Journal of Applied Bacteriology 1992, 72, 357-369

A REVIEW
Biological control of mosquitoes and other biting flies by
Bacillus sphaericus and Bacillus thuringiensis
F.G. Priest
Department of Biological Sciences, Heriot- Watt University, Riccarton , Edinburgh, UK
3681/06/91:received 10 December 1991

1. Introduction, 357 5.2 Plasmids and toxin genes of Bacillus sphaericus,

2. The targets, 358 362
3. Chemical vs biological control, 358 5.3 Mechanism o f action of Bacillus sphaericus
4. Bacillus thuringiensis toxins, 362
4.1 T a x o n o m y o f Bacillus thuringiensis, 359 5.4 Fermentation, 362
4.2 Plasmids a n d toxin genes of Bacillus 6. Formulations a n d applications, 363
thuringiensis, 360 7. F u t u r e prospects, 364
4.3 Mechanism of action of B . thuringiensis subsp. 8. Acknowledgements, 365
israelensis toxins, 361 9. References, 365
4.4 Fermentation, 361
5. Bacillus sphaericus
5.1 T a x o n o m y o f Bacillus sphaericus, 361

1. INTRODUCTION mulation of dead nematodes in the eye (Walsh 1986).

Biting midges (Ceratopogonidae) have also been implicated
Blood sucking insects such as mosquitoes and blackflies
in the transmission of nematodes and viral pathogens
have plagued man since prehistoric times. They are vectors
including Bluetongue virus of ruminants in most of the
of a multitude of diseases of man and animals through
USA (Jones & Foster 1978; Linley et al: 1983).
transmission of pathogenic viruses, bacteria, protozoa and
At least 90% of the world’s malaria, yellow fever, dengue
nematodes (although suggestions that mosquitoes might
and filariasis occurs in the tropics where the environmental
transmit the AIDS virus are fortunately unfounded). Some
conditions favour vector and pest insects responsible for the
of the more important of these diseases are listed in Table
transmission of the diseases (Rawlins 1989). In temperate
1. Historically, malaria has been the greatest single killer of
climates, haematophagous insects transmit fewer diseases
humanity and has an annual incidence of 200-300 million
but remain of considerable nuisance value. Bites from mos-
cases. Viral diseases transmitted by various mosquito
quitoes, black flies or midges can vary in effect from slight
species include yellow fever, dengue and several forms of
annoyance to severe discomfort and itching. Secondary
encephalitis. Wucheria bancroftti is probably the most wide-
infection is also possible. Mosquitoes seldom occur in huge
spread of filarial worm parasites of humans. T h e World
numbers but clouds of blackflies or midges can become
Health Organization (WHO) estimates that almost 300
intolerable and cause severe nuisance and economic prob-
million individuals are infected with this nematode, which
lems (Hendry 1989). In a recent survey of Local Health
in severe cases gives rise to gross deformity as elephantiasis.
Authorities in the British Isles, 22 reported that they had
The human filarial parasite, Onrhorerca uolvulus is
implemented control measures against mosquitoes in 1985
restricted to parts of Africa and Central America where it is
as a result of public demand (Snow 1987). This problem is
transmitted by black flies of the genus Simultium. This
therefore not restricted to the warmer climates.
nematode is responsible for river blindness caused by accu-
In this review I will summarize recent developments in
Correspondence to : F.G. Priest, Department of Biolopcal Sciences, the use of bacteria to control populations of biting flies.
Heriot- Watt University, Rirmrton, Edinburgh. EH14 4.4S, UK. Before we consider ways to control these flies, however, it
358 F . G . P R I E S T

Table 1 Some important vectors of

Insect vector Disease organism Disease Resistance spectrum human diseases and their resistance to
Mosquitoes chemical insecticides
.4nopheles spp. Protozoa Malaria DDT, OPs, Cs
(about 50)
Aedes spp. Virus Dengue DDT, OPs, Cs
Virus Yellow fever Pyrethr
.4edes and Virus Encephalitides DDT, OPs, Cs
Culex spp.
Culex quinquefusciatus, Nematode Filariasis DDT, OPs, Cs,
24edes, Anopheles and including
Mansoniu spp. elephantiasis
Blackflies
Simulium dumnosum Nematode Onchoserciasis OPs
and other spp.

Data from Rawlins (1989) and Walsh (1986). OP, Organophosphates; Cs, carbamates.

will be useful to cover briefly the principal characteristics
and life cycles of these insects.
2. THE TARGETS
Flies with one pair of functional wings are placed in the
order Diptera. Within this group there are several taxa
whose members may be intermittent vertebrate blood
feeders. These may be divided into two groups. T h e
‘primitive’ group comprises flies in which the female takes
a blood meal in order to provide nutrient necessary for egg
development. This includes the mosquitoes (Family
Culicidae), blackflies (Simuliidae) and a few midges
(Ceratopogonidae, in particular members of the genus
Culzcozdes). In several instances, females can produce one
batch of eggs without a blood meal although blood is neces-
sary for the development of a second set. T h e ‘advanced’
group of diptera contains some members which feed on
blood. These include the tsetse fly in which both males and
females feed on vertebrate blood. Here blood is not essen-
tial for egg development and these files are not included in
this review.
