Research Report
In Vitro Effects of Some Plant Extracts on the Development of Culex quinquefasciatus in the Laboratory 
Author Correspondence author
Journal of Mosquito Research, 2016, Vol. 6, No. 36 doi: 10.5376/jmr.2016.06.0036
Received: 29 Nov., 2016 Accepted: 19 Dec., 2016 Published: 22 Dec., 2016
El Maghrbi A.A., 2016, In vitro effects of some plant extracts on the development of Culex quinquefasciatus in the laboratory, Journal of Mosquito Research, 6(36): 1-7 (doi: 10.5376/jmr.2016.06.0036)
Control mosquito vectors concentrated mainly on the application of broad spectrum insecticides but causes development of resistance in vector species and environmental pollution. Ethanol and acetone extracts of five species of plants, Euphorbia cotinofolia, Melia azedarach, Ocimum canum, Ricinus common and Tagetes erecta were tested in respect to their influence on the development of Cx. quinquefasciatus in the laboratory. The plants extracts were prepared by agitating the dried and ground in ethanol and acetone separately for 24 hours followed by filtration and later recuperation of solvent using rotavapor. Ten experiments were conducted, four replications in each case. For each experiment, concentration of 100 mg/L, 10 mg/L and 1 mg/L in distilled water of each plant extract was used (150 ml). 25 first stage larvae were putted in each dish of experiment and control after removing the water through the filter paper to avoid alteration the concentration of extracts. The developments of larvae were daily observed and all changes in the experimental dishes and controls were recorded. The duration of the development of pre-imaginal stages of Cx. quinquefasciatus were prolonged by both of extracts of all plants at high concentration. Also acetone extract of E. cotinofolia and ethanol extract of R. communis principally at 10 mg/L in L3 and L4. In addition, Acetone extract of R. communis and T. erecta were more effective at 10 mg/L and 1 mg/L. Mortality of larvae during development was increased in 100 mg/L of ethanol extract of E. Cotinofolia and both extracts of M. azedarach. Acetone extract of R. communis at 100 mg/L killed all larvae of first stage. The extracts of T. erecta caused high mortality in ethanol and acetone extracts 95% and 100% respectively. The results indicate that the natural insecticides could be taken in the place of synthetic insecticides and save our environment. Due to diversity of response presented, suggesting details on level of chemical of these products, for the possibility of isolation the real substances that have value for control of mosquitoes.
1 Introduction
Mosquitoes act as a vector for most of the life threatening diseases like malaria, yellow fever, dengue fever, filariasis, encephalitis, etc. These diseases produce significant morbidity and mortality in humans and livestock around the world. Mosquito-borne diseases have an economic impact, including loss in commercial and labor outputs, particularly in countries with tropical and subtropical climates; however, no part of the world is free from vector-borne diseases. The continuous application of synthetic insecticides causes development of resistance in vector species, biological magnification of toxic substances through the food chain and adverse effects on environmental quality and non-target organisms including human health. Application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times. Plant extracts may be alternative source to control mosquito larvae, many researchers have reported on the effectiveness of plant extracts or plant oils against mosquito larvae (Rasheed et al., 2005; Sharma et al., 2006). The larvicidal, ovicidal, and repellent potential of the ethanolic crude extracts from the medicinal plants were investigated by Dhanasekaran et al. (2013) against the medically important mosquito vectors. They concluded that among the five plant extract, Gnetumula and Spermacoce hispida have significantly higher larvicidal, ovicidal and repellent activity against selected human vector mosquitoes Anopheles stephensi, Aedes aegypti and Culex tritaeniorhynchus. Sukumar et al. (1991) listed and discussed 344 plant species that only exhibited mosquitocidal activity. Biologically active plant extracts are therefore being studied for their potential efficacy to minimize the extent of pollution and for its activities as larvicidal, ovicidal, influence on oviposition behavior and on the growth of pre-imaginal stages. The acetone extracts of Ageratum conyzoides, Cleome icosandra, Tagetes erectes, and Tridax procumbens showed growth inhibitory and juvenile hormone mimicking activity to the treated larvae of Cx. quinquefasciatus (Saxena et al., 1992). The acetone, chloroform, and methanol leaf extracts of Ocimum canum, O. sanctum, and Rhinacanthus nasutus (Kamaraj et al., 2008); and the latex and stem bark of Euphorbia tirucalli (Yadav et al., 2002) have been tested against the larvae of Cx. quinquefasciatus.
