Performance of microencapsulated Bacillus thuringiensis Cry pesticidal proteins

Performance of microencapsulated Bacillus thuringiensis Cry pesticidal proteins | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Performance of microencapsulated Bacillus thuringiensis Cry pesticidal proteins Jhones Luiz de Oliveira, Isabel Gómez, Jorge Sánchez, Mario Soberón, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1949207/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The entomopathogenic bacteria Bacillus thuringiensis (Bt) produce parasporal-crystal inclusions composed of different pesticidal proteins such as Cry, that show insecticidal activity against insect pests. Cry toxins are highly susceptible to degradation when exposed to adverse temperature and continuous sun-UV-light. Thus, encapsulation techniques are designed to improve their biopesticide performance and shelf-life. However, the effects of polymeric matrix encapsulation on the mechanism of action of Cry toxins produced by Bt kurstaki and Bt aizawai has not been evaluated. Here, we analyzed the solubilization, activation and the binding of Bt insecticidal Cry proteins to their receptors after microencapsulation and compared with commercial non-encapsulated Bt biopesticides. We show that solubilization is one step in the mechanism of action of these proteins that could limit Cry toxin action, the microencapsulation of Bt biopesticides did not alter protein profiles solubilization compared to those non-encapsulated, showing a 130 kDa (corresponding to Cry1 protoxin) and 70 kDa (corresponding to Cry2 protoxin) proteins. Activation with trypsin, chymotrypsin and larval midgut juice was analyzed, showing that this step is highly efficient, and proteins were cleaved producing similar ~ 55 to 65 kDa activated toxins in microencapsulated and non-encapsulated formulations. Binding assays with receptors that are present in brush border membrane vesicles (BBMV) of Manduca sexta and Spodoptera frugiperda larvae showed similar binding curves for conventional and microencapsulated formulations. Finally, LC 50 bioassays against these pests, showed no significant differences among the treatments. However, when these formulations were subjected to UV radiation, we observed for the microencapsulated Bt formulations provided higher mortality against S. frugiperda larvae, supporting a higher protective effect against degradation. Overall, our results show that microencapsulation of Bt biopesticides did not affect the mechanism of action of their pesticidal proteins while enhanced protection to UV radiation. These data will contribute to the development of more efficient Bt biopesticide formulations. Bt Microencapsulation Mechanism of Action Spodoptera frugiperda Manduca sexta Figures Figure 1 Figure 2 Figure 3 Figure 4 Key Messages Microencapsulation of Bt biopesticides did not alter protein profiles solubilization Binding assays with receptors in BBMV of Manduca sexta and Spodoptera frugiperda larvae showed similar binding curves for conventional and microencapsulated formulations Microencapsulated Bt formulations provided higher mortality against S . frugiperda larvae under UV radiation exposition 1. Introduction Agricultural crop production is basic for the economy of many countries. The expanding human population, combined with the environmental changes, rise the pressure to increase agricultural food production, in order to satisfy its greater demand (Bruinsma 2017 ). The green revolution was based mainly on the use of synthetic pesticides and fertilizers, which led to significant changes in the agricultural sector. However, over time, many insect pests have developed resistance to the chemicals used for their control (Tabashnik et al. 2014 ; Brevik et al. 2018 ; Richardson et al. 2020 ). In addition, the excessive use of these products has caused harmful effects in the environment, leading to severe soil, surface waters, and groundwater contamination. Currently it is highly accepted that crop protection from insect damage using ecologically safe strategies are urgently needed (Shiva 2016 ; Nicolopoulou-Stamati et al. 2016 ; Fernandes et al. 2020 ). In recent decades, the research concerning biological control agents has increased, with new products reaching the marketplace, among them pesticides based on microorganisms (Lacey et al. 2015 ; Wakefield 2018 ). Among these microorganism-based products, the entomopathogenic bacteria Bacillus thuringiensis (Bt) have shown excellent results in insect control (Chattopadhyay et al. 2017 ). Bt is a gram positive, spore-producing, rod-shaped bacterium that has been isolated from multiple ecosystems including water, soil, insects, dust, and tree leaves (Paulino-Lima et al. 2013 ; Gutiérrez et al. 2019 ). Bt produce parasporal crystal inclusions with insecticidal activity against different insect pests. The production of such crystals, differentiate these bacteria from other Bacillus species. These crystals are essentially composed of δ-endotoxins proteins that were initially named Cry, Cyt and Vip, which are responsible for the primary virulence effects of Bt pathogens (Knowles 1994 ; Adang et al. 2014 ). These Bt proteins are active against different insect orders including Lepidoptera, Coleoptera, Hymenoptera, and Diptera and also against nematodes (Frankenhuyzen 2009 ). Different protein families are produced by Bt bacteria, and a new classification of these proteins was recently updated (Crickmore et al. 2021 ). Thus from now on the Cry name will be used only for the Bt proteins that are composed of three domains and are classified accordingly to their primary sequence (Crickmore et al. 2021 ). Microbial control agents such as Bt-biopesticides are susceptible to degradation when applied to plants in the field. Most of the negative effects are due to exposure to adverse temperature and continuous sun UV-light, which reduce the effectiveness of these pesticidal proteins (Fernández-Chapa et al. 2019 ). It is in this context that studies in the formulation technology have increased, especially those analyzing encapsulation techniques. Such strategies have the primary objective of improving the performance of the biopesticides for insect control. Mainly, these studies have the following aims: (i) to protect the formulation and shelf life during storage; (ii) to ensure stability under field conditions, protecting against sunlight damage and adverse temperatures; (iii) to increase the residual activity after application; (iv) to increase the contact with target larvae after application; and (v) to improve compatibility with other management strategies (Kala et al. 2020 ). However, the adverse effects that different matrices used for microencapsulation on the different steps of the mechanism of action of Bt toxins such as solubilization, activation and binding to their specific receptors located in the larval gut of susceptible insects, has never been analyzed. In this work we determined all those steps of the mechanism of action, comparing commercial and microencapsulated formulations. Overall, our results show that microencapsulation of Bt formulations did not affect the mechanism of action of these formulations while enhanced protection to UV-radiation. 2. Material And Methods 2.1 Preparation of Bt microencapsulated systems B. thuringiensis aizawai (Bta) and B. thuringiensis kurstaki (Btk) organisms were obtained after pasteurization and growth in Luria broth medium from commercial products Xentari® and Dipel®, respectively. For the encapsulation procedure we used: gum arabic (10%) and maltodextrin (10%). The conditions for spray-drying were inlet temperature 90°C, outlet temperature 50°C, drying flow of 1.8 m 3 .min − 1 , feeding flow of 0.3 L.h − 1 , spray flow of 40 L. h − 1 and double sprayer of 1mm. 2.2 Solubilization and activation conditions of Cry proteins We used 50 mg of each formulated non-encapsulated products (Xentari®, Dipel®) and a mixture 60/40 of Dipel®/Xentari®, as well as the newly designed microencapsulated products (MP_Bta, MP_Btk) and a mixture 60/40 of MP_Btk/MP_Bta and dissolved them in 10 mL deionized water for 5 min. Spore/crystal mixtures were recovered by centrifugation (10 min at 12,000 xg ) and the pellet was suspended in 2 mL ddH 2 0 and stored at − 70°C. These pellets were considered the “total protein samples” as stated below. A sample of 10 µl from each formulation was mixed with 10 µL sample loading Laemmli buffer 2X (0.125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, and 0.01% bromophenol blue), heated 3 min at 100°C, and analyzed by loading in 10% SDS-PAGE. Protein concentration of the final spore/crystal mixtures suspended in ddH 2 0 was estimated by Bradford method (1976) (Bradford 1976 ) using a bovine serum albumin (BSA) standard curve as reference. As controls the Bt kurstaki and Bt aizawai strains used for the microencapsulation preparation and Bt kurstaki (HD-1 strain) were grown 72 h at 30°C in three different mediums: HCT, SP and Embrapa (Lecadet et al. 1980 ; Monnerat et al. 2007 ; Soberón et al. 2007 ) to produce the parasporal-crystals. After bacterial sporulation, spore/crystal mixtures were recovered and washed three times with wash solution (300 mM NaCl, 10 mM EDTA) and three times with 1 mM PMSF. After these washing steps the spore/crystal mixture was then suspended in ddH 2 0 and analyzed in SDS-PAGE as described above. For solubilization total protein samples were incubated 15 min in solubilization buffer (50 mM NaOH and 0.2% b-mercaptoethanol) at 4°C. After incubation the samples were centrifuged (12,000 xg for 10 min) and the soluble fraction (supernatant) and the insoluble fraction (pellet) were separated and analyzed in 10% SDS-PAGE. The toxin activation was performed by using trypsin, chymotrypsin and midgut juice from S. frugiperda larvae. The midgut juice sample containing the intrinsic proteases that are present in the insect gut lumen was obtained after dissection of the intestinal tissue and centrifugation at 12,000 xg for 10 min. Before activation, the pH of the solubilized samples was adjusted to pH 8.5 by adding ¼ volume of 1M Tris-HCl pH 8, and the activation kinetics analyses were performed at different incubation times (15 min, 30 min, 1 h and 2 h) with trypsin (1:20 w/w, enzyme/substrate ratio); with chymotrypsin (1:5, 2:1 and 5:1 ratio w/w, enzyme/substrate ratio) and with Sf -midgut juice (5% v/v). All digestions were carried out at 37 ºC and incubations were stopped by the addition of 1 mM PMSF (final concentration). Activated toxins were loaded onto 10% SDS-PAGE gels to verify the extent of digestion and the protein concentration was determined by Bradford assays. The solubilization efficiency and activation efficiency was calculated for the non-encapsulated and encapsulated formulations. The solubilization efficiency was obtained by analyzing the protein concentration of the soluble fraction divided by the total protein concentration in the sample using the following formula: Solubilization efficiency (%) = (µg protein in soluble fraction / µg protein in the Total protein sample) x 100. The activation efficiency was obtained by analyzing the protein concentration of the activated sample divided by the protein concentration in the solubilized sample using the following formula: Activation efficiency (%) = (µg protein in activated fraction / µg protein in solubilized fraction) x 100. To evaluate the solubilization of the formulations in the presence of midgut juice, the different samples were incubated with midgut juice at different concentrations as indicated in the text for 1 h and 24 h and centrifugated 10 min at 12,000 xg . The solubilized proteins were analyzed in 10% SDS-PAGE gels. 2.3 Preparation of brush border membrane vesicles (BBMV) BBMVs were prepared from third instar S. frugiperda and M. sexta larvae according to the method of Wolfersberger et al., ( 1987 ) (Wolfersberger et al. 1987 ), as modified by Reuveni and Dunn, ( 1991 )(Reuveni and Dunn 1991 ). The midgut tissue was dissected from the larvae in storage buffer (300 mM mannitol, 20 mM 2-mercaptoethanol, 5 mM EGTA, 1 mM EDTA, 0.1 mM PMSF, 150 µg.mL -1 pepstatin A, 100 µg.mL -1 leupeptin, 1 µg. mL -1 soybean trypsin inhibitor, 10 mM HEPES 2.4 µg.mL -1 neomycin sulfate, pH 7.5) immediately frozen and stored at -80 ºC until used. For BBMV preparation, frozen midguts were mechanically homogenized in homogenization buffer (200 mM mannitol, 10 mM ascorbic acid, 5 mM EDTA, 0.03% w/v PMSF, 1% mM PVPP, 0.2 mM leupeptin, 2 mM DTT, 10 mM HEPES pH 7.4) for 10 sec. One volume of 24 mM MgCl 2 was added, and the mixture was incubated for 10 min at 4 ºC. Following centrifugation of the mixture (10 min, 6,000 xg at 4 ºC), the supernatant was further centrifuged (30 min, 30,000 xg at 4 ºC) and the final pellet was suspended in 200 mM mannitol, 1 mM DTT, 1 mM Hepes –Tris, pH 7.4, and stored at − 80 ºC until used. The APN and ALP activities in Sf BBMVs and Ms BBMVs were measured. APN activity was assayed using L-leucine-p-nitroanilide as substrate, and ALP activity was assayed using p -nitrophenyl phosphate as substrate (Arenas et al. 2010 ). Protein content was measured by the DC protein dye method (Bio-Rad) using BSA as a standard (Pierce). The initial rate at 405 nm (Ultrospec II spectrophotometer; GE Healthcare) was used to calculate specific enzymatic activity of both enzymes. The absorption coefficient of p -nitroanilide used was 9.9 × 10 − 3 mol 1 − 1 . One unit of specific APN activity was defined as the amount of enzyme catalyzing the hydrolysis of 1 µmol of L-leucine- p -nitroanilide min − 1 mg of protein − 1 at 25°C. One unit of specific ALP activity was defined as the amount of enzyme producing 1 µmol of nitrophenol min − 1 mg of protein − 1 at 25°C. Nitrophenol concentration was calculated by using a standard curve of 4-nitrophenol in 0.5 mM MgCl 2 , 100 mM Tris, pH 9.5. The APN and ALP specific activities showed enrichment factors values between 2.5 and 4.2-fold in the purified BBMV when compared with their initial homogenate. 