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Isolation and characterization of the insect growth regulatory substances from actinomycetes
Comparative Biochemistry and Physiology, Part C 228 (2020) 108651
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Isolation and characterization of the insect growth regulatory substances from actinomycetes
T
Jong Hoon Kima, Jae Young Choia, Dong Hwan Parka, Dong-Jin Parkb, Min Gu Parka, So Young Kimb, Yoon Jung Jub, Jun Young Kima, Minghui Wanga, Chang-Jin Kimb, ⁎ Yeon Ho Jea,c, a
Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, Seoul 08826, Republic of Korea Industrial Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea c Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Streptomyces Secondary metabolites Juvenile hormone antagonist Insecticidal activity Antimycins
Insect growth regulators (IGRs) are attractive alternatives to chemical insecticides. Since it has been reported that secondary metabolites from actinomycetes show insecticidal activities against various insect pests, actinomycetes could be a potential source of novel IGR compounds. In the present study, insect juvenile hormone antagonists (JHANs) were identified from actinomycetes and their insect growth regulatory and insecticidal activities were investigated. A total of 363 actinomycetes were screened for their insect growth regulatory and insecticidal activities against Aedes albopictus and Plutella xylostella. Among them, Streptomyces sp. AN120537 showed the highest JHAN and insecticidal activities. Five antimycins were isolated as active compounds by assay-guided fractionation and showed high JHAN activities. These antimycins also exhibited significant insecticidal activities against A. albopictus, P. xylostella, F. occidentalis, and T. urticae. Moreover, dead larvae treated with these antimycins displayed morphological deformities that are similar to those of JH-based IGR-treated insects. This is the first report demonstrating that the insecticidal activities of antimycins resulted from their possible JHAN activity. Based on our results, it is expected that novel JHAN compounds potentially derived from actinomycetes could be efficiently applied as IGR insecticides with a broad insecticidal spectrum.
1. Introduction Actinomycetes are filamentous gram-positive bacteria with a high G + C content and widely distributed in various terrestrial and aquatic environments (Sharma et al., 2014). Actinomycetes represent one of the most studied classes of bacteria because they produce a broad spectrum of biologically active compounds (Solecka et al., 2012; Mahajan and Balachandran, 2014). Approximately 10,000 bioactive secondary metabolites are reported to be produced by actinomycetes, which represents 45% of all bioactive microbial metabolites discovered (Jackson et al., 2018). Actinomycetes have also been identified as potential biological pest control agents (Montesinos, 2003; Omura, 2011). It has been reported that secondary metabolites derived from actinomycetes show insecticidal activities against a variety of insect pests belonging to Lepidoptera and Diptera (Dhanasekaran et al., 2010; El-Khawagh et al., 2011; Karthik et al., 2011; Ababutain et al., 2012; Vijayabharathi et al.,
2014). Furthermore, recent studies have shown that actinomycetes produce secondary metabolites with insect growth regulatory activities (Arasu et al., 2013; Kaur et al., 2014; Samri et al., 2017). Insect growth regulators (IGRs) are promising alternatives to conventional chemical insecticides because of their selectivity and relatively low toxicity (Dhadialla et al., 2009; Pener and Dhadialla, 2012). Among them, juvenile hormone (JH)-based IGRs such as JH agonists (JHA) and JH antagonists (JHAN) can promote abnormal development, premature molting, and supernumerary larval stages by fatally disrupting the endocrine system because JH regulates diverse aspects of insect physiology including metamorphosis, reproduction, polyphenism, and immunity (Hartfelder and Emlen, 2012; Lee et al., 2015). We previously developed an effective in vitro JH-based IGR screening system using yeast cells transformed with the Aedes aegypti JH receptors methoprene-tolerant (Met) and Ftz-F1-interacting steroid receptor coactivator (FISC) (Lee et al., 2015). Using this screening system, various substances with JHA or JHAN activity have been
⁎ Corresponding author at: Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea. E-mail address: [email protected] (Y.H. Je).
https://doi.org/10.1016/j.cbpc.2019.108651 Received 27 August 2019; Received in revised form 21 October 2019; Accepted 26 October 2019 Available online 31 October 2019 1532-0456/ © 2019 Elsevier Inc. All rights reserved.
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identified from plant essential oils and chemical libraries (Lee et al., 2018a, 2018b, 2018c). It was assumed that actinomycetes could be another source of natural JHAs and JHANs because they produce a variety of secondary metabolites with insecticidal and insect growth regulatoryactivities. In this study, novel potential JHAN compounds were identified from actinomycetes by screening 363 actinomycete isolates for their JHA/JHAN activities; their insecticidal activities were also evaluated.
2.4. Yeast growth inhibition tests The growth inhibition test for yeast treated with corresponding concentrations (1 mg/mL for crude extract and 2 μg/mL for purified compound) was performed as previously described (Lee et al., 2018a). The inhibition tests were performed in triplicate. 2.5. Bioassays Thirty A. albopictus 3rd instars were treated with actinomycete extract by adding 1 mg/mL of crude extract or 2 μg/mL of purified compound in 5 mL tap water with food mixtures. In the case of P. xylostella, thirty 3rd instars were soaked in the corresponding concentrations (1 mg/mL for crude extract and 2 μg/mL for purified compound) for 30 s and the treated larvae were provided Chinese cabbage leaf discs (3 cm diameter). Female adult F. occidentalis were collected using a custom-made aspirator. Thirty females were treated with 10 μg/mL of purified compound by soaking for 30 s. The treated females were transferred onto kidney beans. For the bioassay against T. urticae, kidney bean leaf discs (15 mm diameter) were permeated with 10 μg/ mL of purified compound for 5 min. Thirty 3rd instar nymph T. urticae were inoculated on the permeated leaf disc and placed onto a Petri dish with a water-soaked thin layered cotton. The number of dead insects was checked at 24 h intervals and calculated after treatment for 3 days. All experiments were performed in triplicate.
2. Material and methods 2.1. Isolation of actinomycetes and fermentation A total of 363 actinomycetes were isolated from soil samples collected from various sites in Korea. One gram of soil samples was serially diluted with distilled water and homogenized by vortexing for 10 min. An aliquot of the soil suspension was plated on humic acid-vitamin agar (1 g humic acid, 0.5 g Na2HPO4, 1.71 g KCl, 0.05 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 1 g CaCl2, B vitamins (0.5 mg each of thiamine-HCl, riboflavin, niacin, pyridoxine, Ca-pantothenate, inositol and p-aminobenzoic acid, and 0.25 mg of biotin), 15 g agar and 1 L water, pH 7.2) and incubated at 28 °C for 7 days. Selected isolates were maintained as a spore suspension in glycerol (20%, v/v) at −80 °C. All 363 isolates were fermented in a 500 mL baffled Erlenmeyer flask containing 100 mL of glucose and soybean meal. Soluble starch broth was used to maintain and cultivate Actinomycete spp. (main ingredients included: glucose, soluble starch, soybean meal, beef extract, yeast extract, NaCl, K2HPO4 and CaCO3), which were incubated on a rotary shaker (150 rpm) at 30 °C for 7 days. After fermentation, the cells were separated from the culture broth by centrifugation at 10,000 rpm for 15 min at 4 °C. The separated cells were dissolved in an equal volume of acetone and mixed for 3 h. The acetone phase was concentrated in a rotary vacuum and the crude extracts were redissolved in dimethyl sulfoxide (DMSO) for activity tests.
