A novel ferritin subunit gene from Asian green mussel, Perna viridis (Linnaeus, 1758).

Iron sequestration through ferritin forms a major part of innate immune response in molluscs and detailed understanding of ferritin gene and its functions can be directly applied in infection and disease management studies. Accordingly, identification and detailed molecular characterization of a ferritin subunit gene from a commercially significant marine mussel Perna viridis was targeted. Molecular screening using degenerate primers in total mantle RNA resulted in the amplification of a novel ferritin gene fragment having <87% identity to the reported ferritin gene sequences. Rapid amplification of cDNA ends-PCR was followed to generate complete cDNA sequence of P.viridis ferritin (PvFer). The complete cDNA was found to be 798 bp, containing an open reading frame of 522 bp, 5' untranslated region (UTR) of 112 bp and 3' UTR of 165 bp. The 5' UTR and 3' UTR were shown to contain an iron response element (IRE) and a polyadenylation signal (767AATAAA772) with poly (A) tail, respectively. Prediction of stem loop structure revealed that, PvFer-IRE can be folded into a typical secondary stem loop structure, having 5-CAGUGA-3' loop, proximal stem of five paired bases followed by a bulged cysteine, and six nucleotide bottom stem, indicating that expression of PvFer is regulated by iron at the translational level. ORF was found to encode 175 amino acid protein with calculated molecular mass of 19.97 kDa and isoelectric point of 4.97. Examination for signal peptide and phylogenetic analysis confirmed that PvFer belonged to cytosolic ferritins of molluscs. Conserved domain analysis showed that PvFer contained both ferroxidase diiron center and ferrihydrite nucleation center, analogous to ferritin M subunit of bony fishes and amphibians. However, amino acid sequence and glycosylation site showed more homology to vertebrate ferritin H subunits. Predicted 3D models of PvFer resembled the typical spatial features of ferritin proteins. The study forms the first comprehensive identification of a ferritin subunit gene in a true/common mussel (Order: Mytilida). Further, the detailed molecular phylogeny conducted through the present study revealed certain thought provoking insights on ferritin genes of the phylum Mollusca.

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

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

Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution.

The mussel Perna perna is an intertidal bivalve that is widely distributed, cultivated and consumed in South Africa, Brazil and Venezuela. Among marine resources, bivalve mollusks are one of the most impacted by anthropogenic pollution, as they can accumulate pathogenic bacteria and water pollutants. Hemocytes are molluscan defense cells, and their abundance and functions can be affected in response to contaminants, such as bacterial load. However, no previous study has investigated the immune response of P. perna hemocytes. The aim of this study was to evaluate several immune parameters in P. perna as indicators of fecal pollution in mussel hemolymph and in seawater. We collected mussels and adjacent seawater from beaches with different levels of fecal contamination in Rio de Janeiro state (Brazil): Vermelha Beach (VB); Icaraí Beach (IB); Urca Beach (UB); and Jurujuba Beach (JB). Hemocyte parameters (density, morphology, phagocytic activity and production of Reactive Oxygen Species - ROS) were evaluated using flow cytometry. We quantified Fecal Indicator Bacteria (FIB) in seawater by the multiple tubes technique for each beach and for hemolymph by the spread-plate technique. In agreement with historical evaluation of fecal contamination levels, UB presented the highest FIB abundance in seawater (thermotolerant coliforms, TEC = 1600 NMP 100 mL-1), whereas VB exhibited the lowest (TEC = 17 NMP 100 mL-1). UB mussels had six and eight times higher hemocyte density and phagocytic activity, respectively, than mussels from VB. Mussels from VB and IB presented a significantly lower number of total coliforms in hemolymph and a significantly higher relative internal complexity of hemocytes than those from UB and JB (p ≤ 0.01, PERMANOVA). ROS production by hemocytes was significantly lower in mussels from VB compared to those from JB (p = 0.04, ANOVA). Our results indicate a significant relationship between the level of fecal contamination in aquatic environments and the immune response of mussel hemocytes. Immune-related parameters may therefore be useful as indicators of bivalve health and environmental quality. Our flow cytometric analysis of P. perna hemocytes represents a new approach for studying Perna perna biology and might represent a novel tool for measuring organic pollution and water quality.

Identification and characterization of a novel defensin from Asian green mussel Perna viridis.

