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tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin
YPEST-03861; No of Pages 5 Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
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Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin Mariam Dammak a,⁎, Saoussen Ben Khedher a, Hanen Boukedi a, Ikbel Chaib b, Asma Laarif b, Slim Tounsi a a b
Team of Biopesticides (LPIP), Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3018 Sfax, Tunisia Unit of Entomology (UR13A-GR09), Regional Research Center on Horticulture and Organic Agriculture, University of Sousse, Chott-Mariem, 4042, Tunisia
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Article history: Received 14 March 2015 Received in revised form 13 September 2015 Accepted 15 September 2015 Available online xxxx Keywords: Cry1Aa Lepidopteran larvae Toxicity Activation Binding Histopathological effect
a b s t r a c t Bacillus thuringiensis (Bt) Cry1A toxins are known for their effectiveness against lepidopteran insects. In this study, the entomopathogenic activity of Cry1Aa was investigated against two lepidopteran larvae causing serious threat to various crops, Spodoptera littoralis and Tuta absoluta. Contrarily to S. littoralis, which showed low susceptibility to Cry1Aa (40% mortality with 1 μg/cm2), T. absoluta was very sensitive to this delta-endotoxin (LC50 of 95.8 ng/cm2). The different steps in the mode of action of this toxin on the two larvae were studied with the aim to understand the origin of their difference of susceptibility. Activation of the 130 kDa Cry1Aa protein by T. absoluta larvae juice generated a 65 kDa active toxin, whereas S. littoralis gut juice led to a complete degradation of the protoxin. The study of the interaction of the brush border membrane vesicles (BBMV) with purified biotinylated Cry1Aa toxin revealed six and seven toxin binding proteins in T. absoluta and S. littoralis BBMV, respectively. Midgut histopathology of Cry1Aa fed larvae demonstrated approximately similar damage caused by the toxin in the two larvae midguts. These results suggest that the activation step was strongly involved in the difference of susceptibility of the two larvae to Cry1Aa. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis produces during its sporulation phase of growth insecticidal proteins which are accumulated into regular crystal inclusions. The insecticidal Cry proteins are critical components of Bt biopesticides which are widely used as bioinsecticides since they are specific to target insects and are generally considered safe for mammals and birds. In spite of their different insect specificity (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera and Mallophaga orders) [1], Cry proteins share similar structure and mode of action. Three-dimensional resolved structures of some Cry toxins showed that the majority of B. thuringiensis toxins are composed of three domains [2] despite their different structures. The primary action of B. thuringiensis toxins is to lyse midgut epithelial cells by inserting into the target membrane and forming pores [3,4]. But to become active, crystal inclusions are dissolved in the alkaline environment of the gut, and the solubilized protoxins are cleaved by midgut proteases yielding 60–70 kDa protease resistant proteins [5]. This processing involves the removal of 600 amino acids corresponding to the C-terminal end for large Cry proteins and a peptide from the N-terminal end (20–60 amino acids). Solubilization and activation of Cry proteins are followed by another important step determining the specificity to insects. In fact the activated toxin interacts with specific membrane receptors on the ⁎ Corresponding author. E-mail address: [email protected] (M. Dammak).
apical membrane of midgut cells [6]. This is the most significant event since subsequent to binding to receptors, pre-pore oligomeric structure formation is initialized followed by insertion into the apical membrane and leading to epithelial cells lysis [7]. Toxicity of Cry proteins to lepidoptera was largely investigated and several species showed different susceptibilities to the same toxin [8]. Some factors such as variability of midgut proteases [9,10] and variability in cell membrane receptors have been proposed to account for this toxin sensitivity. In the present paper, we studied the susceptibility of two lepidopteran species, the tomato leaf miner Tuta absoluta (Gelechiidae family) and the Egyptian cotton leaf worm Spodoptera littoralis (Noctuidae family), towards Cry1Aa11 that differs from the prototype Cry1Aa1 in two amino acids [11]. We investigated also the involvement of the different steps of the mode of action of this delta-endotoxin in the difference of susceptibility of the two studied insects. 2. Materials and methods: 2.1. Preparation of Cry1Aa protoxins Recombinant cells DH5α(pBScry1Aa) of E. coli transformed with plasmid pBScry1Aa11 [11] were grown in LB medium supplemented with ampicillin (60 μg/ml) at 37 °C in a shaking incubator (200 rpm) for 16 h. Pre-culture was used to inoculate 200 ml of LB media containing ampicillin (60 μg/ml). Cultures were agitated until the OD600
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Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
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M. Dammak et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
reached 0.6. Cry protein expression was then induced by the addition of IPTG at a final concentration of 0.4 mM. The culture was maintained at 37 °C for four additional hours with agitation (200 rpm). Protein expression was verified by observing inclusion bodies by light microscopy. After harvesting by centrifugation, cell pellets were washed with cool water, pelleted by centrifugation, and frozen at − 20 °C. Then the cell pellet was suspended in PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4(7H2O), 1.8 mM KH2PO4) and sonicated as described by Dammak et al. [12]. Inclusion bodies were harvested by centrifugation for 10 min at 6000 rpm, and washed twice with 1 M NaCl cold solution, then four times with cold distilled water. Inclusion bodies containing Cry1Aa δ-endotoxins were solubilized in 50 mM sodium carbonate buffer (pH 9.5).
2.5. Toxin purification Solubilized protoxins were digested with trypsin at a trypsin/ protoxin ratio 1:20 (w/w) at 37 °C for 2 h with agitation then dialyzed against Na2CO3 20 mM, pH 9.6 and concentrated with PEG (Poly Ethylene Glycol). Activated toxins were purified by FPLC (Fast Protein Liquid Chromatography) using a Mono Q anion exchange column equilibrated with 20 mM Na2CO3, pH 9.6. Toxin was eluted with a linear gradient of 1 M NaCl in 20 mM Na2CO3, pH 9.6. Eluted fractions containing the active toxin were pooled and separated by electrophoresis in 10% polyacrylamide gel and purified Cry1Aa toxin was used for binding assays.
2.6. Cry1Aa labeling 2.2. Insects The T. absoluta larvae used in the bioassays were reared, in the laboratory of Entomology (CRRHAB), on tomato plants inside insect proof cages and maintained under controlled environment conditions (28 °C, 75 ± 5% relative humidity and 12 h photoperiod). S. littoralis larvae were reared as described by BenFarhat-Touzri et al. [13] on an artificial semi-solid diet [14], in climatic room at 23 °C, 65% relative humidity and with 16:8 light/dark photoperiods.
