Mosquito Larvicidal Activity of the Essential Oils of Erechtites Species Growing Wild in Vietnam

Mosquito-borne infections are a constant problem in Vietnam, and mosquito vector control is a primary approach to control these infections. Essential oils represent environmentally friendly alternatives to synthetic pesticides for mosquito control. The essential oils of two weedy species in Vietnam, Erechtites hieraciifolius and E. valerianifolius, have been obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry. The essential oils have been screened for mosquito larvicidal activity against Aedes albopictus, Ae. aegypti, and Culex quinquefasciatus. The essential oil from the aerial parts of E. hieraciifolius was rich in α-pinene (14.5%), limonene (21.4%), and caryophyllene oxide (15.1%), while E. valerianifolius essential oil was dominated by myrcene (47.8%) and α-pinene (30.2%). Both essential oils showed good larvicidal activity against Ae. albopictus (24-h LC50 10.5 and 5.8 μg/mL, respectively) and Ae. aegypti (24-h LC50 10.6 and 12.5 μg/mL, respectively). The essential oil of E. valerianifolius also showed good activity against Cx. quinquefasciatus larvae (24-h LC50 = 40.7 μg/mL). Thus, Erechtites essential oils may serve as low-cost vector control agents for mosquito-borne infections.


Introduction
Aedes aegypti (L.) and Ae. albopictus (Skuse) (Diptera: Culicidae) are important vectors of arboviral infections, including yellow fever, dengue, Zika, and chikungunya [1][2][3]. Vietnam is classified as a hyperendemic dengue country, with all four dengue serotypes present throughout the year [4]. In the last half century, dengue fever epidemics have increased in frequency, corresponding to a median annual incidence of 232 cases per 100,000 people [4]. Furthermore, chikungunya is expected to become a major health threat in Vietnam in the near future [4,5].
Vector control is one of the primary approaches to reduce the spread of arboviral infections. However, current methods for controlling Aedes mosquitoes have been largely ineffective [6]. Botanical insecticides in general [7,8] and essential oils in particular [9,10] have emerged as promising, environmentally friendly alternatives to synthetic pesticides for mosquito control.
Erechtites hieraciifolius is used traditionally in Venezuela (a plant decoction is used as a bath to reduce fever) and in El Salvador (a decoction is used to treat coughs) [19]. In Bolivia, the Tacana people use an oil extract of E. hieraciifolius to treat wounds and pimples [20]. An ethanol extract of E. hieraciifolius showed in vitro antileishmanial activity against promastigotes of Leishmania (Leishmania) amazonensis Lainson & Shaw and L. (Viannia) braziliensis Vianna [20]. In North America, E. hieraciifolius was previously used to treat hemorrhages, wounds, skin diseases, and as a topical treatment for poison ivy (Toxicodendron radicans (L.) Kuntze, Anacardiaceae) and poison sumac (T. vernix (L.) Kuntze) rash [21].
As part of our ongoing research on identifying the potential utility of invasive plant species in Vietnam, we have obtained the essential oils from E. hieraciifolius and E. valerianifolius and have examined their mosquito larvicidal activities. In order to assess the potential environmental impact of using Erechtites essential oils as a larvicidal control agent, we have carried out lethality assays on the non-target aquatic species. As far as we are aware, there have been no previous investigations on the larvicidal activities of Erechtites essential oils.

Plant Material
Aerial parts of E. valerianifolius were harvested from plants growing in Dong Giang district, Quang Nam Province (15 • 58 9.8 N, 107 • 55 4.7 E; sample Quang Nam), Hoa Vang district, Da Nang city (16 • 01 0.6 N, 108 • 4 25.6 E;), while aerial parts of E. hieraciifolius were harvested from plants growing in Hoa Vang district, Da Nang city (16 • 2 22.0 N, 108 • 3 33.0 E), in April 2018. The plants were identified by Dr. Do Ngoc Dai, and voucher specimens (LTH127 and LTH128, respectively) have been deposited in the Pedagogical Institute of Science, Vinh University. Fresh plant materials (leaves, stems, and flowers) were kept at room temperature (≈25 • C), and 2 kg samples of each of the plant materials were shredded and hydrodistilled for 4 h using a Clevenger type apparatus.

