1 Introduction

Vector-borne diseases viz., malaria, filaria, Japanese encephalitis, dengue and chikungunya are transmitted by mosquitoes and cause severe morbidity and mortality among the affected human population (WHO, 2014). Vector mosquito control is vital to prevent transmission and contain the spread of these diseases. Synthetic insecticides have been widely used to control vector mosquito population but the development of resistance to these insecticides has necessitated search for alternate tools. Plants are reported to possess a plethora of secondary metabolites, many of which are considered promising tools for mosquito control. Alkaloids, terpenoids, steroids, etc., are some of the phytocompounds that exhibit mosquitocidal activity (Kishore et al., 2014). Plant secondary metabolites and other phytochemicals are reported to be biodegradable, environmentally safe and specific in action. In order to determine the presence of bioactive compounds that are detrimental to vector mosquitoes, many plants have been screened for insecticidal activity against varied stages of mosquitoes viz.,egg, larvae, pupae and adult (Shaalan et al., 2005; Ghosh et al., 2012; Tennyson et al., 2012a; Benelli, 2015; Shaalan and Canyon, 2015).

Plants belonging to the genus Cassia (Family: Caesalpiniaceae) are widely distributed in the tropics and constitute about 600 species of herbs, shrubs and trees (Dave and Ledwani, 2012). Many plants of this genus are well known for their medicinal properties and have been used in traditional medicine in many countries (Singh et al., 2013). In addition, phytocompounds that has insecticidal activity have also been reported in plants of this genus. Some species, viz., Cassia auriculata (Kamaraj et al., 2009; 2010), Cassia fistula (Govindarajan, 2009), Cassia holosericea (Qureshi et al., 1986), Cassia nigricans (Georges et al., 2008), Cassia obtusifolia (Rajkumar and Jebanesan, 2009), Cassia siamea (Kamaraj et al., 2011) and Cassia tora (Amerasan et al., 2012) have been screened for mosquitocidal activity. Cassia occidentalis (Synonym: Senna occidentalis)⁺, a plant commonly found in India and used in Indian traditional medicine has been screened for its activity against immature and adult stages of vector mosquitoes (Yadav et al., 2010; Kumar et al., 2014; Venkatesan et al., 2014; Murugan et al., 2015). In the present study, the oviposition determinant and ovicidal activity of crude leaf extracts of Cassia occidentalis against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti, the urban vectors of malaria, filaria and dengue/chikungunya respectively were evaluated.

⁺Presently placed under Family Fabaceae.

2 Materials and Methods

2.1 Preparation of crude extracts

Cassia occidentalis plants were collected from foothill regions of Javardhu hills, Tiruvannamalai district, Tamil Nadu, India and taxanomical identity confirmed at the Department of Plant Biology and Biotechnology, Loyola College, Chennai, Tamil Nadu, India. The fresh and healthy leaves were removed from the plants, washed with dechlorinated tap water, shade dried at room temperature and powdered using an electric blender. The powdered leaves were then sequentially extracted using the solvents (polarity index) viz., hexane (0.1), ethyl acetate (4.4) and methanol (5.1) for a period of 72 hours and then filtered. A rotary vacuum evaporator was used to prepare a concentrate of the filtered content, which was weighed and stored at 4^°C in sterilized amber coloured bottles (Venkatesan et al., 2014).

2.2 Laboratory bioassay

2.2.1 Oviposition determining activity

The oviposition determinant nature, viz., attractant or deterrent nature of hexane, ethyl acetate and methanol crude leaf extracts of Cassia occidentalis on the ovipositing vector mosquitoes was studied at a concentration of 0.1%. Gravid mosquitoes were simultaneously provided ovitraps containing the said concentration of different extracts to oviposit. The number of eggs laid in each of the ovitraps was used to determine the oviposition determinant nature of the crude extracts. Five ovitraps, plastic bowls of 6.0 cm and 12.0 cm in height and diameter, each lined with filter paper and containing 200 ml of 0.1% of hexane, ethyl acetate, methanol crude leaf extracts and one containing Tween 80 dissolved in distilled water (treated control) and another only distilled water (untreated control) were placed in the four corners and at the centre of a two feet mosquito cage. Twenty laboratory colonized fully gravid vector mosquitoes of F₁ generation were introduced into the cage for egg laying. The number of eggs laid in each ovitrap was counted 24 hours after introduction in tests with Anopheles stephensi and Aedes aegypti and after 96 hours with Culex quinquefasciatus. Culex quinquefasciatus was observed to delay egg laying in clean water. The position of ovitraps was changed in different trials to prevent selection bias due to position. A total of 25 trials were carried out for each species of vector mosquitoes and a trial was considered void when no eggs were laid. All trials were carried out at room temperature. The Effective deterrence (ED) and the Oviposition active index (OAI) were calculated using the following formulae to assess the effect of each extract on oviposition.

