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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.

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.

The novel antimicrobial gene Hirudomacin (Hmc), with a 249-bp cDNA, encodes a mature protein of 61 amino acids and a 22-amino acid signal peptide. Hmc exhibits the highest similarity, at 90.1%, with macin family members found in the salivary gland of the leech Hirudo nipponica Whitman. A mature Hmc protein concentration of 219 μg/mL was detected using the Bradford method. The mature Hmc protein is 6862.82 Da and contains 8 cysteine residues. Antimicrobial assays showed a minimum bactericidal concentration and 50% lethal dose of 1.56 μg/mL and 0.78 μg/mL, respectively, for Staphylococcus aureus and 0.39 μg/mL and 0.195 μg/mL, respectively, for Bacillus subtilis. Transmission electron microscopy revealed membrane integrity disruption in S. aureus and B. subtilis, which resulted in bacterial lysis. The level of Hmc mRNA in the salivary gland during three blood meal stages indicated a remarkable trend of increase (P .05), and western blotting demonstrated that among the three blood meal stages, expression of the mature Hmc protein was highest in both the salivary gland and intestine at the fed stage (P .05). Immunofluorescence further showed the mature Hmc protein to be localized outside the cell nucleus, with the signal intensity in the salivary gland peaking at the fed stage (P .05). In conclusion, the mature Hmc protein exhibits broad-spectrum antimicrobial effects against gram-positive and gram-negative bacteria, and a blood meal upregulates Hmc gene and protein expression in H. nipponica.

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, 17β-estradiol(17β-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; 17β-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.

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.

Light energy is an important factor affecting plant growth. The hypothesis ofquot;light-cold and heat propertyquot; 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(Plt;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(Plt;0.05). The vitality of caspase-3 and caspase-9 was negatively correlated with light intensities(Plt;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 ofquot;light-cold and heat propertyquot;.

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-1β, 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-1β, 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(Plt;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 thequot;light-cold and heat propertyquot; hypothesis,The V. yedoensis should be planted under full light according.

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.

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

Whitmania pigra is an important medicinal resource that is widely farmed in aquaculture in Asia, and a decrease in body weight occurring during hibernation and hibernation lasting long time has serious impacts on production efficiency in aquaculture system. We examined energy metabolic and antioxidant enzymes of intestine from a hibernator (W. pigra) over cycles of hibernation-arousal. Results of the study demonstrated that hibernation in W. pigra was characterized by a profound decrease in energy metabolic during deep hibernation that was interrupted by rewarming arousal. And energy metabolic increased significantly during the rewarming arousal. Regulated suppression of energy metabolism probably contributes to energy savings. Oxidative stress decreased during deep hibernation along with a reduction in oxidative metabolism, but increased during entrance into hibernation and arousal from hibernation. This up-regulation of antioxidant defense (AD) during arousal was interpreted as protection of the intestine against oxidative damage to come with the enormous increase in metabolic activity during arousal from hibernation, and the up-regulation of AD during entrance into hibernation was interpreted that leech are exposed to significant stresses (cold acclimation) that must be dealt with appropriately to avoid irreversible tissue damage. It can be concluded that W. pigra has a strong AD system that protects it from the injurious effects of free radicals either during periods of entrance into hibernation and arousal. These results indicate the adaptive mechanism of hibernation that may be applied to increase production efficiency of leech by interrupting or shortening hibernation.

The present study was conducted to explores the effects of short-term addition of 17β-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 17β-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 17β-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)17β-E2 were significantly higher than those of the other groups( P0. 05),oocyte development and endogenous estrogen levels were significantly higher than those of other groups at the concentration of 50 μg·L~(-1)( P0. 05). When the concentration of exogenous 17β-E2 was higher than 50 μg·L~(-1),the levels of male gonad index,spermatocyte development,endogenous androgen and progesterone were significantly inhibited( P0. 05). There was no significant difference in endogenous corticosteroid levels among the groups. In conclusion,short-term addition of exogenous 17β-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.