2.3. Bioassays Bioassays were carried out using washed Cry1Aa inclusion bodies. Proteins concentrations were estimated using Bradford method [15] with bovine serum albumin (BSA, Amersham) as a protein standard. Bioassays were carried out using first instars larvae of S. littoralis. Ten larvae were transferred to sterile petri dishes containing a 1 cm3 of artificial diet impregnated with inclusions of Cry proteins at desired concentrations. The plates were incubated for 6 days in the insect culture room under controlled conditions of temperature 23 °C, relative humidity of 65% and a photoperiod of 18 h light and 6 h dark. For T. absoluta bioassay, first instar larvae were used. Tomato leaves were immersed in different Cry1Aa inclusions concentrations. The leaves were subsequently placed in Petri dishes. Ten larvae were placed in each petri dish, which was placed at 25 ± 0.5 °C, 75 ± 5% relative humidity and 12 h photoperiod. Mortality was recorded after 5 days. A control set devoid of the inclusions but containing the buffer solution was maintained in the same conditions of each test and used as negative control. Experiments were replicated three times. Larval mortality was scored during 6 days. Fifty percent lethal concentrations (LC50) were calculated by probit analysis using programs written in the R. language [16].
2.4. Gut juice preparation and proteolysis assays Tuta absoluta larvae were chilled on ice during 30 min. Then, in each 1.5 ml eppendorf tube, 10 whole larvae were collected in 100 μl MET buffer (300 mM Mannitol, 5 mM EDTA, 20 mM Tris pH 7.2) as described by Dammak et al. [12]. The gut juice of S. littoralis is collected by regurgitation induced by applying an electric current at 20–30 V to the larvae. After centrifugation at 13,000 g for 10 min at 4 °C, the supernatant was recovered. Protein concentration in the gut extracts was determined by the method of Bradford [15]. Solubilized inclusion bodies (50 μg) were mixed with soluble proteins in T. absoluta larvae extracts, S. littoralis gut extract, or with bovine pancreas trypsin (Amersham Pharmacia Biotech, France) in a final volume of 50 μl. The mixtures were incubated at 30 °C for 5–180 min. Samples were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS–PAGE) and stained with Coomassie blue dye.
Activated pure toxin was diluted in bicarbonate buffer (40 mM, pH 8.6) in order to obtain a final concentration of about 1 mg/ml. Then, 40 μl of biotinylation substrate (ECL™ protein biotinylation module: Amersham Pharmacia Biotech, France) were added and the mixture was incubated at room temperature with constant agitation for 1 h. Purification of the biotinylated toxin was performed by loading the mixture on G25 column and elution using PBS 1 × (pH 7.5). Protein concentration was estimated by measuring the optical density of the eluted fractions at 280 nm.
2.7. BBMV collection and preparation BBMVs were extracted according to the method described by Wolfersberger et al. [17]. Intestines were dissected from last-instar S. littoralis and T. absoluta larvae, washed in cold MET buffer (250 mM Mannitol, 17 mM Tris–HCl and 5 mM EGTA pH 7.5), frozen in liquid nitrogen and stored at −80 °C. One g of larval midgut was homogenized in MET buffer by a potter then the homogenate was diluted with an equal volume of ice-cold 24 mM MgCl2. A low speed centrifugation (4500 rpm for 15 min at 4 °C) was applied and the supernatant from the initial centrifugation was further centrifuged at 13,000 rpm for 45 min at 4 °C then the resulting pellet was suspended in the MET buffer, diluted with an equal volume of MgCl2 and centrifuged again at 4500 rpm for 15 min at 4 °C. The supernatant was recovered and also centrifuged at 13 000 rpm for 45 min at 4 °C. The resulting pellet (corresponding to the BBMVs preparation) was suspended in MET buffer (0.5×), flash-frozen in liquid nitrogen and stored at −80 °C.
2.8. BBMV ligand-blotting assay Ligand-blotting was performed in accordance with the procedures reported by Abdelkefi-Mesrati et al. [18]. BBMV (40 μg) prepared from S. littoralis and T. absoluta were separated in SDS–PAGE and blotted onto a nitrocellulose membrane by electrotransfer (Bio-Rad, France). The membranes were blocked with 5% milk for 1 h then reacted with trypsinized and biotinylated Cry1Aa (40 nM) for 2 h at room temperature. Membranes were then, incubated for 1 h with peroxidase (HRP)conjugated streptavidin (1:1500 dilution) supplied in ECL protein biotinylation module. Binding was visualized using luminol according to manufacturer's protocol (ECL; Amersham Pharmacia Biotech, France).
2.9. Preparation and sectioning of insect tissues After exposure to the B. thuringiensis Cry1Aa toxin, killed larvae were placed in 10% formol then dehydrated in increasing ethanol concentrations, rinsed in 100% toluene, and embedded in paraffin wax. Sectioning of larvae tissues and preparation of slides were accomplished as described by Abdelkafi-Mesrati et al. [18].
Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
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First instar larvae of T. absoluta showed a high mortality in toxin treatment. The LC50 after 72 h exposure to Cry1Aa inclusions was 95.8 ng/cm2 with 95% confidence limits of (62.98–128.62). First instar larvae of S. littoralis were also sensitive to Cry1Aa inclusions but showed lower mortality comparing to T. absoluta. After 72 h of toxin treatment only 40% of S. littoralis larvae died in response to the higher tested concentration of Cry1Aa (1 μg/cm2). However, surviving larvae from the toxin treatment showed an inhibition of growth proportional to administrated concentrations. So, Cry1Aa was over ten times more toxic to T. absoluta than to S. littoralis. Exposing the larvae from this species to the negative controls did not cause death or growth inhibition. The potential of B. thuringiensis insecticidal proteins in controlling T. absoluta was not well studied, only some toxins were studied such as Vip3Aa16 (LC50 = 335 ± 17 ng/cm2) [19] and hole delta-endotoxin crystals of HD1 (LC50 of 1.7 μg/cm2) [20]. Compared with these results Cry1Aa is highly toxic to T. absoluta larvae. So, we consider that this toxin could be a good candidate to the construction of transgenic plants resistant to T. absoluta. Against S. littoralis, Vip3Aa16 causes mortality with an LC50 of about 305 ng/cm2 [18], Cry1Da showed toxicity with an LC50 of 224.4 ng/cm2 [13] and against the same larvae, the LC50 of Cry1C is 93 ng/cm2 [21]. Hence, we could confirm the low susceptibility of S. littoralis to Cry1Aa. This difference in the efficacy of Cry1Aa against the two lepidopteran strains could be attributed to the activation step, due to the variability of proteases in the two gut juices, and/or to the binding step, due to the presence or absence of specific midgut receptors.
