Objective: To investigate in-vitro larvicidal property of botanical oils of brassica, Brassica campestris L., mustard, Brassica juncea (L.) Czern., cinnamon, Cinnamomum zeylanicum Blume, clove, Syzygium aromaticum (L.) Merr. et L.M.Perry, eucalyptus, Eucalyptus grandis W. Hill ex Maiden, turpentine Pinus sylvestris L., neem Azadirachta indica A.Juss., and mineral kerosene oils against 4th instar larvae of Culex quinquefasciatus Say, 1823 and test their time dependent repellency/attractancy in the field ovitraps.
Methods: Larvicidal properties of various plant oils and mineral kerosene oil was investigated using 4th instar larvae of Cx. quinquefasciatus in dose dependent experiments in the water media while their attractancy/repellency was tested in the field ovitraps over time.
Results: The fiducial limits for LC90 values at the 48 h for cinnamon, turpentine and kerosene oils were overlapping and therefore, not significantly different (p <0.05) from each other. Equal toxicity of these oils was indicative that kerosene oil can be replaced with environment friendly botanical oils for the control of Cx. quinquefasciatus larvae. The positive oviposition activity index (OAI) of traps treated with eucalyptus, cinnamon, and turpentine oils indicated that these oils were attractive after two weeks of application while neem was repellent to Culex mosquitoes.
Conclusion: Oils of cinnamon, eucalyptus and turpentine are fatal to the larvae of Cx. quinquefasciatus and act as attractant to the adults for oviposition and therefore, may be good candidates for using in the “attract and kill” strategy of mosquitoes control programs.
KeywordsCulex quinquefasciatus, attract and kill, ovitraps, botanical oils, mosquitoes
1) Nuclear Institute for Food and Agriculture (NIFA), Pakistan
2) Department of Entomology, University of Haripur, KPK, Pakistan
RecievedOct 30 2014 AcceptedApr 13 2015 PublishedMay 26 2015
Citation Khan I, Badshah T, Saeed M, Khan GZ (2015) Testing efficacy of botanical and mineral kerosene oils on Culex quinquefasciatus mortality and their repellency in field ovitraps. Science Postprint 1(2): e00050. doi:10.14340/spp.2015.05A0001.
Copyright©2014 The Authors. Science Postprint published by General Healthcare Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 2.1 Japan (CC BY-NC-ND 2.1 JP) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
FundingThis research was partly supported by the International Atomic Energy Agency (IAEA) Vienna, Austria under Coordinated Research Project CRP-13292 and Pakistan Atomic Energy Commission Islamabad, Pakistan.
Competing interest The authors declare that they have no competing interests.
Ethics statementThe NIFA research evaluation committee approved the research methodology and use of mosquitoes in this study.
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Corresponding authorInamullah Khan
AddressNuclear Institute for Food and Agriculture (NIFA), G.T Road Tarnab Peshawar, Pakistan
Peer reviewers Dr. Sarita Kumar1 and Reviewer A
1 Associate Professor, Zoology, Acharya Narendra Dev College (University of Delhi), New Delhi, India.
Mosquitoes represent one of the challenging groups of insects to mankind due to their established role as vectors in the transmission of wide range of human diseases such as malaria, lymphatic filariasis, yellow fever and dengue fever. Culex quinquefasciatus Say, 1823 ( Diptera: Culicidae) is night time active, opportunistic blood feeder and vector of many pathogens, including St. Louis encephalitis virus (SLEv), West Nile virus (WNv), and a major vector of lymphatic filariasis throughout the tropics. Cx. quinquefasciatus is present throughout the tropics and the lower latitudes of temperate regions. It is found in North America, South America, Australia, Asia, Africa, Middle East, and New Zealand. In Pakistan Cx. quinquefasciatus is the major annoying wide spread mosquito species causing severe irritation 1-3 and has been incriminated in the transmission of West Nile virus in India and Pakistan 4.
