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Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab. Rajamanickam Chandrasekaran 1, Kannan Revathi 1, Selvamathiazhagan Nisha, Suyambulingam Arunachalam Kirubakaran, Subbiah Sathish-Narayanan, Sengottayan Senthil-Nathan ⇑ Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Center for Excellence in Environmental Sciences (SPKCES), Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli, Tamil Nadu, India a r t i c l e i n f o Article history: Received 5 January 2012 Accepted 2 July 2012 Available online xxxx Keywords: Bacillus Chitinase Enzyme Molecular masses Cutworm Dietary utilization Mortality a b s t r a c t An extracellular chitinase was purified from Bacillus subtilis. The lethal concentration (LC50) was determined by using chitinase in first, second, and third instars of Spodoptera litura Fab. Chitinase showed the highest insecticidal activity at 6 lM concentration within 48 h. The nutritional indices were also significantly affected by the 6 lM concentration (P < 0.05). Food consumption, efficiency of conversion of ingested and digested food, relative growth rate, and consumption values declined significantly while approximate digestibility was increased. Our study indicates that treatment of host plant leaves with the chitinase can regulate (reduce) larval growth and weight, and enhance the mortality. This may serve as an effective biocide and alternative to Bt toxin. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Pesticides are applied in agricultural systems for protecting crops from damage by insects and disease. The use of pesticides has also resulted in significant benefits effects to public health and the environment. In general the amount of pesticides released into the environment has risen significantly in the last five decades [1,2]. However scientists constantly seek for an effective and environmentally friendly method of controlling pests and diseases [3–5]. Control of crop pests by the use of biological agents holds great promise as an alternative to the use of chemicals. Secondary metabolites and crude enzyme from microorganisms have been used to control crop pest population [6]. Chitin is a long, unbranched polysaccharide of an amino sugar N-acetyl-b-D-glucosamine linked together by b-1,4-glycosidic linkages [7]. It is abundant in nature as a structural compound in cuticle and integument of animals, especially in insects [8]. Chitin is metabolized by various chitinases that are found in insects, bacteria, fungi and higher plants [9]. Chitinases belong to glycosyl hydrolase families 18 and 19 according to the classification made ⇑ Corresponding author. Fax: +91 4634 283270. E-mail addresses: [email protected], (S. Senthil-Nathan). 1 These authors contributed equally to this work. [email protected] by Henrissat and Bairoch [10]. The family 18 chitinases have been determined to possess a common (a/b) 8-barrel domain, comprising of eight a-helices and eight b-strands. The catalytic reaction of the family 18 enzymes takes place through a retaining mechanism, in which b-anomer is generated by hydrolysis of b-l,4-glycosidic linkages. The chitinases of the family 19 have been described as being similar to lysozyme and chitosanase in its mode of action [11]. Insect growth and development are strongly dependent on the construction and remodeling of chitinous structures [12]. Chitinase induced damage to the peritrophic membrane in the insect gut causes a significant reduction in nutrient utilization and consequently in insect growth [13]. Due to this, chitinase present in the insect diet can decrease insect growth [14,15]. Chitinolytic microorganisms have many potential applications as biocontrol agents [16]. Over-expression of a chitinase in an entomopathogenic organism can increase insect mortality [17]. Chitinase plays a vital role in the control of plant pathogenic fungi and raising plant disease tolerance. In the microbial degradation of chitin using chitinase, chitooligosaccahrides are produced, which are further degraded to N-acetylglucosamine by chitobiase [18]. Consequently, microbial chitinases have been isolated for the production of chitooligosaccharides [19]. Bacillus species are well-known as producers of extra-cellular enzymes, including cellulase, glucanase, amylase, proteinase and chitinase, secondary metabolic products, and they are used 0048-3575/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2012.07.002 Please cite this article in press as: R. Chandrasekaran et al., Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab., Pestic. Biochem. Physiol. (2012), http://dx.doi.org/10.1016/j.pestbp.2012.07.002 2 R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx frequently in industrial