Larvicidal and adulticidal effects of some Egyptian oils against Culex pipiens

Mosquitoes and mosquito-borne diseases represent an increasing global challenge. Plant extract and/or oils could serve as alternatives to synthetic insecticides. The larvicidal effects of 32 oils (1000 ppm) were screened against the early 4th larvae of Culex pipiens and the best oils were evaluated against adults and analyzed by gas chromatography-mass spectrometry (GC mass) and HPLC. All oils had larvicidal activity (60.0–100%, 48 h Post-treatment, and their Lethal time 50 (LT50) values ranged from 9.67 (Thymus vulgaris) to 37.64 h (Sesamum indicum). Oils were classified as a highly effective group (95–100% mortalities), including Allium sativum, Anethum graveolens, Camellia sinensis, Foeniculum vulgare, Nigella sativa, Salvia officinalis, T. vulgaris, and Viola odorata. The moderately effective group (81–92% mortalities) included Boswellia serrata, Cuminum cyminum, Curcuma aromatic, Allium sativum, Melaleuca alternifolia, Piper nigrum, and Simmondsia chinensis. The least effective ones were C. sativus and S. indicum. Viola odorata, Anethum graveolens, T. vulgaris, and N. sativa provide 100% adult mortalities PT with 10, 25, 20, and 25%. The mortality percentages of the adults subjected to 10% of oils (H group) were 48.89%, 88.39%, 63.94%, 51.54%, 92.96%, 44.44%, 72.22%, and 100% for A. sativum, An. graveolens, C. sinensis, F. vulgare, N. sativa, S. officinalis, T. vulgaris, and V. odorata, respectively. Camellia sinensis and F. vulgare were the most potent larvicides whereas V. odorata, T. vulgaris, An. graveolens and N. sativa were the best adulticides and they could be used for integrated mosquito control.


Results
The larvicidal effect of 32 oils was screened against the early 4th larvae, Cx. pipiens. The results showed that all plant oils had larvicidal activity ( Table 2).
The least effective group (L group) included the other 17 oils, and the least effective ones were C. sativus, and S. indicum, providing 62. 33 (Table 3).
Furthermore, the Kruskal-Wallis test was performed to compare the mean differences of more than two groups, followed by the Mann-Whitney test to compare the mean differences between groups. Whereas Kruskal-Wallis and Friedman's tests showed there are significant indications between the three groups at different times (P = 0.001) ( Tables 4 and 5).

