J Arthropod-Borne Dis, March 2017, 11(1): 147–155 EA El Zayyat et al.: Bioefficacy of Some … 147 http://jad.tums.ac.ir Published Online: March 14, 2017 Short Communication Bioefficacy of Some Egyptian Aromatic Plants on Culex pipiens (Diptera: Culicidae) Adults and Larvae Elham A El Zayyat 1, Mohammed I Soliman 2, *Noha A Elleboudy 1, Shaimaa E Ofaa 1 1Parasitology Department, Faculty of Medicine, Ain-Shams University, Cairo, Egypt 2Research Institute of Medical Entomology, Dokki, Egypt (Received 8 Jan 2015; accepted 28 Dec 2015) Abstract Background: Protecting the environment from chemical hazards of synthetic insecticides along with offering of new breeding areas for vectors by urbanization indicate the trial of natural insecticides. Methods: The acetone extracts of Anethum graveolens, Ocimum basilicum and Thymus vulgaris were tested for their insecticidal effect on Culex pipiens adults and larvae in different concentrations depending on the technique used. Results: The extracts were significantly effective in all models used with basil being the best in all tested three tech- niques (LC50= 0.064) in larval feeding, (LC50= 0.330) in CDC bottle assay and (LC50= 13.148) in adults feeding (P< 0.05). Conclusion: The results recommend the eco-friendly studied extracts as candidates for controlling Cx. pipiens the lymphatic filariasis vector. Keywords: Culex pipiens, Anethum graveolens, Ocimum basilicum, Thymus vulgaris, Insecticides Introduction Climatic changes can cause expanding the range of geographical distribution and dynam- ics of vector-borne diseases. Global warming and changing sea level may lead to increase in transmission intensity and extending spa- tial distributions of these diseases (Hongoh et al. 2012) by influencing the development rate, longevity of insects and vector reproduc- tion rates, creating new challenges in these insects control in areas where they were nev- er recorded before. Anthropogenic factors with changes in landscaping, rising in hous- ing and settlements, extensive tropical urban- ization, colonization, increase in environmen- tal pollution and changing lifestyle patterns offer new breeding areas for vectors devel- opment (Tatem et al. 2006, Weaver and Rei- sen 2010, Carvalho et al. 2014). Insecticides overuse led to several ecolog- ical drawbacks over the past years. The toxicity, development of resistance phenomenon and the residual effects of these insecticides are the main concern of scientists. The urging need for developing environmental friendly insecticides is rising (Kebede et al. 2010). Several studies on botanicals potential as in- secticides are ongoing with the rise of green insecticides concepts and awareness of these safe, specific, biodegradable, eco-compatible components (Park et al. 2005, Khater and Shalaby 2008, Kumar et al. 2012). Mosquitoes are a major public health threat as they play a vital role in transmitting seri- ous human diseases to hundreds million peo- ple annually (Akinkurolere et al. 2011). Mos- quitoes were reported up to 2551–2528 BC also the invention of mosquito nets were at- tributed to the ancient Egyptians as Herod- otus reported in 445–440 B.C. (Arnold 1995). Culex pipiens is a worldwide mosquito caus- ing nuisance and transmitting many danger- ous diseases as West Nile virus, St Louis en- *Corresponding author: Dr Noha A. Elleboudy, E-mail: nohaleboudy@gmail.com J Arthropod-Borne Dis, March 2017, 11(1): 147–155 EA El Zayyat et al.: Bioefficacy of Some … 148 http://jad.tums.ac.ir Published Online: March 14, 2017 cephalitis virus, filarial worms and avian ma- laria (Farajollahi et al. 2011). With the emer- gence of Cx. pipiens resistance to many in- secticides, control is becoming more difficult providing a flourishing chance to these dis- eases (Zayed et al. 2006, Knio et al. 2008) Essential oils are volatile oils with strong aroma giving