Suitable habitat of Lepidochelys olivacea and the changes under climate change.

As a vulnerable species identified by the International Union for Conservation of Nature (IUCN), Lepidochelys olivacea has attracted extensive attention in recent years. To examine its current distribution and that under future climate change scenarios, we compiled the occurrence data of L. olivacea. With eight predictor variables, including depth, offshore distance, mean primary productivity, minimum primary productivity, mean sea surface temperature, minimum sea surface temperature, mean sea surface salinity, and minimum sea surface salinity, we predicted its distribution in an ensemble species distribution model. The accuracy of the model was evaluated with the parameters of areas under curves (AUC) and true skill statistics (TSS). The results showed that the AUC and TSS values were 0.96 and 0.81, respectively, indicating a good predictive performance of the ensemble model. Sea surface temperature and salinity were the two most important variables determining the distribution of L. olivacea, with the suitable temperature ranging from 23 to 29 ℃ and salinity below 34. The current distribution range of L. olivacea was between 30° N-25° S. Under future climate scenarios, its distribution range would decrease, especially under the RCP85 scenario in the 2100s (with a 28% reduction in the suitable survival range). The results of model validation showed that it had high accuracy and could make accurate predictions of the distribution. This study would provide references for the development of more rational conservation measures and management strategies.

Cloning, promoter analysis and expression of the tumor necrosis factor receptor-associated factor 6 (TRAF6) in Japanese scallop (Mizuhopecten yessoensis).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key adaptor molecule for the tumor necrosis factor superfamily and Toll-like/interleukin-1 receptor superfamily. It plays an important role in innate and adaptive immunity. The TRAF6 of Japanese scallop Mizuhopecten yessoensis (designated as MyTRAF6) was identified and characterized in this study. The full-length cDNA of MyTRAF6 was 2,407 bp, which consisted of 239-bp 5'-terminal untranslated region, 1,974-bp open reading frame encoding a polypeptide of 657 amino acids, 194-bp of 3'-terminal untranslated region followed by a canonical polyadenylation signal sequence AATAAA and a poly (A) tail. The predicted amino acid sequence of MyTRAF6 contained the characteristic motifs of TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, and a MATH (meprin and TRAF homology) domain. It had an overall identity of 43-96% with those of other TRAF6s, the highest identity (96%) with Chlamys farreri TRAF6, and the least identity (43%) with Meleagris gallopavo TRAF6. Phylogenetic analysis classified MyTRAF6 as a true TRAF6 ortholog. In addition, the promoter of MyTRAF6 was also identified by genome walking. It contained several potential transcription factor-binding sites and three single nucleotide polymorphisms. qRT-PCR analysis revealed that MyTRAF6 was highly expressed in hemocytes of adult M. yessoensis. MyTRAF6 transcript level in the hemocytes reached a maximum 6 h after Vibrio anguilarum challenge. The results indicated that MyTRAF6 may fulfill an important function during M. yessoensis bacterial infection. It could be a key effector molecule involved in the innate defense of molluscs.

Identification and expression of a putative LPS-induced TNF-α factor from Asiatic hard clam Meretrix meretrix.

LPS-induced TNF-α (LITAF) is a novel transcriptional factor that mediates the expression of inflammatory cytokines in LPS-induced processes. In the present study, the full-length cDNA encoding LITAF (designated as Mm-LITAF) was identified from Asiatic hard clam, Meretrix meretrix, by expressed sequence tag and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Mm-LITAF was 1653 bp, consisting of a 5' untranslated region (UTR) of 91 bp, a 3'UTR of 1166 bp with one cytokine RNA instability motif (ATTTA) and one polyadenylation signal (AATAAA), and an open reading frame (ORF) of 396 bp encoding a polypeptide of 131 amino acids with a theoretical isoelectric point of 7.49, and predicted molecular weight of 14.47 kDa. The deduced amino acid of Mm-LITAF shared 29-63% similarity with the LITAFs from other species, indicating that Mm-LITAF should be a member of the LITAF family. Two highly conserved CXXC motifs forming a compact Zn(2+)-binding structure were also identified in Mm-LITAF. A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the expression of Mm-LITAF mRNA in different tissues, and the temporal expression of Mm-LITAF in clams challenged with Vibrio anguillarum. The mRNA transcript of Mm-LITAF could be detected in all the examined tissues with the highest expression level in the gill. Mm-LITAF expression was up-regulated significantly at 16 h in the gill and at 8 h in haemocytes after bacterial challenge, respectively. These results suggest that the Mm-LITAF is a constitutive and inducible acute-phase protein that perhaps involved in the innate immune response of hard clam.

Identification of host-defense genes and development of microsatellite markers from ESTs of hard clam Meretrix meretrix.

The hard clam Meretrix meretrix is an economically important shellfish in China. However, genomic research on this species is still at early stage, and few genomic resources are available. The objective of the present study was to generate expressed sequence tags (ESTs), and identify host-defense genes and microsatellite markers for M. meretrix. Three cDNA libraries for intestine, mantle and hepatopancreas were constructed using highly efficient SMART (Switching Mechanism At 5' end of the RNA Transcript) method. A total of 3224 random clones were single-pass sequenced from 5'-ends, resulting in 3129 high-quality (>100 bp) ESTs averaging 734 bp. All the ESTs were assembled by software Cap 3, producing 1796 unigenes-1490 singletons and 306 contigs. All the unigenes were compared to the public protein database using tblastx, and 696 (38.8%) were homologues to known genes while the remaining 1100 (61.2%) appeared to be novel sequences. A total of 31 EST clusters were related to immune and defense functions. They included immune recognition receptors, proteases and protease inhibitors, and other immune-related genes. The screening of 1796 unigenes identified 55 (3.1%) microsatellite-containing sequences, with 20 having sufficient flanking sequences for primer design. Polymerase chain reaction amplification was successful for 12 primer pairs and 7 of them showed polymorphic. The EST collection and microsatellite markers obtained in this study provide a useful resource for further gene discovery and population genetic analysis in M. meretrix.

