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Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae
Accepted Manuscript Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae Saoussen Ben Khedher, Hanen Boukedi, Mariam Dammak, Olfa Kilani-Feki, Tahya Sellami-Boudawara, Lobna Abdelkefi-Mesrati, Slim Tounsi PII: DOI: Reference:
S0022-2011(17)30015-0 http://dx.doi.org/10.1016/j.jip.2017.01.006 YJIPA 6908
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
30 June 2016 3 January 2017 12 January 2017
Please cite this article as: Khedher, S.B., Boukedi, H., Dammak, M., Kilani-Feki, O., Sellami-Boudawara, T., Abdelkefi-Mesrati, L., Tounsi, S., Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.01.006
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Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae
Authors: Saoussen Ben Khedhera, Hanen Boukedia, Mariam Dammaka, Olfa KilaniFekia, Tahya Sellami-Boudawarab, Lobna Abdelkefi-Mesratic,* and Slim Tounsia
a
Laboratory of Biopesticides, Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3018 Sfax, Tunisia. b
Laboratory of Anatomy and Pathological Cytology, Sfax, Tunisia, P.O. Box 1507, 3029 Sfax, Tunisia
c
Department of Biology, Faculty of Sciences and Arts-Khulais, University of Jeddah, Saudi Arabia
* Corresponding author: L. Abdelkefi-Mesrati: [email protected], address: Department of Biology, Faculty of Sciences and ArtsKhulais, University of Jeddah, Saudi Arabia.
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Abstract Spodoptera littoralis, one of the most serious and destructive agricultural pests in the world, is very susceptible to Vip3 toxin. In order to develop a new efficient bioinsecticide and to prevent the development of resistance by the target pest, insecticidal activity of biosurfactant produced by Bacillus amyloliquefaciens AG1 was evaluated against S. littoralis. Bioassays revealed the susceptibility of the first instar larvae of this pest to AG1 biosurfactant with an LC50 of 245 ng/cm2. Moreover, the histopathology examination of the larval midgut treated by AG1 biosurfactant showed vacuolization, necrosis and disintegration of the basement membrane. Binding experiments revealed that the AG1 biosurfactant recognized three putative receptors located in the brush border membrane vesicles of S. littoralis with sizes of 91, 72 and 64 kDa. Competition assays using biotinylated metabolites indicated that AG1 biosurfactant and Vip3Aa16 toxin did not compete for the same S. littoralis receptors. When combined, AG1 biosurfactant and Vip3Aa16 showed an additive effect against S. littoralis larvae. These findings suggested that B. amyloliquefaciens AG1 biosurfactant could be a promising biocontrol agent to eradicate S. littoralis and to prevent resistance development by this pest.
Keywords: Spodoptera littoralis; Bacillus amyloliquefaciens AG biosurfactant; Vip3Aa16; toxicity; histology; putative binding receptors.
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1. Introduction Spodoptera littoralis (Lepidoptera: Noctuidae) is one of the most serious agricultural pests that causes severe damages to a variety of crops within tropical and subtropical range (Korrat et al., 2012; Bergamasco et al., 2013). It can attack numerous economically important crops and causes considerable damage on vegetables throughout the year in Africa, Asia and Europe (Pineda et al., 2007). Chemical insecticides are extensively used to control this pest (Sammour et al., 2008; Korrat et al., 2012). Nevertheless, this procedure has resulted in resistance to major classes of insecticides (Abo-El-Ghar et al., 1986; Haq et al., 2004) and serious toxicological problems to the human health and the environment safety (Costa et al., 2008; Relyea, 2009). To overcome these problems, biological control methods are available. The use of Bacillus thuringiensis as a biocontrol agent is the most reputed method as it has been shown to be extremely valuable (Dutton et al., 2005; AlOtaibi, 2013; Bergamasco et al., 2013). A wide range of lepidopteran larvae are susceptible to various Cry toxins of B. thuringiensis. However, S. littoralis larvae are sensitive to only few of them including Cry1C and Cry1D delta-endotoxins produced by B. thuringiensis subsp. aizawai (Sanchis et al., 1989; Ben Farhat-Touzri et al., 2013). Moreover, some cases of resistance have been developed by this polyphagous insect pest (Tabashnik et al., 2009; Storer et al., 2010; Tiewsiri and Wang, 2011). Resistance to B. thuringiensis Cry toxins can be developed by mutations in the insect pests that affect any of the steps of the mode of action of Cry toxins (Bravo et al., 2011). The most common mechanism of toxin resistance until now is the reduction in toxin binding to midgut cells, in different insect species (Herrero et al., 2005; Zhang et al., 2009; Gahan et al., 2010). To counter this resistance, it is necessary to find other efficient biological agents for S. littoralis control. Vip3 proteins, produced by B. thuringiensis during the vegetative growth phase, do not share homology with Cry proteins and are also active against several species of Lepidoptera (S.
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littoralis, Agrotis segetum, S. frugiperda and S. exigua) (Estruch et al., 1996; AbdelkefiMesrati et al., 2011; Ben Hamadou-Charfi et al., 2013). Vip3 proteins have a similar mode of action as that of Cry proteins, with the formation of ion channels in midgut epithelial cells. However, they recognize receptors different to those recognized by Cry proteins (Lee et al., 2006; Abdelkefi-Mesrati et al., 2009; Sena et al., 2009; Liu et al., 2011).
Kosaric et al., 1987; Georgiou et al., 1992) Biosurfactants are typical examples of microbial metabolites that show biological activity against pest insects and pathogens. They have many advantages such high level of activity and specificity at extreme temperatures, pH and salinity, and high degree of biodegradability (Banat et al., 2010). The use of biosurfactant in pest management is gaining interest because of its biodegradability and environmental safety. Many models have been studied such as sophorolipid, a biosurfactant produced by Candida bombicola, that has showed antimicrobial activity against plant pathogenic fungi (Yoo et al., 2005) and biosurfactant of Pseudomonas strains having antagonistic effects on Phytophthora capsci and Colletorichum orbiculare (Kim et al., 2000). However, few studies have investigated the insecticidal activity of biosurfactants produced by microorganisms, like Orfamide A and rhamnolipid, two biosurfactants derived from Pseudomonas strains displaying insecticidal activity against green peach aphid (Kim et al., 2011; Jang et al., 2013). Biosurfactants produced by B. subtilis have been reported as an efficient biological control agent against S. littoralis and mosquitoes (Abd El-Salam et al., 2011; Ghribi et al., 2012; Revathi et al., 2013). Moreover, lipopeptides biosurfactant produced by B. amyloliquefaciens strains have also insecticidal property against Green peach aphid (Yun et al., 2013) and different mosquito species (Geetha et al., 2011). Therefore, the use of
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biosurfactant could be a possible alternative to overcome resistance of pest insects to B. thuringiensis Cry toxins. In a previous work, we showed that biosurfactant extracted from B. amyloliquefaciens AG1 was effective in controlling tomato leaf miner, Tuta absoluta. The strain AG1 harbored genes involved in the biosynthesis of lipopeptides including iturin, fengycin, surfactin and bacyllomicin, and polyketides mainly difficidin, macrolactin and bacillaene (Ben Khedher et al., 2015). In the present study, we evaluated the toxicity of AG1 biosurfactant against a lepidopteran insect susceptible to just a few B. thuringiensis delta-endotoxins, S. littoralis. Its combination with B. thuringiensis Vip3Aa16, as well as its histopathologic effect, and its ability to bind to receptors in the brush border membrane vesicles (BBMV) of S. littoralis larvae, were investigated and discussed.
