Biochemical studies of Bacillus Thuringiensis var . kurstaki , Serratia marcescns and Teflubenzurone on cotton leafworm , Spodoptera littoralis ( Boisd . ) ( Lepidoptea : Noctuidae )

Two biopesticides Serratia marcescens [Eubacteriales: Enterobacteria] (used at MC50, concentration caused 50% malformation) and Bacillus thuringiensis Var. kurstaki (used at LC50) and insect growth regulator Teflubenzuron (used at LC 50 value) were used for treatment of 2 nd instar larvae of cotton leafworm, Spodoptera littoralis (Boisd.). Sequential combined Effect was carried out by treating 2 nd instar larvae with LC50 value of B. thuringiensis or Teflubenzuron then the larvae allowed to pupate on sawdust treated with S. marcescens at MC50 .The effect of these three agents were assessed by toxicity The obtained LC50 values were 0.113 and 165.64 ppm, for Teflubenzuron and B. thuringiensis Var. kurstaki. (protecto), respectively. Moreover, the activity of Chitinase, protease, trehalase, and the main components (total lipids, proteins and carbohydrates) were significantly fluctuated during the different periods of pupal stage.


INTRODUCTION
The cotton leafworm, S. littoralis (Boisd.) is a highly destructive insect pest.The extensive use of insecticides to control S. littoralis larvae has led to several problems and hazards such as development of resistance and residual effects (Frank et al., 1990).Thus, it is important to search for alternative control agents with new modes of action.Among these agents are insect growth regulators (IGR's) and microbial control agents.The bacterium B. thuringiensis, proved to be a highly successful weapon for fighting some agricultural pests and it offer many advantages over chemical insecticides.B. thuringiensis is known to be one of the most pathogenic species of bacteria, which induce larval mortality after a course of infection stages.The interest of using such agent as a microbial bioinsectcide was increased during the past decade (Dulmage and Co-operators, 1981).As well as, the bacteria of the genus Serratia are often associated with insects and have the behavior of a facultative pathogen.(Trevor et al., 2004).In general, S. marcescens is not pathogenic to insects when present in the digestive tract in small numbers, but once it enters the hemocoel it multiplies rapidly and causes death in one to three days (Sikorowski, 1985).Furthermore, chitinase producing bacteria S. marcescens caused significant physiological and morphological effects on pupal and adult stages where it caused a significant increase in adult malformation % and also affected some enzymes activity El-Sheikh (2006).

The aim of this study:
Evaluate the effectiveness of the insect growth regulator Teflubenzuron as a chitin synthesis inhibitor (No moult), B. thuringiensis (protecto), chitinase producing bacteria S. marcescens and their sequential combined effect through following treatments for controlling S. littoralis.

Rearing technique
The stock culture of the cotton leafworm, S. littoralis (Boisd.)was obtained from a laboratory strain maintained in the Cotton Pest Research Dept, Plant Protection Research Institute, Agricultural Research Centre, Dokki, Giza, for several generations without any insecticidal or microbial pressure.The insect was reared on castor-oil leaves, Ricinus communis, under laboratory conditions at 25 ± 2 °C and 60 ± 5 % R.H. 2 nd and late 6 th instars larvae were used in the current work.