2.6. Statistical analysis One-way ANOVA was performed using the SPSS software (version 24, SPSS, Inc., Chicago, IL, USA). Comparison of the mean values was performed using Scheffé's method, and P values < 0.05 were considered statistically significant. 2.7. Taxonomic characteristics of the selected strain The cultural characteristics of strain AN120537 were determined on various International Streptomyces Project (ISP) media. The colony color was evaluated using the Inter-Society Color Council-National Bureau of Standards (ISCC-NBS) color chart (Kelly, 1964). PCR amplification and nucleotide sequence analysis of the 16S rRNA gene of the strain AN120537 was performed as reported by Li et al. (2007). The 16S rRNA sequences from closely related actinomycetes were retrieved from the National Center for Biotechnology Information (http://www.ncbi. nlm.nih.gov/GenBank/index.html) and aligned using the CLUSTAL X program (Thompson et al., 1997). Molecular phylogeny of strain AN120537 16S rRNA was inferred using the neighbor-joining method under the Jones-Taylor-Thornton (JTT) matrix-based model incorporated in the MEGA7 software (Jones et al., 1992).
2.2. Insects A. albopictus (Skuse) (Diptera: Culicidae) was maintained in breeding chambers at 28 °C and 70% relative humidity with a 12 h light/12 h dark cycle in aged tap water. The larvae were fed a diet of TetraMin fish flakes, and adults were reared using 10% sucrose solution. The diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) and the western flower thrips Frankliniella occidentalis (Pergande) were reared on rape sprouts and kidney bean cotyledons, respectively, and maintained at 25 °C and 70% relative humidity with a 16 h light/8 h dark cycle. The mite Tetranychus urticae Koch was reared on kidney bean at 25 °C and 60% relative humidity with a 16 h light/8 h dark cycle.
2.8. Isolation of JHAN compounds from strain AN120537 cultures Strain AN120537 mycelia were extracted with an equal volume of acetone as described in Section 2.1. The dried acetone extract was dissolved in distilled water and sequentially extracted with equivalent volumes of n-hexane, ethyl acetate and n-butanol. The JHAN and insecticidal activities of the extracted substances were determined as described in Sections 2.3 and 2.5. The concentrated hexane extract, which showed the highest JHAN and insecticidal activities, was separated using a Biotage Isolera (Charlottesville, USA) automated purification system equipped with a UV detector at 254 nm and a SNAP column cartridge (100 g silica gel). Separation was carried out with a chloroform/methanol (95:5) isocratic solution to give eight fractions, among which only one fraction showed JHAN and insecticidal activities at a concentration of 1 mg/mL. The active fraction was separated by preparative reverse-phase HPLC (solvent: methanol/water 70:30, flow rate: 1.0 ml/min) using an Inno C18 column (5 μm, 150 × 4.6 mm;
2.3. Yeast two-hybrid binding tests The yeast two-hybrid β-galactosidase assay for quantitative determination of JHA/JHAN activities was performed as previously described (Lee et al., 2018a). One mg/mL of crude extract or 2 μg/mL of purified compound was applied to yeast cells transformed with A. aegypti Met-FISC to estimate JHA/JHAN activities. The obtained OD420 values were converted to an arbitrary unit representing JHAN activity. Yeast two-hybrid binding tests for JHA/JHAN activities were performed in triplicate.
JHAN activity OD420 of pyriproxyfen (0.033 ppm) − OD420 of treated sample = OD420 of pyriproxyfen (0.033 ppm) 2
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Young Jin Biochrom, Korea) to give five compounds, which were further purified using an U-VDSpher PUR 100 C18E column (1.8 μm, 50 × 2.0 mm VDS Optilab, Germany) with acetonitrile/water (70:30) containing 0.2% formic acid at a flow rate of 0.3 mL/min.
and was selected for further studies. 3.3. Taxonomic identification of strain AN120537 The cultural and physiological characteristics of strain AN120537 observed at 14 days after growing on ISP media suggest that this strain belongs to the genus Streptomyces (Table 1). For further phylogenetic profiling of strain AN120537, the nucleotide sequence of its 16S rRNA gene was compared with those of representative Streptomyces strains. A phylogenetic tree constructed using the neighbor-joining method showed that strain AN120537 was most closely related to Streptomyces celluloflavus NRRL B-2493(T), with 99.03% 16S rRNA nucleotide sequence similarity (Fig. 3).
2.9. Structural determination of the active compounds The 1H and 13C nuclear magnetic resonance (NMR) (600 MHz) spectra were obtained using a high-resolution Avance 600 NMR spectrometer (Bruker, Germany) with methanol as the solvent. Electrospray ionization-mass spectrometry (ESI-MS) was performed using a Q-TOF 5600 (AB Sciex, Canada) high-resolution liquid chromatographytandem mass (LC/MS/MS) spectrometer. The chemical structure of each compound was determined by comparing the NMR and MS data with published literature values.
3.4. Purification of JHAN compounds from strain AN120537
3. Results
Based on bioassay-guided monitoring, the hexane extract of strain AN120537 was fractionated, and five compounds were obtained. The molecular weight and formula of compound 1, which was isolated as a white amorphous solid, was determined as C24H32N2O9 by ESI-MS ([M + H]+, m/z 493.21) and 1D NMR spectra (1H and 13C NMR) (Figs. S1 and S2). The planar structure of the compound was further analyzed by 2D NMR (1H-1H COSY, HSQC, and HMBC) (Fig. S3). The structure of compound 1 was readily identified as antimycin A5 (Fig. 4) by comparison with previously reported structures (Kluepfel et al., 1970; Schilling et al., 1970; Haegele and Desiderio, 1973). The ESI-MS measurements and 1H NMR spectra of compounds 2–5 were significantly similar to those of compound 1 (Figs. S4–S7), suggesting that they are also antimycin derivatives. Based on comparison of their spectroscopic data with those in the literature (Abidi and Adams, 1987; Ha et al., 1989; Barrow et al., 1997; Inai et al., 2011), compounds 2–5 were identified as antimycins A4, A3, A2, and A1, respectively (Fig. 4).
