Mosquitocidal toxins, genes and bacteria: The hit squad

Reviews

Mosquitocidal Toxins, Genes and Bacteria: The Hit

Squad

A.G. Porter Certain entomopathogenic species of bacilli and Clostridium produce one or more toxins that kill mosquito larvae even at concentrations in the picomolar range. Altogether, 19 distinct genes are knozon that encode mosquitocidal toxins, which vary in their potency, species specificity and mode of action. Unlike chemical insecticides, mosquitocidal bacilli used as larvicides are safe for animals and the environment, and do not affect non-pest insects. Mosquitocidal bacteria are effective to varying degrees against Culex, Anopheles and Aedes mosquito larvae, but their rapid sedimentation from the larval feeding zone, UV-light sensitivity and narrow host range have hampered their development. Nezo genetic engineering approaches are being investigated that could overcome these limitations and allow stable expression of broad host range combinations of toxins in UV-resistant, buoyant recombinant bacteria, as discussed here by Alan Porter. Mosquitoes transmit some of the world's most lifethreatening and debilitating parasitic and viral diseases 1-.~,including malaria (Anopheh's), filariasis (Culex, Mansonia and some Anopheles spp) and dengue fever (principally Aedes aGe,ypti). Alarmingly, these diseases are on the rise in many tropical and sub-tropical areas t,,~. Approaches to reducing the incidence of malaria have focused largely on controlling mosquito populations with chemical insecticides2 and by physical barrier methods (impregnated nets), or by using drugs to prevent infection with ma!arial parasites (Plasmodium spp). Limited trials of a candidate malaria vaccine have received much attention, but it may be some time before this type of vaccine is adopted 4. Likewise, various candidate dengue virus vaccines are being developed, but it is not known when an effective vaccine will be availablea,5. Biological control of mosquito larvae with naturally occurring bacteria that synthesize potent mosquitocidal toxins~'-8 has received much less attention, despite the fact that these bacteria have been used safely in the field for many years 2. Commercial control of lepidopteran and coleopteran pests with entornopathogenic strains of Bacillus thuringiensis (Bt) is now well accepted and its usefulness established'L Moreover, control of the aquatic larvae of blackflies (the vector of the filarial parasite Onchocerca volvulus) with B. thuringiensis subsp, israelensis (Bti) in West Africa has been hugely successful, eradicating onchocerciasis from many areas2,"L It is the frightening emergence of drug-resistant malaria parasites and insecticide-resistant mosquitoes 2 and the lack of a malaria vaccine4, together with an appreciation of the tox~.c effects of chemicals to flora and fauna, that has brought the mosquitocidal bacteria firmly back into the spotlight. Alan G. Porter is at the Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore II 9260, Republic of Sip~apore. Tel: +65 772 3761, Fax" +65 779 I 117, e-maih [email protected] Parasitology Today, vol. 12, no. 5, 1996

The potential of naturally occurring and genetically engineered mosquitocidal bacteria for the control of mosquitoes is here evaluated, taking into account the toxicity, mode of action and host range of the protein toxins (the 'warheads'), as well as the properties, suitability and safety of different bacterial hosts as toxin-delivery vehicles (the 'missiles'). Properties of mosquitocidal bacteria The mosquitocidal bacteria (eg. Bacillus sphaericus and Bti) ¢'-s are among the many pathogens and parasites of vector mosquitoes. Certain strains of B. sphaericus and Bt produce mosquitocidal protoxin crystals during sporulation. These crystals are deposited as inclusions alongside the spore and are highly toxic to susceptible species which ingest the spores as food. The protoxins are solubilized in the alkaline pH of the larval midgut, where they are proteolytically activated and bind to specific receptors located on the brush border epithelial cell membranes. The cells are lysed by one of several mechanisms, and the larva stops feeding and dies. Bacillus sphaericus and Bt are among bacteria known to produce mosquitocidal toxins (Table 1), and field trials and laboratory tests of certain high toxicity strains of these bacilli against various species of Culex, Anopheles and Aedes larvae have demonstrated their safety and potential for controlling mosquitoes. Bacillus sphaericus has been used successfully in the control of Culex quinquefasciatus and Culex pipie,s It.t2, whereas Bti, although useful against Culex mosquitoes, is the strain of choice for controlling A. aegyt,ti I~. Both B. sphaericus and Bti show some toxicity to Anopheles, but the effect is rnarginal and has proved variable in the field. In general, operational success with these bacilli in controlling mosquito populations has been confined mainly to temperate regions of the world where these insects are merely a nuisance 7. Toxicity, mode of action and host range of toxins A summary of the known mosquitocidal toxins produced by entomopathogenic bacteria is given in Table 1. In most cases, the toxins are produced during sporulation. The toxins of B. sphaericus (of 100 and 32kDa) are both synthesized during the vegetative phase of growth 14,15. Purification of the natural toxin proteins, together with the gene cloning, expression and purification of recombinant toxins, has built up a picture of the potencies and mosquito host range of the major toxin proteins ot Bti and B. sphaericus 0,7. The potency of the toxins is evaluated in live bacteria by deriving the lethal concentration of cells that, theoretically, kills 50% of larvae (LCso), or by deriving the LC~ of the purified toxin protein in nanograms per millilitre. Together, these approaches have shown that some of the toxins are effective in the picomolar range against certain species of mosquitoes.

