Oxidation and cross-link of tyrosine-rich proteins are involved in the periostracum formation of the green mussel Perna viridis (Linnaeus).

Ocean acidification causes severe shell dissolution and threats the survival of marine molluscs. The periostracum in molluscs consists of macromolecules such as proteins and polysaccharides, and protects the inner shell layers from dissolution and microbial erosion. Moreover, it serves as the primary template for shell deposition. However, the chemical composition and formation mechanism of the periostracum is largely unknown. In this study, we applied transcriptomic, proteomics, physical, and chemical analysis to unravel the mysteries of the periostracum formation in the green mussel Perna viridis Linnaeus. FTIR analysis showed that the periostracum layer was an organic membrane mainly composed of polysaccharides, lipids, and proteins, similar to that of the shell matrix. Interestingly, the proteomic study identified components enriched in tyrosine and some enzymes that evolved in tyrosine oxidation, indicating that tyrosine oxidation might play an essential role in the periostracum formation. Moreover, comparative transcriptomics suggested that tyrosine-rich proteins were intensively synthesized in the periostracum groove. After being secreted, the periostracum proteins were gradually tanned by oxidation in the seawater, and the level of crosslink increased significantly as revealed by the ATR-FTIR. Our present study sheds light on the chemical composition and putative tanning mechanism of the periostracum layer in bivalve molluscs. SIGNIFICANCE: The periostracum layer, plays an essential role in the initiation of shell biomineralization, the protection of minerals from dissolution for molluscs and especially ocean acidification conditions in the changing global climate. However, the molecular mechanism underlying the periostracum formation is not fully understood. In this study, we revealed that the oxidation and cross-link of tyrosine-rich proteins by tyrosinase are involved in periostracum formation in the green mussel Perna viridis. This study provides some insights into the first step of mussel shell formation and the robust adaptation of P. viridis to diverse habitats. These findings also help to reveal the potential acclimation of bivalves to the projected acidifying seawater.

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.

Pathogenicity and virulence of bacterial strains associated with summer mortality in marine mussels (Perna canaliculus).

The occurrence of pathogenic bacteria has emerged as a plausible key component of summer mortalities in mussels. In the current research, four bacterial isolates retrieved from moribund Greenshell࣪ mussels, Perna canaliculus, from a previous summer mortality event, were tentatively identified as Vibrio and Photobacterium species using morpho-biochemical characterization and MALDI-TOF MS and confirmed as V. celticus, P. swingsii, P. rosenbergii, and P. proteolyticum using whole genome sequencing. These isolates were utilized in a laboratory challenge where mussels were injected with cell concentrations ranging from 105 to 109 CFU/mussel. Of the investigated isolates, P. swingsii induced the highest mortality. Additionally, results from quantitative polymerase chain reaction analysis, focusing on known virulence genes were detected in all isolates grown under laboratory conditions. Photobacterium rosenbergii and P. swingsii showed the highest expression levels of these virulence determinants. These results indicate that Photobacterium spp. could be a significant pathogen of P. canaliculus, with possible importance during summer mortality events. By implementing screening methods to detect and monitor Photobacterium concentrations in farmed mussel populations, a better understanding of the host-pathogen relationship can be obtained, aiding the development of a resilient industry in a changing environment.

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.

Solution structure of recombinant Pvfp-5ß reveals insights into mussel adhesion.

Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5ß, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5ß has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5ß is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials.

Effects of sizes and concentrations of different types of microplastics on bioaccumulation and lethality rate in the green mussel, Perna viridis.

Microplastics (MPs) can be defined as small pieces of plastics that are less than five millimetres in diameter. MPs can be consumed and may be accumulated by filter-feeding organisms such as mussels. Therefore, this study aimed to determine the acute effects of different types, sizes and concentrations of artificially synthesized MPs on the mortality rate and MP accumulation of the green mussel Perna viridis. The samples were exposed to 66, 333, 666, and 1333 items/L of small MPs (<30 µm), medium MPs (30-300 µm), and large MPs (300-1000 µm) polystyrene (PS), polypropylene (PP), and polybutylene succinate (PBS) for 96 h. MPs accumulation in the soft tissue of mussels and mortality effects from MPs ingestion were assessed. There was no mortality observed in the control group. Small PP particles can lead to more mortality than PS and PBS particles of the same size. However, medium- and large PS caused a higher mortality percentage than the same size particles of PP and PBS. Large PS, PP, and PBS showed higher mortality potential than other sizes. MPs largely accumulated in the soft tissues rather than in gill tissues following the 96-hour exposure period. Increased accumulation of the three types of MPs was accompanied by an increase in the percentage of mussel mortality. The study highlights how particle size and type are key factors in plastic particulate toxicity.

