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

Abstract

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.

1. 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.

There are around 12 species of Erechtites (Asteraceae), and they are native to North America, West Indies, South America, New Zealand, and Australia [11]. Erechtites hieraciifolius (L.) Raf. ex DC. (syn. Erechtites hieracifolia (L.) Raf., Erechtites hieraciifolia (L.) Raf. ex DC.,) is native to North America, South America, and the West Indies, but it has been introduced to Europe, Hawaii, and Asia [12,13,14,15,16]. Erechtites valerianifolius (Wolf) DC. (syn. Erechtites valerianifolia (Link ex Wolf) Less. ex DC., Erechtites valerianaefolia (Wolf) DC.) is native to Central and South America, but this species has also has been introduced to Asia [13,14,15,17,18].

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.

2. Materials and Methods

2.1. 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.

2.2. 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 CH₂Cl₂ 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].

2.3. 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.

2.4. 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.

2.5. 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 LC₅₀ values, LC₉₀ values, 95% confidence limits, and chi square values using Minitab^® 18 (Minitab Inc., State College, PA, USA). For comparison, LC₅₀ values were also determined using the Reed–Muench method [25].

3. 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.

3.1. Essential Oil Compositions

The chemical compositions of the essential oil of E. hieraciifolius and E. valerianifolius are presented in

Table 1. Chemical compositions of Erechtites hieraciifolius essential oils from Vietnam.

RI	Compound	Area %
		Leaves & Stems	Flowers
921	Tricyclene	---	tr
924	α-Thujene	---	tr
932	α-Pinene	14.5	11.8
948	Camphene	---	0.1
971	Sabinene	0.6	0.7
976	β-Pinene	0.4	0.4
988	Myrcene	2.7	4.4
1006	α-Phellandrene	---	0.3
1016	α-Terpinene	---	tr
1024	p-Cymene	0.4	0.1
1028	Limonene	21.4	29.8
1031	β-Phellandrene	---	0.5
1034	(Z)-β-Ocimene	---	1.2
1044	(E)-β-Ocimene	---	2.3
1057	γ-Terpinene	---	0.1
1084	Terpinolene	---	0.1
1108	Unidentified	0.8	---
1120	trans-p-Mentha-2,8-dien-1-ol	0.8	---
1124	Cycloctanone	0.6	---
1125	α-Campholenal	0.6	---
1127	allo-Ocimene	---	tr
1135	cis-p-Mentha-2,8-dien-1-ol	0.9	---
1140	trans-Pinocarveol	0.7	---
1140	cis-Verbenol	0.3	---
1144	trans-Verbenol	3.5	---
1179	Terpinen-4-ol	0.4	---
1185	Cryptone	1.4	---
1194	Myrtenol	0.8	---
1197	Dodecane	---	0.1
1198	cis-Piperitol	0.8	---
1205	Verbenone	1.4	---
1209	Unidentified	0.5	---
1214	Unidentified	1.1	---
1217	trans-Carveol	3.5	---
1225	Unidentified	0.7	---
1230	cis-Carveol	1.1	---
1242	Carvone	2.0	---
1270	Unidentified	0.8	---
1284	Bornyl acetate	---	0.2
1287	Limonene dioxide	0.9	---
1297	Tridecane	---	0.2
1309	Unidentified	2.2	---
1317	3-Hydroxycineole	0.4	---
1343	Limonene-1,2-diol	4.7	---
1345	α-Cubebene	---	0.1
1357	Neryl acetate	---	0.1
1367	Cyclosativene	---	0.1
1374	α-Copaene	0.6	1.9
1378	trans-p-Menth-6-en-2,8-diol	4.1	---
1386	β-Cubebene	---	0.7
1387	β-Elemene	0.6	3.5
1397	Tetradecane	---	0.2
1402	α-Gurjunene	---	1.1
1419	β-Caryophyllene	3.0	22.1
1450	(E)-β-Farnesene	---	2.0
1454	α-Humulene	0.5	1.8
1470	trans-Cadina-1(6),4-diene	---	0.1
1472	γ-Gurjunene	---	0.2
1473	γ-Muurolene	---	0.1
1480	Germacrene D	---	2.6
1482	(Z,Z)-α-Farnesene	---	0.7
1486	Valencene	---	0.7
1488	Viridiflorene	---	0.7
1490	trans-Muurola-4(14),5-diene	---	0.3
1494	epi-Cubebol	---	0.5
1496	α-Muurolene	---	1.2
1501	(E,E)-α-Farnesene	---	0.1
1514	Cubebol	---	0.2
1516	δ-Cadinene	---	1.4
1549	Isocaryphyllene oxide	1.2	---
1559	(E)-Nerolidol	---	0.3
1582	Caryophyllene oxide	15.1	1.6
1607	Humulene epoxide II	0.9	---
1622	Cyperotundone A	---	0.1
1627	1-epi-Cubenol	---	0.2
1637	Caryophylla-4(12),8(13)-dien-5β-ol	0.6	0.1
1641	τ-Cadinol	---	0.4
1643	τ-Muurolol	---	0.2
1644	Cubenol	0.5	---
1646	α-Muurolol (Torreyol)	---	0.1
1654	α-Cadinol	0.7	0.3
1658	Selin-11-en-4α-ol	---	0.1
1667	14-Hydroxy-9-epi-(E)-caryophyllene	1.4	---
1700	Heptadecane	---	0.2
1831	Neophytadiene	---	0.3
1900	Nonadecane	---	0.2
2103	(E)-Phytol	---	0.4
	Monoterpene hydrocarbons	40.0	51.7
	Oxygenated monoterpenoids	28.2	0.3
	Sesquiterpene hydrocarbons	4.7	41.3
	Oxygenated sesquiterpenoids	19.2	4.1
	Others	0.6	1.5
	Total Identified	92.7	99.0

