| Tegillarca granosa or blood clam belong to Lamellibranchiata, was a traditional farmed shellfish and was distributed widely in Shan Dong, Zhe Jiang, Fu Jian, Guang Dong etc. Now, Zhe Jiang Province dominates the blood clam farming in China, however, blood clam cultivation has suffered seriously from high mortality resently. Understanding the immunity of blood clam is crucial for managing diseases and developing sustainable blood clam culture.It has been proved that one important immune defense reaction of mollusca hemocytes is phagocytosis when the organism is attacked by microorganisms or viruses. Zhou and Pan thought that hemocytes play an vital role in the mollusc immunology for the mollusc lack of the specific immunity, thus the hemocytes were the main protection for pathogens defense. The potential pre-polyphenoloxidase activity of HB was studied, and Jiang etc. argued that HB could defense the infection of many bacteria e.g. Staphylococcus aureus and Pseudo monas aeruginosa. The anti-bacterial function of hemocyanin from invertebrates e.g. Limulus polyphemus, Pacifastacus leniusculus, Octopus vulgaris and Rapana venosa. was already reported.First, the blood smears of five kinds of aquatic animals, bloody clam (T. granosa, Scapharca subcrenatd), Carassius auratus, Rana catesbeiana and Pelodiscus sinensis, were stained by Hemacolor stain. The surface structure and morphological characteristics of erythrocytes were examined by Light Microscope (LM) and Scanning Electronic Microscope (SEM). The results indicated that the erythrocytes of bloody clams (T. granosa&S. subcrenata) have basophil granular or/and eosinophilic granular, but none was found in vertebrates erythrocytes. The size of erythrocytes increased from the two bloody clams to Bull frog(Rana catesbeiana); while the size decreased afterwards; the variation of aspect ratio and nucleocytoplasmic ratio of erythrocytes meet this trend likewise. The undulate fold and depression on the erythrocytes of the two bloody clams were observed apparently, and the depression was deep and wide. The surface depression was also obvious in the erythrocytes of vertebrates, although the depression died away gradually, and no distinct surface fold was examined. Analysis of these results indicated that the changes of animal erythrocytes resulted from the evolution of animals, activities and habitats, in order to obtain the biggest gas exchange efficiency and the stability of the cells. This paper focuses on the the morphological characteristics and microstructure of erythrocytes of five aquatic animals, in order to study the morphological diversity and evolution of erythrocytes.Secondly, the cDNA of the bloody clam (T. granosa) Hb dimer (designated Tg-HbI) was cloned and was found to be748bp in length, consisting of an open reading frame of441bp encoding a polypeptide of147amino acids. The deduced amino acid sequence of Tg-Hbl shared81.6%similarity with HbI from two species of the genus Scapharca and46-51%similarity with the Hb proteins from other mollusks. The Tg-HbⅡA cDNA was cloned and then the bioinformatics, expression and immune defense analysis of Tg-HbⅡA cDNA were carried out. The full length of Tg-HbⅡA cDNA was731bp, which consists of an open reading frame of450bp encoding a polypeptide of150amino acids, a5’-UTR of63bp and a3’-UTR of246bp; the deduced amino acid sequence of Tg-HbⅡA shared89%and88%similarity with HbⅡA from two species of the genus Scapharca respectively. The structure prediction of Tg-HbⅡA showed the hemoglobin domain and the ten potential heme binding sites. The cDNA of the bloody clam (T. granosa) HbⅡB (designated Tg-HbⅡB) was cloned and was found to be698bp in length, consisting of an open reading frame of456bp encoding a polypeptide of152amino acids. The deduced amino acid sequence of Tg-HbⅡB shared low similarity (45%) with HbⅡB from two species of the genus Scapharca and57%similarity with the Hb proteins from Barbatia lima which might indicated the unique evolution position and process of Tg-HBⅡB.Then, the intronic regions from three hemoglobin genes were amplified by PCR and were analyzed. The genomic length of the Tg-HBI, Tg-HBIIA, Tg-HBⅡB gene was about2031bp,1797bp,3203bp, respectively, which all containing3exons and2introns. The three genes showed the typical two intron/three exon organization found in T. granosa and were compared with other Hbs. And many sequence repeats indicated that they may be involved in the expression regulation, DNA retrotransposition and recombination, the repeats located in introns could be used as the markers for gene screen and SNP analysis.Finally, the mRNA expression of Tg-Hb in haemocytes were both detected highest in all the clam cells/tissues including haemocytes, adductor muscle, foot, hepatopancreas, gill and mantle. However, the differences among three genes are that Tg-HBI was expressed higher in gill than in mantle. The gene expression of two HBII genes showed the other way from Tg-HBI. The Quantitative Real-time PCR (qRT-PCR) showed that the mRNA transcript level of Tg-Hb, significantly up-regulated after Vibrio parahaemolyticus, Lipopolysaccharide and Peptidoglycan challenge which indicated that Tg-Hb was involved in the immune defense responses against bacterial infection and exposure to bacterial pathogenic factors. And the rapid expression indicated that the Tg-HB were an rapid inducible proteins. However, the temporal and level of expression were different in three challenge groups. There is an interesting thing that three HB genes showed different different levels of sensitivity to V. parahaemolyticus, Lipopolysaccharide and Peptidoglycan challenge, which indicated the multiple immune paths involved in HB immune response. |
PDF Full Text Request