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Temperature is a fundamental element determining the distribution patterns of ectotherms, and it is generally recognized that physiological tolerances of ectotherms change according to thermal gradients in their habitats. However, little is known about the variation in cold tolerance of marine invertebrates among local populations of the same species. In this study, three experiments were conducted to demonstrate inter-population variation in the cold tolerance of the intertidal gastropod Littorina brevicula, and to explore factors driving the variation. First, seasonal changes in the freezing temperature (the supercooling point, SCP) and the median lower lethal temperature (LLT50) of snails collected from a single population were examined to identify the cold tolerance strategies of this species. Seasonal fluctuation was clear in only the LLT50 which was lower than the SCP in winter, indicating that cold tolerance strategy of this species was freeze tolerant. Second, the cold tolerance of snails from four populations was compared. These populations were distributed at latitudinally different locations, and the comparisons were performed before winter when their cold tolerance was not required and in winter when their cold tolerance increased. In this experiment, while the cold tolerance of snails collected in winter was higher than that of snails collected before winter in all four populations, there were differences in tolerance among the populations. Furthermore, to estimate their ability of acclimation to cold environments, snails that were collected from the northernmost and southernmost populations in the four populations before winter were acclimated to a constant low temperature in the laboratory for a long period (120–130 days). This long period of acclimation led to an increase in the cold tolerance of both populations, but the latitudinal difference was maintained. Additionally, after imposing the long period of acclimation to snails that were collected at 12 locations before winter, factors that might cause local variation in their cold tolerance were explored. This experiment clearly showed that local variation in cold tolerance was explained by the lowest local air temperature. Our study demonstrated inter-population variation in the cold tolerance of an intertidal mollusc, and that the variation is explained by thermal gradients in winter.

The genetic structure of marine animals that inhabit the seashore is affected by numerous factors. Of these, gene flow and natural selection during recruitment have strong influences on the genetic structure of seashore-dwelling species that have larval periods. Relative contributions of these two factors to the genetic structure of marine species would be determined mainly by the duration of larval stage. The relationship between larval period and genetic structure of population has been rarely studied in Korea. In this study, genetic variations of cytochrome oxidase subunit I (COI) were analyzed in two dominant species on rocky shore habitats in the Korean peninsula: periwinkle Littorina brevicula and acorn barnacle Fistulobalanus albicostatus. Both species are not strongly structured and may have experienced recent population expansion. Unlike periwinkle, however, barnacle populations have considerable genetic variation, and show a bimodal pattern of mismatch distribution. These results suggest that barnacle populations are more affected by local adaptation rather than gene flow via larval migration. The bimodal patterns of barnacle populations observed in mismatch distribution plots imply that they may have experienced secondary contact. Further studies on seashore-dwelling species are expected to be useful in understanding the evolution of the coastal ecosystem around Korean waters.

Highlights: • Wound activated prostaglandin production was quantified in Gracilaria vermiculophylla. • Specimens from non-native populations contained higher prostaglandin concentrations. • Prostaglandins and related compounds deter animal consumers. • Activated defense through Prostaglandins was selected during Gracilaria's invasion history. • As a consequence specimens from invasive populations are not safe for human consumption. Abstract: The capacity of the East Asian seaweed Gracilaria vermiculophylla (“Ogonori”) for production of prostaglandin E2 from arachidonic acid occasionally causes food poisoning after ingestion. During the last two decades the alga has been introduced to Europe and North America. Non-native populations have been shown to be generally less palatable to marine herbivores than native populations. We hypothesized that the difference in palatability among populations could be due to differences in the algal content of prostaglandins. We therefore compared the capacity for wound-activated production of prostaglandins and other eicosatetraenoid oxylipins among five native populations in East Asia and seven non-native populations in Europe and NW Mexico, using a targeted metabolomics approach. In two independent experiments non-native populations exhibited a significant tendency to produce more eicosatetraenoids than native populations after acclimation to identical conditions and subsequent artificial wounding. Fourteen out of 15 eicosatetraenoids that were detected in experiment I and all 19 eicosatetraenoids that were detected in experiment II reached higher mean concentrations in non-native than in native specimens. Wounding of non-native specimens resulted on average in 390% more 15-keto-PGE2, in 90% more PGE2, in 37% more PGA2 and in 96% more 7,8-di-hydroxy-eicosatetraenoic acid than wounding of native specimens. Not only PGE2, but also PGA2 and dihydroxylated eicosatetraenoic acid are known to deter various biological enemies of G. vermiculophylla that cause tissue or cell wounding, and in the present study the latter two compounds also repelled the mesograzer Littorina brevicula. Non-native populations of G. vermiculophylla are thus more defended against herbivory than native populations. This increased capacity for activated chemical defense may have contributed to their invasion success and at the same time it poses an elevated risk for human food safety.

