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. 2. 3. 4.

Introduction, 357 The targets, 358 Chemical vs biological control, 358

Bacillus thuringiensis 4.1 T a x o n o m y o f Bacillus thuringiensis, 359 4.2 Plasmids a n d toxin genes of Bacillus thuringiensis, 360 4.3 Mechanism of action of B . thuringiensis subsp. israelensis toxins, 361 4.4 Fermentation, 361 5. Bacillus sphaericus 5.1 T a x o n o m y o f Bacillus sphaericus, 361

1. INTRODUCTION

Blood sucking insects such as mosquitoes and blackflies have plagued man since prehistoric times. They are vectors of a multitude of diseases of man and animals through transmission of pathogenic viruses, bacteria, protozoa and nematodes (although suggestions that mosquitoes might transmit the AIDS virus are fortunately unfounded). Some of the more important of these diseases are listed in Table 1. Historically, malaria has been the greatest single killer of humanity and has an annual incidence of 200-300 million cases. Viral diseases transmitted by various mosquito species include yellow fever, dengue and several forms of encephalitis. Wucheria bancroftti is probably the most widespread of filarial worm parasites of humans. T h e World Health Organization (WHO) estimates that almost 300 million individuals are infected with this nematode, which in severe cases gives rise to gross deformity as elephantiasis. The human filarial parasite, Onrhorerca uolvulus is restricted to parts of Africa and Central America where it is transmitted by black flies of the genus Simultium. This nematode is responsible for river blindness caused by accuCorrespondence to : F.G. Priest, Department of Biolopcal Sciences, Heriot- Watt University, Rirmrton, Edinburgh. EH14 4.4S, UK.

6. 7. 8. 9.

5.2 Plasmids and toxin genes of Bacillus sphaericus, 362 5.3 Mechanism o f action of Bacillus sphaericus toxins, 362 5.4 Fermentation, 362 Formulations a n d applications, 363 F u t u r e prospects, 364 Acknowledgements, 365 References, 365

mulation of dead nematodes in the eye (Walsh 1986). Biting midges (Ceratopogonidae) have also been implicated in the transmission of nematodes and viral pathogens including Bluetongue virus of ruminants in most of the USA (Jones & Foster 1978; Linley et al: 1983). At least 90% of the world’s malaria, yellow fever, dengue and filariasis occurs in the tropics where the environmental conditions favour vector and pest insects responsible for the transmission of the diseases (Rawlins 1989). In temperate climates, haematophagous insects transmit fewer diseases but remain of considerable nuisance value. Bites from mosquitoes, black flies or midges can vary in effect from slight annoyance to severe discomfort and itching. Secondary infection is also possible. Mosquitoes seldom occur in huge numbers but clouds of blackflies or midges can become intolerable and cause severe nuisance and economic problems (Hendry 1989). In a recent survey of Local Health Authorities in the British Isles, 22 reported that they had implemented control measures against mosquitoes in 1985 as a result of public demand (Snow 1987). This problem is therefore not restricted to the warmer climates. In this review I will summarize recent developments in the use of bacteria to control populations of biting flies. Before we consider ways to control these flies, however, it

358 F . G . P R I E S T

Insect vector

Disease organism

Disease

Resistance spectrum

Mosquitoes .4nopheles spp. (about 50)

Protozoa

Malaria

DDT, OPs, Cs

Virus

Dengue

Virus

Yellow fever

DDT, OPs, Cs Pyrethr

.4edes and Culex spp.

Virus

Encephalitides

DDT, OPs, Cs

Culex quinquefusciatus, 24edes, Anopheles and Mansoniu spp.

Nematode

Filariasis including elephantiasis

DDT, OPs, Cs,

Nematode

Onchoserciasis

OPs

Aedes spp.

Blackflies Simulium dumnosum and other spp.

Table 1 Some important vectors of

human diseases and their resistance to chemical insecticides

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 necessary 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 essential 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 following 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, biological control is directed at the larval stages of these insects and the diversity of habitats and feeding habits is an important 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 agriculture and public health. DDT provided excellent, wideranging control at low cost but persistence in the environment together with accumulation in animal fats led to it being banned. Organophosphates (parathion, malathion, 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

B I O L O G I C A L CONTROL OF FLIES 359

environment. Carbamates are particularly useful against adult mosquitoes and synthetic pyrethroids are effective against both larvae and adult mosquitoes. T h e main deficiency 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 intensive 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 duration 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 countries encourages biological control. It is hoped that integrated pest management combining chemicals with locally produced biological pesticides together with medical treatment will give widespread relief from some of the more serious diseases listed in Table 1. Indeed, such a programme, 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 sections 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 extensive trials during the 1980s conducted by W H O have indicated that it has csnsiderable potential as a mosquitocidal control agent.

