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Chapter 2: REVIEW OF LITERATURE
11
The microbial control of mosquitoes is a very promising and rapidly developing
area for the management of mosquito borne diseases. The growing interest in the
microbial control of mosquitoes in the recent times is primarily due to various limitations
posed by chemical insecticides viz., resistance in mosquitoes, prohibitive cost of alternate
insecticides and growing environmental concerns. To cut down dependence on these
chemicals, Bacillus thuringiensis israelensis and Bacillus sphaericus, being biological
control agents of mosquitoes, provide an excellent alternative and cutting edge over
chemical insecticides being environmentally safe and target specific besides being reliable
and efficient tools. Obviously the journey from the first isolation to recent
commercialization and field application has been quite long and indeed challenging.
Winston Churchill once said 'those who ignore history are doomed to repeat it'.
Edward Steinhaus, the father of modern insect pathology, while expressing a similar view,
stressed the importance of understanding the thoughts and philosophies that guided our
predecessors in the research on microbial insecticides as we develop our own programmes
on them. In this chapter, therefore, the available literature has been reviewed on all round
progress made in the research on mosquito-pathogenic bacilli, especially from the earlier
days of their search, isolation, characterization and identification to the more recent
research on bio-efficacy trials, their persistence in the environment, safety profile and large
scale field application in the mosquito control programmes.
2.1 Isolation of mosquito-pathogenic bacilli
2.1.1 The Source: The earliest report on isolation of Bacillus sphaericus comes from
Kellen and Meyers (1964). Kellen, during routine surveillance of rock holes near Big
Creek, Fresno, California, collected several moribund fourth instar larvae of Culiseta
12
incidens. From these larvae, he isolated several bacteria besides B. sphaericus. On the
other hand, Goldberg and Margalit (1977) isolated B. thuringiensis israelensis from larvae
obtained from the stagnant ponds located in a dry desert in Israel. The silt and water
samples containing these larvae were taken to the laboratory and refrigerated, and
processed for isolation and assayed for larvicidal activities. The one which was extremely
toxic to mosquito larvae was designated as ONR -60A from which was derived the now
widely used B. thuringensis israelensis serotype H-14 (de Barjac, 1978).
_ Menon et al. (1982) isolated rapidly larvicidal spores bearing Bacilli (ISPC-5)
from diseased Culex fatigans larvae and identified them as B. sphaericus. Weiser (1984)
isolated mosquito larvicidal Bs strain 2362 from blackfly. Subsequently, Lysenko et al.
(1985) isolated 5 new strains of B. sphaericus, possessing high larvicidal activity from
non-mosquito sources ie. Catterpillars and grasshoppers against which, they were
ineffective.
Manonmani et al. (1987) collected soil, water and larval samples from the
transient pools, coconut gardens, Casuarina plantations, paddy fields, garden lands, river
beds, polluted habitats, irrigation canals, ponds, estuaries and deer sanctuaries. From these
they isolated 101 strains of B. thuringiensis H-14 and 11 non H-14 serotypes showing
activity against Culex quinquefasciatus larvae. They discovered a subgroup of serotype H-
20 and designated it as B. thuringiensis var. pondicherriensis. Martin and Travers (1989)
using acetate selection method examined soil samples for mosquito-pathogenic bacilli from
five continents, Africa, Asia, Europe, North and South America and their associated
islands. They isolated Bt in 785 out of 1115 soil samples and found that insect control
agent Bt was a ubiquitous soil microorganism.
13
In 1991, Gupta et al. isolated a highly toxic strain of B. sphaericus H5a(9001)
from the diseased mosquito larvae and soil samples from Kheda, Gujarat state, India.
Manonmani et al. (1991) also isolated 86 B. sphaericus and 23 B. thuringiensis strain
active against Cx. quinquefasciatus larvae from the root surface of hydrophytes using a
selective medium, nutrient agar containing 0.01% streptomycin. Novel variants of B.
thuringiensis were isolated from the phylloplanes of deciduous and coniferous trees as
well as other plants by Smith and Couch (1991). Hastowa et al. (1992), have isolated B.
thuringiensis from the soils of sericulture areas and natural environments form various
regions of Indonesia. Ohba (1996) examined mulberry leaves and found many B.
thuringiensis strains and a few of these isolates were toxic for silkworm Bombyx mori and
Aedes aegypti. Damgaard et al. (1996 & 1997) have recently isolated strains of Bt from
the food items (pasta, pita, bread and milk) and from the phylloplane of organically grown
cabbage leaves in Slangerup, Denmark.
2.1.2 Methodology for isolation of bacilli: Goldberg and Margalit (1977) and many
other workers isolated mosquito larvicidal bacilli by heat treatment of the samples and
plated them on Nutrient Agar from which the bacterial colonies were picked up. Since
then many media and newer techniques have been devised for efficient and selective
isolation & growth of these bacilli. White and Lotay (1980) discovered that B. sphaericus
NCTC 9602, grew and sporulated well in a simple chemically defined medium with pH 7.2
containing only KH2PO4/ NaHPO4 buffer, Ammonium Sulphate, inorganic salts and
Sodium Acetate as sole source of carbon. They also discussed about minimal nutritional
requirements of 26 other strains of the species.
14
B. sphaericus ISPC-5 isolated by Menon et al. (1982) were found to grow well on
simple media like nutrient broth alone or when supplemented with 0.3% molasses but
showed poor and delayed sporulation when cultured on brain heart infusion broth. Smith
(1982) demonstrated the effect of strain and medium variation on mosquito toxin
production by Bti. He showed that both insect mortality and degree of sporulation were
dependent on the media as well as strain and time of exposure. He further observed that
toxin production was associated with sporulation and the spore count was generally not
- proportionate to toxins produced for those strains and media used. Obeta and Okafor
(1984) formulated five types of media from the seeds of legumes, dried cow blood and
mineral salts, and assessed the growth and production of insecticidal toxins of Bti which
were found effective against Ae. aegypti, Cx. quinquefasciatus and An. gambiae. The
powder containing ground seeds of Voandzeia subterranean was most effective which
also compared well with the standard (IP578). Yousten et al. (1985) defined a selective
medium known as BATS. They reported that this medium containing Streptomycin
Sulphate and Arginine as the sole carbon and nitrogen source allowed the growth of 18
strains of mosquito-pathogenic B. sphaericus but inhibited the growth of 68% of the non-
mosquito pathogenic B. sphaericus strains as well as other bacilli species and aquatic
bacteria.
Travers et al. (1987) devised a selective process for efficient isolation of soil
Bacillus spp. In this process, germination of B. thuringiensis spores was selectively
inhibited by sodium acetate whilst most of the undesired spore-formers germinated. In the
next step, all the non-sporulated microbes were eliminated by heat treatment for 3 min at
15
80°C. The surviving spores were then plated on the rich agar medium and allowed to grow
till sporulation occurred.
Eziofor & Okafor (1988 & 1989) reported the production of Bacillus sphaericus
2362 using fermented cowpea (Vigna unguiculata) medium containing mineral substitutes
from Nigeria. They have shown that entomo-pathogenic Bs can be grown on a medium
compounded entirely from locally available materials and advocated that larvicidal Bt H-14
should also be produced on a similar medium.
Earlier Okafor (1987) had shown that by controlling pH (above 8.7) the growth of
naturally occurring lactic acid bacteria could be enhanced. This in turn actively converted
much of the proteins present in the cowpeas to smaller peptides and amino acids. These
along with minerals that leached out of the cowpeas were actively utilized by Bacillus
sphaericus for growth.
Russel et al. (1989) used NYSM (Nutrient Yeast Sporulating Medium) to study
the carbohydrate metabolism in B. sphaericus. Using BATS and acetate medium,
Carboulec and Priest (1989) isolated 40 strains of B. sphaericus and examined their ability
to use carbon and other energy sources. They tried to establish the correlation between
DNA homology group and nutritional profiles. Gupta et al. (1991) maintained B.
sphaericus 9001 isolated from Gujarat in laboratory at 30 °C in Polymedium in liquid
cultures. Carozzi et al. (1991) have reported the use of modified version of acetate
selection protocol of Travers et al. (1987) for isolating spore forming Bacillus strains
from soils and insect cadavers. Asimeng and Mutinga (1992) developed a baiting
technique for recovering bacteria from the environment. Using the healthy mosquito larvae
16
held in fully permeable plastic bottles, they were able to isolate toxic bacilli from mosquito
breeding habitats.
2. 2 Identification and characterization of mosquito-pathogenic bacilli
The rod shaped bacteria that aerobically form endospores are assigned to the genus
Bacillus. According to Bergey's Manual of Systematic Bacteriology (1989) they are the
endospore forming gram positive rod shaped bacteria which are mostly catalase positive.
Bacillus thuringiensis produces oval spores and its most distinctive characteristic is the
presence of a parasporal body. The parasporal body consists of insecticidal proteins that
are produced during sporulation (Angus, 1965; Heimpel, 1967; Hofte and Whitley 1989).
Since the discovery of Bti (H-14) by Goldberg and Margalit (1977) various authors have
proposed different schemes for their identification.