2. Materials and Methods 2.1. Bacterial strains The strain AG1, a biosurfactant producing bacterium, belonging to the B. amyloliquefaciens strain collection of our laboratory (Laboratory of Biopesticides), was isolated from soil. It was selected on the basis of its biosurfactant ability to reduce surface tension of the water from 70 mN/m to 28.6 mN/m and its good emulsification activities. It was identified as B. amyloliquefaciens by morphological, biochemical and 16S rDNA sequence analysis. The 16S rRNA sequence of the strain AG1 was deposited in the GeneBank nucleotide sequence databases under the accession number KT192073. BL21 (pET-14b-vip3Aa16) is an E. coli recombinant strain expressing vip3Aa16 gene (Abdelkefi-Mesrati et al., 2009).
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2.2. Media and culture conditions For activation, the B. amyloliquefaciens AG1 strain taken from storage was grown on LB (Luria Broth) agar at 30°C for 24 h. The inoculum was prepared as follows: one isolated colony was added to 3 ml of LB medium and incubated overnight at 30°C. 0.5 ml were used to inoculate a 250 ml erlenmeyer flask containing 50 ml LB medium which were incubated at 30°C in a rotatory shaker set at 200 rpm, for 10 h to prepare the inoculum. The culture broth was used to inoculate the culture medium described by Mezghanni et al. (2012). Cultures were grown in 250 ml Erlenmeyer flask with an initial O.D.600 of 0.15 at 30°C and 200 rpm for 48 h.
2.3. Extraction of biosurfactant Extraction of biosurfactant was performed according to the method previously described by Liu et al. (2008). The biosurfactant was extracted from the culture medium after removal of the bacterial cells by centrifugation at 8,000 × g for 10 min at 4°C. The supernatant pH was adjusted to 2 with 6 M HCl and incubating during overnight at 4°C. The precipitant was collected by centrifugation at 8,000 × g for 10 min, washed with acidified water at pH 2, neutralized by dissolving in 6 M NaOH solution and lyophilized. The lyophilized powder was then extracted three times with methanol for 3 h. The methanol was removed with a rotary evaporator under reduced pressure, yielding a brown-colored crude biosurfactant extract. After drying, the yield of biosurfactant production by B. amyloliquefaciens AG1 was 2 g/l. The extract was solubilized in sterile distilled water prior to use in experiments.
2.4. Vip3Aa16 protein preparation and purification vip3Aa16 gene cloned in the pET-14b vector was heterogously expressed in E. coli strain BL21 (Abdelkefi-Mesrati et al., 2009). Strain BL21 was grown in LB medium at 37°C. After
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harvesting by centrifugation, cell pellet was suspended in PBS buffer (1×, pH 7.5) and sonicated as described by Abdelkefi-Mesrati et al. (2009). Lysates containing Vip3A proteins were purified on His-Trap column (Amersham) previously prepared according to the manufacturer’s instructions. The bound proteins were eluted with elution buffer (50 mM phosphate buffer, pH 8.0; 0.5 M NaCl and 500 mM imidazole).
2.5. Bioassay The bioassays were carried out using the method described by Abdelkefi-Mesrati et al. (2011) for neonatal S. littoralis larvae. Treatments were applied to the surface of an artificial diet (Poitout and Bues, 1970) contained in 24 well polystyrene plates. To determine the fifty percent lethal concentration (LC50), seven concentrations were tested by applying 100 µl of each dilution to the artificial diet. Each bioassay was repeated three times, with 24 first instar larvae per replication. AG1 biosurfactant and Vip3Aa16 toxin were tested individually and at different ratios (1:0.25, 1:0.5 and 1:1). Distilled water, used as negative control was included in the bioassay. The plates were incubated at 23°C, 65% relative humidity and a photoperiod of 18 h light and 6 h dark. Mortality was recorded after 5 days and the LC50 was calculated by Probit analysis using programs written in the R. language (Venables and Smith, 2004). Synergism was evaluated according to Tabashnik’s method (Tabashnik, 1992). The theoretical LC50 value is the mean of the intrinsic LC50 values of each component weighted by the ratio used in the mixture: LC 50 (AG1 biosurfactant : Vip3Aa16)Theoretical = [rAG1 biosurfactant/ LC 50(AG1 biosurfactant) + rVip3Aa16/ LC 50(Vip3Aa16)]-1 where rAG1
biosurfactant
and
rVip3Aa16 are the AG1 biosurfactant and Vip3Aa16 protein
proportions used in the final ratio of the mixture. LC50 (AG1 biosurfactant) and LC50 (Vip3Aa16) are the LC50 values for each individual toxin. The synergism factor (SF) was
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calculated by dividing the theoretical toxicity by the observed toxicity of the mixture in bioassays. SF values greater than 1 indicate synergism.
2.6. Histopathological effect of biosurfactant in the midgut of S. littoralis The treated and control first instar larvae of S. littoralis were fixed in formol buffer solution (10%) at 4°C. After that, samples were dehydrated in increasing ethanol concentrations, washed by toluene (100%) then impregnated and embedded in paraffine wax. Ultrathin sections (5 µm) were placed in carriers loaded with in a mix 1.5% egg albumin and 3% glycerol in distilled water. After that, sections already de-paraffinated in 100% toluene were stained with hematoxylineosin as described by Ruiz et al. (2004). Images were observed and photographed using a light microscope (Olympus Optical Co. LTD) operating at Olympus DP70 camera.
2.7. BBMV preparation BBMV were extracted according to the method described by Wolfersberger et al. (1987). Briefly, midguts were extracted from S. littoralis larvae (fourth instar L4), suspended in the ice-cold MET (300 mM mannitol, 17 mM Tris-HCl, pH 7.5 and 5 mM EGTA) buffer and then washed twice with MET buffer before being flash-frozen in liquid nitrogen and stored at -80°C. One g of midgut larvae was homogenized in MET buffer then the homogenate was diluted with an equal volume of ice-cold 24 mM MgCl2. A low speed centrifugation (4,500 × g for 15 min at 4°C) was applied and the supernatant from the initial centrifugation was further centrifuged at 13,000 × g 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 4,500 × g for 15 min at 4°C. The supernatant was recovered and also centrifuged at 13,000 × g for 45 min
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at 4°C. The resulting pellet (corresponding to the BBMV preparation) was suspended in MET buffer (0.5 ×), flash-frozen in liquid nitrogen and stored at -80°C.
2.8. Biosurfactant binding to BBMV and competition assays Biosurfactant was biotinylated by using biotinylation reagent (ECL™ protein biotinylation module: Amersham Pharmacia Biotech, France) according to the manufacturer’s instructions. Binding of biotinylated biosurfactant was carried out as described by Abdelkefi-Mesrati et al. (2011). Briefly, 20 µg of BBM were separated via SDS-PAGE (10%), transferred onto a nitrocellulose membrane before being conjugated with the biotinylated biosurfactant for 2 h. Then the membrane was treated with streptavidin- peroxidase conjugate (1:1500 dilutions) for 1 h to visualize the biotinylated biosurfactant that bound to BBMV. The specific interaction was visualized using luminol according to manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, France). For competition assays, BBMV (40 µg protein) was incubated with labelled (100 nM) and unlabelled biosurfactant (200 nM), in PBS buffer (1×, pH 7.6) for 1 h at room temperature. Samples were then centrifuged for 10 min at 13,000 × g at 4°C to remove unbound biotinylated and unbiotinylated biosurfactant. The pellets were washed twice with PBS buffer (1×, pH 7.6) before being suspended in 20 µl of the same buffer, loaded in a SDS-PAGE and electrotransferred to a nitrocellulose membrane. The biotinylated proteins that were bound to BBMVs were visualized by incubation with streptavidin-peroxydase conjugate (1:1500 dilutions) supplied in ECL protein biotinylation module for 1 h, followed by three 15 min washing cycles. Binding was visualized using luminol according to manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, France). For heterologous competition assay, the same protocol was followed except the biotin-labeled biosurfactant was incubated with the unlabeled Vip3A16 at 100, 200 and 400 nM in the presence of BBMV.
