Parasitology Research

, Volume 110, Issue 2, pp 669–678

Bioefficacy of larvicdial and pupicidal properties of Carica papaya (Caricaceae) leaf extract and bacterial insecticide, spinosad, against chikungunya vector, Aedes aegypti (Diptera: Culicidae)

Authors

    • Division of Entomology, Department of Zoology, School of Life SciencesBharathiar University
  • Kadarkarai Murugan
    • Division of Entomology, Department of Zoology, School of Life SciencesBharathiar University
  • Arjunan Naresh Kumar
    • Division of Entomology, Department of Zoology, School of Life SciencesBharathiar University
  • Savariar Vincent
    • Tamil Nadu State Council for Science and Technology, DOTE Campus, Guindy
  • Jiang-Shiou Hwang
    • Institute of Marine BiologyNational Taiwan Ocean University
Original Paper

DOI: 10.1007/s00436-011-2540-z

Cite this article as:
Kovendan, K., Murugan, K., Naresh Kumar, A. et al. Parasitol Res (2012) 110: 669. doi:10.1007/s00436-011-2540-z

Abstract

The present study was carried out to establish the properties of Carica papaya leaf extract and bacterial insecticide, spinosad on larvicidal and pupicidal activity against the chikungunya vector, Aedes aegypti. The medicinal plants were collected from the area around Bharathiar University, Coimbatore, India. C. papaya leaf was washed with tap water and shade-dried at room temperature. An electrical blender powdered the dried plant materials (leaves). The powder (500 g) of the leaf was extracted with 1.5 l of organic solvents of methanol for 8 h using a Soxhlet apparatus and then filtered. The crude leaf extracts were evaporated to dryness in a rotary vacuum evaporator. The plant extract showed larvicidal and pupicidal effects after 24 h of exposure; however, the highest larval and pupal mortality was found in the leaf extract of methanol C. papaya against the first- to fourth-instar larvae and pupae of values LC50 = I instar was 51.76 ppm, II instar was 61.87 ppm, III instar was 74.07 ppm, and IV instar was 82.18 ppm, and pupae was 440.65 ppm, respectively, and bacterial insecticide, spinosad against the first to fourth instar larvae and pupae of values LC50 = I instar was 51.76 ppm, II instar was 61.87 ppm, III instar was 74.07 ppm, and IV instar was 82.18 ppm, and pupae was 93.44 ppm, respectively. Moreover, combined treatment of values of LC50 = I instar was 55.77 ppm, II instar was 65.77 ppm, III instar was 76.36 ppm, and IV instar was 92.78 ppm, and pupae was 107.62 ppm, respectively. No mortality was observed in the control. The results that the leaves extract of C. papaya and bacterial insecticide, Spinosad is promising as good larvicidal and pupicidal properties of against chikungunya vector, A. aegypti. This is an ideal eco-friendly approach for the control of chikungunya vector, A. aegypti as target species of vector control programs.

Introduction

Mosquitoes constitute a major public health problem as vectors of serious human like malaria, filariasis, Japanese encephalitis, dengue fever, chikunkunya, and yellow fever cause substantial mortality and morbidity among people living in tropical and subtropical zones (Jang et al. 2002). The container-breeding mosquito, Aedes aegypti L. thrives in urban and peridomestic environments where it transmits the dengue virus to humans (Gubler 1998). A. aegypti L. is generally known as a vector for an arbovirus responsible for dengue and chikunkunya, which is endemic to South Asia, the Pacific island area, Africa, and the Americas. This mosquito is also a vector of yellow fever in Central and South America and West Africa.

Dengue fever has become an important public health problem as the number of reported cases continue to increase, especially with more severe of the disease, dengue hemorrhagic fever and dengue shock syndrome, or with unusual manifestations such as central nervous system involvement (Pancharoen et al. 2002). It is estimated that there are between 50 and 100 million cases of dengue fever (DF) and about 500,000 cases of dengue hemorrhagic fever (DHF) each year which require hospitalization (Maheswaran et al. 2008). Dengue fever is spread through the bite of an infected A. aegypti mosquito. The mosquito gets the virus by biting an infected person. The first symptom of the disease appears in about 5–7 days after the infected mosquito bites a healthy person. It is possible to become infected by dengue multiple times because the virus has four different serotypes. The dengue symptoms of dengue fever include high fever, rash, and a severe headache. Additional of chickungunya fever symptoms include severe joint and muscular pain (breakbone fever), nausea, vomiting, and eye pain. Although dengue fever itself is rarely fatal, it can be an extraordinary painful and disabling illness and may become epidemic in a population following the introduction of a new serotype (Morena–Sanchez et al. 2006).