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ReV~ililew s Bti toxins 1~. One of these proteins is a highly rnosquitocidal, 81.29 kDa toxin with sequence homology to Approximate molecular Mosquito host the 72 kDa toxin of Bti ~9. Clostridium mass of protoxin(s) (kDa) range of bacterium ~ Refs Bacterial Strain bifermentans ser. Malaysia is the first !34, 128, 78, 72, 27 Aedes aegypti, Culex 7,8 Bacillus thuringiensis anaerobic bacterium with significant > Anopheles subsp, israelensis (Bti) mosquitocidal activity 2°. During 65 Aedes aegypti 7.8 sporulation, it produces three major Bacillus thuringiensis subsp, kurstakf° proteins, which are probably involved in toxicity, but neither the Anopheles stephensi :> 81, 70-72, 65, 37, 26, 16 Bacillus thuringiensis 19 mode of action of the toxin(s), nor Culex pipiens > subsp,jegathesan whether they function separately or Aedes aegypti in a complex is known 21. 6.7.15 The toxins of Bti, like those of the Culex > Anopheles Bacillus sphaericus 100, 5 I, 42, 32 Bt strains specific for Lepidoptera or > > Aedes ae~pti Coleoptera, appear to kill cells by 20.21 colloid osmotic lysis 2,s,22. Binding of Clostridium 66, 18, 16 Anopheles bifermentans Aedes detritus, a toxin to larval midgut receptors ser. Malaysia Aedes caspius > Culex, leads to pore formation in the cells, Aedes aegypti allowing a rapid inflow of water. Consequently, the cells swell and ~>, order of decreasingsensitivity. bBacillusthuringiensissubsp, kurstaki produces other crystal proteins (not shown). lyse. The 27 kDa toxin of Bti has both larvicidal and haemolytic activity, but it is unclear what role this protein plays in the The isolated crystal proteins of Bti and Bacillus overall toxicity and pathogenicity of Bti 7,s. thuringiensis subsp, kurstaki (Table 1) are toxic to The 32 kDa toxin of B. sphaericus 15shows limited but A. aegypti mosquitoes to varying degrees, and the significant homology to (1) the Clostridium perfringens 72kDa protein of Bti has moderate activity against C. pipiens mosquitoes. In contrast, neither the 134 kDa e-toxin and (2) the Pseudomonas aeruginosa cytotoxin, nor the 78 kDa proteins of Bti are by themselves toxic both of which are specific for mammalian cells, and to C. pipiens 7. Individually, none of the crystal proare believed to form pores on the cell surface. The teins of mosquito larvicidal Bt strains are as toxic as 100kDa toxin of B. sphaericus belongs to a different the spore-crystal complex, suggesting that synergy class of toxin which appears to kill cells by inactivation between toxins (or synergy between toxins and unof certain cellular proteins via ADP-ribosylation 7,14,1~, known s,:~ore components) is an important factor in whereas the mode of action of the binary toxin is untoxicity" known". Thus, the three known toxins of B. sphaericus The toxins of Bti are partially homologous to the may well have distinct modes of action, and it will be 65 kDa toxin of