Inhibition of Diarrheal Shellfish Toxins Accumulation in the Mussel Perna viridis by Curcumin and Underlying Mechanisms.

Diarrheal shellfish toxins (DSTs) are among the most widely distributed phytotoxins, and are associated with diarrheal shellfish poisoning (DSP) events in human beings all over the world. Therefore, it is urgent and necessary to identify an effective method for toxin removal in bivalves. In this paper, we found that curcumin (CUR), a phytopolylphenol pigment, can inhibit the accumulation of DSTs (okadaic acid-eq) in the digestive gland of Perna viridis after Prorocentrum lima exposure. qPCR results demonstrated that CUR inhibited the induction of DSTs on the aryl hydrocarbon receptor (AhR), hormone receptor 96 (HR96) and CYP3A4 mRNA, indicating that the CUR-induced reduction in DSTs may be correlated with the inhibition of transcriptional induction of AhR, HR96 and CYP3A4. The histological examination showed that P. lima cells caused severe damage to the digestive gland of P. viridis, and the addition of curcumin effectively alleviated the damage induced by P. lima. In conclusion, our findings provide a potential method for the effective removal of toxins from DST-contaminated shellfish.

Beyond relaxed: magnesium chloride anaesthesia alters the circulatory metabolome of a marine mollusc (Perna canaliculus).

BACKGROUND: The New Zealand Green-lipped mussel industry is well-established providing vastly to aquaculture exports. To assess mussel health and reproduction status, visual examination of organs and/or collection of haemolymph is commonly applied. Anesthetics, such as magnesium chloride (MgCl2) can be utilized to prevent muscle contraction and keep shells open during sampling. The specific effects of muscle relaxing agents on baseline metabolism in invertebrates is unknown, but it is evident that molecular, cellular and physiological parameters are altered with these chemical applications. To this end, metabolomics approaches can help elucidate the effects of relaxing agents for better assessment of their use as a research tool. METHODS: Adult Green-lipped mussels were anaesthetized for 3 h in a MgCl2 bath, whereafter haemolymph samples were collected and analyzed via gas chromatography-mass spectrometry applying methyl chloroformate alkylation derivatization. RESULTS: Anesthetized mussels were characterized as non-responsive to manual manipulation, with open valves, and limited siphoning function. Metabolite profiling revealed significant increases in the abundances of most metabolites with an array of metabolic activities affected, resulting in an energy imbalance driven by anaerobic metabolism with altered amino acids acting as neurotransmitters and osmolytes. CONCLUSION: This research is the first to use a metabolomics approach to identify the metabolic consequences of this commonly used bivalve relaxing technique. Ultimately the use of MgCl2 anesthetization as a sampling strategy should be carefully evaluated and managed when performing metabolomics-related research.

Ingestion of microplastics and its potential for causing structural alterations and oxidative stress in Indian green mussel Perna viridis- A multiple biomarker approach.

The present study has investigated the distribution of microplastics in sediment and its impact on histological, ultrastructural, and oxidative stress mechanisms in Perna viridis (P. viridis) from Kasimedu, Chennai, India. The results confirmed that fibers were the predominant type of microplastics observed, followed by spheres, flakes, sheets, and fragments. The observed microplastics were confirmed as polyester, polypropylene, polyethylene, cellophane, and rayon using µ-FT-IR. Microplastic particles entangled in gills caused abrasion of ciliated structure and hemocyte infiltration in the hemolymph vessels. The digestive gland showed a shrunken nucleus, dark inclusions, and damage in the nucleoid core structure. Enlarged vacuoles and the presence of clusters of vesicles presumably represented the transformed golgi cisternae. Further, the results confirmed that oxidative stress markers were significantly high in gills and digestive diverticula of P. viridis. Overall, the results indicated that microplastics induced different toxic physiological and structural alterations in gills and digestive diverticula of P. viridis. These findings highlighted the necessity to focus on exposure studies to understand the absolute magnitude of the problem due to microplastic pollution in the urban estuarine ecosystems of Chennai, Tamil Nadu, India.