The female mosquito lays her eggs singly (e.g. Aedes and
Anopheles) or in batches or rafts (e.g. Culex) either in moist
areas where they may be flooded at a later date (e.g. A d & )
or on the surface of water (Culex, .4nopheles). Larvae hatch
from the eggs under suitable conditions and feed on minute
particulate food such as algae and detritus. Larvae proceed
through four growth stages (instars) before pupating. The
adult mosquito generally emerges within 1-3 days, the
body hardens, the wings expand and quickly the insect is
able to fly and mate (Pratt 1959).
Female blackflies lay their eggs on partly submerged
stones in running water. In warm weather eggs hatch in
5-7 days and the larvae fasten themselves to stones with a
piece of salivary silk. Blackfly larvae are filter feeders and
pass through six or seven instars in 7-10 days before
pupation. Adults emerge 2-10 days later (Jamnbeck 1973).
Eggs of Culzrozdes species are often laid during summer
in batches of 50 or more in a wide range of aquatic sites
including bogs and peat land, tidal marshes and decaying
vegetation (Mullen & Hribar 1988). After about 1&14 days
larvae hatch and proceed through four or five instars, often
over-wintering as late instar larvae before emerging the fol-
lowing spring. As larvae, the insects burrow through the
top 5 cm of the soil-eating protozoa, algae, fungal mycelia
and probably bacteria. During this time the larvae increase
in length about six-fold before pupating close to the surface
of the soil. Adults emerge in spring and females mate with
swarms of males before depositing eggs.
Because of the nature of the pathogenic bacteria, bio-
logical control is directed at the larval stages of these insects
and the diversity of habitats and feeding habits is an impor-
tant consideration in the formulation and application of the
control agent.
3. C H E M I C A L YS B I O L O G I C A L CONTROL
DDT, the first organochlorine insecticide, was such an
effective pest control agent that it revolutionized both agri-
culture and public health. DDT provided excellent, wide-
ranging control at low cost but persistence in the
environment together with accumulation in animal fats led
to it being banned. Organophosphates (parathion, mala-
thion, etc.) are widely used in mosquito and blackfly
control programmes and have proved useful for midges
(Kline et al. 1985). Despite their high toxicity to humans
they are readily degraded to harmless products in the

environment. Carbamates are particularly useful against
adult mosquitoes and synthetic pyrethroids are effective
against both larvae and adult mosquitoes. T h e main defi-
ciency of these compounds is insect resistance which is
appearing at such an alarming rate that many chemicals are
now useless (Table 1).
It was once thought that microbial control agents might
be immune from the problem of resistance amongst target
populations but this is now known to be incorrect and
resistance to Bacillus thurtngtensts (B.t.) crystal toxin has
been observed in several lepidoptera (McGaughey 1985;
McGaughey & Beeman 1988; Stone et al., 1989; Tabashnik
et al. 1990) and diptera (Goldman et al. 1986). Moreover, a
cell line of Culex qutnquefasctatus quickly became resistant
to added toxin from B . sphaencus (B.s.) 2362 (Schroeder et
al. 1989). As the application of B.t. products increases, it
seems likely that resistance will become more prevalent but
at present it is fortunately rare. Other benefits of bacterial
control agents have been outlined by MacDonald (1989)
and include: (i) low estimated research and development
costs-about 8 % of an equivalent chemical product
(calculated at $20 million); (ii) requirement for less inten-
sive toxicological testing; (iii) more selective killing of
target insects ; (iv) low operator risk from toxic chemicals;
(v) low environmental impact. On the negative side, the
market for biological control agents is small, the speed and
extent of kill of the target insect is often poor and the dura-
tion of control can be limited. Nevertheless, in the USA
mosquito and blackfly control represents about $4 million
in a $14 million dollar annual market for B.t. products
(Lethbridge 1989).
T h e special situation of vector control in tropical coun-
tries encourages biological control. It is hoped that inte-
grated pest management combining chemicals with locally
produced biological pesticides together with medical treat-
ment will give widespread relief from some of the more
serious diseases listed in Table 1. Indeed, such a pro-
gramme, which has been spectacularly successful in
reducing river blindness in West Africa (Walsh 1986),
would have been impossible without B . thurtngtensts subsp.
tsraelensts (B.t.i.) when Stmultum damnosum (the vector
blackfly) became resistant to first temephos (an
organophosphate) and then chlorphoxim. In the next sec-
tions I shall review briefly the two major bacteria we have
in our armoury against these flies. Both are Gram-positive,
aerobic, endospore-forming bacteria of the genus Bactllus.
B.t.i. has been used for at least a decade in many countries;
its success in controlling mosquitoes in Europe and Asia is
well established. T h e second bacterium, B . sphaertcus, has
recently been registered for use in the USA although exten-
sive trials during the 1980s conducted by W H O have indi-
cated that it has csnsiderable potential as a mosquitocidal
control agent.
