Larvicidal and Pupicidal activity of Clerodendrum philippinum Schauer Leaf Extracts against Anopheles stephensi and Aedes aegypti

Objective: The purpose of this study is to investigate the larvicidal and pupicidal activity of Clerodendrum philippinum leaf extracts against disease causing vectors Anopheles stephensi and Aedes aegypti. Methods: Five different concentrations (200, 300, 400, 500 and 600 ppm) of leaves were prepared by using aqueous (distilled water), ethanol, methanol, n-hexane, chloroform and tested for both the activity. The LC50 and LC90 values of leaf extracts were determined with the help of probit analysis. Results: Among the different extracts of leaf screened, the ethanol extract of C. philippinum was recorded the highest larvicidal and pupicidal activity of 100 ± 1.9 % (1st instar) and 58 ± 0.8 % at 600 ppm concentration for controlling An. stephensi, respectively. Similarly for Ae. aegypti, 97 ± 1.2 (1st instar) and 51 ± 0.5 (pupa) percentage of inhibition were achieved for the same solvent extracts of the leaf which were maximum than others. Moreover, the values of LC50 and LC90 clearly indicate that the activity of both larvicidal and pupicidal are not only solvent extracts dependant but also depend on their concentration. Conclusion: The obtained data highlight the potential role of ethanolic extracts of C. philippinum for controlling An. stephensi and Ae. aegypti mosquitoes at their larval and/ pupal stages of development.


INTRODUCTION
8] In other hands, more than 70,000 and 18,000 cases of dengue and chikungunya, respectively are reported in India caused by Ae. aegypti. 9any control majors (environmental sanitation, epide miological, surveillance, laboratory and research support and education) are available to check against the vectors, those are responsible for spreading the different life threatening disease. 10Moreover, synthetic insecticides are applied to control the agents but have resistant and that might be due to repeated use of such products.It has also caused the environmental threat by destroying non-target organisms and raised adverse effects on both environmental quality and food chain. 4,11For which, Integrated Mosquito Management (IMM) emphasizes the application of alternative strategies to control the larva or pupa stages of mosquito.Under such conditions, plants or plant based products showed their effectiveness as controlling agents of mosquitoes which are safer from environment point of view and target specific.They possess rich source of secondary metabolites which having insecticidal activity (antibacterial, antifungal and larvicidal potential) and is possible due to presence of active phenols, alkaloids, terpenoids, coumarins, polysaccharides and flavonoids etc. [12][13] In fact, many studies have been conducted to find out the effectiveness of different plant extracts against larvicidal and pupicidal activity of mosquito, 4,12,[14][15][16][17]  Clerodendrum philippinum Schauer is an important medicinal plant, which belongs to the family Verbenaceae.9] It is a semi-woody perennial shrub found in Southern part of Asia and in India, abundantly available in various states like Karnataka, Kerala, Tamil Nadu and Odisha.It grows up to a height of 10 ft tall.Leaves are broad, up to 1 ft cm long and wide, margins toothed, somewhat lobed.5] Hence, the present study is undertaken to evaluate the larvicidal and pupicidal activity of leaf extracts as well as flower essential oil against two vectors, Anopheles stephensi and Aedes aegypti.

Collection and preparation of leaf extracts
Healthy leaves of C. philippinum were collected from the botanical garden of Post Graduate Department of Botany, Utkal University and were washed and air dried under the environmental temperature of 27°C-37°C.The dried leaves were powdered by using a mortar and pestle.The powdered sample was eluted using a Soxhlet apparatus with different solvents [aqueous, ethanol, methanol, n-hexane, chloroform) sequentially in a ratio of 1:10 for a period of 72 h each and filtered.The diluted extracts were concentrated (200, 300, 400, 500 and 600 ppm) at low temperature using a rotary evaporator and stored at -4°C for further analysis.

Collection of eggs and larval culture
The eggs of An. stephensi and Ae.aegypti were provided by Department of Entomology, Regional Medical Research Centre, Bhubaneswar, Odisha, India.The eggs were brought to the laboratory and kept in plastic and enamel tray containing normal tap water for hatching.The larvae were fed on a diet having fine a mixture of dog biscuits and dry yeast in a ratio of 3:1, till the pupal stage.

