POTENTIAL OF CITRUS SEED EXTRACTS AGAINST DENGUE FEVER MOSQUITO, AEDES ALBOPIC TUS (SKUSE) (CULICIDAE: DIPTERA)

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1 Pak. J. Bot., 42(4): , POTENTIAL OF CITRUS SEED EXTRACTS AGAINST DENGUE FEVER MOSQUITO, AEDES ALBOPIC TUS (SKUSE) (CULICIDAE: DIPTERA) WASEEM AKRAM 1*, HAFIZ AZHAR ALI KHAN 1, FAISAL HAFEEZ 1, HAZRAT BILAL 1, YEON KOOK KIM 2 and JONG-JIN LEE 2 1 Department of Entomology, University of Agriulture, Faisalaad, Pakistan 2 Chonuk National University, Chonju, South Korea * Corresponding author s areea14@yahoo.om Astrat Citrus seeds and peel ontain ertain ompounds with varied level of itterness. These ompounds have een tested against insets and proved to e effetive. The present study was therefore arried out to test the itrus seed extrats from 10 varieties against 4 th instar larvae of dengue fever mosquito, Aedes alopitus (Skuse). The results indiate that the extrats from rough lemon and lemon were more effetive as larviides with lowest LC 50 ( and ppm respetively, after 24h of exposure and and ppm respetively, after 48h of exposure) and LT 50 values (2.51 and 4.91h, respetively). Seed extrats from remaining itrus varieties were less ative at lower doses; however at higher doses these were iologially ative against Ae. alopitus. Our study has suggested that itrus-seed extrat are environment friendly and an e used for managing Ae. alopitus larvae. Introdution Mosquitoes are important arthropods, transmitting diseases like malaria, filariasis, Japanese enephalitis and dengue (Servie, 1983) and have potential to feed on more than one individual, during a single gonotropi yle (Makenzie et al., 2004). Proper ontrol of mosquitoes lies in personal protetion and puli awareness as the most eonomial method in eradiating reeding sites and ontrolling these through environment friendly larviides (Corel et al., 2004). Aedes alopitus whih is an inreasingly important disease vetor (Gratz, 2004) inluding dengue fever (Yang et al., 2009), has spread with the growth of towns and poor sanitation (Anon., 1982). The drasti effets of syntheti insetiides in the environment have reeived wide puli apprehension (St. Lager et al., 1996), however its misuse in agriulture and puli health programs has aused many prolems like insetiide resistane, resurgene of pest speies, environmental pollution, toxi hazards to humans and other non-target organisms (Sarwar et al., 2009). To alleviate these prolems, major emphasis has een on the use of natural plant ased produts as larviides whih an provide an alternate to syntheti insetiides (Junwei et al., 2006). In general, plant essential oils are important natural alternatives to insetiides (Golade et al., 2000). Many plants have een found to ontain hemials whih are helpful for the ontrol of insets (Roert, 2001) and are useful for field appliations in mosquito ontrol programmes (Kalyanasundaram & Das, 1985). Limonoids from Rutaeae partiularly itrus (Kloke & Kuo, 1982) have attrated greater apprehension due to their growth regulating ativities (Champage et al., 1992). Citrus whih inludes limonin, nomilin, oaunone, epilimonol and limonin diosphenol (Liu et al., 1990) an readily e extrated from itrus seeds, availale in large quantities as waste produts of the itrus industry (Murray et al., 1995). Citrus limonoids

2 3344 WASEEM AKRAM ET AL., work oth as toxiant and feeding deterrents. In struture-ativity studies of limonin, it has een determined that the furan ring and epoxide groups in the itrus limonoid struture are ritial for the antifeedant ativity of the limonoids (Mendel et al., 1991). Limonoids affet the egg laying proess of insets due to nutritional disruption whih ultimately indue antifeedant effets (Murray et al., 1995). With the growing environmental onerns and regular out reak of disease sine the first reord of dengue from Karahi in 1994 (Chan et al., 1995), the present study as therefore een onduted to find est options. Citrus farming is perhaps one of the most important ommerial and industrial agriultural ativities of the world and Pakistan is one of the most important ountries amongst itrus fruit produer. So we seleted different itrus varieties and their seed extrats were tested against one of the important dengue fever vetor, Aedes alopitus. Materials and Methods Colletion of mosquitoes: Immature mosquitoes were olleted from Faisalaad y a standard 375ml dipper (Anon., 1975) during The larvae were kept at 28±2 o C temperature and 65±5% relative humidity in the laoratory for adult emergene. The Ae. alopitus were later mass reared and their larval stages were fed on Tetra-Min Tropial (Tetra TM). Adults were kept in plasti ages where they were provided 10% surose solution (Osghai et al., 2003). Females were fed on lood of white rats (Shaalan et al., 2006). The gravid females were allowed to lay eggs on lak plasti gauze that was plaed in Petri dishes. The eggs were separated and on emergene of larvae these were shifted to rearing trays. Colletion of fruits and extration of itrus oil: Citrus varieties viz., Brazilian sour orange (Citrus aurantium), hakutra (Citrus grandis), galgal (Citrus pseudolimon), grape fruit (Citrus paradisi), kinnow (Citrus retiulata), lemon (Citrus limon), musami (Citrus sinensis), narangi (Citrus mitis), red lood orange (Citrus sinensis) and rough lemon (Citrus jamhiri) were olleted from Punja. The seeds were separated manually and washed with distilled water to remove the pulp and then dried for 48 hours at 60 o C and later ground in an eletri grinder (Anex Germany). The grounded material was put in Soxhlet apparatus for the extration of oil y steam distillation method (Vogel, 1978), using di-ethyl ether as a solvent. Preparation of solution: Oil was olleted in small vials and the quantity was measured. Stok solutions were prepared y adding 1 ml of oil from eah variety in 100ml of aetone that was onsidered as 1% stok solution from whih series of onentrations (ppm) were prepared (Murugan et al., 2007). Bioassay: The extrated oils were used in six different onentrations (300, 400, 500, 600, 700, 800 ppm). For ontrol treatments, 1 ml of aetone was mixed with 199 ml of distilled water. There were three repliates for eah treatment; eah repliate ontaining 200ml of the oil solution plaed in 250ml glass eakers. Thirty early fourth instar larvae of the Ae. alopitus were exposed in eah eaker ontaining oil solution (Mohtar et al., 1999). The experiment was onduted using CRD under la onditions of 28± 2 o C and 65± 5% relative humidity. The numers of dead larvae were ounted after 24, 48 and 72 hours of exposure.

