[Effect of soil moisture on efficacy to eliminate dampness and relieve jaundice and flavonoid content of Sedum sarmentosum].

The growth environment of medicinal plants plays an important role in the formation of their medicinal quality. However, there is a lack of combined analysis studying the close relationship between the growth environment, chemical components, and related biological activities of medicinal plants. Therefore, this study investigated the effect of different soil moisture treatments on the efficacy to eliminate dampness and relieve jaundice and the flavonoid content of Sedum sarmentosum, and explored their correlation. The flavonoid content in the decoction of S. sarmentosum growing under field conditions with soil moisture levels of 35%-40%(T1), 55%-60%(T2), 75%-80%(T3), and 95%-100%(T4) was compared. The effects of these treatments on liver function parameters, liver inflammation, and oxidative damage in mice with dampness-heat jaundice were evaluated, and the correlation between pharmacological indicators and flavonoid content was analyzed. The results showed that the total flavonoid and total phenolic acid content in the decoction of S. sarmentosum were highest in the T1 treatment, followed by the T3 treatment. The content of quercetin, kaempferol, and isorhamnetin was highest in the T2, T1, and T3 treatments, respectively. Among the different moisture treatments, the T3 group of S. sarmentosum effectively reduced the levels of serum ALT, AKP, TBIL, DBIL, TBA, as well as hepatic TNF-α and IL-6 in mice with jaundice, followed by T2 treatment, especially in reducing AST level. The T4 treatment had the poorest effect. Correlation analysis showed a significant negative correlation between AST, ALT, AKP levels in mice and the total content of quercetin and the three flavonoids. MDA showed a significant negative correlation with the total flavonoid content and kaempferol. TNF-α exhibited a significant negative correlation with the content of isorhamnetin. In conclusion, S. sarmentosum growing under field conditions with a soil moisture level of 75%-80% exhibited the best efficacy to eliminate dampness and relieve jaundice. This study provides insights for optimizing the cultivation mode of medicinal plants guided by pharmacological experiments.

[Cloning and expression analysis of wpHSP90 gene from Whitmania pigra at different temperatures].

HSP90 is a widely distributed molecular chaperone that participates in a variety of cellular processes and plays an important role in the meiosis of germ cells. However, its role in the gonadal development of hermaphroditic Whitmania pigra is not yet clear. To explore the effect of HSP90 on the germ cell development of Wh. Pigra, this study cloned the wpHSP90 gene, performed bioinformatics analysis, and measured its expression levels. The results showed that the cloned wpHSP90 was 2 592 bp in length, with an open reading frame(ORF) of 2 373 bp, encoding 790 amino acids. Prediction analysis revealed 85 phosphorylation modification sites on serine, threonine, and tyrosine residues of the wpHSP90 protein. Structural domain prediction and multiple sequence alignment results showed that wpHSP90 contained two conserved domains of HSP90 and exhibited the highest homology with Helobdella robusta, with a sequence similarity of 80.72%. RT-qPCR results showed that the relative expression level of wpHSP90 in the gonads of 5-month-old Wh. pigra was positively correlated with temperature within the range of 12 ℃ to 28 ℃. The expression level in the female gonads was significantly higher than in the male gonads and correlated with the trend of germ cell development in the ovaries and testes. In conclusion, wpHSP90 may be involved in regulating the development of germ cells, particularly oocytes, in Wh. pigra. This study provides a reference for further research on the gonadal development mechanism in Wh. pigra.

[Effects of shading on photosynthetic physiology and energy metabolism of Asarum forbesii].