3.1. Insect growth regulatory activities of actinomycete strains Acetone extracts from 363 actinomycete strains were screened for their JHA and JHAN activities using in vitro yeast two-hybrid β-galactosidase assays. Whereas none of the 363 actinomycete extracts tested showed JHA activities (Table S1), 8 extracts showed high levels of JHAN activity at a final concentration of 1 mg/mL (Fig. 1A). Additionally, actinomycete extracts with high JHAN activities showed no anti-yeast activities when they were subjected to yeast growth inhibition tests (Fig. 1B), demonstrating that their JHAN activities originated from direct disruption of JH receptor complex formation. 3.2. Insecticidal activities of actinomycete strains with JHAN activity The insecticidal activities of the actinomycete extracts with high JHAN activities were investigated at a concentration of 1 mg/mL against an important medical pest (A. albopictus) and an economically important agricultural pest (P. xylostella). Among the 8 extracts, extracts from strains AN120537, AN120590, and AN120867 showed high levels of mosquito larvicidal activities against A. albopictus 3rd instar larvae, with mortalities of 100% (Fig. 2A). Against P. xylostella 3rd instar larvae, the extracts from strains AN120537 and AN120590 showed high insecticidal activities, with mortalities > 80% (Fig. 2B). Strain AN120537 exhibited the highest JHAN and insecticidal activities
3.5. JHAN activities of the strain AN120537 antimycins To investigate the JHAN activities of the antimycins purified from strain AN120537, these compounds were subjected to a yeast two-hybrid β-galactosidase assay. All the antimycins tested displayed increasing JHAN activities with increasing concentrations, demonstrating that these compounds interfere with the pyriproxyfen-mediated binding of A. aegypti Met-FISC in a concentration-dependent manner (Fig. 5). Among them, specifically, antimycin A5 exhibited the highest JHAN
Fig. 1. Screening of actinomycete extracts with JHAN activity. (A) To estimate the JHAN activity, 0.033 μg/ml of pyriproxyfen and 1 mg/mL of each actinomycete extract were applied to a yeast two-hybrid β-galactosidase assay. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05). (B) Actinomycete extracts with JHAN activities were tested for their anti-yeast activities to examine whether the reduced β-galactosidase activity resulted from JHAN activity or yeast toxicity. 3
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Fig. 2. Insecticidal activities of actinomycete extracts with JHAN activities against A. albopictus (A) and P. xylostella (B) larvae. A. albopictus and P. xylostella 3rd instar larvae were treated with 1 mg/mL of each actinomycete extract and the mortality was calculated every 24 h for 3 days after treatment. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05).
activity at a concentration > 2 μg/mL. None of the antimycin derivatives showed anti-yeast activities in growth inhibition tests (Fig. S8).
Table 1 Cultural characteristics of Streptomyces sp. strain AN120537 on various ISP media. Medium
ISP ISP ISP ISP ISP ISP
2 3 4 5 6 7
Growth
Good Good Moderate Moderate Good Moderate
Soluble pigment
– – – – Pinkish purple Pale yellow
Colony color
3.6. Insecticidal activities of the strain AN120537 antimycins
Aerial mycelium
Substrate mycelium
Whitish gray Pale yellow Pale yellow Pale yellow Dark brown Yellow
Pale yellow Pale brown Whitish gray Pale yellow Dark grayish brown Pale yellow
To evaluate the insecticidal activities of strain AN120537 antimycins, A. albopictus and P. xylostella 3rd instar larvae were treated with each compound at a concentration of 2 μg/mL. All five compounds caused 100% larval mortality against A. albopictus (Fig. 6A). Typical morphological deformations were observed from the dead larvae compared to untreated larvae. These deformations were manifested as body segment contraction (Fig. 6C), deformed heads with elongated neck regions and black pigmentation on the entire body (Fig. 6D). In the case of P. xylostella larvae, antimycins A1 and A5 showed the highest
Fig. 3. Phylogenetic relationship of strain AN120537 based on its 16S rRNA gene sequence. The nucleotide sequences of 16S rRNA gene from the genus Streptomyces were compared by the neighbor-joining method. Numbers at each branch node indicate the bootstrap percentage of 1000 replications. 4
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Fig. 4. The chemical structures of the antimycins purified from strain AN120537.
their old cuticles (Fig. 7C and D). The insecticidal spectrum of these antimycins was further investigated against serious sucking pests including F. occidentalis and T. urticae at a concentration of 10 μg/mL. In the case adult female F. occidentalis, antimycins A3, A4, and A5 exhibited
insecticidal activities, with mortalities > 80% (Fig. 7A). Most of the dead P. xylostella larvae treated with antimycins also showed morphological abnormalities such as disordered setae and an inability to withdraw from the head capsule causing failure to completely shed
Fig. 5. Concentration-dependent JHAN activities of antimycins from strain AN120537. To estimate the JHAN activity, 0.033 μg/mL of pyriproxyfen and corresponding concentrations (0.1, 1, 2, 5, and 10 μg/mL) of each antimycin were applied to a yeast two-hybrid β-galactosidase assay. 5
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Fig. 6. Insecticidal activities of the antimycins from strain AN120537 against A. albopictus. (A) A. albopictus 3rd instar larvae were treated with 2 μg/mL of each antimycin and the mortality was calculated every 24 h for 3 days after treatment. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05). (B) Morphological characteristics of untreated A. albopictus larvae. HE, Head; TH, thorax; AB, abdomen; AP, anal papillae; RS, respiratory siphon. (C) and (D) Morphological deformations of dead larvae after antimycin treatment.
Fig. 7. Insecticidal activities of the antimycins from strain AN120537 against P. xylostella. (A) P. xylostella 3rd instar larvae were treated with 2 μg/mL of each antimycin and the mortality was calculated every 24 h for 3 days after treatment. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05). (B) Morphological characteristics of untreated P. xylostella larvae. (C) and (D) Morphological abnormalities of dead larvae after antimycin treatment.
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Fig. 8. Insecticidal activities of the antimycins from strain AN120537 against F. occidentalis (A) and T. urticae (B). F. occidentalis adult females and T. urticae 3rd instar nymphs were treated with 10 μg/mL of each antimycin and the mortality was calculated every 24 h for 3 days after treatment. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05).
the JH receptor complex by competing with JH for binding to Met, their mode of action as JHANs is different from that of precocenes. Additionally, the high insecticidal activities of these compounds suggested that actinomycetes might be plentiful sources of novel IGR compounds, which could be exploited for the development of biopesticides. Antimycins (A1-A5) were identified as active compounds from strain AN120537, which was assigned to Streptomyces celluloflavus by physiological characterization and phylogenetic analysis using its 16S rRNA gene sequence. Antimycins are a family of depsipeptides with structural skeletons of a 9-membered dilactone ring connected to 3formamidosalicylic acid by an amide linkage (Liu et al., 2014). Since their first report from Streptomyces sp. NRRL 2288 in 1949, a variety of natural antimycins have been identified from various actinomycete species (Liu et al., 2016). This family of compounds has been previously reported to exhibit antifungal, insecticidal, and nematocidal properties (Hosotani et al., 2005; Shiomi et al., 2005; Yan et al., 2010; Liu et al., 2019). These compounds have also been known to inhibit the mitochondrial electron transport chain by binding to the quinone reduction site Qi in the cytochrome bc1 complex (Xia et al., 1997; Gao et al., 2003; Huang et al., 2005). In insects, antimycins may act as JHANs and have shown insecticidal activities against A. albopictus. Furthermore, antimycins exhibit a significantly broad insecticidal spectrum, encompassing biting-type (P. xylostella) and sucking-type (F. occidentalis and T. urticae) pests. The dead larvae of A. albopictus showed morphological deformities similar to growth-disrupting effects reported from A. aegypti larvae due to application of some actinomycete extracts (Zebitz, 1984; Sakthivadivel and Thilagavathy, 2003; Kabir et al., 2013; Mahyoub et al., 2016). Moreover, morphological abnormalities observed from dead P. xylostella larvae were comparable to those of P. xylostella treated with another IGR, pyriproxyfen (Alizadeh et al., 2012). Taken together, these results demonstrated that antimycins may disrupt the formation of the JH receptor complex in target insects, which might disturb JH-based endocrine regulation of insects and thereby cause insect mortality.
