Zhihua Lin

Population Genetic Structure of the Blood Clam, Tegillarca granosa, Along the Pacific Coast of Asia: Isolation by Distance in the Sea

ABSTRACT The blood clam, Tegillarca granosa, is widely distributed along the coasts of the Indo-Pacific region, providing an excellent opportunity to study gene flow in sessile marine mollusks. In the present study, amplified fragment length polymorphism (AFLP) DNA markers were used to analyze the genetic structure of five blood clam populations. Genetic differentiation (Gst) and Neis genetic distances between population pairs were found to range from 0.0245 to 0.0785 and 0.0398 to 0.1125, respectively. An AMOVA analysis showed that 89.09% of the genetic variation occurred within populations and only 10.92% occurred among populations, indicating a high degree of genetic homogeneity, probably due to considerable gene flow (average Nm = 7.1853) among populations. Cluster analysis using the unweighted pair group method average (UPGMA) showed that geographically closer populations were clustered, suggesting isolation by distance, which was further supported by a significant positive correlation between Gst and logit transformed geographic distance through a Mantel test and reduced major axis regression (r = 0.77, p < 0.05). The evident isolation by distance observed among blood clam populations along the Pacific coast of Asia is likely to be a consequence of both the passive larval dispersal carried by the South China Sea Warm Current and the genetic homogeneity caused by aquaculture-associated human activities.

Identification and comparative analysis of the Tegillarca granosa haemocytes microRNA transcriptome in response to Cd using a deep sequencing approach.

Background MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group. Results Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR. Conclusion Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.

Identification of a regulation network in response to cadmium toxicity using blood clam Tegillarca granosa as model.

Clam, a filter-feeding lamellibranch mollusk, is capable to accumulate high levels of trace metals and has therefore become a model for investigation the mechanism of heavy metal toxification. In this study, the effects of cadmium were characterized in the gills of Tegillarca granosa during a 96-hour exposure course using integrated metabolomic and proteomic approaches. Neurotoxicity and disturbances in energy metabolism were implicated according to the metabolic responses after Cd exposure, and eventually affected the osmotic function of gill tissue. Proteomic analysis showed that oxidative stress, calcium-binding and sulfur-compound metabolism proteins were key factors responding to Cd challenge. A knowledge-based network regulation model was constructed with both metabolic and proteomic data. The model suggests that Cd stimulation mainly inhibits a core regulation network that is associated with histone function, ribosome processing and tight junctions, with the hub proteins actin, gamma 1 and Calmodulin 1. Moreover, myosin complex inhibition causes abnormal tight junctions and is linked to the irregular synthesis of amino acids. For the first time, this study provides insight into the proteomic and metabolomic changes caused by Cd in the blood clam T. granosa and suggests a potential toxicological pathway for Cd.

Characterization of 34 polymorphic EST-SSR markers in Tegillarca granosa and their transferability in Anadara craticulata

EST-SSR is one of important methods to isolate microsatellite loci,which is low cost,high transferability among phylogenetically related species and a probability of being associated with functional regions of the genome.34 polymorphic EST-SSR markers were isolated and characterized in the blood clam,Tegillarca granosa,from the ESTs generated by 454 sequencing.The number of alleles for the 34 SSR markers in 30 individuals of Zhejiang Fenghua population varied from 2 to 7,with an average of 3.59 alleles per locus.The observed heterozygosity ranged from 0.000 to 0.600,while the expected heterozygosity varied from 0.078 to 0.771.Polymorphic information content ranged from 0.106 to 0.718,and 13 SSR loci deviated from Hardy-Weinberg equilibrium.25 EST-SSR identified from annotated genes were expected to be useful for mapping these genes in linkage maps.Interspecific transferability of the 34 markers revealed that 8 were polymorphic in Anadara craticulata,resulting in a transferability rate of 23.53%.To our knowledge,this is the first report on the survey of SSR transferability in blood clams.These novel polymorphic EST-SSR markers should be particularly useful for comparative mapping,gene tagging and QTL mapping among blood clams.