Xiangshan bay is a narrow semi-closed bay and situated on the northwestern coast of the East China Sea. Over past decades, it has become to a major bay with intensive human activities, dense urbanized area, and poor water quality. The aim of this paper was to reveal the ecological status through the elucidation of the species composition, abundance, biomass and diversity of macrobenthos in this bay. Six intertidal sections were surveyed from January 2007 to November 2008 quarterly. Sections TG, HD and XH are located in the three inner bays, sections QJ and WS are located near the thermal power plants, and section XX is located at the outer part of Xiangshan Bay. Great variations in macrobenthos community were indentified, and the species composition of the community in the present study showed the dominance in the order of molluscs (bivalves and gastropods), crustaceans and others, and only few Polychaeta were recorded. Only three dominant species, Littorina brevicula, Ilyplax tansuiensis, and Cerithidea cingulata were collected in all the sections, and a total of 19 dominant species were recorded only in one section. Two-way ANOVA analyses of abundance indicated that there were significant differences among sections or seasons. Shannon-Wiener diversity index (H’) had its maximum (2.45) in section QJ, and minimum (1.76) in section TG. Multiple irregular k-dominance plots clearly showed that the study area was polluted and the macrobenthos community was under stress. We conclude that the macrobenthos of Xiangshan Bay have been disturbed by human activities, especially at the interior bay.

The aim of this study was to quantify the effects of preservation methods and preservation times on the stable carbon and nitrogen isotope values in the tissues of six mollusk species (Mytilus edulis, Crassostrea gigas, Ruditapes philippinarum, Acanthochiton seulschnochilon, Littorina brevicula, and Rapana venosa). To identify potential preservation effects on δ13C and δ15N values and to examine temporal changes in the effects of preservation, repeated analyses were carried out after 1 day, 5 days, 15 days, 1 month, 3 months, 6 months, and 12 months of preservation. The results showed that drying preservation was the most suitable method for preserving samples, while freezing and chemical preservation significantly affected the stable isotope values compared with those of the controls. The effects of preservatives on the tissues of different mollusk species were statistically significant for both δ13C and δ15N values. Shifts in the δ13C and δ15N values, due to freezing and chemical preservation, were high...

Differences with respect to anti-herbivore defense were investigated in invasive and native populations of the seaweed Gracilaria vermiculophylla. Specimens from 6 native populations in East Asia and from 8 populations invasive in Europe and the Mexican Pacific coast were maintained under identical conditions and offered to herbivorous snails from both the native range (Littorina brevicula) and Europe (L. littorea) in no-choice feeding assays. L. brevicula consumed in total significantly larger amounts of G. vermiculophylla tissue than did L. littorea. Further, both snail species least consumed the seaweed specimens originating from either non-native populations or from populations native to the Korean East Sea/Sea of Japan. The Korean East Sea/Sea of Japan had previously been identified as putative donor region of all the invasive populations of G. vermiculophylla. Thus, populations in the donor region as well as non-native populations in different invaded realms feature an increased capacity to resist feeding pressure. Differences in nutrient content did not account for the observed patterns of consumption, as palatability and carbon to nitrogen (C:N) ratio were not significantly correlated. Thus, mechanical or chemical defenses or the content of feeding cues influenced the behavior of the snails. We suggest that low palatability contributed to the invasion success of the species.

