Fig 1.
Structural comparison of representative drimane sesquiterpenes.
CDIAL, CFRAG, and CMOS were the focus of the present investigation. The conjugation of Ald1 with the double-bond at C-7 and C-8 makes CDIAL a strong electrophile (S1 Fig). The presence of Ald2 attached to a quaternary carbon (C-9) bearing a hydroxyl group also contributes to the electrophilic nature of the molecule (S1 Fig).
Fig 2.
Toxicity of CINEX and isolated sesquiterpenes to Ae. aegypti (LVP strain).
A,C) Concentration-toxicity relationships in 1st instar larvae 24 h after adding CINEX or indicated compound to the rearing water. Efficacy was defined as the percentage of larvae that were dead within 24 h. Values are means ± SEM based on 3–8 independent replicates of 5 larvae per concentration. The mean efficacy of the acetone controls was 0.43 ± 0.43% (N = 46 independent replicates of 5 larvae). B,D) Dose-toxicity relationships in adult females 24 h after applying CINEX or indicated compound to the thoracic cuticle. Efficacy was defined as the percentage of adults that were incapacitated (dead or flightless) within 24 h. Values are means ± SEM based on 3–8 independent replicates of 10 females per dose. The mean efficacy of the acetone controls was 0.67 ± 0.37% (N = 45 independent replicates of 10 females). See text or Table 1 for the specific potency values (EC50 or ED50) and Hill slopes.
Table 1.
Dose-toxicity parameters of sesquiterpenes isolated from CINEX on adult females of Ae. aegypti (LVP strain).
* = significantly different from CDIAL as determined by a F-test (P < 0.05).
Fig 3.
Comparative toxic resistance of a pyrethroid-resistant strain of Ae. aegypti (Puerto Rican strain, PR) to CDIAL (A, B) vs. cypermethrin (C,D). The concentration/dose-toxicity relationships of CDIAL against the LVP strain of Ae. aegypti are from Fig 2 (data points are omitted for clarity). A,C) Concentration-toxicity relationships in 1st instar larvae 24 h after adding indicated compound to the rearing water. Efficacy was defined as the percentage of larvae that were dead within 24 h. Values are means ± SEM based on 3–12 independent replicates of 5 larvae per concentration. The EC50 of CDIAL in PR (97.1 μM; 95% CI = 82.4–114.5 μM) was slightly (1.4-fold), but significantly (P < 0.05; F-test), greater than that in LVP (70 nM; 95% CI = 53.8–91.0 μM). The EC50 of cypermethrin in PR (88.0 nM; 95% CI = 46.2–167.7 nM) was dramatically (131-fold) and significantly (P < 0.05; F-test) greater than that in LVP (0.68 nM; 95% CI = 0.34–1.34 nM). B,D) Dose-toxicity relationships in adult females 24 h after applying indicated compound to the thoracic cuticle. Efficacy was defined as the percentage of adults that were incapacitated (dead or flightless) within 24 h. Values are means ± SEM based on 3–8 independent replicates of 10 females per dose. The ED50 of CDIAL in PR (0.45 nmol/mg; 95% CI = 0.34–0.58 nmol/mg) was slightly (1.5-fold), but significantly, (P < 0.05; F-test) greater than that in LVP (0.29 nmol/mg; 95% CI = 0.25–0.34 nmol/mg). The ED50 of cypermethrin in PR (2,685.0 fmol/mg; 95% CI = 1315.0–5484.0 fmol/mg) was dramatically (84-fold) and significantly (P < 0.05; F-test) greater than that in LVP (27.3 fmol/mg; 95% CI = 12.1–61.4 fmol/mg).
Fig 4.
Antifeedant activity of CINEX and isolated sesquiterpenes as determined via choice CAFE assays in adult female Ae. aegypti (LVP strain).