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.

Molecular and biochemical effects of the antifouling DCOIT in the mussel Perna perna.

Biological fouling is an unwanted phenomenon that results in economic losses to the shipping industry. To prevent fouling, antifouling paints are used. DCOIT (4,5- dichloro-2-n-octyl-4-isothiazolin-3-one) is a biocide present in many antifouling paint formulations, and is toxic to a wide range of organisms. The aim of the present study was to evaluate the effects of DCOIT on oxidative stress indicators of the brown mussel, Perna perna. Molecular (SOD-like, GSTO-like and MGST-like mRNA levels) and biochemical (activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), and levels of glutathione (GSH), reactive oxygen species (ROS) and protein carbonyls (PCO)) components were evaluated. Further, levels of biomarkers were assessed in the gills and digestive glands of mussels. Bivalves were exposed to DCOIT (control, 0.1 µg/L and 10 µg/L) for up to 96 h. DCOIT exposure decreased GSH content in gills. Moreover, exposure to DCOIT also decreased CAT activity in the gills and digestive glands of mussels. GST activity increased in digestive gland after exposure for 24 h to both concentrations of DCOIT tested. SOD activity, ROS levels and PCO content were not affected by exposure to the contaminant. Regarding the molecular biomarkers evaluated, DCOIT exposure altered mRNA levels of SOD-like in both tissues after 24 and 96 h of exposure, and decreased MGST-like mRNA levels in the digestive gland after 96 h of exposure to the chemical. These findings suggested that exposure to DCOIT may alter the biochemical and molecular functioning of P. perna, which may harm the species.

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.

Biomarker responses in New Zealand green-lipped mussels Perna canaliculus exposed to microplastics and triclosan.

Microplastics (MPs) are of increasing concern for filter feeding marine and freshwater species. Additionally MPs can sorb hydrophobic contaminants from the water, potentially providing an additional pathway of exposure of aquatic species to contaminants. An acute 48 h laboratory study was conducted to investigate the effects of microplastics and triclosan, both individually and combined, on New Zealand's green-lipped mussel, Perna canaliculus. Biomarkers included clearance rate, oxygen uptake, byssus production; and superoxide dismutase (SOD) activity, glutathione-S-transferase (GST) activity and lipid peroxidation in the gill tissue. Microplastics and triclosan, both individually and combined significantly decreased oxygen uptake and byssus production. These physiological responses were not observed when the microplastics were spiked with triclosan. Triclosan, both alone and spiked to microplastics, increased mussel oxidative stress markers including SOD activity and lipid peroxidation. An enhanced effect was observed on the SOD enzyme activity when mussels were exposed to microplastics spiked with triclosan. No effects on the biochemical biomarkers were observed for mussels exposed to microplastic only. Microplastics enhanced the uptake of triclosan in mussel tissue compared with triclosan only treatments indicating that microplastics potentially provide an additional pathway of exposure to hydrophobic contaminants.

First evidence of transcriptional modulation by chlorothalonil in mussels Perna perna.

Gills are considered a key player in organism defenses against environmental pollutants. Since it is the major site of uptake of waterborne chemicals, the modulation of important cellular defenses is expected in this tissue. Chlorothalonil, a fungicide presented in herbicides and antifouling paints, might be responsible for toxicity in marine biota. In this context, mussels were exposed to 0.1 µgL-1 and 10 µgL-1 of chlorothalonil for 24 h and 96 h. Genes from biotransformation and antioxidant defense pathways were investigated. Overall, we report, for the first time, an increase in the transcripts of the AhR-like, SULT1A1-like, CYP1A2-like, GSTO-like, MGST-like and SOD-like genes in the gills of the brown mussel Perna perna. This up-regulation was observed mostly after 96 h of exposure to chlorothalonil. Those results reinforce the important role of gills in xenobiotic metabolism and suggest the involvement of the mentioned genes in the detoxification of the compound. Throughout biotransformation and antioxidant defenses pathway, mussels exposed to chlorothalonil are activating mechanisms of defense against this contaminant.

De novo transcriptome analysis of the mussel Perna viridis after exposure to the toxic dinoflagellate Prorocentrum lima.