The coastal environment is affected by Cu and Zn contamination through diverse routes, including antifouling agents, industrial wastewater, and domestic activities. In this study, Crassostrea gigas and Littorina brevicula were used to examine the distributions of Cu and Zn, which are related to the increase in the use of new antifouling agents that have been used for ships since the TBT ban in 2003. The average Cu and Zn concentrations in the samples were 231.8 μg/g and 492.6 μg/g, respectively. Both the Cu and Zn concentrations were highest in the shipyard samples, followed by the harbor sample for Cu and the industrial area sample for Zn. The results indicated that the Cu concentration was affected by the antifouling agents, and the Zn concentration was affected by both the antifouling agents and industrial wastewater. The areas that need intensive control to address the Cu and Zn contamination in major Korean coasts were identified.

Alginate lyase (EC 4.2.2.3) is an enzyme that splits glycosyl linkages of alginate chain via β-elimination producing unsaturated oligoalginates. This enzyme is widely distributed in herbivorous marine mollusks, brown algae, and marine and soil bacteria. In the present study, we determined the general properties and partial amino-acid sequences of alginate lyases from three Archeogastropoda, i.e., Haliotis discus hannai, H. iris, and Omphalius rusticus, and one Mesogastropoda, i.e., Littorina brevicula, in order to enrich the information about functional and structural diversity in gastropod alginate lyases. The alginate lyases were extracted from hepatopancreas of these animals and purified by ammonium sulfate fractionation followed by conventional column chromatography. Single alginate lyases with molecular masses of approximately 28 kDa, 34 kDa, and 34 kDa were isolated from H. discus, H. iris, and O. rusticus, respectively. While three alginate lyases with molecular masses of 35 kDa, 32 kDa, and 28 kDa were isolated from L. brevicula. These enzymes were identified as poly(M) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate. Western blot analysis using an antiserum raised against H. discus enzyme suggested that H. iris, and O. rusticus enzymes shared similar primary/higher order structure with H. discus enzyme, but the L. brevicula enzymes did not. H. discus, H. iris, and O. rusticus enzymes were classified to polysaccharide-lyase family-14 by the analysis of partial amino-acid sequences, while the L. brevicula enzymes were not.

Meso-herbivores can strongly affect biomass accrual and macrophytobenthic commu- nity structure. To counter grazing effects, constitutive and induced anti-herbivory responses evolved in seaweeds. Feeding-assayed induction experiments were conducted in the laboratory to investigate whether 14 d grazing periods by either the periwinkle Littorina brevicula or the abalone Haliotis dis- cus induced anti-herbivory defences in the green alga Ulva pertusa and the brown alga Laminaria japonica. Where appropriate, assays were repeated with artificial food containing lipophilic algal extracts, using dichloromethane (DCM) as a solvent, to test whether or not snails induced chemical defences. While exposure to periwinkles reduced palatability of fresh L. japonica pieces for naive conspecific grazers, no such effect was displayed by abalone-exposed L. japonica pieces. Patterns of grazer specificity in the palatability of fresh algae were not confirmed when using artificial food con- taining algal DCM extracts. This suggests that L. japonica either induced morphological defences or periwinkle-deterrent substances originated from the non-lipophilic fraction of defensive chemicals. Grazer-specific induced responses were not apparent in U. pertusa assays. This study revealed that the induction of chemical anti-herbivory responses depends not only on the type of grazer but also on the type of alga on which the grazer feeds.

The structure and state of the reproductive system were studied in the periwinkles Littorina brevicula and L. mandshurica from the coastal area of Vladivostok and in the dogwhelk Nucella heyseana from the protected area of Vostok Bay and from contaminated sites in Amursky Bay. These species were investigated for the presence of imposex and intersex (pathological changes caused by endocrine disruption under the influence of toxic substances). The pathological changes revealed in the gonads of Littorina were oocyte resorption and lipofuscin accumulation; an intersex condition was not found. Imposex specimens (with penis formation) were found among Nucella females from Amursky Bay. The relative penis length in females was 25% of the penis length in males. This kind of pathology was not found in Nucella from clean waters of Vostok Bay.

