Anti-cyclooxygenase effects of lipid extracts from the New Zealand green-lipped mussel, Perna canaliculus

Total lipid extracts of P. canaliculus (a bivalve marine mollusc native to New Zealand, commonly called the green-lipped mussel) and Mytilus edulis (commonly called the common blue mussel) moderately inhibited ovine COX-1 and COX-2 pure enzymes in vitro. The inhibition was increased after the mussel extracts were saponified by KOH hydrolysis. Protease- and protease-lipase-hydrolysed lipid extracts of P. canaliculus exhibited similarly strong COX inhibition as the KOH-hydrolysed extract. Lyprinol(R) (a commercial extract from P. canaliculus) also exhibited strong inhibition of both COX isoforms, an effect that was increased 10-fold upon subsequent hydrolysis. In contrast, fish oil was not as anti-COX active as Lyprinol. The Lyprinol free fatty acid fraction, and to a lesser extent the Lyprinol triglyceride fraction, were the only lipid classes of Lyprinol to exhibit strong inhibition of the COX isoforms. The purified PUFA extracts were all bioactive, potently inhibiting COX-1 and COX-2. Incubation of Lyprinol in the absence of exogenous arachidonic acid (AA) showed the appearance of alternate prostaglandin metabolites, confirming Lyprinol PUFA as a competitive substrate inhibitor of AA metabolism.     

Novel anti-inflammatory omega-3 PUFAs from the New Zealand green-lipped mussel, Perna canaliculus

The present study has identified in the marine mollusc, Perna canaliculus, an homologous series of novel omega 3 polyunsaturated fatty acids (ω-3 PUFA) with significant anti-inflammatory (AI) activity. The free fatty acid (FFA) class was isolated from a supercritical-CO₂ lipid extract of the tartaric acid-stabilised freeze-dried mussel powder by normal phase chromatography, followed by reversed-phase high performance liquid chromatography (RP–HPLC). The RP–HPLC involved separation based on carbon numbers, followed by argentation–HPLC (Ag–HPLC) of the methyl esters based on degree of unsaturation. Identification of the FFA components was performed using gas chromatography (GC) with flame ionisation detection, and individual structures were assigned by GC-mass spectroscopy (GC-MS). Inhibition of leukotriene production by stimulated human neutrophils was used as an in vitro screening method to test the AI activity of the purified PUFAs. A structurally related family of ω-3 PUFAs was identified in the most bioactive fractions, which included C18:4, C19:4, C20:4, and C21:5 PUFA. The C20:4 was the predominant PUFA in the extract, and was a structural isomer of arachidonic acid (AA). The novel compounds may be biologically significant as AI agents, as a result of their in vitro inhibition of lipoxygenase products of the AA pathway.     

Induction of settlement in mussel (Perna canaliculus) larvae by vessel noise

Underwater sound plays an important role in the settlement behaviour of many coastal organisms. Large steel-hulled vessels are known to be a major source of underwater sound in the marine environment. The possibility that underwater sound from vessels may promote biofouling of hulls through triggering natural larval settlement cues was investigated for the mussel, Perna canaliculus. The mussel larvae showed significantly faster settlement when exposed to the underwater noise produced by a 125-m long steel-hulled passenger and freight ferry. Median time to attachment on the substrata (ie settlement) was reduced by 22% and the time taken for all experimental larvae to settle was reduced by 40% relative to a silent control. There was no difference in the survival of the mussel larvae among the various noise treatments. The decrease in settlement time of the mussel larvae appeared to correlate with the intensity of the vessel sound, suggesting that underwater sound emanating from vessels may be an important factor in exacerbating hull fouling by mussels.     

Efficacy of marinades against Listeria monocytogenes cells in suspension or associated with green shell mussels (Perna canaliculus).

