| Traditional Chinese medicinal material Leech has a long history of use in China and is often used to treat a variety of cardiovascular and cerebrovascular diseases caused by blood stasis.Whitmania pigra Whitman is the main medicinal variety of leech and is used in most of the Chinese patent drugs whose raw materials include leeches.Recently,the wild resources of W.pigra Whitman has declined year by year,the price is constantly rising,and the demand for breeding research has increased.With huge commercial and scientific values,W.Whitman has become an emerging economic species in China.1.The Population Investigation and Breeding Leeches Selection of W.pigra Whitman and Quality Evaluation of Its Medicinal Materials after Cocoon ProductionWeishan Lake area is the traditional production area of W.pigra Whitman,and many breeding leeches for artificially cultivation are wild varieties which are collected by the fishermen from the Weishan Lake area.In this research,the population characteristics of the W.pigra Whitman in Weishan Lake area were investigated through the indicators like the body weight and the developmental condition of clitellum.It was found that the wild species of W.pigra Whitman in Weishan Lake area generally had lower body weight,and the color and area of the reproductive annulus of W.pigra Whitman varied with body weight.The rule of embryonic development of W.pigra Whitman was observed and summarized,which would help to select the appropriate development phase to investigate the number of leech larvae hatched from each cocoon and lay the foundation for the in-depth embryonic development research.The experiment for the selection of breeding leeches was divided into three groups,10 g group,15 g group and 20 g group.The effects of body weight on the reproductive performance of W.pigra Whitman were investigated by some indexes,such as the number of cocoons produced,cocoon volume,and the average number of leech larvae hatched from each cocoon and so on.It was found that the reproductive performance of W.pigra Whitman increased significantly as its body weight increased.However,considering the weight distribution range of wild populations in Weishan Lake area,it was recommended to use the leeches of 15 g and over for breeding,and to use the leeches of 20 g and over for elite breeding researches.Finally,the quality evaluation of medicinal materials before and after the cocoon production were carried out by the physical characteristics and antithrombin activity according to the 2015 edition of the Chinese Pharmacopoeia.The thickness,taste and crispness of the medicinal material after cocoon production were all reduced.The changes of antithrombin activity before and after cocoon production were not significant.The results showed that the product specifications and grades of medicinal materials after producing cocoons were lower,and were not suitable for direct use in clinical.2.Preliminary Construction of Anesthesia Method for W.pigra WhitmanAt present,there is still no suitable anesthesia method for W.pigra Whitman,which is not conducive to further in-depth study.The laboratory management of W.pigra Whitman was preliminarily established in this research based on its growth and reproduction habits,which laid the foundation for the subsequent experimental management of the W.pigra Whitman.Through the response of Tricaine as an anesthetic to W.pigra Whitman,the anesthesia process was divided into four stages:sedation period,imbalance period,anesthesia period,and deep anesthesia period.After 24 hours of resuscitation in clear water after deep anesthesia,the survival rate of W.pigra Whitman was 100%.The safety of Tricaine anesthesia was high.The average number of cocoons before and after anesthesia were 4.10 and 3.38,respectively.The effects of Tricaine anesthesia on the reproductive performance of W.pigra Whitman were small.It was recommended that 2 g·L-1 Tricaine was suitable for non-precision operation,such as sedation and transportation,and Tricaine of 4 g·L-1 and above was suitable for precise operations,such as delicate surgery.Moreover,RNA-seq technology was used to study the gene expression differences of W.pigra Whitman before and after Tricaine anesthesia in this research.On the one hand,the scope of the influence of anesthesia on gene expression was investigated to determine whether Tricaine was suitable for those sampling operations related to gene expression.On the other hand,the anesthetic mechanism of W.pigra Whitman was investigated.It was found that the effects of Tricaine anesthesia on the RNA expression level of most genes of W.pigra Whitman were small,and a total of 29 genes were expressed differentially.The analysis of GO enrichment and KEGG pathway enrichment showed that several genes were expressed differentially and were related to the molecular function of ion binding,including seven calcium binding-related genes,with six upregulated and one downregulated.This indicated that Tricaine anesthesia could be used in gene expression regulation experiments,and would not have a significant impact on the experimental results.Tricaine anesthesia for W.Whitman might be a combinatory effect of several genes,and finally achieved the anesthesia effect by influencing the signal transduction,affecting the release of the neurotransmitter,and changing the concentration of sodium ion.3.Study On the High Stress Resistance of Starvation Stress of the W.pigra WhitmanAt the same time,in the natural environment or artificial farming,W.pigra Whitman is often subject to starvation stress in some processes such as ihibernation,breeding cocoons and so on.High resistance to starvation stress will help to increase the survival rate of the leech.In this study,RNA-seq technology was used to perform transcriptome analysis on the W.pigra Whitman under different periods of starvation stress.In different experiment groups,136 overlapped significantly differentially expressed genes were found,of which 65 genes were up-regulated and 71 genes were down-regulated;199 overlapped GO terms were found,with 189 up-regulated GO terms and 10 down-regulated GO terms;42 overlapped KEGG pathways were obtained,of which 22 pathways were up-regulated and 20 pathways were down-regulated.Under long-term starvation stress,the differential genes of W.pigra Whitman were mostly expressed in glycolysis and gluconeogenesis,fatty acid degradation and metabolism,degradation and metabolism of various amino acids,citric acid cycle,lysosome,glutathione metabolism and other biological process.This indicated that the supply of energy for W.pigra Whitman might be achieved by regulating the metabolism of multiple metabolic pathways,to carry out the metabolic transformation of important substances such as sugars,fatty acids,amino acids,and so on.While at the same time,the risen expression of the genes Lysoz and Gpx7,Gstp,and Ptgds might help increase the body’s immunity and antioxidant capacity,respectively,and then the purpose of improving the body’s resistance to starvation stress was achieved.This will point the way for future research on the mechanism of the resistance to hunger stress,and lay the foundation for subsequent artificial breeding to improve the resistance of W.pigra Whitman. |
