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BASIC RESEARCH
Year : 2018  |  Volume : 8  |  Issue : 4  |  Page : 217-225

Efficacies of four plant essential oils as larvicide, pupicide and oviposition deterrent agents against dengue fever mosquito, Aedes aegypti Linn. (Diptera: Culicidae)


Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang, Bangkok, Thailand

Date of Submission10-Feb-2018
Date of Decision25-Mar-2018
Date of Acceptance15-Apr-2018
Date of Web Publication30-Apr-2018

Correspondence Address:
Aksorn Chantawee
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang, Bangkok
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2221-1691.231284

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  Abstract 


Objective: To evaluate larvicidal, pupicidal and oviposition deterrent activities of four plant essential oils from Alpinia galanga (L.) Willd rhizome, Anethum graveolens L. (An. graveolens) fruit, Foeniculum vulgare Mill. fruit, and Pimpinella anisum L. fruit against Aedes aegypti (Ae. aegypti). Methods: Four essential oils at 1%, 5% and 10% concentrations were assessed for insecticidal activity against larvae and pupae of Ae. aegypti, following the procedure of a dipping method assay. Oviposition deterrent activity of four essential oils was evaluated on gravid female of Ae. aegypti by a dual-choice oviposition bioassay. Results: The results revealed that An. graveolens oil provided the strongest larvicidal activity against Ae. aegypti among four tested plant essential oils with the highest mortality rate of 100% and LC50 value of -0.3%. From the pupicidal experiment, An. graveolens also showed the highest toxicity against Ae. aegypti pupae with the highest mortality rate of 100% at 72 h and LC50 value of 2.9%. In addition, 10% An. graveolens had an oviposition deterrent effect against Ae. aegypti with effective repellency of 100% and an oviposition activity index of –1.0. Conclusions: An. graveolens oil has a good potential as a larvicidal, pupicidal and oviposition deterrent agent for controlling Ae. aegypti.

Keywords: Aedes aegypti Plant essential oils Larvicide Pupicide Oviposition deterrent


How to cite this article:
Chantawee A, Soonwera M. Efficacies of four plant essential oils as larvicide, pupicide and oviposition deterrent agents against dengue fever mosquito, Aedes aegypti Linn. (Diptera: Culicidae). Asian Pac J Trop Biomed 2018;8:217-25

How to cite this URL:
Chantawee A, Soonwera M. Efficacies of four plant essential oils as larvicide, pupicide and oviposition deterrent agents against dengue fever mosquito, Aedes aegypti Linn. (Diptera: Culicidae). Asian Pac J Trop Biomed [serial online] 2018 [cited 2018 Oct 18];8:217-25. Available from: http://www.apjtb.org/text.asp?2018/8/4/217/231284

Foundation project: This study was sponsored in part by the National Research Council of Thailand, (Grant no. GRAD6006 KMITL) and by the Faculty of Agricultural Technology, King Mongkut′s Institute of Technology Ladkrabang (KMITL) (Grant no. 01-04-001).





  1. Introduction Top


Mosquitoes (Culicidae: Diptera) are a serious worldwide threat for humans and animals. They are vectors of many serious pathogens and parasites including dengue, Zika virus, malaria and filariasis[1]. Aedes aegypti (L.) (Ae. aegypti) that inhabits the tropical and subtropical zones carries arbovirus, and is generally known to be a vector of dengue and chikungunya. Ae. aegypti females are anthropophilic, humans are their preferred hosts and thus at risk of being attacked by them[2],[3]. Studies have suggested that most females of Ae. aegypti may spend all of their lives in or around the houses that they have emerged as an adult. Ae. aegypti transmits dengue virus to susceptible humans[4]. It has been estimated recently that 3.9 billions of people in 128 countries are at risk of acquiring dengue and 390 million dengue infections occur every year, of which 294 millions clinically manifest the symptoms[5]. Severe dengue is a relatively rare but serious complication of dengue infection is manifested as plasma leaking, fluid accumulation, respiratory distress, severe bleeding or organ impairment[4],[6]. Therefore, it is of global public health concern to be able to control mosquitoes effectively[7]. Most mosquito control programs aim to control the larvae and pupae with larvicides and pupicides because adulticides may work well only for a temporary period[8],[9],[10].

