Manila clam Venerupis philippinarum as a biomonitor to metal pollution

The Manila clam Venerupis philippinarum is a good biomonitor/bioindicator to marine metal pollution and is frequently used in aquatic toxicology. Two dominant pedigrees (white and zebra) of clam are distributed in the Bohai Sea; however, little attention has been paid to potential biological differences between these two pedigrees. In this study, we tested the sensitivity of both pedigrees to marine metal (cadmium and zinc) pollution biomonitoring and marine environmental toxicology. Results demonstrate significant biological differences in gills of white and zebra clams based on metabolic profiles and antioxidant enzyme activities. In addition, we found that hypotaurine, malonate and homarine were relatively high in white clam gills, while alanine, arginine, glutamate, succinate, 4-aminobutyrate, taurine and betaine were high in zebra clam gills. Zebra clam gills were also more sensitive to a mixture of Cd and Zn, as shown by antioxidant enzyme activities and metabolic profiles, but white clam gills could accumulate more Zn. Therefore, we suggest that the white pedigree can be used as a biomonitor to marine Zn pollution, whereas the zebra pedigree can be used for toxicology studies on Cd and Zn mixed pollution.

Larval and Juvenile Growth Performance of Manila Clam Hybrids of Two Full-Sib Families

In order to determine whether growth performance could be improved by hybridizing full-sib families of Manila clam （Ruditapes philippinarum）, crosses between two full-sib families including self and reciprocal crosses were carried out. The effects of heterosis, combining ability and interaction on the growth of shell length were estimated. The results showed that the growth of hybrid larvae was intermediate between parents on days 6 and 9. Heterosis on shell length was observed, which varied at juvenile stage. The cross of ♂A×♀B （Hp varied between 10.41% and 68.27%） displayed larger heterosis than ♂A×♀B （Hp varied between 1.89% and 32.33%） did, suggesting that ♂A×♀B was an ideal hatchery method of improving the growth performance of Manila clam. The variances of general combining ability （GCA）, special combining ability （SCA） and interaction （I） were significant in shell length （P〈 0.05）, indicating that both additive and non-additive genetic factors were important contributors to the growth of larvae and juveniles. The GCA for shell length of ♂A×♀B was higher than that of ♂A×♀B at both larval and juvenile stages. This con- firmed that the cross between ♂A and ♀B showed great growth in shell length. In summary, the growth of Manila clam seeds could be improved by hybridizing selected parents from large numbers of full-sib families.

Effect of Epinephrine on the Settlement and Metamorphosis of Manila Clam Larvae

Chemical inducement and DDRT-PCR （differential display reverse transcription PCR） are adopted to investigate the effect of epinephrine （EPI） on the settlement and metamorphosis of Manila clam larvae. Chemical inducement shows that EPI has an effect to some extent on the metamorphosis of Manila clam larvae at all concentrations and in all treatments designed. The most significant result of inducement is obtained at the concentration of 10^-6 tool L^-1 and for 4 h. DDRT-PCR using six primer pairs shows that the gene expression pattern is quite different between EPI treatment and the control. Three hundred and forty-three amplification bands are obtained in total, among which, 67 （19.53%） are differentially appeared. Therefore, EPI has an effect on the gene expression of the eye spot larval Manila clam. It can be hypothesized that EPI is a settlement and metamorphosis inducer for Manila clam. EPI may lead to larvae settlement and metamorphosis by binding to the receptors on the membrane and then changing the gene expression of larvae cells.

Identification of Perkinsus-like parasite in Manila clam, Ruditapes philippinarum using DNA molecular marker at ITS region

