Repellent activity of Cnidoscolus phyllacanthus Mart. and Ricinus communis L. extracts against Aedes aegypti L. oviposition behavior

Female Aedes aegypti lay their eggs on nearly any moist substrate. Methods with potential to repel oviposition may reduce infestation, thereby contributing to control of epidemics. We evaluated the inluence of Cnidoscolus phyllacanthus and Ricinus communis plant extracts on the oviposition behavior of A. aegypti. Lethal concentrations were irst determined in experiments with larvae after 24 h of exposure, after which LC50 and LC90 were used to test oviposition repellency. The experiment consisted of an oviposition preference test based multiple-choice and no-choice assays. The Oviposition Activity Indices (OAIs) from the multiple-choice test using both R. communis and C. phyllacanthus were negative, suggesting oviposition repellent and deterrent


Introduction
The control of mosquitoes remains a challenge even after continuous use of synthetic insecticides by the public health sector.The main challenge is development of insecticidal resistance in mosquito vectors, which enables mosquitoes to continue transmitting vector borne diseases in endemic areas (SINGH;MITTAL, 2014).Aedes aegypti has epidemiological importance as the main vector of causative agents of yellow fever and dengue.These viral diseases afect many humans, and are serious public health problems throughout the world (FORATTINI, 2002).
Aedes aegypti is a species found in close association with human habitats, and readily enters buildings to feed and to rest.Adult females preferentially feed on humans; other vertebrate species constitute only a small proportion of their blood meals.Unlike many other mosquito species, A. aegypti is a day-biting mosquito (JANSEN;BEEBE, 2010).Aedes aegypti feeds more than once between successive oviposition events.Moreover, the eggs are the most resistant form in the life cycle and may become quiescent in inhospitable environments (SILVA et al., 2004).Females preferentially lay eggs in man-made or artiicial containers including water tanks, lower vases, potted plant bases, discarded tires, buckets, or other containers typically found around or inside the home (JANSEN;BEEBE, 2010).This aspect has made dengue increasingly common, including the occurrence of an "epidemic mosaic", which includes simultaneous outbreak of numerous viral serotypes (NATAL, 2002).
Vector control strategies typically include the use of synthetic insecticides.The intense use of insecticides has caused substantial numbers of resistant populations which have been described in several Brazilian cities, including Campinas-SP (CAMPOS; ANDRADE, 2001), the Federal District (CARVALHO et al., 2004) Ceará (LIMA et al., 2006) and Paraíba (BESERRA et al., 2007).Thus, alternative methods for control of insects of medical importance deserve greater attention (SIMAS et al., 2004).
Cnidoscolus phyllacanthus is a plant belonging to the Euphorbiaceae family.Structurally, it is composed of several chemical compounds, including favelina methyl ester, which according to Endo et al. (1991a;1991b) has cytotoxic activities.Ricinus communis (Euphorbiaceae), popularly known as the castor oil plant, among other substances contains the alkaloid ricinine, a potentially insecticidal substance (LEITE et al., 2005).The present study evaluated the potential of C. phyllacanthus and R. communis to repel A. aegypti oviposition behavior.

Materials and Methods
Samples of A. aegypti populations were collected in the Monte Santo neighborhood, Campina Grande, Paraíba State, Brazil.Eggs were collected twice with one month between collections, using 50 ovitraps placed both inside and outside of ive houses per block, for a total of ten blocks.

Establishment and maintenance of Aedes aegypti in the laboratory
Laboratory bioassays were conducted in a temperature-controlled room (26 ± 2 °C and 12 h Oviposition behavior of Aedes aegypti front of plant extracts photoperiod) in the entomology laboratory at the Center for Insect Systematics and Bioecology the State University of Paraíba (UEPB).Eucatex wood sheet vanes containing A. aegypti eggs from the ield were dried for 48 h after collection, then placed in white plastic trays (40 x 27 x 7.5 cm) and illed one third of capacity with distilled water.After hatching, 100 mg / tray of ornamental ish food (Goldish growth) was added.Adults were kept in cages (~200 per cage) made of wooden frames covered with organza type fabric (40 cm x 40 cm x 30 cm).Mosquitoes were fed a 20% of honey solution, and females were blood fed using quails (Coturnix japonica) three times a week for 30 min per feeding.After feeding, a disposable cup containing 200 mL distilled water covered with a plastic funnel coated with a paper ilter was places inside of each cage as oviposition substrate.