B. thuringiensis subsp, kurstaki ~, but are interesting to determine whether two or more of these distinct from the toxins of B. sphaericus. The three toxins toxins act sy,~erqi.sticaiJ)i., of B. sphaericus (Table l) are also structurally distinct, Mosquito host range of the toxins (Table 1) may be lacking even short regions of homology 7,1'l,l,~. In most determined, at least in part, by the presence of recepstudies, both the 5'1 and 42kDa components of the tors in the insect midgut. The few studies that have binary toxin of B. sphaericus have been found to be been carried out suggest a correlation between the required for toxicity to mosquito larvae, although the presence or distribution of receptors and suscepti42 kDa protein will efficiently destroy some mosquito bility of the species to killing by the toxin 7,2,~-25. Resiscells in tissue culture f,. One report has shown that tant mosquitoes may lack receptors for one or more the cloned 42 kDa protein, by itself, is toxic to larvae toxins7.2.~. following its purification from a normally nontoxic, recombinant Bt strain ~7. However, toxicity is much Drawbacks of mosquitocidal bacteria lower in the absence of the 51kDa componenU 7. It If the mosquitocidal toxins are effective in the picoseems that both the 42 and 51 kDa components can molar range, why have the mosquitocidal bacteria not independently bind larval gut receptors, and both enjoyed widespread commercial use, as have the Bt subunits contribute to toxicity ~,.7. formulations in crop protection'~? The several reasons The 100kDa toxin of B. sphaericus (Table 1) is unmainly reflect cost and the properties of both the stable when produced by the natural strain SSII-1, but bacteria and the toxins: a purified, engineered 97kDa version of this toxin (1) The spores of the bacilli sediment rapidly frorn (lacking some N-terminal sequences) derived from the larval feeding zone, limiting the duration of conrecombinant Escherichia coli is as toxic to Culex mostrol 2". This problem is particularly severe in the case quitoes as the binary toxin 18 (LC~0, ~15 ngml-1). The of Anopheles larvae, which feed at the surface and do 100kDa toxin of B. sphaericus is moderately toxic to not dive 2. A. aegypti (120-290ng m1-1) unlike most of the binary (2) The spore-crystal complex is sensitive to UV toxins, which are only slightly toxic to these mosquilight 27. toes 6. The 32 kDa toxin of B. sphaericus is toxic to Culex (3) The spores probably do not germinate and probut is otherwise poorly characterized15. duce fresh toxin-producing cells outside the proteinBacillus thuringiensis subsp, jegathesan synthesizes rich larval cadaverL at least seven different crystal proteins (Table 1) which (4) The rate of killing with spores is slow compared display little immunological crossreactivity with the with the chemical insecticides. 176 Parasitolo~ Today, vol. 12, no. 5, 1996 Table I. Mosquitocidal bacteria, their toxins and mosquito host range