Heimpel (1966) described some 32 varieties of 2 species of crystalliferous bacteria
and proposed a taxonomic key for differentiating them. According to him the serological
classification coincides with the gross finding of biochemical and bioassay investigations.
They have given several morphological and biochemical tests for the differentiation of
several varieties of Bacillus thuringiensis. deBarjac and Bonnefoi (1968) proposed a new
key for the identification of Bacillus thuringiensis Berliner based on serological and
biochemical characterization, supported by the analysis of 161 isolates. They recognised 9
serotypes of Bt with 12 type strains or varieties. According to them the production of
thermostable toxin does not justify the criteria for naming new varieties.
Ohba and Aizawa (1978) investigated the distribution of Bt and related spore-
forming bacteria in Japan and performed serological identification of these new isolates.
They have described the preparation of H-antisera and H-factor sera and the methodology
17
for H & 0 agglutination tests. Logan & Berkley (1984) have described a system using a
matrix of results from the tests in the API 20 E and API 50 CHB strips and from the
supplementary tests for rapid and accurate identification of Bacillus isolates. According to
them, the API tests are more reproducible than the other methods and the taxonomy based
on them is also in good agreement with that of other methods. According to Berkely et al.
(1984) pyrolysis mass spectroscopy is the most promising method of general application
to the genus. They have also modified a tentative key for identification of the common
Bacillus species, devised earlier by Gordan (1973) which is based on the biochemical
tests.
de Barjac (1990) reported the classification of all Bt strains according to H-
antigens into 27 groups and 7 subgroups. Recently, Lecadet et al. (1999) have devised
and updated H-antigen classification of Bacillus thuringiensis. According to them, 63
serotypes and 13 sub antigenic groups of this species had been identified giving 82
serovars among the 3500 Bt Isolates available at the IEBC collection Centre at Pasteur
Institute, Paris, France.
Unlike Bt, B. sphaericus was recovered as saprophytic organism. The B.
sphaericus vegetative cells are rod-shaped, straight and motile, 0.6-1 pm wide and 1.5-5
pm long. They form nearly spherical heat resistant spores (Holt et al., 1975). The earlier
studies on B. sphaericus failed to demonstrate the presence of a paraspore in some strains
(c.r. from Yousten & Davidson, 1990)). But Yousten (1984) and Payne & Davidson
(1984) opined that the B. sphaericus synthesizes a parasporal inclusion that contained
proteins toxic to the mosquito larvae.
18
According to Bergeys Manual of Systematic Bacteriology (1986), there were some
40 valid and recognized species of B. sphaericus. Microbiological identification of this
species is presently done on the basis of morphological characteristics and rounded spores
located terminally in swollen sporangia. According to Claus and Berkeley (1986), B.
sphaericus can be differentiated by several phenotypic characteristics. Briefly, B.
sphaericus is a strict round spore former and is unable to use sugars as carbon source for
growth (Russel et al., 1989).
Various other schemes are noteworthy for the identification of B. sphaericus.
Yousten et al. (1980) recommended bacteriophage typing, while de Barjac et al. (1985)
proposed H-serotyping for identification and differentiation. Alexander and Priest (1990)
proposed the use of numerical classification and identification of Bs based on DNA
homologies. They used a combination of carbon-source utilization tests and other
physiological and biochemical characteristics in their study for phenotypic classification.
They results obtained were found to be completely compatible with those based on DNA
homologies. This enabled separation of several clusters that contained strains that were
combined in the previous studies under B. sphaericus (Priest et al. 1988).
Besides these schemes of identification, according to Frachon et al. (1991), the
fatty acid patterns revealed by gas chromatography, is another method to recognize
mosquito larvicidal Bs strains.
2.3 Bacilli toxins and their mode of action
Since the discovery of mosquito-pathogenic bacilli, attempts have been made by
various workers to understand the very nature and characteristics of the toxins, their mode
of action and pathogenesis. As stated earlier the most distinctive characteristic of Bti is the
19
parasporal body produced during sporulation that contains insecticidal proteins (Angus,
1965; Heimpel, 1967; Hofte & Whitley, 1989). According to Luthy & Ebersold (1981), in
most sub-species of Bt, the parasporal body is bi-pyramidal crystal containing one or more
similar proteins of about 135 kDa that are toxic to Lepidopterous larvae. When ingested
by the larvae, this toxin containing inclusion dissolves in the gut juices. The midgut
proteases cleave the protoxin, yielding an active peptidoglycan of 60-70 kDa, the delta-
endotoxin. The intoxication occurs as a result of osmotic imbalance across the midgut
epithelial cell membrane leading quickly to hypertrophy and lysis of midgut cells. Lysis is
followed by the disruption of basement membrane and leakage of digestive juices into the
haemocoel which is followed by larval death (Fig.2.1).
The majority of the toxins of Bti have been cloned and sequenced. Attempts have
also been made to express them in other organisms (Aronson, 1993). Kawalek et al.
(1995) by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
have revealed that the crystals of B. thuringiensis subsp. jegathesan were composed of
77, 74, 72, 68, 55, 38, 27 &23 kDa and were found to be distinct from that of Bti.
Schnepf et al. (1998) have discussed in details the ecology, genetics, toxin structure,
mechanism of action and biotechnology of Bt in their review "Bacillus thuringiensis and
its pesticidal crystal proteins."
Thomas & Ellar (1983) and Chilcott et al. (1990) obtained three major proteins
from the Bti crystals by FPLC on a mono Q column. They also determined the relative
toxicity of all the three proteins by estimating their LC50 value (i.e. concentration required
to kill 50% of mosquito larvae) and found that 65 kDa protein component was the most
toxic.
20
Invasion of haemolymph and haemocoel by bactena
Alkaline gut contents in haemolymph & haemocoel
A
Ingestion of biolarvicide with food
Ilir
0
Spore
Bacterial multiplication
Fig. 2.1: Mechanism of action of bacilli toxins in the mosquito larval gut.
21
Various toxic proteins of Bt are however reported to have a synergistic
interaction by several workers (Wu & Chang, 1985; Ibarra & Federici, 1986 and Chilcott
& Ellar, 1988). They concluded that all the major parasporal body proteins are
mosquitocidal but none alone is as toxic as the parasporal body per se. Hoftey and Whitely
(1989) have classified these endotoxins into several classes based on their toxicity Cry I
(Cry 1)- Lepidoptera specific; Cry II (Cry2)- Lepidoptera and Diptera specific; Cry III
(Cry 3,Cry 7 and Cry8)- Coleoptera specific and Cry IV (Cry 4, Cry 10, Cry 11)- Diptera
specific.
According to Hofmann et al. (1988 a & b) and van Rie et al. (1990) the delta-
endotoxins are synthesized as inactive protoxins that are solublized and proteolytically
activated in the insect gut. They suggested that the active toxin binds to specific, high
affinity receptors in the apical microvilli of epithelial cells in the insect midgut. It was
earlier stated by Knowles & Ellar (1987) that this bound toxin inserts irreversibly into the
plasma membrane to form pores or lesions leading to colloid-osmotic lysis. There is a
general accepted view that Bt toxins interact with the midgut membrane causing
physiological changes which lead to larval paralysis, cessation of feeding and eventually
death (Ellar et al. 1986; Knowles & Ellar, 1987; WHO Expert Committee on
Onchocerciasis, 1987; Gill et al. 1987; Hofte & Whitley, 1989, Drobniewsky & Ellar,
1988; 1989 and Chillcott et al. 1990).
Fast (1981) also attributed insecticidal activity of Bt to the production of toxin
protein crystals (the delta-endotoxin) which according to him account for 20-30% of dry
weight of sporulating culture. Jarret (1985) has concluded that the genes coding for the
production of toxins or for their expression may be located on both the plasmids or on the
22
chromosomes. Various theories proposed to explain the mode of action of Bt are summed
up in the Table 2.3. From these theories, it can be assumed that Bt toxins have detergent
like activity as they finally cause neurotoxicity or colloid-osmotic lysis. Neurotoxicity of
Bt has been reported by different workers (Chilcott et al. 1984; Cheung et al. 1985 &
1987; Singh and Gill, 1985 and Singh et al. 1986).
As mentioned earlier the first studies on Bacillus sphaericus failed to demonstrate
the presence of a parasporal body (c.r. from Davidson & Yousten, 1990). However later
studies revealed the presence of a paraspore in some strains, which are produced in the
course of sporulation and contain protein toxic to mosquito-larvae (Payne & Davidson
1984; Kalfon et al., 1984 and Yousten, 1984).
Baumann et al. (1985) and Broadwell et al. (1990) reported extraction of the toxic
proteins of 100kDa, 110kDa & 125kDa from the spores using various isolation
techniques. They discovered that these proteins further split into 63kDa & 43kDa and
51.4kDa & 41.9kDa. It is now accepted that of these, two proteins of molecular weights
51.4 and 41.9kDa are toxic to mosquito-larvae and both are required for the toxicity.
The pathology of B. sphaericus infections in susceptible larvae has been
studied both by light and electron microscopy. Kellen et al. (1965) who discovered
the first mosquitocidal strain of Bs also described its pathology. According to them
the death of larvae required up to 7 days and appeared to result in the progressive
degeneration of the gut cells. Davidson et al. (1975) found that after ingestion of
Bs strain SS11-I toxins, the epithelial cells of the intoxicated larvae show visible
ultra structural changes. These were separated from one another at the base and
also resulted in swelling of the gut. Large cytolysosomes appeared in the posterior
23
Table 2.3: Theories for Mechanism of Action of Bacillus thuringiensis Toxins
(source: Chilcott et al., 1990).