2.4 Binding of Cry1 toxins to S. frugiperda and M. sexta BBMV Binding assays of Cry1 activated toxins to BBMV from 3rd instar S. frugiperda ( Sf BBMV) and M. sexta ( Ms BBMV) larvae were performed as follows. In these assays different concentrations (0.152–2.27 µM) of activated Cry proteins from the different samples were incubated with 10 µg BBMV protein for 1 h at room temperature in 100 µl of binding buffer (PBS, 0.1%, BSA, 0.1% Tween 20, pH 7.6). A control of Sf BBMV and Ms BBMV without toxin incubation was included in these assays. After incubation, the unbound toxin was removed by centrifugation for 10 min at 12,850 ×g . The pellet containing Sf BBMV or Ms BBMV and bound toxin was washed twice with 100 µL binding buffer, suspended in 10 µL of PBS, and mixed with 10 µL sample loading Laemmli buffer 2X. Samples were boiled 3 min, loaded in 10% SDS-PAGE gels and electro transferred to polyvinylidene difluoride membrane (PVDF) (Immobilion-P, Bio-Vin). The PVDF membrane was blocked with 0.5% milk powder and 0.1% Tween 20 for 1 h under agitation, and bound Cry1A toxins was revealed by western blot using anti-Cry1A polyclonal antibody (1/30,000 dilution; 1 h) as primary antibody. As secondary antibody, a goat anti-rabbit antibody coupled to horseradish peroxidase (HRP) enzyme was used (Santa Cruz Biotechnology, Dallas, TX, USA) (1/10,000 dilution; 1 h), followed by luminol (Santa Cruz Biotechnology Inc.) treatment, according to the manufacturer’s instructions. 2.5 ELISA binding assays Sf BBMV and Ms BBMV proteins were used to coat 96-well ELISA plates (2.5 µg/well for Sf BBMV and 1.0 µg/well Ms BBMV) (Rochester, NY, USA). The different activated protein samples at different concentrations (0.152–2.27 µM) were incubated with the BBMV-coated ELISA plates. Unbound toxins were removed by washing with PBS buffer, followed by three washes with PBS supplemented with 0.1% Tween 20. Bound toxins were detected using anti-Cry1A polyclonal antibody (1:20,000 dilution) and secondary goat anti-rabbit antibody conjugated with HRP enzyme (1:20,000 dilution). Finally, o -phenylenediamine (Sigma) and H 2 O 2 were used as substrates for peroxidase activity detection. Reaction was stopped by adding 50 µL of 5 M HCl and OD 490 was measured using an ELISA microplate reader (PerkinElmer, Waltham, MA, USA). Negative controls were performed in parallel, where the BBMV proteins were not used to coat the ELISA plate wells. All experiments were done in triplicate and plotted using GraphPad Prism 9. 2.6 Toxicity bioassays Toxicity bioassays of non-encapsulated products (Xentari®, Dipel® and Dipel®/Xentari®) and microencapsulated products (MP_Bta, MP_Btk and MP_Btk/Bta) were performed against neonate S. frugiperda and M. sexta larvae. We used the surface contamination method. Different concentrations of the formulations (0.5 to 20 µg of formulation/cm 2 of artificial diet) were applied to the diet surface contained in 128-well polystyrene plates (Bio-BA-128 bioassay trays; C-D International, Inc.). A total of 72 larvae per formulation concentration were used (one larva per well). The mortality was recorded after 7 days, larvae were considered dead if no movement was apparent. The medium lethal concentration (LC 50 ) was estimated by Probit analysis (Polo-PC LeOra Software) and the fiducial limits in each LC 50 value were estimated. 2.7 Effect of UV radiation of the Bt formulations on the insecticidal activity against S. frugiperda larvae. To evaluate the effect of UV exposure of the Bt formulations on the toxicity against S. frugiperda , the bioassay-plates containing 14 µg of formulation/cm 2 of the different formulations in the surface of the diet were subjected to UV exposure (254 nm) in a Laminar Flow for 15 min. After UV exposure, one neonate S. frugiperda larva was added to each plate well. 3. Results 3.1 Protein profile of Bt strains compared to Bt formulations The samples containing Bt aizawai control strain grown in different sporulation media showed the expected Cry1 protoxin size of 130 kDa, while for the samples of Bt kurstaki control strains showed the presence of two mayor protoxin protein bands of 130 and 70 kDa (Figure S1). These molecular masses correspond to the typical sizes of proteins that belong to the Cry1 and Cry2 classes (Crickmore et al. 1998 ; Lereclus et al. 2000 ), respectively. The non-encapsulated and the microencapsulated formulations showed similar protein profiles to the corresponding Bt kurstaki and Bt aizawai control strains (Figure S1). However, it is clear that non-encapsulated commercial products Xentari® and Dipel® showed a higher concentration of total protein than the MP_Bta and MP_Btk microencapsulated formulations after suspension of similar water volume. Analysis of the images, by using ImageJ program, indicated that Xentari formulation has 2.2-fold more protein that MP-Bta and Dipel formulation has 1.9-fold more protein than MP-Btk. 3.2 Analysis of solubilization efficiency Figure 1 shows the protein profile after solubilization in 50 mM NaOH and 0.2% β-mercaptoethanol at 4°C. The protoxin proteins of 130 and 70 kDa were solubilized under these conditions. The protein concentration in each band was estimated by using the corresponding BSA control curve as shown in the figure and the corresponding values of the solubilized formulations are shown in Table 1 . Table 1 Protein quantification of formulations and solubilization efficiencies Formulations Formulations in Water (µg) Water Supernatant Discarded (µg) Solubilized - Pellet (µg) Solubilized – Supernatant (µg) Solubilization efficiency (%) 1 – Xentari ® .2.13 0.28 µg 0.178 0.49 26.50 2 – Dipel ® 1.33 0.15 0.069 0.66 55.93 3 – Dipel ® /Xentari ® 1.43 0.13 0.014 0.62 47.69 4 – MP_Bta 1.49 0.06 0.020 0.34 23.86 5 – MP_Btk 1.16 0.10 0.021 0.50 47.28 6 – MP_Btk/Bta 1.52 0.12 0.009 0.62 44.22 These data indicate that solubilization is not an efficient step, since solubilization efficiency values ranged from 23–55%. We selected to work with this extreme condition since it was more efficient for solubilization of Cry2 protein than solubilization in 50 mM bicarbonate buffer pH 10.5 supplemented with 0.2% β-mercaptoethanol where the 70 kDa protein from Dipel® formulation corresponding to Cry2Ab was not observed as a solubilized protein (data not shown). These data show that solubilization efficiency of Xentari® and Dipel® formulation was just slightly higher than MP_Bta (0.11-fold higher) and MP_Btk (0.18-fold higher), respectively. Also, that the solubilization of protoxins from formulations made with Bt kurstaki was approximately 2-fold (1.92- 2.11-fold) more efficient than formulations with Bt aizawai . We also analyzed the solubilization process using midgut juice from S. frugiperda . For this step, a standardization of gastric juice concentration was initially performed (data not shown). Different concentrations ranging from 0.5 to 50% v/v of midgut juice were tested, with a concentration of 30% v/v being selected for the following tests. The solubilization was carried out during 1 h and 24 h. It is worth mentioning that the gastric juice was extracted from 3rd instar larvae of S. frugiperda and it showed a pH of a 9.5. The addition of water for the dilution of midgut juice did not change the pH of the solution. Figure 2 shows the protein profile in SDS-PAGE gel after solubilization with the larval midgut juice. When the formulations were incubated with the larval midgut juice there was no protein solubilization even after 24 h, and the proteins bands were only observed in the pellet samples (Fig. 2 A). These data indicate that solubilization may be a critical step inside the larva. We then made a pH adjustment of the gastric juice, using 50 mM NaOH solution to reach a pH 10.5 value. Under this condition, 1 h incubation was not enough to solubilize the Cry proteins, since the ~ 130 kDa protein band was only observed in the pellet (data not shown). However, when the incubation time was increased up to 24 h (Fig. 2 B) a complete solubilization was observed. We did not observe protein bands in the pellet and the protoxin proteins were found activated in the supernatant, where protein bands of ~ 55 to 60 kDa were observed for all formulations. These proteins were processed by endogenous proteases that are present in the midgut fluid of the insects. The microencapsulation did not influence the solubilization profile compared to the non-encapsulated samples. However, analysis of the image with ImageJ program indicated that the concentration of solubilized-activated toxin in the microencapsulated samples was 0.2-0.5-fold higher than the commercial non-encapsulated sample, indicating that solubilization was improved in the microencapsulated samples. 3.3 Analysis of activation efficiencies In the midgut of insects, the most abundant proteolytic enzymes are serine proteases, such as trypsins and chymotrypsins (Liu et al. 2010 ). Therefore, in vitro activation assays were performed with trypsin, chymotrypsin and also with midgut juice isolated from S. frugiperda . Incubation time kinetics were performed (15, 30 min, 1 h and 2 h), as well as, concentration tests, varying the enzyme/protein ratios for chymotrypsin (1:5, 2:1 and 5:1). For trypsin, we used a ratio of 1:20 (enzyme/protein m/m) and midgut juice was used at a concentration of 5% v/v. The standardization results showed that for all tested enzymes, the process of toxin activation occurred after 15 min incubation, and for the subsequent tests the incubation times were done only for 15 min and 1 h. For chymotrypsin, the 2:1 and 5:1 ratio (enzyme/protein) did not show differences in the protein profile, and the 2:1 ratio was selected for the following tests. Figure 2 S shows the SDS-PAGE gels with the protein profiles after activation of the the different formulations with trypsin (Fig. 2 S-A), chymotrypsin (Fig. 2 S-B) and midgut juice (Fig. 2 S-C). We did not observe differences in the protein profiles of the activated toxins from microencapsulated when compared with the non-encapsulated formulated products (Fig. 2 S). Table 2 shows the activation efficiencies after analysis of bands intensity by ImageJ® software. These data showed that the activation process is more efficient than solubilization, since up to 96% efficiency in activation for some samples was observed. Also, that both types of formulations ,microencapsulated and non-encapsulated, showed rather similar values. In general, the non-encapsulated products were slightly less efficiently activated than the microencapsulated formulations when treated with single commercial proteases such as trypsin and chymotrypsin. For example, when trypsin was used as protease, a 0.1-fold higher activation efficiency of was observed for MP_Bta when compared with Xentari® formulation and MP_Btk showed a 0.18-fold higher activation efficiency than Dipel®. However, when midgut juice was used the microencapsulated formulations showed a slightly lower activation efficiency than the commercial non-encapsulated products (0.10-fold lower for MP-Bta vs Xentari® and 0.18-fold lower for MP-Btk vs Dipel®). Table 2 Protein quantification of formulations and calculation of activation efficiencies. Formulations Solubilized (µg) Trypsin (µg) Chymotrypsin (µg) Midgut juice (µg) Trypsin Activation Efficiency (%) Chymotrypsin Activation Efficiency (%) Midgut juice Activation Efficiency (%) 1 – Xentari ® 0.49 0.42 .2g 0.28 0.35 84.83 58.16 72.20 2 – Dipel ® 0.66 0.54 0.40 0.44 81.86 60.30 66.11 3 – Dipel ® / Xentari ® 0.62 0.53 0.32 0.38 86.23 51.93 62.08 4 – MP_Bta 0.34 0.32 0.19 0.24 93.80 55.59 66.74 5 – MP_Btk 0.50 0.48 0.29 0.28 96.73 58.00 55.98 6 – MP_Btk/Bta 0.62 0.48 0.30 0.33 88.18 47.74 53.07 3.4 Binding of toxins from conventional and microencapsulated formulations to BBMV from S. frugiperda and M. sexta Figure S3 shows the results of binding the formulations of Xentari ® and Dipel ® and their respective microencapsulated formulations (MP_Bta and MP_Btk) with BBMVs from S. frugiperda and M. sexta . For these assays, the activated samples of the formulations were incubated with BBMV from the different insects. After incubation, the bound toxin to the BBMV was recovered by centrifugation, washed and analyzed in the western-blot analysis. Binding of activated toxins from the different formulations to Sf BBMV (Figure S3-A and B) and to Ms BBMV (Figure S3-C and D) directly correlated with the concentration of toxin used. The binding analysis after densitometry of the bands using the ImageJ Software is shown in Fig. 3 . The binding with the receptor was calculated by performing a normalization with the intensity of the band corresponding only to the formulation, without the presence of BBMV (data not shown). In these binding experiments, the medium effective concentration (EC 50 ) value is the concentration of ligand at which half of the target is present in the bound state (Table 3 ). Table 3 EC 50 values for conventional and microencapsulated formulations evaluated with M. sexta and S. frugiperda BBMV. EC 50 values (µM) Manduca Sexta Spodoptera frugiperda Xentari ® 0.08 0.29 Dipel ® 0.32 0.94 MP_Bta 0.14 0.67 MP_Btk 0.75 1.12 The lower EC 50 values found for the assays with BBMV of M. sexta indicate a greater binding affinity than to S. frugiperda BBMV, which corroborates with previous data (Gómez et al. 2018 ) that indicate greater susceptibility of M. sexta to Cry1A toxins when compared to S. frugiperda . These results also show that the Cry1A proteins from conventional formulations have higher affinity than to microencapsulated formulations, since they showed lower EC 50 values. However, it is important to take into account that the activated toxins samples used in these assays contain a mixture of proteins, thus the affinity values are apparent values that cannot be adjudicated to a single protein. In addition, Fig. 3 shows that the biding curves for both types of formulations were highly similar, supporting that there were no significant variations in the binding process to BBMV. To confirm these results, ELISA binding assays were also performed between the different formulations and Sf BBMV and Ms BBMV (Figure S4). The data show similar binding curves confirming that samples from microencapsulated products have similar binding to BBMV than samples from non-encapsulated formulations. 