Introduction

Mosquitoes are the principal pest of public health importance and out of 3492 mosquito species more than hundred species are capable of transmitting various diseases to human beings (Arivoli and Tennyson, 2012). Filariasis, dengue fever, malaria, yellow fever, Japanese Encephalitis and many more dreadful diseases are transmitted by different species of mosquitoes (Rahuman et al., 2008; Mallick and Chandra, 2015a). Culex quinquefasciatus Say, 1823 is a vector of lymphatic filariasis which is widely distributed in the tropical countries. Worldwide 1.10 billion people in 55 countries remain get affected by filariasis. In 2000, 120 million people became infected and near about 40 million people became incapacitated and disfigured by the disease. 5.63 billion treatments have been provided to eradicate infection through chemotherapy. Strategies may be needed in about 28 countries to achieve the goal of elimination of the disease and stop treatments by 2020 (WHO, 2016). Larval control is more effective because mosquito larvae are restricted to a small space due to their low mobility in nature (Howard et al., 2007; Bhattacharya and Chandra, 2014). Use of synthetic insecticides to control different microorganisms, pests and vectors are effective but they create many problems like insecticide resistance, environment pollution, and toxic side effects on human beings and other non targets (Liu et al., 2005; Lixin et al., 2006; Mukherjee et al., 2015).  Easily degradable plant products are the best alternatives to control vectors and thus preventing vector borne diseases (Mallick et al., 2014; Singha Ray et al., 2014). Therefore, the researchers experimented with many plants which involved larvicidal activities, and reported accordingly (Mallick et al., 2015a; Mallick et al., 2015b; Mallick and Chandra, 2015b; 2015c; 2016; Singh et al., 2015). Citrus maxima Merr. (synonyms: Citrus grandis; Citrus decumana) belongs to the family Rutaceae and is ancestor of grape fruit. In Philippines and South East Asia, leaves of C. maxima is used to treat cough, fever, epilepsy, ulcer, etc. (Haja et al., 2014).  Its native land is South Eastern China. In West African countries, it is cultivated as ornamental tree for its large fruits. Fruits of C. maxima is also known as shaddock. The colour of pulp is either white or pink. It is heart stimulant, appetizer, and also acts as a stomach tonic and antitoxic. Essential oil of the peel of C.maxima has the antibacteial activity against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) (Oyedepo, 2012).

The present study was done to establish the larvicidal activity of fruit peel extracts of C maxima against Cx. quinquefasciatus mosquito and examine the presence of different phytochemicals on fruit peel extracts.

1 Material and Methods

1.1 Collection of plant materials

After proper identification of the plant, fresh mature fruits of C. maxima were collected from the premises of MUC Women’s College, Burdwan, West Bengal, India (23^◦16^ꞌ N, 87^◦54^ꞌ E) during the month of  September, 2014. A herbarium sheet of stem with flowers and small fruits of the plant was deposited (voucher specimen number: GCSM-05) to Mosquito, Microbiology and Nanotechnology Research Units, Parasitology Laboratory. Fruits were washed initially with running tap water and then rinsed with distilled water; and subsequently dried on paper towel. The fruit peel of C. maxima was separated by steel knife, thereby discarding spongy tissues.

1.2 Test mosquito

Larvae of Cx. quinquefasciatus were collected for larvicidal bioassay experiments from laboratory mosquito colony. The mosquito colonies were well maintained and devoid of any insecticides, repellents, and exposure to pathogens. The larvae were fed with artificial food of mixture of dog biscuits and dried yeast powder (3:1 ratio).

1.3 Preparation of crude extract

5 g cleaned fruit peel (without spongy tissues) of C. maxima was crushed by electrical grinder, and the fluid was filtered through muslin cloth. The filtrate was collected on a beaker, and the filtrate was used as a crude stock solution (100% concentration). From stock crude solution, different concentrations (0.1- 0.5%) were prepared through dilution with tap water for larvicidal bioassay experiments.

1.4 Preparation of solvent extracts

Fruit peel of C. maxima were cut into very small pieces and dried in shade for 13- 15 days. 200 g dried fruit peel of C. maxima was packed in column of soxhlet apparatus, and the extract was prepared treating solvents, namely n-hexane, ethyl acetate, and methanol (2000 ml each), using fresh sample for each extraction with the said solvents.  Extraction period was 72 h for each solvent extract preparation, and the temperature was below 40º C. Each solvent extractive was collected, filtered through Whatman No. 1 filter paper and was concentrated through rotary evaporator. After evaporation of each solvent, semisolid extractive was obtained and stored at 4º C in a refrigerator for further bio assay experiments. 