S. No Target of Action Reference
1. Plasma membrane (Ionophore) Angus, 1968 Nickerson and Schnell, 1983
2. Mitochondria Travers et al., 1976
3 Plasma membrane (General breakdown)
Luthy and Ebersold 1981 Nishitsusuji-Uwo, Endo, and Himeno, 1979
4 Goblet cell K+ pump Griego et al. 1979, Harvey and Wolfersberger, 1979
5 Plasma membrane (B. t. i.) (detergent like action )
Thomas and Ellar, 1983b
6 Neuromuscular system Chilcott et al. 1984 Cheung et al. 1985, 1987 Singh et al. 1986
7 Na+ and /or K+ transport Himeno et al. 1985 Gupta et al. 1985 Sacchi et al. 1986
8 Plasma membrane (colloid-osmotic lysis)
Knowies and Ellar, 1987 Haider and Ellar, 1987 Drobniewski and Ellar, 1988a
24
midgut cells and eventually these cells were observed to slough off from the basement
membrane. Invasion of the larval haemocoel by gut bacteria did not occur until after the
death of larvae (Davidson, 1979). The larvae experienced tremors, became sluggish and
eventually died while hanging from the water surface. According to Davidson (1981 &
1984), the mortality in larvae that fed on toxins may begin as early as 4 hrs after injestion
but generally 48 hrs are required for full expression of mortality in bioassays. Davidson &
Titus (1987) reported rapid swelling of the mitochondrial cristae and endoplasmic
reticulum which was noticed within 5 minutes of the treatment of the cultured cells.
Charles (1987) also observed pathological changes in the larvae following
ingestion largely involving the midgut cells. Lakshmi and Gopinathan (1988) reported that
the toxins inhibited the activity of choline acetyl transferase and oxygen uptake by the
mitochondria. Some workers have observed that following the death of the larvae the Bs
cells multiply in the larval cadavers and form new spores, indicating re-infection which
may be crucial in the recycling capability of Bs in the environment (Davidson et al. 1984;
Des Rochers & Garcia, 1984 and Charles & Nicholas, 1986)
2.4 Application of biocides: Formulations and testing of toxic strains
2.4.1 Formulations: For the successful control of vectors, it is necessary that the
biolarvicide is in such a form that is readily available to the larvae in their feeding zone and
is easily ingested by them. Besides, it should be economical to use and safe to the non-
target insects. The activity of the Bti and Bs toxic strains can also be enhanced through
appropriate formulation strategies (Lacey, 1984).
The earlier formulations were liquid concentrates of the bacterial cells and spores
or the primary lyophilized powders. The concentrated liquid cultures are dispersible and
25
simpler in preparation but they are bulkier and hence difficult to transport (Mulla, 1985).
According to Duimage et al. (1970) the primary powders could be obtained by
lyophilization or freeze drying, but this method resulted in the significant loss of spores
and cells. They devised a protocol for obtaining spore crystal complexes in dry form by
Lactose-Acetone Co-precipitation method.
According to Lacey et al. (1984) formulation that provides an adequate level of
toxin for the initial control of older mosquito larvae and continuous subsequent release of
toxin for the control of younger larvae would be optimal. They compared aqueous
suspension of formulated Bacillus thuringiensis (H-14) (Bactimos ® WP) and B.
sphaericus (1593) acetone precipitated spore powder with slow release floating pellet
formulation of each pathogen for the control of Culex quinquefasciatus breeding in urban
settings and found that the pellet formulations gave prolonged activity.
The Bti formulations (Table.2.4.1A) have been commercialized by many firms viz.,
Sandoz (Teknar ®), Abbot (Vectobac ®), Solvay (Bactimos) and used on a large scale in the
bio-control of mosquitoes and blackflies in many countries (de Barjac, 1990). More
recently, WP formulation of Bti named bacticide has been field tested in a multi-centric
trial in India. This is Russian formulation and marketed by Biotech International Limited,
New Delhi in India and in many other countries. The Bs formulations produced for
commercial purpose include Solvay liquid 2362, Abbot granules 2297, HIL-8, HIL-9 &
HIL-10 (M/s Hindustan Insecticides Ltd.), Spherix (Berdesk Plant in Russia marketed by
Biotech International Ltd., New Delhi), etc (Table 2.4.1B).
26
Table 2.4.1A Formulations of B. thuringiensis israelensis (H-14) tested against
various mosquito species.
S. No. Formulation Trade name Mosquito Species
Reference
1. Flowable concentrate
Bactimos® Vectobac®
An. arabiensis Mulla, 1990 Romi et al., 1993
2. Suspension concentrate
Teknar, SAN 402-1 SC
Mansonia spp. Foo & Yap, 1983
3. Corncob Vectobac-G® Culiceta spp., Psorophora spp., Aedes spp.
Charbonneau et al., 1994
4. Granular Floating granules Sinking granules Fine powdery granules
ABG-6108IIG® ABG-61081IG® Bactisand®
Cx. quinquefasciatus -do- -do-
Mulla,1990
5. Powder Bactoculicide/ Bacticide
Bactimos®WP Bactoculicide/ Bacticide Vectobacl2AS
An. stephensi An. subpictus An. culicifacies Ae. albopictus Ae. aegypti Cx. quiquefasciatus Cx. quinquefasciatus
An. stephensi Cx. sitiens Ae. vigila:c
An. stephensi An. culicifacies Ae. aegypti Cx. quinquefasciatus
Dua et al.,1993
Lacey et al., 1984 Kumar et al., 1995
Brown et al., 1998 Brown et al., 1999
Mittal et al., 1993
6. Briquetts Bactimos® Aedes spp. Culex Spp.
Logan & Linthicum, 1992
7. Tablets NM Ae. aegypti Becker et al., 1991
8. Sustained release formulation.
NM Cx. quinquefasciatus Lacey et al., 1984
NM Not menlicrvecl.
27
Table 2.4.1B Bacillus sphaericus formulations tested against various mosquito
species.
S. No.
Strain Formulation Trade Name Mosquito species
Reference
1. 1593 Acetone precipitated spore powder
NM Cx. quinquefasciatus Lacey et al., 1984
2. 1593 Wettable powder
NM An. annulipes, Cx. annulirostis, Cx. quinquefasciatus
Davidson et al., 1981
1593I1 -do- IIIL- 8 An. culicifacies, Cx. quinquefasciatus
Mittal et al., 1985
1593-4 -do- Stauffer WP 4920-34-3
Cx. quinquefasciatus, Cx. tarsalis
Mulligan et al., 1978; 1980
3. 1593 Sustained release pellets
.
Cx. quinquefasciatus, Cx. restuans
Lacey et al., 1984; 1988
2362 Cx. restuans Theobald, Cx. nigripalpus Theobald, Cx quinquefasciatus Say, Cx. salinarus Coquillett
Lord, 1991
Cx. territanus Walker Ps. Columbiae
4. 2362 Briquettes NM Cx. quinquefasciatus Lacey et al., 1988
5. NM Granules NM Cx. peus Mulla et al., 1988
NM Cx. pipiens, Cx. pipiens, Ae. trivittatus Berry et al., 1987 Ps. Columbiae
Abbot granules 2297
Vectolex
An. culicifacies, An. stephensi, An. subpictus, Cx. quinquefsciatus
Ansari et al., 1989
Karch et al., 1992 An. gambiae, Culex spp., Mansonia spp.
6. 2362 Flowable concentrate
NM
NM
An. quadrimaculatus, Ps. Columbiae, Cx. peus
Lacey et al., 1986
Mulla et al., 1988 Vectolex 2.5AS Spherimos
Cx. quinquefasciatus, An. stephensi, An. culicifacies
Ansari et al., 1995
7. 1593M Dust formulation
HIL-9 & HIL- 10 Cx. quinquefasciatus, An. culicifacies
Mittal et al.,1985
8. NM Floating type Spherix I'm Mansonia spp. Rajendran et al., 1991
9. 101 Powder Spherix ® An. stephensi Kumar et al., 1994 Cx. quinquefasciatus Kumar et al., 1996
ISM.- t\k-E 118nca_ 28
2.4.2 Assays of the biocides: The lab evaluation of biolarvicides is necessary before their
field trials and use in the vector control programmes. Assaying microbial insecticides is
very much difficult as compared to chemical insecticides. In the case of latter, the assayer
already knows the quantity and purity of the test insecticide and hence assay is an
accessory to the production process and it is confirmatory in nature. But in the case of Bt
or Bs larvicides, the assay is a measure of all the stages of the production process viz.,
quality of the fermentation; losses of activity occurring during the recovery process; the
quality of the product recovered and the characteristics of the final formulations. All these
must be known accurately and this knowledge could be achieved through bioassays at
different levels.
