Novel Fermentation media for the production of mosquito pathogenic

bacilli in mosquito control

SubbiahPOOPATHI

Unit of Microbiology and Immunology, Vector Control Research Centre (Indian Council of Medical Research), Medical

complex, Indira Nagar, Pondicherry – 60 5006, India.

E-mail address: Subbiahpoopathi@rediffmail.com; Tel: 91-9443957479; 91-413-2272396; Fax: 91-413-2272041

Human industrial activities, inevitably, generate industrial wastes, consisting of, inorganic and organic materials,

discharged from factories, fisheries, poultries and food processing industries. Degrading or handling these wastes, as

unused disposals, without acquiring any additional benefits has led to an idea to develop a suitable technology to utilize

bio-organic wastes. Chicken feathers have been discarded in bulk as waste from poultry processing industries, poultry

farms and shops, globally. They normally accumulate structural proteins (keratins) that are resistant to biodegradation.

Deoiled rice bran is also an agricultural waste which contains protein, fibre and carbohydrate content. Considering the

abundant supply of these bioorganic wastes, we have successfully produced the biopesticides by culturing Bacillus

sphaericus (Bs) and B. thuringiensis serovar israelensis (Bti) strains to synthesize mosquitocidal toxins. Biochemical

studies indicate that the mosquitocidal spore/crystal toxins produced from the experimental culture medium (bird feather

waste, BFW cum de-oiled rice bran, RBW) are higher than the conventional medium (Nutrient Yeast Extract Salt

Medium, NYSM). The bacteria produced in these media (NYSM and BF, RB, RB+BF) were bioassayed against the

mosquito vectors (Culex quinquefasciatus, Anopheles stephensi, Aedes aegypti) and the toxic effect was found to be the

highest for the combination medium (BF+RB). Cost-effective analysis indicates that the use of bird feather waste and

deoiled rice bran waste as culture medium is highly economical for the industrial production of these mosquito pathogenic

bacilli. This study is, therefore, very important as it possesses the dual benefit of effective utilization of bio-organic waste

materials from the environment and for the production of mosquitocidal biopesticides as well.

Keywords: Bird feather waste; Deoiled rice bran waste ; Bacillus species; Culture medium; Crystal toxins; Mosquito

control; Cost-effectiveness

1. Introduction

Mosquitoes cause great nuisance to human beings and pose threats to public health as vectors of diseases like malaria,

filariasis, dengue, Japanese encephalitis, West Nile fever. Annually 300 million people are estimated to be affected by

malaria, transmitted by Anopheles mosquitoes with more than one million deaths (Kabilan, 1997, Sharma 1998, WER

1999). The world burden of lymphatic filariasis is estimated to be 250 million people (Ottesen and Ramachandran,

1995). Approximately 20 million people are infected every year by dengue viruses transmitted by Aedes mosquitoes

with ~24,000 deaths (WHO1997). Several strategies have been adopted to control these dipteran pests and to reduce

vector-borne diseases. Synthetic insecticides have been effectively used during the past several decades for mosquito

control operations. But the chemical approach has demerits, such as the development of insecticide resistance,

environmental pollution, bioamplification of contamination of food chain and harmful effects to beneficial insects.

Hence, there has been an increased interest in recent years in the use of biological control agents for mosquito control.

The discovery of bacteria like Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis which are highly toxic

to dipteran larvae opened up the possibility of the use of these biolarvicides in mosquito eradication programmes

(Goldberg and Margalit,1977, deBarjac and Larget-Thiery 1984). Mosquito pathogenic bacilli have some advantages

over conventional insecticides in mosquito control operations because they have a broader host spectrum, are safer for

non-target organisms (including humans) and are more environment friendly. Bacillus thuringiensis subsp. israelensis

synthesizes intracellular crystal inclusions by sporulation that contains multiple protein components of 134 kDa, 125

kDa, 67 kDa, and 27 kDa.(Sekar 1986,hofte and Whitely 1989. Federici et al. 1990, Wirth et al.1998).These proteins

have been cloned individually and are toxic to mosquito larvae (Sekar and Carlton 1985; Delecluse et al., 1991, 1993)