Collection and preparation extracts of solutions
The seeds used for extraction of the C. phyllacanthus oil were provided by the Agricultural and Technical School of the State University of Paraiba, Lagoa Seca-PB.Ricinus communis seeds were collected in the city of Esperança-PB.The seeds were washed, milled, and subjected to hydraulic cold pressing to extract oils.The ixed oils were stored in glass bottles covered with aluminum foil, and stored in the refrigerator until the beginning of the experiments.
The repellent efect of C. phyllacanthus and R. communis extracts was evaluated using the following lethal doses: LC 50 =0.28μL/mL; LC 90 =1.48 μL/mL and LC 50 = 0.26 μL/mL; LC 90 = 0.029 μL/mL.Doses were veriied with a larvicidal activity bioassay (CANDIDO et al., 2013).Dilutions of the oils were made in a 1:1 ratio (extract: emulsiier).Tween 20 with water was used for controls.The preference test for oviposition of A. aegypti was based on a) a multiple choice test and b) a no-choice test, as follows.

Multiple-choice test
Oviposition repellency activity of C. phyllacanthus and R. communis extracts were evaluated separately in two identical experiments using a randomized block experimental design with three treatments LC 50 and LC 90 , and water replicated six times.Each replicate consisted of a cage made of a wooden screened frame (40.0 cm x 40.0 cm width x 30.0 cm depth) containing ten male and ten female A. aegypti.Mosquitoes were ofered a 20% honey solution and females were blood fed using quails for a period of 30 minutes, for three days in a row prior to initiating oviposition behavior assays.After the third blood feeding, each of the three treatment solutions (LC 50 , LC 90 or distilled water) in 200 mL plastic cups were placed inside each cage.In each cup, a funnel plastic coated with a ilter paper served as oviposition substrate.Cages were checked daily for a period of 72 h, at which time the ilter paper was removed and the number of eggs was counted using a stereomicroscope.

No-choice test
In this test we evaluated oviposition preference following a randomized experimental design, with four replicates per treatment, each replicate consisting of a wooden screened cage (20 cm 3 ) containing ten male and ten female A. aegypti.For each replicate there was only one oviposition substrate available for female mosquitoes.We used the LC50 and LC90 from C. phyllacanthus and R. communis oil as experimental substrates, and water as control.Cages were checked daily following the same procedures as in the multiplechoice test.

Data analysis
Attractiveness or repellency of the solutions for A. aegypti oviposition and preference in multiple-choice test were compared using a Friedman test (P < 0.05), and a Kruskal-Wallis test (P < 0.05) was used to compare results for the no-choice test.The oviposition activity index (OAI) was determined with the following formula (KRAMER; MULLA, 1979).
L. P. Candido e E. B. Beserra in which Nt = number of eggs in the test solution and Nc = number of eggs in the control solution.According to these authors the OAI + ≥ 0.3 indicates attractiveness, while OAI ≤ 0.3 indicates that the solution is repellent to oviposition (TIKAR et al., 2014).

Results
In the multiple-choice test with R. communis and C. phyllacanthus, we observed a clear preference for oviposition in the control solution, where 457 eggs (91%) were deposited in the R. communis test, and 108 eggs (73%) were deposited in the C. phyllacanthus test (Figure 1).In the test using R. communis extract, females select oviposition site based on substrate type (LC 50 , LC 90 or water).In this treatment 12 eggs were laid in one other group, in the LC 50 extract; though the egg number seems small comparatively, this solution repellency with an OAI of -0.9 (Table 1).For all treatments the LC 90 of R. communis showed oviposition deterrent activity against of A. aegypti, which was confirmed by an OAI = -1, where egg-laying was not observed (Figure 1).