distinctive odor, flavor and scent to a plant. They are secondary metabolites char- acterized by being complex mixtures of organ- ic compounds (Okunowo et al. 2013). Usage of essential oils as a whole usually have higher efficacy than using its separate metabolites (Mourey and Canillac 2002, Bassole´ and Juliani 2012) as synergism of all compounds of the essential oil seems to be important (Zoubiri and Baaliouamer 2011). Despite being safe for domestic insect control strategies, few products based on plant essential oils are com- mercially available (Koul et al. 2008, Sut- thanont et al. 2010). From the ancient Pharaohs, Egyptians were familiar with aromatic plants and included their use in religion, cosmetics, embalming and me- dicinal purposes. In addition, the first record of essential oil distillation was from Egypt (Fakhry 2004). Egypt has a variety of flora, among which Anethum graveolens (Dill), Oci- mum basilicum (Basil) and Thymus vulgaris (Thyme) were chosen to conduct this study being available in local market, cheap and widely used in traditional medicine. This study aimed at determining the insecti- cidal effect of three indigenous plant extracts, commercially available in Egypt for domestic use, to control Culex pipiens adults and larvae. Materials and Methods Collection of plants materials Anethum graveolens, Oc. basilicum and Th. vulgaris were purchased from local mar- ket, washed with distilled water to remove dust particles and identified by Department of Botany, Faculty of Science, Ain-Shams University staff members. Preparation of plant extracts The whole plants including leaves and stems were dried for 7V10 days in the shade at 27–37 °C. The dried plants were powdered using a commercial electric blender then soaked in acetone 99% (Biochem. Company, Egypt) at a ratio of 1:4 for three days. Then the ex- tracts were sieved and filtered through a Buchner funnel with sterile Whatman filter paper number one, acetone was evaporated using Rotator evaporator apparatus (Hs-2005s, Jisico Co., Ltd. Korea) in Central laborato- ries, Faculty of Science, Ain-Shams Univer- sity, Egypt. The extracts were concentrated under reduced pressure 22–26mm Hg at 45 °C and the crude extracts residue were kept in dark bottles, labeled and preserved in the refrigerator at 4 °C until further use (Akinku- rolere et al. 2011). Insects rearing Laboratory reared colony of Cx. pipiens free from insecticides and pathogens obtained from Mosquitoes Research Department, Re- search Institute of Medical Entomology, Min- istry of Health and Population, Dokki, Giza was maintained starting from egg rafts. Lar- vae used in the tests were reared in a plastic cup containing dechlorinated water, under the standard conditions of 28±2 °C tempera- ture and 70±5% RH and L12/D12 photoper- iod and fed daily on tetramine (tropical fish food) until the larvae transformed into the pupal stage. The larvae were reared until adults in insectaries. Adults were maintained on a 10% sugar solution and females were allowed to feed periodically on guinea pig blood for 2–3h every two days to obtain pro- tein used principally for egg production (Kovendan et al. 2012). Larvicidal bioassay The assay was done on healthy late third instar larvae, three replicates for each test were used. Larvae were collected with a pas- ture pipette, placed on a filter paper to re- J Arthropod-Borne Dis, March 2017, 11(1): 147–155 EA El Zayyat et al.: Bioefficacy of Some … 149 http://jad.tums.ac.ir Published Online: March 14, 2017 move excess water then placed in cups con- taining 250mL dechlorinated tap water to which different concentrations of the tested extracts were added (0.02g/L, 0.05g/L, 0.1 g/L, 0.2g/L and 0.3g/L) then readjusted ac- cording to obtained results. Three controls cups containing water were used in the pres- ence of the larvae semi synthetic diet. All cups