fermentation [20,21]. Bacillus thuringensis is notable as a source of insect toxins [9]. Bacillus subtilis (B. subtilis), is a gram-positive, non-pathogenic, spore forming bacterium with notable chitinase activity [22]. Insects systematically synthesize and degrade chitin in a highly controlled manner to allow ecdysis and regeneration of the peritrophic matrices. Some of the chemical compounds that disrupt chitin metabolism, such as diflubenzuron, have been of special interest for the control of agricultural pests [12]. Spodoptera litura Fab. (Lepidoptera: Noctuidae) is a polyphagous insect, and worldwide it infests crops like cotton, groundnut, crucifers, tomato, tobacco, potato and soybean [23,24]. S. litura has 150 host species [25]. S. litura causes serious damage during early seedling stage [25]. Farmers usually make several applications of synthetic insecticides for the control of this pest during the growing season in India [26]. We have purified a B. subtilis isolate which secretes to a large amount of extracellular chitinase shows high levels of pathogenicity against S. litura. In the present study, a chitinase was purified and characterized from B. subtilis, and tested against S. litura. 2. Materials and methods 2.1. Isolation and screening of chitinase producing microorganism Bacterial isolates were obtained from the soil in and around Namakkal (11°7333300 latitude and 78°0666700 longitude) and Tirunelveli (08°800 and 09°2300 latitude and 77°0900 and 77°5400 longitude) district, Tamil Nadu, India. From 25 soil samples (Table 1), we isolated chitinolytic bacteria, which produced a zone of clearance in chitin media. The clear zones occur due to the hydrolysis of colloidal chitin from the medium. We selected two chitinase producing organisms (B. subtilis) for the purification of chitinase. The soil organisms were screened on colloidal chitin agar plates containing (g/l): chitin, 5.0; yeast extract, 0.5; (NH4)2SO4, 1.0; Table 1 Isolation of chitinolytic bacteria from soils from Namakkal and Triunelveli district in of Tamil Nadu, India. S. No. Soil samples collected Chitinase producing bacteria Presence of zone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Namakkal Triunelveli Triunelveli Triunelveli Triunelveli Triunelveli Triunelveli Triunelveli Triunelveli Triunelveli Triunelveli Triunelveli Triunelveli Bacillus sphericus Bacillus thuringiensis Bacillus sphericus Bacillus popilliae Streptomyces Bacillus subtilis Bacillus megaterium Bacillus thuringiensis Bacillus thuringiensis Bacillus thuringiensis Bacillus cereus Bacillus cereus Streptomyces Pseudomonas fluroscence Pseudomonas fluroscence Bacillus subtilis Baciillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus thuringiensis Bacillus cereus Bacillus cereus Bacillus subtilis Bacillus subtilis Bacillus sphericus Bacillus thuringiensis + + + + + + + + + + + +++ +++ + + + + + + + +, Presence of zone in chitin agar plate; , represents absence of zone in chitin agar plate, +++, used for chitinase enzyme purification. MgSO47H2O, 0.3; and KH2PO4, 1.36, agar 20 pH 7.2 at 30 °C for 72 h [22]. Well separated colonies that showed the clear zone in the chitin agar plates were selected. The identified cultures were transferred into pure agar slants and the morphological, and the physiological characters of microbes were analyzed according to the Bergey’s manual determinative bacteriology [27]. Colonies that showed a large zone of clearance were selected and used for chitinase production. 2.2. Insect rearing S. litura pupal cultures were collected from Project Directorate of Biological Control, Bangalore, Karnataka, India. Emerging adult moths were transferred to cages and fed on a 10% sucrose solution to enhance oviposition. Moths were transferred at a ratio of one male: two females to oviposition cages containing castor plant, Ricinus communis L. (Euphorbiaceae) leaves and covered with sterilized muslin cloth for egg laying. The muslin cloths containing eggs were removed daily and eggs present were surface-sterilized in situ by dipping in 10% formaldehyde solution for 2–5 min, then washing with distilled water for 2 min. The muslin cloths containing eggs were moistened and kept in plastic containers (500 356 200 mm) to allow hatching. Larvae were reared in the laboratory on R. communis leaves. All experiments and cultures were carried out at 28 ± 2 °C, 65% relative humidity, with a 14:10 light/dark cycle. 2.3. Preparation of colloidal chitin Colloidal chitin was modified from the method of Lee et al. [28]. Twenty grams of chitin powder (RM1356-500G, Hi-media, India) was added slowly into 350 ml of concentrated HCl and left at 4 °C overnight with vigorous stirring. The mixture was added to 2 L of ice-cold 95% ethanol with rapid stirring and kept overnight at 35 °C. The precipitate was collected by centrifugation (EltekRC4100F) at 5000g for 20 min at 4 °C. The precipitate was washed with sterile distilled water until the colloidal chitin became neutral (pH 7.0). 