Defensin is one of the most diversified groups of antimicrobial peptides in invertebrate. In the present study, a novel defensin member referred as Pv-Def was identified and characterized from Asian green mussel Perna viridis. Using in silico survey of several EST databases released from diverse tissues of P. viridis, a single peptide referred as Pv-Def was predicted as defensin homologue with Mytilus counterparts. Further analysis on gene structure revealed that Pv-Def was 1001 nt in length and consisted of 3 exons and 2 introns. The precursor of Pv-Def was composed of a signal peptide of 19 amino acids and a mature peptide of 45 amino acids. The mature Pv-Def peptide contains 6 cysteines which formed 3 disulfide bonds at 27C1- 54C4, 40C2- 60C5 and 44C3- 62C6. Like most of the defensin family members, mature Pv-Def peptide included an alpha helix and 2 beta strands. Pv-Def showed significantly tissue-specific expression pattern, while highest transcription level was observed in hepatopancreas, which was about 900 folds to that in hemocytes. Moreover, the expression of Pv-Def mRNA in hemocytes was significantly and accurately up-regulated at different time intervals by Vibrio parahaemolyticus challenge. Interestingly, phylogenetic analysis suggested that the Pv-Def possesses closest relationships with arthropods counterparts rather than other mollusk defensins. To our knowledge, this is the first time that a defensin member was reported in Asian green mussel P. viridis.

Metabolic and immunological responses of male and female new Zealand Greenshell™ mussels (Perna canaliculus) infected with Vibrio sp.

Massive mortalities due to pathogens are routinely reported in bivalve cultivation that have significant economic consequences for the global aquaculture industry. However, host-pathogen interactions and infection mechanisms that mediate these interactions are poorly understood. In addition, gender-specific immunological responses have been reported for some species, but the reasons for such differences have not been elucidated. In this study, we used a GC/MS-based metabolomics platform and flow cytometry approach to characterize metabolic and immunological responses in haemolymph of male and female mussels (Perna canaliculus) experimentally infected with Vibrio sp. Sex-based differences in immunological responses were identified, with male mussels displaying higher mortality, oxidative stress and apoptosis after pathogen exposure. However, central metabolic processes appeared to be similar between sexes at 24 h post injection with Vibrio sp. DO1. Significant alterations in relative levels of 37 metabolites were detected between infected and uninfected mussels. These metabolites are involved in major perturbations on the host's innate immune system. In addition, there were alterations of seven metabolites in profiles of mussels sampled on the second day and mussels that survived six days after exposure. These metabolites include itaconic acid, isoleucine, phenylalanine, creatinine, malonic acid, glutaric acid and hydroxyproline. Among these, itaconic acid has the potential to be an important biomarker for Vibrio sp. DO1 infection. These findings provide new insights on the mechanistic relationship between a bivalve host and a pathogenic bacterium and highlight the need to consider host sex as a biological variable in future immunological studies.

Proteomic profile in the mussel Perna viridis after short-term exposure to the brown tide alga Aureococcus anophagefferens.

Blooms of Aureococcus anophagefferens, referred to as brown tides are responsible for massive mortalities and recruitment failure of some bivalves. However, the molecular mechanisms underlying the toxicity remain elusive despite its biological significance, and the information currently available on the molecular effects is still insufficient. In this study, to evaluate the toxicity and associated mechanism of A. anophagefferens on bivalves, we analyzed the protein expression profiles in digestive glands of the A. anophagefferens-exposed Perna viridis by using iTRAQ. A total of 3138 proteins were identified in the digestive glands of A. anophagefferens-exposed P. viridis based on iTRAQ. Amongst, a repertoire of 236 proteins involved in cell, cell part, catalytic activity, metabolic process, biological regulation, immune system process, and response to stimulus were found to be differentially expressed. Functional analysis of the differentially expressed proteins demonstrated that innate immune system of P. viridis was activated, and some proteins associated with stress response and lipid metabolism were induced after exposure to A. anophagefferens. Additionally, MDA content, SOD activity and GSH-Px activity was increased significantly in the digestive gland of A. anophagefferens-exposed P. viridis. Taken together, our results indicated that the A. anophagefferens could induce oxidative stress, activate complement system and alter fat acid metabolism of P. viridis.

P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins.