Control of Cx. quinquefasciatus population attains strategic importance in public health, particularly in the case of communicable diseases against which effective vaccines have not been evolved. Pesticides continue to play a significant role in vector pest control activities around the world, and this trend will be sustained for many years to come. Pesticides are poisons by definition and the majorities of people recognize the advantage of their use in view of their benefits derived in the control of disease vectors. Unfortunately, conventional pesticides are misused in many instances giving rise to growing public concern about the development of resistance in many vectors 5, 6. An alternative to conventional insecticides are products from plants sources that has been highlighted by many researchers as larvicides, fungicides or as repellents for anti parasitic activity 7-11 with their little harmful effects on the non target organisms 12, 13. Many plant materials have been investigated as larvicides, oviposition attractants, insect growth hormone regulators and as deterrent agents 14-17. Antifeeding activity of neem against the larvae of Cx. tarsalis and Cx. quinquefasciatus has been reported by Mulla and Su (1999) 18. Commercially available pine (Pinus longifolia) oil and cinnamon (Cinnamomum zeylanicum) oil against the early 4th instar larvae of Aedes aegypti, has shown larvicidal properties 18. Phytochemical effects of various plant derived materials have been extensively studied 19 and recommended for vector control. Current studies report on the comparative toxicity of commonly available plant oils and kerosene mineral oils and their repellency/attractancy in ovitraps against of Cx. quinquefasciatus mosquitoes in their attract and kill strategy.
Larvae of Cx. quinquefasciatus were reared in the round plastic tubs of 30 cm diameter containing 1.5 L tap water and fed with combination larval diet composed of powder of wheat rice, chickpea, bean, corn and bovine liver 20 till adult emergence. Pupae were collected and placed in small plastic cups inside an adult cage for emergence. Adults were maintained in transparent Plexiglas cages (40 x 30 x 30 cm) with resting places and provided with a 10% (W/V) sugar solution through a feeding apparatus as described by Khan et al. (2013) 20. The rearing conditions of the room were 27 ± 2оC, 60 ± 5% RH; and 12 h: 12 h (light period: dark period).
Commercial grade oils of brassica, Brassica campestris L., cinnamon, Cinnamonum zeylanicum Blume, clove, Syzygium aromaticum (L.) Merr. et L.M.Perry, eucalyptus, Eucalyptus grandis W. Hill ex Maiden, turpentine, Pinus sylvestris L., mustard, Brassica juncea (L.) Czern., neem, Azadirachta indica A.Juss, and mineral kerosene oils were tested for their toxicity on early 4th instar larvae of Cx. quinquefasciatus mosquitoes. In order to know activity range of oils under examination and to establish dose response lines, initially a wide range of concentrations (8–10) was tested. Based on this, a narrow range of concentrations yielding mortality between 10 to 95% were selected. Batches of 25 early 4th instar larvae were transferred from larval rearing trays by means of plastic droppers to small disposable test cups (7.8 cm diameter) each containing 100 ml of water giving a depth of 5 cm. Small, unhealthy or damaged larvae were replaced with uniform size larvae. The appropriate volume of oils was added to the test cups with automated pipette. Each concentration was replicated four times for all oils with an equal number of controls. All tests were run three times on different days and the test containers were held at 27 ± 2oC, 60 ± 5% RH; and12 h: 12 h (light period: dark period). After 24 and 48 h exposure, larval mortality was recorded. Dead larvae were recorded dead if they were unable to move when probed with a needle in the siphon or the cervical region. The test was discarded and repeated if more than 10% of control mortality was observed.
Bioassay on the repellency/attractantcy of selected oils was evaluated in ovitraps constructed from one liter plastic mugs wrapped in black polyethylene bags. Each trap was filled in with 500 ml of tap water. A 500 µl of plant oil was added to the water in each trap except control in which plain tap water was used. All traps were placed at a height of 2 feet from ground surface in the close vicinity of fields and irrigation channels. Each oil was replicated in three traps. Data on oviposition assessment and larval mortality within the traps was recorded weekly for up to one month period at the end of which trap water was tested for its mortality against laboratory colony of Cx. quinquefasciatus L4 stage larvae. The oviposition was expressed as mean number of eggs laid and 1st or 2nd instar larvae of Cx. quinquefasciatus in each treatment. Oviposition activity index (OAI) was calculated using the formula OAI = (NT - NC)/(NT + NC) adopted from Kramer and Mulla (1979) 21.