Though the high efficacy and specificity of Bs and Bti are useful in controlling mosquitoes, the cost to grow and

produce Bs or Bti formulations through a highly refined laboratory bacterial culture medium is exorbitant. In the past,

several efforts have been made to develop bacterial formulations using different culture media with varying degrees of

efficacy (Salma et al., 1983; Obeta & Okafor 1984, Kuppusamy, 1990; Adams et al., 1999; Adams et al., 2002; Montiel

et al., 2001, Vidyarthi et al., 2001). An inexpensive medium is needed to produce bacteria for large scale use in

developing countries. Obeta and Okafor (1984) formulated five media from the seeds of legumes, dried cow blood, and

mineral salts and assessed growth and production of insecticidal toxins of Bti that were effective against Aedes aegypti,

Culex quinquefasciatus, and Anopheles gambiae. Similarly, other culture media containing fish-meal, soybean and

cornsteep liquor for the production of Bti and Bacillus sphaericus (Neide) (Bs) have also been reported to be effective

and compared well with the standard (Salma et al., 1983, Kuppusamy 1990, Kumar et al., 2000). But the cost of culture

media for the production of these biopesticides was not studied. Besides bulk of organic and in-organic waste products

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are discarded into the environment without extracting maximum benefits. Moreover they pose problem towards environmental waste management. Hence this has led to novel ideas for effective utilization of biowaste products as bacterial culture media through simple fermentation technology. The poultry processing industries produce enormous quantities of chicken feathers as a waste byproduct, which is an environmental menace. Several approaches are in vogue for disposing of bulk feather waste, including land filling, burning, natural gas production and treatment for animal feed (Sangali and Brandelli, 2000; Boothe and Arnold, 2002; Suntornsuk and Suntornsul, 2003; Balint et al., 2005; Bertsch and Coello, 2005; Werlang and Brandelli, 2005). Bird feathers generally accumulate proteins, mainly keratins, which are the major class of structural proteins, that are highly resistant to biological degradation (William et al., 1990; Ichida et al., 2001; Riffel et al., 2003; Ramnani et al., 2005). However, keratinase produced from bacteria (Bacillus licheniformis) degrades the keratin from feathers and the degraded material was used for the production of animal feed and fertilizer (Lin et al., 1992; Cheng et al., 1995; Zaghloul et al., 1998; Lin et al., 1999; Riffel and Brandelli, 2002; Ramnani and Gupta, 2004; Wang et al., 2005). Keratins are made up of long chains of various amino acids and the two major types of keratin are known as alpha-keratin and beta-keratin (Lin et al., 1996; Kim et al., 2002). Bird feathers are largely composed of beta-keratin (Takahashi et al., 2004; Gupta and Ramnani 2006). The present study focuses on degradation of the keratin based chicken feathers by the bacterial strains of Bacillus sphaericus (Bs) and B. thuringiensis serovar israelensis (Bti) which are useful in the production of endotoxins to kill the mosquito larvae. The by-products of rice milling are used for a variety of purposes. Rice bran is the most valuable by-product of rice milling industry. It is obtained from the outer layers of the brown rice. Generally, rice bran consists of pericarp, aleurone layer, germ and a part of endosperm. Bran removal amounts to 4 to 9 per cent of the weight of paddy milled and is abundant in oil. Raw rice bran contains about 18 to 20 per cent oil whereas parboiled rice bran contains about 22 to 25 per cent oil. The de-oiled bran, which is a rich source of protein (about 17%) and vitamins (Vitamins A and E), is used as cattle feed and poultry feed. Studies on Aspergillus oryzae mutants for the production of single cell proteins from deoiled rice bran. Food Technol. Biotechnol. 41(3)243-246 (2003) R. Ravinder, Linga Venkateshwar Rao, Pogaku Ravindra. It is a good source of foreign exchange earnings. In the current study, we have successfully developed a culture media based on rice bran and bird feather waste for the growth of Bti and tested the efficacy of the produced toxin against three mosquito species(Culex quinquefasciatus, Anopheles stephensi and Ae.aegypti).We also discussed the feasibility of these media for the production of Bti based biopesticide in large quantities. Bacillus sphaericus is a gram positive, aerobic, spore forming soil bacterium that has been used in recent years as a microbial larvicide, to control mosquito vector populations (Kalfon et al., 1983; Payne and Davidson, 1984; Bauman et al., 1985; Davidson, 1988). It shows major toxic effect against larvae of filariasis (Culex) and malaria (Anopheles) vectors (Poopathi et al., 2002a). Its main larvicidal effect is due to two kinds of toxins (crystal (Cry) and mosquitocidal (Mtx) toxins), which differ in composition and time of synthesis. Among these two toxins, crystal toxin is the main toxic factor in the highly larvicidal strains. The crystal toxin is made up of two polypeptides with molecular weight of about 51 and 42 kDa (Charles et al., 1997). The genes encoding these crystal toxins have been cloned and characterized from several Bs strains (i.e. 1593, 2297 and 2362). The amino acid sequences of these two polypeptides are not similar to those of other bacterial or larvicidal toxins, including Bti. However, the 51 and 42 kDa proteins share four segments of sequence similarity (Baumann et al., 1988). Studies conducted with recombinant bacteria expressing these polypeptides individually have revealed that the 42 kDa protein could be toxic at high dosage in the absence of 51 kDa protein, but this was not the case for the 51 kDa protein alone. However, the presence of both proteins in equimolar amounts results in high toxicity to the larvae, as they seem to act in synergy (Nicolas et al., 1993). B.thuringiensis serovar israelensis is the most effective microbial control agent active against mosquitoes that is available to date (Goldberg and Margalit, 1977; deBarjac 1978; Tyrell et al., 1979; deBarjac and Thiery, 1984; Federici et al., 1990; Mahmood, 1998; Su and Mulla, 1999). It synthesizes intracellular crystal inclusions containing multiple protein components with molecular weights of 134, 125, 67 and 27 kDa (Sekar, 1986; Hofte and Whiteley, 1989; Wirth et al., 1998; Poopathi and Tyagi, 2006). These proteins have also been cloned individually and are shown to be toxic to mosquito larvae (Sekar and Carlton, 1985; Delecluse et al., 1991, 1993). However, a combination of these proteins seems to exhibit much higher toxicity than any of the individual components. Biopesticide application in mosquito control operations has gained much importance, during the last two decades, in view of, environmental protection. However, production of Bs or Bti formulations using existing fermentation technology (using conventional culture media) incurs heavy expenditure. Therefore, the use of these biopesticides has limitations. Hence, it is imperative to develop a cheaper media for culturing Bs /Bti which would facilitate the production of biopesticides in a cost effective manner. Obeta and Okafor (1984) formulated five media from the seeds of legumes, dried cow blood and mineral salts and assessed the production of the insecticidal toxins from Bti which were effective against Culex, Anopheles and Aedes mosquito species. Similarly, other media containing fish meal, soyabean and corn steep liquor, for the production of Bs and Bti, have also been reported (Saalma et al., 1983; Kuppusamy, 1990; Kumar et al., 2000). Poopathi and co-workers have also reported cost-effective culture media, using potatoes, for the growth of mosquitocidal bacterial toxins (Poopathi et al., 2002b, 2003; Poopathi and Anupkumar 2004). Recently, we have successfully developed a cost-effective culture medium from poultry industry waste, i.e.,

