Bioactivity of plant extracts on the larval and pupal stages of Aedes aegypti ( Diptera , Culicidea ) Lafayette

Introduction: Aedes aegypti is responsible for the transmission of the dengue and yellow fever viruses. This study evaluated the effects of extracts from Cnidosculos phyllacanthus, Ricinus communis, and Coutarea hexandra on the developmental periods of A.aegypti larvae and pupae. Crude extracts of C. phyllacanthus and C. hexandra and oil from R. communis and C. phyllacanthus were used. Methods: Bioassays of the larvicidal and pupicidal effects of these products at different concentrations and times of exposure were evaluated. The lethal and sublethal effects were determined using different concentrations in larvicidal tests. Mortality data were evaluated by Probit analysis to determine the LC50 and LC90 values. Results: The vegetable oils from C. phyllacanthus and R. communis demonstrated greater effi ciency for larval control with an LC50=0.28μl/mL and an LC90=1.48μl/ mL and LC50=0.029μl/mL and a LC90=0.26μl/mL, respectively. In pupal tests toxic effects for all insects were verifi ed after exposure to the products at signifi cant LC50 and LC90 values for 24 and 48h. The effects of sublethal concentrations of C. phyllacanthus (oil) were more effective on the insects. Conclusions: The vegetables oils from C. phyllacanthus and R. communis demonstrated greater potential from the control of different developmental periods in the life cycle of this insect.

Aedes aegypti, Diptera, Culicidae (Linnaeus, 1762); can be found in human dwellings where it obtains food, copulates and spawns.As ubiquitous insect, this species exhibits a high ability to adapt to artifi cial habitats allowing for their occupation and expansion and, consequently, emergence of epidemias 1 .The highest epidemiologic importance of this study is that mosquitoes play a role as transmitters of yellow fever and dengue virus 2 .
The control of the dengue vector and important insect species related to public health has been managed in the last decades mainly with synthetic chemical insecticides.The intense use of these synthetic compounds has many exposed mosquito populations to intense selective pressure and hence the prevalence of some populations are resistant to products used to control these vectors.Thus, the use of secondary metabolites of plants that have insecticidal potential is being studied to minimize the impact of synthetic compounds on the environment and human health 3 .
The close relationship between plants and insects and the possible coevolution of plants led to the development of strategies to attack these invertebrates that, are physical or chemical in nature 4 .Thus, Cnidoscolus phyllacanthus, Ricinus communis (Euphorbiaceae) and Coutarea hexandra (Rubiaceae) are chemical compounds whose biological activities have been described [5][6][7][8] .
In view of the operational and economic difficulties generated by increased mosquito resistance to synthetic insecticides, alternative methods have gained increasing attention 9 .Several plant species have been investigated and tested with potential larvicides directed against several insect species including Aedes aegypti, Magonia pubescens 10 , Atlantia monophylla 11 , Cybistax antisyphilitica 12 , Anacardium humile 13 and Spathelia excelsa 14 .In this study we report the insecticidal activity (larval and pupal) of C. phyllacanthus, C. hexandra and R. communis on Aedes aegypti.

METHODS
The population of A. aegypti was collected in the District of Monte Santo, Campina Grande, State of Paraíba, Brazil.The collection and establishment of strains in the laboratory occurred between December 2009 and August 2010.Eggs were collected with 50 ovitraps that were installed inside and outside homes distributed in ten blocks where fi ve traps were installed per block.

Aedes aegypti in the laboratory
The laboratory bioassays were conducted in a temperaturecontrolled room (26±2°C and 12h light).Vanes eucatex containing A. aegypti eggs from the fi eld were dried for 48h and then packed in white plastic trays (40x27x7.5cm)with a

RESULTS
third of its capacity fi lled with non chlorinated water.After the outbreak, ornamental fi sh food (Goldfi sh growth) was added (100mg/tray).Adults were maintained in cages with a wooden frame coated with organza (40cm x 40cm x 30cm) containing 200 individuals (100 males and 100 females).Adults were fed with a 20% honey solution and the females were fed blood from the quail Coturnix japonica three times a week for 30min.After meals were introduced inside each cage, a disposable cup containing 200ml of distilled water with a plastic funnel coated with fi lter paper served as a substrate for oviposition.

