Biological material and toxicity assays
The extracts used for the toxicity assays were flour of the M. truncatula seeds, solvant-extracted flour and purified saponin. All these fractions have been tested on S. oryzae, whereas all other organisms were assayed with the purified saponin only.
Rice weevils (
, Coleoptera) were reared on wheat seeds at 27.5°C and 70% RH. Tests and survival analysis were performed on adults feeding on food pellets (composed of wheat flour and water) incorporating the tested fraction, as described in detail in . LT50 values were calculated using the SIMFIT software (http://www.simfit.man.ac.uk). The tests of the juvenile stages of Sitophilus oryzae could not be done because the larvae of weevils live inside the wheat grain and could not be grown outside, so we do not have an artificial diet where we can incorporating the toxin.
The red flour beetle, Tribolium castaneum (Coleoptera)
Tests were performed, by mixing the saponin into the standard diet (wheat flour 95%, yeast extract 5%). Then, three groups of 20 adults were deposited on cages, and the mortality was recorded every day.
The aphid Acyrthosiphon pisum (Hemiptera)
Growth and toxicity assays were carried out according to . Briefly, UV-sterilized Parafilm sachets enclosing 500 μL of an artificial diet were made under sterile conditions and placed on a PVC ring. A group of 20 neonate larvae were deposited on day 0 on diet containing or not the tested molecule (three groups per condition). The mortality was then recorded every day.
Aedes aegypti was assayed on two strains: the laboratory strain Bora-Bora, susceptible to all insecticides, and a strain selected from Bora-Bora which is tolerant to Bti Cry toxins (LiTOX strain, ). Mosquitoes were reared in standard insectary conditions (27°C, 16 h/8 h light/dark period and 80% relative humidity). Larvae were reared in tap water and fed with standard larval food (hay pellets). Bioassays were performed, in triplicate, in a final volume of 200 μL on 10 calibrated 2nd-instar larvae, with saponin concentrations of 0, 25, 250 and 1000 μg/mL. Mortality was recorded at 24 h and 48 h. Because data were not normally distributed, non-parametric Kruskal-Wallis ranked tests were used to test the strain and dose effects on larval mortality. In addition, Mann–Whitney one-tailed tests were used to compare the mortality, at each dose and each time, with that of the control using R software version 2.5 (R Development Core Team 2005).
Sf9 cells were grown at 27°C in Grace’s culture medium, supplemented with 10% foetal calf serum (FCS) and with 10 mg.ml-1 gentamicin. Sf9 cells were seeded, in 96-well plates, 24 h prior to the experiments (10 000 cells / well) and were exposed to increasing saponin concentrations for another 24 h or 48 h. Cell viability was determined using the CellTiter-Blue Viability Assay (Promega), according to the manufacturer’s instructions. After addition of the dye, the cells were incubated at 27°C for 4 h. The absorbance, at 570 and 600 nm, was then measured using a microplate reader (MR 7000, Dynatech Laboratories Inc., USA).
worms, from the N2 wild type strain, were cultured in liquid growth medium (KH2PO4 17.2 mM; Na2HPO4 16.8 mM; NaCl 85.6 mM; MgSO4 1 mM, cholesterol 26 μM, with OP50 E. coli as the food source) in 96-well plates, under constant agitation. Two adult worms were dispensed in each well using a COPAS BIOSORT robot from Union Biometrica (Massachussets, USA), which allows sorting and dispensing of worms according to their size and optical density. They were grown for 7 days at 15°C. The wells also contained increasing concentrations of the tested molecule. The offspring were observed on each day of the growing period to evaluate the effects of the molecule in terms of growth retardation and toxicity.
DH5α was grown in LB media at 37°C. For toxicity assays, the saponin was added directly to 1 mL of the media and bacterial growth was monitored by recording the OD at 600 nm for 10 h, starting at t = 0 with an OD = 0.01.
, strain BY4742, was grown on liquid media (YNB; 20 g.L-1 glucose; 0.02 g.L-1 His; 0.06 g.L-1 Leu; 0.04 g.L-1 Lys) at 30°C. For toxicity assays, the saponin was added directly to 1 mL of the media and yeast growth was monitored by recording the OD at 600 nm for 30 h, starting at t = 0 with an OD = 0.01.
Purification of the saponin
We used seeds from Medicago truncatula cv. Jemalong. Seeds were crushed in a Warring blender and sieved through a 0.4 mm mesh to separate the cuticles from the flour. The flour was submitted to successive extractions: first, it was extracted in H2O/EtOH (80/20, 10 mL for 1 g of flour) for 2 h, at room temperature and with stirring, and then centrifuged for 10 min at 10 000 × g. The supernatant was dried under vacuum in a Buchi Rotavapor. The resulting powder was resuspended in H2O/ACN (40/60, 10 mL for 0.1 g of powder), and immediately centrifuged for 10 min at 10 000 × g. The supernatant was again dried under vacuum.
