Endophytic actinobacteria and culture conditions
Seventy actinobacteria strains, previously isolated from six wild medicinal plant species (F. Compositae) in South Sinai, Egypt (El-Shatoury et al. 2013), were studied. The strains were provided by Actinobacteria Lab, Botany Department, Faculty of Science, Suez Canal University. Their selection was based on their published enzymatic activity, plant growth promotion and antimicrobial activity, as shown in Supplementary (Table S1). The generic identity of the strains and their host plants is illustrated in Table 1. The strains were provided as spore suspensions in 20 % v/v glycerol at – 15°C, and were refreshed on starch casein agar, as described by (Kieser et al. 2000).
Table 1 Illustration of the endophytic actinobacteria strains and their host plants.
The origin of the seven endophytes that showed the highest effects on the laboratory S. littoralis strain in the initial screen are highlighted in grey
a indicates the origin of the four endophytes that showed potent effects on the field S. littoralis strains.
S. littoralis larvae rearing
The laboratory S. littoralis strain was reared under constant laboratory conditions at the Plant Protection Research Institute, Agricultural Research Center, Zagazig, Egypt. It was reared in an incubator at a temperature of 26 ± 2°C and a relative humidity of 65 ± 10 % RH with a 16 L: 8 D photoperiod, according to (El-Defrawi et al., 1964). The details are shown in Supplementary (Table S2). The field strain S. littoralis larvae were collected from the local open field at Sharqia Governorate. They were transferred to the laboratory and reared for two successive generations as described above. The laboratory strain is reared in the laboratory for several successive generations away from any contamination with pesticides; so, it is guaranteed that it is free from any resistance to the action of pesticides. When experiments are conducted on it, the results of any treatment are assured of confidence; unlike the field strain that is randomly brought from the field where exposure to different pesticides is present. It is available and it is uncertain whether the field strain may enhance the resistance genes for the action of the treated substance or not; therefore, conducting experiments on the two strains gives additional dimensions in evaluating the tested toxicants.
Actinobacteria fermentation and metabolites extraction
The actinobacterial strains were cultured (2 µL of spore suspension) in 50 mL sterilized starch casein broth and incubated at 28 ± 2°C for 21 days with continuous shaking at 100 rpm. The mycelia were separated by centrifugation at 5000 rpm. The filtrates were extracted three successive times using equal volumes of ethyl acetate. The solvent layers were combined, concentrated, and evaporated to dryness using a rotary evaporator (HS-2005S-N, HAHN SHIN Scientific Co., Korea) at 40°C. The dried extracts were redissolved in ethyl acetate to prepare a stock concentration of 100 mg/mL and stored at 4°C for bioactivity screening tests.
Toxicity of ES2 metabolite to S. littoralis larvae
The effects of the ES2 metabolite on the growth and development of the S. littoralis 4th instar larvae were evaluated at different concentrations. Toxicity was compared to that of spinetoram (Radiant® 12% SC 100 cm3/ feddans (4200m2), a 2nd generation of the spinosyns, at LC50 0.5 mL/ liter (i.e. 0.05% concentration). The commercial Radiant SC 12% (Dow Agros ciences; CAS Number: 187166-40-1) was obtained from the PPRI, Ministry of Agriculture, Egypt. The experimental design included normal group (untreated, receiving distilled water) and negative control group (receiving ethyl acetate). All bioassay assessments were duplicated (each replicate included four larvae) and performed under constant laboratory conditions. A detailed schema for the laboratory and field S. littoralis treatments is presented in Supplementary (Figure S1).
Toxicity to 4th instar larvae of the laboratory S. littoralis strain (L-larvae)
A series of four concentrations (0.6, 6, 60, 100 mg/mL) of crude metabolites from the 70 actinobacteria strains were prepared. Newly molted 4th instar L-larvae were starved for 3 – 4 hours prior to the treatment to clear their alimentary canal and assure quick ingestion of treated leaves. Groups of larvae were transferred to 350 mL sterilized clean glass jars, and all jars were supplied with 7.0 cm filter paper to absorb any surplus moisture. Healthy, untreated leaves of castor, Ricinus communis L., were collected from the experimental field of the Plant Protection Research Institute. The leaves were washed, cut into equal discs using a cork borer, and impregnated with 50 µL of the corresponding metabolite concentration (i.e. equivalent to 0.03, 0.3, 3 and 5 µg / disc) using a leaf dipping technique. The lethal effects (mortality %) were recorded, daily, and corrected according to Abbott's formula (Abbott 1925). The treated insects were followed up, until the pupation stage.
Toxicity to 4th instar larvae of the Field S. littoralis strain (F-larvae)
Metabolic extracts from seven actinobacterial strains, that showed potent activities against the laboratory 4th instar larvae, were further investigated for toxicity to the 4th instar F-larvae, at 100 mg/mL concentration. Lethality was recorded as detailed above.
Phylogenetics of the promising actinobacteria strain
The partial 16S rRNA gene sequence of Kitasatospora ES2 (which produced the metabolite of highest toxicity to the laboratory and field strains) was analyzed. DNA extraction was performed based on the salting-out method (Kieser et al. 2000), with an additional purification step using phenol/chloroform. The 16S rRNA gene of the strain was amplified using the universal primer set 27F (5´-AGA GTT TGA TCC TGG CTC AG-3´) and 1492R (5´-GGT TAC CTT GTT ACG ACT T-3´). Amplification conditions were according to (Trujillo et al. 2010). Briefly: an initial denaturation step was performed for 9 min at 94°C, followed by 30 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 55°C and extension for 2 min at 72°C. A final extension step was performed for 10 min at 72°C.