were covered with muslin cloth for pro- tection. The observed mortality was recorded at the end of 72h, larvae were considered dead if there is no sign of any movement even after mild touch with a glass rod (Ku- mar et al. 2014). Adulticidal bioassay Adaptation of CDC Bottle Bioassays technique was done to evaluate insecticidal properties of the extracts as follows: 250ml Wheaton bottles with screw lids were proper- ly cleaned and dried then they were coated with 1ml of (0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L) of the tested extracts by swirling assuring complete coating of the bottle and its cap. One ml of absolute alcohol was added to the control bottle handled as before. Forty mos- quitoes, divided among four replicate bottles were introduced using an aspirator into each bottle with the tested extract concentrations (Brogdon 2014). Each bioassay included a control bottle with 10 mosquitoes. Mortality was assessed after 2h then after 24h after the mosquitoes' introduction and mosquitoes were considered dead if they could no longer stand (Aïzoun et al. 2013). The experiment was done under normal room conditions. Feeding toxicity was done according to Allan (2011) for Cx. pipiens adults using ran- domly selected male and female, 3–6 days after emergence, starved for 24h, and placed in cages 0.5× 0.5× 0.5m (10/cage), Baits were presented in a 1cm length of cotton wick soaked with 200μl of 10% sucrose contain- ing the tested extracts doses (10g/L, 20g/L, 40g/L) and placed on top of the mesh of each cage and removed after 2h. The insects with distended abdomens indicating inges- tion of a sugar meal were transferred to a new cage (10/cage) and maintained. A cotton pad soaked with glucose only solution was used as a control for each extract. Mortality was assessed up to 72h after the treatment per- formance. Adults that did not respond to pin prick were be considered dead. Percentage of mortality was corrected us- ing of Abbott’s formula (Abbott 1925) if needed. X100 All statistical tests were performed using The SPSS version 22, 2013 (14· 0, Chicago, IL, USA). A probit regression model to pre- dict the probability of percentage of insect mortality and logarithmic concentration of the extracts was done. Z-scores were estimated. Pearson Goodness-of-Fit of Chi-square test to determine the applicability of the resultant regression models was done. LC50, LC90 with their 95 per cent confidence limits were de- termined. The Toxicity index was calculated as follows, lLC50 of the most effective com- pound ×100 divided by the lLC50 of the com- pound used and the most toxic compound was given 100 units on the toxicity index scale according to Rawi et al. (2011). The probability of error at 0.05 was considered significant, while at 0.01 and 0.001 were con- sidered highly significant throughout the whole study. Results Evaluation of insecticidal properties of three indigenous plants was done using dif- ferent An. graveolens, Oc. basilicum and Th. vulgaris crude extracts concentrations on both larvae and adults of Cx. pipiens mosquito. The results revealed the effectiveness of the used extract different doses to obtain mortal- ity and suggested the potentiality of their usage in Cx. pipiens control. The results of J Arthropod-Borne Dis, March 2017, 11(1): 147–155 EA El Zayyat et al.: Bioefficacy of Some … 150 http://jad.tums.ac.ir Published Online: March 14, 2017 larvicidal activity of extracts by feeding tech- nique after 72h are shown in Table 1 and 2). Adulticidal activity of the tested extracts by feeding technique after 72h is shown in Ta- ble 3 and 4 and by CDC bottle bioassay after 24 h is shown in Table 5 and 6. Plate (1) shows scanning electron microscopy of Cx. pipiens larva induced by basil extract after 72h while plate (2) for Cx. pipiens larva induced by basil extract by CDC technique after 24h. Table 