Light is the main source for plants to obtain energy.Asarum forbesii is a typical shade medicinal plant, which generally grows in the shady and wet place under the bushes or beside the ditches.It can grow and develop without too much light intensity.This experiment explores the effects of shading on the growth, physiological characteristics and energy metabolism of A.forbesii, which can provide reference and guidance for its artificial planting.In this experiment, A.forbesii was planted under 80%, 60%, 40%, 20% and no shade.During the vigorous growth period, the photosynthetic physiological characteristics such as fluorescence parameters, photosynthetic parameters, photosynthetic pigment content and ultrastructure, as well as the content of mitochondrial electron transport chain(ETC) synthase and nutrients were measured.The results showed that the photosynthetic pigment content, chlorophyll fluorescence parameters and net photosynthesis rate(P_n) decreased with the decrease of shading.Under 20%-40% shading treatment, the plants had damaged ultrastructure, expanded and disintegrated chloroplast, disordered stroma lamella and grana lamella, and increased osmiophi-lic granules and starch granules.The activities of nicotinamide adenine dinucleotide dehydrogenase(NADH), succinate dehydrogenase(SDH), cytochrome C oxidoreductase(CCO) and adenosine triphosphate(ATP) synthasewere positively related to light intensity.With the reduction of shading, the content of total sugar and protein in nutrients increased first and then decreased, and the content was the highest under 60% shade.In conclusion, under 60%-80% shading treatment, the chloroplast and mitochondria had more complete structure, faster energy metabolism, higher light energy-conversion efficiency, better absorption and utilization of light energy and more nutrient synthesis, which was more suitable for the growth and development of A.forbesii.

[Comparison of internal quality and nutrients between male and female Eupolyphaga sinensis].

According to the Chinese Pharmacopoeia(2020 edition), Eupolyphaga Steleophaga is prepared from female Eupolyphaga sinensis. To explore the quality difference between male and female E. sinensis, the present study determined indexes in the Chinese Pharmacopoeia, anticoagulant activity, and content of primary metabolites, amino acids, and trace elements in male and female E. sinensis before and after sexual maturity. The results showed that the content of ash, water, extract, and aflatoxin of the larvae and female adults of E. sinensis met the Chinese Pharmacopoeia standards, while the content of ash and extract of male adults failed to meet the standards. After sexual maturity, the content of water-soluble proteins, crude fat, and extract of female E. sinensis was higher than that of the other three groups(P<0.05). Although the content of total protein, total amino acid(TAA), essential amino acid(EAA), non-essential amino acid(NEAA), branched-chain amino acid(BCAA), functional amino acid(FAA), and delicious amino acid(DAA) of male adults was higher than that of the other groups(P<0.05), the anticoagulant activity was the weakest(P<0.05). Before sexual maturity, the content of Fe in E. sinensis larvae was significantly higher than that in adults(P<0.05). The content of K and Mg in female larvae was the highest, while the content of Ca was the lowest(P<0.05). The content of Cu in male adults was higher than that in the other three groups(P<0.05). In conclusion, except for male adults, the larvae and female adults of E. sinensis meet the Chinese Pharmacopoeia standards. There is no difference in the internal quality of male and female E. sinensis larvae. It is recommended to collect female adults as much as possible. Male adults are rich in proteins and amino acids with high fibrinolytic activities, which can be developed in the future.

[Screening and validation of RT-qPCR reference genes of Viola yedoensis under different light intensities].

Screening the reference genes that were stably expressed under different light intensities for Viola yedoensis could provide reference for the related molecular research. In this study, 11 candidate reference genes were detected by RT-qPCR for tissues of V. yedoensis treated with different light intensities. Ge Norm, Norm Finder, Best Keeper, and Ref Finder website were used to comprehensively evaluate the reference genes, and verify the stability of the reference gene based on CAT1. Finally, the ideal reference gene was determined. The results showed that CYP, Actin, and SAMDC had small Ct value ranges and stable expression. Ge Norm demonstrated that CYP, SAMDC, and Actin were suitable reference genes. Norm Finder showed that the expression of α-TUB was the most stable. Best Keeper recommended CYP, Actin, and SAMDC as reference genes. Ref Finder assessed that SAMDC, CYP, α-TUB, and Actin had better stability, while GAPDH had the worst stability. The expression trend of CAT1 gene was consistent when calibrated with SAMDC, CYP, and Actin, while it was quite different from that calibrated with GAPDH. In summary, SAMDC, CYP, and Actin can be used as ideal reference genes for the gene expression profiling of V. yedoensis under different light intensities.

