Isolation and characterisation of Streptomyces sp. KSF103
Streptomyces sp. KSF103 (Kuala Sat Forest number 103) was isolated from bulk soils collected from a primary forest in Jerantut, Pahang, Malaysia. Morphological characterisation of the strain was carried out according to Goodfellow and Cross (1984)18. The formation of aerial and substrate mycelia, and the arrangement of spores were observed under a light microscope. The strain was then purified using the streak method on yeast-malt extract agar medium (ISP Medium No. 2, ISP2) and also maintained at − 80 °C in glycerol suspension (30%, v/v).
Taxonomic identification of isolated actinobacterium
Molecular characteristics of Streptomyces sp. KSF103
At the stationary phase, Streptomyces sp. KSF103 was harvested by centrifugation at 2600 rcf for ten minutes. Following that, the pellet was subsequently collected for DNA isolation. The genomic DNA was isolated using the Quick DNA Fungal/Bacterial Kit (Zymo Research, USA) according to the manufacturer’s protocol. The amplification of 16S rRNA was performed using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′). Taq polymerase was used to amplify the 16S rRNA gene using exTEN 2X PCR Master Mix (1st Base, Singapore). The amplification reaction was performed with the extracted genomic DNA as a template following the conditions: initial denaturation at 94 °C for 4 min, 35 cycles of amplification (denaturation at 94 °C for 1 min, annealing at 54 °C for 1 min, extension at 72 °C for 1 min), and a final extension at 72 °C for 10 min. The PCR product was purified using NucleoSpin Gel and PCR Clean-up (Macherey–Nagel, Germany) as per the manufacturer’s protocol.
The forward and reverse 16S rRNA gene sequences were assembled and analysed using the BioEdit Sequence Alignment Editor19 and compared to those of the type strains available on the EzBioCloud database20. Multiple alignments of the sequences were conducted using the CLUSTAL-W tool in MEGA-X. The phylogenetic tree was constructed using the MEGA-X software21, employing the neighbour-joining method22. Kimura’s two-parameter model was used to calculate evolutionary distances23. The topology of the resultant neighbour-joining trees was evaluated by bootstrap analysis after 1000 replications24.
Physiological characteristics of Streptomyces sp. KSF103
The formation of aerial and substrate mycelium, and arrangement of the spore were observed under a light microscope. Physiological characterizations, including growth temperature (20–50 °C), salt concentration (0–10%) and pH (4–10), were performed.
Fermentation and extraction of bioactive compound
The fermentation and extraction of bioactive compounds from Streptomyces sp. KSF103 in this study was performed following the methods of Ganesan, et al.25 and Thenmozhi, et al.26 with suitable modifications. Streptomyces sp. KSF103 was cultivated on yeast-malt extract (ISP2) agar plates for five days at 28 °C. Chunks of the corresponding agar with bacterial colonies were added to two 50 mL centrifuge tubes containing 30 mL of ISP2 medium. After three days of incubation at 28 °C with agitation at 120 rpm, the seed cultures were used to inoculate nine 1000 mL flasks containing 800 mL of ISP2 broth media. A total of 7.2 L of Streptomyces sp. KSF103 was fermented at 28 °C for 14 days at 120 rpm agitation. The resulting culture broth was separated from the mycelium by centrifugation at 2600 rcf for 30 min at 4 °C. The culture filtrates were collected and subjected to SMs extraction with ethyl acetate, an organic solvent with semi-polarity. For 4–6 days, the culture filtrates were soaked in ethyl acetate. The aqueous organic phase was separated and subsequently concentrated at 40 °C with a vacuum rotary evaporator until the eluant was colourless. The supernatant was extracted with an equal volume of ethyl acetate, and the resulting organic layers were evaporated to get the ethyl acetate extract (EA extract). The resulting EA extract was stored at − 20 °C in a sterile vial. Prior to each bioassay, stock solutions were made fresh. In the current study, two equivalent batches of EA extract tested, previously submitted to quality control, were used.
