Journal of Vector Borne Diseases

RESEARCH ARTICLE
Year
: 2022  |  Volume : 59  |  Issue : 3  |  Page : 228--235

Larvicidal potential and residual activity effect of kinnow peel oil against Aedes aegypti L.


Arshkamaljot Kaur1, Devinder Kaur Kocher1, Rajender Kumar2,  
1 Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, Punjab, India
2 School of Organic Farming, College of Agriculture, Punjab Agricultural University, Ludhiana, Punjab, India

Correspondence Address:
Prof. Devinder Kaur Kocher
Prof. Devinder Kaur Kocher, Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana 141004, Punjab
India

Abstract

Background & objectives: Transmission of dengue virus by Aedes aegypti mosquito is one of the major global health concerns. The present study was aimed to explore the larvicidal potential of oil extracted from kinnow peel waste to be used as an efficient, economic and safe agent against Ae. aegypti. Methods: Kinnow peel oil was extracted and its five concentrations at 40, 50, 60, 70 and 80 ppm were tested against 4th instar larvae of Ae. aegypti. Larval mortality (%) and LC50 and LC90 values of toxicity were determined followed by evaluation of the residual activity effect of its leftover effective concentration on larval mortality, development and emergence. Effect of storage (2, 4 and 6 months) on larvicidal potential of kinnow peel oil was also determined. Results: Out of the tested concentrations, 70 ppm of kinnow peel oil was found to be the effective concentration against 4th instar larvae of Ae. aegypti. LC50 and LC90 toxicity values were 47.26 and 61.56 ppm, respectively. No residual activity effect in terms of larval mortality was found, however a significant delay in development (L4 to adult) was observed after placing new larvae in the leftover effective oil concentration. No effect of storage on larvicidal potential of 2, 4 and 6 months old kinnow peel oil in comparison to freshly extracted oil was observed. Interpretation & conclusion: Kinnow peel oil proved to have a good potential as a biolarvicide against Ae. aegypti and could be used as an effective and eco-friendly mosquito control agent in the future.



How to cite this article:
Kaur A, Kocher DK, Kumar R. Larvicidal potential and residual activity effect of kinnow peel oil against Aedes aegypti L. J Vector Borne Dis 2022;59:228-235


How to cite this URL:
Kaur A, Kocher DK, Kumar R. Larvicidal potential and residual activity effect of kinnow peel oil against Aedes aegypti L. J Vector Borne Dis [serial online] 2022 [cited 2023 Feb 6 ];59:228-235
Available from: http://www.jvbd.org//text.asp?2022/59/3/228/337508


Full Text

 INTRODUCTION



The mosquito borne diseases like malaria, dengue, Chikungunya, Japanese encephalitis (JE), and lymphatic filariasis are present in tropical areas, and out of these, transmission of dengue virus by Aedes aegypti mosquito is one of the major global issues as every year 50–100 million dengue infections occur worldwide[1]. The control and prevention of dengue disease entirely depends on the effective strategies and measures towards the supervision of these dreadful vectors. Ae. aegypti is abundantly present in small freshwater collections like roadside ditches, earthen pots, desert coolers, gardens, and small containers lying in peri-domestic areas[2]. According to the recent report of National Vector Borne Disease Control Programme (NVBDCP) in India, 14,044 dengue cases have been reported along with 2764 chikungunya cases till July 2021[3]. Because of the lack of adequate medication and vaccines, the only measure to control the dengue is still based on vector control.

Chemical insecticides have been extensively used which either kill the vectors or prevent them from biting or by eradicating the larvae at breeding sites. DEET (N, N-Diethyl-meta-toluamide), picaridin, permethrin, allethrin and malathion are some synthetic repellents used for mosquito control. But frequent application of these chemicals causes insecticidal resistance in the mosquito species, adverse effects on environmental quality and biological magnification of toxic substances through the food chain resulting in harm to non-target organisms including humans[4]. Due to their potential virulent effects, high operational cost and environmental pollution, there is an urgent need for developing and implementing alternate approaches to control vector borne diseases. Thus the current scenario encourages development of other insecticides which are effective, economical and have negligible impact on the environment[5].