Identification of 30 microsatellite markers in Meretrix meretrix and their transferability in Meretrix lamarckii and Meretrix lyrata

30 microsatellite loci of Mereteix meretrix were developed using microsatellite-enhanced genomic library and expressed sequence tags(ESTs).Genetic diversity was analyzed in a population consisting of 30 individuals.8 G-SSRs and 22 EST-SSRs were found to be polymorphic.In these 30 microsatellite loci,a total of 140 alleles were identified,and the number of alleles varied from 3 to 6,with a mean of 4.67 alleles per locus.Polymorphic information content(PIC)ranged from 0.172 to 0.744.The observed heterozygosity(Ho)was from 0.040 to 0.720,while the expected heterozygosity(He)was from 0.187 to 0.793.The mean values of number of alleles,PIC,Ho,and He of G-SSRs were higher than those of the EST-SSRs.24 of the 30 loci deviated from the Hardy-Weinberg equilibrium after Bonferroni correction.11 EST-SSR identified from annotated genes were expected to be useful for mapping these genes in linkage maps.Interspecific transferability of the 30 markers revealed that 12 and 9 were polymorphic in M.lamarckii and M.lyrata,respectively.These loci obtained will lay a foundation for the study of genetic diversity,germplasm resources evaluation,marker-assisted selection(MAS)and other relevant research in M.meretrix,and these novel polymorphic SSR markers can be used for comparative mapping,gene tagging and QTL mapping among M.meretrix,M.lamarckii and M.lyrata.

Genetic analysis among four strains of different shell colors and decorative patterns of Meretrix meretrix using microsatellite markers

Genetic variation of four strains of Meretrix meretrix,thin checkered(TC),black spot(BS),dark fringe(DF)and red shell(RS)was studied.Thirty randomly selected individuals from each strain and nine polymorphic microsatellite loci were used for genetic analysis.A total of 105 alleles were identified,the most alleles were 21(WG07)and the least locus were 7(WG11).The distribution of the alleles for the same loci was different in different strains and specific allele types were detected in each strains.All the microsatellites were highly polymorphic,and various genetic variability measures,including observed heterozygosity(0.486 to 0.867),expected heterozygosity(0.110 to 1.000),and polymorphism information content(0.433 to 0.834),Chi-square tests showed that most of the cases in four strains deviated from Hardy-Weinberg equilibrium.Pairwise FST ranged from 0.005 to 0.269.Genetic distances among strains ranged from 0.052 0 to 0.098 0.The results of cluster analysis indicated that TC strain had higher genetic difference than other strains,then was BS strain,and the genetic variation between DF and RS strains was relatively lower.

Nfu1 Mediated ROS Removal Caused by Cd Stress in Tegillarca granosa

The blood clam Tegillarca granosa, a eukaryotic bottom-dwelling bivalve species has a strong ability to tolerate and accumulate cadmium. In our previous study, Nfu1 (iron-sulfur cluster scaffold protein), which is involved in Fe-S cluster biogenesis, was shown to be significantly up-regulated under Cd stress, as determined by proteomic analysis. To investigate the function of Nfu1 in cadmium (Cd) detoxification, the function of blood clam Nfu1 (designated as Tg-Nfu1) was investigated by integrated molecular and protein approaches. The full-length cDNA of Tg-Nfu1 is 1167 bp and encodes a protein of 272 amino acid residues. The deduced Tg-Nfu1 protein is 30 kDa contains a conserved Nfu-N domain and a Fe-S cluster binding motif (C-X-X-C). qRT-PCR analysis revealed that Tg-Nfu1 was ubiquitously expressed in all examined tissues; it was up-regulated in the hepatopancreas and gill, and kept a high level from 9 to 24 h after Cd exposure (250 μg/L). Western blot analysis further revealed that the Tg-Nfu1 protein was also highly expressed in the hepatopancreas and gill after 24 h of Cd stress. Further functional analysis showed that the production of ROS was increased and Cu/ZnSOD activity was inhibited in blood clam, treated with the specific Nfu1 siRNA and Cd stress, respectively. These results suggest that Tg-Nfu1 could protect blood clam from oxidative damage caused by Cd stress.

