Recombinant bacteria for mosquito control
Department of Entomology, University of California Riverside, Riverside, CA 92521, USA
* Author for correspondence (e-mail: brian.federici{at}ucr.edu)
Accepted 18 July 2003
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Summary |
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Key words: Bacillus thuringiensis subsp. israelensis, Bacillus sphaericus, Cyt protein, Cry protein, B. sphaericus binary toxin, transcript stabilization, chaperone, recombinant bacterial larvicide
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Introduction |
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Since World War II, disease control methods have relied heavily on
broad-spectrum synthetic chemical insecticides to reduce vector populations.
However, synthetic chemical insecticides are being phased out in many
countries due to insecticide resistance in mosquito populations. Furthermore,
many governments restrict chemical insecticide use owing to concerns over
their environmental effects on non-target beneficial insects and especially on
vertebrates through contamination of food and water supplies. As a result, the
World Health Organization
(1999b) is facilitating the
replacement of these chemicals with bacterial insecticides through the
development of standards for their registration and use.
Vector control products based on bacteria are designed to control larvae. The most widely used are VectoBac® and Teknar®, which are based on Bacillus thuringiensis subsp. israelensis (Bti). In addition, VectoLex®, a product based on Bacillus sphaericus (Bs), has come to market recently for the control of mosquito vectors of filariasis and viral diseases. These products have achieved moderate commercial success in developed countries, but their high cost deters use in many developing countries. Moreover, concerns have been raised about their long-term utility due to resistance, which has already been reported to B. sphaericus in field populations of Culex mosquitoes in several different countries.
The insecticidal properties of these bacteria are due primarily to insecticidal proteins produced during sporulation. In Bti, the key proteins are Cyt1A, Cry11A, Cry4A and Cry4B, whereas Bs produces a single binary toxin commonly referred to as Bin. Of particular interest among these proteins is Cyt1A, which synergizes and delays resistance to mosquitocidal Cry proteins and can be used to overcome resistance to Bs, as well as extend its spectrum of activity to, for example, the yellow fever mosquito Aedes aegypti (Fig. 1). In addition to Bti and Bs, mosquitocidal proteins have been identified in other species, such as B. thuringiensis subsp. jegathesan; these also offer promise for use in new types of larvicide.
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Aside from this variety of mosquitocidal proteins, several genetic elements have been identified that, when used in combination with strong Bt promoters, can be used to improve efficacy by increasing endotoxin synthesis significantly. The most important of these are the STAB/SD sequence, a nine-nucleotide polypurine sequence that improves transcript stability and thus endotoxin synthesis, and a 20-kDa protein that occurs naturally in the Cry11A operon. This protein enhances net synthesis of Cry11A and other proteins and apparently acts as a molecular chaperone.
The biochemical and toxicological differences between mosquitocidal Bt and Bs toxins prompted several attempts during the late 1980s and 1990s to construct recombinant bacteria that combined the best properties of these species. However, none of the resultant recombinants had efficacy sufficiently improved over wild-type strains to warrant commercial development. The prospects for developing recombinant bacteria with high efficacy suitable for commercial development have improved recently due to the availability of genetic elements for improving endotoxin synthesis, a greater range of mosquitocidal proteins and the development of a better understanding of the toxicological properties of Cyt1A. In this overview, we first describe the properties of Bti and Bs and summarize previous research on improving bacteria for mosquito control. We then go on to show how new knowledge and technologies have been used to create recombinant bacteria that have much better potential for use in operational mosquito control programs owing to their very high efficacy and built-in resistant management properties based on Cyt1A. The literature on Bt, Bti and Bs is extensive and thus, in this overview, we cite review papers to guide interested readers to the original literature.
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Properties of Bti |
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Despite our uncertainty about Cyt1A's mode of action, it is an extremely
important protein with respect to mosquito control. Numerous studies have
revealed that Bti's high toxicity is due to synergistic interactions among its
Cry proteins and especially between Cyt1A and the Cry proteins
(Wu and Chang, 1985;
Ibarra and Federici, 1986
;
Crickmore et al., 1995
; Wirth
et al.,
2000a
,b
).
Even more importantly, recent studies suggest that Cyt1A can delay resistance
to Cry proteins in mosquitoes (Georghiou
and Wirth, 1997
) and overcome resistance to these if it develops.
