jear2012 Abstract Intervention measures to control the transmission of vector-borne diseases include control of the vector population. In mosquito control, synthetic insecticides used against both the larvae (larvicides) and adults (adulticides) create numerous problems, such as environmental pollution, insecticide resistance and toxic hazards to humans. In the present study, a bacterial pesticide, Bacillus sphaericus (Bs G3-IV), was used to control the dengue and filarial vectors, Aedes aegypti and Culex quinquefasciatus. Bacillus sphaericus (Bs G3-IV) was very effective against Aedes aegypti and Culex quinquefasciatus, showing significant larval mortality. Evaluated lethal concentrations (LC50 and LC90) were age-dependent, with early instars requiring a lower concentration com- pared with later stages of mosquitoes. Culex quinquefasciatus was more susceptible to Bacillus sphaericus (Bs G3-IV) than was Aedes aegypti. Fecundity rate was highly reduced after treatment with differ- ent concentrations of Bacillus sphaericus (Bs G3-IV). Larval and pupal longevity both decreased after treatment with Bacillus sphaericus (Bs G3-IV), total number of days was lower in the B. sphaericus treatments compared with the control. Our results show the bacterial pesticide Bacillus sphaericus (Bs G3-IV) to be an effective mosquito control agent that can be used for more integrated pest management programs. Introduction Mosquitoes are insect vectors responsible for the transmission of many diseases. Mosquito-borne diseases include yellow fever, dengue fever and Chikungunya, transmitted mostly by Aedes aegypti; malaria, carried by the genus Anopheles, and Culex serves as a vector of impor- tant diseases such as West Nile virus, filariasis, Japanese encephalitis, St. Louis encephalitis, and avian malaria. Insect-transmitted disease remains a major source of illness and death worldwide. Mosquitoes alone transmit disease to more than 700 million people annually and are responsible for several million deaths every year (WHO, 2012; Taubes, 2000; Kessler & Guerin, 2008). Management of these vectors is a serious concern in a developing country like India, due to development of pesticide resistance and for socio-economic reasons. Every year, a large part of the population is affected by one or more vector-borne diseases. Vector control, which includes both anti-larval and anti-adult measures, constitutes an impor- tant aspect of any mosquito control program. Mosquito control using synthetic insecticides is an effective vector control strategy used exten- sively in daily life. Synthetic insecticides are still at the forefront of mos- quito-controlling efforts. However, the environmental threat that these chemicals pose affects on non-target organisms, and resistance of mos- quitoes to insecticides have all increased during the last five decades (Wattanachai & Tintanon, 1999; Amer & Mehlhorn, 2006a, 2006b). In recognition of these facts, it is necessary to develop new insecti- cides for controlling mosquitoes that are environmentally safer, biodegradable, and more target-specific against the mosquitoes. Recent negative consumer perceptions concerning the use of chemi- cals as larvicides have shifted research efforts towards the develop- ment of alternatives that the public perceives as natural products, such as bacterial pesticides, predators, and plant extracts. Consequently, the present work deals with the insecticidal activities of natural products, such as bacterial pesticides. Bacillus sphaericus is an aerobic, mesophilic, spore-forming bac- terium with terminal swollen sporangia and spherical spores. As a con- sequence of the specific toxicity to mosquito larvae of binary toxin (Bin) and mosquitocidal toxins (Mtxs) produced during the sporula- tion and vegetative stages, respectively, some toxic strains have been widely used for many years as biopesticides in the field in mosquito control programs (Bei et al., 2007). Bacillus sphaericus is a naturally occurring soil bacterium that can Journal of Entomological and Acarological Research 2012; volume 44:e15 Correspondence: Kadarkarai Murugan, Department of Zoology, Bharathiar University, Coimbatore, 641046, India. E-mail: kmvvkg@gmail.com Key words: Bacillus sphaericus (Bs G3-IV), Aedes aegypti, Culex quinquefas- ciatus, fecundity, mosquito longevity. Acknowledgments: we are very thankful to the authorities of the Defence Research and Development Laboratory (DRDL), Defence Research and Development Organization, Ministry of Defence, Government of India, Tezpur for providing funds plus their isolate of Bacillus sphaericus, for the successful completion of this project. Received for publication: 20 August 2012. Revision received: 20 November 2012. Accepted for publication: 20 November 2012. ©Copyright A. Nareshkumar et al., 2012 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2012; 44:e15 doi:10.4081/jear.2012.e15 This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncom- mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited. Larvicidal potentiality, longevity and fecundity inhibitory activities of Bacillus sphaericus (Bs G3-IV) on vector mosquitoes, Aedes aegypti and Culex quinquefasciatus A. Nareshkumar,1 K. Murugan,1 I. Baruah,2 P. Madhiyazhagan,1 T. Nataraj1 1Department of Zoology, Bharathiar University, Coimbatore; 2Defence Research and Development Organization, Defence Research Laboratory, Assam, India [Journal of Entomological and Acarological Research 2012; 44:e15] [page 79] No n- co mm er cia l u se on ly effectively kill mosquito larvae present in water. B. sphaericus has the unique property of being able to control mosquito larvae in water that is rich in organic matter. Depending on the formulation and environ- mental conditions, B. sphaericus is generally effective 1-4 weeks after application (Charles et al., 1996). The larvicidal binary toxin produced by B. sphaericus is composed of two chains, BinA (42 kDa) and BinB (51 kDa) that are deposited as parasporal crystals during sporulation (Baumann et al., 1991). These crystals bind to a specific receptor pres- ent on midgut brush-border membranes, resulting in damage to the midgut cells, which further leads to the mosquito’s death (Neilsen- Leroux & Charles, 1992). Bacillus sphaericus is specific and toxic against the genera Culex and Anopheles. It is harmless to humans, animals and the environment, and its use is recommended by the World Health Organization in pub- lic health programs worldwide (WHO, 1987). Many strains of B. sphaer- icus have been used as a toxicant in vector control programs all over the world (Regis et al., 2001). In a recent study in Brazil, da Silva Pinto et al. (2012) cloned the BinA and BinB genes of Bacillus sphaericus, pro- duced recombinant BinAB protein in three strains of Escherichia coli, and used these recombinant strains in toxicity assays against Culex quinquefasciatus larvae. Research on Bacillus sphaericus in South Asian countries and India includes a number of strains, such as Bacillus sphaericus strain SI-1 (Hossain et al., 2007), Bacillus sphaericus strain B-101 (serotype H5a, 5b) (Yadav et al., 1997), Bacillus sphaericus H5a5b (VCRC B42) (Prabhakaran et al., 2007), Bacillus sphaericus H-5a5b (Manonmani & Hoti, 1999), Bacillus sphaericus (Bs) 2362 SPH-88 (serotype: H5a5b) (Poopathi et al., 2009), Bacillus sphaericus, C3-41, 2362, and IAB59 (Pei et al., 2002) for mosquito control. Mosquito larvicidal activity of B. sphaericus was assessed by isolating it from ecologically different soil habitats in South India (Surendran & Vennison, 2011). The present study was conducted to test the larvicidal and pupacidal activities of the microbial insecticide Bacillus sphaericus (Bs G3-IV) on Aedes aegypti and Culexquinquefasciatus in the laboratory as well as in direct breeding sites. We also report the effects of Bacillus sphaericus (Bs G3-IV) on longevity and fecundity of Aedes aegypti and Culex quinquefasciatus. Materials and methods Collection of eggs and mosquitoes Eggs of Aedes aegypti were collected using oviposition traps placed in shaded areas at a height of less than 1.2 m. Traps were filled with water plus a few dried leaves placed at the bottom of the container, with a muslin strip placed vertically inside the container and half-submerged in the water. Culex quinquefasciatus egg rafts were collected from sewage water bodies in Coimbatore