Carica papaya, belongs to the family of Caricaceae, and several species of Caricaceae have been used as remedy against a variety of diseases (Mello et al. 2008; Munoz et al. 2000). Originally derived from the southern part of Mexico, C. papaya is a perennial plant, and it is presently distributed over the whole tropical area. In particular, C. papaya fruit circulates widely, and it is accepted as food or as a quasi drug. Many scientific investigations have been conducted to evaluate the biological activities of various parts of C. papaya, including fruits, shoots, leaves, rinds, seeds, roots or latex. The leaves of papaya have been shown to contain many active components that can increase the total antioxidant power in blood and reduce lipid peroxidation level, such as papain, chymopapain, cystatin, à-tocopherol, ascorbic acid, flavonoids, cyanogenic glucosides, and glucosinolates (Seigler et al. 2002).

Fruit and seed extracts have pronounced bactericidal activities (Emeruwa 1982). Leaves have been poulticed into nervous pains, elephantoid growths, and it has been smoked for asthma relief amongst tropical tribal communities. The hypoglycemic effect of ethanolic extract of unripe, mature fruits has been reported by Olagunju et al. (1995). Moreover, C. papaya leaf juice is consumed for its purported anti-cancer activity by people living on the Gold Coast of Australia, with some anecdotes of successful cases being reported in various publications. C. papaya leaf extracts have also been used for a long time as an aboriginal remedy for various disorders, including cancer and infectious diseases.

C. papaya contains two important biologically active compounds vis: chymopapain and papain which are widely used for digestive disorders (Huet et al. 2006). It showed that papaya-derived papain, caricain, chymopain, and glycerin endopeptidase can improve acidic pH conditions and pepsin degradation. Other active compounds of C. papaya are lipase, or CPL, a hydrolase, which is tightly bonded to the water-insoluble fraction of crude papain and is thus considered as a “naturally immobilized” biocatalyst (Dominguez et al. 2006). According to the folk medicine, papaya latex can cure dyspepsia and also applicable for external burns and scalds. Seeds and fruits are excellent anthelminthic and anti-amoebic (Okeniyi et al. 2007). Dried and pulverized leaves are sold for making tea; also the leaf decoction is administered as a purgative for horses and used for the treatment of genetic-urinary system. Unripe and semi-ripe papaya fruits are ingested or applied on the uterus to cause absorption. However, the consumption of unripe and semi-ripe papaya fruits could be unsafe during pregnancy causes no risk (Adebowale et al. 2002).

The larvicidal properties of crude extracts of three plants, viz. C. papaya, Murraya paniculata, and Cleistanthus collinus against Culex quinquefasciatus as target species. The relative efficacy of the plant extracts in vector control was as follows: C. papaya seed extract>M. paniculata fruit extract>M. paniculata leaf extract>C. collinus leaf extract. To examine the potential role of C. papaya as anti-cancer therapy, we analyzed in this report the anti-tumor activity of the aqueous extract of the leaves of C. papaya (CP) against various cancer cell lines, as well as its potential immunomodulatory effects, and attempted to identify the active components (Rawani et al. 2009).

In fact, many researchers have reported on the effectiveness of plant extracts or essential oils against mosquito larvae (Mehlhorn et al. 2005; Amer and Mehlhorn 2006a, b; Rawani et al. 2009 a, b). Govindarajan (2009) reported that the leaf methanol, benzene, and acetone extracts of Cassia fistula were studied for the larvicidal, ovicidal, and repellent activities against A. aegypti. Daniellia oliveri is traditionally used to reduce numbers of mosquitoes indoors at night (Curtis et al. 1991).