superior activities, with mortalities > 60% (Fig. 8A). In the T. urticae bioassay, application of antimycins A2 and A3 promoted the highest mortality, which was > 70% (Fig. 8B). 4. Discussion During the last several decades, studies searching for eco-friendly insecticidal compounds have been extensively carried out to overcome the undesirable effects caused by synthetic insecticides such as toxicity to the environment and the development of resistant pests. Many natural products originating from plants and microorganisms have been successfully utilized as environmentally benign alternatives to synthetic insecticides (Cantrell et al., 2012). Specifically, actinomycetes have been regarded as plentiful sources for natural products as they have been reported to produce various secondary metabolites with a diverse range of biological activities, including insecticidal, antifeedant, and insect growth inhibitory activities (Omura, 2011; Arasu et al., 2013; Vijayabharathi et al., 2014). In this study, among 363 actinomycetes isolated from Korean soil samples, extracts from eight strains showed high JHAN activities, with the strain AN120537 extract causing 100% mortality against both A. albopictus and P. xylostella larvae at a concentration of 1 mg/ml, demonstrating that the strain might produce novel JHAN compounds with high insecticidal activities. Several endophytic actinomycetes have been reported to play a role as beneficial symbionts with plants, and diverse bioactive metabolites from symbiotic actinomycetes could have been utilized by plants to endure environmental stresses, such as plant diseases and insect pests, over the course of plant and actinomycete coevolution (Qin et al., 2011; Kinkel et al., 2012). Thus, potential JHAN compounds derived from endophytic actinomycetes might be adopted as effective defense mechanisms for symbiotic plants because IGRs are insect-specific, and insects may have difficulty acquiring resistance to these IGRs (Bowers, 2012). These results provided novel insights into the interactions between actinomycetes, plants, and insects. Botanical precocenes isolated from Ageratum houstonianum and their synthetic analogues have also been reported to show JHAN activities (Banerjee et al., 2008). They cause precocious metamorphosis in several insect species by inducing irreversible degeneration of the corpora allata and inhibiting JH production (Azambuja and Garcia, 1991). However, precocenes have not been commercialized as insecticides because of their low insecticidal activities and potential carcinogenic effects in mammals (Alzogaray and Zerba, 2017). Because the JHAN compounds from actinomycetes identified in this study interrupt the formation of
5. Conclusions In this study, the Streptomyces strain AN120537, which displayed high levels of JHAN and insecticidal activities, was selected through in vitro screening of 363 actinomycete strains. Antimycins isolated from strain AN120537 showed possible JHAN activities in a concentrationdependent manner and a broad spectrum of insecticidal activities against A. albopictus, P. xylostella, F. occidentalis, and T. urticae. These 7
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results suggest that actinomycetes are promising sources of novel IGR compounds when exploring natural products with effectiveness and environmental safety. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpc.2019.108651.
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Springer, Berlin, Heidelberg, pp. 37–58. Mahyoub, J.A., Hawas, U.W., Al-Ghamdi, K.M., Aljameeli, M.M., Shaher, F.M., Bamakhrama, M.A., Alkenani, N., 2016. The biological effects of some marine extracts against Aedes aegypti (L.) mosquito vector of the dengue fever in Jeddah Governorate, Saudi Arabia. J. Pure Appl. Microbiol. 10, 1949–1956. Montesinos, E., 2003. Development, registration and commercialization of microbial pesticides for plant protection. Int. Microbiol. 6, 245–252. Omura, S., 2011. Microbial metabolites: 45 years of wandering. wondering and discovering. Natural Product Updates 67, 6420–6459. Pener, M.P., Dhadialla, T.S., 2012. An overview of insect growth disruptors; applied aspects. Adv. Insect Physiol. 43, 1–162. Qin, S., Xing, K., Jiang, J.-H., Xu, L.-H., Li, W.-J., 2011. Biodiversity, bioactive natural products and biotechnological potential of plant-associated endophytic actinobacteria. Appl. Microbiol. Biotechnol. 89, 457–473. Sakthivadivel, M., Thilagavathy, D., 2003. Larvicidal and chemosterilant activity of the acetone fraction of petroleum ether extract from Argemone mexicana L. seed. Bioresour. Technol. 89, 213–216. Samri, S.E., Baz, M., Ghalbane, I., El Messoussi, S., Zitouni, A., El Meziane, A., Barakate, M., 2017. Insecticidal activity of a Moroccan strain of Streptomyces phaeochromogenes LD-37 on larvae, pupae and adults of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae). Bull. Entomol. Res. 107, 217–224. Schilling, G., Berti, D., Kluepfel, D., 1970. Antimycin A components. II. Identification and analysis of antimycin A fractions by pyrolysis-gas liquid chromatography. J. Antibiot. 23, 81–90. Sharma, M., Dangi, P., Choudhary, M., 2014. Actinomycetes: source, identification, and their applications. Int. J. Curr. Microbiol. App. Sci. 3, 801–832. Shiomi, K., Hatae, K., Hatano, H., Matsumoto, A., Takahashi, Y., Jiang, C.-L., Tomoda, H., Kobayashi, S., Tanaka, H., Ōmura, S., 2005. A new antibiotic, antimycin A9, produced by Streptomyces sp. K01-0031. J. Antibiot. 58, 74–78. Solecka, J., Zajko, J., Postek, M., Rajnisz, A., 2012. Biologically active secondary metabolites from Actinomycetes. Open Life Sci. 7, 373–390. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Vijayabharathi, R., Kumari, B.R., Sathya, A., Srinivas, V., Abhishek, R., Sharma, H.C.,