Studies have revealed plant extracts or botanicals as safe, eco-friendly alternative for synthetic insecticides and the richest source as anti-mosquito agents. In several countries, phyto-products have been used as traditional methods to solve the different types of mosquito-borne parasitic diseases by using plant-based products extracted from different parts of plants like rhizome, leaf, bark, flower, fruit, and seed. Botanicals are user-friendly and promising alternatives for mosquito control because of lower toxicity to non-target organisms and their innate biodegradation ability[6]. Therefore, researchers are nowadays exploring botanical extracts from weeds and fruit wastes which have been investigated as mosquito control agents and found to be an eco-safe and effective remedy.

Citrus fruits are the most widely cultivated fruits worldwide, containing several important phytochemicals. Out of these, limonene is present in high concentration in citrus fruits which acts as an organic insect repellent and larvicidal agent[7]. Among citrus fruits, kinnow has been established as a high yield mandarin hybrid variety (a cross between Citrus nobilis and C. deliciosa) being cultivated in Punjab, India. The peels of the fruit which are about 30–34%[8], are generally considered as waste but are more significant due to the presence of many active phytochemicals having potential larvicidal properties for their usage. Their byproducts which are treated as waste, are an important source of different bioactive compounds and are found to have insecticidal properties[7],[8]. Thus, after knowing the importance of such properties the present study was planned to assess the larvicidal potential of oil extracted from kinnow peel waste against Ae. aegypti.

 Material & Methods



Collection of Ae. aegypti larvae

For collection of Ae. aegypti larvae, firstly, water samples were taken from various small fresh water collections such as desert coolers, roadside ditches, rubber tires, plate under pots, plastic containers and earthen pots lying in peri-domestic areas of urban regions of Ludhiana district of Punjab state in India, from October to November 2020 and June to September 2021 and brought to laboratory. From the collected water samples, Ae. aegypti L. larvae were identified and separated from any other types of larvae (if present) on the basis of their morphological characters by following the standard keys[9].

Collection of kinnow peels

Fresh kinnow peels were collected from various local fruit vendors of Ludhiana city from February to April 2020 and January to April 2021. The collected peels were cleaned, separated from the pulp, leaves and stem of the fruit and kept for shade drying. Infected peels were discarded.

Extraction of kinnow peel oil

The collected cleaned fresh peels of kinnow fruit were cut into small pieces and spread on newspapers to wither for about 2–3 days at room temperature under shade. The newspapers were replaced daily to avoid any bacterial/ fungal growth. After slight withering, 250 g of withered kinnow peels were placed in round bottom flask with 5 L capacity and fitted in Clevenger apparatus for extraction of oil by hydro-distillation technique. The flask was filled up-to half with water and allowed to heat over a heating mantle for about 4–5 h at 60°C. The oil vapours released by the heating process got condensed with the cold water steam and collected in the recuperating channel. The extracted volume (ml) of oil was carefully collected in a clean glass vial and stored at 4°C. The oil content/yield (%) of extracted kinnow peel oil was calculated by the following formula:

[INLINE:1]

GC-MS analysis

The composition of extracted fresh kinnow peel oil was determined by Gas Chromatography-Mass Spectrometry analysis at Central University of Punjab, Bath-inda by GC program GCMS-QP2010 Ultra, using helium as carrier gas flowing at a rate of 1 ml/min, with split ratio 10:0, injection temperature 250°C and oven temperature programming from 40 to 250°C. GC was equipped with a capillary column Rtx-5MS (30 mtr). The volume injected was 1μl of the oil which was diluted in 1 ml of methanol.

