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Antioxidant effect of Tryptophan on biochemical parameters in the haemolymph and fat body of final instar larvae of silk insect, Bombyx mori
Abstract
Ageing is a natural life process whose manifestations are familiar and unambiguous. Oxidative modification of cellular molecules by reactive oxygen species and impaired antioxidant mechanism play unique role in a variety of age-associated degenerations. As a defence, cells have developed antioxidant defence system of a group of enzymes including catalases and peroxidases destroying toxic molecules. The natural antioxidant mechanism of an organism may be insufficient and external dietary administration of anti oxidant compounds play vital role in defence against ageing. In the present study, the antioxidant effects of reducing amino acid tryptophan in the final instar larvae of silkworm, Bombyx mori. The turnover of total protein, amino acid and glucose was evaluated. The total haemolymph protein of treated larvae showed 34-94% increase when compared to normal and the pattern of the changes in the levels of fat body protein was same but with a change of 12 fold. The total content of free amino acids in the haemolymph of normal and treated larvae increased gradually from the period of 0 h to 96 h with a peak value at 96 h and then decreased. The total content of free amino acids in the fat body is much less than that found in the haemolymph. The peak glucose levels in the total larval haemolymph were almost 28 times to that found at the early stage in normal larvae and approximately 12 times in tryptophan treated larvae. The fat body glucose level showed a consistent reduction in the treated larvae.
... Like other holometabolous insects, the silkworm cannot synthesize tryptophan itself. Bhaskaran et al. (2015) found that tryptophan administration on F I G U R E 6 A schematic diagram of sugar metabolism and main biosynthesis pathway for the aromatic amino acids (L-tryptophan, tyrosine and phenylalanine) identified in the genome of Ent. casseliflavus ECB140. ...
Aims:
L-tryptophan is an essential aromatic amino acid for the growth and development of animals. Studies about enteric L-tryptophan-producing bacteria are scarce. In this report, we characterized the probiotic potential of Enterococcus casseliflavus ECB140, focusing on its L-tryptophan production abilities.
Methods and results:
ECB140 strain was isolated from the silkworm gut and can survive under strong alkaline environmental conditions. Bacterial colonization traits (motility and biofilm) were examined and showed that only ECB140 produced flagellum and strong biofilms compared with other Enterococcus strains. Comparative genome sequence analyses showed that only ECB140 possessed a complete route for L-tryptophan synthesis among all 15 strains. High-performance liquid chromatography and qRT-PCR confirmed the capability of ECB140 to produce L-tryptophan. Besides, the genome also contains the biosynthesis pathways of several other essential amino acids, such as phenylalanine, threonine, valine, leucine, isoleucine and lysine. These results indicate that ECB140 has the ability to survive passage through the gut and could act as a candidate probiotic.
Conclusions:
The study describes a novel, natural silkworm gut symbiont capable of producing L-tryptophan. Enterococcus casseliflavus ECB140 physical and genomic attributes offer possibilities for its colonization and provide L-tryptophan for lepidopteran insects.
For the past few years, DNA barcoding has become an efficient method for the identification of species. In this study we test the efficiency of DNA barcoding for true bug, Zicrona caerulea (Hemiptera: Heteroptera), an ecologically and economically important as well as morphologically diverse insect taxon. These bugs are useful predators of leaf beetles of the genus Altica, larvae of various beetles and caterpillars of moths, and it also feeds on plants. The study emphasizes the use of DNA barcodes for the identification of Zicrona caerulea and to represent an important step in building-up a comprehensive barcode library for the true bugs. As part of our study we analyzed DNA barcodes of Zicrona caerulea isolated from Kerala and its phylogenetic status with other related taxonomic groups. The PCR amplified cytochrome oxidase subunit I gene (COI) partial sequence of Zicrona caerulea has 1.11% difference to that of Zicrona caerulea (GenBank Accession: GQ292256) of Seoul, Korea and 1.68% difference to Zicrona caerulea (KM023126) Bremen, Germany.
The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of pro-oxidant endobiotics and xenobiotics. The possible involvement of beetle non-enzymatic [α-tocopherol, glutathione (GSH), and ascorbic acid] and enzymatic [catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), and polyphenol oxidase (PPO)] antioxidant defense system on the insecticidal activity of synthetic insecticides (cypermethrin, 2,2-dicholorovinyl dimethyl phosphate, and λ-cyhalothrin) and ethanolic plant extracts of Tithonia diversifolia, Cyperus rotundus, Hyptis suaveolens leaves, and Jatropha Curcas seeds was investigated. 2,2-Dicholorovinyl dimethyl phosphate (DDVP; 200 ppm, LC₅₀ = 13.24 ppm) and T. diversifolia (20,000 ppm) resulted in 100% beetle mortality at 96-hour post-treatment. The post-treatments significantly increased the beetle α-tocopherol and GSH contents. Activities of CAT, SOD, POX, and PPO were modulated by the synthetic insecticides and bioinsecticides to diminish the adverse effect of the chemical stresses. Quantitative and qualitative allelochemical compositions of bioinsecticides and chemical structure of synthetic insecticides possibly account and for modulation of their respective enzyme activities. Altogether, oxidative stress was enormous enough to cause maladaptation in insects. This study established that oxidative imbalance created could be the molecular basis of the efficacy of both insecticides and bio-insecticides. Two, there was development of functional but inadequate antioxidant defense mechanism in the beetle.
Aging is the accumulation of deleterious changes occurring in an organism over time. It causes as a result of free radical deposition in the body which is fought out by the antioxidants. Present study mainly focuses on the dietary supplementation of antioxidants, ascorbic acid and tyrosine in the aging of Bombyx mori larvae. Total protein, hydrogen peroxide and catalase activity of haemolymph and fat body have been estimated at time interval of 24 hrs for a period of 4 days. The results obtained were statistically analysed. It was found that ascorbic acid and tyrosine acted as a potential dietary antioxidant supplement which caused significant changes in the organism's body, by scavenging the free radicals formed in the body as a result of metabolic reactions.
