Soil sampling
Approximately 30 grams of sandy soil (Figure 1) was collected thoroughly with a sterile spatula at a depth of approximately 10 cm. Then, it was placed in a sterile urine collection vial and stored at room temperature during transport. After arriving at the laboratory (IHU-Méditerranée Infection, Marseille, France), the samples were stored at -80°C until further experiments.
Bacterial isolation
The sample was thawed immediately before isolation, homogenized then two times 1 gram was taken and mixed in 10 ml of sterile distilled water. Then, the contents were incubated for 24 hours at room temperature. Thereafter, the supernatant was recovered and homogenized, and ten serial 1/10 dilutions were performed. The inoculum (50 μL) was seeded on Columbia agar supplemented with 5% sheep blood (bioMérieux, Marcy l'Etoile, France) in actinomycetes isolation agar (Merck, Darmstadt, Germany) and then incubated under aerophilic conditions at 32°C for at least 48 hours for Bacillus spp. isolation and for at least 1 week for Streptomyces spp. isolation.
Strains identification
Bacterial species were directly identified from each bacterial colony using matrix-assisted laser desorption ionization-time mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) as previously described 21. A score of > 2 allowed identification at the species level, and a score of < 1.7 did not allow any identification.
To obtain the MALDI-TOF spectra of Streptomyces species, first, treatment of hard stained colonies on agar was performed with Tissue Lyser (QIAGEN, Maryland, USA) and tungsten beads for at least 3 minutes to obtain isolated bacteria, which were suspended in distilled water. Liquid cultures (5 ml) were then generated using the bacterial suspension. Two millilitres of liquid culture were centrifuged at 13,000 rpm for 5 minutes. The supernatant was discarded, and the bacterial pellets were recovered. Subsequently, we performed the extraction of proteins by using an ethanol-formic acid extraction procedure 22. Briefly, we aliquoted 300 μl of the suspended bacteria in distilled water and added 900 μl ethanol. Subsequently, the cell suspension was centrifuged at 17,000 × g for 2 minutes, and the supernatant was discarded. The centrifugation was repeated, and the residual ethanol was discarded. The pellet was air-dried and thoroughly resuspended in 5 to 50 μl formic acid-water (70:30 [vol/vol]), depending on its size, and finally, an equal volume of acetonitrile was added. After centrifugation at 17,000 × g for 2 minutes, 1 μl of the supernatant was transferred to a polished steel MSP 96 target plate (Bruker Daltonics) and allowed to dry at room temperature before being overlaid with 1 μl of a saturated a-cyano-4-hydroxy-cinnamic acid (HCCA) matrix solution in 50% acetonitrile-2.5% trifluoroacetic acid (Bruker Daltonik). Finally, the matrix sample was crystallized by air-drying it at room temperature for 5 minutes.
The spectra generated by MALDI-TOF MS without initial identification were recovered to control their quality. After validation of the spectra by using the Bruker software, the MALDI Biotyper 3.0 software was used to build dendrograms that allowed the comparison of the different isolates. To identify the Streptomyces strains, the 23S rRNA 23 and atpD 24 genes were amplified, and the amplicons were sequenced. The obtained electropherograms were assembled and edited using ChromasPro 1.7.7 software (Technelysium Pty Ltd., Tewantin, Australia), and then the obtained sequences were compared with those available in the GenBank database by NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Fractions preparation
Bacterial culture
The strains were stored at -80°C. The cultures were grown on solid Columbia agar medium with 5% sheep blood (bioMérieux) under aerobic conditions at 32°C. Thereafter, several colonies were transferred in sterile conditions into 1 liter of liquid Tryptic soy broth medium (Sigma-Aldrich, France), which was incubated for 3 days for Bacillus spp. and for 14 days for Streptomyces spp. at 32°C and 110 rpm in a shaker incubator under aerobic conditions. A negative control containing only culture medium was prepared and regularly checked for evidence of contamination.
The supernatant-pellet separation was carried out by centrifugation at 8000 g for 20 minutes at 4°C using an A98813 J-Lite PP bottle assembly with a JLA-8.1000 rotor (Beckman Coulter, Villepinte, France). After centrifugation, the supernatant was immediately filtered with a 0.45 μm filter and placed into 75 ml flasks, after which it was frozen horizontally overnight at -80°C and then lyophilized the next day. The lyophilizate was stored at -20°C prior to the assays.