Laboratory evaluation revealed that the treatment of larvae of Ae. aegypti and Cx. quinquefasciatus with ethanol and aqueous extract of Pongamia glabra seed kernels significantly increased the larval mortality and developmental period proportionately with increase in the extract concentrations (Sagar and Sehgal 1996; Sagar et al., 1999). In addition, Pelargonium citrosa (Jeyabalan et al., 2003), and Mentha piperita (Ansari et al., 2000) were shown to contain larvicidal and growth inhibitory activity against An. stephensi. Cx. quinquifasciatus is one of the species essentially domestic, per domestic and the principle vector on Wuchereria bancrofti in the world (Pessoa and Martins 1988).The present investigation was undertaken to study the activities of some plant extract on the development of Cx. quinquefasciatus in the laboratory.
2 Materials and Methods
Adult male and female mosquitoes (Cx. quinquifasciatus) were maintained in cages transparent and screen (40×40×40 cm) in the laboratory containing recipient with dechlorinated water for the oviposition of females and 5% honey solution. Female had sucking blood on pigeon. Five species of plants, Euphorbia cotinofolia (leaves and steam), Melia azedarach (leaves, steam and roots), Ocimum canum (leaves and steam), Ricinus common (fruits and seeds) and Tagetes erecta (fruits and branches) were selected on the basis of its biological activity on mosquito or other organism, facility of obtaining and plenty in nature and identified according to Marbberly (1987). Plant extracts were prepared by agitating the dried and ground plant parts in ethanol and/or acetone separately for 24 h followed by filtration through filter paper and later recuperation of solvent using rotary evaporator. For each plant extract (ethanol or acetone), concentration of 100 mg/L, 10 mg/L and 1 mg/L in distilled water were used. Four replications were conducted in each experiment. For each replication, a dish of control was used contained distilled water. Twenty five first stage larvae were putted in each dish of experiment and control (150 ml/9.5 cm in diameter) after removing the water through the filter paper to avoid alteration the concentration of extracts. The developments of larvae and pupae were followed daily and all changes in the different concentrations and control were recorded until the adult emerged. The dead larvas and pupae were removed. The Medias which contain the larvae were changed completely every three days. The developed larvae were feeding on grinding, sieved and autoclaved ration of cats with equal quantity according to AMCA (1970).
3 Results
The activities of ethanol and acetone extracts in different concentrations of five plants tested on the development of pre-imaginal stages of Cx. quinquefasciatus are summarized in Figure 1; Figure 2; Figure 3; Figure 4; Figure 5; Figure 6; Figure 7; Figure 8; Figure 9; Figure 10. (The different letters indicating the differences significantly occurred inside each stage evolution) and the mortalities occurred during the same experiments (Table 1; Table 2; Table 3; Table 4; Table 5; Table 6; Table 7; Table 8; Table 9; Table 10). The duration of the development of pre-imaginal stages of Cx. quinquefasciatus were prolonged by both of extracts of E. cotinofolia, principally acetone extract (10 mg/L) in L3 and L4. Also, ethanol extract at concentration 100 mg/L increase mortality of larvae during development (Figure 1; Figure 2) and (Table 1; Table 2). M. azedarach in both extract of high concentrations, the time of the growth of Cx. quinquefasciatus was increased beside the concentrations 10 and 1 mg/L showed capacity to interfere in some stages. Ethanol and acetone extracts of this plant in 100 mg/L caused high mortality of larvae during development (Figure 3; Figure 4) and (Table 3; Table 4). In both extracts of O.canum, the development was delayed in 100 mg/L and still at 10 mg/L showed real differences in various stages. The two extract of this plant was not shown significant mortality during development of Cx. quinquefasciatus (Figure 5; Figure 6) and (Table 5; Table 6).