C. phyllacanthus
Total number of eggs Cnidoscolus phyllacanthus had lower A. aegypti oviposition repellant activity than did R. communis (Figure 1).However, even though a greater number of eggs were laid in the C. phyllacanthus LC50 solution, the extracts still signiicantly reduced A. aegypti oviposition compared to controls.
Eggs were also laid in the C. phyllacanthus LC50 oviposition substrate, though only in two blocks.Despite diferences found in the average numbers of eggs via a Friedman test (Table 1) for C. phyllacanthus, the deterrent bioactivity of the product against A. aegypti oviposition is not invalidated because there were more eggs in this treatment compared to controls (OAI = -0.7).
Similar behavior was observed for the LC 90 substrate, which showed repellent activity for all experimental blocks (OAI = -1) as in the R. communis tests (Table 1).
The OAI values were negative in the multiplechoice test for both R. communis and C. phyllacanthus.Both extracts showed repellent effects against A. aegypti oviposition, with OAI values lower than -0.3 (Table 1).There were no significant differences between treatments in tests with no choice (x 2 = 0.77, P ≤ 0.05).Oviposition was observed for all concentrations (Figure 2), demonstrating that females vectors may oviposit in environments less favorable to larval development when there is no clean water available.Although oviposition did occur in R. communis and C. phyllacanthus extract solution treatments, these

Total number of eggs
Oviposition behavior of Aedes aegypti front of plant extracts treatments also showed repellent activity since LC 50 and CL 90 values were both negative (Table 2), in agreement with prior results for choice tests.
Ricinus communis oil at LC 90 had higher repellent activity than did the LC 50 despite the increased number of eggs, as indicated by OAI values: LC 50 = -0.5, and LC 90 = -0.4(Table 2).
Aedes aegypti was indiferent to solution type in the no-choice test (p = 0.77 > 0.05) (Table 2).The deterrent efect of the lethal concentrations (LC 50 and LC 90 ) of R. communis and C. phyllacanthus were similar to controls (Figure 2).aegypti (-0.1 and -0.4) for LC50 and LC90 respectively (Table 2).Data from the multiple-choice vs. no-choice tests demonstrate that, when given a choice, A. aegypti avoided treatment solutions and preferred no-treatment controls (Figures 1 and 2).

Discussion
The OAIs in both multiple-choice and no-choice Similar results were observed for Earias vittella (Fab) (Lepidotera, Noctuidae) subjected to methanol extracts from Azadirachta indica and Melia azedarach seeds in the multiple-choice test; females preferred to place a greater number of eggs in the control solution substrate (GAJMER et al., 2002).Chen et al. (1996) also observed that oviposition of Plutella xylostella (L.) (Lepidoptera, Acrolepiidae) was reduced by the presence of aqueous extract from M. azedarach fruits, with a reduction of 49.6%, 86.6% and 93.5% in free-  (TORRES et al., 2006).In this study, R. communis deterred oviposition by A. aegypti to a greater extent than did C. phyllacanthus.
The use of oviposition deterrents and attractants to modify mosquito oviposition behavior has an important role in mosquito control programs.The selection of an oviposition site by gravid mosquito females is a critical factor that determines species proliferation and population densities, and dispersion in diferent geographical areas (TIKAR et al., 2014).Plant tritaeneorynchus.The role of essential oils as oviposition-altering compounds has also been in studied in A. aegypti (WARIKOO et al., 2011).
Although the number of eggs in the nochoice test was signiicantly higher than in the multiple-choice test, the deterrent efect of these extracts on adult A. aegypti is not disregarded.In one study with diferent types of oviposition substrates in which mosquitoes were conined in cages containing only one substrate, there were also a greater number of eggs in the unfavorable treatments compared with multiple-choice assays (BESERRA et al., 2010).When only one choice is available, Tauil (2002) suggest that the adaptive capacity of A. aegypti in diferent environmental situations, including those considered unfavorable for production of ofspring (e.g., the LC 50 and LC 90 plant extracts used in this study) female vectors adapt to the conditions available.
According to Lopes et al. (1993), A. aegypti shows no preference for container type, but prefers environments with water that does not contain high levels of pollutants.In the present work, the females preferred to oviposit in the control solution containing only water.An environment considered favorable for oviposition is associated with conditions that the medium presents that wind velocity may also be inluential.However, the domestic nature of this species probably exerts more inluence on its distribution than either environmental factor (JANSEN;BEEBE, 2010).
Generally speaking, the greater the repellency the lower the infestation, thus reduction or elimination of egg-laying causes a reduction in the number of insects.Our study concludes that C. phyllacanthus and R. communis repel oviposition by A. aegypti.
These results should encourage the search for new active natural compounds, ofering an alternative to synthetic repellents and insecticides from other medicinal plants.

