Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera: Studies on Refractory Behaviour and its Sustenance across Gonotrophic Cycles Manju Singhi, Vinod Joshi#, R.C. Sharma and Keerti Sharma Desert Medicine Research Centre (Indian Council of Medical Research), New Pali Road, Jodhpur 342 005, India

Abstract Dengue fever associated with dengue haemorrhagic fever is gaining endemicity in India. Due to lack of any chemotherapy against this arboviral infection, the control of the disease depends largely on preventive measures against Aedes mosquito vectors. A wild shrub, Calotropis procera, commonly growing in the desert areas of Rajasthan has shown a remarkable effect as a larvicide against Aedes aegypti. However, different water concentrations of this biocide have also brought forward very important observations on the ovipositioning behaviour of Aedes aegypti. At 0.7% concentration of latex, the ovipositiong was avoided by the gravid female mosquitoes and this behaviour continued till three gonotrophic cycles. However, at lower concentrations (0.2% and 0.1%) of the larvicidal latex, the refractory behaviour of ovipositioning could not be retained up to the third gonotrophic cycle. The concentration of latex such as 0.7% and 0.2% were observed as ovicidal also and this effect continued across all the gonotrophic cycles. The behavioural observations reported in the present study may serve as significant information on choosing bio-larvicides for vector control against dengue. Keywords: Dengue, Aedes aegypti, Calotropis procera, ovipositioning, refractory behaviour, gonotrophic cycle.

Introduction Dengue fever and dengue haemorrhagic fever (DF/DHF) is gaining endemicity in many states in India[1] . In the absence of chemotherapy and vaccines, vector control, largely based on larval control, is the only option available. In Rajasthan, a number of epidemics of dengue associated with DHF #

have been reported[2-4] . It has also been established that dengue virus undergoes transovarial transmission across generations of Aedes aegypti under natural as well as experimental conditions[5,6] . Latex of Calotropis procera, a milky weed plant growing all across the desert areas in the state has shown promising larvicidal properties in a series of laboratory

E-mail: [email protected]

184

Dengue Bulletin – Vol 28, 2004

Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera

experiments carried out against Aedes aegypti (unpublished data, Desert Medicine Research Centre). In an attempt to assess the value of this agent as an oviposition attractant for use in ovitraps, it was discovered that the agent showed oviposition refractoriness instead. The present communication incorporates the findings of these studies.

The experiments were conducted at 2530 °C (room) temperature and at relative humidity of about 60-70%. The eggs of each generation were counted under a dissecting microscope in the control as well as experimental sets. All the eggs were immersed in plain water to observe whether exposure to larvicide had any ovicidal effect on them.

Materials and methods

Results and discussion

Six experimental sets were designed for the study. Cages of 30 cm3 size, with wooden frame and iron mesh with muslin cloth on one side, were used as units of present experiments. In each cage, 16 gravid females of Aedes aegypti were released. The concentration of latex in water, which showed the highest (0.7%), moderate (0.2%) and no mortality (0.1%) effect in the larvicidal efficacy experiments, were prepared and put in beakers in the cages A, B, C, D and E while cage F was kept without latex solution and contained only water. A beaker containing plain water was also placed in each of the six cages to serve as control. While in cage D all the experimental concentrations were placed along with the control, in cage E all the concentrations were placed without choice of a control. In the sixth cage (cage F) two beakers with plain water were placed without any experimental lethal concentration. The eggs laid by female Aedes aegypti were counted after 48 hours in each of the experimental cages. Second and third blood meals were provided to facilitate G2 and G3 (gonotrophic cycles) to mosquitoes.

Table 1 shows relative observations on different concentrations of latex of Calotropis procera on the ovipositioning behaviour of Aedes aegypti. In experimental cage A, while in the control set, 65 eggs were laid, in the corresponding lethal concentration of 0.7% of the same cage, no eggs were laid. In this cage all the mosquitoes showed persistent refractiveness of ovipositioning across all gonotrophic cycles from G1-G3. In cage B where 0.2% concentration was offered along with control, refractive behaviour up to two cycles only was observed while in the third cycle (G3), 127 eggs were laid. Similar observations were made in cage C. It was interesting to note that in cage D where all the experimental concentrations were offered to mosquitoes along with choice of control, even in G1 cycle no refractiveness was shown. In experiment E where choice of control (plain water) was not offered, maximum preference for ovipositioning was shown in lowest concentration (0.1%). The different preference among different concentrations continued across all the gonotrophic cycle (Table 1).