Hibernation is an energy-saving and adaptive strategy adopted by leech, an important medicinal resource in Asia, to survive low temperature. Reversible protein phosphorylation (RPP) plays a key role in the regulation of mammalian hibernation processes but has never been documented in freshwater invertebrate such as leech. In this study, we detected the effects of hibernation on the proteome and phosphoproteome of the leech Whitmania pigra. A total of 2184 proteins and 2598 sites were quantified. Deep-hibernation resulted in 85 up-regulated and 107 down-regulated proteins and 318 up-regulated and 204 down-regulated phosphosites using a 1.5-fold threshold (P0.05). Proteins involved in protein digestion and absorption, amino acid metabolism and N-glycan biosynthesis were significantly down-regulated during deep-hibernation. However, proteins involved in maintaining cell structure stability in hibernating animals were up-regulated. Differentially phosphorylated proteins provided the first global picture of a shift in energy metabolism, protein synthesis, cytoprotection and signaling during deep hibernation. Furthermore, AMP-activated protein kinase and protein kinase C play major roles in the regulation of these functional processes. These data significantly improve our understanding of the regulatory mechanisms of leech hibernation processes and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals. SIGNIFICANCE: The leech Whitmania pigra as an important medicinal resource in Asia is an excellent model freshwater invertebrate for studies of environmentally-induced hibernation. The present study provides the first quantitative proteomics and phosphoproteomic analysis of leech hibernation using isobaric tag based TMT labeling and high-resolution mass spectrometry. These data significantly improve our understanding of the regulatory mechanisms when ectotherm animals face environmental stress and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals.

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(P0.05).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(P0.05).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(P0.05).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.

Yellow catfish (Pelteobagrus fulvidraco) has become a commercially important fish species in China and eastern Asia. High-density aquaculture has led to congestion and excessive stress and contributed to bacterial infection outbreaks that have caused high mortality. We investigated the effects of dietary supplementation with astaxanthin and emodin alone and in combination on the growth and stress resistance of yellow catfish. After 60 days of feeding, each group of fish (control, astaxanthin, emodin, and astaxanthin plus emodin (combination) groups) was exposed to acute crowding stress for 24 h, and a subsample of fish from the four groups was challenged with the bacterial septicemia pathogen Proteus mirabilis after the end of the crowding stress experiment. Compared with the control, the astaxanthin and emodin groups showed increases in serum total protein (TP), hepatic superoxide dismutase (SOD) activity and hepatic heat shock proteins 70 (HSP70) mRNA levels at 12 and 24 h after the initiation of crowding stress. The combination group exhibited increases in alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, serum TP, hepatic SOD activity and hepatic HSP70 mRNA levels within 24 h after the initiation of crowding stress. However, decreases relative to the control were observed in the serum cortisol and glucose contents in the three treatment groups at 12 and 24 h after the initiation of crowding stress, in ALT and AST activity in the astaxanthin and emodin group at 24 h after the initiation of crowding stress, and in the serum lysozyme activity, serum alkaline phosphatase (ALP) activity, and hepatic catalase (CAT) and malondialdehyde (MDA) activity in the combination group at 24 h after the initiation of crowding stress. Additionally, the cumulative mortality after P. mirabilis infection was lower in all three treatment groups (57.00%-70.33%) than in the control (77.67%). Dietary supplementation with astaxanthin and emodin decreased the specific growth rate (SGR) and weight gain (WG) of healthy yellow catfish, although significant differences in mortality were not observed. These results indicate that dietary supplementation with 80 mg/kg astaxanthin and 150 mg/kg emodin can improve the anti-oxidative capabilities, hepatic HSP70 levels, and resistance to acute crowding stress of yellow catfish. Finally, an appropriate strategy for enhance yellow catfish stress resistance and disease resistance is proposed.

We aimed to assess the effect of Radix Isatidis dietary supplementation on resistance to Proteus mirabilis infection in Poecilobdella manillensis . Six groups of leeches housed in tanks were randomly divided into five treatment groups (fed a basal diet supplemented with 2.5, 5.0, 7.5, 10.0, or 12.5 g/kg of Radix Isatidis ) and one control group (fed a basal diet). Compared with the control group, the specific growth rate (SGR) and weight gain (WG) were significantly decreased in the treatment groups supplemented with Radix Isatidis . However, supplementation with 2.5 to 10.0 g/kg of Radix Isatidis did not produce an effect on mortality. Mortality was recorded after the leeches were challenged with P. mirabilis , and changes were observed in the serum cortisol, glucose, alanine aminotransferase (ALT), 3,5,30-triiodothyronine (T3), thyroxin (T4), alkaline phosphatase (ALP), total protein (TP), globin, and lysozyme contents. In the ingluvies, changes were observed in the total anti-oxidative capacity, superoxide dismutase (SOD) and catalase (CAT) activity, malondialdehyde (MDA) content and heat shock protein 70 (HSP70) gene expression over a period of 7 d. Treatment with 5.0 to 10.0 g/kg of Radix Isatidis increased the following parameters: total anti-oxidative capacity of the ingluvies prior to infection and at 1, 2 and 7 d after infection; serum ALP activity and serum globin concentrations prior to infection and at 1 and 2 d after infection; T4 concentrations prior to infection; serum lysozyme activity at 1 d after infection; SOD and CAT activity in the ingluvies prior to infection and 7 d after infection; T3 concentrations at 1 and 2 d after infection; and the relative level of HSP70 mRNA expression in the ingluvies at 2 d after infection. However, treatment with 5.0 to 10.0 g/kg of Radix Isatidis increased the following parameters: ALT contents prior to infection and 1, 2 and 7 d after infection; serum cortisol concentrations and serum glucose at 1, 2 and 7 d after infection; MDA contents of the ingluvies at 1 and 2 d after infection; and T4 concentrations at 2 and 7 d after infection. Compared with the control group, significantly lower mortality was observed in the groups supplemented with 5.0 to 10.0 g/kg of Radix Isatidis . These results suggest that supplementation with 5.0 to 10.0 g/kg of Radix Isatidis in the basal diet could enhance resistance to P. mirabilis infection in P. manillensis. Statement of relevance We believe that this manuscript is appropriate for publication in Aquaculture because it provides a natural method of controlling disease in P. manillensis , thereby avoiding the use of antibiotics and the development of antibiotic resistance. Furthermore, the results of our study provide new information on the physiological and molecular mechanisms that underlie the protective effect of Radix Isatidis .

