Oviposition-stimulant and ovicidal activities of Moringa oleifera lectin on Aedes aegypti

Abstract

Background: Natural insecticides against the vector mosquito Aedes aegypti have been the object of research due to their high level of eco-safety. The water-soluble Moringa oleifera lectin (WSMoL) is a larvicidal agent against A. aegypti. This work reports the effects of WSMoL on oviposition and egg hatching of A. aegypti.

Methodology/principal findings: WSMoL crude preparations (seed extract and 0-60 protein fraction), at 0.1 mg/mL protein concentration, did not affect oviposition, while A. aegypti gravid females laid their eggs preferentially (73%) in vessels containing isolated WSMoL (0.1 mg/mL), compared with vessels containing only distilled water (control). Volatile compounds were not detected in WSMoL preparation. The hatchability of fresh eggs deposited in the solutions in the oviposition assay was evaluated. The numbers of hatched larvae in seed extract, 0-60 protein fraction and WSMoL were 45 ± 8.7 %, 20 ± 11 % and 55 ± 7.5 %, respectively, significantly (p<0.05) lower than in controls containing only distilled water (75-95%). Embryos were visualized inside fresh control eggs, but not within eggs that were laid and maintained in WSMoL solution. Ovicidal activity was also assessed using stored A. aegypti eggs. The protein concentrations able to reduce the hatching rate by 50% (EC50) were 0.32, 0.16 and 0.1 mg/mL for seed extract, 0-60 protein fraction and WSMoL, respectively. The absence of hatching of stored eggs treated with WSMoL at 0.3 mg/mL (EC99) after transfer to medium without lectin indicates that embryos within the eggs were killed by WSMoL. The reduction in hatching rate of A. aegypti was not linked to decrease in bacteria population.

Conclusions/significance: WSMoL acted both as a chemical stimulant cue for ovipositing females and ovicidal agent at a given concentration. The oviposition-stimulant and ovicidal activities, combined with the previously reported larvicidal activity, make WSMoL a very interesting candidate in integrated A. aegypti control.

Figure 1. Top view of glass vessels used in oviposition assay.

A filter paper cone was placed covering the inner of each vessel to provide a support for oviposition. Next, the vessels were filled with distilled water (A, control) or WSMoL at 0.1 mg/mL in distilled water (B).

Figure 2. Effect of M. oleifera seed preparations (0.1 mg/mL of protein) on oviposition and egg hatching of Aedes aegypti.

(A) Mean of eggs laid by A. aegypti gravid females in distilled water and M. oleifera seed preparations. The oviposition response was evaluated by double-choice bioassays. Three distinct assays (“control vs. seed extract”, “control vs. 0–60 protein fraction” and “control vs. WSMoL”) were performed separately, each one with its respective control. The oviposition response was expressed as: % oviposition  = 100× [(number of eggs in sample vessel) / (number of eggs in sample and control vessels)]. (B) Hatching rate (%) of eggs laid by gravid females during the oviposition assays in distilled water and M. oleifera seed preparations. (*) indicates significant differences (p<0.05) between control and test groups.

Figure 3. Percentage of unhatched stored eggs after incubation with seed extract (A), 0–60 protein fraction (B) and WSMoL (C) for 72 h.

Figure 4. Visualization of A. aegypti fresh and stored eggs exposed or not to WSMoL in stereomicroscope (x80) after clearing of the heavily pigmented chorion with 5% sodium hypochlorite.

(A) Fresh egg laid and maintained in distilled water (control). (B) Fresh egg laid and maintained in WSMoL (0.1 mg/mL). (C) Stored egg which was not treated with any solution. (D) Stored egg that did not hatch after incubation with WSMoL at EC₉₉ (0.3 mg/mL) for 72 h. (h) head; (a) abdomen.