the temporal and geographic scales of ecosystem decline, identifying areas for intervention, and establishing clear metrics of success.

Abstract The global loss in ecosystem engineers has initiated calls for restoration, which includes the UN declaration of 2021–2030 as the decade of ecosystem restoration. As researchers dive into this decade it is important to consider the current state of an ecosystem to ensure restoration success. Pilot‐scale restoration has been recommended by global guidelines and standards as an effective starting point for restoration to provide valuable initial information to increase efficiency and success of subsequent larger‐scale restoration. To test habitat suitability, 4 t of green‐lipped mussels were placed onto three 2.25 m2 plots at five locations with differing benthic environments and assessed over 2 years. This study had a mean of 85% mussel survival at four of the five locations, while the restored shellfish in one location were completely extirpated, most likely by sea star predation. This pilot‐scale restoration revealed location‐specific differences that have implications for ensuring larger‐scale restoration success, including mussel survival, density, and health, and recommends a timeframe of at least 18 months to properly assess habitat suitability. Overall, the results validated the efficacy of using small‐scale pilot experiments to test habitat suitability and optimize location selection to maximize success and efficiency of larger‐scale restoration efforts.

Providing benthic substrate is the most common method used for oyster reef restoration. The physical relief from the seabed, increased habitat complexity, and attachment surfaces have been shown to improve oyster health, recruitment, and survival. While the addition of shell material is an effective substrate for oyster restoration, its usefulness in mussel restoration has been understudied. This study tested the effectiveness of adding shell substrate to two different types of soft sediment for the restoration of adult green-lipped mussels. Over 10 t of shell was used to create a 10 cm layer on the seabed in replicated experimental plots at the two locations. 10 t of live adult mussels were deployed onto the shell substrate and an additional 10 t of mussels onto adjacent soft sediment control plots. A year after deploying the live mussels, mussel survival across all plots was 80.6 ± 6.5%, with no differences between mussel plots with or without the added shell substrate for either of the two locations. This study emphasizes the importance of context-dependency, revealing promising avenues for future research, and indicates that for adult green-lipped mussels the addition of a shell substrate appears to provide little advantage for adult mussel restoration at high deployment densities. [ABSTRACT FROM AUTHOR]

Temperature is considered to be a major abiotic factor influencing aquatic life. Marine heatwaves are emerging as threats to sustainable shellfish aquaculture, affecting the farming of New Zealand’s green-lipped mussel [Perna canaliculus (Gmelin, 1791)]. In this study, P. canaliculus were gradually exposed to high-temperature stress, mimicking a five-day marine heatwave event, to better understand the effects of heat stress on the metabolome of mussels. Following liquid chromatography-tandem mass spectrometry analyses of haemolymph samples, key sugar-based metabolites supported energy production via the glycolysis pathway and TCA cycle by 24 h and 48 h of heat stress. Anaerobic metabolism also fulfilled the role of energy production. Antioxidant molecules acted within thermally stressed mussels to mitigate oxidative stress. Purine metabolism supported tissue protection and energy replenishment. Pyrimidine metabolism supported the protection of nucleic acids and protein synthesis. Amino acids ensured balanced intracellular osmolality at 24 h and ammonia detoxification at 48 h. Altogether, this work provides evidence that P. canaliculus has the potential to adapt to heat stress up to 24 °C by regulating its energy metabolism, balancing nucleotide production, and implementing oxidative stress mechanisms over time. The data reported herein can also be used to evaluate the risks of heatwaves and improve mitigation strategies for aquaculture.

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 Vibrio sp. 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. • 24 h post-infection, Vibrio sp. Proliferated in mussel haemolymph at 16 and 24 °C. • Haemocyte immune responses were altered by infection and temperature. • Acclimating mussels to 24 °C did not exacerbate the impact of Vibrio sp. infection. • Mussels held at 24 °C activated additional energy production pathways. • Mussels injected with Vibrio sp. showed decreased metabolic activity. [ABSTRACT FROM AUTHOR]