Spinosad is a natural fermentation product produced by an actinomycete, Saccharopolyspora spinosa Mertz and Yao. This compound is a mixture of spinosyns A and D. It has shown activity against Lepidoptera, Thysanoptera, and other insect orders such as Diptera. This naturally derived insecticide has been reported to have no adverse effects on predatory insects such as ladybirds, lacewings, big-eyed bugs, or minute pirate bugs (Kirst et al. 1992; DeAmicis et al. 1997; Copping and Menn 2001; Williams et al. 2003). Spinosad acts as a stomach and contact poison and degrades rapidly in the environment (Cisneros et al. 2002). An immediate effect of ingestion is the cessation of feeding, followed 24 h later, by paralysis and death. This compound is a neurotoxin with a novel mode of action involving the nicotinic acetylcholine receptor and GABA receptors (Watson 2001). Spinosad has little toxicity to birds and mammals (Bret et al. 1997; Breslin et al. 2000). There is no cross-resistance to synthetic and traditional biological insecticides (Salgado 1997, 1998). Moreover, spinosad is classified by the US Environmental Protection Agency as an environmentally and toxicologically reduced risk material (Saunders and Bret 1997).

In insects, the mode of action of spinosad is associated with the excitation of the insect nervous system (Salgado 1998). Although the exact site of action of spinosyns is still under investigation, spinosad uniquely alters the function of nicotinic and GABA-gated ion channels at the synapse between nerve cells. However, spinosad does not interact with known binding sites for other nicotinic or GABA insecticides such as neonicotinoids, fiproles, avermectins, and cyclodienes. These data indicate that spinosad acts through a unique insecticidal mechanism.

Spinosad is a novel insecticide produced from a family of natural products derived from fermentation of the actinomycete Saccharopolyspora spinosa (Snyder et al. 2007). These two bacterial biocides have been evaluated in various formulations against mosquito vectors worldwide (Lacey and Lacey 1990). Bacillus thuringiensis produces four key insecticidal proteins, and Bacillus sphaericus produces a single binary toxin effective against JE vector (Federici et al. 2003). B. sphaericus and B. thuringiensis H-14 showed larvicidal activity up to 91–99% against Culex tritaeniorhynchus and Anopheles subpictus; however, activity did not subsist beyond a few days (Kramer 1984). B. thuringiensis H-14 showed 100% reduction with doses of 27 × 105 spores per milliliter, but first and second instars reappeared 3 days after application (Balaraman et al. 1983).

Bond et al. (2004) reported that the naturally derived insecticide spinosad is highly toxic to Aedes and Anopheles mosquito larvae. Cetin et al. (2005) worked on the Evaluation of the naturally derived insecticide spinosad against Culex pipiens L. (Diptera: Culicidae) larvae in septic tank water in Antalya. Additional studies have reported the larvicidal properties of spinosad in this and other mosquito species (Liu et al. 2004a, 2004b; Cetin et al. 2005a; Darriet et al. 2005; Darriet and Corbel 2006; Romi et al. 2006), or as an adulticide in a sugar bait formulation (Muller and Schlein 2006).

During the present study, an attempt was made to establish the larvicidal and pupicidal properties of methanol leaf crude extract of C. papaya and bacterial insecticide, spinosad against chikungunya vector, A. aegypti as target species.

Materials and methods

Collection of eggs and maintenance of larvae

The eggs of A. aegypti were collected from National Centre for Disease Control (NCDC) field station of Mettupalayam, Tamil Nadu, India, using an“O” type brush. These eggs were brought to the laboratory and transferred to 18 × 13× 4 cm enamel trays containing 500 ml of water for hatching. The mosquito larvae were fed with pedigree dog biscuits and yeast at 3:1 ratio. The feeding was continued until the larvae transformed into the pupal stage.

Maintenance of pupae and adults

The pupae were collected from the culture trays and transferred to plastic containers (12 × 12 cm) containing 500 ml of water with the help of a dipper. The plastic jars were kept in a 90 × 90 × 90-cm mosquito cage for adult emergence. Mosquito larvae were maintained at 27°C + 2°C, 75–85% RH under a photoperiod of 14 L/10 D. A 10% sugar solution was provided for a period of 3 days before blood feeding.

Blood feeding of adult A. aegypti

The adult female mosquitoes were allowed to feed on the blood of a rabbit (a rabbit per day, exposed on the dorsal side) for 2 days to ensure adequate blood feeding for 5 days. After blood feeding, enamel trays with water from the culture trays were placed in the cage as oviposition substrates.