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3. Results 3.1. Insecticidal activity of AG1 biosurfactant against S. littoralis larvae The bioassay of biosurfactant produced by B. amyloliquefaciens AG1 was performed against the first instar larvae of S. littoralis. The obtained results showed that the AG1 biosurfactant exhibited insecticidal activity against S. littoralis larvae with an LC50 of 0.245 pmol/cm2 which is more active than Vip3Aa16 (approximately 13-fold) (Table 1). However, when toxicity is expressed in weight, AG1 biosurfactant (LC50 value of 245 ng/cm2) was efficient as Vip3Aa16 (LC50 of 305 ng/cm2) (Table 1). Exposure of larvae to water, used as negative control, did not cause any mortality.
3.2. Hisopathological effect of AG1 biosurfactant in S. littoralis midgut larvae The histopathological study was conducted on S. littoralis larvae treated by AG1 biosurfactant. As shown in Fig. 1B, the AG1 biosurfactant caused enlargement of epithelial cells, cytoplasm vacualization, appearance of vesicles at the apical part of the cells toward the midgut lumen and disruption of the basement membrane. In contrast, the midgut section of the untreated larvae showed habitual structure organisation with a well-developed brush border, a clear cytoplasm and a normal adhesive basement membrane (Fig. 1A).
3.3. Toxicity of AG1biosurfactant combined to Vip3Aa16 against S. littoralis Combinations between AG1 biosurfactant and Vip3Aa16 were tested against S. littoralis larvae. The mortality observed and the LC50 values obtained were shown in Fig. 2 and Table 2. According to Fig. 2, AG1 biosurfactant was active as Vip3Aa16 for S. littoralis larvae. Error bars on 1:0.25 ratio mortality almost overlap with those of AG1 biosurfactant and Vip3Aa16, suggesting no significant synergism. The AG1 biosurfactant-Vip3Aa16 ratios (1:1) and (1:0.5) were significantly more active than AG1 biosurfactant and Vip3Aa16 tested
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individually (Fig. 2). An SF value larger than 1 indicates a synergistic interaction, a value of 1 indicates additive toxicity, and a value lower than 1 indicates an antagonistic interaction. The bioassays performed with the combination AG1 biosurfactant-Vip3Aa16 showed lower LC50 values than that expected, for the (1:0.5) and (1:0.25) ratios with SF slightly higher than 1, indicating an additive effect. The ratio (1:1) particularly indicated a slight synergism(Table 2).
3.4. Binding of AG1 biosurfactant to S. littoralis BBMV In order to investigate putative receptors from S. littoralis BBMV to which the biosurfactant binds, interactions between biotinylated-AG1 biosurfactant and S. littoralis BBMV were investigated. Ligand blot analysis indicated three binding proteins of approximately 91 kD, 72 kDa and 64 kDa (Fig. 3; Lane 1). A BSA blot, used as negative control, showed no interaction with the biotinylated-biosurfactant of B. amyloliquefaciens AG1 (Fig. 3; Lane 2). This interaction property seems to be specific since the incubation of BBMV without the labeled ligand but only with streptavidin-peroxidase conjugate did not show any interactions (Fig. 3; Lane 3). The specificity of AG1 biosurfactant interaction with S. littoralis BBMV was confirmed by homologous competition assay. As shown in Fig. 4, an excess of unlabeled biosurfactant reduced biotinylated-biosurfactant binding to BBMV (Fig. 4; Lane 3). Moreover, heterologous competition with an excess of unlabeled Vip3A16 did not reduce AG1 biosurfactant binding, confirming that it did not compete with Vip3Aa16 for the same receptors in S. littoralis midgut (Fig. 4; lanes 4-6). Fig. 4 (Lane 7and 8) showed homologous competition with an excess of an unlabeled Vip3Aa16, substantially reducing the labeled Vip3Aa16 binding to S. littoralis BBMV. Otherwise, heterologous competition assays demonstrated that an excess of unlabeled AG1 biosurfactant did not inhibit the binding of
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Vip3Aa16 (Fig. 4; Lane 10-13). Overall, these studies support the conclusion that AG1 biosurfactant and Vip3Aa16 act independently.
4. Discussion Biosurfactants are surface active molecules that are produced by a variety of microorganisms including bacteria, yeast and filamentous fungi (de Faria et al., 2011). These amphiphilic biomolecules are increasingly used in agriculture for indirect plant growth promotion as these biosurfactants have antimicrobial and antifungal activities (Touré et al., 2004; Romero et al., 2007) and to increase the microbe-plant interaction (Ongena et al., 2007; Jourdan et al., 2009). Thus, they have received much attention for the control of plant pathogens, but few studies have investigated their insecticidal activity. In the present study, we reported the insecticidal activity of biosurfactant produced by B. amyloliquefaciens AG1 against S. littoralis. This is the first report showing that AG1 biosurfactant (LC50 value of 245 ng/cm2) was as efficient as Vip3Aa16 (LC50 of 305 ng/cm2) when tested individually on S. littoralis (Table 1). SPB1 biosurfactant derived from B. subtilis (Ghribi et al., 2012) and Cry1D of strain HD133 (Ben Farhat-Touzri et al., 2013) were also active against S. littoralis with LC50 of 250 ng/cm2 and 224.4 ng/cm2, respectively. Therefore, B. amyloliquefaciens AG1 biosurfactant could be used as efficient lipopeptides metabolites for S. littoralis control. Interestingly, no antagonist effect was observed when AG1 biosurfactant and Vip3Aa16 where combined against S. littoralis. Moreover, this combination resulted in an improvement of the toxicity (Fig. 2). This improvement could be due to the pore formation by these two different types of metabolites, accelerating the induction of osmotic breakdown in the cells, and consequently the mortality rate. Several studies reported that combination of Cyt1Aa and B. thuringiensis dipteran specific toxins (Cry4Aa, Cry4Ba, Cry11Aa and Cry2Aa) increased
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their insecticidal activities (Crickmore et al., 1995; Wirth et al., 1997, 2003; Park et al., 2013). Lee et al. (1996) found that Cry1Ab-Cry1Ac and Cry1Aa-Cry1Ac combinations showed increase in toxicity against Lymantria dispar. The target and the mode of action of biosurfactant inside the insect remain unknown and are not well documented. One of the possible modes of action could be the significant reduction in the activities of acetylcholinesterse, α-carboxylesterase and acid phosphatases of larvae, as reported by Revathi et al. (2013). AG1 Biosurfactant was suggested to affect the larvae midgut and led to membrane perturbation, as evidenced by the histopathological examination of S. littoralis midgut. These effects could be attributed to its composition on lipopeptides (surfactin, fengycin, iturin and bacyllomicin) and polyketides (bacillaene, difficidin and macrolactin) (Ben Khedher et al., 2015) which are amphiphilic compounds with surfaceactive properties. It was reported that surfactin produced by B. subtilis was toxic to mosquitoes, causing heamolysis (Revathi et al., 2013) and mainly bactericidal activity. Iturin and fengycin displayed fungicidal activity, specifically fengycin targeted filamentous fungi (Ongena and Jacques, 2008). Lipopeptides are surface-active molecules, comprising a hydrophilic protein moiety attached to a hydrophobic lipid chain, which act to destabilise the phospholipid membrane of the target phytopathogen resulting in cell lysis (Heerklotz and Seelig, 2007). Similar effects were described in midguts of T. absoluta larvae treated with AG1 biosurfactant (Ben Khedher et al., 2015) and S. littoralis larvae treated with SPB1 biosurfactant (Ghribi et al., 2012). On the basis of the multiple midgut membrane proteins reported to bind Cry and Vip3A toxins from B. thuringiensis in different insects, we suggested to examine if biosurfactant metabolites could have the same mode of action as Cry toxins. Ligand blot analysis revealed that biotinylated AG1 biosurfactant specifically bound to three putative receptors of about 91, 72 and 64 kDa from S. littoralis BBMV. These putative receptors differ from those