Larviciding and pupiciding are common methods for reducing mosquito population and preventing dengue and chikungunya diseases. Larvicidal activity is very important in vector management because larvae that are in the growth stage are the easiest to kill. In particular, larvae control usually depends on extended application of organophosphates or other growth regulators such as diflubenzuron and methoprene[11]. Temephos is one of an organophosphate registered and produced commercially that has been extensively used for controlling Ae. aegypti larvae[12].

Today, synthetic chemical insecticides used for controlling mosquito vectors are being seriously questioned because of the irreversible damages they cause to the ecosystem and the various patterns of their mosquito resistance. In recent years, it has been suggested that plant essential oils (EOs) and their constituents can be good alternative larvicidal and pupicidal agents for mosquito control, mainly because their bioactive chemicals usually cause only inconsequential side effects on other organisms and the agricultural environment[12],[13]. EOs from Apiaceae and Zingiberaceae plants have been reported to have potent repellent, larvicidal, pupicidal and adulticidal activities against Culex pipiens, Culex quinquefasciatus, Ae. aegypti, and Anopheles stephensi[14],[15],[16],[17],[18].

In the present study, larvicidal and pupicidal activities of EOs from three Apiaceae species, namely, Anethum graveolens L. (An. graveolens), Foeniculum vulgare Mill. (F. vulgare), and Pimpinella anisum L. (P. anisum) as well as from a Zingiberaceae species, Alpinia galanga (L.) Willd (Al. galanga), were examined against Ae. aegypti (Linn.).


  2. Materials and methods Top


2.1. Plant materials

Plant materials from fresh rhizomes of Al. galanga, dried fruits of An. graveolens, dried fruits of F. vulgare, and dried fruits of P. anisum were investigated in this study. The plants were identified positively by a herbal taxonomist at the Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang (KMITL), Thailand. The rhizomes and fruits were cut into small pieces and distilled with water to obtain the EOs[10]. The plant materials were added with water at a ratio of 1:2 (plant:water) and placed in a distillation column connected to a round-bottomed distillation flask[10]. The flask was heated to about 100 °C and the distillation process began. It was stopped after 6 h. The EOs were dried with anhydrous sodium sulfate[10] and kept in a refrigerator at 4 °C until further use. Each EO was diluted to 1%, 5% and 10% in ethyl alcohol and kept in an airtight bottle at 4 °C for later uses[15].

2.2. Mosquito rearing

A number of Ae. aegypti (L.) were raised by the Department of Plant Production Technology, Faculty of Agricultural Technology, KMITL, Bangkok. The laboratory colony was kept under the following conditions: (29.5±2.0) °C with (75.5±2.0) relative humidity, and a photoperiod of 12-h light and 12-h dark (12L:12D). Eggs were hatched in plastic boxes (18 cm × 28 cm × 10 cm in size), each containing 1 500 mL of tap water. The larvae were fed with fish food pellets (HIPRO®) until pupation occurred. Pupae were collected and transferred to an insect cage (30 cm × 30 cm × 30 cm) and adult mosquitoes were provided with 5% glucose on cotton wool. On day 5, blood meals were given to the female adults following an artificial membrane feeding method. For egg collection, after the females were fed with blood meals for 2-3 d and ready to spawn, moist filter papers were placed on the surface of the water in a cup where they could lay eggs on. For 7 d, the eggs were kept wet and then put on a pan to hatch. The early 4th instar larvae and pupal stages of Ae. aegypti were tested in this experiment.

2.3. Larvicidal and pupicidal bioassay

Larvicidal activity and pupicidal activity were each determined according to a test dipping assay by Soonwera and Phasomkusolsil[10]. For each experimental treatment, 1 mL of a plant essential oil solution was added to 99 mL of distilled water in a 200 mL glass cup and shook lightly to ensure homogeneity. Ten Ae. aegypti in an immature stage (early fourth instar larvae or pupae stage) were put into the glass cups containing 100 mL of EOs in prepared water mentioned above. For ten larvae susceptibility test, all larvae were exposed to EOs until pupation, and mortality was observed for 24 h[10]. For ten pupae susceptibility test, the pupae were exposed to EOs until some were grown into adults, and mortality was observed for 72 h. Thirty replicates for each concentration of essential oil were performed. For comparison, a commercial formulation of temephos was used as a positive control and ethyl alcohol served as a negative control. During the period of the experiment, the larvae were offered no food[10]. They were considered dead if at the end of a 24-h period, they did not swim or move even after getting prodded by a rod. The dead and moribund larvae that showed sluggishness or abnormal movement were recorded after 24 h. Also, the pupae were recorded at 72 h and considered dead if they did not swim or move even after getting prodded by a rod[10],[19].