Where NT represents total number of eggs in the test solution, and NC is the total number of eggs in the control solution and OAI ranges from -1 to +1, with 0 indicating neutral response. The positive index values indicate that more eggs were deposited in the test cups than in the control cups, and that the test solutions were attractive. Conversely, more eggs in the control cups than in the test cups result in negative index values and the test solutions were deterrent to the adults. The percent effective repellency (ER% = (NC - NT)/NC x 100) and percent effective attractancy (EA% = NT - NC)/NT x 100) for each essential oil was calculated using formula adopted from Rajkumar and Jebanesan (2005) 22. The whole experiment continued for 4 weeks at the end of which all larvae from three replicate traps along with trap solutions was pooled together and brought to laboratory for mortality assessment of 4th instar Culex larvae.
Bioassays from all treatment replicates were pooled together and analyzed using POLOPlus 23 (LeOra computer software L) for probit regression analysis to estimate the dosage response of exposed larvae 24. The LC50 and LC90 confidence limits of each lethal level and slopes were determined for each group. For ovitraps the mean number of eggs deposited in test and control cups were analyzed using a completely randomized design, followed by Tukey’s honest significant difference (HSD) for mean separation in Statistix 8.1 (Analytical software, Tallahassee, FL) 25. All levels of statistical significance were determined at p <0.05.
Twenty four hour toxicity data of Cx. quinquefasciatus to the various oils is shown in Table 1. The lowest LC50 (in mg/L) for these oils was recorded for kerosene followed by eucalyptus and turpentine. The LC90 values (mg/L) for these oils as recorded were lowest for eucalyptus followed by kerosene and turpentine. The 24 h LC50 values for cinnamon, eucalyptus, and turpentine oil did not differ at p <0.05. However these values were higher than kerosene indicating the acute toxicity of kerosene. At the LC90 eucalyptus and turpentine were not different from each other but the LC90 value for kerosene was lower than eucalyptus showing acute action of kerosene. Turpentine and eucalyptus were equally toxic at the LC90 values but the LC90 value of eucalyptus was higher than obtained with kerosene indicating the delayed acute action of eucalyptus.
Table 2 shows the 48 h toxicity of Cx. quinquefasciatus mosquitoes. The lowest LC50 for these oils as recorded (mg/L) was achieved with kerosene followed by that obtained with turpentine and eucalyptus oil. The LC50 values at 48 h for cinnamon, eucalyptus and turpentine oils were not significantly different (p <0.05) from each other indicating equal toxicity. The lowest LC90 value at 48 h post observation time was revealed with kerosene followed by turpentine and cinnamon. The fiducial limits at LC90 for 48 h exposure time to eucalyptus, turpentine and kerosene oils were overlapping and therefore, not significantly different (p <0.05) from each other. Thus equal toxicity of these oils was indicative that kerosene oil can be replaced with environment friendly botanicals (eucalyptus and turpentine) for the control of Cx. quinquefasciatus mosquitoes.
Results on the repellency/attractancy of various oils in field ovitraps are shown in Table 3. Data indicated that during the first two weeks no mosquito egg or larvae was present in the ovitraps including control. During the 3rd week, unhatched eggs and in 4th week, larvae and hatched eggs were recorded from all ovitraps. Positive oviposition index (OAI) was recorded for cinnamon, eucalyptus, turpentine and kerosene oils indicating that these oils were attractive to Culex mosquitoes. Their attractantcy during the 3rd week was in the order of kerosene < turpentine < eucalyptus < cinnamon and in the 4th week as turpentine < kerosene < eucalyptus < cinnamon. Only neem oil showed negative (OAI), and repellency indicating the repellent effect of neem oils in ovitraps.
* The OAI ranges from -1 to +1; the positive index values (+) indicated that the test solutions were
attractants; the negative index values (–) indicated that the test solutions were deterrents and 0
indicating neutral response.
OAI = oviposition active index; ER = effective repellency; EA = effective attractancy
Plant products have been used in many parts of the world for killing or repelling mosquitoes either as extracts, oils or as whole plant. Some investigations 26, 27 have indicated that some chemical constituents of plant products interfere with the octopaminergic nervous system in insects. As this target site is not shared with mammals, most essential oils and plant extracts are relatively non-toxic to mammals and fish in toxicological tests, and meet the criteria for “reduced risk” pesticides.
