Evaluation of Cellulolytic Endo-1,4-β-D-Glucanase Activity in the Digestive Fluid of Adult Phytophagous Beetle Hoplasoma unicolor

Insects of the taxonomic order Coleoptera are recognised for considerable cellulolytic activity in their digestive fluid. The cellulolytic activity of the gut fluid in Hoplasoma unicolor, a member of Coleoptera, however, remains unexplored. In this study, we, for the first time, report the qualitative and quantitative analysis of cellulolytic activity in the digestive fluid of this insect. The cellulolytic endo-1,4-β-D-glucanase activity was confirmed in the supernatant of the insect’s digestive fluid by agar plate assay using carboxymethyl cellulose as the substrate. To determine the optimum pH, enzyme activity was further assessed in an acidic pH range of 5 to 6, and the highest activity was observed at pH 5.3. For quantitative analysis, endoglucanase activity was measured using 3,5-dinitrosalicylic acid method which revealed that the specific activity of the gut sample was 0.69 (±0.01) units per mg of protein. For further characterisation of the cellulases in the sample, SDS-PAGE and zymogram analysis were carried out. Two active cellulolytic bands were detected on the zymogram suggesting the presence of two distinct endoglucanases which completely disappeared upon heating the sample at 55°C. Our study, therefore, highlights prospect of the gut fluid of H. unicolor as an important source of cellulase enzymes that merits further investigations into their extensive characterisation for potential industrial applications.

Effects of Three Types of Organic Fertilizers on Greenhouse Gas Emissions in a Grassland on Andosol in Southern Hokkaido, Japan

Reduction of chemical fertilizers and effective use of livestock excrement are required for the realization of sustainable agriculture and reduction of greenhouse gas (GHG) emissions. The purpose of this study was to estimate the reduction rate of GHG emissions represented by comparing global warming potential (GWP) using organic fertilizers instead of chemical fertilizers. The study was conducted in a managed grassland on Andosol in southern Hokkaido for 3 years from May 2017 to April 2020. There were five treatment plots: no fertilizer, chemical fertilizer, manure, slurry, and digestive fluid. Organic fertilizers were applied such that the amount of NPK did not exceed the recommended application rate, and the shortage was supplemented with chemical fertilizers. Fluxes in CO2 caused by heterotrophic respiration (RH), CH4, and N2O were measured using the closed chamber method. Net ecosystem carbon balance (NECB) was obtained as net primary production + organic fertilizer application—RH—harvest. The GWP was estimated by CO2 equivalent NECB and CH4 and N2O emissions in each treatment. Chemical fertilizer nitrogen application rates in the organic fertilizer treatments were reduced by 10% for manure, 19.7% for slurry and 29.7% for digestive fluid compared to chemical fertilizer only, but the grass yields were not significantly different among the fertilizer treatments. The 3-year NECB showed significantly smallest carbon loss in manure treatment, and smaller carbon loss in the organic fertilizer treatments than in the chemical fertilizer only. The reduction rate in the GWP with use of organic fertilizers relative to that of chemical fertilizer was 16.5% for slurry, 27.0% for digestive fluid, and 36.2% for manure. The NECB accounted for more than 90% of the GWP in all treatments. CH4 emissions were < 0.1% of the GWP. On the other hand, N2O emissions accounted for more than 5% of the GWP, and was larger in the order of slurry > chemical fertilizer only > digestive fluid > manure. As a conclusion, these organic fertilizers can be used without no reduction of crop yield instead of chemical fertilizer, however, manure is the best way to increase soil carbon and to decrease GWP, followed by digestive fluid.

Pactacin is a novel digestive enzyme in teleosts

Generally, animals extract nutrients from food by degradation using digestive enzymes. Trypsin and chymotrypsin, one of the major digestive enzymes in vertebrates, are pancreatic proenzymes secreted into the intestines. In this investigation, we report the identification of a digestive teleost enzyme, a pancreatic astacin that we termed pactacin. Pactacin, which belongs to the astacin metalloprotease family, emerged during the evolution of teleosts through gene duplication of astacin family enzymes containing six cysteine residues (C6astacin, or C6AST). In this study, we first cloned C6AST genes from pot-bellied seahorse (Hippocampus abdominalis) and analyzed their phylogenetic relationships using over 100 C6AST genes. Nearly all these genes belong to one of three clades: pactacin, nephrosin, and patristacin. Genes of the pactacin clade were further divided into three subclades. To compare the localization and functions of the three pactacin subclades, we studied pactacin enzymes in pot-bellied seahorse and medaka (Oryzias latipes). In situ hybridization revealed that genes of all three subclades were commonly expressed in the pancreas. Western blot analysis indicated storage of pactacin pro-enzyme form in the pancreas, and conversion to the active forms in the intestine. Finally, we partially purified the pactacin from digestive fluid, and found that pactacin is novel digestive enzyme that is specific in teleosts.

