Introduction

Mosquitoes constitute a major public health problem as vectors of serious human and animal diseases, such as malaria, filariasis, Japanese encephalitis, dengue fever, chikungunya, yellow fever, and – more recently – Zika virus. These diseases cause high mortality and morbidity among people living in tropical and sub-tropical zones (Benelli 2015a; Mehlhorn 2015; Benelli et al. 2016; Yakob and Walker 2016). Nowadays, the effective and environmentally sustainable control of mosquitoes is a challenge of essential importance. This is due to the fact that some mosquitoes, with special reference to the Aedes genus, are particularly difficult to control, due to their high ecological plasticity. A good example is the Asian tiger mosquito, Aedes albopictus, which is now ranked among the one hundred most invasive organisms worldwide (Benedict et al. 2007; Becker 2008; Becker et al. 2013; Benelli and Mehlhorn 2016). In addition, the indiscriminate overuse of synthetic insecticides resulted in undesirable effects on human health and non-target organisms, as well as resistance development in targeted vectors, with consequent loss of efficacy (see Hemingway and Ranson 2000, as well as Naqqash et al. 2016 for dedicated reviews). This current scenario is worsened by the lack of access to modern and costly mosquito control tools by rural populations of developing countries, which are the most afflicted by mosquito-borne diseases (Benelli 2015a).

Therefore, the use of plant derivatives with multiple mechanisms of action and eco-friendly features has been proposed as alternative tools against Culicidae, ticks, and other vectors (Benelli 2015b; Pavela and Benelli 2016a, b). In latest years, plant essential oils (EOs), plant extracts, plant extraction byproducts (e.g. neem cake), as well as botanical-fabricated capped mosquitocidal nanoparticles have been evaluated as mosquito control agents (Amer and Mehlhorn 2006a, b, c, d; Dinesh et al. 2015; Subramaniam et al. 2015, 2016; Murugan et al. 2016a, b; Panneerselvam et al. 2016; Benelli 2016a, b, 2017; Benelli and Govindarajan 2017).

In particular, a wide number of EOs extracted from aromatic plants have been tested as mosquitocides, ovideterrents and/or repellents, against different mosquito species (Benelli 2015a; Pavela 2015; Pavela and Govindarajan 2016; et al. 2016a, b, c). Good examples include Origanum majorana (El-Akhal et al. 2014), Ocimum gratissimum (Pratheeba et al. 2015), Lavandula stoechas (El Ouali et al. 2016), Corymbia citriodora (Santi and Simone 2014), Myristica fragrans (Carolina and Maman 2016), Crataeva magna (Veni et al. 2016), Laurus nobilis (Verdian-Rizi 2009), Apium graveolens (Kumar et al. 2014), Clausena anisata (Jayaraman et al. 2015), Melissa officinalis (Baranitharan et al. 2016) and Coleus aromaticus (Govindarajan et al. 2013b; Baranitharan et al. 2017). Results underlined that the plant EOs may be an alternative source of mosquito larval control agents, since are rich in bioactive compounds that show multiple mechanisms of action, are biodegradable into non-toxic products, and potentially suitable for use in IPM programs (Pavela and Benelli 2016a, b).

However, most of the studies focused on routine testing of EOs without digging deep in their chemical composition (i.e. no GC-MS, HPLC-MS, HPTLC and NMR analyses have been performed) and evaluating the bioactivity of selected pure constituents, formulated alone or in synergistic blends (Benelli et al. 2017a, b, c). Most importantly, as recently reviewed by Pavela (2015), the majority of tested EOs achieved LC50 higher than 50 ppm on mosquitoes, highlighting the value of systematic screening endemic flora of tropical and sub-tropical countries searching for effective mosquitocidal and antiplasmodial  products (Benelli and Mehlhorn 2016).

Blumea is a genus of shrubs and small trees, comprising about 80 species distributed in tropical and subtropical Asia, Africa, and Oceania (Liang et al. 2011). This genus includes some key medicinal plants largely used in traditional medicine. For example, Blumea membranacea EO led to blood pressure reduction (Mehta et al. 1986). Besides the ethno-pharmacological potential of Blumea species, the EOs from B. mollis (Senthilkumar et al. 2008), Blumea perrottetiana (Owolabi et al. 2010) and B. densiflora (Zhu and Tian 2011) have been reported for their insecticidal activity, while B. membranacea EO shows antifungal activity (Mehta et al. 1986).