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chicken feathers, enabling the growth of Bs and Bti (Poopathi 2005, 2006). The findings from our experiments are presented in this paper and this is the first report on the cost-effective utilization of chicken feather waste for the efficient production of mosquitocidal bacterial toxins.

2. Materials and methods

2.1. Bacteria

Cultures of Bacillus sphaericus (IAB-59) and B. thuringiensis serovar israelensis (IPS-82) were provided by Dr. Jean Francois Charles, Institute Pasteur, Paris, France.

2.2. Bacterial culture medium

The conventional laboratory culture broth (Nutrient Yeast Extract Salt Medium, NYSM), used as reference medium in the present study was prepared by mixing glucose (5 g), peptone (5 g), NaCl (5 g), beef extract (3 g), yeast extract (0.5 g), mineral solutions (MgCl2, CaCl2, MnCl2) (10 ml) in an appropriate volume (1liter) of double distilled water (pH 7.5). Bird feathers (BF) was collected from local poultry farm and brought to the laboratory, washed in tap water, air-dried and stored at room temperature. A known quantity of these dried feathers (100 g /liter) was boiled in ordinary tap water for 15 minutes. After cooling, the feather extract was filtered and the pH of the filtrate was adjusted (pH 7.5). One liter volume of the feather extract medium was dispensed in each of the three conical flasks (2 liter capacity each) for culturing Bs and Bti and plain medium (control, without Bs and Bti) respectively. Similar procedure was followed for rice bran collected from local rice mill. But for the combination medium (BF+RB) a ratio of 3:1 was followed. Similar flasks were kept for conventional medium (NYSM) also. All the culture media were autoclaved (at 120 0C / 20 lb / in2 / 20 min).