Collection and preparation of solutions
The ethanolic fraction was obtained from cauline tissue from C. phyllacanthus and C. hexandra, which was placed in an incubator at 40°C for 48h.When dry, the shells were crushed and sieved.The powder was moistened with 90% ethanol and subsequently percolated for cold extraction for 48h.After this period, the ethanolic fractions were collected and concentrated by spin evaporation to yield extracts.Seeds from C. phyllacanthus and R communis were used for the extraction of vegetable oil.These were fi rst crushed and macerated and then subjected to a hydraulic press in the cold.The fi xed oils were stored in glass bottles covered with aluminum foil and kept in the refrigerator.

Larvicidal and pupicidal bioassays
Larvicidal and pupicidal test were performed according to the methodology of the World Health Organization 15 with some adjustments.The biological activity of the extracts of the specimens used in the laboratory was found in late L 3 and/ or early L 4 larval and pupal stages of populations of A. aegypti to obtain of the lethal concentrations (LC 50 and LC 90 ) values.We used 30, 60, 125, 250 and 500mg/mL of ethanolic fractions diluted in 1mL of dimethyl sulfoxide (DMSO).For vegetable oils, a 1:1 ratio with tween 20 was made at the following concentrations: 100, 190, 380, 750 and 1.5µl/mL.DMSO and tween and water were used as controls.For each concentration there were four replicates with 25 individuals per replicate, and mortality assessed was after 24 and 48h of exposure.For pupal tests, the occurrence of adults, if they were dead, was counted as pupae.
Effect of sublethal (lethal concentrations of cumulative effect), plant extracts were established via the LC 50 and LC 90 values determined with the test larvicide after 48h of exposure to plant products.There were four replicates with each containing 30 third instar A. aegypti larvae.Daily readings were made with verifi cation of the larval stage, behavior changes, the presence of exuvia, adult emergence, possible mortality of the larvae, pupae and adults, and the water temperature.The experiment was conducted until the last pupa or adult died or until the last adult completely emerged.
Mortality data for the larvae and pupae of the studied population were subjected to Probit analysis using the Polo-PC program to determine the lethal concentrations (LC 50 and LC 90 ).There was no need to correct the data with the Abbott formula 16 .
With respective to the larvicidal activity of the plant products analyzed, it was observed that after 24 and 48h of exposure, only vegetable oils demonstrated relevant results against L3 A. aegypti larvae.Tests including vegetable oils from R. communis and C. phyllacanthus on L3 A. aegypti larvae proved to be the most promising for the control of the mosquito larvae, which was confi rmed by the LC 50 =0.029µl/mLand LC 90 =0.261µl/mL and LC 50 =0.288µl/mLand LC 90 =1.485µl/mL values obtained after 24h of exposure.These tests indicated that lower concentrations are needed for these products to obtain a larval mortality of 50-90% of the population (Table 1).Among the species studied, oil from R. communis was the most effective with regard to lethal concentrations (LC 50 and LC 90 ) and had lower values compared to other species studied.The X 2 values indicated the adequacy of the data for the entire experiment using the Probit model (p<0.95).
Seed oil from C. phyllacanthus also showed larvicidal potential after 24h of exposure.However, crude extract made from the bark of this vegetable demonstraded activity after 24 and 48h of exposure with larvae of this vector when the values from the LC 50 and LC 90 levels were obtained (Table 1).
The results obtained from a crude extract from the stem of C. hexandra allowed us to deduce that these dosages treat yourself a non-toxic plant on L3 stage A. aegypti because they could not be estimated after 24h of exposure lethal concentrations LC 50 and LC 90 (Table 1).
After exposing the larvae to vegetable products for 24 and 48h, it was observed that oil extract from the seeds of R. communis had the lowest lethal dose (LC 50 =0.016μl/mLand LC 90 =0.082μl/mL).However, for fractions extracted from the stems of C. phyllacanthus and C. hexandra after 48h of treatment, it was noted that despite a considerable reduction in the LC 50 and LC 90 values compared with the test performed after 24h, the levels were still considered high for this mosquito larval control (Table 1).
To evaluate potential overlaps in the larvicide behavior induced by the studied extracts was performed principal component analysis (PCA).The analysis demonstrated that in PC1/PC2, there was a split into three groups (C.hexandra with C. phyllacanthus (stem), R. communis and C. phyllacanthus (oil)) which explained 98% of the variance in the data.This result alloweds us to infer that there were differences in the behavior induced by larvicidal oils and extracts from the stem but not from the extracts of C. phyllancanthus (stem) and C. hexandra (stem) which formed a single group (Figure 1).
