RESUMO
The present study cloned wpGuamerin gene from a non-bloodsucking leech (Whitmania pigra), and the bioinformatics îanalysisî 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.
Assuntos
Hormônios de Invertebrado/genética , Sanguessugas/genética , Animais , Clonagem Molecular , Músculos/metabolismo , Estrutura Secundária de Proteína , Glândulas Salivares/metabolismoRESUMO
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.
Assuntos
Hormônios Esteroides Gonadais/análise , Gônadas/química , Sanguessugas/química , Animais , Cromatografia Líquida , Estradiol , Progesterona , Espectrometria de Massas em Tandem , TestosteronaRESUMO
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.
Assuntos
Astrágalo/química , Medicamentos de Ervas Chinesas/farmacologia , Sanguessugas/efeitos dos fármacos , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Sanguessugas/crescimento & desenvolvimento , Sanguessugas/imunologia , Raízes de Plantas/químicaRESUMO
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 esterï¼p-NPPï¼colorimetry, 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⻹ 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.
Assuntos
Nível de Alerta , Hibernação , Sanguessugas/fisiologia , Animais , Trato Gastrointestinal/enzimologia , TemperaturaRESUMO
The present study explored the effects of Cu²âºenrichment 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 Cu²âº was added to the Wh. pigra for 50 days, and stopped adding for another 50 days. The growth index of Wh. pigra, Cu²âº 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⻹ of Cu²âº 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 Cu²âº 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 Cu²âº 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 Cu²âº in different tissues of Wh. pigra was digestive tract>muscle>skin. The ability of release Cu²âº was muscle>digestive tract>skin. There was no significant difference in the internal quality and hirudin gene expression between Cu²âº group and control group. In conclusion, Cu²âº 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 Cu²âº within 30 days.
Assuntos
Cobre/farmacologia , Sanguessugas/crescimento & desenvolvimento , Sanguessugas/genética , Animais , Regulação da Expressão GênicaRESUMO
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 (P<0.05). The activity of antithrombin in muscle tissue increased initially and then decreased with the prolongation of the time after feeding, and the peak was observed on the 5th day after feeding (P<0.05). The activity of antithrombin in the salivary glands, gluvies and intestine were found the highest on the 1st day after feeding(P<0.05), and then gradually decreased with the prolongation of the time of stopping the diet. The total amount of antithrombin activity on the 5th day was increased by 49.5%, 73.5%, 69.1% and 126.0% comparing with the 1st, 3rd, 7th and 11th day after feeding, respectively (P<0.05). In summary, both the feeding behavior and the food can induce the secretion of anticoagulant substances in the salivary glands and the digestive tract. The total amount of antithrombin activity was the highest on the 5th day after feeding and the 5th day after feeding was suggested as harvesting time.
Assuntos
Antitrombinas/química , Comportamento Alimentar , Sanguessugas/química , Glândulas Salivares/química , Animais , AnticoagulantesRESUMO
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.
Assuntos
Variação Genética , Sanguessugas/genética , Repetições de Microssatélites/genética , Filogenia , Animais , China , Espécies em Perigo de ExtinçãoRESUMO
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 Uâ¢g⻹) was significantly highest than the other groups (P<0.05), and that processed with baking under 90 â (15.44 Uâ¢g⻹) was the lowest. The anti-thrombin activity of the cephalon of P. manillensis (226.42 Uâ¢g⻹) was the highest, and that of the pygidium (102.12 Uâ¢g⻹) was lowest; the anti-thrombin activities for different body weights were significantly different (P<0.05); and among the five groups, the body weight of ≤10 g (328.86 Uâ¢g⻹) was the highest (P<0.05), and the body weight of ≥40 g (87.71 Uâ¢g⻹) was the lowest. In conclusion, harvest and different processing methods had a significant impact on the anti-thrombin activities of P. manillensis. In the study, for the optimal processing method for P. manillensis, the body weight between 20-30 g is recommended, and the vacuum freeze drying is preferred, which is followed by the drying under sunlight.
Assuntos
Química Farmacêutica/métodos , Fibrinolíticos/farmacologia , Sanguessugas , Animais , Dessecação , Liofilização , Luz SolarRESUMO
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 esterï¼ρ-NPPï¼colorimetry 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 â, 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 â, began to stabilize below 4 â and doesn't change with the decrease of temperature since then. During the 40 days treatment at 4 â, 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 â and the crescent shape can be considered as a symbol of hibernation.