Dose response bioassay for larvicidal potential of kinnow peel oil against Ae. aegypti

Preliminary testing of kinnow peel oil derived by hydro-distillation technique was carried out against 4th instar Ae. aegypti larvae by random selection of higher and lower concentrations of kinnow peel oil. On the basis of preliminary screening, five different concentrations of kinnow peel oil at 40, 50, 60, 70 and 80 ppm were prepared by mixing the required volume of kinnow peel oil in 1 ml of a non-polar and non-toxic emulsifying agent dimethyl sulphoxide (DMSO) and total volume was made upto 250 ml with de-chlorinated water. De-chlorinated water was prepared by keeping the tap water in open buckets for 24 h. Twenty 4th instar Ae. aegypti larvae were exposed to the above prepared five different concentrations of kinnow peel oil in plastic beakers of 250 ml capacity covered with muslin cloth using a rubber band. A control set (having 250 ml of de-chlorinated water) and a vehicle-control set (having 1 ml DMSO and 249 ml of de-chlorinated water) containing twenty 4th instar larvae in each beaker were also run simultaneously. All experimental sets were run in triplicate and larvae were fed adequately with dog biscuits and yeast ground in ratio 3:1 (2mg/100gm) till their transformation to next non-feeding pupal stage. Larval feed was provided after every 2–3 days, when the earlier feed provided was consumed by larvae. Larval mortality after 3, 6, 12, 24, 36 and 48 h of treatment was recorded in oil treated, control and vehicle-control sets. The larvae were considered dead if they were unable to respond or move when stimulated using a brush and the dead larvae found in each set were counted. Out of the tested concentrations, the minimum concentration of the oil showing maximum mortality within lesser time duration was considered as the effective concentration for its usage in further experiments. For calculating LC50 and LC90 after 24 hours of post-exposure, the log concentration-mortality regression was worked out by log probit technique[10] employing the computer programme POLO[11].

Residual activity effect of kinnow peel oil

For determining the residual activity effect of kinnow peel oil treatment on the larval mortality, fresh twenty 4th instar Ae. aegypti larvae were kept in the leftover tested solution of effective concentration of kinnow peel oil (assessed during larvicidal bioassay) after removing all the dead larvae from the beakers. After 3, 6, 9, 12, 24, 36 and 48 h, dead larvae (if any) were counted and replenished with the same number of the new larvae so as to have total number of larvae as twenty. Control and vehicle-control sets were also kept simultaneously along with treatment trials in triplicate.

In another experimental trial, fresh twenty 4th instar Ae. aegypti larvae were introduced in properly covered beakers having the leftover tested solution of the effective concentration of kinnow peel oil and were further monitored to test its residual activity effect (after removing all the dead larvae of the larvicidal bioassay) on their development from 4th instar to adult emergence. When larvae got transformed into pupae, the muslin cloth cover was removed from the beakers and then these beakers were placed in mosquito rearing cages. Emerged adults were fed on sugary juice of deseeded water-soaked raisins kept in a sterilized petri plate already placed inside mosquito rearing cages. A moist cotton swab was placed on the top of each cage to provide them water. After the completion of experiments, adult mosquitoes were killed by keeping chloroform dipped cotton swabs inside the mosquito rearing cages. Control and vehicle-control sets were also kept simultaneously along with treatment trials in triplicate. The time taken for each transformation (i.e., from 4th larval instar to pupa and pupa to adult formation) was recorded along with recording of adult emergence (%) in all sets.

Effect of storage on larvicidal potential of kinnow peel oil

Extracted kinnow peel oil was stored in clean vials covered with aluminum foil at 4°C in the refrigerator for 2, 4 and 6 months. Freshly prepared and stored (2, 4 and 6 months old) kinnow peel oils were tested again (at already determined effective concentration) to determine the effect of storage on the larvicidal potential against Ae. aegypti by following the same procedure as mentioned during dose response bioassay. Mortality of larvae after 3, 6, 12, 24, 36 and 48 h of treatment with stored kinnow peel and freshly extracted oil was recorded.

Statistical analysis

Data obtained was statistically analyzed by comparing the larval mortality (during larvicidal bioassay) and developmental duration (during residual activity effect determination experiments) recorded in kinnow peel oil treated sets with control and vehicle-control sets by one way analysis of variance (Duncan multiple range test). The larvicidal potential data of stored kinnow peel oil treated sets were statistically analyzed by comparing it with that of fresh oil by one way analysis of variance (Duncan multiple range test).