Reviews (5) In general, Bt spore formulations are -1.5-3 times more expensive to produce than chemical insecticides ~, and many tropical countries do not have the funds to pay for them. (6) The toxins have a narrower mosquito host range than the chemicals (Table 1). Typically, the specific activity of the individual toxins on different mosquito species varies by an order of magnitude or mo~e °,7. Some mosquitoes are resistant to the t o x i n s 7,23'28. Thus, the relatively slow rate of knockdown, coupled with the lack of long-term efficacy, higher cost and narrow host range of the mosquitocidal bacilli are factors which have limited their use. Nevertheless, these bacteria have many positive attributes compared to the chemical insecticides, pa~rticularly in terms of their environmental friendliness and safety 2 (Box 1). But to compete with the chemical insecticides, the drawbacks of the mosquitocidal bacilli m u s t be overcome, and one solution is the development of genetically engineered toxin producing bacteria. Genetically engineered bacteria

The cloning of mosquitocidal toxin genes from Bti and different B. sphaericus strains has allowed the re-expression of a combination of toxins from both species in one recombinant cell in an attempt to broaden the host range of the toxins and obtain beneficial synergistic effects 7.29-31.For example, B. sphaericus shows little or no toxicity to A. aegypti unless toxins of Bti are expressed in addition to the endogenous binary toxin genes 3°m. The converse has been achieved: the expression of B. sphaericus binary toxins in Bti2'L While satisfactory levels of recombinant toxins were expressed, long-term plasmid stability and useful synergistic effects were not adequately demonstrated 2'~-3'. There are several other examples of efficient expression of cloned toxin genes in heterologous bacteria, including Bacillus subtilis, Bacillus megaterium and nontoxic strains of Bt ",7,1". The principle of expanding insect host range with combinations of toxins with different, complementary insecticidal specificities has been established in the case of Bt toxins active against caterpillars and beetles 32-34. A strain of Bt toxic only to Lepidoptera was transformed with a plasmid expressing a Coleopteraspecific toxin gene, which was integrated into the large resident plasmid by homologous recombination without antibiotic resistance markers. The recombinant strain stably expressed high amounts of both Lepidoptera- and Coleoptera-active toxins 32. A likely consequence of expressing combinations of toxins with overlapping specificities is that insect resistance 2~,34,35 to the toxins will be delayed or prevented. Expression of toxin genes located on stable, resiclent plasmids or on the chromosome is preferred, as small plasmids may be structurally a n d / o r segregationally unstable and express an antibiotic resistance gene 7, which is undesirable for environmental release. Homologous recombination can be applied to Bti or B. sphaericus (or other bacteria) to integrate novel combinations of mosquitocidal toxin genes into the bacterial chromosome and obtain their expression during the vegetative or sporulation phase of cell growth. Bacterial spores are used to control mosquito larvae, but spores generally sediment more rapidly from the larval feeding zone than vegetative cells. A novel Pdrdsitology Toddy, vol. 12, no. 5. 1996

Box 1. Attributes of Mosquitocidal Bacteria and Chemical Insecticides Mosquitocidal bacteria a

• Safe for animals and non-pest insects. • Safe to handle. • No long-term persistance (minimizes resistance development). • Resistance does not develop easily. • More selective killing of target insects. • Effective in polluted water (B. sphaericus}. • Stable over a range of temperatures. • Toxins potent in picomolar range. • Less intensive toxicological testing required (cf. chemicals). Chemical insecticides

• • • • •

Rapid killing of insects. Wide range of mosquito species controlled. Chemicals remain in larval feeding zone. Chemicals not rapidly inactivated by UV light. Some chemicals (eg. organophosphates) eventually degrade to harmless products. • Effective under varied environmental conditions.

~YRefs2.7.261. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . approach to controlling crop pests utilizes heat-killed, encapsulated vegetative cells of Pseudomonas fluorescens that are genetically engineered to express high levels of Bt toxins 9. In another technique, a beetle-active toxin was overproduced in vegetative 'ghost' cells of a sporulation-defective mutant of Bt 36. Much effort has been put into evaluating engineered vegetative bacteria such as cyanobacteria, Caulobacter and Ancylobacter aquaticus as delivery vehicles for mosquitocidal toxins 7,37-4°. These bacteria are ubiquitous and exist at or near the water surface. Cyanobacteria and A. aquaticus are buoyant and float quite well, in part because they produce hollow gas vesicles .aT,~. Caulobacters do not produce gas vesicles, but they do inhabit the upper layer of water where mosquito larvae feed 3'~. Being s~arface dwellers, cyanobacteria, ca,.dobacters and A. aquaticus are able to resist rapid inactivation by UV light. These bacteria are non-p.athogenic to animals and grow in simple, inexpenswe media. Moreover, the reproduction of these bacteria occurs naturally in environments that are low in nutrient levels, unlike the spores of bacilli which depend on protein-rich larval cadavers for their germination. Various toxin genes from Bti and B. sphaericus have been transferred into different cyanobacteria, Caulobacter crescentus or A. aquaticus, and the recombinant cells have been shown to be significantly toxic to Culex and Aedes larvae 7,37-4°. Although toxin expression levels remain disappointingly low, the characteristic buoyancy of the cells may prolong their larvicidal action37, 3~. Deliberate release of mosquitocidal bacteria

By 1993, approximately 27 recombinant microorganisms had been approved for release into the environment in several countries 41, but so far no genetically modified mosquitocidal bacteria have been released. Before seeking approval to release engineered mosquitocidal bacteria, a number of issues must be raised and experiments performed in order 177