Where NC is the total number of eggs laid in control and NT is the total number of eggs laid in treatment.

Where NT is the total number of eggs laid in test solution and NS is the total number of eggs laid in control solution.

OAI determines the ‘attractant’ or ‘deterrent’ nature of the extract. Oviposition active index of +0.3 and above are considered as attractants while those with -0.3 and below are considered as deterrents (Kramer and Mulla, 1979). Positive value indicates that more eggs were deposited in treated solutions, and negative value, more eggs in control.

2.2.2 Ovicidal activity

The ovicidal activity of the crude leaf extracts was studied at concentrations of 0.25%, 0.50%, 1.00% and 2.00%. Tween 80 dissolved in distilled water and only distilled water was used as treated and untreated control. Single tubing method was followed (Tennyson et al., 2015). A known quantity (100 mL) of respective crude extract prepared was taken in an ovitrap, a plastic bowl of aforementioned dimensions, which was covered with transparent muslin cloth to hold the experimental vector mosquito. The inner portion of the ovitrap was lined with filter paper strips at the water surface level to prevent drying of laid eggs. One fully gravid vector mosquito was introduced in each of these ovitraps for oviposition. The ovitraps were checked daily for eggs. The adult vector mosquitoes were removed from the ovitrap after egg laying. The total number of eggs laid, number hatched and unhatched were recorded on the fourth day after egg laying in Anopheles stephensi and Culex quinquefasciatus and on the tenth day in Aedes aegypti mosquitoes. A total of three trials with three replicates per trial were carried out. Assessment of ovicidal activity was based on the Egg mortality rate (EMR) using the following formula.

2.3 Data analysis

SPSS version 17.0 was used for statistical analysis (SPSS, 2008). ANOVA and Karl Pearson’s correlation tests were performed wherever necessary.

3 Results

3.1 Oviposition determinant activity

A total of 25 trials were undertaken to study the preference of ovipositing female mosquitoes to lay eggs in ovitraps containing the experimental solutions viz., hexane, ethyl acetate and methanol crude leaf extracts, treated and untreated control. The mean noon laboratory temperature and relative humidity during the study period was 26.7 ± 1.1^°C and 84.3 ± 4.9%.

In studies with Anopheles stephensi, eggs were found laid in five, nine and eight trials with hexane, ethyl acetate and methanol treated extracts. In treated control, eggs were found in 20 and in untreated control in all trials. The minimum number of eggs that was laid in all extract treated ovitraps was five and in treated and untreated control, it was 15 and 79. The maximum number of eggs laid was 66, 84, 96, 75 and 225 respectively. Mean number of eggs in each of the experimental solutions were 4.6 ± 14.0, 11.4 ± 22.9, 9.6 ± 24.8, 32.3 ± 23.0 and 152.5 ± 40.6 respectively. The ED to oviposition was 97.0%, 92.5%, 93.7% and 78.2% and OAI was–0.9 in all crude leaf extracts tested and – 0.6 in treated control (Table 1; Figure 1).

Culex quinquefasciatus mosquitoes laid egg rafts in nine, seven, fifteen, twenty and twenty five of the trials with hexane, ethyl acetate, methanol, treated and untreated control. The minimum number of egg rafts that were found laid was one in all the test solutions and the maximum was four, two, seven, four and ten respectively. Mean number of egg rafts found were 0.6 ± 0.9, 0.4 ± 0.8, 1.2 ± 1.7, 1.6 ± 1.2 and 4.6 ± 2.2 respectively. ED based on untreated control was 87.8%, 90.4%, 73.0% and 66.1% and OAI -0.8, -0.8, -0.5 and -0.5 respectively (Table 1; Figure 1).

In the case of Aedes aegypti mosquitoes, eggs were laid in ten, six and six of the trials with hexane, ethyl acetate and methanol crude leaf extracts. In treated and untreated water, eggs were found laid in 22 and 25 of the total trials. The minimum number of eggs found laid in the test solutions was five, six, five, twenty one and sixty and the maximum was 79, 70, 14, 110 and 325. Mean number of eggs observed was 9.9 ± 19.5, 6.4 ± 16.0, 1.7 ± 3.9, 51.4 ± 31.1 and 166.6 ± 73.9 respectively. ED was 94.0%, 96.2%, 99.0% and 69.0% and OAI, -0.8, -0.9, -0.9 and -0.5 respectively (Table 1; Figure 1).

