2.1. Source of the strains

Reference strains (Bt kurstaki HD73 and HD1) used for this study were kindly provided by the Sciences Center of Sinaloa. Native strains were isolated from dead insects (Spodoptera frugiperda) collected from the fields and labeled as Bt-A, B, C, D, 5, 15, 22 and 64. Insects were macerated in 0.85% sterile saline solution and subjected to thermic shock (85 ◦C for 10 min and 4 ◦C for 2 min). Subsequently, a loopful of the solution was streaked in nutrient agar and incubated at 30 ◦C for 24 h. The colonies that showed typical Bacillus morphology (Gram-positive, opaque, whitish and irregular) were recultured in nutrient agar at 30 ◦C for 48 h. Then, bacteria were stained with Scheaffer-Fulton endospore stain and visualized in a light microscope at 100X. Cultures that showed spore-forming bacilli and parasporal crystals production were selected.

2.2. Molecular analysis

For DNA extraction, vegetative cells were obtained by culturing the strains in nutrient broth at 150 rpm and 37 ◦C for 24 h. DNA was extracted using heat and LiCl-precipitation.¹² Briefly, cells were lysed with extraction buffer (100 mM Tris-HCl, pH 8; 10 mM EDTA, pH 8; 1% SDS; 100 mM LiCl) and chloroform at 80 ◦C. Samples were centrifuged at 13,000 rpm for 10 min, and the upper aqueous phase was recovered, mixed with an equal volume of 4 M LiCl and incubated at 4 ◦C overnight. Samples were centrifuged, and the supernatant was mixed with isopropanol and centrifuged again. DNA pellet was recovered, washed with 70% ethanol and resuspended in sterile distilled water. DNA integrity was visualized on a 1.5% agarose gel and quantified by spectrophotometry.

Amplification of the 16S rDNA fragment was carried out in a total volume of 50 l containing PCR buffer [50 mM KCl, 20 mM Tris-HCl (pH 8.3), 2.5 mM MgCl₂], 0.2 M each primer, 0.2 mM dNTPs, 1.25 U AmpliTaq DNA polymerase (Applied Biosystems) and 10 ng of DNA. The reactions were cycled 35 times under the following parameters: 95 ◦C for 40 s, 55 ◦C for 40 s and 72 ◦C for 90 s with 5′-AGAGTTTGATCMTGGCTCAG-3′ and 5′-TACGGYTACCTTGTTACGACTT-3′ oligonucleotides as primers.

Sequencing was performed by Macrogen Inc (using an ABI3730XL DNA analyzer). The sequences obtained were analyzed using Chromas Lite software, and alignment and neighbor-joining tree method were performed using CLC Sequence Viewer software. The sequences were compared with the ones in GeneBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the algorithm BLAST to confirm they belonged to Bacillus thuringiensis.

2.3. Bioassays

We performed preliminary bioassays with Galleria mellonella larvae to confirm that the strains were pathogenic for Lepidopteran insects. Based on these results, we selected the most pathogenic strain (Bt-D). For the bioassays with D. considerata Heinrich, we used first-instar larvae. Larvae were fed with an artificial diet¹³ inoculated with the selected Bt strain since proteins have to be ingested to achieve a toxic effect. Experimental groups were fed with the following diets: alone (group 1), diet with sterile Bt medium (group 2), culture with 5.7 x 10⁹ CFU/g (50% sporulation, group 3), lyophilized culture with 5.7 x 10⁹ CFU/g (50% sporulation, group 4), culture with 3.51 x 10⁹ CFU/g (> 90% sporulation, group 5), lyophilized culture with 3.51 x 10⁹ CFU/g (>90% sporulation, group 6), and culture with 5.37 x 10⁶ CFU/g (only vegetative cells, group 7). Each group contained 30 larvae, and the survival rates were monitored every 24 h for four days.

Bacteria were cultured in soybean medium¹⁴ using a bioreactor with constant conditions (30 ◦C, 600-800 rpm, > 20% dissolved oxygen, pH 7.4). Fresh as well as lyophilized cultures were used to assess the importance of the formulation in the efficacy of the biological agent.