and

Table 2. Chemical compositions of Erechtites valerianifolius essential oils from Vietnam.

RI	Compound	Quang Nam	Quang Nam	Da Nang
		Leaves & stems	Flowers	Flowers
922	Tricyclene	tr	tr	tr
924	α-Thujene	tr	0.1	tr
933	α-Pinene	30.2	32.5	30.6
949	Camphene	0.1	0.1	0.1
952	Thuja-2,4(10)-diene	tr	tr	tr
971	Sabinene	0.7	1.0	0.9
977	β-Pinene	0.3	0.4	0.3
990	Myrcene	47.8	57.0	60.6
1006	α-Phellandrene	0.3	tr	tr
1016	α-Terpinene	tr	tr	tr
1024	p-Cymene	0.1	tr	tr
1028	Limonene	1.4	2.5	1.5
1030	β-Phellandrene	0.1	0.2	0.2
1034	(Z)-β-Ocimene	0.3	0.1	tr
1044	(E)-β-Ocimene	1.4	0.4	0.2
1057	γ-Terpinene	0.1	0.1	0.1
1084	Terpinolene	tr	0.1	0.1
1100	Undecane	---	tr	tr
1101	Perillene	0.1	tr	tr
1102	Linalool	tr	tr	tr
1112	(E)-4,8-Dimethylnona-1,3,7-triene	tr	tr	tr
1128	α-Campholenal	0.1	---	---
1146	trans-Verbenol	---	tr	tr
1181	Terpinen-4-ol	0.1	tr	tr
1229	Thymol methyl ether	tr	---	---
1333	δ-Elemene	0.1	0.1	0.1
1374	α-Copaene	0.1	tr	tr
1380	cis-β-Elemene	0.1	tr	tr
1382	β-Bourbonene	tr	tr	tr
1386	β-Cubebene	---	tr	0.3
1387	β-Elemene	2.4	0.2	0.1
1400	Methyl eugenol	tr	---	---
1401	α-Gurjunene	0.1	---	---
1411	Dimethoxy-p-cymene	0.2	---	---
1418	β-Caryophyllene	5.4	2.7	2.2
1427	γ-Elemene	0.1	tr	tr
1428	β-Copaene	0.1	tr	tr
1450	(E)-β-Farnesene	0.2	tr	tr
1454	α-Humulene	0.7	0.3	0.3
1471	γ-Selinene	0.2	---	---
1473	γ-Muurolene	0.1	tr	tr
1480	Germacrene D	3.3	1.8	1.8
1486	Viridiflorene	0.3	---	---
1488	β-Selinene	0.2	tr	tr
1491	trans-Muurola-4(14),5-diene	0.1	tr	tr
1494	α-Selinene	0.4	---	---
1494	Bicyclogermacrene	---	0.1	0.2
1496	α-Muurolene	0.1	0.1	tr
1501	(E,E)-α-Farnesene	0.7	tr	0.1
1511	γ-Cadinene	tr	tr	tr
1516	δ-Cadinene	0.2	0.1	0.1
1558	Germacrene B	0.1	tr	tr
1576	Spathulenol	0.1	tr	tr
1582	Caryophyllene oxide	0.7	0.1	0.1
1609	Humulene epoxide II	0.1	---	---
1622	Cyperotundone A	0.1	---	---
1627	iso-Spathulenol	tr	---	---
1642	τ-Cadinol	0.1	tr	tr
1643	τ-Muurolol	0.1	tr	tr
1655	α-Cadinol	0.1	tr	tr
1659	Selin-11-en-4α-ol	0.1	---	---
1684	Germacra-4(15),5,10(14)-trien-1α-ol	---	---	tr