3.5 Toxicity bioassays The biological activity of the different formulations was assayed against S. frugiperda and M. sexta larvae. Table 4 shows the medium lethal concentration (LC 50 ) values. Table 4 Insecticidal activity of different biological formulation (conventional and microencapsulated) against first instar S. frugiperda and M. sexta larvae. Formulations a LC 50 values in µg/cm 2 (fiducial limits) Biological Spodoptera frugiperda Manduca sexta Xentari® 0.72 (0.49–1.20) 0.74 (0.54–1.04) Dipel® 1.11 (0.68–1.82) 0.68 (0.51–0.95) Dipel®/Xentari® 1.01 (0.68–1.47) 1.09 (0.82–1.56) MP_Bta 0.61 (0.44–0.85) 0.84 (0.61–1.20) MP_Btk 1.16 (0.82–1.64) 0.47 (0.33–0.63) MP_Btk/Bta 1.06 (0.69–1.62) 0.80 (0.58–1.14) a Values are means of at least 3 repetitions; fiducial limits are 95% confidence intervals The results shown in Table 4 indicate that for the biological formulations, no significant differences were observed between commercial products and microencapsulated products. Although for M. sexta the MP-Btk showed higher potency than MP-Bta, while for S. frugiperda larvae the opposite was observed since MP-Bta showed higher potency than MP-Btk. These tests were carried out on a diet, and the same condition was applied to all treatments without exposure to external factors. However, it has been explained that formulated products may improve effectiveness under environmental conditions, such as UV radiation, high temperature, among others. It was in this context that we decided to carry out tests applying an external factor such as UV light to evaluate the effect of these formulations. The formulations at 14 µg formulation/cm 2 were exposed to UV radiation, and the mortality tests were performed. Figure 4 show the S. frugiperda mortality data evaluated after 2, 4 and 7 days. It is worth noting that the Bt aizawai strain in the commercial product Xentari® provide effective control of S. frugiperda . Our data showed that Xentari® and its respective microencapsulated formulation MP-Bta, showed similar insecticidal activity after UV irradiation. However, we can observe differences between the Dipel® formulation and its respective microencapsulated formulation MP-Btk (Fig. 4 B). For these formulations, a great difference after 4 days of application was observed, and the samples with higher concentration of microencapsulated product had higher mortality up to 75% mortality than Dipel which induced 20.8% mortality. After 7 days these values were 87.5% and 66.7%, respectively, indicating a much better UV damage protection of MP-Btk microencapsulation resulting in higher mortality. We can also highlight the treatment containing the proportion of 60% of MP-Btk and 40% of commercial Dipel that after 2, 4 and 7 days showed mortality values of 4.2%, 87.5% and 95.8%, respectively, being the most effective combination of formulations. 4. Discussion In this study we analyzed the different steps in the mechanism of action of de Cry toxins to determine if the formulation itself may affect performance of Bt Cry toxins. Specifically, we compared commercial non-encapsulated products with microencapsulated products named MP-Bta and MP- Btk. The same Bt strains were used as the basic material to make Xentari and MP-Bta, or to make Dipel and MP-Btk. According to the manufacturer, Dipel® is formulated with Bt kurstaki , containing the proteins Cry1Aa (15%), Cry1Ab (39%), Cry1Ac (23%), Cry2Aa (22%), while Xentari® is formulated with from Bt aizawai , containing Cry1Aa (21%), Cry1Ab (53%), Cry1Ca (20%) and Cry1Da (6%) (Valent 2022 ). Our first result showed that non-encapsulated commercial products showed a higher concentration of total protein than the microencapsulated formulations after suspension of similar water volume. Similar protein concentrations of these formulations were then subjected to solubilization analysis. We did not observe changes in the profile of the solubilized protoxin bands of the SDS-PAGE analysis, indicating that the matrices used in the microencapsulation process did not influence the solubilization of Cry proteins. However, the results presented in Table 1 show that none of the tested formulations have 100% solubilization efficiency. It important to mention that these formulations (conventional and microencapsulated) present a mixture of Cry toxins, which may influence their solubilization process. Aronson et al. ( 1991 ), tested different solubilization buffers for Bta and Btk strains, obtaining solubilization efficiencies that varied from 8 to 70%, and these authors concluded that the solubilization differences between these strains may be related to the protoxin composition of the crystals. Our data confirmed that Btk strain showed higher solubilization efficiency in relation to the Bta strain. In relation to conventional and microencapsulated formulations, variations in solubilization values were observed, with microencapsulated formulations showing lower solubilization values in in vitro conditions. However, when midgut juice was used to solubilize these samples, it was evident that microencapsulated formulations showed a better solubilization than the conventional formulations. The solubilization of Bt parasporal crystals is a fundamental step to initiate the intoxication process. It was described that the solubilization process is facilitated by the physicochemical conditions (mainly pH and reducing conditions) that are found in the host's digestive fluids (Gill et al. 1992 ; Bravo and Soberón 2008 ; Deist et al. 2014 ). In the work by Du et al. ( 1994 ), the authors compared two strains (Bta and Btk) in relation to the solubility of their parasporal crystals. The authors showed that for both strains, the solubilization started with pH values of 9.5, reaching a complete solubilization only when pH reaches values above pH 11, and it was proposed that similar high pH conditions could be found inside the midgut lumen of the lepidopteran larvae. The authors describe that the different insecticidal crystals produced present distorted and destabilized disulfide bonds, which can also influence the processes of solubilization and toxicity. In another study, Naimov et al. ( 2008 ), evaluated the solubilization of crystals of Bt thompsoni HD542 composed of Cry15Aa toxin. The authors tested different buffers and pH values (ranging from 6.0 to 11.0) with or without the reducing agent DTT. The authors obtained complete protein solubilization only at pH 11.0 in sodium hydrogen carbonate buffer and CAPS buffer, respectively. In the presence of the reducing agent, the reduction of disulfide bonds allowed solubilization at pH 10. Other buffers (ethanolamine, Tris, borate-buffered saline) did not solubilize a significant amount of protein, with or without the addition of DTT. Here we show that the midgut lumen on S. frugiperda has a pH of 9.5, and solubilization is extremely low under these conditions. Only after increasing the pH up to 10.5 we were able to observe complete solubilization and activation of Cry proteins. However, our results also demonstrate that incubation time is also important for solubilization. We reinforce the importance of studying additional factors, which can contribute to altering the pH values of the midgut, consequently altering the solubility and toxicity of these proteins, such as, for example, the different instars and also the geographic region where the pest may be found (Bravo and Soberón 2008 ). It could be worth to isolate in the future some Cry mutants with improved solubilization, specially at lower pH values that may correlate with improved toxicity. During the activation step, approximately 40–60 amino acids from the N-terminus are removed by proteases for 70 kDa and 130 kDa protoxins. For the 130 kDa protoxins, in addition to N-terminal processing, about 500–600 amino acids are also cleaved out from the C-terminus, in both cases the active Cry toxins resulted in activate toxin proteins of ~ 55 to 65 kDa (Bravo et al. 2002 ; Bergamasco et al. 2013 ; Gómez et al. 2014 ). Our results corroborate the proteins found in Bta and Btk were correctly activated, since there was no great difference in the proteolytic patterns of the different formulations. It was possible to observe a double band of ~ 70 and ~ 65 kDa for the formulations containing mixture of Cry1 and for the formulations containing Cry2 it was possible to observe a band of ~ 55 kDa. Previously, Liu et al. ( 2020 ), obtained similar proteolytic profiles when they evaluated activation of Cry1Ac and Cry2Ab protoxins by proteases from the midgut juice of Helicoverpa armigera larvae. We also noticed that the profile observed for the cleavage with chymotrypsin differs from the other treatments, being possible to observe with more precision the bands of ~ 55kDa for the formulations containing Cry2 protoxin. However, the protein profiles obtained after incubation with trypsin and midgut juice were very similar, suggesting that it is possible that midgut juice contains high levels of trypsin and low levels of chymotrypsin. Actually, Saadaoui, Rouis and Jaoua ( 2009 ) showed that trypsin-like activity is predominant in the midgut homogenate of Ephestia kuehniella . However, it is worth noting that the activation step has been related to resistance mechanisms. Improper activation such as insufficient processing or over digestion can result in insect resistance to Cry protoxin action (Domínguez-Arrizabalaga et al. 2020 ). After the activation process, the active toxins bypass the peritrophic matrix to interact with the brush border membrane (BBM) of the midgut tissue. This membrane is considered the main target for toxins. The toxin then undergoes a complex sequential binding steps with the different receptors present in BBM, which results in its insertion into the membrane, with the consequent formation of pores, and osmotic lysis, leading to insect death (Lu et al. 2013 ). In the literature, we found several works that report that Cry toxin receptors are located in BBM, including cadherin-like (Aimanova et al. 2006 ; Zhang et al. 2020 ; Jin et al. 2021 ), aminopeptidase N (APN) (Wei et al. 2016 ; Shao et al. 2018 ) and alkaline phosphatase (ALP) (Likitvivatanavong et al. 2011 ; Stalinski et al. 2016 ). Our studies concluded that activated toxin samples obtained from the different formulated products showed similar binding to BBMV from two lepidopteran insects. Bel et al. ( 2017 ) studied the binding of different Cry toxins (Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry2Ab) with BBMVs from different insects ( S. exigua, S. litura, A. ipsilon and H. armigera ) reporting different binding curves for these toxins, supporting that these toxins display different affinity values to the BBMVs from the different larval species. In our studies we are analyzing a mixture of activated toxins and we were not able to provide specific affinity values for each protein. Bioassay data against M. sexta and S. frugiperda larvae supported that the different procedures that were used in the formulation of these products did not affect the mechanism of action of Cry toxins, since no differences in LC 50 values between commercial and microencapsulated products were observed. It is worth mentioning that these tests were carried out on a diet, under the same condition such as been reported by Eski et al. ( 2019 ) where microencapsulated formulations of an indigenous strain of B. thuringiensis (Se13) was evaluated against S. exigua. showing similar LC 50 values compared to the commercial formulations on laboratory conditions. However, we show here that when Dipel® formulation was treated with UV and analyzed against S. frugiperda larvae, the toxicity was lower than MP-Btk formulation. The microencapsulation helped to maintain the insecticidal effect of Btk, most likely due to an improved protection of crystals and spores. Another point that is worth to mention is that the results showed that the combination of conventional and microencapsulated formulations can be an important management strategy. Khorramvatan et al. (2014), compared the effect of three polymers (starch, gelatin and sodium alginate) as wall materials for the production of a microencapsulated Bt formulation. The authors observed that for the alginate polymer, the viability of the spores was 90% after exposure to long−term UV radiation (UVB 385 nm), while the viability of non−microencapsulated spores under this condition was only 40%. In another study,) Jalali et al. (2020) used the Pickering−emulsion technique to perform the microencapsulation of Bt. The authors tested different materials such as latex particles, graphene oxide nanosheets and olive oil as protective materials. The authors evaluated toxicity of these formulations against E. kuehniella larvae after UV−A radiation. The results showed that the combination of matrices at a concentration of 0.045% allowed an effective control and greater protection against radiation, supporting the effectiveness of microencapsulation. Overall our results provide important information for the development of future more efficient biopesticide formulations. 