Cry1Aa is completely converted into a major band of expected size 65 kDa corresponding to the active toxin. This band coincides with the 65 kDa toxin obtained by activation with trypsin (Fig. 1, lane 15). While digestion of Cry1Aa by T. absoluta intestinal proteases yields a resistant active protein, proteolysis by S. littoralis proteases leads to complete degradation of the protoxin since the early minutes of incubation. Hence, Cry1Aa seems to be very sensitive to the type of S. littoralis intestinal proteases or to their high concentrations. Using a protease:toxin ratio of 1:100, the degradation of Cry1Aa was attenuated. The 130 kDa protein is converted into a major band of 65 kDa after 20 min of incubation with S. littoralis proteases (Fig. 2). This active form disappears progressively during the time course processing. With a mass ratio of 1:1000, proteolyses of Cry1Aa yields a 65 kDa active form stable until 3 h of incubation. These results confirm the sensitivity of Cry1Aa to relatively high concentrations of intestinal proteases of S. littoralis. So, a similar processing in vivo in the midgut of insects could explain the low sensitivity of S. littoralis to Cry1Aa toxins. Our results correlate in part with those of Keller et al. [25], who reported that complete degradation of Cry1C with late instars intestinal extracts is associated with the reduced susceptibility of fifth instar S. littoralis larvae to the toxin. In another example, the low toxicity of Cry1Ab toxin to S. frugiperda could be attributed in part to a rapid degradation of the toxin on the midgut of insect [22]. Loseva et al. [26] reported, in another hand, that an increased expression of midgut proteases could explain the reduced susceptibility of Colorado potato beetle to Cry3Aa toxin, although enhanced degradation of this toxin by midgut protease extracts was not found. Our finding, suggests that an over expression of midgut proteases leads to toxin degradation while in vitro incubation of Cry1Aa with low concentrations of proteases yields attenuation of toxin degradation.
3.2. In vitro Cry1Aa protoxin activation
3.3. Binding of Cry1Aa to proteins in T. absoluta and S. littoralis BBMV
Proteolytic processing of Cry protoxins is a critical step involved not only on toxin activation but also on specificity [22] and insect resistance [23,24]. In order to study the processing of Cry1Aa, this protein was incubated with intestinal larvae juice using a larvae juice: protoxin ratio of 1:20 and proteolysis by trypsin was used as control. Using T. absoluta larvae juice, the 130 kDa Cry1Aa protoxin is converted into intermediate proteolysis products with molecular weights between approximately 80 and 65 kDa (Fig. 1). After 3 h incubation,
Receptor identification is essential because interaction with specific receptors on the insect gut is the major determinant of toxin selectivity. To identify putative receptors of Cry1Aa toxin on BBMV proteins, binding experiments were conducted with biotinylated-Cry1Aa and isolated gut BBMV. Our results showed that trypsinated, purified and biotinylated Cry1Aa toxin binds to 6 proteins in the BBMV of T. absoluta: 150, 80, 65, 44, 30 and 20 kDa. However, on the BBMV of S. littoralis, it binds strongly to 3 proteins of 126, 90 and 65 kDa and weakly to 4 other ones of 46, 40, 33 and 20 kDa (Fig. 3). A BSA blot, used as negative control, showed no interaction with Cry1Aa toxin of B. thuringiensis.
3. Results and discussion 3.1. Bioassays
Fig. 1. In vitro activation of Cry1Aa by T. absoluta and S. littoralis gut proteases. Gut proteases:protoxin ratio used is 1:20. Processed samples at the indicated times were subjected to SDS-10%-PAGE. Lane 1, Cry1Aa protoxin; lanes 2–7, In vitro time course processing of Cry1Aa by S. littoralis gut extract for respectively: 5 min, 20 min, 30 min, 1 h, 2 h and 3 h; Lane 8, molecular weight markers (LMW: phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), α-lactalbumin (14.4 kDa); Amersham, France); lanes 9–14, In vitro time course processing of Cry1Aa by T. absoluta larvae juice for respectively: 5 min, 20 min, 30 min, 1 h, 2 h and 3 h; lane 15, In vitro processing of Cry1Aa by trypsin after 3 h (Protease:protoxin ratio used is 1:20).
Fig. 2. Effect of the decrease of S. littoralis larvae juice extract concentration on the in vitro proteolysis of Cry1Aa protoxin. Lane 1, Cry1Aa toxin; lanes 2–7, time course processing of Cry1Aa using a larvae juice:toxin ratio 1:100 for respectively 5 min, 20 min, 30 min, 1 h, 2 h and 3 h; lanes 8–14, time course processing of Cry1Aa using a larvae juice:toxin ratio 1:1000 for respectively 5 min, 20 min, 30 min, 1 h, 2 h and 3 h; lane 15, molecular weight markers (HMW, Amersham, France).
Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
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Fig. 3. Cry1Aa binding to T. absoluta and S. littoralis gut BBMVs. Lane 1, T. absoluta BBMV ligand blotting with biotinylated Cry1Aa toxin; lane 2, containing bovine serum albumin (BSA) used as negative control; lane 3, S. littoralis BBMV ligand blotting with biotinylated Cry1Aa toxin.
The binding of Cry1Aa to multiple proteins on a blot of BBMV from S. littoralis, reinforces the idea that the low susceptibility of this insect to Cry1Aa is not due to lack of receptors, but possibly to some other factors such as protoxin processing or instability of toxin in the lumen environment.
