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Activity of Kaffir Lime (Citrus Hystrix) Essential Oil Against Blow Flies and House Fly

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Activity of Kaffir Lime (Citrus Hystrix) Essential Oil Against Blow Flies and House Fly SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH ACTIVITY OF KAFFIR LIME (CITRUS HYSTRIX) ESSENTIAL OIL AGAINST BLOW FLIES AND HOUSE FLY Suttida Suwannayod1,2, Kabkaew L Sukontason1, Pradya Somboon1, Anuluck Junkum1, Ratana Leksomboon3, Tarinee Chaiwong3, Malcolm K Jones4, Banchob Sripa5, Suwit Balthaisong5, Chitsakul Phuyao5, Theeraphap Chareonviriyaphap6 and Kom Sukontason1 1Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai; 2Gradulate School, Chiang Mai University, Chiang Mai; 3College of Medicine and Public Health, Ubon Ratchathani University, Ubon Ratchathani, Thailand; 4School of Veterinary Sciences, The University of Queensland, Brisbane, Australia; 5Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen; 6Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand Abstract. Blow flies and the house fly are not only pests but can be carriers of -hu man pathogens. We aimed to determine the activity of the essential oil (EO) of the peel of Kaffir limeCitrus ( hystrix) against 3 species of blow flies (Chrysomya mega- cephala, Chrysomya rufifacies and Lucilia cuprina) and the house fly Musca( domestica) in order to develop a plant derived method to control these pests. Larvicidal and adulticidal efficacy of C. hystrix’s EO were evaluated by dipping method and topi- cal application, respectively. The EO studied gave lethal concentration 50 (LC50) =38.93 g/l against M. domestica, a LC50=61.00 g/l against L. cuprina, a LC50=66.39 g/l against C. rufifacies and a LC50=71.00 g/l against C. megacephala. Among female µ flies studied EO gave a lethal dose 50 (LD50) =83.50 g/fly againstM. domestica, a µ µ LD50=124.03 g/fly againstC. megacephala, a LD50=210.46 g/fly againstL. cuprina µ and a LD50=408.63 g/fly againstC. rufifacies. Scanning electron microscopy of the studied flies showed the studied EO resulted in a swollen, corroded integument with bleb formation. Light microscopy revealed a deformed midgut and hindgut and the fat cells having a vacuolated appearance. There was also a decrease in the number of nuclei in the fat cells and there were degeneration of the nuclei. Gas chromatography-mass spectrometry (GC-MS) evaluation of the studied EO revealed twenty-one compounds obtained by steam distillation. The major constituents were β-pinene (24.62%), sabinene (22.06%), limonene (19.29%), and citronellal (10.58%). Kaffir lime EO appears to be a potential candidate for further development as a plant derived method to control medically important fly species. Keywords: Citrus hystrix, essential oil, blow flies, house fly, toxicity Correspondence: Dr Kom Sukontason, De- INTRODUCTION partment of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Blow flies and house flies are not only Thailand. pests but can spread human pathogens, Tel: +66 (0) 53 935342 such as bacteria, viruses, protozoa and E-mail: [email protected] helminth eggs (Greenberg, 1973; Sukon- 32 Vol 49 No. 1 January 2018 ACTIVITY OF KAFFIR LIME AGAINST BLOW FLIES AND HOUSE FLY tason et al, 2007). Pathogenic bacteria found to be carried by these flies include Salmonella sp, Shigella sp and Escherichia coli O157:H7 (Chaiwong et al, 2014). The larvae of blow flies can also cause myiasis (Ferraz et al, 2010). In Thailand, 3 spe- cies of blow flies known to be medically important are Chrysomya megacephala, Chrysomya rufifacies and Lucilia cuprina and the house fly Musca( domestica) is also medically important (Ngoen-klan et al, 2011). These species can be found in and around human dwellings, especially dur- ing day-light hours. Since these flies live in or near human dwellings, they may be Fig 1–Photograph of Kaffir limeCitrus ( hystrix) exposed to insecticides. fruit. Conventional chemical pesticides are effective in fly control but continued, aimed to: 1) determine the efficacy of the uncontrolled use of pesticides can lead EO of C. hystrix against C. megacephala, C. to resistance (WHO, 1986; Kristensen et rufifacies, L. cuprina and M. domestica; 2) al, 2000). Chemical pesticides may have a determine the histopathological effects of negative effect on the environment, persist this EO against the adults and larvae of in the food chain and threaten nontarget the studied flies using a scanning electron organisms (Kristensen and Jespersen, microscope (SEM); and 3) determine the 2003; Kumar et al, 2012b). This has re- chemical composition of the studied EO. sulted in a search for alternative botanical insecticides. MATERIALS AND METHODS The essential oils (EO) of plants have been studied for their efficacy against flies Flies (Kumar et al, 2012a; Morey and Khan- The flies used in this study C.