In order to determine the listericidal efficacies of three marinades used in the production of marinated green shell mussels (Perna canaliculus), decimal reduction times (D values) were determined for a mixture of seven strains of Listeria monocytogenes exposed to marinades in the presence and absence of mussels. With an acetic acid (1.5%, wt/vol) marinade, calculated D values in the presence and absence of mussels were 77.3 and 33.3 h, respectively. Likewise, for an acetic acid (0.75%)-lactic acid (0.75%) marinade and an acetic acid (1.5%)-Glucono Delta-Lactone (0.2%)-based marinade, the D values in the presence and absence of mussels were 125.5 and 26.9 h and 86.3 and 19.3 h, respectively. Various increases in decimal reduction times in the presence of mussels indicated that there was no simple relationship between the listericidal natures of these marinades and the presence of mussels. This result suggests that difficulties may occur in trying to relate acid inhibition studies carried out in model broth systems to "real food" systems.     

Field-to-laboratory transport protocol impacts subsequent physiological biomarker response in the marine mussel,Perna canaliculus

The transfer of mussels from field to laboratory, or transplantation between clean and contaminated field settings, is a common protocol in ecotoxicology. However, collection and transport of mussels could lead to stress that may impact biomarker responses, and thus confound interpretation of results. Physiological responses (clearance rate, absorption efficiency, excretion rate, respiration rate and scope-for-growth) of green-lipped mussels (Perna canaliculus) exposed to four different transportation protocols were investigated. These protocols included immersion in site seawater (SSW), immersion in artificial seawater (ASW), and emersion (aerial transport; EMS) at two temperatures (15 °C and 5 °C). Physiological measurements were conducted after a simulated 24 h “transport” phase and a 48 h “recovery” phase. Clearance rates were significantly inhibited by the EMS 5 °C and ASW protocols relative to SSW treatment, although the clearance rate of the latter recovered after 48 h. A similar pattern was observed for excretion and respiration rates for ASW. Decreased excretion rates for EMS 15 °C and respiration rates for EMS 5 °C were also recorded relative to values for SSW following “recovery”. Negative scope-for-growth was observed for all treatments except EMS 15 °C. These data suggest transport emersed at ambient air temperatures is the best method to maintain physiological health of green-lipped mussels.     

A serine protease inhibitor from hemolymph of green mussel, Perna viridis

Bioactivity guided fractions of cell-free hemolymph of bacterially challenged marine mussel, Perna viridis led to the isolation of a novel quaternary alkaloid 1, which was identified by its spectral data. The isolated molecule 1 has been found to be a potent serine protease inhibitor (SPI) showing IC₅₀ and K_i values of 102.5 and 97.1–104.68 μM, respectively. The E_t/K_i value of SPI is 6.3, whereas E_t/K_m value is 1.04. The Van’t Hoff analysis showed that the value of K_i decreases with increase in temperature, and the binding of the inhibitor is entropically driven.     

A glycosylated byssal precursor protein from the green mussel Perna viridis with modified dopa side-chains

Foot tissue of the green mussel Perna viridis contains a variety of byssal precursor proteins with the unusual redox-active amino acid, Dopa (-beta-3,4-dihydroxyphenyl-alpha-alanine). Eight proteins were detectable in acidic extracts of the Perna foot by a redox cycling assay with nitroblue tetrazolium. In one of these, however, P. viridis foot protein-1 (Pvfp-1), activity was not due to Dopa, but to another redox-active derivative. Based on specific colorimetric derivatization with Arnow's reagent, ninhydrin and phenylisothiocyanate (Edman), mass spectrometry, the redox-active derivative in Pvfp-1 is not consistent with any known modification. Another uncommon modification of Pvfp-1 involves O-glycosylation of threonine by mannose, glucose or fucose. As in previously characterized fp-1s, the primary sequence of the Pvfp-1 (apparent mass 89 kDa) has two consensus decapeptide motifs; one is APPKPX1TAX2K and the other is APPPAX1TAX2K, where P is Pro/Hyp, and X1 and X2 are difucosylated threonine and a redox sensitive derivative of tyrosine or Dopa, respectively. Of these two unusual residues, X2 is unique to Pvfp-1, whereas O-glycosylated Thr has been previously detected in freshwater mussel fp-1. The sequence homology of Pvfp-1 with the common structural motifs of the fp-1 protein family strongly suggests that the Pvfp-1 functions as the byssal coating (lacquer) protein.     

