Application of Environmental DNA (eDNA) Metabarcoding Method to Identify Threatened Sulawesi Mammal Based on 12S rRNA Gene

Species detection and identification is a crucial steps in biodiversity assessment. Traditional methods are often invasive and resource intensive. The number of studies demonstrating successful of eDNA metabarcoding approach in species identification has increased rapidly in recent years. Some of large terrestrial mammals have reportedly utilize natural salt licks as a source of minerals in the diet and its genetic material left in the environment can be used to identify species from this site. An eDNA metabarcoding protocol had been carried out to identify Sulawesi mammals from Adudu natural salt-licks, Nantu Wildlife Reserve, Gorontalo. Environmental DNA were extracted from water samples, Amplicon libraries were prepared by PCR amplification and Illumina MiSeq high throughput sequencing. Reads processing and taxonomic assignment carried out in two bioinformatics packages, PipeCraft-1.0 and OBITools-2.11. Two endangered Sulawesi mammals species had been identified, i.e. lowland anoa (Bubalus depressicornis) and babirusa (Babyrousa babyrussa). The accuracy of mammal species identification using eDNA metabarcoding is affected by rigorous experimental procedures, DNA marker reliability, and availability of reference sequence database.

Quality Improvement of White Pepper with Processing through Optimizing the Ratio of Starter Culture from Acetobacter sp., Bacillus subtilis, and Bacillus cereus

Fermentation process using known microbial species can be exploited for the processing of white pepper. It is expected to generate quality of white pepper in a short period soaking time. This research aimed to study characteristics of white pepper through a fermentation process by addition of combination isolates of Acetobacter sp., B. subtilis, and B. cereus. After threshing, 2 kg of fresh pepper berries was soaked in water mixed with starter culture. The experiment used a completely randomized design, two replications. The treatment consisted of: A) starter culture ratio of Acetobacter sp.: B. subtillis: B. cereus (A1 = 0:0:0; A2 = 1:1:1; A3 = 2:1:1; A4 = 1:2:1; and A5 = 1:1:2) and soaking time (B1 = 5 and B2 = 7 days). Fermented pepper was decorticated, washed, and dried. The best treatment was fermented for 7 days with the combination isolates of Acetobacter sp., B. subtilis, and B. cereus with ratio 2:1:1. This condition produced white pepper in fulfilling in requirement of SNI standards with piperine and essential oil contents and TPC of 5.95%, 2.95% and 1.1 x 102 CFU/g, respectively. This process is expected to generate high quality of white pepper in a short soaking time.

In Vitro Pancreatic Lipase Inhibition by Marine Fungi Purpureocillium lilacinum Associated with Stylissa sp. Sponge as Anti-obesity Agent

This study aimed to evaluate the potential of marine fungus Purpureocillium lilacinum isolated from an Indonesian marine sponge Stylissa sp. as an anti-obesity agent through pancreatic lipase inhibition assay. The fungus was identified as P. lilacinum through morphological and molecular characteristics. The fungal extract’s inhibition activity and kinetics were evaluated using spectrophotometry and Lineweaver-Burk plots. Ethyl acetate and butanol were used for extraction. Both extracts showed pancreatic lipase inhibition in a concentration-dependent manner. Both crude extracts were then fractionated once. All fractionated extracts showed inhibitory activity above 50%, with the highest activity found in fraction 5 of ethyl acetate at 93.41% inhibition. The best fractionated extract had an IC50value of 220.60 µg.mL-1. The most active fraction of P. lilacinum had a competitive-type inhibitor behavior as shown by the value of Vmax not significantly changing from 388.80 to 382.62 mM pNP.min-1, and the Michaelis-Menten constant (KM) increased from 2.02 to 5.47 mM in the presence of 500 µg.mL-1 fractionated extract. Metabolite identification with LC-MS/MS QTOF suggested that galangin, kaempferol, and quercetin were responsible for the observed lipase inhibition.