Declaration of competing interest The authors have declared that no conflict of interest exist. Acknowledgements This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agro-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number 316030-3). J. H. Kim, D. H. Park, M. G. Park, J. Y. Kim, and M. Wang were supported by the Brain Korea 21 plus project, Seoul National University, Republic of Korea. References Ababutain, I.M., Abdul Aziz, Z., AL-MeshheRn, N.A., 2012. Lincomycin antibiotic biosynthesis produced by Streptomyces sp. isolated from Saudi Arabia soil II - extraction, separation and purification of lincomycin. Canadian Journal of Pure and Applied Sciences 6, 1905–1911. Abidi, S., Adams, B., 1987. 1H and 13C resonance designation of antimycin A1 by twodimensional NMR spectroscopy. Magn. Reson. Chem. 25, 1078–1080. Alizadeh, M., Karimzadeh, J., Rassoulian, G., Farazmand, H., Hoseini-Naveh, V., Pourian, H., 2012. Sublethal effects of pyriproxyfen, a juvenile hormone analogue, on Plutella xylostella (Lepidoptera: Plutellidae): life table study. Arch. Phytopathol. Plant Protect. 45, 1741–1763. Alzogaray, R.A., Zerba, E.N., 2017. Rhodnius prolixus intoxicated. J. Insect Physiol. 97, 93–113. Arasu, M.V., Al-Dhabi, N.A., Saritha, V., Duraipandiyan, V., Muthukumar, C., Kim, S.-J., 2013. Antifeedant, larvicidal and growth inhibitory bioactivities of novel polyketide metabolite isolated from Streptomyces sp. AP-123 against Helicoverpa armigera and Spodoptera litura. BMC Microbiol. 13, 105. Azambuja, P., Garcia, E.S., 1991. Effects of proallatotoxins (precocenes) on the development and reproduction of Rhodnius prolixus: some data. Mem. Inst. Oswaldo Cruz 86, 113–115. Banerjee, S., Kalena, G.P., Banerji, A., Singh, A.P., 2008. New synthetic precocenoids as potential insect control agents. J. Environ. Biol. 29, 951–957. Barrow, C.J., Oleynek, J.J., Marinelli, V., Sun, H.H., Kaplita, P., Sedlock, D.M., Gillum, A.M., Chadwick, C.C., Cooper, R., 1997. Antimycins, inhibitors of ATP-citrate lyase, from a Streptomyces sp. J. Antibiot. 50, 729–733. Bowers, W.S., 2012. Insect hormones and antihormones in plants. In: Rosenthal, G.A., Berenbaum, M.R. (Eds.), Herbivores: Their Interactions with Secondary Plant Metabolites. Academic Press, New York, pp. 431–456. Cantrell, C.L., Dayan, F.E., Duke, S.O., 2012. Natural products as sources for new pesticides. J. Nat. Prod. 75, 1231–1242. Dhadialla, T.S., Retnakaran, A., Smagghe, G., 2009. Insect growth-and developmentdisrupting insecticides. In: Gilbert, L.I. (Ed.), Insect Development: Morphogenesis, Molting and Metamorphosis. Academic Press, London, pp. 679–740. Dhanasekaran, D., Sakthi, V., Thajuddin, N., Panneerselvam, A., 2010. Preliminary evaluation of Anopheles mosquito larvicidal efficacy of mangrove actinobacteria. Int. J. Appl. Biol. Pharm. Technol. 1, 374–381. El-Khawagh, M., Hamadah, K.S., El-Sheikh, T., 2011. The insecticidal activity of actinomycete metabolites against the mosquito Culex pipiens. Egypt. Acad. J. Biol. Sci. 4, 103–113. Gao, X., Wen, X., Esser, L., Quinn, B., Yu, L., Yu, C.-A., Xia, D., 2003. Structural basis for the quinone reduction in the bc1 complex: a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the Qi site. Biochemistry 42, 9067–9080. Ha, S.T., Wilkins, C.L., Abidi, S.L., 1989. Analysis of antimycin A by reversed-phase liquid chromatography/nuclear magnetic resonance spectrometry. Anal. Chem. 61, 404–408. Haegele, K.D., Desiderio, D., 1973. Structural elucidation of minor components in the antimycin A complex by mass spectrometry. J. Antibiot. 26, 215–222. Hartfelder, K., Emlen, D., 2012. Endocrine control of insect polyphenism. In: Gilbert, L.I. (Ed.), Insect Endocrinology. Academic Press, London, pp. 464–522. Hosotani, N., Kumagai, K., Nakagawa, H., Shimatani, T., Saji, I., 2005. Antimycins A10~A16, seven new antimycin antibiotics produced by Streptomyces spp. SPA-10191 and SPA-8893. J. Antibiot. 58, 460–467. Huang, L.-s., Cobessi, D., Tung, E.Y., Berry, E.A., 2005. Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc1 complex: a new crystal structure reveals an altered intramolecular hydrogen-bonding pattern. J. Mol. Biol. 351, 573–597. Inai, M., Nishii, T., Tanaka, A., Kaku, H., Horikawa, M., Tsunoda, T., 2011. Total synthesis of the (+)-antimycin A family. Eur. J. Org. Chem. 2011, 2719–2729.
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Yan, L.-L., Han, N.-N., Zhang, Y.-Q., Yu, L.-Y., Chen, J., Wei, Y.-Z., Li, Q.-P., Tao, L., Zheng, G.-H., Yang, S.-E., 2010. Antimycin A18 produced by an endophytic Streptomyces albidoflavus isolated from a mangrove plant. J. Antibiot. 63, 259–261. Zebitz, C.P., 1984. Effect of some crude and azadirachtin-enriched neem (Azadirachta indica) seed kernel extracts on larvae of Aedes aegypti. Entomol. Exp. Appl. 35, 11–16.
Gopalakrishnan, S., 2014. Biological activity of entomopathogenic actinomycetes against lepidopteran insects (Noctuidae: Lepidoptera). Can. J. Plant Sci. 94, 759–769. Xia, D., Yu, C.-A., Kim, H., Xia, J.-Z., Kachurin, A.M., Zhang, L., Yu, L., Deisenhofer, J., 1997. Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277, 60–66.
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Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Isolation and characterization of the insect growth regulatory substances from actinomycetes
T
Jong Hoon Kima, Jae Young Choia, Dong Hwan Parka, Dong-Jin Parkb, Min Gu Parka, So Young Kimb, Yoon Jung Jub, Jun Young Kima, Minghui Wanga, Chang-Jin Kimb, ⁎ Yeon Ho Jea,c, a
Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, Seoul 08826, Republic of Korea Industrial Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea c Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Streptomyces Secondary metabolites Juvenile hormone antagonist Insecticidal activity Antimycins
Insect growth regulators (IGRs) are attractive alternatives to chemical insecticides. Since it has been reported that secondary metabolites from actinomycetes show insecticidal activities against various insect pests, actinomycetes could be a potential source of novel IGR compounds. In the present study, insect juvenile hormone antagonists (JHANs) were identified from actinomycetes and their insect growth regulatory and insecticidal activities were investigated. A total of 363 actinomycetes were screened for their insect growth regulatory and insecticidal activities against Aedes albopictus and Plutella xylostella. Among them, Streptomyces sp. AN120537 showed the highest JHAN and insecticidal activities. Five antimycins were isolated as active compounds by assay-guided fractionation and showed high JHAN activities. These antimycins also exhibited significant insecticidal activities against A. albopictus, P. xylostella, F. occidentalis, and T. urticae. Moreover, dead larvae treated with these antimycins displayed morphological deformities that are similar to those of JH-based IGR-treated insects. This is the first report demonstrating that the insecticidal activities of antimycins resulted from their possible JHAN activity. Based on our results, it is expected that novel JHAN compounds potentially derived from actinomycetes could be efficiently applied as IGR insecticides with a broad insecticidal spectrum.