[Effects of Wp-PKC on gonadal development of Whitmania pigra].

Protein kinase C(PKC) is a kind of kinase which is widely involved in cell proliferation and development. PKC(Wp-PKC) in Whitmania pigra body belongs to classic PKC. In order to investigate the effect of Wp-PKC on the development of Wh. pigra germ cells, 17ß-estradiol(17ß-E2)(100 ng·mL~(-1)) and methyltestosterone(MT)(150 µg·L~(-1)), 150 µg·L~(-1)(MT)+0.5 mg·L~(-1) PKC, 0.5 mg·L~(-1) PKC inhibitor were added to Wh. pigra culture water, and no addition group(control group) was added, and the effects on the development of Wh. pigra germ cells and the expression of Wp-PKC were observed. The results showed that: Wp-PKC in male gonads was always higher than that in female gonads; MT promoted the development of male gonads in Wh. pigra, while the expression of Wp-PKC was significantly higher than that in the control; 17ß-E2 promoted the development of female gonads in Wh. pigra and Wp-PKC expression significantly lower than that of the control; while the development of the female and male gonads in the PKC inhibitor group was inhibited, the expression of Wp-PKC was significantly lower than that of the control. In summary, Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads.

[Cloning and expression analysis of protein kinase C gene from Whitmania pigra].

Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.

[Effects of light intensity on cleaning away heat property of Viola yedoensis].

The current study was conducted to explore the effects of light intensity in cultivating environment on the cleaning away heat property of Viola yedoensis. In the present study, we established the acute inflammation model of ICR mice by injecting carrageenan. We compared the effects of V. yedoensis grown under different light intensities(100%, 80%, 50%, 35% and 5% of full sunlight) on mice body temperature, thermal radiation and the swelling degree of foot tissue before and after modeling observing by thermal infrared imaging technique and weighing method. The changes of energy metabolism related enzymes in liver were detected by enzyme-linked immunosorbent assay(ELISA). In addition, the effects of V. yedoensis grown under different light intensities on human lung cancer cell A549 proliferation were explored with MTT method. The results showed that the body temperature of all groups of mice in V. yedoensis group were significantly lower than that of the blank group, except 5% full sunlight group, and the body temperature declined in positive proportion to light intensity. V. yedoensis group could alleviate foot swelling, reduce SDH activity in liver tissue(especially 100% full sunlight group and 80% full sunlight group were significantly lower than model group), and the degree of alleviating and reducing was positively correlated with light intensity. There was no significant difference in the activity of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase in liver tissue among treatments. The contents of IL-1ß, IL-6, TNF-α, PGE_2 in foot tissue of mice in V. yedoensis groups were significantly lower than those in model group. Among them, the lowest levels of IL-1ß, IL-6, TNF-α, PGE_2 were found in 80% full sunlight group, and there was no significant difference in TNF-α among different groups. The effects of V. yedoensis aqueous extract on A549 cell line proliferation inhibition rate increased with the light intensities of V. yedoensis cultivating environment. And the effects of V. yedoensis grown under 100% of full sunlight showed significantly higher A549 cell line proliferation inhibition rate compared with other groups(P<0.05). In summary, the light intensity of V. yedoensis cultivating environment is positively correlated with the cleaning away heat property of V. yedoensis, which conforms to the "light-cold and heat property" hypothesis,The V. yedoensis should be planted under full light according.

[Effects of light intensity on physio-biochemical characteristics of Chrysanthemum indicum].