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.

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.

The present study explored the effects of Cu²⁺enrichment 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 Cu²⁺ was added to the Wh. pigra for 50 days, and stopped adding for another 50 days. The growth index of Wh. pigra, Cu²⁺ 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 Cu²⁺ group was significantly higher than that in control group during the experimental period (

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 (

The current study was conducted to explore the relationship between property of traditional Chinese medicines (TCM) and the light conditions of their original plant in habitats and advance a "light-cold and heat property" hypothesis under the guidance of theory of TCM. In the present study, we selected 127 common Chinese herbs. Association rules mining method was used to find out the association relationship between the cold-heat properties of different TCM and the habitats of their original plant, while using statistical methods to analyze the correlation between light condition and cold-heat property of TCM based on the description of the habitats in the Flora of China and investigation. The results showed that the habitats associated to TCM with cold property are in the order of grasslandcurbhillsidewastelandgullyfarmlandriverside. The habitats that associated to TCM with heat property are: undergrowthshrubgullyjungle. For 68 TCMs with cold property, 81.2% of their original plants are light-demanding, 8.7% are shade-demanding, and 8.7% of them showed no obvious light preference. For 59 TCMs with heat property, 62.7% of their original plants are shade-demanding, 20.3% are shade-demanding, and 17.0% of them showed no obvious light preference. Meanwhile, Chi-square test showed that there was a significant difference between the cold-heat property and light preference (

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.

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 (

The objective of this study was to obtain an overview of the genetic relationships within Perinereis aibuhitensis using Inter-Simple Sequence Repeat (ISSR) and Sequence-Related Amplified Polymorphism (SRAP) markers that were derived from related populations residing in the Chinese coasts. The percentage of polymorphic bands, Nei's gene diversity and Shannon's information index revealed a high level of genetic diversity at the species level. The analysis of molecular variance revealed that 81.22% (ISSR) and 76.29% (SRAP) of variability were partitioned among individuals within populations, which indicated the coherent trend by Nei's genetic differentiation (Gst) (0.2568/0.2876). The gene flow number (Nm) was 1.4470/1.2385, which indicated that there was limited gene exchange between populations. The phylogenetic tree of the ten P. aibuhitensis populations was separated into four major clusters using the neighbor-joining (NJ) method. These results provide a simple and useful basis for P. aibuhitensis germplasm research and aquaculture breeding.

To study the effects of ingestion on antithrombin activities in different tissues of Whitmania pigra, the salivary glands, ingluvies, intestine and muscle of adult leeches were weighted on the 1st d, 3rd d, 5th d, 7th d and 11th d after feeding, respectively, and meanwhile antithrombin activity was measured by antithrombin titration method. The results showed that the antithrombin activity of salivary glands in different stages was significantly higher than that in other tissues (

The reproductive system and gonad development and germ cell occurrence of Whitmania pigra have been studied by using tissue section electron microscope techniques. W. pigra has completely independent male and female reproduction system, which lasts 11 months. The development of spermary started before the development of ovary. When egg cell is only a primordial germ cell, sperm has an initially complete form. Meanwhile, sperm cells and egg cells orderly development and synchronously mature. According to the development of sperm cells and egg cells, the development of cycle of the spermary could be divided into 6 stages: proliferating stage (1-3 months of age), growing stage (4-5 months of age), resting stage (6-8 months of age), maturing stage (9 months of age), spawning stage (10 months of age) and degradation stage (11 months of age). The development of cycle of the ovary could be divided into 6 stages: forming stage (1-2 months of age), proliferating stage (3-4 months of age), growing stage (5-8 months of age), maturing stage (9 months of age), spawning stage (10 months of age) and resting stage (11 months of age). W. pigra is a synchronous hermaphrodite animal, the development of cycle of the spermary and ovary each has six stages, sperm cells and egg cells orderly development and synchronously mature.

