J Arthropod-Borne Dis, September 2017, 11(3): 427–432 H Bilal et al.: Citrus Seed Oils Efficacy … 427 http://jad.tums.ac.ir Published Online: September 08, 2017 Short Communication Citrus Seed Oils Efficacy against Larvae of Aedes aegypti *Hazrat Bilal 1, Waseem Akram 2, Soaib Ali Hassan 1, Sadrud Din 3 1Medical Entomology and Disease Vector Control, Health Services Academy, Islamabad, Pakistan 2Department of Agriculture Entomology, University of Agriculture, Faisalabad, Pakistan 3Water, Agriculture and Technology Transfer Program, Kabul, Afghanistan (Received 21 Sep 2012; accepted 29 Nov 2016) Abstract Background: Dengue fever is a serious public health issue in Pakistan for many years. Globally plants have been reported to contain compounds with insecticidal properties. These properties have been demonstrated more recently on the larval stages of mosquitoes. Therefore, Citrus cultivar seeds were evaluated for larvicidal potential against the primary dengue vector Aedes aegypti. Methods: Extraction of oil was done by a steam distillation method and oils were evaluated according to WHO guidelines for larvicides 2005 for evaluation of insecticidal properties of citrus seed extracts against mosquito larvae. Result: Among the Citrus cultivar seed oil, rough lemon (Citrus jambhiri) had the lowest LC50 value (200.79ppm), while musambi (C. sinensis var musambi) had the highest LC50 value (457.30ppm) after 24 h of exposure. Conclusion: Citrus cultivars have some larvicidal potential but C. jambhiri had the greatest potential against A. ae- gypti larvae. Further small-scale field trials using the extracts of C. jambhiri will be conducted to determine opera- tional feasibility. Keywords: Citrus cultivars, Aedes aegypti larvae Introduction Dengue fever (DF) and dengue hemorrhag- ic fever (DHF) are serious public health con- cerns in many developing countries, includ- ing Pakistan. Over 2.5 billion people over 40% of the world's population are now at risk from dengue. WHO currently estimates 50–100 million dengue infections occurred worldwide every year (1). In Pakistan, the first case of DHF was ob- served in Karachi in 1994, (2) and 11,024 confirmed cases of DF including 40 deaths were reported in 2010 (3). In 2011, a subse- quent dengue outbreak occurred with 22,778 confirmed cases and 353 deaths (4). The ma- jority of the cases and deaths occurred in La- hore, Punjab Province while there was also epidemic in Khyber Paktunkhua (KPK) dur- ing 2013 that resulted in 23 deaths Apart from the KPK province, sporadic cases have also been reported from the provinces of Punjab, Sindh, and Balochistan (5). Unlike yellow fever, there is no vaccine for dengue fever. However Osorio et al. (6) is developing a vaccine, which is still in the preclinical stage. Therefore, the only current effective approach to control dengue is through vector control. This is done mainly through integrated vector management pro- grammes using insecticides, environmental management and public awareness (7, 8). Due to the toxic effects and resistance to syn- thetic insecticides (9), vector control managers are facing problems in controlling mosqui- toes. Therefore, it is necessary to develop safe alternative insecticides, which require mini- mum care (10). To overcome these problems, concentra- tion has been shifted steadily to the use of bi- *Corresponding author: Mr Hazrat Bilal, E-mail: hazratbilal@hsa.edu.pk J Arthropod-Borne Dis, September 2017, 11(3): 427–432 H Bilal et al.: Citrus Seed Oils Efficacy … 428 http://jad.tums.ac.ir Published Online: September 08, 2017 opesticides as adulticides (11), larvicides (12), and repellents (13) providing alternatives to synthetic chemicals. Many investigators have reported on the efficiency of biopesticides (plant extracts) against mosquito larvae (14). Recent studies have encouraged the investi- gation of insecticidal properties of botanicals. Muthukrishnan and Puspalatha (15) evaluated the larvicidal effects of extracts from Rhina- canthus nasutus (Acanthaceae), Solanum su- ratense (Solanaceae), Calophyllum inophyllum (Clusiaceae), Samadera indica (Simaroubace- ae) and Myriophyllum spicatum (Haloragaceae) against Aedes aegypti, Culex quinquefascia- tus and Anopheles stephensi and concluded that they are environmentally safe, degradable and target specific (16). In the view of an increased interest in the development of plant-based insecticides as al- ternatives to synthetic insecticides, this study was conducted to assess the larvicidal poten- tial of citrus cultivars against the dengue vec- tor A. aegypti. Materials and Methods Collection and Rearing of mosquitoes Aedes aegypti larvae were collected from old tire shops of Sargodha (32.0836° N, 72.6711° E) and, reared in 3-inch deep steel trays, and were reared in the insectary of bi- osystematics Lab University of Agriculture Faisalabad-Pakistan. Larvae were fed on Tet- ra-min® fish feed until adult emergence (17). Adults were maintained in well-aerated cages (70× 35× 35cm), where the mosquitoes were provided with cotton wicks soaked in 10% sucrose solution. Females were also fed blood from white mice every alternate day (18). A Petri dish with landing pad was provided to lay their eggs. The population was maintained at set conditions of 27±2 oC, 75±5% RH and L14:D10 photoperiod. Extraction of oil The seeds of citrus cultivars were washed to remove the pulp, oven dried for 48 hours