Maintenance of pupae and adults
Developed pupae were relocated in to separate plastic cup containing tap water and were kept under periodical check-up for emerging into adults.Larvae/ pupae were maintained throughout at 27 ± 2°C and 75-85 % of relative humidity with a photoperiod of 14 h/ 10 h light and dark cycles.After pupation, they were allowed inside a mosquito cage for becoming adults.Prior to blood feeding, 10 % sucrose solution was provided for 3 days and finally, adult female mosquitoes were maintained by providing a blood meal from rabbit (exposed in dorsal side).

Larvicidal and pupicidal activity test
The bioassay was done as per the standard method of World Health Organization by taking different solvent extracts of leaf. 26Five different concentrations (200, 300, 400, 500 and 600 ppm) of each extract were tested against freshly moulted first (1 st ), second (2 nd ), third (3 rd ) and fourth (4 th ) instar larvae and pupae of An. stephensi and Ae.aegypti.Twenty numbers of first to fourth instar larvae and pupae were introduced into 500 ml thermocol glass containing 249 ml dechlorinated water and 1 ml of desired concentrations of plant extract (dilution was done by dechlorinated water).At each tested concentration, three trials were made and each trial consisted of five replicates.The control was set up by providing 250 ml of dechlorinated water however the larval food was added to all tested and control cups.The larval mortality was observed and recorded after 24 h of post treatment.The mortalities were recorded by using Abbott's formula (Abbott, 1925).

Statistical analysis
Results of larval and pupal mortality were the mean of three independent experimental replicates (n = 5) and were subjected to probit analysis 27 for calculating LC 50 , LC 90 and other statistics at 95 % fiducial limits of upper fiducial limit (UFL) and lower fiducial limit (LFL) and chi-square values were calculated using the SPSS 16.0 version (software package).Values with P<0.05 were considered to be statistically significant.

Larvicidal and pupicidal activity against An. stephensi
Larvicidal and pupicidal mortality responses from various leaf extracts (aqueous (distilled water), ethanol, methanol, n-hexane, chloroform) of C. philippinum, against malaria causing vector An.stephensi are presented in Tables 1-5.The different concentrations (200, 300, 400, 500 and 600 ppm) of each extract were tested against 1 st , 2 nd , 3 rd , 4 th instar and pupal stages of An. stephensi.Increasing trends of mortality were found against both larval and pupal stages of An. stephensi with raising concentrations (200 to 600 ppm) of each leaf extracts.A significant mortality was noticed in all applied solvent systems however, best mortality was observed in ethanolic extracts.At 600 ppm, the ethanolic extract of leaf exhibited 100 % of results in 1 st instar stage of larva and subsequently the percentage of mortality was decreased to 97 ± 0.8 %, 85 ± 0.7 %, 73 ± 0.9 % (2 nd , 3 rd and 4 th instar, respectively).A similar trend has been noticed for the pupal stage that the maximum mortality (58 ± 0.8 %) was achieved in 600 ppm of etanolic extract (Table 2).Furthermore, the LC 50 and LC 90 values for ethanol extract of 1 st instar and pupa were found to be 257.14, 811.11 and 582.04, 1185.71ppm respectively with the chi square values of 1.92 and 0.85.The values of LC 50 and LC 90 of other extracts against instars and pupae are shown in Tables 1-5, where the chi square values were statistically significant at P<0.05 level.

Larvicidal and pupicidal activity against Ae. aegypti
The same set up was tried against dengue and chikungunya causing mosquito, Ae. aegypti and the results of larvicidal and pupicidal activity of different leaf extracts of C. philippinum were tabulated (Tables 6-10).Similar findings were detected as occurred in An. stephensi that the ethanol extract of leaves gave maximum mortality in comparison to others.97 ± 1.2 and 51 ± 0.5 % of larval and pupal mortality, respectively were recorded at 600 ppm of ethanol extract, which was maximum to others (Table 6).When the values of larvicidal activity were noticed in all solvent extracts, the effective results were obtained in 1 st instar and then followed by 2 nd , 3 rd and 4 th against individual concentration.Moreover from the solvent efficacy point of view, the hierarchy of mortality (both larval and pupal) was found to be ethanol > methanol > chloroform > n-hexane > aqueous.The LC 50 and LC 90 values for different extracts against larval and pupal mortality of An. stephensi are represented in Tables 6-10.The tables indicating chi square values were significant at P<0.05 (Tables 6-10).