2.3. Bacterial growth

A small amount of Bs and Bti lyophilized primary powder was inoculated separately in 2 ml each NYSM medium and allowed to grow for 12 h at 37 0C as pre-culture. A small volume of these pre-cultures (50µl each) were inoculated into sterile culture media.The cultures were allowed to grow under constant agitation (120 rev / min) under room temperature (37 0C) in an orbital shaker. Culture samples (2 ml) were drawn from each culture medium at 6 hr intervals from 0 to 72 hours. The pH and culture turbidity were measured using a digital pH meter (Genei, India) and SP75 UV-VIS spectrophotometer (Sanyo, UK). These were also examined microscopically for the presence of spores/crystals.

2.4. Spore/crystal toxin recovery from culture media

As soon as the cultures were fully sporulated, the spore/crystal toxin complex was recovered by centrifugation (10,000 x g / 30 min / 40 C) using SORVALL Evolution RC super speed centrifuge (Kendro, USA) and the spore/crystal free supernatants were discarded (Payne and Davidson, 1984). The spore/crystal mixtures (from Bs /Bti cultures) were thoroughly washed three times each with 0.1 M NaCl and sterile double distilled water (10,000 x g / 15 min / 40 C). Finally, these were washed with protease inhibitor, phenyl methyl sulphonyl fluoride (PMSF, 1 mM, Sigma), resuspended in the required volume of double distilled water and stored at – 200C, until further use, for biochemical studies and toxicity bioassays.

2.5. Protein estimation

A small volume of the stored spore/crystal sample was centrifuged (10,000 x g / 15 min / 4 0C) and the pellets were solubilized in solubilization buffer (50 mM NaHCO3, 10 mM dithiothreitol, pH 10) and incubated for 2-3 h, at 25-30 0C. After centrifugation and extraction (same rpm as above), the pure solubilized protein (from Bs /Bti) was quantified for protein estimation (Lowry et al., 1951) with bovine serum albumin (BSA, Sigma) as standard.

2.6. SDS-PAGE

A total of 5µg protein equivalent samples from Bs and Bti spores/crystals (NYSM, BF, RB, BF+RB) was mixed with an equal volume of sample loading buffer and boiled for 5 minutes and separated on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) unit (Genei, India), according to Laemmli (1970). The protein bands were stained with Coommasie Brilliant Blue R-250 and visualized.

2.7. Toxicity studies

Bioassays, for toxicity studies, were conducted with the laboratory colonies of three mosquito species (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) that have been received from the Division of Mosquito

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Rearing and Colonization (R & C), Vector Control Research Centre, Pondicherry.The larvae were reared as per the

standard procedure

(Poopathi et al., 1999). The bioassay procedures followed for Bs and Bti spore/crystal toxins were essentially the

same as that recommended by the World Health Organization (Anonymous, 1982, 1985). Aliquots of 2 fold dilutions

ranging from 0.25 to 64 µg / liter of the toxin solution from Bs and 0.01 to 2.56 µg / liter from Bti were used. Bioassays

were conducted in disposable wax coated paper cups (350 ml capacity). Test media were prepared by adding

appropriate volumes of Bs or Bti in 300 ml of water and 20 early third instar larvae from all the three mosquito species

were introduced separately, in each of the test concentrations. While food supplement (dog biscuit and yeast mixture,

ratio 2:1, respectively) was provided for larvae under Bs treatment, no food was provided for Bti treated larvae as

recommended by WHO. The bioassays were conducted at room temperature (37 0C) and larval mortality was assessed

(after 24 and 48 hours for Bti and Bs treatment respectively). If the mortality in control larvae was between 5 to 20 %,

Abbott’s (1925) formula was used to correct the mortality. Moribund larvae in the replicates were counted as dead.

2.8. Statistical analysis

The data were subjected to students ‘t’ test to analyze the significance of any difference between the growth of Bs and

Bti cultured from the test media (BF,RB, BF+RB) with control (NYSM) (P≤ 0.05). Analysis was carried out using the

SPSS package for window version 13.0 (SPSS 2001). For larval bioassays, Probit regression analysis was carried out to

calculate LC50 and LC90 values as well as 95 % fiducial limits using the software package ‘ASSAY’ (courtesy of Dr.

C.F. Curtis, London School of Tropical Medicine and Hygiene, UK).