For example, resistance levels to Cry11A of >900-fold in laboratory
populations of Culex quinquefasciatus were suppressed completely when
Cry11A was combined with Cyt1A in a 3:1 ratio
(Wirth et al., 1997
). In
addition, Cyt1A can overcome very high levels of resistance to the Bin toxin
of B. sphaericus 2362 when combined with this species
(Wirth et al., 2000a
) and can
extend its target spectrum to A. aegypti
(Wirth et al., 2000b
). Recent
studies using fluorescent dyes have shown that the lack of sensitivity in
B. sphaericus-resistant C. quinquefasciatus is due to the
absence of the Bin toxin receptor in the midgut microvillar membrane
(Darboux et al., 2002
).
Studies in our laboratory have shown that Cyt1A forms lesions in this membrane
that enable the Bin toxin to enter these cells and exert toxicity
(Fig. 3).
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The high efficacy that Bti showed in laboratory and field trials during the
early 1980s led rapidly to its development as a commercial bacterial larvicide
for control of mosquito and blackfly larvae
(Mulla, 1990;
Becker and Margalit, 1993
).
Four commercial products, VectoBac® (Valent Biosciences, Libertyville, IL,
USA), Teknar® (Valent Biosciences, Libertyville, IL, USA), Bactimos®
(Bayer Research, Triangle Park, NC, USA) and Acrobe® (Becker Microbial
Products, Plantation, FL, USA) are used in many countries for the control of
vector and nuisance mosquitoes and blackflies. Teknar® and VectoBac®
proved to be particularly important for the World Health Organization's
Onchocerciasis Control Program in West Africa, where they have been used for
almost two decades to control the blackfly vectors of Onchocerca
volvulus, which causes river blindness in humans
(Becker, 2000
).
Despite its intensive use in numerous mosquito and blackfly ecosystems and
the development of resistance under intensive selection in the laboratory,
resistance to Bti has not been reported in the field
(Becker and Ludwig, 1993).
Laboratory studies suggest that this lack of resistance is due primarily to
the presence of Cyt1A in the parasporal body
(Georghiou and Wirth, 1997
;
Wirth et al., 1997
). Cyt1A's
capacity to synergize endotoxin proteins, including the B. sphaericus
Bin toxin against resistant and non-sensitive mosquitoes
(Wirth et al., 2000a
), and to
delay resistance are important properties for the improvement of mosquito
larvicides.
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Properties of Bs |
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In addition to the binary toxin, many strains of Bs produce other
mosquitocidal toxins during vegetative growth that are referred to as Mtx
toxins. Two of these have been well studied - Mtx (100 kDa) and Mtx2 (30.8
kDa) - but are not as toxic as the Bin toxin
(Delécluse et al.,
2000).
The target spectrum of Bs is more limited than that of Bti, being
restricted to mosquitoes, and its highest activity is against Culex
and certain Anopheles species
(Delécluse et al.,
2000). Some important species of Aedes, such as A.
aegypti, are not very sensitive to Bs, whereas others, for example,
Aedes atropalpus and Aedes nigromaculis, appear to be quite
sensitive (Delécluse et al.,
2000
). Moreover, although strain 2362 was isolated from a blackfly
(Simulium damnosum) adult, Bs strains have little or no activity
against nematoceran flies other than mosquitoes. Nevertheless, Bs does appear
to have better initial and residual activity than Bti against mosquitoes in
polluted waters. As a result, a commercial formulation, VectoLex® (Abbott
Laboratories), based on strain 2362 is marketed in many countries, especially
to control Culex larvae in polluted waters. A disadvantage of Bs
strains is that the Bin toxin is, in essence, a single toxin. Laboratory
studies have shown that it is much more likely to result in resistance than
Bti. In fact, resistance to Bs has already been reported in field populations
of Culex mosquitoes in Brazil, China, France and India
(Sinègre et al., 1994; Rao et al.,
1995
; Silva-Filha et al.,
1995
; Yuan et al.,
2000
), with resistance levels in some areas of China reported as
>20 000-fold.
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Other mosquitocidal bacteria |
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Initial recombinant mosquitocidal bacteria |
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Bti endotoxins in Bs |
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In one of the first sets of Bs/Bti recombinants, a Bti DNA fragment
encoding the Cry11A- and Cyt1A-encoding genes was cloned into pPL603E and
introduced into Bs 2362 by protoplast transformation
(Bar et al., 1991). One
recombinant produced Cyt1A, Cry11A and the Bs Bin toxin and was 10-fold more
toxic to A. aegypti than parental Bs 2362 but was not nearly as toxic
to this species as Bti. Initially, this recombinant appeared to be stable, but
it was eventually found to be unstable (Bar
et al., 1998
).