By analyzing the effects of light intensity on the growth, physiological and biochemical characteristics of Chrysanthemum indicum, the light intensity suitable for the growth of Ch. indicum was revealed, which provided a reference for the planting of Ch. indicum. There were five treatment groups of Ch. indicum, which was planted under 100%, 80%, 60%, 40% and 20% all natural light intensity conditions, respectively. In the meantime, these indicators of photosynthetic physiology, such as relative growth, photosynthetic pigment content, photosynthetic parameters, and chlorophyll fluorescence parameters etc. were measured in the peak period of growth of Ch. indicum as well as related indicators of the protective enzyme system, and the ultrastructure of chloroplast was observed with the use of transmission electron microscope technique. In our study, the results showed that the leaves of Ch. indicum occurred yellow phenomenon in different degrees when Ch. indicum was placed at more than 80% of the total natural light intensity condition, the height and stem diameter of plant reached the maximum at 60% of the total natural light intensity. More importantly, the number of branches of Ch. indicum was significantly increased under the total natural light intensity of more than 60%. Both the content of photosynthetic pigment and net photosynthetic rate were negatively correlated with light intensity, while photosynthetic parameters and chlorophyll fluorescence parameters showed a trend of rising first and then decreasing with the decrease of light intensity. The physiological indexes of Ch.indicum including stomatal conductivity, intracellular CO_2 concentration, transpiration rate, water use efficiency and actual photochemical quantum yield of PS Ⅱ had been determined, and the results showed that all of them were at the highest level under 60% total natural light intensity condition. The chloroplast structure of Ch. indicum was not obviously abnormal under 60% and 80% total natural light intensity treatments, but the stroma lamella was broken under 100% total natural light intensity, and not only the number of chloroplast, but also the number and volume of starch grains were decreased significantly under 20% and 40% total natural light intensity. With the decrease of light intensity, the enzymes activities of SOD and CAT decreased, the activity of POD increased in the early stage and decreased in the later stage, and the content of MDA showed a decreasing trend. The analysis of results indicated that the Ch. indicum can grow under 20%-100% total natural light intensity, but the best growth condition was under 60% total natural light intensity.

[Effects of constant temperature on growth and gonad development of overwintering Whitmania pigra].

The present study was conducted to explore the effect of constant temperature overwintering on the growth, gonadal development and internal quality of the Wh. pigra which were in overwintering period. Wh. pigra which in overwintering period were placed in light incubator at 21 ℃, and the Wh. pigra that overwintered under natural conditions were used as control. That the changes of growth performance, gonad index, internal quality of two groups of Wh. pigra were measured at the end of the overwintering. Simultaneously the tissue slice technique was used to observe the morphological structure of the spermary and ovary of the two groups. The results showed that the body weight of constant temperature overwintering Wh. pigra which were placed in light incubator at 21 ℃ was 2.35 times that of natural overwintering Wh. pigra which overwintered under natural conditions, and the weight of female gonads of the Wh. pigra which were placed in light incubator at 21 ℃ was 11.54% higher than that of Wh. pigra which overwintered under natural conditions, and the weight of male gonads of the Wh. pigra which were placed in light incubator at 21 ℃ was 48.33% higher than that of the Wh. pigra which overwintered under natural conditions. At the same time, that vitellogenesis cells and film forming cells which in ovarian vesicles were significantly higher than those of the Wh. pigra which overwintered under natural conditions, and that spermatogonia and primary spermatocytes which in seminal vesicle were significantly higher than those of Wh. pigra which overwintered under natural conditions. Most important of all, the anti-thrombin activity of Wh. pigra which were placed in light incubator at 21 ℃ increased by 27.85% compared with the Wh. pigra that overwintered under natural conditions. In conclusion, that constant temperature can promote the growth, the development of sperm and egg cells, and the increase of anti-thrombin activity of Wh. pigra which were in overwintering period.

[Effects of light intensity on material and energy metabolism of Viola yedoensis].