3 CITRUS SEED EXTRACTS AGAINST DENGUE FEVER MOSQUITO 3345 Data analysis: Mortality data, if needed, was orreted y Aot s formula (Aot, 1925) and the data was analyzed y Proit analysis (Finney, 1971), using POLO-PC software (LeOra Software, 1987) for dose and time mortality regression lines. Signifiant differenes were onluded y non-overlapping of 95% fiduial limits. Data on perent mortalities were analyzed with Statistix version 8.1 (Anon., 2005) and means were ompared with least signifiant differene (LSD) test at 5% level of signifiane. Results and Disussion In our experiment, different itrus seed extrats were sujeted to laoratory ioassay against Ae. alopitus larvae. These extrats provided satisfatory results as far as the suseptiility and time limit is onerned. Oils from all the itrus varieties showed strong larviidal effets. Tale 1 shows that out of the total itrus seed extrats tested, rough lemon and lemon had the lowest LC 50 values ( and ppm, respetively), partiularly after 24 hours of exposure, followed y red lood orange ( ppm), hakutra ( ppm), galgal ( ppm), razilian sour ( ppm) and kinnow ( ppm). Narangi had the highest LC 50 value ( ppm) after 24h of exposure followed y grape fruit ( ppm) and musami ( ppm). After 48 hours of treatment, rough lemon and lemon had the lowest LC 50 values ( and ppm respetively), followed y narangi ( ppm), galgal ( ppm), red lood orange ( ppm), razilian sour ( ppm) and hakutra ( ppm). Musami had the highest LC 50 values ( ppm), followed y kinnow ( ppm) and grape fruit ( ppm). The χ 2 values for larval mortality tests show no heterogeneity in all the experiment. Tale 1. LC 50 values of itrus seed extrats against 4 th instar larvae of Aedes alopitus Citrus extrats Oservation LC 50* χ 2 95% FL Slope ± S.E (h later) (ppm) (df=2) Brazilian sour ± (Citrus aurantium) a 1.75 ± Chakutra a 4.07 ± (Citrus grandis) a 6.71 ± Galgal ± (Citrus pseudolimon) a 2.43 ± Grape fruit d 1.88 ± (Citrus paradisi) ± Kinnow ± (Citrus retiulata) ± Lemon a 2.17 ± (Citrus limon) a 2.83 ± Musami ± (Citrus sinensis) ± Narangi d 2.08 ± (Citrus mitis) a 2.65 ± Red lood a 4.52 ± (Citrus sinensis) a 7.86 ± Rough lemon a 3.03 ± (Citrus jamhiri) a 2.53 ± *LC 50 i.e., lethal onentration (ppm) to kill 50% population of the sujeted organism