B I O L O G I C A L CONTROL OF FLIES 359
4. BACILLUS THURINGIENSIS
Bacillus thuringiensis is best known as a pathogen of lepi-
dopterous larvae. First isolated at the beginning of this
century from diseased silkworms in Japan, and shortly
thereafter from larvae of the Mediterranean flour moth in
Germany (for historical review see Rogoff 1966), com-
mercial production of pesticides based on B.t. began in the
USA in 1958. Today, B.t. products are the most widely
used biological agents for the control of lepidopteran pests
on various food crops and forest trees (Rowe & Margaritis
1987; Jutsum 1988). More recently, strains of B.t. with tox-
icity towards Diptera (mosquitoes and blackflies; Goldberg
& Margalit 1977; Margarlit & Dean 1985) and Coleoptera
(various beetles; Krieg et al. 1983; Herrnstadt et al. 1986)
have been isolated thus widening the scope of biological
control with this bacterium. Moreover, reports of strains
containing unusual crystal proteins of unknown toxicity
(Ohba et al. 1987; Rodriguez-Padella et al. 1990) suggest
that toxins of other insects remain to be characterized.
4.1 Taxonomy of Bacillus thuringiensis
Since the systematics of B.t. is surrounded by much confu-
sion a brief overview will be useful, especially for compari-
son with B . sphaericus (see below). B.t. is a large,
rod-shaped bacterium that under appropriate conditions
differentiates into an ellipsoidal spore. It is facultatively
anaerobic but is fairly restricted in the range of sugars cata-
bolized (Priest et al. 1988). B.t. is closely related to B .
anthracis and B . cereus to the extent that in the absence of
pathogenicity they would be considered a single species.
Total DNA sequence homology between strains of these
three species is high, generally greater than 50% (reviewed
by Priest 1981), with no consistent grouping of the taxa.
Other approaches such as enzyme electrophoresis (Zahner
et al. 1989) and numerical phenetics (Priest et al. 1988)
have failed to provide good evidence for separation of these
taxa although pyrolysis G C showed some potential
(O’Donnell et al. 1980). Nevertheless, the pathogenic
properties of B . anthracis and B.t. are sufficient reason to
maintain species status and to distinguish them from B .
cereus (Claus & Berkeley 1986).
B.t. has been divided into at least 34 serotypes or sero-
vars on the basis of flagellar (H) antigens (de Barjac &
Frachon 1990). These serotypes form the basis of the vari-
etal or subspecies names given to B.t. Although this system
has provided an invaluable classification for ordering the
ever-increasing number of B.t. strain, it has little predictive
value with regard to toxicity. Since the majority of crystal
protein genes are plasmid encoded, readily transmissible
and often associated with transposon-like elements
(Aronson et al. 1986; Jarrett & Stephenson 1990), lack of
360 F . G . PR I ES T
correlation between the serotype and toxin type is perhaps
not so surprising and indicates that a more useful classi-
fication might be based on toxin gene structures (Hofte &
Whiteley 1989) or pathogenicity profiles (Knowles & Ellar
1988).
Nevertheless, mosquitocidal strains of B.t. are commonly
associated with serotype 14 (subsp. isruelensis (B.t.i.); de
Barjac 1978) and more rarely with serotype 8 (subsp. morri-
soni; Padua et al. 1984; Ibarra & Frederici 1986). For
example, since the original isolation of B.t.i. by Goldberg &
Margalit (1977), mosquitocidal strains identified as serotype
14 have been isolated from Egypt (Abdel Hameed et al.
1990a), India (Balaraman et al. 1981), China (Zhang et al.
1984) and Israel (Brownbridge & Margalit 1986). On the
other hand, serotype 14 strains isolated from Japanese soil
proved to be non-toxic (Ohba et al. 1988) while the mos-
quitocidal strain PG-14 (serotype 8a, 8b) was isolated from
this country (Padua et al. 1984). It may be that the preva-
lence of mosquito toxicity in serotype 14 indicates that this
variety may be closely associated with dipteran breeding
grounds. Similarly, Yorris (1969) noted an association of
serotype 4b with storage products and Ohba & Aizawa
(1989) concluded from a survey of serotype 3 strains that
selection of particular groups of B.t. may occur in particu-
lar environments related to insect distribution.
4.2 Plas.mids and toxin genes of B. fhuringiensis
subsp. lsraelensis
Genes specifying crystal protein synthesis in B.t.i. are
located on plasmids. Himeno et al. (1985) screened numer-
ous strains of B.t.i. and showed that toxicity was inyariably
associated with the presence of a large (ca 70 mD) plasmid
although numerous smaller and one larger plasmid were
present in both crystalliferous and acrystalliferous B.t.i.
strains. Toxicity was also attributed to a 72 m D plasmid by
extensive curing studies (Ward & Ellar 1983) and conjuga-
tion experiments (Gonzalez & Carlton 1984) in which it
was shown that transmission of a 72 m D plasmid from
B.t.i. into a cured, acrystalliferous strain by a natural,
conjugation-like process was accompanied by delta-
endotoxin synthesis.