2.4. Proteomic analysis

The strains were grown in nutrient broth at 37 ◦C and 150 rpm until they reached Abs₆₀₀ = 0.2-0.3. Subsequently, bacteria were inoculated into soybean medium in a 1:20 volume ratio in shaken flasks with 1:4 liquid-air ratio. Samples were then incubated at 30 ◦C and 150 rpm for 96 h or until they reached > 90% sporulation. Cultures were centrifuged at 5,000 rpm for 15 min; the supernatant was discarded, and the pellet containing the spores and proteins was resuspended and solubilized in an alkaline buffer¹⁵ (50 mM NaHCO₃, 50 mM Na₂CO₃, 50 mM EDTA, pH10) at 37 ◦C and 150 rpm for 2 h. Samples were centrifuged again at 5,000 rpm for 15 min, and supernatants were recovered as well. The total amount of protein was quantified by the Bradford protein assay.¹⁶ The proteomic profile was obtained by SDS-PAGE.

Once one of the local strains (Bt-D) was selected, Western blot analysis was performed to select the protein bands that would be processed for mass spectrometry analysis. For this purpose, a commercial rabbit polyclonal antibody against Cry1Ab toxin (Abcam, ab51586) was used. An HRP-conjugated goat anti-rabbit IgG was used as a secondary antibody.

The spots cut from the gels (~1 mm²) were processed following Bruker’s standard protocol for in-gel protein digestion. Briefly, gel particles were washed four times with 50 mM NH₄HCO₃ and acetonitrile. The proteins were then reduced and alkylated with 10 mM DTT (56 ◦C for 45 min) and 55 mM iodoacetamide (20 min at room temperature in the dark). Samples were washed again with acetonitrile and NH₄HCO₃. The supernatant was removed, and gel particles were air-dried. For the digestion of the proteins, trypsin (25 ng/ l in 25 mM NH₄HCO₃) was used. The samples were incubated at 37 ◦C overnight. The supernatant was recovered and stored at -20 ◦C. The particles were then incubated with 50 mM NH₄HCO₃ at 37 ◦C overnight, and the supernatant was stored at -20 ◦C. Peptides were extracted with trifluoroacetic acid 0.1%/acetonitrile (1:1) for 30 min at room temperature. Finally, all supernatants were mixed and dried in a vacuum centrifuge.

Peptides were resuspended in trifluoroacetic acid and MALDI-TOF (MS/MS). For their identification, an Ultra-flextreme mass spectrometer (Bruker, USA) with LIFT fragmentation was performed. An AnchorChip target and α-Cyano-4-hydroxycinnamic acid (HCCA) matrix were used, following the dried droplet protocol suggested by the manufacturer. The laser intensity was adjusted to 50% for the acquisition of parent masses and 60-70% for fragment masses, with three or four repetitions. For the analysis, spectra with 1x10³-1x10⁴ intensity peaks were considered. The identity of the protein was matched using the eubacteria Swiss-Prot database and the Mascot search engine with the following parameters: enzyme, trypsin; missed cleavages, 1; fixed modification, carbamidomethyl C; variable modification, oxidation M; parent tolerance, 0.2 Da; fragment tolerance, 0.5 Da. Positive protein identifications were those rendering a MASCOT score higher than 30. The toxins of both Bt kurstaki HD73 and HD1 reference strains were used as positive controls.

2.5. Statistics

For the bioassays with first-instar larvae, data were analyzed by one-way ANOVA with IBM SPSS v.20 software. p values < 0.05 were considered as statistically significant.

3.1. Molecular analysis

The sequences of the amplified segments (16S rDNA) were compared with the ones available in the NCBI database, which demonstrated that the strains were all Bacillus thuringiensis (Figure 1). The phylogenetic tree showed an aggrupation between the strains Bt-A, 5, 15 and 22, and another branch for the strains Bt-D, B, HD73, and C (Figure 1). The strain Bt-64 showed a major divergence from the others, which matches with its macroscopic and microscopic morphological characteristics (data not shown).