Genomic DNA was extracted from hypnospores of Perkinsus-like parasite of Manila clam Ruditapes philippinarum collected at the fishing grounds in Huanghai Sea coast Shicheng Island and East China Sea coast Ningbo, China. The internal transcribed spacer（ITS） in rDNA was PCR-amplified, cloned, sequenced, and compared with that of five Perkinsus species in GenBank. The fragment amplified from DNA of parasite of either Shicheng Island or Ningbo contained 649 bp, including partial ssrRNA（51 bp） and ITS（＋5.8 S） （598 bp） regions. The ITS（＋5.SS） sequences of Perkinsus-like parasite of both Shicheng Island and Ningbo were all 99% identical to those ofPerkinsis atlanticus, and were not more than 95% identical to those of other four Perkinsus species including P. marinus, P. andrewsi, P. qugwadi and P. medierraneus.The ITS （＋5.8S） sequence of Perkinsus-like parasite of Shicheng Island was 99% identical to that of Ningbo. These facts about nucleotide sequences suggested that the Perkinsus-like parasite in Manila clam, Ruditapes philippinarum collected from either the Huanghai Sea coast or the East China Sea coast was P. atlanticus, and might reflect P. atlanticus strains of distinct geographic distribution.

Annual Variations of Biogenic Element Contents of Manila Clam(Ruditapes philippinarum) Bottom-Cultivated in Jiaozhou Bay, China

Manila clam （Ruditapes philippinarum） was monthly sampled from its benthic aquaculture area in Jiaozhou Bay from May 2009 to June 2010.The annual variations of major elemental composition,organic content,fatness and element ratio of Manila clam were examined.The element removal effect of clam farming in Jiaozhou Bay was analyzed based on natural mortality and clam harvest.The results indicated that the variation trend of carbon content in shell （Cshell） was similar to that in clam （Cclam）.Such a variation was higher in summer and autumn than in other seasons,which ranged from 9.10±0.13 to 10.38 ± 0.09 mmol g^-1 and from 11.28 ±0.29 to 12.36±0.06 mmol g 1,respectively.Carbon content of flesh （Cflnesh） showed an opposite variation trend to that of shell in most months,varying from 29.42 ± 0.05 to 33.64 ± 0.62 mmol g^-1.Nitrogen content of shell （Nshell） and flesh （Nflesh） changed seasonally,which was relatively low in spring and summer.Nshell and Nflesh varied from 0.07 ±0.009 to 0.14±0.009 mmol g^-1 and from 5.46 ± 0.12 to 7.39 ± 0.43 mmol g^-1,respectively.Total nitrogen content of clam ranged from 0.50 ± 0.003 to 0.76 ± 0.10 mmol g^-1 with a falling tend except for a high value in March 2010.Phosphorus content of clam （Nclam） fluctuated largely,while phosphorus content of shell （Pshell） was less varied than that of flesh （Pflesh）.Pshell varied from 0.006±0.001 to 0.016±0.001 mmol g^-1; while Pflesh fluctuated between 0.058±0.017 and 0.293±0.029mmolg^-1.Pclam ranged from 0.015±0.002 to 0.041 ±0.006mmolg^-1.Carbon and nitrogen content were slightly affected by shell length,width or height.Elemental contents were closely related to the reproduction cycle.The removal amounts of carbon,nitrogen and phosphorus from clam harvest and natural death in Jiaozhou Bay were 2.92× 10^4t,1420t and 145 t,respectively.The nutrient removal may aid to reduce the concentrations of nitrogen and phosphorus,the main causes of eutrophication,and to maintain the ecosystem health of Jiaozhou Bay.

Impact of seawater acidification on shell property of the Manila clam Ruditapes philippinarum grown within and without sediment

Although the impact of ocean acidification on marine bivalves has been previously investigated under mainly controlled laboratory conditions,it is still unclear whether the impact of acidification on sediment-burrowing species differs between those within or without sediment.In order to fill this gap in our knowledge,we compared shell properties of the infaunal Manila clam(Ruditapes philippinarum)exposed to three pH concentrations(7.4,7.7,and 8.0),within and without sediments.In the first experiment(140 d),clams were exposed to seawater in an acidification system without sediment.A decrease in shell weight corresponding to the increase in dissolution rate was observed in the group ofpH 7.4,at which shell color disappeared or whitened.SEM observations confirmed the changes of the external shell surface.In the second experiment(170 d),sediment was placed at the bottom of each exposure chamber.The effects were found obvious in shell dissolution rate and shell color in the shell specimens exposed to overlying seawater but not found in the shell specimens exposed to sediment.Although the experimental period was longer in the second experiment,shell specimens in the first experiment were more seriously damaged than those in the second experiment under acidic seawater conditions.Our results,in relation to the defense function of the shell,show that marine bivalves in burrowing behavior are more adaptable to seawater acidification than those who do not burrow into sediment.