Essentially, a bioassay measures the interaction between the test insect and the
toxin being assayed. According to Dulmage et al. (1990) the most dramatic response of an
insect to the microbial insecticide and the one which is easiest to observe is the death. The
killing power of a biolarvicide, like any other insecticide, is expressed in terms of LC
(Lethal concentration), LD (Lethal Dose) or LT (Lethal Time), related to the
concentration, dose and the time of lethality respectively. But the most accurate and
accepted expression is the LC 50 and LC90 value i.e. a concentration of the sample that
causes 50% and 90% mortality of the test mosquito species larvae respectively. The major
steps in the bioassay are exposing the larvae to the biolarvicide, incubating them for a set
period of time, determining the percentage mortality at each concentration and then
plotting the Probit regression curve to determine the LC 50 and LC90 values. It is however
very important to perform bioassay following accurate and recommended procedure.
29
Several workers have proposed different methods, of which the two most popular ones
are those of de Barjac (1983) and McLaughlin et al. (1984).
The larvicidal activity of the very first strain of Bt was studied by the Goldberg &
Margalit in 1976. They found this strain to be very effective against 5 species of
mosquitoes viz. Culex pipiens (LC 50 = 6 X 103 spores/ml), C. univittatus (LC 50 = 2 X 104),
Aedes aegypti (LC 50 = 1 X 104), Uranotaenia unguiculata (LC50= 3 X 104) and Anopheles
sergentii (LC50= 5 X 105).
Ramoska et al. (1977) bioassayed B. sphaericus strains 1593, 1404 and SS11 -I for
infectivity against field collected Psorophora columbiae, Culex nigripalpis and Aedes
taeniorhynchus. They found that Aedes larvae were less susceptible to all the three strains
but most susceptible to strain 1593. They also observed that 43% of the larvae that were
to die in the course of experiment, died within 24hrs of incubation with the bacteria.
Tyrell et al. (1979) described the toxicity of Bacillus thuringiensis israelensis
parasporal crystals in larvae of three medically important mosquito vector species. The
number of larvae killed was in relation to crystal dry weight. The LC 5o values obtained
were: Aedes aegypti Linnaeus, 1.9 X 1ettg/m1; Culex pipiens var. quinquefasciatus
(now Culex quinquefasciatus) Say, 3.7 X 10 -4 µg/m1 and Anopheles albimanus
Weidmann, 8.0 X le pg/ml.
Myers et al. (1979) performed bioassay of two strains of B. sphaericus against
second instar larvae of Culex quinquefasciatus. The bacterial cells were removed from
growth medium by centrifugation and re-suspended in the equal volume of sterile de-
chlorinated tap water. They tested three cups each containing 10 larvae in 20m1 de-
chlorinated tap water per 10-fold dilution of bacterial cells and recorded the larval
30
survival. The strain SSII-1 had LC 50 at 2.5 X 108 spores/m1 while LC5 0 of strain 1593 was
2.9 X 102 spores/ml. Smith (1982) performed bioassays with Bti (H-14) against fourth
instar larvae ofAedes aegypti. As differences among log dilutions were detected, they did
not calculate LC 50 values. Instead they devised a rating system based on 10-fold dilution
and on the number of surviving larvae at the lowest effective sample dilution to define the
effectiveness of the samples. Balaraman et al. (1983) cultured Bt H-14 (VCRC B-17) and
using different additives prepared six different formulations. They then determined their
potency against early fourth instar Cx. quinquefasciatus larvae using IPS-78 as the
standard. Many other workers conducted lab bioassays using different commercial
formulations viz., Mittal et al. (1985 & 1993); Rettich (1987); Balaraman & Hoti (1987);
Ansari et al. (1989 & 1995); Klein et al. (1989) and Theoduloz et al. (1997).
Brown et al. (1998) conducted laboratory bioassays based on the standard methods
to determine effectiveness of Vectobac® 12 AS. The larvae were exposed to serial
dilutions of formulations in filtered water habitats. They maintained 5 replicates each of 20
late III/early IV instar larvae in 250m1 glass beakers containing 200m1 of the test
concentration. Initially they conducted a number of range-finding assays with widely
spread exposure concentrations. Based on these, they then conducted the main bioassays
with a narrow effective range of concentrations and calculated LC 50 and LC95 values.
Rodrigues et al. (1998) conducted lab bioassays to test 10 strains of Bs isolated from
Brazilian soils against third instar larvae of Anopheles species. They used lyophilized
bacteria for the bioassays in six or seven concentrations ranging from 0.5ppb to 50ppb.
Twenty larvae from each Anopheline species were used per concentration tested in one
31
single bioassay. They calculated Relative Activity (RA) of the isolates using the following
formula for 48 hrs. results:
LC50 Standard RA =
LC50 Sample
Extensive literature is also available on the successful small and large scale field
trials of biolarvicides in mosquito control from all over the world. Reports are available on
testing of various preparations and formulations of Bs and Bti against different species of
mosquitoes in a variety of habitats. Field trials of Bs as a mosquito larvicide were first
reported by Ramoska et al. (1978), who found it to be a promising biological control
agent of mosquitoes. Mulligan et al. (1980) conducted field trials to test the efficacy and
persistence of Bs strain 1593-4 and Bt (H-14) against mosquitoes. They found that both
were more active in raw and sewage effluent than in tap water. They observed that an
aerial application of Bt (H-14) at 1 kg/ha could bring about C. tarsalis control apparently
without adverse effects on predator populations.
Davidson et al. (1981) conducted comparative field trials to test the efficacy of Bs
strain 1593 and Bti commercial formulations in test ponds and found them to be very
similar in efficacy. Foo & Yap, 1983 conducted field trials on the use of Bt serotype H-14
against Mansonia mosquitoes in Malaysia. They tested a suspension concentrate Teknar ®
SAN 402-1 SC against lab reared as well as naturally occurring Mansonia mosquito larvae
using a Hudson knapsack sprayer in small plots in swampy ditches on Penang Island. They
obtained comparable dosage/response values for natural and introduced populations and
32
suggested the use of caged populations in bioassay methods for Mansonia larvae in the
field tests.
Mulla et al. (1984) reported excellent initial control of Culex tarsalis and Cx. peus
using primary powders of two strains of BS 2362 (IF-97) and 1593 (IF-94) at the rates of
0.1 and 0.2 lb/ha and also found them to be safer towards non-target organisms. Sandoski
et al. (1986) applied Bt (H-14) against Anopheles quadrimaculatus larvae in rice fields
using Beecomist® spray head and affirmed that this method of application is a cost
effective for high volume ground application in large areas. Bhalwar et al. (1993) and
Kumar et al. (1996) carried out field trials with B. sphaericus (Spherix formulation) in
Delhi and Goa, India respectively and at a dose of 1g/sq. m. demonstrated effective
control of Culicine breeding in the polluted water habitats such as drains, septic tanks,
cesspits, cesspools, etc.
Mittal et al. (1993) from their field trials concluded that Spherix formulations of
Bs @ 0.25-2 g/sq. m. produced 90-95% reduction in the larval densities of both An.
stephensi and Cx, quinquefasciatus within 48hrs. in different habitats and the larvicidal
activity persisted for 2-4 weeks in these trials. Dua et al. 1993 from their field trials with
Bactoculicide (Bti) sprayed @0.5g/sq. m. against Culex, Aedes and Anopheles larvae
breeding in industrial scrap observed that mosquito breeding was controlled from 96-100
% up to 5 weeks.
Ansari et al. (1995) evaluated the efficacy of two flowable formulations of B.
sphaericus i.e. Spherix and Vectolex 2.5AS and found that they produced control for
longer duration in deep wells than in shallow ponds and also recommended their use in
polluted habitats. Shulda et al. (1997) observed in Haldwani, District Nainital, India, that
33
B. sphaericus gives better control of Anopheline breeding in cemented tanks and small
ponds than B. thuringiensis.
Kumar et al. (1994 & 1997) demonstrated control of Malaria utilizing B.
thuringiensis israelensis against Anopheles stephensi in major field trials in Goa, India.
Brown et al. (1998) conducted field trials with Vectobac ® 12AS (active ingredient: 1200
ITU/mg Bti) against late third/fourth instar field specimens of Cx. sitiens in floating mesh
cylinders introduced in salt marsh pools near Coomera Marina, Southeast Queensland,
Australia. They achieved 100% mortality of Cx. sitiens larvae at 24hr post treatment at
the rate of 0.5 litre/ha.
Brown et al. (1999) evaluated in the field efficacy of four insecticides including 2
organophosphate compounds (temephos & pirimiphos methyl), a growth regulator (delta-
methoprene) and Bti against Aedes vigilax (Skuse). Based on high selectivity ratios
excellent field efficacy and lack of influence on abiotic water characteristics, they
suggested that delta-methoprene and Bti were ideal for insecticidal control of Ae. vigilax
in Australia.
Many more reports are available on the successful small and large field trials
with biolarvicides from all over the world (Lacey et a/. 1986; Rettich, 1987; Ansari et al.
1989; Rajendran et al. 1990; Logan & Linthicum, 1992; Karch et al. 1992; Romi et al.
1993; Rettich & Ryba, 1993 and All et al. 1994).