Blumea eriantha is an annual aromatic herb, which grows abundantly along roadsides and degraded forestlands. Common names are “Nimurdi” in Marathi and “Kukronda” in Hindi. B. eriantha is distributed in Bihar, Karnataka, Madhya Pradesh, Maharashtra, Orissa, Uttar Pradesh and Southern India (Singh et al. 2011). The juice extracted from this herb has been reported as a carminative, while the warm leaf infusion is used as sudorific, and the cold infusion is considered as a diuretic and herbal emmenagogue. The EO extracted from B. eriantha is traditionally recognized for its antibacterial and antifungal uses in folk medicine (Khare 2007). A recent study focused on the antimicrobial efficacy of B. eriantha EO (Pednekar et al. 2012).

However, the toxicity of B. eriantha EO against insect vectors of medical and veterinary relevance is unknown. In the present research, we investigated the environmentally sustainable use of B. eriantha EO and its main chemical components for the development of new products to combat the spread of the mosquito-borne diseases, with special reference to dengue and Zika virus. We tested the B. eriantha EO larvicidal activity on six key mosquito vectors, i.e. Anopheles stephensi, An. subpictus, Ae. aegypti, Ae. albopictus, Culex quinquefasciatus, and Cx. tritaeniorhynchus. Furthermore, B. eriantha EO was analyzed using gas chromatography-mass spectrometry (GC-MS). The major components of B. eriantha EO, i.e. (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate, were also tested against the six mosquito vectors. Lastly, to shed light on non-target effects of the B. eriantha EO as well as (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate, we evaluated them in acute toxicity tests on four non-target predators of mosquito larvae.

Materials and methods

Extraction, GC and GC-MS of the B. eriantha essential oil

Fresh leaves of B. eriantha were collected in the Munnar mountains, India (10°05’21”N 77°03’35”E, 1700 m a.s.l.) in May 2016. Blumea eriantha EO was hydro-distilled using 3 kg of fresh leaves, then analyzed by GC and GC-MS as described by Govindarajan and Benelli (2016a, b). Compound identification was carried out comparing retention indices and mass spectra with those available in NIST 98.1, Mass Finder 3.1 and Adams (2007).

Larvicidal activity of (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate

The B. eriantha EO as well as (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate, were tested against 3rd instar larvae of An. stephensi, Ae. aegypti, Cx. quinquefasciatus, An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus following the protocol by WHO (2005), slightly modified by Govindarajan and Benelli (2016c). For each tested product, 5 replicates of each dose were prepared. 10 3rd larvae were transferred into each beaker. Mortality was assessed after 24 h of exposure.

Toxicity on non-target predators

The effect of B. eriantha EO as well as (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate, on non-target aquatic predators was assessed following the method by Sivagnaname and Kalyanasundaram (2004) modified Govindarajan et al. (2016d, e). The toxicity of the B. eriantha EO, (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate was tested against adults of the non-target biological control agents of mosquito young instars, including adult backswimmers and water bugs i.e. Anisops bouvieri and Diplonychus indicus , and larvivorous fish Gambusia affinis and Poecilia reticulata. The non-target species were reared as reported by Govindarajan and Benelli (2016d). The B. eriantha EO, (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate were evaluated at doses higher than 50xLC50 calculated on the six mosquito species. 10 replicates were performed for each dose. 4 control replicates were also done (where no B. eriantha EO, (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate were added to the water). Mortality of each non-target predator was assessed after 48 h of exposure (Govindarajan and Benelli 2016b; Benelli et al. 2017c).

Data analysis

Mortality data were analyzed by probit analysis (Benelli 2017). LC50 and LC90 were calculated following Finney (1971). Concerning non-target predators, the Predator Safety Factor (PSF) was calculated as described by Deo et al. (1988). Data were analyzed by SPSS version 16.0.