In other studies, the putative midgut receptors described in S. littoralis are 55 kDa, 100 kDa and 65 kDa for Vip3Aa [10,27], 65 kDa for Cry1Da [13], 40 kDa for Cry1C [28,29], 65 kDa for Cry1Ia [27] and 120 kDa for Cry1Ac [30].This large variety of recognized midgut binding proteins in lepidopteran larvae strongly suggests that there is a difference in receptors specificity to these toxins. We noticed that 120, 65 and 40 kDa S. littoralis toxin binding proteins are identified in our ligand blot. In similar studies, Yaoi et al. [31, 32] identified a 120 kDa protein as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN) in the BBMV of B. mori specific to Cry1Aa toxin; they also reported that this toxin is bound faintly to 220 kDa, 180 kDa, 55 kDa and 30 kDa proteins from BBMV of the same larvae. Forever, other specific receptors for Cry1Aa were described in different species such as a 210 kDa cadherin-like glycoprotein in L. dispar and in M. sexta [33,34] and a 205 kDa protein in O. nubilalis [35]. So specific BBMV binding proteins to Cry1Aa differ from specie to other. This explains the differences in protein sizes identified within the two blots of S. littoralis and T. absoluta, whereas they share two binding proteins of the same size of 65 and 20 kDa. The nature of these binding proteins was not identified, whereas the most identified receptors specific to Cry1A toxins identified in the midguts of susceptible insect larvae from the Lepidoptera order belong to cadherin, aminopeptidase N and alcaline phosphatase families. In fact, Bravo et al. [36] presented two models explaining binding of Cry1A toxins to specific receptors; the toxin binds a cadherin-like receptor and forms oligomers that are believed to insert into the cell membrane after binding glycosyl-phosphatidylinositol anchored receptors such as aminopeptidase N (APN) and alkaline phosphatase (ALP). The smaller bands observed in the two blots could be degradation products resulting from digestion of larger proteins by endogenous proteases in the BBMV suspension as reported by Yaoi et al. [33].
Fig. 4. Histopathological effects of Cry1Aa on T. absoluta and S. littoralis midguts. (A) and (D) Sections through the midgut epithelium of control larvae not exposed to toxins of respectively T. absoluta and S. littoralis. (B) and (C) Sections through the midgut epithelium of T. absoluta larvae fed with Cry1Aa-containing diet. (E) and (F) Sections through the midgut epithelium of S. littoralis larvae fed with Cry1Aa-containing diet. (B) and (E) First changes in midgut epithelium. (C) and (F) Extensive damage of the midgut epithelium. LU, lumen; AM, apical membrane; BM, basal membrane; V, vacuole formation; L, lysis of columnar cells; Black arrow shows vesicle formation and white arrow indicates detachment of epithelial cells from basal membrane. (A), (B) and (C) Magnification: 40×; (D), (E) and (F) Magnification: 100×.
Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
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3.4. Histopathological effects of Cry1Aa toxin on T. absoluta and S. littoralis larvae Both non treated S. littoralis and T. absoluta control midguts showed preserved layer of epithelial cells with regular placed microvilli bordering the midgut lumen (Fig. 4A and Fig. 4D). Structural changes were observed in the midgut of T. absoluta when larvae were exposed to Cry1Aa toxin. First changes include hypertrophy of the epithelial cells, degeneration of brush border membrane microvilli, vacuolisation of the cytoplasm, vesicles formation and the beginning of the degradation of the peritrophic membrane (Fig. 4B). Then an extensive damage of the epithelium midgut occurred with brush border membrane lysis and disintegration of the apical membrane (Fig. 4C). Similar changes were observed in intoxication of T. absoluta with HD1 δ-endotoxins [20]. Histopathological observations of the effects of Cry1Aa δ-endotoxin on S. littoralis midgut showed also irregularly structured brush border membrane with beginning of epithelial cells detachment from the basement membrane and lysis of columnar cells (Fig. 4E and Fig. 4F). When comparing the two treatments, it can be seen that T. absoluta is more sensitive to Cry1A, because vacuolisation of the cytoplasm and vesicles formation in the apical region were more intensive in T. absoluta midgut than in S. littoralis one. Moreover, almost all epithelial cells were damaged in the midgut of treated T. absoluta larvae whereas only some columnar cells were affected in S. littoralis larvae. These observations could confirm our first hypothesis, in fact, the low susceptibility of S. littoralis to Cry1Aa is due to toxin degradation by midgut proteases. When using high concentrations of Cry1Aa some toxins overcome, in some way, this degradation and could be bound to specific receptors and lead to cell lysis. The present report shows that treatment of tomato crops with B. thuringiensis Cry1Aa toxin could offer an effective control of T. absoluta larvae. It also clearly demonstrates that proteolysis of this toxin in the larvae midgut could be a key step determining their potency against different susceptible pests. Further studies could be conducted to analyze the ability of Cry1Aa to oligomerize and to forming pores in the BBMV of S. littoralis. Acknowledgement This work is financially supported by the “Tunisian Ministry of Higher Education and Scientific Research”. References [1] E. Schnepf, N. Crickmore, J. van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler, D.H. Dean, Bacillus thuringiensis and its pesticidal crystal proteins, Microbiol. Mol. Biol. Rev. 62 (1998) 772–806. [2] M. Adang, N. Crickmore, J.L. Jurat-Fuentes, Diversity of Bacillus thuringiensis crystal toxins and mechanism of action. Insect Midgut and Insecticidal Proteins, Adv. Insect Physiol. 47 (2014) 39–87. [3] A.I. Aronson, Y. Shai, Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action, FEMS Microbiol. Lett. 195 (2001) 1–8. [4] A. Bravo, S.S. Gill, M. Soberón, Mode of action of Bacillus thuringiensis cry and Cyt toxins and their potential for insect control, Toxicon 49 (2007) 423–435. [5] A. Bravo, S.S. Gill, M. Soberón, Comprehensive Molecular Insect Science, Bacillus thuringiensis Mechanisms and Use, Elsevier BV, 2005 175–206. [6] C. Hofmann, H. Vanderbruggen, H. Hofte, H. Van Mellaert, Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high affinity binding sites in the brushborder membrane of the insect membrane of target insect midguts, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 7844–7848. [7] I. Gómez, J. Sánchez, R. Miranda, A. Bravo, M. Soberón, Cadherin-like receptor binding facilitates proteolytic cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin, FEBS Lett. 513 (2002) 242–246. [8] H. Höfte, H.R. Whiteley, Insecticidal crystal proteins of Bacilllus thuringiensis, Microbiol. Rev. 53 (1989) 242–255. [9] B. Oppert, Protease interactions with Bacillus thuringiensis insecticidal toxins, Arch. Biochem. Physiol. 42 (1999) 1–12. [10] L. Abdelkefi-Mesrati, H. Boukedi, M. Chakroun, F. Kamoun, H. Azzouz, S. Tounsi, S. Rouis, S. Jaoua, Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin, J. Invertebr. Pathol. 107 (2011) 198–201.
