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.

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.

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.