In this study, four light color treatments (red blue, white, natural light) were tested to determine effects of light spectrum on the growth of larval Whitmania pigra (30 days old) at the same intensity. The experiment lasted for 50 days. The specific growth rate (SGR) under blue light condition was significantly higher during 30 days to 40 days. At the end of the experiment, specific growth rate (SGR) and weight gain rate under red light condition were higher (P0.05) than those under other groups. Meanwhile, the enzymes activities of digestive enzymes (protease, amylase and lipase) were higher under red light condition. However, the enzymes activities of antioxidant enzymes (SOD, CAT, ALP) were lower under white light condition compared with the other treatments（P＜0.05）. In addition, no significant changes were observed for water content, total ash, acid insoluble ash, pH and antithrombin activity in the all treatments. After 50 days, the digestive enzymes and growth rate were higher under red light condition but antioxidant enzymes were lower under white light condition. Furthermore, light spectrum has no significant effect on the internal quality of Wh. pigra.

Transcriptome sequencing data (6.5 Gb) of the salivary glands of the haematophagous leechHirudo nipponiawas obtained by using the BGIseq-500 platform. After identification and analysis, one transcript (Unigene5370) was annotated to hirudin HV3 fromHirudo medicinaliswith ane-value of 1e-29 and was named hirudin-HN. This transcript was a new thrombin inhibitor gene belonging to the proteinase inhibitor I14 (hirudin) family. Hirudin-HN, with a 270-bp cDNA, encodes an 89-aa protein containing a 20-aa signal peptide. The mature hirudin-HN protein contains the typical structural characteristics of hirudin, e.g., three conserved disulfide bonds and the PKP and DFxxIP motifs. Proteins (HirandM-Hir) were obtained via prokaryotic expression, and the mature hirudin-HN protein was shown to have anticoagulant activity and thrombin affinity by using the chromogenic substrate S2238 and surface plasmon resonance (SPR) interaction analysis, respectively. The N-terminal structure of the mature hirudin-HN protein was shown to be important for anticoagulant activity by comparing the activity and thrombin affinity ofHirandM-Hir. The abundances of Hirudin-HN mRNA and protein were higher in the salivary glands of starving animals than in those of feeding or fed leeches. These results provided a foundation for further study on the structure-function relationship of hirudin-HN with thrombin.

The objective of this study was to isolate microsatellite loci to analyze the genetic diversity of Whitmania pigra . Four new microsatellite markers of W. pigra were developed from an enriched library and ten from a modified SAMPL assay. A total of 127 alleles were detected, with an average of 9.1 alleles per locus. The expected heterozygosity (He) of each microsatellite locus varied from 0.451 to 0.857, with an average of 0.688. The polymorphism information content (PIC) of each microsatellite locus ranged from 0.361 to 0.838, with an average of 0.640. Analysis of molecular variance showed that the main variation component existed within the populations (81.64%) rather than among the populations (18.36%). Phylogenetic tree for 15 populations of Hirudo using the NJ method by MEGA 5.1 software were divided into two major clusters. These microsatellite markers will contribute to research on the individual identification, genetic diversity, population structure, genome mapping and conservation biology of Hirudo .

The objective of this study was to obtain an overview of the genetic relationships among and within Whitmania pigra and Hirudo nipponica using the ISSR and SRAP markers that were derived from related accessions, 11 W. pigra and 4 H. nipponica, from 8 provinces in China. All populations were uniquely fingerprinted by two markers. Mean genetic similarities were estimated at 0.76 and 0.79 using the ISSRs and SRAPs. Two main clusters that separated the W. pigra and H. nipponica populations were produced using UPGMA analysis. The Mantel test resulted in a good fit (SRAP = 0.80609) to the fit (ISSR = 0.76006) of the cophenetic values. Comparing the two marker systems, the ISSR and SRAP similarity indices were correlated (r = 0.8564). Finally, an appropriate strategy for conserving the Hirudo germplasm is proposed.