district, Tamil Nadu, India, using CDC gravid traps (Reiter 1983, 1987). These eggs were brought to the laboratory and transferred to 18¥13¥4 cm enamel trays (with separate trays for each species) containing 500 mL of water, and held for larval hatch. Maintenance of larvae The mosquito larval culture was maintained in our laboratory at 27+2°C, 75-85% RH. The mosquito larvae were fed with dog biscuits and yeast (Scottlabs Pvt. Ltd., Hyderabad, India) at a 3:1 ratio. The feed- ing was continued until the larvae transformed into pupae. Maintenance of pupae and adults The pupae were collected from the culture trays and transferred into plastic jar containers (12¥12 cm) containing 500 mL of water. These plastic jars were kept in a 90¥90¥90 cm mosquito cage for adult emer- gence. The emerged adults were maintained at 27±2°C, 75-85% RH, under 14 light (L):10 dark (D) photoperiod cycles. Adults were fed with 10% sugar solution for a period of three days before they were given an animal for blood feeding. Blood feeding of adult mosquitoes The female mosquitoes were allowed to feed on the blood of a rabbit (exposed on the dorsal side) for two days. The males were provided with 10% glucose solution on cotton wicks. The cotton was kept moist with the solution and changed every day. An egg trap (cup) lined with filter paper containing water was placed in a corner of the egg collection cage. Collection and preparation of Bacillus sphaericus Bacillus sphaericus (Bs G3-IV) with improved toxicity was collected from Defence Research Laboratory, Tezpur, Assam, India. To assure good suspensions for selection and bioassay procedures, stock suspen- sions (1 ppm) of the primary powders were prepared in distilled water by vigorously shaking 1 g of the powder in 1000 mL of water in a screw- cap glass vial. Required concentrations (0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm, and 10 ppm) were prepared by serial dilution of the stock solu- tion in distilled water. All stocks and dilutions were kept refrigerated at -4°C for no more than four months. Larval/pupal assays Laboratory colonies of mosquito larvae/pupae (F1 generation) were used for the larvicidal/pupacidal activity. Twenty-five individual I-IV- instar larvae and pupae were introduced into a 500 mL glass beaker containing 250 mL of dechlorinated water with the desired concentra- tions of biopesticide. Larval food was provided for the test larvae. At each tested concentration, 2-5 trials were run and each trial consisted of 3 replicates. The larvae/pupae exposed to dechlorinated water with- out biopesticide served as a control. The control mortalities were cor- rected using Abbott’s formula (Abbott, 1925) where: Fecundity studies The fecundity experiments were conducted by taking an equal num- ber of male and female mosquito larvae that had emerged from the con- trol and treated sets. These were placed in individual 30¥30 cm cages for each concentration. Three days after the blood meal, eggs were col- lected daily from small plastic bowls containing water kept in an ovit- rap in the cages. Fecundity was calculated from the number of eggs laid in ovitraps divided by the number of mated females. Death of adults in these experiments was taken into account. Longevity test The adult longevity of male and female mosquitoes (F1 generation) was also recorded. This was calculated as the number of days lived by the adult. Total number of days from adult emergence to death was recorded and the means were calculated to give the mean longevity in days. Field trial Field applications of plant extracts were made uniformly with a knap- sack sprayer on the surface of the water in each habitat. Sampling of larvae was undertaken before treatment and 24, 48, 72 and 96 h after treatment by dipper sampling and counting. A separate sample was taken to determine the species composition of each larval habitat. Six trials were conducted for each area with similar temperature and alti- Article [page 80] [Journal of Entomological and Acarological Research 2012; 44:e15] No n- co mm er cia l u se on ly tude. The required quantity of biopesticide was determined by calculat- ing the total surface area, and the required concentration