In two other early recombinants, a plasmid containing cry4B was
transformed into Bs strains 1593 and 2297 by protoplast transformation.
Parental Bs 1593 and Bs 2297 strains had low toxicity to A. aegypti.
However, production of Cry4B in the transformants increased toxicity to this
species 100-fold (Trisrisook et al.,
1990), making Bs transformants as toxic to A. aegypti as
Bti. Against Anopheles dirus and C. quinquefasciatus, the
Cry4B Bs transformants were similar in toxicity to the parental strains, being
slightly more or less toxic depending on the recombinant strain and mosquito
species tested.
In a related study, the cry4B or cry11A genes of Bti were
transferred into Bs 2297 by electroporation using the shuttle vector pMK3
(Poncet et al., 1994). The
parental Bs 2297 strain was non-toxic to A. aegypti, whereas the Bs
Cry4B and Bs Cry11A 2297 transformants were both moderately toxic to this
species but not as toxic as Bti. In this study, it was found that the Cry4B
transformant was approximately 10-fold more toxic to A. aegypti than
the Cry11A transformant, and the authors suggested that the higher toxicity of
the former was due to synergism between the Cry4B and the Bs binary toxin.
A more recent attempt to improve Bs used the transposon Tn917 to insert the
major Bti toxin-encoding genes or fragments thereof into the chromosome of Bs
2362 (Bar et al., 1998). A
series of recombinants was obtained that produced one or more of the Bti
proteins in Bs 2362 along with the Bs binary toxin. As in previous studies,
although not as toxic as Bti, many of the Bs 2362 recombinants obtained in
this study were as much as 10-fold more toxic to A. aegypti than the
parental Bs 2362 strain. However, against C. quinquefasciatus and
A. gambiae, the recombinant toxicity was only in the range of
parental Bs 2362 or Bti. In another study, integrative plasmids were used to
introduce the cry11A gene into Bs 2362, resulting in recombinants
that produced both the Bs binary toxin and Cry11A
(Poncet et al., 1997
). These
recombinants were much more toxic to A. aegypti than parental Bs 2297
and were similar in toxicity to the parental strain against C.
quinquefasciatus. However, one of the Cry11A Bs 2297 recombinants
[2297(::pHT5601)] had toxicity to Anopheles stephensi that was
comparable with that of Bti. In addition, the recombinant
2297(::cry11A) partially suppressed resistance to Bs 2297 in a strain
of C. quinquefasciatus from India resistant to Bs 1593. Similar
results were obtained when either the cry11A or cry11B (from
B. thuringiensis subsp. jegathesan) or both of these genes
were inserted into the chromosome of Bs 2297 using integrative plasmids
(Servant et al., 1999
). The
production of Cry11A and/or Cry11B along with the Bs 2297 binary toxin
increased the toxicity of this strain against A. aegypti, depending
on the specific recombinant, from 5-fold to 11-fold. Against Culex
pipiens, most recombinants were similar in toxicity to parental Bs 2297,
although one (2297pro::cry11Ba) was about twice as toxic.
Recombinants producing Cry11A and/or Cry11B were able to partially suppress
resistance to Bs 2297 in different populations of C. pipiens.
A different type of Bs/Bt recombinant was constructed by using the shuttle
vector pMK3 to insert the cyt1Ab1 gene from B. thuringiensis
subsp. medellin into Bs 2297
(Thiéry et al., 1998).
The production of the Bs 2297 binary toxin together with Cyt1Ab did not
improve toxicity to A. aegypti or C. pipiens. However, the
recombinant strain was able to restore the sensitivity of Bs-resistant
populations of C. pipiens and C. quinquefasciatus by
10-20-fold.
