Abstract
Background
Mosquito-borne diseases continue to pose major public health challenges, necessitating the development of environmentally safe biological control agents. Although several bacterial larvicides are currently used, novel gut-associated bacteria with high specificity and low non-target toxicity remain under-explored. This study (This is the first report demonstrating mosquito larvicidal activity of Alcaligenes faecalis against Culex quinquefasciatus larvae.) reports, for the first time, the mosquito larvicidal potential of Alcaligenes faecalis against Culex quinquefasciatus larvae.Methods
A bacterial (This is the first report demonstrating mosquito larvicidal activity of Alcaligenes faecalisagainst Culex quinquefasciatus larvae.) isolate, obtained from the midgut of naturally dead and moribund Cx. quinquefasciatus larvae, was identified as Alcaligenes faecalis strain BUMCn01 (GenBank accession: PX526002) based on morphological, biochemical, physiological, and 16S rRNA gene analyses. Larvicidal bioassays using A. faecalis strain BUMCn01 formulation were performed against all four larval instars. Bacterial localization and aggregation in the larval midgut following exposure to A. faecalis strain BUMCn01 were examined by scanning electron microscopy. Field efficacy was evaluated in sewage drains of different depths. Laboratory evaluations of the A. faecalis strain BUMCn01 formulation was also conducted on non-target aquatic organisms (Chironomus circumdatus larvae, Daphnia spp., Poecilia reticulata, and tadpoles of Bufo spp.). Furthermore, bacterial proteins were isolated and assessed for larvicidal efficacy against 3rd instar larvae, to identify the bacterial component responsible for the bioactivity.Results
The isolate was identified as Alcaligenes faecalis strain BUMCn01 using a polyphasic approach including morphological, biochemical, physiological and 16S rRNA gene sequence analysis (GenBank accession: PX526002).The Alcaligenes faecalis BUMCn01 formulation resulted in clear dose-response relationship. 100% mortality was recorded in 1st instar larvae after 24 h and in 2nd and 3rd instars Cx. quinquefasciatus larvae after 48-72 h at 2.0 mg/mL. 24 h LC₅₀ values ranged from 395 µg/mL to 490 µg/mL across all larval instars, with 1st instar larvae being the most susceptible. Scanning electron microscopy revealed bacterial proliferation and dense aggregation witin the treated larval midgut, comparison with control indicating oral ingestion and possible gut mediated toxicity. Field trials demonstrated complete larval elimination within 7-9 days at an application rate of 100 ml/m2, with moderate residual activity. In non-target aquatic organisms, the A. faecalis BUMCn01 formulation exhibited no mortality in tadpoles of Bufo spp. and only mild effects on C. circumdatus, Daphnia spp. and guppy (P. reticulata). The isolated proteins were found to possess potent larvicidal activity (24 h LC50 of 491 µg/mL) against 3rd instar larvae.Conclusions
This study provides the first evidence that (The isolate was identified as Alcaligenes faecalis strain BUMCn01 using a polyphasic approach including morphological, biochemical, physiological, and 16S rRNA gene sequence analysis (GenBank accession: PX526002).) Alcaligenes faecalis strain BUMCn01 exhibits significant larvicidal activity against Culex quinquefasciatus. Bacterial proteins appear to be the primary toxic agent. The Alcaligenes faecalis BUMCn01 formulation showed significant efficacy in laboratory and field trials with minimal non-target toxicity, supporting its potential use as a microbial larvicide in integrated mosquito management.