The mechanistic approach and the potential predictive power of Dynamic Energy Budget (DEB) models are making this modelling framework increasingly popular in aquaculture management. The potential to mechanistically simulate life-history traits of a species in a changing environment has already been applied to many commercial species, including the New Zealand Greenshell™ mussel Perna canaliculus. However, the previous P. canaliculus model parameterisation, available in the Add-my-Pet (AmP) collection, has been carried out on a limited data set. We used data obtained through targeted laboratory experiments and combined it with literature data, to derive a new version of the model which included information on additional processes – such as feeding, respiration, and reproduction – and life stages: larvae and adults. We compared the performance of the previous version of the model, termed AmP-2018 with the revised model (AmP-2024), to predict the whole life-cycle of the mussel and life-history traits of interest in the context of aquaculture. The entry completeness score (0−10), which provides an indication of data quality and variety as well as model comprehensiveness and applicability, increased dramatically from 2.5 to 5.5, suggesting P. canaliculus is now the most comprehensively DEB-parameterised bivalve, while the model increased in accuracy of the predictions. The endeavour highlights several conclusions: (i) reliable temperature response data are required to establish the thermal response curve; the presence/absence of this type of data should be taken into account when assessing the completeness of a model and its applicability to predict thermal responses of a species; (ii) species-specific parameter values are preferred over general (higher taxonomic level) specific values, e.g. for tissue organics and water content, as they increase the accuracy of predictions; (iii) data pertaining to laboratory trials and to wild populations both need to be taken into account when calibrating a mechanistic model at a species level; and (iv) calculating energy requirements of any organism, including bivalves, requires data on characteristics and products often overlooked in an experimental setting. We discuss the new version of the model in the context of the knowledge and predictive power potentially gained by adding more information and data during model calibration. The obtained results and conclusions are especially relevant in aquaculture, whether for commercial rearing of mussels, or their potential application in multitrophic aquaculture systems. [Display omitted] • A DEB model for Perna canaliculus was reparameterised with targeted experimental and literature data. • The model can realistically predict the growth, development (including larval stage), and reproduction of the mussel. • Quality thermal response data is crucial for assessing thermal response curves in DEB models. • Both ex-situ and in-situ data should be considered for species-level calibration in DEB models. • We discuss calculations for closing an energy and/or mass budget in an experimental setting. [ABSTRACT FROM AUTHOR]

The restoration of shellfish reefs to soft‐sediment environments often relies on the translocation of donor stock, typically from aquaculture, to the seafloor. In New Zealand, subadult green‐lipped mussels (Perna canaliculus) grown on subtidal aquaculture long‐lines are being considered over adult conspecifics to increase restoration efficiency but are currently limited by predation and hydrodynamic dislodgment following transfer to the seabed. In this study, the survival of subadult mussels from five different sources representing a range of growth conditions (i.e. subtidal aquaculture long‐lines 1 and 2, subtidal shellfish aquaculture baskets 1, wild intertidal reefs 1 and 2) were compared across separate translocations (1 and 2) to see if careful consideration of stock source could improve subadult mussel survival following transfer to the seafloor. Mussel morphology (shell strength, attachment thread structure) and clumping behavior (perimeter:area ratios, clump densities, clump complexity) were compared among populations to explain relationships among prior stock growth conditions, mussel size, and survival. Following experimental translocations, high survival (>90%) was closely related to decreases in perimeter:area ratios of mussel clumps and increases in clump complexity. High survival groups of mussels were selected from stock with high shell compression strength (subtidal aquaculture long‐line 2, 95.5% survival), greater attachment thread number and thickness (aquaculture basket 1, 92.3% survival), or some combination of the two (wild intertidal reef 2, 99.3% survival). This study supports further consideration for incorporating subadult mussels into restoration provided they are selected from sources that produce individuals with the resistant characteristics outlined in this study. [ABSTRACT FROM AUTHOR]

• A protocol to measure the concentration and viability of Perna canaliculus hemocytes was validated. • The validated assay, using a simple user interface flow cytometer, was compared to 3 other assays. • Using physiologically-compatible media with commercially-available assay kits is important. [ABSTRACT FROM AUTHOR]