2.4. Crude enzyme preparation The chitinase producing B. subtilis strains were isolated from the soil. These isolates were preserved in the colloidal chitin medium. For the crude enzyme preparation, the isolates were grown in the medium containing chitin, colloidal chitin-2 g/L; K2HPO4-1 g/L; NaCl-5 g/L: MgSO47H2O-0.04 g/L; CaCl2-0.02 g/L [29] and cultured in the 100 ml medium in 500-ml conical flasks and incubated under shaking condition for about 8 days. The cultured fluids were centrifuged at 10,000g for 20 min. The supernatant was allowed for ammonium sulphate precipitation, allowed to stand overnight. The pellet was centrifuged at 10,000g for 20 min. Then the precipitates were dissolved in a small amount of 20 mM citrate phosphate buffer (pH 7.8) and extensively dialyzed against the same buffer. The dialysate was used for further purification. Dialysates were subjected to ion-exchange chromatography on DEAE-Cellulose. The peak fractions from this column containing the purified enzymes were confirmed to contain 48 kDa chitinases by SDS–PAGE analysis, along with standard molecular weight markers. The recovery of the yield and specific activity at various steps of the purification procedure were summarized in the Table 2. 2.5. Enzyme purification All the purification steps were carried out at 4 °C. The dialysate sample was loaded directly into the DEAE-Cellulose column equilibrated with 20 mM citrate phosphate buffer (pH 7.5). The enzyme Please cite this article in press as: R. Chandrasekaran et al., Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab., Pestic. Biochem. Physiol. (2012), http://dx.doi.org/10.1016/j.pestbp.2012.07.002 R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx 3 2.9. Food utilization, consumption and nutritional indices Table 2 Purification of Chitinase from B. subtilis. S. No. Steps involved Total protein (mg) Total activity (U) Specific activity (U/mg) 1 2 3 Culture supernatant Dialysate DEAE-cellulose 525 230 60 2954 2785 1150 6.52 11.2 42 1U = 1 lmol of reducing sugar per hour. was eluted with a linear gradient from the citrate phosphate buffer and eluted using the same buffer at a flow rate of 1 ml/h. One milliliter fractions were collected and assayed for chitinolytic activity. 2.6. Determination of molecular masses SDS–PAGE was performed on 12% gels according to Lemmli [30]. A standard protein ladder was used as a protein standard marker. The molecular masses were also determined by chromatography (The same column as under the condition for the purification of the enzymes) using a standard curve obtained from proteins with known molecular weights. The standard proteins were BSA and cytochrome C. 2.7. Enzyme assay Chitinase activity was assayed, based on dinitrosalicylic acid (DNS) method [31]. The reaction mixture contained 70 ll of enzyme solution and 70 ll of 1% colloidal chitin in 0.1 M citrate phosphate buffer (pH 5.0). After incubation at 50 °C, 120 rpm for 30 min the mixture was heated in a boiling-water bath for 5 min and then centrifuged at 3000 rpm for 5 min to remove extra chitin. An equal aliquot vial containing 90 ll of the supernatant and equal volume of DNS solution were mixed and heated in water bath for 15 min simultaneously, 30 ll of potassium sodium tartarate solution (400 g/L) was added. After cooling at 4 °C to room temperature, the absorbance at 540 nm (Systronic106) was measured. The activity was calculated from a standard curve obtained using known concentration of N-acetylglucosamine [32]. One enzyme unit was defined as the amount of enzymes that produces 1 lmol of reducing sugar per hour under reaction conditions. The protein estimation was done by using Lowry et al. [33]. The S. litura third-instar larvae were starved for about 3 h, before that the initial weight of the larvae was measured. The larvae were (10 larvae per concentration) allowed to feed on weighed leaf treated with chitinase. After 24 h the remaining uneaten leaves in the container were measured and replaced with fresh leaves daily. Food consumption, weight gain and fecal weights were measured using an electronic balance (Shimadzu, Japan). Each larva was weighed, oven-dried and re-weighed to determine a percentage dry weight of the experimental larvae after 24 h. The food ingestion was estimated by subtracting the diet remaining at the end of the experiment from the total dry weight of the leaves. Fecal pellets were collected, weighed and then oven dried, and reweighed to estimate the dry weight of