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recognized by the same biosurfactant in T. absoluta midgut and having molecular weights of about 68, 63, 44, 30 and 19 kDa (Ben Khedher et al., 2015). Although SPB1 biosurfactant exhibited the same toxicity and effects on S. littoralis midgut larvae, it recognized a single putative receptor of 45 kDa in S. littoralis BBMV (Ghribi et al., 2012). This difference in the number and molecular weights of putative receptors recognized by biosurfactants suggested variability in their (AG1 and SPB1 biosurfactants) lipopeptides and polyketides content. These properties make AG1 biosurfactant very promising to fight resistance in target pest insect. Abdelkefi-Mesrati et al. (2011) detected two putative Vip3Aa16 receptors at 55 kDa and 100 kDa in BBMV from S. littoralis. Despite multiple putative receptors from S. littoralis BBMV have been reported to bind Cry and Vip3Aa16 toxins (Oddou et al., 1993; Sanchis and Ellar, 1993; Abdelkefi-Mesrati et al., 2011; Ben Farhat-Touzri et al., 2013), the size of the three putative receptors shown in this study was reported for the first time. According to the heterologous competition assays, AG1 biosurfactant and Vip3Aa16 toxin did not compete for the same receptors. This would decrease cross resistance development by insect larvae, if resistance is due to receptor alteration. Similar results were found by Lee et al. (2006), who reported that there was no competition between Vip3Aa and Cry1Ac in Heliothis virescens and Helicoverpa zea BBMV. Sena et al. (2009) also showed that Cry1 and Vip3A occupy distinct binding sites on S. frugiperda BBMV. However, Cry1Fa occupies the same binding site as Cry1Ab, on S. frugiperda BBMV. Similarly, Vip3Af1 and Vip3Aa1 compete for the same receptor, probably because of their structure similarity (Sena et al., 2009). Therefore, the combination or rotation of AG1 biosurfactant and Vip3Aa16 toxin should be considered a strategic approach to delay resistance evolution against S. littoralis. Further studies should be done to identify and purify lipopeptides biosurfactant metabolites and to investigate their mode of action in S. littoralis midgut larvae. The identification of biosurfactant interacting proteins in the midgut of target
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insects, not yet reported, might provide tools for enhancing biosurfactant action against major crop pests.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgement This work was financially supported by the “Tunisian Ministry of Higher Education and Scientific Research”.
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Ruiz, L.M., Segura, C., Trujillo, J., Orduz, S., 2004. In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae). Mem. Inst. Oswaldo Cruz 99, 73-79. Sammour, E.A., Kandil, M.A., Abdel-Aziz, N.F., 2008. The reproductive potential and fate of chlorfluazuron and leufenuron against cotton leafworm Spodoptera littoralis (Boisd.). American – Eurasian. J. Agric. Environ. Sci. 4, 62-67. Sanchis, V., Ellar, D.J., 1993. Identification and partial purification of a Bacillus thuringiensis Cry1C d-endotoxin binding protein from Spodoptera littoralis gut membranes. FEBS 316, 264-268. Sanchis, V., Lereclus, D., Menou, G., Chaufaux, J., Guo, S., Lecadet, M.M., 1989. Nucleotide sequence and analysis of the N-terminal coding region of the Spodoptera-active δendotoxin gene of Bacillus thuringiensis var. aizawai 7.29. Mol. Microbiol. 3, 229238. Sena, J.A., Hernández-Rodríguez, C.S., Ferré, J., 2009. Interaction of Bacillus thuringiensis Cry1 and Vip3Aa proteins with Spodoptera frugiperda midgut binding sites. Appl. Environ. Microbiol. 75, 2236-2237. Storer, N.P., Babcock, J.M., Schlenz, M., Meade, T., Thompson, G.D., Bing, J.W., Huckaba, R.M., 2010. Discovery and characterization of field resistance to Bt Maize: Spodoptera frugiperda (Lepidoptera: Noctuidae) in Puerto Rico. J. Econ. Entomol. 103, 1031-1038. Tabashnik, B.E., 1992. Evaluation of synergism among Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 58, 343-346. Tabashnik, B.E., Van Rensburg, J.B., Carrière, Y., 2009. Field-evolved insect resistance to Bt crops: Definition, theory, and data. J. Econ. Entomol. 102, 2011-2025.
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Zhang, S., Cheng, H., Gao, Y., Wang, G., Liang, G., Wu, K., 2009. Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin. Insect Biochem. Mol. Biol. 39, 421-429.
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Figures legends Fig. 1. Histopathological effects of Bacillus amyloliquefaciens AG1 biosurfactant on S. littoralis midgut: (A) untreated S. littoralis larvae showing regular epithelial cell and welldeveloped lumen content. (B) S. littoralis larvae treated with AG1 biosurfactant showed vacuolization,
demolished
epithelial
layer
undistinguished
basement
membrane.
Magnification 40×. Lu: Lumen, Bm: basal membrane, Am: apical membrane, V: vacuole.
Fig. 2. Mortality of AG1 biosurfactant and Vip3Aa16 at different ratios mixture against the first instar larvae of S. littoralis. (∆) AG1 biosurfactant alone, (■) Vip3Aa16 alone, (◊) AG1 biosurfactant-Vip3Aa16 ratio (1:0.25), (○) AG1 biosurfactant-Vip3Aa16 ratio (1:0.50), (▲) AG1 biosurfactant -Vip3Aa16 ratio (1:1).
Fig. 3. Detection of the three putative receptors (91, 72 and 64 kDa) for biotinylated Bacillus amyloliquefaciens AG1 biosurfactant in the midgut of S. littoralis by ligand blot. Lane 1: S. littoralis BBMV, Lane 2: BSA used as negative control and Lane 3: BBMV without incubation with the biotinylated AG1 biosurfactant but only with the detection reagents, also used as negative control.
Fig. 4. AG1 biosurfactant and Vip3Aa16 toxin binding to S. littoralis BBMV. Homologous competition: Lanes: 1- BBMV without incubation with the biotinylated AG1 biosurfactant but only with the detection reagents, used as negative control, 2- Biotinylated AG1 biosurfactant bound to BBMV, 3- Biotinylated AG1 biosurfactant and unlabeled AG1 biosurfactant (2× excess). Heterologous competition: 4- Biotinylated AG1 biosurfactant and unlabeled Vip3Aa16 (1× excess), 5- biotinylated AG1 biosurfactant and unlabeled Vip3Aa16 (2× excess), 6- biotinylated AG1 biosurfactant and unlabeled Vip3Aa16 (4× excess). Homologous
24
competition: 7- biotinylated Vip3Aa16 bound to BBMV, 8- biotinylated Vip3Aa16 and unlabeled Vip3Aa16 (2× excess). Heterologous competition: 9- BBMV without incubation with the biotinylated Vip3Aa16 but only with the detection reagents, used as negative control, 10- Biotinylated Vip3Aa16 bound to BBMV, 11- Biotinylated Vip3Aa16 and unlabeled AG1 biosurfactant (1 × excess), 12- biotinylated Vip3Aa16 and unlabeled AG1 biosurfactant (2× excess), 13- biotinylated Vip3Aa16and unlabeled AG1 biosurfactant (4× excess).
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Table 1. Toxicity of AG1 biosurfactant and Vip3Aa16 toxin against the first instar larvae of S. littoralis. Toxins
LC50a (pmol/cm2)
LC50 (ng/cm2)
AG1 Biosurfactant
0.245 (0.17-0.31)
245 (179.53-312.43)
Vip3Aa16
3.39 (2.48-4.30)
305 (223.00-387.00)
a
Mean lethal concentration (LC50) was estimated by Probit analysis. 95% confidence limits
are given in parentheses.