Results

Yield and composition of B. eriantha essential oil

The yield of B. eriantha leaf EO was 2.5 ml/kg of leaf fresh weight. Table 1 showed a total of 34 chemical constituents, representing 94.2% of the B. eriantha leaf EO. The major constituents of B. eriantha EO were (4E,6Z)-allo-ocimene (12.8%), carvotanacetone (10.6%) and dodecyl acetate (8.9%). The chemical structures of (4E,6Z)-allo-ocimene, carvotanacetone and dodecyl acetate are reported in Figure 1. The amount of remaining 31 molecules ranged from 0.7 % to 4.2 % (Table 1).

Table 1 Chemical composition of Blumea eriantha essential oil
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Fig. 1
figure 1

Chemical structures of the three major constituents of the Blumea eriantha essential oil. a (4E,6Z)-Allo-ocimene. b Carvotanacetone. c Dodecyl acetate

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Larvicidal activity of (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate

The B. eriantha EO showed acute toxicity against third instar larvae of An. stephensi, Ae. aegypti, Cx. quinquefasciatus, An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus, with LC50 of 41.61, 44.82, 48.92, 51.21, 56.33 and 61.33 μg/ml, respectively (Table 2).

Table 2 Larvicidal activity of Blumea eriantha essential oil on six mosquito vectors
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Furthermore, the three major pure compounds extracted from the B. eriantha EO, (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate, were tested individually against six mosquito vector larval populations. We observed that (4E,6Z)-allo-ocimene, carvotanacetone and dodecyl acetate were extremely toxic to the six mosquito species. (4E,6Z)-allo-ocimene LC50 values estimated on An. stephensi, Ae. aegypti, Cx. quinquefasciatus, An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus, were 4.06, 4.52, 4.92, 6.14, 6.70 and 7.26 μg/ml, respectively (Table 3). Carvotanacetone LC50 values were 6.20, 6.77, 7.38, 8.43, 9.21 and 10.02 μg/ml, respectively (Table 4). Dodecyl acetate LC50 values were 10.22, 11.18, 12.16, 12.31, 13.45 and 14.68 μg/ml, respectively (Table 5). No mortality was recorded in controls.

Table 3 Larvicidal activity of (4E,6Z)-allo-ocimene from Blumea eriantha essential oil on six mosquito vectors
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Table 4 Larvicidal activity of carvotanacetone from Blumea eriantha essential oil on six mosquito vectors
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Table 5 Larvicidal activity of dodecyl acetate from Blumea eriantha essential oil on six mosquito vectors
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Toxicity on non-target predators

The acute toxicity of B. eriantha EO was tested on the four non-target predators A. bouvieri, D. indicus, P. reticulata and G. affinis. Results are reported in Table 6 . B. eriantha EO LC50 values were 4139.79, 6285.59, 10251.51 and 11431.04 μg/ml, respectively.

Table 6 Toxicity of Blumea eriantha essential oil on four non-target predators of mosquitoes
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Furthermore, the three major pure compounds extracted from the B. eriantha EO, (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate, were tested individually against four important non-target predators of mosquito larvae. We observed that (4E,6Z)-allo-ocimene, carvotanacetone and dodecyl acetate were scarcely toxic to the non-target predators. (4E,6Z)-allo-ocimene LC50 estimated on A. bouvieri, D. indicus, P. reticulata and G. affinis were 519.97, 845.65, 1656.78 and 1854.25 μg/ml, respectively (Table 7). Carvotanacetone LC50 were 631.59, 1051.39, 1863.86 and 2075.07 μg/ml, respectively (Table 8). Dodecyl acetate LC50 were 823.94, 1483.11, 2065.56 and 2369.78 μg/ml, respectively (Table 9). No mortality was recorded in control treatments.

Table 7 Toxicity of (4E,6Z)-allo-ocimene from Blumea eriantha essential oil on four non-target predators of mosquitoes
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Table 8 Toxicity of carvotanacetone from Blumea eriantha essential oil on four non-target predators of mosquitoes
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Table 9 Toxicity of dodecyl acetate from Blumea eriantha essential oil on four non-target predators of mosquitoes
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The estimated PSF indicated that the B. eriantha EO and its main constituents showed little toxicity on A. bouvieri, D. indicus, P. reticulata and G. affinis (Table 10).

