Background

Mosquitoes play a major role as vectors that transmit parasitic and viral diseases worldwide, especially in tropical and subtropical countries. Mosquito borne diseases not only affect humans but they also affect livestock in many parts of the world. Phytochemicals are widely used as biocontrol agent against vector mosquitoes. Present study was that the seaweeds, which were collected from south east coast of India, showed enormous resources to find out the new marine product with mosquito larvicidal activities. These results suggest that the effective plant crude extracts have the potential to be used as an ideal eco-friendly approach for the control of disease vectors.

Human death occurs in millions every year due to mosquito bites. Since conventional insecticides are prone to resistance to many mosquitoes, it is high time that we find an alternative source to control mosquito. Therefore plants can be utilized as a source are controlling mosquitoes as they possess highly bioactive compounds which are non-toxic and are biodegradable in nature. Many vectors belonging to the genera Aedes, Anopheles and Culex are the causative agents for many dreadful diseases viz, Dengue Fever, Dengue Hemorrhagic Fever (DHF), Malaria, Japanese Encephalitis (JE), leads to the death of millions worldwide.

The vector of the Dengue, Aedes aegypti is widely distributed in the tropical and sub-tropical zones. The first incident of the Dengue has been notifiable in India around 1996. The National Vector Borne Disease Control Programme reported an annual average (± Standard Deviation (SD)) of 20,474 (± 13,760) Dengue cases and 132 (± 57) deaths caused by Dengue among 2006 to 2012.

Plants and their products have been tradionally used by humans for treating diseases early 15^th century. Many plants have been exploited for their medicinal values in and around surroundings. But very little consideration was given to the marine plants viz Mangrove and its associates. Although many works have been done recently in this area for various diseases still many mangroves have been left un-explored. Mangroves extracts can be used as anti-fungal, anti-viral, anti-cancer and mosquito larvicides, possibly due to the presence of many bioactive compounds in them (Kathiresan and Thangam, 1991; Kathiresan et al., 2006; Nabeel et al., 2010).

Sessuvium portulacastrum is a perennial herb belonging to a family Aizoaceae that grows in coastal area throughout the world. It is commonly known as sea purslane are shore line purslane. It is often used as an ornamental plant (Rabi et al., 2010). In Zimbabwe and South Africa this plant is used to treat various infections and kidney disorders (Lokhande et al., 2011).

Rhizophora mucronata, commonly known as red mangroves belongs to the family Rhizophoraceae is the medicinally important mangroves. It is used for the treatment of ulcer, diarrhea, hemorrhage, hematoma, elephantiasis, angina, febrifuge and hepatitis. It has been reported that Rhizophora mucronata contains a diverse group of secondary metabolites with high medicinal values.

The literature revealed that the plant possesses hepatoprotective, antioxidant (Ravikumar and Gnanadesigan, 2012), anti-HIV (Premanathan et al., 1999), antiplasmodial (Ravikumar et al., 2011) activity. Hence, the present study was made an attempt to find out the larvicidal efficacy of fractionated methanolic extract of Sesuvium portulacastrum and Rhizophora mucronata, mangrove and associated mangrove respectively, against dengue causing vector Aedes aegyptii larvae.

1 Materials and Methods

1.1 Plant material

Sesuvium portulacastrumleaves were taken from the backwaters of Muthukadu Lake, Chennai, Tamil Nadu, India and Rhizophora mucronata stilt root was collected from Parangipettai mangrove forest. All the samples were washed thrice with normal tap water, then with lukewarm water once and twice with distilled water in order to remove the adhering salts and other debris.

1.2 Extract preparation

The collected plant material was shade dried for about 7-10 days for complete removal of moisture from the plant parts and extracted in methanol. After 21 days of dark incubation, the filtrate was concentrated by rotary vacuum evaporation (>45ºC) and then lyophilized at -80ºC. The percentage of extraction was calculated by using the following formula:

% of extraction= wt of the extract/wt of the plant material X 100

1.3 Mosquito larval culture

The mosquito larvae were obtained from the stagnant water and sewerage of Shantha Nagar, Selaiyur, Chennai, Tamil Nadu. The larvae was maintained at (28±2ºC), 75-85% relative humidity under 14:10 light and dark photo period cycle. The larvae were given yeast powder mixed with dog biscuits in the ratio of 1:3 and the water in the tray were changed regularly.

