Protease Mediated Resistance Mechanism to Cry 1 C and Vip 3 A in Spodoptera litura

The present study was undertaken to understand the protease mediated resistance mechanism of Cry1C and Vip3A in S. litura. Proteases play an important role for convertion of protoxins to active toxin. Hence laboratory selection of Spodoptera litura (Fab.) with Cry1C and Vip3A was done for eight generations to develop resistance, which developed 30.32 and 285.47 fold resistance respectively. Fourth instar larvae from nine generation of S. litura were dissected in an ice-cold 20 mM Tris-HCl buffer (pH7). Major midgut proteases viz, azocaseinolytic, tryptic and chymotryptic were quantified by using azocasein, trypsin and chymotrypsin as substrate. Results revealed that lowest azocaseinolytic activity 0.896 U/gut was observed in Cry1C resistant strain of S. litura which was 2.57 fold less than susceptible. Similarly, Vip3A resistant strain recorded 1.08 U/gut azocaseinolytic activities, which was 2.13 fold less than susceptible strain of S. litura. There was 2.26 and 3.35 fold decrease in activity respectively in Cry1C resistant and Vip3A resistant S. litura over susceptible strains. Chymotryptic activity in midgut of susceptible stain of S. litura was 1.13 U/gut, whereas in Cry1C resistant strain it was 0.512 U/gut which was 2.20 fold less over susceptible strain. Similarly, in Vip3A resistant strain, lower chymotryptic activity was recorded as compared to susceptible, which was 1.66 fold less than susceptible strain. Maximum five protease isoforms were identified in the electrophoretic profile of susceptible S. litura. Remarkable variation was observed between susceptible and resistant S. litura for protease isoform. The Cry1C resistant S. litura homogenate showed three bands viz., Pro3, Pro4 and Pro5 while only two bands viz., Pro3 and Pro5 in Vip3A resistant homogenate of S. litura whereas five bands were observed in susceptible strain of S. litura.


INTRODUCTION
Mode of action of B.thuringiensis in the gut is a complex process, involving many steps in the conversion of ICPs to toxins (Gill et al. 1992).ICPs interact through hydrogen bonding, disulfide linkages, and hydrophobic interactions.In lepidopteran insects, ICPs are released in the alkaline gut and hydrolyzed to toxins by proteases.Toxin binds to brush border membrane cells in the midgut, receptor toxin aggregation leads to pore formation, ionic imbalance, cell lysis and septicemia.Traditionally Bt toxicity has been attributed mainly to its ICPs.Therefore, most studies of Bt over the past two decades have focused on the discovery of new ICPs and elucidation of their mode of action in the insect midgut by proteases.Proteases involved in protoxin activation are described as trypsin or chymotrypsin like proteases in several insect species.Proteases are defined as peptide hydrolases and include all enzymes that hydrolyze peptide bonds (Beynon and Bond, 1993).Proteinases refer to a specific class of proteases and are synonymous with the term endopeptidases, which cleave internal bond in a peptide.Most of the proteases that degrade Bt ICPs are Proteinases.Proteases are involved in crystal dissolution and protoxin activation and contribute to toxin specificity.Reduced protoxins processing due to decreased activities of proteinases may be associated with resistance to Bt toxin in Ostrinia nubilalis (Huraong et al., 2004).Platella interpunctella resistant to B.thuringiensis subsp.entomocidus HD-198 was found to process Bt protoxin at a slower rate (more than 300 times slower) than the parental susceptible strain (Oppert et al., 1994).Serine proteinases, such as trypsin, chymotrypsin and elastase, are important in both solublization and activation of Bt protoxins (Dai and Gill, 1993).Proteinase mechanism was responsible for about 90% of the total resistance to Cry1Ac (Herrero et al., 2001).Extensive investigations on mechanism of resistance in insects have helped in developing diagnostic tools and appropriate resistance management tactics.