Plant bioassay

C. papaya was collected in and around Bharathiar University, Coimbatore, India. The voucher specimen has been deposited at the Zoology Department, Bharathiar University, Coimbatore, Tamil Nadu, India. C. papaya leaf was washed with tap water and shade-dried at room temperature. An electrical blender powdered the dried plant materials (leaves). The powder (500 g) of the leaf was extracted with 1.5 l of organic solvents of methanol for 8 h using a Soxhlet apparatus (Vogel 1978). The extracts were filtered through a Buchner funnel with Whatman number 1 filter paper. The crude plant extracts were evaporated to dryness in rotary vacuum evaporator. One gram of the plant residue was dissolved in 100 ml of acetone (stock solution) and considered as 1% stock solution. From this stock solution, different concentrations were prepared ranging from 100 to 500 ppm, respectively.

Microbial bioassay

Spinosad was obtained from T-Stanes & Company Limited, Research and Development Centre, Coimbatore, Tamil Nadu, India. The required quantity of spinosad was thoroughly mixed with distilled water to prepare at various concentrations ranging from 20 to 100 ppm, respectively.

Larval/pupal toxicity test

Laboratory colonies of mosquito larvae/pupae were used for the larvicidal/pupicidal activity. Twenty-five numbers of I to IV instar larvae and pupae were introduced into 500-ml glass beaker containing 249 ml of dechlorinated water and 1 ml of desired concentrations of plant extract, and spinosad were added. Larval food was given for the test larvae. At each tested concentration, two to five trials were made, and each trial consisted of three replicates. The control was set up by mixing 1 ml of acetone with 249 ml of dechlorinated water. The larvae and pupae which were exposed to dechlorinated water without acetone served as control. The control mortalities were corrected by using Abbott's formula (Abbott's 1925). The LC50 and LC90 were calculated from toxicity data by using probit analysis (Finney 1971).
$$ {\text{Corrected mortality}} = \frac{{{\text{Observed mortality in treatment - Observed mortality in control}}}}{{{\text{100 - Control }}\,{\text{mortality}}}} \times 100 $$
$$ {\text{Percentage mortality}} = \frac{{{\text{Number of dead larvae/pupae}}}}{{{\text{Number of larvae/pupae introduced }}}} \times 100 $$

Statistical analysis

All data were subjected to analysis of variance; the means were separated using Duncan's multiple range tests by Alder and Rossler (1977). The average larval mortality data were subjected to probit analysis, for calculating LC50 and LC90, values were calculated by using the Finney (1971) method. SPSS software package 9.0 version was used. Results with P < 0.05 were considered to be statistically significant.

Results

Larval and pupal mortality of A. aegypti after the treatment of methanolic extract of C. papaya leaf was observed. Table 1 provides the larval and pupal mortality of A. aegypti (I to IV instars) after the treatment of A. aegypti at different concentrations (100 to 500 ppm). Thirty-four percent mortality was noted at I instar larvae by the treatment of C. papaya at 100 ppm, whereas it has been increased to 92% at 500 ppm of C. papaya leaf extract treatment. Similar trend has been noted for all the instars of A. aegypti at different concentration of C. papaya treatment. The LC50 and LC90 values were represented as follows: LC50 value of I instar was 227.15 ppm, II instar was 277.16 ppm, III instar was 335.99 ppm, and IV instar was 375.88 ppm, respectively. The LC90 value of I instar was 536.36 ppm, II instar was 593.90 ppm, III instar was 679.51 ppm, and IV instar was 715.75 ppm, respectively. The LC50 value of pupae was 440.65 ppm, and the LC90 value of pupae was 796.59 ppm, respectively.
Table 1

Larval and pupal toxicity effect of C. papaya leaf extract against the chikungunya vector, A. aegypti

Mosquito larval instars and pupae

Larval and pupal mortality

LC50 (LC90)

95% Confidence limit

x2 (df = 4)

Concentration of C. papaya (ppm)

LFL

UFL

100

200

300

400

500

LC50 (LC90)

LC50 (LC90)

I

34a

45a

56a

73a

92a

227.15280 (536.36209)

192.98940 (484.34574)

256.16186 (612.86015)

5.015*

II

29b

35b

47b

68b

86b

277.16385 (593.90556)

246.74353 (534.82633)

306.10427 (681.63126)

5.006*

III

22c

31b

40c

54c

79c

335.99208 (679.51541)

304.96606 (604.56073)

370.39636 (795.48488)

4.570*

IV

18cd

26c

34d

47d

75c

375.88635 (715.75632)

306.16292 (567.78724)

490.40852 (1,150.40125)

