The Diapause Induction Of Parthenogenetic Artemia-studies On Combined Effects Of Environmental Factors, Sensitive Stages, And Expression Of Related Proteins

Artemia is a small crustacean living in in hypersaline and saline lakes, ponds, lagoons and man-made salterns. Artemia cyts is an important commodity in aquaculture and its use in larviculture of fish and shellfish brought about a substantial development in the aquaculture industry.Whether bisexual or parthenogenetic Anemia, development of a given cluth of offsprings follows one of two paths:nondiapause mode, resulting in nauplius larvae, or diapause mode resulting in encysted gastrula embryos (cysts). Till now, there is no technique to control the reproductive mode of Artemia due to lacking of adequate information about the effective of "token stimuli" on Artemia diapause induction and the sensitive stage to receive the "token stimuli" To explore these questions, four chapters of works was conducted in this thesis.1. Combined effects of photoperiod, temperature and salinity on diapause induction are assayed using a diploid clone of the parthenogenetic Artemia population from Barkol Lake (Xinjiang, China) under laboratory conditions. With regard to the obtained results, the modes of offspring in the parthenogenetic clonal Artemia are significantly influenced by photoperiod and temperature. Nondiapause mode happened predominatly in longer photoperiod or higher temperature. Meanwhile, there is an interaction effect between salinity and photoperiod. The effect of the longer photoperiod can be diminished by higher salinity, while the influence of higher temperature cannot be determined by increasing salinity. The photoperiod and temperature also show interaction effect. The photoperiod show predominant when its effect conflict with temperature.2. To determine the sensitive stage for diapause induction of Artemia, shift-culture experiments are conducted between 27℃, LD18:6 and 19℃, LD6:18 by using clonal population BLK.53. The result shows that Artemia indeed have a sensitive stage for diapause induction, the sensitive stage lies between post-larvaⅢand post-larvaⅣ. In this stage, the ovisac of the female hangs freely, surpassing the length of the genital segments and reaching to the half of the third abdomen segment under lateral view. Based on the histology of the genital segments, the ovaries are during previtellogenesis stage.3. To test the relationship of ArHsp22 with Artemia diapause induction, partial CDS of ArHsp22 gene is cloned and with q RT-PCR, the expressions of this gene are analysed at three developmental stages(before sensitive stage, sensitive stage, after sensitive stage for diapause induction) under two reproductive mode. The results show that:under nondiapause reproductive mode, there are no significant difference of ArHsp22 mRNA among the three stages, but under diapause reproductive mode, the expression of ArHsp22 mRNA at the stage-after sensitive stage for diapause induction is higer that the other two stages (P<0.05). Beacause there is no significant different at the sensitive stage for diapause induction under two reproductive mode, it suggests that ArHsp22 only participate in diapause maintenance of Artemia embryos and the survival of adult animals experiencing environmental insult, no relatiship between ArHsp22 and Artemia diapause induction.4. To test the relationship of GnRH receptor (GnRHR) orthologue with Artemia diapause induction, the distribution of GnRHR immunoreactve in Artema is studied by immunohistochemistry, and the differential express of GnRHR orthologue at three developmental stages(before sensitive stage, sensitive stage, after sensitive stage for diapause induction) under two reproductive mode were studied by western blot. The immunohistochemistry results show that. GnRHR immunoreactve mainly located in the several part of the esystalk, brain, maxillary gland and shell gland of genital segments.The western blot results show that no significant difference of the expression of GnRHR orthologue is found among three stages under nondiapause reproductive mode, but under diapause reproductive mode, the expression of GnRHR orthologue at sensetive stage of diapause induction is significant higher than the other two stages. And the expression of GnRHR orthologue at sensetive stage of diapause induction under diapause reproductive mode is similar with the three stages under nondiapause reproductive mode, it meas both before and after the sensetive stage of diapause induction, the expression of GnRHR orthologue were suppressed by the shorter photoperiod and lower temperature, only at the sensetive stage of diapause induction, the suppression can fade away. All the results from the immunohistochemistry and western blot suggest this GnRHR orthologue have relationship with diapause induction of Artemia.