The Potency of Anadara nodifera Shell as Natural Testosterone Booster for Male Canary (Seriunus canaria)

Anadara clam shell contains high Zinc. This microelement is recognised as acting natural aromatase blocker (NAB) that capable of boosting testosterone level. The aim of this study was to evaluates the testosterone level of Canaries following Anadara nodifera administration. Adult male Canaries (n = 27) were group into 3 with 9 repetition of each i.e T0 [control, was given by Carboxymethyl Cellulose Sodium (Na-CMC)], T1 (Zinc Sulphate 0.013 mg/30 g BW), and T2 (NAB 0.3 mg/30 g BW). The T2 treatment was NAB or shell powder of Anadara nodifera which was diluted 1:1 in Na-CMC. The birds were treated orally for 21 days. The complete blood cell count was performed in a haemo analyser except the erythrocyte was calculated manually in the Improved Neubauer counting chamber. Testosterone level was measured in ELISA. Subsequently gen CYP19 aromatase in syrinx, brain and testis were identified employing IHC and descriptively analysed. The result indicated significant elevated level of testosterone (p<0.05). The expression of CYP19 aromatase receptor was depressed in syrinx, brain and testis. However there was no significant changes on the blood profile (p>0.05). To conclude, Anadara nodifera at 0.3 mg/30 g BW was capable of acting as NAB to promote testosterone level. This was confirmed by the reduction of CYP19 aromatase gene receptors in syrinx, brain and testis. However NAB treatment was not significantly affecting routine blood profile.

Characteristics and Activity Anti Qourum Sensing Bacillus spp. Isolated from Penaeus vannamei Shrimp Ponds

Certain strains of V. parahaemolyticus carry a gene that encodes a toxin that causes Acute hepatopancreatic necrosis disease (AHPND) in P. vannamei. AHPND attacks shrimp post larvae within 20-30 days after stocking causing up to 100% mortality. The expression of these virulent genes is controlled by the quorum sensing system. This system is inhibited by an anti-quorum sensing (AQS) mechanism. Several Bacillus strains have AQS mechanism by producing AHL-Lactonase enzyme. Therefore, this study aimed to obtain Bacillus spp. having AQS activity for controlling AHPND. The study was conducted from isolation and selection of Bacillus isolates, as well as determination of AQS activity. From 22 samples consisting of shrimp intestines, water and pond sediment samples, a total of 151 isolates of Bacillus spp. were isolated. The screening test for AQS activity obtained 11 isolates that showed AQS activity on Cromobacterium violaceum. Determination of violacein pigment in liquid cultures of C. violaceum showed the index value of the pigment formation was between 0.025-0.166 and 0.026-0.567 at 24-hour and between 48-hour incubations, respectively. The quantitative analysis of violacein production showed that there were six isolates of Bacillus could inhibit the pigment production more than 75%. The isolates were identified as Bacillus cereus (four isolates), Bacillus thuringiensis (one isolate), and Bacillus velezensis (one isolate), respectively. The molecular analysis had confirmed that the isolates have aiiA genes encoding AHL-lactonase enzyme. These Bacillus isolates have potential application for controlling AHNPD disease.

Carotenoid Assessments and Antioxidant Activities from Flower Petals

The objectives of this study were to evaluate carotenoid pigment profile and the antioxidant activity from 14 commonly grown flowers in Thailand. The result found that orange marigold showed the highest total carotenoid content at as 2,209±75.58 µg/g, followed by deep yellow chrysanthemum at 551.27±47.72 µg/g (P<0.01). The next lower total carotenoid content group was found in yellow silk cotton, yellow trumpetbush, yellow marigold and yellow golden shower with total carotenoid content of 447.42±27.56, 429.46±28.34, 409.85±34.58 and 363.88±12.74 µg/g, respectively. The highest antioxidant activities against DPPH and ABTS radicles were found from orange marigold, which were 32.34±2.16 and 50.08±0.87%, respectively. Deep yellow chrysanthemum, yellow silk cotton, yellow trumpetbush, yellow marigold and yellow golden shower also showed significantly higher antioxidant activities than other flowers (P<0.01). Total carotenoid contents well correlated with antioxidant activities against DPPH and ABTS radicals (r = 0.6924 and r = 0.8270, respectively) at P<0.01. TLC result elucidated that orange marigold, yellow silk cotton and yellow golden shower were a good source of β-carotene, while deep yellow chrysanthemum and yellow marigold were a good source of lutein and/or zeaxanthin. The result indicated that flower petals would be useful as natural carotenoid source and provide antioxidants for food industry.