Ethical statement: Not applicable

 RESULTS



General characteristics and GC-MS analysis of kinnow peel oil

Oil extracted from the fresh peels of kinnow fruit by hydro-distillation technique was transparentin colourwith citrus like odour, insoluble in water and soluble in organic solvents and an average oil content/yield of 0.748±0.17% (v/w) was obtained. The chromatogram obtained by GC-MS analysis of kinnow peel oil showed 81 peaks indicating the presence of 81 compounds in the prepared kinnow peel oil [Figure 1]. Based on the percent area, limonene was the most abundant compound (64.82%) and ten other major constituents of the oil with >1% area were elemol (8.28%), β-citronellal (4.34%), geraniol (3.58%), viridifloral (2%), N, N-dimethylacetamide (DMA) (1.91%), β-elemene (1.64%), β-citronellol (1.52%), β-myrcene (1.34%) and germacra-1,4,5-triene (1.07%) [Table 1].{Table 1}{Figure 1}

Dose response larvicidal bioassay of kinnow peel oil against Ae. aegypti

Exposure of 4th instar Áe, aegypti larvae to 40 ppm concentration of kinnow peel oil resulted in 6.67±2.88 per cent larval mortality after 3 h and this mortality was found to increase with exposure time and after 12 h 23.33±2.88 percent mortality was observed. However, after 12 h no further larval mortality was found. Similar mortality trend was observed after treatment with 50 and 60 ppm ofkinnow peel oil. But when the larvae were exposed to 70 ppm oil, 100 per cent mortality was observed after 9 h and further exposure of larvae to 80 ppm resulted in 100 percent larval mortality within 3 h [Table 2]. Percent mortality values recorded in all the treated sets were found to be statistically higher as compared to that of control and vehicle-control sets as no larval mortality was found in these sets. During the present study, 70 ppm was found to be the effective larvicidal concentration as it resulted in maximum larval mortality (100%) within lesser duration of treatment (9 h) and with minimum concentration (70 ppm) in comparison to that of all the tested concentrations [Figure 2]. Various toxicity values i.e., LC50 and LC90 of kinnow peel oil computed for 4th instar Ae. aegypti larvae based on record of mortality till 24 hours after exposure to kinnow peel oil worked out to be 47.26 and 61.56 ppm against Ae. aegypti larvae, respectively [Table 3].{Figure 2}{Table 2}{Table 3}

Residual activity effect of kinnow peel oil

When fresh 4th instar Ae. aegypti larvae were introduced in the leftover effective concentration (70 ppm) of kinnow peel oil (determined during larvicidal bioassay in [Table 2]), no larval mortality was observed after 3, 6, 9, 12, 24, 36 and 48 h in any of the triplicate sets. This revealed no residual activity effect of kinnow peel oil due to its highly volatile nature. However, the exposure of leftover effective concentration of kinnow peel oil resulted in significant delay in development of freshly introduced larvae from their stage L4 to pupa, as it took longer duration i.e., 4.83±0.28 days in treated set in comparison to control and vehicle-control sets where average time reported was 3.50±0.50 and 3.33±0.57 days respectively [Table 4]. But the transformation of pupa to adult stage took statistically similar time in treated, control and vehicle-control sets. Overall development from L4 stage till adult formation in treated set was found to take statistically longer duration i.e., 6.67±0.28 days as compared to control and vehicle-control sets. However, there was no effect of kinnow peel oil treatment on the adult emergence, as 100% adult emergence was recorded in all the sets [Table 4].{Table 4}

Effect of storage on larvicidal potential of kinnow peel oil

When effective concentration (70 ppm) of two and four months stored kinnow peel oils was tested for the larvicidal potential, 100.00±0.00 per cent mortality after 9 h was observed, which was statistically similar with that of freshly prepared kinnow peel oil. Storage of oil for six months resulted in 91.67±5.77 per cent larval mortality after 12 h which was also statistically similar to that of freshly prepared one [Table 5], indicating no effect of storage on the larvicidal potential of kinnow peel oil.{Table 5}