Discussion
EOs could serve as suitable alternatives to synthetic insecticides because they are relatively safe, available, and biodegradable 15  In this study, the most effective oils against adults were An. graveolens and V. odorata followed by T. vulgaris then N. sativa. The data revealed that F. vulgare is a highly potent larvicide. Similarly, its oil controlled Anopheles atroparvus, Culex quinquefasciatus 23,24 , and Aedes aegypti 25 . Despite its effectiveness as larvicide in this study, F. vulgare was the least effective adulticide. In contrast, it induced adulticidal properties against Cx. quinquefasciatus 23 .
Our data indicated that C. sinensis was a highly effective larvicide and the less effective adulticide. Comparatively, the chemical extracts of C. sinensis induced larvicidal and adult repellent effects against Cx. pipiens providing the highest protection (100%) from the bites of starved females at the dose of 6 mg/cm 226 . Moreover, its leaf extract showed larvicidal effect against Anopheles arabiensis and Anopheles gambiae (s.s.) 27 .
Thymus vulgarisd An. graveolens showed potent larvicidal and adulticidal effects in this work. Likewise, T. vulgaris has both effects against Cx. quinquefasciatus 28 and Ae. aegypti 29 . Thymus vulgaris exhibited larvicidal properties, 100% mortality, against Cx. pipiens larvae, at 200 ppm, whereas the LC 25 and LC 50 vlalues indicated no effect on AChE activity, activation of the detoxification system, as indicated by an increase in GST activity and a decrease in GSH rate 30 .
Our findings agree with another study found that the most potent EOs out of 53 oils against larvae were F. vulgare, T. vulgaris, Citrus medica (lime), and C. sinensis (LC 50 = 27.5, 31.6, 51.3, 53.5 ppm, respectively). C. sinensis was the most efficient EOs enhancing the efficacy of deltamethrin, co-toxic factor = 316.67, over than PBO, the positive control, co-toxic factor = 283.35) 31 .
Some oils applied in this study showed a similar larvicidal effect against Cx. pipiens as N. sativa 32,33 and S. officinalis 34 . Some essential oils such as T. vulgaris, S. officinalis, C. sempervirens and A. graveolens had a larvicidal effect against mosquito larvae and their LC 90 values were < 200-300 ppm. This result may be due to several   39,40 .
Allium sativum showed high potency against larvae in this study. A similar finding was recorded for Cx. pipiens and Culex restuans (LC 50 = 7.5 and 2.7 ppm, respectively) 41 . Argania spinosa oil showed a low larvicidal effect in this study. A similar effect was recorded against Cx. quinquefasciatus larvae 42 .     Zingiber officinale and Syzygium aromaticum were less effective. In contrast, they were effective against Cx. pipiens (LC 50 = as 71.85 and 30.75, respectively) 45 .
Sesamum indicum is one of the L group in this study. In contrast, petroleum ether extract showed larvidcidal, antifeedant and repellent action against Cx. pipiens 33 . Furthermore, EOs of N. sativa, Allium cepa, and S. indicum, induced larvicidal effect and their LC 50 values against both field and laboratory strains of Cx. pipiens were 247.99 and 108.63; 32.11 and 2.87; and finally, 673.22 and 143.87 ppm, respectively. They influenced the pupation and adult emergence rates besides developmental abnormalities at sublethal concentrations 46 .
Boswellia serrata (M group) and Brassica carinata (L group) showed relative larvicide against Cx. pipiens in this study. A similar result was reported 47,48 49 .
A similar larvicidal effect was recorded for Rosmarinus officinalis, hexane extract (80 and 160 ppm), reduced 100% mortality against 3rd and 4th instars larvae of Cx. pipiens and the toxicity increased in the pupal and adult stages 50 .
The results showed that A. sativum, and S. officinalis oils were effective against mosquito larvae, maybe due to the presence of a number of active secondary compounds such as ISOCHIAPIN B%2 < (sesquiterpene lactone) and 9-Octadecenamide, (Z)-that are anti-inflammatory activity 57 , also, Terpinen-4-ol and Camphor in Sage oil that these are excellent natural insecticide 58 , but these oils garlic and Sage did not show the required efficacy against adult mosquitoes.
The phytochemical analysis of this study revealed the major activated compounds of the analyzed oils. Green tea oil is a highly effective larvicide in this study contains a high amount of polyphenols that have antioxidant activity. A similar finding was reported 59 . Our data indicated that green tea oil also contains polyphenols as Gallic acid, Catechin, Methyl gallate, Coffeic acid, Coumaric acid, Naringenin, and Kaempferol which might aid in its insecticidal effect.
This study indicated that F. vulgare contains Estragole (70.36%) and Limonene (8.96%). Similarly, Limonene as a cyclic monoterpene has a viable insecticidal effect 60 . Besides, Estragole induced toxicity to adult fruit flies, Ceratitis capitata 61 . Moreover, An. graveolens contains thiosemicarbazone (32.13%) in this study. Likewise, thiosemicarbazide is a major component An. graveolens with insecticidal effect 62 . Also, Dauco and carotol are essential oils documented for An. graveolens in this work have repellent activity against adult Ae. aegypti, Ae. albopictus, and Anopheles quadrimaculatus Say 63 . Furthermore, V. odorata in the present analysis contains alphaionone, which revealed anti-inflammatory and analgesic effects 64 . Thymus vulgaris showed good alpha-pinene and pyridine derivatives that play an important role as larvicidal and adulticidal effects against Ae. aegypti and growth regulator, respectively 65,66 . In addition, the combination of all constituents may promote their individual larvicidal and adulticidal effects.
The biochemical compositions showed that T. vulgaris oil affected the energy reserves with a marked effect on proteins and lipids 30 . The differences between our findings and those of the others could be attributed to the biological activities and the chemical composition for EOs, which could vary between plant age, tissues, geographical origin, the part used in the distillation process, distillation type, and the species. Therefore, types and levels of active constituents in each oil may be responsible for the variability in their potential against pests 16 .

Conclusions
Diseases transmitted by mosquitoes represent global concerns. Our findings demonstrate the potential of F. vulgare and C. sinensis as the most potent larvicides and N. sativa, V. odorata, and An. graveolens as the most effective adulticides as they contain good command of different essential oils. EOs could be used for integrated mosquito control programs as larvicides or synergists for enhancing the efficacy of current adulticides 31 . Further