Influence of soil environmental factors on N2O fluxes just after application of three types of organic fertilizers - 4 years study in a grassland on Andosol in southern Hokkaido, Japan

<p>Appropriate application of organic fertilizer is required to reduce environmental impact from grassland and to achieve sustainable livestock production. However, N<sub>2</sub>O fluxes from soil increase mainly due to changes in soil environmental factors such as temperature, moisture, soil pH and soil mineral nitrogen content, immediately just after fertilization, and it may be different among the types of fertilizer. In this study, we investigated that how N<sub>2</sub>O fluxes are influenced by the application of three types of organic fertilizer (manure, slurry, and digestive fluid) for 4 years in a grassland on Andosol in southern Hokkaido, Japan. Five treatment plots: no fertilizer, chemical fertilizer, manure, slurry, and digestive fluid were established in a managed grassland in Shizunai Livestock farm, Hokkaido University. Fertilizers were applied in late April every year from 2017 to 2020. Organic fertilizers were applied such that the NPK not exceed the regional recommendation rate, and the shortage was compensated by chemical fertilizer. N<sub>2</sub>O flux was measured by using a closed chamber method. At the same time of the flux measurements, soil temperature at 5 cm soil, and soil moisture (WFPS), soil pH, NO<sub>3</sub>-N contents in 0-5 cm soil were measured to see the relationship with N<sub>2</sub>O fluxes.</p><p>In 2017, a large peak of N<sub>2</sub>O flux was observed in slurry plot (195.8μg m<sup>-2</sup>h<sup>-1</sup>) and digestive fluid plot (347.8 μg m<sup>-2</sup>h<sup>-1</sup>), whereas in 2018 and 2019, there were no large peak after the fertilization at all plots, however, in 2020, a large peak of N<sub>2</sub>O flux was observed in manure plot (472.7 and 475.7μg m<sup>-2</sup>h<sup>-1</sup>) and slurry plot (194.9μg m<sup>-2</sup>h<sup>-1</sup>). These peaks of N<sub>2</sub>O flux were significantly larger than those in no fertilizer and chemical fertilizer plots. All N<sub>2</sub>O flux peaks were observed when the soil temperature ranged 10-14 ℃. In 2017 and 2020, a large peak of N<sub>2</sub>O flux was observed although WFPS was always above 80% which is the soil moisture level leading to the complete denitrification. There was a negative relationship between N<sub>2</sub>O flux and soil pH. Low soil pH might reduce the N<sub>2</sub>O reductase activity, leading to the large peak of N<sub>2</sub>O flux at high WFPS above 80%. In addition, there was a positive relationship between N<sub>2</sub>O flux and soil NO<sub>3</sub><sup>-</sup>-N contentin 2017 and 2020. However, in 2018 and 2019, when WFPS was below 80% in most days, there was no positive relationship between N<sub>2</sub>O flux and soil NO<sub>3</sub><sup>-</sup>-N content. In conclusion, the peak of N<sub>2</sub>O flux was different depending on the year and fertilizer, In order to reduce N<sub>2</sub>O flux just after fertilization, it is especially important not to lower the soil pH and not to increase the WFPS.</p><div> </div>

Using Digestive Fluid Biomarkers to Predict Acute Gastrointestinal Injury in Critically Ill Patients: A Pilot Study

Background Acute gastrointestinal injury is associated with significantly increased mortality in critically ill patients. However, markers for measuring acute gastrointestinal injury are neither sensitive nor specific. Objective To determine whether enzymes in digestive fluid are predictive of the severity of acute gastrointestinal injury. Methods A prospective observational study was conducted between June 2015 and December 2015 in a surgical intensive care unit. Enrolled patients were classified by acute gastrointestinal injury grade according to the 2012 European Society of Intensive Care Medicine system. Digestive fluid was collected through nasointestinal tubes and analyzed 24 hours after the diagnosis of acute gastrointestinal injury. Intestinal markers of injury (pH, interleukin 6, interleukin 10, tumor necrosis factor α, and secretory immunoglobulin A) were measured in digestive fluid. Results Of the 76 patients included, acute gastrointestinal injury was grade I in 41, grade II in 20, grade III in 8, and grade IV in 7. Secretory immunoglobulin A was an independent predictor of grade III acute gastrointestinal injury. When data from patients with grades I and II injury and patients with grades III and IV injury were grouped together, analysis revealed that pH, interleukin 10, and secretory immunoglobulin A were independent predictors of acute gastrointestinal failure. Conclusions Secretory immunoglobulin A was predictive of grade III acute gastrointestinal injury. Digestive fluid markers of injury (pH, interleukin 10, and secretory immunoglobulin A) were predictors of acute gastrointestinal failure. Further study is required to determine if other markers are specific or sensitive for acute gastrointestinal injury.