J Arthropod-Borne Dis, June 2017, 11(2): 278–285 H Bilal et al.: Bio-Pesticides: New Tool … 278 http://jad.tums.ac.ir Published Online: May 27, 2017 Original Article Bio-Pesticides: New Tool for the Control of Aedes (Stegomyia) albopictus (Culicidae: Diptera) in Pakistan *Hazrat Bilal 1, Sumrin Sahar 2, Sadrud Din 3 1Medical Entomology and Disease Vector Control, Health Services Academy, Islamabad, Pakistan 2University of Copenhagen, Copenhagen, Denmark 3Water, Agriculture and Technology Transfer Program, Kabul, Afghanistan (Received 9 Nov 2015; accepted 9 July 2016) Abstract Background: Application of plant extracts as mosquito control strategy was practiced from centuries. These are eas- ily available, non-toxic, biodegradable and exhibit broad-spectrum target specific activities against larval stages of mosquitoes. Methods: Different potential parts of locally grown plants, seeds of nutmeg (Myristica fragrans), peel of musambi (Citrus sinensis), leaves of babuna (Matricaria chamomilla), mint (Mentha spicata) and ginger rhizome (Zingiber officinale) selected and evaluated for their larvicidal properties against Aedes (Stegomyis) albopictus. Oils were ex- tracted through steam distillation process and extracts were evaluated as per WHO 2005 guidelines for testing of insecticides against larvae of mosquitoes. Results: Among the five plant extracts, C. sinensis had the lowest LC50 (400.81ppm) while M. fragrans had the highest LC50 value (710.30ppm) respectively after 24h of exposure. In terms of % age mortality, a series of con- centrations (300–800ppm) gave high % mortality in case of C. sinensis while M. fragrans gave low % age mortality. Conclusion: All the five plant species have larvicidal effects to certain extant and C. sinensis had great potential. Further small-scale field trials with the extracts of the most promising one (C. sinensis) shall be conducted to deter- mine operational feasibility. Keywords: Mosquitoes, Plant extracts, Larvicide Introduction Dengue, malaria, filariasis, yellow fever and Japanese encephalitis are the most important diseases transmitted by mosquitoes (Rozen- daal 1997). Fifty million cases of dengue oc- cur globally every year (WHO 2009). Dengue has now emerged in many countries, especial- ly in Pakistan where occurred outbreaks af- fected the socio-economic development in the Region (Savioli and Velayudhan 2014). Dengue epidemic in Pakistan (2011) is being observed where, more than 22,778 cases con- firmed and 353 deaths reported (Anonymous 2011). In 2013, a Punjab and Khyber-Pakh- tunkhwa (KPK) provinces hit by second epi- demic, in northern areas of Pakistan the human mobilization from dengue endemic regions, geographic expansion of dengue fever vector due to importation, climatic change, all are the factors, which resulted in the emergence of dengue in northern areas (Ali 2013). In dis- trict Swat (KPK) 6,000 dengue cases with 47 deaths were reported (Khan and Khan 2013). Worldwide mosquito control depends on the application of synthetic insecticides as a part of Integrated Vector Control (IVM) Pro- grammes (Becker et al. 2010). Toxic effects and resistance to synthetic insecticides are barriers in controlling mosquitoes. Therefore, it is necessary to develop safe alternative insecticides, which required minimum care (Mittal and Subbarao 2003). *Corresponding authors: Mr Hazrat Bilal, E-mail: hazratbilal@hsa.edu.pk http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 278–285 H Bilal et al.: Bio-Pesticides: New Tool … 279 http://jad.tums.ac.ir Published Online: May 27, 2017 Plant based insecticides may be the best option for mosquito control as they have bi- ologically active chemicals that are easily de- composed into products which are not toxic to other species (Sanjay and Tiku 2009) and potentially suitable for use in control of mosquito larvae (Yang et al. 2004). In fact, many researchers have reported the effectiveness of plant extracts or essential oils against mosquito larvae (Rahuman et al. 2008). Research on insecticidal properties of botan- icals inferred that they are bio-degradable, environmentally safe and target specific (Go- vindarajan et al. 2008). (Muthukrishnan 2012) and (Bilal et al. 2012) evaluated the larvicidal effects of extracts from Cinnamomum cassia, Citrus sinensis Linnaeus var musambi, Tribulus terrestris, Eucalyptus camaldulensis, Piper ni- gera, Ricinus communis, Allium sativum, Li- num usitatissimum and Citrus sinensis L var succari against Aedes albopictus. A number of other researchers which have used plant prod- ucts for the mosquito control like (Komalamis- ra et al. 2005) reported