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Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
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Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin Mariam Dammak a,⁎, Saoussen Ben Khedher a, Hanen Boukedi a, Ikbel Chaib b, Asma Laarif b, Slim Tounsi a a b
Team of Biopesticides (LPIP), Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3018 Sfax, Tunisia Unit of Entomology (UR13A-GR09), Regional Research Center on Horticulture and Organic Agriculture, University of Sousse, Chott-Mariem, 4042, Tunisia
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Article history: Received 14 March 2015 Received in revised form 13 September 2015 Accepted 15 September 2015 Available online xxxx Keywords: Cry1Aa Lepidopteran larvae Toxicity Activation Binding Histopathological effect
a b s t r a c t Bacillus thuringiensis (Bt) Cry1A toxins are known for their effectiveness against lepidopteran insects. In this study, the entomopathogenic activity of Cry1Aa was investigated against two lepidopteran larvae causing serious threat to various crops, Spodoptera littoralis and Tuta absoluta. Contrarily to S. littoralis, which showed low susceptibility to Cry1Aa (40% mortality with 1 μg/cm2), T. absoluta was very sensitive to this delta-endotoxin (LC50 of 95.8 ng/cm2). The different steps in the mode of action of this toxin on the two larvae were studied with the aim to understand the origin of their difference of susceptibility. Activation of the 130 kDa Cry1Aa protein by T. absoluta larvae juice generated a 65 kDa active toxin, whereas S. littoralis gut juice led to a complete degradation of the protoxin. The study of the interaction of the brush border membrane vesicles (BBMV) with purified biotinylated Cry1Aa toxin revealed six and seven toxin binding proteins in T. absoluta and S. littoralis BBMV, respectively. Midgut histopathology of Cry1Aa fed larvae demonstrated approximately similar damage caused by the toxin in the two larvae midguts. These results suggest that the activation step was strongly involved in the difference of susceptibility of the two larvae to Cry1Aa. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis produces during its sporulation phase of growth insecticidal proteins which are accumulated into regular crystal inclusions. The insecticidal Cry proteins are critical components of Bt biopesticides which are widely used as bioinsecticides since they are specific to target insects and are generally considered safe for mammals and birds. In spite of their different insect specificity (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera and Mallophaga orders) [1], Cry proteins share similar structure and mode of action. Three-dimensional resolved structures of some Cry toxins showed that the majority of B. thuringiensis toxins are composed of three domains [2] despite their different structures. The primary action of B. thuringiensis toxins is to lyse midgut epithelial cells by inserting into the target membrane and forming pores [3,4]. But to become active, crystal inclusions are dissolved in the alkaline environment of the gut, and the solubilized protoxins are cleaved by midgut proteases yielding 60–70 kDa protease resistant proteins [5]. This processing involves the removal of 600 amino acids corresponding to the C-terminal end for large Cry proteins and a peptide from the N-terminal end (20–60 amino acids). Solubilization and activation of Cry proteins are followed by another important step determining the specificity to insects. In fact the activated toxin interacts with specific membrane receptors on the ⁎ Corresponding author. E-mail address: [email protected] (M. Dammak).
apical membrane of midgut cells [6]. This is the most significant event since subsequent to binding to receptors, pre-pore oligomeric structure formation is initialized followed by insertion into the apical membrane and leading to epithelial cells lysis [7]. Toxicity of Cry proteins to lepidoptera was largely investigated and several species showed different susceptibilities to the same toxin [8]. Some factors such as variability of midgut proteases [9,10] and variability in cell membrane receptors have been proposed to account for this toxin sensitivity. In the present paper, we studied the susceptibility of two lepidopteran species, the tomato leaf miner Tuta absoluta (Gelechiidae family) and the Egyptian cotton leaf worm Spodoptera littoralis (Noctuidae family), towards Cry1Aa11 that differs from the prototype Cry1Aa1 in two amino acids [11]. We investigated also the involvement of the different steps of the mode of action of this delta-endotoxin in the difference of susceptibility of the two studied insects. 2. Materials and methods: 2.1. Preparation of Cry1Aa protoxins Recombinant cells DH5α(pBScry1Aa) of E. coli transformed with plasmid pBScry1Aa11 [11] were grown in LB medium supplemented with ampicillin (60 μg/ml) at 37 °C in a shaking incubator (200 rpm) for 16 h. Pre-culture was used to inoculate 200 ml of LB media containing ampicillin (60 μg/ml). Cultures were agitated until the OD600
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Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
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reached 0.6. Cry protein expression was then induced by the addition of IPTG at a final concentration of 0.4 mM. The culture was maintained at 37 °C for four additional hours with agitation (200 rpm). Protein expression was verified by observing inclusion bodies by light microscopy. After harvesting by centrifugation, cell pellets were washed with cool water, pelleted by centrifugation, and frozen at − 20 °C. Then the cell pellet was suspended in PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4(7H2O), 1.8 mM KH2PO4) and sonicated as described by Dammak et al. [12]. Inclusion bodies were harvested by centrifugation for 10 min at 6000 rpm, and washed twice with 1 M NaCl cold solution, then four times with cold distilled water. Inclusion bodies containing Cry1Aa δ-endotoxins were solubilized in 50 mM sodium carbonate buffer (pH 9.5).
2.5. Toxin purification Solubilized protoxins were digested with trypsin at a trypsin/ protoxin ratio 1:20 (w/w) at 37 °C for 2 h with agitation then dialyzed against Na2CO3 20 mM, pH 9.6 and concentrated with PEG (Poly Ethylene Glycol). Activated toxins were purified by FPLC (Fast Protein Liquid Chromatography) using a Mono Q anion exchange column equilibrated with 20 mM Na2CO3, pH 9.6. Toxin was eluted with a linear gradient of 1 M NaCl in 20 mM Na2CO3, pH 9.6. Eluted fractions containing the active toxin were pooled and separated by electrophoresis in 10% polyacrylamide gel and purified Cry1Aa toxin was used for binding assays.
2.6. Cry1Aa labeling 2.2. Insects The T. absoluta larvae used in the bioassays were reared, in the laboratory of Entomology (CRRHAB), on tomato plants inside insect proof cages and maintained under controlled environment conditions (28 °C, 75 ± 5% relative humidity and 12 h photoperiod). S. littoralis larvae were reared as described by BenFarhat-Touzri et al. [13] on an artificial semi-solid diet [14], in climatic room at 23 °C, 65% relative humidity and with 16:8 light/dark photoperiods.
