Molecular Detection and Phylogenetic Analysis of the alkB Gene in Klebsiella oxytoca Strains Isolated from the Gut of Tenebrio molitor.

The challenge in polystyrene disposal has caused researchers to look for urgent innovative and ecofriendly solutions for plastic degradation. Some insects have been reported to use polystyrene as their sole carbon source, and this has been linked to the presence of microbes in their guts that aid in plastic digestion. Thus, this study focuses on the molecular detection and phylogenetic analysis of the alkane-1-monooxygenase (alkB) gene in Klebsiella oxytoca strains isolated from the gut of Tenebrio molitor. The alkB gene encodes for alkane-1-monooxygenase, an enzyme involved in the oxidation of inactivated alkanes. This gene can be used as a marker to assess bacteria's ability to biodegrade polystyrene. Three bacterial strains were isolated from the guts of T. molitor mealworms and were confirmed using polymerase chain reaction (PCR) of the 16S ribosomal RNA gene. The primers used in the amplification of the 16S ribosomal RNA region were designed using NCBI, a bioinformatics tool. To detect the presence of the alkB gene in the isolated bacterial strains, a set of primers used in the amplification of this gene was manually designed from the conserved regions of the alkB nucleotide sequences of eleven bacterial species from GenBank. TCOFFE online tool was used to align the alkB sequences of the bacteria, while Jalview and ConSurf were used to view the alignment. The amplified alkB gene was then sequenced using the Sanger sequencing technique, blasted on NCBI to look for similar sequences, and a phylogenetic tree was constructed. Based on the 16S ribosomal RNA gene sequences, the isolated bacterial strains were confirmed to be Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca ATCC 13182. The alkB gene sequence identical to fourteen alkB gene sequences derived from Actinobacteria whole genome was detected in Klebsiella oxytoca for the first time to the best of our knowledge. The novel nucleotide sequence was published in the NCBI database under accession number OP959069. This gene sequence was found to be for the enzyme alkane-1-monooxygenase and may be one of the enzymes responsible for polystyrene degradation by the putative Klebsiella oxytoca ATCC 13182 in T. molitor.

Sub-chronic toxicity determination of powdered Tenebrio molitor larvae as a novel food source.

Tenebrio molitor larvae are the first insect species to be given a favorable assessment by the European Food Safety Authority (EFSA) as a novel food source, enabling consumption of whole insect larvae or larvae that have been powdered and processed into a variety of food products. Pressure from economic hardships and increase in population growth have paved a way for the realization of an alternative food source in Zimbabwe. This study focused on determining the potential toxicity of Tenebrio molitor larvae powder as an alternative food source for humans. To determine the sub-chronic toxicity of Tenebrio molitor, the powder was administered daily by oral gavage to Sprague-Dawley rats at dose levels of 0, 300, 1000 and 3000 mg/kg for 70 days. A toxicological assessment which included mortality, appearance of clinical symptoms, food consumption, organ and body weight changes were performed. There were no treatment-related mortalities, clinical signs, changes in food consumption, body and organ weights observed during the treatment period. The study's findings suggest Tenebrio molitor larvae to be a good alternative as it did not appear to affect the rats' normal physiological and metabolic processes hence can be considered safe for human consumption. However, further studies on hematological, histological and biochemical markers may be necessary for confirmation of these current results.

Screening of mushrooms from the woodlands of Zimbabwe: Occurrence of lectins and partial purification of a mucin specific lectin from Boletus edulis.

Mushrooms are known to possess a diversity of bioactive compounds that include lectins, which are proteins or glycoproteins that bind specifically to cell surface carbohydrates, culminating in cell agglutination. The present study describes the screening of lectin activity from ten local mushrooms, namely, Amanita zambiana, Boletus edulis, Cantharellus heinemannianus, Cantharellus miomboensis, Cantharellus symoensii, Lactarius kabansus, Amanita sp., Coprinus sp., Ganoderma lucidum and Trametes strumosa. The lectin content was detected by the haemagglutination activity of mushrooms against sheep and goat erythrocytes. Among the different mushrooms screened Amanita sp., Boletus edulis and Lactarius kabansus showed high lectin activity (39, 617 and 77 HAU/mg mushroom, respectively). Boletus edulis was used for the haemagglutination inhibition assay. A total of twenty sugars and sugar derivatives, namely, α-lactose, D-glucose, D-mannose, D-raffinose, N-acetyl glucosamine, maltose, melibiose, D-ribose, porcine mucin, D-cellobiose, D-arabinose, α-methyl-D-glucoside, methyl-α-D-mannopyranoside, D-trehalose, L-arabinose, L-sorbose, L-lyxose, ß-lactose, DL-xylose, and D-galactose, were used for the haemagglutination inhibition assay. Of the various carbohydrates tested, only porcine mucin was found to be the most potent inhibitor of Boletus lectin. The lectin from Boletus mushroom was partially purified using ammonium sulphate precipitation. The highest lectin activity was observed in the 30%-60% fraction. This study revealed for the first time the occurrence of lectins in the local Zimbabwean mushrooms studied as well as isolation of a novel mucin-specific lectin. The information obtained can be used for further investigation of cell surface sugars, purification and characterisation of glycoproteins and their contribution towards the medicinal properties of local mushrooms.