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

Molecular Biology Reports - Perna viridis (Linnaeus, 1758), the Asian green mussel, belonging to the family Mytilidae is widely distributed along the Indian coast. The species is majorly found in...     

Investigation of early mussel (Perna canaliculus) development using histology,SEM imaging,immunochemistry and confocal microscopy

A comprehensive study, incorporating histology, light microscopy, scanning electron microscopy, immunochemistry and confocal microscopy, was performed to investigate embryogenesis and larval development of the New Zealand Greenshell? mussel, Perna canaliculus. Detailed observations with this multi-technique approach revealed a gastrula stage at 18 hours post-fertilization, with the appearance of a blastopore, apical sense organ and enclosing vegetal pole. Early D-stage larvae showed limited alimentary organogenesis and clear initiation of a developing nervous system. Shell morphology of D-larvae was characterized by a flat, hinged, pitted–punctate prodissoconch I shell, followed closely by commarginal growth lines within the prodissoconch II shell. Early umbo larvae had a protruding functioning velum, and well-developed posterior adductor and velar retractor muscles. Significant progression in neuronal development occurred just before the umbo stage with noticeable paired cerebral, pedal and visceral ganglia. Shell morphology was characterized by further prodissoconch II secretion with a more rounded umbonate appearance. During the transition through the pediveliger stage, rapid development of the gill rudiment, eye spot and functioning foot was observed with ongoing neuronal development. The first appearance of the dissoconch shell layer took place during this transition, at which point the nervous system was highly distinct with innervations extending throughout muscle regions and between ganglia. This study provides the first comprehensive documentation of the developmental stages of P. canaliculus larvae from fertilization to settlement. The study highlights the advantages of using a combination of techniques to understand larval development and provides crucial information to identify larval performance during larval rearing.     

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.     

Pigment alterations in the brown mussel Perna perna

Potential sex and/or gametogenic stage differences in the metabolism of chlorophyll-a and carotenoids in the brown mussel Perna perna of southern Brazil were studied using high performance liquid chromatography (HPLC). Carotenoids derived directly from diet (phytoplankton) were fucoxanthin plus diatoxanthin (diatoms), alloxanthin (cryptophytes) and zeaxanthin (mainly cyanobacteria). Females accumulated carotenoid-diols and epoxides (~ 3–4 mg/g-dry wt.) while males had much lower concentrations (~ 0.7 mg/g-dry wt.). An antioxidant/free radical scavenging role is proposed for carotenoids in females. Mean ratios of chlorophyll plus derivatives (Chlns-a) to carotenoids for male and female P. perna were 50:1 and 4:1, respectively. The higher ratio in males relates to both higher carotenoid contents in females plus higher total Chlns-a in males (~ 22 mg/g-dry wt.), relative to the females (~ 4 mg/g-dry wt.). Chlorophyll-a metabolism in both sexes followed two distinct pathways. First, cyclization of pyropheophorbide-a gave 13², 17³-cyclopheophorbide-a-enol (CPPaE) which was further oxidized to hydroxy-chlorophyllone. Second, chlorophyll-a derivatives retaining the 13²-carbomethoxy moiety were oxidized to purpurin-18 which was hydrolyzed to chlorin-p₆. In both cases, metabolism of dietary chlorophyll-a was oxidative and derivatives could either serve as antioxidants or merely be the results of non-specific digestive processes.     

Settlement and growth of the green mussel Perna viridis (L.) in coastal waters: influence of water velocity

Green mussels Perna viridis were observed to be a major foulant in the seawater intake tunnel of a coastal power station. Field experiments were carried out to ascertain what factors were responsible for the successful colonisation by mussels. Two adjacent stations (25 m apart) were selected, one representing the coastal waters and the other representing the intake screens (with higher water velocity). Gonadal activity, larval abundance, spat settlement and growth rate of the mussels were monitored at monthly intervals for a total period of two years. The results showed that the breeding activity of the mussels at the study area is influenced largely by temporal distribution of seawater temperature. However, ensuing larval availability in the coastal waters is more dependent on food availability. On the other hand, spat settlement and growth rate are predominantly influenced by water flow, probably as a result of increased propagule and food flux rate at higher water velocities. Higher water velocity at the intake screens also contributed to mussel dominance by preventing settlement of many potential competitors.     