5. Conclusions The results presented in this study demonstrate that after ingestion, the solubilization of the different formulations (conventional and microencapsulated) formulations is the critical point and in vivo it seems to be relatively low. The microencapsulated products showed a slightly higher solubilization efficiency. However, these differences were not significant when toxicity was analyzed, since both types of formulations displayed similar toxicity values. The final conclusion is that the initial steps in the mechanisms of action of these pesticidal proteins (solubilization, activation and binding with receptors present in BBMVs) were not significantly affected due to the microencapsulation procedure. The results also demonstrate the importance of studying other factors, such as the pH change of the midgut, which influences the solubilization process. In addition, our data showed that when subjected to external factors such as UV radiation, the microencapsulated formulations showed greater control efficiency in shorter times and we propose that the mixture of conventional and microencapsulated formulations may be an important management strategy for the development of future biopesticide formulations. Declarations Author’s contributions AB, MS, RAP and JO designed experiments. JO, IG and JS collected, prepared material, conducted experiments and collected data. AB, MS and RAP supervised experiments. JO analyzed data. JO, AB, MS and RAP wrote the manuscript. All authors read and approved the manuscript. Acknowledgements and Funding The authors are grateful for financial support provided by the São Paulo State Research Foundation (FAPESP, Grants 2017/21004-5, 2018/21142-1, and 2020/12779-6) and the National Council for Scientific and Technological Development (CNPq) Data availability All data are available from the corresponding author upon reasonable request. Conflict of interest: The authors declare that they have no relevant financial or non-financial interests to disclose. Ethics approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. References Adang MJ, Crickmore N, Jurat-Fuentes JL ( 2014 ) Chapter Two - Diversity of Bacillus thuringiensis Crystal Toxins and Mechanism of Action . In : Dhadialla TS , Gill SS ( eds ) Advances in Insect Physiology . Academic Press , pp 39 – 87 Aimanova KG, Zhuang M, Gill SS ( 2006 ) Expression of Cry1Ac cadherin receptors in insect midgut and cell lines . 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Springer, Berlin, Heidelberg , pp 143–168 Wei J, Zhang M, Liang G, et al ( 2016 ) APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea . Sci Rep 6 :19179. https://doi.org/10.1038/srep19179 Wolfersberger M, Luethy P, Maurer A, et al ( 1987 ) Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae) . Comp Biochem Physiol A Physiol 86 :301–308. https://doi.org/10.1016/0300-9629(87)90334-3 Zhang J, Jin M, Yang Y, et al ( 2020 ) The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda . Toxins 12 :375. https://doi.org/10.3390/toxins12060375 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1949207","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":128023864,"identity":"a2cc18a2-07f2-45f2-b4ac-781282b05c2a","order_by":0,"name":"Jhones Luiz de Oliveira","email":"","orcid":"","institution":"Faculty of Agronomy and Veterinary Sciences, São Paulo State University (UNESP)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jhones","middleName":"Luiz","lastName":"de Oliveira","suffix":""},{"id":128023867,"identity":"d3a701d0-89ba-4485-b3df-4c59039842d8","order_by":1,"name":"Isabel Gómez","email":"","orcid":"","institution":"Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Gómez","suffix":""},{"id":128023869,"identity":"bc265704-c24b-45d2-bb95-8838834a1e7f","order_by":2,"name":"Jorge Sánchez","email":"","orcid":"","institution":"Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"","lastName":"Sánchez","suffix":""},{"id":128023871,"identity":"5ca1c6a6-b6bf-4397-9f09-8c4d954aa8bb","order_by":3,"name":"Mario Soberón","email":"","orcid":"","institution":"Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Soberón","suffix":""},{"id":128023874,"identity":"d4df9a92-dad6-4c1e-8ae4-319c2da76993","order_by":4,"name":"Ricardo Antonio Polanczyk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYLCCB0DMz8DABuOz4VYKAwlALNlAshaDA8RqMW8/e/BBQs0deeMbyc8efKhgkOcXO8D2uAKPFpkzeckGCceeGW67kWZuOOMMg+HM2QnshmfwaJFgyDGTSGA7nGB2I8FMmreNIcHgdgIb0GN4tPC/AWr5dzjBeEb6NyK1SABtSWw7nGAAZBCr5V2yQWLfYaA33pRJzjgjAfRLYrshfoflHnzw4dthef729G0SHyps5Pmlk489xKeFgYEHSgskgI0AYkb8GhBa+A8QUDgKRsEoGAUjFgAATjhJW+Sr9qMAAAAASUVORK5CYII=","orcid":"","institution":"Faculty of Agronomy and Veterinary Sciences, São Paulo State University (UNESP)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"Antonio","lastName":"Polanczyk","suffix":""},{"id":128023876,"identity":"d9ddf358-c9d6-4b01-9c3a-895799be8fbc","order_by":5,"name":"Alejandra Bravo","email":"","orcid":"","institution":"Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alejandra","middleName":"","lastName":"Bravo","suffix":""}],"badges":[],"createdAt":"2022-08-10 12:59:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1949207/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1949207/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25151689,"identity":"f3c9696c-5a1e-4337-bf52-ab0f41c902f4","added_by":"auto","created_at":"2022-08-12 16:58:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":469118,"visible":true,"origin":"","legend":"\u003cp\u003eA) Protein profile of the supernatant from formulations after solubilization in NaOH Buffer (50mM); B) BSA control curve. Formulations: 1, Xentari\u003csup\u003e®\u003c/sup\u003e; 2, Dipel\u003csup\u003e®\u003c/sup\u003e; 3, Dipel\u003csup\u003e®\u003c/sup\u003e/Xentari\u003csup\u003e®\u003c/sup\u003e; 4, MP_Bta; 5, MP_Btk; and 6, MP_Btk/Bta.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1949207/v1/0fcbe45f52911bec2d5f276f.png"},{"id":25151690,"identity":"d1a379ec-ff09-4daa-9870-8f0d2b5ab9b1","added_by":"auto","created_at":"2022-08-12 16:58:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":414228,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the protein profiles on SDS-PAGE of different formulations after solubilization in midgut juice (30% v/v) isolated from \u003cem\u003eS. frugiperda\u003c/em\u003e larvae. A) Protein profile of formulations incubated in midgut juice pH 9.5 for 24 h; B) Protein profile of formulations incubated in midgut juice pH 10.5 for 24 h.\u0026nbsp;Formulations: 1, Xentari\u003csup\u003e®\u003c/sup\u003e; 2, Dipel\u003csup\u003e®\u003c/sup\u003e; 3, Dipel\u003csup\u003e®\u003c/sup\u003e/Xentari\u003csup\u003e®\u003c/sup\u003e; 4, MP_Bta; 5, MP_Btk; and 6, MP_Btk/Bta.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1949207/v1/da04f31181a1a404d2c93eba.png"},{"id":25152351,"identity":"4f5e5956-94d4-4f4c-9e92-e6c2f38401ae","added_by":"auto","created_at":"2022-08-12 17:03:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31928,"visible":true,"origin":"","legend":"\u003cp\u003eBinding interaction of activated toxin samples from the different formulations to BBMV from \u003cem\u003eS. frugiperda\u003c/em\u003e (A and B) and \u003cem\u003eM. sexta\u003c/em\u003e (C and D). A sample of 10 μg BBMV was incubated with different concentrations of activated toxins present in different formulations. Plots were constructed after densitometry analysis of the 65 kDa band shown in figure S3.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1949207/v1/2ce0df32d09dbb3737e68dfd.png"},{"id":25151692,"identity":"e35741b9-d7d9-4b81-aeea-9c270e730741","added_by":"auto","created_at":"2022-08-12 16:58:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":114558,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of UV radiation on the toxicity of different Bt formulations against \u003cem\u003eS. frugiperda \u003c/em\u003elarvae\u003cem\u003e.\u003c/em\u003e A) Xentari\u003csup\u003e®\u003c/sup\u003e formulation and mixtures with different proportions of MP_Bta; B) Dipel\u003csup\u003e®\u003c/sup\u003e formulation and mixtures with different proportions of MP_Btk; C) Dipel\u003csup\u003e®\u003c/sup\u003e/Xentari\u003csup\u003e®\u003c/sup\u003e formulation and mixtures with different proportions of MP_Btak/Bta.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1949207/v1/e47d46fb58554908e174cc35.jpg"},{"id":25152352,"identity":"de278a4d-b8be-4915-8eb1-3ee562fcdbab","added_by":"auto","created_at":"2022-08-12 17:03:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2596817,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1949207/v1/89fb2c42-42dd-4108-ba9c-1c32222e8066.pdf"},{"id":25151693,"identity":"61334286-82b5-4fe5-8496-4840278018f0","added_by":"auto","created_at":"2022-08-12 16:58:28","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":736101,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPEMENTARYMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-1949207/v1/08a15a280e337db38e9b9664.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Performance of microencapsulated Bacillus thuringiensis Cry pesticidal proteins","fulltext":[{"header":"Key Messages","content":"\u003cul\u003e\n \u003cli\u003eMicroencapsulation of Bt biopesticides did not alter protein profiles solubilization\u003c/li\u003e\n \u003cli\u003eBinding assays with receptors in BBMV of \u003cem\u003eManduca sexta\u003c/em\u003e and \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e larvae showed similar binding curves for conventional and microencapsulated formulations\u003c/li\u003e\n \u003cli\u003eMicroencapsulated Bt formulations provided higher mortality against S\u003cem\u003e. frugiperda\u003c/em\u003e larvae under UV radiation exposition\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eAgricultural crop production is basic for the economy of many countries. The expanding human population, combined with the environmental changes, rise the pressure to increase agricultural food production, in order to satisfy its greater demand (Bruinsma \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The green revolution was based mainly on the use of synthetic pesticides and fertilizers, which led to significant changes in the agricultural sector. However, over time, many insect pests have developed resistance to the chemicals used for their control (Tabashnik et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Brevik et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Richardson et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, the excessive use of these products has caused harmful effects in the environment, leading to severe soil, surface waters, and groundwater contamination. Currently it is highly accepted that crop protection from insect damage using ecologically safe strategies are urgently needed (Shiva \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nicolopoulou-Stamati et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fernandes et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In recent decades, the research concerning biological control agents has increased, with new products reaching the marketplace, among them pesticides based on microorganisms (Lacey et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wakefield \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Among these microorganism-based products, the entomopathogenic bacteria \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) have shown excellent results in insect control (Chattopadhyay et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Bt is a gram positive, spore-producing, rod-shaped bacterium that has been isolated from multiple ecosystems including water, soil, insects, dust, and tree leaves (Paulino-Lima et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Guti\u0026eacute;rrez et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBt produce parasporal crystal inclusions with insecticidal activity against different insect pests. The production of such crystals, differentiate these bacteria from other \u003cem\u003eBacillus\u003c/em\u003e species. These crystals are essentially composed of δ-endotoxins proteins that were initially named Cry, Cyt and Vip, which are responsible for the primary virulence effects of Bt pathogens (Knowles \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Adang et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These Bt proteins are active against different insect orders including Lepidoptera, Coleoptera, Hymenoptera, and Diptera and also against nematodes (Frankenhuyzen \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Different protein families are produced by Bt bacteria, and a new classification of these proteins was recently updated (Crickmore et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thus from now on the Cry name will be used only for the Bt proteins that are composed of three domains and are classified accordingly to their primary sequence (Crickmore et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicrobial control agents such as Bt-biopesticides are susceptible to degradation when applied to plants in the field. Most of the negative effects are due to exposure to adverse temperature and continuous sun UV-light, which reduce the effectiveness of these pesticidal proteins (Fern\u0026aacute;ndez-Chapa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is in this context that studies in the formulation technology have increased, especially those analyzing encapsulation techniques. Such strategies have the primary objective of improving the performance of the biopesticides for insect control. Mainly, these studies have the following aims: (i) to protect the formulation and shelf life during storage; (ii) to ensure stability under field conditions, protecting against sunlight damage and adverse temperatures; (iii) to increase the residual activity after application; (iv) to increase the contact with target larvae after application; and (v) to improve compatibility with other management strategies (Kala et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the adverse effects that different matrices used for microencapsulation on the different steps of the mechanism of action of Bt toxins such as solubilization, activation and binding to their specific receptors located in the larval gut of susceptible insects, has never been analyzed.