1700	Heptadecane	0.1	0.1	0.1
1832	Neophytadiene	0.2	---	tr
1900	Nonadecane	---	tr	0.1
1944	α-Springene	0.1	0.1	0.1
2100	Heneicosane	---	tr	tr
	Monoterpene hydrocarbons	82.9	94.3	94.6
	Oxygenated monoterpenoids	0.3	tr	tr
	Sesquiterpene hydrocarbons	14.9	5.4	5.1
	Oxygenated sesquiterpenoids	1.3	0.1	0.1
	Others	0.4	0.1	0.2
	Total Identified	99.9	100.0	100.0

, 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.

The essential oil from the aerial parts (stems and leaves) of E. valerianifolius was dominated by the monoterpene hydrocarbons myrcene (47.8%) and α-pinene (30.2%), with a lesser quantity of the sesquiterpene β-caryophyllene (5.4%) (Table 2). The floral essential oils of E. valerianifolius were also rich in myrcene (57.0 and 60.6%) and α-pinene (32.5 and 30.6%).

Erechtites hieraciifolius and E. valerianifolius essential oils from other geographical locations have shown wide variations in chemical composition (

Table 3. Major chemical components (>5%) of Erechtites essential oils.

Erechtites Species	Geographical Location	Major Components	Ref.
E. hieraciifolius	Pacoti-Ceara, Brazil	α-phellandrene (41.3%), p-cymene (22.2%), β-caryophyllene (7.4%), camphor (5.4%)	[26]
E. hieraciifolius	Chimoré area, Chapare Province, Bolivia	α-pinene (48.0%), (E)-β-ocimene (13.9%), myrcene (13.7%)	[31]
E. hieraciifolius	“Private Reservation of Natural Heritage”, Parana State, Brazil	dillapiole (33.8%), α-pinene (33.0%), β-pinene (14.7%), limonene (9.7%)	[27]
E. valerianifolius	Mérida, Venezuela	limonene (56.7%), myrcene (12.7%), (E)-β-farnesene (10.2%), α-phellandrene (8.7%)	[32]
E. valerianifolius	“Private Reservation of Natural Heritage”, Parana State, Brazil	α-pinene (25.8%), sabinene (17.0%), myrcene (16.7%), β-pinene (13.3%), limonene (12.6%)	[27]

). 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].

3.2. 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 (

Table 4. Mosquito larvicidal activity of Erechtites hieraciifolius aerial parts (leaves and stems) essential oil.