Population genetics of introduced and native populations of the green mussel, Perna viridis: determining patterns of introduction

Genetic variation can be used to determine routes of introduction of non-native species and whether introduced populations lost variation during establishment. The present study sought to determine whether multiple, geographically isolated non-native populations of the green mussel, Perna viridis, were the product of a stepping stone expansion of a single introduction or from multiple independent introductions from the native range. Measurements of genetic variation were compared among five introduced populations and three populations from within the native range. We sequenced 650 bp of the mitochondrial gene cytochrome oxidase I from 280 samples from five introduced populations and another 190 samples from three native populations. Haplotype frequencies of all introduced populations were not significantly different from each other, but virtually all populations differed from samples taken from the native range. Measurements of genetic variation tended to suggest that introduced populations had less variation than most native populations and there was no evidence for admixture in any of the introduced populations. The genetic data and Monte Carlo simulations both provide compelling evidence of a stepping-stone pattern of introduction of P. viridis from the native range to Trinidad, and from Trinidad to other locations in the Caribbean and United States. The lack of genetic variation in introduced populations suggests that the initial introduction was relatively small and the lack of admixture suggests a single original source population.     

Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis.

The primary structure of the cDNA and metallothionein (MT) genomic sequences of the tropical green mussel (Perna viridis) was determined. The complete cDNA sequences were obtained using degenerate primers designed from known metallothionein consensus amino acid sequences from the temperate species Mytilus edulis. The amino acid sequences of P. viridis metallothionein deduced from the coding region consisted of 72 amino acids with 21 cysteine residues and 9 Cys-X-Cys motifs corresponding to Type I MT class of other species. Two different genomic sequences coding for the same mRNA were obtained. Each putative gene contained a unique 5'UTR and two unique introns located at the same splice sites. The promoters for both genes were different in length and both contained metal responsive elements and active protein-binding sites. The structures of the genomic clones were compared with those of other species. J. Exp. Zool. 284:445-453, 1999.     

Eleven novel polymorphic microsatellite DNA markers from the green-lipped mussel Perna viridis

We report on the characterization of 11 polymorphic microsatellite loci in P. viridis, the first set of such markers developed and characterized for this species. The number of alleles per locus ranged from 2 to 7, whereas the observed heterozygosity ranged from 0.0447 to 0.4837. These markers should prove useful as powerful genetic markers for this species.     

Bioavailability of sediment-bound Cd, Cr and Zn to the green mussel Perna viridis and the Manila clam Ruditapes philippinarum

We assessed the degree to which Cd, Cr and Zn bound with sediment were assimilated by the green mussel Perna viridis and the Manila clam Ruditapes philippinarum. The influences of the metal concentration in the sediment, the presence of phytoplankton, and the oxidation condition of the sediment on metal assimilation were examined. No major difference was found for metal assimilation efficiency (AE) in sediment with different metal concentrations, except for Cd in the green mussels, in which the AE increased by 1.7x when the Cd concentration in sediment was elevated to 15x the natural background level. The higher assimilation of Cd with increasing Cd load in ingested sediment may be due to the higher desorption of Cd in the acidic gut of the bivalves. Both mussels and clams assimilated metals at a higher efficiency from the diatom diet (Thalassiosira pseudonana) than from inorganic sediment particles. The presence of algal particles had little influence on metal assimilation from ingested sediment, and conversely, the presence of sedimentary particles had little effect on metal assimilation from ingested diatom (except for Cd in the mussels). In the mussels, AEs were higher from oxic sediment than from anoxic sediment by 3.1x for Cd, 2.0x for Cr, and 1.4x for Zn, and in the clams AEs were higher from oxic sediment by 2.8x for Cd, 2.0x for Cr, and 2.0x for Zn. Our study suggested that metals associated with anoxic sediment can be potentially available to marine bivalves, and that metal AEs determined for a single diet were probably not affected by the presence of other food particles.     