Rev,ew, s to determine, as far as possible, that the modified organism will only kill mosquito larvae and not disturb the ecosystem 42. Both known and conjectural hazards must be considered 41.42. Natural strains of Bti and B. sphaericus have proved safe for mammals and have been used in biocontrol for over 20 years without any reports of untoward effects2,2". The spores settle and presumably degrade over time. There are no known hazards 2. Conjectural hazards are harder to evaluate. Questions that must be asked are: Will the organism persist or transfer the toxin gene to different species? If it persists and continues to p,,~oduce the toxin(s), will this encourage resistance development in the continuously exposed mosquito population? Does the toxin kill beneficial insects which are predators of mosquitoes? Will the organism form 'blooms' and pollute the aquatic environment? Chromosomal integration will minimize the risk of transfer of toxin genes to heterologous microorganisms, but the risk is still there as no genetic material is considered completely stable. The most likely event is that the toxin gene will not persist as it will be inactivated by mutation, assuming that continuous high-level production of a toxin is a drain on cellular metabolism and does not provide the cell with a selective advantage. This would probably be the case for the normally nontoxic cyanobacteria, C. crescentus and A. aquaticus, which are engineered to produce toxins. There are powerful arguments both for and against the release of genetically manipulated microorganisms 7,41,'12. Protagonists point out that there is an urgent need for a new approach to control vectors of parasitic and viral diseases. Over 400 million people live in highly malarious areas, and malaria kills over one million children annually in Africa alone. Genetic recombination between species is going on all the time in the environment, so why worry if toxin genes are transferred to different species? Unfortunately, there is a lack of understanding of the biological niche and survival of natural mosquitocidal bacteria, and the consequences of release cannot necessarily be extrapolated from laboratory tests. Once released, an organism cannot be withdrawn. The decision whether or not to release a genetically modified organism must be taken on a case-by-case basis and made after very careful consideration. C o n c l u s i o n s and p e r s p e c t i v e s

The number and diversity of mosquitocidal toxins and the bacteria that produce them have increased gradually since their discovery, and this increase is accelerating. Currently, 19 distinct mosquitocidal toxin genes are known, and novel genes will undoubtably be found and cloned. The toxins appear to vary in their species specificity and mode of action, making it likely treat particular combinations cloned in recombinant rr,,roorganisms can be chosen so as to enlarge insect host range and delay or prevent the development of resistance. The potency of mosquitocidal toxins towards susceptible species is generally excellent, but the naturally occurring bacilli have several drawbacks that have hampered their widespread use. The mosquito host range of the toxins is rather narrow and difficult to predict. The spores do not multiply significantly outside the larval cadaver; the spore--crystal 178

complex is sensitive to UV light and the spores settle rapidly, limiting the duration of control. Insecticidal bacteria are costlier to produce than chemical insecticides. However, a combination of novel genetic manipulation approaches (high-level expression of toxin combinations; chromosomal integration of toxin genes), coupled with existing formulation technology or the use of engineered vegetative bacteria which can exist in the upper layer of water, may well overcome these problems. The phasing out of chemical insecticides, together with the emergence of pesticide-resistant mosquitoes and drug-resistant parasites, will act as a strong incentive to develop effective mosquitocidal bacteria. A~ " ; '~.' " edgements ,~,, :~ _ ~,~ankArmelle Del6cluse (Pasteuc Institute) foc urpublished data, M,s Rina Wati, Jian Wei Liu and Thicumacan Thanabalu fo~ comme'~ts on the manuscript and Peady Aw fo~-excellent typing. This wock wa~. funded by the Institute of Moleculac and Cell Biology, National Unl ~ersity of Singapoce. Reference I Miller, L.H. (1¢)92)The challenge of malaria. Science 257, 36-37 2 Priest, F.G. (1992) Biological control of mosquitoes and other biting flies by B a c i l l u s sphaericus and B a c i l l u s thuringiensis.

I. Appl. Bacteriol.72, 357-369 3 Monath, T.P 0994) Dengue: the risk to developed and developing countries. Proc. Natl Acad. Sci. USA 91, 2395-2400 4 Maurice, J. (1995) Malaria vaccine raises a dilemma. Sch'nc,' 267, 320-323 5 Brandt, W.E. (1990) From the World Health Organization. Development of Dengue and Japanese Encephalitis vaccines. I. hq'ect. Dis. 102,577-583 6 Baumann, P. et al. (Igt)l) Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxin. Microbiol.