5.729*

Pupa

14d

20d

27e

35e

67d

440.65630 (796.59968)

350.71643 (597.86625)

701.11584 (1,683.05935)

7.991*

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT

Control-Nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom

*P < 0.05 level

Table 2 illustrates the larval and pupal mortality of A. aegypti (I to IV instars) after the treatment of spinosad at different concentrations (20–100 ppm). Thirty-one percent mortality was noted at I instar larvae by the treatment of spinosad at 20 ppm, whereas it has been increased to 87% at 100 ppm of spinosad treatment and 12% mortality was noted at pupae by the treatment of spinosad at 20 ppm and it has been increased to 64% at 100 ppm. Similar trend has been noted for all the instars of A. aegypti at different concentrations of spinosad treatment. The LC50 and LC90 values were represented as follows: LC50 value of I instar was 51.76 ppm, II instar was 61.87 ppm, III instar was 74.07 ppm, and IV instar was 82.18 ppm, respectively. The LC90 value of I instar was 117.60 ppm, II instar was 139.27 ppm, III instar was 149.03 ppm, and IV instar was 155.50 ppm, respectively. The LC50 value of pupae was 93.44 ppm and the LC90 value of pupae was 162.66 ppm, respectively.
Table 2

Larval and pupal toxicity effect of bacterial insecticide, spinosad against the chikungunya vector, A. aegypti

Mosquito larval instars and pupae

Larval and pupal mortality

LC50 (LC90)

95% Confidence limit

x2 (df = 4)

Concentration of spinosad (ppm)

LFL

UFL

20

40

60

80

100

LC50 (LC90)

LC50 (LC90)

I

31a

39a

52a

68a

87a

51.76882 (117.60759)

45.15904 (105.51486)

57.74529 (135.82611)

3.563*

II

26b

33b

44b

55b

80b

61.87610 (139.27069)

44.34864 (108.38719)

81.06919 (240.91736)

5.534*

III

20c

29b

37c

48c

73c

74.07166 (149.03313)

67.19039 (130.75840)

82.51930 (178.61302)

3.931*

IV

17c

23c

31d

42d

69c

82.18527 (155.50594)

74.89372 (136.34454)

91.89465 (186.53804)

5.135*

Pupa

12d

16d

24e

30e

64d

93.44808 (162.66591)

73.60570 (119.98824)

167.99669 (397.58642)

9.474*

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT

Control-Nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom

*P < 0.05 level

Table 3 shows the considerable larval and pupal mortality after the combined treatment of spinosad and C. papaya leaf of methanolic extract for all the larval instars and pupae. The concentration at 50 + 100 ppm combined treatment of spinosad and C. papaya for I instar larval mortality was 95%, respectively. The LC50 and LC90 values were represented as follows: LC50 value of I instar was 55.77 ppm, II instar was 65.77 ppm, III instar was 76.36 ppm, and IV instar was 92.78 ppm. The LC90 value of I instar was 151.17 ppm, II instar was 175.79, III instar was 190.98 ppm, and IV instar was 202.94 ppm. The LC50 value of pupae was 107.62 ppm, and the LC90 value of pupae was 222.92 ppm, respectively.
Table 3

Combined treatment of larval and pupal toxicity effect of C. papaya leaf extract and bacterial insecticide, spinosad against the chickugunya vector, A. aegypti

Mosquito larval instars and pupae

Larval and pupal mortality

LC50 (LC90)

95% Confidence limit

x2 (df = 4)

Concentration of C. papaya (ppm) + Spinosad (ppm)

LFL LC50 (LC90)

UFL LC50 (LC90)

20 + 10

40 + 20

60 + 30

80 + 40

100 + 50

I

42a

51a

60a

78a

95a

55.77193 (151.17382)

6.41336 (119.41368)

78.84710 (253.41303)

7.563*

II

39a

45b

56b

70b

89b

65.77360 (175.79516)

53.04977 (155.84897)

76.00133 (207.29224)

5.253*

III

30b

41bc

52b

65b

83c

76.36812 (190.98989)

64.59055 (168.32860)

86.63494 (227.48104)

2.813*

IV

24c

37cd

46c

59c

78c

92.78842 (202.94203)

82.76361 (179.35220)

103.12543 (240.36802)

1.545*

Pupa

19 c

32d

40d

55c

69d

107.62874 (222.92774)

97.21988 (195.14688)

