Abstract
An ethanolic extract of Pedilanthus tithymaloides L. (Euphorbiaceae) leaves was tested for biological activity against the eggs, larvae, and pupae of Culex quinquefasciatus. Significant mortality effects were observed in each life stage. In eggs, these effects ranged from 7% to 14% at the 0.013–0.040% concentrations, respectively. In larvae, the same concentration range induced, respectively, 30–95% and 23–88% mortality in first to fourth instars. In pupae, 0.013–0.040% concentrations of leaf extract induced between 18% and 42% mortality. Fitted probit–mortality curves for larvae indicated the median and 90% lethal concentrations (LC50/LC90) of extract for instars 1–4 to be 0.024/0.042, 0.025/0.043, 0.026/0.045, and 0.028/0.047, respectively. Qualitative analyses of the extract revealed the presence of flavonoids, phenols, and steroids but the absence of alkaloids, glycosides, resins, saponins, and tannins. The results indicate that Pe. tithymaloides leaf extract exhibits significant biological activity against immature stages of Cx. quinquefasciatus and warrants further study for development and use as a natural product–based biocide in disease vector control.
Introduction
One method for managing the incidence of mosquito-borne disease is to reduce vector densities through the use of synthetic insecticides. These chemicals are fast-acting and easy to apply, and their toxicity to a spectrum of mosquito species is well known (Rajavel et al. 1987, Saxena and Kaushik 1988). Malathion, for example, is effective for mosquito control, is relatively inexpensive, and has lower mammalian toxicity than many organochlorine insecticides (Gopalan et al. 1996). Other synthetic insecticides can be expensive and toxic to humans and animals. These limitations, and the development of resistance to insecticides by many mosquito species have encouraged research efforts to develop alternative vector control technologies that use products of natural origin derived from locally available plants (Tawatsin et al. 2001).
Botanical insecticides are generally considered safe, decompose quickly, and are locally available in many parts of the world (Sivagnaname and Kalyanasundaram 2004). More than 2000 plant species are known to have insecticidal constituents (Rawls 1986, Sukumar et al. 1991) that include pyrethrum, rotenone, and nicotine (Balandrin 1985). Today, limnoids from Azardiachtia spp. (Meliaceae) and Gedunin spp. (Rutaceae) are used as insecticides in many areas of the world (Dua et al. 1995, Nagpal et al. 1996).
Extracts from a single plant species, such as neem (Azadirachta indica A. Juss), can manifest a range of activities that include antifeedant, antioviposition, repellent, and growth-regulating properties (Schmutterer 1995). Similarly, extracts of basil (Ocimum basilicum L. and Oc. americanum L.), citronella grass (Cymbopogon nardus [L.] Rendle), clove (Syzygium aromaticum L.), and thyme (Thymus vulgaris L.) have been studied for repellency to mosquitoes (Sharma et al. 1993, Chokechaijaroenporn et al. 1994, Boonyabancha et al. 1997, Barnard 1999).
Extracts from plants of the family Piperaceae possess insecticidal properties (Tawatsin et al. 2001) as do leaf extracts of Atlantia manophylla L. (Rutaceae) against Aedes aegypti L., Anopheles stephensi Liston, and Cx. quinquefasciatus larvae (Sivagnaname and Kalyanasundaram 2004). Oviposition-deterrent and skin-repellent activity has been observed for leaf extracts of Solanum trilobutum L. (Solanaceae) against An. stephensi (Rajkumar and Jebanesan 2005).
Leaf and twig extracts of plants in the family Euphorbiaceae contain chemical constituents, such as monoterpenes, sesquiterpenes, triterpenoids, and polyphenols, that have antimicrobial properties and are used to treat gastrointestinal disorders, fungal infections, and skin disease (Banjo et al. 2006, Nehra 2007). In the present study, we evaluated a range of concentrations of leaf extract from Pedilanthus tithymaloides L., a euphorb native to southern India, for insecticidal activity against immature and adult Cx. quinquefaciatus (Diptera: Culicidae).