Table 10 Predator safety factors calculated on four non-target predators over six key mosquito species exposed to Blumea eriantha essential oil, (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate
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Lastly, focal observations conducted daily until 10 days from the exposure to B. eriantha EO and its main constituents, formulated at the LC50 and LC90 calculated on the targeted six mosquito vectors, indicated that the survival and swimming activity of the non-target predators were not damaged.

Discussion

Wide chemical diversity of Blumea essential oils

Results from GC and GC-MS analyses showed that 34 compounds were identified in the B. eriantha EO, with (4E,6Z)-allo-ocimene, carvotanacetone and dodecyl acetate as main components. This highlighted a quite different chemical composition, if compared to other EOs extracted from close-related Blumea species. Currently, the EOs of several species of the genus Blumea have been examined. Good examples are B. balsamifera (Sakee et al. 2011), Blumea lacera (Khair et al. 2014), B. balsamifera (Norikura et al. 2008), and B. lacera (Jahan et al. 2014). At variance with our results on B. eriantha, it is worthy to note that B. perrottetiana aerial part EO was mostly composed by 2,5-dimethoxy-p-cymene (30.0 %), 1,8-cineole (11.0 %) and sabinene (8.1 %) (Owolabi et al. 2010). Blumea balsamifera leaf EO was mostly composed by borneol (33.22 %), caryophyllene (8.24 %) and ledol (7.12 %) (Bhuiyan et al. 2009). Blumea mollis leaf EO was mainly composed by linalool (19.43 %) and γ-elemene (12.19 %) (Senthilkumar et al. 2008). Blumea brevipes EO main chemicals were terpinen-4-ol (27.6 %) and germacrene-D (15.4 %) (Mwangi et al. 1994), while B. lanceolaria EO mainly contained methyl thymol (Dung et al. 1991). Lastly, B. lacera leaf EO was composed by thymoquinol di-mether, β-caryophyllene, α-humulene, and (E)-β-farnesene (Laakso et al. 1989). Main compounds in B. densifora EO were borneol (11.43%), germacrene D (8.66%) and β-caryophyllene (6.68%) (Zhu and Tian 2011). Based on the presence of (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate in our GC-MS analysis of B. eriantha EO, we selected these molecules for further toxicity screenings on mosquito vectors.

Blumea essential oils really work against mosquitoes!

The use of plant EOs in vector control may represent a cheap alternative method to minimize the side effects of chemical pesticides on human health and the environment (Benelli 2015b; Govindarajan et al. 2013a, b, 2016a, b, c; Pavela 2015). Although a number of compounds of botanical origin have been currently reported (Wang et al. 2006; Cheng et al. 2009; Pavela and Benelli 2016b), the discovery of more effective plant products is of paramount importance to improve insecticide formulation and develop environmentally acceptable insecticides (Alkofahi et al. 1989).

In our experiments, the EO extracted from the leaves of B. eriantha showed high toxicity against 3rd instar larvae of Anopheles, Aedes and Culex species, including Zika virus vectors, with LC50 ranging from 41.61 to 61.33 μg/ml. Our results fit the criteria of EO larvicidal efficacy outlined by Pavela (2015). Earlier, it has been reported that two other EOs from the Blumea genus are highly toxic to anopheline vectors. Indeed, the B. densiflora EO tested on Anopheles anthropophagus larvae showed a LC50 of 10.55 ppm after 24 h, and 22.32 ppm after 12 h (Zhu and Tian 2011), and Senthilkumar et al. (2008) showed the larvicidal effectiveness of B. mollis EO on Cx. quinquefasciatus, with LC50 of 52.2 ppm.

Concerning the larvicidal activity of other EOs and extracts from the Asteraceae family, Macêdo et al. (1997) evaluated the toxicity of Tagetes minuta extract on Ae. fluviatilis (1.0 mg/l). The ethyl acetate leaf extract of Eclipta prostrata achieved a LC50 of 119.89 ppm on Cx. tritaeniorhynchus (Elango et al., 2009), while Achilea millefolium methanolic stem extract led to LC50 of 120.0 ppm on Cx. quinquefasciatus (Pavela 2008), Tanacetum vulgare methanolic flower extract (LC50 = 178.0 ppm) and methanolic stem extract of Otanthus maritimus (LC50 = 195.0 ppm) were also toxic to Cx. quinquefasciatus (Pavela et al. 2009).