3. Results

In the present study, the production of Bs and Bti spore/crystal toxins from the experimental media (bird feather waste

(BFW), deoiled rice bran (RB), combination (BF+RB)) was measured and compared with the toxins produced from the

conventional medium (NYSM). The results showed that in all the culture media, after a lag phase of about an hour,

there was a rapid multiplication of bacterial cells and maturation of spores. With increasing culture time, culture density

(measured by optical density at 650 nm) increased and reached a plateau in the range 2 to 2.5. This process of

multiplication lasted for 48hrs followed by lysis of the cells, which released the spore/crystal toxin complex

(endotoxins) into the medium. It is understood that, unlike Bti, Bs does not fully lyse the sporangium at sporulation. The

maximum growth and endotoxin release was completed after 72 hours.

Thus, the overall growth and production of Bs and Bti in RB was less than NYSM but that of BF was same as

NYSM, whereas, the combination medium RB + BF was found to be higher than NYSM and the mean values (3 to

3.2) were significantly different (P>0.05) (Figs. 1 and 2). Biomass production of Bti spore crystal toxins were also

found to be very in combinational medium i.e. RB + BF (Fig. 3). Microscopic observation of Bs and Bti spores / crystal

complex obtained from NYSM and BF after 72 h growth indicates that the sporulation in all the medium was high in

the following order BF+RB>NYSM= BF>RB (Fig. 4 to 7).

Fig. 1. Growth pattern of Bacillus thuringiensis serovar israelensis (IPS-82) in different culture media

0

0.5

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0 6 12 18 24 30 36 42 48 54 60 66 72

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Optical density (

650 n

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NYSM CFW DRB DRB+CFW

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Fig. 2 Protein concentration of Bti cultured from different media

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Fig. 3 Biomass of Bti spore/crystals in different culture media

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Fig: 4 to 7. Photomicrograph of Bs and Bti produced from different medium (Fig: 4 & 5 = Bs from BF+RB and NYSM respectively;Fig 6 & 7 = Bti from BF+RB and NYSM respectively).

The protein profile of Bs and Bti spore/crystal complex produced from NYSM, BF, RB and BF+RB were analyzedby SDS-PAGE and the results were compared. Five microgram protein equivalent samples were taken for this purpose.The major polypeptides present in the spore/crystals complex of Bs and Bti (Bs: 51 and 42 kDa and Bti: 134, 125, 67and 27 kDa proteins) produced from all the culture media were clear and conspicuous. There is no variation in theprotein pattern between the toxins produced from the conventional and test media for BF but RB showed a lesser trendthan NYSM whereas the combination medium of BF+RB indicated the maximum biomass, protein concentration andtoxin production. The protein profiles as indicator of binary and multiple toxins, in the respective Bs and Btispore/crystal complex were correspondingly related to their larvicidal activity.

Bioassays (larval toxicity tests) were performed with Bs and Bti spore/crystal complex produced from conventional(NYSM) and test (BF, RB, CF+RB) media. The lethal concentrations were expressed in micrograms of toxins per liter.The laboratory reared mosquito species of Cx. quinquefasciatus, An. stephensi and Ae. aegypti were used for bioassays.The comparative toxicities of Bs toxins produced from all the culture media (BF, RB, BF+RB and NYSM) were shownin Table 1 and 2.

Table. 1 Toxicity of Bacillus sphaericus (IAB-59) produced from media formulated from agro/poultry waste materials againstvarious mosquito species

Mosquito species Intercept Slope ± SE LC50 (mg/l) LC90 ( mg/l) χ 2 (df)

Culex quinquefasciatus

A 7.75 0.65 ± 0.09 0.014 (0.017 – 0.01)* 0.20 (0.25 – 0.069) 2.53 (3)

B 7.69 0.67 ± 0.07 0.018 (0.022 -0.015) 0.123 (0.183 -0.08) 4.26 (3)

C 7.62 0.63 ± 0.09 0.016 (0.019 – 0.01) 0.12 (0.18 – 0.08) 4.82 (3)

D 7.80 0.69 ± 0.08 0.017 (0.02 – 0.015) 0.11 (0.16 – 0.076) 4.0 (3)

Anopheles Stephen’s

A 7.03 0.54 ± 0.10 0.023 (0.028 – 0.02) 0.24 (0.40 – 0.16) 6.99 (3)

B 6.86 0.51 ± 0.10 0.026 (0.032 – 0.021) 0.32 (0.55 -0.18) 7.18 (3)