Cloning studies confirmed not only the location of the
B.t.i. toxin genes but also the complexity of the crystal.
T h e common lepidopteran-specific crystals encoded by cryI
genes (see Hofte & Whitely 1989 for details of gene
classification) comprise accumulations of a single or several
proteins of 130-140 kD in a bipyrimidal inclusion. The
cryll genes encode 65 kD proteins which form cuboidal
inclusions toxic to both Lepidoptera and Diptera. cryIII
genes are responsible for 72 kD proteins assembled in a
rhomboidal crystal that is pathogenic towards Coleoptera.
T h e crystal of B.t.i., however, comprises several proteins of
135, 128, 78 and 72 kD as predicted from the four cryIV
genes, cryIV,4, B, C and D. Together with the 27 kD cyt.4
product, these proteins form an ovoid crystal complex in
sporulating cells. All five genes reside on the 72 m D
plasmid of B.t.i. (reviewed by Hofte & Whitely 1989). In
brief, the large crtIVA and B genes resemble the lepidop-
teran cryl genes. T h e C-terminal portions of these proteins
are almost identical. T h e N-terminal halves show more
variation but retain five conserved domains common to all
cryI, cryIII and cryIVA, B, and C specified proteins. This
suggests that the toxic portions of these proteins lay in the
variable N-terminal regions, a prediction that has been con-
firmed by deletion analysis (Chunjatupornchai et al. 1988;
Delicluse et al. 1988). The cryIVA and B proteins are con-
verted by proteolytic enzymes in the larval gut to toxic
fragments of 53 kD (Chilcott & Ellar 1988) or 68-78 kD
(Chunjatupornchai et al. 1988) which are selectively toxic
towards Anopheles and Culex mosquito cells. The cryIVC
gene appears to be a disrupted version of a full 130 kD
toxin-specifying gene (Thorne et al. 1986) and the 78 kD
product is a minor constituent of the crystal. The cryZVD
gene encodes a 72 kD protein which is referred to com-
monly in the literature as the 65 kD protein. It is proteo-
lytically converted to toxins of 3&35 kD which are active
against a variety of dipteran cell lines (Chilcott & Ellar
1988). Finally, the cytA gene is totally unlike the other
genes and its product is cytolytic towards various vertebrate
and invertebrate cells. T h e 27 kD product is the most
abundant protein in the crystal. However, the processed 25
kD form has lower mosquitocidal activity than would be
expected from a primary toxin.
There has been considerable controversy over the tox-
icity and roles of the individual components of the crystal.
As noted above, the four major proteins are mosquitocidal
but none is as toxic as the complete parasporal body. Orig-
inally the problem was the preparation of pure crystal com-
ponents for toxicity testing but cloned gene products have
enabled individual proteins to be prepared and all are less
toxic than the complete crystal (reviewed by Frederici et al.
1990). This led to the suggestion that synergism between
crystal proteins was responsible for high toxicity, particu-
larly between the 27 kD protein and each of the other
major parasporal body proteins. For example, high activity
against Ae. aegypti was achieved with a 0.75/1.0 (w/w) ratio
of the 27 and 65 kD proteins (Chilcott & Ellar 1988).
However, the role of the 27 kD protein in toxicity has
recently been questioned by the preparation of strains
deleted for the cytA gene. The toxicity of the crystals from
these strains did not differ significantly from wild type
crystals when tested on .4e. aegypti and C. pipiens larvae
(Delecluse et al. 1991). T h e role of the cytA protein, the
major component of the crystal, therefore remains open to

debate although it may be involved in toxicity towards
blackflies.
4.3 Mechanism of action of B.t.i. toxins
Larvae of mosquitoes and blackflies feed on small particu-
late matter in their breeding grounds. Upon ingestion, the
crystal protein dissolves at the high p H of the insect gut,
proteolytic action releases toxic fragments, the epithelial
cells of the gut swell and lyse and death rapidly ensues. At
the molecular level, the model for toxicity proposed by
Knowles & Ellar (1987) has a large amount of experimental
support. Processed toxin binds to a specific receptor
(probably a glycoprotein) on the plasma membranes of sus-
ceptible cells in the midgut epithelium. This initial binding
could account for the specificity of the toxin and it follows
that resistance to the toxin would result from structural
changes in the receptor. This has in fact been observed in
laboratory-selected, resistant cells of Plodia interpunctella
(Indian mealmoth) (Van Rie et al. 1990). Initial binding is
followed by the creation of small pores in the membrane
leading to colloid-osmotic lysis in which equilibration of
ions across the pores leads to a net influx of ions, an accom-
panying influx of water, cell swelling and lysis (Knowles et
al. 1989). Disruption of the epithelial lining kills the larvae
rapidly. This model is based on studies with the processed
cytA protein but the same mechanism of action can account
for thc toxicity of other B.t. toxins (Chilcott et al. 1990).