The biological characteristics, oxygen consumption, oxygen consumption rate, and activities of amylase, lipase and protease of Whitmania pigra at different temperature were studied by using direct observational method, the still water method and 3, 5-dinitrosalicylic acid colorimetry, right-nitrophenyl palmitate ester（ρ-NPP）colorimetry and folin-phenol method.The results revealed that with decreasing water temperature, the daily activity and the daily feeding ration were decreased. As the temperature was lowered to 4 ℃, the head and tail of Wh.pigra curved, showing a crescent-shape without feeding and daily activity. Oxygen consumption, oxygen consumption rate and digestive enzyme activities reduced along with temperature drops. The downward trend slowed below 10 ℃, began to stabilize below 4 ℃ and doesn't change with the decrease of temperature since then. During the 40 days treatment at 4 ℃, the changes of amylase were not significant, the lipase and protease activity decreased at the 20th day, and the lipase showed an slightly increase after the decrease and finally remained at a low level.In conclusions, the pivotal temperature of hibernation of Wh.pigra is 4 ℃ and the crescent shape can be considered as a symbol of hibernation.

The oxygen consumption, oxygen consumption rate and suffocation point of different quality Whitmania pigra and Bellamya purificata were determined by hydrostatic breathing room method. The effects of feeding modes on growth of W.pigra were determined by biomass. The results showed that the oxygen consumption correlated positively with the weight of W.pigra and B. purificata（P

The effects of harvest and different processing methods on the anti-thrombin activity of Poecilobdella manillensis were respectively studied. The indicators included processing methods (vacuum freeze drying, fresh homogenate, drying under sunlight, freezing, scalding, baking under different temperatures), different parts (entire body, cephalon, pygidium, exudate) and body weights (≤10, 10-20, 20-30, 30-40, ≥40 g). The anti-thrombin activities of P. manillensis with different processing methods were evaluated by direct anti-thrombin titration. The results indicated that the processing methods significantly affected the anti-thrombin activities of P. manillensis. Among the 11 groups, the anti-thrombin activity of P. manillensis processed with vacuum freeze drying (1 303.56 U•g⁻¹) was significantly highest than the other groups (P

Studies on the variation of amylase, lipase and lrotease activity of Whitmania pigra in 0-6 months old using 3, 5-dinitro- salicylic acid colorimetry, right-nitrophenyl palmitate ester (ρ-NPP) colorimetry and folin-phenol method. The results showed that pro- tease activity remained low before 1.5 months old and with the highest activity in 2 months old, but after showing a small peak in 4 months, alkaline protease rapid declined. Amylase was low at born, then gradually increased the activity of the highest in 2.5 months old. Lipase with a strong vitality at birth, then 1 month with minimum and 2 months peaked, but appeared a small peak in 4 months old. In summary, only lipase exhibits strong activity at birth, lipase with the strongest activity in the digestive tract during develop- ment. Protease, lipase and amylase with the strongest activity at 2-3 months old, but were decreased after 4 months old.

The performance of the single-stage anaerobic digestion of kitchen wastes was investigated in an anaerobic sequencing batch reactor(ASBR) with gas-phased absorb of CO2. The ASBR was operated at four chemical oxygen demand(COD) loading rates, 2.8, 5.1, 6.2 and 8.4 g/(L x d) respectively. The COD loading rate was increased with the TS concentration and HRT changing. At maximum COD loading rate of 8.4 g/(L x d), the COD, total solid(TS) removal rate and methane gas yield were 69%, 68% and 2.5 L/(L x d) respectively. The operation of the reactor with gas-phased absorb of CO2 was stable in spite of the low pH (2.6-3.9) and high concentration of TS (142 g/L) of input mixture. The output volatile fatty acid(VFA) concentration was between 2.7-4.7 g/L and had no inhibition on the methanogenic microorganism. The reactor without gas-phased absorb of CO2 became acidified when the total COD loading rate was increased to 5.1 g/(L x d). Stoichiometry of the methanogenesis for kitchen wastes showed a considerable amount of alkaline will be required to keep pH in the appropriate range for the methanogenic microorganism based on theoretical calculation. Gas-phased absorb of CO2 effectively reduced the alkaline consumption, hence avoided excessive cation into the reactor.