Mosquito colonization and maintenance
Both Ae. aegypti and Ae. albopictus were long-established laboratory colonies that were reared under controlled conditions (26 ± 1 °C, 70 ± 5% relative humidity (RH) with 12 h day/night cycle) and were free from the exposures of insecticides, repellents, and pathogens for over 90 and 70 generations, respectively. The rearing method was adopted from Amelia-Yap et al.27. The same larval densities, diets and environmental conditions were maintained for both of these strains, provided with ground beef liver powder and yeast ad libitum. After the formation of pupae, they were transferred daily from larval trays to cups containing dechlorinated water and introduced into the rearing cage [30 cm (L) × 30 cm (W) × 30 cm (H)] where adults emerged and mated freely. Adult mosquitoes were maintained on a ten percent w/v sugar solution supplemented with vitamin B complex and blood-fed twice per week with human blood from the group of researchers (tested negative for several mosquito-borne diseases) drawn in EDTA tube using Hemotek® membrane-feeding system to promote egg production.
Anopheles cracens has been incriminated as the main vector of Plasmodium knowlesi which is the ethiological agent of human knowlesi malaria in Southeast Asia, with more than 1000 sporozoites in positive mosquitoes and this revealed that they are efficient vectors28. A susceptible laboratory reference strain that has been surviving well in the insectary was used for all the laboratory-based experiments in this study. The eggs of the An. cracens self-mating colony were obtained from the Department of Parasitology laboratory, Chiang Mai University, Thailand. The mosquito colony was maintained as described by Choochote and Saeung29 in an insectarium at a temperature of 26 °C with a RH 80 ± 5% and an alternation of 12 h day/night cycle. A few female mosquitoes were isolated in a cup and fed with human blood drawn in EDTA tube using Hemotek® membrane-feeding system to promote egg production.
The laboratory reference strain of Culex quinquefasciatus was maintained in the Insectary unit of Arthropod Research Laboratory, TIDREC, Universiti Malaya, which has been cultured under insecticide-free conditions for 75 generations (26 ± 1 °C, 70 ± 5% RH with 12 h day/night cycle). Freshly laid egg rafts were incubated in distilled water to ensure complete hatching. The hatched-out larvae were reared following standard techniques in plastic trays (30 cm × 25 cm × 5 cm), at the rate of 100 larvae/tray of distilled water. A pinch of finely ground TetraBits Complete® was provided to the larvae every other day. However, the mosquito colony collapsed before carrying out the ovicidal and adulticidal tests.
Larvicidal bioassays
The test concentrations were chosen following preliminary experiments against early fourth instar larvae at a range of dosages ranging from 0.010 to 10 mg/mL. The WHO protocol for evaluating larvicidal bioassays was adopted with suitable modifications30. The EA extract’s acute toxicity was determined by measuring mortality after 24 h of exposure in early fourth instar larvae of Ae. aegypti, Ae. albopictus, An. cracens and Cx. quinquefasciatus. Using a wide-bore plastic transfer pipette, batches of twenty-five larvae were transferred into disposable test containers. Excess water was gently removed, followed by the addition of 100 mL of dechlorinated water. To prepare the test solutions, the EA extract was diluted in dimethyl sulfoxide (DMSO) to a 200 mg/mL concentration after weighing. The appropriate volumes of dilutions were added into the disposable test containers (350 mL) to obtain the desired target concentrations, beginning with the lowest concentration. Based on the preliminary screening results, the EA extract of Streptomyces sp. KFS103 were subjected to concentration–response bioassay for larvicidal activity against the larvae again. The LC50 and LC90 values were determined using a restricted range of five concentrations yielding 10–95% mortality rate. Since all bioassays could not be performed concurrently, treatments were blocked across time, with a separate 1% DMSO control treatment added in each block. Each block of the bioassay was performed using freshly prepared extract solutions.