Interest in relationship between diet and ageing is growing. Research has shown that dietary calorie restriction and some antioxidants extend lifespan in various ageing models. On the one hand, oxygen is essential to aerobic organisms because it is a final electron acceptor in mitochondria. On the other hand, oxygen is harmful because it can continuously generate reactive oxygen species (ROS), which are believed to be the factors causing ageing of an organism. To remove these ROS in cells, aerobic organisms possess an antioxidant defense system which consists of a series of enzymes, namely, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). In addition, dietary antioxidants including ascorbic acid, vitamin A, vitamin C, α -tocopherol, and plant flavonoids are also able to scavenge ROS in cells and therefore theoretically can extend the lifespan of organisms. In this connection, various antioxidants including tea catechins, theaflavins, apple polyphenols, black rice anthocyanins, and blueberry polyphenols have been shown to be capable of extending the lifespan of fruit flies. The purpose of this review is to brief the literature on modern biological theories of ageing and role of dietary antioxidants in ageing as well as underlying mechanisms by which antioxidants can prolong the lifespan with focus on fruit flies as an model.
If aging is due to or contributed by free radical reactions, as postulated by the free radical theory of aging, lifespan of organisms should be extended by administration of exogenous antioxidants. This paper reviews data on model organisms concerning the effects of exogenous antioxidants (antioxidant vitamins, lipoic acid, coenzyme Q, melatonin, resveratrol, curcumin, other polyphenols, and synthetic antioxidants including antioxidant nanoparticles) on the lifespan of model organisms. Mechanisms of effects of antioxidants, often due to indirect antioxidant action or to action not related to the antioxidant properties of the compounds administered, are discussed. The legitimacy of antioxidant supplementation in human is considered.
The biosynthesis of the amino acid taurine has been studied in the locust Schistocerca americana gregaria. Tissue concentrations of putative precursors of taurine, and taurine itself were estimated in haemolymph, nervous tissue and muscle. Following this locusts were injected with 20 μCi of [35S]cysteine and tissue samples were taken at times thereafter, and taurine and its precursors were extracted and separated by thin-layer chromatography (TLC). Taurine, cysteine sulphinic acid (CSA) were quantified by scintillation counting. Cysteic acid and hypotaurine were qualitatively detected by autoradiography of TLC plates. Results indicate that in haemolymph and muscle the biosynthetic pathway for taurine is cysteine then CSA, cysteic acid and thereafter taurine. In nervous tissue both cysteic acid and hypotaurine were detected suggesting two possible pathways for taurine since both are immediate precursors of this amino acid. Taurine biosynthesis was markedly greater in juvenile compared with mature adult locusts. It was also observed that picrotoxin treatment and prolonged flying, which have both previously been found to cause a redistribution of taurine in the locust, increased the biosynthesis of this amino acid.
The amount of protein, carbohydrate, lipids, and free amino acids were examined in the spinning stage in the fat body, haemolymph, skeletal muscle, and gut of Rhynchosciara americana. Protein and lipids increase in the fat body soon after the animal stopped feeding, probably at the expense of the digestion of the gut contents and of the reserves of the gut wall. Afterwards there is a fall in protein and lipids in the fat body. Haemolymph protein rises a little at the beginning of spinning and then decreases steadily during cocoon production. Carbohydrate and free amino acids decrease from the beginning of spinning in all tissues studied. Quantitatively, the most important decrease of carbohydrate during spinning occurs in the fat body whereas that of free amino acids occurs in the haemolymph. Lipid increases during spinning in the skeletal muscle, probably due to enlargement of the lateral fat body which occurs as a contaminant in the skeletal muscle preparation. The Malpighian tubules contain a large amount of calcium carbonate, which is eliminated during spinning. A correlation of our chemical data with histochemical data recently published is presented and the physiological implications of our findings are discussed in comparison to other insects.
The relationship between the proteins of the haemolymph and fat body was examined during metamorphosis of the large white butterfly, Pieris brassicae. The protein analysis results showed that about 4·9 mg protein was lost from the haemolymph of an individual insect between the prepupal and newly ecdysed pupal stages, while during the same period the fat body gained about 5·5 mg protein. From the change in distribution of 14C protein between these two tissues it was concluded that the fat body was storing the lost haemolymph protein at pupation. A more detailed examination using disk electrophoresis of labelled and unlabelled proteins from these two tissues showed that the fat body was in fact selectively storing two major haemolymph proteins. It was found that these proteins accumulated in the haemolymph during the fourth and fifth instars before being transferred to the fat body at the pharate pupal stage.No changes were detected in either haemolymph or fat body proteins during pupal diapause. However, the adult moulting cycle was marked by partial histolysis and release of degraded fat body proteins and the loss of another major protein from the haemolymph, not into the fat body, but presumably into the differentiating adult tissues.The changing rôles that the fat body and haemolymph play in the synthesis, storage, translocation, or degradation of proteins during development are discussed.
The gut β-galactosidase activity of Locusta migratoria has been characterised with respect to the substrate lactose. Lactase activity is apparently due to a single component with a molecular weight of approximately 110,000. The enzyme exhibits a pH optimum of 5.25 and value of 31.4 mm. The temperature coefficients for enzymic catalysis are: , kJ/mole; the enzyme is relatively thermostable. The absence of any obvious physiological function for the lactase, its low catalytic efficiency and its similarities to L. migratoria gut cellobiase lead to the hypothesis that both activities result from the presence of a single enzyme.