The number of eggs laid by the vector mosquitoes viz., Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti varied in both experimental and control solutions and the results of ANOVA showed significant difference at P<0.01 in all the vector mosquito species studied.

3.2 Ovicidal activity

All vector mosquito species laid eggs during the three trials in both treated and untreated ovitraps. The number of eggs laid and those that were left unhatched varied within species and extracts (Table 2). In Anopheles stephensi, the mean number of eggs laid in ovitraps containing 0.25%, 0.50%, 1.00% and 2.00% of hexane, ethyl acetate and methanol treated crude leaf extracts was 57.1 ± 30, 54.8 ± 47.5, 48.8 ± 4.7 and 47.4 ± 33.0; 99.1 ± 20.2, 95.7 ± 17.2, 87.8 ± 6.0, 56.8 ± 58.0; and 76.3 ± 11.9, 67.9 ± 28.3, 57.9 ± 50.4 and 53.1 ± 47.9 respectively. The mean number of eggs laid in treated and untreated control was 100.2 ± 37.7 and 121.1 ± 30.8. The eggs that remained unhatched varied and at the lowest concentration of 0.25%, the unhatchability per cent or EMR ranged between 58.2% and 74.5% among the extracts. At the highest concentration, it was between 85.0% and 94.9%. The order of increasing activity at the lowest and highest concentration was methanol>ethyl acetate>hexane and hexane>methanol>ethyl acetate crude leaf extracts respectively. In treated control, the EMR was 6.7% and in untreated control, all eggs hatched out successfully.

In Culex quinquefasciatus, the mean number of egg rafts laid in both extract treated and untreated ovitraps was one and the mean number of eggs in the egg rafts was 150. EMR at the lowest (0.25%) and highest (2.00%) concentration ranged from 62.8% to 66.5% and 95.7% to 98.7% in the different extract treated ovitraps. The order of increasing activity at the lowest and highest concentration was ethyl acetate>methanol>hexane and hexane>ethyl acetate>methanol crude leaf extracts respectively. In treated and untreated control, EMR was 36.2% and 7.0%.

In trials with Aedes aegypti, the mean number of eggs laid was 30.9 ± 37.2, 40.0 ± 11.7, 24.8 ± 9.1 and 17.2 ± 6.8; 47.0 ± 14.5, 32.8 ± 7.1, 32.6 ± 12.7 and 30.5 ± 26.5; and 37.2 ± 19.9, 42.1 ± 4.8, 43.3 ± 24.2 and 31.6 ± 17.3 in hexane, ethyl acetate and methanol extracts. EMR ranged from 57.7% to 92.5% and 89.7% to 95.3% at the lowest and the highest concentration and the order of increasing activity was hexane>ethyl acetate>methanol in both the concentrations. In treated control, EMR was 19.3%. Egg mortality was not observed in untreated control.

The effect of concentration on the EMR of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was analyzed using Karl Pearson’s correlation test. The results showed r-value to be 0.68, 0.79 and 0.69 and the correlation was significant at P<0.01 level. EMR caused by the hexane, ethyl acetate and methanol leaf extracts at different concentrations was analyzed using ANOVA and the results showed no significant difference.

4 Discussions

Crude extracts of various parts of the plant, Cassia occidentalis has been screened for its effect as a larvicide, growth regulator, adulticide, repellent and ovicide against vector mosquitoes viz., Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti (Nazar et al., 2009; Dhandapani and Kadarkarai, 2011; Kumar et al., 2012; Panneerselvam and Murugan, 2013; Kumar et al., 2014; Venkatesan et al., 2014; Murugan et al., 2015). In the present study, the oviposition determinant and ovicidal activity were evaluated.

4.1 Oviposition determinant activity

Gravid mosquitoes, attracted to a preferential breeding habitat are dependent on many factors, of which one is the quality of water. The dissolved substance present in the aquatic habitat, the gaseous exchange that takes place on the habitat surface and the odour that is emitted may attract or deter oviposition in the habitat. Plant extracts, which contain many secondary metabolites, can alter the nature of habitat and impact oviposition.

In the present study, the number of eggs laid by all the vector mosquito species studied was found to be highest in ovitraps with distilled water (untreated control). In Anopheles stephensi, the mean number of eggs laid in untreated control was 33.1, 13.4, 15.9 and 4.7 times higher when compared to those laid in ovitraps with hexane, ethyl acetate, methanol crude leaf extract and Tween 80 (treated control) indicating deterring nature of the crude leaf extracts. Similarly, the number of egg rafts laid was 7.7, 11.5, 3.8 and 2.9 times more in Culex quinquefasciatus and 16.8, 26.0, 98.0 and 3.2 times more in Aedes aegypti confirming the same. There was significant difference in the number of eggs laid in untreated control and other extract treated ovitraps but within the extract treated ovitraps there was no significant difference in the number of eggs laid showing that hexane, ethyl acetate and methanol crude leaf extracts to exert similar activity against ovipositing mosquitoes.