4 3346 WASEEM AKRAM ET AL., 24 h 48 h 72 h Mortality (%) g e a e d h g f f f d a a h g e i a d a a a 0 Brazilian Sour Chakutra Galgal Grape Fruit Kinnow Citrus varieties Lemon Musami Narangi Red Blood Rough Lemon Fig. 1. Bars are mean perent mortality (± S.E.) at different time intervals. Bars sharing the same letters are not signifiantly different at the 5% level of signifiane (Least signifiant differene [LSD] test, Statistix 8.1). Rough lemon and lemon showed highest perent mortalities (95.6 and 88.9% respetively), after 24 hours of exposure, followed y hakutra (84.4%) and red lood orange (82.8%) while narangi gave lowest perent mortality (11.7%). Rough lemon and lemon also showed highest perent mortalities (99.6 and 97.7%, respetively) after 48 hours of exposure followed y hakutra (91.6%) while musami and grape fruit gave lowest perent mortalities (46.7 and 47.2%, respetively). After 72 hours of exposure, hakutra, galgal, lemon, narangi, red lood orange and rough lemon showed almost 100% mortalities (Fig. 1). In terms of lethal time to kill 50% population of sujeted organism, rough lemon and lemon took minimum time to kill 50% population i.e., 2.51 and 4.91hours, followed y red lood, hakutra and galgal (8.52, and hours, respetively). Kinnow and musami took almost two days (50.25 and hours respetively) to kill 50% population while grape fruit took longest time (67.18 hours) to ause 50% mortality (Tale 2). The χ 2 values for larval mortality tests show no heterogeneity in all the experiment. The results of the present study are in agreement with the sientists who worked with different plant extrats against Ae. alopitus. The extrats from peel of different itrus varieties (Mwaiko, 1992) also had good larviidal potential with lemon peel oil as the est (Mwaiko & Savaeli, 1994) and are safe for human eings Murgun et al., (2007) traed out the larviidal and repellent ativities of Alizzia amara and Oimum asilium against Ae. alopitus at different onentrations. The extrats of five mediinal plants i.e., Autilon indium, Aegle marmelos, Euphoria thymifolia, Jatropha gossypifolia and Solanum torvum were prepared separately y using ethyl aetate, aetone, rude hexane, petroleum ether and methanol and were heked for their toxiity against the early fourth instar larvae of Culex quinquefasiatus. Petroleum ether extrat of Autilon indium gave the highest larval mortality (Chansang et al., 2005). The plant derivatives are proale soures of some iologially ative agents for mosquito ontrol in the future (Mathur, 2003). Our results indiate that itrus seed extrats have good larviidal potential against Ae. alopitus larvae with rough lemon and lemon eing the most effetive in terms of LC 50, LT 50 and perent mortalities. So we suggest that itrus seed extrats as well as other plant extrats should e investigated for the ontrol of dengue mosquitoes. This study, however, was onduted in the laoratory and the effiay of itrus extrats against Ae. alopitus should now e examined in the field.

5 CITRUS SEED EXTRACTS AGAINST DENGUE FEVER MOSQUITO 3347 Tale 2. Time mortality response of Aedes alopitus larvae against itrus seed extrats. Citrus extrats LT 50 Time mortality response* 95 % FL Slope ± SE χ 2 Brazilian sour (Citrus aurantium) ± Chakutra (Citrus grandis) a 2.18 ± Galgal (Citrus pseudolimon) ± Grape fruit (Cirus paradisi) d 2.40 ± Kinnow (Citrus retiulata) d 2.17 ± Lemon (Citrus limon) a 1.6 ± Musami (Citrus sinensis) d 2.87 ± Narangi (Citrus mitis) ± Red lood (Citrus sinensis) a 1.72 ± Rough lemon (Citrus jamhiri) a 1.34 ± *LT 50 data reorded within an extrat treatment denoting the same letters are not different at the 5% level of signifiane. FL, fiduial limit is the upper and lower limits of respetive LT 50 values. Referenes Aot, S.W A method of ompeting the effetiveness of an insetiide. J. Eon. Entomol., 18: Anonymous Manual on pratial Entomology in Malaria, World Health Organization. Prepared y the WHO Division of Malaria and other parasites diseases. Part 1-2. Offset puliation No.13. World Health Organization, Geneva. Anonymous Data sheet on the iologial ontrol agent Baillus thuringiensis serotype H-1 (de Barjae, 1978). WHO Mimeographed douments. WHO/VBC/ Anonymous POLO-PC. A user s guide to proit or logit analysis. LeOra Software, Berkeley, CA, USA. Anonymous Statistix for Windows. Analytial Software, Tallahassee, Florida. Champage, D.E., O. Koul, M.B. Isman, G.G.E. Sudder and G.H.N. Towers Biologial ativity of limonoids from rutales. Phytohem., 31: Chan Y.C., N.I. Salahuddin, J. Khan, H.C. Tan, C.L. Seah and J. Li Dengue haemorragi fever out reak in Karahi Pakistan Trans. Roy. Soi. Trop. Med. Hyg., 89: Chansang, U., N. Zahiri, S. Bansiddhi, Jaree, B. Thidarat, T. Ratom, M. Jiranuh, B. Nipa, Mulla and S. Mir Mosquito larviidal ativity of aqueous extrats of long pepper (Piper retrofratum vahl) from Thailand. J. vet. Eol., 30: Corel, V., S. Duhon, M. Zainm and J.M. Hougand Dinotefuran: A potential neoniotinoid insetiide against resistant mosquitoes. J. Med. Entomol., 41: Finney, D.J Proit analysis, 3 rd ed., Camridge Great Britain University Press, UK. Golade, A.A., A.D. Dyedele, M.B. Sosan, F.B. Adewayin and O.I. Soyela Mosquito repellent ativities of essential oils from two Nigerian Oimum speies. J. Trop. Med. Plants, 1: Gratz, N.G Critial review of the vetor status of Aedes alopitus. J. Med. Vet. Entomol., 18:

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