Insecticidal efficacy of Rubus steudneri and Rubus apetalus against Aedes (Diptera: Culicidae) and Culex (Diptera: Culicidae) mosquitoes

Introduction and Aim: Rubus steudneri Schweinf. and Rubus apetalus Poir. belonging to the family Rosaceae is one of the ethnomedicinal plants used widely in Ethiopia as food, for construction and as medicine. To the best of our knowledge, insecticidal activity of R. steudneri and R. apetalus has not been investigated so far. In this study, we report insecticidal efficacy of R. steudneri and R. apetalus in terms of larvicidal effect against II and III instar larvae of Aedes and Culex mosquitoes. Materials and Methods: The shade-dried and powdered leaves were extracted using methanol by maceration process. Insecticidal activity of leaf extract was determined by larvicidal assay against II and III instar larvae of Aedes and Culex species. LC50 and LC90 values were calculated. Results: The leaf extract of both Rubus species exhibited concentration dependent larvicidal effect. II instar larvae were shown to be highly susceptible than III instar larvae. Among leaf extracts, extract of R. steudneri exhibited marked insecticidal activity when compared to extract of R. apetalus as revealed by lower LC50 and LC90 values. Culex larvae displayed marked susceptibility to leaf extracts when compared to Aedes larvae. Conclusion: Marked larvicidal effect was observed against larvae of Culex species when compared to Aedes species as indicated by lower LC50 and LC90 values. It is evident that the leaf extracts of both Rubus contain insecticidal principles.


INTRODUCTION
osquitoes are one among the important vectors that transmit human diseases. Since development, chemical insecticides have shown to be promising with respect to control of insect vectors that transmit dreadful diseases such as dengue, malaria, chicken gunya, Filariasis and Japanese encephalitis. However, their indiscriminate usage results in deleterious effects on environment, emergence of resistant races of insect vectors and toxic effects on humans. Botanical extracts are promising with respect to their potent activity against eggs, larvae, pupae, and adult stages of mosquito vectors (1)(2)(3). Rubus steudneri Schweinf. is a scandent shrub belonging to the family Rosaceae. The plant is commonly known as Gora and is characterized by deeply furrowed greyish-tomentose stems that are covered with stellate hairs. The fruits of the plant are edible. The plant has got ethnobotanical significance (4)(5)(6)(7)(8) and the studies revealed that R. steudneri displayed pharmacological activities such as antimicrobial, antioxidant, antiproliferative and antidiabetic activities (9-12).
Rubus apetalus Poir. (Family: Rosaceae) is a scrambling shrub growing up to a height of 2.5m, characterized by hairy branches and scattered hooked spines on the stem. The plant is traditionally grown as living fences and as an ornamental plant. The fruits are edible and are eaten. R. apetalus possess immense therapeutic properties. It is shown that R. apetalus display biological activities such as antimicrobial, antioxidant, and antidiabetic activity (12)(13)(14)(15). To the best of our knowledge, no work has been done on insecticidal properties of R. steudneri and R. apetalus. Hence, in this study, we report the insecticidal efficacy of extract of R. steudneri and R. apetalus leaves against larvae of Aedes and Culex mosquitoes.

Collection of plant material
The leaves were collected at Nekemte, Oromia region, Ethiopia and identified by Dr. Tesfaye Awas and authenticated at the Ethiopian Biodiversity Institute, Addis Ababa, Ethiopia (12).

Extraction
Maceration process was employed to extract the shade dried and powdered leaves of R. steudneri and R. apetalus. Methanol was used as menstruum. The leaf powder was left in methanol in stoppered container for 48 hours followed by filtration and evaporation of the filtrate at room temperature (16). The leaf extract thus obtained was used to assess larvicidal activity.
Larvicidal activity of R. steudneri II and III instar larvae of Aedes and Culex species were subjected for screening their susceptibility to leaf extract of R. steudneri and R. apetalus (Fig. 1). The protocol employed by Kamaraj et al. (3) was used to determine larvicidal potential of leaf extract. The II and III instar larvae were exposed to different concentrations of extract (0.1 to 5.0mg extract/ml of water) for 24 hours. The number of dead larvae were counted after 24 hours of exposure. DDT (0.1% in water) was used as positive control. DMSO was used as negative control.