Gas Chromatographic-Mass Spectral Analysis
Each of the Erechtites essential oils was analyzed by gas chromatography-mass spectrometry (GC-MS) using a Shimadzu GCMS-QP2010 Ultra operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software. The GC column was a ZB-5 fused silica capillary column with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm. The carrier gas was helium with a column head pressure of 552 kPa and flow rate of 1.37 mL/min. The injector temperature was 250 • C and the ion source temperature was 200 • C. The GC oven temperature program was programmed to have an initial temperature of 50 • C, and the 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 literature [22], and stored in our in-house Sat-Set library [23].

Mosquito Larvicidal Assay
Laboratory-reared larvae of Ae. aegypti and Ae. albopictus were collected from a mosquito colony maintained at the Laboratory of Parasitology and Entomology of Duy Tan University, Da Nang Vietnam. Wild larvae of Ae. albopictus and Culex quinquefasciatus (Say) were collected from Hoa Khanh Nam district (16 • 3 14.9" N, 108 • 9 31.2" E). For the assay, aliquots of the aerial parts (leaves and stems) and essential oils of E. hieraciifolius and E. valerianifolius (Quang Nam stems & leaves) dissolved in dimethylsulfoxide (DMSO) (1% stock solution of essential oil in DMSO) were placed in 500 mL beakers and added to water that contained 25 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 at 25 ± 2 • C. Each test was conducted with four replicates with six concentrations (100, 80, 50, 25, 12.5, and 5 µg/mL). Permethrin was used as a positive control.

Non-Target Lethality Assays
For the assay against Daphnia magna Straus (Cladocera: Daphniiidae), aliquots of the essential oil of E. hieraciifolius and E. valerianifolius (Quang Nam stems and leaves), dissolved in DMSO (1% stock solution), were placed in 250 mL beakers and added to water that contained 20 larvae (fourth instar). Mortality was recorded after 24 h and 48 h of exposure, during which no nutritional supplement was added. The experiments were carried out at 25 ± 2 • C. Each test was conducted with four replicates with five concentrations (12, 6, 3, 1.5, and 0.75 µg/mL). The assay against Chiromonus tentans Fabricius (Diptera: Chironomidae) larvae was carried out as above using four replicates with five concentrations (100, 50, 25, 12.5, and 6 µg/mL). For the assay against Danio rerio Hamilton (Cypriniformes: Cyprinidae), young, immature fish around 2-3 cm in size were selected for the experiment. Twenty fish were separated in 2.5 L plastic containers with 1.0 L of tap water, with a temperature of 25 ± 2 • C and external relative humidity of 85%. For each dose (100, 50, 25, 12.5, and 6 µg/mL), four repetitions of the experiment were performed. The mortality of organism non-target was calculated following an exposure period of 24 h. With each experiment, a set of controls using DMSO was also run for comparison.

Data Analysis
The mortalities were recorded 24 h and 48 h after treatment. The data obtained were subjected to log-probit analysis [24] to obtain LC 50 values, LC 90 values, 95% confidence limits, and chi square values using Minitab ® 18 (Minitab Inc., State College, PA, USA). For comparison, LC 50 values were also determined using the Reed-Muench method [25].

Results and Discussion
The essential oils from the aerial parts of E. valerianifolius and E. hieraciifolius were obtained in 1.53% and 1.47% yields, respectively.

Essential Oil Compositions
The chemical compositions of the essential oil of E. hieraciifolius and E. valerianifolius are presented in Tables 1 and 2, respectively. The essential oil from the aerial parts (leaves and stems) of E. hieraciifolius was rich in the monoterpene hydrocarbons α-pinene (14.5%) and limonene (21.4%), as well as the oxygenated sesquiterpenoid caryophyllene oxide (15.1%). The floral essential oil of E. hieraciifolius was also rich in α-pinene (11.8%) and limonene (29.8%), but β-caryophyllene (22.1%) was the dominant sesquiterpene.
Erechtites hieraciifolius and E. valerianifolius essential oils from other geographical locations have shown wide variations in chemical composition (Table 3). Thus, α-phellandrene (41.3%) and p-cymene (22.2%) dominated the essential oil of E. hieraciifolius from Pacoti-Ceara, Brazil [26], while these compounds were only minor components in the sample from Vietnam. Likewise, dillapiole (33.8%) was the major component in E. hieraciifolius from Parana State, Brazil [27]; this compound was not observed in the essential oils from Vietnam. The essential oil compositions of E. valerianifolius from Vietnam were qualitatively similar to those reported by do Amaral and co-workers from southern Brazil [27], but with major quantitative differences.
It is not clear why there is so much variation in the essential oils of Erechtites species. The phytochemical variations may be due to genetic variation. For example, the Missouri Botanical Garden [28] lists six varieties of H. hieraciifolius native to the Americas: var. cacalioides (Fisch. Ex Spreng.) Griseb (West Indies, Central and South America), var. carduifolius (Cass.) Griseb (West Indies), var. hieraciifolius (North America and West Indies), var. intermedia Fernald (North America), var. megalocarpus (Fernald) Cronquist (North America), and var. praealtus (Raf.) Fernald (North America). In addition, climatic and edaphic factors, maturity, and phenology can also be responsible for phytochemical variations, particularly in wide-ranging species. For example, several chemotypes of Artemisia absinthium L. (Asteraceae) are known, based largely on geographical location [29]. The essential oil of Peperomia pelucida (L.) Kunth (Piperaceae) also shows wide variation depending on the geographical source of material [30].