2.3. Bioassays Bioassays were carried out using washed Cry1Aa inclusion bodies. Proteins concentrations were estimated using Bradford method [15] with bovine serum albumin (BSA, Amersham) as a protein standard. Bioassays were carried out using first instars larvae of S. littoralis. Ten larvae were transferred to sterile petri dishes containing a 1 cm3 of artificial diet impregnated with inclusions of Cry proteins at desired concentrations. The plates were incubated for 6 days in the insect culture room under controlled conditions of temperature 23 °C, relative humidity of 65% and a photoperiod of 18 h light and 6 h dark. For T. absoluta bioassay, first instar larvae were used. Tomato leaves were immersed in different Cry1Aa inclusions concentrations. The leaves were subsequently placed in Petri dishes. Ten larvae were placed in each petri dish, which was placed at 25 ± 0.5 °C, 75 ± 5% relative humidity and 12 h photoperiod. Mortality was recorded after 5 days. A control set devoid of the inclusions but containing the buffer solution was maintained in the same conditions of each test and used as negative control. Experiments were replicated three times. Larval mortality was scored during 6 days. Fifty percent lethal concentrations (LC50) were calculated by probit analysis using programs written in the R. language [16].
2.4. Gut juice preparation and proteolysis assays Tuta absoluta larvae were chilled on ice during 30 min. Then, in each 1.5 ml eppendorf tube, 10 whole larvae were collected in 100 μl MET buffer (300 mM Mannitol, 5 mM EDTA, 20 mM Tris pH 7.2) as described by Dammak et al. [12]. The gut juice of S. littoralis is collected by regurgitation induced by applying an electric current at 20–30 V to the larvae. After centrifugation at 13,000 g for 10 min at 4 °C, the supernatant was recovered. Protein concentration in the gut extracts was determined by the method of Bradford [15]. Solubilized inclusion bodies (50 μg) were mixed with soluble proteins in T. absoluta larvae extracts, S. littoralis gut extract, or with bovine pancreas trypsin (Amersham Pharmacia Biotech, France) in a final volume of 50 μl. The mixtures were incubated at 30 °C for 5–180 min. Samples were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS–PAGE) and stained with Coomassie blue dye.
Activated pure toxin was diluted in bicarbonate buffer (40 mM, pH 8.6) in order to obtain a final concentration of about 1 mg/ml. Then, 40 μl of biotinylation substrate (ECL™ protein biotinylation module: Amersham Pharmacia Biotech, France) were added and the mixture was incubated at room temperature with constant agitation for 1 h. Purification of the biotinylated toxin was performed by loading the mixture on G25 column and elution using PBS 1 × (pH 7.5). Protein concentration was estimated by measuring the optical density of the eluted fractions at 280 nm.
2.7. BBMV collection and preparation BBMVs were extracted according to the method described by Wolfersberger et al. [17]. Intestines were dissected from last-instar S. littoralis and T. absoluta larvae, washed in cold MET buffer (250 mM Mannitol, 17 mM Tris–HCl and 5 mM EGTA pH 7.5), frozen in liquid nitrogen and stored at −80 °C. One g of larval midgut was homogenized in MET buffer by a potter then the homogenate was diluted with an equal volume of ice-cold 24 mM MgCl2. A low speed centrifugation (4500 rpm for 15 min at 4 °C) was applied and the supernatant from the initial centrifugation was further centrifuged at 13,000 rpm for 45 min at 4 °C then the resulting pellet was suspended in the MET buffer, diluted with an equal volume of MgCl2 and centrifuged again at 4500 rpm for 15 min at 4 °C. The supernatant was recovered and also centrifuged at 13 000 rpm for 45 min at 4 °C. The resulting pellet (corresponding to the BBMVs preparation) was suspended in MET buffer (0.5×), flash-frozen in liquid nitrogen and stored at −80 °C.
2.8. BBMV ligand-blotting assay Ligand-blotting was performed in accordance with the procedures reported by Abdelkefi-Mesrati et al. [18]. BBMV (40 μg) prepared from S. littoralis and T. absoluta were separated in SDS–PAGE and blotted onto a nitrocellulose membrane by electrotransfer (Bio-Rad, France). The membranes were blocked with 5% milk for 1 h then reacted with trypsinized and biotinylated Cry1Aa (40 nM) for 2 h at room temperature. Membranes were then, incubated for 1 h with peroxidase (HRP)conjugated streptavidin (1:1500 dilution) supplied in ECL protein biotinylation module. Binding was visualized using luminol according to manufacturer's protocol (ECL; Amersham Pharmacia Biotech, France).
2.9. Preparation and sectioning of insect tissues After exposure to the B. thuringiensis Cry1Aa toxin, killed larvae were placed in 10% formol then dehydrated in increasing ethanol concentrations, rinsed in 100% toluene, and embedded in paraffin wax. Sectioning of larvae tissues and preparation of slides were accomplished as described by Abdelkafi-Mesrati et al. [18].
Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
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First instar larvae of T. absoluta showed a high mortality in toxin treatment. The LC50 after 72 h exposure to Cry1Aa inclusions was 95.8 ng/cm2 with 95% confidence limits of (62.98–128.62). First instar larvae of S. littoralis were also sensitive to Cry1Aa inclusions but showed lower mortality comparing to T. absoluta. After 72 h of toxin treatment only 40% of S. littoralis larvae died in response to the higher tested concentration of Cry1Aa (1 μg/cm2). However, surviving larvae from the toxin treatment showed an inhibition of growth proportional to administrated concentrations. So, Cry1Aa was over ten times more toxic to T. absoluta than to S. littoralis. Exposing the larvae from this species to the negative controls did not cause death or growth inhibition. The potential of B. thuringiensis insecticidal proteins in controlling T. absoluta was not well studied, only some toxins were studied such as Vip3Aa16 (LC50 = 335 ± 17 ng/cm2) [19] and hole delta-endotoxin crystals of HD1 (LC50 of 1.7 μg/cm2) [20]. Compared with these results Cry1Aa is highly toxic to T. absoluta larvae. So, we consider that this toxin could be a good candidate to the construction of transgenic plants resistant to T. absoluta. Against S. littoralis, Vip3Aa16 causes mortality with an LC50 of about 305 ng/cm2 [18], Cry1Da showed toxicity with an LC50 of 224.4 ng/cm2 [13] and against the same larvae, the LC50 of Cry1C is 93 ng/cm2 [21]. Hence, we could confirm the low susceptibility of S. littoralis to Cry1Aa. This difference in the efficacy of Cry1Aa against the two lepidopteran strains could be attributed to the activation step, due to the variability of proteases in the two gut juices, and/or to the binding step, due to the presence or absence of specific midgut receptors.