Evaluation of Tuberculosis Vaccine Candidate, pcDNA3.1-rpfD using Mycobacterial Growth Inhibition Assay (MGIA)

Resuscitation-promoting factor D (RpfD) is a protein involved in the resuscitation of dormant bacteria. A new tuberculosis vaccine carrying the rpfD gene has been successfully constructed, pcDNA3.1-rpfD. It was demonstrated that this vaccine exhibits cellular and humoral immune responses. Therefore, within this study, the efficacy of this new vaccine candidate was evaluated using mycobacterial growth inhibition assay (MGIA). MGIA is a functional assay that measures the complex host immune response, peripheral blood mononuclear cell (PBMC) and splenocyte from BALB/c mice against mycobacteria. With BACTECTM MGITTM 960 automated system, the effect of vaccination on bacterial growth was reported as a time to positivity (TTP) in hours. The mean of TTP from the vaccinated group (both pcDNA3.1-rpfD and BCG) was higher than the negative control group. These results suggest that pcDNA3.1-rpfD may be effective in controlling tuberculosis growth and may provide a clue for the development of the tuberculosis vaccine. In addition, despite previous evidence that IFNγ was essential for tuberculosis immunity, IFNγ (interferon gamma) production was found not to be correlated with mycobacterial inhibition. Therefore, these findings offer an alternative method to evaluate vaccine candidates than the assessment using IFNγ only.

Serum HMGB1, DKK1, and ACE2 as a Function of Lung Injuries in COVID-19 Patients

There is a need for a biomarker for lung injury in COVID-19 patients. In the present study, an attempt was carried out to examine the role of Dickkopf-related protein 1 (DKK1), High-mobility group box 1 protein (HMGB1), angiotensin-converting enzyme 2 (ACE2) as a function for the lung abnormalities in CT-scan (LACTS). To perform the goals, DKK1, HMGB1, and ACE2 were measured in patients and controls using the ELISA technique. In contrast, other parameters were measured spectrophotometrically. The results showed decreased SpO2 and albumin and an increase in the serum biochemical parameters (glucose, urea, creatinine, D-dimer, ACE2, DKK1, and HMGB1) in COVID-19 patients compared with the control group. In COVID-19 patients, the percentages of the lung abnormalities in CT-scan% are 40.67±11.84. The results showed that those patients with LACTS patients are slightly older and have lower SpO2 than the patients without the LACTS group. ACE2 shows a significant correlation with SpO2 (ρ = 0.336, p<0.01) and a negative correlation with albumin (ρ = -0.197, p<0.05). Other parameters showed no significant correlation with the measured biomarkers. In conclusion, COVID-19 patients have higher ACE, DKK1, and HMGB1 indicating the involvement of the pathways of these biomarkers in the disease progression including lung injury.

The Acute Single and Mixture Toxicity of Paraquat Dichloride and Deltamethrin to Guppy (Poecilia reticulata)

Although herbicide and insecticide contamination of surface waters normally occurs in the form of mixtures, the toxicity interactions displayed by such mixtures have only rarely been characterized. This study evaluated the acute effects of single pesticides (paraquat dichloride and deltamethrin, tested in their commercial formulations Gramoxone 276 SL and Decis 25EC) and their equitoxic mixtures on the survival of adult male guppy fish (Poecilia reticulata). Mixture toxicity was evaluated against the reference models of Concentration Addition (CA) and Independent Action (IA). In the single treatments, the 72h LC10 and LC50 values were 1.5 and 6.0 mg a.s. L-1 and 0.53 and 3.6 µg a.s. L-1 for paraquat dichloride and deltamethrin, respectively. The equitoxic mixtures showed significant paraquat dichloride-deltamethrin antagonism, both based on the CA and the IA model, without significant dose-level dependent deviations.

A Behavioural Study of Free Ranging Ceylon Spotted Deer (Axis axis ceylonensis) in Trincomalee, Sri Lanka

Axis axis ceylonensis (Ceylon spotted deer) is a sub species endemic to Sri Lanka. Ecological observations in wild populations of this subspecies have been reported but there is no published research on its behaviour. We report here a behavioural study on a free ranging population of A. a. ceylonensis inhabiting a temple surroundings in Trincomalee, Sri Lanka. Behaviour was quantified by focal sampling on sex age groups in three time zones: 6:30 to 7:30, 12:30-13:30, and 17:00 to 18:00 hours. The deer were more active in the hour after dawn and an hour before dusk. The main activities were feeding and play, the latter common in juveniles. There was a preference to graze on grasses and browse on Ficus sp. leaves. Resting was highest in the afternoon across all groups. This bimodal activity pattern is similar to that of both wild Ceylon and Indian A. axis subspecies despite the absence of predators in the study area. The bimodal activity may be related to thermoregulatory functions while grazing in open grass areas.