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Mortality responses of spodoptera litura following feeding on BT- sprayed plants.

Byline: Saad A. AlOtaibi

Abstract: Bacillus thuringiensis delta-endotoxins are safe biological insecticidal proteins whose usefulness has long been recognized. The first commercialized Bt insecticidal formulations were composed of spore-crystal preparations derived from wild-type strains. These products generally have a limited insecticidal host range and several genetically modified strains have, therefore, been constructed in this study using conjugation procedure. However, addition of a new plasmids to Bt strains already harboring other genes often resulted in broader - spectrum.

Bacillus thuringiensis serovar Kurstaki, Bacillus subtilis and four of their transconjugants were used in this study as a biocontral agents against lepidopterous cotton pest. Bacterial transconjugants were evaluated for their hybrid vigor in relation to the mid parents and better parent. This evaluation was related to survival and mortality percentages induced in Spodoptera littoralis larvae. Two groups of bioinsecticides; crystals, crystals + endospores were used to be evaluated in this study. The results appeared that bioinsecticides containing crystals + endospores was more effective than crystals for increasing mortality percentage and reducing survival percentage.

This effective was including reduction in the mean number of Spodoptera littoralis larvae feeding on leaves sprayed with crystals + endospores. Increasing mortality percentage of crystals + endospores was due to higher toxicological effects than that of crystals. This recommended bioinsecticide biologists to use crystals + endospores in all bioinsecticides formulations. Higher positive efficiency was appeared at 168 h of treatments. Recombinant Bacillus thuringiensis was more effective as biocontrol agents against lepidopteran pests at the early instars, because susceptibility was decreased with larval development. This indicated that the first instars were more susceptible to Bt sprayed plants than the later instar stages. The combined effects of crystals + endospores produced higher mortality. This factor was important to be considered in designing resistance management strategies.

Keywords: Crystals, Endospores, Hybrid vigor, Mortality, Recombinant bioinsecticides, survival.

INTRODUCTION

Each year billions of dollars are spent worldwide on insect control in agriculture [1]. Despite this expenditure, up to 40% of a crop can be lost to insect damage, particularly in developing countries [2]. Some of the most damaging insect species belong to the Lepidoptera, the second largest insect order comprised of moths and butterflies. The larval stage of Spodoptera littoralis cause major damage to an array of economically valuable crops including cotton, tomato, corn, sorghum and Lucerne [3]. Until recently, broad spectrum chemical insecticides have been the primary control agent for agricultural pests, with about40% targeted to the control of lepidopteran insects [4].

Over the years the widespread use of pesticides has led to pesticide resistant insects, a reduction in beneficial insect populations and harmful effects to humans and the environment [5]. These problems have led researchers to develop different insect control strategies using both synthetic and natural molecules that are more environmentally friendly.

Bacillus thuringiensis is a naturally occurring gram - positive bacterium commonly present in soil. Various strains of B. thuringiensis (Bt) are capable of producingcrystal (Cry) proteins (delta - endotoxins) or inclusion bodies that have selective insecticidal effects against different groups of insects [6]. Microbial preparations containing Bt cry proteins (as well as cell bodies and spores) have been used as foliar sprays in agricultural and forest settings for several decades. Partly because of their selectivity and short half - life, Bt-based microbial insecticides are generally considered to have fewer adverse impacts on the environment than synthetic chemicals [5].

Environmental concerns about the extensive use of chemical pesticides, together with the rising cost of discovering useful new molecules, has stimulated interest in the development of environmentally safe and cheap biopesticides such as microbial insecticides. One example of the successful development of such alternative products is Bucillus thuringiensis (Bt) which today accounts for more than 90% of the biopesticides used worldwide.

The insecticidal properties of this entomopathogenie bacterium are mainly due to the production, during sporulation, of larvicidal proteins that accumulate (up to 25% of the dry weight) as parasporal crystalline inclusions (also called crystals) within the cell [7]. At the end of the sporulation the cells lyse, spores and crystals are liberated. The inclusions produced by Bt subspecies are generally composed of several proteins (designated as delta - endotoxins or Cry proteins) each having a narrow activity spectrum.