Growth Performance of Larval and Juvenile Manila Clam(Ruditapes philippinarum) from Divergently Selected Individuals of a Full-Sib Family

Abstract In this study, the method of divergent selection was employed to test the larval and juvenile growth performance within a full-sib family of Manila clam Ruditapes philippinarum. The 10% largest and 10% smallest clam individuals （on the basis of shell length） of a full-sib family were selected as parents for the fast and slow growing lines, respectively. The difference in shell length was significant among the three lines （fast, control, and slow） tested. The sequence of shell length were fast line 〉 control line 〉 slow line. The responses to selection, realized heritability, and genetic gain were 0.06%-0.81%, 0.04%-0.47% and 0.58%-18.89% in the fast direction, respectively; and were 0.14%-1.27%, 0.08%-0.73%, and 0.31%-49.03% in the slow direction, respectively. The re- suits suggested that there was a large portion of additive genetic variance affecting the growth in the full-sib family. Selection in the fast direction within the full-sib family would greatly improve the growth ofR. philippinarum.

First molluscan antimicrobial peptide hydramacin in Manila clam:molecular characterization and expression analysis

Objective:To investigate molecular characterization and the immune responses of Manila clam hydramacin(Rp-hdmc).Methods:cDNA sequence of hydramacin was isolated from Manila clam transcriptome database.Molecular characterization of hydramacin cDNA was performed by BLAST and SWISS-MODEL bioinformatics programs.Tissue-specific expression and transcriptional regulation after Vibrio tapetis challenge was done by quantitative real time PCR.Results:Rp-hdmc has 291 bp open reading frame(ORF),encoding 97 amino acids with a mature hydramacin consisting of 77 amino acid residues.In un-challenged clam,Rp-hdmc was constitutively expressed in all tested tissues and the highest expression level was detected in gill.After pathogenic bacteria Vibrio tapetis challenge,Rp-hdmc mRNA was up-regulated in gill and hemocytes.Conclusions:We identified hydramacin cDNA(Rp-hdmc)from mollusk Manila clam that shows the characteristic features of hydramacin sequence.It has eight cysteine residues with four disulfide linkages,three helices and two β-strands in secondary structure.Expression results after V.tapetis challenges suggest that Rp-hdmc is involved in immune response against pathogenic bacteria.

Using lysosomal membrane stability of haemocytes in Ruditapes philippinarum as a biomarker of cellular stress to assess contamination by caffeine, ibuprofen, carbamazepine and novobiocin

Although pharmaceuticals have beendetected in the environment only in the range from ng/L to μg/L, it has beendemonstrated that they can adversely affect the health status of aquatic organisms. Lysosomal membrane stability （LMS） has previously been applied as an indicator of cellularwell-being todetermine health status in bivalve mussels. The objective of this study is to evaluate LMS in Ruditapes philippinarum haemolymph using the neutral red retention assay （NRRA）. Clamswere exposed in laboratory conditions to caffeine （0.1, 5, 15, 50 μg/L）, ibuprofen （0.1, 5, 10, 50 μg/L）, carbamazepine and novobiocin （both at 0.1, 1, 10, 50 μg/L） for35days. Results show adose-dependent effect of the pharmaceuticals. The neutral red retention time measured at the end of the bioassaywas significantly reduced by 50% after exposure to environmental concentrations （p 〈 0.05） （caffeine = 15 μg/L; ibuprofen = 10 μg/L; carbamazepine = 1 μg/L and novobiocin = 1 μg/L）, compared to controls. Clams exposed to these pharmaceuticalswere considered to present adiminished health status （retention time 〈45 min）, significantlyworse than controls （96 min） （p 〈 0.05）. The predicted no environmental effect concentration （PNEC） results showed that these pharmaceuticals are very toxic at the environmental concentrations tested. Measurement of the alteration of LMS has been found to be a sensitive technique that enables evaluation of the health status of clams after exposure to pharmaceuticals under laboratory conditions, thus representing a robust Tier-1 screening biomarker.