Groups of 5 mosquitoes were allowed to feed equally on two capillaries of 10% sucrose with 0.01% trypan blue; the control capillary included 1% acetone (the solvent), and the treatment capillary included 1% acetone and CINEX (0.48 μg/μl) or a sesquiterpene (0.75 or 1.5 mM). In ‘Mock’ experiments, both capillaries included 1% acetone alone. The difference in volume consumed between the capillaries was used to calculate the antifeedant activity (see Methods for details). Values are means ± SEM; N = number of independent replicates of 5 mosquitoes each. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA and Holm-Sidak’s posttest (P < 0.05).
Fig 5.
Repellency of DEET, CINEX, and isolated sesquiterpenes in adult female Ae. aegypti (LVP strain) as determined via a non-choice membrane blood-feeding bioassay.
Cages of 20 mosquitoes were allowed to feed on a blood source (defibrinated rabbit blood) for 1 h; the feeding membrane was treated with DEET (20.8 μg/cm2 = 108.75 nmol/cm2), CINEX (20.8 μg/cm2), or a sesquiterpene (67.45 nmol/cm2) dissolved in 100% acetone. Control membranes were treated with 100% acetone. The number of mosquitoes that fed from each cage was determined. The percent reduction in the number of mosquitoes that fed from the treatment cage (relative to the mean feeding percentage of the control cages) was calculated to determine ‘percent mosquitoes repelled’. Values are means ± SEM; N = number of independent replicates of 20 mosquitoes each. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA and Holm-Sidak’s posttest (P < 0.05).
Fig 6.
Effects of isolated sesquiterpenes on the electrophysiological activity of Xenopus oocytes expressing AgTRPA1 as determined by two-electrode voltage clamping.
A) Representative traces of membrane current (Im) in AgTRPA1-expressing (TRPA1) or H2O-injected oocytes. Horizontal bars indicate the addition of 10 μM CDIAL, CFRAG, CMOS, or ruthenium red (RR) to the extracellular bath. The bidirectional arrows show the peak changes in membrane current (ΔIm). The dashed lines indicate the activation (1) and deactivation (2) rates (ΔIm/Δt). For H2O-injected oocytes, the addition of CDIAL (shown), CFRAG (S3 Fig), or CMOS (S3 Fig) did not noticeably elicit a change in Im. B) Summary of ΔIm elicited by each sesquiterpene (10 μM) in TRPA1-expressing oocytes. Values are means ± SEM; N = number of oocytes measured. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA and Holm-Sidak’s posttest (P < 0.05). C) Summary of ΔIm/Δt during periods ‘1’ and ‘2’ shown in panel A for CDIAL and CFRAG. Values are means ± SEM; N = number of oocytes measured. The mean ΔIm/Δt during period ‘1’ for CDIAL was significantly greater than that for CFRAG as determined by an unpaired t-test (P < 0.01). ‘***’ and ‘**’ indicate significant difference (P < 0.001 and P < 0.01, respectively) between activation (‘1’) and deactivation (‘2’) rates for each molecule as determined by a paired t-test.
Fig 7.
Effects of CDIAL on TRPA1-/- Ae. aegypti.
A) Antifeedant activity of CDIAL (1.5 mM) in adult females of the parental ORL and TRPA1-/- strains of A. aegypti, as determined via the CAFE choice bioassay. Antifeedant activity was calculated as described in Fig 4. Values are means ± SEM; N = number of independent replicates of 5 mosquitoes each. P value indicates significant difference from ORL strain as determined via an unpaired t-test. B) Dose-toxicity relationship of CDIAL in adult females of ORL and TRPA1-/- Ae. aegypti. Efficacy was defined as the percentage of adults that were incapacitated (dead or flightless) within 24 h. Values are means ± SEM, based on 3–6 independent replicates of 10 mosquitoes per dose. The ED50 values and Hill slopes for the two strains were not significantly different from each other (P > 0.05; F-test). ORL: ED50 = 0.33 nmol/mg (95% C.I. = 0.28–0.38 nmol/mg); Hill slope = 4.77 (95% C.I. = 2.70–6.83). TRPA1-/-: ED50 = 0.30 nmol/mg (95% C.I. = 0.24–0.37 nmol/mg); Hill slope = 2.87 (95% C.I. = 1.16–4.58).