1. Introduction Actinomycetes are filamentous gram-positive bacteria with a high G + C content and widely distributed in various terrestrial and aquatic environments (Sharma et al., 2014). Actinomycetes represent one of the most studied classes of bacteria because they produce a broad spectrum of biologically active compounds (Solecka et al., 2012; Mahajan and Balachandran, 2014). Approximately 10,000 bioactive secondary metabolites are reported to be produced by actinomycetes, which represents 45% of all bioactive microbial metabolites discovered (Jackson et al., 2018). Actinomycetes have also been identified as potential biological pest control agents (Montesinos, 2003; Omura, 2011). It has been reported that secondary metabolites derived from actinomycetes show insecticidal activities against a variety of insect pests belonging to Lepidoptera and Diptera (Dhanasekaran et al., 2010; El-Khawagh et al., 2011; Karthik et al., 2011; Ababutain et al., 2012; Vijayabharathi et al.,
2014). Furthermore, recent studies have shown that actinomycetes produce secondary metabolites with insect growth regulatory activities (Arasu et al., 2013; Kaur et al., 2014; Samri et al., 2017). Insect growth regulators (IGRs) are promising alternatives to conventional chemical insecticides because of their selectivity and relatively low toxicity (Dhadialla et al., 2009; Pener and Dhadialla, 2012). Among them, juvenile hormone (JH)-based IGRs such as JH agonists (JHA) and JH antagonists (JHAN) can promote abnormal development, premature molting, and supernumerary larval stages by fatally disrupting the endocrine system because JH regulates diverse aspects of insect physiology including metamorphosis, reproduction, polyphenism, and immunity (Hartfelder and Emlen, 2012; Lee et al., 2015). We previously developed an effective in vitro JH-based IGR screening system using yeast cells transformed with the Aedes aegypti JH receptors methoprene-tolerant (Met) and Ftz-F1-interacting steroid receptor coactivator (FISC) (Lee et al., 2015). Using this screening system, various substances with JHA or JHAN activity have been
⁎ Corresponding author at: Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea. E-mail address: [email protected] (Y.H. Je).
https://doi.org/10.1016/j.cbpc.2019.108651 Received 27 August 2019; Received in revised form 21 October 2019; Accepted 26 October 2019 Available online 31 October 2019 1532-0456/ © 2019 Elsevier Inc. All rights reserved.
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identified from plant essential oils and chemical libraries (Lee et al., 2018a, 2018b, 2018c). It was assumed that actinomycetes could be another source of natural JHAs and JHANs because they produce a variety of secondary metabolites with insecticidal and insect growth regulatoryactivities. In this study, novel potential JHAN compounds were identified from actinomycetes by screening 363 actinomycete isolates for their JHA/JHAN activities; their insecticidal activities were also evaluated.
2.4. Yeast growth inhibition tests The growth inhibition test for yeast treated with corresponding concentrations (1 mg/mL for crude extract and 2 μg/mL for purified compound) was performed as previously described (Lee et al., 2018a). The inhibition tests were performed in triplicate. 2.5. Bioassays Thirty A. albopictus 3rd instars were treated with actinomycete extract by adding 1 mg/mL of crude extract or 2 μg/mL of purified compound in 5 mL tap water with food mixtures. In the case of P. xylostella, thirty 3rd instars were soaked in the corresponding concentrations (1 mg/mL for crude extract and 2 μg/mL for purified compound) for 30 s and the treated larvae were provided Chinese cabbage leaf discs (3 cm diameter). Female adult F. occidentalis were collected using a custom-made aspirator. Thirty females were treated with 10 μg/mL of purified compound by soaking for 30 s. The treated females were transferred onto kidney beans. For the bioassay against T. urticae, kidney bean leaf discs (15 mm diameter) were permeated with 10 μg/ mL of purified compound for 5 min. Thirty 3rd instar nymph T. urticae were inoculated on the permeated leaf disc and placed onto a Petri dish with a water-soaked thin layered cotton. The number of dead insects was checked at 24 h intervals and calculated after treatment for 3 days. All experiments were performed in triplicate.
2. Material and methods 2.1. Isolation of actinomycetes and fermentation A total of 363 actinomycetes were isolated from soil samples collected from various sites in Korea. One gram of soil samples was serially diluted with distilled water and homogenized by vortexing for 10 min. An aliquot of the soil suspension was plated on humic acid-vitamin agar (1 g humic acid, 0.5 g Na2HPO4, 1.71 g KCl, 0.05 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 1 g CaCl2, B vitamins (0.5 mg each of thiamine-HCl, riboflavin, niacin, pyridoxine, Ca-pantothenate, inositol and p-aminobenzoic acid, and 0.25 mg of biotin), 15 g agar and 1 L water, pH 7.2) and incubated at 28 °C for 7 days. Selected isolates were maintained as a spore suspension in glycerol (20%, v/v) at −80 °C. All 363 isolates were fermented in a 500 mL baffled Erlenmeyer flask containing 100 mL of glucose and soybean meal. Soluble starch broth was used to maintain and cultivate Actinomycete spp. (main ingredients included: glucose, soluble starch, soybean meal, beef extract, yeast extract, NaCl, K2HPO4 and CaCO3), which were incubated on a rotary shaker (150 rpm) at 30 °C for 7 days. After fermentation, the cells were separated from the culture broth by centrifugation at 10,000 rpm for 15 min at 4 °C. The separated cells were dissolved in an equal volume of acetone and mixed for 3 h. The acetone phase was concentrated in a rotary vacuum and the crude extracts were redissolved in dimethyl sulfoxide (DMSO) for activity tests.
2.6. Statistical analysis One-way ANOVA was performed using the SPSS software (version 24, SPSS, Inc., Chicago, IL, USA). Comparison of the mean values was performed using Scheffé's method, and P values < 0.05 were considered statistically significant. 2.7. Taxonomic characteristics of the selected strain The cultural characteristics of strain AN120537 were determined on various International Streptomyces Project (ISP) media. The colony color was evaluated using the Inter-Society Color Council-National Bureau of Standards (ISCC-NBS) color chart (Kelly, 1964). PCR amplification and nucleotide sequence analysis of the 16S rRNA gene of the strain AN120537 was performed as reported by Li et al. (2007). The 16S rRNA sequences from closely related actinomycetes were retrieved from the National Center for Biotechnology Information (http://www.ncbi. nlm.nih.gov/GenBank/index.html) and aligned using the CLUSTAL X program (Thompson et al., 1997). Molecular phylogeny of strain AN120537 16S rRNA was inferred using the neighbor-joining method under the Jones-Taylor-Thornton (JTT) matrix-based model incorporated in the MEGA7 software (Jones et al., 1992).
2.2. Insects A. albopictus (Skuse) (Diptera: Culicidae) was maintained in breeding chambers at 28 °C and 70% relative humidity with a 12 h light/12 h dark cycle in aged tap water. The larvae were fed a diet of TetraMin fish flakes, and adults were reared using 10% sucrose solution. The diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) and the western flower thrips Frankliniella occidentalis (Pergande) were reared on rape sprouts and kidney bean cotyledons, respectively, and maintained at 25 °C and 70% relative humidity with a 16 h light/8 h dark cycle. The mite Tetranychus urticae Koch was reared on kidney bean at 25 °C and 60% relative humidity with a 16 h light/8 h dark cycle.
2.8. Isolation of JHAN compounds from strain AN120537 cultures Strain AN120537 mycelia were extracted with an equal volume of acetone as described in Section 2.1. The dried acetone extract was dissolved in distilled water and sequentially extracted with equivalent volumes of n-hexane, ethyl acetate and n-butanol. The JHAN and insecticidal activities of the extracted substances were determined as described in Sections 2.3 and 2.5. The concentrated hexane extract, which showed the highest JHAN and insecticidal activities, was separated using a Biotage Isolera (Charlottesville, USA) automated purification system equipped with a UV detector at 254 nm and a SNAP column cartridge (100 g silica gel). Separation was carried out with a chloroform/methanol (95:5) isocratic solution to give eight fractions, among which only one fraction showed JHAN and insecticidal activities at a concentration of 1 mg/mL. The active fraction was separated by preparative reverse-phase HPLC (solvent: methanol/water 70:30, flow rate: 1.0 ml/min) using an Inno C18 column (5 μm, 150 × 4.6 mm;
2.3. Yeast two-hybrid binding tests The yeast two-hybrid β-galactosidase assay for quantitative determination of JHA/JHAN activities was performed as previously described (Lee et al., 2018a). One mg/mL of crude extract or 2 μg/mL of purified compound was applied to yeast cells transformed with A. aegypti Met-FISC to estimate JHA/JHAN activities. The obtained OD420 values were converted to an arbitrary unit representing JHAN activity. Yeast two-hybrid binding tests for JHA/JHAN activities were performed in triplicate.