For both PCs, the natural products analyzed were completely separate from the toxicity of the plant extracts.When evaluating PC2, which apresented only 5% of the variance the data, the plants had a dimension of differential larvicidal behavior.In agreement with this result the LC 50 and LC 90 values were not the same between treatments, they quantitatively differed in the dose-response (Figure 1).When assessing the effect of plant extracts on the pupal stage of A. aegypti, all results were signifi cant with LC 50 and LC 90 values thet were more promising than those obtained by the larvicide test.The larvicide test with vegetable oils R. communis and C. phyllacanthus demonstrated greater effi ciency in controlling the pupae of this vector, which was confi rmed by the (LC 50 and LC 90 values) obtained after 24h of exposure (Table 2).Oil extracted from the seeds of C. phyllacanthus was among the products evaluated, which demonstrated greater pupicidal activity with lower LC 50 and LC 90 value after 24 and 48h of exposure.
A greater interaction between the plant products and pupae of A. aegypti was observed when evaluating extracts made after 48h of exposure, which resulted in lower lethal doses capable of killing 50 and 90% of the population.Thus, lethality was enhanced with increasing time of exposure to plant products.
With regard to the species evaluated after 48h of treatment R. communis extract effi ciently killed mosquito pupae, resulting in a mortality rate ranging from 50 to 90% for the lower stage and with small LC 50 and LC 90 values (Table 2).During this period of exposure (48h), it was found that adults at all concentrations and treatments were susceptible to the extracts tested verifying mortality in all replicates.This newly emerged adult mortality might be related to the action of contact with plant extracts present in solutions for the adult mosquito.Thus, one can conclude that the solutions tested negatively affected adult emergence.
Assessment of the activity pupicidal of plant extracts via analysis of PC1 versus PC2 with 95% of variance shows that there were no differences in the mortality response between treatments after 24h of exposure.In Figure 2, all samples were blended.The toxicity of the products was the same for the pupal stage of this vector.Therefore, at this stage of the life cycle of A. aegypti, both of the crude vegetable oils demonstrated effi ciency in controlling this vector.
The sublethal effects of C. phyllacanthus (oil) LC 50 =0.067μl/mLlasted 28 days with a larvicide cumulative mortality of ± 84.1% (Table 3).During the experiment it was observed that the larvae were able to develop and reach the adult stage.In addition, the temperature of the water throughout the experiment was verifi ed to be approximately 22ºC.
The concentration that signifi cantly reduced the rate of adult emergence of A. aegypti was the LC 90 (0.39μl/mL) of C. phyllacanthus (oil).For this extract, the duration of the experiment was 14 days, and for the 120 larvae exposed to this concentration, there was a mortality rate of ± 88.3%.The effi ciency in reducing adult emergence helped to confi rm that at this dose ± 11.7% of the larvae developed to the pupal stage and only 0.9% became viable adults (Table 3).With regard to a treatment with an LC 50 (0.067µl/mL) of C. phyllacanthus, of the larvae that did could develop, 19 turned into pupae, but there was a 5% mortality rate even at this stage, and only ± 10.9% reached adulthood (± 3.1% died in this stage).
The sublethal effects of R. communis at an LC 50 of 0.016μl/mL lasted approximately 33 days, and the LC 90 concentration lasted 24 days.At the end of the 33 day exposure to 120 larvae, the mortality rate was ± 87.5% LC 50 R. communis.Between those that managed to develop, 10 became pupae with only one death.The highest number of emerged adults (11.7%) and ± 1.6% mortality was recorded of the (LC 50 ) of the R. communis extract.This was the most effective treatment for the larvae of A. aegypti with a mortality rate of 87.5%.The highest larval  mortality observed for the larvicide test was obtained with a LC 90 of 0.082μl/mL, where at the end of 24 days, the larval mortality was ± 93.3%.The remaining ± 6.7% developed into pupae, only one death was recorded, and 5.9% emerged into adults and ± 2.3% died at this stage.
The control experiment lasted 35 days with only fi ve larval mortalities (± 4.1%).If the mortality of the control group was between 5 and 20%, it was be adjusted using the Abbott formula or the bioassay would have been repeated according to recommendations of the World Health Organization 17,18 (Table 3).Moreover, there was no mortality for pupae and adults, verifying the emergence of 95.9% viable adults.