1.4 Larvicidal activity

The larvicidal effect of methanolic extracts of the two mangrove species viz., leaf of Sesuvium portulacastrum and stilt root of Rhizophora mucronata against dengue larvae was conducted with varying grades of concentration such as 20, 40, 60, 80, 100 mg/ml of DMSO and the samples were diluted with 99ml of distilled water. DMSO (1 ml) and Distilled water (99 ml) was used as control. Ten larvae of same stage (i.e. 4^th instar) were put into beakers containing the test solution of each above mentioned concentration. Each experiment was conducted in three replicates. The larval mortality was observed and recorded after 24 hrs. The larvae were considered dead if, at the end of 24 h, they showed no sign of swimming movements even after gentle touching with a glass rod or a brush. The percentage of mortality was calculated by using Abbott’s formula:

% of mortality= [(% of test mortality - % of control mortality) / (100 - % of control mortality)] X100.

1.5 Statistical analysis

The average larval mortality data were subjected to probit analysis to calculate LC₅₀, LC₉₀ and 95% fiducial limits of upper confidence limit (UCL) and lower confidence limit (LCL), regression equation, Chi-square and analysis variation values were calculated using the Stat plus 2009 software. Results with P<0.05 were considered to be statistically significant.

2 Results

From the column chromatography conducted, it revealed that, the leaf extract of Sesuvium portulacastrum yielded 5 fractions viz, SF1 to SF5, similarly the stilt root of Rhizophora mucronata also yielded 5 fractions viz, RF1 to RF5. The LC₅₀ and LC₉₀ values of both the fractionated extracts against Aedes aegyptii were listed in Table 1 and Table 2 respectively. The fractionated leaf extracts showed maximum larvicidal activity fractionated extract 3 showed minimum level of LC₅₀ value (0.051±0.2 (0.41-0.602). Simultaneously, the fractionated stilt root extract of Rhizophora mucronata showed maximum larvicidal activity and (RF-2) showed minimum level of LC₅₀ value (0.51±0.29 (0.45-0.65). The regression equations of the leaf extract of Sesuvium portulacastrum and stilt root of Rhizophora mucronata for 4^th in star respectively. The Chi square value was significant at P < 0.05 level (Table 1; Table 2).

3 Discussion

Synthetic insecticides can be replaced by plant products as these are relatively cheap, safe and readily available in many areas of world. Secondary metabolites obtained from different parts of the plants contains various bioactive compounds with insecticidal and mosquitocidal activity. In order to reduce the resistance developed using the conventional insecticide we can employ the crude extract as the mosquito killing agent due to its natural synergism. Many mangrove plants viz Derris trifoliate, Heritieralittoralis, Rhizophoraapiculata, Xylocarpusgranatum, Rhizophoramucronata etc., contains insecticidal or mosquitocidal properties.

In the present study mangrove and associated mangrove plants was the most fraction for all the mosquito species. This should be due to the presence Terpenoids and Steroids revealed within the fraction. Earlier, it was proven that as typical lipophiles, the essential oil passed through the cell wall and cytoplasmic membrane, disrupted the structure of their different layers of polysaccharides, fatty acids and phospholipids and permeabilized them. Alkaloids are nitrogenous compounds that show insecticidal properties at low concentration and are often toxic to Vertebrates. Nicotine and Anabasine are common Alkaloids used as pesticides. The mode of action of Alkaloids on insect vectors varies with the structure of their molecules, but many are reported to affect acetylcholinesterase or sodium channels (Rameshwar, 2010). Devan et al. (2013) observed that Carbohydrates, Tannins, Saponins, Flavonoids, Alkaloids, Quinones, Terpenoids, Triterpenoids, Phenols, Coumarins, Proteins, Cyanin, Cardiac Glycosides screened from Tridaxprocumbens extracts exhibited larvicidal activity in fourth instar larvae of Anophelesstephensi, Aedesaegypti and Culexquinquefasciatus.

Thus, it can be concluded that the two mangroves and its associate showed potent larvicidal activity and can be used as alternative mosquito control agent. Further investigations are necessary for the isolation of active ingredients of the extract responsible for larvicidal activity. Further clinical applications will bring more endeavors to the study.

Authors’ contributions

All the authors have been actively participated in the study in order to complete it successfully.

Acknowledgements

The authors are thankful to the Authorities of the Mohamed Sathak College of Arts & Science, Sholinganallur, and Chennai for providing the required facilities to perform the study.

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