Essential Oils from Vietnamese Asteraceae for Environmentally Friendly Control of Aedes Mosquitoes

Mosquitoes, in addition to being a biting nuisance, are vectors of several pathogenic viruses and parasites. As a continuation of our work identifying abundant and/or invasive plant species in Vietnam for use as ecologically friendly pesticidal agents, we obtained the essential oils of Blumea lacera, Blumea sinuata, Emilia sonchifolia, Parthenium hysterophorus, and Sphaeranthus africanus; analyzed the essential oils using gas chromatographic techniques; and screened the essential oils for mosquito larvicidal activity against Aedes aegypti and Aedes albopictus. The most active larvicidal essential oils were B. sinuata, which was rich in thymohydroquinone dimethyl ether (29.4%), (E)-β-caryophyllene (19.7%), α-pinene (8.8%), germacrene D (7.8%), and α-humulene (4.3%), (24-h LC50 23.4 and 29.1 μg/mL) on Ae. aegypti and Ae. albopictus, respectively, and Emilia sonchifolia, dominated by 1-undecene (41.9%) and germacrene D (11.0%), (24-h LC50 30.1 and 29.6 μg/mL) on the two mosquito species. The essential oils of P. hysterophorus and S. africanus were also active against mosquito larvae. Notably, B. sinuata, P. hysterophorus, and S. africanus essential oils were not toxic to the non-target water bug, Diplonychus rusticus. However, E. sonchifolia essential oil showed insecticidal activity (24-h LC50 48.1 μg/mL) on D. rusticus. Based on these results, B. sinuata, P. hysterophorus, and S. africanus essential oils appear promising for further investigations.


Introduction
The Asteraceae is the largest family of flora in the world, comprising about 1550 genera and about 23,000 species [1]. In Vietnam, there are about 126 genera and 379 species from this family [2]. Many species are used as medicines, for isolation of essential oils, or as ornamentals [2].

Blumea sinuata
The fresh aerial parts of B. sinuata were hydrodistilled using a Clevenger apparatus to obtain the essential oil in 0.16% yield. The essential oil composition of B. sinuata is shown in Table 2. The major components in the essential oil of B. sinuata were thymohydroquinone dimethyl ether (29.4%), (E)-β-caryophyllene (19.7%), α-pinene (8.8%), germacrene D (7.8%), and α-humulene (4.3%). As far as we are aware, there is only one previous report on the essential oil of B. sinuata (as B. laciniata, from Dapoli region, Maharashtra, India) [47]. The GC-MS analysis, however, is not reliable, so a meaningful comparison of the compositions is not possible.

Emilia sonchifolia
Hydrodistillation of the fresh aerial parts of E. sonchifolia gave a 0.51% yield of essential oil. A total of 43 compounds were identified, accounting for 93.2% of the total composition (see Table 3). Gas chromatographic analysis of E. sonchifolia essential oils revealed the oil to be dominated by 1-undecene (41.9%) and germacrene D (11.0%). The essential oil composition of E. sonchifolia from Vietnam is in marked contrast to the essential oils from Belagavi, Karnataka, India [48] or Ojo State, Nigeria [49]. The E. sonchifolia sample from India was rich in the sesquiterpene hydrocarbons, (E)-β-caryophyllene (22.7%) and γ-muurolene (32.1%). The essential oil from Nigeria was also rich in sesquiterpene hydrocarbons, namely (E)-β-caryophyllene (15.7%), γ-gurjunene (8.6%), and γ-himachalene (25.2%). The differences in essential oil compositions may be due to genetic or environmental factors. RI calc. = Retention indices determined with reference to a homologous series of n-alkanes on a ZB-5 ms column. RI db = Retention indices from the databases. tr = trace (<0.05%). % = percent of total essential oil composition.