the fecal material. Differences in average weight of the larvae recorded at the beginning and at the end of the period gave the gain in body weight while the mean larval body weight gain was calculated using the formula: mean weight = final weight initial weight [36]. The evaluations of nutritional indices of S. litura were done according to Waldbauer [36]. Relative consumption rate RCR = dry weight of food eaten/duration of feeding (days) mean dry weight of the larva during the feeding period, the relative growth rate RGR = dry weight gain of the larva during the period/duration of feeding (days) mean dry weight of the larva during the feeding period, approximate digestibility AD = 100 (dry weight of food eaten dry weight of feces produced)/dry weight of food eaten, Efficiency of conversion of ingested food ECI = 100 dry weight gain of larva/dry weight of food eaten, and efficiency of conversion of digested food ECD = 100 dry weight gain of the larva/(dry weight of food eaten dry weight of feces produced). Larval growth and food utilization were calculated after 24 h. 2.10. Larval weight versus larval duration Emerged second instar larvae of S. litura were taken from a laboratory culture. Larvae which have been reared on the fresh castor leaves that had been treated with different concentrations of chitinase (0.5, 1, and 2 lM). The uneaten leaves were removed after 24 h, and replaced with fresh leaves. Control leaves were treated with distilled water and air-dried. A treated and control groups were maintained at 28 ± 2 °C, 65% relative humidity, with a 14:10 light/dark cycle. Records were made daily of weight of living and dead individuals. 2.8. Insect bioassay 2.11. Data analysis Bioassays were performed with first, second- and third-instars S. litura larvae using whole castor leaves amended with 2, 4 and 6 lM of chitinase. Control leaves received distilled water only. A group of 10 larvae per concentration was used for all the treatments with five replicates. The uneaten diets were removed after 24 h and replaced with fresh untreated diets for control and treated diets for treatments respectively. Deformities in the larvae, pupae and emerged adults were recorded. Mortality was recorded every 24 h, and final mortality was recorded after 12 days. The percentage mortality was calculated by using the formula (1). Data from mortality and nutritional indices were expressed as the mean of five replications and normalized by arcsine-square root transformation of percentages. The transformed percentages were subjected to analysis of variance (ANOVA) and were fitted with linear regression using MinitabÒ16 software package. Differences between the treatments were determined using the Tukey–Kramer HSD test (P 6 0.05) via the MinitabÒ16 software package. The lethal concentrations LC50 was calculated using probit analysis [35]. Percentage of mortality ¼ Number of dead larvae 100 Number of larvae introduced ð1Þ The percent mortality data after corrections [34] were subjected to probit analysis [35] to calculate the mean of lethal concentration (LC50). From the mean lethal concentration, the effective doses (EC) were selected for biological studies. 3. Results 3.1. Molecular masses The molecular weight of the protein eluted from the DEAE column was determined to be 48 kDa by SDS–PAGE. SDS–PAGE analysis of the purified enzyme revealed the presence of only one protein band (chitinase) compared with a standard marker 116, Please cite this article in press as: R. Chandrasekaran et al., Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab., Pestic. Biochem. Physiol. (2012), http://dx.doi.org/10.1016/j.pestbp.2012.07.002 4 R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx Fig. 1. SDS–PAGE of purified chitinase and standard marker proteins (116, 66, 45, 29 and 20 kDa). After electrophoresis, the gel was stained with Commassie blue. L1 and L2 lanes are pooled material of B. subtilis and L3 and L4 are peak material from the DEAE column. Fig. 2. Effects of chitinase on S. litura first (1), second (2) and third instar (3) larva (6 lM) [C-Control, T-treated]. 66, 45, 29 and 20 kDa. The molecular weight of the chitinase isolated from the bacteria was 48 kDa (Fig. 1). 