1.5 Larvicidal bioassay

The larvicidal bioassays were done following the standard protocol of World Health Organization (2005), with slight modification. All instars larvae were used in bioassay experiments with crude extract, and only early 3^rd instar larvae were used in bioassay experiments with each solvent extract. Thirty larvae were put in plastic bowls of 225 ml capacity and 9 cm diameter, each containing 100 ml of test solution of different concentrations of crude (0.1-0.5%) and different concentrations of each of  n-hexane, ethyl acetate and methanol fruit peel extract (i.e. 200, 400, 600, 800 and 1000 ppm) after initial trialing. 2.5 g of each of different solvent extracts were initially dissolved on 2.5 ml of ethanol separately and then added 22.5 ml of distilled water to the same to prepare 25 ml stock test solution. So stock solution (10⁵ ppm concentration) of each different solvent extracts were made on 10% ethanol. From stock test solutions, different test concentrations for each solvent extract were prepared. Fresh stock test solution of crude and each solvent extract were prepared on the same day of larvicidal bioassay experiments. 100 ml of tap water with 0.5 ml of ethanol was used as positive control and only 100 ml of tap water was used as negative control. Larval percent mortality was recorded after 24, 48, and 72 h of post exposures cumulatively. Dead larvae were identified when they did not move after being touch with a fine brush in the siphon or cervical region. Besides control experiments, all experiments were replicated three times on separate three days under laboratory conditions (25º- 30º C and 80- 90% relative humidity).

1.6 Phytochemical analyses

Phytochemical analyses of the aqueous and ethanol extracts (charcoal filtered) of fruit peel were carried out through standard protocol of Trease and Evans (1989) and Harborne (1984) with some modifications.

1.6.1 Test for presence of alkaloids (Mayer’s test)

1.36g mercuric chloride was  dissolved in 60 ml of H₂O (solution 1) and 5 g potasium iodide dissolve in 100ml of water (solution 2). These two solutions were mixed together and the new solution is named as Mayer’s reagent. After preparation of Mayer’s reagent, 2 ml of ethanol peel extract was taken in a clean test tube, and  few drops of 2N HCL and subsequently 2-3 drops of Mayer’s reagent were added to it. Cream or pale yellow colour precipitation gives the indication of presence of alkaloids.

1.6.2 Test for presence of terpenoids (Salkowski test)

2 ml of ethanol extract was taken in a test tube and then 5 ml of chloroform was added to it. Then 1 ml of concentrated sulphuric acid was added carefully to the interior wall of the test tube. Reddish brown colour at the interface is the indication of presence of terpenoids.

1.6.3 Test for presence of flavonoids

2 ml of aqueous extract was treated with 1 ml of NaOH solution. Intence colour was formed and it becomes colourless on addition of dilute HCL. It is the indication of presence flavonoids.

1.6.4 Test for the presence of tannins and phenolic compounds (Ferric chloride test)

2 ml of aqueous extract was taken in a test tube and 4-5 drops of ferric chloride were added to it. Appearence of blue green colouration is the indication of tannins and phenolic compounds.

1.6.5 Test for presence of steroids

2 ml of the ethanolic extract was taken in a test tube and added 6 ml of chloroform and then added 3 ml of concentrated sulphuric acid was carefully added to the interior side wall of the test tube. The upper layer appeared red and sulphuric acid layer yellow with green florescence. This is the indication of the presence of steroids.

1.7 Application of n-hexane solvent extract on non target organisms

Median lethal concentration (LC₅₀) of n-hexane fruit peel extract against early 3^rd instar larvae after 24 h exposure was used against non-target organisms like Chironomus circumdatus larvae and Diplonychus annulatum nymphs.

1.8 Statistical analyses

‘MS EXCEL 2007’ and SPSS 16.0 were used to calculate percent mortalities and standard error, LC₅₀, LC₉₀ values (95% confidence level), regression equations, R² (Co-efficient of determination) values and ANOVA (for testing statistical significance).