( mega- dagle, 2012). Kaffir limeCitrus ( hystrix cephala, C. rufifacies, L. cuprina and M. DC; family Rutaceae), known in Thailand domestica) were obtained from laboratory as “Ma-krud”, has been studied for its ef- strains maintained for 9 years at ambient ficacy against mosquitoes Aedes( aegypti, temperature in the Department of Parasi- Anopheles dirus, Culex quinquefasciatus tology, Faculty of Medicine, Chiang Mai (Tawatsin et al, 2001), Anopheles minimus University, Thailand. Fresh pork liver was (Nararak et al, 2017), cockroaches (Peri- provided for oviposition of the flies as planeta americana (L.), Blattella germanica described previously (Bunchu et al, 2008). (L.), Neostylopyga rhombifolia (Stoll) (Tha- vara et al, 2007)), the larvae of Ae. aegypti Essential oil preparation (Sutthanont et al, 2010) and tobacco army C. hystrix (Fig 1) was purchased from worms (Spodoptera litura) (Loh et al, 2011). a local market and identified by botanists The efficacy of the EO ofC. hystrix against in the Department of Biology, Faculty of medically important flies has not yet Science, Chiang Mai University, Thailand. been studied in Thailand. Therefore, we Voucher specimens were deposited at the Vol 49 No. 1 January 2018 33 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH Department of Parasitology, Faculty of rate for each box was recorded at 24 hours Medicine, Chiang Mai University, Thai- after exposure; mortality was determined land. The peels were manually removed, by touching each larva with a soft paint shade dried at ambient temperature for brush (No.0) and those not responding 5-10 days and ground to coarse powder. were considered dead. The experiment The ground C. hystrix powder was was conducted in triplicate for each then distilled following the protocol of group. The lethal concentrations (LC50, Champakaew et al (2015). About 250-300 LC90 and LC99) were calculated with LdP g of ground C. hystrix powder was placed line Software (Ehab Mostafa Bakr, Dokki, in an extraction column connected to a Cairo, Egypt). distillation flask containing ~1,600 ml Adulticidal bioassay of distilled water and 10-15 glass beads. The lethal dose of the EO of C. hystrix The distilled water was heated to 100°C against the studied adult flies followed in an immersion heater to produce steam the method of the WHO Expert Commit- to pass through the plant materials. The tee (1980). Approximately two hundred distillate mixture of essential oil and water 3-5 day-old adult flies of each studied was collected and allowed to settle into 2 species were transferred from a rear- layers over 3-5 days with the EO on top of ing cage (30×30×30 cm) into a smaller the water. The water was slowly released cage (16×16×16 cm). The smaller cages so only the EO remained. This was then were then placed in a large transparent dried with anhydrous sodium sulfate plastic bag and CO was then palced in (Na SO ) for 24 hours and then kept at 2 2 4 the plastic bag to anesthetize the flies. 4°C until used. After being anesthetized for 15 minutes, Larvicidal toxicity assay the small cage was removed from the The lethal concentration of the EO of plastic bag and all 200 flies were poured C. hystrix against the third instar larvae of into a small plastic box and placed on an C. megacephala, C. rufifacies, L. cuprina and iceplate. Thirty flies were then randomly M. domestica was determined following selected and placed on a Petri dish on an the method of Matsumura (1985). Five iceplate and then the studied solution was concentrations of the EO were prepared applied to each fly using an autopipette using 80% ethyl alcohol to prepare the (Sartorius®, Goettingen, Germany). The dilutions. A negative control consisted flies were then placed back in the smaller of 80% ethyl alcohol without the EO. cage again and given water and sugar Thirty third instar larvae of each fly spe- ad libitum and kept at 28-32°C with 70- cies tested (3 days after hatching from 80% relative humidity. Each fly was then the same egg batch) were divided into 6 examined 24 hours after exposure to the groups. Each group was placed in a fine studied EO to determine mortality. Each net (each pore 334 µm×522 µm) and the experiment was conducted in triplicate. net was then dipped into the respective The lethal dose (LD50, LD90 and LD99) was studied solutions for 30 seconds. The calculated using LdP line Software (Ehab dipped larvae by group were then placed Mostafa Bakr, Dokki, Cairo, Egypt). The in a tightly-sealed transparent plastic box EO preparations were made by diluting (6.0×8.5×3.2 cm) containing pork liver (10 the EO with acetone to form 5 different g cut into 2 small pieces). The mortality concentrations. 34 Vol 49 No. 1 January 2018 ACTIVITY OF KAFFIR LIME AGAINST BLOW FLIES AND HOUSE FLY Preparation of larvae for scanning electron of C. hystrix was determined by gas chro- microscopic examination matography-mass spectrometry (GC/MS) About 10-15 dead larvae of each using a Hewlette-Packard GC-MS system studied fly species exposed to the high- (Model 7890), following the method of est dose of EO were randomly selected Champakaew et al (2015).
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