2.4.3. Factors affecting biolarvicide activity: The efficacy of the biolarvicide is
influenced by various biotic and abiotic factors including the target species, type of
formulation, feeding behaviour of the larvae and ecological factors like temperature, pH,
34
exposure to UV radiation in the sunlight, stream discharge, presence of micro-organisms,
etc.
2.4.3.1. Feeding behaviour: Ramoska & Pacey (1979) reported that the efficacy of Bs
Neide. strain 1593 was inversely related to the amount of food available to the mosquito
larvae and that the larvae of Culex quinquefasciatus consumed lethal quantities rapidly
after bacterial application whereas those of Aedes aegypti did so less rapidly, resulting in
low mortality even after 90 minutes of incubation at high dosage (7 X 10' cells/m1)
2.4.3.2 Temperature & pH: The effect of temperature and pH at which the mosquito
larvae are exposed to Bs and Bt formulations has been reported by Muligan et al. (1980);
Wraight et al. (1981); Subramonian et al. (1981); Molloy et al. (1981) & Mittal et al.
(1993).
2.4.3.3. Exposure to sunlight: Mulligan et al. (1980) reported that the active ingredient
of B. sphaericus was readily degraded by exposure to direct sunlight, while that of Bt. H-
14 was not even after 5 hours exposure to direct sunlight. Burke et al. (1983) reported
that the UV light from a germicidal lamp reduced the viability of B. sphaericus 1593
spores but insecticidal activity was resistant to inactivation after continuous exposure to
UV light for 4 hour.
2.4.3.4. Pollutants: Lab and field trials with Bs and Bti have suggested the efficacy of
biolarvicide is lowered in waters which are turbid or contain sewage with high chloride
content or other waste effluents (Mulligan et al.1 1980; Ramoska et alb 1982 and Car,
1984).
35
2.4.3.5. Other Factors: A number of other factors known to affect the efficacy of the
biolarvicides include strain of the biocide & media used for growth (Smith, 1982), larval
instars (Ramoska et al. 1977; Wraight et al. 1981), larval breeding habitat (Huang et al.,
1993; Romi et aL, 1993; Ansari et al., 1995), species of the mosquito (Habib, 1983) and
the stream discharge.
2.5. Persistence and recycling of biolarvicides in the environment
One of the most desirable qualities of a biocide is that it should be able to produce
long lasting control with one or few treatments. This is important to compensate for the
high cost of production and applications of these bio-control agents. Therefore it is
desirable to use only those agents that are able to persist or recycle in the treated habitats
for reasonably long period and provide satisfactory control apart from possessing the
other desired qualities.
Soon after the discovery of the two entomo-pathogenic bacteria (Bs and Bti) and
their use as biolarvicide, various researchers made efforts to study their persistence and
recycling potential in the host, soil, mud and water (Hertlein et al., 1979; Chilcott et al.,
1983; Davidson, 1984; Hoti & Balaraman, 1991; Yousten et al., 1991& 1992; Khawaled
et aL, 1992 and Becker et al., 1995).
Saleh et al. (1970) reported that B. thuringiensis spores can remain viable for long
periods of time and that the organisms can compete successfully under conditions
favouring the Bacillus component of soil microbial populations. Ignoffo & Garcia (1978)
however reported that the UV light inactivates the cells and causes their death. West et al.
(1984) studied the persistence of viable and heat killed vegetative cells, parasporal crystals
36
and the spores of B. thuringiensis in the soil monitoring them by immuno-fluorescence and
found that the number of spores remains unaltered throughout 91 days incubation at 25 °C.
Persistence and the residual activity can be enhanced through proper formulation.
Lacey et al. 1984 had reported that some slow release formulations provided persistent
release of toxins and control of larvae.
Menon & Mestral (1985) conducted field and laboratory studies to assess the
survival of Bt var. kurstaki in waters and reported that Btk could survive for relatively
long periods in fresh water and marine environment at 20 °C.
According to Aly et al. (1985), Bti sporulates and produces crystals in cadavers of
mosquito larvae and thus helps in the recycling of the organism in the environment. Petras
& Casida (1985) reported good survival of B. thuringiensis spores in the soils. However
Aronson et al. (1986) opined that the rather sluggish germination of B. thuringiensis
spores may contribute to the lack of persistence of these organisms. According to Mulla
(1990), most of the commercial formulations give high levels of initial control but young
mosquito larvae soon appeared after a few days of the treatment. This according to them
shows that Bti fails to recycle in nature.
Recently Mansherob et al. (1998) reported bio-encapsulation of the spores of
Bti in protozoan, Tetrahyemna pyriformis as the new mode of Bti recycling in nontarget
organisms. They have observed that spores indeed germinated, grew and sporulated in
excreted food vacuole of T. pyriformis forming new active ICPs during this cycle.
The first reports of the recycling potential of Bacillus sphaericus came from
Hertlein et al. (1979). They reported that Bs remained viable and infective even 9 months
after applications as a larvicide agent of mosquitoes in a roadside ditch. Burke et al.
37
(1983) have demonstrated the effect of UV light from a germicidal lamp on Bacillus
sphaericus, 1593. They found that due to this exposure spore viability was considerably
reduced but insecticidal activity was resistant to the inactivation even after continuous
exposure to UV light for four hours. Thus the sprayed habitats which have low light
intensity would probably exhibit longer larval control.
Mulligan, et al. (1980) found that larvae were continuously killed by exposure to
water taken from the bottom of the same basins for up to one month after treatment with
Bs suggesting its settlement and the prolonged activity. On the contrary, Karch & Charles
in 1987 reported that spores that had settled at the bottom of cesspools rapidly lost their
toxicity against Culex pipiens larvae.
There are, on the other hand, several reports suggesting that B. sphaericus cells
multiply in the larval cadaver and formed fresh spores (Silapanuntakul et al., 1983;
Davidson et al., 1984; Des Rochers & Garcia, 1984; Charles & Nicolas, 1986). The
recycling potential of Bs has been attributed to the ingestion of spores by non-target
invertebrates to the role of cadavers (Yousten et al., 1991; Becker et al., 1995 and
Carvalho-Pinto et al., 1995)
2.6 The Safety of Bacillus species as mosquito control agents
With the discovery of a number of larvicidal strains of B. sphaericus and B.
thuringiensis there emerged a need to test these for safety against man, fish, wildlife and
other non-target biota before they could be recommended for the biocontrol of disease
vectors. Since one member of the genus Bacillus i.e B. anthrax is a known mammalian
pathogen, it has become necessary to evaluate the mosquitocidal strains of Bs and Bti for
their safety to the man and environment.
38
There is a large volume of information on the impact of these two
entomopathogens on non-target biota. As early as in 1975, Kingsbury had reported that
the aerial applications of Bti had no adverse effect on aquatic insects. Ever since then,
various authors have shown Bs and Bti to be safer to most of the non-target fauna
(Mulligan et al. 1978 & 1980; Molloy & Jamnback 1981; Walton & Ladle, 1983; Car &
Moor, 1984; Pistrang & Burger, 1984; Mulla et al. 1984; Rettich, 1987; Majori et al.
1987; Molley, 1992; Richardson & Perrin. 1994; Chui et al. 1993; Wipelli & Merrit, 1994
and Roberts, 1995)
A few other reports are briefly discussed here. Moulinier et al. (1981) had
conducted experiment with Bti to show its innocuity against Oyster larvae and hence
suggested its possible use near the areas of Oyster culture. Davidson (1982) reported that
Bti itself has no effect on aquatic organisms such as water mites, shrimps and oysters.
Bslararnan et al. (1983) found it to be safer for fishes, copepods, notonectids and other
aquatic fauna. Lacey and Mulla, 1990 have written a comprehensive review on the safety
of Bti and Bs on the non-target organisms. Charbonneau et al. 1994 conducted field and
laboratory studies to determine the effects of Bti (Vectobac® G) on water fowl macro-
invertebrate food resources at Minnesota valley National Wild life Refuge Bloomington,
Minnesota. They concluded that chironomids (a major water fowl food source) were
adversely affected by Vectobac G under controlled laboratory situation. However,
environmental factors reduce the efficacy of the biolarvicide in the field. Hershey et al.
(1995) studied the effects of methoprene and Bti on non-target insects in USA and did not
observe any negative effects of biolarvicide treatment on density or biomass of
invertebrate groups nor any decrease in richness of benthic invertebrate taxa. Brown et al.
39
(1999) from their study concluded that Bti was an ideal insecticide for the control ofAe.
vigilax in Australian salt marsh pools based on its high selectivity ratio, excellent field
efficacy, and lack of influence on abiotic water characteristics.
Several reports are available on biolarvicide safety to humans as well as other
mammals. Burges (1981) reported the testing of several species of B. sphaericus and B.
thuringiensis israelensis against mammals including human volunteers and concluded
them to be safe for use as insecticides. Shuddack et al. (1980) could recover Bs from
rodents 14-18 days after intra-peritoneal and intra-cerebral injections. Seigel et al. (1987)
reported that B. thuringiensis israelensis could be recovered for as long as 7 weeks after
injection. Seigel & Shadduck (1990) summarized that oral, intra-peritoneal and aerosol
exposure to Bti produced no significant illness or mortality. According to them the
entomo-pathogens disappeared rapidly from the lungs of rats, and there was no evidence
of their multiplication. Subcutaneous injections of Bti produced abscesses but these
abscesses appeared following injection with autoclaved material as well. Abscesses most
likely arose from the presence of heat stable foreign material in the injection site. The
ocular irritation to Bti depended upon the physical state of the preparation; fine powders
produced no irritation and large clumps caused considerable corneal irritation, conjuctival
swelling, congestion and discharge. Intra-cerebral injection of 10 7CFU resulted in
mortality of rats. As cited by these workers, the toxicological studies performed by
experimental exposure of various animals (mice, rats, guinea pigs and rabbits) showed the
absence of acute and prolonged toxicity.