2 Results

Fruit peel extract of C. maxima was found to be effective as mosquito larvicidal agent against all the larval instars of Cx. quinquefasciatus. Cent percent mortality was found after 24h of exposure against 1^st, 2^nd and 3^rd instar of larvae to crude extract of fruit peel (Table 1). Table 2 depicts the mortality percent of different solvent extracts (n-hexane, ethyl acetate, and methanol) of fruit peel of C. maxima among which n-hexane extract showed best larvicidal efficacy. Mortality percent increased with increase in concentration of both crude and solvent extracts along with time of exposure. No mortality was observed on negative control experiments and ethanol treated control. Table 3 and Table 4 represented the calculated LC₅₀ and LC₉₀ values, regression equations, and R² values of larvicidal activity of crude and different solvent extracts of the plant respectively. With the increase of exposure periods LC₅₀ and LC₉₀ values gradually decreased. Regression analyses revealed that mortality percentages (Y) were positively correlated with the concentrations of time of exposures (X) as coefficient of determination values (R²) are close to 1 in each case (Table 3 and Table 4). LC₅₀ and LC₉₀ values of different solvent extract after 24, 48, and 72 h of exposure were lower in order of solvent extracts n- hexane< methanol< ethyl acetate (Table 4). Presences of different secondary metabolites were detected after preliminary phytochemical analyses of aqueous and ethanol dried fruit peel extracts (Table 5).

The three-way factorial ANOVA (Table 6) analysis of larvicidal bioassay of crude fruit peel extract was carried out with respect to three parameters (concentrations,  times of exposure and instars) and significant larval mortality (p<0.05) was obtained in terms of concentrations of extract, times of exposure and instars of larvae. No sluggishness or anomalous activity was observed on tested non target organisms like, Chironomus circumdatus larvae and Diplonychus annulatum.

3 Discussions

Crude as well as different solvent extracts of C. maxima fruit peels has good larvicidal potency against Cx. quinquefasciatus mosquito. 1^st instar larvae showed 100% mortality only at 0.2% concentration of crude extract. Among tested three solvent extracts, n-hexane extract was the most potent to kill late 3^rd instar larvae of Cx. quinquefasciatus followed by methanol and ethyl acetate extracts. 100% mortality was observed at 400 ppm concentration of n-hexane extract having LC₅₀ value 204.60 ppm after 24 h of exposure. Respective LC₅₀ value of methanol and ethyl acetate extracts were 599.44 and 946.28 ppm after 24 h of exposure. Many researchers worked with fruit peel extracts of different plants and showed larvicidal activity against different mosquito species. Murugan et al. (2012) worked with ethanol peel extract of Citrus sinensis having larvicidal, pupicidal, repellent, and adulticidal activity against An. Stephensi, Ae. aegypti, and Cx. quinquefasciatus mosquito species upto 24 h of exposure. Respective median lethal concentration (LC₅₀) for 1^st- 4^th instar larvae of Cx. quinquefasciatus were 244.70, 324.04, 385.32, and 452.78 ppm. Velu et al. (2015) assessed the larvicidal activity of chloroform, acetone, ethanol, methanol and aqueous peel extracts of Arachis hypogaya against 4^th instar larvae of Ae. aegypti and An. stephensi upto 24 h of post exposure and they showed that methanol extract was most potent for larvicidal activity against Ae. aegypti and An. Stephensi. They reported that Ae. aegypti and An. stephensi exhibited 100% mortality at dose 150 ppm having LC₅₀ values 45.75 and 45.98 ppm respectively. Arivoli et al. (2012) reported seven plants having larvicidal activity against early 3^rd instar larvae of Cx. quinquefasciatus upto 24 h of exposure period. Among the tested seven plants, hexane extract of arial part of Hyptis suaveolens was most potent with LC₅₀ value 203.37 ppm. Singh et al. (2006) reported larvicidal activity of hexane extract of Momordica charantia against 4^th instar larvae of Cx. quinquefasciatus, An. stephensi, and Ae. aegypti having respective LC₅₀ values of 66.05, 96.11, and 122.45 ppm after 24 h of exposure. The findings of the present investigation revealed that fruit peel extract of C. maxima show good larvicidal activity against Cx. quinquefasciatus mosquito. Phytochemicals like alkaloids, terpenoids, steroids, and flavonoids are present in the fruit peel extract of C. maxima, of which any one or a combination of them is responsible for larvicidal activity. Further study was needed to elucidate its activity against other mosquito species and to identify the active ingredient of the extract responsible for mosquitocidal activity. Fruit peel extract of C. maxima is harmless to non-target organisms and fruit peel is a waste product, biodegradable having no cost, i.e. the product will be cost effective.  

Conflict of interest

We have no conflict of interest.

Acknowledgement

Authors are thankful to Professor Dr. A. Mukhopadhyay, Department of Botany, The University of Burdwan, Burdwan, West Bengal, India for identification of the plant species. We are also grateful to UGC DRS, DST-INSPIRE, DST-WB for providing financial support.

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