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.     

In situ settlement behaviour of damselfish (Pomacentridae) larvae

Settlement‐stage damselfish (Pomacentridae) larvae of 13 species in seven genera were obtained from light traps at Lizard Island, Great Barrier Reef, Australia. Behaviour, observed in situ by SCUBA divers, of 245 larvae (6–13 mm, L_S; 5–60 individuals per species) released individually within a few m of reefs during the day differed markedly among species. From 0–28% (range among 13 species) of individuals of each species swam away from the adjacent reefs without swimming to the reefs. Of those that swam to a reef, 0–75% settled. For three species, sufficient data were available to test the hypothesis that these percentages did not differ amongst reefs: the hypothesis was rejected in one species. From 0–75% of larvae that reached the reef were eaten, 0–63% subsequently left the reef and 0–60% were still swimming over the reef at the end of the observation period. Swimming speeds of all but one species were greater when swimming away from the reef than toward it. Most species exceeded average current speeds when swimming away from reefs, but not when swimming toward and over them. Average swimming depths were in the upper half of the water column for most species, and were somewhat greater where the water depths were greater. The time the larvae swam over the reef before settling and the distance swum varied greatly among species from 0 to a mean of 5.5 min and 43 m. Settlement habitats chosen differed amongst species, and in some species, they were very specific. Average settlement depth varied among species from 6–13.5 m. In one species, settlement depth varied between reefs. About half of the 53 observed interactions between larvae and reef resident fishes were predation attempts: fishes of eight species (six families) attacked larvae. The other interactions were aggressive approaches by 11 species of resident fishes, all but one of which were pomacentrids. Many of these aggressive interactions discouraged settlement attempts. Larvae of some species experienced no predatory or aggressive interactions, whereas in other species interactions averaged >0.6 per released larva. Species that swam more‐or‐less directly to settlement sites near the reef edge experienced more interactions. Even within the same family, settlement behaviour differed among species in nearly all measures.     

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.     

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.     

Coating proteins: structure and cross-linking in fp-1 from the green shell mussel Perna canaliculus

The protein family known as fp-1 provides mussel byssus with a protective outer coating and has drawn much attention for its water resistant bioadhesive properties in vitro. A new fp-l isolated from the green shell mussel Perna canaliculus (pcfp-1) reveals a composition dominated by only four amino acids: 3,4-dihydroxyphenyl-L-alanine (dopa), lysine, proline, and valine at approximately 20 mol % each. SDS-PAGE and MALDI-TOF mass spectrometry detected size variants at 48 and 52 kDa in preparations of purified Pcfp-1. The N-terminal sequence enabled construction of oligonucleotide primers for PCR and RACE-derived cDNAs from which the complete sequence of four variants was deduced. pcfp-1 deviates from all known homologues in other mussels in several notable respects: its mass is half, most of its sequence is represented by 75 tandem repeats of a tetrapeptide, i.e., PY*VK, in which Y* is dopa, prolines are not hydroxylated, and thiolate cysteines are clustered in homologous sequences at both the amino and carboxy termini. Amino acids in the repeat sequence show a striking resemblance to proline-rich cell wall proteins with tandemly repeated PPVYK pentapeptides [Hong, J. C., Nagao, R. T., and Key, J. L. (1987) J. Biol. Chem. 262, 8367-8376]. Cysteine plays a key role in cross-linking pcfp-1 by forming adducts with dopaquinone. Significant 5-S-cysteinyldopa and smaller amounts of 2-S-cysteinyldopa were detected in hydrolysates of the byssal threads of P. canaliculus. The cross-links could also be formed by oxidation of pcfp-1 in vitro using mushroom tyrosinase. Cysteinyldopa cross-links were present in trace amounts only in the byssus of other mussel species.     

In situ observation of settlement behaviour in larvae of coral reef fishes at night

The swimming behaviour of 534 coral reef fish larvae from 27 species was explored at Moorea Island (French Polynesia) while they searched for a suitable settlement habitat, on the first night of their lagoon life. Most larvae swam actively (74%) and avoided the bottom (77%). A significant relationship was highlighted between the vertical position of larvae in the water column and the distance they travelled from lagoon entrance to settlement habitat: larvae swimming close to the surface settled farther away on the reef than bottom-dwelling larvae.     

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.     

Population genetic subdivision in the New Zealand greenshell mussel (Perna canaliculus) inferred from single-strand conformation polymorphism analysis of mitochondrial DNA

Single-strand conformation polymorphism (SSCP) analysis of the NADH IV region of the mitochondrial DNA (mtDNA) molecule in greenshell mussels (Perna canaliculus) indicated strong population genetic structuring in this endemic New Zealand species. A northern and a southern group were differentiated by frequency shifts in common haplotypes and by the occurrence of a unique southern haplotype at approximately 20% frequency. This split occurred south of Cook Strait (the body of water between the North and the South Island) at approximately 42 degrees S latitude. Northern populations were less genetically diverse than southern populations and mussels from the west coast of the South Island were most distinct from northern mussels. We hypothesize that the unique haplotype VIII originated in the lower South Island, and that its spread northwards was obstructed by the opening of Cook Strait approximately 15 000-16 000 years ago and the subsequent establishment of present-day surface water circulation patterns in Greater Cook Strait. We suggest that present-day strong tidal flows and turbulent mixing of water masses in Cook Strait, and intense up-welling on the east and west coasts in this region, represent a barrier to gene flow between mussels located in the North Island and northern South Island vs. mussels in most of the South Island and Stewart Island.     