\u003c/p\u003e \u003cp\u003eIn this work we determined all those steps of the mechanism of action, comparing commercial and microencapsulated formulations. Overall, our results show that microencapsulation of Bt formulations did not affect the mechanism of action of these formulations while enhanced protection to UV-radiation.\u003c/p\u003e"},{"header":"2. Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of Bt microencapsulated systems\u003c/h2\u003e \u003cp\u003e \u003cem\u003eB. thuringiensis aizawai\u003c/em\u003e (Bta) and \u003cem\u003eB. thuringiensis kurstaki\u003c/em\u003e (Btk) organisms were obtained after pasteurization and growth in Luria broth medium from commercial products Xentari\u0026reg; and Dipel\u0026reg;, respectively. For the encapsulation procedure we used: gum arabic (10%) and maltodextrin (10%). The conditions for spray-drying were inlet temperature 90\u0026deg;C, outlet temperature 50\u0026deg;C, drying flow of 1.8 m\u003csup\u003e3\u003c/sup\u003e.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, feeding flow of 0.3 L.h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, spray flow of 40 L. h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and double sprayer of 1mm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2 Solubilization and activation conditions of Cry proteins\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eWe used 50 mg of each formulated non-encapsulated products (Xentari\u0026reg;, Dipel\u0026reg;) and a mixture 60/40 of Dipel\u0026reg;/Xentari\u0026reg;, as well as the newly designed microencapsulated products (MP_Bta, MP_Btk) and a mixture 60/40 of MP_Btk/MP_Bta and dissolved them in 10 mL deionized water for 5 min. Spore/crystal mixtures were recovered by centrifugation (10 min at 12,000 \u003cem\u003exg\u003c/em\u003e) and the pellet was suspended in 2 mL ddH\u003csub\u003e2\u003c/sub\u003e0 and stored at \u0026minus;\u0026thinsp;70\u0026deg;C. These pellets were considered the \u0026ldquo;total protein samples\u0026rdquo; as stated below. A sample of 10 \u0026micro;l from each formulation was mixed with 10 \u0026micro;L sample loading Laemmli buffer 2X (0.125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, and 0.01% bromophenol blue), heated 3 min at 100\u0026deg;C, and analyzed by loading in 10% SDS-PAGE. Protein concentration of the final spore/crystal mixtures suspended in ddH\u003csub\u003e2\u003c/sub\u003e0 was estimated by Bradford method (1976) (Bradford \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1976\u003c/span\u003e) using a bovine serum albumin (BSA) standard curve as reference. As controls the Bt \u003cem\u003ekurstaki\u003c/em\u003e and Bt \u003cem\u003eaizawai\u003c/em\u003e strains used for the microencapsulation preparation and Bt \u003cem\u003ekurstaki\u003c/em\u003e (HD-1 strain) were grown 72 h at 30\u0026deg;C in three different mediums: HCT, SP and Embrapa (Lecadet et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Monnerat et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sober\u0026oacute;n et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) to produce the parasporal-crystals. After bacterial sporulation, spore/crystal mixtures were recovered and washed three times with wash solution (300 mM NaCl, 10 mM EDTA) and three times with 1 mM PMSF. After these washing steps the spore/crystal mixture was then suspended in ddH\u003csub\u003e2\u003c/sub\u003e0 and analyzed in SDS-PAGE as described above.\u003c/p\u003e \u003cp\u003eFor solubilization total protein samples were incubated 15 min in solubilization buffer (50 mM NaOH and 0.2% b-mercaptoethanol) at 4\u0026deg;C. After incubation the samples were centrifuged (12,000 \u003cem\u003exg\u003c/em\u003e for 10 min) and the soluble fraction (supernatant) and the insoluble fraction (pellet) were separated and analyzed in 10% SDS-PAGE.\u003c/p\u003e \u003cp\u003eThe toxin activation was performed by using trypsin, chymotrypsin and midgut juice from \u003cem\u003eS. frugiperda\u003c/em\u003e larvae. The midgut juice sample containing the intrinsic proteases that are present in the insect gut lumen was obtained after dissection of the intestinal tissue and centrifugation at 12,000 \u003cem\u003exg\u003c/em\u003e for 10 min. Before activation, the pH of the solubilized samples was adjusted to pH 8.5 by adding \u0026frac14; volume of 1M Tris-HCl pH 8, and the activation kinetics analyses were performed at different incubation times (15 min, 30 min, 1 h and 2 h) with trypsin (1:20 w/w, enzyme/substrate ratio); with chymotrypsin (1:5, 2:1 and 5:1 ratio w/w, enzyme/substrate ratio) and with \u003cem\u003eSf\u003c/em\u003e-midgut juice (5% v/v). All digestions were carried out at 37 \u0026ordm;C and incubations were stopped by the addition of 1 mM PMSF (final concentration). Activated toxins were loaded onto 10% SDS-PAGE gels to verify the extent of digestion and the protein concentration was determined by Bradford assays.\u003c/p\u003e \u003cp\u003eThe solubilization efficiency and activation efficiency was calculated for the non-encapsulated and encapsulated formulations. The solubilization efficiency was obtained by analyzing the protein concentration of the soluble fraction divided by the total protein concentration in the sample using the following formula: Solubilization efficiency (%) = (\u0026micro;g protein in soluble fraction / \u0026micro;g protein in the Total protein sample) x 100. The activation efficiency was obtained by analyzing the protein concentration of the activated sample divided by the protein concentration in the solubilized sample using the following formula: Activation efficiency (%) = (\u0026micro;g protein in activated fraction / \u0026micro;g protein in solubilized fraction) x 100.\u003c/p\u003e \u003cp\u003eTo evaluate the solubilization of the formulations in the presence of midgut juice, the different samples were incubated with midgut juice at different concentrations as indicated in the text for 1 h and 24 h and centrifugated 10 min at 12,000 \u003cem\u003exg\u003c/em\u003e. The solubilized proteins were analyzed in 10% SDS-PAGE gels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of brush border membrane vesicles (BBMV)\u003c/h2\u003e \u003cp\u003eBBMVs were prepared from third instar \u003cem\u003eS. frugiperda\u003c/em\u003e and \u003cem\u003eM. sexta\u003c/em\u003e larvae according to the method of Wolfersberger et al., (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) (Wolfersberger et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), as modified by Reuveni and Dunn, (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1991\u003c/span\u003e)(Reuveni and Dunn \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). The midgut tissue was dissected from the larvae in storage buffer (300 mM mannitol, 20 mM 2-mercaptoethanol, 5 mM EGTA, 1 mM EDTA, 0.1 mM PMSF, 150 \u0026micro;g.mL\u003csup\u003e-1\u003c/sup\u003e pepstatin A, 100 \u0026micro;g.mL\u003csup\u003e-1\u003c/sup\u003e leupeptin, 1 \u0026micro;g. mL\u003csup\u003e-1\u003c/sup\u003e soybean trypsin inhibitor, 10 mM HEPES 2.4 \u0026micro;g.mL\u003csup\u003e-1\u003c/sup\u003e neomycin sulfate, pH 7.5) immediately frozen and stored at -80 \u0026ordm;C until used. For BBMV preparation, frozen midguts were mechanically homogenized in homogenization buffer (200 mM mannitol, 10 mM ascorbic acid, 5 mM EDTA, 0.03% w/v PMSF, 1% mM PVPP, 0.2 mM leupeptin, 2 mM DTT, 10 mM HEPES pH 7.4) for 10 sec. One volume of 24 mM MgCl\u003csub\u003e2\u003c/sub\u003e was added, and the mixture was incubated for 10 min at 4 \u0026ordm;C. Following centrifugation of the mixture (10 min, 6,000 \u003cem\u003exg\u003c/em\u003e at 4 \u0026ordm;C), the supernatant was further centrifuged (30 min, 30,000 \u003cem\u003exg\u003c/em\u003e at 4 \u0026ordm;C) and the final pellet was suspended in 200 mM mannitol, 1 mM DTT, 1 mM Hepes \u0026ndash;Tris, pH 7.4, and stored at \u0026minus;\u0026thinsp;80 \u0026ordm;C until used.\u003c/p\u003e \u003cp\u003eThe APN and ALP activities in \u003cem\u003eSf\u003c/em\u003eBBMVs and \u003cem\u003eMs\u003c/em\u003eBBMVs were measured. APN activity was assayed using L-leucine-p-nitroanilide as substrate, and ALP activity was assayed using \u003cem\u003ep\u003c/em\u003e-nitrophenyl phosphate as substrate (Arenas et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Protein content was measured by the DC protein dye method (Bio-Rad) using BSA as a standard (Pierce). The initial rate at 405 nm (Ultrospec II spectrophotometer; GE Healthcare) was used to calculate specific enzymatic activity of both enzymes. The absorption coefficient of \u003cem\u003ep\u003c/em\u003e-nitroanilide used was 9.9 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e mol 1\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. One unit of specific APN activity was defined as the amount of enzyme catalyzing the hydrolysis of 1 \u0026micro;mol of L-leucine-\u003cem\u003ep\u003c/em\u003e-nitroanilide min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg of protein\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 25\u0026deg;C. One unit of specific ALP activity was defined as the amount of enzyme producing 1 \u0026micro;mol of nitrophenol min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg of protein\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 25\u0026deg;C. Nitrophenol concentration was calculated by using a standard curve of 4-nitrophenol in 0.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 100 mM Tris, pH 9.5. The APN and ALP specific activities showed enrichment factors values between 2.5 and 4.2-fold in the purified BBMV when compared with their initial homogenate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Binding of Cry1 toxins to \u003cem\u003eS. frugiperda\u003c/em\u003e and \u003cem\u003eM. sexta\u003c/em\u003e BBMV\u003c/h2\u003e \u003cp\u003eBinding assays of Cry1 activated toxins to BBMV from 3rd instar \u003cem\u003eS. frugiperda\u003c/em\u003e (\u003cem\u003eSf\u003c/em\u003eBBMV) and \u003cem\u003eM. sexta\u003c/em\u003e (\u003cem\u003eMs\u003c/em\u003eBBMV) larvae were performed as follows. In these assays different concentrations (0.152\u0026ndash;2.27 \u0026micro;M) of activated Cry proteins from the different samples were incubated with 10 \u0026micro;g BBMV protein for 1 h at room temperature in 100 \u0026micro;l of binding buffer (PBS, 0.1%, BSA, 0.1% Tween 20, pH 7.6). A control of \u003cem\u003eSf\u003c/em\u003eBBMV and \u003cem\u003eMs\u003c/em\u003eBBMV without toxin incubation was included in these assays. After incubation, the unbound toxin was removed by centrifugation for 10 min at 12,850 \u003cem\u003e\u0026times;g\u003c/em\u003e. The pellet containing \u003cem\u003eSf\u003c/em\u003eBBMV or \u003cem\u003eMs\u003c/em\u003eBBMV and bound toxin was washed twice with 100 \u0026micro;L binding buffer, suspended in 10 \u0026micro;L of PBS, and mixed with 10 \u0026micro;L sample loading Laemmli buffer 2X. Samples were boiled 3 min, loaded in 10% SDS-PAGE gels and electro transferred to polyvinylidene difluoride membrane (PVDF) (Immobilion-P, Bio-Vin). The PVDF membrane was blocked with 0.5% milk powder and 0.1% Tween 20 for 1 h under agitation, and bound Cry1A toxins was revealed by western blot using anti-Cry1A polyclonal antibody (1/30,000 dilution; 1 h) as primary antibody. As secondary antibody, a goat anti-rabbit antibody coupled to horseradish peroxidase (HRP) enzyme was used (Santa Cruz Biotechnology, Dallas, TX, USA) (1/10,000 dilution; 1 h), followed by luminol (Santa Cruz Biotechnology Inc.) treatment, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 ELISA binding assays\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSf\u003c/em\u003eBBMV and \u003cem\u003eMs\u003c/em\u003eBBMV proteins were used to coat 96-well ELISA plates (2.5 \u0026micro;g/well for \u003cem\u003eSf\u003c/em\u003eBBMV and 1.0 \u0026micro;g/well \u003cem\u003eMs\u003c/em\u003eBBMV) (Rochester, NY, USA). The different activated protein samples at different concentrations (0.152\u0026ndash;2.27 \u0026micro;M) were incubated with the BBMV-coated ELISA plates. Unbound toxins were removed by washing with PBS buffer, followed by three washes with PBS supplemented with 0.1% Tween 20. Bound toxins were detected using anti-Cry1A polyclonal antibody (1:20,000 dilution) and secondary goat anti-rabbit antibody conjugated with HRP enzyme (1:20,000 dilution). Finally, \u003cem\u003eo\u003c/em\u003e-phenylenediamine (Sigma) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were used as substrates for peroxidase activity detection. Reaction was stopped by adding 50 \u0026micro;L of 5 M HCl and OD\u003csub\u003e490\u003c/sub\u003e was measured using an ELISA microplate reader (PerkinElmer, Waltham, MA, USA). Negative controls were performed in parallel, where the BBMV proteins were not used to coat the ELISA plate wells. All experiments were done in triplicate and plotted using GraphPad Prism 9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Toxicity bioassays\u003c/h2\u003e \u003cp\u003eToxicity bioassays of non-encapsulated products (Xentari\u0026reg;, Dipel\u0026reg; and Dipel\u0026reg;/Xentari\u0026reg;) and microencapsulated products (MP_Bta, MP_Btk and MP_Btk/Bta) were performed against neonate \u003cem\u003eS. frugiperda\u003c/em\u003e and \u003cem\u003eM. sexta\u003c/em\u003e larvae. We used the surface contamination method. Different concentrations of the formulations (0.5 to 20 \u0026micro;g of formulation/cm\u003csup\u003e2\u003c/sup\u003e of artificial diet) were applied to the diet surface contained in 128-well polystyrene plates (Bio-BA-128 bioassay trays; C-D International, Inc.). A total of 72 larvae per formulation concentration were used (one larva per well). The mortality was recorded after 7 days, larvae were considered dead if no movement was apparent. The medium lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) was estimated by Probit analysis (Polo-PC LeOra Software) and the fiducial limits in each LC\u003csub\u003e50\u003c/sub\u003e value were estimated.