Bivalves naturally exposed to toxic algae have mechanisms to prevent from harmful effects of diarrhetic shellfish poisoning (DSP) toxins. However, quite few studies have examined the mechanisms associated, and the information currently available is still insufficient. Multixenobiotic resistance (MXR) is ubiquitous in aquatic invertebrates and plays an important role in defense against xenobiotics. Here, to explore the roles of P-glycoprotein (P-gp) in the DSP toxins resistance in shellfish, complete cDNA of P-gp gene in the mussel Perna viridis was cloned and analyzed. The accumulation of okadaic acid (OA), a main component of DSP toxins, MXR activity and expression of P-gp in gills of P. viridis were detected after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins in the presence or absence of P-gp inhibitors PGP-4008, verapamil (VER) and cyclosporin A (CsA). The mussel P. viridis P-gp closely matches MDR/P-gp/ABCB protein from various organisms, having a typical sequence organization as full transporters from the ABCB family. After exposure to P. lima, OA accumulation, MXR activity and P-gp expression significantly increased in gills of P. viridis. The addition of P-gp-specific inhibitors PGP-4008 and VER decreased MXR activity induced by P. lima, but had no effect on the OA accumulation in gills of P. viridis. However, CsA, a broad-spectrum inhibitor of ABC transporter not only decreased MXR activity, but also increased OA accumulation in gills of P. viridis. Together with the ubiquitous presence of other ABC transporters such as MRP/ABCC in bivalves and potential compensatory mechanism in P-gp and MRP-mediated resistance, we speculated that besides P-gp, other ABC transporters, especially MRP might be involved in the resistance mechanisms to DSP toxins.

Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis.

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis.

Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature.

The green mussel, Perna viridis, is a bivalve mollusk native to Asia and was recently introduced to Florida, USA. Since its first observation in 1999 in Tampa Bay, Florida, green mussel population has expanded considerably, to reach the Atlantic coast of Florida, Georgia and South Carolina. Most of currently available studies about the ecology and biology of green mussels were performed in the Indian and Pacific oceans. Very recently, it has been suggested that due to a weak low temperature resistance, green mussels might have already reached the Northern edge of their distribution in the USA. However, there is currently an obvious lack of data about the adaptation capacities of Perna viridis to environmental conditions in Florida, especially at the physiological and cellular levels. In the present work, we determined and characterized the populations of circulating hemocytes, and the cellular components of hemolymph involved in various physiological functions, including immunity. Two main populations were characterized, hyalinocytes and granulocytes. Granulocytes accounted for 60% of circulating cells, and displayed higher phagocytic capacities, lysosomal content and basal oxidative metabolism than hyalinocytes. Hemocyte parameters were not influenced by the size of green mussels. In addition, hemocytes were subjected to acute temperature challenges (10, 20 and 30 °C) and their immune-related functions and metabolism analyzed. Our results showed that 10 °C represent a stressful condition for the Floridian green mussels, as depicted by a low phagocytosis capacity and an increase of oxidative metabolism.

Effects of DCOIT (4,5-dichloro-2-octyl-4-isothiazolin-3-one) to the haemocytes of mussels Perna perna.

Bivalve molluscs rely only on an innate immune system to execute cellular and humoral processes. Haemocytes, the haemolymph circulating cells, play a major role in this type of immunity, principally regarding cellular defences. Considering that environmental pollutants can affect the immune system of invertebrates, this work evaluated the effects of the antifouling biocide 4,5-dicloro-2-n-octil-4-isotiazolin-3-ona (DCOIT) on the haemocytes of mussels Perna perna. Individuals were exposed to 0 (control), 0.1 µg L-1 and 10 µg L-1 of DCOIT for up to 96 h. The analysed parameters included: total (THC) and differential (DHC) haemocyte count, cellular viability, adhesion capacity, phagocytic activity, levels of reactive oxygen species and DNA damage. Moreover, the stress on stress (SOS) response of mussels was analysed as a general stress index. The results show that DCOIT increased the haemocyte adhesion capacity and caused a decrease in THC and in the haemocyte viability after 24 h of exposure. After 96 h of exposure, DCOIT only affected the haemocyte adhesion capacity, which was decreased by biocide exposure. Moreover, exposure to DCOIT for 96 h did not affect the capacity for air survival of mussels. These results indicate that DCOIT interferes in important parameters associated with the innate immunity of P. perna, mainly after 24 h of exposure. It is suggested that the animals were able to develop some compensatory response strategy, making them more resistant to the biocide.