DISCUSSION
The larvicidal activity of the R. communis oil can be attributed to its chemical components which include the a derivative of ricin ricinoleic acid, a toxic substance that is present in abundance in the seed oil of this plant 7 .This constituent had a negative effect on the larval of this vector.No studies have reported on the effects of R. communis on A. aegypti; however, the insecticidal effect of this vegetable was evaluated in other species of insects.Hebling 19 , assessed the bioactivity of R. communis on ants and demonstrated its effi cacy in combating Hymenopteran, Burg and Mayer 20 tested the effect of R. communis seed oil on aphids and different species of lice, describing how promising this oil was for the control of these insects.In a study of the effects of aqueous extract from the green fruits of castor on the larvae and pupae of Spodoptera frugiperda, Santiago 21 observed a reduction in the lifetime of these stages, and Rother 22 identifi ed toxic effects of leaf extracts of R. communis on Apis mellifera worker larvae.
The plant extracts studied here showed greater effi ciency in the control A. aegypti pupae.The promising effects of these products on the pupal stage of A. aegypti are related to the morphological differences between the pupae and larvae of this insect, indicating that the mode of action of these products is by contact or choking and not swallowing, because the pupal stage does not involve ingestion.A curious observation was made with crude extracts from C. hexandra and C. phyllacanthus the concentrations studied were nontoxic to the L3 larvae of this vector, but were lethal to the pupae.Furthermore, there was similarity between the pupal mortality of products C. phyllacanthus (stems and seeds) after 24h.According to Santos 23 despite being a source of extracts from different parts of the plant chemicals biosynthesis is not restricted to one part of the plant; thus, the products are accumulated in the whole plant or different organs because of an intercellular system.
There are several studies of the activity of plant extracts on A. aegypti, mainly examining its larvicidal action.However, there are few studies of the effect of natural products on the pupal stage.Thus, we found no studies in the literature involving these species and their effects on the pupal stage of A. aegypti.However, studies of the pupicidal activity of other plant species have been reported by Macchioni et al. 24 with Condonopsis javanica on the dipteran Aedes albopictus, demonstrating 75% mortality of pupae at a concentration of 60.000ppm.Using the extracts of leaves and seeds from Melia azedarach on Anopheles stephensi, Nathan 25 found a 92.3 and 90.9% mortality rate, respectively, at 20,000ppm.Nathan 26 also tested Eucalyptus tereticornis on Anopheles stephensi and found a 160ppm 88% mortality for pupae.Although different material was used in this study, the results discussed above only corroborate the need for development studies using plant extracts to evaluate their effects on different stages of A. aegypti.
Regarding the effects of the sublethal plant extracts studied, we observed that although there was a small immediate response for some of the products analyzed, when evaluating larval development, these products interfered with the in timing and number of hatched adult insects.The sublethal effects of chemical compounds on natural specimens of A. aegypti were investigated by Shaalan 27 who evaluated the effect of synthetic insecticides and Callitris glaucophyllam extracts and various chemical insecticides Adanan et al. 28 and Kamaraj 29 showed that, while not fatal, these sublethal concentrations lead to a disorder in insect biology, both morphologically and physiologically, interfering with the duration of the larval period and the pupae exposed to these concentrations, refl ected in the emergence of adults.
These results need corroboration further, in addition to the more detailed study on the insecticidal properties with phytochemicals present in R. communis and C. hexandra and C. phyllacanthus.The next challenge consists identify chemical compounds that were responsible for the larval and pupal mortality and control programs in the future serve as a tool to control of the actions to A. aegypti.

FIGURE 1 -
FIGURE 1 -Graph scores for PC1 versus PC2 for the fi ve tested concentrations of plant extracts after 24h of exposure to Aedes aegypti.PC: principal component.

FIGURE 2 -
FIGURE 2 -Graph scores for PC1 versus PC2 for the fi ve tested concentrations of plant extracts after 24h of exposure for Aedes aegypti larvae.PC: principal component.

TABLE 3 -
Duration of the experiments for sublethal effects of the vegetable oils Cnidoscolus phyllacanthus and Ricinus communis at the lethal LC 50 and LC 90 concentrations for Aedes aegypti.Total of was 120 L 3 larvae of this vector were in each treatment.