4.4 Fermentation
B.t., like B . cereus, is a facultative anaerobe which can also
use nitrate as an electron acceptor under anaerobic condi-
tions. Carbohydrates are catabolized predominantly
through the E M P pathway and TCA cycle to generate
energy (reviewed by Rowe & Margaritis 1987). Most strains
grow optimally at about 30°C.
It seems that toxin biosynthesis might be subject to cata-
bolite repression, either directly or indirectly in association
with sporulation. Thus Pearson & Ward (1988) noted
slightly higher toxin biosynthesis in media based on starch
compared with mollasses. Moreover, replacement of
glucose by glycerol consistently results in a higher yield of
crystal protein (Smith 1982; Faloci et al. 1990). Various
complex nitrogen sources are used in B.t.i. fermentations
but it may be advisable to supplement these with ammon-
ium sulphate (Avignone-Rossa et al. 1990). Oxygen has a
profound effect on spore yield and entomotoxicity and a
combination of vigorous aeration and strong buffering was
found to be very effective by Foda et al. (1985). Fermenta-
tion conditions for B.t. have been reviewed in detail by
Rowe & Margaritis (1987) and Priest & Sharp (1989).
Most effort in B,t.i. fermentation is currently aimed at
formulating effective production media for use in
BIOLOGICAL CONTROL OF FLIES 361
developing countries. Ideally, these media should use local
agricultural products to keep costs minimal. Starchy
materials are generally not a problem but nitrogen sources
are often scarce or expensive. Thus the use of various legu-
minous seeds, fermented casava and maize was evaluated
for B.t.i. production in Africa (Obeta & Okafor 1984;
Ejiofor & Okafor 1989) and Abdel Hameed et al. (1990b)
have reported the formulation of an effective medium based
on molasses and soya for use in Egypt. The versatile
metabolism of B.t.i. should be beneficial in allowing the
exploitation of this valuable insecticide in many developing
countries.
5 . BACILLUS S P H A E R I C U S
Isolation of mosquito pathogenic strains of B. sphaericus
(B.s.) predated the isolation of B.t.i. by some 10 years
(Kellen et al. 1965) but these early strains showed low tox-
icity. Following an intensive isolation and screening pro-
gramme organized by WHO, more highly toxic strains were
recovered and these, together with several recently isolated
strains, have considerable potential as control agents
(reviewed by Singer 1988). In contrast to B.t.i., B.s. strains
are non-toxic towards Simulium larvae but are toxic
towards many mosquito larvae, in particular, those of the
genus Culex. Toxicity against Anopheles, Mansonia and Pso-
rophora is variable depending on species and against Aedes
larvae is generally very low. Important attributes of B.s.
seem to be its persistence in the environment following
application and activity in heavily polluted areas (see below)
which have promoted its use as a biocontrol agent.
5.1 Taxonomy of Bacillus sphaericus
Virtually all mesophilic, aerobic rod-shaped bacteria that
differentiate into spherical endospores are placed in the
species B. sphaericus. These strictly aerobic bacteria prefer
organic acids to sugars as soures of carbon and energy and
indeed, most carbohydrates are not metabolized by these
organisms. Thus, the usual taxonomic tests, which rely
heavily on acid production from sugars, are of no value in
characterizing and classifying this group (de Barjac et al.
1980; Baumann et al. 1984). The groundwork of B.s.
systematics was established by Krych et al. (1980) who allo-
cated 62 strains to five homology groups using DNA reass-
ociation. Inter-group sequence homology was sufficiently
low to equate each homology group with species status
although this has not been formalized. Homology group I
represented B.s. sensu stricto. T h e insect pathogens were all
allocated to group 111which was divided into group IIA,
which contained strains pathogenic to mosquitoes, and
group IIB which comprised non-toxic strains. Sequence
homology between these subgroups was consistently inter-
362 F . G . PRIEST
mediate (6&70°/0) but sufficiently low to justify the sub-
groupings. We have recently substantiated the distinction
of groups IIA and IIB using D N A homology (Geurineau et
al. 1991) and numerical phenetic analysis (Alexander &
Priest 1990). Given the D N A homology data, the most
appropriate classification would be to consider these two
groups as subspecies (Johnson 1989). Moreover, we have
separated by numerical phenetics all five DNA homology
groups, supporting the view that each represents a separate
species (Alexander & Priest 1990).
The mosquito pathogens in group IIA may be divided
into high toxicity and low toxicity types. Only the former
make parasporal crystals responsible for acute larval toxicity
and contain toxin genes responsible for the crystal proteins
(Geurineau et al. 1991). T h e strains of groups IIA have
been typed using phage (Yousten 1984), antisera (de Barjac
et al. 1985) and, in a preliminary study, DNA finger-
printing (Abadjieva et al. 1990). Highly toxic strains have
been assigned to phage types 2 (strain 1593), 3 (strain 2362)
and 4 (strain 2297) which correspond to serotypes 6, 5a, 5b
and 25 respectively. Of these, phage type 3 (serotype 5a,
5b) contains most of the highly toxic strains. One highly
toxic strain from India was not lysed by the phage and was
allocated to serotype 45 (Manonmani et al. 1990). Despite
variations in phage- and serotype, all these strains have the
same spectrum of activity with high toxicity towards Culex
and low toxicity towards Aedes larvae.