Cry1Aa is completely converted into a major band of expected size 65 kDa corresponding to the active toxin. This band coincides with the 65 kDa toxin obtained by activation with trypsin (Fig. 1, lane 15). While digestion of Cry1Aa by T. absoluta intestinal proteases yields a resistant active protein, proteolysis by S. littoralis proteases leads to complete degradation of the protoxin since the early minutes of incubation. Hence, Cry1Aa seems to be very sensitive to the type of S. littoralis intestinal proteases or to their high concentrations. Using a protease:toxin ratio of 1:100, the degradation of Cry1Aa was attenuated. The 130 kDa protein is converted into a major band of 65 kDa after 20 min of incubation with S. littoralis proteases (Fig. 2). This active form disappears progressively during the time course processing. With a mass ratio of 1:1000, proteolyses of Cry1Aa yields a 65 kDa active form stable until 3 h of incubation. These results confirm the sensitivity of Cry1Aa to relatively high concentrations of intestinal proteases of S. littoralis. So, a similar processing in vivo in the midgut of insects could explain the low sensitivity of S. littoralis to Cry1Aa toxins. Our results correlate in part with those of Keller et al. [25], who reported that complete degradation of Cry1C with late instars intestinal extracts is associated with the reduced susceptibility of fifth instar S. littoralis larvae to the toxin. In another example, the low toxicity of Cry1Ab toxin to S. frugiperda could be attributed in part to a rapid degradation of the toxin on the midgut of insect [22]. Loseva et al. [26] reported, in another hand, that an increased expression of midgut proteases could explain the reduced susceptibility of Colorado potato beetle to Cry3Aa toxin, although enhanced degradation of this toxin by midgut protease extracts was not found. Our finding, suggests that an over expression of midgut proteases leads to toxin degradation while in vitro incubation of Cry1Aa with low concentrations of proteases yields attenuation of toxin degradation.
3.2. In vitro Cry1Aa protoxin activation
3.3. Binding of Cry1Aa to proteins in T. absoluta and S. littoralis BBMV
Proteolytic processing of Cry protoxins is a critical step involved not only on toxin activation but also on specificity [22] and insect resistance [23,24]. In order to study the processing of Cry1Aa, this protein was incubated with intestinal larvae juice using a larvae juice: protoxin ratio of 1:20 and proteolysis by trypsin was used as control. Using T. absoluta larvae juice, the 130 kDa Cry1Aa protoxin is converted into intermediate proteolysis products with molecular weights between approximately 80 and 65 kDa (Fig. 1). After 3 h incubation,
Receptor identification is essential because interaction with specific receptors on the insect gut is the major determinant of toxin selectivity. To identify putative receptors of Cry1Aa toxin on BBMV proteins, binding experiments were conducted with biotinylated-Cry1Aa and isolated gut BBMV. Our results showed that trypsinated, purified and biotinylated Cry1Aa toxin binds to 6 proteins in the BBMV of T. absoluta: 150, 80, 65, 44, 30 and 20 kDa. However, on the BBMV of S. littoralis, it binds strongly to 3 proteins of 126, 90 and 65 kDa and weakly to 4 other ones of 46, 40, 33 and 20 kDa (Fig. 3). A BSA blot, used as negative control, showed no interaction with Cry1Aa toxin of B. thuringiensis.
3. Results and discussion 3.1. Bioassays
Fig. 1. In vitro activation of Cry1Aa by T. absoluta and S. littoralis gut proteases. Gut proteases:protoxin ratio used is 1:20. Processed samples at the indicated times were subjected to SDS-10%-PAGE. Lane 1, Cry1Aa protoxin; lanes 2–7, In vitro time course processing of Cry1Aa by S. littoralis gut extract for respectively: 5 min, 20 min, 30 min, 1 h, 2 h and 3 h; Lane 8, molecular weight markers (LMW: phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), α-lactalbumin (14.4 kDa); Amersham, France); lanes 9–14, In vitro time course processing of Cry1Aa by T. absoluta larvae juice for respectively: 5 min, 20 min, 30 min, 1 h, 2 h and 3 h; lane 15, In vitro processing of Cry1Aa by trypsin after 3 h (Protease:protoxin ratio used is 1:20).
Fig. 2. Effect of the decrease of S. littoralis larvae juice extract concentration on the in vitro proteolysis of Cry1Aa protoxin. Lane 1, Cry1Aa toxin; lanes 2–7, time course processing of Cry1Aa using a larvae juice:toxin ratio 1:100 for respectively 5 min, 20 min, 30 min, 1 h, 2 h and 3 h; lanes 8–14, time course processing of Cry1Aa using a larvae juice:toxin ratio 1:1000 for respectively 5 min, 20 min, 30 min, 1 h, 2 h and 3 h; lane 15, molecular weight markers (HMW, Amersham, France).
Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
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Fig. 3. Cry1Aa binding to T. absoluta and S. littoralis gut BBMVs. Lane 1, T. absoluta BBMV ligand blotting with biotinylated Cry1Aa toxin; lane 2, containing bovine serum albumin (BSA) used as negative control; lane 3, S. littoralis BBMV ligand blotting with biotinylated Cry1Aa toxin.
The binding of Cry1Aa to multiple proteins on a blot of BBMV from S. littoralis, reinforces the idea that the low susceptibility of this insect to Cry1Aa is not due to lack of receptors, but possibly to some other factors such as protoxin processing or instability of toxin in the lumen environment.