Mosquito Species	Treatment Time	LC50, μg/Ml a (Fiducial Limits)	LC90, μg/Ml a (Fiducial Limits)	Regression Equation	χ2	p
Ae. Albopictusb	24 h	10.47 (9.12–11.70) 10.06 ± 0.92	21.11 (19.28–23.59)	y = −1.764 + 0.1443x	17.6	< 0.001
Ae. Albopictusb	48 h	5.49 (1.99–7.87) 6.50 ± 2.38	18.64 (15.95–22.92)	y = −0.177 + 0.0782x	12.68	0.002
Ae. Aegyptib	24 h	10.58 (9.42–11.68) 10.43 ± 1.93	19.47 (17.82–21.76)	y = −2.078 + 0.172x	14.34	0.001
Ae. Aegyptib	48 h	8.83 (7.76–9.79) 8.65 ± 1.56	16.27 (14.89–18.21)	y = −2.073 + 0.206x	35.49	< 0.001

^a There was no mortality in the dimethylsulfoxide (DMSO) controls; LC₅₀ values in italics are from Reed–Muench analysis. ^b Laboratory-reared mosquito larvae.

and

Table 5. Mosquito larvicidal activity of Erechtites valerianifolius aerial parts (leaves and stems) essential oil.

Mosquito Species	Treatment Time	LC50, μg/Ml a (Fiducial Limits)	LC90, μg/Ml a (Fiducial Limits)	Regression Equation	χ2	p
Ae. Albopictusb	24 h	6.07 (5.44–6.73) 6.38 ± 0.72	11.10 (10.11-12.42)	y = −2.110 + 0.306x	1.02	0.599
Ae. Albopictusb	48 h	4.65 (4.11–5.25) 5.32 ± 1.11	9.01 (7.96–10.67)	y = −1.892 + 0.352x	2.26	0.323
Ae. Albopictusc	24 h	38.01 (33.56–43.39) 40.71 ± 8.44	75.84 (65.43–94.11)	y = −1.796 + 0.041x	5.83	0.016
Ae. Albopictusc	48 h	38.57 (34.47–43.73) 35.59 ± 6.58	67.80 (59.41–81.64)	y = −1.691 + 0.044x	5.36	0.021
Ae. Aegyptib	24 h	12.56 (11.21–13.84) 12.64 ± 2.25	23.72 (21.78–26.34)	y = −1.981 + 0.137x	7.69	0.006
Ae. Aegyptib	48 h	9.60 (7.97–11.01) 9.40 ± 1.55	22.22 (20.15–25.07)	y = −1.422 + 0.122x	22.53	< 0.001
Cx. Quinquefasciatusc	24 h	40.06 (37.08–42.64) 40.00 ± 4.92	55.19 (51.92–59.82)	y = −4.316 + 0.101x	5 × 10−7	0.999
Cx. Quinquefasciatusc	48 h	39.48 (36.73–42.23) 37.53 ± 5.26	53.18 (49.70–58.00)	y = −3.697 + 0.094x	1.2 × 10−6	0.999

^a There was no mortality in the DMSO controls; LC₅₀ values in italics are from Reed–Muench analysis. ^b Laboratory-reared mosquito larvae. ^c Wild mosquito larvae.

). Larvicidal activity of permethrin (positive control) is shown in

Table 6. Mosquito larvicidal activity of permethrin (positive control).

Mosquito Species	Treatment Time	LC50, μg/Ml a (Fiducial Limits)	LC90, μg/mL a (Fiducial Limits)	Regression Equation	χ2	p
Ae. Albopictusb	24 h	0.0023 (0.0021–0.0026) 0.0022 ± 0.0003	0.0042 (0.0038–0.0049)	y = −1.628 + 686.9x	4.73	0.030
Cx. Quinquefasciatusb	24 h	0.0167 (0.0152–0.0183) 0.0148 ± 0.0011	0.0294 (0.0270–0.0326)	y = −2.292 + 121.6x	26.62	< 0.001

^a There was no mortality in the DMSO controls; LC₅₀ values in italics are from Reed-Muench analysis. ^b Wild mosquito larvae.