Light energy is an important factor affecting plant growth. The hypothesis of "light-cold and heat property" holds that the original plants of traditional Chinese medicines(TCM) with cold property can obtain more energy to maintain growth in high light intensity environment, whereas the original plants of TCM with heat property prefer weak light environment. The current experiment investigated the effects of different light intensities on primary metabolites levels, energy levels, cell apoptosis, and leaves ultrastructure of Viola yedoensis, the original plants of TCM Violae Herba with cold property. There were five treatment groups of V. yedoensis, which was planted under Li1(8 500 lx),Li2(7 250 lx),Li3(6 000 lx),Li4(4 750 lx),Li5(3 500 lx)LEDs light intensity conditions, respectively. After harvest, primary metabolites levels, contents of ATP, ADP, AMP, activities of ATP synthesis and hydrolysis related enzyme, as well as cell apoptosis activation degree were measured, and transmission electron microscopy technique was used to observe leaves ultrastructure. The results showed that the total sugar, total protein, contents of ATP, ADP and AMP, activities of NADH dehydrogenase, cytochrome C reductase, ATP synthase and ATP hydrolase were positively correlated with light intensities(P<0.05). The crude fat content, activities of SDH and CCO enzyme showed a trend of increasing first and then decreasing, the highest value were found in Li2 group and Li3 group respectively(P<0.05). The vitality of caspase-3 and caspase-9 was negatively correlated with light intensities(P<0.05). The structure of chloroplast and mitochondria were normal and intact in Li1-Li3 groups, and the damage degree of Li4 and Li5 groups increased with the decrease of light intensities. The analysis of results indicated that the structure of chloroplast and mitochondria of V. yedoensis is normal under the light intensity of 6 000-8 500 lx, which can obtain more energy to maintain its growth and metabolism. When the light intensity is lower than 4 750 lx, the chloroplast morphology and mitochondrial membrane are damaged, affecting the metabolism of material and energy. There was no significant difference in energy charge of V. yedoensis in the light intensity range of 3 500～8 500 lx. The effect of light intensity on energy metabolism of V. yedoensis accords with the hypothesis of "light-cold and heat property".

[Effects of MT on growth and gonad development of natural overwintering Whitmania pigra].

The objectives of study were to explore the effects of exogenous methyltestosterone( MT) on the growth and gonadal development of overwintering Whitmania pigra. Before overwintering,0. 1,1. 0,10. 0,100. 0,150. 0 µg·L-1 of MT were added to the aquaculture water for 6 weeks. The changes of growth performance,gonad index,endogenous steroid hormones level and internal quality were measured after hibernate for 60 days. Then the tissue slice technique was used to observe the spermary( ovary) of Wh. pigra.The results showed that the body weight,survival rate and gonadal index increased first and then decreased with the increase of exogenous MT concentration; the male gland index was found the highest at the concentration of MT 10. 0 µg·L-1 and the female gland index was the highest at the concentration of MT 1. 0 µg·L-1. The survival rate of Wh. pigra peaked at the concentration of MT 10. 0 µg·L-1.The weight reaches a peak at a concentration of MT 100. 0 µg·L-1( P<0. 05). The number of primary spermatocytes in the testis was negatively correlated with the concentration of exogenous MT. The number of secondary spermatocytes and sperm cells increased first and then decreased. The concentration of secondary spermatocytes was the highest when the concentration of MT was 100. 0 µg·L-1.The number and volume of oocytes in the ovary and the yolk granules increased first and then decreased with the increase of exogenous MT concentration,and the highest was observed at the MT concentration of 100. 0 µg·L-1. The endogenous steroid hormone of Wh.pigra increased first and then decreased with the increase of exogenous MT concentration. The concentration of androgen and progesterone was the highest in MT 100. 0 µg·L-1 treatment( P<0. 05),and the concentration of estrogen was found the highest in MT 10. 0 µg·L-1 treatment( P<0. 05). After adding exogenous MT,Wh. pigra moisture content,acid-insoluble ash content,p H and anti-thrombin activity met the quality criterion of medicinal Wh. pigra in Chinese Pharmacopoeia( 2015 edition). In conclusion,the short-term addition of 1. 0-100. 0 µg·L-1 exogenous MT before hibernation can promote the growth,the development of sperm cells and the antithrombin activity of Wh. pigra.

[Effects of 17ß-E2 on growth and gonad development of overwintering Whitmania pigra].