was prepared by multiplying ten times the observed laboratory LC50 values. Percent reduction of the larval density was calculated using the formula: where C - total number of mosquitoes in control T - total number of mosquitoes in treatment Statistical analysis The data obtained from the bioassay were subjected to statistical analysis using SPSS (Version 14.0) software (IBM Corp., Armonk, NY, USA). Lethal concentrations (LC), LC50 and LC90, DMRT (Duncan Multiple Range Test) and c2 tests were used. Results Table 1 illustrates the larval (I-IV) and pupal mortality data of the dengue vector, Aedes aegypti, after treatment with Bacillus sphaericus (Bs G3-IV) at different concentrations. The maximum mortality observed was 100% at 10 ppm concentration in I and II instar larvae. The observed mortality rate was greatly reduced in late instar larvae and pupae. Pupae showed high resistance to Bacillus sphaericus (Bs G3-IV), with low mor- tality rates of 2%, 2%, 3%, 12% and 30% at 0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm and 10 ppm, respectively. Duncan’s Multiple Range Test proved that the observed mortality rates were significant at P<0.05. LC50 values for I, II, III and IV instar larvae were 0.60 ppm, 0.72 ppm, 2.45 ppm and 3.76 ppm, respectively, and, for pupae, 14.08 ppm. Percentages of larval and pupal mortality of the filarial vector, Culex quinquefasciatus, after treatment with Bacillus sphaericus (Bs G3-IV) at different concentrations (0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm and 10 ppm) are shown in Table 2. Mortality values ranged from 22% to 100% for the larval stages, but were greatly reduced for the pupal stage. At the highest concentration (10 ppm), no larvae were found alive and 100% mortality was recorded. Duncan’s Multiple Range Test proved that the observed mortality rates were significant at P<0.05. The calculated LC50 and LC90 values are 0.07 ppm and 0.56 ppm, 0.15 ppm and 0.87 ppm, 0.29 ppm and 1.14 ppm, 0.41 ppm and 1.32 ppm, and 10.84 ppm and 25.14 ppm for I, II, III, IV instar larvae and pupae, respectively. Adult longevity and fecundity of the dengue vector, Aedes aegypti, after treatment with Bacillus sphaericus (Bs G3-IV) is shown in Table 3. A significant reduction in adult longevity and fecundity was record- ed in this experiment when compared with the control. Longevity and fecundity after treatment with different concentrations of Bacillus Table 1. Larvicidal and pupacidal activity of Bacillus sphaericus on the dengue vector, Aedes aegypti. Larval and % Larval and pupal mortality (mean±SD) Standard LC50 95% Confidence limit c2 value pupal stages Concentration (ppm) error (LC90) LC50 LC90 0.001 0.01 0.1 1 10 (ppm) LCL-UCL LCL-UCL (ppm) (ppm) I 22±1.6d 26±0.7cd 38±0.4c 65±1.1b 100a 0.16 0.60 0.46-0.80 1.48-2.56 4.07* (1.86) II 20±1.2d 23±0.8cd 34±1.5c 60±0.7b 100a 0.16 0.72 0.56-0.97 1.61-2.85 3.35* (2.04) III 15±1.1d 17±1.6cd 26±2.1c 47±1.1b 98±0.5a 0.04 2.45 0.85-12.13 3.71-37.20 15.35* (6.69) IV 12±1.6d 15±0.4cd 22±2.2c 43±0.8b 90±0.4a 0.02 3.76 1.36-10.58 5.82-30.13 19.06* (9.64) Pupae 2±1.2c 2±0.7c 3±1.1c 12±1.5b 30±1.5a 0.02 14.08 8.46-93.08 14.51-186.15 11.30* (24.52) Means±standard deviation (SD) followed by same letter within rows indicate no significant difference (Duncan’s multiple range test, P<0.05). LC50, LC90, lethal concentration; LCL, lower confidence limits; UCL, upper confidence limits. *Significant at P<0.001 (heterogeneity factor used in calculation of confidence limits). Table 2. Larvicidal and pupacidal activity of Bacillus sphaericus on the filarial vector Culex quinquefasciatus. Larval and % Larval and pupal mortality (mean±SD) Standard LC50 95% Confidence limit c2 value pupal stages Concentration (ppm) error (LC90) LC50 LC90 0.001 0.01 0.1 1 10 (ppm) LCL-UCL LCL-UCL (ppm) (ppm) I 38±0.4cd 43±1.1c 58±0.7b 99±0.4a 100a 0.42 0.07 0.02-0.13 0.43-0.79 2.00* (0.56) II 35±0.9cd 39±1.6c 53±2.1b 93±0.5ab 100a 0.21 0.15 0.07-0.23 0.72-1.10 2.68* (0.87) III 27±1.2de 32±0.7d 48±1.2c 85±1.2b 100a 0.18 0.29 0.08-0.54 0.79-2.13 5.49* (1.14) IV 22±0.9de 28±1.1d 42±2.2c 79±0.9b 100a 0.17 0.41 0.19-0.72 0.92-2.48 5.48* (1.32) Pupae 11±1.5d 13±0.5cd 18±0.6c 28±1.6b 46±1.5a 0.02 10.84 