Insect
S. litura eggs, larvae were collected from the field of soybean, castor and sunflower in and around Akola and reared in the laboratory under controlled conditions of temperature 25 ± 2 °C, 75 ± 5 per cent relative humidity and photo period of 13 hrs light : 11 hrs dark.The larvae were reared in the plastic jars on fresh castor leaves washed with water, and fed up to last instar larvae.Final instar larvae were transferred to jars containing sand for pupation.The pupae were collected and disinfected with 0.02 per cent sodium hypochloride solution.Disinfected pupae were separated by sex determination (Krickpatrick, 1961) and transferred to adult emergence chamber.The adults emerged were transferred into mating chamber by maintaining male female ratio (1:1) and were provided with adult diet.Two pairs of moths were released in each mating chamber.The neonates emerged from the eggs were transferred on fresh castor leaves.In this way continuous rearing was done up to 12 generations for bioassay studies.Toxin of Cry1C was prepared from recombinant E. coli strains as per Lee et al. (1992).Serial dilutions of Cry1C (in distilled water) were prepared and the bioassays were carried out using leaf dipping method.About 5 concentrations of toxin with 3 replications were used for each bioassay.One sq.cm. of castor leaf disc dipped in Cry1C toxin solution and allowed to air dry.Leaf disc was kept in petriplate.Ten neonate larvae were released on treated leaf disc per replication.The control consisted of leaves dipped in distilled water (without toxin).A minimum 180 neonate larvae were used for each bioassay.All the bioassays were carried out at 25 0 ±2 o C and 60 to 80% RH.To maintain the leaf turbid, moist tissue paper was placed at the bottom of the plate.Moribund larvae not responding to probing were considered as dead.Observations on mortality of larvae were recorded after 72 hours post treatment.

Toxin
Toxin of Cry1C and Vip3A3A was prepared from recombinant E. coli strains as per Lee et al. (1992).Cells were grown in Luria broth containing 50 µg/ml ampicillin for 72 hrs., harvested by centrifugation at 4500 g at 4 °C and the pellet suspended in lysis buffer (50 mM Tris, pH 8; 50 mM EDTA, 15 % sucrose, lysozyme @ 2 mg/ml) and incubated for 4 hr.After incubation, lysis buffer was replaced with crystal wash I (0.5 M sodium chloride and 2% Triton X-100) and sonicated for 3 min on ice.The pellet was collected by centrifugation at 4500 g and washed thrice with crystal wash II (0.5 M sodium chloride) and then with distilled water.Finally the pellet was dissolved in solubilizing buffer (50 mM sodium carbonate 10 mM dithiothreitol, pH 10.5) at 37 °C for 6 hrs.Supernatant containing toxin was collected after centrifugation at 4500 g for 10 minutes and stored at -20 °C till further use.The proteins in the supernatant were quantified by Bradford method.

Selection of resistant strain to Cry 1C and Vip3A
Field collected population of S. litura were reared for four generations.Fifth generation homogenized S. litura neonates were exposed to a dose corresponding to the LC 50 .ofCry1C and Vip3A toxin by using leaf dip method.Surviving larvae were allowed to complete their life cycle upto adult and placed in a mating chamber for oviposition.Progeny larvae and subsequent generations were reexposed to Cry1C and Vip3A toxins to select for resistance.After twelve generations, the strain exhibiting high resistance level was considered as resistant strain and use for further study.The strain which was found to be most susceptible to Cry1C and Vip3A having the lowest LC 50 was maintained in the laboratory for several generations without exposure to Cry1C and Vip3A toxin and considered as susceptible population

Protease activity assays Azocaseinolytic activity
The midgut azocaseinolytic activity was measured by azocasein digestion method (Marcheti et al. 1998).Midgut homogenate was mixed with 130 µl of Tris HCl buffer (pH 9).To the above mixture, 100 µl of 2% azocasein was added and incubated for 1 hr at 37 °C.The reaction was stopped by adding 500 µl of 5% ice-cold trichloroacetic acid (TCA).The mixture was centrifuged at 14000 rpm for 15 minutes at 4 °C.Supernatant (50 µl) was mixed with 50 µl of 1 N NaOH and absorbance was estimated at 420 nm by using microplate reader.The protease activity of sample was calculated using trypsin standard curve in terms of tryptic unit (TU).Increasing OD by one unit was considered as one unit activity (Chandrashekar and Gujar, 2003).