Spatial and temporal variations of benthic fauna over a wide region (>1,000 km) that includes complex ocean currents have seldom been quantitatively studied. The present study revealed malacofaunal variations on Japanese rocky shores affected by the Soya, Tsushima, Oyashio, and Kuroshio currents, and in the Inland Sea. On 29 shores in seven regions in these sea areas, previous researchers have recorded the number of molluscs in quadrats in spring and summer of 1978 and 1984–1986. Reanalyses of these records revealed the following patterns. (1) Shore-to-shore variation in malacofauna was correlated with shore-to-shore geographical distance less clearly for more southern regions. This might show that in southern regions many taxa with low relative abundance are haphazardly recorded on some shores and blur the effect of shore-to-shore distance on malacofaunal dissimilarity. (2) Region-to-region variation in malacofauna was significantly correlated with region-to-region geographical distance. This suggests the influences of environments and paleontological events specific to each current surrounding each region. (3) The pattern of dissimilarity between the Inland Sea and the other sea areas greatly changed from the 1970s to 1980s. This was due in part to the following density fluctuations on some shores in the Inland Sea: increases in the non-indigenous mussel Mytilus galloprovincialis, cultured oyster Crassostrea gigas, and littorinid Littorina brevicula reportedly abundant in sheltered areas; and decreases in the littorinid Nodilittorina radiata reportedly abundant in exposed areas and muricid gastropods Thais spp. showing imposex. This suggests an increase of anthropogenic perturbations during the study period in the Inland Sea which is surrounded by large cities and ports.

Species composition and community structure of marine benthic community were studied in the intertidal zone of Jijindo Island, Gwangyang Bay. A total of 85 species of benthic marine plants including the 5 Cyanophyta, 9 Chlorophyta, 13 Phaeophyta, 56 Rhodophyta and 2 Magnoliophyta that were listed. The community structure was represented by Gelidium divaricatum in the upper zone, Celidium divalicatum and Ulva pertusa in the middle, and Ulva pertusa, Chondria crassicaulis, Chondracanthus intermedia and Sargassum thunbergii in the lower intertidal zone. The dominant species in the rnscro-zoobenthic community were Chthamalux challengeri in the upper and middle, Littorina brevicula between the upper and middle, and Mytilus edulis and Crassostrea gigas in the middle and lower intertidal zones. The economic benthic organisms such as Undaria pinnatifida, Mytilus edulis, and Crassostrea gigas found in the lower intertidal zone were frequently disturbed due to their collection by local resident. Therefore, it is necessary to record the correct information pertaining to these cases. The species diversity indices estimated kom different sources were quite different. They were 2.22 derived from frequency, 1.67 based on coverage, 2.17 based on sum of frequency and coverage and 2.04 derived from importance value. Species diversity and number of algal species in Gwangyang Bay have noticeably decreased, compared with their previously reported status. It is estimated that their decreases were caused by changes in the marine environment, especially pertaining to the polychaete community resulting from reclamation and dredging activity undertaken for the industrial development.

The fauna associated with mussel (Mytilus galloprovincialis) beds and vertical distribution of macrobenthos were studied on the Goijeong rocky shore in Jinhae Bay, Korea. Sampling was conducted seasonally using a quadrate from January to October, 1998. In the first experiment, to reveal the community structure of macrobenthos in mussel bed, a total of 62 species were identified, which included polychaetes molluscs (16 spp.), crustaceans (10 spp.), and others. The density-dominant species were Pseudopolydora antennata, Nereis heterocirrata, Littorina brevicula and Balanus kondakovi. And the density of associated fauna was correlated with that of mussel (r

Because Korean waters are influenced by different minor current systems, this may lead us to expect certain genetic differentiation among populations of marine invertebrates around Korean coastlines. To test this idea, we examined the genetic differentiation of 14 populations of a widely distributed marine gastropod, Littorina brevicula, using mtDNA markers, cyt b and ND6. Sequencing analyses of 500 bp fragment of the cyt b and 501 bp of the ND6 mtDNA revealed the existence of 14 and 24 haplotypes, respectively. Geographic association with population genetic variability was tested using a nested clade analysis (NCA). However, no significant association between geography and haplotype distribution was found, which suggests the existence of a high level of gene flow of L. brevicula around Korean waters. The lack of genetic structure and likely high gene flow among these populations, even under influences of different minor current systems, may be accounted for long periods of dispersal of the planktonic larvae from this gastropod species within major currents on the north western Pacific Ocean.