Absence of population genetic differentiation in the New Zealand greenshell mussel Perna canaliculus (Gmelin 1791) as assessed by allozyme variation

Genetic variation in the endemic New Zealand greenshell mussel, Perna canaliculus (Gmelin 1791), was examined using starch-gel electrophoresis at seven protein-coding loci (Idh; Acon-1; Acon-2; Gpd; Pgi; Pgm; Pgd) in 35 populations (N=1038 mussels). For all loci and all populations, Fisher's exact tests indicated highly significant departures from Hardy-Weinberg equilibrium (HWE), but this overall result was caused by significant heterozygote deficiencies at only two loci (Pgm and Pgd), and in only three northern populations (Kuaotunu, Te Haumi and Days Bay). Allelic and genotypic differentiation between population pairs at individual loci and across all loci were nonsignificant, and genotypic disequilibrium at each locus pair was also nonsignificant for all populations. Genetic variation in all populations was high (mean heterozygosity, 0.210+/-0.113), while Nei's D among populations was very low (0.002+/-0.002). Low population subdivision (θ=-0.001+/-0.002) and high levels of gene flow (Nm(p)=10.18; Nm(θ)=infinity) also indicated that the single panmictic unit model best explains population genetic homogeneity in P. canaliculus over a north-south distance >2000 km. Lack of genetic subdivision in this species is discussed in light of two previous allozyme studies, with differing results: one suggested that a north-south division exists between greenshell mussel stocks, and the other suggested that population structure in this species can be explained through isolation by distance model modified by local hydrology.     

Pernin: a novel, self-aggregating haemolymph protein from the New Zealand green-lipped mussel, Perna canaliculus (Bivalvia: Mytilidae)

A protein, designated pernin, found in the New Zealand green-lipped mussel, comprises almost all of the protein in cell-free haemolymph. It occurs as large, aggregate structures of several hundred units resembling small virus-like particles. Pernin is a non-pigmented, glycosylated protein, composed of 497 amino acids, which has an estimated molecular mass of 60 kDa. It is exceptionally rich in histidine (13.7%) and aspartic acid (12.3%), amino acids both known to participate in the binding of divalent metal cations. In addition, pernin has serine protease inhibitor activity, likely due to a sequence of eight N-terminal amino acid residues, separated from the remainder of the protein via a histidine–aspartate spacer. The pernin monomer comprises three regions of obvious sequence duplication. These make up approximately 95% of the pernin molecule and have sequences clearly homologous to the active-site domain of Cu–Zn SODs (superoxide dismutases). Despite several of the metal ion co-ordinating histidine residues being retained, pernin contains no Cu or Zn. It is, however, associated with Fe with an apparent stoichiometry of 1 atom of Fe to 6 molecules of pernin. Since pernin has no demonstrable SOD activity, these SOD-derived sequences presumably have been modified for another function.     

A Raman and infrared spectroscopic investigation of biological hydroxyapatite

Raman spectra were acquired on ox femur samples treated with hydrazine to remove the organic components of bone. A large increase in the signal-noise ratio of the mineral spectrum resulted from the exposure of the mineral surface and the removal of fluorescent components of the organic matrix. The effect of hydrazine treatment of the mineral matrix has been reinvestigated and shown to be slight on the basis of second derivative FTIR data. This is the first time that this high resolution technique has been applied to biological minerals.     

Effects of near‐bed turbulence on the suspension and settlement of freshwater dreissenid mussel larvae

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Survival,growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays

Abstract

Straight-hinge veliger and pediveliger larvae of the mussel Mytilus galloprovincialis were refrigerated for varying periods for use in bioassays. Straight-hinge veliger larvae grew to the umbo-veliger stage after 2 months in the refrigerator, but no pediveligers were observed during the 3-month refrigeration period. The average survival rate of larvae in the refrigerator was 79% after 1 month, but gradually decreased with the refrigeration period, and was as low as 22% after 3 months. All refrigerated larvae grew to the pediveliger stage in the incubator at 17°C at the same rate as that of the control larvae that were not refrigerated. Settlement and metamorphosis of pediveligers from both refrigerated and control groups were facilitated by microbial film and epinephrine and inhibited by phentolamine. Thus, refrigeration can be used as an effective method of storing larvae of M. galloprovincialis for use in assays to assess candidate settlement inducers and antifouling substances.     