FIGURE 1 :
FIGURE 1: Oviposition response of Aedes aegypti females with in multiple-choice tests with lethal concentrations LC 50 and LC 90 of Ricinus communis and Cnidoscolus phyllacanthus.

FIGURE 2 :
FIGURE 2: Oviposition response of female Aedes aegypti in no-choice tests using lethal concentrations LC50 and LC90 of Ricinus communis and Cnidoscolus phyllacanthus.
L. P. Candido e E. B. Beserra choice tests, and 46.2%, 72.1% and 80.2% in no-choice tests.Therefore, the results indicate similarity of the oviposition behavior of P. xylostella and E. vittella, in both tests who underwent to plants extracts.Nathan et al. (2005) evaluated repellent or deterrent activity of plants on other groups of insects and veriied a strong oviposition repellent activity in M. azedarach seed and leaf methanol extracts on Anopheles stephensi Liston (Diptera, Culicidae) in the laboratory.Medeiros et al. (2005) found 100% deterrent efects of diferent aqueous extracts of Sapindus saponaria L. (Sapindaceae) and Enterolobium contortisiliquum Vell.(Fabaceae) fruits, and Trichilia pallida Sw (Meliaceae) leaves on P. xylostella oviposition in kale leaf discs.The efect of aqueous extracts of Aspidosperma pyrifolium Mart.Bark showed higher repellency than M. azedarach and Azadirachta indica (almond) fruit extracts on P. xylostella oviposition extracts and oils have been reported by several authors to act as mosquito oviposition deterrents.The action of various oils have been studied for use against A. aegypti , C. quinquefasciatus, and Anopheles stephensi (PRAJAPATI et al., 2005); Elango et al. (2010) observed that leaf extracts in diferent solvent deterred oviposition by C.
foster development and survival of immature mosquitoes.Factors such as the color of the vivarium, organic matter, and other substances that favor the development of the immature mosquitoes may serve as stimuli to the female when choosing a location for oviposition (CONSOLI; LOURENÇO-DE-OLIVEIRA, 1994).Humidity and rainfall have overt impacts on mosquito survival and ecology, and other climatic factors such as photoperiod and Oviposition behavior of Aedes aegypti front of plant extracts

TABLE 1 :
Comparison of the mean number Aedes aegypti eggs laid in substrates with plant extracts in multiple-choice test.Analyzed using Friedman test (α = 0.05), with respective chi-square values (x 2 ), degrees of freedom (gl), means values and OAIs.

TABLE 2 :
Comparison of the mean number Aedes aegypti eggs laid in substrates with plant extracts in no choice tests.Analyzed using Kruskall-wallace test (α = 0.05), with respective chi-square values (x2), degrees of freedom (gl), mean values and OAIs.