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.
Animals ; Female ; Histology ; Leeches ; anatomy & histology ; chemistry ; classification ; growth & development ; Male ; Mouth ; anatomy & histology ; chemistry ; Mucous Membrane ; chemistry

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.
Age Factors ; Amylases ; metabolism ; Animals ; Leeches ; enzymology ; Lipase ; metabolism ; Peptide Hydrolases ; metabolism

This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.
Amino Acid Sequence ; Animals ; Betaretrovirus ; chemistry ; classification ; genetics ; physiology ; Cell Transformation, Viral ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Phylogeny ; Retroviridae Infections ; veterinary ; virology ; Sequence Alignment ; Sheep ; Sheep Diseases ; virology ; Transformation, Genetic ; Tumor Virus Infections ; veterinary ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

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).
Animal Feed ; analysis ; Animals ; Arginine ; analysis ; metabolism ; Body Weight ; Feeding Behavior ; Leeches ; growth & development ; physiology

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 (P < 0.05). 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.
Amino Acids ; analysis ; Animals ; Gastropoda ; chemistry ; Leeches ; chemistry ; Medicine, Chinese Traditional

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 (P<0.05) 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.

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<0.05）, suffocation point increased with the increases of the weight（P<0.05）.Oxygen consumption correlated negatively with the weight of W. pigra, the oxygen consumption rate of B.purificata first increased and then decreased with the increasing of the weight. Feeding modes had no significant effects on the finial weight, SGR, WGR, death rates of W. pigra. Feeding modes had significant effects on eating ratio. It suggested that the optimum feeding frequency of W. pigra was once every three days. Scientific and reasonable feeding amount of B. purificata should be calculated based on oxygen consumption and suffocation point of W.pigra and B.purificata at every period. Meanwhile, stocking density, water area and water exchanging frequency should be taken into consideration.

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.
Animals ; Female ; Gonads ; cytology ; Leeches ; growth & development ; Male ; Ovary ; cytology ; Ovum ; cytology ; Reproduction ; Spermatocytes ; cytology

Objective:To analyze the causes of skin necrosis after penis lengthening surgery and corresponding treatment meas-ures,and observe the clinical effect of free skin graft repair in the treatment of penile skin defects.Methods:We retrospectively an-alyzed the clinical data on 12 cases of extensive penile skin necrosis and defect after penis lengthening surgery performed in our depart-ment from January 2017 to January 2022.The patients underwent free skin graft repair with medium-or full-thickness skin grafts from the thigh after wound preparation.Results:The skin grafts survived well in all the 12 patients and the incisions healed in the first stage without any complications.At 6 months after surgery,skin sensation was mostly recovered in the area of penis skin grafting,no obvious skin ulceration or edema was observed,and the appearance of the penis was satisfactory.The IIEF-5 scores,Erectile Hardness Scale(EHS)scores,and the results of penile hardness tests of the patients all indicated normal erectile function.Conclusion:Free skin graft repair with autologous medium-or full-thickness skin grafts is a safe and effective surgical option for extensive penile skin necrosis after penis lengthening surgery.

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.