.

The essential oils from the aerial parts of both E. hieraciifolius and E. valerianifolius showed excellent larvicidal activity against Ae. aegypti. The 24 h LC₅₀ 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 LC₅₀ 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 LC₅₀ = 42.1 μg/mL). Likewise, wild Culex quinquefasciatus showed less susceptibility than the laboratory-reared mosquitoes.

Mosquito larvicidal activities (LC₅₀) of essential oils against Cx. quinquefasciatus have generally ranged between 25.6 μg/mL and 225 μg/mL [36,37]. Thus, the Cx. quinquefasciatus larvicidal activity of E. valerianifolius (LC₅₀ = 40.65 μg/mL) was good compared to other essential oils.

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. Mosquito larvicidal activities (24 h LC₅₀) of essential oil components against various mosquito species.

Compound	Mosquito Species	LC50 (μg/mL)	Ref.
β-caryophyllene	Aedes aegypti	88.30	[38]
β-caryophyllene	Aedes aegypti	38.58	[39]
β-caryophyllene	Aedes albopictus	44.77	[40]
β-caryophyllene	Aedes albopictus	39.52	[39]
β-caryophyllene	Anopheles subpictus	41.66	[40]
β-caryophyllene	Culex pipiens pallens	93.65	[38]
β-caryophyllene	Culex pipiens pallens	47.79	[39]
β-caryophyllene	Culex tritaeniorhynchus	48.17	[40]
β-caryophyllene	Ochlerotatus togoi	97.90	[38]
caryophyllene oxide	Aedes aegypti	125	[41]
caryophyllene oxide	Aedes aegypti	113.00	[39]
caryophyllene oxide	Aedes albopictus	107.62	[39]
caryophyllene oxide	Culex pipiens pallens	126.28	[39]
limonene	Aedes aegypti	19.4	[42]
limonene	Aedes aegypti	18.1	[43]
limonene	Aedes albopictus	15.0	[42]
limonene	Aedes albopictus	32.7	[43]
(+)-limonene	Aedes aegypti	27	[44]
(+)-limonene	Aedes aegypti	24.47	[38]
(+)-limonene	Aedes aegypti	71.9	[45]
(+)-limonene	Aedes aegypti	37	[41]
(+)-limonene	Aedes aegypti	15.31	[39]
(+)-limonene	Aedes albopictus	35.99	[46]
(+)-limonene	Aedes albopictus	41.2	[45]
(+)-limonene	Aedes albopictus	10.77	[39]
(+)-limonene	Aedes albopictus	19.15	[47]
(+)-limonene	Aedes albopictus	41.75	[48]
(+)-limonene	Culex pipiens pallens	13.26	[38]
(+)-limonene	Culex pipiens pallens	10.76	[39]
(+)-limonene	Culex quinquefasciatus	40	[49]
(+)-limonene	Ochlerotatus togoi	19.20	[38]
(-)-limonene	Aedes aegypti	30	[44]
(-)-limonene	Aedes albopictus	34.89	[46]
(-)-limonene	Aedes albopictus	15.01	[47]
myrcene	Aedes aegypti	35.8	[43]
myrcene	Aedes aegypti	27.9	[42]
myrcene	Aedes aegypti	66.42	[38]
myrcene	Aedes aegypti	39.51	[39]
myrcene	Aedes albopictus	27.0	[43]
myrcene	Aedes albopictus	23.5	[42]
myrcene	Aedes albopictus	35.98	[39]
myrcene	Aedes albopictus	37.76	[47]
myrcene	Culex pipiens pallens	66.28	[38]
myrcene	Culex pipiens pallens	41.31	[39]
myrcene	Culex quinquefasciatus	167	[49]
myrcene	Ochlerotatus togoi	64.76	[38]
α-pinene	Aedes aegypti	15.4	[50]
α-pinene	Aedes aegypti	79.1	[43]
α-pinene	Aedes albopictus	74.0	[43]
α-pinene	Aedes albopictus	34.09	[40]
α-pinene	Anopheles subpictus	32.09	[40]
α-pinene	Culex quinquefasciatus	95	[49]
α-pinene	Culex tritaeniorhynchus	36.75	[40]
(+)-α-pinene	Aedes aegypti	50.92	[38]
(+)-α-pinene	Aedes aegypti	51.28	[39]
(+)-α-pinene	Aedes albopictus	68.68	[46]
(+)-α-pinene	Aedes albopictus	55.65	[39]
(+)-α-pinene	Culex pipiens molestus	47	[51]
(+)-α-pinene	Culex pipiens pallens	53.96	[38]
(+)-α-pinene	Culex pipiens pallens	60.84	[39]
(+)-α-pinene	Ochlerotatus togoi	47.25	[38]
(-)-α-pinene	Aedes aegypti	64.80	[38]
(-)-α-pinene	Aedes aegypti	39.98	[39]
(-)-α-pinene	Aedes albopictus	72.30	[46]
(-)-α-pinene	Aedes albopictus	28.61	[39]
(-)-α-pinene	Culex pipiens molestus	49	[51]
(-)-α-pinene	Culex pipiens pallens	70.36	[38]
(-)-α-pinene	Culex pipiens pallens	31.98	[39]
(-)-α-pinene	Ochlerotatus togoi	57.93	[38]