5.2 Piasmids and toxin genes of Baciiius sphaericus
There are conflicting reports concerning both the number
and sizes of plasmids in B.s. strains (reviewed by Singer
1988). Both high and low toxicity strains of group IIA as
well as members of other homology groups contain plas-
mids but they are probably not involved in crystal forma-
tion. The toxin genes are located on the chromosome in
strains 2362, 2297 and BSE 18 (unpublished).
Toxin genes were initially cloned and sequenced from
strains 1593 (Hindley & Berry 1987) and 2362 (Baumann et
al. 1987). T h e sequences now available (Berry et al. 1989;
Berry unpublished), including strain IAB59 (serotype 6),
strains 1593, 2362, 2317.3 and BSE18 (serotype 5a, Sb),
and strain 2297 (serotype 25), show remarkable conserva-
tion and the genes from the four serotype 5a, 5b strains are
identical despite originating from geographically wide-
spread locations. This contrasts the families of toxin genes
found in B.t, but, as more strains are isolated, variation in
toxin specificity and structure may be discovered.
T h e crystal comprises proteins of 51.4 (51) and 41.9 (42)
kD which are transcribed from adjacent genes. T h e 42 kD
protein is toxic for larvae of Culex pipiens (Baumann et al.
1987; Sgarella & Szulmajster 1987) but the 51 kD protein
shows no toxicity. Interestingly, when the cloned gene for
the 42 kD protein is expressed in the absence of the 51 kD
protein, toxicity is lost (Baumann et al. 1987; de la Torre et
al. 1989). Mixing of the two proteins recovers the activity
of the 42 kD protein. Approximately equimolar amounts of
the two proteins are required for maximal toxicity, perhaps
in the form of a binary toxin (Broadwell et al. 1990b).
Upon ingestion of the crystal protein, larval gut pro-
teases, which resemble chymotrypsin and trypsin slowly
degrade the 42 kD protein to a toxic product of 39 kD by
removal of short peptides at the N- and C-termini
(Broadwell & Baumann 1987; Aly et al. 1989). The 51 kD
protein is also processed in the larval gut to a product of
about 43 kD (Aly et al. 1989; Broadwell et al. 1990a). Both
processed forms are required for toxicity to mosquito
larvae.
The gene rntx encoding the toxin from a low toxicity
strain (SSII-1) has recently been cloned and sequenced.
The 100 kD protein showed regional homology to ADP-
ribosyltransferase-type toxins such as those of Vibrio chol-
erae and enterotoxigenic Escherichia colt’ which transfer
ADP-ribose from NAD to regulatory G proteins with the
resulting activation of adenyl cyclase. The cloned gene
product was active against C. guinquefasciatus and ‘4e.
aegypti. All highly toxic and most low toxicity strains
(except those of serotype H26a, 26b) contained the rntx
gene (Thanabalu et al. 1991).
5.3 Mechanism of action of Bacillus sphaericus toxin
Bacillus sphaericus toxin has high activity towards larvae of
Culex and Psorophora, variable toxicity to ‘4nopheles
depending on the species and is inactive on Aedes larvae
(Ramoska et al. 1977). Aedes larvae, like those of Culex,
ingest the toxin and process the 42 kD protein to the
smaller form. However, fluorescent-labelled toxin binds to
midgut epithelial cells of Culex larvae with high specificity,
poorly to ‘4nopheles cells and not to those of Ae. aegypti or
A e . triseriatus (Davidson 1988, 1989). Interestingly, cultured
cells of C. quinquefasciatus induced to toxin resistance by
exposure to the toxin, bound and internalized labelled toxin
(Schroeder et al. 1989). Pathological changes in the larvae
following ingestion of toxin largely involve the midgut cells.
Large vacuoles or cytolysosomes appear in the posterior
midgut cells accompanying general swelling of the gut.
Eventually these cells separate from one another and slough
from the basement membrane (Charles 1987). In cultured
cells, there was very rapid swelling of the mitochondria1
christae and endoplasmic reticulum within 5 min of treat-
ment (Davidson & Titus 1987). T h e detailed mode of
action of the B . sphaericus toxin(s) is unknown.
5.4 Fermentation
Bacillus sphaericus does not use carbohydrates for growth
and lacks many of the enzymes of sugar metabolism

(Russell et al. 1989). Instead, B.s. grows best with organic
acids such as acetate, succinate, arginine and glutamate as
sources of carbon and energy (White & Lotay, 1980; Car-
boulec & Priest 1989) although gluconate and glycerol can
be used as sole carbon source (Russell et al. 1989). This
feature of B.s. physiology restricts the use of agricultural
products in fermentation media to those rich in protein/
amino acids and prevents the use of surplus, agricultural
starchy materials. Nevertheless, effective media based on
byproducts from a monosodium glutamate factory
(Dharmsthitji et al. 1985) and fermented cowpea (Ejiofor &
Okafor 1988) have shown that local production from inex-
pensive ingredients is possible.