Bioassay
Preliminary tests were carried out using series of concentrations (in water) for each of the bio-agent, Serratia marcescens (10 4 , 10 5 , 10 6 , 10 7 , 10 8 .10 9 ) colony forming unit/ml (cfu/ml), B. thuringiensis (44.187, 88.375, 176.75, 352.5, 705, 1410 ppm) and the chitin synthesis inhibitor Teflubenzuron (0.02, 0.04, 0.08, 0.16, 0.32, 0.64 ppm) were prepared using the commercial formulation.Sawdust was treated with each concentration of S. marcescens in glass jars and offered to late 6 th instar larvae to pupate on it.The offered treated sawdust was in a wettable form, while, in case of Teflubenzuron and B. thuringiensis, the use of leaf-dipping technique was carried out according to Abo El-Ghar et al., 1994.Castor bean leaves, R. communis, were dipped in each concentration then left to dry at room temperature and these were offered to the newly moulted 2 nd instar larvae.Larvae were allowed to feed for 24 hrs.Then, they were provided with fresh, clean and untreated castor bean leaves until pupation.Larvae that fed on untreated castor bean leaves were used as control for Teflubenzuron and B. thuringiensis treatments whereas larvae kept in untreated sawdust were considered as control for Serratia marcescens.In all treatments, three replicates were carried out for each concentration; each replicate consisted of 20 larvae.The larval mortality and adult malformation percentages were determined.The data were then subjected to probit analysis (Finney, 1971) to obtain the LC 50 values of both Teflubenzuron and B. thuringiensis as well as the concentration which causes 50% adult malformation (MC 50 ) for S. marcescens.The combined effect was studied by treatment of 2 nd instar larvae with LC 50 of Teflubenzuron or B. thuringiensis then the larvae were allowed to pupate on sawdust treated with S. marcescens at MC 50.The Toxicity index of the tested compounds was determined according to Sun (1950) as follows:

Biochemical determinations
Preparation of samples for biochemical analysis:

Toxicity index =
LC 50 of the most toxic compound X 100 LC 50 of other compounds Pupal samples were collected after 2 nd , 4 th , 6 th , 8 th , 10 th , 12 th and 14 th days of prepupation and homogenized in distilled water using a Teflon homogenizer surrounded with a jacket of crushed ice for 3 minutes.Homogenates were centrifuged at 6000 r.p.m. for 10 minutes at 5°C.The supernatant was divided into small aliquots (0.5 ml) and stored at-20°C until analysis.Three replicates were used for each biochemical determination.

Determination of Chitinase activity:
Chitinase was determined according to the method described by Ishaaya and Casida (1974).

Determination of Protease activity:
The proteolytic activity was determined by the casein digestion method described by Ishaaya et al. (1971).

Determination of trehalase activity:
The trehalase was determined according to the method described by Ishaaya and Swiriski (1976).

Determination of total carbohydrates
Total carbohydrates were determined as described by Singh and Sinha (1977).

Total soluble protein content:
Total soluble protein content was determined by the method of lowery et al. (1951) Total lipid content: Total lipid content was estimated according to Knight et al. (1972).

Statistical analysis
Data were analyzed using Proc.ANOVA in SAS (SAS Institute, 1998).

RESULTS AND DISCUSSION Toxicological effects:
Table (1) shows the susceptibility of the 2 nd instars of S.littoralis towards the B. thuringiensis and Teflubenzuron compounds.The LC 50 of Teflubenzuron is 0.113ppm, whereas, it is 165.64ppm in case of treatment with B. thuringiensis.Based on LC 50 values, it is obvious that both compounds caused considerable toxic effects against the 2 nd larvae of S. littoralis particularly in case of Teflubenzuron which had drastical toxic effects comparing to B. thuringiensis toxicity.On the other hand, S. marcescence had very low toxic effects against larvae and high tendency to induce malformation.Thus, because of its ability to cause a high malformation percentage to Spodoptera littoralis, so, the concentration which causes 50 % adult malformation (3.09x10 8 cfu.) was used instead of LC 50 that S. marcescens failed to achieve.The LC 50 value of chitin synthesis inhibitor Teflubenzuron was similar to that obtained by Thabit (2011) who recorded LC 50 of 0.177ppm for Teflubenzuron towards 2 nd instar larvae of S. littoralis.Teflubenzuron in the present study had drastical toxic effect comparing to B.thuringiensis, this was similar to Abd El-Aziz (2007) who found that lufenuron had drastical toxic effect comparing to B. thuringiensis on 2 nd instar larvae of S. littoralis.On the other hand, the concentration which causes 50% adult malformation to S. littoralis was similar to Tolba(2006) and EL-Sheikh et al.(2005) working on Agrotis ipsilon.Abd El-Aziz (2000) stated that the crystal toxins from most of B. thuringiensis serotypes are toxic to larvae of Lepidoptera up on ingestion and is quickly activated by a combination of the alkaline gut P H and proteolytic enzymes present in the mid gut of the insect.