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Figure 1 Effect of ethanol extract concentrations of Euphorbia cotinofolia on the development of Culex quinquefasciatus |
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Figure 2 Effect of acetone extract concentrations of Euphorbia cotinofolia on the development of Culex quinquefasciatus |
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Figure 3 Effect of ethanol extract concentrations of Melia azedarach on the development of Culex quinquefasciatus |
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Figure 4 Effect of acetone extract concentrations of Melia azedarach on the development of Culex quinquefasciatus |
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Figure 5 Effect of ethanol extract concentrations of Ocimum canum on the development of Culex quinquefasciatus |
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Figure 6 Effect of acetone extract concentrations of Ocimum canum on the development of Culex quinquefasciatus |
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Figure 7 Effect of ethanol extract concentrations of Ricinus communis on the development of Culex quinquefasciatus |
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Figure 8 Effect of acetone extract concentrations of Ricinus communis on the development of Culex quinquefasciatus |
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Figure 9 Effect of ethanol extract concentrations of Tagetes erecta on the development of Culex quinquefasciatus |
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Figure 10 Effect of acetone extract concentrations of Tagetes erecta on the development of Culex quinquefasciatus |
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Table 1 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of ethanol extract of Euphorbia cotinofolia and in control (0) |
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Table 2 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of acetone extract of Euphorbia cotinofolia and in control (0) |
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Table 3 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of ethanol extract of Melia azedarach and in control (0) |
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Table 4 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of acetone extract of Melia azedarach and in control (0) |
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Table 5 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of ethanol extract of Ocimum canum and in control (0) |
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Table 6 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of acetone extract of Ocimum canum and in control (0) |
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Table 7 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of ethanol extract of Ricinus communis and in control (0) |
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Table 8 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of acetone extract of Ricinus communis and in control (0) |
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Table 9 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of ethanol extract of Tagetes erecta and in control (0) |
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Table 10 Mortality, males and females of Cx. quinquefasciatus developed in various concentrations (mg/l) of acetone extract of Tagetes erecta and in control (0) |
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The development of Cx. quinquefasciatus was delayed in ethanol extract of R. communis at 100 mg/L and still with 10 mg/L interfered in maturation of L3 and L4. Acetone extract, however, was more effective: at 10 and 1 mg/L interfered in maturation of all stages and a concentration 100 mg/L killed all larvae of the first stage (Figure 7; Figure 8) and (Table 7; Table 8). The growth Cx. quinquefasciatus was prolonged in two high concentrations of ethanol extracts of T. erecta. The same happened in 10 and 1 mg/L of acetone extract. The mortality of larvae was high during development (94% and 100% in 100 mg/L in two extracts respectively) (Figure 9; Figure 10) and (Table 9; Table 10). None of vegetable extracts utilized in testes the development resulted real differences between number of males and females emerged.
4 Discussions
The environmental safety of an insecticide is considered to be of paramount concern. Botanical derivatives have drawn attention as potential insect control agents and as a source of new pesticides. The influence of vegetable extracts on the population of mosquitoes were experimented by number of authors with different forms; Supavaran et al. (1974) reported that between 36 methanol extracts, 12 reduced the emerging of the adults, 11 inhibited significantly the development of larvae and 10 prolonged the time of growth of the larvae of Ae. aegypti. The aqueous extract obtained with organic solvent of A. indica caused extreme prolongation of larval period, when L1 exposed to these extracts, the author suggested that the component of Azadirachtin and others presented in this vegetable being capable the interfere in hormonal equilibrium or effect on neuroendocrine control of ecdisteroides (Zebitz, 1984). The water extract of M. volkensii inhibited the growth of second stage larvae of Ae. aegypti in concentrations higher or equal 2 microgrames/ml and LC50 was 50 microgrames/ml (Mwangi and Rembold, 1988). The methanol extract of R. communis was capable to kill all larvae (L4) of Ae. aegypti in three days in concentration of 1 000 ppm (Supavaran et al., 1974). The interference in developmental time or inhibition of the growth from plant chemicals could be also species specific. Sujatha et al. (1988) reported that extracts of Acorus calamus producing greater malformations in An. Stephensi than in Cx. quinquefasciatus and Ae. Aegypti but Madhuca longifolia induced greater development inhibition in Cx. quinquefasciatus. Also, developmental time affected by the solvent used in the method of extract. Sivagnaname and Kalyanasundaram (2004) recorded that methanolic leaf extracts of Atlantia monophylla manifesting insect regulating effectiveness on immature stages of Cx. quinquefasciatus, An. stephensi and Ae. Aegypti. Zebitz (1984) found that growth inhibition and prolongation of larval developmental period effected by azadirachtin as an anti-ecdysteroid.