To ensure a homogeneous mixture of the chemicals, the solutions were resuspended using a micropipette and the cup was gently swirled. All bioassays were conducted under the same environmental conditions to guarantee the reproducibility between experiments. For each assay, five concentrations were tested in triplicates, and each assay was repeated three times. The mortality data was recorded 24 h post-treatment. No food was offered to the larvae. Larvae were graded as “dead” if they did not respond to tapping or prodding with a sterile micropipette tip. Larval mortality was calculated for each concentration. Mortality was converted into percent mortality. Each concentration’s corrected percentage mortality value was subjected to subsequent data analysis.
Adult topical bioassays
All experimental assays were conducted on non-blood-fed adult Ae. aegypti, Ae. albopictus, and An. cracens females aged four to five days, following previously described procedures of direct topical application31 with slight modifications to precisely determine the toxicity of the EA extract to mosquitoes. Direct topical application also permits accurate plotting of a dose–response curve to assess lethal dose (LD50 and LD90) values, the statistically derived dose required to score 50% and 90% mortality rates. Briefly, the EA extract was dissolved to a concentration of 200 mg/mL in acetone with vigorous vortexing. Different ranges of concentrations of the solution for the tests were created by further diluting the 200 mg/mL stock in acetone. The mosquitoes were first aspirated into separate clear cups. After purging the glass syringe with acetone, it was filled with the diluted EA extract and secured in the microapplicator (Hamilton PB600-1 Repeating Syringe Dispensers) that delivered a volume of 0.2 μL. Adult female mosquitoes were cold-anaesthetized in batches of five and were then placed on ice in a Petri dish. A single female was taken from the cup with a fine tweezer, and 0.2 μL of the test solution was administered to the dorsal thorax using the syringe microapplicator. To confirm the delivery of the EA extract and ensure all mosquitoes receive a given volume of the test solution, each mosquito was observed using a dissecting microscope. Cohorts of five mosquitoes were treated with a specific compound until the amount reached twenty-five females per concentration in each assay.
Subsequent dose–response assays were conducted based on screening mortality that produced a 10–95% range in initial screening to calculate LD50 and LD90. Involving five concentrations of the EA extract, twenty-five adult female mosquitoes were dosed per concentration. Females treated with 0.2 μL of acetone alone were used as the negative control. After dosing, cups were covered with a screen mesh and secured with a rubber band. Cotton balls saturated with ten percent sucrose solution were provided for each cup. All the mosquitoes were kept at 26 ± 1 °C, 70 ± 5% RH with 12 h day/night cycle during the 24-h recovery period. Mortality was recorded at 24 h post-exposure. Mosquitoes that appeared dead or lacked movements with no flying ability were scored as “dead”. For each assay, five concentrations were tested in triplicates, and each assay was repeated three times.
Ovicidal bioassays
The protocol for ovicidal bioassays was adopted from Prajapati et al.32 with slight modifications. To facilitate embryonation of the freshly laid eggs, the oviposition papers were stored in rearing conditions for 48 h of the drying period. Eggs were examined for any deformities following drying, and then groups of twenty-five eggs were treated separately with the extracts at 0.250, 0.500, 1, 2, 4 mg/mL concentrations in a six-well plate. Eggs treated with 1% of DMSO in water were used as the negative control groups. For each assay, five concentrations were tested in triplicates, and each assay was repeated three times. The ovicidal activity was evaluated up to 168 h post-treatment and subsequently, both treated and negative control eggs were observed under the microscope. The non-hatched eggs with unopened opercula were counted in each treatment. The percent mortality was calculated using the following formula:
$${text{Percentage,, of ,,egg ,,mortality}}= frac{{text{No.,, of,, unhatched eggs}}}{{text{Total ,, no ,,of,, eggs ,,introduced}}} times 100%$$
Environmental toxicity to non-target organisms