Littorina brevicula (Philippi) is an abundant herbivorous gastropod species inhabiting intertidal zones of the north-western Pacific and has internal fertilization with planktonic eggs and a larval stage. A population of L. brevicula on a shore in Amakusa, Japan, splits into an upper and a mid shore sub-population during the mating season in winter, while all individuals occurred in the upper zone in summer. Transplant and recapture experiments have shown that snails from each sub-population have a distinct pattern of seasonal migration, suggesting that the snail population consists of two behavioral morphs. To explore the relationship between migration pattern and life history characteristics in the snails of the two migration morphs, monthly quantitative samplings and a mark-release and recapture survey were carried out for the population of L. brevicula over a 1-year period. In autumn, some of the adults migrated downward to the mid shore and formed the lower sub-population from October to April. Migration between the upper and lower sub-populations was minimal from December to February, when L. brevicula copulate actively. The shell growth rates of the snails in the lower zone were slightly higher than those in the upper zone, but the shell width of females was not a good predictor of the number of mature ova they contained. The number of mature ova was not significantly different between the females of the two migration morphs. In addition, the mortality of marked snails did not differ significantly between the upper and lower zones. Thus, no evidence was obtained to indicate a difference in the reproductive fitness between the two migration morphs.

To elucidate the differences between the detoxification mechanisms of essential metal (Zn) and non-essential metal (Cd) in Littorina brevicula that is highly resistant to a wide range of heavy metal concentrations, Asian periwinkles were exposed to Cd (400 microg/L), Zn (3000 microg/L) and a mixture of both metals. We examined metal accumulation, elimination and subcellular distribution for binding to proteins. The metal concentration in L. brevicula increased gradually with exposure time (up to 70 days), following which accumulated levels reached saturation point. The accumulated Zn content was increased in the presence of Cd, while Cd uptake was decreased when Zn was present. During the depuration period (42 days), Cd was not removed from periwinkles, while Zn was eliminated in a rate of 2.19 microg Zn g(-1) day(-1). This elimination rate was particularly high on exposure to the metal mixture (3.80 microg Zn g(-1) day(-1)). Subcellular distribution studies on Cd and Zn revealed that most Cd (80%) was bound to cytosolic ligand, while more than 75% Zn was distributed in the membrane (insoluble) fraction. An additional difference in sequestering of metal in the cytosol was noted between Cd and Zn; most Cd in the cytosol was bound to metallothionein-like cadmium binding protein, MBP-1 (9.8 kDa), while the profile for Zn distribution revealed the presence of four Zn-binding ligand peaks, specifically, HMW (60 kDa), MBP-1 (9.8 kDa), MBP-2 (5 kDa) and LMW (1 kDa). Our data confirm that metallothionein-like cadmium binding protein, MBP-1, has the same affinity for Zn, while MBP-2 displays comparatively higher affinity for Zn than Cd.

Changes in genetic variability and allele frequency can be responses from natural populations when encountering a novel contaminated environment. The genetic diversity and population structuring of natural populations of the gastropod Littorina brevicula from heavy-metal polluted and unpolluted environments along the southeast coast of Korea were examined using two mtDNA markers, cyt b and ND6. This study applied a nested clade analysis to test the existence of structuring association of haplotype distribution with environments (polluted and unpolluted). No genetic differences within cyt b mtDNA were detected between environments. On the other hand, differences in population haplotype diversity and structuring were found within ND6 mtDNA between polluted and unpolluted environments. The ND6-mtDNA haplotype (= genetic) diversity was significant lower in polluted environments. This decreased genetic diversity along with differences in the haplotype distribution within heavy-metal polluted environments compared to those unpolluted ones stand out as emergent effects from pollution at a population level. In this study, we propose the use of different approaches, such as the NCA, that takes into account the rare haplotypes, when assessing the effects of pollution on population genetic structuring. (C) 2002 Elsevier Science Ltd. All rights reserved.

