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Aim: The present study aimed to select a powerful tool to evaluate the antibiotic susceptibility of endosymbionts of papaya mealybug. Methodology: Antibiotic Ofloxacin (0.25µg) was used to disrupt the endosymbionts of mealybug. The antibiotic susceptibility of endosymbionts was studied by conventional method; isolation of symbionts in Luria Bertani Agar and Nutrient Agar and also metagenomic analysis was conducted using nanopore sequencing of 16S rRNA gene. Results: Molecular identification of isolates showed Gram-positive bacteria i.e., Bacillus cluasii, B. altitudinis, B. siamensis and Gram-negative bacteria, i.e., Serratia marcescens and Stenotrophomonas maltophilia. Metagenomic analysis using 16S amplicon sequencing resulted in identification of Candidatus Tremblaya Princeps (55%) was more abundant followed by Candidatus Mikella endobia (23%) in untreated mealybug population. Their population was reduced to 7% in Candidatus Tremblaya Princeps and below 1% in Candidatus Mikella endobia. Interpretation: The current study confirms that the metagenomic analysis acts as a power lens to evaluate the antibiotic elimination of endosymbionts, hence with this method future works on developing aposymbiotic mealybugs can be done.

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