Mosquito Larvicidal Activities
The essential oils from the aerial parts of E. hieraciifolius and E. valerianifolius collected from Vietnam were screened for mosquito larvicidal activity (Tables 4 and 5). Larvicidal activity of permethrin (positive control) is shown in Table 6.   The essential oils from the aerial parts of both E. hieraciifolius and E. valerianifolius showed excellent larvicidal activity against Ae. aegypti. The 24 h LC 50 values were 10.6 and 12.5 µg/mL, respectively, which compare very favorably with other essential oils reported in the literature against this species [33][34][35]. Similarly, the larvicidal activities for the two Erechtites essential oils against Ae. albopictus were also very encouraging, with 24 h LC 50 values of 10.5 and 5.8 µg/mL for E. hieraciifolius and E. valerianifoliu, respectively. Notably, the laboratory-reared Ae. albopictus larvae were more susceptible, based on the 95% confidence limits, to E. valerianifolius essential oil than the larvae obtained from the wild (24 h LC 50 = 42.1 µg/mL). Likewise, wild Culex quinquefasciatus showed less susceptibility than the laboratory-reared mosquitoes.
The major components of E. hieraciifolius aerial parts essential oil were α-pinene, limonene, and caryophyllene oxide. Both α-pinene and limonene have shown good larvicidal activities against Ae. aegypti and Ae. albopictus (see Table 7). The LC 50 values for (+)-limonene average 35.1 and 29.8 against Ae. aegypti and Ae. albopictus, respectively. Caryophyllene oxide, however, has not shown good larvicidal activity, with LC 50 values > 100 µg/mL against all mosquito species reported (Table 7).  The larvicidal activities of E. hieraciifolius and E. valerianifolius essential oils can be attributed to the high concentrations of α-pinene and limonene in E. hieraciifolius oil and α-pinene, myrcene, and β-caryophyllene in E. valerianifolius oil. However, synergy between essential oil components may also be important [49,52]. Scalerandi and coworkers have demonstrated that Musca domestica preferentially metabolizes the major components in an essential oil while leaving the components of lower concentrations to act as toxicants [53].
In order to assess the potential environmental impact of using Erechtites essential oils as a larvicidal control agent, we have carried out lethality assays on non-target aquatic species: the water flea, Daphnia magna Straus (Cladocera: Daphniidae); non-biting midge larvae, Chironomus tentans Fabricius (Diptera: Chironomidae); and zebrafish, Danio rerio Hamilton (Cypriniformes: Cyprinidae) ( Table 8). Unfortunately, the Erechtites essential oils also show toxicity to representative non-target organisms, with LC 50 values against the midge larvae (C. tentans) and the zebrafish (D. rerio) comparable to those for laboratory-reared mosquito larvae. The small crustacean (D. magna) was particularly susceptible to the Erechtites essential oils. Therefore, care must be taken if these essential oils are to be used in broad applications. Local application of Erechtites essential oils (e.g., urban areas) may prove useful as controls for container-breeding mosquitoes, however.

Conclusions
Erechtites hieraciifolius and E. valerianifolius are introduced weedy species that grow prolifically in Vietnam, particularly where forests have been cleared; acquisition of abundant quantities of plant material should not be a problem. Mosquito larvicidal screening of these two species indicates good larvicidal activity, which can be attributed to their major components. Thus, this work provides evidence that otherwise noxious introduced weeds might provide low-cost vector control agents to prevent the spread of arboviral infections in Vietnam.