A lipid extract of Perna canaliculus affects the expression of pro-inflammatory cytokines in a rat adjuvant-induced arthritis model.

As published initially in this same journal in 2000, the lipid extract of Perna canaliculus (New Zealand green-lipped mussel; Lyprinol) is known for its anti-inflammatory effects in animal models and in human controlled studies (arthritis; asthma). As a follow-up of its effects on pain in a rat model of adjuvant-induced arthritis (ALA), we studied its effects on the production of cytokines known to be associated with inflammation (IL-6, IL-1alpha TNF-alpha, IFN-gamma). Feeding with Lyprinol was associated with significantly decreased expression levels of TNF-alpha and IFN-gamma when compared to Naproxen (positive control) and, even more when compared with sham and extra-virgin olive oil (negative control). When compared to Naproxen, sham and extra-virgin olive oil, the levels of IL-6 and IL-1alpha were also marginally decreased in rats fed with Lyprinol. This study demonstrates that AIA rats fed with Lyprinol had decreased production ofcytokines associated with inflammation.

Metabolomics Study of Immune Responses of New Zealand Greenshell™ Mussels (Perna canaliculus) Infected with Pathogenic Vibrio sp.

Vibrio coralliilyticus is a bacterial pathogen which can affect a range of marine organisms, such as corals, fish and shellfish, with sometimes devastating consequences. However, little is known about the mechanisms involved in the host-pathogen interaction, especially within molluscan models. We applied gas chromatography-mass spectrometry (GC-MS)-based metabolomics to characterize the physiological responses in haemolymph of New Zealand Greenshell™ mussels (Perna canaliculus) injected with Vibrio sp. DO1 (V. coralliilyticus/neptunius-like isolate). Univariate data analyses of metabolite profiles in Vibrio-exposed mussels revealed significant changes in 22 metabolites at 6 h post-infection, compared to non-exposed mussels. Among them, 10 metabolites were up-regulated, while 12 metabolites were down-regulated in infected mussels. Multivariate analyses showed a clear distinction between infected and non-infected mussels. In addition, secondary pathway analyses indicated perturbations of the host innate immune system following infection, including oxidative stress, inflammation and disruption of the TCA cycle, change in amino acid metabolism and protein synthesis. These findings provide new insights into the pathogenic mechanisms of Vibrio infection of mussels and demonstrate our ability to detect detailed and rapid host responses from haemolymph samples using a metabolomics approach.

Copper-induced immunomodulation in mussel (Perna canaliculus) haemocytes.

Copper is a common contaminant in aquatic environments, which may cause physiological dysfunction in marine organisms. However, the toxicity mechanisms of copper in marine bivalves is not fully understood. In this study, we applied an integrated approach that combines flow cytometry and Gas Chromatography-Mass Spectrometry (GC-MS)-based metabolomics to characterize cellular and molecular mechanisms of copper immunotoxicity in New Zealand Greenshell™ mussel (Perna canaliculus) haemolymph. Flow cytometric results showed significant increases in haemocyte mortality, production of reactive oxygen species and apoptosis (via alteration of caspase 3/7 and mitochondrial membrane potential) of haemocytes exposed to increasing total concentrations of Cu2+ (62.5, 125.0 and 187.5 µM) compared to a low Cu2+ concentration (25.0 µM) and control (0.0 µM). In addition to flow cytometric data, our metabolomics results showed alterations of 25 metabolites within the metabolite profile of Cu2+-exposed haemolymph (125 µM) compared to those of control samples. Changes in levels of these metabolites may be considered important signatures of oxidative stress (e.g., glutathione) and apoptosis processes (e.g., alanine, glutamic acid). This study provides insights into the cellular and molecular mechanisms of oxidative stress and apoptosis in marine bivalves and highlights the applicability and reliability of metabolomic techniques for immunotoxicological studies in marine organisms.

Immunotoxicity in green mussels under perfluoroalkyl substance (PFAS) exposure: Reversible response and response model development.