The present study was conducted to explores the effects of short-term addition of 17ß-E2 on the growth,gonad development and internal quality of overwintering Whitmania pigra. Before overwintering,0. 0,1. 0,10. 0,25. 0,50. 0,100. 0 µg·L~(-1) of 17ß-E2 were added to the aquaculture water for 6 weeks and then hibernated for 60 days. The changes of growth performance,gonad index,morphological structure of spermary( ovary),endogenous steroid hormones level and internal quality were measured. The results showed that the body weight,weight gain rate,specific growth rate,female gonad index,oocyte development and endogenous estrogen level of the leech increased first and then decreased with the increase of the concentration of exogenous 17ß-E2,which were higher than those of the control group. The body weight,weight gain rate and specific growth rate of the leech at the concentration of 25 µg·L~(-1)17ß-E2 were significantly higher than those of the other groups( P<0. 05),oocyte development and endogenous estrogen levels were significantly higher than those of other groups at the concentration of 50 µg·L~(-1)( P<0. 05). When the concentration of exogenous 17ß-E2 was higher than 50 µg·L~(-1),the levels of male gonad index,spermatocyte development,endogenous androgen and progesterone were significantly inhibited( P< 0. 05). There was no significant difference in endogenous corticosteroid levels among the groups. In conclusion,short-term addition of exogenous 17ß-E2 of 10-25 µg·L~(-1) could promote the growth of overwintering leeches,oocyte development and antithrombin activity without inhibiting the development of male gonads.

[Effects of light intensities on growth,physiological characteristic and chemical composition of Viola yedoensis].

The study is aimed to investigate the effects of light intensities on growth,photosynthetic physiology,antioxidant systems and chemical composition of Viola yedoensis and provide cultivation references for V.yedoensis.Five groups of V.yedoensis were planted under five light intensities conditions,namely 100%,80%,50%,35%,5%of full sunlight,and then morphological index,growth,chlorophyll fluorescence parameters,photosynthetic parameters and antioxidant enzyme system indexes were measured during harvest.The results showed that there was no significant difference in the biomass of V.yedoensis among 35% -100%full sunlight,but the biomass of those were significantly higher than that in the 5%full sunlight treatment(P<0.05).The net photosynthetic rate,transpiration rate,stomatal conductance,intercellular CO_2 concentration and water use efficiency increased firstly and then decreased with the decrease of light intensity;F_m,F_v/F_mand Yield in 5% full sunlight treatment were significantly lower than those in the other four groups(P<0.05).The structure of chloroplast was normal under light intensity ranged from 50%to 100% full sunlight.The lamellar concentration of chloroplast matrix decreased and the starch granules decreased in 35% full sunlight treatment,and the margin of lamellar layer of chloroplast and substrate were blurred,and the starch granules were small and the number of starch granules decreased significantly under 5% full sunlight.MDA content in 5%full sunlight treatment was significantly higher than those in the other four groups(P<0.05).The total coumarin content and total flavonoid content decreased with the decrease of light intensity.In summary,the light in-tensity range suitable for the growth of V.yedoensis is wide(ranging from 35% to 100% full sunlight).The content of flavonoids and coumarins is positively correlated with light intensity.

[Cloning of Guamerin gene in Whitmania pigra and its spatio-temporal expression analysis after ingestion].

The present study cloned wpGuamerin gene from a non-bloodsucking leech (Whitmania pigra), and the bioinformatics analysis of the sequence was performed. Additionally, the effects of feeding duration on the expression profile of the wp Guamerin gene were explored. The results showed that its sequence consisted of 295 nucleotides encoding a peptide of 83 amino acids(Genbank: KX768545), and its relative molecular weight is 9 145.95 Da. wp Guamerin does not encode proteins with a signal peptide, belonging to the hydrophilic protein. Its secondary structure is mainly composed of α-helix, extending chain, folding and random curl. Its similarity with other blood-sucking leeches ranges from 29% to 65%. The results revealed that wpGuamerin mRNA was detected higher expression in muscle than in salivary glands of Wh. pigra, and did not expressed in ingluvies and intestine. Its expression in muscle and salivary glands showed a single peak curve after feeding and the peak was observed in the 1st and 3rd after feeding, respectively. In summary, wp Guamerin in Wh. pigra is a small molecule polypeptide protein and is different from the Guamerin in blood-sucking leeches. wpGuamerin does not express in the digestive tract of Wh. pigra, and mainly express in muscle. Feeding behavior would stimulate the expression of wpGuamerin gene in muscle and salivary glands, but not in digestive tract.