Diarrheic shellfish poisoning (DSP) toxins are produced by harmful microalgae and accumulate in bivalve mollusks, causing various toxicity. These toxic effects appear to abate with increasing DSP concentration and longer exposure time, however, the underlying mechanisms remain unclear. To explore the underlying molecular mechanisms, de novo transcriptome analysis of the digestive gland of Perna viridis was performed after Prorocentrum lima exposure. RNA-seq analysis showed that 1886 and 237 genes were up- and down-regulated, respectively after 6 h exposure to P. lima, while 265 genes were up-regulated and 217 genes were down-regulated after 96 h compared to the control. These differentially expressed genes mainly involved in Nrf2 signing pathways, immune stress, apoptosis and cytoskeleton, etc. Combined with qPCR results, we speculated that the mussel P. viridis might mainly rely on glutathione S-transferase (GST) and ABC transporters to counteract DSP toxins during short-term exposure. However, longer exposure of P. lima could activate the Nrf2 signaling pathway and inhibitors of apoptosis protein (IAP), which in turn reduced the damage of DSP toxins to the mussel. DSP toxins could induce cytoskeleton destabilization and had some negative impact on the immune system of bivalves. Collectively, our findings uncovered the crucial molecular mechanisms and the regulatory metabolic nodes that underpin the defense mechanism of bivalves against DSP toxins and also advanced our current understanding of bivalve defense mechanisms.

Occurrence and congener profiles of polybrominated diphenyl ethers in green mussels (Perna viridis) collected from northern South China Sea and the associated potential health risk.

Polybrominated diphenyl ether (PBDE) contamination has become a major concern over the effects on human health. In the present study, we collected widely consumed green mussels (Perna viridis) samples from the northern South China Sea (NSCS) to investigate the occurrence, spatial distribution, congener profiles as well as potential risk of 18 PBDEs. All the target PBDEs were detected in green mussel samples, indicating their ubiquitous distribution. The concentrations of the total 18 PBDES (ΣPBDEs) in all samples varied from 6.96 to 55.6 ng/g lipid weight (lw), with BDE-47 and BDE-209 being the predominant PBDE congeners. Overall, the ΣPBDEs pollution in green mussels from NSCS was at a moderate to high level in comparison with the PBDEs pollution worldwide. The dietary exposure of the local population in South China to PBDEs via consuming green mussels was estimated to be 0.30-0.80 ng/kg body weight (bw)/day. Evaluation of the exposure risk for BDE-47, 99, 153 and 209 indicated that health risks due to green mussel consumption are substantially lower than the U.S. EPA minimum concern level.

Antioxidant and antiinflammatory secondary metabolites from the Asian green mussel Perna viridis.

The Asian green mussel, Perna viridis is a nutritious health food in the estuarine and coastal sea beds of the Arabian Sea along the west coast of India. In the present study, bioactivity-guided purification of the chloroform fraction of the methanolic extract of P. viridis was carried out. The isolated secondary metabolites were characterized by spectroscopic experiments, and their antioxidative/antiinflammatory properties were evaluated. The titled compounds were characterized as 3-hydroxy-13-vinyl-dodecahydro-11-phenanthrenone (1), 4,4,9-trimethyl-13-vinyl-dodecahydro-2-phenanthrenone (2), 11,20-dihydroxy-6,6-dimethyl-decahydro-5H-benzo[h]naphtho[1,2-c]chromene-16-carbaldehyde (3), 16-acetyl-20-hydroxy-6,6-dimethyl-dodecahydro-5H-benzo[h]naphtho[1,2-c]chromen-12-one (4), cholest-5-en-3ß-3-yl-(30-hydroxy-3-methyl-36-methyleneundeca-30E,34E-dienoate) (5), and cholest-5-en-3ß-3-yl-((E)-33-oxooct-31-enoate) (6). No significant differences in the antioxidant activities of the compounds with chromene-16-carbaldehyde (3) and chromen-12-one (4) functionalities (IC50 0.52-0.68 mg/ml) vis-à-vis the positive control, α-tocopherol (IC50 0.65-0.76 mg/ml) were registered. The studied compounds, 1-4, displayed potential antiinflammatory activities against pro-inflammatory 5-lipoxygenase (5-LOX) (IC50  < 1 mg/ml). The balanced hydrophilic-lipophilic properties and lower steric values of the studied compounds, 1-4, were correlated with their bioactive potentials. PRACTICAL APPLICATIONS: The edible bivalve green mussels, P. viridis, are broadly available in the estuarine and coastal regions of the Indian Peninsula. The sequential chromatographic purification of the chloroform fraction of the methanolic extract of P. viridis led to the identification of six pure secondary metabolites. The metabolites with substituted chromene-16-carbaldehyde and chromen-12-one functionalities displayed potential antioxidative and antiinflammatory activities compared to other studied compounds. These bioactive metabolites could be used in functional food formulations and as antioxidant leads in medicinal food applications.