The immunotoxicity of 4 commonly detected perfluoroalkyl substances (PFASs), namely, perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) was investigated by measuring biomarkers of the immune profile of green mussels, Perna viridis. The biomarkers included neutral red retention, phagocytosis, and spontaneous cytotoxicity, all of which were tested on mussel hemocytes. Hemocytes are an important component of the invertebrate immune system. We found that exposure to PFASs could lead to reduced hemocyte cell viability and suppress immune function by up to 50% of normal performance within the experimental exposure range. The results indicate that PFASs have an immunotoxic potential and thus could pose severe health risks to aquatic organisms. The reported immunotoxicity is likely to result from the compounds' direct and indirect interactions with the hemocyte membrane, and therefore likely to affect the functionality of these cells. The immunotoxic response was found to be related to the organism's burden of PFASs, and was reversible when the compounds were removed from the test organisms. Based on this relationship, models using an organism's PFAS concentration and bioaccumulation factor (BAF) as the independent variables were established to quantify PFAS-induced immunotoxicity. The models help us to gain a better understanding of the toxic mechanism of PFASs, and provide a tool to evaluate adverse effects for the whole group of compounds with one mathematical equation. Environ Toxicol Chem 2018;37:1138-1145. © 2018 SETAC.

Use of a suite of biomarkers to assess the effects of carbamazepine, bisphenol A, atrazine, and their mixtures on green mussels, Perna viridis.

The present study investigated the toxicity of several emerging contaminants: the pharmaceutical drug carbamazepine (CBZ), the plasticizer bisphenol A (BPA), and the herbicide atrazine (ATZ) in a marine bivalve. Green mussels (Perna viridis) were exposed to different concentrations of CBZ, BPA, and ATZ, either individually or as mixtures over a 7-d period, and a suite of molecular and cellular biomarkers were analyzed: biomarkers of immunotoxicity (total hemocyte count, phagocytosis, extracellular lysozyme), genotoxicity (Comet assay), neurotoxicity (inhibition of acetylcholinesterase [AChE]), endocrine disruption (vitellin-like proteins), and detoxification enzymes (cytochrome P4501A [CYP1A], 7-ethoxyresorufin O-deethylase [EROD], and glutathione-S-transferase [GST]). Results of the single-chemical exposure tests highlighted the relatively low toxicity of CBZ because most biomarker responses observed were recorded at concentrations well above environmental levels. Bisphenol A exposure at environmentally realistic concentrations resulted in clear immunomodulatory, genotoxic, and endocrine-disruptive effects. Similarly, 3 of the 10 biomarkers tested on green mussels (genotoxicity, inhibition of AchE, and EROD) responded after exposure to ATZ at environmentally relevant doses or below, and confirmed the potency of this herbicide to marine bivalves. Exposure tests using mixtures of CBZ, BPA, and ATZ also revealed that these 3 substances were generally acting in an additive manner on the selected biomarkers, at environmental doses, with some exceptions (antagonism and/or synergy) at low and high concentrations. The present study also confirms that most of the biomarkers used are suitable for biomonitoring studies with green mussels. Environ Toxicol Chem 2017;36:429-441. © 2016 SETAC.

Hypoxia effects on oxidative stress and immunocompetence biomarkers in the mussel Perna perna (Mytilidae, Bivalvia).

This study investigated the effects of hypoxia on oxidative stress response and immune function in mussels Perna perna exposed to air for 6, 12, 24 and 48 h. In air-exposed mussels, the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione reductase (GR) activities were lower in gill tissues (24-48 h) and digestive gland (12 h), while the glutathione peroxidase and GR activities were increased in the digestive gland (48 h). In both tissues, aerial exposure promoted a rapid (6 h) and persistent (up to 48 h) increase of glutathione levels. Decreased hemocyte count and viability, as well as increased phagocytic activity and cellular adhesion capacity were detected after prolonged aerial exposure (>12 h). In summary, induction of thiol pools, altered antioxidant enzyme activities, and activation of immune responses were detected in hypoxia exposed brown mussels, indicating hypoxia induced tissue-specific responses in both antioxidant and immune systems.

Antifouling biocides: Impairment of bivalve immune system by chlorothalonil.