4. BACILLUS THURINGIENSIS

Bacillus thuringiensis is best known as a pathogen of lepidopterous 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), commercial 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 toxicity 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 confusion a brief overview will be useful, especially for comparison 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 catabolized (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 serovars on the basis of flagellar (H) antigens (de Barjac & Frachon 1990). These serotypes form the basis of the varietal 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 classification 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. morrisoni; 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 mosquitocidal strain PG-14 (serotype 8a, 8b) was isolated from this country (Padua et al. 1984). It may be that the prevalence 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 particular 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 numerous 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 conjugation 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 deltaendotoxin 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 lepidopteran 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 confirmed by deletion analysis (Chunjatupornchai et al. 1988; Delicluse et al. 1988). The cryIVA and B proteins are converted 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 commonly in the literature as the 65 kD protein. It is proteolytically 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 toxicity 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. Originally the problem was the preparation of pure crystal components 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, particularly 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

BIOLOGICAL CONTROL OF FLIES 361

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 particulate 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 susceptible 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 accompanying 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 conditions. 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 catabolite 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 ammonium 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). Fermentation 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

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 leguminous 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 toxicity. Following an intensive isolation and screening programme 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 Psorophora 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 allocated 62 strains to five homology groups using DNA reassociation. 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 subgroupings. 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 fingerprinting (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 plasmids but they are probably not involved in crystal formation. 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 conservation and the genes from the four serotype 5a, 5b strains are identical despite originating from geographically widespread 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 proteases, 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 ADPribosyltransferase-type toxins such as those of Vibrio cholerae 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 treatment (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

B I O L O G I C A L C O N T R O L OF FLIES

(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; Carboulec & 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 inexpensive 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 neutrality 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 application 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 blackfly larvae have resulted in the development of various formulations of B.t.i. and B.s. Wettable powders (which give an even aqueous suspension) and liquid flowable concentrates (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 particularly 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 briquettes 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 formulation 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

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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 variable 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 disappearance of the larvicidal effects of B.t.i. seems to be adsorbtion 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 multiply 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 posttreatment) and remained substantial up to 21 days posttreatment, 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 bacterium 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 insecticides with biological types will come from two directions : increasing resistance to chemicals amongst target populations, and the reduced ecological impact of biological control programmes. However, several development areas must be addressed before biological insecticides gain widespread acceptance.

Spectrum of activity. T h e toxicity range of B.t.i. is generally 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 toxicity 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 processing 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 bacterial 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 biological 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 established worldwide, presumably in conjunction with air travel (Raymond et al. 1991). T h e use of integrated pest management programmes together with other biocontrol 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 formulations are addressing this problem but environmental 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 engineered 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 production of both B.t:i. and B.s. Effective products at a realistic price will assure a bright future for bacterial insecticides.

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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. 9. R E F E R E N C E S A B A D J I E V AA, . N . , G R I G O R O V A R,. T . & M I T E V A ,V . I . (1990) DNA fingerprinting of the mosquito pathogen Bacillus sphaericus with M13 DNA as probe. Letters in Applied Microbiology 10, 141-143. A B D E L - H A M B EA D , , C A R L B E R GG, . & E L - T A Y E B O , .M. (1990a) Studies on Bacillus thuringiensis H-14 strains isolated in Egypt-1. Screening for active strains. World Journal of Microbiology and Biotechnology 6, 299-304. ABDEL-HAMEED A, , CARLBERG G,. & E L - T A Y E BO , .M. (1990b) Studies on Bacillus thuringiensis H-14 strains isolated in Egypt-111. Selection of media for &endotoxin production. World Journal of Microbiology and Biotechnology 6, 3 13-217. A L E X A N D E R ,B. & P R I E S T , F . G . (1990) Numerical classification and identification of Bacillus sphaericus including some strains pathogenic for mosquito larvae. Journal of General Microbiology 136, 367-376. ALY, C., MULLAM , . S . & F E D E R I C IB.A. , (1989) Ingestion dissolution and proteolysis of the Bacillus sphaericus toxin by mosquito larvae. Journal of Invertebrate Pathology 53, 12-20. A L Y , C . , M U L L A ,M . S., S C H N E T T EW R ., & Z u , B-Z. (1987) Floating bait formulations increase effectiveness of Bacillus thuringiensis var. israelensis against Anopheles larvae. Journal of the American Mosquito Control .4ssociation 3, 583589. ARONSON, A . I . , B E C K M A NW. , & D U N N P. , (1986) Bacillus thuringiensis and related insect pathogens. Microbiological Reviews 50, 1-24. A V I G N O N E R O S S A , C.A., Y A N T O R N OO , . M . , ARCAS, J . A . & E R T O L AR, . J . (1990) Organic and inorganic nitrogen source ratio effects on Bacillus thuringiensis var. israelensis deltaendotoxin production. World Journal of Microbiology and Bioter hnology 6, 27-3 1. I ,. M . B A L A R A M AK N ., , H O T I , S . L . & M A N O N M A N L (1981) An indigenous virulent strain of Bacillus thuringiensis highly pathogenic and specific to mosquitoes. Current Science 50, 199-200. B A U M A N N P, . , B A U M A N N L., , B O W D I T C H ,R . D . & B R O A D W E LA L ,. H . (1987) Cloning of the gene for the larvicidal toxin of Bacillus sphaericus 2362: evidence for a family of related sequences. Journal of Bacteriology 169, 4061-4067. B A U M A N NL,. , O K A M O T OK, . , U N T E R M A NB,. , L Y N C H , M.J. & BAUMANN P ,. (1984) Phenotypic characterization of Bucillus thuringiensis and Bacillus cereus. Journal of Invertebrate Puthology 44, 329-341. B E R R YC , . , J A C K S O N - J A P P , J . , O E I , C . & H I N D L E YJ, . (1989) Nucleotide sequence of two toxin genes from Bacillus sphaericus IAB59, sequence comparisons between five highly toxigenic strains. Nucleic .4cids Research 18, 75 16. B O U R G O U I N MC,. . DELECLUSE,A., D E L A T O R R E F ., & S Z U L M A J S TJ E . R (1990) , Transfer of the toxin protein genes

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