Biochemical Effects: Effects on chitinase activity:
The obtained data in Table (2) show the change in chitinase activity in pupal stage of S. littoralis treated with S.marcescence at concentration caused 50% malformation, LC 50 either of B. thureinginsis or Teflubenzuron and their sequential following treatments at which sawdust was treated with S. marcescence after larvae treatment either with B. thureinginsis (Bt/serr) or with Teflubenzuron (Teflu /Serr).Results confirmed that the treatments with S. marcescens, Bt/Serr, Teflubenzuron and Teflu /Serr caused a significant increases in the chitinase activity through the pupal stage of S. littoralis at all times except the last time in Bt/Serr, with respect, the highest induction was obvious in case of Teflu /Serr followed by Teflubenzuron, and S. marcescens in which there were gradually increases reached the maximum of 901.1 and 664.99, 785.27 and 572.15 and 324.24 and 245.58 % at 10 th and 12 th days of pupation, respectively, then there were decreased at 14 th day to 255.79, 232.25 and 28.52%, respectively.The individual treatment with B. thureinginsis revealed decreases through pupal stage except at 2 nd and 10 th days of pupation in which significant increases were recorded (41.61 and 41.96%, respectively).The obtained results in S. marcescens and Teflubenzuron treatments were in agreement with the findings obtained by Tolba (2006) who found high increases in chitinase activity during pupal stage of A. ipsilon treated either with S. marcescens or flufenoxuron.Also El-Sheikh (2006) reported that both S. marcescens and lufenuron cause significant increases in chitinase activity during pupal stage of S. littoralis.As well as similar results were reported by Lee et al. (1994) who found an increase in the chitinase activity of larvae of Hyphantria cunea treated with Diflubenzuron and Chlorofluazuron (chitin synthesis inhibitors).The increase in chitinase activity in pupae of S. littoralis treated with S. marcescens could be attributed to its ability to secrete chitinase, which facilitated penetration of bacteria within pupal skin, multiplication within tissues and killing insect by septicemia El-Sheikh (2006).In addition, the increase in chitinase activity of pupae treated with Teflubenzuron could be attributed to the secondary effect of chitin synthesis inhibitor.The primary effect involves blocking of incorporation of uridine 5'diphospho-N-acetylglucose-amine into chitin.Chitin synthetase carried out through this polymerization step (Verloop, 1977).Moreover, the increase in chitinase activity may be a secondary effect for the reduced activity of -ecdysone metabolizing enzymes, followed by ecdysone accumulation which resulted in hyper chitinase activity (Yu and Terriere, 1977).On the other hand, Abdel-Aal et al. (2009) found that some chitin synthesis inhibitors (CSI) increased chitinase activity of the late 6 th instar larvae of S.littoralis and recorded that chitinase and protease are essential for digestion of old endocuticle in the moulting process.So, any changes in these enzyme activities may attribute to the interference of the (CSI) in moulting process.

Effects on protease activity:
Results in Table (3) show that S. marcescence caused significant increases in protease activity at all most pupation times recording the maximum increase (25.29% at 12 th day) moreover, treatment with B. thureinginsis gave higher increases (maximum of 96.47 % at the last time) through the pupal stage, while recorded -58.51 %,-71.39%and -66.72% at 6 th , 8 th and 12 th day of pupation time.However, Bt/Serr treatment exhibited higher significant effects in protease activity at 2 nd and 4 th days -.of pupation time (-32.00% and 81.31%, respectively) than that of treatment with B. thureinginsis (27.47% and 35.21%, respectively).While, there were less effect at other times, as compared with control.Treatment with Teflubenzuron revealed significant decreases (maximum of -70.06 % at 8 th day) as compared to control.Moreover, the treatment with Teflu /Serr caused significant increases from 6 th to 14 th days of pupation time.Treatment with Teflu /Serr had more effect in protease activity at last three times (60.33 %, 37.43% and 59.62%, respectively) than either S. marcescence (22.10%, 25.29% and19.71%,respectively) or Teflubenzuron (25.72%,-28.71%and -14.58%, respectively) treatments, as compared to control .
The increase in protease activity was similar to the results obtained by El-Sheikh ( 2006) who observed significant increases in protease activity after 3, 8 (logarithmic phase) and 13 (stationary phase) days of S. littoralis pupation in sawdust treated with S. marcescens.On the other hand, lufenuron significantly decreased protease activity at the 3 rd , 8 th and 13 th days of pupation.Also, Abdel-Aal et al. (2009) recorded that Teflubenzuron decreased the porotease activity of the late 6 th instar larvae of S.littoralis.