Bacillus sphaericus is an obligate aerobe and an adequate
air supply is needed for growth, initiation of sporulation
and toxin synthesis (Yousten & Wallis 1987). Growth of
B.s. normally causes the p H to rise from neutrality to about
pH 8.5 in stationary phase, controlling the p H near neutral-
ity is detrimental to toxin synthesis in strain 2362 (Yousten
& Wallis 1987) but for some unknown reason beneficial for
strain 1593 (Yousten et al. 1984).
6. FORMULATIONS AND APPLICATIONS
The preparation of an insecticide for a particular applica-
tion method is called a formulation. I n the case of Bacillus
insecticides, the formulation of spore/toxin material and
carrier is devised to present a suitable amount of the crystal
protein to larvae in an acceptable form for ingestion. Other
considerations are ease of handling (mixing, compatibility
with application equipment), stability both during storage
and in the field, and cost (see Lacey 1984 for details). T h e
different habitats and feeding habits of mosquito and black-
fly larvae have resulted in the development of various for-
mulations of B.t.i. and B.s. Wettable powders (which give
an even aqueous suspension) and liquid flowable concen-
trates (both aqueous and oil based) are generally used in
conventional aerial and ground sprays to unobstructed
breeding sites. Granules (size range 0.25-1.0 mm) using
plant, such as corn (maize) grits or clay carriers are particu-
larly useful in aerial application to breeding sites with dense
foliage such as salt marshes or rice fields (Lacey & Inman
1985). Sustained release formulations such as floating bri-
quettes or semi submersible pellets are designed to provide
long-lasting larvicidal activity in containers or small ponds
(Lacey 1984; Lacey et al. 1988).
The feeding habits of mosquito larvae influence formula-
tion design. Larvae of Culex and Aedes species are filter
feeders. Ingestion of the toxin depends on the rate of
feeding, the rate at which the toxin falls to the bottom of
the pool and becomes inaccessible, and competition to
ingestion from other suspended organic materials. Thus in
B I O L O G I C A L C O N T R O L OF FLIES 363
turbid and polluted waters the rate of application of both
B.t.i. or B.s. needs to be at least two-fold greater than in
clear water (reviewed by Mulla 1985). .4nopheles larvae, on
the other hand, are surface feeders and ingest particulate
material from the water surface such as yeast or flour and
filter feed poorly. This may explain at least in part the vari-
able results from field trails of B.s. against anopheline
larvae. This has led to the development of formulations
that present the toxin at, or just below, the water surface
and such B.t.i. preparations are particularly effective
against certain Anopheles larvae (Cheung & Hammock
1985; Aly et al. 1987).
One of the major drawbacks in the use of B.t.i. is its
rapid inactivation (24-48 h) in the environment. Thus
larval populations of stagnant water mosquitoes recover
within 5-7 days following treatment. Since there is little
persistence of the toxin and the bacterium does not recycle,
further applications are necessary to effect continuous
control (the more recent sustained release formulations help
in this matter). T h e principal reason for the rapid disap-
pearance of the larvicidal effects of B.t.i. seems to be adsorb-
tion of toxin and bacteria to soil/mud particles (Ramoska et
al. 1982; Van Essen & Hembree 1982). Under simulated
field conditions, B.t.i. was unable to germinate and multi-
ply in mud at the bottom of pools although it did remain
viable for up to 22 days (Ohana et al. 1987).
Bacillus sphaericus toxicity is more persistent than B.t.i.
In field trails of strains 1593 and 2362 against Culex tarsalis
and C. peus initial control was excellent (2 days post-
treatment) and remained substantial up to 21 days post-
treatment, 2362 being the superior strain (Mulla 1985).
However, on drying and reflooding the ponds, activity was
lost, presumably due to inactivation by sunlight.
After application, B.s. spores accumulate at the bottom
of the pool within 24-48 h (Matanmi et al. 1990; Karch et
al. 1990) where, protected from sunlight, they remain
viable for many months (Hertlein et al. 1979). Recycling
may occur through germination and growth of the bacte-
rium in the larval gut followed by formation of new spores
which are released as the larval cadaver disintegrates (Des
Rochers & Garcia 1984; Davidson et al. 1984) but this does
not explain the prolonged presence of B.s. compared with
B.t.i. since the latter is also able to germinate, grow and
sporulate in mosquito larvae (Pantuwatana & Sattabongkot
1990). Recent evidence (Karch et al. 1990) shows that B.s.
is able to grow at the water surface (although other studies
contradict this; Matanmi et al. 1990) and that non-target
insects may play an important role since the bacterium was
able to germinate in the larval guts of Chironomus spp.
