Larvicidal Potential of the Halogenated Sesquiterpene (+)-Obtusol, Isolated from the Alga Laurencia dendroidea J. Agardh (Ceramiales: Rhodomelaceae), against the Dengue Vector Mosquito Aedes aegypti (Linnaeus) (Diptera: Culicidae)

Abstract

Dengue is considered a serious public health problem in many tropical regions of the world including Brazil. At the moment, there is no viable alternative to reduce dengue infections other than controlling the insect vector, Aedes aegypti Linnaeus. In the continuing search for new sources of chemicals targeted at vector control, natural products are a promising alternative to synthetic pesticides. In our work, we investigated the toxicity of a bioactive compound extracted from the red alga Laurencia dendroidea J. Agardh. The initial results demonstrated that crude extracts, at a concentration of 5 ppm, caused pronounced mortality of second instar A. aegypti larvae. Two molecules, identified as (-)-elatol and (+)-obtusol were subsequently isolated from crude extract and further evaluated. Assays with (-)-elatol showed moderate larvicidal activity, whereas (+)-obtusol presented higher toxic activity than (-)-elatol, with a LC50 value of 3.5 ppm. Histological analysis of the larvae exposed to (+)-obtusol revealed damage to the intestinal epithelium. Moreover, (+)-obtusol-treated larvae incubated with 2 µM CM-H₂DCFDA showed the presence of reactive oxygen species, leading us to suggest that epithelial damage might be related to redox imbalance. These results demonstrate the potential of (+)-obtusol as a larvicide for use against A. aegypti and the possible mode of action of this compound.

Keywords: (+)-obtusol; (−)-elatol; Aedes aegypti; Laurencia dendroidea; larvicide; oxidative stress; sesquiterpenes.

Figure 1

Larvicidal activity of crude extracts from Laurencia. dendroidea collected from two different localities against Aedes aegypti, Rockefeller strain. Extract 1 was derived from seaweed collected at “Azeda” beach in Búzios and the Extract 2 was derived from seaweed collected at “Vermelha” beach in Parati. Five parts per million of each extract were used in the experiments. Results are means of three independent experiments (ANOVA, followed by Tukey’s multiple comparisons test; **** p < 0.0001; ns, denotes no significant difference).

Figure 2

Larvicidal activity of different concentrations of Laurencia dendroidea crude extract collected at Vermelha beach in Parati and tested against Aedes aegypti. The results are means of three independent experiments (ANOVA, followed by Tukey’s multiple comparisons test; * p ≤ 0.012; ** p ≤ 0.003; **** p < 0.0001; ns, denotes no significant difference).

Figure 3

Larvicidal activity of the sesquiterpenes (+)-obtusol and (−)-elatol against Aedes aegypti (Rockefeller strain) second instar larvae. Ten larvae were incubated in distilled water in the presence of different concentrations of either obtusol or elatol and mortality was evaluated after 24 h. The control consisted of 0.1% DMSO in water. The results represent mean ± SEM of three independent experiments (ANOVA, followed by Bonferroni’s multiple comparisons test; **** p ≤ 0.0001; ns, denotes no significant difference) (see Figure S1).

Figure 4

Larvicidal activity of (+)-obtusol against Aedes aegypti (Rockefeller strain) forth instar larvae. Ten larvae were incubated in distilled water in the presence of 5 ppm (+)-obtusol and mortality evaluated following 24 h. The controls consisted of water and water containing 0.1% DMSO. The experiment was carried out in three replicas and the results represent mean ± SEM of three independent experiments (ANOVA, followed by Tukey’s multiple comparisons test; **** p ≤ 0.0001).

Figure 5

Structures of the isolated sesquiterpenes [27].

Figure 6

Larvicidal activity of two different concentrations of (+)-obtusol against wild strain Aedes aegypti. Ten second instar larvae were incubated in water containing two different concentrations of (+)-obtusol (5 and 10 ppm). The controls consisted of water and water +0.1% DMSO. Mortality was evaluated after 24 h. The results represent mean ± SEM of three independent experiments (ANOVA, followed by Tukey’s multiple comparisons test; **** p ≤ 0.0001; ns, no significant difference).

Figure 7

Light micrographs showing the midgut epithelium of second instar Aedes aegypti larvae, which had been exposed to 5 ppm (+)-obtusol: (A) control larval gut after incubation with water; (B) control larval gut after incubation with 0.1% DMSO in distilled water; (C) A. aegypti (Rockefeller strain) larval gut after incubation with 5 ppm (+)-obtusol in distilled water; and (D) wild strain A. aegypti larval gut after incubation with 5 ppm (+)-obtusol in distilled water. Lu: Lumen. Arrows point to regions with altered morphology.

Figure 8

(+)-Obtusol increases ROS level in the larvae. Second instar Aedes aegypti larvae (Rockefeller and wild strains), were incubated in water under different conditions; (A) bright field image of larvae incubated in distilled water; (B) fluorescent control image of larvae incubated in distilled water alone; (C) bright field image of larvae incubated in distilled water containing 0.1% DMSO; (D) fluorescent control image of larvae incubated in distilled water containing 0.1% DMSO; (E) bright field image of second instar larvae (Rockefeller strain), incubated with water containing 5 ppm (+)-obtusol; (F) fluorescent image of second instar larvae (Rockefeller strain), incubated with water containing 5 ppm (+)-obtusol; (G) bright field image of second instar larva (wild strain), incubated in water containing 5 ppm (+)-obtusol; (H) fluorescent image of second instar larva (wild strain), incubated in water containing 5 ppm (+)-obtusol. Scale bars: 100 µm.

Figure 8

(+)-Obtusol increases ROS level in the larvae. Second instar Aedes aegypti larvae (Rockefeller and wild strains), were incubated in water under different conditions; (A) bright field image of larvae incubated in distilled water; (B) fluorescent control image of larvae incubated in distilled water alone; (C) bright field image of larvae incubated in distilled water containing 0.1% DMSO; (D) fluorescent control image of larvae incubated in distilled water containing 0.1% DMSO; (E) bright field image of second instar larvae (Rockefeller strain), incubated with water containing 5 ppm (+)-obtusol; (F) fluorescent image of second instar larvae (Rockefeller strain), incubated with water containing 5 ppm (+)-obtusol; (G) bright field image of second instar larva (wild strain), incubated in water containing 5 ppm (+)-obtusol; (H) fluorescent image of second instar larva (wild strain), incubated in water containing 5 ppm (+)-obtusol. Scale bars: 100 µm.

Figure 9

Increase in ROS level in larvae incubated in water in the presence of (+)-obtusol. Second instar larvae of Aedes aegypti (Rockefeller strain), previously incubated in water containing 5 ppm (+)-obtusol. The integument was then pricked and larvae incubated in 2 µM CM-H₂DCFDA in PBS for 20 min. After that time, larvae were homogenized in probe-free PBS and centrifuged at 20,000× g for 10 min at 4 °C. The supernatant was analyzed in a Spectrofluorometer (Excitation: 563 nm; Emission: 587 nm). The results represent mean ± SEM of two independent experiments. Each replica consisted of ten larvae (ANOVA, followed by Sidak’s multiple comparisons test; **** p ≤ 0.0001).