Abstract: This study aimed to evaluate the effects of dexatrol antibiotic and peppermint oil with different concentrations as a disinfectants on infected silkworm Bombyx mori larvae with bacteria by studying their effects on some biochemical characters of larvae as the protein enzymes activity; aspartate transaminase (AST), alanine transaminase (ALT), Total Soluble Protein (TSP) and free radical biomarkers; lipid peroxidation (MDA) and Protein Carbonyl Content (PCC). It found that, most concentrations enhanced the enzymes activity and decreased the effects of free radical on lipid and protein of larval haemolymph compared with the control group, especially dexatrol concentration (0.4%) and peppermint oil concentrations (0.5 and 0.25%) revealed the best results significantly.
INTRODUCTION
Silkworm is prone to a number of diseases during its larval stage. The mortality rates become very high during infection spread. These diseases cause very serious problem that face silkworm rearing as they cause quantitative and qualitative reduction of natural silk crop production. Therefore, the produced cocoons by survivors have the depleted economic characters. Disease control can only be affected by prophylactic use of certain disinfectants or antibiotics (Saha et al., 1995; Sanakal et al., 1996; Deehu et al., 1997; Bali et al., 2008; Saad et al., 2012).
Antibiotics are widely used in sericulture industry as a component of bed disinfectants and as therapeutic applications against bacterial diseases, they are approved for four different purposes: Disease treatment, disease prevention, disease control and for health maintenance or growth promotion (Phillips et al., 2004; Subramanian et al., 2009).
The control of infectious diseases is seriously threatened by the continuous increase in the number of microorganisms that are resistant to the chemical antimicrobial drugs (Nenaah and Ahmed, 2011) and in view of high cost of chemicals and antibiotics and their hazardous consequences, plant extracts has been on the top priority for controlling diseases, recently there has been a concerted effort to promote the use of botanicals as possible alternatives to treat infectious diseases. These natural products were found to possess promising antimicrobial activities when applied alone or in combination with conventional antimicrobial drugs (Kumar et al., 1999; Jazani et al., 2007; Mohsenzadeh, 2007; Jazani et al., 2009; Chanda et al., 2011).
The components in essential oils vary not only with plant species but also in relation to climate, soil composition, part of the plant and age of the plant. Many essential oils are composed of a variety of terpenoid compounds. These substances are usually volatile and can be detected by the antennae or tarses of insects. The major terpenoids contained in essential oils are monoterpenoids (citronellal, linalol, menthol, pinene, mentona, carvona and limonene) (Paula et al., 2004; Simoes and Spitzer, 2003).
Peppermint, is an aromatic and medicinal plant widely used in the food industry, perfumery and cosmetic, pharmacy and traditional medicine. Its essential oil displays antimicrobial activity against a range of bacteria and fungi. It has a high menthol content, contains also menthone and menthyl esters, particularly menthyl acetate and small amounts of many additional compounds including limonene, pulegone, caryophyllene and pinene (Bruneton, 1995).
Oxidant stress may lead to cellular injury and subsequent organ dysfunction (oxidative damage) by such mechanisms as oxidative damage to essential proteins, lipid peroxidation, DNA strand breakage (Darley-Usmar and Halliwell, 1996).
The process of uncontrolled lipid peroxidation in biological system may be associated with the loss of essential polyunsaturated fatty acids which represented the high content of cellular membranes constituents. This process leads to the formation of toxic hydroperoxide or other secondary products and causes extensive disturbance to the fine structure of biological membranes, thus affects the permeability and function of the membrane and extensive oxidation may lead to rupture of cellular membranes and concomitant release of destructive lysosomal enzymes (Abdel-Rahmaan, 1999).
Proteins constitute the major working force for all forms of biological work. Their exact conformation and pattern of folding are tightly connected to their activity and function. Reactive oxygen species are formed during normal metabolism and in higher fluxes under pathological conditions. They cause cellular damage, an important part of which is the oxidation of amino acid residues on proteins, forming protein carbonyls. The addition of carbonyl groups to amino acid residues in proteins is an oxidative modification of protein and involved in many physiological and pathological processes (Fagan et al., 1999; Chevion et al., 2000).
The present study aimed to evaluate the effects of dexatrol antibiotic and peppermint oil as a disinfectants on some biochemical aspects of the haemolymph of infected silkworm larvae Bombyx mori.
MATERIALS AND METHODS
The present study was carried out during the spring season of 2012 in the laboratory of Sericulture Research Department of Plant Protection Research Institute, Sharkia Branch, Agriculture Research Center.