The powder was resuspended in H2O/ACN (40/60), at approximately 15 mg.mL-1, and filtered on a 0.45 μm sterile filter. The molecules of the extract were separated by RP-HPLC. The extract was injected into a C18 column (250 × 25 mm, 5 μm, Phenomenex) on an Agilent 1200 HPLC apparatus. The flow was 3.5 mL.min-1. The gradient was H2O + 0.04%TFA (solvent A) / ACN +0.04% TFA (solvent B) 90/10 for 5 min, then 60% solvent B for 25 min. The elution was monitored using a diode array detector at 210 nm. Each fraction harvested was lyophilized.
The fraction containing entomotoxic activity was resuspended in H2O, and then injected in the same column with an elution under isocratic conditions (solvent B 30%). Each fraction harvested was lyophilized and stored dry at −20°C until required.
All of the mass spectra were obtained using a Thermo LCQ advantage ion –trap spectrometer equipped with an electrospray ionization source. Both positive and negative-ion mass spectra were acquired. Positive-ion ESI was performed using an ion source voltage of −4.0 kV and a capillary offset voltage of 42 V. Nebulization was aided by a coaxial nitrogen sheath gas provided at a pressure of 60 psi and desolvation was aided by the use of a nitrogen counter current gas at a pressure of 12 psi. The capillary temperature was set at 200°C.
Negative-ion ESI was performed using an ion source voltage of 4.0 kV and a capillary offset voltage of −86 V. Again, nebulization was aided by a coaxial nitrogen sheath gas provided at a pressure of 60 psi and desolvation was aided by the use of a nitrogen counter current gas at a pressure of 12 psi. The capillary temperature was set at 200°C.
Mass spectra were recorded over the range 50–2000 m/z. Tandem mass spectra were obtained using automated MS/MS and MS3. MS/MS was performed by isolating the base peak (parent ion) above m/z 1087 and using an isolation width of 2.0, a fragmentation amplitude of 0.6, a threshold set at 15,000 and the ion charge control switched on with the maximum acquired time set at 100 ms. The MS3 was performed by isolating, in the same conditions, the parent ion, initially at m/z 1087 and then we performed the isolation and the fragmentation of the product ion at m/z 911.
1 and 13C NMR spectra were recorded on a 500-MHz Brucker Avance NMR spectrometer equipped with a z axis field gradient unit, using CD3OD as the solvent for measurement. Conventional 2D 1H-1H experiments DQF-COSY (double quantum filtered correlation spectroscopy), HOHAHA (homonuclear Hartman Hahn) NOESY (nuclear overhauser effect spectroscopy) and 2D inverse detected 1H-13C experiments HSQC (heteronuclear single quantum coherence) , HMBC (heteronuclear multiple bond coherence)  and HMQC (heteronuclear multiple quantum coherence)  were all performed at 293K. The data were processed and analyzed using the Topsin software package.
Acid hydrolysis of saponin
The saponin (4 mg) was treated with 2 mL of 2 N HCl (methanol-H2O, v/v 1:1) under conditions of reflux, at 90°C, for 3 hours. The mixture obtained was extracted with CH2Cl2 three times to separate the agylcone part. The CH2Cl2 layer was dried, then the powder was resuspended in H2O/ACN (20/80). The aglycone was purified by RP-HPLC. The extract was injected into a C18 column (250 x 4.1 mm, 3 μm, Phenomenex). The flow was 1 mL.min-1. The gradient was H2O + 0.04%TFA (solvent A) / ACN +0.04% TFA (solvent B) 70/30 to 0/100 for 40 min. 1.25 mg of the aglycone part were retained in order to perform biological assays.
Protein was measured by the bicinchoninic acid procedure, developed by Pierce, with BSA as a reference.
1 mL of anthrone solution (200 mg in 100 mL H2SO4) was added to 0.5 mL of the tested solution on ice. The tubes were covered and vortexed, then the reaction mixture was boiled for 10 min. After cooling the tubes, the OD was read at 585 nm and glucose was used as a reference.
1–2 mg of the dried compound were dissolved in 2 mL of CHCl3, followed by the addition of 2 mL of concentrated H2SO4. After a few minutes, the CHCl3 fraction becomes red if sterols are present.
The CMC (Critical Micelle Concentration) was determined according to . Briefly, 1 μL of 1,6-diphenyl – 1,3,5 – hexatriene (DPH), solubilized in THF, was added to 2 mL of increasing doses of the tested compound in 10 mM MES pH 6. After a 30 min incubation in the dark, fluorescence was determined with an excitation wavelength of 358 nm and an emission wavelength of 430 nm.