Essential Oil Compositions of Three Invasive Conyza Species Collected in Vietnam and Their Larvicidal Activities against Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus

Mosquito-borne infectious diseases are a persistent problem in tropical regions of the world, including Southeast Asia. Vector control has relied principally on synthetic insecticides, but these have detrimental environmental effects and there is an increasing demand for plant-based agents to control insect pests. Invasive weedy plant species may be able to serve as readily available sources of essential oils, some of which may be useful as larvicidal agents for control of mosquito populations. We hypothesize that members of the genus Conyza (Asteraceae) may produce essential oils that may have mosquito larvicidal properties. The essential oils from the aerial parts of Conyza bonariensis, C. canadensis, and C. sumatrensis were obtained by hydrodistillation, analyzed by gas chromatography–mass spectrometry, and screened for mosquito larvicidal activity against Aedes aegypti, Ae. albopictus and Culex quinquefasciatus. The essential oils of C. canadensis and C. sumatrensis, both rich in limonene (41.5% and 25.5%, respectively), showed notable larvicidal activities against Ae. aegypti (24-h LC50 = 9.80 and 21.7 μg/mL, respectively) and Ae. albopictus (24-h LC50 = 18.0 and 19.1 μg/mL, respectively). These two Conyza species may, therefore, serve as sources for alternative, environmentally-benign larvicidal control agents.

In order to assess the potential detrimental impact of the Conyza essential oils on beneficial aquatic species, the insecticidal activity was assessed against the water bug, Diplonychus rusticus, an insect predator of mosquito larvae [66]. Both C. canadensis and C. sumatrensis essential oils were substantially less toxic to D. rusticus than they were to the mosquito larvae. Other components in the Conyza essential oils likely contribute to the mosquito larvicidal effects. Conyza bonariensis was rich in (E)-caryophyllene (13.3%) and caryophyllene oxide (12.2%), but both of these compounds have been found to have weak larvicidal activities against Ae. aegypti (24-h LC50 = 70.8 and 137 μg/mL, respectively (Table 3). On the other hand, β-pinene, a major component of C. canadensis essential oil (8.8%), has shown larvicidal activity against Ae. aegypti (24-h LC50 = 23.6 μg/mL), Cx. quinquefasciatus (24-h LC50 = 30.5 μg/mL) (Table 3), and Ae. albopictus [61]. In addition, synergy between essential oil components may also be important [63,64]. Scalerandi and coworkers have found that the housefly (Musca domestica) metabolizes the major components in an essential oil, but leaves the minor components to act as toxicants [65].
In order to assess the potential detrimental impact of the Conyza essential oils on beneficial aquatic species, the insecticidal activity was assessed against the water bug, Diplonychus rusticus, an insect predator of mosquito larvae [66]. Both C. canadensis and C. sumatrensis essential oils were substantially less toxic to D. rusticus than they were to the mosquito larvae.

Plant Material
The three Conyza species were collected from Bach Ma National Park, Thue Thien Hue province (16 •

Gas Chromatography-Mass Spectrometry
The Conyza essential oils were analyzed by GC-MS as previously described [67]: Shimadzu GCMS-QP2010 Ultra, electron impact (EI) mode, electron energy = 70 eV, scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, ZB-5 fused silica capillary column (30 m × 0.25 mm, 0.25 µm film thickness), He carrier gas, 552 kPa column head pressure, and 1.37 mL/min flow rate. Injector temperature was 250 • C and the ion source temperature was 200 • C. The GC oven temperature program was programmed for 50 • C initial temperature, temperature increased at a rate of 2 • C/min to 260 • C. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared and 0.1 µL was injected with a splitting mode (30:1). Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the databases [36][37][38][39].

Mosquito Larvicidal Assay
Mosquito larvicidal activity was carried out on Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus as previously described [67]: For the assay, 1% stock solutions of each essential oil in dimethylsulfoxide (DMSO) were prepared, and aliquots of the stock solutions were placed in 500-mL beakers and added to water that contained 20 larvae (fourth instar). With each experiment, a set of controls using DMSO was also run for comparison. Mortality was recorded after 24 h and again after 48 h of exposure during which no nutritional supplement was added. The experiments were carried out 25 ± 2 • C. Each test was conducted with four replicates with three concentrations (50, 25, and 12.5, µg/mL for C. canadensis and C. sumatrensis; 150, 100, and 50 µg/mL for C. bonariensis). Permethrin was used as a positive control.

Non-Target Insecticidal Assay
The Diplonychus rusticus adults were collected in the field and maintained in glass tanks (60 cm long × 50 cm wide) containing water at 25 • C with a water depth of 20 cm. The essential oils were tested at concentrations of 200, 150, 100, 75, 50, and 25 µg/mL. Four replicates were performed for each concentration. Twenty D. rusticus adults were introduced into each solution. The non-target organism was observed for mortality after 24 h and 48 h exposure.

Conclusions
Invasive plant species are generally considered to be ecologically and detrimental with potential economic impacts, and the control or eradication of invasive plant species can be prohibitively costly. However, identification of beneficial uses of invasive plants could be economically advantageous and aid in the control of the species. Conyza spp., as well as Erechtites spp. [34], Crassocephalum crepidioides [35], and Severinia monophylla [33], are invasive weeds in Vietnam, and essential oils from these plants have demonstrated promising mosquito larvicidal activities. The plant materials are readily available and harvesting of these weeds may provide economically valuable "cash crops" as well as serve as a means for ecological remediation. Note that C. bonariensis [69], C. canadensis [70], and C. sumatrensis [71] have all shown resistance to the commonly used herbicide glyphosate, so herbicidal control of these weeds is impractical as well as environmentally detrimental. Further research on potential formulations (e.g., nanoemulsions or essential oil-loaded nanoparticles) [72] for field use of these promising essential oils is warranted.