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.7 Effect of UV radiation of the Bt formulations on the insecticidal activity against\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eS. frugiperda\u003c/span\u003e \u003cb\u003elarvae.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the effect of UV exposure of the Bt formulations on the toxicity against \u003cem\u003eS. frugiperda\u003c/em\u003e, the bioassay-plates containing 14 \u0026micro;g of formulation/cm\u003csup\u003e2\u003c/sup\u003e of the different formulations in the surface of the diet were subjected to UV exposure (254 nm) in a Laminar Flow for 15 min. After UV exposure, one neonate \u003cem\u003eS. frugiperda\u003c/em\u003e larva was added to each plate well.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Protein profile of Bt strains compared to Bt formulations\u003c/h2\u003e \u003cp\u003eThe samples containing Bt \u003cem\u003eaizawai\u003c/em\u003e control strain grown in different sporulation media showed the expected Cry1 protoxin size of 130 kDa, while for the samples of Bt \u003cem\u003ekurstaki\u003c/em\u003e control strains showed the presence of two mayor protoxin protein bands of 130 and 70 kDa (Figure S1). These molecular masses correspond to the typical sizes of proteins that belong to the Cry1 and Cry2 classes (Crickmore et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Lereclus et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), respectively. The non-encapsulated and the microencapsulated formulations showed similar protein profiles to the corresponding Bt \u003cem\u003ekurstaki\u003c/em\u003e and Bt \u003cem\u003eaizawai\u003c/em\u003e control strains (Figure S1). However, it is clear that non-encapsulated commercial products Xentari\u0026reg; and Dipel\u0026reg; showed a higher concentration of total protein than the MP_Bta and MP_Btk microencapsulated formulations after suspension of similar water volume. Analysis of the images, by using ImageJ program, indicated that Xentari formulation has 2.2-fold more protein that MP-Bta and Dipel formulation has 1.9-fold more protein than MP-Btk.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Analysis of solubilization efficiency\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the protein profile after solubilization in 50 mM NaOH and 0.2% β-mercaptoethanol at 4\u0026deg;C. The protoxin proteins of 130 and 70 kDa were solubilized under these conditions. The protein concentration in each band was estimated by using the corresponding BSA control curve as shown in the figure and the corresponding values of the solubilized formulations are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtein quantification of formulations and solubilization efficiencies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFormulations in Water (\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWater Supernatant Discarded (\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSolubilized -\u003c/p\u003e \u003cp\u003ePellet (\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolubilized \u0026ndash; Supernatant (\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSolubilization efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 \u0026ndash; Xentari\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e.2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.28 \u003cb\u003e\u0026micro;g\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e26.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 \u0026ndash; Dipel\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e55.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3 \u0026ndash; Dipel\u003csup\u003e\u0026reg;\u003c/sup\u003e/Xentari\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 \u0026ndash; MP_Bta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e23.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 \u0026ndash; MP_Btk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6 \u0026ndash; MP_Btk/Bta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese data indicate that solubilization is not an efficient step, since solubilization efficiency values ranged from 23\u0026ndash;55%. We selected to work with this extreme condition since it was more efficient for solubilization of Cry2 protein than solubilization in 50 mM bicarbonate buffer pH 10.5 supplemented with 0.2% β-mercaptoethanol where the 70 kDa protein from Dipel\u0026reg; formulation corresponding to Cry2Ab was not observed as a solubilized protein (data not shown).\u003c/p\u003e \u003cp\u003eThese data show that solubilization efficiency of Xentari\u0026reg; and Dipel\u0026reg; formulation was just slightly higher than MP_Bta (0.11-fold higher) and MP_Btk (0.18-fold higher), respectively. Also, that the solubilization of protoxins from formulations made with Bt \u003cem\u003ekurstaki\u003c/em\u003e was approximately 2-fold (1.92- 2.11-fold) more efficient than formulations with Bt \u003cem\u003eaizawai\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe also analyzed the solubilization process using midgut juice from \u003cem\u003eS. frugiperda\u003c/em\u003e. For this step, a standardization of gastric juice concentration was initially performed (data not shown). Different concentrations ranging from 0.5 to 50% v/v of midgut juice were tested, with a concentration of 30% v/v being selected for the following tests. The solubilization was carried out during 1 h and 24 h. It is worth mentioning that the gastric juice was extracted from 3rd instar larvae of \u003cem\u003eS. frugiperda\u003c/em\u003e and it showed a pH of a 9.5. The addition of water for the dilution of midgut juice did not change the pH of the solution.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the protein profile in SDS-PAGE gel after solubilization with the larval midgut juice. When the formulations were incubated with the larval midgut juice there was no protein solubilization even after 24 h, and the proteins bands were only observed in the pellet samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These data indicate that solubilization may be a critical step inside the larva. We then made a pH adjustment of the gastric juice, using 50 mM NaOH solution to reach a pH 10.5 value. Under this condition, 1 h incubation was not enough to solubilize the Cry proteins, since the ~\u0026thinsp;130 kDa protein band was only observed in the pellet (data not shown). However, when the incubation time was increased up to 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) a complete solubilization was observed. We did not observe protein bands in the pellet and the protoxin proteins were found activated in the supernatant, where protein bands of ~\u0026thinsp;55 to 60 kDa were observed for all formulations. These proteins were processed by endogenous proteases that are present in the midgut fluid of the insects. The microencapsulation did not influence the solubilization profile compared to the non-encapsulated samples. However, analysis of the image with ImageJ program indicated that the concentration of solubilized-activated toxin in the microencapsulated samples was 0.2-0.5-fold higher than the commercial non-encapsulated sample, indicating that solubilization was improved in the microencapsulated samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Analysis of activation efficiencies\u003c/h2\u003e \u003cp\u003eIn the midgut of insects, the most abundant proteolytic enzymes are serine proteases, such as trypsins and chymotrypsins (Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, \u003cem\u003ein vitro\u003c/em\u003e activation assays were performed with trypsin, chymotrypsin and also with midgut juice isolated from \u003cem\u003eS. frugiperda\u003c/em\u003e. Incubation time kinetics were performed (15, 30 min, 1 h and 2 h), as well as, concentration tests, varying the enzyme/protein ratios for chymotrypsin (1:5, 2:1 and 5:1). For trypsin, we used a ratio of 1:20 (enzyme/protein m/m) and midgut juice was used at a concentration of 5% v/v.\u003c/p\u003e \u003cp\u003eThe standardization results showed that for all tested enzymes, the process of toxin activation occurred after 15 min incubation, and for the subsequent tests the incubation times were done only for 15 min and 1 h. For chymotrypsin, the 2:1 and 5:1 ratio (enzyme/protein) did not show differences in the protein profile, and the 2:1 ratio was selected for the following tests.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS shows the SDS-PAGE gels with the protein profiles after activation of the the different formulations with trypsin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS-A), chymotrypsin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS-B) and midgut juice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS-C). We did not observe differences in the protein profiles of the activated toxins from microencapsulated when compared with the non-encapsulated formulated products (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the activation efficiencies after analysis of bands intensity by ImageJ\u0026reg; software. These data showed that the activation process is more efficient than solubilization, since up to 96% efficiency in activation for some samples was observed. Also, that both types of formulations ,microencapsulated and non-encapsulated, showed rather similar values. In general, the non-encapsulated products were slightly less efficiently activated than the microencapsulated formulations when treated with single commercial proteases such as trypsin and chymotrypsin. For example, when trypsin was used as protease, a 0.1-fold higher activation efficiency of was observed for MP_Bta when compared with Xentari\u0026reg; formulation and MP_Btk showed a 0.18-fold higher activation efficiency than Dipel\u0026reg;. However, when midgut juice was used the microencapsulated formulations showed a slightly lower activation efficiency than the commercial non-encapsulated products (0.10-fold lower for MP-Bta vs Xentari\u0026reg; and 0.18-fold lower for MP-Btk vs Dipel\u0026reg;).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtein quantification of formulations and calculation of activation efficiencies.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolubilized\u003c/p\u003e \u003cp\u003e(\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003cp\u003e(\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChymotrypsin (\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMidgut juice\u003c/p\u003e \u003cp\u003e(\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003cp\u003eActivation Efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChymotrypsin Activation Efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMidgut juice\u003c/p\u003e \u003cp\u003eActivation Efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 \u0026ndash; Xentari\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.42\u003cb\u003e.2g\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e84.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e58.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e72.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 \u0026ndash; Dipel\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e81.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e60.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e66.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3 \u0026ndash; Dipel\u003csup\u003e\u0026reg;\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eXentari\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e86.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e51.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 \u0026ndash; MP_Bta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e55.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e66.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 \u0026ndash; MP_Btk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e96.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e58.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e55.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6 \u0026ndash; MP_Btk/Bta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e47.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e53.