Investigation On Bioactive Metabolites Produced By Three Insect Symbionts

Microorganisms engaged in a long-term relationships with insect hosts, are important mediators in complex ecological interactions. The insect species are large, and their habitats are multifarious, indicating the symbionts associated with them are extensive and their metabolic pathways are distinctive. Insect symbionts are regarded as sources of new species and novel bioactive metabolites. However, only a small percentage of such diversity microorganisms group have been cultivated and chemically studied.In this dissertation, three symbionts including one fungus and two actinomyces were isolated from the gut of Longhorned grasshoppers and the body of Odontotermes formosanus, respectively. The strains were classified by morphological characteristics and analysis of rDNA sequence. From the culture filtrates of above strains,10compounds were isolated using chromatography techniques including silica gel column chromatography, sephadex LH-20, reversed phase C18and HPLC. The structures of these metabolites were elucidated by spectral experiments such as ESI-MS,1D-and2D-NMR. Tn addition, unreported bioactivities of the new and known compounds were investigated in our study. The specific were detailed as follows:One bioactive fungus was isolated from the gut of L. grasshoppers. The strain was identified as Fusarium proliferatum by the observation of morphological characteristics and analysis of ITS rDNA sequence. Six fungal metabolites, including a new polyketide derivate named cercophorins E (2) and five known compounds cercophorins D (1), rhodolamprometrin (3), radicinin (4), dehydroallogibberic acid (5) and3-methyl-6,8-dihydroxyisocoumarin (6) were isolated from the fermentation broth of F. proliferatum ZS07. Cercophorins E and radicinin showed good phytotoxic activity against the growth of Amaranthus retroflexus L. at the concentration of100μg/mL, with the inhibition rate of83.0%and65.2%, respectively. The antibacterial activity of compounds were evaluated against selected bacteria and the results showed that cercophorins D, E and rhodolamprometrin possess potent antimicrobial activity against Bacillus subtilis (ATCC6633), with the MIC values of3.1-12.5μg/mLOne actinomyces, identified as Streptomyces canus was isolated from the body of O. formosanus using spread plate method. Further study of the metabolites of this strain led to the isolation of two compounds, which were elucidated as resistomycin (7) and tetracenomycin (8). Resistomycin had strong antifungal activities against mycelial growth of Valsa mali (IC50=1.1μg/mL) and Magnaporthe grisea (IC50=3.8μg/mL), which were comparable to those of referenced cycloheximide, with IC50values of2.3and0.3μg/mL, respectively. The test of spore germination showed that resistomycin exhibited potent reduction in spore germination of M. grisea (IC50=5.55μg/mL). Finally, the antifungal activity experiment in vivo showed that resistomycin possessed promising preventive efficacy against rice blast, which was more potent than that of referenced carbendazim, with control efficacies of66.8%and58.7%, respectively. To our best knowledge, it is the first report that resistomycin possessed potent antifungal activities against phytopathogens.Another actinomyces, identified as S. davawensis, was isolated from the body surface of queen termite (O. formosanus) using spread plate method. Further study of the metabolites led to the isolation of two compounds roseoflavin (9) and8-methylamino-8-demethyl-D-riboflavin (10). Roseoflavin exhibited potent antibacterial activity against gram-positive bacterium B. subtilis (ATCC6633)(MIC=1.56μg/mL) and Staphylococcus aureus (ATCC8739)(MIC=3.125μg/mL), which were comparable to that of referenced gentamicin, with MIC value of1.56μg/mL.The discovery of our study provided additional evidence that poorly or neglected insect symbiontic microbes may inspire the discovery of chemical agents with interesting biological activity.