120.05786 (268.31690)

0.486*

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT

Control-Nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom

*P < 0.05 level

Discussion

Dengue is an arboviral disease mainly transmitted by the mosquito A. aegypti, and in the last years has become a major international public health concern (WHO 1998). It is found in tropical and subtropical regions around the world, predominantly in urban and semi-urban regions. The WHO published a global map of the distribution of the dengue epidemic activity during the year 2006 that shows whole India in red color. More than 50 million people are at risk of dengue virus exposure worldwide. Annually, there are two million infections, 500,000 cases of dengue hemorrhagic fever, and 12,000 deaths (Guha-Sapir and Schimmer 2005). Conventional control of A. aegypti is based on treatment of water containers with larvicides and on the use of adulticides (Gubler and Clark 1995; Gubler 2004).

The main objective of the current study is to investigate the potential of C. papaya leaves extract against dengue fever. The secondary metabolite of plant origins makes up a vast repository compounds with a wide range of biological activities. There have been many reports of higher plant extracts possessing relatively good potential to inhibit viruses (Van Den Berghe 1978). Laboratory bioassay of latex from the unripe fruits of C. papaya was carried out against A. stephensi, and the LC50 and LC90 value was 0.013% and 0.062%, respectively (Thomas et al. 2004).

Roark (1947) described approximately 1,200 plant species, whilst Sukumar et al. (1991) listed and discussed 344 plant species that exhibited mosquitocidal activity. Shaalan et al. (2005) reviewed the current state of knowledge on larvicidal plant species and listed the growth and reproduction inhibiting phytochemicals, botanical ovicides, synergistic, additive and antagonistic joint action effects of botanical mixtures, residual capacity and effects on nontarget organisms, and appearance of resistance. Usually, it has been found that secondary metabolites produced by plants are responsible for their chemical defense and toxicity to other animals. Several secondary metabolites such as steroids (Ghosh et al. 2008; Chowdhury et al. 2008; Rahuman et al. 2008; Zolotar et al. 2002), phenolics (Tripathi and Rathore 2001), essential oils (Amer and Mehlhorn 2006).

Sharma et al. (2005) reported that the acetone extract of Nerium indicum and Thenus orientalis have been studied with LC50 values of 200.87, 127.53, 209.00, and 155.97 ppm against III instar larvae of A. stephensi and C. quinquefasciatus, respectively. Earlier authors reported that the methanol leaf extracts of Vitex negundo, Vitex trifolia, Vitex peduncularis, and Vitex altissima were used for larvicidal assay with LC50 values of 212.57, 41.41, 76.28, and 128.04 ppm, respectively, against the early fourth-instar larvae of C. quinquefasciatus (Kannathasan et al. 2007). The larvicidal activity of the essential oil aqueous solutions of the stalks and leaves of Croton argyrophylloides, Croton nepetaefolius, Croton sonderianus, and Croton zehntneri showed 100% mortality at 50 ml against A. aegypti (Lima et al. 2006); Morais et al. (2006) also reported that the main components methyleugenol and alpha-copaene for C. nepetaefolius (LC50 of 84 ppm); alpha-pinene and beta-pinene for C. argyrophyloides (LC50 of 102 ppm); and alpha-pinene, beta-phelandrene, and transcaryophyllene for C. sonderianus (LC50 of 104 ppm) and Croton zenhtneri exhibited higher larvicidal activity with an LC50 of 28 ppm against A. aegypti. The toxicity of Euphorbia milii molluscicidal latex and niclosamide showed toxic affect to Anopheles albitarsis, A. aegypti, and Aedes fluviatilis larvae (Filho and Paumgartten 2000). Likewise, the crude extracts of a few indigenous plants showed moderate larvicidal effects against larvae of Culex quinquefasciatus Say with LC50 value ranging from 41.75 to 709.51 ppm (Rahuman et al. 2009a).

Recently, studies stimulated that the larval and pupal mortality of A. stephensi after the treatment of methanolic extract of C. inerme leaf extract is shown; results of 22% mortality was noted at I instar larvae by the treatment at 20 ppm, whereas it has been increased to 81% at 100 ppm of C. inerme leaf extract of larval and pupal mortality of A. stephensi (I to IV instars) after the treatment of methanolic extract of A. ilicifolius at different concentrations (20–100 ppm). Twenty-three percent mortality was noted at I instar larvae by the treatment of A. ilicifolius at 20 ppm, whereas it has been increased to 89% at 100 ppm of A. ilicifolius leaf extract treatment (Kovendan and Murugan 2011). Kovendan et al. (2011a, b) recently have reported that the leaf extract of methanol Jatropha curcas against C. quinquefasciatus and Leucas aspera leaf extract against A. stephensi, respectively.