Tryptic activity
Homogenized midgut supernatant containing soluble gut enzymes was used in assays.Samples were diluted 1:100 in buffer containing 200 mM Tris, CaCl 2 and 50 µl were added to microplate well.N-α-Benzyl-L-arginine p nitroanilide (BApNA, Sigma 100 mg /ml in dimethyl sulfoxide) was diluted 1:100 in buffer and 50 µl were added to each well to initiate the reaction.After 30 second incubation at 37 °C, absorbance was monitored at 405 nm (Oppert et al., 1997).Increasing OD by one unit was considered as one tryptic unit activity.

Chymotryptic activity
Midgut supernatant were diluted (1:100) in buffer containing 200 mM Tris, pH 8.0, 20 mM CaCl 2 and 50 µl were added to microplate well N-succiyl-ala-ala-proleucine p-nitranilide (SAAPLpNA), Sigma 100 mg / ml in dimethyl sulfoxide) was diluted 1:100 in buffer A and 50 µl were added to each well to initiate the reaction.After 30 second incubation at 37 o C absorbance was monitored at 405 nm (Oppert et al 1997).Increasing OD by one unit was considered as one chymotryptic unit activity.

Qualitative protease activity profile
Midgut protease of test insects was subjected to SDS-PAGE analysis on 12% gels under non denaturing condition and processed for zymogram analysis.Midgut homogenate with known protease activity was dissolved in non reducing sample buffer without boiling (2% SDS, 25% glycerol, 60 mM Tris-HCl, pH 6.8, 0.1% bromophenol blue), and electrophoresis was carried out at 4 °C.Thereafter, the gel was washed for 10 min in 2.5% Tritox X-100 to remove SDS.Gel was incubated with 100 mM Glyicine NaOH buffer (pH10) containing 2% casein for 1hr.Then gel was stained with 0.5% commassie brilliant blue R250.Protease activity was revealed as zone of white clearing in dark blue background (Garcia-Carreno et al., 1993).

Total protease activity (Azocaseinolytic activity) in S. litura midgut
The data regarding the azocaseinolytic activity are presented in Table1.Lowest azocaseinolytic activity 0.896 U/gut was observed in Cry1C resistant strain of S. litura which was 2.57 fold less over susceptible.Similarly, Vip3A resistant strain recorded 1.08 U/gut azocaseinolytic activity, which was 2.13 fold less over susceptible strain of S. litura.

Tryptic activity in S. litura midgut
Data presented in Table 2 showed that specific activity of trypsin like proteases in the soluble fraction of Cry1C resistant and Vip3A resistant S. litura larvae was significantly lower than that of susceptible larvae.There was 2.26 and 3.35 fold decrease in activity respectively in Cry1C resistant and Vip3A resistant S. litura over susceptible.

Chymotryptic activity in S. litura midgut
Chymotryptic activity in midgut of susceptible stain of S. litura was 1.13 U/gut, whereas in Cry1C resistant strain it was 0.512 U/gut which was 2.20 fold less over susceptible strain.Similarly, in Vip3A resistant strain, lower chymotryptic activity was recorded as compared to susceptible, which was 1.66 fold less than susceptible strain.

Electrophoretic profile of midgut protease activity in S. litura
The electrophoretic pattern of proteases from midgut homogenate of Cry1C resistant, Vip3A resistant and susceptible strains of S. litura larvae is presented in Table 4 and Plate 1.In this study five protease isoforms (Pro1 to Pro5) were observed having molecular weight 295.43, 40.66, 32.44, 27.09, 23.01 kDa according to its mobility towards anode.Variation in protease activity profile of Cry1C resistant, Vip3A resistant and susceptible strains of S. litura were observed.In midgut homogenate of susceptible S. litura showed five protease isozymes (Pro1 to Pro5) with Rf values 0.20, 0.38, 0.44, 0.52 and 0.58 respectively, were observed.This differed significantly from electrophoretic profile of midgut homogenate from Cry1C resistant and Vip3A resistant S. litura.Three protease isozymes viz, Pro3, Pro4 and Pro5 were present in Cry1C resistant strain, out of which Pro3 and Pro5 showed medium intensity and pro4 showed light intensity.In Vip3A resistant strain, only two protease isozymes viz, Pro3 and Pro5 were observed having Rf value 0.44 and 0.58, respectively.