The seeding of small juvenile green‐lipped mussels (Perna canaliculus) onto coastal farms is associated with high losses. These losses can be reduced by seeding larger juveniles; however, the nursery culture of juveniles is unviable because of the high cost of producing live microalgal feeds. In this study, we compared the ability for two diets, a formulated microparticulate feed, MySpat and liposomes fabricated with mussel extract to replace live microalgae at different proportions for feeding small (1.9 mm shell length ±0.02 SE) green‐lipped mussels. The experimental diets consisted of a mix of live Tisochrysis lutea, Diacronema lutheri and Chaetoceros muelleri that were replaced with increasing proportions of MySpat (25%, 50% and 75%), and liposomes (25% and 50%). There were no significant differences in mortality of mussels relative to the control (100% microalgae) among any of the diet treatments. However, mussel growth tended to decrease with increasing substitution of the microalgal component of the diets. The results from this study suggest that microparticulate and liposome formats provide a viable mode of food delivery to juvenile mussels, with improved formulation of these formats having the potential to lead to the substitution of live microalgal diets which make nursery culture unviable at present. [ABSTRACT FROM AUTHOR]

In the past decade, the use of marine mussels as seafood is being more popular. They considered being a rich source of various nutritional bioactive compounds that aroused the scientific community's interest. This study investigated the antioxidant and the antithrombotic consequences on Sprague‐Dawley male rats after adding dried New Zealand mussel Perna canaliculus in their diet. The biochemical, hematological and histopathological changes were also observed. Forty rats were divided into four groups according to the amount of dried mussels in their diet, in addition to a control group that consumed a basal diet only. Group 1 consumed 25% dried mussels in its basal diet; Group 2 consumed 35% dried mussels in its basal diet, and Group 3 was consumed 45% dried mussels in its basal diet. The biochemical parameters showed improvements in liver function. Interestingly, the lipid profile decreased, especially the low‐density lipoprotein cholesterol (LDL‐C) levels which were reduced significantly in Group 3 (p <.01). These observations were accompanied by a decrease in iron levels significantly as the amount of dried mussels increased (p <.01). Furthermore, the noticed changes in the hematological profile prove that there is an increase in antithrombotic activity. Dried mussels had potent antioxidant effects in the liver as shown by increased lipid peroxide (p <.05), reduced glutathione (p <.05), and glutathione peroxidase (GSH‐Px; p <.05). Additionally, antioxidant activity in the kidney was shown to increase through GSH‐Px activity (p <.01). In conclusion, these results indicate that consuming dried mussels resulted in improved biochemical and antioxidants activities and could be used as an antithrombotic agent. [ABSTRACT FROM AUTHOR]

The New Zealand Greenshell™ mussel (Perna canaliculus) is an economically important aquaculture species. Prolonged increases in seawater temperature above mussel thermotolerance ranges pose a significant threat to mussel survival and health, potentially increasing susceptibility to bacterial infections. Using challenge experiments, this study examined the combined effects of increased seawater temperature and bacterial (Photobacterium swingsii) infection on animal survival, haemocyte and biochemical responses of adult mussels. Mussels maintained at three temperatures (16, 20 and 24 °C) for seven days were either not injected (control), injected with sterile marine broth (injection control) or P. swingsii (challenged with medium and high doses) and monitored daily for five days. Haemolymph and tissue samples were collected at 24, 48, 72, 96, 120 h post-challenge and analysed to quantify bacterial colonies, haemocyte responses and biochemical responses. Mussels infected with P. swingsii exhibited mortalities at 20 and 24 °C, likely due to a compromised immune system, but no mortalities were observed when temperature was the only stressor. Bacterial colony counts in haemolymph decreased over time, suggesting bacterial clearance followed by the activation of immune signalling pathways. Total haemocyte counts and viability data supports haemocyte defence functions being stimulated in the presence of high pathogen loads at 24 °C. In the gill tissue, oxidative stress responses, measured as total antioxidant capacity and malondialdehyde (MDA) levels, were higher in infected mussels (compared to the controls) after 24h and 120h post-challenge at the lowest (16 °C) and highest temperatures (24 °C), indicating the presence of oxidative stress due to temperature and pathogen stressors. Overall, this work confirms that Photobacterium swingsii is pathogenic to P. canaliculus and indicates that mussels may be more vulnerable to bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios. [Display omitted] • Higher mussel mortality with combined temperature and bacterial stressors. • Reduction of bacterial colonies in haemolymph with high temperature exposure. • Haemocyte parameters fluctuated with changes in temperature and over time. • Bacterial infection elicits oxidative stress at 16 and 24 °C. [ABSTRACT FROM AUTHOR]

