1 Introduction

Amongst the sanguinivorous insects, mosquitoes are most harmful to human as well as other animals (WHO, 1996). These tiny creatures act as biological vectors and normally transmit most of life threatening diseases like malaria, filariasis, yellow fever, dengue fever, encephalitis etc. in almost all tropical and subtropical parts of the world (Borah et al., 2010; Ghose et al., 2012). Culex quinquefasciatus is responsible for spreading filarial nematode, Wuchereria bancrofti that causes lymphatic filariasis (Rahuman et al., 2009). It can also spread pathogen of avian malaria, and some arbovirus like St. Louis Encephalitis Virus, Western Equine Encephalitis Virus and west Nile Virus (Hossain et al., 2011; Singh Ray et al., 2015). In tropical countries, the causative parasites of lymphatic filariasis have infected over 120 million people throughout the world and common chronic manifestations occurred in about 44 million people (Otten et al., 1997; Bernhard et al., 2003). It is necessary to prevent the mosquito borne diseases to improve public health as well as environment. To control the mosquito population, larval killing is an easy way because larvae are the most susceptible to any treatment and restricted to their common aquatic habitats (Bhattacharya et al., 2013). The conventional method to control mosquito is the application of synthetic insecticides. Nevertheless, application of these chemicals causes harmful effects on environment including humans and other non-target organisms. These insecticides are non-biodegradable and cause resistance development due to their long-term use (Rawani et al., 2009; Singh et al., 2015). To protect the environment with its biotic components, alternative methods have been applied to avoid the use of synthetic insecticides (Singha et al., 2011a; Singha et al., 2011b; Singh Ray et al., 2014). One of the most effective and easy alternative approaches is the use of phytochemicals of botanical origin (Rawani et al., 2010; Singha et al., 2012,). The use of phytochemicals is given a priority due to their eco-friendly nature, easy biodegradability and easy availability (Bhattacharya et al., 2014a; Bhattacharya et al., 2014b; Mallick et al., 2015).

Tinospora crispa, a common climbing shrub of Menispermaceae family, is disseminated throughout the tropical and subtropical countries including Philippines, Malaysia, Indonesia, Thailand, India, China and Vietnam (Mohd et al., 2012). It is used for treatment of type II diabetes. In rural areas, it is used as a traditional medicine to treat fever, cholera, jaundice, snake bite and rheumatism (Quisumbing, 1978). They also have immunomodulatory, antimalarial, antibacterial, antiviral, antiallergic, antiproliferative and antioxidant properties (Nidhi et al., 2013). The objective of this study was to evaluate the larvicidal activity of crude and solvent extracts of mature fruits of T. Crispa against Cx. quinquefasciatus as the target species.  Phytochemical analyses were also carried out to determine possible active ingredients in T. crispa that contribute to the larval toxicity.

2 Materials and Methods

2.1 Collection of plant materials

Fresh and mature fruits of T. crispa were collected during April-May 2015, from its growing area located at the outlying areas of Burdwan (23°16´N, 87°54´E), West Bengal, India. The plant was identified and a voucher specimen (Voucher No. GCZJKP-03) was submitted as a herbarium specimen at the Mosquito, Microbiology and Nanotechnology Research Units, Department of Zoology, The University of Burdwan.

2.2 Collection of larvae and colony set up

Larvae of Cx. quinquefasciatus were originally collected from cemented drains around the campus of the University of Burdwan and maintained in the laboratory, of the Mosquito, Microbiology and Nanotechnology Research Units (Sharma and Saxena, 1994). A mixture of dog biscuits, dried Brewer’s yeast powder and algae in the ratio of 1:3:1 was provided as a complementary food at 27 (± 2)°C temperature and 80% - 85% relative humidity under a photoperiod of 14:10 light and dark cycles in a day. Transformed pupae were transferred manually into a beaker (500 ml) containing tap water using a glass dropper. The beaker was placed in a mosquito rearing cage (24 x 20 x 18 inches) for emergence of adult mosquitoes. A cotton ball soaked in 10% glucose solution and multivitamin syrup was used for meal of adult mosquitoes. At the 5^th day of rearing an immobilized pigeon was provided for periodical blood meal of females. Water filled Petri dish and crumpled filter papers were kept in the cage for egg laying of adult females. Eggs were allowed to hatch in a separate container and maintained under similar laboratory conditions to obtain F₁ generation which were used during experiment.