Effect on trehalase activity:
The obtained data in Table ( 4) show the change in trehalase activity in pupal stage of S. littoralis treated with S. marcescence at concentration causes 50% malformation ,LC 50 of B. thureinginsis, Teflubenzuron and their sequential following treatments at which sawdust was treated with S. marcescence after larvae treatment either with B. thureinginsis Bt/serr or with Teflubenzuron (Teflu /Serr).The results revealed potentiality of S. marcescens and B. thureinginsis to cause significant decreases in trehalase activity in pupal stage of Spodoptera littoralis by -66.32-43.83and -23.67-18.76 at 8 th and 10 th day of pupation time, respectively, in comparison with control.On the other hand, significant increases in trehalase activity were found during intervals from 4 th , 6 th , 8 th and 10 th days by 26.96,30.07,38.88 and 52.74% for Teflubenzuron and by 26.84,30.57,56.50 and 57.41% for Bt/Serr, respectively.Treatment with S. marcescence, Teflubenzuron and Teflu /Serr exhibited significant decreases at 12 th and 14 th days of pupation time by (-37.37, -53.16, and -66.68%) and (-35.72, -22.13 and -80.27), respectively, as compared with control.The reduction in trehalase activity of pupae treated with S. marcescens was similar to that published by Tolba ( 2006) who revealed potentiality of S. marcescens to cause significant decrease in trehalase activity during pupal stage of A. ipsilon.In addition, the effect of Teflubenzuron on trehalase activity in the present study may be in harmony with Tolba ( 2006) who recorded significant increases in trehalase activity at the most times during pupal stage of A. ipsilon treated with Flufenoxuron.It is known that in insects, trehalase degrades trehalose to glucose for internal energy supply and generation of glucose needed for chitin build-up (during moulting), so the inhibition of trehalase observed in the present work might affect chitin build-up.The mode of action of CSIs revealed that these compounds affect the integument composition of insect, especially that of chitin (Ishaaya and Casida, 1974).The reduced level of chitin synthesis in the cuticle formation is due to the inhibition of biochemical processes leading to chitin build-up (Post and Vincent, 1973).Trehalase has the important function for liberating glucose for energy, and is activated during moulting to generate glucose for chitin build up (Meisner et al., 1978).In addition, trehalase played a significant role in the supply of energy to the insect and the activity of trehalase might serve as an indicator of energy reserves resulting from availability of carbohydrate nutrients (Wyatt 1967).During moulting cycles, the trehalose-trehalase system is activated to generate glucose needed, probably, for chitin build-up in the newly synthesized cuticle (Candy and Kilby, 1962).