Roy,55, 425-,130 7 Porter, A.G. et al. (1t;93) Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes. Microbiol. Rev. 57, 838-801 116fte, II. and Whiteley, I I.R. (I98~)) Insecticidal crystal proteins of Bacillus thuringiensis. MicrobiN. Rc~,.53, 2.12--255 ~) Feitelson, ].S. et al. (!~)~)2)Bacilhts thuringiensis: insects and beyond. Biofl'ech,oh~gy 10, 271-275 I(1 Itougard, I-M. and Back,C. (1992)Perspectiveson the bacterial control of insects in Ihe tropics. Parasilofi~gyToday 8, 364-366 l l Yap, H.H. (1990) in Bacterhil Control OffMosquitoes trod Blackflies: Biochemistry, Genetics amt Applia~tious of Bacillus thuringiensis and Bacillussphaericus (de Barjac, H. and Sutlaerland, D, ed% pp 307-320,Rutgers UniversityPress 12 Hougard, J-M. et al. (1993)Campaign against Culex quinquefasciatus using Bacillus sphaericus: results of a pilot project in a large urban area of equatorial Africa. Bull. WHO 71,367-375 13 Mulla, M. (1090) in Bacterhll Cotttrol of Mosquitoes and Bhwkflics: Biochemista3t, Gt'm'tics and Applh'athms of Bacillus thuringiensis

amt Bacillussphaericus (de Barjac, H. and Sutherland, D., eds), pp 134-100,Rutgers UniversityPress 14 Thanabalu,T. et al. (1991)Cloning, sequencing and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1. [. Bacteriol. 173,2775-2785 15 Thanabalu,T. and Porter, A.G. Bacillus sphaericus gene encoding a novel type of mosquitocidal toxin of 31.8 kilodaitons.

Gt'm'.(inpress) io Tabashnik, B.E.0992) Evaluation of synergism among Bacillus thuringiensis toxins. Appl. E~wirou. Microbiol. 58, 3343-3346 17 Nicolas, L. et al. (1993) Respeclive roles of the 42- and 51-kDa components of the BaciUus sphaericus toxin overexpressed in Bacillus thuringiensis. FEMS MicrobhK Lett. 106,275-280 18 Thanabalu, T. et al. (1992) Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1. 1. BacterhK 174, 5051-5056 19 Del~cluse,A. el al. (1995)Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp, jegathesan encoding a highly mosquitocidal protein. Appl. Envi~vn. Microbh~l. 61, 42304235 20 Thiery, I. et al. (1992) Host range of Clostridium bifermentans Parasitology Today, vol, 12, no. 5, 1996

Reviews 21 22 23 24

25 26 27 28

29 30 31

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serovar. Malaysia, a mosquitocidai anaerobic bacterium. [. Am. Mosq. Control Assoc. 8, 272-277 Nicolas, L. et al. (1993) Clostridium bifermentans serovar Malaysia: characterization of putative mosquito larvicidal proteins. FEMS Microbiol. Lett. 113, 23-28 Aronson, A.I. (1993) The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival. Mol. Microbh)l. 7, 489-496 Davidson, E.W. (1989) Variation in binding of Bacillus sphaericus toxin and wheat germ agglutinin to larval midgut cells of six species of mosquitoes. J. hlvertebr. Pathol. 53, 251-259 Oei, C. et al. (1992) Binding of purified Bacillus sph~ericus binary toxin and its deletion derivatives to Cuh, x quh:,qn,,fasciatus gut: elucidation of functional binding domai~as. ]. Gem Miclvbiol. 138, 1515-1526 Haider, M.Z. and Ellar, D.J. (1987) Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal 8-endotoxin. Bh)chem. J. 248,197-201 Berry, C. et al. (1991) in Bh)technoh)gy for Bh)h)gical Control of Pests a,d Vectol"s (Maramorosch, K., ed.), pp 35-51, CRC Press Benoit, T.G. et al. (1990) Plasmid-associated sensitivity of Bacillus thuringiensis to UV light. Appl. Environ. Microbiol. 56, 2282-2286 Rao, D.R. et al. (1995) Development of a high level of resistance to Bacillus sphaericus in a field population of Culex quinquefasciatus from Kochi, India. J. Am. Mosq. Control Assoc. 11,1-5 Bourgouin, C. et al. (1990) Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp, israelensis and their expression. Appl. Era,iron. Microbiol. 56, 340-344 Bar, E. et al. (1991) Cloning and expression of Bacillus thuringiensis 8-endotoxin DNA in B. sphaericus. J. hwertebr. Pathol. 57, 149-158 Trisrisook, M. et al. (1990) Molecular cloning of the 130kilodalton mosquitocidal 8-endotoxin gene of Bacillus thuringiensis subsp, israelensis in Bacillus sphaericus. Appl. Environ. M icrobh)l. 56, 1710-1716 Lereclus, D. et al. (1992) Expansion of insecticidal host range of Bacillus thuringiensis by in vivo genetic recombination. Bh)/T('chnoh)gy 10, 418-421