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Binding of toxins from conventional and microencapsulated formulations to BBMV from\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eS. frugiperda\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eM. sexta\u003c/span\u003e\u003c/p\u003e \u003cp\u003eFigure S3 shows the results of binding the formulations of Xentari\u003csup\u003e\u0026reg;\u003c/sup\u003e and Dipel\u003csup\u003e\u0026reg;\u003c/sup\u003e and their respective microencapsulated formulations (MP_Bta and MP_Btk) with BBMVs from \u003cem\u003eS. frugiperda\u003c/em\u003e and \u003cem\u003eM. sexta\u003c/em\u003e. For these assays, the activated samples of the formulations were incubated with BBMV from the different insects. After incubation, the bound toxin to the BBMV was recovered by centrifugation, washed and analyzed in the western-blot analysis. Binding of activated toxins from the different formulations to \u003cem\u003eSf\u003c/em\u003eBBMV (Figure S3-A and B) and to \u003cem\u003eMs\u003c/em\u003eBBMV (Figure S3-C and D) directly correlated with the concentration of toxin used. The binding analysis after densitometry of the bands using the ImageJ Software is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The binding with the receptor was calculated by performing a normalization with the intensity of the band corresponding only to the formulation, without the presence of BBMV (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn these binding experiments, the medium effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) value is the concentration of ligand at which half of the target is present in the bound state (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e values for conventional and microencapsulated formulations evaluated with \u003cem\u003eM. sexta\u003c/em\u003e and \u003cem\u003eS. frugiperda\u003c/em\u003e BBMV.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e values (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eManduca Sexta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXentari\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDipel\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMP_Bta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMP_Btk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe lower EC\u003csub\u003e50\u003c/sub\u003e values found for the assays with BBMV of \u003cem\u003eM. sexta\u003c/em\u003e indicate a greater binding affinity than to \u003cem\u003eS. frugiperda\u003c/em\u003e BBMV, which corroborates with previous data (G\u0026oacute;mez et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) that indicate greater susceptibility of \u003cem\u003eM. sexta\u003c/em\u003e to Cry1A toxins when compared to \u003cem\u003eS. frugiperda\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThese results also show that the Cry1A proteins from conventional formulations have higher affinity than to microencapsulated formulations, since they showed lower EC\u003csub\u003e50\u003c/sub\u003e values. However, it is important to take into account that the activated toxins samples used in these assays contain a mixture of proteins, thus the affinity values are apparent values that cannot be adjudicated to a single protein. In addition, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the biding curves for both types of formulations were highly similar, supporting that there were no significant variations in the binding process to BBMV.\u003c/p\u003e \u003cp\u003eTo confirm these results, ELISA binding assays were also performed between the different formulations and \u003cem\u003eSf\u003c/em\u003eBBMV and \u003cem\u003eMs\u003c/em\u003eBBMV (Figure S4). The data show similar binding curves confirming that samples from microencapsulated products have similar binding to BBMV than samples from non-encapsulated formulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Toxicity bioassays\u003c/h2\u003e \u003cp\u003eThe biological activity of the different formulations was assayed against \u003cem\u003eS. frugiperda\u003c/em\u003e and \u003cem\u003eM. sexta\u003c/em\u003e larvae. Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the medium lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) values.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInsecticidal activity of different biological formulation (conventional and microencapsulated) against first instar \u003cem\u003eS. frugiperda\u003c/em\u003e and \u003cem\u003eM. sexta\u003c/em\u003e larvae.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e LC\u003csub\u003e50\u003c/sub\u003e values in \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e (fiducial limits)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBiological\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSpodoptera frugiperda\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eManduca sexta\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXentari\u0026reg;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.72 (0.49\u0026ndash;1.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.74 (0.54\u0026ndash;1.04)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDipel\u0026reg;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.11 (0.68\u0026ndash;1.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.68 (0.51\u0026ndash;0.95)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDipel\u0026reg;/Xentari\u0026reg;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.01 (0.68\u0026ndash;1.47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.09 (0.82\u0026ndash;1.56)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMP_Bta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.61 (0.44\u0026ndash;0.85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.84 (0.61\u0026ndash;1.20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMP_Btk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.16 (0.82\u0026ndash;1.64)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.47 (0.33\u0026ndash;0.63)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMP_Btk/Bta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.06 (0.69\u0026ndash;1.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.80 (0.58\u0026ndash;1.14)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003ea\u003c/sup\u003e Values are means of at least 3 repetitions; fiducial limits are 95% confidence intervals\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results shown in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e indicate that for the biological formulations, no significant differences were observed between commercial products and microencapsulated products. Although for \u003cem\u003eM. sexta\u003c/em\u003e the MP-Btk showed higher potency than MP-Bta, while for \u003cem\u003eS. frugiperda\u003c/em\u003e larvae the opposite was observed since MP-Bta showed higher potency than MP-Btk. These tests were carried out on a diet, and the same condition was applied to all treatments without exposure to external factors. However, it has been explained that formulated products may improve effectiveness under environmental conditions, such as UV radiation, high temperature, among others. It was in this context that we decided to carry out tests applying an external factor such as UV light to evaluate the effect of these formulations.\u003c/p\u003e\u003cp\u003eThe formulations at 14 \u0026micro;g formulation/cm\u003csup\u003e2\u003c/sup\u003e were exposed to UV radiation, and the mortality tests were performed. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e show the \u003cem\u003eS. frugiperda\u003c/em\u003e mortality data evaluated after 2, 4 and 7 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is worth noting that the Bt aizawai strain in the commercial product Xentari\u0026reg; provide effective control of \u003cem\u003eS. frugiperda\u003c/em\u003e. Our data showed that Xentari\u0026reg; and its respective microencapsulated formulation MP-Bta, showed similar insecticidal activity after UV irradiation. However, we can observe differences between the Dipel\u0026reg; formulation and its respective microencapsulated formulation MP-Btk (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). For these formulations, a great difference after 4 days of application was observed, and the samples with higher concentration of microencapsulated product had higher mortality up to 75% mortality than Dipel which induced 20.8% mortality. After 7 days these values were 87.5% and 66.7%, respectively, indicating a much better UV damage protection of MP-Btk microencapsulation resulting in higher mortality. We can also highlight the treatment containing the proportion of 60% of MP-Btk and 40% of commercial Dipel that after 2, 4 and 7 days showed mortality values of 4.2%, 87.5% and 95.8%, respectively, being the most effective combination of formulations.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study we analyzed the different steps in the mechanism of action of de Cry toxins to determine if the formulation itself may affect performance of Bt Cry toxins. Specifically, we compared commercial non-encapsulated products with microencapsulated products named MP-Bta and MP- Btk. The same Bt strains were used as the basic material to make Xentari and MP-Bta, or to make Dipel and MP-Btk. According to the manufacturer, Dipel\u0026reg; is formulated with Bt \u003cem\u003ekurstaki\u003c/em\u003e, containing the proteins Cry1Aa (15%), Cry1Ab (39%), Cry1Ac (23%), Cry2Aa (22%), while Xentari\u0026reg; is formulated with from Bt \u003cem\u003eaizawai\u003c/em\u003e, containing Cry1Aa (21%), Cry1Ab (53%), Cry1Ca (20%) and Cry1Da (6%) (Valent \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur first result showed that non-encapsulated commercial products showed a higher concentration of total protein than the microencapsulated formulations after suspension of similar water volume. Similar protein concentrations of these formulations were then subjected to solubilization analysis. We did not observe changes in the profile of the solubilized protoxin bands of the SDS-PAGE analysis, indicating that the matrices used in the microencapsulation process did not influence the solubilization of Cry proteins. However, the results presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e show that none of the tested formulations have 100% solubilization efficiency. It important to mention that these formulations (conventional and microencapsulated) present a mixture of Cry toxins, which may influence their solubilization process. Aronson et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), tested different solubilization buffers for Bta and Btk strains, obtaining solubilization efficiencies that varied from 8 to 70%, and these authors concluded that the solubilization differences between these strains may be related to the protoxin composition of the crystals. Our data confirmed that Btk strain showed higher solubilization efficiency in relation to the Bta strain. In relation to conventional and microencapsulated formulations, variations in solubilization values were observed, with microencapsulated formulations showing lower solubilization values in \u003cem\u003ein vitro\u003c/em\u003e conditions. However, when midgut juice was used to solubilize these samples, it was evident that microencapsulated formulations showed a better solubilization than the conventional formulations.\u003c/p\u003e \u003cp\u003eThe solubilization of Bt parasporal crystals is a fundamental step to initiate the intoxication process. It was described that the solubilization process is facilitated by the physicochemical conditions (mainly pH and reducing conditions) that are found in the host's digestive fluids (Gill et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Bravo and Sober\u0026oacute;n \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Deist et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the work by Du et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), the authors compared two strains (Bta and Btk) in relation to the solubility of their parasporal crystals. The authors showed that for both strains, the solubilization started with pH values of 9.5, reaching a complete solubilization only when pH reaches values above pH 11, and it was proposed that similar high pH conditions could be found inside the midgut lumen of the lepidopteran larvae. The authors describe that the different insecticidal crystals produced present distorted and destabilized disulfide bonds, which can also influence the processes of solubilization and toxicity. In another study, Naimov et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), evaluated the solubilization of crystals of Bt \u003cem\u003ethompsoni\u003c/em\u003e HD542 composed of Cry15Aa toxin. The authors tested different buffers and pH values (ranging from 6.0 to 11.0) with or without the reducing agent DTT. The authors obtained complete protein solubilization only at pH 11.0 in sodium hydrogen carbonate buffer and CAPS buffer, respectively. In the presence of the reducing agent, the reduction of disulfide bonds allowed solubilization at pH 10. Other buffers (ethanolamine, Tris, borate-buffered saline) did not solubilize a significant amount of protein, with or without the addition of DTT. Here we show that the midgut lumen on \u003cem\u003eS. frugiperda\u003c/em\u003e has a pH of 9.5, and solubilization is extremely low under these conditions. Only after increasing the pH up to 10.5 we were able to observe complete solubilization and activation of Cry proteins. However, our results also demonstrate that incubation time is also important for solubilization. We reinforce the importance of studying additional factors, which can contribute to altering the pH values of the midgut, consequently altering the solubility and toxicity of these proteins, such as, for example, the different instars and also the geographic region where the pest may be found (Bravo and Sober\u0026oacute;n \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It could be worth to isolate in the future some Cry mutants with improved solubilization, specially at lower pH values that may correlate with improved toxicity.