Tenebrio molitor: possible source of polystyrene-degrading bacteria.

BACKGROUND: The excessive use of polystyrene as a packaging material has resulted in a rise in environmental pollution. Polystyrene waste has continually increased water pollution, soil pollution and the closing of landfill sites since it is durable and resistant to biodegradation. Therefore, the challenge in polystyrene disposal has caused researchers to look for urgent innovative and eco-friendly solutions for plastic degradation. The current study focuses on the isolation and identification of bacteria produced by the larvae of beetle Tenebrio molitor (yellow mealworms), that enable them to survive when fed with polystyrene foam as their sole carbon diet. MATERIALS AND METHODS: The biodegradation of polystyrene by Tenebrio molitor was investigated by breeding and rearing the mealworms in the presence and absence of polystyrene. A comparison was made between those fed with a normal diet and those fed on polystyrene. The mealworms which were fed with polystyrene were then dissected and the guts were collected to isolate and identify the bacteria in their guts. The viability and metabolic activity of the isolates were investigated. The polymerase chain reaction (PCR) followed by sequencing was used for molecular identification of the isolates. The PCR products were directly sequenced using Sanger's method and the phylogenetic tree and molecular evolutionary analyses were constructed using MEGAX software with the Neighbour Joining algorithm. The evolutionary distances were computed using the Maximum Composite Likelihood method. RESULTS: The decrease in mass of the polystyrene as feedstock confirmed that the mealworms were depending on polystyrene as their sole carbon diet. The frass egested by mealworms also confirmed the biodegradation of polystyrene as it contained very tiny residues of polystyrene. Three isolates were obtained from the mealworms guts, and all were found to be gram-negative. The sequencing results showed that the isolates were Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665. CONCLUSION: Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665 maybe some of the bacteria responsible for polystyrene biodegradation.

Is neutral genetic diversity related to quantitative variation in semen traits in bulls?

Conservation decisions based on neutral genetic diversity have been observed to promote retention of useful quantitative variation in biological populations. An experiment was undertaken to determine the association between microsatellite marker polymorphisms and phenotypic variation in semen production and cryosurvival traits in bulls. Thirty-five ejaculates were collected from ten bulls of two breeds and evaluated before and after cryopreservation for several semen traits. The bulls were also genotyped using a set of sixteen bovine-specific microsatellite marker loci. Fixation indices (FST ), heterozygosity and Nei's genetic distance measures were computed from allele frequency data for each of the bulls. Molecular and phenotypic data were used to compute tri-distance matrices for the ten bulls and correlated using Mantel's test in GenAIEx 6.5. The study revealed extensive heterogeneity in semen traits, heterozygosity and FST values among the bulls. Large pairwise phenotypic and genetic distances were also observed. Correlation between pairwise genetic distances and phenotypic distances was significant and highly positive for sperm viability (r = .61, p < .001) and moderately positive for sperm motility (r = .40-42, p < .05) variables. For sperm morphology, ejaculate volume and sperm concentration, correlation with genetic distances was positive, low and not significantly different from zero (p > .05). A tendency for a triangular-shaped relationship between genetic and phenotypic distances for post-thaw motility and viability traits was also observed. Accordingly, association with neutral genetic diversity was absent for semen production traits and moderate to highly positive for sperm cryosurvival traits. Given these findings, conservation decisions based on neutral genetic diversity may capture variation in some adaptive traits, but not others.

Variation in sperm cryosurvival is not modified by replacing the cryoprotectant glycerol with ethylene glycol in bulls.

Breed and sire differences in sperm cryosurvival have been noted, with negative implications for sperm cryobanking and assisted reproduction programmes. This study hypothesized that these differences could be modified by using lower molecular weight cryoprotectants. Therefore, the effect of replacing glycerol (GLY) with ethylene glycol (EG) on differential cryosurvival of semen from two Sanga cattle breeds (Mashona vs. Tuli) was determined. Three to five ejaculates were collected from each of ten bulls (3-8 years) by electro-ejaculation, diluted in three Tris-egg yolk extenders (Triladyl® , 7% GLY-based and 7% EG-based) and evaluated for sperm motility, viability and morphology at three time periods (fresh - 0 hr, pre-freeze - 4 hr and post-thaw). Tuli bulls produced larger (11.8 ± 0.31 ml vs. 8.5 ± 0.38 ml) and more concentrated ejaculates of lower fresh semen quality. Breeds differed across time for motility and morphology, but not viability. Mashona bull semen had significantly higher motility and normal morphology values at each sampling time. Bulls classified as poor freezers had lower concentration (0.70 ± 0.09 × 109  sperm/ml vs. 1.37 ± 0.10 × 109  sperm/ml), sperm motility index (SMI, 35.0 ± 3.4 % vs. 67.8 ± 2.1 %) and viability (69.7 ± 1.1 % vs. 75.7 ± 1.0 %) compared to good freezers. Maintenance of semen quality by GLY and EG did not differ between breeds, poor and good freezers, or age groups. The interaction breed by extender across time did not reach statistical significance for all variables. The study revealed that bull and breed variation in sperm quality and cryosurvival is not modified by replacing GLY with EG, suggesting that cryostress tolerance of sperm may be under control of mechanisms other than differential response to GLY cytotoxicity.