In situ biochemical and bacterial variation of sediments enriched with mussel biodeposits

In order to estimate the qualitative variation of sedimented mussel deposits, biochemical and microbial measurements were undertaken after a sediment enrichment with fresh faeces and pseudo-faeces collected in a mussel farming area (Carteau, Gulf of Fos).During two months, cores sampled were investigated at three stations: a first station enriched with mussel deposits, a second considered as a reference station without mussel deposits and occasionally a third corresponding to a continuously enriched sediment under a rope culture.Carbohydrate, organic carbon and nitrogen contents in the sediments gave evidence of a short term variation after sedimentation.Bacterial production increased rapidly at the enriched station and returned to its initial level 8 days later. At this station, exoglucosidasic activity of bacteria, low at the beginning, presented a maximum two weeks after enrichment whereas exoproteolytic activity, which was high in the biodeposits, decreased in the course of the first week. These exoenzymatic activities were significantly higher at the enriched station than at the reference stations. Carbohydrate measurements were in agreement with these results.Degradation rate of biodeposits is discussed on the basis of exoenzymatic activity, organic carbon, nitrogen and free amino acid content at the three stations.     

Survival, growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays

Straight-hinge veliger and pediveliger larvae of the mussel Mytilus galloprovincialis were refrigerated for varying periods for use in bioassays. Straight-hinge veliger larvae grew to the umbo-veliger stage after 2 months in the refrigerator, but no pediveligers were observed during the 3-month refrigeration period. The average survival rate of larvae in the refrigerator was 79% after 1 month, but gradually decreased with the refrigeration period, and was as low as 22% after 3 months. All refrigerated larvae grew to the pediveliger stage in the incubator at 17 degrees C at the same rate as that of the control larvae that were not refrigerated. Settlement and metamorphosis of pediveligers from both refrigerated and control groups were facilitated by microbial film and epinephrine and inhibited by phentolamine. Thus, refrigeration can be used as an effective method of storing larvae of M. galloprovincialis for use in assays to assess candidate settlement inducers and antifouling substances.     

Topographically generated fronts, very nearshore oceanography and the distribution and settlement of mussel larvae and barnacle cyprids

Flow patterns adjacent to shore may prevent or aid shorewardmigration of benthic invertebrate larvae. We hypothesized thata front at the mouth of Sunset Bay, Oregon, prevents shorewarddispersal of larvae, significantly altering settlement of mussellarvae and barnacle cyprids. Settlement was measured at threesets of moorings (three moorings per site) distributed acrossthe front at Sunset Bay. From 6 July to 4 September 2000, sampleswere collected roughly every other day. Concurrently, we madevertical zooplankton tows adjacent to each mooring site andcollected physical oceanographic data. During upwelling-favorablewinds, the front was always present at the bay mouth, separatingsignificantly cooler, saltier and denser offshore water fromthat within the bay. During downwelling winds, the front brokedown and we found no significant difference in the surface physicaloceanographic parameters across the bay mouth. During upwelling,the concentration of mussel larvae was higher seaward of thefront than landward, but there was no significant differencein concentration during downwelling, suggesting that the frontmay act as a barrier to the shoreward dispersal of mussels.Mussel settlement was too low and sporadic to allow statisticalanalysis. There was no difference in cyprid concentrations acrossthe bay mouth whether the front was present or not. Cyprid settlementwas, however, nearly an order of magnitude lower at mooringsseaward of the front than at those landward. A significant cross-correlationwas found between settlement at the offshore mooring and tidalrange (r = 0.464, lag = 0 days) and between settlement at themid and inner moorings and downwelling winds (r = 0.532 midbay, r = 0.532 inner bay, lag = 0 days). Seaward of the front,settlement varied with tidal range, while landward of the front,most settlement occurred as brief pulses during downwellingwinds, periods when the front was not present. We found largedifferences in the distribution of cyprids, and mussel larvaeand cyprid settlement relative to the front; larval distributionsand settlement varied with upwelling versus downwelling windsand was due to differences in the very nearshore (i.e. within100–1000 m of shore) coastal oceanography.     

Toxicity of mosquitocides on freshwater mussel larvae

The effect of four insecticides also used for mosquito-killing (Fyfanon ULV, K-Othrin ULV, Unitox 7 ULV and Unitox 20 ULV) was studied on the larvae (glochidia) of two freshwater mussel species, Anodonta cygnea and Anodonta anatina. In addition to the determination of the 24-96 h LC50 values of the insecticides, studies were also performed on their effect exerted on a physiological process, the tryptamine-(TA)-induced activation of the larvae in normal and 24 h test. On the basis of the LC50 values, Fyfanon and Unitox 20 were found to be the most toxic to the studied mussel larvae, however, the TA-induced adductor muscle activity, as physiological process, was also inhibited by Unitox 7 in relatively low concentrations. This effect may reduce the chance of further development of the larvae, by exerting harmful effect on the number of freshwater mussels.