the ether extracts of Trigonostemon reidioides, Rhinacanthus nasu- tus, Derris elliptica, Homalomena aromatica, Stemona tuberose, Acorus calamus and Piper nigrum (Siddiqui et al. 2004), Artemisia annua, Sonchus oleraceus and Chenopodium album (Sharma et al. 2006) Solanum xanthocarpum (Mohan et al. 2005) Argemone mexicana (Sak- thivadivel and Thilagavathy 2003). In the view of increased interest in devel- opment of plant-based insecticides as an al- ternative to synthetic insecticide, this study was planned and conducted to assess the lar- vicidal potential of five medicinal plants against the medically important mosquito (Aedes (Stegomyia) albopictus). Materials and Methods Collection of Plants Nutmeg (M. fragrans Houtt) seeds, mu- sambi (C. sinensis (L) Osbeck) peel, babuna (M. chamomilla L) leaves, Mint (M. spicata L) leaves and ginger (Z. officinale Roscoe) rhizome were collected from botanical gar- den of University of Agriculture Faisalabad (31.4339° N, 73.0649° E) and local market of Faisalabad (31.4181° N, 73.0776° E). Extraction of oil The seeds, peel, leaves and rhizomes were washed, then dried and later pulverized in an electric grinder (Anex Germany). The pul- verized material was placed in thimble and kept in extraction tube of Soxhelt apparatus with extractor ID 38mm, extractor volume 85ml and flask volume 250ml (Vogel 1978) for the extraction of oil by steam distillation method using ether as solvent (250ml/20g sam- ple). The cycle time for one sample was 4– 5h. Solvent was evaporated at room tempea- ture, leaving oil, collected in flask. Stock so- lution was prepared by adding 1ml of oil from each plant in 99ml of ether and consid- ered as 1% stock solution from which series of concentrations (%) were prepared (Akram et al. 2010). Collection and Rearing of Mosquitoes Mosquito larvae were collected from po- tential breeding sites of Aedes around Islam- abad (33.7167° N, 73.0667° E) with a stand- ard pipette while adults were collected by battery-operated aspirator. Larvae were reared for mass population in the insectary of Med- ical Entomology and Disease Vector Control department, Health Services Academy, Islam- abad. The first instar larvae were fed with fat free milk powder while other instars larvae were fed with Tetra-Min fish feed powder at 28±2 ˚C and 75±5% humidity. Adults were reared in screened cages by providing 10% sucrose solution while female mosquitoes were also fed on the blood of albino rats (Imam et al. 2014). Larvae of Ae. (Stg.) albopictus were identified using identification key of Leo- poldo (Leopoldo 2004). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 278–285 H Bilal et al.: Bio-Pesticides: New Tool … 280 http://jad.tums.ac.ir Published Online: May 27, 2017 Larvicidal Bioassay The extracted oils were used in six differ- ent concentrations along control with three replicates for each treatment, each replicate containing 200ml of the oil solution in 250 ml Pyrex glass beakers. A batch of fifteen 3rd instar larvae of the Ae. (Stg.) albopictus were exposed in each beaker containing oil solu- tion (WHO 2005), while control was treated with ether only. Mortality of larvae was count- ed after 24 hours. The experiment performed under lab conditions at 28±2 ˚C and 75±5% relative humidity. Statistical Analysis Abbot’s formula (Capinera 2008) was used for correction of mortality and the data so obtained was analyzed by probit analysis (WHO 2005) by using MANITAB-15 soft- ware for dose mortality regression line and % age mortality graph were prepared using Microsoft office 2007. Results The crude ether extracts of nutmeg (M. fra- grans) seeds, musambi (C. sinensis) peel, ba buna (M. chamomilla) leaves, mint (M. spicata) leaves and ginger (Z. officinale) rhizome had been evaluated as potential source of insecti- cides. Results on the larvicidal activities of extracts were reported in the present study (Table 1) confirms their potential for the con- trol of Ae. (Stg.) albopictus larval popula- tion. All extracts showed moderate larvicidal effects however, the highest larval mortality was found in musambi peel with 400.81ppm LC50 value, followed by babuna (438.60ppm), ginger (502.55ppm) while mint and nutmeg had the highest LC50 value (596.94ppm and 710.30ppm) respectively after 24h of exposure. In terms of % mortality musambi had the high mortality (64.25%) followed by babuna and ginger (58.51 and 56.48%) respectively, while nutmeg and mint had the lowest % mortality (33.51 and 50.92%) respectively af- ter 24h of exposure as shown in Fig. 1. The percentage of mortality was directly propor- tional to concentration of the extract (Table 1). After exposure to the test concentrations, the treated larvae exhibited restlessness, tremors, sluggishness and convulsions fol- lowed by paralysis at the bottom of the bowl. Table 1. Larvicidal activity different plant extracts at different concentrations against 3rd instar larvae of Aedes (Stg.) albopictus Plants Con. % mortali *LC50 (ppm) 95% FL (LFL–UFL) 2 p Musambi 300 32 400.81 359.82–435.22 4.62 0.32 C. sinensis 400 46 500 54 600 61 700 70 800 82 Nutmeg 300 10 710.30 654.35–793.80 4.60 0.33 M. fragrans 400 16 500 25 600 29 700 43 800 58 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 278–285 H Bilal et al.: Bio-Pesticides: New Tool … 281 http://jad.tums.ac.ir Published Online: May 27, 2017 Babuna 300 27 438.60 394.18–477.52 0.18 0.99 M. chamomilla 400 41 500 51 600 61 700 66 800 70 Mint 300 31 596.94 521.21–719.96 1.11 0.89 M. spicata 400 38 500 42 600 47 700 55 800 62 Ginger 300 35 502.55 437.55–570.33 1.05 0.90 Z. officinale 400 39 500 47 600 56 700 61 800 67 *LC50 ie, lethal concentration (%age) to kill 50% population of the subjected organism Fig. 1. Percentage mortality of different plant extracts against Aedes (Stg.) albopictus larvae after 24 hours of expo- sure Discussion Recently Ae. (Stg.) aegypti along with Ae. (Stg.) albopictus played havoc in different parts of Pakistan. Different control measures have been adopted with the major focus on chemical control. Resultantly, occurrence of insecticide resistance in mosquitoes and other public health pests have been reported (Khan et al. 2011, 2013). Their residues in the envi- ronment and effects on humans and non- target organism are major problems due to which investigators are now directing their attentions towards the development of plant Table 1. Continued… http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 278–285 H Bilal et al.: Bio-Pesticides: New Tool … 282 http://jad.tums.ac.ir Published Online: May 27, 2017 based insecticides (Biopesticides). Various compounds, including phenolics, terpenoids, and alkaloids, exist in plants (Wink 1993, Swin 1977, Kim et al. 2001) and may jointly or independently contribute to the gen- eration of larvicidal activities of mosquito (As- sabugi et al. 1997, Hostettmann and Potterat 1997). Outcome of five different plant oils, which were, used against the 3rd instar larvae of Ae. (Stg.) albopictus are shown in Table 1. Re- sults were satisfactory and showed efficacy. Larval mortality increased with increase in dose of plant oil and at higher doses, it gave more than 80% mortality without any pupal or adult emergence. While in control, there was less than 5% mortality after 24h. Citrus sinensis peel extract gave reasonably good results (400.81ppm) against the larvae of Ae. (Stg.) albopictus when compared with other tested plant oils, like findings of Warikoo et al. (2012) they concluded that C. sinensis leaf extracts had 446.84ppm LC50 value against the larvae of Aedes aegypti as Citrus plants contain limonoids which work both as toxicant and feeding deterrents (Akram et al. 2010) and also has insecticidal effects (Bilal et al. 2012). Thus, the larvicidal activity of Citrus sinensis is due to limonoids, in addi- tion to alkaloids, saponins, steroids, flavo- noids and tannins. While on the other side, previous workers (Michaelakis et al. 2009, Din et al. 2011) reported LC50 values for some cit- rus peel and seed oils against the larvae of Ae. albopictus and Cx. pipiens different than the obtained values in the present investigation. In our findings, M. fragrans had least ef- fectiveness and gave 50% mortality after 24h of exposure is not in agreement with the studies of Senthilkumar et al. (2009). They tested M. fragrans, Eucalyptus globulus, Ar- temisia annua, Cymbopogan citratus, Justi- cia gendarussa, Annona squamosa and Cen- tella asiatica and found that all gave 80– 100% mortality against larvae of Anopheles stephensi. It is well documented that toxicity values of a substance may be largely varied due to several factors (Busvine 1971) attribut- ed to the test conditions (temperature, light, humidity, exposure period and solvent); test- ed species (age, stage and susceptibility) and tested plant material (season, location, extrac- tion method and used part) and there may be different constituents in a botanical extract may interact with each other’s, leading to synergistic or antagonistic effects (Mansour et al. 2000, 2003). Conclusion Out of the 5 plants extracts, C. sinensis has good larvicidal potential against larvae of Ae. (Stg.) albopictus in terms of LC50 and % age mortality. 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