2.4. Morphological aberrations observed

A stereomicroscope was used to determine and categorize the morphological aberrations of the dead Ae. aegypti, and a method described by Soonwera and Phasomkusolsil was used to categorize dead specimens[10].

Normal larvae (NL): This group represented the larvae that died after reaching the pre-pupal stage of development.

Deformed larvae (DL): This group represented the larvae that died abnormally. Dorsal splitting (arrow) of thoracic cuticle was observed in dying and dead larvae.

Pre-pupae and pupa that did not completely shed off its exoskeleton (PP): This group represented the larvae that died before they came out of their exoskeleton. Some specimens died when their heads were still enclosed in their exoskeleton.

White pupa (WP): This group represented the pupae that came out of their larval exoskeleton completely. The white cuticle made it known as “albino”.

Deformed pupae (DP): This group represented the pupae that died abnormally. In some cases, the dead pupa had an appearance of a tiny elephant and was designated “elephantoid”.

Dead normal brown pupae (BP): pupae in this group were brown and normal in appearance.

Adults attached to pupal case (PA): This group represented the adults that died when they were emerging from their exoskeleton; For example, their tarsi, legs, wing and abdomen were still enclosed in their exoskeleton.

Normal adult (NA): This group represented the adults that emerged completely from their exoskeleton with normal appearance.

2.5. Oviposition deterrent assay

The oviposition deterrent activity was conducted in a laboratory using the method of Reegan et al[20] and a dual-choice oviposition bioassay was performed on gravid females of Ae. aegypti. Fifteen gravid females (5 days old) of Ae. aegypti were introduced into an insect cage (30 cm × 30 cm × 30 cm) under room conditions of (29.5±2.0) °C, (75.5 ±2.0) relative humidity and 12L:12D. The adults were provided with 5% glucose solution which was available at all time. Two 200-mL plastic cups for oviposition were filled with 99 mL distilled water, one for untreated cup and the other for treated cup. One milliliter of 1%, 5% and 10% of a plant essential oil solution and temephos was added to one cup to make up the preparation for a treatment, while the untreated cup was added with 1 mL ethyl alcohol. A support for oviposition was provided by placing a piece of filter paper (Whatman® No.1) on the inner surface of each plastic cup so that the lower half of it was submerged in the treated solution or untreated solution in order for the whole paper to get moistened while the upper half of it was above the solution where the mosquitoes would lay their eggs on. The untreated and treated cups were placed at alternate diagonally opposite locations for each replicate so as to nullify any effect of their locations on oviposition. After 3 d, the number of eggs laid in the treated and untreated cups were counted under a stereomicroscope[21].

2.6. Statistical analysis

The LT50 and LC50 values were calculated using probit analysis [10]. The mortality rates that were the results of using different EOs at different concentrations were statistically analyzed by Duncan's multiple range test to compare their different efficacies.



The oviposition activity index (OAI) was calculated using a formula used by Tikar et al[22]:



Where Ntreated was the number of eggs laid in the treated cups and Nuntreated was the number of eggs laid in the untreated cups. OAI was in the range of -1 and +1. Negative OAI values indicated that more eggs were laid in the untreated cup than in the treated cup and the treated solutions were a deterrent, whereas positive OAI values indicate that more eggs were laid in the treated cup than in the untreated cup, and that the treated solutions were attractive. Treatments of each concentration of EOs were replicated in six different cages. For oviposition deterrent assay, the percent effective repellency (ER) at each concentration was calculated by the following formula:

ER(%)=(Nuntreated-Ntreated)/Nuntreated × 100

Where Nuntreated was the number of eggs found in the untreated, and Ntreated was the number of eggs found in the treated.

The mean numbers of eggs deposited in the treated and untreated cups were statistically analyzed by a paired t-test and they were analyzed by a t-test and one-way analysis of variance with SPSS software (version 23.0).