Because they contained broad-spectrum mosquitocidal Cry proteins, the Bs recombinants described above were typically considerably more toxic to A. aegypti than were parental Bs strains. However, none of these recombinants was better than Bti against this species, and only a few were more toxic to Culex and Anopheles species than the parental Bs strains. Nevertheless, these studies were very valuable because they resulted in techniques for constructing recombinants and showed that the various proteins of Bti and other Bt subspecies could be produced in substantial quantities in different strains of Bs. Moreover, they showed that producing Bt Cry and Cyt proteins in Bs extended its target spectrum to A. aegypti and partially suppressed Bs resistance in Culex species. Although not tested under field conditions, based on laboratory studies with Bti, it is probable that Bs strains containing Cry and/or Cyt toxins, even if not as effective as Bti, would be less prone to resistance and therefore useful for Culex control in polluted waters.
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Bs binary toxin in Bti |
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In the first study where Bs toxins were produced in Bti, the binary toxin
of Bs 1593 was cloned into the shuttle vector pBU4, yielding pGSP10
(Bourgouin et al., 1990). This
plasmid was then transformed into the 4QS-72 strain of Bti, a strain that only
contains the large plasmid encoding the Cyt and Cry endotoxins typically found
in this subspecies. Analysis of the recombinant Bti strain showed that it
produced the standard Bti toxins in normal amounts along with the 51.4-kDa and
41.9-kDa peptides of the Bs binary toxin. When tested against A. aegypti,
C. pipiens and A. stephensi, the toxicity of the recombinant was
no better than that of either the parental Bti or Bs strains.
In the above study, Bs promoters were used to express the Bs binary toxin in Bti, and none of the enhancing elements identified after this study was published were present in the plasmid used to produce the Bs binary toxin in Bti. Electron microscopy indicated that only small crystal of the binary toxin was produced in the Bti transformants. This could account for the lack of improved toxicity.
More recently, we have taken a different strategy in which we use Bt
promoters and genetic elements that enhance toxin synthesis in Bt to produce
both Bs and Bt proteins in Bti. Using this strategy, we have achieved
significant improvement in the levels of Bt and Bs endotoxins synthesized in
Bti and improvements in toxicity that were correspondingly higher against
C. quinquefasciatus. Examples of two improved Bti strains will be
used to illustrate this strategy. In the first, we constructed a strain of Bti
that produced a combination of Cyt1A, Cry11B and the Bs Bin toxin, all in
large quantities (Park et al.,
2003). To engineer this strain, we constructed two plasmids, each
of which contained a different selectable marker for expression in Bti
(Fig. 4). The first plasmid
(P45S1) contained as its key elements the Bs Bin toxin operon, under the
control of cyt1A promoters and the STAB/SD sequence, along with the
cyt1A gene and the 20-kDa chaperone-like protein, under the control
of the cry1Ac promoters (Wu and Federici,
1993
,
1995
;
Park et al., 1998
). This
plasmid had, as its selectable marker, erythromycin resistance. The second
plasmid (pPFT11Bs-CRP) contained the cry11B gene under the control of
cyt1A promoters and the STAB/SD sequence. The first plasmid was
transferred into a Bti crystal minus strain (4Q7) by electroporation, and the
transformants were selected on brain heart infusion agar containing
erythromycin. Subsequently, the second plasmid (pPFT11BS-CRP) was transferred
into this transformant by electroporation and was selected for on plates
containing chloramphenicol. Analysis of this double transformant by microscopy
and SDS-PAGE showed that it produced large quantities of all three endotoxins
(Figs 5,
6). Bioassays of this double
transformant against fourth instars of C. quinquefasciatus showed
that it had an LC50 of 1.7 ng ml-1, making it
approximately fourfold as toxic as Bti (LC50=7.9 ng
ml-1) and approximately sixfold as toxic as Bs 2362
(LC50=12.6 ng ml-1). While the activity of this
recombinant was much better against this species than Bti or Bs, against
A. aegypti the toxicity was much lower than Bti
(Table 1).
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As the second example, we transformed the p45S1 plasmid described above, which produces large amounts of the Bs Bin toxin, into the acrystalliferous strain of Bti (4Q7) as well as into IPS-82, a strain of Bti that produces the normal complement of Bti toxins. Both of these strains produced very large quantities of the Bs Bin toxin, as assessed by SDS-PAGE analysis (Fig. 7). Both of these transformants had markedly improved toxicity in comparison with wild-type strains of Bti or Bs 2362, each being at least 10-fold more toxic against fourth instars of C. quinquefasciatus as assessed by comparison of their LC50s per unit fermentation medium (Table 2). However, there was no substantial improvement in toxicity against fourth instars of A. aegypti.
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Assessment and future prospects |
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Acknowledgments |
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References |
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