Larvicidal activity of (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate

Interestingly, two of the main B. eriantha EO components, (4E,6Z)-allo-ocimene and carvotanacetone, achieved LC50 lower than 10 μg/ml on all tested mosquito species. As mentioned above, a wide number of plant EOs have been tested against mosquitoes. Indeed, screening the abundance of research products using “essential oil mosquito” as keywords on Scopus database (accessed: January 2017) we found more than 900 studies on this topic.

Unfortunately, only a very limited number of them considered testing single molecules identified in the EO (Pavela 2015; Benelli et al. 2017a). Some recent and noteworthy exceptions with larvicidal LC50 lower than 50 ppm are reviewed here. For example, the leaf EO from Clausena anisata contained β-pinene, sabinene, germacrene-D, estragole and linalool, which achieved LC50 of 23.17, 19.67, 16.95, 11.01 and 35.17 ppm on An. stephensi, LC50 of 27.69, 21.20, 18.76, 12.70 and 38.64 ppm on Ae. aegypti, and LC50 of 32.23, 25.01, 21.28, 14.01 and 42.28 ppm on Cx. quinquefasciatus (Govindarajan 2010). The LC50 of germacrene D-4-ol from Zanthoxylum monophyllum EO ranged from 6.12 to 7.26 μg/mL, while the LC50 for α-cadinol ranged from 10.27 to 12.28 μg/mL (Pavela and Govindarajan 2016). ar-curcumene and epi-β-bisabolol from Hedychium larsenii EO were toxic to An. stephensi (LC50 = 10.45 and 14.68 μg/ml), Ae. aegypti (LC50 = 11.24 and 15.83 μg/ml) and Cx. quinquefasciatus (LC50 = 12.24 and 17.27 μg/ml) (AlShebly et al. 2017). Artemisia absinthium EO-isolated (E)-β-farnesene, (Z)-en-yndicycloether, and (Z)-β-ocimene were toxic to An. stephensi (LC50 = 8.13, 16.24 and 25.84 μg/ml), An. subpictus (LC50 = 10.18, 20.99, and 30.86 μg/ml), Ae. aegypti (LC50 = 8.83,17.66, and 28.35 μg/ml), Ae. albopictus (LC50 = 11.38, 23.47, and 33.72 μg/ml), Cx. quinquefasciatus (LC50 = 9.66, 19.76, and 31.52 μg/ml), and Cx. tritaeniorhynchus (LC50 = 12.51, 25.88, and 37.13 μg/ml) (Govindarajan and Benelli 2016a). Lavandulyl acetate and bicyclogermacrene from Heracleum sprengelianum EO were toxic to An. subpictus (LC50 = 4.17 and 10.3 μg/ml), Ae. albopictus (LC50 = 4.60 and 11.1 μg/ml) and Cx. tritaeniorhynchus (LC50 = 5.11 and 12.5 μg/ml) (Govindarajan and Benelli 2016b). from Syzygium zeylanicum EO was a source of α-humulene and β-elemene, which were toxic to An. subpictus (LC50 = 6.19 and 10.26 μg/ml), Ae. albopictus (LC50 = 6.86 and 11.15 μg/ml), and Cx. tritaeniorhynchus (LC50= 7.39 and 12.05 μg/ml) (Govindarajan and Benelli 2016c). Eugenol, α-pinene and β-caryophyllene were identified in the Plectranthus barbatus EO, and they showed toxicity to An. subpictus (LC50 = 25.45, 32.09 and 41.66 μg/ml, respectively), Ae. albopictus (LC50 = 28.14, 34.09 and 44.77 μg/ml) and Cx. tritaeniorhynchus (LC50 = 30.80, 36.75 and 48.17 μg/ml) (Govindarajan et al. 2016a). Carvacrol and terpinen-4-ol isolated from the Origanum vulgare EO showed toxicity to An. stephensi (LC50 = 21.15 and 43.27 μg/ml), An. subpictus (LC50 = 24.06 and 47.73 μg/ml), Cx. quinquefasciatus (LC50 = 26.08 and 52.19 μg/ml) and Cx. tritaeniorhynchus (LC50 = 27.95 and 54.87 μg/ml) (Govindarajan et al. 2016d). δ-cadinene, calarene and δ-4-carene from the Kadsura heteroclita EO acted as larvicides on An. stephensi (LC50 = 8.23, 12.34 and 16.37 μg/ml), Ae. aegypti (LC50 = 9.03, 13.33 and 17.91 μg/ml) and Cx. quinquefasciatus (LC50 = 9.86, 14.49 and 19.50 μg/ml) (Govindarajan et al. 2016e).