100 a I instar II instar a III instar The results showed that the purified enzyme hydrolyzed colloidal chitin. Our result indicates the presence of chitinases in purified enzyme preparation. The pure chitin prepared from colloidal was used for the experiment. This hydrolysis of colloidal chitin has showed the activity. During hydrolysis the enzyme released reducing sugars from the colloidal chitin. Percentage of mortality a 3.2. Substrate specificity 80 c 3.4. Food utilization, consumption and nutritional indices Chitinase treatment greatly reduced the relative consumption rate and relative growth rate of the larval stage of S. litura. The growth reduction was dependent on the concentration of chitinase. The larval growth was at maximal level in control larvae. The chitinase containing diets altered growth parameters; the growth rate is affected more than the relative consumption rate (Table 3). After treatment with chitinase, low level of food consumption was resulted with less growth rate. Approximate digestibility of treated larvae was gradually increased. The efficiency of food conservation of chitinase treated larvae was significantly lower than control larvae. Significant reduction of all nutritional indices was observed (F = 49.91; df = 3.69; P = 0.001) in 2, 3 and 4 lM concentrations. Regression between the relative growth rate and the relative consumption c c 20 d d 0 Cont 2uM 4uM 6uM The peak fractions from the DEAE column were used for application. Chitinase-treated leaves significantly reduced the growth of the larvae. Mortality rate was gradually increased in first, second and third instar due to the treatment of chitinase. Control larval mortality was reached maximum of 3.8% (±0.6) in third instar. In all the larval instar, treatment with chitinase caused mortality and the percentage of mortality decreased slightly but not significantly in third instar. The lowest (2 lM) concentration of the chitinase produced the lowest mortality in third instar larvae, but it caused several abnormalities in larvae (Fig. 2). We noted that the treated larvae were physiologically affected and malformed in larval to adult stage when compared to control. At concentrations of 4 lM and 6 lM, larval mortality were significantly different from the control in the first (F = 49.12; df = 3.11; P = 0.001), second (F = 40.43; df = 3.11; P = 0001) and third instar larvae of S. litura (F = 43.18; df = 3.11; P = 0.001) (Fig. 3). b 40 d 3.3. Mortality and growth of S. liutra after treatment with chitinase b b 60 Cont 2uM 4uM 6uM Cont 2uM 4uM 6uM Fig. 3. Percentage mortality of first, second and third instar of S. litura after treatment with chitinase (Con-Control, 2, 4 and 6 lM). Means (SEM±) followed by the same letters above bars indicate no significant difference (P < 0.05) according to a Tukey test. rate were plotted to determine the effects of chitinase against S. litura and the regression of RCR–RGR for control (r2 = 0.897), and the chitinase treated (r2 = 0.953) were significantly different in treated compared to control. The regression lines represent the reduced growth level of the larvae which fed on the chitinase treated leaves (Fig. 4). 3.5. Larval weight versus larval duration Compared to the control larvae weight, chitinase exposed larvae lost body weight. At high concentration it produced mortality and also larvae did not survive as long as the controls. The third instar larva fed with chitinase treated leaves at different concentrations (0.5, 1 and 2 lM) for 1 h and then they were allowed to feed on untreated leaves, and several effects were noted. Larvae lost weight and consumed significantly less diet than those exposed to the lowest concentration (0.5 lM) and controls (Fig. 5). Survival periods of the larvae were calculated up to third to fourth instar and the survival of larvae when treated with chitinase (1 and 2 lM) were significantly different (F = 8.56, df = 3.36; P = 0.0001). The longest survival periods were obtained in the control. 4. Discussion Chitinase has been used previously as an insecticide and a fungicide [3]. Chitinase disrupts the peritrophic membrane and enables microorganisms and pathogens to invade the hemocoel [37]. Bacteria produce chitinase for the use of chitin as nitrogen Please cite this article in press as: R. Chandrasekaran et al., Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab., Pestic. Biochem. Physiol. (2012), http://dx.doi.org/10.1016/j.pestbp.2012.07.002 5 R. Chandrasekaran et al. / Pesticide Biochemistry and Physiology xxx (2012) xxx–xxx Table 3 Nutritional indices of third instar larvae of S. litura after treatment with chitinase. Chitinase (lM) S. No. 1 2 3 4 RGR (mg/mg/day) RCR (mg/mg/day) b 2 3 4 Control b 0.045 ± 0.0021 0.040 ± 0.0010c 0.022 ± 0.0006d 0.066 ± 0.0010a 0.94 ± 0.0057 0.87 ± 0.0058c 0.73 ± 0.0115d 1.04 ± 0.0051a ECI (%) ECD (%) b 25.28 ± 0.85 20.96 ± 0.59c 18.22 ± 0.51d 30.42 ± 1.10a AD (%) b 32.700 ± 0.72 24.600 ± 0.74c 19.680 ± 0.73d 38.740 ± 0.65a 44.36 ± 0.25c 46.26 ± 0.39b 47.60 ± 0.79a 41.48 ± 0.57d Means (±SE) followed by the same letters within columns of indicate no significant difference (P < 0.05) in a Tukey test. 0.09 µ 0.08 2 Relative growth rate (mg/mg/day) y = 0.0738x - 0.00123; R=0.897 0.07 0.06 0.05 0.04 0.03 0.02 2 0.01 y = 0.0505x-0.00272; R=0.953 0.00 0.0 0.2 0.4 0.6 0.8 1.0 Relative consumption rate (mg/mg/day) Fig. 4. Regression equation and correlation between relative consumption rate and relative growth rate of S. litura fed on leaves containing chitinase with different concentration and larvae with different quantities of control leaves. 