Cloning and functional identification of pmKPI cDNA in Poecilobdella manillensis.

BACKGROUND: Kazal-type serine protease inhibitors play a role in physiological processes such as blood coagulation and fibrinolysis. The amino acid residues at the P1 site are different, and they inhibit different types of proteases. The inhibitory mechanism of the protease in the salivary glands of Poecilobdella manillensis is still unclear. METHODS AND RESULTS: Based on cloning, prokaryotic expression and bioinformatics analysis, we studied the role of Kazal-type serine protease inhibitors in P. manillensis and analyzed their expression by quantitative real-time PCR. The results suggested that the recombinant protein was successfully expressed in the supernatant when a prokaryotic expression vector was constructed and induced with 0.2 mmol/L IPTG at 37 °C for 4 h, and the enzymatic activity was determined. The mature protein encodes 91 amino acids and has a relative molecular weight of 9929.32 Da, and after removing the signal peptide, the theoretical isoelectric point was 8.79. It is an unstable protein without a transmembrane domain. The mature protein contains two Kazal-type domains, in which all P1 residues are Lys, consisting of an α helix and three antiparallel ß sheets. The upregulated expression of the mRNA was induced after a meal was provided, and the results showed an increasing and then decreasing trend. CONCLUSIONS: Taken together, the results indicate that mature proteins from P. manillensis inhibit thrombin activity, laying the foundation for the subsequent in-depth study of the function of genes encoding Kazal-type serine protease inhibitors.

[Optimization of Extraction Methods and Distribution Characteristics of Antibiotics and Metabolites in Sediments of a River Water-Groundwater Interaction Zone].

The river water-groundwater interaction zone is an important area for the enrichment, degradation, and transformation of pollutants and other biogeochemical processes. The distribution characteristics of antibiotics, as organic pollutants of wide concern, in the interaction zone is essential for understanding the migration and transformation process of pollutants in the environment. Due to the sensitive changes in the redox conditions and special composition of sediments in the interaction zone, this study established an effective pretreatment method for extracting 22 antibiotics and four sulfonamide metabolites in the interaction zone, and optimized the initial state of the samples, extraction temperature, pH value of the extraction solution and organic extraction solvent. The content of antibiotics in the sediments of the river water-groundwater interaction zone and lower reaches of the Hanjiang River was also analyzed. The results show that the best recovery of the target compounds is obtained by using pH 3 acetonitrile/Na2EDTA-Mcllvaine buffer(1:1, volume ratio) to digest and extract the unoxidized sediment samples at 40℃ three times using a microwave. A total of 11 antibiotics are detected in the sediments of the interaction zone in the lower reaches of the Hanjiang River, among which oxytetracycline and ofloxacin are the main compounds with the highest concentrations of 6.77 ng·g-1 and 5.81 ng·g-1, respectively. The vertical distribution of antibiotics in different sediment profiles is significantly different, which may be related to the lithology of sediments, physicochemical properties of antibiotics, and interaction between surface water and groundwater.

[Studies on effects of water temperature, stocking density and feeding cycle on growth and feeding in Hirudo nipponica].

Effects of different water temperature, stocking density and feeding cycle on growth, feeding and survival of Hirudo nipponica have been studied, six temperature gradients were set: 18, 22, 26, 30, 34 and 38 ℃, five stocking density gradients were set: 30, 60, 120, 180 and 240 leech/L, four feeding cycle gradients were set: 2, 5, 10 and 20 d, respectively. The results showed that there exists a significant regression relationship between water temperature and specific growth rate: y=-0.016 5x²+0.836 9x-6.847 5（R²=0.990 8）（P<0.05）, a regression analysis indicated that specific growth rate reached the maximum (3.76) at 25.36 ℃. When water temperature was beyond 30 ℃, the survival rate significantly decreased as water temperature increased （P<0.05）. The specific growth rate and survival rate decreased as stocking density increased. A linear relationship exists between the feeding cycle and the SGR: y=-0.094 1x+3.832 9（R²=0.992 7）. From this study, it can be concluded that the optimal water temperature and stocking density for the growth of H. nipponica is 22-26 ℃ and 30-120 leech/L, respectively.

Genetic variation in Whitmania pigra, Hirudo nipponica and Poecilobdella manillensis, three endemic and endangered species in China using SSR and TRAP markers.

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.

[Effects of harvest and different processing methods on anti-thrombin activity of Poecilobdella manillensis].

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<0.05), and that processed with baking under 90 ℃ (15.44 U•g⁻¹) was the lowest. The anti-thrombin activity of the cephalon of P. manillensis (226.42 U•g⁻¹) was the highest, and that of the pygidium (102.12 U•g⁻¹) 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 U•g⁻¹) was the highest (P<0.05), and the body weight of ≥40 g (87.71 U•g⁻¹) 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.

[Effect of water temperature, stocking density and feeding cycle on growth of juvenile Poecilobdella manillensis].

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).