Research On Development Of Digestive Tract And Hg²⁺ Toxic Effect Of The Tachypleus Tridentatus Larvae

In this paper,on the basis of captive breeding of T.tridentatus,the path analysis and multiple regression analysis of the morphological traits and body mass of 1-year-old T.tridentatus were carried out.And the multiple linear regression equation of the main morphological traits and body mass was established to provide theoretical basis for the germplasm evaluation and seedling screening of T.tridentatus.Then through the paraffin section and H-E staining techniques for T.tridentatus histological study on digestive tract development.To compare different development phase of the difference of shape and structure of digestive tract is conducive to improve technology and enhance the survival rate of seedling raising.Finally,to investigate the toxic effects of mercury on juvenile T.tridentatus,an acute toxicity test of Hg²⁺at various concentrations to the first instar juvenile T.tridentatus was carried out at laboratory.The results are as follows:?1?We conducted a preliminary study on artificial breeding of horseshoe crabs T. tridentatus in Zhanjiang,Guangdong Province from May to September 2018.When the water temperature was 28?32?,salinity was 26?30,and p H was 7.4?7.8,9 pairs of T.tridentatus laid 87130 eggs with a fertilization rate of 56%to 68%.After 45 days,the embryos hatched into the first instars of juvenile T.tridentatus.The hatching rate was 92%,and 48,268 T.tridentatus larvae were obtained.The path coefficients of various morphological traits for body mass reached a highly significant level?P<0.01?,and the largest path coefficient was used in analyzing the width of the head and chest armor.This indicated that the width of the head and chest armor had the greatest direct effect on body mass.The trend of determination coefficient analysis was comparable to that of path analysis.The elevated total determination coefficient??d=0.863?between phenotypic features and body weight indicated that the selected traits were practically useful.The multiple regression equation for estimating body weight was Y=-0.228+0.248X₁+0.073X₂+0.333X₃+ 0.073X₄.?2?Paraffin sections and H-E staining were used to study the individual development of alimentary canal of T.tridentatus at 15 days old,30 days old,60 days old,2-year-old and adult.The results showed that the structure of digestive tract of T.tridentatus at 15 days old has not been clearly distinguished.The esophagus,stomach,midgut and rectum are similar in structure.Only mucous membrane is clear,and mucous folds are relatively short.When the age of T.tridentatus was 30 days old,the mucous epithelium of esophagus and stomach appeared dark bands,the muscle fibers of esophagus extended in a ring,and the muscle bundles of stomach were thickened.The mucous epithelium of the middle intestine and rectum has a striate border at the top,and the muscular fiber bundles are thickened.When the age of T.tridentatus was 60 days old,the dark bands at the top of the mucosa epithelium of the esophagus and stomach develop into striate margins,with increased esophageal muscle fibers and thickened gastric myofilament bundles.The mucosa fold of the midgut and rectum increased in height and number,and the pigmentation of the striate border deepened.The digestive tract of the second instar T.tridentatus is composed of mucous layer,submucous,muscle layer and serosal layer,and the tissue structure is basically well-developed.With the gradual improvement of tissue structure,the number of folds of esophagus and gastric mucosa increased significantly to 9?15,and the striate border at the top of the mucosal epithelium developed into chitin layer.The number of folds in the mucosa of the middle intestine and rectum increased rapidly to 10?12,and the epithelial striatum of the mucosa of the middle intestine thickened,with little tin layer,and the epithelial striatum of the rectum developed into chitin layer.The structure of digestive tract of adult T.tridentatus is complete,and the mucous layer,submucous layer,muscle layer and serous membrane of digestive tract are obviously thicker than that of early stage.The results showed that the tissue structure of the alimentary canal of T. tridentatus was basically developed at age 2,and the tissue structure of each layer was thickened with age,which was suitable for its living habit.?3?To investigate the toxic effects of mercury on Marine animals,an acute toxicity test of Hg²⁺at various concentrations to the first instar juvenile T.tridentatus was carried out at water temperature of 26?,Salinity of 27?30 and p H of 7.6?7.8 by an acute toxicity method.And the effects of low concentration of Hg²⁺on MT content in for the first instar juvenile T.tridentatus at 24 h,48 h,72 h and 96 h were 15.013, 7.084,4.008 and 2.683mg·L^-1respectively.And the safe mass concentration was 0.473 mg·L^-1,Hg²⁺is medium toxic to T.tridentatus larvae.MT content is positively correlated with Hg²⁺concentration within a certain range of exposure concentration and exposure time.In the safe concentration range,different concentrations of Hg²⁺ can significantly induce the production of MT?P<0.05?,MT content is 1.152?1.716 times of the control group.Among them,the highest MT induction was at0.025 mg·L^-1,which was 8.93±0.16 ng·L^-1.This study provides theoretical basis for safety assessment of juvenile T.tridentatus in natural water environment and establishment of water quality standard by fishery department.?4?Transcriptome reveals involvement of immune defense in Hg²⁺stress response of the T.tridentatus larvae.A comparative transcriptome analysis identified 641,203 and 163 differentially expressed genes in different treatment days of Hg²⁺:control?HG0?vs the 15th day?HG15?,control?HG0?vs the fourth day?HG4?and control?HG4?vs the 15th day?HG15?.Then 422,81 and 142 of which were up-regulated,and 219,122 and 21 of which were down-regulated.During the process of Hg²⁺stress,the genes related to substance anabolism and immunity changed significantly,especially those related to amino acid metabolism and antioxidant enzyme activity.Compared with the control group,the gene expression of material catabolism and oxidoreductase activity was significantly different in Hg²⁺treatment on the fourth day.And there was a significant difference in the expression of Hg²⁺on the 15th day. Differentially expressed genes of the fourth day and the 15th day treated by Hg²⁺ were enriched in the function of material synthesis.These changes reflect that T. tridentatus larvae in Hg²⁺stress can resist the harm of mercury stress by adjusting the body's substance anabolism and antioxidant enzyme activity.