Mammalian safety of Bacillus sphaericus has been discussed in detailed by Siegal
and Shadduck (1990). They concluded that exposure to Bs isolates 1404, 1593 and NCTC
40
11025 did not result in clinical illness or death. Occular irritancy of B. sphaericus was
minimal and although this organism persisted for 8 weeks in the conjuctival cul-de-sac of
rabbits, there was no evidence of infection. B. sphaericus 1404 and 1593 did produce
lesions when injected intra-ocularly and intra-cerebrally. The safety data of strain 2362
however are more variable, although dose greater than 10 7 CFU proved toxic to mice.
Clearance studies conducted by them and those by de Barjac et al. (1987) indicated that B.
sphaericus disappeared from blood within two weeks following parenteral injection. They
concluded that these biolarvicides could be used safely in the environment where exposure
to human beings might occur.
Recently some workers have indicated the risk of gastroenteritis outbreak due to
B. thuringiensis in humans (CarLston et al. 1994; Damgaard, 1995; Damgaard et a1.1996).
A review by Drobniewsky in 1994 entitled 'The safety of Bacillus species as vector control
agents' sums up the safety profile of Bti and Bs stating that these species may cause
disease in man but the reported cases are very less. In comparison to their benefits to the
community the risk to the public health is very small and at present there is no good
evidence to discontinue their use.
2.7 The problem of resistance to biolarvicides
The first reports of the insect resistance to Bacillus thuringiensis came from
McGaughey, 1985 who found that Plodia interpunctella, a major lepidopteran pest can
develop resistance to the insecticide within few generations. According to him the strains
of P. interpunctella collected from the treated grain bins were more resistant than the
strains from the untreated bins indicating that the resistance can develop quickly in the
field. They also found that resistance was inherited as a recessive trait. van Rei et al. 1990
41
studied the mechanism of insect resistance to B. thuringiensis that was due to the
alteration in toxin membrane binding. Resistance to Cry I A(b) toxin was found to be
correlated with reduction in the affinity of Cry I A(b) toxin binding, whereas increased
sensitivity to Cry I(c) toxin is reflected in an apparent increase of the Cry I (c) binding site
correlation. Georghiou et al. (1991) reported that the cross resistance between Bs strains
harbouring different gene sequences has not been found to occur, which suggest that the
toxins may be binding to different receptors.
Adak et al. (1995) for the first time reported the B. sphaericus resistance to Culex
quinquefasciatus. According to them initially B. sphaericus was very effective but within a
year i.e. after 20-25 rounds of application, field populations of Cx. quinquefasciatus
developed resistance up to 150 fold. Through genetic studies they found that resistance
was recessive autosomal chromosome linked and was controlled by more than one gene.
Several authors have tried to explain the mechanism of insect resistance and have
suggested methods to overcome the problem. McGaughey & Whalon (1992) have
mentioned several theories for managing the development of resistance to Bt toxins but
their utility has not been assessed in the field. Among their recommendations is the
rotation or the alteration of Bt toxins with other toxins. They have also suggested three
other ways to manage resistance viz. application of low dose, high dose and ultra high
dose. According to them the low dose tactics include the reduction in the rate of toxin
application and reduced frequency of application and they aim at reducing the pest
populations slightly or slowing down the pest larval development to the point that the
number of generations per year is reduced. Probably this reduces the selection pressure.
High dose kills heterozygotes, which are the most abundant carriers of insect resistance
42
genes, whereas, the 'ultra high dose' is sufficient to kill both the heterozygous and
homozygous resistant insects. Ferre et al. (1995) found that the insect resistance to Bt
insecticidal proteins is inherited as a single recessive or partially recessive major gene.
According to them the binding site modification seems to be the most significant resistance
mechanism under field conditions. Moar et al. (1995) suggested that the use of individual
protoxins or toxins especially expressed in transgenic organisms induce resistance in the
field populations more readily than the formulated materials containing multiple Cry
proteins and spores. Charles et al. (1996) reported that the resistance in field seems to
decline very quickly when the treatment is suspended. They have also suggested methods
to overcome the problem of resistance through the use of Bs strains belonging to different
serotypes that display no cross-resistance. Besides, according to them the best way to
prevent or retard resistance may be to produce bacterial strains that simultaneously
express different toxins binding to different receptors. Thus the diversification and the
combination of Bt and Bs strains could help in the management of resistance in target
mosquito species.
van Frankenhuyzen et al. (1997) discovered that toxin Cry 9Ca I binds to different
receptors than do other ICP's that are currently used in conventional sprays or transgenic
plants. Such toxins may become valuable tools to manage resistance problem. Koskella &
Stotzy (1997) reported that the clay bound toxins become resistant to degradation and
retain insecticidal activity for long, thus the toxins from transgenic plants and microbes
may accumulate in soils and thereby enhance the development of insect resistance. They
have recommended that this mechanism of persistence of toxins in soil should be
incorporated in the future management plans to reduce the development of resistance to
43
biolarvicides. Georghiou & Wirth (1997) studied the influence of single versus multiple
toxins of Bacillus thuringiensis subsp. israelensis on the development of resistance in
Culex quinquefsciatus and concluded that the usage of natural mixture of four Bti toxins
would be advantageous in suppressing or delaying the resistance in Cx. quinquefasciatus.
Liu et al. (1998) opined that the absence of spores in Bt toxin-expressing transgenic plants
and bacteria would accelerate the evolution of pest resistance. Cross-resistance to B.
sphaericus has been reported by Poopathi et al., 1999. They have indicated the judicial
use of Bti for the management of resistance in Bs resistance Cx. quinquefasciatus and
have suggested the use of other alternate mosquito control strategies in the microbial
resistance management. It may therefore be concluded that insect resistance to B.
thuringiensis and B. sphaericus can occur and to overcome this serious problem and for
the judicious deployment of these tools in insect control, the resistance management
strategies recommended by experts should be adopted.
2.8 Genetic manipulation of B. sphaericus and B. thuringiensis
The narrow target range of mosquitocidal bacilli, lack of persistence in the
environment for a long time, high cost of production especially that of B. sphaericus are
the major limitations of naturally occurring strains being used extensively in biocontrol.
The cost of production can be reduced considerably using locally available cheaper raw
materials that support good growth and toxin production (Eziofor &Okafor, 1988 and
1989). The problem of narrow host range can be addressed by genetic engineers and new
strains expressing the new and existing combination of mosquitocidal toxins could be
obtained. The lack of persistence of the biolarvicides in the environment for a long time
has been attributed to the settling of the spore -crystal complexes (Davidson et al. 1984
44
and Ohana et al. 1987). This settling problem can be solved by the improvement of
formulation technology and with the use of genetically engineered organisms in which the
mosquitocidal toxin genes of Bacillus sphaericus and B. thuringiensis, could be expressed
in those micro-organisms that do not settle at the bottom of ponds and lakes and rather
suspend in feeding zones of larvae.
Porter et al. (1993) summed up recent advances on new type of recombinant
microorganisms and new cloning strategies in their review. According to them these
genetically transformed microorganisms have the potential to provide effective control of a
wider range of mosquito species for a longer duration than the naturally occurring bacilli.
A few other important research articles on this topic are briefly reviewed here.
According to Huber (1986), attempts have been made to express these
mosquitocidal toxins in E. coli and Baculoviruses. But these new organisms are of not
much importance because viruses take a long time for activity and E. coli. is a mammalian
pathogen. Angsuthanasombat and Panyim (1989) have reported the introduction of 130
kDa mosquitocidal toxin gene cloned from Bti into the Cynobacterium, Agmenellum
quadruplicatum PR-6 by plasmid transformation and the synthesis of mosquitocidal toxin
by the transformed cells. They demonstrated the use of engineered Cynobacterium for
biological control of mosquitoes. The Cynobacteria are widely distributed in the upper
layer of water and thus the produced toxin would reach the target mosquito larvae more
effectively. Thanabalu et al. (1992), reported the production of recombinant Caulobacter
crescentus by transformation with broad host range plasmids carrying genes encoding
larvicidal toxins from either B. sphaericus or B. thuringiensis subsp. israelensis. They
further demonstrated the mosquito larvicidal activity of the intact recombinant C.
45
crescentus. Bacteria belonging to Caulobacter species are found in almost every aquatic
habitat predominantly near water surface.
Liu et al. (1996) expressed the binary toxin of Bs in the gram-negative bacterium
Asticacaulis excentricus and reported that the plasmid transformed bacterium exhibited
toxicity to Culex and Anopheles larvae. A. excentricus has the potential advantage as a
larvicide compared with the bacilli, especially persistence in the larval feeding zone,
resistance to UV light, lack of toxin degrading proteases and low production cost.
