Characterization of novel expressed sequence tag-simple sequence repeat markers and analysis of genetic diversity in four geographic populations of Thais luteostoma.

In this study, the genetic diversity in four geographic populations (Yushan Island, Zhoushan, Wenzhou, and Xiamen) of Thais luteostoma was analyzed using 21 microsatellite loci. The results of this study showed that the alleles obtained from different populations ranged from 2 to 8. The average number of alleles and effective alleles were 4.59 and 3.16, respectively. The observed heterozygosity and expected heterozygosity values were in the range 0.338-0.372 and 0.452-0.495, respectively. The polymorphism information content ranged from 0.273 to 0.785. We observed a high level of genetic diversity at 9 of the 21 microsatellite markers in these populations. The genetic differentiation indices of the four geographic populations ranged from 0.0312 to 0.0565, showing a medium level of genetic differentiation. The genetic distances among populations ranged from 0.158 to 0.465. The UPGMA tree indicated that the Yushan Island and Zhoushan populations clustered first, and these subsequently clustered with the Wenzhou and Xiamen populations, indicating that the Xiamen shellfish population was least related to the other populations. The information regarding the shellfish population structure obtained in this study would facilitate the genetic breeding and conservation of T. luteostoma.

Development of novel polymorphic microsatellite markers for the blood clam Tegillarca granosa by pyrosequencing.

Large amounts of expressed sequence tags (ESTs) generated using next-generation sequencing technologies provide a cost-effective and valuable genomic resource for the development of microsatellite markers. In this study, we isolated 115 novel polymorphic microsatellite markers for the blood clam Tegillarca granosa from ESTs in 454 sequencing data. All the loci were characterized in 30 individual clams from a natural population in Xiangshan (Zhejiang Province, China). The number of alleles per locus varied from 2 to 10, with an average of 3.78. The observed and expected heterozygosities ranged from 0 to 1 and from 0.040 to 0.799, respectively. The polymorphic information content (PIC) ranged from 0.038 to 0.825, and 29 highly polymorphic loci (PIC ≥ 0.5) and 42 moderately polymorphic loci (0.25 < PIC < 0.5) were identified. Thirty-eight of the 115 loci deviated significantly from the Hardy-Weinberg equilibrium (P < 0.01) after a Bonferroni correction. A BlastX search revealed that 46 (40%) of the polymorphic loci identified were from transcript regions of known genes. The microsatellite markers developed in the present study will greatly enrich the microsatellite resources of T. granosa, and are available for further population genetic analysis, genetic trait mapping, and molecular-assisted selection.

Structure and immune expression analysis of hemoglobin genes from the blood clam Tegillarca granosa.

Hemoglobin (Hb) is the major protein component of erythrocytes in animals with red blood, although it can serve additional functions beyond the transport of oxygen. The blood clam (Tegillarca granosa) is one of the few mollusks that has Hb, although the structure and function of molluskan Hbs remain unclear. We characterized two unique and highly compartmentalized blood clam hemoglobin genes, Tg-HbIIA and Tg-HbIIB, at the molecular level. The full-length cDNA of Tg-HbIIA was 731 bp with a 450-bp open reading frame encoding 150 amino acids; that of Tg-HbIIB was 698 bp, with a 456-bp open reading frame encoding 152 amino acids. Their intronic regions were amplified by PCR. The two genes showed the typical 2 intron/3 exon organization found in T. granosa. The 3-D structures of the three blood clam Tg-Hbs were predicted using the SWISS-MODEL Protein Modeling Server, and a phylogenetic analysis was conducted to investigate its evolution. As quantified by qRT-PCR, the expression levels of Tg-HbIIA and Tg-HbIIB were significantly upregulated upon challenge by Vibrio parahaemolyticus, lipopolysaccharides, and peptidoglycans. Three Hb isoforms, Tg-HbI, Tg-HbIIA, and Tg-HbIIB, were found. Specific structures and evolutionary features were found in these molluskan Hb genes. Challenge experiments indicated that Tg-Hbs are involved in immune defense responses against bacterial infection and bacterial pathogenic factors. As this is the first functional research on Hb genes in the blood clam, our findings provide new insight into the innate immune defense mechanisms of T. granosa.

Expression of glutamine synthetase in Tegillarca granosa (Bivalvia, Arcidae) hemocytes stimulated by Vibrio parahaemolyticus and lipopolysaccharides.

The blood cockle, Tegillarca granosa, is a widely consumed clam in the Indo-Pacific region. Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. We identified the GS of T. granosa (Tg-GS) from hemocytes by 3'- and 5'-rapid amplification of cDNA ends (RACE)-PCR. The full-length cDNA consisted of 1762 bp, with a 1104-bp open reading frame encoding 367 amino acids. Sequence comparison showed that Tg-GS has homology to GS of other organisms, with 79.78% identity with GS from the Pacific oyster Crassostrea gigas, 71.98% identity with GS from the zebrafish Danio rerio, and 68.96% identity with human Homo sapiens GS. A C-beta-Grasp domain and an N-catalytic domain were identified in Tg-GS, indicating that Tg-GS should be classified as a new member of the GS family. A quantitative RT-PCR assay was used to detect mRNA expression of Tg-GS in five different tissues. Higher levels of mRNA expression of GS were detected in the tissues of hemocytes and the mantle. Up-regulation of GS by challenge with the bacteria Vibrio parahaemolyticus and with bacterial wall lipopolysaccharides showed that GS plays a role in anti-bacterial immunity. We conclude that pathogen infection significantly induces expression level of Tg- GS, and that activation of GS influences the immune response of T. granosa by increasing glutamine concentration.

Feasibility of disposing waste glyphosate neutralization liquor with cement rotary kiln.

The waste neutralization liquor generated during the glyphosate production using glycine-dimethylphosphit process is a severe pollution problem due to its high salinity and organic components. The cement rotary kiln was proposed as a zero discharge strategy of disposal. In this work, the waste liquor was calcinated and the mineralogical phases of residue were characterized by scanning electron microscope (SEM) and X-ray diffraction (XRD). The mineralogical phases and the strength of cement clinker were characterized to evaluate the influence to the products. The burnability of cement raw meal added with waste liquor and the calorific value of waste liquor were tested to evaluate the influence to the thermal state of the kiln system. The results showed that after the addition of this liquor, the differences of the main phases and the strength of cement clinker were negligible, the burnability of raw meal was improved; and the calorific value of this liquor was 6140 J/g, which made it could be considered as an alternative fuel during the actual production.