  3. Results Top


3.1. Larvicidal and pupicidal activity

The outcomes of the larvicidal bioassay on the early fourth instars of Ae. aegypti treated with the four EOs and the statistical data of mortality, LT50 and LC50 were shown in [Table 1]. All EOs at 10% concentration showed more toxicity than those at 1% and 5% concentrations. After 24 hours of exposure, it was found that An. graveolens EO at 1% concentration produced the mortality rate against Ae. aegypti at 91%, while the oil achieved 100% mortality at 5% and 10% concentrations. The result showed that LT50 decreased with increased concentration. Both 5% and 10% concentrations of An. graveolens exhibited LT50 values of 0.8 against Ae. aegypti larvae. Among the four EOs tested, the oil from An. graveolens exhibited the strongest larvicidal effect with the lowest lethal concentration, LC50 value of -0.3% (Y=109.8×X+ 90.71, χ 2=77.2), followed by the essential oil from F. vulgare with LC50 of 0.5% (Y=95.9×X+91.89, χ2=155.9), P. anisum with LC50 of 0.6% (Y=362.3×X+69.34, χ2=173.0) and Al. galanga with LC50 of 7.8% (Y=931.9×X+11.63, χ 2=289.0). On the other hand, temephos at 1% concentration (positive control) showed 94.3% mortality at 24 h with LT50 value of 255.7 h. Larvicidal activity of four EOs showed a positive relationship between mortality rates and exposure periods which were significant [Figure 1]. The results of the pupicidal activity against Ae. aegypti pupae were shown in [Table 2]. An. graveolens oil at 10% concentration showed more toxicity than the oil at 1% but the same toxicity to the oil at 5%. At 1% concentration, no EOs produced any larvae mortality during the observation period. As for the results for 5% concentration, An. graveolens oil produced the highest mortality against Ae. aegypti pupae with 100% mortality at 72 h and LT50 value of 10.3 h, followed by F. vulgare with 94.0% mortality at 72 h and LT50 of 14.6 h. At 10% concentration, An. graveolens showed the highest pupicidal activity against Ae. aegypti pupae with 100% mortality at 72 h, LT50 of 6.7 h and LC50 of 2.9% (Y=1 066×X+9.836, χ 2=0.02), followed by the essential oil from F. vulgare with 99.7% mortality, LT50 of 7.5 h and LC50 of 3.5% (Y=1 062×X+7.769, χ 2=63.9), P. anisum with 98.3% mortality and LC50 of 3.84% (Y=1 052×X+7.67, χ 2=106.0) and Al. galanga with 92.0% mortality and LC50 of 6.3% (Y=1 028×X-12.71, χ 2=317.0). Pupicidal activity of four EOs showed a positive relationship between mortality rates and exposure periods which were significant [Figure 2]. The low value of LC50 for An. graveolens oil demonstrated its good larvicidal and pupicidal activity against Ae. aegypti. In contrast, temephos (positive control) showed only 4.8% mortality at 72 h with LT50 of 98.7 h. Not surprisingly, ethyl alcohol, the negative control, did not produce any mortality of pupae during the observation period. Therefore, cultivated in the negative control, all larvae were active and exhibited normal movement. Conversely, cultivated in the treatments, larvae were observed to have restless movements. After 1 h of treatment, all treated larvae started to have tremor and convulsion, and dead larvae started to settle to the bottom of the cup.
Figure 1:. Relationship of mortality rate and exposure periods of larvicidal activity of essential oils against Ae. aegypti larvae expressed as regression.

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Figure 2: Relationship of mortality rate and exposure periods of pupicidal activity of essential oils against Ae. aegypti pupae expressed as linear regression.

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Table 1: Effect of herbal EOs at three concentrations (1%, 5% and 10%) against 4th instar larvae of Ae. aegypti at 24 h.

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Table 2: Effect of herbal EOs at three concentrations (1%, 5% and 10%) against pupae of Ae. aegypti at 72 h.

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3.2. Morphological aberrations

