Indigenous Bacillus Species Isolated From Aedes Aegypti Larvae: Isolation, Larvicidal Toxicity Screening, Phenotypic Characterization, and Molecular Identification

Abstract

Vector-borne diseases transmitted by mosquitoes are considered a significant public health problem worldwide. Aedes aegypti is one of the mosquito species responsible for transmitting these diseases. One environmentally friendly method of vector control is the use of microbial agents such as Bacillus species. This study aimed to explore investigate indigenous entomopathogenic bacteria of Bacillus species isolated from A. aegypti larvae. Larvae samples were collected from breeding sites of A. aegypti. All isolates underwent screening and affirmation confirmation tests to assess their larvicidal toxicity against A. aegypti larvae. Phenotypic characterizations and molecular identifications were conducted to determine the species of the Bacillus isolates based on similarity index and percent identity (%ID). Phylogenetic trees were used to compare the isolates with other Bacillus species. The results revealed 120 isolates of Bacillus species from A. aegypti larvae samples. Among them, three isolates (LS3.3, LS9.1, and LSD4.2) exhibited the highest larvicidal toxicity in the confirmation test, resulting in larval mortality rates of 100%, 96.7%, and 100%, respectively, after 48 hours of exposure. Molecular identifications, showed that LSD4.2 had a 99.16% ID with Bacillus velezensis, LS3.3 had a 98.22% ID with Bacillus mojavensis, and LS9.1 had a 99.93% ID with Bacillus subtilis. These three bacteria from the Bacillus genus have been reported to offer significant benefits to humans.

Prospects of Indigenous Bacillus subtilis Strain LS9.1 as a Potential Biocontrol Agent against Aedes aegypti Larvae

“…and the BLAST analysis has a 99.98% similarity with B. subtilis subsp. inaquosorum (Salamun et al 2023). Dunlap et al (2020) have promoted that B. subtilis currently includes four subspecies, B. subtilis subsp.…”

“…Genomic DNA of B. subtilis strain LS9.1 was isolated using DNA Purification by Promega Kit (Tamura et al 2013). The process of gene amplification, isolation of the Cry-IV DNA gene, and preparation of samples for electrophoresis was carried out as for the 16S rRNA gene (Ausubel et al 2003;Salamun et al 2023). The Cry-IV gene primers are self-designed, which were forward (5'-GCATATGATGTAGCGAAACAAG-3') and reverse (5'-GCGTGACATACCCATTTCC-3') (Aramideh et al 2016).…”

“…B. thuringiensis has been reported to successfully kill mosquito larvae of species of the genera Aedes, Culex and Anopheles. Studies on the isolation and identification of Bacillus are still being carried out, because of the numerous of disease vectors are transmitted by mosquitoes (Katak et al 2021;Salamun et al 2023). Manonmani et al (2011) recommended to increase mosquito cyclic lipopeptide production from B. subtilis subtilis.…”

“…Bacillus subtilis strain LS9.1 was one of the indigenous bacteria isolated from larvae of A. aegypti in the breeding sites of Surabaya city, East Java, Indonesia (Salamun et al 2023). In the screening and confirmation test whole culture, a mixture of pellets and supernatant against larvae of A. aegypti with a larval mortality percentage of 90% at 24 hours exposure and 96.7% at 48 hours exposure (Salamun et al 2023).…”

“…Bacillus subtilis strain LS9.1 was one of the indigenous bacteria isolated from larvae of A. aegypti in the breeding sites of Surabaya city, East Java, Indonesia (Salamun et al 2023). In the screening and confirmation test whole culture, a mixture of pellets and supernatant against larvae of A. aegypti with a larval mortality percentage of 90% at 24 hours exposure and 96.7% at 48 hours exposure (Salamun et al 2023). This research the larvicidal toxicity test of whole culture, ultrastructure cells and molecular weight of parasporal inclusion protein, the coding gene of protein toxins and hemolytic activity test to detect mechanisms of actions the toxicity of B. subtilis strain LS9.1 against A. aegypti larvae.…”