 DISCUSSION



Citrus species are remarkably known for their economic importance as these plants exhibit a wide range of biological activities including insecticidal and mosquitocidal, and especially as larvicidal property due to which they arrest the metabolic activities of larvae[7],[12]. Peel extracts of C. sinensis and C. grandis have been analyzed and found advantageous in the control of Ae. aegypti larvae[13]. Decoction of C. aurantifolia leaves exhibited larvicidal activity against larvae of Ae. aegypti[14]. Byproducts of citrus plants are an important source of different bioactive compounds like saponin, terpenoid, alkaloid flavonoid, and cardiac glycoside. Out of these, limonene is found in higher amounts and considered as a naturally occurring mosquito larvicidal agent[8],[15],[16]. In the present study, GC-MS analysis showed limonene as the most abundant compound (64.82%) in the prepared kin-now peel oil along with other chemical compounds [Table 1] which were responsible for the morphological, physiological and biochemical alterations in Ae. aegypti larvae resulting in their mortality[16]. In citrus, two major components responsible for the larvicidal action: monoterpene limonene and β-myrcene[12]. In the extracted kinnow peel oil, β-myrcene was also found to be present, though in small quantity i.e., 1.34% [Table 1]. Earlier it has been reported that limonene is a cyclic monoterpene found in abundance i.e., 80.57%, while β-myrcene, a noncyclic monoterpene, which constitutes about 5.59% of the essential oil in the fruit rind of C. sinensis[15]. In C. limon peel essential oil, limonene (55.40%) and neral (10.39%) have been identified as major compounds followed by trans-verbenol (6.43%) and decanal (3.25%)[16]. These chemical compounds especially terpenes and a smaller quantity of sesquiterpenes in the citrus oils/extracts serve as carriers for a larger class of oxygenated compounds that are usually the bearers of the oil’s distinctive odour[12] which provides the peculiar citrus like odour to extracted kinnow peel oil along with larvicidal action[16].

Kinnow peel oil was found to have a highly volatile nature which was clear from the larvicidal bioassay test, as during treatment trials with its lower concentrations (40 to 60 ppm) no larval mortality was seen after the initial 12 h of treatment [Table 2]. Also, no residual activity effect in terms of larval mortality was observed in the leftover effective concentration of kinnow peel oil (70 ppm). This might be due to its high volatility and quick degradability which is a big limitation of kin-now peel oil. To overcome this volatility problem and to enhance its residual activity time, proper formulation is needed to be prepared either by blending it with appropriate fixatives or combining with other stable plant based oils or following some advance techniques like synthesizing its nanoemulsions or microencapsulation. Literature also shows that different plant-based extracts/oils show different residual activity time for different plant-based products. Like the residual activity effect of essential oil extracted from Chenopdium ambrosioides may last only a few hours[17], while in case of neemarin (a neem product), the residual toxicity persisted up to 7 days against Anopheles stephensi and Culex quinquefasoiatus larvae[18]. The decrease in the larval mortality rate with the increase in the post-treatment time could be due to the degradation of active compounds of plant based extracts by light exposure[19]. During the present study, overall development (L4 to adult) was found to take statistically longer duration in the leftover effective concentration of kinnow peel oil [Table 4], revealing its residual activity effect in terms of delaying the developmental period. Plant-based products show growth inhibiting effects onthe various developmental stages of different mosquito species and out of these a range of pre-emergent effects can occur like prolongation of instar and pupae duration, inhibition of larval and pupal molting, morphological abnormalities and mortality due to toxicity during the developmental phases. Delayed development after treatment of Ae. aegypti larvae with a non-lethal concentration of Adenanthera pavonina have been reported, the reason for which may be improper functioning of proteolysis in the midgut of the larvae[20]. Treatment with seaweed extracts resulted in inhibiting the development of Ae. aegypti larvae along with histological alterations and morphological aberrations[21]. Recent studies have shown larval delay in Ae. aegypti when exposed to sublethal concentrations of curcumin/d-mannitol[22] and Euoalyptus oil nanoemulsion[23].