Materials
| • | Mulberry silkworm, B. mori eggs (Egyptian hybrid Giza) |
| • | Dexatrol antibiotic consists of dexamethasone (1 mg/1 mL), Neomycin {as sulphate} (3.5 mg/1 mL) and polymyxin-B-sulphate (6000 IU/1 mL) from Egyptian International Pharmaceutical Industries Company (E.I.P.I.Co.) |
Four milliliter of antibiotic dissolved in 1 L distilled water to prepare a concentration of 0.4% and 3 mL of antibiotic to prepare a concentration of 0.3% and so the remaining concentrations to 0.1%.
| • | Peppermint oil (Cap. Pharm. Company) was used with 4 concentrations prepared by Harvey and John (1898) |
Methods
Silkworm rearing technique: Rearing of silkworm was carried out in laboratory under the hygro-thermic conditions 28±1°C and 75±5% RH, according to the technique of Krishnaswami (1978). The larval bed was cleaned daily. Cleaning net was used for removing the remained dried food and feces. Chicken egg cartons plates were used as montages for cocoon spinning (Zannoon and Shadia, 1994). Larvae under investigation were divided into two groups. Each group was divided into four subgroups. One group feed on mulberry leaves treated with dexatrol antibiotic concentrations and the other group feed on mulberry leaves supplemented with peppermint oil concentrations using 3 replicates (100 larvae) for each concentration. Mulberry leaves were dipped in the concentrations of both disinfectants for 5 min and left to dry then offered to larvae. Larvae were treated with disinfectants two times in both the 4th and 5th larval instars, all feeds/day at the first day and the middle of both instars. The control group of leaves was treated only with distilled water.
Infecting of silkworm larvae with bacteria: Bacterial pathogens were collected and isolated from diseased larvae (Aneja, 2003). After bacterial culture prepared using luria agar medium (Suparna et al., 2011), Bombyx mori artificially infected by spraying mulberry leaves with the concentration (15 ppm) of bacterial flacherrie (Streptococcus pneumoniae) and fed one time in the 2nd day of the 4th instar larvae.
Biochemical assays: Samples were made by removing one of the thoracic legs of the 5th instar larvae and bending the body to expose the sternum at the position of the removed leg. This ensured proper drainage of the haemolymph and avoided any risk of internal organs to be destructed. The haemolymph of each treatment was collected in eppendorf tubes 1.5 mL with small crystal of phenyl thiourea (PTU) to prevent melanization of sample (Mahmoud, 1988). The tubes were kept at-20°C. The blood samples were centrifuged at 10000 rpm for 10 min at 5°C. The supernatant was immediately assayed to determine aspartate transaminase (AST), alanine transaminase (ALT) activities according to the method of Reitman and Frankel (1957), Total Soluble Protein (TSP) as described by Gornall et al. (1949), lipid peroxidation (MDA) according to the method of Sharma and Wadhwa (1983) and Protein Carbonyl Content (PCC) as described by Levine et al. (1990).
Statistical analysis: The obtained results were subjected to statistical analysis of variance and the data were presented as means according to Snedecor and Cochran (1982) methods using software COSTAT program.
RESULTS AND DISCUSSION
Alanine transaminase (ALT): Data exhibited a significant increase as shown in Fig. 1a of ALT means of larvae fed on mulberry leaves treated with dexatrol antibiotic and peppermint oil with different concentrations than control (0.90 μg Pyruvate/mL, SE = ±0.07) especially the 4th concentration (0.4%) of dexatrol (5.17 μg Pyruvate/mL, SE = ±0.15) and the 2nd and 3rd concentrations (0.5%, 0.25%) of peppermint oil (4.56 μg Pyruvate/mL, SE = ±0.34 and 4.56 μg Pyruvate/mL, SE = ±0.15) respectively, LSD 0.05 (conc.) = 0.863*** for each one.
| Fig. 1(a-c): | Effect of dexatrol and peppermint oil on (a) Alanine transaminase (ALT), (b) Aspartate transaminase and (c) Total Soluble Proteins (TSP) of infected larval haemolymph |
Aspartate transaminase (AST): Obtained data in Fig. 1b, cleared that larvae fed on mulberry leaves treated with dexatrol antibiotic and peppermint oil with different concentrations recorded higher means of AST than control (3.68 μg Pyruvate/mL, SE = ±0.35) especially the 4th concentration (0.4%) of dexatrol (14.21 μg Pyruvate/mL, SE = ±0.48) and the 3rd concentration (0.25%) of oil (14.21 μg Pyruvate/mL, SE = ±0.51), LSD 0.05 (conc.) = 1.905*** for each treatment. According to the obtained results, using the disinfectants and antibiotic to the infected mulberry silkworm, B. mori increased the protein content as compared to the infected control group. These results may suggest that the disinfectants may stimulate the enzyme activity which influences the biochemical contents of the haemolymph of the silkworm, B. mori. The results are in partial accordance with El-Sayed et al. (1990) who stated that, disinfectants increased the amount of haemolymph protein of larval instars and in agreement with those of Ghada (2009, 2012) evaluated the effects of mulberry leaves supplemented with the botanical oils on silkworm Bombyx mori. The results proved that oils exhibited the highest activity of ALT and AST. Also, it was found that the activity of AST enzyme was higher than ALT enzyme.