Mosquito Larvicidal Activity
The essential oils of B. lacera, B. sinuata, E. sonchifolia, P. hysterophorus, and S. africanus were screened for mosquito larvicidal activity against Aedes aegypti (the yellow fever mosquito) and Aedes albopictus (the Asian tiger mosquito), as previously described [34,56]. The essential oils were also screened for possible insecticidal activity against the nontargeted water bug, D. rusticus, as previously reported [33,36]. The larvicidal and insecticidal activities for the essential oils are summarized in Table 6. According to Dias and Moraes [39], essential oils and their components are considered to be active with larvicidal LC 50 values less than 100 µg/mL. However, we have recently amended the activity definition: essential oils with 24-h LC 50 < 10 µg/mL are considered "exceptionally active", those with 24-h LC 50 between 10 and 50 µg/mL are "very active", those with 24-h LC 50 between 50 and 100 µg/mL are "moderately active", and LC 50 >100 µg/mL are "inactive" [58]. Thus, B. lacera leaf essential oil was only marginally active against Ae. aegypti and inactive against Ae. albopictus.
Although E. sonchifolia essential oil showed moderately active mosquito larvicidal activity (24-h LC 50 = 30.1 and 29.6 µg/mL against Ae. aegypti and Ae. albopictus, respectively), it was also insecticidal to the non-target insect, Diplonychus rusticus with a 24-h LC 50 of 48.1 µg/mL. Thus, the E. sonchifolia essential oil is not selectively toxic and should not be considered further for this purpose.
The essential oil of S. africanus showed moderate larvicidal activity with 24-h LC 50 values of 50.7 and 36.9 µg/mL, respectively, on Ae. aegypti and Ae. albopictus. In a previous study, the S. africanus (as S. indicus) essential oil from India was screened for mosquito larvicidal activity against Culex quinquefasciatus and Ae. aegypti [62]. The larvicidal activities were very modest, however (24-h LC 50 = 130 and 140 µg/mL, respectively). Unfortunately, the essential oil characterization in this study is not reliable.

Plant Material
The details of plant material collection and hydrodistillation are summarized in Table 7. During this process, botanical identification and confirmation was conducted by Dr. Huong, L.T., Faculty of Biology, College of Natural Science Education, Vinh University, Vietnam. In addition, voucher specimens with codes LTH 881, LTH 284, LTH 286, LTH 327, and LTH 332 were preserved in the plant specimen room, Vinh University, Vietnam. Aerial parts were shredded and hydrodistilled for 5 h using a Clevenger-type apparatus (Witeg Labortechnik, Wertheim, Germany). Essential oil isolation yields of three consecutive replicates were used to calculate the average yield. The essential oils were dried over anhydrous Na 2 SO 4 and stored in sealed glass vials at 4 • C until use in analysis and bioactivity assays.

Gas Chromatography-Mass Spectral Analysis
Gas chromatography-mass spectral analyses (GC-MS) of B. lacera, B. sinuata, E. sonchifolia, P. hysterophorus, and S. africanus essential oils were carried out using the instrumentation and protocols previously published [36,56,63]. A Shimadzu GCMS-QP2010 Ultra, with a ZB-5 ms fused silica capillary column (60 m length, 0.25 mm diameter, 0.25 µm film thickness) was used, He carrier gas, 2.0 mL/min flow rate, injection and ion source temperatures of 260 • C, and a GC oven program of 50 to 260 • C at 2.0 • C/min. Injection volumes of 0.1 µL of 5% (w/v) samples of essential oil in CH 2 Cl 2 were injected in split mode, with a 24.5:1 split ratio. Identification of the essential oil components was carried out with a comparison of MS fragmentation and retention indices (RI) with those available in the databases [64][65][66][67]. The peak areas were corrected for response using external standards of representative compounds from each compound class.

Mosquito Larvicidal Activity Screening
Mosquito larvicidal activity screening against Ae. aegypti and Ae. albopictus was carried out as previously described [34,56]. Quadruplicate assays using 20 fourth-instar mosquito larvae and five essential oil concentrations (100, 75, 50, 25, and 12.5 µg/mL) and a permethrin positive control. Mortality was recorded after 24 h and again after 48 h of exposure. Lethality data were subjected to log-probit analysis to obtain LC 50 values, LC 90 values, and 95% confidence limits using Minitab ® version 19.2020.1 (Minitab, LLC, State College, PA, USA).

Conclusions
The essential oils of B. sinuata, rich in thymohydroquinone dimethyl ether, (E)-βcaryophyllene, α-pinene, and germacrene D; P. hysterophorus, rich in germacrene D, myrcene, and (E)-β-caryophyllene; and S. africanus, dominated by 1-decen-3-ol and α-pinene, all showed good mosquito larvicidal activities without toxicity to a non-target aquatic species. Based on these encouraging results, B. sinuata, P. hysterophorus, and S. africanus essential oils should be further investigated for use as eco-friendly botanical pesticides. Field trials and formulations are needed to enhance the environmental lifetime of the essential oils and determine whether they are a viable alternative pest-control agents in aquatic systems.