6.01-90.25 14.15-243.62 8.72* (25.14) Means±standard deviation (SD) followed by same letter within rows indicate no significant difference (Duncan’s multiple range test, P<0.05). LC50, LC90, lethal concentration; LCL, lower confidence limits; UCL, upper confidence limits. *Significant at P<0.001 (heterogeneity factor used in calculation of confidence limits). Article [Journal of Entomological and Acarological Research 2012; 44:e15] [page 81] No n- co mm er cia l u se on ly sphaericus (Bs G3-IV) were 26.4 days (d) in males and 37.9 d in females at 0.001 ppm concentration, 21.8 d in males and 35.7 d in females at 0.01 ppm concentration, 18.9 d in males and 31.2 d in females at 0.1 ppm concentration, 15.8 d in males and 27.3 d in females at 1.0 ppm concentration, and 11.6 d in males and 21.1 d in females at 10 ppm con- centration. Fecundity was also reduced after treatment with Bacillus sphaericus (Bs G3-IV). A total of 178 eggs were recorded in the control, and the number of eggs recorded in the B. sphaericus treatments were 170, 155, 137, 116 and 82 at 0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm and 10 ppm, respectively. Adult longevity and fecundity of the filarial vector, Culex quinquefas- ciatus, after treatment with Bacillus sphaericus (Bs G3-IV) is shown in Table 4. Significant reduction in adult longevity and fecundity was recorded compared with the control. Longevity and fecundity recorded after treatment with different concentrations of Bacillus sphaericus (Bs G3-IV) were 30.9 d in males and 44.1 d in females at 0.001 ppm concen- tration, 25.8 d in males and 38.4 d in females at 0.01 ppm concentra- tion, 18.2 d in males and 25.9 d in females at 0.1 ppm concentration, 11.8 d in males and 14.6 d in females at 1.0 ppm concentration, and 4.7 d in males and 7.1 d in females at 10 ppm concentration. The fecundi- ty was also highly reduced after treatment with Bacillus sphaericus (Bs G3-IV). A total of 270 eggs were recorded in the control, compared with 249, 215, 178, 119 and 63 at 0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm and 10 ppm, respectively, of B. sphaericus. Table 5 shows the effect of Bacillus sphaericus (Bs G3-IV) on the dengue vector, Aedes aegypti, in their breeding sites. The field trial was conducted in stagnant water bodies at Vadavalli. The surface areas of the selected breeding sites were 0.6¥0.7¥0.5 m. A total of 481 larvae were found. After treatment with Bacillus sphaericus (Bs G3-IV), the percentage of reduction in larval density was 39.70%, 59.87%, 82.74% and 96.25% at 24 h, 48 h, 72 h and 96 h, respectively. Field application of Bacillus sphaericus (Bs G3-IV) in the sewage water systems in Vadavalli (breeding sites of the filarial vector, Culex quinquefasciatus), is given in Table 6. The surface areas of the selected breeding sites were 0.4¥1.7¥0.28 m. Required quantity and concentra- tion of biopesticide were calculated as 0.19 L and 2.88 ppm, respective- ly. A total of 788 larvae were found. After treatment with Bacillus sphaer- icus (Bs G3-IV), the percentage of reduction in larval density was 65.1%, 87.6%, 97.5% and 100% at 24 h, 48 h, 72 h and 96 h, respectively. Discussion Mosquitoes breed in varied habitats, such as ponds, marshes, ditch- es, pools, drains, water containers and other similar collections of water (Rozendaal, 1997). Mosquitoes such as Anopheles, Culex and Aedes are vectors responsible for diseases such as malaria, filariasis, Japanese encephalitis, dengue, dengue hemorrhagic fever, yellow fever and chikungunya. The increase in mosquito vectors and incidence of mosquito-borne diseases such as malaria, dengue, and chikungunya is rising in India due to climate change and water contamination. Unclean water bodies act as temporary and permanent breeding sites of mosquitoes, which tend to spread mosquito-borne diseases, along with cholera, dysentery, typhoid, etc. Understanding the ecology of mosquitoes and the mechanism of dis- ease management is a prerequisite to adopting any type of control. In general, the population of vector species must be of sufficient size so as to promote the transmission of vector-borne diseases. If the vector population falls below a critical