26
Table 2. Combination of AG1 biosurfactant and Vip3Aa16 toxin against the first instar larvae of S. littoralis. (AG1 biosurfactant:Vip3Aa16)
Predicted LC50(ng/cm2)
Experimental LC50 (ng/cm2)
SFb
1 : 0.25
255.03
190.65 (139.01-242.29)a
1.33
1 : 0.50
264.85
150.13 (122.75-177.51)
1.76
1:1
271.73
91.84 (63.02-120.66)
2.95
a
95% confidence limits.
b
Synergism factor (predicted LC50/experimental LC50).
27
28
29
30
31
32
Graphical abstract
33
Highlights
Spodoptera littoralis is one of the most serious agricultural pest in the world.
B. amyloliquefaciens AG1 biosurfactant was active against S. littoralis larvae.
AG1 biosurfactant caused histological damages in the larval midgut of S. littoralis.
AG1 biosurfactant recognized midgut putative receptors of about 91, 72 and 64 kDa.
High toxicity and synergism between AG1 biosurfactant and Vip3Aa16 in S. littoralis.
34
S0022-2011(17)30015-0 http://dx.doi.org/10.1016/j.jip.2017.01.006 YJIPA 6908
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
30 June 2016 3 January 2017 12 January 2017
Please cite this article as: Khedher, S.B., Boukedi, H., Dammak, M., Kilani-Feki, O., Sellami-Boudawara, T., Abdelkefi-Mesrati, L., Tounsi, S., Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.01.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae
Authors: Saoussen Ben Khedhera, Hanen Boukedia, Mariam Dammaka, Olfa KilaniFekia, Tahya Sellami-Boudawarab, Lobna Abdelkefi-Mesratic,* and Slim Tounsia
a
Laboratory of Biopesticides, Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3018 Sfax, Tunisia. b
Laboratory of Anatomy and Pathological Cytology, Sfax, Tunisia, P.O. Box 1507, 3029 Sfax, Tunisia
c
Department of Biology, Faculty of Sciences and Arts-Khulais, University of Jeddah, Saudi Arabia
* Corresponding author: L. Abdelkefi-Mesrati: [email protected], address: Department of Biology, Faculty of Sciences and ArtsKhulais, University of Jeddah, Saudi Arabia.
1
Abstract Spodoptera littoralis, one of the most serious and destructive agricultural pests in the world, is very susceptible to Vip3 toxin. In order to develop a new efficient bioinsecticide and to prevent the development of resistance by the target pest, insecticidal activity of biosurfactant produced by Bacillus amyloliquefaciens AG1 was evaluated against S. littoralis. Bioassays revealed the susceptibility of the first instar larvae of this pest to AG1 biosurfactant with an LC50 of 245 ng/cm2. Moreover, the histopathology examination of the larval midgut treated by AG1 biosurfactant showed vacuolization, necrosis and disintegration of the basement membrane. Binding experiments revealed that the AG1 biosurfactant recognized three putative receptors located in the brush border membrane vesicles of S. littoralis with sizes of 91, 72 and 64 kDa. Competition assays using biotinylated metabolites indicated that AG1 biosurfactant and Vip3Aa16 toxin did not compete for the same S. littoralis receptors. When combined, AG1 biosurfactant and Vip3Aa16 showed an additive effect against S. littoralis larvae. These findings suggested that B. amyloliquefaciens AG1 biosurfactant could be a promising biocontrol agent to eradicate S. littoralis and to prevent resistance development by this pest.
Keywords: Spodoptera littoralis; Bacillus amyloliquefaciens AG biosurfactant; Vip3Aa16; toxicity; histology; putative binding receptors.
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1. Introduction Spodoptera littoralis (Lepidoptera: Noctuidae) is one of the most serious agricultural pests that causes severe damages to a variety of crops within tropical and subtropical range (Korrat et al., 2012; Bergamasco et al., 2013). It can attack numerous economically important crops and causes considerable damage on vegetables throughout the year in Africa, Asia and Europe (Pineda et al., 2007). Chemical insecticides are extensively used to control this pest (Sammour et al., 2008; Korrat et al., 2012). Nevertheless, this procedure has resulted in resistance to major classes of insecticides (Abo-El-Ghar et al., 1986; Haq et al., 2004) and serious toxicological problems to the human health and the environment safety (Costa et al., 2008; Relyea, 2009). To overcome these problems, biological control methods are available. The use of Bacillus thuringiensis as a biocontrol agent is the most reputed method as it has been shown to be extremely valuable (Dutton et al., 2005; AlOtaibi, 2013; Bergamasco et al., 2013). A wide range of lepidopteran larvae are susceptible to various Cry toxins of B. thuringiensis. However, S. littoralis larvae are sensitive to only few of them including Cry1C and Cry1D delta-endotoxins produced by B. thuringiensis subsp. aizawai (Sanchis et al., 1989; Ben Farhat-Touzri et al., 2013). Moreover, some cases of resistance have been developed by this polyphagous insect pest (Tabashnik et al., 2009; Storer et al., 2010; Tiewsiri and Wang, 2011). Resistance to B. thuringiensis Cry toxins can be developed by mutations in the insect pests that affect any of the steps of the mode of action of Cry toxins (Bravo et al., 2011). The most common mechanism of toxin resistance until now is the reduction in toxin binding to midgut cells, in different insect species (Herrero et al., 2005; Zhang et al., 2009; Gahan et al., 2010). To counter this resistance, it is necessary to find other efficient biological agents for S. littoralis control. Vip3 proteins, produced by B. thuringiensis during the vegetative growth phase, do not share homology with Cry proteins and are also active against several species of Lepidoptera (S.
3
littoralis, Agrotis segetum, S. frugiperda and S. exigua) (Estruch et al., 1996; AbdelkefiMesrati et al., 2011; Ben Hamadou-Charfi et al., 2013). Vip3 proteins have a similar mode of action as that of Cry proteins, with the formation of ion channels in midgut epithelial cells. However, they recognize receptors different to those recognized by Cry proteins (Lee et al., 2006; Abdelkefi-Mesrati et al., 2009; Sena et al., 2009; Liu et al., 2011).
Kosaric et al., 1987; Georgiou et al., 1992) Biosurfactants are typical examples of microbial metabolites that show biological activity against pest insects and pathogens. They have many advantages such high level of activity and specificity at extreme temperatures, pH and salinity, and high degree of biodegradability (Banat et al., 2010). The use of biosurfactant in pest management is gaining interest because of its biodegradability and environmental safety. Many models have been studied such as sophorolipid, a biosurfactant produced by Candida bombicola, that has showed antimicrobial activity against plant pathogenic fungi (Yoo et al., 2005) and biosurfactant of Pseudomonas strains having antagonistic effects on Phytophthora capsci and Colletorichum orbiculare (Kim et al., 2000). However, few studies have investigated the insecticidal activity of biosurfactants produced by microorganisms, like Orfamide A and rhamnolipid, two biosurfactants derived from Pseudomonas strains displaying insecticidal activity against green peach aphid (Kim et al., 2011; Jang et al., 2013). Biosurfactants produced by B. subtilis have been reported as an efficient biological control agent against S. littoralis and mosquitoes (Abd El-Salam et al., 2011; Ghribi et al., 2012; Revathi et al., 2013). Moreover, lipopeptides biosurfactant produced by B. amyloliquefaciens strains have also insecticidal property against Green peach aphid (Yun et al., 2013) and different mosquito species (Geetha et al., 2011). Therefore, the use of
4
biosurfactant could be a possible alternative to overcome resistance of pest insects to B. thuringiensis Cry toxins. In a previous work, we showed that biosurfactant extracted from B. amyloliquefaciens AG1 was effective in controlling tomato leaf miner, Tuta absoluta. The strain AG1 harbored genes involved in the biosynthesis of lipopeptides including iturin, fengycin, surfactin and bacyllomicin, and polyketides mainly difficidin, macrolactin and bacillaene (Ben Khedher et al., 2015). In the present study, we evaluated the toxicity of AG1 biosurfactant against a lepidopteran insect susceptible to just a few B. thuringiensis delta-endotoxins, S. littoralis. Its combination with B. thuringiensis Vip3Aa16, as well as its histopathologic effect, and its ability to bind to receptors in the brush border membrane vesicles (BBMV) of S. littoralis larvae, were investigated and discussed.