[Preliminary study on changes of sex steroid hormones in periodic development of Whitmania pigra gonad].

The present study was conducted to investigate the rule of sex steroid hormones dynamic in the periodic development of Whitmania pigra gonad. The dynamic of sex steroid hormones in different age of Wh. pigra were detected by enzymolysis, extraction and UPLC-MS/MS. The results showed that the concentrations of estrone, estriol, testosterone and progesterone in the Wh. pigra showed M-type curve and peaked in 6-month-old Wh. pigra. The concentrations of above-mentioned four steroid hormones were the lowest in 9-month-old and then increased slightly. The another peaks of testosterone and progesterone were found in 2-month-old and the another peaks of estrone and estriol were obtained in 8-month-old. The concentrations of testosterone and progesterone are slightly higher than estriol during 1-month-old to 4-month-old, and thereafter strone and estriol showed higher concentration than testosterone and progesterone. In summary, the concentrations of four sex steroid hormones in Wh. pigra increased gradually with the maturation of gonads and decreased rapidly after the discharge period, which indicated that the ratio between estrogen, androgen and progesterone may be greatly related to the specificity of gonad development.

[Effect of Astragali Radix on growth, immunity and related gene expression of Whitmania pigra].

To explore the effect and mechanism of Astragali Radix on growth and immunity of Whitmania pigra, 0, 0.01%, 0.03%, 0.05%, 0.07%, 0.09% of Astragali Radix were added to the daily feeding of Wh. pigra. After 60 days of feeding, the growth performance, activities of digestive enzyme and anti-reverse enzyme, inner quality, the expression levels of GH, IGF-1 and digestive enzyme-related genes were measured. Meanwhile, the effects of heat stress on the living conditions of Wh. pigra were observed and counted, and the expression levels of HSP70 and immune related genes were measured. The results showed that the final weight, weight gain rate, specific growth rate, activities of digestive enzyme and anti-reverse enzyme, the expression levels of GH, IGF-1 and digestive enzyme-related genes in the Astragali Radix group were higher than those in the control group, and with the increase of Astragali Radix concentration, the above-mentioned indexes increased initially and then decreased, and significantly higher in the 0.05% of Astragali Radix group than in the other groups (P<0.05). There was no significant difference in the inner quality of Wh. pigra between the Astragali Radix and control groups. The survival rate of Wh. pigra was negatively correlated with heat stress treatment duration. With the prolongation of heat stress treatment duration, the expression levels of HSP70 and immune related genes were increased first and then decreased, and peaked at 24 h. The survival rate and the expression levels of HSP70 and immune related genes in the Astragali Radix group were higher than those in the control group, and was significantly higher in the 0.05% of Astragali Radix group than in the other groups (P<0.05). In conclusion, Astragali Radix can increase the activities of digestive enzyme and anti-reverse enzyme, the expression levels of related genes, growth performance, and immunity to heat stress of Wh. pigra. It is suggested to add 0.05% of Astragali Radix in the actual production of Wh. pigra to improve the production profit.

[Different arousal modes of hibernating Whitmania pigra].