2.3 Preparation of crude extract

The collected fresh and mature fruits of T. crispa were rinsed well with tap water, followed by distilled water and soaked on a paper towel. Unspotted clean fruits were crushed with mechanical grinder and then filtered. The filtrate was used as a stock solution (100% concentration) for further bioassays. The concentrations (0.1%, 0.2%, 0.3%, 0.4% and 0.5%) were prepared by the addition of distilled water to the stock solution.

2.4 Preparation of solvent extract with petroleum ether

Mature fruits of T. crispa were dried in shed for few days at room temperature. The dried fruits were cut in small pieces and 200 gm of them were put into the thimble of Soxhlet apparatus. Two thousand (2 000) ml of petroleum ether was then loaded into the still pot. The extraction period was 72 hours and maximum 8 hours per day was fixed. The extract was collected in a beaker and then evaporated using a rotary evaporator. The concentrated extract was kept at 4°C in a refrigerator until being used.

2.5 Dose-response larvicidal bioassay

Larvicidal bioassays were performed according to the standard protocol of WHO (WHO, 2005). Batches of 25 larvae of different instars (1^st, 2^nd, 3^rd and 4^th) were transferred using droppers into sterilized glass Petri dishes (9 cm diameter, 150 ml capacity), each containing 100 ml of tap water. Small, unhealthy or damaged larvae were removed and replaced. Graded concentrations of crude extract ranging from 0.1% to 0.5% were added into different Petri dishes. Similarly, graded concentrations of petroleum ether extract (from 80 ppm to 160 ppm) were also prepared from stock extract and applied in different Petri dishes. Four replicates were set for each concentration and an equal numbers of control were set up. Each test was run three times on different days at room temperature. Larval mortalities were recorded after exposure of 24 h, 48 h and 72 h. Larvae were counted as dead when they failed to move as they were pinned with a needle in the siphon or the cervical region.

2.6 Phytochemical analysis of the plant extracts

A phytochemical analysis of crude extract of the fruits of T. crispa was performed according to the standard methodologies of Trease and Evans (1989), Sofowara (1993) and Harborne (1973). In our study, crude extract was tested for the presence or absence of some secondary biochemicals like tannins, flavonoids, alkaloids and steroids.

2.7 Larvicidal bioassay on a non-target organism

Chironomus circumdatus larvae sharing similar habitat with mosquito larvae were selected as the non-target organism. C. circumdatus larvae were exposed to the median lethal concentration (LC₅₀) of 24 hours for 3^rd instar larvae of Cx. quinquefasciatus of the crude and petroleum ether extracts of T. Crispa fruits. Larval physiological or behavioural abnormalities or mortalities were recorded after exposure of 24 h, 48 h and 72 h. Larvae were counted as dead when they failed to move.

2.8 Statistical analyses

Abbott’s formula (Abbott, 1925) was used to correct the percent mortality (% M) throughout the observation. Estimations of LC₅₀ and LC₉₀ values through Log-probit and regression analyses were carried out using the “STAT PLUS 2009 (Trial version)” and “MS EXCEL 2007” respectively. Multivariate ANOVA was carried out using “SPSS 16.0 version”.

3 Results

The percent mortality of all instars with crude extract of different concentrations is presented in Table 1. Maximum mortality was observed at 0.5% concentration of the crude extract that showed highest mortality in 1^st and 2^nd instars larvae than that of 3^rd and 4^th instars larvae after 24 h, 48 h and 72 h of exposure. Similarly at 0.1%, 0.2%, 0.3% and 0.4% concentration the highest mortality observed in 1^st and 2^nd instars larvae after 72 h of exposure. In petroleum ether extract the highest mortality was recorded at 160 ppm against 1^st instar larvae followed by 2^nd, 3^rd and 4^th instars after 72 h of exposure. 1^st instar larvae exhibited 100% mortality at 140 ppm concentrations while 2^nd and 3^rd instars larvae exhibited 100% mortality at 160 ppm after 72 h of exposure. For 4^th instar larvae, 82.68% mortality was recorded at 160 ppm following 72 h of exposure (Table 2). LC₅₀ with their LCL and UCL values and LC₉₀ values (at 95% level of confidence) are given in Table 3 and Table 4. There was positive relation between concentration (X) and mortality (Y) with a regression coefficient (R²) close to 1 in each case (Table 3 and Table 4). No abnormality or mortality was recorded in the larvicidal bioassays conducted on C. circumdatus. The larvicidal activity was found statistically significant (p < 0.05) through completely randomized ANOVA analyses by using instars, hours and concentrations as variable. All parameters i.e. instars, hours and concentrations are correlated significantly at p < 0.05 degree (Table 5 and Table 6). The phytochemical analysis on crude extract of T. Crispa mature fruits revealed the presence of steroid and tannin. No flavonoid and alkaloid were detected in the extract.