Release of inclusions and the main cell components
After centrifugation, the pellet was resuspended in PBS, and 700 μl of the resuspension was distributed into 2 ml cryotubes (Bio-One GmbH, Rainbach im Mühlkreis, Austria). In order to exclude the effects of live bacteria on future experiments, we disintegrated the bacteria by consecutive freezing/thawing and sonication treatments. Each tube was subjected to 3 freeze-thaw cycles for 5 minutes each using liquid nitrogen and a hybridization incubator heated to 50°C. The tubes were centrifuged at 13,000 g for 10 seconds, and the contents were transferred into 1.5 ml Eppendorf Safe-Lock tubes (Eppendorf, Montesson, France) and then subjected to 3 sonication cycles at an amplitude of 50 Hz for 30 seconds. Subsequently, ultracentrifugation was carried out at 20,000 g for 20 minutes at 4°C. Thereafter, the supernatant was recovered, directly filtered with a 0.45 μm filter and stored at -20°C prior to the assays. All fractions were regularly cultured on Columbia sheep blood agar plates (bioMérieux) after filtration to ensure their sterility.
Fractions used in larval assays
Once the larvae were ready (3rd and early 4th instar), the sterile fractions (pellet and supernatant) already prepared of a bacterium were thawed at room temperature, then, the Bradford protein assay was carried out. Subsequently, the volume of the fraction that was used was adjusted to have two concentrations, 2 mg/l and 6 mg/l, which were administered to the larvae. We tested the supernatant and pellet fractions of each stain separately at concentrations of 2 mg/l and 6 mg/l. We also tested the mixture of the two fractions at 6 mg/l to determine whether there was a synergetic effect of the secreted and cell constituent compounds.
We used Bacillus thuringiensis subsp. israelensis AM65-52 (Bti), which was isolated from a commercial granular formulation (VectoBacGR, Valent Bioscience, Libertyville, USA), to validate our protocol and to assess the insecticidal activity of the strain used as a positive control. We used this strain as a control for several reasons. It produces different Cry toxins that, after ingestion, are able to form pores in the plasma membrane of midgut epithelial cells in susceptible insects 25. Thus, in both the studied bacterial strains and in positive controls, we eliminated the effect of live bacteria on mosquito larvae. We administered the bacterial fractions 24 hours before feeding the larvae to give them enough time to ingest the bacterial fraction compounds.
Screening for insecticidal activity
The Aedes albopictus laboratory colony was maintained at 27 ± 0.5°C and in 80 ± 5% relative humidity. Adult mosquitoes were maintained with constant exposure to 10% sterile sucrose on cotton balls that were changed daily. For egg production, adult female mosquitoes were given defibrinated human blood (French Blood Agency, France) via the Haemotek membrane feeding system (Haemotek Ltd, Blackburn, United Kingdom). Larvae were fed Tetra-Min (Spectrum Brands, Fennimore, USA) fish food in clear water until the nymphae stage.
Seventy-five milliliter flasks were used for the insecticide screening assays. Only 3rd and early 4th instar larvae were used in the insecticidal activity assays. All tests of each fraction were performed with 25 larvae (N=4), and a total of 100 larvae for each fraction were tested as recommended by the WHO 26. Immediately after separating the larvae in flasks containing 100 ml of clean distilled water, we added the calculated volume of insecticide. Larvae were not fed until the 24th hour. Dead larvae were counted at 24, 48 and 72 hours. In each assay, 100 larvae were used as negative controls, which did not receive any fraction, to assess natural mortality. We considered that a strain had a good insecticidal activity if it induced >20% of mortality.
Statistical analysis
The Epi Info version 7 program (http://www.cdc.gov/epiinfo/index.html) (Addinsoft, 2019) and the XLSTAT statistical and data analysis solution (Paris, France, https://www.xlstat.com) were used to compare the mortality rates recorded at 72 hours after administration of 6 mg/l of each supernatant fraction. The Kruskal-Wallis test, the comparison of k proportions test and the pairwise comparison test were also performed. A difference was statistically significant when the p-value was <0.05. The Dunn procedure (bilateral test) was performed to separate groups of strains according to their efficiencies.