2. Materials and Methods 2.1. Bacterial strains The strain AG1, a biosurfactant producing bacterium, belonging to the B. amyloliquefaciens strain collection of our laboratory (Laboratory of Biopesticides), was isolated from soil. It was selected on the basis of its biosurfactant ability to reduce surface tension of the water from 70 mN/m to 28.6 mN/m and its good emulsification activities. It was identified as B. amyloliquefaciens by morphological, biochemical and 16S rDNA sequence analysis. The 16S rRNA sequence of the strain AG1 was deposited in the GeneBank nucleotide sequence databases under the accession number KT192073. BL21 (pET-14b-vip3Aa16) is an E. coli recombinant strain expressing vip3Aa16 gene (Abdelkefi-Mesrati et al., 2009).
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2.2. Media and culture conditions For activation, the B. amyloliquefaciens AG1 strain taken from storage was grown on LB (Luria Broth) agar at 30°C for 24 h. The inoculum was prepared as follows: one isolated colony was added to 3 ml of LB medium and incubated overnight at 30°C. 0.5 ml were used to inoculate a 250 ml erlenmeyer flask containing 50 ml LB medium which were incubated at 30°C in a rotatory shaker set at 200 rpm, for 10 h to prepare the inoculum. The culture broth was used to inoculate the culture medium described by Mezghanni et al. (2012). Cultures were grown in 250 ml Erlenmeyer flask with an initial O.D.600 of 0.15 at 30°C and 200 rpm for 48 h.
2.3. Extraction of biosurfactant Extraction of biosurfactant was performed according to the method previously described by Liu et al. (2008). The biosurfactant was extracted from the culture medium after removal of the bacterial cells by centrifugation at 8,000 × g for 10 min at 4°C. The supernatant pH was adjusted to 2 with 6 M HCl and incubating during overnight at 4°C. The precipitant was collected by centrifugation at 8,000 × g for 10 min, washed with acidified water at pH 2, neutralized by dissolving in 6 M NaOH solution and lyophilized. The lyophilized powder was then extracted three times with methanol for 3 h. The methanol was removed with a rotary evaporator under reduced pressure, yielding a brown-colored crude biosurfactant extract. After drying, the yield of biosurfactant production by B. amyloliquefaciens AG1 was 2 g/l. The extract was solubilized in sterile distilled water prior to use in experiments.
2.4. Vip3Aa16 protein preparation and purification vip3Aa16 gene cloned in the pET-14b vector was heterogously expressed in E. coli strain BL21 (Abdelkefi-Mesrati et al., 2009). Strain BL21 was grown in LB medium at 37°C. After
6
harvesting by centrifugation, cell pellet was suspended in PBS buffer (1×, pH 7.5) and sonicated as described by Abdelkefi-Mesrati et al. (2009). Lysates containing Vip3A proteins were purified on His-Trap column (Amersham) previously prepared according to the manufacturer’s instructions. The bound proteins were eluted with elution buffer (50 mM phosphate buffer, pH 8.0; 0.5 M NaCl and 500 mM imidazole).
2.5. Bioassay The bioassays were carried out using the method described by Abdelkefi-Mesrati et al. (2011) for neonatal S. littoralis larvae. Treatments were applied to the surface of an artificial diet (Poitout and Bues, 1970) contained in 24 well polystyrene plates. To determine the fifty percent lethal concentration (LC50), seven concentrations were tested by applying 100 µl of each dilution to the artificial diet. Each bioassay was repeated three times, with 24 first instar larvae per replication. AG1 biosurfactant and Vip3Aa16 toxin were tested individually and at different ratios (1:0.25, 1:0.5 and 1:1). Distilled water, used as negative control was included in the bioassay. The plates were incubated at 23°C, 65% relative humidity and a photoperiod of 18 h light and 6 h dark. Mortality was recorded after 5 days and the LC50 was calculated by Probit analysis using programs written in the R. language (Venables and Smith, 2004). Synergism was evaluated according to Tabashnik’s method (Tabashnik, 1992). The theoretical LC50 value is the mean of the intrinsic LC50 values of each component weighted by the ratio used in the mixture: LC 50 (AG1 biosurfactant : Vip3Aa16)Theoretical = [rAG1 biosurfactant/ LC 50(AG1 biosurfactant) + rVip3Aa16/ LC 50(Vip3Aa16)]-1 where rAG1
biosurfactant
and
rVip3Aa16 are the AG1 biosurfactant and Vip3Aa16 protein
proportions used in the final ratio of the mixture. LC50 (AG1 biosurfactant) and LC50 (Vip3Aa16) are the LC50 values for each individual toxin. The synergism factor (SF) was
7
calculated by dividing the theoretical toxicity by the observed toxicity of the mixture in bioassays. SF values greater than 1 indicate synergism.
2.6. Histopathological effect of biosurfactant in the midgut of S. littoralis The treated and control first instar larvae of S. littoralis were fixed in formol buffer solution (10%) at 4°C. After that, samples were dehydrated in increasing ethanol concentrations, washed by toluene (100%) then impregnated and embedded in paraffine wax. Ultrathin sections (5 µm) were placed in carriers loaded with in a mix 1.5% egg albumin and 3% glycerol in distilled water. After that, sections already de-paraffinated in 100% toluene were stained with hematoxylineosin as described by Ruiz et al. (2004). Images were observed and photographed using a light microscope (Olympus Optical Co. LTD) operating at Olympus DP70 camera.
2.7. BBMV preparation BBMV were extracted according to the method described by Wolfersberger et al. (1987). Briefly, midguts were extracted from S. littoralis larvae (fourth instar L4), suspended in the ice-cold MET (300 mM mannitol, 17 mM Tris-HCl, pH 7.5 and 5 mM EGTA) buffer and then washed twice with MET buffer before being flash-frozen in liquid nitrogen and stored at -80°C. One g of midgut larvae was homogenized in MET buffer then the homogenate was diluted with an equal volume of ice-cold 24 mM MgCl2. A low speed centrifugation (4,500 × g for 15 min at 4°C) was applied and the supernatant from the initial centrifugation was further centrifuged at 13,000 × g 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 4,500 × g for 15 min at 4°C. The supernatant was recovered and also centrifuged at 13,000 × g for 45 min
8
at 4°C. The resulting pellet (corresponding to the BBMV preparation) was suspended in MET buffer (0.5 ×), flash-frozen in liquid nitrogen and stored at -80°C.
2.8. Biosurfactant binding to BBMV and competition assays Biosurfactant was biotinylated by using biotinylation reagent (ECL™ protein biotinylation module: Amersham Pharmacia Biotech, France) according to the manufacturer’s instructions. Binding of biotinylated biosurfactant was carried out as described by Abdelkefi-Mesrati et al. (2011). Briefly, 20 µg of BBM were separated via SDS-PAGE (10%), transferred onto a nitrocellulose membrane before being conjugated with the biotinylated biosurfactant for 2 h. Then the membrane was treated with streptavidin- peroxidase conjugate (1:1500 dilutions) for 1 h to visualize the biotinylated biosurfactant that bound to BBMV. The specific interaction was visualized using luminol according to manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, France). For competition assays, BBMV (40 µg protein) was incubated with labelled (100 nM) and unlabelled biosurfactant (200 nM), in PBS buffer (1×, pH 7.6) for 1 h at room temperature. Samples were then centrifuged for 10 min at 13,000 × g at 4°C to remove unbound biotinylated and unbiotinylated biosurfactant. The pellets were washed twice with PBS buffer (1×, pH 7.6) before being suspended in 20 µl of the same buffer, loaded in a SDS-PAGE and electrotransferred to a nitrocellulose membrane. The biotinylated proteins that were bound to BBMVs were visualized by incubation with streptavidin-peroxydase conjugate (1:1500 dilutions) supplied in ECL protein biotinylation module for 1 h, followed by three 15 min washing cycles. Binding was visualized using luminol according to manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, France). For heterologous competition assay, the same protocol was followed except the biotin-labeled biosurfactant was incubated with the unlabeled Vip3A16 at 100, 200 and 400 nM in the presence of BBMV.