Leeches are not only important medicinal animals worldwide but also are endangered. We aimed to (i) explore the level of genetic diversity within/among populations of three leeches, (ii) assess genetic differentiation among these three leeches, and (iii) discuss an appropriate strategy for conserving leech germplasm. A total of 315 individuals of Whitmania pigra, Hirudo nipponica and Poecilobdella manillensis from 21 populations were collected in China and Vietnam. The genetic structure and genetic diversity among and within the 21 populations were evaluated using target region amplified polymorphism (TRAP) and simple sequence repeat (SSR) markers. Sixteen pairs of TRAP primers generated a total of 398 fragments, of which 396 (99.50%) were polymorphic; fourteen pairs of SSR primers generated a total of 60 fragments, of which 59 (98.33%) were polymorphic. Shannon's index (I) and Nei's gene diversity index (H) for the three leeches were high at the species level (I=0.4980 and H=0.3323 for TRAPs, I=0.4487 and H=0.2969 for SSRs in W. pigra; I=0.4147/0.3769, H=0.2788/0.2566 for H. nipponica; and I=0.4616/0.4717, H=0.3099/0.3203 for P. manillensis). However, low genetic diversity was determined at the population level; the average genetic diversity measures within populations were H=0.1767/0.1376, I=0.2589/0.2043 for W. pigra, H=0.2149/0.2021, I=0.3184/0.3000 for H. nipponica and H=0.2850/0.2724, I=0.4152/0.3967 for P. manillensis. We conclude that there was limited gene exchange within/among populations and species, as the gene flow number (Nm) was 0.5493/0.5807. However, for all three species, the genetic diversity was different at the population level. Gene differentiation (Gst) and Nm were 0.4682 /0.5364 and 0.5678/0.4321 for W. pigra, 0.2294/0.2127 and 1.6797/1.8512 for H. nipponica and 0.1214/0.1496 and 3.6202/2.8412 for P. manillensis. STRUCTURE analysis, Unweighted Pair-Group Method with Arithmetic means (UPGMA) cluster analysis and Principal Coordinates Analysis (PCOA) all yielded similar results. The isolation-by-distance pattern was not significant for any of the three species by the Mantel test. These data emphasize the need for management, conservation, and rehabilitation of this animal species. Finally, an appropriate strategy for conserving leech is proposed.

The effects of stocking density and exchanging water frequency on growth, digestive enzyme activity, anti-oxidative enzyme and inner quality of Whitmania pigra Whitman were evaluated with corresponding measures. The results showed that the eventual biomass, specific growth rate, gained weight rate, activities of amylase, lipase, protease, SOD, CAT, and ALP correlated positively with stocking density and negatively with exchanging water frequency (P

The effect of water temperature, stocking density and feeding cycle on the growth of Poecilobdella manillensis juvenile was conducted P. manillensis was conducted respectively under different conditions: water temperatures(18, 22, 26, 30,34, 38 degrees C and CT), stocking density (75, 125, 200, 275, 350 individual/L) and feeding cycle(2, 4, 6, 8, 12, 16 d). After 30 days, survival rate, weight gain rate, specific growth rate were measured. There was a significant correlation between water temperature and specific growth rate (γ = -0.066x2 + 3.543 1x -38.09, R2 = 0.837 9). Based on the regression equation, the specific growth rate of P. manillensis achieved the maximum (9.461 4) at 26.84 degrees C. And the most optimal water temperature was 26-30 degrees C. Meanwhile, the survival rates of P. manillensis was 0 at 38 degrees C in 3 d. There was significant negative correlation between density and specific growth rate (γ = -0.005 7x + 9.197 3, R2 = 0.998 3) and between feeding cycle and specific growth rate (γ = -0.468 2x + 10.574, R2 = 0.998 8).

The effects of the feed attractants on Whitmania pigra were studied. The average weight of Wh. pigra were 5.0 g. Arginine was selected as feed attractants, xanthan gum was selected as feed substrate. The times of Wh. pigra going into the inducing room were recorded. The water temperature was 22-25 degrees C during the whole experiment. Arginine that had better inducing effect was chosen to carry on in the gradient experiment. The results showed that the best inducing effect was found when the added amount of arginine was 0.3%, which was close to the arginine content of the natural body fluid of Wh. Pigra and Bellamya purificata, 2.97 mg x g(-1).

