Oviposition Deterrent Activity and Ovicidal Effect of Pometia pinnata Leaves Extract against Dengue Vector, Aedes aegypti (Diftera: Culicidae)

. Aedes aegypti is the main vector of dengue fever that most commonly occurred in Indonesia. The application of botanical insecticides is being developed as an alternative vector control. Botanical components have been reported as the potential alternative to inhibit the process of oviposition and fecundity suppression. This study aims to determine the oviposition deterrent activity and ovicidal effects of ethanol extract of Pometia pinnata leaves against Aedes aegypti. Oviposition deterrent assay was conducted on twenty blood-fed females in a cage (40 cm x 40 cm x 40 cm). One treated oviposition trap and one control oviposition trap were placed in the opposite corner of the cage. Four replicates were performed for each concentration. The eggs were counted and analyzed after 72 hours of trial. Ethanol extract of Pometia pinnata leaves have anti-oviposition effect on female mosquitoes Aedes aegypti at 400 ppm (effective repellency / ER = 56.38%; oviposition active index/OAI = -0.39) and 500 ppm (ER = 66.4%; OAI = -0.49). In the ovicidal assay, twenty-five eggs are used for determination ovicidal effect and five replicates were performed for each concentration. The hatched larvae were collected and counted daily. The ethanolic extract of Pometia pinnata leaves at 0.05%, 0,1%, 0.15%, 0.2%, and 0.25% concentration have ovicidal activity of 11%, 28%, 87%, 94%, and 98% against Aedes aegypti eggs, respectively. This study revealed that the ethanol extract of Pometia pinnata leaves could be a potential alternative in controlling the dengue vector.


Introduction
Aedes aegypti is the primary vector of dengue fever in tropical countries, such as Indonesia. Dengue hemorrhagic fever remains one of the most important diseases worldwide with 500.000 cases occurred and caused 22.000 people deaths. One of the strategies to reduce the number of dengue vectors is by inhibiting its life cycle [1]. The use of chemical insecticides at mosquitos breeding had been proven to reduce the mosquito populations, but leading to adverse environmental and non-target organism effects and cause resistance [2]. The outspread and evolution of resistance of insecticide is a big problem for the dengue vector controls [3]. The awareness that the use of chemical insecticides has negative impacts encourages research to look out for environment-friendly innovative strategies to target mosquitoes [2,4]. Phytochemicals from plant extract have been reported potential as alternative methods against vector mosquitoes due to their ability to control mosquitoes in various ways such as to cause growth inhibition, ovicidal activity, oviposition deterrence, and fecundity inhibition.
Pometia pinnata is a native plant in Indonesia and commonly called "Matoa" plants. Pometia pinnata leaves extracted with ethanol, N-hexane, and ethyl acetate contain several metabolites such as alkaloid, flavonoid, terpenoid, tannin, saponin, and coumarin [5]. The purpose of this study was to assess the oviposition deterrent and ovicidal activities of Pometia pinnata leaves extract against Aedes aegypti mosquitoes.

Collection of plant material
The matured of Pometia pinnata leaves were collected from surrounding area in Universitas Islam Indonesia, Sleman City, Indonesia. The healthy leaves were washed with tap water and dried using cabinet drying. The dried leaves were crushed and using a miller to get a fine powder.

Preparation of leaves extracts
Dried Pometia pinnata powder was extracted using ethanol 96% and allowed to soak for 5 days at room temperature. The supernatant was filtered. A rotary evaporator was used to separate the solvent from its extract and the green residue was obtained. The stock solution was used to prepare the various concentration of the extract.

Mosquito rearing
The mosquito and eggs used were obtained from the colony maintained at the Parasitology Laboratory of Medical Faculty, Universitas Islam Indonesia. The mosquito eggs were collected and used to asses ovicidal activity. The blood-fed female mosquitos were collected and used to asses oviposition deterrent activity.

Oviposition deterrent assay
For the oviposition deterrent assay, ethanol extact of Pometia pinnata was prepared at concentrations of 100 ppm, 200 ppm, 300 ppm, 400 ppm and 500 ppm based on a preliminary test. Five cages (40cm x 40cm x 40cm) weres also prepared and twenty blood-fed female of Ae.aegypti was transferred into each cage. One treated oviposition trap and one control oviposition trap were placed in the opposite corner of each cage. Four replicates were performed for each concentration. The eggs were counted and analyzed after 3 days of trial [1]. The identification of eggs was carried out using a light microscope with a magnification of 10x. The average numbers of eggs were calculated using a standard formula to assess Effective Repellency (ER) and Oviposition Activity Index (OAI) as follows Where NC is the number of eggs in the control trap and NT is the number of eggs in the treated trap.
The oviposition activity index with a result of +3 and above is considered as an attractant, while a result of -0,3 and below is considered as an anti-oviposition [6,7].

Ovicidal assay
The various concentrations of Pometia pinnata extracts were prepared using DMSO as the solvent. Twenty-five eggs of Ae. aegypti were soaked in five different concentrations (0.05%, 0.1%, 015%, 0.2%, and 0.25%). The distilled water was used as a negative control for comparison. Percentage of ovicidal effects after being treated with the ethanol extract of Pometia pinnata was calculated with the following formula:

100%
where A is the number of eggs did not hatch during the treatment and B is the total of eggs before the treatment

Results and Discussion
In oviposition deterrent assay, all of the female Ae. aegypti were put their eggs in each oviposition trap either containing Pometia pinnata leaves extract or containing only distilled water. The average numbers of mosquito eggs in the treated oviposition trap were less than the control oviposition trap (Table 1). This result showed that there was a disruption in the process of laying mosquito eggs in contact with Pometia pinnata leaves extracts. The oviposition is an important event in the life cycle of a mosquito. If this stage is inhibited, it will disrupt the life cycle of mosquitoes and will decrease the mosquito population [1,8].
Data are mean ± standard error (SE) of four replicates. ppm = parts per millions. Different means result with control are tested by Independent samples T-test (p <0.05, level of significance).
The lowest ER values at 100 ppm concentration was 21.71% and the highest at 500 ppm concentration was 66.4%. There is no statistically difference (p>0.05) in the number of eggs in treated oviposition trap with control. In another study showed that Vitex negundo L. extracts against Ae.aegypti, Anopheles stephensi and Culex quinquefasciatus obtained significant results with concentrations of 0.1%, 0.075%, 0.05%, 0.025% and 0.01% [9]. Ethanol extract of Pometia pinnata leaves have an anti-oviposition effect on female mosquitoes Aedes aegypti at 400 ppm (effective repellence/ER = 56.38%; oviposition active index/OAI = -0.39) and 500 ppm (ER  In this study, the ethanol extract of Pometia pinnata showed the highest ovicidal effects of 98% at 0.25% concentration against Ae.aegypti. When expose to the higher concentration of Pometia pinnata leaves extract, non-hatching eggs showed increase (Figure 1). Another investigator also conducted a study using plants extract to assess ovicidal activities. The crude extract of C.copticum showed zero hatchability (100% mortality) at 150 µm/ml for Culex pipiens [12]. Wariko and Kumar [2] reported that the petroleum ether and hexane root extract causing only 4.9% and 8.9% hatch respectively at highest concentration against Ae.aegypti.

Conclusion
In the present study, Pometia pinnata leaves extract show potential effects as ovicidal and anti-oviposition against Aedes aegypti mosquitoes. Based on these results, the ethanol extract of Pometia pinnata leaves could be a potential alternative for use in dengue vector control. The phytochemical compound of this extract should be investigated in the future. . .