Ontogenesis of Main Organs and Cloning and Expression of Two Immune-related Genes in Litopenaeus Vannamei

【摘要】：White leg shrimp Litopenaeus vannamei is a prime aquaculture species, and itcovers90%of western hemisphere culture and is also extensively farmed in Asia. Theestimated annual production from Asia during2002was316000mt. In China, exoticspecies (L. vannamei) had introduced during1988for experimental purposes and by1998is being successfully cultured in commercial scale; over1000hatcheries are inoperation. The300000tons and210000tons harvested during2003in marine andfreshwater farming respectively. To give more understanding for the white leg shrimp,we have investigated the genesis of important systems and organ, including digestivesystem, nervous system and haematopoetic tissue, furthermore dealed with twoimportant immune related genes. Our investigation on ontogenesis of digestive system in Litopenaeus vannamei,revealed that in non-feeding stages the yolk mass is the indigenous source of food anduntil the fifth nauplius (N5) stage retained in foregut cavity, absorption is gradual innauplius (N) stages while disappearance occurs at N6stage. The anterior midgutcaecum generates at N3and it reaches maximum size in Z3; thereafter in successivestages it reduces in size and becomes a single structure in post-larval stage (P3-P4),named the anterior diverticulum. The lateral midgut caecum first appears at N6stageand lengthens along both sides of the midgut and then ramifies to form lobes as larvaldevelopment. Finally, these lobes become a compact structure, the hepatopancreas.The midgut lumen is quite apparent in N stages and only becomes wider in successivestages; it is without a cuticle lining and there is no difference among all investigatedstages. The foregut in N stages is a simple tube and pyloric and cardiac chamber occurs in the third mysis (M3) stage. In the cardiac chamber of post-larval stages,folds were noted and number of that increased gradually. The gastric mill is absent inall investigated larval stages (P1-P10). The pyloric chamber is smaller than the cardiacchamber and possesses a gland filter, which first appears in M3stage and forms amore complete shape in forthcoming stages. The hindgut gland first appears in P2andin successive stages it shows more protuberance and complexity. The hindgut orrectum is a simple tube. Cells of the digestive system in all feeding stages show thepresence of acid mucins and general protein, while the inner margins of the foregutand connective tissues of the gland filter are positive for collegen. The nervous system plays pivotal role in co-ordination of body activities andcoping with the task of controlling the behavioral activities during larval development.In present study, we give a basic description in the early stage morphology of nervoussystem in L. vannamei by classical histological techniques. Results revealed thatneuropil first emerged in N1stage and the front part of nervous system started gettingstructural shape in N3where the ganglions were coalesced in front part of head. Thecomplete structure of central nervous system including proto-, deuto-andtritocerebrum was observed in N6stage. The structural expansion of nervous systemin successive stages was continued and protocerebrum differentiated into anterior andposterior median protocerebrum at Z3stage. The optic ganglions, sub-esophagealganglions and pleon ganglions were distinct in zoea stage. Furthermore, we discussthe relativity between the development of olfactory lobes and the voluntary movementof larva. It is believed that haematopoietic tissue (HPT) is the source of haemocytesproduction in crustaceans and defensive role of haemocytes is progressing butinvestigations on structure of HPT are limited hence this study has been designed inwhich we investigated the genesis and morphology of haematopoietic tissue (HPT)and types of haemocyte based on its morphology and cytochemical feature usinghistological and histochemistical methods. The results revealed that HPT in L.vannamei is not visible histologically in mysis and early two post larvae (P1P2), and emerges in P3which locates in epigastric region. The HPT showed a continuouspropagation of cells in successive post larval stages. Four types of cells can beidentified in the HPT from adult specimen and mitotic activity is visible on occasionin the HPT. The haemocytes has been differentiated into five types and some of thesetypes are co-related with the HPT cells. The cytochemical studies suggested that typeI and type II cells of haemocytes are PAS positive while scanty presence ofprophenoloxidase was observed in type I cells similarly the type I cells are SudanBlack B positive and rest of the cells showed weak activity against the lipid detectivestain. Best of knowledge this is the first effort towards the genesis of HPT and itsrelation with circulating haemocytes in L. vannamei. The second part of the study focuses two important immune related genesCyclophilin A and Proliferating cell nuclear antigen (PCNA); the obtained results areas below respectively. Cyclophilins are ubiquitous proteins with an enzymatic activity of peptidyl-prolylcis-trans isomerase (PPlase), which play important roles in a variety of stressresponses. In present investigation a Cyclophilin A gene has been cloned from whiteleg shrimp (Litopenaeus vannamei) by rapid amplification of cDNA (RACE) andanchored PCR method. The full length of the shrimp L. vannamei CypA cDNA(LvCypA) is855bp, containing495bp of open reading frame (ORF), encoding164amino acids with an estimated molecular mass of17.6kDa. The LvCypA has highhomology with that of other known organisms and it has four β-strands. The real timePCR analysis revealed that its expression is ubiquitous and expression level is higherin muscle, gill, lymphoid organ and hepatopancrease respectively. The tissueschallenged with Vibrio angullarium revealed that in hepatopancrease it was the lowestat6h and highest in48h. The results revealed that the LvCypA might involve inpotential role of innate immunity. The proliferating cell nuclear antigen (PCNA) has important role in nucleic acidmetabolism and its immune responses have also been reported. In present investigation the PCNA was cloned from L. vannamei (LvPCNA). The full lengthcDNA sequence of LvPCNA encodes261amino acids showing high identity withPCNAs reported in other species. Tissue distribution of the LvPCNA revealed that itsexpression is obvious in gill and muscle. In order to understand the response ofbacterial challenge, mRNA level of LvPCNA in hepatopancrease, was analyzed afterchallenged by V. anguillarum. The obtained expression profile of LvPCNA in tissue isvariable in different time point. The data enhance the existing knowledge of defensemechanisms of shrimp against bacteria.