at 60 ˚C and later ground in an electric grinder. A thimble was used to hold the grounded material and kept in an extraction tube of Soxhelt apparatus with extractor ID 38mm, extractor volume 85ml and flask volume 250ml (19) for the extraction of oil by steam distillation method using Diethyl-ether as solvent (250ml/ 20g sample). The cyclic time of extraction for each sample was 4–5h. Bioassay Six different concentrations increasing by 100ppm from 300–800ppm of extracted oils were used with three replicates for each treat- ment, each replicate containing 200ml of the oil solution placed in 250ml glass beakers. Batches of 30 late 3rd and early 4th instar lar- vae of the A. aegypti were placed in each beaker (20). Control beakers were treated with diethyl-ether only. The experiment was con- ducted using Completely Randomized De- sign (CRD) under lab conditions at 27±2 ˚C and 70±5% relative humidity. Data analysis Abbot’s formula (21) was used to correct for mortality, and this data was analyzed by probit analysis (22) using Minitab® Statistical Software (23) software to create a dose mor- tality regression line. In the control treat- ment, if mortality rates were between 5% to 20% then percent mortality was corrected by Abbot’s formula as follows: % corrected mortality= % observed mortality- % control mortality × 100 100-% control mortality Results The efficacy of citrus seed oils against the late 3rd and early 4th instar larvae of A. aegypti was expressed in terms of LC50 as shown in Table 2. Among the oils tested, rough lemon had the lowest LC50 value (200.79ppm) which is highly significant (0.001), followed by valencia late (213.02ppm), chakutra (221.40) J Arthropod-Borne Dis, September 2017, 11(3): 427–432 H Bilal et al.: Citrus Seed Oils Efficacy … 429 http://jad.tums.ac.ir Published Online: September 08, 2017 and narangi (248.16ppm). Musambi had the highest LC50 value (457.30ppm) followed by freutrall early (337.63ppm), kinnow (321.60 ppm), succari (316.60ppm) and red blood orange (286.41ppm) after 24h of exposure. Table 1. Following Citrus cultivars were collected from Sargodha Sr.# Common Name Botanical Name 1 Chakutra (Citrus grandis) 2 Kinnow (Citrus reticulate) 3 Musambi (Citrus sinensis var musambi) 4 Narangi (Citrus mitis) 5 Red blood orange (Citrus sinensis) 6 Rough lemon (Citrus jambhiri) 7 Feutrell (Citrus reticulate) 8 Valencia late (Citrus sinensis var valencia late) 9 Succari (Citrus sinensis var succari) Table 2. LC50values of citrus seed extract against late 3 rd and early 4th instar larvae of Aedes aegypti after 24 hours of exposure Citrus extracts LC50* (ppm) 95% FL (LFL**-UFL***) Slope ± S.E χ2 P Chakutra (Citrus grandis) 221.40 155.29-268.40 1.20±0.23 5.86 0.19 Kinnow (Citrus reticulate) 321.60 266.89-379.10 1.09±0.15 1.15 0.76 Musambi (Citrus sinensis) 457.30 412.25-512.18 1.22±0.16 1.10 0.77 Narangi (Citrus mitis) 248.16 195.98-275.87 1.38± 0.25 6.74 0.12 Red blood orange (Citrus sinensis) 286.41 262.64-305.54 3.38±0.30 5.44 0.23 Rough lemon (Citrus jambhiri) 200.79 167.62-230.80 1.75±0.22 10.72 0.001 Feutrell early (Citrus reticulate) 337.63 298.24-397.30 1.06±0.14 0.55 0.87 Valencia late (Citrus sinensis) 213.02 138.20-161.42 1.01±0.16 2.20 0.43 Succri (Citrus sinensis) 316.60 267.00-351.10 1.04±0.14 2.04 0.60 *LC50 ie, lethal concentration (ppm) to kill 50% population of the subjected organism ** Lower Fiducial Limit *** Upper Fiducial Limit Discussion Mosquito borne diseases are one of the most important public health problems in the developing countries like Pakistan. Use of synthetic insecticides are the best option to control mosquito larvae but resistance, envi- ronmental problems etc are the some con- cerned problems which can be overcome by the use of Plant essential oils and extracts as a part of Integrated Vector Management (IVM). A variety of plants is well known to contain chemicals with bioactive potential (24) as de- terrents and attractants (25). The ether oils of different citrus cultivars (Table 2) have been studied as natural insecticides against A. ae- gypti larvae instead of synthetic insecticides as they are reported resistant to the mosqui- toes especially A. albopictus (26). Rough lem- on (C. jambhiri) had the lowest LC50 value (200.79ppm) against A. aegypti larvae. This is well supported by the findings of Akram et al. (17) in A. albopictus larvae. Other citrus seed oils also have some larvicidal effect as reported by Hafeez at al. (27) Din et al. (28) against A. albopictus. Sumroiphon et al. (29) J Arthropod-Borne Dis, September 2017, 11(3): 427–432 H Bilal et al.: Citrus Seed Oils Efficacy … 430 http://jad.tums.ac.ir Published Online: September 08, 2017 reported the effects of citrus seed extract against the larvae of A. aegypti and Culex quinquefasciatus. Extracts of C. sinensis (30) and C. bergamia (31) have been analyzed for the toxicity against the larvae of A. aegypti and found quite effective. Bagavan et al. (32) reported the peel chloroform extract of C. sinensis, leaf ethyl acetate extracts of Oci- mum sanctum, O. canum and leaf chloroform extract of Rhinacanthus nasutus as possible insecticides against the larvae of Anopheles subpictus and methanol extract of Citrus sinensis peel, methanol extract of O. canum leaves, and ethyl acetate extracts of R. nasu- tus and O. sanctum against the larvae of Cu- lex tritaeniorhynchus. 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