Dengue Bulletin – Vol 28, 2004

185

Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera

Table 1. Ovipositing preference of Aedes aegypti in different larvicidal concentrations of latex of Calotropis procera Latex concentration (%) in experimental cages

Cage

A

B

C

D

Eggs laid within 48 hrs

C

0.7

C

0.2

C

0.1

C

G1

65

0

156

0

180

0

109 18

G2

93

0

156

1

149

0

100

G3

275

0

E

0.1 0.2 0.7 0.1

F

0.2 0.7

C

C

18

6

101

51

11 102 110

0

0

0

33

10

230 127 275 52 270 98

31

0

215 150 20 233 197

7

96

83

C: Control G1-G3: Gonotropic cycles

Table 2. Effect of latex on the percentage viability of eggs of Aedes aegypti Experimental cages

Cage

A

B

C

D

E

Latex (%)

C

0.7

C

0.2

C

0.1

C

Eggs immersed

65

0

156

0

180

0

109

18

18

6

Eggs hatched

55

0

78

0

126

0

78

0

0

0

50

0.0

70

0.0

73

% eggs hatched

84.6 0.0

F

0.1 0.2 0.7 0.1 0.2 0.7 101 51 6

2

C

C

11

102

110

0

96

103

0.0 0.0 0.0 5.9 4.0 0.0 93.1 93.6

C: Control

Table 2 shows the results of the experiment carried out to study the viability of eggs that were exposed to different larvicidal concentrations and to control groups. The eggs laid in experiments A, B, C and D showed no hatching, while in experimental cage E where no control had been kept, the eggs laid up to a concentration 0.2% showed some viability and in cage F where only controls were kept the laid eggs showed 93% viability. 186

Dengue fever associated with DHF has become a problem of public health importance. Available evidence shows that the virus undergoes vertical transmission across generations of mosquitoes[6] . Larval control, therefore, is the most effective approach to restrict the vector and virus sustenance in nature. The wildly grown plant, Calotropis procera, has shown very encouraging results in its different lethal concentrations. However, if this plant Dengue Bulletin – Vol 28, 2004

Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera

species is to be used as a material of choice, its other aspects such as the ovipositioning behaviour of gravid females towards the larvicide can also become known. The significance of the reported observations is that the refractiveness developed by the species is sustained across two gonotrophic cycles when one larvicidal concentration was offered with a corresponding control (Table 1). However, when all the concentrations viz. 0.1 %, 0.2 % and 0.7% were of fered with one control, such avoidance was not shown. Similarly, in the experimental set E where all concentrations of latex were made available without any control, the maximum egg laying was preferred in 0.1 % of latex. The data suggest fine chemo-sensation in Aedes aegypti where the ovipositioning female could distinguish to choose the least larvicidal concentration for its egg laying. The observation showed that if in the various types of domestic containers where water is stored a non-lethal concentration of latex (0.1%) is used, the refractiveness of ovipositioning will not be there and the

domestic mosquito fauna in such premises will lay the eggs but they will lose their viability to hatch into larvae (Table 2). The role of Calotropis procera as a larvicide has been reported by other workers from India[7] . However, contrary results have been reported by some workers[8] where insect growth regulators actually enhanced ovipositioning. The observations reported by us not only present the results of the ovipositioning behaviour induced by a bio-larvicide, but also add the information on relative preference in ovipositioning and its sustenance across gonotrophic cycles when different larvicidal concentrations were offered.

Acknowledgements The authors gratefully acknowledge the guidance and inspiration received from Mr N.L. Kalra, Member, Scientific Advisory Committee of the Desert Medicine Research Centre, Jodhpur, India.

References [1] Dengue alert in South-East Asia Region. New Delhi: World Health Organization, Regional Office for South-East Asia, 2004 (http://w3.whosea.org/index.htm, accessed 25 August 2004). [2] Ghosh SN and Sheikh BH. Investigations on the outbreak of dengue fever in Ajmer city, Rajasthan. Part II: Results of serological tests. Indian Journal of Medical Research, 1974, 62: 523-533. [3] Padbidri VS, Dandawate CN, Goverdhan MK, Bhat UK, Rodrigues FM, D'Lima LV, Kaul HN, Guru PY, Sharma R and Gupta NP. An investigation of the etiology of the 1971 Dengue Bulletin – Vol 28, 2004

outbreak of febrile illness in Jaipur city, India. Indian Journal of Medical Research, 1973, 61(12): 1737-1743. [4] Chouhan GS, Rodrigues FM, Shaikh BH, Ilkal MA, Khangaro SS, Mathur KN, Joshi KR and Vaidhye NK. Clinical and virological study of dengue fever outbreak in Jalore city, Rajasthan, 1985, Indian Journal of Medical Research, 1990, 91: 414-418. [5] Joshi V, Singhi M and Chaudhary RC. Transovarial transmission of dengue 3 virus by Aedes aegypti. Transaction of Royal Society of Tropical Medicine and Hygiene, 1996, 90: 643-644. 187

Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera [6] Joshi V, Mourya D and Sharma RC. Persistence of vertical transmission of dengue-3 virus through vertical transmission passage in successive generations of Aedes aegypti mosquitoes. American Journal of Tropical Medicine and Hygiene, 2002, 67(2): 158-161.

188

[7] Girdhar G, Deval K, Mittal PK and Vasudevan P. Mosquito control by Calotropis latex. Pesticides, 1984, 26-29. [8] Moore CG. Insecticide avoidance by ovipositioning Aedes aegypti. Mosquito News, 1977, 37: 291-293.

Dengue Bulletin – Vol 28, 2004