The mortality and morphological aberrations of larvae of Ae. aegypti were observed after 24 h of exposure to 1%, 5% and 10% concentrations of EOs which were shown in [Table 3]. At these concentrations, all EOs caused morphological aberrations at the time of death of the larvae. The results showed the morphological aberrational changes of Ae. aegypti larvae from NL to DL and deformed PP. The death of larvae at the highest mortality was usually as NL. One percent concentration of Al. galanga, An. graveolens, F. vulgare and P. anisum oils caused no morphological change (NL) at 11.6%, 84.0%, 47.7% and 56.0% NL mortality rate [Figure 3]A. They caused some changes at the time of death at 1.0%, 5.3%, 39.0%, and 6.0% DL mortality rate [Figure 3]B,[Figure 3]C,[Figure 3]D. The absence of underlying epithelium in the dead larvae from EO treatment might indicate that lectin larvicidal activity was probably due to damage in the Ae. aegypti midgut. The deformations were such as damaged anal papillae, distorted body, darken body and shrunken cuticle. They also caused abnormal PP with deformed cephalothorax and posterior abdominal segment at 0.7%, 1.6%, 3.0% and 4.0% PP mortality rate [Figure 3]E. At the concentrations of 5% and 10%, all EOs caused the greatest NL mortality rate.
Figure 3: Morphological aberration of larvae Ae. aegypti after treatment with essential oils.
A: normal larvae of Ae. aegypti (NL); B-D: deformed larvae at death at the larval stage (DL); E: the abdomen of the pre-pupa did not come completely out of the larval exoskeleton (PP); F: a dead brown pupa (BP); G-I: deformed pupa (DP); J-K: a partially emerged, tarsi-deformed adult (PA); L: normal adult (NA).


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Table 3: The effect of four EOs on morphology and mortality of Ae. aegypti larvae.

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All of the EOs caused some morphological aberration in the specimens during pupation after 72 hours of exposure as shown in [Table 4]. The main characteristic of death from EOs was DP: some abnormal pupae died with enlarged cephalothorax and wing pads were not appressed to the body; their head and body also turned black [Figure 3]G,[Figure 3]H,[Figure 3]I. Ten percent concentration of Al. galanga, An. graveolens, F. vulgare and P. anisum caused major changes almost found at the time of death at 83.0%, 75.3%, 99.0% and 97.7% DP mortality rate, respectively. However, some pupae were found dead as normal BP, their cephalothorax and abdomen had normal brown color [Figure 3]F. Ten percent concentration of Al. galanga, An. graveolens and P. anisum oils caused no morphological change (BP) at 5.7%, 24.7% and 0.3% BP mortality rate. Some adults died while they were emerging (PA). Their tarsi, legs, wings, and abdomen were still attached to the pupal exoskeleton [Figure 3]J,[Figure 3]K. They also caused partially emerged, tarsi-deformed adult (PA) at 3.3% (Al. galanga), 0.7% (F. vulgare) and 0.3% (P. anisum) PA mortality rate.
Table 4: The effect of four EOs on morphology and mortality of Ae. aegypti pupae.

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3.3. Oviposition deterrent activity assay

The resulted obtained from the oviposition deterrent assay of four EOs at all three concentrations against Ae. aegypti were shown in [Table 5]. The results showed that all concentrations of all EOs were able to reduce the number of deposited eggs by gravid Ae. aegypti compared to the number of eggs from the gravid females treated with the ethyl alcohol. All EOs at three concentrations tested were observed to repel mosquitoes from oviposition and repellency of four EOs increased with the increase of concentration. The range of the mean number of eggs laid in the cups with the four EOs at three different concentrations 1%, 5% and 10% were 6.8-162.4. In addition, there was also a marked difference in the amount of the eggs laid. An. graveolens exhibited the most effective repellency activity against gravid female mosquitoes. The mean number of laid eggs in the cups with 1%, 5% and 10% of An. graveolens oil were 145.6, 22.6 and 6.8 eggs per cup, respectively, while the untreated cups gave a mean number of 392.0, 385.4 and 355.8 eggs per cup. A paired t-test confirmed that these results were significantly different (P<0.05). The percentage of ER caused by An. graveolens against oviposition were 62.9%, 94.1% and 98.1% for 1%, 5% and 10% concentrations, respectively. The range of OAI of An. graveolens at three concentrations was from -0.5 to -1.0. The results showed that gravid females Ae. aegypti preferred to lay eggs in the untreated cups rather than in the treated cups, thus it was demonstrated An. graveolens oil had potential to repel mosquito females for laying eggs. The present results indicated that the oviposition deterrent activity depended on concentrations as 10% An. graveolens oil exhibited the strongest deterrent effect. On the other hand, temephos provided a mean number of 249.8 eggs laid per cup, while the untreated cups gave a mean number of 319.8 eggs per cup. The OAI value of temephos was -0.1; there was no significant difference in the number of eggs laid in the treated and untreated cups in this case. Therefore, temephos showed a lowest oviposition deterrent activity against Ae. aegypti females than those of plant EOs.
Table 5: The effect of four EOs on oviposition deterrent activities against Ae. aegypti.