The ED to oviposition expressed in terms of per cent also shows the deterrent activity of the extracts. The minimum ED to ovipositing Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti mosquitoes was 92.5%, 73.0% and 94.0% in ethyl acetate, methanol and hexane extracts. The maximum ED of 97.0%, 90.4% and 99.0% was observed in hexane, ethyl acetate and methanol crude leaf extracts respectively. Among the extracts, no single extract showed similar activity across the different vector species studied. OAI of -8 and above in all extracts against all species except methanol extract against Culex quinquefasciatus indicate the crude leaf extracts of Cassia occidentalis to be a deterrent to oviposition.

The crude leaf extracts of various species of plants including hydrophytes have been screened for their influence on the ovipositing mosquitoes. The plant species screened viz., Schoenoplectus acutus (Du and Millar, 1999), Melia azedarach (Coria et al., 2008), Ricinus communis (Elimam et al., 2009), Cassia obtusifolia (Rajkumar and Jebanesan, 2009), Ocimum kilimandscharicum and Ocimum suave (Kweka et al., 2010), Pongamia pinnata, Coleus forskohlii and Datura stramonium (Swathi et al., 2010), Ageratum houstonianum (Tennyson et al., 2012b), Artimesia annua (Cheah et al., 2013), Ipomoea cairica (Ahbirami et al., 2014), Alstonia scholaris, Callistemon viminalis, Hyptis suaveolens, Malvastrum coromandelianum, Prosopis juliflora and Vernonia cinerea (Yadav et al., 2014), Aegle marmelos, Limonia acidissima, Sphaeranthus indicus, Sphaeranthus amaranthoides and Chromolaena odorata (Reegan et al., 2015) and Bryopsis pennata (Yu et al., 2015) have all been reported to cause varied level of deterrence. Besides deterrent property, attractant quality has been reported in other species to ovipositing mosquitoes viz., Chara globularis, Ranunculus aquatilis (Angerilli, 1980), Chara zeylanica, Cupressus semipervirens, Coriandum sativu, Foeniculumn vulgare, Spatodea campanulata (Consoli et al., 1989) and Schoenoplectus acutus (Du and Millar, 1999).

An extract can be considered to possess a potential oviposition deterrent activity if at a very low concentration the ED obtained is high. The crude leaf extracts of Cassia occidentalis, even at a higher concentration of 0.1% (1 000 mg/L), did not show 100% ED and OAI of -1.0 was not obtained in any of the crude leaf extracts. When compared to the results of the present study, crude leaf extracts of other plant species have shown potential oviposition deterrent activity and at lower concentrations. The ethanolic leaf extract of Cassia obtusifolia showed effective repellency of 92.5% at 400 mg/L to ovipositing Anopheles stephensi mosquitoes (Rajkumar and Jebanesan, 2009). Reegan et al. (2015) studied the oviposition deterrent activities of hexane, ethyl acetate and methanol crude leaf extracts of five medicinal plant extracts viz., Aegle marmelos, Limonia acidissima, Sphaeranthus indicus, Sphaeranthus amaranthoides and Chromolaena odorata against Culex quinquefasciatus and Aedes aegypti mosquitoes and their results indicated that the hexane extract of Limonia acidissima to show maximum ED of 100% against both vector mosquito species at 62.5 ppm which was the lowest concentration studied. In another study, the methanol and chloroform extract of the leaves of the seaweed, Bryopsis pennata was reported to show ovipositional repellent activity and the OAI at 400 µg/mL was -1.0, indicating potential deterrent activity against Aedes aegypti mosquitoes (Yu et al., 2015). In the present study, though the hexane, ethyl acetate and methanol crude leaf extracts of Cassia occidentalis showed oviposition deterrence activity, there is lack of potent oviposition deterrent activity against the vector mosquitoes studied.