density, the transmission of these dis- eases will not be very effective. Effective mosquito control is often a complex, expensive task, fre- quently requiring the cooperative efforts of communities as well as industry, agriculture, and state and local governments. We must be con- cerned with the harmful effects of synthetic pesticides on the environ- ment and living organisms, and reports have emerged on the resur- gence of several mosquito-borne diseases in the world as a conse- quence of increasing resistance of mosquitoes to commercial insecti- cides (Becker et al., 2003). This has necessitated the need for research and development of environmentally safe, biodegradable, indigenous methods for vector control. We found Bacillus sphaericus (Bs G3-IV) to be significantly effective against Aedes aegypti and Culex quinquefasciatus. This may have been due to the presence of binary toxin (Bin) and mosquitocidal toxins (Mtxs). The Bin toxin produced by Bacillus sphaericus targets mosquito larval midgut epithelial cells, where it binds to Cpm1 (Culex pipiens mal- tase 1), a digestive enzyme, and causes severe intracellular damage, including dramatic cytoplasmic vacuolation (Opota et al., 2011). Culex quinquefasciatus was much more susceptible to Bacillus sphaericus (Bs G3-IV), showing 100% mortality at a 10 ppm concentration against I-IV instars. Median lethal concentrations (LC50) observed were relatively low (0.07 ppm, 0.15 ppm, 0.29 ppm, 0.41 ppm, and 10.84 ppm for I, II, III, IV instar larvae and pupae, respectively) when compared with Aedes aegypti. Earlier, Yousten and Davidson (1982) and Davidson (1983) reported that Bacillus sphaericus, a spore-forming, entamopathogenic bacterium, possess potent larvicidal activity against several species of mosquito larvae. As a consequence of the specific toxicity to mosquito larvae of binary toxin (Bin) and mosquitocidal toxins (Mtxs) produced during the sporulation and vegetative stages, respectively, some toxic strains have been widely used for many years as biopesticides in the field in mosquito control programs (Bei et al., 2007). Table 3. Effect of Bacillus sphaericus on fecundity and longevity of dengue vector Aedes aegypti in the laboratory. Treatment Adult longevity Fecundity (ppm) (Ins) Male Female Control 29±0.2a 39±0.5a 178±0.4a 0.001 26.4±0.9b 37.9±0.6ab 170±1.6ab 0.01 21.8±0.9c 35.7±1.1b 155±1.1b 0.1 18.9±0.6d 31.2±0.6c 137±0.9c 1 15.8±0.2e 27.3±0.9d 116±0.6d 10 11.6±0.9f 21.1.±0.2e 82±1.1e Means±standard deviation (SD) followed by same letter within rows indicate no significant difference (Duncan’s multiple range test, P<0.05). Table 4. Effect of Bacillus sphaericus on fecundity and longevity of the filarial vector Culex quinquefasciatus in the laboratory. Treatment Adult longevity Fecundity (ppm) (Ins) Male Female Control 34±0.2a 47±0.2a 270±0.6a 0.001 30.9±1.5ab 44.1±1.9ab 249±2.8b 0.01 25.8±1.5b 38.4±1.6b 215±3.2c 0.1 18.2±2.1c 25.9±1.1c 178±1.9d 1 11.8±0.9d 14.6±1.5d 119±1.6e 10 4.7±1.0e 7.1±0.6e 63±0.9f Means±standard deviation (SD) followed by same letter within rows indicate no significant difference (Duncan’s multiple range test, P<0.05). Article [page 82] [Journal of Entomological and Acarological Research 2012; 44:e15] No n- co mm er cia l u se on ly In the present study, Aedes aegypti showed a slight reduction in mor- tality when compared with Culex quinquefasciatus after treatment with Bacillus sphaericus (Bs G3-IV). This may be due to its breeding habi- tats, characterized by high organic matter and oxygen content, which promotes the growth and development of Bacillus sphaericus. A previ- ous study showed Bacillus sphaericus exhibited only a low level of tox- icity against fourth instar larvae of Aedes aegypti (Mulla et al., 1984), but the present isolate was more toxic when compared with those in earlier studies. Usually B. sphaericus is minimally toxic to A. aegypti because this species either lacks a specific receptor for the binary toxin of B. sphaericus or has an extremely low concentration of such recep- tors (Nielsen-Le Roux & Charles, 1992), whereas the isolate B. sphaer- icus (Bs G3-IV) used in the present study was improved with necessary qualities for targeting