The toxic effects of heavy metals on organisms have received considerable scientific interest in recent years. Heavy metal ions are waste products of many industrial processes and be released into the ocean, causing environmental pollution. Environmental physiological stresses allow us to subject a polymorphic population which would be easily affected by these stresses, and determine the differential fitness of allozyme genotypes in the field. A lot of evidence drawn from both natural and laboratory populations indicate that the distribution of allozyme polymorphism is nonrandom and varies in spatio-temporal environmental variation (Bryant 1974; Nelson and Hedgecock 1980). In laboratories, the effects of pollution on the genetic structure of marine organisms has been studied. It has been reported that the marine animals selected the specific genotypes on the effect of heavy metal (de Nicola et al. 1993; Benton et al. 1992 a, b; Lavie and Nevo 1982, 1986; Ben-Shlomo and Nevo 1988; Nevo et al. 1981). These experiments have shown both tolerance and sensitivity of allozyme genotypes to the pollutants. They have also shown that heavy metals affect the enzyme functions and the sex ratio of the species. Unfortunately, most of the studies were performed to determine the effects of pollutants on marine organisms in the laboratory, and they did not determine allozyme variance of natural population in the field.

Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28 kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5′- and 3′-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129 bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43–53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.

Relationships between community structure and environmental factors were sought through multivariate analysis (quantification analysis and correspondence analysis) of data obtained in a benthic macrofauna survey of a rocky low-tide platform located at Usujiri, southern Hokkaido, Japan. A total of 21 species were found. However, their annual mean abundance was dominated by only a few species. Three dominant species of the platform (Chthamalus challengeri, Littorina brevicula andSeptifer virgatus) accounted for an average of 92% of total abundance. Three environmental factors with associated species groups were examined. Among them, micro-topographic characteristics and wave action defined the main correspondence variables.L. brevicula was a characteristic member of the boulder and sheltered field.S. virgatus was the dominant species of sloped surfaces.C. challengeri, S. virgatus, andMytilus edulis were characteristic of ledge or bench environments. Finally,C. challengeri was characteristic of nip and high wave-exposure environments, given its exclusive appearences in these locations. However, as the degree of exposure depends on the scale of wash through the platform, it is directly limited by the microtopographic properties of low-tide platforms. Specific environmental influences such as exposure and height above the datum-plane were rather disapparent. Any overall effects on the community structure were synthesized by those of microtopography. After complete consideration, it was concluded that the community structure of the macrobenthos in this low-tide platform was found to be primarily under the influence of microtopographic characteristics.

1 Plants must either tolerate consumption or defend themselves against grazer attacks. Selection for phenotypically plastic antiherbivory responses has been suggested for many plants, including a few species of seaweed, but little is known about its specificity or seasonality. 2 Multi-factorial experiments tested the effects of consumer identity (Littorina brevicula vs. Haliotis discus) and season (summer vs. autumn) on the induction of antiherbivory defences in the brown seaweed Ecklonia cava. Following a grazer-free acclimation phase, algae were incubated with grazers (treatment phase) and, subsequently, without grazers (recovery phase). Feeding preference assays, were used to assess differences in consumption rates between grazer-exposed and control plants. 3 In summer, Littorina, but not Haliotis, induced defence in Ecklonia. This defence vanished by the end of the recovery phase. In autumn, neither exposure to direct attack nor to waterborne cues induced defensive responses. 4 Both consumer identity and season of consumption can influence the ability of a given macroalgal species to induce antiherbivory defences. Tailoring such responses to spatial and temporal variation in grazer pressure could have profound ecological implications, for example changing food webs and community structure.

The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 μM CuCl 2 ) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 μg/l CuCl 2 ). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C 18 reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula . The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.

Although mussels and oysters in the ocean are known to act as bioconcentrators for contaminants such as heavy metals, their ability to survive in heavily polluted water is relatively limited. The Asian periwinkle, Littorina brevicula, is one species that can accumulate a variety of environmental heavy metals, and the expression of its metal binding protein (MBP) is induced by cadmium. To better characterize this protein and its detoxification mechanism against cadmium, the present work examined the induction of a cadmium binding protein (Cd-BP) in Littorina brevicula exposed to 400 μg/l CdCl2 for 30 days. The induced Cd-BP was purified by chromatography from the supernatants of homogenized organs (digestive gland, gonad, gill and kidney). This Cd-BP was found to consist of 103 amino acids, was rich in Cys (21 residues), and partial C-terminal sequence obtained by MALDI-TOF MS analysis revealed a Cys-XXX-Cys motif, which resembles a typical feature of mollusc metallothionein (MT). The Cd-BP molecular weight of 9.8 kDa is a little larger than that of other MTs.