Statistical analysis
The experiment was conducted in triplicates and the data presented as mean±SD. The Median lethal concentrations, LC50 (dose that kill 50% of the exposed larvae) and LC90 (dose that kill 90% of the exposed larvae) were determined by the regression equations (Y= a + bX; where, X=concentrations; Y=% mortality) using MS Excel 2010. Fig. 2 and 3 show the result of larvicidal potential of leaf extract of R. steudneri against Aedes and Culex species. The extract was effective in causing mortality of larvae stages of both the mosquitoes in dose dependent manner. The extract was more effective against larvae of Culex species when compared to Aedes species. A mortality of 50% and higher against II and III instar larvae of Culex as well as Aedes species was recorded at extract concentration 0.25mg/ml and 0.5mg/ml, respectively. At extract concentration 1mg/ml and higher, 100% larval mortality was observed in case of both mosquito species.  Here also, the leaf extract was more effective against larvae of Culex species when compared to Aedes species. A mortality of 50% and higher against II and III instar larvae of Culex species was recorded at extract concentration 0.25mg/ml and 0.5mg/ml, respectively. In case of II and III instar larvae of Aedes species, a mortality of 50% and higher was recorded at extract concentration of 0.5mg/ml and higher. At extract concentration 1mg/ml and higher, 100% larval mortality was observed in case of Culex species while 100% mortality of II instar larvae of Aedes species was recorded at the highest concentration of leaf extract tested.

DISCUSSION
Interest in botanicals with insecticidal activities arose as a consequence of several drawbacks that are associated with the use of synthetic insecticides. It has been shown that crude extracts, as well as purified compounds from higher plants, display marked insecticidal activity in terms of their ovicidal, larvicidal, pupicidal and repellant activities (1,(17)(18)(19)(20). In our study, the leaf extract of R. steudneri and R. apetalus were effective in causing mortality of Aedes and Culex larvae in a dose dependent manner. It is clear from the results that the larvicidal potential observed is influenced by the larval developmental stage as II instar larvae were susceptible to extract to high extent than III instar larvae. In similar studies, the larvicidal effect of extracts of Aloe vera (21), Ficus benghalensis (22), and Couroupita guianensis (23) was marked against lower instar stages than higher instar stages. The positive control i.e., DDT, at tested concentration, was effective in causing 100% mortality of both the larval instars of test mosquitoes. DMSO showed no larvicidal property. Among leaf extracts, the extract of R. steudneri displayed higher insecticidal potential than the leaf extract of R. apetalus as indicated by lower LC50 and LC90 values (Table 1). Our earlier studies have shown the presence of various phytochemicals viz. saponins, alkaloids, flavonoids, and tannins in R. steudneri and R. apetalus (14,24). Insecticidal activities of plant secondary metabolites such as alkaloids, flavonoids and tannins have been documented (25)(26)(27). In our previous study, the butyl isobutyl phthalate and 2pyrrolidinone 5-(cyclohexylmethyl) were identified in chloroform fraction of R. steudneri using GC-MS (28). The dibutyl phthalate isolated from ethyl acetate extract of Ipomoea carnea showed larvicidal activity against 4 th instar larvae of Aedes aegypti and Culex quinquefasciatus (29).

CONCLUSION
The leaf extract of R. steudneri and R. apetalus displayed marked larvicidal property in a dosedependent manner indicating the utilization of the plant species for developing novel insecticidal formulations that can be used to manage mosquitoborne diseases such as malaria, dengue, chikungunya and filariasis. The presence of butyl isobutyl phthalate in R. steudneri and other metabolites such as flavonoids, alkaloids, and tannins in both the plants might be responsible for the observed insecticidal activity. Further studies are warranted to identify the insecticidal principles present in the Rubus species selected in this study.