Marine ecosystems are subjected to a variety of contaminants. Antifouling paints, for example, have been extensively used to protect ship surfaces from marine biofouling, but their toxicity has generated great concern. Thus, we evaluated the effect of the biocide chlorothalonil on the immune system of Perna perna mussels. The mussels were exposed to 0 (control), 0.1µg/L and 10µg/L of chlorothalonil for up to 96h. After 24h and 96h of exposure, the following immune-related parameters were analyzed in the hemolymph of mussels: total hemocyte count, cell adhesion, phagocytic activity, level of reactive oxygen species, cell viability and comet assay. After 24h and 96h of chlorothalonil exposure, cellular adhesion increased and the hemocyte viability reduced. Moreover, an increase in phagocytic activity was also observed after 96h of exposure to cholorothalonil. The exposure to 10µg/L of chlorothalonil for 96h reduced the air survival capacity of mussels. Total hemocyte count, ROS generation and DNA damage were not affected by the contaminant exposure. Our results indicate that chlorothalonil affected important immune responses of the bivalves, demonstrating that this biocide has effects on non-target species. This modulation of immune system reduced the health status of mussels, which could compromise their ability to survive in the environment.

Pollution monitoring in Southeast Asia using biomarkers in the mytilid mussel Perna viridis (Mytilidae: Bivalvia).

Mytilid mussels have been extensively used in marine pollution monitoring programmes in temperate regions of the world although widespread subtropical representatives such as Perna viridis have only comparatively recently been utilised to monitor the sublethal effects of pollution in Southeast Asia. P. viridis is considered a subtropical equivalent of the temperate Mytilus sp. and has considerable potential for pollution monitoring throughout its geographical range. This paper reviews the current status of biomarkers in P. viridis and provides some recommendations on biological-effects monitoring to facilitate the assessment of coastal pollution in Southeast Asia.

Immune toxicity of TiO2 under hypoxia in the green-lipped mussel Perna viridis based on flow cytometric analysis of hemocyte parameters.

The combined effects of DO and TiO2 (mixed rutile/anatase phase, 7/3) on immune responses in Perna viridis were examined. Mussels were exposed to six combinations of oxygen levels (hypoxia: 1.5 mg O2l(-1), normoxia: 6.0 mg O2 l(-1)) and TiO2 concentrations (0, 2.5 mg l(-1) and 10 mg l(-1)) for 216 h. Mussels were sampled after 24h, 48h, 120 h and 216 h, and immune parameters of hemocytes, including mortality, phagocytosis, non-specific esterase, ROS production, lysosomal content and total hemocyte count were investigated using flow cytometric assay. Hemocyte mortality was higher under hypoxia than normoxia, and increased with TiO2 concentrations, but no interaction was found between DO and TiO2. Phagocytosis was reduced under hypoxia and decreased with TiO2 exposure, and the interactive effect between time and TiO2 was observed. The percentage of hemocytes showing non-specific esterase activity was lower under hypoxia, and decreased as TiO2 concentration increased with the significant interactive effect of DO and TiO2. ROS production and lysosomal content were lower under hypoxia and reduced as concentration of TiO2 increased, and interactive effect of DO and TiO2 on ROS was evident. THC was significantly affected by the interactive effect between TiO2 and DO, with higher values under normoxia in the presence of TiO2. The present study demonstrated that immune functions of P. viridis were influenced by both nano-TiO2 and hypoxia with some synergistic effects between the two stressors. This implies that DO has to be considered in the evaluation of the toxicity of nano-materials to bivalves.

Immune responses to combined effect of hypoxia and high temperature in the green-lipped mussel Perna viridis.

Flow cytometry was used to examine immune responses in haemocytes of the green-lipped mussel Perna viridis under six combinations of oxygen level (1.5 mg O2 l(-1), 6.0 mg O2 l(-1)) and temperature (20 °C, 25 °C and 30 °C) at 24 h, 48 h, 96 h and 168 h. The mussels were then transferred to normoxic condition (6.0 mg O2 l(-1)) at 20 °C for further 24 h to study their recovery from the combined hypoxic and temperature stress. Esterase (Est), reactive oxygen species (ROS), lysosome content (Lyso) and phagocytosis (Pha) were reduced at high temperatures, whereas hypoxia resulted in higher haemocyte mortality (HM) and reduced phagocytosis. For HM and Pha, changes were observed after being exposed to the stresses for 96 h, whereas only a 24 h period was required for ROS and Lyso, and a 48 h one for Est. Recovery from the stresses was observed for HM and Pha but not other immune responses.