There is thus a large family of related delta - endotoxins classified as CryI, II, III, IV, V, etc., depending on molecular relatedness and activity against insect larvae [8]. More than 50 cry genes were belonging to more than 20 different classes or subclasses, have now been cloned from different Bt strains. However, despite the demonstrated efficacy of such genetically altered Bt products, the efficiency and economic production of Bt products could still greatly benefit from the construction of engineered Bt strains with a broader activity spectrum and producing larger amounts of each of the crystal delta - endotoxins in the strain. Lepidoptera of the Noctuidae family, such as Spodoptera littoralis, are important agricultural pests which are poorly susceptible to most insecticides. The Bt recombinant strains were constructed in this study via conjugation that allows plasmid DNA transfer between strains that is necessary at the first stages of the construction of the recombinant strains.

Bacillus thuringiensis (Berliner) proteins are becoming ubiquitous, highly bioactive substances in the agro-ecosystems worldwide. This is due to an increase in the use of B. thuringiensis - based insecticides and the large scale release of various, transgenic crop plants expressing B. thuringiensis proteins conferring plant resistance to certain target insect pests. In the currently commercially available transgenic crop plants, B. thuringiensis proteins are present throughout most of the plant during most of the growing period. Further, B. thuringiensis protein is expressed in relatively high concentrations and, in contrast to B. thuringiensis insecticides, in a truncated, activated form.

The current and future trends in plant molecular biology is to increase B. thuringiensis expression levels in plants with the most dramatic example being the expression of Cry1Ac in tobacco chloroplasts [9]. Consequently, most, if not all, herbivores colonizing transgenic B. thuringiensis plants in the field are not lethally affected by B. thuringiensis proteins. However, they will ingest plant tissue containing B. thuringiensis protein which they may pass on to their natural enemies in a more or less processed form.

The ubiquitous and temporally extended availability of B. thuringiensis proteins in the field in addition to its modified form of release, makes it necessary to verify and monitor the compatibility of this new pest management strategy with natural enemies. The long-term, agro-ecological safety of the combined use of trans###20.31374###0.360031###1.772352

###Day 3###20.72452

###Levofloxacin

###Day 1###29.25701

###Day 2###30###29.66766###29.32451###0.314869###1.073740

###Day 3###29.04887

###Day 1###19.47814

###Day 2###20###19.63234###19.65914###0.195786###0.995903

###Day 3###19.86695

###Moxifloxacin

###Day 1###29.30477

###Day 2###30###29.45182###29.32402###0.119345###0.406987

###Day 3###29.21547

Table 6: Determination of Drug Content Found in the Levofloxacin and Moxifloxacin Formulations

###Injected conc.###Mean peak###Intercept###Slope###Recovered conc.

###Drug###Sample code###% Recovery

###(g/mL)###area###(c)###(m)###(g/mL)

###Sample-1###55###8775922###55.31###100.56

###Levofloxacin###Sample-2###55###8690673###672467.95###146515.30###54.73###99.50

###Sample-3###55###8862801###55.90###101.64

###Sample-1###55###12112392###56.46###102.64

###Moxifloxacin###Sample-2###55###12143002###700570###202141.94###56.61###102.92

###Sample-3###55###12126139###56.521###102.77

CONCLUSION

The efficacy of a drug depends on some absolute requirements such as quality, potency etc. It is obvious that a little change in the formulation or variations in the manufacturing process or use of low quality materials including APIs can affect the efficacy of the drugs leading to harmful effects to the patients. Therefore, quality and efficacy assessment and maintenance of proper dosage schedule are strongly needed to ensure the effectiveness of the drug. Hence, we planned to study the potency of most commonly used antibacterial preparations like levofloxacin and moxifloxacin which are frequently prescribed in Bangladesh.

To attain this objective, a rapid and sensitive reversed phase high performance liquid chromato- graphic (RP-HPLC) method has been developed and validated according to the guidelines of FDA, ICH and USP with respect to accuracy, precision, specificity and linearity. The newly developed method has been found to be simpler, accurate, reproducible, efficient and less time consuming, and has been applied successfully for the simultaneous study of levofloxacin and moxifloxacin.

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This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh

2Centre for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh

3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Pakistan Address correspondence to this author at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh; Tel: 880-2- 9661900-73, Extn. 8137; Fax: 880-2-8615583;E-mail: rashidma@du.ac.bd
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Author:Alotaibi, Saad A.
Publication:Journal of Basic & Applied Sciences
Article Type:Report
Date:Dec 31, 2013
Words:1897
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