4.2 Ovicidal activity

Results of the present study showed that many eggs did not hatch indicating the presence of ovicidal activity. The egg hatch rate in control was 100% (EMR is zero), whereas the same was not obtained in any experimental solutions showing that eggs subjected to treatment with plant extracts resulted in unhatchability or mortality of certain number of eggs. Govindarajan et al. (2008) reported 100% mortality of eggs of Culex quinquefasciatus at a low concentration of 100 ppm (0.01%) in methanol extract of Cassia fistula, a plant belonging to the same genera. In the present study, 100% EMR was not obtained in any of the extracts and at the highest concentration of 2.00%, the maximum mortality obtained was 96.1 ± 2.3; 98.7 ± 1.4; 95.8 ± 3.6 in ethyl acetate, methanol and methanol extracts against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti mosquitoes respectively indicating poor ovicidal activity. Maximum EMR rate at a low concentration is an indication of potential ovicidal activity. Ovicidal activity is reported to be influenced by concentration, age of eggs, formulation and mosquito species (Su and Mulla, 1998). Plant extract that can cause effective mortality at a very low dosage should be considered for further studies to develop suitable ovicides that can be used in vector control operations.

The egg mortality caused by hexane, ethyl acetate and methanol leaf extracts at different concentration did not show any significant difference indicating that at same concentrations, the ovicidal activity was similar among the extracts. There was, however, a concentration dependent ovicidal activity in all the tested extract, though not in entirety. This may be attributed to the variations associated with the exposure of eggs to the target concentrations, as the mosquitoes were held captive to lay eggs directly on to the experimental solutions. Elango et al. (2009) observed the per cent hatchability to be inversely proportional to the concentration and the activity was concentration dependent. In their experiments, the laid eggs were subjected to treatment with experimental solution. Further studies are required to understand the ovicidal effect when eggs are allowed to be laid directly or subjected to treatment in the experimental solution.

Many plant species have been screened for their activity against eggs of mosquitoes (Ouda et al., 1998; Govindarajan et al., 2008; 2011a; 2011b; Elango et al., 2009; Govindarajan, 2011; Tennyson et al., 2011; 2015; Govindarajan and Sivakumar, 2014a; 2014b; Govindarajan and Rajeswary, 2015). Though there are reports of effective ovicidal activity, reports on underlying mechanism that results in unhatchability of eggs is wanting. Mosquito eggs are soft and white at oviposition but undergo sclerotization during embryogenesis becoming hard and dark (Monnerat et al., 1999). The embryo within the laid egg is protected by a shell which comprises protease-resistant envelope (Moreira, 2007), namely, the roughly homogenous endochorion and the compound exochorion which is made up of an internal lamellar layer and external protruding tubercles (Monnerat et al., 1999). Metabolites of specific amino acids are critical for the formation and maturation of the egg chorion. Tyrosine, an amino acid obtained through ingestion of blood meal or through hydroxylation of ingested phenylalanine by phenylalanine hydroxylase is a determining factor in the melanization reaction that is responsible for chorion hardening (Li and Christensen, 1993; Li, 1994). Chorion peroxidase is also involved in the hardening of the mosquito egg chorion by catalyzing the formation of ditryrosine through tyrosine residues on structural proteins (Li et al., 1996). It has been reported that endo and exochorion have different biochemical natures (Monnerat et al., 1999) and the exochorion can easily be removed while endochorion remain intact (Mortenson, 1950; Decoursey and Webster, 1952; Forbes and Ingham, 1993). 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. Benserazide, an inhibitor of Dopa Decarboxylase, one of the enzymes needed for mosquito egg shell sclerotization was reported to cause permeability without affecting the embryo and hatching, and it was effectively used to study the ultrastructure of the chorionic membrane (Monnerat et al., 1999). Silencing of the gene for phenylalanine hydroxylase resulted in impairment of egg melanization and hatching (Fuchs et al., 2014). More research, therefore, is required to screen for plant secondary metabolites that impair chorionic membranes, affect embryogenesis and prevent hatching of eggs.

5 Conclusions

Phytoconstituents in crude extracts that have toxic properties are responsible for mosquitocidal activity. Any phytocompound that can either act as a strong oviposition deterrent or an oviposition attractant along with intense ovicidal activity will be a candidate compound for use in mosquito control activities. In view of the aforesaid factors, from the results of the present study, the crude leaf extracts of Cassia occidentalis may be considered as an oviposition deterrent but not as an effective ovicide.

Author’s contribution

VR was involved in maintaining the mosquito colony for experiments and carried out bioassay tests. JR was responsible for the original idea of the study, conceived the experimental design and prepared the manuscript. AE and JW contributed to the development of protocol and supervised the study.

Acknowledgements

Authors are grateful to the Director, National Institute of Malaria Research (ICMR), New Delhi, India for laboratory facilities and thankful to the staff of IDVC Field Unit, National Institute of Malaria Research (ICMR), Chennai, Tamil Nadu, India for their kind assistance. The authors also thank the National Institute of Malaria Research (ICMR) publication screening committee for approval of the manuscript vides approval no. 030/2016.

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