Targeted metabolomics to investigate antimicrobial activity of itaconic acid in marine molluscs.

INTRODUCTION: Itaconic acid (ITA) has recently been identified as an antimicrobial metabolite in mammalian immune cells. The presence of ITA was also reported in different tissues of marine molluscs, indicating its role as an endogenous metabolite of molluscs. In addition, the accumulation of ITA has been observed in different tissues of mussels following pathogen challenges. However, the concentration of ITA in mussel tissues and the possible role of this metabolite in the molluscan innate immune system remain unknown. OBJECTIVES: This study aims to quantitatively measure ITA levels in different tissues of marine mussels following an experimental challenge with Vibrio sp. DO1 isolate, and to identify the antimicrobial role of ITA in the innate immune system through the measurement of metabolic and immune alterations in tissues following the challenge. METHODS: In this study, adult Perna canaliculus mussels were experimentally challenged with a pathogenic Vibrio sp. DO1 isolate. The metabolite profiles of five different tissues, including mantle, gill, muscle, hepatopancreas and haemolymph were obtained, and levels of ITA in each tissue were characterized using a gas chromatography-mass spectrometry (GC-MS) metabolomics approach. Flow cytometry was also employed to measure cell health parameters, including oxidative stress via reactive oxygen species (ROS) production, apoptosis via changes in mitochondrial membrane potential (MMP) and haemocyte viability. RESULTS: The ITA levels in mantle, gill, muscle and hepatopancreas tissues at 18-h post infection (hpi) with Vibrio sp. were 40.31, 41.71, 11.61 and 41.66 ng mg-1, respectively. In haemolymph, the level of ITA was significantly increased from 95.25 ng ml-1 at 6 hpi to 174.36 ng ml-1 at 18 hpi and 572.12 ng ml-1 at 60 hpi. In line with the accumulation of ITA, we observed increased levels of metabolites within the tricarboxylic acid (TCA) cycle, anti-inflammatory metabolites and alterations of other metabolites associated with immune responses of the host. The flow cytometry analyses revealed increases in ROS production, apoptotic cells and decreases in cell viability. CONCLUSIONS: We reported on the production of ITA in different tissues of P. canaliculus mussels challenged with a marine pathogen which confirmed ITA as an antimicrobial metabolite. The findings revealed insights into the biosynthesis of ITA and suggests its role in antimicrobial and anti-inflammatory activities in the innate immune system. This study also provided insights into the innate immune system of bivalves and highlighted the potential use of ITA as a biomarker for shellfish health assessment in aquaculture.

Comparative influence of genetics, ontogeny and the environment on elemental fingerprints in the shell of Perna canaliculus.

The trace elemental composition of biogenic calcium carbonate (CaCO3) structures is thought to reflect environmental conditions at their time of formation. As CaCO3 structures such as shell are deposited incrementally, sequential analysis of these structures allows reconstructions of animal movements. However, variation driven by genetics or ontogeny may interact with the environment to influence CaCO3 composition. This study examined how genetics, ontogeny, and the environment influence shell composition of the bivalve Perna canaliculus. We cultured genetically distinct families at two sites in situ and in the laboratory. Analyses were performed on shell formed immediately prior to harvest on all animals as well as on shell formed early in life only on animals grown in the laboratory. Discriminant analysis using 8 elements (Co, Ti, Li, Sr, Mn, Ba, Mg, Pb, Ci, Ni) classified 80% of individuals grown in situ to their family and 92% to growth site. Generalised linear models showed genetics influenced all elements, and ontogeny affected seven of eight elements. This demonstrates that although genetics and ontogeny influence shell composition, environmental factors dominate. The location at which shell material formed can be identified if environmental differences exist. Where no environmental differences exist, genetically isolated populations can still be identified.