33 l-ion&,,G. ('tal. (1990) A translation fusion product of two dif-

34 35 36 37 38

39

40 41 42 4~

ferent insecticidal crystal protein genes of Bacillus thuringiensis exhibits an enlarged insecticidal spectrum. Appl. Environ. Microbiol. 56, 823-825 Bosch, D. et al. (1994) Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Bio/Technoh)gy 12, 915-918 McGaughey, W.H. and Whakm, M.E. (1992) Managing insect resistance to Bacillus thuringiensis toxins. Sch'nce 258, 1451-1455 Lereclus, D. et al. (1995) Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spoOA mutant. Bio/Technoh)gy 13, 67-71 Yap, W.H. et al. (1994) Expression of mosquitocidal toxin genes in a gas-vaculolated strain of Ancylobacter aquaticus. Appl. Environ. Microbh,L 60, 4199-4202 Murphy, R.C. and Stephens, S.E. (1992) Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp, israelensis in the cyanobacterium Agmeneilum quadruplication PR-6 and its resulting larvicidal activity. Appl. Environ. Microbh)l. 58, 1650-1655 Thanabalu, T. el al. (1992) Expression of the mosquitocidal toxins of Bacilhis sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae. Appl. Environ. Microbh)l. 58, 905-910 Yap, W.H. et al. (1994) Influence of transcriptional and translational control sequences on the expression of foreign genes in Caulobacter crescentus. ]. Bacteriol. 176, 2603-2010 Wilson, M. and Lindow, S.E. (1993) Release of recombinant i~icroorganisms. Atom. Rev. Microbiol. 47, 913-944 Wiliiam ~on, M. (1992) Environmental risks from the release of genetically modified organisms. Mol. Ecol. 1, 3-8 Liu, J-W. (,t al. (1996) Efficient synthesis of mosquitocidal toxins in Asticcacaulis exc,!ntricus demonstrates potential of Gram-negative bacteria in mosquito control. Nat. r;ioteclmol. 14, 345-349

Note added in proof Recently43, the potential of the Gram-negative bacterium, Asticcacaulis excentricus, in mosquito control has been demonstrated.

Malaria and Onchocerciasis: On bILA and Related Matters C.G. Meyer and RG. Kremsner hl recent years, associatiolls of particuhlr factors of the luuna, letda)cyte antigen (ttLA) system with two major infectious diseases (q ta)pical countries have been recognized: coinnton West African HLA antigens ale associated with protecth)n from severe Plasmodium falciparum malaria, and HLA-D alleles are associated with generali~:ed disease, h)calized dL;ease and putative ilnlnunity in Onchocerca volvult~s infection. Here, Christian Meyer and Peter Krentsner slunlnarize current inforlnation on the involvelnellt # HLA hlctors in P. falciparum malaria alld O. volvulus infection, and briefly report Oll HLA-related inlmunoh)gicai characteristics of various couditious in these infectious diseases. Plasmodilml falciparum and Onchocerca voh, ldltS both have a profound impact on the host's immune system and may modify the immune responsiveness I-5. The generation and nature of an adaptive immune reChristian Meyer and Pete)- K1-emsnerare at the Institut for Tropenrnedizin Berlin, Engeldamm 62, 10179 Berlin, Gecmany. Tel" +49 30 2746 317, Fax-" +49 30 2746 736 Parasitology Today, vol. 12, no, 5, 1996

sponse depend initially upon tile genetically controlled recognition of foreign antigenic peptides. This recognition and the discrimination of self and non-self is mediated by human leukocyte antigen (HLA) class I and class II molecules. Unimpaired and accurate recognition is crucial for a regular reaction to foreign antigens. It results in the explicit distinction of antigens requiring a response and those which must be tolerated. HLA class I and I! molecules are encoded by genes located within the major histocompatibility complex (MHC), a complex set of polymorphic genes on the short arm of the human chromosome 6. Common techniques for HLA-typing include traditional serological/cellular assays and recently developed polymerase chain reaction (PCR) based methods. The use of PCR has greatly supported and improw'd the identification of HLA class II specificities and has provided detailed information on many as yet unrecognized allelic variants. The polyrnorphism of MHC genes and their encoded proteins is part of the inherent strategy allowing the presentation of num~:ous different antigens.

Copynoht Q 1996. ElsevleT'Soence Lld All )7~hls rese),~ed 0109 -1758/96 $1b0',)

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