Testing selected chemicals from plant essential oils is really important, since in a number of instances the single molecules are more effective if compared to the raw oil. In addition, testing single products for which the mechanism (s) of action is well known may help to shed light on the precise alterations induced on insect biochemical pathways (Pavela and Benelli 2016b). Lastly, the most effective molecules could be formulated in dedicated blends to shed light on possible synergistic and antagonistic toxicity effects (Benelli et al. 2017b, d). Further research on potential synergic as well as antagonistic effects occurring among the B. eriantha-borne molecules tested in blend is ongoing.

Toxicity of selected Blumea-borne molecules on non-target aquatic predators

The acute toxicity of B. eriantha EO, as well as (4E,6Z)-allo-ocimene, carvotanacetone, and dodecyl acetate on four aquatic predators was limited, with a LC50 range of 519-11.431 μg/ml (Tables 6, 7, 8, and 9). Plant EOs have been recognized as important sources of biopesticides, with limited toxic effects on human health and non-target organisms (Pavela and Benelli 2016b). For instance, recent research showed very limited acute toxicity of Pinus kesiya EO on A. bouvieri, D. indicus and G. affinis, with LC50 ranging from 4135 to 8390 mg/ml. Also in the above-cited research, in agreement with the present results, G. affinis has been found less susceptible to EO-based treatments, if compared to A. bouvieri, D. indicus and P. reticulata (Govindarajan et al. 2016c). Besides size differences, enzymatic assays to shed light on the reasons at the basis of this differential susceptibility are required. Furthermore, Govindarajan et al. (2016b) reported that the Zingiber nimmonii EO was safer towards D. indicus and G. affinis, with LC50 of 3241.53 and 9250.12 μg/ml, respectively. Pavela and Govindarajan (2016) showed that Z. monophyllum EO and its main constituents germacrene D-4-ol and a-cadinol tested on G. affinis had LC50 of 4234, 414 and 635 μg/ml, respectively. H. sprengelianum EO and its two major compounds lavandulyl acetate and bicyclogermacrene tested on A. bouvieri, D. indicus and G. affinis led to LC50 ranging from 414 to 4219 μg/ml. The S. zeylanicum EO (LC50 = 20,374 μg/ml), β-elemene (LC50 = 2073 μg/ml), and α-humulene (LC50 = 1024 μg/ml) from Syzygium zeylanicum are scarcely toxic towards G. affinis (Govindarajan and Benelli 2016c). Taken together, the data reported above underline the eco-friendly features of EO-borne molecules used as mosquito larvicides, allowing us to claim their potential employ as larvicides in urban and rural areas, with special reference to developing countries where mosquito-borne diseases are endemic and people should synergize different control tools in the fight against mosquitoes (see also Benelli 2015a).

Conclusions

Overall, the present research showed the toxicity of B. eriantha EO on six important mosquito vectors. Besides the effective larvicidal potential of the B. eriantha EO, which led to LC50 values lower than 50 ppm for most of the tested mosquitoes (Pavela 2015), is extremely noteworthy the toxicity of two main EO components, (4E,6Z)-allo-ocimene and carvotanacetone, which achieved LC50 lower than 10 μg/ml on all tested mosquito species, including two aedine vectors of Zika virus. Therefore, the extremely high larvicidal activity of (4E,6Z)-allo-ocimene and carvotanacetone far exceed most of the LC50 calculated in current literature on botanical mosquito larvicides, coupled with their eco-friendly features on non-target aquatic predators of mosquito larval instars, allowing us to propose both of them as potentially alternatives for developing eco-friendly mosquito control tools.