Influence Of Different Yeast Cell-wall Mutants On The Protection Against Vibrio Campbellii Infection In Artemia Sinica And Gene Cloning, Expression Analysis Of A Prophenoloxidase From Artemia Sinica In Its Early Developmental Stages

The brine shrimp, Artemia, belong to Phylum Arthropoda, Class Crustacea, is one of the most important live feeds for commercial production of fish and shellfish larvae. This organism is unique in the animal kingdom, because it is extremely euryhaline and can produce cysts (encapsulated embryos), which can be easily stored and transported. Recently, it has been used as an excellent model organism to study the biology of infections in Crustaceans, stress response and feed quality analysis and so on. Similar to the other invertebrates, Artemia can only rely on their non-specific immunology system and have no immunological memory. So the immune effectors in haemolymph such as prophenoloxidase-activating system(proPO-AS), superoxide dismutase (SOD) and lysozyme (LSZ) play a critical role for the immune reaction. At the present time, PO, SOD and LSZ have been used as indicator of health status and pathogen infection in crustacean. Recently, crustacean such as shrimp and crab culture around the world has suffered problems linked to deteriorating environments due to development of intensification, subsequently resulting in stress-induced disease incidence. Bacteria such as vibrio are by far the most serious, causing massive mortalities of cultured organisms worldwild. Diseases have become the major inhibitor of aquaculture production, and lead to the massive financial losses. The use of chemotherapeutic drug have sever negative impact on living organism and the environment, such as inducing the development of resistant pathogens, and moreover, contaminating the environment and accumulating in tissues of seafood, then could be harmful to people's health So its use has been restricted. In order to solve this problems, some new bio-control strategies and environmently friendly prophylactic alternatives were required urgently, and the health of organisms and enhancement of their immunity are of primary concern.In this study, wild type (WT) of Saccharomyces cerevisiae (SC) and its cell wall mutant mnn9 were chose as Artemia larvae feed, effects of WT and mnn9 on protecting Artemia to resist Vibrio campebllii were studied. The survival of Artemia fed with mnn9 was significantly higher than that of Artemia fed with WT after two days incubation with the feed schedule, at the mean time, the same significant difference occured after 24 h of vibrio infection, and this significance between them became bigger in the following incubation.What's more, the average length of Artemia larvae was measured, and the results showed that Artemia larvae fed with mnn9 had longer length of the whole body. Regardless of larval survival and larval length, PO, SOD and LSZ activity of Artemia fed with WT or mnn9 were checked following the Vibrio challenge or non-challenge test, then the similar results were found that these three enzyme activity showed higher in Artemia fed with mnn9 in different incubation time whatever under Vibrio challenge or not. Therefore, mnn9 was deduced to maily have two effects on Artemia larvae. Firstly, mnn9 has higher nutritional quality for Artemia than WT, so it can make Artemia larvae to be more healthier and stronger. Secondly, mnn9 for its mutation in cell wall has moreβ-glucan and chitin which are immunostimulants as known, then these immunostimulants could stimulant the innate immune system of Artemia larvae, and enhance the immune reaction by improving the immune-related enzyme activity such as PO, SOD and LSZ, and improve the survival of Artemia larvae in the end.Larval surval of Artemia fed with WT significantly decreased after 24h challenging with Vibrio campbellii, and decreased quikly in the following incubation. In addition, PO, SOD and LSZ activity of Artemia larvae were checked after Vibrio challenge and also showed significantly decrease in different challenging time. what means that Vibrio campbellii could damage the host immune system, then lead to increasing mortality of Artemia larvae by inhibiting the immune-related enzyme activity.Artemia were fed with heat shocked WT and mnn9 respectively which were induced to produce more Hsp70, then Vibrio challenge test was arranged. The results showed larval survival of Artemia fed with WT significantly increase in 48-72h challenging time. In addition, PO, SOD and LSZ activity of Artemia larvae fed with heat shocked WT showed significantly increased in different vibrio challenge time. So the yeast of Hsps may stimulate Artemia immune response to resist the Vibrio infection by one kind of still unclear mechnism.A full length of 2125 bp AsproPO cDNA was cloned by RT-PCR using oligonucleotide primers based on the proPO sequence of Artemia franciscana. It contains an open reading frame of 2100 bp, and 25 bp of the 3'-untranslated region, and encodes protein of 699 amino acids with a predicted molecular weight of 80.37 kDa and with an estimated pI of 6.48. Analysis of the deduced amino acid sequces of AsproPO shows that The AsproPO has a proteolytic activation site (Arg75-Gly76), two putative copper-binding sites with six conserved histidine residues (CuA:225, 229,251and CuB:379,383,419) and a thiol-ester-like motif (GCGWPEHM) which are highly conserved inarthropods. However, no signal peptide sequence was detected. Analysis of sequence similarity in a range of arthropod taxa suggested that the proPO gene in Artemia was as dissimilar to other crustaceans as it was to insects.Semi-quantification RT-PCR was used to study expression of proPO in Artemia at five different developmental stages (0h,6h, Emergence(E), Instar I and Instar II), while the expression ofβ-actin gene as inner control. And PO enzymatic activity was also checked.The results showed that proPO expression was expressed at E stage, InstarⅠand InstarⅡstages, while significant PO activity was only found in larvae at InstarⅡstage. The expression analysis indicates that proPO system were modulated in Artemia early developing stages.In conclusion, effects of nutritional quality of feed and immunostimulation or vibrio infection and Hsps on Artemia immune response were studied,which suplies the academic groundwork for the still unclear crustacean immune mechanism. And some methods such as immunostimulation or Hsps to enhance Crustacean immune response were explored. For the first time, the expression of proPO and PO was studied and analyzed, which could help to deeply understand the immune fuction and modulation mechanism of proPO system, and make the academic support for disease control in Crustacean.