JHAN activity OD420 of pyriproxyfen (0.033 ppm) − OD420 of treated sample = OD420 of pyriproxyfen (0.033 ppm) 2
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Young Jin Biochrom, Korea) to give five compounds, which were further purified using an U-VDSpher PUR 100 C18E column (1.8 μm, 50 × 2.0 mm VDS Optilab, Germany) with acetonitrile/water (70:30) containing 0.2% formic acid at a flow rate of 0.3 mL/min.
and was selected for further studies. 3.3. Taxonomic identification of strain AN120537 The cultural and physiological characteristics of strain AN120537 observed at 14 days after growing on ISP media suggest that this strain belongs to the genus Streptomyces (Table 1). For further phylogenetic profiling of strain AN120537, the nucleotide sequence of its 16S rRNA gene was compared with those of representative Streptomyces strains. A phylogenetic tree constructed using the neighbor-joining method showed that strain AN120537 was most closely related to Streptomyces celluloflavus NRRL B-2493(T), with 99.03% 16S rRNA nucleotide sequence similarity (Fig. 3).
2.9. Structural determination of the active compounds The 1H and 13C nuclear magnetic resonance (NMR) (600 MHz) spectra were obtained using a high-resolution Avance 600 NMR spectrometer (Bruker, Germany) with methanol as the solvent. Electrospray ionization-mass spectrometry (ESI-MS) was performed using a Q-TOF 5600 (AB Sciex, Canada) high-resolution liquid chromatographytandem mass (LC/MS/MS) spectrometer. The chemical structure of each compound was determined by comparing the NMR and MS data with published literature values.
3.4. Purification of JHAN compounds from strain AN120537
3. Results
Based on bioassay-guided monitoring, the hexane extract of strain AN120537 was fractionated, and five compounds were obtained. The molecular weight and formula of compound 1, which was isolated as a white amorphous solid, was determined as C24H32N2O9 by ESI-MS ([M + H]+, m/z 493.21) and 1D NMR spectra (1H and 13C NMR) (Figs. S1 and S2). The planar structure of the compound was further analyzed by 2D NMR (1H-1H COSY, HSQC, and HMBC) (Fig. S3). The structure of compound 1 was readily identified as antimycin A5 (Fig. 4) by comparison with previously reported structures (Kluepfel et al., 1970; Schilling et al., 1970; Haegele and Desiderio, 1973). The ESI-MS measurements and 1H NMR spectra of compounds 2–5 were significantly similar to those of compound 1 (Figs. S4–S7), suggesting that they are also antimycin derivatives. Based on comparison of their spectroscopic data with those in the literature (Abidi and Adams, 1987; Ha et al., 1989; Barrow et al., 1997; Inai et al., 2011), compounds 2–5 were identified as antimycins A4, A3, A2, and A1, respectively (Fig. 4).
3.1. Insect growth regulatory activities of actinomycete strains Acetone extracts from 363 actinomycete strains were screened for their JHA and JHAN activities using in vitro yeast two-hybrid β-galactosidase assays. Whereas none of the 363 actinomycete extracts tested showed JHA activities (Table S1), 8 extracts showed high levels of JHAN activity at a final concentration of 1 mg/mL (Fig. 1A). Additionally, actinomycete extracts with high JHAN activities showed no anti-yeast activities when they were subjected to yeast growth inhibition tests (Fig. 1B), demonstrating that their JHAN activities originated from direct disruption of JH receptor complex formation. 3.2. Insecticidal activities of actinomycete strains with JHAN activity The insecticidal activities of the actinomycete extracts with high JHAN activities were investigated at a concentration of 1 mg/mL against an important medical pest (A. albopictus) and an economically important agricultural pest (P. xylostella). Among the 8 extracts, extracts from strains AN120537, AN120590, and AN120867 showed high levels of mosquito larvicidal activities against A. albopictus 3rd instar larvae, with mortalities of 100% (Fig. 2A). Against P. xylostella 3rd instar larvae, the extracts from strains AN120537 and AN120590 showed high insecticidal activities, with mortalities > 80% (Fig. 2B). Strain AN120537 exhibited the highest JHAN and insecticidal activities
3.5. JHAN activities of the strain AN120537 antimycins To investigate the JHAN activities of the antimycins purified from strain AN120537, these compounds were subjected to a yeast two-hybrid β-galactosidase assay. All the antimycins tested displayed increasing JHAN activities with increasing concentrations, demonstrating that these compounds interfere with the pyriproxyfen-mediated binding of A. aegypti Met-FISC in a concentration-dependent manner (Fig. 5). Among them, specifically, antimycin A5 exhibited the highest JHAN
Fig. 1. Screening of actinomycete extracts with JHAN activity. (A) To estimate the JHAN activity, 0.033 μg/ml of pyriproxyfen and 1 mg/mL of each actinomycete extract were applied to a yeast two-hybrid β-galactosidase assay. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05). (B) Actinomycete extracts with JHAN activities were tested for their anti-yeast activities to examine whether the reduced β-galactosidase activity resulted from JHAN activity or yeast toxicity. 3
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Fig. 2. Insecticidal activities of actinomycete extracts with JHAN activities against A. albopictus (A) and P. xylostella (B) larvae. A. albopictus and P. xylostella 3rd instar larvae were treated with 1 mg/mL of each actinomycete extract and the mortality was calculated every 24 h for 3 days after treatment. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05).
activity at a concentration > 2 μg/mL. None of the antimycin derivatives showed anti-yeast activities in growth inhibition tests (Fig. S8).
Table 1 Cultural characteristics of Streptomyces sp. strain AN120537 on various ISP media. Medium
ISP ISP ISP ISP ISP ISP
2 3 4 5 6 7
Growth
Good Good Moderate Moderate Good Moderate
Soluble pigment
– – – – Pinkish purple Pale yellow
Colony color
3.6. Insecticidal activities of the strain AN120537 antimycins
Aerial mycelium
Substrate mycelium
Whitish gray Pale yellow Pale yellow Pale yellow Dark brown Yellow
Pale yellow Pale brown Whitish gray Pale yellow Dark grayish brown Pale yellow
To evaluate the insecticidal activities of strain AN120537 antimycins, A. albopictus and P. xylostella 3rd instar larvae were treated with each compound at a concentration of 2 μg/mL. All five compounds caused 100% larval mortality against A. albopictus (Fig. 6A). Typical morphological deformations were observed from the dead larvae compared to untreated larvae. These deformations were manifested as body segment contraction (Fig. 6C), deformed heads with elongated neck regions and black pigmentation on the entire body (Fig. 6D). In the case of P. xylostella larvae, antimycins A1 and A5 showed the highest
Fig. 3. Phylogenetic relationship of strain AN120537 based on its 16S rRNA gene sequence. The nucleotide sequences of 16S rRNA gene from the genus Streptomyces were compared by the neighbor-joining method. Numbers at each branch node indicate the bootstrap percentage of 1000 replications. 4
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Fig. 4. The chemical structures of the antimycins purified from strain AN120537.