In the present investigation the botanical oils especially cinnamon, eucalyptus and turpentine resulted in considerable mortality of the larvae of Cx. quinquefasciatus mosquitoes. The LC values for these oils as recorded were low and in most cases did not differ from kerosene oil. Mortality was dose dependent as shown in other studies 28. Data on the oviposition of Culex and Aedes in oil treated traps showed that these oils were not attractive to the mosquito during the first two weeks. During the 3rd and 4th week, the positive oviposition index (OAI) showed the attractiveness of the oil treated traps which increased with aging of the oil in the traps. Other investigators have also reported the aging effect of plant based infusion. Five- to 20-day old grass infusion was strongly attractive to gravid females of Cx. quinquefasciatus for laying eggs 29. Su and Mulla (1999) 30 have reported similar results from two experimental azadirachtin (AZ) formulations, wettable powder Azad™ WP10 (WP) and emulsifiable concentrate Azad™ EC4.5 (EC). Strong ovipositional responses were observed in the tests with aged suspensions from 1–7 days at 0.5 and 1 mg/L. Azad™ WP was more active in eliciting oviposition responses in Cx. tarsalis than the fresh preparations.
Our data on the use of botanical oils indicated positive oviposition index (OAI) for cinnamon, eucalyptus, turpentine and kerosene oils indicating that these oils were attractive to Culex mosquitoes. Their attractantcy during the 3rd week was in the order of kerosene < turpentine < eucalyptus < cinnamon and in the 4th week as turpentine < kerosene < eucalyptus < cinnamon. Only neem oil showed negative (OAI) indicating the repellent effect of neem oils in ovitraps.
Mulla and Su (1999) 18 have reported significant anti-feeding activity of neem at 5 mg/L and 10 mg/L of neem against the larvae of Cx. tarsalis and Cx. quinquefasciatus and therefore, was recommended to be applied for personal protection against mosquito bites.
Other studies on the oviposition activity index (OAI) of six essential oils namely Cananga odorata, Cymbopogon citratus, Cymbopogon nardus, Eucalyptus citriodora, Ocimum basilicum and Syzygium aromaticum have also shown that they were more deterrent than the control whereas Citrus sinensis oil acted as oviposition attractant 31. Lethal ovitraps (LOs) have shown sustained effect on dengue vector population densities in housing conditions of Brazilian municipalities 32-34, and in Thailand for Ae. aegypti 35. The efficacy of LO may be improved when used as part of an integrated control program for reduction in larval population in their habitats 36. Other studies on a yeast containing attractive contamination device with pyriproxyfen larvicide has shown 100% mortality of larvae in the trap and over 90% in the alternative breeding sites 37. Similarly addition of hay infusion in traps has been reported to increase efficiency of gravid ovitraps under semi and natural conditions 38. Pyriproxyfen is a larvicide and growth inhibitor used in very minute quantities. Its addition to environment friendly attractive lethal ovitraps with cinnamon, eucalyptus and turpentine oils may increase its efficiency in dissemination of pyriproxyfen to adjacent breeding sites. We suggest future studies to be done on the addition of plant based attractants with a growth inhibitor to improve attractancy plus mortality of the mosquitoes in the traps and in adjacent breeding sites.
Oils of cinnamon, eucalyptus and turpentine are fatal to the larvae of Cx. quinquefasciatus and becomes attractive to their adults’ oviposition after some time and therefore, may be good candidates for using in the “attract and kill” strategy of mosquitoes control programs. Moreover, in comparison to synthetic pesticides, (temophos or deltamethrin) botanical oils are effective in very small quantities (mg/L) and the cost to benefit ratio is very low. We recommend the use of environment friendly plant based oils as mosquito larvacides in Pakistan and suggest the addition of pyroprofoxifen in lethal ovitraps as a future strategy in the control of mosquitoes.
Conceived and designed the work: Khan I
Acquired the data: Khan I, Badshah T, Khan GZ
Analyzed and/or interpreted the data: Khan I, Saeed M
Drafted the work: Khan I
Revised and approved the work: Khan I, Badshah T, Khan GZ, Saeed M
All authors approved the final version of the manuscript.
We are thankful to Muhammad Nissar, Senior Scientific Assistant who helped us in maintaining the laboratory colony of mosquitoes and laboratory conditions during this experiment. Special thanks are extended to Mr. Alamzeb and Mr. Muhammad Sulman Shah who helped us in the data analysis.