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C 7.03 0.54 ± 0.10 0.023 (0.029 – 0.02) 0.25 (0.40 – 0.15) 6.98 (3) D 6.61 0.41 ± 0.12 0.020 (0.026 – 0.015) 0.44 (0.92 – 0.22) 5.81 (3) Aedes aegypti A 6.71 0.51 ± 0.10 0.036 (0.044 – 0.03) 0.43 (0.75 – 0.25) 1.61 (3) B 6.84 0.55 ± 0.10 0.035 (0.040 – 0.03) 0.36 ( 0.58 – 0.22) 5.61 (3) C 6.89 0.55 ± 0.10 0.032 (0.039 – 0.03) 0.32 (0.52 – 0.20) 0.19 (3) D 6.80 0.53 ± 0.10 0.034 (0.043 – 0.028) 0.38 (0.62 – 0.23) 5.01 (3)

Bacillus sphaericus (IAB-59) culture medium: A = NYSM; B = de-oiled rice bran (DRB); C = chicken feather waste (CFW); D = DRB + CFW; *95% Fiducial limits of upper and lower at LC50 and LC90 levels.

The LC50 and LC90 values for Bs produced from BF against Cx. quinquefasciatus were 0.46µg / liter and 4.40µg / liter respectively. Similarly, the LC50 and LC90 values for Bs produced from NYSM were 0.45µg / liter and 4.58µg / liter respectively. These toxicity values indicate that they were statistically similar (fiducial limits overlapping). But the LC50 and LC90 values for Bs produced from RB was 0.018 and 0.12 and from BF+RB was 0.017 and 011. This shows that the lethal potential of BF was same as that of NYSM whereas RB had a lower potential in toxicity than NYSM. On the other hand, BF+RB showed the maximum toxic potential against mosquitoes. The other two mosquito species had similar effects of toxicity when tested with the cultures grown in these two media. Table 1 also presents data on the efficacy of Bti toxins produced from BF, RB, CF+RB in comparison with that of NYSM. The Bti toxin produced from BF was highly effective against all the three mosquito species tested and was found to be equally comparable to the toxins produced from NYSM. But the toxin produced from BF+RB was most effective against all the mosquito species and also the highest. It is worth mentioning here that, the Bti toxins produced from all the culture media exhibited a higher lethal effect on the larvae than the Bs toxins, obviously, due to the presence of multiple toxins in Bti.

Table. 2 Toxicity of Bacillus thuringiensis serovar israelensis (IPS-82) produced from media formulated from agro/poultry waste materials against various mosquito species

Mosquito species Intercept Slope ± SE LC50 (mg/l) LC90 (mg/l) χ 2 (df)

Culex quinquefasciatus A 10.14 0.65 ± 0.08 0.00036 (0.0004 – 0.0003)* 0.0026 (0.004 – 0.002) 5.27 (3) B 9.58 0.59 ± 0.09 0.0004 (0.0005 - 0.0003) 0.0038 (0.006 – 0.002) 1.33 (3) C 10.24 0.65 ± 0. 87 0.0003 (0.0004 - 0.0003) 0.0023 (0.003 - 0.002) 5.41 (3) D 9.36 0.57 ± 0.10 0.00047 (0.0006 – 0.0003) 0.004 (0.007 - 0.003) 0.44 (3) Anopheles stephensi

A 7.28 0.43 ± 0.13 0.005 (0.007 – 0.004) 0.097 (0.18 – 0.05) 6.63 (3) B 6.93 0.39 ± 0.13 0.0075 (0.009 – 0.006) 0.095 (0.16 – 0.06) 11.89 (3) C 7.78 0.54 ± 0.10 0.0058 (0.007 – 0.005) 0.062 (0.097 –0.04) 4.72 (3) D 7.50 0.49 ± 0.11 0.0064 (0.008 – 0.005) 0.085 (0.14 – 0.05) 11.88 (3) Aedes aegypti A 8.13 0.63 ± 0.09 0.007 (0.009 – 0.006) 0.054 (0.08 – 0.04) 6.55 (3) B 7.96 0.63 ± 0.07 0.009 (0.01 – 0.008) 0.069 (0.10 – 0.05) 7. 67 (3) C 8.01 0.62 ± 0.09 0.008 (0.009 – 0.007) 0.061 (0.09 – 0.04) 9.88 (3) D 7.88 0.61 ± 0.05 0.008 (0.01 – 0.007) 0.071 (0.11 – 0.05) 3.43 (3)

Bacillus thuringiensis serovar israelensis (IPS-82) culture medium: A = NYSM; B = de-oiled rice bran (DRB); C = chicken feather waste (CFW); D = DRB + CFW; *95% Fiducial limits of upper and lower at LC50 and LC90 levels.