9
3. Results 3.1. Insecticidal activity of AG1 biosurfactant against S. littoralis larvae The bioassay of biosurfactant produced by B. amyloliquefaciens AG1 was performed against the first instar larvae of S. littoralis. The obtained results showed that the AG1 biosurfactant exhibited insecticidal activity against S. littoralis larvae with an LC50 of 0.245 pmol/cm2 which is more active than Vip3Aa16 (approximately 13-fold) (Table 1). However, when toxicity is expressed in weight, AG1 biosurfactant (LC50 value of 245 ng/cm2) was efficient as Vip3Aa16 (LC50 of 305 ng/cm2) (Table 1). Exposure of larvae to water, used as negative control, did not cause any mortality.
3.2. Hisopathological effect of AG1 biosurfactant in S. littoralis midgut larvae The histopathological study was conducted on S. littoralis larvae treated by AG1 biosurfactant. As shown in Fig. 1B, the AG1 biosurfactant caused enlargement of epithelial cells, cytoplasm vacualization, appearance of vesicles at the apical part of the cells toward the midgut lumen and disruption of the basement membrane. In contrast, the midgut section of the untreated larvae showed habitual structure organisation with a well-developed brush border, a clear cytoplasm and a normal adhesive basement membrane (Fig. 1A).
3.3. Toxicity of AG1biosurfactant combined to Vip3Aa16 against S. littoralis Combinations between AG1 biosurfactant and Vip3Aa16 were tested against S. littoralis larvae. The mortality observed and the LC50 values obtained were shown in Fig. 2 and Table 2. According to Fig. 2, AG1 biosurfactant was active as Vip3Aa16 for S. littoralis larvae. Error bars on 1:0.25 ratio mortality almost overlap with those of AG1 biosurfactant and Vip3Aa16, suggesting no significant synergism. The AG1 biosurfactant-Vip3Aa16 ratios (1:1) and (1:0.5) were significantly more active than AG1 biosurfactant and Vip3Aa16 tested
10
individually (Fig. 2). An SF value larger than 1 indicates a synergistic interaction, a value of 1 indicates additive toxicity, and a value lower than 1 indicates an antagonistic interaction. The bioassays performed with the combination AG1 biosurfactant-Vip3Aa16 showed lower LC50 values than that expected, for the (1:0.5) and (1:0.25) ratios with SF slightly higher than 1, indicating an additive effect. The ratio (1:1) particularly indicated a slight synergism(Table 2).
3.4. Binding of AG1 biosurfactant to S. littoralis BBMV In order to investigate putative receptors from S. littoralis BBMV to which the biosurfactant binds, interactions between biotinylated-AG1 biosurfactant and S. littoralis BBMV were investigated. Ligand blot analysis indicated three binding proteins of approximately 91 kD, 72 kDa and 64 kDa (Fig. 3; Lane 1). A BSA blot, used as negative control, showed no interaction with the biotinylated-biosurfactant of B. amyloliquefaciens AG1 (Fig. 3; Lane 2). This interaction property seems to be specific since the incubation of BBMV without the labeled ligand but only with streptavidin-peroxidase conjugate did not show any interactions (Fig. 3; Lane 3). The specificity of AG1 biosurfactant interaction with S. littoralis BBMV was confirmed by homologous competition assay. As shown in Fig. 4, an excess of unlabeled biosurfactant reduced biotinylated-biosurfactant binding to BBMV (Fig. 4; Lane 3). Moreover, heterologous competition with an excess of unlabeled Vip3A16 did not reduce AG1 biosurfactant binding, confirming that it did not compete with Vip3Aa16 for the same receptors in S. littoralis midgut (Fig. 4; lanes 4-6). Fig. 4 (Lane 7and 8) showed homologous competition with an excess of an unlabeled Vip3Aa16, substantially reducing the labeled Vip3Aa16 binding to S. littoralis BBMV. Otherwise, heterologous competition assays demonstrated that an excess of unlabeled AG1 biosurfactant did not inhibit the binding of
11
Vip3Aa16 (Fig. 4; Lane 10-13). Overall, these studies support the conclusion that AG1 biosurfactant and Vip3Aa16 act independently.
4. Discussion Biosurfactants are surface active molecules that are produced by a variety of microorganisms including bacteria, yeast and filamentous fungi (de Faria et al., 2011). These amphiphilic biomolecules are increasingly used in agriculture for indirect plant growth promotion as these biosurfactants have antimicrobial and antifungal activities (Touré et al., 2004; Romero et al., 2007) and to increase the microbe-plant interaction (Ongena et al., 2007; Jourdan et al., 2009). Thus, they have received much attention for the control of plant pathogens, but few studies have investigated their insecticidal activity. In the present study, we reported the insecticidal activity of biosurfactant produced by B. amyloliquefaciens AG1 against S. littoralis. This is the first report showing that AG1 biosurfactant (LC50 value of 245 ng/cm2) was as efficient as Vip3Aa16 (LC50 of 305 ng/cm2) when tested individually on S. littoralis (Table 1). SPB1 biosurfactant derived from B. subtilis (Ghribi et al., 2012) and Cry1D of strain HD133 (Ben Farhat-Touzri et al., 2013) were also active against S. littoralis with LC50 of 250 ng/cm2 and 224.4 ng/cm2, respectively. Therefore, B. amyloliquefaciens AG1 biosurfactant could be used as efficient lipopeptides metabolites for S. littoralis control. Interestingly, no antagonist effect was observed when AG1 biosurfactant and Vip3Aa16 where combined against S. littoralis. Moreover, this combination resulted in an improvement of the toxicity (Fig. 2). This improvement could be due to the pore formation by these two different types of metabolites, accelerating the induction of osmotic breakdown in the cells, and consequently the mortality rate. Several studies reported that combination of Cyt1Aa and B. thuringiensis dipteran specific toxins (Cry4Aa, Cry4Ba, Cry11Aa and Cry2Aa) increased
12
their insecticidal activities (Crickmore et al., 1995; Wirth et al., 1997, 2003; Park et al., 2013). Lee et al. (1996) found that Cry1Ab-Cry1Ac and Cry1Aa-Cry1Ac combinations showed increase in toxicity against Lymantria dispar. The target and the mode of action of biosurfactant inside the insect remain unknown and are not well documented. One of the possible modes of action could be the significant reduction in the activities of acetylcholinesterse, α-carboxylesterase and acid phosphatases of larvae, as reported by Revathi et al. (2013). AG1 Biosurfactant was suggested to affect the larvae midgut and led to membrane perturbation, as evidenced by the histopathological examination of S. littoralis midgut. These effects could be attributed to its composition on lipopeptides (surfactin, fengycin, iturin and bacyllomicin) and polyketides (bacillaene, difficidin and macrolactin) (Ben Khedher et al., 2015) which are amphiphilic compounds with surfaceactive properties. It was reported that surfactin produced by B. subtilis was toxic to mosquitoes, causing heamolysis (Revathi et al., 2013) and mainly bactericidal activity. Iturin and fengycin displayed fungicidal activity, specifically fengycin targeted filamentous fungi (Ongena and Jacques, 2008). Lipopeptides are surface-active molecules, comprising a hydrophilic protein moiety attached to a hydrophobic lipid chain, which act to destabilise the phospholipid membrane of the target phytopathogen resulting in cell lysis (Heerklotz and Seelig, 2007). Similar effects were described in midguts of T. absoluta larvae treated with AG1 biosurfactant (Ben Khedher et al., 2015) and S. littoralis larvae treated with SPB1 biosurfactant (Ghribi et al., 2012). On the basis of the multiple midgut membrane proteins reported to bind Cry and Vip3A toxins from B. thuringiensis in different insects, we suggested to examine if biosurfactant metabolites could have the same mode of action as Cry toxins. Ligand blot analysis revealed that biotinylated AG1 biosurfactant specifically bound to three putative receptors of about 91, 72 and 64 kDa from S. littoralis BBMV. These putative receptors differ from those