During the present study it was observed that the larvicidal potential of stored kinnow peel oil remains statistically similar in comparison to that of the freshly prepared oil extract and it remained unaffected even after storage for 6 months [Table 5]. This is because of their proper storage in cold and dark conditions in closed vials which effectively prevented autoxidation[24]. Larvicidal properties of the essential oils against Ae. aegypti have been observed to remain unchanged even for upto three years of storage[25]. Monitoring the chemical properties of essential oils for 150 days under appropriate storage conditions revealed a slight variation in these indices when the oil was kept away from light and low temperature[26]. In another study it was confirmed that oils extracted from plants are easily degraded when stored at room temperature and to a lesser degree for those kept in the dark[27]. Thus, these oils are stable at low temperatures if they are properly stored to prevent oxidation and polymerization caused by air and light.

 CONCLUSION



The study concluded that kinnow peel oil at 70 ppm was found to have efficient larvicidal potential along with its residual activity effect in terms of delaying the development of Ae. aegypti. No effect of storage (even up to six months) on the larvicidal activity of kinnow peel oil was observed. The present study highlighted the importance of kinnow peel (generally treated as waste) as an important source of different bioactive compounds having larvicidal properties, thus can be exploited in future for managing the problem of dengue transmitting vector, Ae. aegypti. However, the problem ofhighly volatile nature of the kinnow peel oil needs to be resolved by adding some stabilizer/fixative for the preparation of any formulation in future.

Conflict of interest: None

 Acknowledgements



The authors are thankful to the Head, Department of Zoology, Punjab Agriculture University, for providing all the necessary facilities and Department of Science and Technology, Government of India, New Delhi, for providing infrastructural facilities under FIST grant.