(non-biting midges), and other filter feeding arthropods
such as Daphnia (water fleas) are probably responsible for
redistributing spores and bacteria (Karch et al. 1990). Thus
the persistence and recycling of B.s. may be largely influ-
364 F . G . P R I E S T
enced by the activities of other insects in the mosquito
habitat.
An ahernathe possibility is that the crqstal/spore
complex of B.s. is enclosed within an exosporium, whereas
the crystal of I3.t:. is separate from the spore. This could
provide the crystal of B.s. with higher bouyancy and thus
promote the wafting of the toxin into the water column by
passing animals. The toxin of B.t.i. on the other hand
would remain associated with mud particles at the bottom
of the pool. Despite the current lack of understanding in
this area, several studies have shown that the extended
control provided by B.s. offers substantial savings in cost
and labour over repeat applications of chemical larvicides
particularly for the control of Culex larvae (for example see
Jones et al. 1990).
7. FUTURE PROSPECTS
The encouragement to augment or replace chemical insecti-
cides with biological types will come from two directions :
increasing resistance to chemicals amongst target popu-
lations, and the reduced ecological impact of biological
control programmes. However, several development areas
must be addressed before biological insecticides gain wide-
spread acceptance.
Spectrum of activity. T h e toxicity range of B.t.i. is gen-
erally broader than that of B.s. and includes 14edes
species, blackflies and some midges. B.s., on the other
hand, is more active against certain 14nopheles species.
One approach to obtaining strains with different tox-
icity ranges is to intensify screening programmes. This
has resulted in strains with increased toxicity but little
variation in the range of toxicity of both B.s. and B.t.i.
Genetic engineering offers an attractive alternative to
strain improvement and strains of B.s. that contain and
express the B.t.i. 130 kD toxin gene show high toxicity
towards 24e. aegypti (Trisrisook et al. 1990). Similarly,
strains of B.t.i. expressing the B.s. toxin genes have
been constructed but in this case no significant
enhancement of toxicity was observed (Bourgouin et al.
1990). Moreover, improved understanding of the pro-
cessing and mode of action of the toxin genes should
ultimately lead to our ability to design toxins and
strains for specific circumstances in terms of safety,
host range and site of application.
Beyond mosquitoes and blackflies, there may be
important new areas in the control of midges and
houseflies where biological insecticides could replace
chemicals. For example B.t.i. has proved very effective
for the control of Chironomidae (non-biting midges),
Psychodidae (moth flies) and the nuisance fly Syluicola
fenestralis in sewage filter beds in the UK (Coombs et
al. 1091). Flies of agricultural importance such as the
Tipulids also comprise an important application of bac-
terial control.
( 2 ) Persistance and recycling. T h e rapid loss of B.t.i. toxicity
from treated areas is a major drawback. Attempts to
overcome this have involved expressing the B.t.i. toxin
gene in B.s. (Trisrisook et al. 1990) and slow release
formulations. I t is important, however, to keep a
balance between longevity in the environment sufficient
to keep application costs in the same range as chemicals
and yet not sufficiently persistent to accelerate the
selection of resistant insects. It is not clear at present
how quickly the repeated or continual use of a bio-
logical mosquito control agent will lead to resistance
but laboratory and field studies suggest that it could be
far more rapid than first suspected (see Section 3).
Moreover, studies of resistance of C. guinguefasciatus
against organophosphates show that, having developed
in one site, resistant populations quickly become estab-
lished worldwide, presumably in conjunction with air
travel (Raymond et al. 1991). T h e use of integrated
pest management programmes together with other bio-
control agents such as fungi, viruses and larvivorous
fish should help to minimize resistance to B.t.i. and
B.s.
(3) Formulations. Bacterial insecticides are not equally
effective under all environmental situations. New for-
mulations are addressing this problem but environ-
mental sites such as saline habitats will require new
agents and products. Inactivation by sunlight, p H and
chemical composition of the water are additional factors
that must be considered. One of the differences
between bacterial and chemical formulations is that the
former must be presented in an ingestible form. The
ultimate is therefore to include the toxin in the larval
food and to this end the B.s. toxin gene has been cloned
into a cyanobacterium (Tandeau de Marsac et al. 1987)
which is widely distributed in both mosquito and
blackfly larval habitats and is an important food source
for these insects. T h e larvicidal activity of the engi-
neered Anacystis nidulans was inferior to B. sphaericus
but the expression of the toxin genes can presumably
be improved. It is not clear at present if such organisms
will be able to compete and survive in larval breeding
grounds and what effects they will have on mosquito
larvae and non-target insects.
(4) Cost. Finally, implementation of bacterial insecticides
will only occur if the cost of treatment is equal to or
less than the chemical alternatives. There are many
opportunities in the realms of strain improvement and
fermentation development to reduce the cost of pro-
duction of both B.t:i. and B.s. Effective products at a
realistic price will assure a bright future for bacterial
insecticides.

8. ACKNOWLEDGEMENTS
Work in the author's laboratory was supported by a grant
from the German Culture Collection and the ERASMUS
programme of the European Communities.
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