BIO EFFICACY OF SPATHODEA CAMPANULATA P.BEAUV.(BIGNONIACEAE) HEXANE LEAF EXTRACT AGAINST DENGUE AND ZIKA VIRUS VECTOR AEDES AEGYPTI (DIPTERA:CULICIDAE).

Pravin Y and Mohanraj R.S. PG and Research Department of Zoology, Government Arts, College, Coimbatore. 641018, Tamilnadu ,India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 11 December 2018 Final Accepted: 13 January 2019 Published: February 2019

Ae.aegypti play an important role in the transmission of dengue fever, chikungunya, yellow fever, filariasis, zika, Japanese encephalitis, and several diseases which are today the greatest health problems in the world. Dengue fever virus (DENV) belongs to RNA virus of the Flaviviridae family and it was first isolated from Japan in 1942 by Hotta (Izabela et al., 2010). Three structural proteins are present in mature virion: (1) capsid protein C (2) membrane protein M (3) envelope protein E. C protein forms viral nucleocapsid. Chikungunya is an infection caused by the chikungunya virus. Chikungunya virus, also referred to as CHIKV, is a member of the alphavirus genus, and Togaviridae family. It is an RNA virus with a positive-sense single-stranded genome of about 11.6kb (Weaver et al., 2012). Zika virus (ZIKV) is a member of the virus family Flaviviridae and the genus Flavivirus, transmitted by day time-active Aedes mosquitoes, such as Ae. aegypti. It has been estimated recently that 3.9 billions of people in 128 countries are at risk of acquiring dengue and 390 million dengue infections occur every year, of which 294 millions clinically manifest the symptoms. The increase in dengue cases is considered to be a reflection of the rampant development towards massive infrastructure as well as urbanization, which is a favourable factor for breeding of Ae.aegypti (Azami et al., 2011 andGubler, 2002).
Epidemic outbreaks of dengue fever have also been reported in India. More recent and systemic data are now available because of the NVBDCP. The data on the web site of NVBDCP show that dengue has been endemic in 16 states since the beginning: Andhra Pradesh, Goa, Gujarat, Haryana, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Punjab, Rajasthan, Tamil Nadu, Uttar Pradesh, West Bengal, Chandigarh, Delhi and Puduchery (NVBDCP, 2014 At present, no effective vaccine is available for these diseases; therefore, the only way of reducing the incidence of this disease is mosquito control (Sarita et al ., 2012). The control methods should aim at the weakest link of the life cycle of the mosquito, which is the larval stage. Larviciding is a successful way of reducing mosquito densities in their breeding places before they emerge into adults. During the immature stage, mosquitoes are relatively immobile; remaining more concentrated than they are in the adult stage (Rutledge et al., 2003).
Many control strategies for mosquitoes have been suggested since the ancient times. Among the various control measures, viz., mechanical control by source of reduction (Mazzarri and Georghiou, 1995) ; biological control, using endopathogenic bacteria, Bacillus thuringiensis (Seleena et al., 1995 andMulla et al.,1999;Tabashnik et al., 1994); larivorous fish (Gluckman and Hartney, 2000) as well as predatory arthropods (Bohidar and Mohapatra, 2000 ) and chemical control (Laird and Miles, 1983). The chemical insecticides, including organophosphates, organochlorines and pyrethroids are being utilized for the control of vector and mosquito populations (Govindarajan et al., 2013). Repeated use of chemical insecticide resulted in several problems such as environmental hazards, elimination of natural enemies, toxic residues in food, and also produced insecticidal resistance in major vector species (Macedo et al., 1997). These and other pitfalls have compelled scientists to advocate for a refocus on botanicals, in Integrated Mosquito Management (IMM) protocols.
Plants are considered as a rich source of bioactive chemicals and they may be an alternative source of mosquito control agents. Naturally products are generally preferred because of the innate biodegradability. More than 2000 plant species have been known to produce chemical factors and metabolites of value in the pest control programmes (Ahmed et al., 1984) and among these plants, products of some 344 species have been reported to have a variety of activities against mosquitoes (Sukumar et al.,1991). The phytochemicals derived from plant sources possess a complex of chemicals with unique biological activity.The phytochemicals derived from plant resources can act as larvicidal, ovicidal, oviposition deterrence, growth and reproduction inhibitors, repellents, growth regulation, fecundity suppression, male sterility and smoke toxicity (Elimam et al., 2009a,b). Some of the plant leaves extracts are tested for their diverse insecticidal properties on the medically important mosquitoes: methanolic extract of Derris elliptica (Prempree and Sukhapanth,1990); aqueous extract of Senna didymobotrya (Ojewole et al., 2000); aqueous extract of Solanum nigrum (Singh et al., 2001); acetone extract of Solanum trilobatum (Rajkumar and Jebanesan, 2004); aqueous extract of Gymnema sylvestre and Eclipta prostrate (Khanna and Kannabiran, 2007); methanol, benzene and acetone extracts of Cassia fistula (Govindarajan, 2009