The bio-control potentiality of crude extracts of the three plants, C. papaya, M. paniculata, and C. collinus against C. quinquefasciatus have been well-established in the laboratory condition (Rawani et al. 2009). The highest mortality was recorded in C. papaya seed extract. The phytochemical analysis of the plant extracts reveals the presence of several bioactive secondary metabolites that singly or in combinations may be responsible for the larval toxicity. As no mortality occurs in the non-target organisms (invertebrates), it can be assumed that all the plant extracts are safe to use in the aquatic ecosystem, though some toxicity of C. collinus in higher vertebrates have been reported (Sarathchandra and Balakrishnamoorthy 1998; Eswarappa and Benjamin 2007). In conclusion, the crude extracts of C. papaya, M. paniculata, and C. collinus can be recommended in large-scale field trials and can be effectively used as potent larvicides in mosquito control programs. During present study, C. papaya methanolic extract was of considerable and good larvicidal and pupicidal properties against the chikungunya vector, A. aegypti.

Cisneros et al. (2002) said that spinosad acts as a stomach and contact poison and degrades rapidly in the environment. Earlier, Bond et al. (2004) have reported that the spinosad was most effective at the lowest concentrations (0.024–0.025). Romi et al. (2006) studied the efficacy of a spinosad-based product (laser 4.8% emulsifiable concentrate) which was evaluated in laboratory bioassays against laboratory-reared mosquito strains of three species of medical importance: A. aegypti, A. stephensi, and C. pipiens. Spinosad was particularly effective against larval Aedes and Culex, with a less marked activity against Anophelines (24-h median lethal concentration = 0.0096, 0.0064, and 0.039 mg/l, respectively), showing a persistence of the insecticide action of about 6 weeks in laboratory containers.

However, it was clear that spinosad solutions placed in warm, sunny locations lost toxicity tenfold faster than solutions placed in the shade. Because A. aegypti preferentially oviposits in shaded habitats (Fay and Eliason 1966; Vezzani et al. 2005), the ability of spinosad to persist for weeks or months in the shade favors the suppression of mosquito development for periods that extend over the annual peaks of vectorial activity and that often coincide with seasonal fluctuations in rainfall in tropical regions. The insignificant mammalian toxicity and favorable environmental profile of spinosad, involving degradation by photolysis and microbial action (Cleveland et al. 2002; Thompson et al. 2002; Liu and Li 2004), means that bioaccumulation and related ecological problems that arise from persistent xenobiotic compounds are highly unlikely for this product.

High concentrations of organophosphate and pyrethroid insecticides tend to be deterrent for oviposition (Moore 1977; Verma 1986), whereas other compounds, such as methoprene or granular formulations of temephos, are not repellent (Mather and DeFoliart 1983; Beehler and Mulla 1993; Pates and Curtis 2005). In our study, a weak but significant attraction to visit cups containing spinosad was observed at a concentration of 20 ppm but not at 5 ppm. Spinosad has a distinctive aroma of damp earth, characteristic of the presence of actinomycetes that may have proved attractive to gravid females. However, it did not result in an increase in the number of eggs laid in the spinosad treatments, or any other of the treatments that we tested. This finding could have been influenced by the response of A. aegypti to conspecific eggs (Allan and Kline 1998) or by the skip oviposition behavior shown by some, but not all populations, of this species (Corbet and Chadee 1993; Harrington and Edman 2001), and the limited possibility to disperse eggs over various oviposition sites in our caged experiments.