Widespread resource extraction and habitat degradation have severely reduced functionally important subtidal mussel reefs globally. While methods for restoring oyster reefs are becoming increasingly well‐established, the development of techniques for the effective restoration of mussel reefs remain in their infancy and face biological and logistical challenges. This study investigated the potential use of subadult and juvenile green‐lipped mussels (Perna canaliculus) for mussel reef restoration with the aim of understanding factors related to subadult and juvenile mussel survival after transfer to the seafloor. Small‐scale (m2) field experiments were conducted subtidally in a New Zealand harbor to assess subadult and juvenile mussel survival after translocation to soft‐sediment seafloor, the efficacy of biodegradable substrate to support reef development, and whether juvenile mussel survival was related to changes in seeding density. Results demonstrated survival of cultured subadult and juvenile mussels after transfer to soft‐sediment seafloor only when completely protected from mobile predators. Attachment to biodegradable substrate alone was insufficient to prevent the loss of cultured juvenile mussels, while ~80% of wild subadult mussels survived translocation to the seafloor without predator protection—indicating a certain level of resilience. Changes in seeding density failed to prevent loss of cultured juvenile mussels. This study supports further consideration for incorporating cultured subadult and juvenile mussels into restoration, provided subadult and juvenile mussels can be protected until they become established as adults. [ABSTRACT FROM AUTHOR]

Mollusc shells are an abundant resource that have been long used to predict the structures of ancient ecological communities, examine evolutionary processes, reconstruct paleoenvironmental conditions, track and predict responses to climatic change, and explore the movement of hominids across the globe. Despite the ubiquity of mollusc shell in many environments, it remains relatively unexplored as a substrate for molecular genetic analysis. Here we undertook a series of experiments using the New Zealand endemic greenshell mussel, Perna canaliculus, to explore the utility of fresh, aged, beach-cast and cooked mollusc shell for molecular genetic analyses. We find that reasonable quantities of DNA (0.002–21.48 ng/mg shell) can be derived from aged, beach-cast and cooked mussel shell and that this can routinely provide enough material to undertake PCR analyses of mitochondrial and nuclear gene fragments. Mitochondrial PCR amplification had an average success rate of 96.5% from shell tissue extracted thirteen months after the animal’s death. A success rate of 93.75% was obtained for cooked shells. Amplification of nuclear DNA (chitin synthase gene) was less successful (80% success from fresh shells, decreasing to 10% with time, and 75% from cooked shells). Our results demonstrate the promise of mollusc shell as a substrate for genetic analyses targeting both mitochondrial and nuclear genes.

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. Image 1 • Unknown summer mortality causes perturbations in mussel metabolism. • Metabolomics allows classification of mussels from different affected levels. • Accumulation of itaconic acid may indicate the involvement of pathogen infections. • Itaconic acid has potential to be a biomarker for pathogen infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • 3 sub-populations of Perna canaliculus hemocytes were found using flow cytometry. • Granulocytes showed higher immune functions vs hyalinocytes and blast-like cells. • Hemocytes were extremely responsive, aggregating within 2 min of extraction. • Modified Alsever's Solutions delayed cell clumping but influenced immune parameters. • Optimal hemocyte handling, storage and flow cytometric protocols are detailed. [ABSTRACT FROM AUTHOR]

Mollusc shells are an abundant resource that have been long used to predict the structures of ancient ecological communities, examine evolutionary processes, reconstruct paleoenvironmental conditions, track and predict responses to climatic change, and explore the movement of hominids across the globe. Despite the ubiquity of mollusc shell in many environments, it remains relatively unexplored as a substrate for molecular genetic analysis. Here we undertook a series of experiments using the New Zealand endemic greenshell mussel, Perna canaliculus, to explore the utility of fresh, aged, beach-cast and cooked mollusc shell for molecular genetic analyses. We find that reasonable quantities of DNA (0.002 21.48 ng/mg shell) can be derived from aged, beach-cast and cooked mussel shell and that this can routinely provide enough material to undertake PCR analyses of mitochondrial and nuclear gene fragments. Mitochondrial PCR amplification had an average success rate of 96.5% from shell tissue extracted thirteen months after the animal's death. A success rate of 93.75% was obtained for cooked shells. Amplification of nuclear DNA (chitin synthase gene) was less successful (80% success from fresh shells, decreasing to 10% with time, and 75% from cooked shells). Our results demonstrate the promise of mollusc shell as a substrate for genetic analyses targeting both mitochondrial and nuclear genes. [ABSTRACT FROM AUTHOR]