13
recognized by the same biosurfactant in T. absoluta midgut and having molecular weights of about 68, 63, 44, 30 and 19 kDa (Ben Khedher et al., 2015). Although SPB1 biosurfactant exhibited the same toxicity and effects on S. littoralis midgut larvae, it recognized a single putative receptor of 45 kDa in S. littoralis BBMV (Ghribi et al., 2012). This difference in the number and molecular weights of putative receptors recognized by biosurfactants suggested variability in their (AG1 and SPB1 biosurfactants) lipopeptides and polyketides content. These properties make AG1 biosurfactant very promising to fight resistance in target pest insect. Abdelkefi-Mesrati et al. (2011) detected two putative Vip3Aa16 receptors at 55 kDa and 100 kDa in BBMV from S. littoralis. Despite multiple putative receptors from S. littoralis BBMV have been reported to bind Cry and Vip3Aa16 toxins (Oddou et al., 1993; Sanchis and Ellar, 1993; Abdelkefi-Mesrati et al., 2011; Ben Farhat-Touzri et al., 2013), the size of the three putative receptors shown in this study was reported for the first time. According to the heterologous competition assays, AG1 biosurfactant and Vip3Aa16 toxin did not compete for the same receptors. This would decrease cross resistance development by insect larvae, if resistance is due to receptor alteration. Similar results were found by Lee et al. (2006), who reported that there was no competition between Vip3Aa and Cry1Ac in Heliothis virescens and Helicoverpa zea BBMV. Sena et al. (2009) also showed that Cry1 and Vip3A occupy distinct binding sites on S. frugiperda BBMV. However, Cry1Fa occupies the same binding site as Cry1Ab, on S. frugiperda BBMV. Similarly, Vip3Af1 and Vip3Aa1 compete for the same receptor, probably because of their structure similarity (Sena et al., 2009). Therefore, the combination or rotation of AG1 biosurfactant and Vip3Aa16 toxin should be considered a strategic approach to delay resistance evolution against S. littoralis. Further studies should be done to identify and purify lipopeptides biosurfactant metabolites and to investigate their mode of action in S. littoralis midgut larvae. The identification of biosurfactant interacting proteins in the midgut of target
14
insects, not yet reported, might provide tools for enhancing biosurfactant action against major crop pests.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgement This work was financially supported by the “Tunisian Ministry of Higher Education and Scientific Research”.
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Zhang, S., Cheng, H., Gao, Y., Wang, G., Liang, G., Wu, K., 2009. Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin. Insect Biochem. Mol. Biol. 39, 421-429.
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Figures legends Fig. 1. Histopathological effects of Bacillus amyloliquefaciens AG1 biosurfactant on S. littoralis midgut: (A) untreated S. littoralis larvae showing regular epithelial cell and welldeveloped lumen content. (B) S. littoralis larvae treated with AG1 biosurfactant showed vacuolization,
demolished
epithelial
layer
undistinguished
basement
membrane.
Magnification 40×. Lu: Lumen, Bm: basal membrane, Am: apical membrane, V: vacuole.
Fig. 2. Mortality of AG1 biosurfactant and Vip3Aa16 at different ratios mixture against the first instar larvae of S. littoralis. (∆) AG1 biosurfactant alone, (■) Vip3Aa16 alone, (◊) AG1 biosurfactant-Vip3Aa16 ratio (1:0.25), (○) AG1 biosurfactant-Vip3Aa16 ratio (1:0.50), (▲) AG1 biosurfactant -Vip3Aa16 ratio (1:1).
Fig. 3. Detection of the three putative receptors (91, 72 and 64 kDa) for biotinylated Bacillus amyloliquefaciens AG1 biosurfactant in the midgut of S. littoralis by ligand blot. Lane 1: S. littoralis BBMV, Lane 2: BSA used as negative control and Lane 3: BBMV without incubation with the biotinylated AG1 biosurfactant but only with the detection reagents, also used as negative control.
Fig. 4. AG1 biosurfactant and Vip3Aa16 toxin binding to S. littoralis BBMV. Homologous competition: Lanes: 1- BBMV without incubation with the biotinylated AG1 biosurfactant but only with the detection reagents, used as negative control, 2- Biotinylated AG1 biosurfactant bound to BBMV, 3- Biotinylated AG1 biosurfactant and unlabeled AG1 biosurfactant (2× excess). Heterologous competition: 4- Biotinylated AG1 biosurfactant and unlabeled Vip3Aa16 (1× excess), 5- biotinylated AG1 biosurfactant and unlabeled Vip3Aa16 (2× excess), 6- biotinylated AG1 biosurfactant and unlabeled Vip3Aa16 (4× excess). Homologous
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competition: 7- biotinylated Vip3Aa16 bound to BBMV, 8- biotinylated Vip3Aa16 and unlabeled Vip3Aa16 (2× excess). Heterologous competition: 9- BBMV without incubation with the biotinylated Vip3Aa16 but only with the detection reagents, used as negative control, 10- Biotinylated Vip3Aa16 bound to BBMV, 11- Biotinylated Vip3Aa16 and unlabeled AG1 biosurfactant (1 × excess), 12- biotinylated Vip3Aa16 and unlabeled AG1 biosurfactant (2× excess), 13- biotinylated Vip3Aa16and unlabeled AG1 biosurfactant (4× excess).
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Table 1. Toxicity of AG1 biosurfactant and Vip3Aa16 toxin against the first instar larvae of S. littoralis. Toxins
LC50a (pmol/cm2)
LC50 (ng/cm2)
AG1 Biosurfactant
0.245 (0.17-0.31)
245 (179.53-312.43)
Vip3Aa16
3.39 (2.48-4.30)
305 (223.00-387.00)
a
Mean lethal concentration (LC50) was estimated by Probit analysis. 95% confidence limits
are given in parentheses.
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Table 2. Combination of AG1 biosurfactant and Vip3Aa16 toxin against the first instar larvae of S. littoralis. (AG1 biosurfactant:Vip3Aa16)
Predicted LC50(ng/cm2)
Experimental LC50 (ng/cm2)
SFb
1 : 0.25
255.03
190.65 (139.01-242.29)a
1.33
1 : 0.50
264.85
150.13 (122.75-177.51)
1.76
1:1
271.73
91.84 (63.02-120.66)
2.95
a
95% confidence limits.
b
Synergism factor (predicted LC50/experimental LC50).
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29
30
31
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Graphical abstract
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Highlights
Spodoptera littoralis is one of the most serious agricultural pest in the world.
B. amyloliquefaciens AG1 biosurfactant was active against S. littoralis larvae.
AG1 biosurfactant caused histological damages in the larval midgut of S. littoralis.
AG1 biosurfactant recognized midgut putative receptors of about 91, 72 and 64 kDa.
High toxicity and synergism between AG1 biosurfactant and Vip3Aa16 in S. littoralis.
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