The complete mitochondrial genome of the hard clam Meretrix meretrix.

Veneridae is a diverse, commercially important, and cosmopolitan family. Here we present the complete mitochondrial genome of the hard clam Meretrix meretrix (Bivalvia: Veneridae). The entire mitochondrial genome (mitogenome) sequence of M. meretrix is 19,826 bp in length, and contains 37 genes including 12 protein-coding genes, 2 ribosomal RNAs, and 23 tRNAs. All genes are encoded on the heavy strand. In contrast to the typical animal mitochondrial genome, it lacks the protein-coding gene ATP8, and has only one copy of the tRNA(Ser) gene, but three duplications of the tRNA(Gln), which is the first report among the present molluscan mtDNAs. We observed that the gene arrangement between M. meretrix and M. petechialis is same except one more tRNAGln gene in M. meretrix., and the sequence similarity is as high as 99%, indicating that M. petechialis and M. meretrix could be treated as a junior synonym of M. meretrix. Maximum Likelihood and Bayeslan analysis of 12 concatenated protein-coding amino acid sequences place the Unionidae as a sister group to other bivalves, which reflects the general opinion that the Unionidae deverged very early in Bivalvia evolution.

Sixteen polymorphic simple sequence repeat markers from expressed sequence tags of the Chinese Mitten Crab Eriocheir sinensis.

The Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species in China. In this study, we developed and evaluated simple sequence repeat markers from expressed sequence tags of E. sinensis. Among the 40 wild E. sinensis individuals tested, 16 loci were polymorphic. The number of alleles per locus ranged from two to ten. The observed heterozygosity ranged from 0.0667 to 0.9667, whereas the expected heterozygosity ranged from 0.0661 to 0.9051. These markers have the potential for use in genetic studies of population structure and intraspecific variation in E. sinensis.

Genetic differentiation between natural and hatchery stocks of Japanese scallop (Mizuhopecten yessoensis) as revealed by AFLP analysis.

Japanese scallop (Mizuhopecten yessoensis) is a cold-tolerant bivalve that was introduced to China for aquaculture in 1982. In this study, amplified fragment length polymorphism (AFLP) markers were used to investigate levels of genetic diversity within M. yessoensis cultured stocks and compare them with wild populations. Six pairs of primer combinations generated 368 loci among 332 individuals, in four cultured and three wild populations. High polymorphism at AFLP markers was found within both cultured and wild M. yessoensis populations. The percentage of polymorphic loci ranged from 61.04% to 72.08%, while the mean heterozygosity ranged from 0.2116 to 0.2596. Compared with wild populations, the four hatchery populations showed significant genetic changes, such as lower expected heterozygosity and percentage of polymorphic loci, and smaller frequency of private alleles, all indicative of a reduction in genetic diversity. Some genetic structures were associated with the geographical distribution of samples; with all samples from Dalian and Japan being closely related, while the population from Russia fell into a distinct clade in the phylogenetic analysis. The genetic information derived from this study indicated that intentional or accidental release of selected Japanese scallops into natural sea areas might result in disturbance of local gene pools and loss of genetic variability. We recommend monitoring the genetic variability of selected hatchery populations to enhance conservation of natural Japanese scallop resources.

[Comparison of mitochondrial genomes of bivalves].

The structure and organization of mitochondrial genomes of 14 marine bivalves and two freshwater bivalves were analyzed using comparative genomics and bioinformatics methods. The results showed that the organization and gene order of the mitochondrial genomes of these bivalve species studied were different from each other. The size, organization, gene numbers, and gene order of mitochondrial genomes in bivalves at different taxa were different. Phylogenetic analysis using the whole mitochondrial genomes and all the coding genes showed different results-- phylogenetic analysis conducted using the whole mitochondrial genomes was consistent with the existing classification and phylogenetic analysis conducted using all coding genes not consistent with the existing classification.

[Structure analysis of mtDNA control region of spotted halibut (Verasper variegatus) and its related species].

Spotted halibut (Verasper variegatus) is the only species of Genus Verasper in China. The fish was naturally distributed in Yellow Sea and Bohai Sea in northern China and Kyushu in Japan and in Korean sea area. Using PCR product direct sequencing, mitochondrial control region sequences of 24 individuals of spotted halibut was confirmed and analyzed. 4 control region haplotypes, resulting from length heteroplamy of the tandem repeat region, was obtained from these 24 fish. Sequence analysis demonstrated that there were four similar structures in the control region, i.e., extended terminal associated sequences (ETAS), central conserved sequence block (CSB), conserved sequence block (CSB), and repeat region, in V. moseri, Limanda ferruginea, Reinhardtius hippoglossoides, Heppoglossoides platessoides, Paralichthys olivaceous, Solea solea, S. senegalensis, and S. lascari. By comparing with other vertebrates, we found that there were similar repeated sequences immediately after the CSB-3 in all of the anuran species.