To study the suitable arousal modes of hibernating Whitmania pigra, the biological characteristics, activity of amylase, lipase, and protease as well as morphologic structure of digestive tract were investigated by direct observational method and 3, 5-dinitrosalicylic acid colorimetry, p-nitrophenyl palmitate ester（p-NPP）colorimetry, folin-phenol and histological methods. The results revealed that Wh. pigra activity was increased with increases of the water temperature and prolonging treating duration. Except for the feeding groups of direct arousal mode at 24 h and 48 h, none of the other Wh. pigra groups died. Compared with that of normal groups, the digestive tract structure of hibernating Wh. pigra was looser and the mucosal folds of craw were more sparse. No obvious recovery was observed for the structure of the digestive tract before 48 h of direct arousal mode or the 5th day of 15 °C gradient arousal mode. The mucosal folds of craw increased and the muscularis were incrassated after 72 h of direct arousal mode or the 8th day of 20 °C gradient arousal mode, that indicated the tissue structure was approximately restored to the normal state. The digestive enzyme activities were increased with prolonging treating duration. And the feeding groups recovered faster than that of the no feeding groups. Additionally, the enzyme activities of feeding groups were significantly higher than that of no feeding groups (P<0.05) and approximately restored to the normal level after 48 h in the direct arousal mode or 20 °C in the gradient arousal mode. In conclusion, both of the two modes can be applied to the arouse of hibernating Wh. pigra, and it should be fed when the temperature is recovered to 15 °C or 20 °C at 2 °C·d⁻¹ in the gradient arousal mode after 72 h in the direct arousal mode to reduce the death ratio and improve effectively the economic profit of Wh. pigra aquaculture.

[Preliminary study on pathogen of Whitmania pigra disease in high temperature environment].

In this paper, on the contrast of healthy leech, the bacterial diversities were analyzed by 16S rDNA sequence analysis of the bacteria of muscle and intestinal tract of Whitmania pigra, the environment water and sediment of cultivating the diseased Wh. pigra in high temperature by high-throughput sequencing to determine the possible pathogenic bacteria of bacterial diseases of Leech in high temperature. The results showed that the original sequence reached over 83 000, and the effective sequences accounted for more than 87%. The GC contents ranged from 52% to 54% and the bacterial diversities were abundant. Bacterial relative abundance analysis showed that the bacterial content of Proteobacteria, Bacteroidetes, and Firmicutes was the most abundant in all treatments. Compared with healthy leech muscles and intestines, the muscle and intestinal tract of pathogenic leech relative abundance of Bacteroides, Pseudomonas, and Desulfovibrio was significantly increased, and it was abundant in water and sediment of diseased leeches, Lead to the possibility that the pathogenic bacteria of this bacterial disease may be Bacteroides, Pseudomonas, Desulfovibrio.

[Effects of Cu²âº enrichment and release on growth， internal quality and relative gene expression of Whitmania pigra].

The present study explored the effects of Cu²âºenrichment and release on the growth and internal quality of Whitmania pigra, and the regularity of enrichment and release in different tissues of Wh. pigra. In the range of safe concentration(SC), a certain concentration of Cu²âº was added to the Wh. pigra for 50 days, and stopped adding for another 50 days. The growth index of Wh. pigra, Cu²âº content in different tissues, and the internal quality were determined. The results showed that the average body weight of Wh. pigra in 0.014 mg·L⁻¹ of Cu²âº group was significantly higher than that in control group during the experimental period (P<0.05), and the mortality rate was lower than that in the control group. The specific growth rate, digestive enzyme activity, growth and digestive enzyme related gene expression of Wh. pigra in Cu²âº group were significantly higher than that in the control group (P<0.05) during the early 40 days of enrichment, and there was no significant difference in the release period. In Cu²âº group, the expression of immune enzyme and immune enzyme related gene of Wh. pigra increased first and then decreased compared with control group (P<0.05). The ability of enriched Cu²âº in different tissues of Wh. pigra was digestive tract>muscle>skin. The ability of release Cu²âº was muscle>digestive tract>skin. There was no significant difference in the internal quality and hirudin gene expression between Cu²âº group and control group. In conclusion, Cu²âº can improve the expression of digestive enzymes, immune enzyme and related genes of Wh. pigra, promote the growth of Wh. pigra and enhance their immunity, but it doesn't affect the internal quality of Wh. pigra. The Wh. pigra can completely release Cu²âº within 30 days.