PCR product sequencing was performed at Macrogen™ Biotechnology, Ltd. (Korea) (https://dna.macrogen.com/eng/). The sequence obtained and those of its most closely related Streptomycetes spp., retrieved from GenBank, were aligned using BLASTN (Version: 2.9.0+) (Zhang et al. 2000). The maximum identity score sequences were selected and aligned using the multiple alignment program ClustalW (Thompson et al. 1997). The phylogenetic tree was established by the maximum likelihood method, 1000 bootstrap, Tamura 3-parameter model; constructed using MEGA11 (Tamura et al. 2021).
Toxic effects Kitasatospora sp. ES2 crude metabolic extract (ES2)
The 4th larval instar of the laboratory S. littoralis strain (L-larvae) were exposed to ES2, the most potent strain, and investigated for morphological defects and histopathological and biochemical changes.
Histopathological examinations of L-larvae
Samples of the treated L-larvae and controls were collected at 72 hours post treatment. They were preserved in 3 mL 10 % formaldehyde (v/v) in sterilized screw-capped tubes, prior to fixation, dehydration and embedding in paraffin wax. Serial sections, at five microns, were made with a microtome and mounted on clean slides using Mayer’s albumin. The sections were stained with Ehrlich’s hematoxylin-eosin (HE) (Ruiz et al. 2004). The histological longitudinal and transverse sections were examined under a light binocular stereomicroscope (NOVEL; NLCD-120, China) using two magnifications (100-X and 400-X).
Biochemical assessments, Larvae-samples preparation
Samples (groups of four) of the treated and control L-larvae were placed in clean screw-capped tubes and kept frozen overnight. The frozen samples were homogenized for three min in distilled water (50 mg/1 mL) using a chilled glass Teflon tissue homogenizer (ST–2 Mechanic-Preczyina, Poland) surrounded with a crushed ice jacket. Then, they were centrifuged at 8000 rpm for 15 min at 5°C in a refrigerated microcentrifuge (Hettich, Germany). The supernatants, used as enzyme extracts, were stored at – 20°C until use in biochemical assays. All biochemical measurements were performed in triplicates. A double beam UV spectrophotometer (Spectronic 1201, Milton Roy Co., USA) was used to measure the absorbance of colored substances. The total protein concentration was determined according to Bradford's method (Bradford 1976).
Acetylcholinesterase (AchE, EC 22.214.171.124) determination. The biochemical activity of acetylcholinesterase, a detoxification enzyme, was measured according to (Simpson et al. 1964), using acetylcholine bromide (AchBr) as a substrate. The samples were measured at 515 nm absorbance against a blank (ethanol in phosphate buffer, pH 8.0). The activity was expressed as U/mg protein.
Protease (EC 126.96.36.199) determination. Proteolytic activity was measured as described by (Tatchell et al. 1972), with modifications, by measuring the increase in free amino acids split from a substrate protein (albumin) during 1 hour of incubation at 30°C. Amino acids were colorimetrically assayed by ninhydrin reagent. The zero adjustment was performed at 570 nm against the reagent blank (100 µL distilled water). The amino acids were expressed as µg D, L-alanine/min/mg protein.
Lactate dehydrogenase (LDH, EC 188.8.131.52) determination. The LDH activity was performed as described by (Diamantino et al. 2001). The zero adjustment was performed against buffer without substrate. The activity was expressed as U/mg protein (1 U=1 µmol substrate hydrolysed per minute).
Non targeted metabolomics analysis
Liquid chromatography, combined with quadrupole-time-of-flight high-definition mass spectrometry (LC-Q-TOF-MS), was used to investigate the chemical constituents of the metabolites from Kitasatospora sp. ES2 strain. This technique is a powerful tool for the characterization of microbial compounds with similar structures, particularly in the analysis of natural products (Liu et al. 2010).
The study was conducted on a Triple TOF® 5600+, Sciex system, Canada; pre-column (0.5 µm × 3.0 mm; Phenomenex Co., USA) and XBridge C18 column (3.5 µm, 2.1 × 50 mm; Waters Co., USA) with two LC columns, in-line filter discs, at 40°C. Detailed preparation and processing of the sample is provided in the Supplementary (Table S3). Based on their fragments, MasterView was used to define peaks using Build-in databases Data acquisition Analyst TF 1.7.1 software, Sciex). Using the Reaxys ChemDraw software, version 184.108.40.206 (https://www.reaxys.com), the cyromazine compound that can effectively target lethality to the larvae (Table. 1) was drawn.
Molecular docking simulation
Molecular docking aimed to illustrate the virtual mechanism of binding of some reported compounds used as insecticides towards acetylcholinesterase (AchE, PDB=4EY5), lactate dehydrogenase (LDH, PDB=1LDG), and protease (SREBPs, PDB= 5GPD) target proteins, that were freely accessible through the protein data bank. Both proteins and ligands were prepared and optimized according to (Nafie et al. 2019), and the molecular docking study was carried out using MOE 2015-10 as the computational software. Each complex was analyzed for 2D interaction images were taken by using MOE visualizing tool. 3D interaction images were taken by Chimera (UCSF).
All data were formulated as means ± standard error of the mean (SEM). Data was subjected to normality testing using Kolmogorov-Smirnov at 0.05 level. Accordingly, LDH, protease and AchE were parametric and parametric data analysis applied. One-way ANOVA was applied to assess the difference between treatment groups, ANOVA was followed by Duncan’s Multiple Range tests (DMRTs) as a posthoc test at 0.05 level.