In other studies, the putative midgut receptors described in S. littoralis are 55 kDa, 100 kDa and 65 kDa for Vip3Aa [10,27], 65 kDa for Cry1Da [13], 40 kDa for Cry1C [28,29], 65 kDa for Cry1Ia [27] and 120 kDa for Cry1Ac [30].This large variety of recognized midgut binding proteins in lepidopteran larvae strongly suggests that there is a difference in receptors specificity to these toxins. We noticed that 120, 65 and 40 kDa S. littoralis toxin binding proteins are identified in our ligand blot. In similar studies, Yaoi et al. [31, 32] identified a 120 kDa protein as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN) in the BBMV of B. mori specific to Cry1Aa toxin; they also reported that this toxin is bound faintly to 220 kDa, 180 kDa, 55 kDa and 30 kDa proteins from BBMV of the same larvae. Forever, other specific receptors for Cry1Aa were described in different species such as a 210 kDa cadherin-like glycoprotein in L. dispar and in M. sexta [33,34] and a 205 kDa protein in O. nubilalis [35]. So specific BBMV binding proteins to Cry1Aa differ from specie to other. This explains the differences in protein sizes identified within the two blots of S. littoralis and T. absoluta, whereas they share two binding proteins of the same size of 65 and 20 kDa. The nature of these binding proteins was not identified, whereas the most identified receptors specific to Cry1A toxins identified in the midguts of susceptible insect larvae from the Lepidoptera order belong to cadherin, aminopeptidase N and alcaline phosphatase families. In fact, Bravo et al. [36] presented two models explaining binding of Cry1A toxins to specific receptors; the toxin binds a cadherin-like receptor and forms oligomers that are believed to insert into the cell membrane after binding glycosyl-phosphatidylinositol anchored receptors such as aminopeptidase N (APN) and alkaline phosphatase (ALP). The smaller bands observed in the two blots could be degradation products resulting from digestion of larger proteins by endogenous proteases in the BBMV suspension as reported by Yaoi et al. [33].
Fig. 4. Histopathological effects of Cry1Aa on T. absoluta and S. littoralis midguts. (A) and (D) Sections through the midgut epithelium of control larvae not exposed to toxins of respectively T. absoluta and S. littoralis. (B) and (C) Sections through the midgut epithelium of T. absoluta larvae fed with Cry1Aa-containing diet. (E) and (F) Sections through the midgut epithelium of S. littoralis larvae fed with Cry1Aa-containing diet. (B) and (E) First changes in midgut epithelium. (C) and (F) Extensive damage of the midgut epithelium. LU, lumen; AM, apical membrane; BM, basal membrane; V, vacuole formation; L, lysis of columnar cells; Black arrow shows vesicle formation and white arrow indicates detachment of epithelial cells from basal membrane. (A), (B) and (C) Magnification: 40×; (D), (E) and (F) Magnification: 100×.
Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005
M. Dammak et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
3.4. Histopathological effects of Cry1Aa toxin on T. absoluta and S. littoralis larvae Both non treated S. littoralis and T. absoluta control midguts showed preserved layer of epithelial cells with regular placed microvilli bordering the midgut lumen (Fig. 4A and Fig. 4D). Structural changes were observed in the midgut of T. absoluta when larvae were exposed to Cry1Aa toxin. First changes include hypertrophy of the epithelial cells, degeneration of brush border membrane microvilli, vacuolisation of the cytoplasm, vesicles formation and the beginning of the degradation of the peritrophic membrane (Fig. 4B). Then an extensive damage of the epithelium midgut occurred with brush border membrane lysis and disintegration of the apical membrane (Fig. 4C). Similar changes were observed in intoxication of T. absoluta with HD1 δ-endotoxins [20]. Histopathological observations of the effects of Cry1Aa δ-endotoxin on S. littoralis midgut showed also irregularly structured brush border membrane with beginning of epithelial cells detachment from the basement membrane and lysis of columnar cells (Fig. 4E and Fig. 4F). When comparing the two treatments, it can be seen that T. absoluta is more sensitive to Cry1A, because vacuolisation of the cytoplasm and vesicles formation in the apical region were more intensive in T. absoluta midgut than in S. littoralis one. Moreover, almost all epithelial cells were damaged in the midgut of treated T. absoluta larvae whereas only some columnar cells were affected in S. littoralis larvae. These observations could confirm our first hypothesis, in fact, the low susceptibility of S. littoralis to Cry1Aa is due to toxin degradation by midgut proteases. When using high concentrations of Cry1Aa some toxins overcome, in some way, this degradation and could be bound to specific receptors and lead to cell lysis. The present report shows that treatment of tomato crops with B. thuringiensis Cry1Aa toxin could offer an effective control of T. absoluta larvae. It also clearly demonstrates that proteolysis of this toxin in the larvae midgut could be a key step determining their potency against different susceptible pests. Further studies could be conducted to analyze the ability of Cry1Aa to oligomerize and to forming pores in the BBMV of S. littoralis. Acknowledgement This work is financially supported by the “Tunisian Ministry of Higher Education and Scientific Research”. References [1] E. Schnepf, N. Crickmore, J. van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler, D.H. Dean, Bacillus thuringiensis and its pesticidal crystal proteins, Microbiol. Mol. Biol. Rev. 62 (1998) 772–806. [2] M. Adang, N. Crickmore, J.L. Jurat-Fuentes, Diversity of Bacillus thuringiensis crystal toxins and mechanism of action. Insect Midgut and Insecticidal Proteins, Adv. Insect Physiol. 47 (2014) 39–87. [3] A.I. Aronson, Y. Shai, Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action, FEMS Microbiol. Lett. 195 (2001) 1–8. [4] A. Bravo, S.S. Gill, M. Soberón, Mode of action of Bacillus thuringiensis cry and Cyt toxins and their potential for insect control, Toxicon 49 (2007) 423–435. [5] A. Bravo, S.S. Gill, M. Soberón, Comprehensive Molecular Insect Science, Bacillus thuringiensis Mechanisms and Use, Elsevier BV, 2005 175–206. [6] C. Hofmann, H. Vanderbruggen, H. Hofte, H. Van Mellaert, Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high affinity binding sites in the brushborder membrane of the insect membrane of target insect midguts, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 7844–7848. [7] I. Gómez, J. Sánchez, R. Miranda, A. Bravo, M. Soberón, Cadherin-like receptor binding facilitates proteolytic cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin, FEBS Lett. 513 (2002) 242–246. [8] H. Höfte, H.R. Whiteley, Insecticidal crystal proteins of Bacilllus thuringiensis, Microbiol. Rev. 53 (1989) 242–255. [9] B. Oppert, Protease interactions with Bacillus thuringiensis insecticidal toxins, Arch. Biochem. Physiol. 42 (1999) 1–12. [10] L. Abdelkefi-Mesrati, H. Boukedi, M. Chakroun, F. Kamoun, H. Azzouz, S. Tounsi, S. Rouis, S. Jaoua, Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin, J. Invertebr. Pathol. 107 (2011) 198–201.
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Please cite this article as: M. Dammak, et al., Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin..., Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.09.005