Mouthpart developmental histology of Whitmania pigra at different month of age were studied by paraffin section, HE staining combined alcian blue and periodic acid schifts reaction procedure (AB-PAS). The following results was obtained: Change ranges: oral width 0.6 mm (1-3 month), 1.2 mm (34 month); oral diameter 0.3 mm (1-3 month); 1.2 mm (34 month), the oral size reached maximum during 4-6 months and unchanged thereafter. Oral lip had a thin protective film located in the front of the mouthpart. The W. pigra possessed three jaws in oral cavity, the big one was in dorsum, the other two separated on both side of abdomen respectively. Jaws and muscular pharynx were interrelated closely. The jaws were composed by cuticle, epithelial layer, muscularis and jaw cavity from outside to inside. In the front of jaws had mastoid abdomen with function of secreting acidophilic granule from 2 month age. Oral cavity was composed by mucosa, submucosa and muscularis inside and outside. Oral cavity was rich of peristomial nerves. And pharynx was composed of mucosa, muscularis, adventitia from inside to outside. The folds height and width become heighten and thicken. Mucosa epithelium from complex flat epithelium changed into columnar epithelium, muscularis gradually developed into thickened along with growing. Muscular thickness reached maximum at 4 months. Mucous cells of W. pigra were classified into I-IV types based on different staining and two mainly morphological shapes (Tubular, Pear-shaped). Jaws, oral cavity, pharynx by AB-PAS staining showed little changes at different month of age. Mucous cells were few at 1 month age, and type II cells were increased rapidly in 2-3 month age in oral lip. Oral cavity contains more mucous gland cells type I. Under the muscularis there were connective tissues which distributed a few of mucous cells type II.

Prunella vulgaris L (Labiatae) is commonly used as a traditional medicinal herb in some Asian and Europe countries. To date, few studies have been conducted to determine the influence of [Formula: see text] - N/[Formula: see text] - N ratio on growth, physiological development, and bioactive phytochemical accumulation in hydroponically grown P. vulgaris.The current study was conducted to evaluate the effect of five [Formula: see text] - N/[Formula: see text] - N ratios on growth, nitrogen metabolism, photosynthetic efficiency, and bioactive phytochemical production in P. vulgaris.Hydroponically cultivated P. vulgaris were fertilized with five [Formula: see text] - N/[Formula: see text] - N ratios in a greenhouse for 85 d. Dried weight of root, stem, leaf and spica, leaf area, photosynthetic efficiency, activities of nitrate reductase (NR), glutamine synthetase (GS), and the concentrations of N, soluble protein, and free amino acids in the leaves, as well as the contents of rosmarinic acid (RA), ursolic acid (UA), and oleanolic acid (OA) in the spicas were measured.Both [Formula: see text] - N and [Formula: see text] - N as the sole source of nitrogen had inhibitory effects on P. vulgaris growth. P. vulgaris fertilized with the 25/75 ([Formula: see text] - N/NO3 - N) ratio had the highest leaf area, photosynthetic rate, and chlorophyll content. The 25/75 ([Formula: see text]/[Formula: see text]) ratio increased the spica biomass by 1828%, nitrate-reductase (NR) activity by 98%, and soluble protein concentration by 29.45% compared with the 100/0 ([Formula: see text]/[Formula: see text]) treatment. Additionally, 25 [Formula: see text] - N/75 NO3 - N resulted in the highest contents of RA and total flavonoids as well as relatively high contents of UA and OA; therefore, this ratio had the highest yield of RA, UA, OA, and total flavonoids in spicas.The use of 25 [Formula: see text] - N/75 [Formula: see text] - N is recommended to improve biomass production and medicinal quality of P. vulgaris.

The dried Whitmania pigra is used for the treatment of cardiovascular and cerebrovascular diseases in traditional Chinese medicine. Bellamya purificata is widely distributed in the Chang Jiang River basin, it is natural diets of W. pigra. Current study was conducted to compare and analyze the nutritional ingredient in W. pigra, body fluid and flesh of B. purificata. Results showed that the contents of protein, crude fat and total sugar in W. pigra, body fluid and flesh of B. purificata were significantly different (P0.05). Protein content in W. pigra accounts up to 65.01%. The contents of inorganic elements and amino acid were abundant in W. pigra, body fluid and flesh of B. purificata. The content of essential amino acids in them were 32.6, 221.59, 40.78 mg x g(-1), respectively. The content of flavor amino acid in them were 27.51, 14.5, 32.03 mg x g(-1), while the coresponding content of antioxidant amino acid were 8.81, 5.91, 9.73 mg x g(-1), respectively. The individual amino acids of high content in them were Glu, Asp and Leu. Macro elements Ca, P, Mg and trace elements Zn, Si, Fe were abundant. It could be speculated that W. pigra may be a promising novel food, and the present results provide a foundation to develop artificial feed for W. Pigra.