Thiery et al. (1997) isolated a gene, Cyt 1 AbI encoding the 27,490 Da protein
from B. t. var. medellin (H30 serotype) by using an oligonucleotide probe corresponding
to Cyt 1 AbI gene. It was expressed in crystal negative Bt strains. They found that the
large inclusion of cytlAbl protein was twice as haemolytic as that from the wild strain.
Besides, transgenic plants have been obtained using toxin genes from Bt and Bs. (Vaeck et
al. 1987; Hofte & Whitely, 1989 and Peferoen, 1997). These transgenic plants of tomato,
tobacco, cotton, corn and potato are protected from the feeding damage by the insect
pests. Whether the genetically engineered mosquitocidal organisms will possess all the
necessary qualities of biocide is however still a question of concern for many workers.
2.9 Economics of biolarvicide usage in vector management
For the commercialization and wide spread use of biocide in insect control, it is
very important that these should be economical. Many authors have tried to evaluate and
compare their costs and have suggested ways to reduce costs of production or application.
Margalit & Dean (1985) suggested that the newer strain of entomopathogens may be
constructed which grow faster, produce more toxin and grow on cheaper carbon source,
thus reducing the cost of production and formulation (Table 2.9.1).
46
Table 2.9.1: List of potential raw materials for use in formulating fermentation media for local production of B. sphaericus and B. thuringiensis israelensis (Source: B hum iratana, 1990)
Raw Material Comment; Reference
Agricultural Products: These materials are extracted in boiling water and
added to basal medium containing dried cow blood
and mineral salts
Obeta & Okafor, 1983.
Vandekar and Dulmage, 1983
Bambara bean, cowpea,
Groundnut cake, soya bean
Ground corn, Cottonseed meal,
Cassava, Yams
Animal Products: Vandekar and Dulmage 1988.
Hertlein et al' 1981 Blood, beef bones, chicken parts,
Fishmeal, animal dung
Industrial By-products: Dh arm sthiti et al., 1985.
Byproduct from monosodium-
Glutamate factory.
Other Vandekar and Dulmage, 1983
Coconut milk, corn-steep,
Liquor, molasses, whey
Table 2.9.2 Production cost of one batch* of B. thuringiensis H-14 and B. sphaericus strains in laboratory fermenter, of 13 lit. working capacity (Source: Balaraman and Hoti, 1987).
Inputs B. thuringiensis B. sphaericus H5a5b strain H-14 strains
VCRC-B17 VCRC-MB24 VCRC- B42 Us $ Us $ Us $
Raw materials 1.25 1.17 1.25 Equipment 0.42 0.42 0.42 Personnel 6.67 6.67 6.67 Energy 3.17 2.17 4.33 Building 0.25 0.25 0.25 Laboratory Maintenance 0.17 0.17 0.25 Servicing and Maintenance of Equipment 0.17 0.17 0.17 Total 12.10 11.02 13.34
47
For the economical use of a biolarvicides, it is also desirable that the cost of
application of the biocide should be less and affordable. Yates (1984) and Sandoski
et al. (1986) affirm that Beekomist ® spray head as a cheaper alternative to high
volume aerial application of Bti on large areas. According to Sandoski et al.
(1984) if the effective swath width, optimum flight path interval and flow rate are
more precisely determined, the cost of treating large areas of rice for mosquito
control will be further decreased.
Mulla (1990) opined that the lower dosages of entomopathogens should be
sufficient to control rising populations of insects after their first application. Hence
reducing the dose by 50% to 75% after the first application yielded equivalent
reduction in larval populations, while it resulted in significant saving in cost of the
treatment.
Balaraman & Hoti (198'7) not only worked out the comparative cost of
production of one batch of Bti and Bs strains in the laboratory fermenter (Table
2.9.2) but also compared the costs involved in controlling mosquito larvae in an
area of one hectare with the bacilli and chemical larvicides. They concluded from
their study that the cost of larval control with bacilli was more or less equivalent to
that incurred if Fenthion was used but the cost was significantly lesser if malaria
oil, abate or Paris green were used (Table 2.9.3).
48
Table 2.9.3: Comparative cost of larval control (in unpolluted waters) per hectare for four weeks with chemical and microbial larvicides (Source: Balaraman & Hoti, 1987).
Larvicides Unit price US$
Dosage/ Ha
Cost of larvicide (US$)
Cost of labour (US$)
Total cost of larval
control for 4 weeks (US$)
For 1 appli- cation
For 4 applic- ation
For I appli- cation
For 4 appli- cation
Chemical
1. Malaria Oil 0.25 lit 142 lit 35.50 142.00 4.25 17.00 159.00
2. Fenthion 25.00/lit 500 ml 12.50 50.00 4.25 17.00 67.00
3.Paris Green 7.08/ kg 4kg 28.32 113.28 5.42 21.67 134.95
4. Abate (50%)
41.67 lit 400 ml 16.67 66.68 4.28 17.00 83.68
Microbial
1.Bt H-14(B17) 12.10/120 660 brig. 66.55 - 0.83 - 67.38
2.Bt H-14
(MB24)
11.02/120 660 brig. 60.61 - 0.83 - 61.44
3.Bs (B42) 13.34/120 660 brig. 73.37 - 0.83 - 74.20
49
According to Sutherland (1990), the ground application cost per acre treatment
with Bti and Abate® respectively was US$3.2 and US$4.39 and consequently for
economy, Bti was chosen. But for the aerial application cost/acre worked out was US$3.2
and US$2.3 respectively. According to them, suitable formulation development of Bti
through cheaper sources could further reduce this difference.
According to Hougard (1990) despite the high concentration of B. sphaericus
recommended for the treatment of polluted waters (10g/m 2), it was found to be promising.
To substantiate this view, he cited the example of the use of a liquid formulation of the
strain 2362 in Dar-as-salaam (Tanzania) by the sanitary services of the town (Table
2.9.4).
Moffat (1991) reported that the higher insurance premiums were associated with
the use of chemicals than the biocides, due to which the people were switching over to the
latter. According to Priest (1992) the bacterial insecticides will become popular only if
their treatment cost is equal or less than the chemical alternatives. There is surely
possibility of improvement of the strain and fermentation process to reduce the cost of
production of both Bti and Bs. It was also opined that effective products at the realistic
prices could ensure a bright future for the bacterial insecticides.
50
Table 2.9.4: Comparison of the approximate cost (U.S.$) of operational use
for a one-year Period of B. sphaerius (BSP1) at 10g/m 2 and Chlorpyrifos
(Dursban®) at 1 g/m 2 in Dar-as- salaam (Tanzania) for Control of Cx.
quinquesfasciatus (Source: Hougard, 1990).
Parameter B. Sphaericus 2362 (flowable conc.)
Chlorpyrifos (E.C. 480g11)
Dosage 10g/m2 (0.01 lt/m2) 1 g/m2(0.001 lt/m 2)
Frequency of larviciding 9 cycles a year 24 cycles a year
Quantity of formulation 45 liters 25 liters
Staff' $12 x 9 = $108 $12x 24=$288
Equipment b $30 x 9 = $270 $30 x 24 = $720
Insecticide $8 x 45 = $360 $15 x 25 = $ 375
TOTAL $738 $1,383
NOTE : The cost is calculated on the basis of 500 m 2 oftreated sites.
a- About $12 for two technicians for one cycle of larviciding.
b- About $30 for one vehicle for one cycle of larviciding.
51
Becker & Margalit (1993) have stated that the application of Bti can be
cost effective as compared to the conventional insecticides. They have given the
example of the KABS programme of German Mosquito Control Association, by
which it was possible to suppress the mosquitoes from a catchment area of more
than 500km2 including 100km2 of the existing breeding grounds. According to
them a total yearly budget of US $ 1 million protects more than 2 million residents
in the area. They have also compared the relative costs of larvicides in the
Onchocerciasis Control Program and have reported that the cost of Bti treatment is
more or less comparable to the cost of treatment using chemical insecticides such
as temephos or chlorophoxim when the discharge of the streams is low. However
the cost of Bti treatment is slightly higher when the stream discharge is several
hundred m3/sec. Furthermore, they have also stated that the cost of development
and registration of Bti (app. US $ 50,0000) are many times lower than those than
for a conventional chemical insecticides (about US $20 million)
2.10 The test mosquito species
2.10.1 Identification of the mosquitoes: Christopher (1933 & 1934) has given
the key for the identification of the families Culicidae and Anophelini. According to
him the adult anophelines are at once recognised by their spotted wings and from
their characteristic posture of resting on the walls or other objects. He described
43 species and 10 varietal forms collected at that time in India. Barraud (1934) has
given keys to the identification of adults & larvae belonging to family Culicidae
tribes Megharhinini and Culicinae. According to him the characteristics taken into
consideration for the identification of the adults are found chiefly in the pleural
52
chetotaxy and in the venation of the wings. Besides, the other characteristics taken
into consideration for morphological identification were scaling and the length of
palpi. He also used structure of larvae and pupae which often shows more definite
generic characteristics than adults. Puri (1954 & 1960) provided suggested rapid
identification method for the adult and larvae of Anopheline mosquitoes. Rao
(1984) in his book has provided comprehensive information on biology and basic
principles of control of Anopheles mosquitoes. This book also contains a key for
the rapid identification of Indian Anopheles species. Das et al. (1990) proposed a
simplified and easy pictorial key to 54 species of Indian Anopheles mosquitoes and
contains 177 illustrations and 3 tables. Nagpal & Sharma (1994) have also
prepared a key to the identification of all Anopheline mosquitoes reported from
India with illustrations of the various species, their morphological variations,
distribution and zoogeography.