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  4. Discussion Top


EOs derived from plants have a good potential for controlling mosquitoes in their larval and pupal stages. In the present study, 10% An. graveolens oil recorded the highest larvicidal and pupicidal activities of 100% mortality rate against Ae. aegypti immature stages.

Anethum graveolens L., commonly known as dill, is a medicinal plant with tiny yellow flowers that belongs to the plant family Apiaceae[23]. Leaves, stems and fruits of dill are widely used in various applications in the food industry, especially for their unique taste and spicy aroma[24]. Extract of An. graveolens obtained from the seeds have antibacterial, antispasmodic, antioxidant, antimicrobial properties[25]. The EO of An. graveolens exhibited a larvicidal activity among many biological activities. EO of bulk dill (pure, not in a formulation) at different concentrations (10-100 ppm) was evaluated against Anopheles stephensi[26]. Meanwhile, larvicidal activity was observed at the concentration of 20 ppm and increased with increasing concentration of An. graveolens. Lethal concentrations at 50% and 90% of An. graveolens EO were found to be 38.8 and 65 ppm, respectively, against the 3rd and 4th instar larvae of Anopheles stephensi[27]. The EO of An. graveolens at a concentration of 0.1 mg/ mL has also shown a strong larvicidal activity against Asian tiger mosquito, Aedes albopictus (90% mortality)[28]. In another report, An. graveolens EO has also shown an effect against Culex pipiens adult (LC50 =0.495) and larvae (LC50 =16.996)[29]. Moreover, An. graveolens EO is also toxic larvicides to other insect pests. An. graveolens seed essential oil was found from continuous exposure and fumigant toxicity bioassays to be toxic to Periplanata americana L., Musca domestica L. and Tribolium castaneum. The mortality against Periplanata americana ranged from 25% to 100% during the first 3 h in a contact toxicity bioassay and during the first 12 h in a fumigant toxicity bioassay. In case of Musca domestica L., mortality ranged from 33.3% to 70.0% during the first 3 h and from 58.3% to 100.0% during the first 24 h for Tribolium castaneum[23]. In a previous report, the EO of An. graveolens was assessed for insecticidal activity against Callosobruchus maculates L. adults through a fumigant bioassay with LC50 value at 12.75 μ /L air[30]. In another study, the toxicity of An. graveolens (leaves) plant extract at 5 and 10 mg/ mL concentrations against 2-day-old (first instar) and 6-day-old (third instar) larvae of Spodoptera litura was investigated[31]. Many researchers have determined the chemical composition of essential oil of the fruits and seeds of An. graveolens which was extracted by steam distillation and hydrodistillation[32],[33]. The constituents were found to be carvone, limonene, α -phellandrene, dichloromethane, α -terpinene, p-cymene, α - and β -pinene, γ -terpinene, cumin aldehyde, neral, trans-anethole, thymol, carvacrol, myristicin, apiol, and carotol constituents. However, the EO extracted by steam distillation contained higher amounts of limonene and carvone than the oil extracted by hydrodistillation. From the literature, carvacrol, α -pinene, and β -pinene were found to inhibit the activity of Aedes albopictus acetylcholinesterase with LC50 values of 0.057, 0.062, and 0.190 mg/mL, respectively[28]. The insecticidal property of An. graveolens (that contains 59% phellandrene, its most abundant compound) has been evaluated against larvae of the 3rd and early 4th instars of Culex pipiens with LC50 value of 52.74 mg/ L[34]. Rocha et al.[35] reported morphological changes in the anal papillae of Ae. aegypti larvae after they were in contact with some EOs and the major chemical constituents of An. graveolens: (+)-limonene and (-)-limonene. After contact with these two compounds, an accumulation of dark pigmentation was observed all over the chest and at the base of the anal papillae. Structural damages to the larvae exposed to (-)-limonene include destruction of the gut and extrusion of hemolymphatic content etc, while damages to the larvae exposed to (+)-limonene were such as a darkening at the base of the anal papillae extended to the apex region. The present results confirm the previously reported results, revealing similar morphological changes such as changes in head and abdomen pigmentation, distorted body, darkened body and anal papillae and shrunk cuticle. The larvicidal activity of EOs may be according to diverse mechanisms. Mortality may occur at different development stages. Owing to contact effect, mode of action of EOs may act on digestive or neurological enzymes and interact with the insect's integument. Several studies have reported tremors and paralysis of larvae in their assays as well as dying larvae staying at the bottom of the containers[26],[36]. In another study, it was found that several EOs blocked the effects on chemosensory receptors at the mouth parts stimulated by glucose and inositol[37]. The EOs and their constituents disrupted the endocrinological balance of the insects. They induced neurotoxicity via various mechanisms hence disrupting the normal process of morphogenesis. The damages on the muscles caused by the oils might affect the larvae's movement for respiration or feeding and the adults' development and flying ability[38].