Effects on the main components: Effects on total lipids content:
The obtained data in Table (5) revealed significant sharp decreases in total lipid content of untreated pupae at 12 th and 14 th days of pupation.S. marcescens, Teflubenzuron and Teflu /Serr caused significant decreases at all times of pupation, except significant increase of 24.24% at 8 th day for Teflubenzuron.With respect, significant sharp decreases in total lipid content in pupae treated with S. marcescens were found at early time of pupation in which decreased by about -63.25%, -71.17 % and -59.13% after 2 nd , 4 th and 6 th days of pupation time, respectively.
B. thureinginsis exhibited significant decreases from 4 th to 10 th days of pupation, whereas, significant increases at last two times were found.As well as, Bt/Serr caused significant decreases from 2 nd to 10 th days, while, significant increase at last time was recorded.Regarding to all treatments, it is worth to mention that all treatments contained S. marcescens had higher decreasing effect than the others, as compared to control.The reduction in total lipid content of pupal stage after treatment with Teflubenzuron is similar to the data obtained by El-sheikh, (2002) and Abdel-Aal (2003) after using Flufenoxuron against Agrotis ipsilon and S. littoralis larvae, respectively.Also Tolba (2006) reported that Flufenoxuron reduces the total lipid content in Agrotis ipsilon pupal stage and that the sharp reduction in total lipid content at early time of pupation resulted in pupae treated with Serratia marcescens may be due to bacterial growth in the insect body and consuming lipids for energy requirement.Similar reduction in total lipid content of pupae treated with B. thuringiensis was recorded by Abd El-Aziz (2000) who found a sharp decrease in lipid content of S. littoralis larvae treated with B. thuringiensis var.kurstaki.Similarly lipid content in tobacco cutworm Spodoptera litura (fab.)larvae was studied by Tripathi and Singh (2002) who reported that the infection resulted in significant reduction in total lipid content of infected larvae suggesting that the reason for the lower fat content could be due to extended larval period of the treated insects and blocked food ingestion so, the fat reserves might have been utilized for the maintenance during larval period.Also, Abdel-Aal (2006) found that B. thuringiensis var.kurstaki caused a significant reduction in the lipid content of S. littoralis larvae.Bennett and Shotwell (1972) suggested that the infected larvae might produce enzymes that utilize lipids in effort to remove the invading organisms.They also, assumed another possible reason for the decreasing in haemolymph lipid as infection progressed or pathogens may break down haemolymph lipids to simpler moieties that can be utilized as a carbon source for growth and sporulation.

Effects on total soluble protein content:
From results recorded in Table (6) it could be stated significant decreases of-12.45%and -15.05% in total soluble protein content after 8 th and 10 th days of pupation in sawdust treated with Serratia marcescens, respectively.In addition, Teflubenzuron caused significant decreases by about -12.76 and -7.29 in total soluble protein content at 2 nd and 4 th days of pupation.Whereas, Teflu /Serr treatment caused significant decreases in total soluble protein content at all times of pupation (except 14 th day of pupation) comparing to control.Results also revealed significant decreases in total soluble protein content in pupal stage of S. littoralis treated with B. thuringiensis at 6 th and 8 th days (-22.37% and -24.27%, respectively).On contrary, there was a significant increase (6.06%) at the first time of pupation, as compared with control.Once more, Bt/Serr caused significant decreases in total soluble protein content in pupal stage of S. littoralis at all times of pupation with exception at 2 nd and 14 th days of pupation comparing to control.Thus, it could be stated that both Bt/serr and Teflu /Serr had more significant effect on the reduction in total soluble protein content than treatments with B. thuringiensis, Teflubenzuron and S. marcescens.Similar results were obtained by Abdel-Aal (2006) who found that B. thuringiensis var.kurstaki caused a significant reduction in the protein content of S.littoralis larvae.Kamel et al. (2010) observed significant reduction in total protein content of S. littoralis larvae after treatment with B. thuringiensis var.kurstaki.This could be due to the break down of protein into amino acids which help to supply energy for the insect.El-Shershaby et al. (2008) indicated that, B. thuringiensis var.kurstaki resulted in a great reduction in protein content of S. littoralis larvae and these toxins of B. thuringiensis are responsible for the inhibition of protein synthesis by forming a protein complex.Also Tolba ( 2006) reported that treatment with either Flufenoxuron or S. marcescens reduces the protein content of Agrotis ipsilon pupal stage.The same reduction in total soluble protein content due to Teflubenzuron treatment was found by (El-sheikh, 2002 andAbdel-Aal 2003) after using Flufenoxuron against Agrotis ipsilon and S. littoralis larvae, respectively.In addition, Abdel-Aal (2006) reported that treatment with either chlorfluazuron or B. thureinginsis var.kurstaki decreased total soluble protein content of S. littoralis larvae.The decrease in the protein content of the pupae in the present work in all treatments might be due to the protein was binding with foreign compounds as the compounds used.