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. • D-larvae are good life-stage candidates for cryobanking in Perna canaliculus. • D-stage larvae were more resilient to cryopreservation than trochophore larvae. • Organogenesis was delayed in all cryopreserved larvae with no eyespot development. • Neurogenesis was stunted in cryopreserved trochophores and normal in D-stage larvae. • Developmental delays were indicative of cryo-injuries sustained at a cellular level. [ABSTRACT FROM AUTHOR]

[Display omitted] • Mussels were in vivo challenged with live and heat-killed Photobacterium swingsii. • Mortality, haemocyte counts, and haemolymph colony forming units peaked at 48 hpc with live P. swingsii. • qPCR showed highest levels of bacterial DNA at 12 hpc in digestive gland. • Histopathological observations suggested a non-specific inflammatory response in all mussels. 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. [ABSTRACT FROM AUTHOR]

The early stages of intertidal mussels, including the green-lipped mussel, Perna canaliculus, face both direct and indirect environmental threats. Stressors may influence physiological status and, ultimately, survival. An understanding of the nature of stress experienced is critical to inform conservation and aquaculture efforts. Here, we investigated oxidative stress dynamics in juvenile P. canaliculus in relation to emersion duration (1–20 h) and relative humidity (RH, 29–98%) by quantifying oxidative damage (protein carbonyls, lipid hydroperoxides, 8-hydroxydeoxyguanosine) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and reductase). Mussels held in low RH during emersion experienced severe water loss (>70%), high mortality (>80%) and increased oxidative damage (35–45% increase compared to control conditions), while mussels held at high RH were not impacted, even after 20 h of air exposure. Following re-immersion, reoxygenation stress resulted in further increases in damage markers in mussels that had experienced dryer emersion conditions; protective action of antioxidants increased steadily during the 10 h re-immersion period, apparently supporting a reduction in damage markers after 1–5 h of immersion. Clearly, conditions during emersion, as well as duration, substantially influence physiological performance and recovery of juvenile mussels. Successful recruitment to intertidal beds or survival in commercial aquaculture operations may be mediated by the nature of emersion stress experienced by these vulnerable juveniles.

Perna viridis and P. canaliculus are economically and ecologically important species of shellfish. In this study, the complete ribosomal DNA (rDNA) unit sequences of these species were determined for the first time. The gene order, 18S rRNA–internal transcribed spacer (ITS) 1–5.8S rRNA–ITS2–28S rRNA–intergenic spacer (IGS), was similar to that observed in other eukaryotes. The lengths of the P. viridis and P. canaliculus rDNA sequences ranged from 8,432 to 8,616 bp and from 7,597 to 7,610 bp, respectively, this variability was mainly attributable to the IGS region. The putative transcription termination site and initiation site were confirmed. Perna viridis and P. canaliculus rDNA contained two (length: 93 and 40 bp) and one (length: 131 bp) repeat motifs, respectively. Individual intra-species differences mainly involved the copy number of repeat units. In P. viridis, three cytosine-guanine (CpG) sites with sizes of 440, 1,075 and 537 bp were found to cover nearly the entire IGS sequence, whereas in P. canaliculus, two CpG islands with sizes of 361 and 484 bp were identified. The phylogenetic trees constructed with maximum likelihood and neighbour-joining methods and based on ITS sequences were identical and included three major clusters. Species of the same genus were easily clustered together.

Arthritis is a common condition that causes pain and inflammation in a joint. Previously, we reported that the mixture extract (ME) from Agrimonia pilosa Ledeb. (AP) and Salvia miltiorrhiza Bunge (SM) could ameliorate gout arthritis. In the present study, we aimed to investigate the potential anti-inflammatory and antinociceptive effects of ME and characterize the mechanism. We compared the anti-inflammatory and antinociceptive effects of a positive control, Perna canaliculus powder (PC). The results showed that one-off and one-week treatment of ME reduced the pain threshold in a dose-dependent manner (from 10 to 100 mg/kg) in the mono-iodoacetate (MIA)-induced osteoarthritis (OA) model. ME also reduced the plasma TNF-α, IL-6, and CRP levels. In LPS-stimulated RAW 264.7 cells, ME inhibited the release of NO, PGE2, LTB4, and IL-6, increased the phosphorylation of PPAR-γ protein, and downregulated TNF-α and MAPKs proteins expression in a concentration-dependent (from 1 to 100 µg/mL) manner. Furthermore, ME ameliorated the progression of ear edema in mice. In most of the experiments, ME-induced effects were almost equal to, or were higher than, PC-induced effects. Conclusions: The data presented here suggest that ME shows anti-inflammatory and antinociceptive activities, indicating ME may be a potential therapeutic for arthritis treatment.