The influence of cross-linking on protein-protein interactions in a marine adhesive: the case of two byssus plaque proteins from the blue mussel

The interaction between two proteins, Mefp-1 and Mefp-2, from the byssal plaque of the blue mussel, Mytilus edulis, was investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The challenge in using a surface-sensitive technique to investigate the interaction between two strongly adhesive proteins was met by coupling a biotinylated version of one of the proteins (b-Mefp-1) to an inert two-dimensional arrangement of streptavidin (SA) formed on top of a biotin-doped supported phospholipid bilayer. The interaction between Mefp-1 and Mefp-2 was further investigated by addition of Mefp-2 to SA-coupled b-Mefp-1, where the latter was either in the native state or cross-linked using sodium periodate (NaIO(4)), Cu(2+), or mushroom tyrosinase. With this coupling strategy it is shown that a requirement for attraction between the two proteins is that tyrosinase is used as the cross-linking agent of b-Mefp-1. By inhibiting the enzymatic activity of tyrosinase it is also shown that enzymatic activity is required for both efficient binding of tyrosinase to SA-coupled b-Mefp-1 as well as for the subsequent binding of Mefp-2. In contrast, spontaneous adsorption of Mefp-1 to a methyl-terminated (thiolated) gold surface followed by addition of Mefp-2 results in binding of Mefp-2 for all cross-linking agents. This suggests that cross-linking of Mefp-1 adsorbed on a solid surface induces structural changes in the adsorbed protein layer, resulting in exposure of free surface patches on which Mefp-2 binds.     

邻苯二甲酸二乙基己酯对翡翠贻贝(Perna viridis)生化指标的影响

实验条件下,研究邻苯二甲酸二乙基己酯(DEHP)5个浓度组(0、0.38、1.92、9.60和48.00mg.L-1)长时间胁迫下翡翠贻贝(Perna viridis)内脏团和外套膜中抗氧化酶(SOD和CAT)活性和丙二醛(MDA)含量的变化,以及胁迫解除后这些指标的恢复情况。结果表明:在胁迫过程中,翡翠贻贝内脏团SOD活性表现为先显著升高,随后受抑制而逐渐降低(P<0.05),CAT活性则表现为先被抑制后受诱导,15d后恢复到对照组水平,MDA含量呈显著增加的趋势(P<0.05);外套膜中的SOD活性在胁迫初期在低浓度组被抑制,而在高浓度组则被诱导(P<0.05),4d后SOD活性逐渐恢复到对照组水平,各浓度组MDA含量均出现明显的增加(P<0.05);净化阶段,低浓度组(0.38mg.L-1)内脏团SOD活性和CAT活性逐渐恢复到对照组水平,但MDA含量升高;净化7d后,除高浓度组(48.00mg.L-1)外,其余浓度组外套膜中SOD活性均已经恢复到对照组水平,MDA含量也没有出现明显升高的现象。研究表明,DEHP对翡翠贻贝内脏团和外套膜抗氧化防御系统酶具有明显的影响,DEHP诱导引起2种组织内脂质过氧化损伤,并且短期内这种损伤无法消除。     

DNA damage in digestive gland and mantle tissue of the mussel Perna perna

Data concerning the susceptibility of DNA to damage by reactive oxygen and nitrogen species and other endogenous compounds produced by physiological stress in marine organisms is lacking, especially in bivalve mollusks. In this article, we analyzed the background levels of lipid peroxidation (malondialdehyde, MDA), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 1,N2-etheno-2'-deoxyguanosine (1,N2-epsilon dGuo) in digestive gland and mantle tissue of mussels Perna perna collected at a cultivation zone in Florianópolis (Santa Catarina, Brazil). The present data point to the possibility of the use of both 8-oxodGuo and 1,N2-epsilon dGuo as complementary indicators of oxidative stress processes in mussels. A sensitive method coupling high performance liquid chromatography to mass spectrometry was applied for the detection of 1,N2-epsilon dGuo in mussel tissues.