A. aegypti. Production of enhanced mosquitoci- dal toxin by B. sphaericus 2362 and B. sphaericus 14N1 using whey per- meate (WP) under submerged fermentation conditions has resulted in high mosquitocidal activity (El-Bendary et al., 2008). No significant dif- ferences have been found in other factors that could affect the activity of B. sphaericus, such as the rate of ingestion of the toxins or differ- ences in proteolytic activation, between C. quinquefasciatus and A. aegypti (Aly et al., 1989). The present findings also agree with those from earlier studies pointing out the high susceptibility of Culex spp. to B. sphaericus (Singer, 1980; Yousten, 1984; Mulla et al., 1986). B. sphaericus has the unique property of being able to control mosquito larvae in water that is rich in organic matter. B. sphaericus is effective against Culex spp., but is less effective against some other mosquito species (Poopathi & Abidha, 2010). In the present study, Bacillus sphaericus (Bs G3-IV) reduced larval and pupal longevity and inhibited adult emergence in both species. The life span of emerged adults was also very low when mosquito larvae were treated with Bacillus sphaericus (Bs G3-IV), the reduction being more pronounced in Culex quinquefasciatus and less so in Aedes aegyp- ti. Aedes aegypti was generally less susceptible to Bacillus sphaericus (Bs G3-IV), but effects on its longevity and fecundity were comparable with those seen in Culex quinquefasciatus. The affected pupae also resulted in a great reduction in fecundity. Adults emerging from treat- ed larvae were morphologically normal but laid fewer eggs. The num- ber of eggs laid by Culex quinquefasciatus was very low compared with Aedes aegypti. Murugan et al. (2002) reported changes in fecundity after treatment with Bti. Combined treatment of Bti with neem and pongamia showed 76% adult mortality and a reduction in fecundity (Senthil Nathan et al., 2004). Poopathi & Tyagi (2002) reported a reduction in adult longevity (17% in males and 27% in females) in Culex quinquefasciatus after treatment with Bacillus sphaericus (GR strain), which supports the present findings. This study showed that Bacillus sphaericus (Bs G3-IV) was also effective in a field environment. The percentage of reduction in larval density was observed every 24 h, and was seen to increase as the time after treatment increased. This supports the observation that the bac- terial pesticide was not negatively affected by the external environ- ment and exhibits a persistent effect. This pesticide has also been found to be eco-friendly and non-toxic to non-target organisms. Mittal (2003) reported that the mosquitocidal toxins of certain strains of Bacillus sphaericus and Bacillus thuringiensis var israelensis H-14 (Bti) are highly effective against mosquito larvae in their direct breeding sites, even at very low doses, and are also safe to other non- target organisms. He also stated that the biolarvicide formulations of B. sphaericus are useful in the control of Culex and certain Anopheles spp., such as An. stephensi and An. subpictus, but are not very effec- tive against An. culicifacies. Because Bacillus sphaericus distinctly affects the developmental stages of insects, it also has distinct advan- tages over synthetic pesticides. The specificity of the B. sphaericus toxin is in part due to differences in the number of target sites to which it binds (Baumann et al., 1991). The binding of the protein toxin to the gastric caecum and posterior midgut has been observed in Culex pipiens (a susceptible species) but not in resistant Aedes aegypti (Mittal, 2003). References ABBOTT W.S., 1925 - A method of computing the effectiveness of an insecticide. - J. Econ. Entomol. 18: 265-267. ALY C., MULLA M.S., FEDERICI B.A., 1989 - Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae. - J. Invertebr. Pathol. 53: 12-20. 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Appl. 35: 11-14. ZEBITZ C.P.W., 1986 - Effects of three neem seed kernel extracts and azadirachtin on larvae of different mosquito species. - J. Appl. Entomol. 102: 455-463. Article [page 84] [Journal of Entomological and Acarological Research 2012; 44:e15] No n- co mm er cia l u se on ly