水蛭在我国用药历史悠久，常用于治疗多种淤血所致心脑血管疾病。水蛭的主要药用品种为宽体金线蛭，多数中成药也以宽体金线蛭进行投料。近年来，宽体金线蛭的野生资源逐年减少，价格不断攀升，养殖研究需求增加。宽体金线蛭具有巨大的经济价值和科研价值，成为了我国新兴的经济物种。

1. 宽体金线蛭的群体考察、种蛭筛选与产茧后药材质量评价

微山湖是我国宽体金线蛭的传统产区，许多人工养殖的种蛭来自于微山湖地区渔民的野外捕捞作业。本研究通过体重和生殖环带发育等特征对微山湖宽体金线蛭的群体进行考察，发现微山湖地区宽体金线蛭野生种群的体重普遍较小，宽体金线蛭生殖环带的颜色和面积随体重的变化而变化；观察并总结了宽体金线蛭胚胎发育的规律，便于选择合适的发育时相进行出苗数的考察，也为后续深入胚胎发育研究奠定基础；将种蛭分为10、15和20 g三个试验组，通过产茧数、茧体积、出苗数等指标考察体重对宽体金线蛭繁殖性能的影响，发现宽体金线蛭的繁殖性能随种蛭体重的增加而显著提高，但结合野生种群的体重分布范围，建议使用15 g及以上的种蛭进行繁殖生产作业，20 g及以上的种蛭用于良种繁育研究；最后，依照2015版《中华人民共和国药典》对产茧前后水蛭药材的性状和抗凝血酶活性进行质量评价，发现产茧后药材厚度变薄、腥味散失严重、脆度下降，产茧前后抗凝血酶活性变化不大，这表明产茧后药材的商品规格和等级较低，不适宜作为饮片直接用于临床。

2. 宽体金线蛭麻醉方法的初步构建

目前宽体金线蛭的研究尚缺乏合适的麻醉方法，不利于后续宽体金线蛭研究工作的展开。本研究基于宽体金线蛭的生长繁殖习性初步建立了宽体金线蛭的实验室管理方法，为后续所用宽体金线蛭的实验管理奠定基础；通过观察宽体金线蛭对麻醉剂Tricaine的反应，初步将麻醉划分为4个阶段：镇静期、失衡期、麻醉期、深度麻醉期；深度麻醉后在清水中复苏24 h，宽体金线蛭的存活率为100%，安全度高，麻醉前后的平均产茧数分别为4.10和3.38，对繁殖性能的影响较小；建议选择2 g·L^-1的Tricaine用于镇静和运输等常规操作，选择4 g·L^-1及以上的Tricaine用于进行实验室处理等精细操作。此外，我们通过RNA-seq技术对麻醉前后宽体金线蛭的基因表达差异进行研究，一方面考察麻醉对基因表达影响的范围以判断麻醉是否适合与基因表达相关的取样操作，另外一方面尝试探讨宽体金线蛭麻醉的机制；研究发现Tricaine麻醉对大多数基因的表达没有显著影响，一共有29个基因显著差异表达；显著富集的GO term和KEGG pathway多与离子结合功能有关，包括7个钙结合相关基因，其中6个上调，1个下调。这表明Tricaine麻醉可以用于后续深入生化分析实验中，不会对实验结果造成显著影响； Tricaine麻醉宽体金线蛭可能是几个基因的综合作用，可能通过影响信号传导、影响神经递质的释放、改变Na⁺浓度等功能最终实现麻醉的作用。

3. 宽体金线蛭饥饿胁迫的高抗逆性研究

在自然环境或人工养殖中，宽体金线蛭常被迫经历饥饿胁迫，如越冬、繁殖产茧等过程。对饥饿胁迫的高抗逆性将有助于提高宽体金线蛭的养殖成活率。本研究利用RNA-seq技术对经过不同时长饥饿胁迫的宽体金线蛭进行转录组分析，在不同饥饿组中找到了136个共有显著差异表达的基因，其中65个上调基因、71个下调基因；共有的GO term一共199个，其中189个上调GO term、10个下调GO term；共有的KEGG pathway一共42条，其中22条上调通路，20条下调通路。在长期饥饿胁迫下，宽体金线蛭的差异基因主要在糖酵解与糖异生、脂肪酸降解与代谢、多种氨基酸的降解与代谢、柠檬酸循环、溶酶体、谷胱甘肽的代谢等多个生物过程中表达。这表明宽体金线蛭可能是通过调控多个代谢通路，进行糖类、脂肪酸、氨基酸等重要物质的代谢转化，实现机体能量的供给，同时基因Lysoz和Gpx7、Gstp、Ptgds的表达上调可能分别有助于提高机体的免疫能力与抗氧化能力，最终达到提高机体在饥饿胁迫下的抗逆性的目的。这为后续探索饥饿胁迫的高抗逆性的机制研究指明方向，同时为后续人工育种提高宽体金线蛭抗逆性的研究奠定基础。

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. pigra 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 Production

Weishan 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 Whitman

At 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. pigra 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 Whitman

At 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, with189 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.