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Cost-effective analysis from Table 3 shows that the quantity of feather waste required to prepare one liter of BF was only 100g, which is free of cost, being a poultry waste. Preparation of one liter of RB required 100g which is also an agricultural waste, thereby, minimal in cost. Preparation of one litre of BF+RB combination required 70 gm of BF and 30gm of RB, which is again free of cost as both are bio-organic waste products. On the contrary, preparation of one liter of Luria Bertani (conventional) involves a cost of US $ 2.50. Thus, the use of BF+RB was found to be ideal as an efficient and cost-effective bacterial culture medium.

Table: 3 Comparative costing for producing conventional and experimental culture media

4. Discussion

Mosquito borne diseases remain a serious global, public health problem. Mosquito control is an essential component of disease control and relies on the use of chemical insecticides, though they are expensive and toxic to non-target organisms. The discovery of biopesticides (Bs and Bti) has revolutionized over conventional insecticides in mosquito eradication programs. The high cost of conventional media components, to produce these biopesticides on a large scale, make it necessary to utilize cheap and commonly available biological waste materials through simple fermentation technology. Earlier, many Bs and Bti formulations produced from conventional media (Luria Bertani and NYSM) have been tested in the field for mosquito control. Subsequently, cost-effective formulations were utilized for biopesticide production. Obeta and Okafor (1984) cultured Bti on five formulated media from seeds of legumes (groundnut cake, cow pea of white and black varieties, soya bean, bambara beans), dried cow blood and mineral salts. Saalma et al., (1983), Kuppuswamy (1990), Ventosilla and Guerra (1997), Poopathi et al., (2002b, 2003), Poopathi and Anupkumar (2004) and Prabakaran et al., (2007) also have used potatoes, coconuts, fishmeal, cornsteep liquor and soybean for the production of biopesticides. Our fermentation studies with mosquitocidal bacteria produced from poultry waste and agricultural wastes indicates that the growth and production of endotoxins were appreciable and comparable with that of conventional medium. Bioassays of Bs and Bti toxins against mosquito larvae showed considerable toxicity. The toxicity is due to the binding of active toxins (crystals) to specific receptors present in the midgut brush border membrane (MBBM) (Charles et al., 1997). The crystal toxins from spore/crystal complex are ingested along with the food material, by the mosquito larvae and after solubilization and proteolytic cleavage, the activated toxin interacts with the midgut epithelium, leading to the death of the larvae (Poopathi et al., 2002a). Hence, these bioassay studies suggest that the toxins produced from BF are equally efficient as that of NYSM, and a combination of BF and deoiled rice bran is more efficient than NYSM for mosquito control. The efficacy is of the order BF+RB >NYSM =BF>RB. Cost-effective analysis indicated that, using bird feathers and deoiled rice bran as a source of bacterial culture media did not incur any cost, as it is easily available globally, as poultry and agricultural industry wastes. These results, from the present study, permit us to conclude that, bird feathers and deoiled rice bran available as cheap bioorganic wastes, are very useful substrates, for the industrial production of mosquito pathogenic bacilli (Bs and Bti), especially in developing countries, towards the mosquito control program.

Acknowledgements The author thank Department of Science and Technology (DST), Government of India, New Delhi for the project sanctioned (Ref: DST:F.No:SR/SO/HS-02/2008 dated 30/12/2009). The author also thankful to the Director of Vector Control Research Centre (ICMR), Pondicherry for his permission and Smt. R. Sundarammal, Senior Library Information Officer, VCRC, Pondicherry for providing valuable information on the research articles.

Culture medium Main constituent

of the medium

Quantity for

culture

preparation

(gm/litre)

Cost of quantum

used (US $)

Total cost for

culture medium

used (US $)

Net difference in

cost between

Slaughter house

waste and LB (in

ratio)

Conventional:

Luria Bertani (LB)

Experimental:

RB + CFW

(7 : 3)

Peptone + Yeast extract + Sodium

chloride

RB + CFW

10+5+10

70 + 30

1.0 + 1.0 + 0.50

0.01

2.50

0.01

1:250

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