4 Discussions

Although the use of synthetic insecticides showed rapid effectiveness in the control of mosquito populations, it is not recommended for long-term use because of their non-biodegradable nature, persistent in the environment and last but not least the development of resistance in mosquitoes and also their harmful effects on non-target organisms (Campbell et al., 1993). To overcome all these problems and to prevent the spread of mosquito borne diseases, the use of biodegradable, ecofriendly, target specific phytochemicals has become the central focus of the vector control programmes (Rawani et al., 2012; Bhattacharya et al., 2015; Mondal et al., 2016; Singh et al., 2016). These plants are unconventional sources of huge secondary metabolites which are in single or in combination responsible for larval or adult mosquito mortality. Different types of phytochemicals extracted either from the whole plant or from a specific part of the plant using different solvent extracts have been reported to show remarkable larvicidal effects on mosquito larvae.

The present study revealed the efficacy of fruit extract of T. crispa as a mosquito larvicidal agent. Both crude and solvent extract showed the potentiality against all the larval instars of Cx. quinquefasciatus. The lowest LC₅₀ value was at 0.04% against 1^st instar larvae of Cx. quinquefasciatus in crude extract after 72 h of exposure. The lowest LC₅₀ value for 2^nd and 3^rd instars larvae were at 0.06% and for 4^th instar larvae was at 0.07%. In the case of petroleum ether extract the lowest LC₅₀ value calculated was 64.49 ppm for 1^st instar larvae followed by 66.29 ppm, 68.36 ppm and 69.37 ppm for 2^nd, 3^rd and 4^th instars larvae respectively after 72 hours of exposure. The petroleum ether extract would be better to use because in crude extract the activity of the extract may be due to the synergistic effect of two or more active compound that responsible for the larval mortality. But in the petroleum ether extract the active bio compound extract out along with petroleum ether solvent. So in this case the lees concentration of petroleum ether will be more powerful than the crude extract. The extracts were also safe to use in aquatic system as no abnormality was seen in the behaviour of non-target organism, C. circumdatus. The preliminary phytochemical analysis demonstrated the presence of tannin and steroid in the extract of T. crispa mature fruits. These two photochemical either singly or in combination may be responsible for larval toxicity. From different plant parts, several secondary metabolites such as alkaloids, terpenoids, essential oils, phenolics etc. have been reported previously for their mosquitocidal activities (Shaalan et al., 2005). Kumar et al., 2014 reported on larvicidal activity of petroleum ether extract of whole plant of Cassia occidentalis against Cx. quinquefasciatus with LC₅₀ and LC₉₀ values of 98.4 ppm and 198.8 ppm, respectively after 72 h of exposure. The biocontrol efficacy of petroleum ether extract of leaf of Argemone mexicana against Cx. quinquefasciatus was established by Karmegan et al., (1997) with 100% mortality at 250 ppm. Rahuman et al., (2008) also evaluated the efficacy of petroleum ether extract of Citrullus colocynthis against Cx. quinquefasciatus. LC₅₀ values calculated was 88.24 ppm after 24 h of exposure.

In conclusion, fruit extract of T. crispa showed a promising larvicidal activity against all instars of Cx. quinquefasciatus larvae. Further studies are required to determine all active ingredients in detail and their roles in larvicidal activity.

Acknowledgement

The authors are thankful to Professor Dr. A. Mukhopadhyay, Department of Botany, The University of Burdwan, for his kind help in plant species identification. We are grateful to UGC DRS and DST-INSPIRE for providing financial assistance.

Conflict of Interest

The authors declare that they have no conflict of interest.

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