Materials and Methods
Rearing and maintenance of Cx. quinquefasciatus
Initially, egg rafts were collected in and around Coimbatore (11°N, 77°E) from stagnant water bodies. These were transported to the laboratory on tap water in plastic containers and allowed to hatch at 29°C ± 2°C in glass trays (18 cm long × 9 cm wide × 3 cm deep) containing 2 L of tap water.
After 24 h, first instars were transferred to clean plastic trays (dimensions as above) with 2 L of tap water and provided 5 g of ground Pedigree® (Hyderabad, India) fish food daily until pupation. Aliquots of 100 pupae were separated from the rearing medium and placed into net cages (42 cm long × 33 cm wide × 39 cm high) for emergence. Adults were allowed access to 10% sucrose solution ad libitum via cotton wick. Female mosquitoes were fed stored human blood using the methods described by Meola and Readio (1987). A glass beaker (10 cm long × 6 cm diameter) containing tap water for oviposition was placed inside each net cage. Eggs rafts were removed daily, and the eggs allowed to hatch and the larvae reared using the methods described above. Mosquitoes were reared and maintained in all cases at ∼29°C, ∼50% relative humidity (RH), and a 12:12 light:dark (L:D) photoperiod.
Preparation and evaluation of phytochemical extract
Leaves of Pe. tithymaloides were collected from sites in and around Coimbatore, brought to the laboratory, washed first with tap water then with distilled water, and hung indoors at 22°C and 85% RH until dry. The leaves were ground to a fine powder (∼200–250 microns') using an electric blender. One-hundred-gram aliquots of the powder were extracted with 300 mL of ethanol for 8 h using a Soxhlet apparatus. The extract was dark green and of gummy consistency. One milliliter of extract was added to 99 mL of acetone to make a 1% stock solution that was used in mosquito bioassays and for phytochemical screening.
Qualitative analysis of the leaf extract was performed using the methods described by Nkere and Iroegbu (2005). These methods tested for the presence of alkaloids, flavonoids, glycosides, phenols, resins, saponins, steroids, and tannins.
Mosquito bioassays
Eggs (n = 100 per replicate), first to fourth instars (n = 100 per life stage, per replicate), and pupae (n = 100 per replicate) of Cx. quinquefasciatus were exposed to five concentrations of Pe. tithymaloides leaf extract ranging from 0.013 to 0.040%. In each case, we used a 250 mL glass beaker (90 mm long × 65 mm diameter) containing 2–6 mL of leaf extract and 148–144 mL distilled water, respectively, to which was added 50 mg fish food. Tests of each concentration of leaf extract were replicated three times for each life stage and instar. Each replicate test for each life stage included a single control comprising distilled water and the appropriate quantity (2–6 mL) of acetone resulting in a final volume of 150 mL. Mortality in all tests was recorded after 24 h. Mortality responses for each life stage/instar were analyzed using analysis of variance procedures with means separation using Tukey's Honestly Significant Difference test (SAS 2003). We used probit analysis (Finney 1971) to evaluate the Pe. tithymaloides extract concentration–response (toxicity) relationship for each life stage and instar of Cx. quinquefasciatus and corrected for control mortality before data analysis using a modified Abbott's formula (Abbott 1925). The level of significance used in all statistical tests was p = 0.05.
Results
Qualitative analyses
The results of phytochemical screening of Pe. tithymaloides indicated the presence of flavonoids, phenols, and steroids in the ethanolic extract. The results of analyses for the presence of alkaloids, glycosides, resins, saponins, and tannins were negative.
Mosquito bioassays
Pe. tithymaloides leaf extract induced a significant (F 4,14 = 71.1, p < 0.0001) decrease in egg survival (Table 1). Mortality in eggs ranged from 7 (±0.6)% (at 0.013%) to 14 (±0.3)% (at 0.014%). Average mortality in the controls was 3 (±1.0)%. There was a significant difference in mean mortality at the 0.013–0.020% concentrations compared with those at the 0.026 and 0.033–0.040% concentrations (Table 2). The range of extract concentrations tested was too low to reliably estimate a probit–mortality curve for the egg stage.
