Exploration of culturable bacterial associates of aphids and their interactions with entomopathogens.

Aphids shelter several bacteria that benefit them in various ways. The associates having an obligatory relationship are non-culturable, while a few of facultative associates are culturable in insect cell lines, axenic media or standard microbiology media. In the present investigation, isolation, and characterization of the culturable bacterial associates of various aphid species, viz., Rhopalosiphum maidis, Rhopalosiphum padi, Sitobion avenae, Schizaphis graminum, and Lipaphis erysimi pseudobrassicae were carried out. A total of 42 isolates were isolated using different growth media, followed by their morphological, biochemical, and molecular characterization. The isolated culturable bacterial associates were found to belong to the genera Acinetobacter, Bacillus, Brevundimonas, Cytobacillus, Fictibacillus, Planococcus, Priestia, Pseudomonas, Staphylococcus, Sutcliffiella, and Tumebacillus which were grouped under seven families of four different orders of phyla Bacillota (Firmicutes) and Pseudomonata (Proteobacteria). Symbiont-entomopathogen interaction study was also conducted, in which the quantification of colony forming units of culturable bacterial associates of entomopathogenic fungal-treated aphids led us to the assumption that the bacterial load in aphid body can be altered by the application of entomopathogens. Whereas, the mycelial growth of entomopathogens Akanthomyces lecanii and Metarhizium anisopliae was found uninhibited by the bacterial associates obtained from Sitobion avenae and Rhopalosiphum padi. Analyzing persistent aphid microflora and their interactions with entomopathogens enhances our understanding of aphid resistance. It also fosters the development of innovative solutions for agricultural pest management, highlighting the intricate dynamics of symbiotic relationships in pest management strategies.

LC-MS/MS analysis of carcinogenic tobacco-specific nitrosamines in Spodoptera litura using the QuEChERS method.

Nicotine is a highly addictive alkaloid and a neurostimulator found in tobacco that causes addiction in humans and makes tobacco a high-demand commercial product. It is popularly used for recreational purposes and is a harmful substance (Oral LD50 value for rat is 50 mg/kg) and causes addiction. The metabolites of nicotine such as the Tobacco-specific Nitrosamines (TSNAs) are hazardous substances whose metabolites are highly electrophilic and form DNA adducts, which will initiate the process of carcinogenesis. TSNAs are formed during curing, storage and fermentation due to the nitrosation of nicotine and other tobacco alkaloids. TSNAs are used as biomarkers for cancer risk assessment in humans exposed to tobacco and its products. To determine the occasional formation of TSNAs in tobacco-feeding insects, 5th instar larvae of Spodoptera litura and their faeces were analyzed for the presence of N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) along with the stored tobacco leaves (PT-76) using an Agilent 6470B LC-MS/MS system following ISO/DIS 19290:2015 protocol. The larvae are extracted in a buffered acetonitrile-water extraction and the amount of TSNAs are quantified in multiple reaction monitoring (MRM) mode. 20 [Formula: see text]l of each extracted and cleaned up sample was injected into the LC-MS/MS system for quantification. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were 0.001 mg/kg and 0.005 mg/kg for all the tested nitrosamines. NNN was found to be 0.361 mg/kg, 0.340 mg/kg, and 5.66 mg/kg in insect whole-body samples, faeces, and tobacco leaves, respectively. NNK was found to be 0.060 mg/kg, 0.035 mg/kg and 0.93 mg/kg in insect whole body samples, faeces and tobacco leaves, respectively. However, NNAL was not detected in both the insect's whole body and faeces. Recoveries ranged between 95 and 98% for all compounds when spiked at LOD and LOQ. The presence of TSNAs is a biomarker for cancer risk and their presence in insects would point to cancer risk assessment in tobacco feeding insects and any possible TSNA-detoxifying pathways in insects that might prevent mutagenesis caused these compounds.