Using for modifying the oviposition behavior of mosquitoes, oviposition deterrents and attractants play an important role in mosquito control programs. Oviposition site selected by gravid females is a critical factor that determines the proliferation and population density of the species as well as its dispersion in different geographical areas[39],[40]. As female mosquitoes approach an oviposition site, they use a site-specific olfactory cue as a short-range signal for determining its quality. Volatile chemical emanated from an oviposition site is sensed and evaluated by the olfactory receptors located on the antennae, palps, labrum, and tarsi[41],[42]. It has been observed that gravid females of many species of mosquitoes preferred an oviposition site over some others. This preference may be due to the presence of oviposition pheromones or oviposition attractants or repellents at the site[15],[43]. In this study, An. graveolens oil exhibited the highest oviposition deterrent activity against female Ae. aegypti. The strongest activity was produced by the highest concentration of An. graveolens oil tested. It might produce the maximum effective repellency against oviposition by acting as a chemical signal that was detected by the sensory receptors on the antenna of the mosquitoes[44]. Warikoo et al.[45] have reported that pure An. graveolens oil deterred oviposition completely and boasted 75% effective repellency. The EO derived from dried fruits of An. graveolens exhibited a repellent activity against the adults of Ae. aegypti[46]. An. graveolens oil's insecticidal, oviposition deterrent, egg hatching and developmental inhibitory activities were determined against pulse beetle Callosobruchus chinensis[47]. Another study showed that An. graveolens oil reduced Tribolium castaneum's oviposition potential and lengthened its developmental period in comparison with the control group. The oviposition potential of Tribolium castaneum decreased significantly when it was fumigated with An. graveolens oil[48].

The ability of gravid mosquitoes to perceive the presence of organic acids and hence detect unsuitable oviposition sites might have been acquired through evolutionary adaptation. This ability helps mosquitoes to avoid ovipositioning in unsuitable breeding sites that contain harmful toxic compounds[43]. EOs affect mosquito's nervous system; it can affect adult mosquitoes' ability to find the right host to feed on or to find the right oviposition site[38] by possible interference with nerve impulse transmission to the brain, hence changing the mosquito' response to internal and external stimuli.

In Thailand, An. graveolens is a cultivated crop in the Northeastern region. Its aerial part (dill weed) is a popular seasoning agent[33]. An. graveolens fruits in Thailand have already been used as aromatic plants and spices for food preservation and in medicine, alternative medicine and natural therapy for a long time. In this study, An. graveolens oil showed strong larvicidal, pupicidal and oviposition deterrent effects, thus, the good potential of An. graveolens oil for controlling Ae. aegypti has been verified. An. graveolens oil can be used in places where mosquitoes usually breed for controlling larvae and pupae population and for preventing egg-laying by female Ae. aegypti adults. If the oviposition activity of Ae. aegypti is inhibited, its life cycle will be disrupted and its population growth will be reduced.

In conclusion, our results clearly show that An. graveolens oil can be applied as larvicide, pupicide and oviposition deterrent for controlling Ae. aegypti population, and warrant further studies for field application. We believe that this study has demonstrated the usefulness of An. graveolens insecticidal properties against Ae. aegypti. An. graveolens has a full potential to be used as an inexpensive, safe and efficient larvicide on its own as well as a supplement to other larvicides.

Conflict of interest statement

The authors confirm that they have no other conflicts of interest regarding the content of this article.

Acknowledgments

This study was sponsored in part by the National Research Council of Thailand (NRCT), (Grant no. GRAD6006 KMITL) and by the Faculty of Agricultural Technology, KMITL (Grant no. 01-04-001). The authors wish to extend our thanks to the plant taxonomist and entomologist of the Faculty of Agricultural Technology, KMITL, for their help in identifying the species of the four herbs and the mosquito. We also wish to express our gratitude to Mr. Pratana Kangsadal, the KMITL proofreader, for reviewing and giving comments on the manuscript.



 
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