1. Probit analysis of larvicidal activity of the tested extracts against Culex pipiens by feeding technique after 72h Plant Parameter Mortality rate Standard Error Z Sig 95% Confidence Interval Pearson Goodness of Fit Test Lower Bound upper Bound Chi- Square Sig Thyme Slope 1.057 0.228 4.641 .000 0.611 1.503 0.725 0.867 Intercept 1.220 0.204 5.991 .000 1.016 1.424 Dill Slope 1.428 0.192 7.442 .000 1.052 1.804 2.319 0.509Intercept 1.642 0.217 7.554 .000 1.424 1.859 Basil Slope 0.996 0.190 5.257 .000 0.625 1.368 1.589 0.662 Intercept 1.187 0.211 5.615 .000 0.976 1.398 Table 2. Larvicidal activity of the tested doses of extracts against Culex pipiens by feeding technique after 72h Plant Concentration (g/L) Log Concentration Observed Responses Expected Responses Residual Probability Thyme 0.02 -1.699 15 15.935 -0.935 0.398 0.05 -1.301 24 22.590 1.410 0.565 0.1 -1.000 28 27.392 0.608 0.685 0.2 -0.699 30 31.518 -1.518 0.788 0.3 -0.523 34 33.512 0.488 0.838 Dill 0.02 -1.699 14 13.000 1.000 0.217 0.05 -1.301 26 24.883 1.117 0.415 0.1 -1.000 30 35.091 -5.091 0.585 0.2 -0.699 46 44.413 1.587 0.740 0.3 -0.523 50 48.883 1.117 0.815 Basil 0.02 -1.699 12 12.263 -0.263 0.307 0.05 -1.301 17 18.262 -1.262 0.457 0.1 -1.000 26 23.026 2.974 0.576 0.2 -0.699 28 29.893 -1.893 0.747 0.3 -0.523 33 32.500 0.500 0.812 J Arthropod-Borne Dis, March 2017, 11(1): 147–155 EA El Zayyat et al.: Bioefficacy of Some … 151 http://jad.tums.ac.ir Published Online: March 14, 2017 Table 3. Probit analysis of adulticidal activity of the tested extracts against Culex pipiens by feeding technique after 72h Table 4. Adulticidal activity of the tested doses of extracts against Culex pipiens by feeding technique after 72h Plant Concentration (g/L) log Concentra- tion Observed Responses Expected Responses Residual Probability Thyme 10 1.000 9 8.514 0.486 0.284 20 1.301 14 15.016 -1.016 0.501 40 1.602 22 21.514 0.486 0.717 Dill 10 1.000 7 7.201 -0.201 0.240 20 1.301 18 17.573 0.427 0.586 40 1.602 26 26.183 -0.183 0.873 Basil 10 1.000 14 12.883 1.117 0.429 20 1.301 16 18.221 -2.221 0.607 40 1.602 24 22.953 1.047 0.765 Table 5. Probit analysis of adulticidal activity of the tested extracts by CDC bottle bioassay after 24h Plant Parameter Mortality rate Standard Error Z Sig 95% Confidence Interval Pearson Goodness of Fit Test Lower Bound upper Bound Chi- Square Sig Thyme Slope 1.903 0.571 3.334 .001 0.785 3.022 0.215 0.643 Intercept -2.475 0.754 3.281 .001 -3.229 -1.721 Dill Slope 3.066 0.636 4.823 .000 1.820 4.311 0.042 0.837 Intercept -3.772 0.824 4.579 .000 -4.595 -2.948 Basil Slope 1.496 0.568 2.632 .008 0.382 2.610 1.063 0.303 Intercept -1.674 0.741 -2.26 .024 -2.415 -0.933 Plant Parameter Mortality rate Standard Error Z Sig 95% Confidence Interval Pearson Good- ness-of-Fit Test Lower Bound Upper Bound Chi- Square Sig Thyme slope 2.750 0.570 4.825 .000 1.633 3.868 3.383 0.184 Intercept 0.988 0.233 4.246 .000 0.755 1.221 Dill Slope 2.964 0.595 4.984 .000 1.799 4.130 2.465 0.292Intercept 0.906 0.232 3.911 .000 0.674 1.138 Basil slope 3.247 0.592 5.480 .000 2.085 4.408 1.011 0.603 Intercept 1.561 0.262 5.967 .000 1.300 1.823 J Arthropod-Borne Dis, March 2017, 11(1): 147–155 EA El Zayyat et al.: Bioefficacy of Some … 152 http://jad.tums.ac.ir Published Online: March 14, 2017 Table 6. Adulticidal activity of the tested doses of extracts against Culex pipiens by CDC bottle bioassay after 24h Plant Concentration (g/L) log Concen- tration Observed Responses Expected Responses Residual Probability Thyme 0.2 -0.699 7 5.252 1.748 0.175 0.4 -0.398 10 13.729 -3.729 0.458 0.6 -0.222 19 19.418 -0.418 0.647 0.8 -0.097 25 22.941 2.059 0.765 Dill 0.2 -0.699 5 3.654 1.346 0.122 0.4 -0.398 9 11.765 -2.765 0.392 0.6 -0.222 17 17.941 -0.941 0.598 0.8 -0.097 24 21.958 2.042 0.732 Basil 0.2 -0.699 8 7.182 0.818 0.239 0.4 -0.398 16 18.183 -2.183 0.606 0.6 -0.222 25 23.994 1.006 