). The LC₅₀ 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 LC₅₀ 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. Non-target lethality (LC₅₀, μg/mL) of Erechtites hieraciifolius and Erechtites valerianifolius aerial parts (leaves and stems) essential oils.

Erechtites hieraciifolius
Non-Target Species	Treatment Time	LC50, μg/Ml a (Fiducial Limits)	LC90, μg/mLa (Fiducial Limits))	Regression Equation	χ2	P
Daphnia magna	24 h	0.931 (0.808–1.035) 0.909 ± 0.169	1.531 (1.386–1.767)	y = −1.897 + 0.153x	8.2 × 10−4	0.977
Daphnia magna	48 h	0.874 (0.754–0.974) 0.864 ± 0.180	1.431 (1.297–1.644)	y = −2.011 + 2.301x	8.1 × 10−5	0.993
Chironomus tentans	24 h	10.01 (9.18–10.90) 9.37 ± 0.57	14.73 (13.46–16.71)	y = −2.723 + 0.272x	0.0037	0.951
Chironomus tentans	48 h	7.81 (6.27–9.03) 7.64 ± 0.51	15.42 (13.56–18.73)	y = −1.315 + 0.168x	0.370	0.543
Danio rerio	24 h	12.41 (11.11–13.78) 11.21 ± 1.47	21.18 (19.12–24.22)	y = −1.897 + 0.153x	1.34	0.247
Daphnia magna	24 h	0.969 (0.871–1.061) 0.937 ± 0.150	1.471 (1.347–1.656)	y = −2.478 + 2.556x	1.7 × 10−5	0.997
Daphnia magna	48 h	0.917 (0.837–0.999) 0.901 ± 0.119	1.298 (1.190–1.464)	y = −3.081 + 3.361x	0	1.0
Chironomus tentans	24 h	10.12 (8.85–11.40) 10.08 ± 2.58	17.99 (15.97–21.28)	y = −1.650 + 0.163x	1.98	0.159
Chironomus tentans	48 h	5.63 (2.67–7.47) 6.67 ± 0.81	16.31 (14.07–20.35)	y = −0.677 + 0.120x	2.90	0.088
Danio rerio	24 h	18.37 (16.89–20.00) 16.75 ± 1.81	27.77 (25.45–31.04)	y = −2.505 + 0.136x	11.38	0.001

^a There was no mortality in the DMSO controls; LC₅₀ values in italics are from Reed–Muench analysis.

).

Unfortunately, the Erechtites essential oils also show toxicity to representative non-target organisms, with LC₅₀ 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.

4. 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.