DISCUSSION
Total protease activity (Azocaseinolytic activity) Azocaseinolytic activity was less in Cry1C resistant strain of S. litura which was 2.57 fold less over susceptible.Similarly, Vip3A resistant strain recorded 1.08 U/gut azocaseinolytic activity, which was 2.13 fold less over susceptible strain of S. litura.Our findings are in agreement with the previous workers.Bai et al. (1990) analysed the midgut juices of S. littoralis resistant to Bt which contained only 50% of total protease activity, compared with that in gut juice of susceptible strain.Oppert (1996) also studied the protease activity of midgut extract of HD-198 resistant insect which showed lower proteolytic activity toward several substrates than midgut extracts of susceptible insects.

Tryptic activity
Tryptic activity was 2.26 and 3.35 fold decrease respectively in Cry1C resistant and Vip3A resistant S. litura over susceptible.Similar, results were reported by Bai et al. (1990) who analyzed the midgut juices of S. littoralis resistant to Bt which contained about five times less trypsin activity compared with those in the gut juice of susceptible.Houseman and Chin (1995) also quantified the digestive trypsin proteases in the midgut of Ostrinia nubilalis, resistant to B. thuringiensis.They found that trypsin activity was higher in midgut sap of susceptible 5 th instar larvae as compared to resistant population.Huarong et al. (2004) also found trypsin like protease activity in Bt resistant strain compared to that in which was approximately half of that susceptible strain of Ostrinia nubilalis.

Chymotryptic activity
Chymotryptic activity in midgut of susceptible stain of S. litura was 2.20 fold less over susceptible strain.Similarly, in Vip3A resistant strain, lower chymotryptic activity was recorded than susceptible strain.Bai et al. (1990) analyzed the midgut juices in S. littoralis resistant to B. thuringiensis which contained about six times less chymotryptic activity as compared to susceptible.Huang et al. (1999) also reported the reduced chymotryptic activity in resistant strain of European corn borer as compared to susceptible strain.
The present results indicated that there was less total protease, tryptic and chymotryptic activity in S. litura resistant to Cry1C and Vip3A toxins as compared to susceptible strain.These results suggested that there is protease mediated mechanism of resistance against Cry1C as well as Vip3A toxins.Midgut proteases found to play an important role for the proteolytic processing of protoxin.Protease convert the protoxin into toxin by proteolytic processing, which is essential for binding to brush border membrane vesicle.In the present study, there is no conversion of Cry1C as well as Vip3A protoxin into toxin, which is essential for binding to brush border membrane vesicle.This is one of the mechanism of Bt resistance in S. litura.The other mechanism such as alteration in binding site, reduced binding need to be studied for better understanding of Bt resistance mechanism in insect.

Electrophoretic Protease activity analysis
Variation in protease activity profile of Cry1C resistant, Vip3A resistant and susceptible strains of S. litura were observed.In Vip3A resistant strain only three protease isoforms were observed, and in Cry1C resistant strain two protease isoforms detected whereas in susceptible strain five isoforms were detected.Similarly Chandrashekar and Gujar (2003) also studied the mechanism of resistance to Bt endotoxin Cry1Ac in H. armigera.Midgut proteases of H armigera were subjected to SDS-PAGE analysis.It was further observed that six midgut proteases, 2 major bands with molecular weights of approximately 71.6 and 31.6 kDa and 4 minor bands of 44.6, 40.5, 35.7, 29.7 kDa visible in susceptible and resistant populations of H. armigera, while only one protease band was visible with Cry1Ac protoxin, suggesting the possible role of protease in development of resistance to Cry1Ac in H. armigera.Huarong et al. (2004) who studied the comparative analysis of proteases activities of B.thuringiensis resistant and susceptible Ostrinia nubilalis (Lepidoptera) using casein as a substrate.Further zymogram analysis indicates six proteases activities (C1 to C6) in soluble extracts of susceptible larvae.Activities C5 and C6 were the most prominent in susceptible larvae.However, isozyme C6 was not detected in soluble extracts of resistant larvae.The relative intensities of all proteases in the resistant strains were weaker than the corresponding activities in the susceptible strain.

Table 1 :
Azocaseinolytic activity in Spodoptera litura midgut using azocasein as a substrate

Table 2 :
Tryptic activity in Spodoptera litura midgut using BApNA as substrate

Table 3 :
Chymotryptic activity in Spodoptera litura midgut using SAAPLpNA as a substrate