The environmental toxicity of Streptomyces sp. KSF103 EA extract was determined against microalgae and ants. Microalgae, including Chlorella spp., are often used as bioindicators in detecting contaminants as they are sensitive to contaminations33. Ants, including Odontoponera spp., can be chronically exposed to insecticide residues after prolonged applications. This is of great concern because ants play multiple crucial roles for ecosystem functioning34. The current study involved the freshwater microalga Chlorella sp. Beijerinck UMACC 313 and the marine microalga Chlorella sp. Beijerinck UMACC 258 for non-target toxicity tests. Both strains of tropical algae were obtained from the University of Malaya Algae Culture Collection (UMACC)35. The freshwater microalga was grown in Bold’s Basal Medium, while the marine microalga was cultured in Prov Medium. An inoculum size of 20%, standardized at an optical density at of 0.2 at 620 nm (OD620 nm) from exponential phase cultures was used. Each microalga was grown in triplicate in a total volume of 500 mL and added to tubes containing the Streptomyces sp. KSF103 EA extract at the LC90 value (Table 2—6.956 mg/mL) or 1% DMSO (negative control) in a final reaction volume of 10 mL of the medium. At 0, 4, 8 and 12 days of incubation at 25 °C, the growths of both microalgal strains were monitored based on OD620 nm, adopted from Ng et al.36. Other biochemical profiling and photosynthetic performance of the microalgae such as chlorophyll-a (chl-a), carotenoid, maximum quantum yield (Fv/Fm), alpha, maximum relative electron transport rate (rETRmax) and photoadaptive index (Ek) were measured.
Biomass of algal cultures were estimated based on chl-a and carotenoid content. The chl-a and carotenoid concentrations were determined using the spectrophotometric method37. Fluorescence analysis was employed to measure the photosynthetic performance of algal cultures in both Streptomyces sp. KSF103 EA extract and DMSO treatment. Photosynthetic parameters in this study were measured using a pulse amplitude modulation (PAM) fluorometer (Diving PAM, Walz, Germany)38,39.
The effect of Streptomyces sp. KSF103 EA extract was also evaluated on Odontoponera denticulata, a species commonly found in Malaysia. Adults female O. denticulata were obtained from the wild. They were exposed to LD90 concentration (Table 3—274.823 mg/mL) of the extract with acetone. Similar to adult topical bioassays, 0.2 μL of the test solution was administered to the dorsal thorax of the ants using the syringe microapplicator. They were then (20 adults) transferred to a rearing cage with rearing conditions of 26 ± 1 °C, 70 ± 5% RH with 12 h day/night cycle. Acetone was used as a negative control. Mortality was determined after 4 days.
Data analyses
Mosquito mortality in the control groups that ranged from 0 to 10% should be corrected accordingly using Abbott’s formula40. Dataset with mortality rate greater than 10% were not considered. The average larval and egg mortality data were subjected to a probit regression analysis to calculate the lethal concentrations LC50 and LC90 using SPSS (version 26). In the case of adulticidal testing, LD50 and LD90 values were calculated. Other statistics at 95% confidence limits of upper confidence limits (UCL) and lower confidence limits (LCL), degree of freedom (df), and chi-square values were also calculated41. Moreover, GraphPad Prism version 9 (GraphPad Software, La Jolla, CA, USA) was used to analyze dose–response mortality data in best-fit sigmoidal plots with the minimum and maximum constrained to 0 and 100%, respectively. Using the same software, the effect of the EA extract treatment was determined by a one-way analysis of variance (ANOVA). If the analysis of variance showed significant differences between the groups (P ≤ 0.05), a Dunnett’s post-hoc test (P ≤ 0.05) was performed to determine the significant difference between control and multiple treatment groups.
The environmental toxicity of the Streptomyces sp. KSF103 against Chlorella sp. Beijerinck UMACC 313 and Chlorella sp. Beijerinck UMACC 258 was assessed by reading the optical density at 620 nm, and the results were compared with the negative control using a two-way ANOVA from the GraphPad Prism software combined with the Bonferroni test (P < 0.05). All experimental data were expressed as the mean ± standard deviation and analyzed at 5% of significance level (P < 0.05).