Fruit extracts of M. azedarach and A. indica produce a variety of effects in insects such as antifeedant, growth retardation, reduced fecundity and moulting disorders (Schmidt et al., 1998; Hammad et al., 2001; Wandscheer et al., 2004). Under laboratory conditions, A. indica extract, was tested against larvae of Cx. pipiens. After treatment of larval stage, LC50 and LC90 values for Azadirachtin were 0.35 mg/L and 1.28 mg/L in direct effect and 0.3-0.99 mg/L in indirect effect, respectively. In addition, fecundity of adult mosquito was decreased and sterility was increased by the Azadirachtin after treatment of the fourth instar and pupal stage. The treatment also prolonged the duration of the larval stage (Alouani et al., 2009).The development retarding effects on Cx. Pipiens larvae by methanol and ether extracts of A. indica, Rhazya stricta and Syzygium aromaticum were investigated by El Hag et al. (1999). They found that all tested plants showed biological activity, however, only 3.3% of the larvae pupated and no adults emerged even at the lowest concentration (200 ppm) of methanol extract. The methanol extracts of A. indica at concentrations above 800 ppm reduced pupation to 3.3% and completely inhibited adult emergence. Both extracts of S. aromaticum were causing complete inhibition of adult emergence at 200 ppm and 600 ppm concentrations of the methanol and ether extracts, respectively. Govindarajan and Sivakumar (2014) recorded that the crude hexane, ethyl acetate, benzene, chloroform and methanol extracts of root of Asparagus racemosus against three important vector mosquitoes, Cx. quinquefasciatus, Ae. Aegypti and An. Stephensi have moderate ovicidal activity; however, the methanol extract showed the highest ovicidal activity. The methanol extract of A. racemosus against Cx. quinquefasciatus, Ae. Aegypti and An. stephensi exerted 100% mortality (zero hatchability) at 375 ppm, 300 ppm and 225 ppm, respectively. All extracts showed moderate larvicidal effects; however, the highest larval mortality was found in methanol extract of root of A. racemosus against the larvae of Cx. quinquefasciatus, Ae. Aegypti and An. Stephensi. On the other effects of vegetable extracts, El Maghrbi and Hosni (2014) reported that Ethanol/acetone extracts of T. erecta at 100 mg/L; E. cotinofolia and O. canum at 100 and 10 mg/L were proved to be repulsive for ovi-position of Ae. fluviatilis. On the other hand, acetone extracts of M. azederach at 100 and 10 mg/L; O. canum, E. cotinofolia and R. communis at 100 mg/L produced same effect on ovi-position behavior of Ae. fluviatilis. Ethanol extracts of E. cotinofolia, R. communis (100 mg/L) and M. azedarach (100 mg/L and 10 mg/L) were attractive to Cx. quinquifasciatus. Acetone extract of M. azedarach was repulsive for ovi-position at 100 mg/L, 10 mg/L and 1 mg/L maintained the same properties.
The results indicate that the natural insecticides could be taken in the place of synthetic insecticides and save our environment from chemical hazards. Further investigations are needed to clarify this activity against vector mosquito species and other insects, also the active ingredient (s) of the extract responsible for duration of the development of pre-imaginal stages of Cx. quinquefasciatus should be identified and utilized as possible in preparing a commercial product to be used as biological control of mosquitoes.
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