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As far as our literature survey is concerned that there was no information available on larvicidal, ovicidal effects of the hexane leaf extract of the S.campanulata. The present study was therefore carried out to evaluate the larvicidal, ovicidal effects of S.campanulata hexane leaf extract against the vector mosquito, Ae. aegypti.
Spathodea is a monotypic genus in the flowering plant family Bignoniaceae. It contains the single species, Spathodea campanulata, which is commonly known as the African Tulip Tree, Flame-of -the forest in English, Rugtoora in Hindi, Patadi in Tamil. It is a tree that grows between 7-25 m (23-82ft) tall and native to tropical Africa. This tree is planted as ornamental tree throughout the tropics and much appreciated for its very showy reddish argane (or) crimson (rarely yellow), campanulated flowers. It is commonly planted as a street tree in south Tamil Nadu. The tree is considered evergreen but it sheds leaves in dry summers and hence it is a dry season deciduous tree. S.campanulata commonly employed to control epilepsy. This species has many uses in folk medicine. The flowers are employed as diuretic and anti-inflammatory while the leaves are used against kidney diseases, urethra inflammation and as a antidote against animal poisons. The leaves have furnished Spathodol, caffeic acid and other phenolic acids and flavonoids. The plant leaf is used for anti-plasmodial activity, antimicrobial activity and anti -larvicidal activity ( The aim of the present study is therefore to find out: 1. Phytochemical and GC-MS analysis of hexane leaf extract of S.campanulata, 2. Estimate the toxicity of the hexane leaf extract of S.campanulata to the larvae and pupa of Ae.aegypti, 3. Ovicidal activity of the hexane leaf extract S. campanulata on Ae.aegypti egg.

Colonization of Ae.aegypti:
The eggs of Ae.aegypti were collected from National Institute for Communicable Disease (NICD), Mettupalayam, Coimbatore (Dt), Tamil Nadu, India. The eggs were then brought to the laboratory and transferred to enamel trays containing water and kept for larval hatching. They were hatched, reared and have been still maintained for many generations in the laboratory. The eggs and larvae obtained from this stock were used for different experiments. The larvae were reared in plastic cups. They were daily provided with commercial fish food ad libitum (Lymio et al .,1992). Water was changed alternate days. The normal cultures as well as breeding cups used for any experimental purpose during the present study were kept closed with muslin cloth for preventing contamination through foreign mosquitoes. The pupae were collected from culture trays and were transferred into plastic cups containing water with help of a sucker. The pupae containing plastic cups were kept inside mosquito cage for adult emergence. The females were fed by human arm every alternate day (Judson ,1967;Briegel,1990). Both females and males were provided with 10% glucose solution on cotton wicks (Villani et al.,1983). An egg trap (plastic cup) lined with filter paper containing water was always placed at a corner of the cage.

Collection of plant materials
S. campanulata P. Beauv. (Family :Bignoniaceae) leaves were collected from Government Arts college campus, Coimbatore, Southern India. The identification of the plants was authentified at BSI (Botanical Survey of India), Coimbatore.

Preparation of plant extract
The fresh leaves of the plant S. campanulata were collected in our college campus area. Then the leaves brought to the laboratory. The plant leaves were observed carefully for any kind of diseases or infection and if found any, those parts were separated and not used for the experiment. The selected leaves washed with distilled water in order to clean dust or any particle stuck to them. Then the leaves kept for drying under shade at room temperature (27± 2 o C) for about 2 weeks till they dried completely. The leaves were finely powdered using electric blender. 100 g of leaf powder was dissolved in 1000 ml of hexane in airtight wide mouth bottle and kept for 4 days with periodic shaking. After that, the extract was filtered using Whatman No.1filter paper and kept in Petri dishes for drying at room temperature (Kongkathip ,1994). Dried extract was used for the preparation of stock solution.

Preparation of stock solution and different concentrations of leaf extract
1g of the concentrated extract of leaves of S. campanulata was dissolved in 100 ml of acetone and kept as stock solution. This stock solution was used to prepare the desired concentrations of the extract for exposure of the mosquito larvae.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis:
The GC-MS analysis was conducted at South Indian Textile Research Association, Coimbatore. 1 µl of hexane leaf powder was injected into a Thermo GC -Trace ultra ver: 5.0, Thermo MS DSQ 11.The chromatography was performed by using the DB 35-MS capillary standard non-polar column. Helium flow was 1ml/ min. The oven temperature was increased at 70ºC /min to 250ºC.