Current study was conducted to investigate and compare the impact of temperature and pH on the activities of amylase, protease and lipase in alimentary tract of Whitmania pigra.The responses of amylase, protease, and lipase activities were determined over a wide range of temperatures (7-52 degrees C) and pH gradient (2.2-11.2).The highest lipase activity was found under 37 degrees C, pH 8.2, and the highest amylase activity was detected under 37 degrees C, pH 5.2, while protease activity peaked at 42 degrees C, pH 3.2 or pH 9.2.The optimal temperature in alimentary tract of Wh. pigra for lipase and amylase was 37 degrees C, and the responding temperature for protease was 42 degrees C. The optimal pH value in alimentary tract of Wh. pigra for lipase and amylase was pH 8.2 and pH 5.2, respectively. While pH 3.2 or 9.2 seems to be both favorable for high protease activity.

To determine the best spawning temperature and weight of Whitmania pigra and the hatching temperature of its eggs.Observe the state of W. pigra spawning and hatching were observed under different temperatures and weights conditions.25 degrees C is the most appropriate temperature for W. pigra spawning and incubation, the best spawning weight of W. pigra was around 20 g. The temperature influenced the lost weight after spawning significantly and was proportion to the lost weight. The body weight showed no substantial influence to the lost weight after spawning. The egg weight of W. pigra which body weight was below 2.0 g was proportion to the hatching rate. The same relationship was not observed in W. pigra over 2.0 g.

To observe Whitmania pigra growth and intake law in different temperature conditions and patterns.Comparison of W. pigra growth in different weight and different temperature conditions, and observe the intake law of W. pigra within 24 h.The appropriate temperature for W. pigra growth is 15 - 25 degrees C. Its largest weight of intake increas with the weight of W. pigra and temperature rise, but with the intake rate increase, individual intake reduced. W. pigra intake have the two peaks in 24 h.

Through observation of the tissue structures of internal organs and skin of Whitmania pigra, to provide basic method for identification of W. pigra and research of its physiological functions.This experiment adopted the W. pigras as the material and carried on dissection observing,and W. pigra's cavity, swallow, esophagus, crop, bowel, rectum, parasomal sac stomach, testicle, seminal vesicle, vasa deferentia duct, ejaculatory ball, prostate gland and skin was observed by the method of the paraffin organization slice and microphotography.The structures and functions of the skin and the organ of the digestive and reproductive system, as well as the types and distributions of the mucous cells were acquired,which provided abasis for further study on the living behaviour, propagating behaviour and breeding environment of W. pigra.

To give a reference to the safe and standardized production of Whitmania pigra, through the comparative research on residues of heavy metals and pesticide in W. pigra, breeding base soil and breeding water .Residues of heavy metals and pesticide in W. Pigra, soil and water were determined by atomic absorption spectrophotometers, atomic fluorescence spectrophotometers and GC-ECD.With the exclusion of Pb residue, the residues of HCH, DDT and other heavy metals in W. pigra, soil and water in breding base met the requirements of the national standards. We suggest that the residues limits of animal drugs like W. pigra should be listed in a separate category when the national standards are revised in the fature.

To study the genetic diversity of pollen grains morphologies of Pinellia ternata in different populations and supply palynological evidences for further research.Pollen exine ultrastructure were compared for 18 populations growing under in the same environment by means of scanning electron microscope(SEM).Big difference was found in pollen size among populations of P. ternata: the mean value of seventeen populations of P. ternata with bulbils was 17.42 microm x 14.86 microm, the biggest of populations originated from Jangyan city is 20.43 micromx 18.16 microm, but the least originated from Peixian County is only 15.23 microm x 12.82 microm; the size of populations without bulbils originated from Linguo Temple side was only 13.23 microm x 11.98 microm and far less than of all populations with bulbils. The pollen surface of all populations sticks out with spines, varying in size and density, the base and top part of spinules was different among populations. The pollen grains patterns of germination apertures of P. ternata was likely to have colpate cover.

To study the oxygen consumption rate and suffocation point of Whitmania pigra.Using the method of respiratory chamber, the oxygen consumption rate, oxygen consumption volume and suffocation point were obtained.The oxygen consumption rate(OCR) ranged from 0.049 to 0.094 mg x g(-1) x h(-1), the oxygen consumption volume(OCV) from 0.44 to 0.67 mg x p(-1) x h(-1), and suffocation point(SP) from 0.9 to 1.51 mg x L(-1), when the average weight of W. pigra was 10 g and the water temperature varied from 15 to 35 degrees C. OCR ranged from 0.044 to 0.058 mg x g(-1) x h(-1), OCV from 0.19 to 0.77 mg x p(-1) x h(-1) and SP from 1.4 to 1.57 mg x l(-1) when water temperature was 20 degrees C.OCR rose when the water temperature and weight increased, but SP was justly opposite. The weight was negatively correlated with OCR and positively with OCV. OCR had little difference from day to night.