0.800 0.8 -0.097 27 26.811 0.189 0.894 Discussion Natural insecticides are of growing interest worldwide being eco-friendly as with climatic changes and urbanization offering new breed- ing areas for vectors, it is becoming manda- tory to meet the needs for screening of plants potential insecticidal effect. Plants phytochem- ical compounds have demonstrated a prom- ising potential for insecticidal activity. The toxicological safe nature of most essential oils and its availability in developing coun- tries with huge biodiversity and complex mix- tures of its constituents resulting in more slow- ly development of resistance may ultimately have their greatest impact in future insects control programs (Koul et al. 2008). The usage of plant crude extracts as a whole besides being less expensive, it ad- vances the synergistically of its active com- pounds complex mixtures providing greater bioactivity compared to its purified individ- ual constituents (Cavalcanti et al. 2004). Sev- eral studies reported the botanical insecti- cidal effect on Culex mosquito with variation depending on geographical origin of the plant, the plant parts processed, type of sol- vent used and the Culex species tested (Jeya- balan et al. 2003). The mortality effect of plant extracts evaluated in this study varied according to the plant, concentration of the extract and the used technique. Highly significant linear predictions from the resultant probit models were obtained as shown by z-scores. There was a non-signifi- cant difference on comparing the observed and expected response by chi-square good- ness of fitness test (Tables 1, 3 and 5) so as- sumptions made by this study were reasona- ble and the choice of models were appropri- ate and the frequency counts distributed iden- tically across different populations. Dose response relationship of the tested extracts showed increase in the mortality rate with increasing the tested doses of extracts with non-significant difference between the observed value of mortality and the predict- ed value, the residual (Tables 2, 4 and 6) in- dicating the appropriateness of the used mod- els. The mechanism of toxicity of essential oils on insects needs further study to be clar- ified. Comparing these results to other stud- ies on same extracts may vary in results as variation of plant essential oil composition according to its indigenous origin, target mos- quito species tested, method tested, different exposure times and extract concentrations used (Kumar et al. 2011). In larvicidal assay of the tested extracts by feeding technique after 72h, basil was J Arthropod-Borne Dis, March 2017, 11(1): 147–155 EA El Zayyat et al.: Bioefficacy of Some … 153 http://jad.tums.ac.ir Published Online: March 14, 2017 highly effective (LC50= 0.064) followed by thyme (LC50= 0.070) and dill (LC50= 0.071). Concerning adulticidal activity of the tested extracts by feeding technique after 72h basil was highly effective (LC50= 13.148) fol- lowed by dill (LC50= 16.996) and thyme (LC50= 19.967) and regarding adulticidal ac- tivity of the tested extracts by CDC bottle bioassay, no mortality recorded after 2h but after 24h basil was highly effective (LC50= 0.330) followed by thyme (LC50= 0.437) and dill (LC50= 0.495) according to the lowest LC50 and in return the highest toxicity indi- ces. So Ocimum basilicum was the most ef- fective extract tested on Cx. pipiens larvae and adults in accordance to other studies as Aarthi and Murugan (2010), Belong et al. (2013) and Govindarajan et al. (2013). Conclusion The integrated usage of indigenous An. graveolens, Oc. basilicum and Th. vulgaris in control of Cx. pipiens would probably help in reducing the magnitude of vector borne diseases. They provide promising results as insecticides besides being cheap, available and easily handled. 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