Scanning Electron Microscope analysis
Ovicidal changes of the treated egg were studied and recorded and further compared to the control larvae after treatment of the hexane, ethyl acetate , Iso-propanal, dichloromethane, chloroform extracts for Scanning electron microscope study, subsequently spurted with 45nm gold, attached to the stubs and viewed under Scanning Electron Microscope (FEI Quanta 250, Bharathiar University,Coimbatore,Tamilnadu India)

Larvicidal, pupicidal assay test:
Bioassay test are carried out for testing the efficacy of hexane leaf extract of S. campanulata on Ae.aegypti at different stages of development viz I, II, III and IV instars and pupae (Pampana,1963 ). Mortality rates of larvae were recorded after 24 and 48 hours. Five or more concentrations of a test compound giving between 0 and 100% mortality for larvae at different instar stages were tested. In recording the percentage mortalities for each concentration, the moribund and dead larvae in three replicates were combined. The values of LC 50, LC 90 /24, 48hrs and their 95% confidence limit of upper confidence limit (UCL) and lower confidence limit (LCL), regression and chi-square values were calculated using probit analysis (Finney,1971). The SPSS 17.0 (Statistical Package of Social Sciences) used for statistical analysis.

Ovicidal assay:
Effect of hexane leaf extract of S.campanulata on the hatchability of Ae.aegypti eggs were determined (Judson &Gojrati ,1967). Hatching rate was calculated on the basis of non-hatchability of eggs (Sahgal &Pillai ,1993).The data were statistically examined using Student's t-test.

Phytochemical analysis of the hexane leaf extract of S.campalulata:
The qualitative phytochemical analysis revealed the presence of different phytochemicals such as carbohydrates, tannins, flavonoids, alkaloids, terpenoids, phenols, coumarins, and phytosteroids (Table-1  The effectiveness of this plant could be attributed to the presence of phytochemical compounds that act as insecticides (Abayomi,1993). S.alata leaves have furnished carbohydrates, tannins, flavonoids, terpenoids, coumarins, phenols, alkalodis, phytosteroids. These compounds were previously reported to have mosquito larvicidal activity (Farooq et al.,1999). These compounds may jointly (or) independently contribute to larvicidal activity against Ae.aegypti. The phytochemicals interfered with functioning of mitochondria (Usta et al.,2002) and primarily effect the midgut epithelium and secondarily affect the gastric caeca and the malpighian tubules in mosquito larvae (Rey et al.,1999;David et al.,2003). Tannins are toxic by blocking the digesting of foods causing growth disturbances (Lumowa & Nova, 2014), also causing a water-absorption disorder in the larvae, thus causing death to the larvae (Steyaningsih & Pasmiati,2015). Flavonoids have larvicidal effects due to their mechanism of action to inhibit the respiratory system and disrupt the electron transport process in the larval body thus decreasing ATP production and reducing the use of oxygen by mitochondria (David et al.,2003). Alkaloids are nitrogenous compounds that show insecticidal properties at low concentration and the mode of action on insect vectors varies with the structure of their molecules, but many are reported to affect acetylcholinesterase (AChE) or sodium channels as inhibition of acetylcholinesterase activity is responsible for terminating the nerve impulse transmission through synaptic pathway (Rattan,  ; ovicidal activity of methanol leaf extract of Taddalia asiatica was tested against the eggs of Ae. aegypti. It was observed that methanol extract concentration at 400ppm produced 99.3%, 94.00% and 100.00% ovicidal activity against Ae. aegypti (Saranya et al., 2018); in the case of ovicidal activity, exposure of freshly laid eggs was more effective than that of the older eggs (Miura et al.,1976).The hexane leaf extract of S.campanulata treated eggs exhibited an allayed hatchability and this may be due to the action of phytochemicals present in the extract. The extract may inhibit the hatchability of the eggs by interfering with their chorion (Rajkumar et al.,2011). Any compound that can cause permeability or a disruption to the chorionic layers in order to effectively deliver compounds that can terminate embryogenesis can be considered for development of effective ovicides. Our compound disrupted chorionic layers (Plate. 1c,1e,) so, it can be consider for development of effective ovicides. When eggs were directly exposed to high concentrations of the compounds, more chemicals entered the egg shell, which affected the embryogenesis; similarly, longer exposure periods also facilitated the increased penetration of the compounds into the shells, thus increasing their effectiveness (Broadbent & Pree,1984). The mechanism through which the embryos died may not be known as this was not investigated by the present study but it is speculated it could have been due to blackage of the micropyle apparatus (Plate.1c) thereby interfering with gaseous exchange or by interfering with purity of the gases within the operculum preventing the embryo from accessing air and thus ultimately dying (Bhatnagar & Sharma,1994).

Conclusion:-
The present investigation revealed that hexane leaf extract of S.campanulata possesses remarkable larvicidal, ovicidal effects against dengue vector mosquito. These results could encourage the search for new active natural compounds offering an alternative to synthetic insecticides from other medicinal plants. Further investigations about the mode of action of the constituent, effect on non-target organisms and field evaluation are necessary prior to commercialization.