References

1World Health Organisation 2019. Dengue and severe dengue. Available from: https://www.who.int/news-room/faot-sheets/detail/dengue-and-severe-dengue. (Accessed on January 01, 2020)
2Kaur K, Jamwal S, Kocher DK. Monitoring of mosquito larvae from temporary water collections of Ludhiana, Punjab. Indian J Eool 2016; 43: 357–8.
3National Vector Borne Disease Control Programme Dengue situation in India. 2021. Available from: https://nvbdop.gov.in/index4.php?lang=1&level=0&linkid=43J&lid=371. (Accessed on January 01, 2020)
4Thatheyus AJ, Selvam ADG. Synthetic pyrethroids: toxicity and biodegradation. Appl Ecol Environ Sci 2013; 1: 33–6.
5Verle P, Lieu TTT, Kongs A, Stuyft VP, Coosemans M. Control of malaria vectors: cost analysis in a province of northern Vietnam. Trop Med Int Health 1999; 4: 139–45.
6Piplani M, Bhagwat DP, Singhvi G, Sankaranarayanan M, Balana-Fouce R, Vats T, et al. Plant-based larvicidal agents: an overview from 2000 to 2018. Experim Parasitol 2019; 199: 92–103.
7Malar SM, Jamil M, Hashim N, Kiong LS, Jaal Z. Toxicity of white flesh Citrus grandis Osbeck fruit peel extracts against Aedes aegypti (Linnaeus) larvae and its effect on non-target organisms. Int J Mosq Res 2017; 4(4): 49–57.
8Yaqoob M, Aggarwal P, Aslam R, Rehal J. Extraction of bioactives from citrus. In: Inamudin, Asiri AM, Isloor AM, editors. Green sustainable process for chemical and environmental engineering and science. Elsevier, Amsterdam 2020; p. 357–77.
9Becker N, Petriae D, Zgomba M, Boase C, Madon M, Kaiser A, Mosquitoes and their control, 2nd edition. Springer Publication 2010; p. 9–40.
10Finney DJ. Probit analysis, 3rd edition. Cambridge: Cambridge University Press 1971; p. 68–72.
11Robertson JL, Russel RM, Savin NE. POLO: A user’s guide to probit or logit analysis. Pacific South-West Forest and Range Experiment Station, Berkeley, USA 1980.
12Darjazi BB. The effect of seasonal variation on sour orange (Citrus aurantifolia) leaf components. Int J Agrie Biosci 2014; 3(4): 161–5.
13Amusan AA, Idowu AB, Arowolo FS. Comparative toxicity effect of bush tea leaves (Hyptis suaveolens) and orange peel (Citrus sinensis) oil extract on larvae of the yellow fever mosquito Aedes aegypti. Tanzan J Health Res 2005; 7(3): 174–8.
14Muniandy PD, Riswari SF, Ruchiatan K. Larvicidal activity of Citrus aurantifolia decoction against Aedes aegypti Larvae. Althea Med J 2020; 7(1): 35–9.
15Theochari I, Giatropoulos A, Papadimitriou V, Karras V, Balatsos G, Papachristos D, et al. Physicochemical characteristics of four limonene-based nanoemulsions and their larvicidal properties against two mosquito species, Aedes albopietus and Culex pipiens molestus. Insects 2020; 11(11): 740.
16Paw M, Begum T, Gogoi R, Pandey SK, Lal M. Chemical composition of Citrus limon L. burmf peel essential oil from North East India. J Essent Oil Bearing Plants 2020; 23(2): 337–44.
17Chiasson H, Bostanian NJ, Vincent C. Acaricidal properties of a Chenopodium-based botanical. J Eeon Entomol 2004; 97(4): 1378–83.
18Vatandoost H, Vaziri VM. Larvicidal activity of a neem tree extract [Neemarin] against mosquito larvae in the Islamic Republic of Iran. East Mediterr Health J 2004; 10(4-5): 573–81.
19Thiagaletchumi M, Zuharah WF, Rami RA, Fadzly N, Dieng H, Ahmad AH, et al. Assessment of residual bio-efficacy and persistence of Ipomoea eairica plant extract against Culex quinquefasciatus Say mosquito. Trop Biomed 2014; 31(3): 466–76.
20Sasaki DY, Jacobowski AC, de Souza AP, Cardoso MH, Franco OL, Macedo ML. Effects of proteinase inhibitor from Adenanthera pavonina seeds on short-and long term larval development of Aedes aegypti. Biochimie 2015; 112: 172–86.
21Yu KX, Wong CL, Ahmad R, Jantan I. Larvicidal activity, inhibition effect on development, histopathological alteration and morphological aberration induced by seaweed extracts in Aedes aegypti (Diptera: Culicidae). Asian Pac J Trop Med 2015; 8(12): 1006–12.
22Mezzacappo NF, de Souza LM, Inada NM, Dias LD, Garbuio M, Venturini FP, et al. Curcumin/d-mannitol as photolarvicide: induced delay in larval development time, changes in sex ratio and reduced longevity of Aedes aegypti. Pest Manag Sci 2021; 77(5): 2530–8.
23Kaur N, Kocher DK. Effect of nanoemulsified Eucalyptus globulus oil on development, emergence and survival of Aedes aegypti L. Indian J Entomol 2021; e20366.
24Karlberg AT, Shao LP, Nilsson U, Gäfvert E, Nilsson JLG. Hydroperoxides in oxidized d-limonene identified as potent contact allergens. Arch Dermatol 1994; 286(2): 97–103.
25Santos GKN, Dutra KA, Lira CS, Lima BN, Napoleão TH, Paiva PMG, et al. Effects of Croton rhamnifolioides essential oil on Aedes aegypti oviposition, larval toxicity and trypsin activity. Molecules 2014; 19: 16573–87.
26Haddouchi F, Chaouche T, Lazouni HA, Benmansour A. Physicochemical study essential oils of Thymus fontanesii according to its conservation. Der Pharm Chem 2011; 3: 404–10.
27Pibiri M C, Goel A, Vahekeni N, Roulet CA. Indoor air purification and ventilation systems sanitation with essential oils. Int J Aromather 2006; 16(3-4): 149–53.