\u003c/p\u003e \u003cp\u003eDuring the activation step, approximately 40\u0026ndash;60 amino acids from the N-terminus are removed by proteases for 70 kDa and 130 kDa protoxins. For the 130 kDa protoxins, in addition to N-terminal processing, about 500\u0026ndash;600 amino acids are also cleaved out from the C-terminus, in both cases the active Cry toxins resulted in activate toxin proteins of ~\u0026thinsp;55 to 65 kDa (Bravo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Bergamasco et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; G\u0026oacute;mez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our results corroborate the proteins found in Bta and Btk were correctly activated, since there was no great difference in the proteolytic patterns of the different formulations. It was possible to observe a double band of ~\u0026thinsp;70 and ~\u0026thinsp;65 kDa for the formulations containing mixture of Cry1 and for the formulations containing Cry2 it was possible to observe a band of ~\u0026thinsp;55 kDa. Previously, Liu et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), obtained similar proteolytic profiles when they evaluated activation of Cry1Ac and Cry2Ab protoxins by proteases from the midgut juice of \u003cem\u003eHelicoverpa armigera\u003c/em\u003e larvae. We also noticed that the profile observed for the cleavage with chymotrypsin differs from the other treatments, being possible to observe with more precision the bands of ~\u0026thinsp;55kDa for the formulations containing Cry2 protoxin. However, the protein profiles obtained after incubation with trypsin and midgut juice were very similar, suggesting that it is possible that midgut juice contains high levels of trypsin and low levels of chymotrypsin. Actually, Saadaoui, Rouis and Jaoua (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) showed that trypsin-like activity is predominant in the midgut homogenate of \u003cem\u003eEphestia kuehniella\u003c/em\u003e. However, it is worth noting that the activation step has been related to resistance mechanisms. Improper activation such as insufficient processing or over digestion can result in insect resistance to Cry protoxin action (Dom\u0026iacute;nguez-Arrizabalaga et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter the activation process, the active toxins bypass the peritrophic matrix to interact with the brush border membrane (BBM) of the midgut tissue. This membrane is considered the main target for toxins. The toxin then undergoes a complex sequential binding steps with the different receptors present in BBM, which results in its insertion into the membrane, with the consequent formation of pores, and osmotic lysis, leading to insect death (Lu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the literature, we found several works that report that Cry toxin receptors are located in BBM, including cadherin-like (Aimanova et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jin et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), aminopeptidase N (APN) (Wei et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shao et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and alkaline phosphatase (ALP) (Likitvivatanavong et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Stalinski et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our studies concluded that activated toxin samples obtained from the different formulated products showed similar binding to BBMV from two lepidopteran insects. Bel et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) studied the binding of different Cry toxins (Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry2Ab) with BBMVs from different insects (\u003cem\u003eS. exigua, S. litura, A. ipsilon\u003c/em\u003e and \u003cem\u003eH. armigera\u003c/em\u003e) reporting different binding curves for these toxins, supporting that these toxins display different affinity values to the BBMVs from the different larval species. In our studies we are analyzing a mixture of activated toxins and we were not able to provide specific affinity values for each protein.\u003c/p\u003e \u003cp\u003eBioassay data against \u003cem\u003eM. sexta\u003c/em\u003e and \u003cem\u003eS. frugiperda\u003c/em\u003e larvae supported that the different procedures that were used in the formulation of these products did not affect the mechanism of action of Cry toxins, since no differences in LC\u003csub\u003e50\u003c/sub\u003e values between commercial and microencapsulated products were observed. It is worth mentioning that these tests were carried out on a diet, under the same condition such as been reported by Eski et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) where microencapsulated formulations of an indigenous strain of \u003cem\u003eB. thuringiensis\u003c/em\u003e (Se13) was evaluated against \u003cem\u003eS. exigua.\u003c/em\u003e showing similar LC\u003csub\u003e50\u003c/sub\u003e values compared to the commercial formulations on laboratory conditions.\u003c/p\u003e \u003cp\u003eHowever, we show here that when Dipel\u0026reg; formulation was treated with UV and analyzed against \u003cem\u003eS. frugiperda\u003c/em\u003e larvae, the toxicity was lower than MP-Btk formulation. The microencapsulation helped to maintain the insecticidal effect of Btk, most likely due to an improved protection of crystals and spores. Another point that is worth to mention is that the results showed that the combination of conventional and microencapsulated formulations can be an important management strategy.\u003c/p\u003e \u003cp\u003e Khorramvatan \u003cem\u003eet al.\u003c/em\u003e (2014), compared the effect of three polymers (starch, gelatin and sodium alginate) as wall materials for the production of a microencapsulated Bt formulation. The authors observed that for the alginate polymer, the viability of the spores was 90% after exposure to long\u0026minus;term UV radiation (UVB 385 nm), while the viability of non\u0026minus;microencapsulated spores under this condition was only 40%. In another study,) Jalali \u003cem\u003eet al.\u003c/em\u003e (2020) used the Pickering\u0026minus;emulsion technique to perform the microencapsulation of Bt. The authors tested different materials such as latex particles, graphene oxide nanosheets and olive oil as protective materials. The authors evaluated toxicity of these formulations against \u003cem\u003eE. kuehniella\u003c/em\u003e larvae after UV\u0026minus;A radiation. The results showed that the combination of matrices at a concentration of 0.045% allowed an effective control and greater protection against radiation, supporting the effectiveness of microencapsulation.\u003c/p\u003e \u003cp\u003eOverall our results provide important information for the development of future more efficient biopesticide formulations.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe results presented in this study demonstrate that after ingestion, the solubilization of the different formulations (conventional and microencapsulated) formulations is the critical point and \u003cem\u003ein vivo\u003c/em\u003e it seems to be relatively low. The microencapsulated products showed a slightly higher solubilization efficiency. However, these differences were not significant when toxicity was analyzed, since both types of formulations displayed similar toxicity values. The final conclusion is that the initial steps in the mechanisms of action of these pesticidal proteins (solubilization, activation and binding with receptors present in BBMVs) were not significantly affected due to the microencapsulation procedure. The results also demonstrate the importance of studying other factors, such as the pH change of the midgut, which influences the solubilization process.\u003c/p\u003e \u003cp\u003eIn addition, our data showed that when subjected to external factors such as UV radiation, the microencapsulated formulations showed greater control efficiency in shorter times and we propose that the mixture of conventional and microencapsulated formulations may be an important management strategy for the development of future biopesticide formulations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAB, MS, RAP and JO designed experiments. JO, IG and JS collected, prepared material, conducted experiments and collected data. AB, MS and RAP supervised experiments. JO analyzed data. JO, AB, MS and RAP wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements and Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful for financial support provided by the S\u0026atilde;o Paulo State Research Foundation (FAPESP, Grants 2017/21004-5, 2018/21142-1, and 2020/12779-6) and the National Council for Scientific and Technological Development (CNPq)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors declare that they have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdang MJ, Crickmore N, Jurat-Fuentes JL \u003cb\u003e(\u003c/b\u003e2014\u003cb\u003e) Chapter Two\u003c/b\u003e - \u003cb\u003eDiversity of Bacillus thuringiensis Crystal Toxins and Mechanism of Action\u003c/b\u003e. \u003cb\u003eIn\u003c/b\u003e: \u003cb\u003eDhadialla TS\u003c/b\u003e, \u003cb\u003eGill SS\u003c/b\u003e (\u003cb\u003eeds\u003c/b\u003e) \u003cb\u003eAdvances in Insect Physiology\u003c/b\u003e. \u003cb\u003eAcademic Press\u003c/b\u003e, \u003cb\u003epp 39\u003c/b\u003e\u0026ndash;\u003cb\u003e87\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAimanova KG, Zhuang M, Gill SS \u003cb\u003e(\u003c/b\u003e2006\u003cb\u003e) Expression of Cry1Ac cadherin receptors in insect midgut and cell lines\u003c/b\u003e. 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Comp Biochem Physiol A Physiol \u003cb\u003e86\u003c/b\u003e:301\u0026ndash;308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0300-9629(87)90334-3\u003c/span\u003e\u003cspan address=\"10.1016/0300-9629(87)90334-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Jin M, Yang Y, \u003cb\u003eet al (\u003c/b\u003e2020\u003cb\u003e) The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda\u003c/b\u003e. Toxins \u003cb\u003e12\u003c/b\u003e:375. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins12060375\u003c/span\u003e\u003cspan address=\"10.3390/toxins12060375\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bt, Microencapsulation, Mechanism of Action, Spodoptera frugiperda, Manduca sexta","lastPublishedDoi":"10.21203/rs.3.rs-1949207/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1949207/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe entomopathogenic bacteria \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) produce parasporal-crystal inclusions composed of different pesticidal proteins such as Cry, that show insecticidal activity against insect pests. Cry toxins are highly susceptible to degradation when exposed to adverse temperature and continuous sun-UV-light. Thus, encapsulation techniques are designed to improve their biopesticide performance and shelf-life. However, the effects of polymeric matrix encapsulation on the mechanism of action of Cry toxins produced by Bt \u003cem\u003ekurstaki\u003c/em\u003e and Bt \u003cem\u003eaizawai\u003c/em\u003e has not been evaluated. Here, we analyzed the solubilization, activation and the binding of Bt insecticidal Cry proteins to their receptors after microencapsulation and compared with commercial non-encapsulated Bt biopesticides. We show that solubilization is one step in the mechanism of action of these proteins that could limit Cry toxin action, the microencapsulation of Bt biopesticides did not alter protein profiles solubilization compared to those non-encapsulated, showing a 130 kDa (corresponding to Cry1 protoxin) and 70 kDa (corresponding to Cry2 protoxin) proteins. Activation with trypsin, chymotrypsin and larval midgut juice was analyzed, showing that this step is highly efficient, and proteins were cleaved producing similar\u0026thinsp;~\u0026thinsp;55 to 65 kDa activated toxins in microencapsulated and non-encapsulated formulations. Binding assays with receptors that are present in brush border membrane vesicles (BBMV) of \u003cem\u003eManduca sexta\u003c/em\u003e and \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e larvae showed similar binding curves for conventional and microencapsulated formulations. Finally, LC\u003csub\u003e50\u003c/sub\u003e bioassays against these pests, showed no significant differences among the treatments. However, when these formulations were subjected to UV radiation, we observed for the microencapsulated Bt formulations provided higher mortality against S. \u003cem\u003efrugiperda\u003c/em\u003e larvae, supporting a higher protective effect against degradation. Overall, our results show that microencapsulation of Bt biopesticides did not affect the mechanism of action of their pesticidal proteins while enhanced protection to UV radiation. These data will contribute to the development of more efficient Bt biopesticide formulations.\u003c/p\u003e","manuscriptTitle":"Performance of microencapsulated Bacillus thuringiensis Cry pesticidal proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-12 16:58:26","doi":"10.21203/rs.3.rs-1949207/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d626f74-0ca0-4bc4-8357-23a5ee378172","owner":[],"postedDate":"August 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-12T16:58:28+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-12 16:58:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1949207","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1949207","identity":"rs-1949207","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}