Isolation of a Pseudomonas fluorescens metabolite/exotoxin active against both larvae and pupae of vector mosquitoes

Abstract

A formulation was developed from the metabolite(s) of a novel Pseudomonas fluorescens Migula strain (VCRC B426) and tested against 4th-instar larvae and pupae of three species of vector mosquitoes, Anopheles stephensi Liston, Culex quinquefasciatus Say and Aedes aegypti (L). The larvae and pupae of An stephensi were the most susceptible to the formulation, followed by those of C quinquefasciatus and Ae aegypti, in that order, and the dosage requirement for pupal mortality was less than that required for larval mortality. The LC 50 dosage requirements for larvae of these mosquito species were, respectively, 70.4, 511.5 and 757.3 mg protein ml À1 , whereas for pupae they were, respectively, 2.0, 9.4 and 19.2 mg protein ml À1 . The lethal fraction was purified from the culture broth and its molecular mass, as determined by high performance liquid chromatography, was 44 kDa. This is the first report of a microbial formulation acting upon mosquito pupae, a non-feeding stage. Its mode of action and efficacy to control mosquitoes under field conditions need to be studied further.

Binding Sites of Mosquitocidal Toxins ofPseudomonas FluorescensandBacillus Subtilison Pupae and Larvae ofCulex quinquefasciatus

“…The mode of action of the newly isolated pupicidal agent B-426 [7] killing the pupae, a non feeding stage and B-471 killing the larvae [8] is not known yet and in the present study an attempt was made to investigate the binding sites of their toxins onto the pupae and larvae. The possible binding of the toxins (metabolites) may be by the adhesion and not by ingestion.…”

“…Pupae/larvae (IV instar) of the adult mosquitoes of Culex quinquefasciatus were obtained from the rearing and colonization facility of VCRC and exposed to lethal concentration 50 (LC 50) of B-426 (511.5 µg/mL) [7] and B-471 (27 µL/mL). [8] This was done by adding the metabolite containing toxic proteins to disposable cups each containing 25 larvae or 25 pupae with 100 mL of chlorine-free tap water.…”

“…P. fluorescens Migula was grown in shake flask cultures using Glucose Peptone Salt (GPS) medium [7] and after 48 hr the cells were removed by centrifugation at 12,000 rpm for 20 min and the cell-free supernatant was used for testing the mosquitocidal activity. The culture filtrate after confirming the mosquitocidal activity was subjected to Ammonium sulphate (50% w/v) precipitation and the precipitate was collected by centrifugation at 10,000 rpm for 20 min, dialyzed overnight against 0.05 M phosphate buffer and then lyophilized.…”

“…[3,4] Although these agents have been used extensively in the field, report of development of resistance by mosquito larvae to B. sphaericus [5,6] has led to the search of new microbial agents or their metabolites. At the Vector Control Research Centre (VCRC), Pondicherry search for such new microbial agents or their metabolites for mosquitocidal activity has resulted in the identification of two potential bacterial agents viz., Pseudomonas fluorescens (B-426) [7] and Bacillus subtilis (B-471), [8] whose metabolites could kill the mosquito pupae and larvae.…”

In vitro pathogenicity of Northern Peru native bacteria on Phyllocnistis citrella Stainton (Gracillariidae: Phyllocnistinae), on predator insects (Hippodamia convergens and Chrysoperla externa), on Citrus aurantiifolia Swingle and white rats

“…On the other hand, S. marcescens is reported to be a nosocomial pathogen (Carrero et al, 1995) and a facultative anaerobe that multiplies quickly in the gut of many insect species, causing septicaemia and death. It is often isolated from diseased and dead insects (Benoit et al, 1990;Rodríguez, 1995;Escobar et al, 2001;Prabakaran et al, 2002;Green et al, 2005). Other species, such as S. entomophila, induce pathologies in pest insects when the bacterium is ingested (Hurst and Jackson, 2002).…”