2.10.2 Field sampling: For the bioassay studies a table top insectary facility for
the regular supply of healthy mosquito larvae of particular instar, age and species is
a must. Initially, the immature were collected from the field directly by sampling,
brought to the insectary, maintained under suitable light, temperature and rearing
conditions by providing larval food.
A manual of WHO (1975) entitled 'Practical Entomology in Malaria'
provides detailed information on the construction of exit traps, larval collection
methods, maintenance of culture in the lab, etc. Service (1976) has also described
procedures of larval sampling, types of traps, and methods used to collect
53
mosquitoes and methods for the analysis of the results that are of great use for the
field workers.
2.10.3 Mosquito rearing procedure: Ansari et al. (1978) described a method for
the mass production of Anopheles stephensi Liston. For mass production, standard
wooden cages of 70 x 60 x 60cm sizes were used. Adults were held in cages for
oviposition (egg production) using cyclic and non-cyclic population methods.
Mosquitoes were fed on water soaked raisins and offered blood meals at regular
interval. Eggs were collected in ovipositional jars (6 x 8 x 10cm) containing 250m1
water and lined with filter paper and they were held in these jars for 72 hrs. for
hatching. Newly hatched larvae were transferred to plastic trays (63 X 60 X 9 cm).
Larvae were fed on powdered food that consisted of 60% dog biscuit and 40%
brewer's yeast. They have described the establishment of the adult colonies, larval
rearing and pupal collection. They concluded that the maximum of 15000 larvae of
An. stephensi can be reared in a plastic tray (63 x 60 x 9 cm) as against 30,000 of
Cx. p. fatigans or Ae. aegypti. Ramoska & Pacey(1979) maintained mosquito
larvae in the lab for bioassays and fed them on yeast and rabbit chow.
2.10.4 Bioassays using mosquito larvae: This topic has been already reviewed
under applications. Literature on some aspects such as calculation of lethal doses
has been reviewed again here briefly. Ramoska et al. (1977) bio-assayed three
strains of Bs against field collected Psorophora columbiae, Culex nigripalpus and
Aedes taeniorhynchus larvae. They found that nearly 75% of the mortality that
occurred in the course of Bs exposure occurs within 48 hrs post-incubation with
the bacteria and also that susceptibility of P. columbiae larvae decreased to
54
Bacillus with increase in larval age (instar). They calculated Median Lethal Dose
(LDso) by plotting dose vs. mortality for a series of doses on log probit paper.
They then recorded LD so in terms of the reciprocal of the log dilution of the
original culture medium. Mulligan et al. (1978) used lab reared second instar of the
southern house mosquitoes, Culex quinquefasciatus Say. for bioassaying Bs strains
1593-4 and SSII-1 in the lab and in the field. They found strain 1593-4 to be more
virulent. Tyrell et al. (1979) used lab reared II instar larvae of Ae. aegypti , Cx.
pipiens quinquefasciatus and An. albimanus to test the toxicity of the parasporal
crystals of Bti. They calculated LCso in terms of the dry weight of the crystals/ml
and found the relative sensitivities as follows: Ae. Aegypti (LCs0=1.9 X 10-4
14,/m1) > Cx. pipiens var. quinquefasciatus (LCso= 3.7 X 10-4 µg/m1) > An.
albanimus (LCso= 8.0 X le RAW).
Wraight et al. (1981) used field collected I, II & III instar Aedes stimulans
larvae to study the effect of temp. and instar on the efficacy of Bs strain 1593.
They recorded larval mortality after 12hr interval and considered the larvae dead if
they were unable to return to the water surface after being forced to the tray
bottom.
Smith (1982) studied the effect of strain & medium variation on mosquito
toxin production by Bti and performed the bioassays against IV instar Ae. aegypti
(Linneaus). After the treatment the containers with larvae were incubated at 30 °C
and surviving larvae were counted. As he detected differences among log dilutions,
he did not determine LC so instead proposed a rating system based upon the 10-fold
dilutions and on number of surviving larvae at the lowest effective sample dilution.
55
Foo & Yap (1983) obtained comparable dosage response values for the
caged and introduced populations in the field trials ofBti serotype 11-14 against
Mansonia mosquitoes and suggested that lab reared populations can be used in
bioassay methods in the field.
Mulla et al. (1984) tested the larvicidal and field activity of Bs strains
against II and III instar larvae of the 5 species of mosquitoes. They calculated the
percentage reduction as per following formula (Mulla et al., 1971):
Ci X T2 %Reduction (R) = 100 X 100
C2x -ri Where
C 1 = No. of larvae in control pre-treatment
C2= No. of larvae in control post-treatment
T1 = No. of larvae in treated pre-treatment
T2= No. of larvae in treated post-treatment
Obeta & Okafor (1984) used lab reared third instar larvae ofAe. aegypti
and Cx. quinquefasciatus and field collected third instar larvae of An. gambiae to
test the powder formulation of Bti produced from 5 local media. For this they
suspended 100 mg of the powder in 1000m1 distilled water containing 1% (vol/vol)
Tween 80.Then they added 20 mosquito larvae to each 250 ml capacity plastic cup
containing 150m1 distilled water. They kept three replicates and maintained
simultaneously controls for the comparison. The Aedes and Culex larvae were fed
with small portions of ground oat flakes mixed with dried yeast, while the
Anopheles larvae were fed with baby cereal. The observations were made for
56
paralysis and knockdown effect and the mortality counts were made after 24 & 48
hrs post-exposure. The larvae that did not move on touching were presumed dead
and moribund larvae were counted among the survivors. They used Abbot's
formula for correction of the mortality and rejected the assays if mortality in the
control exceeded 20% in any of the experiments.
Ansari et al (1989) lab evaluated Bs formulations viz. Solvay liquid and
Abbott granules (ABG6185) against colonized larvae of An. culicifaciens, An.
stephensi, An. subpictus and Cx. quiquefasciatus. They recorded larval mortality
after 24 & 48 hrs and corrected it by Abbot's formula given below:
% test mortality - % control mortality Corrected % mortality = X 100
100 - % control mortality
Mittal et al. (1993) used lab reared larvae of An. stephensi, Cx.
quinquefasciatus and Ae aegypti to test two biolarvicides Spherix (B. sphaericus)
and Bactoculicide (B. thuringiensis) and observed the effect of temperature on
their toxicity. They exposed the II instar larvae to serially diluted concentration of
biolarvicide suspension in water and kept three replicates of each concentration.
They used Abbot's formula for the % mortality correction and calculated LC so and
LC90 values.
Brown et al. (1998) conducted lab and field trials to evaluate the
efficacy of Vectobac ® 12AS (Bti) against Cx. sitiens larvae. Initially they
conducted a large number of range-finding bioassays with widely spread exposure
concentrations. Based on these they obtained a narrow range of concentrations in
57
which lay the effective concentration. They conducted bioassays at 25 ° C under
12hr light and dark cycles and used probit analysis to calculate LC 50 & LC95 values.
The field trials were conducted by suspending field collected mosquito larvae in
salt marsh pools treated with biocide.
Rodrigues et al. (1998) recently carried out studies on the Bs larvicidal
activity against Malaria vector species in Amazonia. They conducted bioassays
with the 20 III instar larvae per concentration for each Anopheline species tested.
They calculated LC soin each period of observation (24 & 48hrs) using POLO-PC
programme. They also calculated relative activity (RA) using B. sphaericus 2362
as standard, according to the following formula:
LC50 standard RA=
LC50 sample The values of RA were calculated for 48 hrs results.
2.11 Conclusion
From all the literature reviewed in this chapter, it is evident that it has
taken decades of research from the point of initial isolation of mosquito-pathogenic
B. sphaericus and B. thuringiensis israelensis to their current status of commercial
availability for the vector control programmes. Their efficacy and safety aspects
have been studied and they provide a viable operational alternative to chemical
larvicides. Depending upon their suitability to the local ecological conditions and
vector breeding behaviour, these biolarvicides can be judiciously deployed either
individually or with other vector control agents in an integrated manner. Their
large-scale and prolonged use may however, pose problems like development of
58
resistance in the target vectors in the long run. Therefore suitable resistance
management strategies are being proposed and adopted. Various studies to
enhance their recycling potential are also being carried out. At the same time, there
is felt need for the isolation of potent indigenous strains for the better compatibility
between the local vector and the pathogenic bacilli which when commercialised
and indigenously mass produced is expected to be cost effective. Efforts are also
being made to express toxin genes of virulent strains in other organisms like algae
and cynobacteria in order to overcome their limitations like settling to ensure ready
availability in the feeding zones of larvae. Continued research on mosquito-
pathogenic bacilli therefore is a rewarding enterprise.
59