Effects on Total Carbohydrate contents:
Table (7) shows that all treatments cause significant decreases in total carbohydrate content at all times except treatment with Teflubenzuron in which there were significant increases at 2 nd and 10 th days .The highest decrements were observed in case of treatments with S. marcescence, Bt/Serr and Teflu /Serr.The decrements for S. marcescence were gradually pronounced with time reaching drastical decreases at 10 th and 12 th days of pupation time.These decrements were of -27.60, -32.00,-40.14, -55.28, -80.93, -81.57 and -79.1 % at 2 nd , 4 th , 6 th , 8 th , 10 th , 12 th and 14 th .Whereas, they were -46.40, -46.84, -58.27, -74.95, -82.11, -66.25 and -51.36 % for Bt/Serr at the same times, respectively, which were significantly higher than those of B. thureinginsis.On the other hand, the decrements were -33.33, -31.48, -50.45,-60.31,-42.47,-50.26 and -52.74 % in case of Teflu /Serr at all times, respectively, which were significantly higher than those of treatment with Teflubenzuron, as compared to control .Thus, it could be stated that all treatments contained Serratia marcescence induced more reducing effect on total carbohydrate content of S. littoralis pupae than those of Teflubenzuron or B. thuringiensis.S. marcescens caused a high decrease in total carbohydrates because of their requirements to glucose as energy source for propagation and growth so bacteria utilize carbohydrates as carbon source for energy and built a new cell, this may decrease the available carbohydrates in treated insect especially glucose which plays an important role in energy supply, adult maturation and builds up a new chitin (Tolba, 2006;El-sheikh et al. 2005 andEl-sheikh 2006) that may explain the high malformation% obtained in the present study.

Table 4 :
Changes in trehalase activity in pupal stage of Spodoptera littoralis treated with Serratia marcescence at MC 50 , LC 50 of B. thureinginsis, Teflubenzuron and their sequential effects.*Means with the same letter(s) is not significantly different.** The reduction and induction percentage in the enzyme activity with compared to control.

Table 7 :
Changes of total carbohydrates concentration in pupal stage of Spodoptera littoralis treated with Serratia marcescence at MC 50 , LC 50 of B. thureinginsis, Teflubenzuron and their sequential effects.*Means with the same letter(s) is not significantly different.** The reduction and induction percentage in the enzyme activity with compared to control.

Table 1 :
Toxicity of B. thuringiensis and Teflubenzuron against 2 nd instar larvae of Spodoptera littoralis.

Table 2 :
Changes in chitinase activity in pupal stage of Spodoptera littoralis treated with Serratia marcescence at MC 50 , LC 50 of B. thureinginsis, Teflubenzuron and their sequential effects.

Table 3 :
Changes in protease activity in pupal stage of Spodoptera littoralis treated with Serratia marcescence at MC 50 , LC 50 of B. thureinginsis, Teflubenzuron and their sequential effects.
*Means with the same letter(s) is not significantly different.** The reduction and induction percentage in the enzyme activity with compared to control.

Table 5 :
Changes in total lipids in pupal stage of Spodoptera littoralis treated with Serratia marcescence at MC 50 , LC 50 of B. thureinginsis, Teflubenzuron and their sequential effects.
*Means with the same letter(s) is not significantly different.** The reduction and induction percentage in the enzyme activity with compared to control.

Table 6 :
Changes of total protein in pupal stage of Spodoptera littoralis treated with Serratia marcescence at MC 50 , LC 50 of B. thureinginsis, Teflubenzuron and their sequential effects.
*Means with the same letter(s) is not significantly different.** The reduction and induction percentage in the enzyme activity with compared to control.