Concentrations of Al, Fe, Mn, Cu, Zn, V, Ni, Co, Cr, Cd and Pb in the Asian periwinkle Littorina brevicula were determined from 39 sampling locations along the whole Korean coast. Metal concentrations in the periwinkle varied highly with sampling stations, but the variations could be well explained by the body size after eliminating the data exceeding the 'High' criteria. Among the stations exceeding the 'High' criteria corresponding to mean plus one standard deviation of the logarithms of the individual site mean, some stations exhibited values higher than expected from the relationship between metal body burden and body size, indicating that these locations were polluted with metals. However, several 'High' stations followed the trends of the curve, suggesting that they resulted from the effect of their small body size, not from environmental contamination. Periwinkles of similar body sizes exhibited same range of metal concentrations in their body, independent of geographical position.

Cadmium (Cd), lead (Pb), copper (Cu) and zinc (Zn) concentrations in the marine gastropod, Littorina brevicula Philippi, were determined to assess the metal pollution in Onsan Bay, Korea. Samples of L. brevicula employed as a biomonitor and seawater were collected from 12 to 20 stations of Onsan Bay in November 1997, respectively. Dissolved metal concentrations in surface seawater were highest at the station near Onsan Non-ferrous Industrial Complex: 1.15 micrograms l-1 for Cd, 2.49 micrograms l-1 for Pb, 3.75 micrograms l-1 for Cu and 23.98 micrograms l-1 for Zn. These values were 1-2 orders higher than those shown at outer regions of the Bay. Metal concentrations in the soft body of periwinkles were highly variable at different sampling locations: 0.48-27.11 micrograms g-1 for Cd, 1.41-24.91 micrograms g-1 for Pb, 57-664 micrograms g-1 for Cu and 83-246 micrograms g-1 for Zn. The values from stations near the industrial complex were higher than those expected from relationships between body sizes and metal body burdens in periwinkles collected from the whole Korean coast. Spatial distribution of metal concentrations in the periwinkle and seawater indicated that Onsan industrial complex near the Bay is the input source of these metals. Especially, Cd and Pb concentrations in the periwinkle and seawater were distinctly decreased with distance from the Onsan industrial complex. Non-essential metals such as Cd and Pb in the periwinkle showed a strong correlation with dissolved metal concentrations in seawater. Conversely, essential Cu and Zn in the periwinkle were hardly explained by those in seawater, except at the most contaminated sites.

An endo-1,4-beta-D-mannanase (EC 3.2.1.78) was purified from viscera of a marine mollusc Littorina brevicula. The purified enzyme, with a molecular weight of 42,000, was homogeneous by SDS-PAGE. The amino-terminal sequence starting with Gly was analyzed up to the 30th amino acid. The enzyme was stable from pH about 4.0 to about 9.0 and had its maximum activity at pH about 6.5. The purified enzyme produced M2, M3, M4, and M5 from Codium beta-1,4-mannan. The enzyme activity was greatly inhibited by Ag+, HG2+, Cu2+, and N-bromosuccinimide at 1 mM concentration.

Several carbohydrase activities were determined on the crystalline styles and the hepatopancreata from 50 species of shellfish.Laminarinase activity was the highest of other carbohydrase activities in most species. The activity was generally higher in bivalves than in gastropods, especially the crystalline style of certain species such as Gomphina melanaegis, surf clam Mactra chinensis, egg cockle Fulvia mutica have remarkable high activities, reducing sugars produced by those species during 2h at 37°C were 50-70% of the initial substrate weigh. On the contrary, the degradation activities on structural polysaccharides were rather high in small herbivorous gastropods; especially Acmaeidae on alginic acid, Patellidae on xylan, and Littorina brevicula, Batillus cornutus, Patelloida sp. on microcrystalline cellulose, respectively. Although the degradation activity on fucoidan was widely distributed, it was not so strong in most of the species. Degradation activities on the Kombu powder and the Kombu extraction residue were also determined in shellfish, and compared with a commercial cellulase preparations. The activity of chiton Liolophura japonica was close to that of the commercial enzyme on the degradation of the Kombu powder, while sugars produced due to both enzymes were quite different.