Hemoglobins Likely Function as Peroxidase in Blood Clam Tegillarca granosa Hemocytes

Hemoglobins are a group of respiratory proteins principally functioning in transport of oxygen and carbon dioxide in red blood cells of all vertebrates and some invertebrates. The blood clam T. granosa is one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity of T. granosa hemoglobins (Tg-Hbs) was characterized and the associated mechanism of action was deciphered via structural comparison with other known peroxidases. We detected that purified Tg-Hbs catalyzed the oxidation of phenolic compounds in the presence of exogenous H2O2. Tg-Hbs peroxidase activity reached the maximum at pH 5 and 35°C and was inhibited by Fe2+, Cu2+, SDS, urea, and sodium azide. Tg-Hbs shared few similarities in amino acid sequence and overall structural characteristics with known peroxidases. However, the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (HRP) and myeloperoxidase (MPO). This research represented the first systemic characterization of hemoglobin as a peroxidase.

Genetic analysis assessed by microsatellites for a diallel mating design of two geographical stocks of the blood clam Tegillarca granosa

To determine the potential for productive efficiency and genetic improvement in the blood clam Tegillarca granosa, four offspring populations (ZZ, ZK, KZ and KK) were produced from a diallel mating of two different geographical stocks (Z and K). The levels of genetic diversity and population structures of four populations were analyzed using 14 polymorphic microsatellites. The results showed that the mean observed heterozygosities (Ho) of reciprocal cross populations (ZK and KZ) was higher than those of pure populations(ZZ and KK). The largest values of genetic differentiation coefficient (Fst = 0.067) and Nei’s unbiased genetic distance (Dc = 0.263) were between ZK and KZ, and the smallest (Fst = 0.020, Dc = 0.116) were between ZZ and KK, which revealed that the largest genetic divergence was between the two reciprocal cross populations and the smallest was between two pure populations. This study demonstrated that the reciprocal cross populations of T. granosa had an extensive genetic difference and improvement, which may be advantageous for future breeding studies.

DPEP1 balance GSH involve in cadmium stress response in blood clam Tegillarca granosa

The blood clam, Tegillarca granosa, is a benthic filter feeder with a strong capacity to accumulate and tolerate cadmium (Cd). In our previous study, DPEP1 was shown to be significantly up-regulated under Cd stress based on proteomic analysis. To investigate whether DPEP1 is involved in Cd-induced response, the function of DPEP1 in T. granosa was investigated by integrated molecular and protein approaches. Rapid amplification cDNA end (RACE) assay was established to achieve the complete cDNA sequence of DPEP1 from T. granosa. The full-length cDNA of DPEP1 was 1811 bp, and it contained a 1359-bp open reading frame (ORF), including a 22-amino acid signal peptide. qRT-PCR analysis revealed that DPEP1 was expressed in all examined tissues with the highest expression in gills. At the same time, we investigated DPEP1 gene expression changes after Cd stress at different time points over 96 h. We found that the expression of DPEP1 increased upon initial Cd stress, then it was inhibited, and finally, it was maintained at a low level. Moreover, recombinant DPEP1 showed that higher glutathione (GSH) hydrolysis activity in the temperature range of 30–40°C, and its maximum activity was at pH = 6. Additionally, the results of immunohistochemistry also confirmed that DPEP1 protein was expressed in all test tissues with the highest expression in gills. In addition, there was a positive correlation between QRT-PCR and immunohistochemistry. These results suggested that DPEP1 is probably involved in Cd-induced response by balancing GSH.