their old cuticles (Fig. 7C and D). The insecticidal spectrum of these antimycins was further investigated against serious sucking pests including F. occidentalis and T. urticae at a concentration of 10 μg/mL. In the case adult female F. occidentalis, antimycins A3, A4, and A5 exhibited
insecticidal activities, with mortalities > 80% (Fig. 7A). Most of the dead P. xylostella larvae treated with antimycins also showed morphological abnormalities such as disordered setae and an inability to withdraw from the head capsule causing failure to completely shed
Fig. 5. Concentration-dependent JHAN activities of antimycins from strain AN120537. To estimate the JHAN activity, 0.033 μg/mL of pyriproxyfen and corresponding concentrations (0.1, 1, 2, 5, and 10 μg/mL) of each antimycin were applied to a yeast two-hybrid β-galactosidase assay. 5
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Fig. 6. Insecticidal activities of the antimycins from strain AN120537 against A. albopictus. (A) A. albopictus 3rd instar larvae were treated with 2 μg/mL of each antimycin and the mortality was calculated every 24 h for 3 days after treatment. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05). (B) Morphological characteristics of untreated A. albopictus larvae. HE, Head; TH, thorax; AB, abdomen; AP, anal papillae; RS, respiratory siphon. (C) and (D) Morphological deformations of dead larvae after antimycin treatment.
Fig. 7. Insecticidal activities of the antimycins from strain AN120537 against P. xylostella. (A) P. xylostella 3rd instar larvae were treated with 2 μg/mL of each antimycin and the mortality was calculated every 24 h for 3 days after treatment. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05). (B) Morphological characteristics of untreated P. xylostella larvae. (C) and (D) Morphological abnormalities of dead larvae after antimycin treatment.
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Fig. 8. Insecticidal activities of the antimycins from strain AN120537 against F. occidentalis (A) and T. urticae (B). F. occidentalis adult females and T. urticae 3rd instar nymphs were treated with 10 μg/mL of each antimycin and the mortality was calculated every 24 h for 3 days after treatment. Different letters above the error bars indicate significant differences by Scheffé's test (P < 0.05).
the JH receptor complex by competing with JH for binding to Met, their mode of action as JHANs is different from that of precocenes. Additionally, the high insecticidal activities of these compounds suggested that actinomycetes might be plentiful sources of novel IGR compounds, which could be exploited for the development of biopesticides. Antimycins (A1-A5) were identified as active compounds from strain AN120537, which was assigned to Streptomyces celluloflavus by physiological characterization and phylogenetic analysis using its 16S rRNA gene sequence. Antimycins are a family of depsipeptides with structural skeletons of a 9-membered dilactone ring connected to 3formamidosalicylic acid by an amide linkage (Liu et al., 2014). Since their first report from Streptomyces sp. NRRL 2288 in 1949, a variety of natural antimycins have been identified from various actinomycete species (Liu et al., 2016). This family of compounds has been previously reported to exhibit antifungal, insecticidal, and nematocidal properties (Hosotani et al., 2005; Shiomi et al., 2005; Yan et al., 2010; Liu et al., 2019). These compounds have also been known to inhibit the mitochondrial electron transport chain by binding to the quinone reduction site Qi in the cytochrome bc1 complex (Xia et al., 1997; Gao et al., 2003; Huang et al., 2005). In insects, antimycins may act as JHANs and have shown insecticidal activities against A. albopictus. Furthermore, antimycins exhibit a significantly broad insecticidal spectrum, encompassing biting-type (P. xylostella) and sucking-type (F. occidentalis and T. urticae) pests. The dead larvae of A. albopictus showed morphological deformities similar to growth-disrupting effects reported from A. aegypti larvae due to application of some actinomycete extracts (Zebitz, 1984; Sakthivadivel and Thilagavathy, 2003; Kabir et al., 2013; Mahyoub et al., 2016). Moreover, morphological abnormalities observed from dead P. xylostella larvae were comparable to those of P. xylostella treated with another IGR, pyriproxyfen (Alizadeh et al., 2012). Taken together, these results demonstrated that antimycins may disrupt the formation of the JH receptor complex in target insects, which might disturb JH-based endocrine regulation of insects and thereby cause insect mortality.
superior activities, with mortalities > 60% (Fig. 8A). In the T. urticae bioassay, application of antimycins A2 and A3 promoted the highest mortality, which was > 70% (Fig. 8B). 4. Discussion During the last several decades, studies searching for eco-friendly insecticidal compounds have been extensively carried out to overcome the undesirable effects caused by synthetic insecticides such as toxicity to the environment and the development of resistant pests. Many natural products originating from plants and microorganisms have been successfully utilized as environmentally benign alternatives to synthetic insecticides (Cantrell et al., 2012). Specifically, actinomycetes have been regarded as plentiful sources for natural products as they have been reported to produce various secondary metabolites with a diverse range of biological activities, including insecticidal, antifeedant, and insect growth inhibitory activities (Omura, 2011; Arasu et al., 2013; Vijayabharathi et al., 2014). In this study, among 363 actinomycetes isolated from Korean soil samples, extracts from eight strains showed high JHAN activities, with the strain AN120537 extract causing 100% mortality against both A. albopictus and P. xylostella larvae at a concentration of 1 mg/ml, demonstrating that the strain might produce novel JHAN compounds with high insecticidal activities. Several endophytic actinomycetes have been reported to play a role as beneficial symbionts with plants, and diverse bioactive metabolites from symbiotic actinomycetes could have been utilized by plants to endure environmental stresses, such as plant diseases and insect pests, over the course of plant and actinomycete coevolution (Qin et al., 2011; Kinkel et al., 2012). Thus, potential JHAN compounds derived from endophytic actinomycetes might be adopted as effective defense mechanisms for symbiotic plants because IGRs are insect-specific, and insects may have difficulty acquiring resistance to these IGRs (Bowers, 2012). These results provided novel insights into the interactions between actinomycetes, plants, and insects. Botanical precocenes isolated from Ageratum houstonianum and their synthetic analogues have also been reported to show JHAN activities (Banerjee et al., 2008). They cause precocious metamorphosis in several insect species by inducing irreversible degeneration of the corpora allata and inhibiting JH production (Azambuja and Garcia, 1991). However, precocenes have not been commercialized as insecticides because of their low insecticidal activities and potential carcinogenic effects in mammals (Alzogaray and Zerba, 2017). Because the JHAN compounds from actinomycetes identified in this study interrupt the formation of
5. Conclusions In this study, the Streptomyces strain AN120537, which displayed high levels of JHAN and insecticidal activities, was selected through in vitro screening of 363 actinomycete strains. Antimycins isolated from strain AN120537 showed possible JHAN activities in a concentrationdependent manner and a broad spectrum of insecticidal activities against A. albopictus, P. xylostella, F. occidentalis, and T. urticae. These 7
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results suggest that actinomycetes are promising sources of novel IGR compounds when exploring natural products with effectiveness and environmental safety. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpc.2019.108651.
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