The efficient seeding of juvenile mussels is critical to the sustainability and profitability of mussel aquaculture. However, seeding mussels is highly inefficient, with many juveniles being lost in the following few months. One possible cause of these losses could be the development of biofouling assemblages. Therefore, the relationships between biofouling accumulation and losses of juveniles were assessed. Losses of juvenile mussels were initially high (42.9–49.1% over approximately one to two weeks), with lower rates of loss over the following four to five months. Biofouling development followed a successional pattern beginning with colonisation by amphipods, subsequent establishment of macroalgae, and the formation of an assemblage dominated by mussels and sessile invertebrates. However, biofouling development did not play a major role in the loss of juveniles. Rather, large-scale losses of mussels occurred shortly after seeding when biofouling was scant, suggesting alternative causes of loss were in operation. [ABSTRACT FROM AUTHOR]

Predation by sea stars has the potential to cause elevated levels of mortality in reestablished populations of bivalves relative to levels of recruitment. Recent efforts to restore beds of the nearly extirpated green‐lipped mussel within the Hauraki Gulf, New Zealand, resulted in high abundances of sea stars occupying those beds and it is unknown whether predation poses a potential limitation to the success of restoration in this bivalve species. The contribution of predation by sea stars to the mortality of mussels across four experimental mussel beds over a 2‐year period was estimated using data from regular assessments of sea star abundance and an experimentally determined consumption rate of sea stars upon mussels. In addition, the potential size selection of mussels by sea stars was assessed to determine if large sea stars selected for recent settlers. Sea stars' abundance within the mussel beds grew to an average of 0.57 sea stars/m2 within 9 months, remaining at similar levels throughout the study. These predominantly large sea stars were estimated to have consumed 30.1% of the mussels over a 25‐month period, representing a contribution of 40.4% of the mussel mortality. Sea stars predominantly selected for larger mussels, and their predation likely contributes little to any lack in mussel recruitment. Sea star predation is clearly a limiting factor on the survival of transplanted adult mussels and the present study highlights the need to continually assess predation risk to determine if remediation is necessary for the persistence of the restored beds. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Thermal history and current temperature interact to influence survival at 24 °C. • Aerobic and anaerobic energy supplies are vital for defence mechanisms and survival. • Oxidative stress is managed by antioxidant metabolites and haemocyte functions. • Mussel condition index decreased with an increase in temperature. • Marine heatwave duration and rate of onset determine net physiological impact. [ABSTRACT FROM AUTHOR]

Seafood represents a significant part of the human staple diet. In the recent years, the identification of emerging lipophilic marine toxins has increased, leading to the potential for consumers to be intoxicated by these toxins. In the present work, we investigate the presence of lipophilic marine toxins (both regulated and emerging) in commercial seafood products from non-European locations, including mussels Mytilus chilensis from Chile, clams Tawerea gayi and Metetrix lyrate from the Southeast Pacific and Vietnam, and food supplements based on mussels formulations of Perna canaliculus from New Zealand. All these products were purchased from European Union markets and they were analyzed by UPLC-MS/MS. Results showed the presence of the emerging pinnatoxin-G in mussels Mytilus chilensis at levels up to 5.2 µg/kg and azaspiracid-2 and pectenotoxin-2 in clams Tawera gayi up to 4.33 µg/kg and 10.88 µg/kg, respectively. This study confirms the presence of pinnatoxins in Chile, one of the major mussel producers worldwide. Chromatograms showed the presence of 13-desmethyl spirolide C in dietary supplements in the range of 33.2–97.9 µg/kg after an extraction with water and methanol from 0.39 g of the green lipped mussels powder. As far as we know, this constitutes the first time that an emerging cyclic imine toxin in dietary supplements is reported. Identifying new matrix, locations, and understanding emerging toxin distribution area are important for preventing the risks of spreading and contamination linked to these compounds.