Perez et al. (2007) have reported that the naturally derived insecticide spinosad is a reduced-risk material that is neurotoxic to Diptera. The 24-h 50% lethal concentration by laboratory bioassay in third instars of A. aegypti (L.) (Diptera: Culicidae) (Rockefeller strain) was estimated at 0.026 ppm. Water containers treated with 1 or 5 ppm spinosad suspension concentrate (Tracer, DowAgrosciences) were as effective in preventing the development of Aedes spp. (mostly A. aegypti) as temephos granules during both trials, whereas the bacterial insecticide VectoBac 12AS performed poorly. The half-life of aqueous solutions of spinosad (10 ppm) placed in a warm sunny location was 2.1 days, compared with 24.5 days for solutions in a shaded location. Spinosad was as effective as temephos granules in eliminating the immature stages of Aedes spp., mostly A. aegypti, in an urban cemetery during the wet and dry seasons in southern Mexico. Persistence and oviposition response studies indicated that spinosad could retain its insecticidal properties for periods of several months in shaded conditions preferred by A. aegypti, and it was not repellent for mosquito oviposition. The combination of the toxicological properties and favorable environmental profile means that spinosad deserves detailed evaluation as a mosquito larvicide in domestic and urban vector control programs.

In earlier study, the microbial pesticide spinosad against the malarial vector A. stephensi Liston showed 85% mortality (Aarthi and Murugan 2010). The observed mortality rate suggests that the above extract can be used as biopesticides. The LC50 of second, third, and fourth-instar larvae of A. stephensi were 0.276%, 0.285%, and 0.305%, respectively. In the present study, spinosad treatment reduced the larval and pupal properties of microbial insecticides development of growth control.

Garcia and Desrochers (1979) observed appreciable mortality only with high concentrations (1 × 107 cells per milliliter) of B. thuringiensis var. israelensis. The biocide at 1 to 10 Kg/ha (0.25–2.5 ppm) caused 18% to 88% mortality of midges during a 4-week evaluation period. Younger instars are more susceptible than older ones as shown by C. quinquefasciatus. Exposure periods longer than 48 h in the laboratory may produce better activity results of the B. thuringiensis var israelensis formulations against the midges' species (Ali 1981). Similarly, the alternative use of plant extracts as an additive to B. thuringiensis var. israelensis may be a promising approach because larval feeding and subsequent defoliation would be reduced greatly without interference with bacterial activity.

Ludlum et al. (1991) have reported that aromatic compounds and plant allelochemicals increase B. thuringiensis var. israelensis activity. The addition of B. thuringiensis var. israelensis with the plant extracts had an adverse effect upon the larval mortality. When combined with plant extracts, B. thuringiensis var. israelensis increased the percentage of larval mortality and decreased the time to kill when compared with treatment containing only B. thuringiensis var. israelensis. The addition of B. thuringiensis var. israelensis with plant extracts caused a significant mortality due to the avoidance of treated diet and may be due to increased toxicity (Gould et al. 1991). Since the discovery of the agent and its lethal effects against species of Anopheles, Aedes, Culex, Ochlerotatus, and Uranotaenia larvae by Goldberg and Margalit (1977), Kovendal et al. (2011) recently reported that B. thuringiensis israelensis against the first to fourth-instar larvae of values LC50 = 9.332%, 9.832%, 10.212%, 10.622%, and LC90 = 15.225%, 15.508%, 15.887%, and 15.986% larvae of C. quinquefasciatus, respectively.

In the present study, 31% mortality was noted at I instar larvae by the treatment of spinosad at 20 ppm, whereas it has been increased to 87% at 100 ppm of spinosad treatment and 12% mortality was noted at pupae by the treatment of spinosad at 20 ppm, and it has been increased to 64% at 100 ppm. Similar trend has been noted for all the instars of A. aegypti at different concentrations of spinosad treatment. LC50 value of I instar was 51.76 ppm, II instar was 61.87 ppm, III instar was 74.07 ppm, and IV instar was 82.18 ppm, respectively. The LC90 value of I instar was 117.60 ppm, II instar was 139.27 ppm, III instar was 149.03 ppm, and IV instar was 155.50 ppm, respectively. The concentration at 50 + 100 ppm combined treatment of spinosad and C. papaya for I instar larval mortality was 95%, respectively

The finding of the present investigation revealed that the leaf extract of C. papaya and bacterial insecticide, spinosad has good larvicidal and pupicidal properties of against chikungunya vector, A. aegypti as target species of vector control programs.

Acknowledgments

The authors are thankful to the Department of Science and Technology (DST), New Delhi, India and Tamil Nadu State Council for Science and Technology (TNSCST), Chennai, Tamil Nadu for providing financial support for the present work. The authors are grateful to Dr. K. Sasikala, Professor and Head, Department of Zoology, Bharathiar University for the laboratory facilities provided.

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© Springer-Verlag 2011