500 Control 0.5 µM 1 µM 2 µM Larval weight in mg 400 300 200 100 0 2 4 6 8 10 12 14 16 Number of days after treatment Fig. 5. Larval weight of S. litura after treatment with chitinase (0.5, 1 and 2 lM) (values are means of five replicates). and carbon sources [38]. Most of the chitinases used as insecticides are purified from the bacteria like, Bacillus [39,40], Pseudomonas [41], and Streptomyces spp. [42]. Bacillus thuringiensis also produce several chitinases with different molecular weights: 66, 60, 47 and 32 kDa [40]. This type of the chitinolytic enzyme is used as an antagonist and moreover, used as a defense mechanism against chitin containing insects [43]. Bacterial chitinolytic enzymes have been used to enhance the activity of microbial insecticides including Bacillus thuringiensis and a baculovirus. Larvae of spruce budworm, Choristoneura fumiferana, died more quickly when exposed to a chitinase-Bacillus mixture than when exposed to the enzyme or bacterium alone [44]. The chitinase was caused perforations in the gut peritrophic membrane, facilitating entry of the pathogens into the hemocoel of susceptible insects [45]. The degradation of chitin requires more than one enzyme type. Endochitinases produce chitololigomers converted into monomers by exochitinase N-acetylglucosaminidases and then later the enzyme cleaves N-acetylglucosamine from non-reducing ends. It favors smaller substrates than chitinases [46,47]. Chitinolytic enzymes associated with the ecdysial cycle have been established to act synergistically in cuticular chitin degradation [48]. In a previous study [49], chitinase was purified from the Bacillus spp, and has been analyzed as insecticides. Previously chitinase has been combined with an insecticidal protein, Bt toxin against S. litura neonate larvae [50]. Also it was shown that the crude chitinase from B. circulans could increase the efficacy of B. thuringiensis spp. kurstaki against diamondback moth larvae [51]. Chitinase was used as insecticide in bioassays that decreased the LC50 value of crystal protein produced by Bacillus thuringiensis against S. exigua and H. armigera [52]. Apparently, chitinases found in the gut have the ability to decrease digestive function in addition to their role in breaking down the chitin present in the gut lining or peritrophic membrane [9]. An enhanced toxic effect towards S. littoralis also resulted when a combination of low concentrations of a truncated recombinant Bt toxin and a bacterial endochitinase were incorporated into a semisynthetic insect diet [50]. Crude chitinase preparations from B. circulans enhanced the toxicity of Bt kurstaki towards diamondback moth larvae [53]. The chitinase tested in our study was similar to that reported by Smirnoff and Valero [51], Sneh et al. [54,55] and Wiwat et al. [53]. Microbial chitinases may partially digest the peritrophic membrane, assisting the microbes and their toxins in penetration of the peritrophic membrane [54]. Compared to the above results our present study with B. subtilis, chitinase has more effectively controlled S. litura. The relative growth rate and the relative consumption rate (RGR and RCR) of Spodoptera strains were decreased significantly when treated with toxins and secondary metabolites identified from Bacillus [56]. Further the growth rate of third instar Spodoptera strains were reduced, and the developmental periods were extended in the treated secondary metabolites from Bacillus [57,58]. Decreased larval growth was coupled with lower RGR, because the food was retained in the gut of the larvae for maximum of approximate digestibility to increase the nutrition. The regression coefficients of the RCR–RGR relations for control and treated animals were significantly different; showing that the reduction in growth of larvae fed on chitinase containing leaves was not entirely a result of lower food intake. The chitinase reduced RGR and RCR with significant change in the ECI. 5. Conclusion Bacterial chitinases emerge with properties that make them exclusively useful for pest control. Our present study reveals that purified chitinase from B. subtilis is effective against the S. litura Please cite this article in press as: R. Chandrasekaran et al., Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab., Pestic. Biochem. Physiol. (2012), http://dx.doi.org/10.1016/j.pestbp.2012.07.002 6 R. 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