Determination of Total Soluble Protein (TSP): As illustrated in Fig. 1c, larvae fed on mulberry leaves treated with the 4th concentration (0.4%) of dexatrol (28.43 mg mL-1 of serum, SE = ±0.68) and the 3rd concentration (0.25%) of peppermint oil (31.77 mg mL-1 of serum, SE = ±0.65) recorded the highest TSP values compared with the other concentrations and control (1.77 mg mL-1 of serum, SE = ±0.18), LSD 0.05 (conc.) = 2.656***.
The infection with bacterial diseases decreased the total protein content. These results explained with Azab (2003) found that, the infection with B. thuringiensis caused marked reduction in the total protein contents at different concentrations in the black cutworm. Ross et al. (1980) stated that, peppermint oil inhibited the growth in vitro of many kinds of bacteria such as Staphylococcus aureus and fungi. In parallel direction, Rietschel and Fowler (2001) reported that, neomycin is an antibacterial that is used widely in medical treatments. It is also commonly found in combination preparations with other antibacterials and corticosteroids, it has excellent activity against Gram-negative bacteria and has partial activity against Gram-positive bacteria.
Lipid peroxidation (MDA): The levels of lipid peroxidation of haemolymph of the infected larvae reared on mulberry leaves enriched with investigated antibiotic and oil with different concentrations compared with control were shown in Fig. 2a. A significant decrease were noticed in MDA contents associated with increased concentrations of dexatrol; the concentrations 0.3% (25.11 nol mL-1 of serum, SE = ±0.62) and 0.4% (20.78 nol mL-1 of serum, SE = ±0.41) compared with control (41.11 nol mL-1 of serum, SE = ±0.62), also, the concentrations of oil 0.5% (26.11 nol mL-1 of serum, SE = ±0.67) and 0.25% (23.89 nol mL-1 of serum, SE = ±0.52) revealed a significant decrease in MDA content compared with control, (LSD 0.05 (conc.) = 2.946***).
Niesink et al. (1995) reported that free radicals can induce a wide range of effects such as membrane damage, inactivation of enzymes and cell death. These results supported by the study of Andallu and Varadacharyulu (2003) observed that mulberry leaves significantly decreased lipid peroxidation and increased the activity of almost all the antioxidant enzymes in normal rats and Bouari et al. (2014) stated that peppermint could represent a potential source of natural antimicrobial products. Dexatrol combines two antibiotics, neomycin and polymyxin B which highly active against a wide range of Gram-positive and Gram-negative bacteria including Staphylococcus aureus, E. coli, Klebsiella sp., Neisseria sp., Proteus sp., Enterobacter aerogenes and Pseudomonas aeruginosa.
| Fig. 2(a-b): | Effect of dexatrol and peppermint oil on (a) Lipid peroxidation (MDA) and (b) Protein Carbonyl Content (PCC) of infected larval haemolymph |
It also contains dexamethasone, a potent and effective corticosteroid with anti-inflammatory action, thus controlling the undesirable phases of inflammation associated with bacterial infections (E.I.P.I.Co.).
Protein Carbonyl Content (PCC): As shown in Fig. 2b, all antibiotic and oil concentrations exhibited a significant decrease in the level of protein carbonyl content of infected larval haemolymph compared with control, especially larvae fed on mulberry leaves supplemented with the concentration 0.4% of dexatrol (0.003 nol mL-1 of serum, SE = ±0.0003) and the concentration 0.25% of peppermint oil (0.005 nol mL-1 of serum, SE= ±0.0003) which revealed significantly the least values compared with control group (0.082 nol mL-1 of serum, SE = ±0.004), LSD 0.05 (conc.) = 0.014***.
These results are in agreement with those of Sarker et al. (1995) who found that, the total protein content of silk gland increased by feeding larvae on mulberry leaves supplemented with different nutrients and Murugan et al. (1998) stated that, the extracts of T. terrestris, B. diffusa and P. niruri plants revealed more protein fractions and a higher level of proteins in the silk glands of silkworm. Alagumalai et al. (1991) found that, the antibiotics increased the larval weight and silk gland weights (Fig. 3).
| Fig. 3: | A comparison among dexatrol antibiotic, peppermint oil as a disinfectants and control on biochemical aspects |
CONCLUSION
Conclusively, the mechanism of increased silk production in silkworm larvae depending on amines transfer which involved in the uptake of used compounds from leaves by the body tissues and silk glands which resulted in the subsequent promotion of silk protein synthesis (Enas, 2014). So, the treated mulberry leaves with disinfectants, dexatrol antibiotic and peppermint oil have benefit effect on diseased silkworm larvae, especially with higher concentrations; increased the protein enzymes activity (ALT and AST) and Total Soluble Proteins (TSP) and decreased free radicals biomarkers of lipid and protein (MDA and PCC). So it could be recommended that, the use of the disinfectants, dexatrol antibiotic and peppermint oil inhibit the symptoms of bacterial diseases as compared to the infected control group.