Row mean values followed by the same letter are not significantly different (p = 0.05, Tukey's Honestly Significant Difference test).
Used in Abbott's formula to correct for control mortality before probit analysis.
Significant at p = 0.001 (heterogeneity factor used in calculation of confidence limits).
SEM, standard error of the mean; LC, lethal concentration; LCL, lower confidence limit; UCL, upper confidence limit.
For larvae, the mortality response trend to increasing leaf extract concentration was consistent among instars. This effect was significant for first instars (F 4,14 = 3574.9, p < 0.0001), second instars (F 4,14 = 7142.0, p < 0.0001), third instars (F 4,14 = 3277.9, p < 0.0001), and fourth instars (F 4,14 = 8197.8, p < 0.0001). In addition, mean responses to concentration within instar were each significantly different (Table 1). Probit analyses indicated a significant response to the concentration of extract for each instar (Table 2). The lethal concentration 50 (LC50) ranged from 0.024 to 0.028% for first to fourth instars and the LC90 for same from 0.042 to 0.047%.
Pupae were relatively less affected by Pe. tithymaloides leaf extract than larvae, although the overall effect of concentration was significant (F 4,14 = 643.1, p < 0.0001) as were differences in the mean response to each concentration (Table 1). Mean percent mortality of pupae ranged from 18 (±0.0)% (at 0.013%) to 42 (±0.6)% (at 0.040%). The range of extract concentrations tested against pupae was too low to reliably estimate a probit–mortality curve.
Discussion
Protection against mosquito bites can be achieved by avoiding infested habitats, by wearing protective clothing, and by applying repellent (Fradin 2001). At present, the main threat to effective mosquito control is resistance to insecticide in the mosquito (Chandra et al. 1998). Botanical insecticides provide an alternative to synthetic insecticides because they are generally considered safe, are biodegradable, and can often be obtained from local sources (Prabhakar and Jabanesan 2004). In addition, the use of medicinal plants for mosquito control is likely to generate local employment, reduce dependence on expensive imported products, and stimulate efforts to enhance public health (Bowers et al. 1995).
In the present study, we sought to determine whether an ethanol extract from Pe. tithymaloides could be used for mosquito control. We observed a functional response by all immature life stages of Cx. quinquefasciatus to the ethanolic extract of leaves of this plant species. This biological activity is attributed to the compounds in the leaves, including flavonoids, phenols, and steroids that together or independently produce morbidity and mortality effects in Cx. quinquefasciatus.
Other studies have shown phytochemical compounds to have potential for insect control. For example, saponin-induced mortality in mosquitoes may result from alteration of the larval cuticle (Morrissey and Osbourn 1999, Farid et al. 2002). Pizarro et al. (1999) estimated the LC50 and LC90 for the saponin fraction of Agave sisalana against third instars of Cx. quinquefasciatus to be 183 and 408 ppm, respectively. For the Pe. tithymaloides extract we tested against Cx. quinquefasciatus, the estimated LC50 and LC90 for instars 1–4 were 240, 256, 262, and 281; and 421, 429, 448, and 470, respectively.
Botanical insecticides can adversely impact development and reproduction in mosquito populations. These morbidity effects are important, given that high mortality in the vector population may not be a prerequisite to the acceptability of a biocide (Kabaru and Gichia 2001). More than 2000 plant species are known to contain chemicals that have pest control properties. Of these, 344 species have biologically active phytochemicals that affect mosquitoes (Sukumar et al. 1991). Thousands of plant-based compounds have been tested for insect repellent activity (Tawatsin et al. 2001).
The results of our study show that Pe. tithymaloides extract has a toxic effect on mosquito larvae. In this regard, we believe that Pe. tithymaloides phytochemicals warrant further study for use as natural product–based compounds for mosquito control and as an adjunct and/or alternative to the use of synthetic pesticides for vector control.
Footnotes
Acknowledgment
We thank the Head, Department of Zoology, Bharathiar University, Coimbatore, for providing the facilities necessary to conduct these experiments.
Disclosure Statement
No competing financial interests exist.