DISCUSSION
In the present study, probit analysis reveals that LC 50 and LC 90 of leaf extracts of C. philippinum were effective against An.stephensi and Ae.aegypti during the larval and pupal stage.In general, the mortality rate from 1 st to 4 th instar were cumulatively decreasing which indicate that the extracts created a more toxic environment during the very beginning stages of mosquitoes (that have tried).Leaf extracts produced enhanced mortality with increasing concentrations of extracts and further, it has been noticed that ethanol extracts possessed strong activity than others (Tables 1-10).Globally, the people are always searching the eco-friendly alternative options to control the mosquitoes.For that, exploring of floral biodiversity is preferred, as they contain vast repository secondary metabolites.The tested plant, C. philippinum has larvicidal and pupicidal activity against An.stephensi which may be due to the presence of active biological compounds including terpenoids, flavonoid, alkaloids and phenolics etc. [28][29] The above mentioned compounds may jointly or independently contribute to impact on larvicidal and pupicidal activity against An.stephensi and a similar report was given by Subarani et al. in which aqueous and solvent leaf extracts of Catharanthus roseus is able to impact on An. stephensi. 4The other effective results for malarial vector An.stephensi were also reported against different plant extracts such as Euphorbia hirta, 11 Leucas aspera, 30 Citrus sinensis, 31 Gliricidia sepium. 32] The immature stages of mosquitoes are more susceptible to control as not only evidenced by An. stephensi but also from Ae. Aegypti against leaf extracts of C. philippinum (Tables 6-10).The synthetic toxins have been proved to be effective, but they pose some hazardous substances.Due to aquatic condition, they cause adverse effects on the environment and human health 33 and hence, this finding not only helps to control the spreading of mosquito but also biodegradable and easily available in low cost. 34Plants possessing bioactive compounds are main the culprit against mortality of Ae.Aegypti which was suggested by Gandhi et al. after experimented on roots of Rubia cordifolia with LC 50 and LC 90 values of 3.86 and 8.28 ppm for larvae, and 3.92 and 8.05 ppm for pupae of A. aegypti. 35Further, these group isolated alizarin from roots of R. cordifolia, which is the main source to destroy the larva/ pupa of mosquito.Larvicidal and pupicidal actions of methanol leaf extract of Tephrosia purpurea was also observed against A. aegypti and found that the LC 50 values of 1st instar to 4 th larval instars were 139.24, 176.24, 219.28, 256.27, and 326.29 ppm, respectively whereas LC 50 value of pupa was 326.29 ppm. 15he results so obtained also having similarities to Amir et al. who have taken the direct leaf and stem extracts of Parthenium hysterophorus against A. aegypti and confirmed as potential natural larvicidal agent. 12n our present study, the ethanol and methanol extracts of C. philippinum possessed higher larvicidal and pupicidal activities against An.stephensi and Ae.Aegypti if compare to aqueous, n-hexane and chloroform.This variation was attributed with dissolving properties of the bioactive compounds in respect to solvent system or the polarity of the solvents. 36owever, the mode of action of plant extracts on mosquito larvae are still unknown and up to some extent, it is postulated that the active phytochemicals interfering with the proper functioning of mitochondria more specifically at the proton transferring sites. 37[40]

CONCLUSION
The finding of the current investigation revealed that the leaf extracts of C. philippinum possess potential mosquito larvicidal and pupicidal activity against An.stephensi and Ae.aegypti.Since there is no availability of previous larvicidal and pupicidal activity of the selected plant species, this investigation serves as first-hand information.However, these results need corroboration further by characterizing, size estimating and determining the mode of action of individual bioactive compounds from leaf against both larval and pupal stages of development.In the future, this attempt may be recommended as an alternative tool to control the actions of An. stephensi and Ae.aegypti at their early life cycle processes.