Figure 1 Neighbor-joining phylogenetic tree of the Bacilus thuringiensis isolates. 

3.2. Bioassays

The survival rates of first-instar larvae were drastically decreased in the groups that contained a significant sporulation percentage, therefore, high levels of parasporal crystals (Figure 2). Since day 2, first-instar larvae groups 4 and 5 had a significant decrease in survival rates (Table 1). The following days all groups had statistically significant differences versus the control group, except from group 7. Since group 7 treatment consisted of vegetative cells (no sporulation and crystal production), those results emphasize the role that parasporal crystals have over the entomopathogenic effect. Although there are strains that produce VIP toxins during the vegetative growth,⁵ it does not seem to be the case for this strain in particular.

Figure 2 Survival rates in the bioassays with first-instar larvae. 

Table 1 Survival percentage for bioassays with first-instar larvae. 

All groups were compared to group 2 (ST), which was considered as the negative control.

SD, standard deviation; M, diet alone; ST, sterile medium.

For cultures with 50% sporulation, treatments with lyophilized spore-crystal mixture were more efficient than fresh cultures (group 3 versus 4, Figure 2), which underlines the importance of formulation when an agrobiological product is applied. However, in the 90% sporulation cultures, lyophilization did not improve insecticidal activity (group 5 versus 6, Figure 2). The high standard deviation (SD) values obtained in some days (especially day 1) could be due to the inner variation among individuals. Although all larvae belong to the same population, there is a natural diversity among them, which causes a higher susceptibility in some individuals. Moreover, the controlled variable in the experiment was the concentration of Bt inoculated to the diet; however, larvae fed at libitum, so the amount of spore-crystal complex ingested by each larva was not controlled.

3.3. Proteomics analysis

For the Bt kurstaki HD73 strain, a protein band with a molecular weight of ~65 kDa was identified as Crystal protein Cry1Ac. In the case of the Bt kurstaki HD1 strain, a protein band with a molecular weight of ~65 kDa was identified as Crystal protein Cry1Aa (Figure 3), according to reported data.^17,18 These results were confirmed by MALDI-TOF analysis, where the protein rendered a > 40 score.

Figure 3 Reference strains protein profiles. A) Bacillus thuringiensis kurstaki HD73 protein profile (15% SDS-PAGE gel). Lane 1 was loaded with 9.6 μg of protein. B) Bacillus thuringiensis kurstaki HD1 protein profile (10% SDS-PAGE gel). Lane 2 was loaded with 11.1 μg of protein. 

The proteomic analysis of our local strain Bt-D showed that it produces a protein with a calculated molecular weight of 67 kDa (Figure 4), identified as a Cry1Ac toxin by Western blot analysis, which may be the responsible for the insecticidal activity observed in the bioassays. It is known that Cry proteins are originally produced as protoxins, which are then solubilized and cleaved in the midgut of the Lepidopteran insects when they are ingested, becoming active toxins.¹⁹ Since an alkaline buffer solution was used to solubilize the proteins in the samples,²⁰ the bands observed as ~67 kDa proteins are the active forms of the toxin. Two bands detected by Western blot were observed on lanes 3 and 4 maybe because the native strain produced more than one toxin, whose epitope is similar or equal to Cry1Ac, and causes the protein to bind to the primary antibody.

Figure 4 Bacillus thuringiensis strain Bt-D protein profile. A) 12% SDS-PAGE. M, molecular weight marker; lane 1, 12 h culture (vegetative cells only); lane 2, 24 h culture; lane 3, 48 h culture (50% sporulation); lane 4, 96 h culture (> 90% sporulation). B) Western blot membrane for Bt-D strain. M, molecular weight marker; lane 1, 12 h culture (vegetative cells only); lane 2, 24 h culture; lane 3, 48 h culture (50% sporulation); lane 4, 96 h culture (> 90% sporulation). Each lane was loaded with 30 μg of protein.