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Bacteriological larvicides of dipteran disease vectors

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 Review TRENDS in Parasitology Vol.17 No.8 August 2001
Bacteriological larvicides of dipteran
disease vectors
Lêda Regis, Maria Helena Silva-Filha, Christina Nielsen-LeRoux and Jean-François Charles
The apparent success in vector control observed between 1950 and 1970 was
followed by worldwide resistance to organosynthetic insecticides wherever
they were used intensively. Insect resistance to one or more categories of
insecticides has limited the effectiveness of these compounds, and their non-
selective mode of action adversely affects non-target organisms. This scenario
highlights the need for selective agents in integrated vector control programs.
This article gives an overview of the main fundamental and applied research
topics on entomopathogenic bacteria in relation to their role in vector control.
Vector-borne diseases are still rife because of the
recrudescence and re-emergence of diseases
transmitted by mosquitoes. Malaria is the most
dramatic case because it affects around 250 million
people in the world. Control of insects such as
mosquitoes and blackflies is complex because of their
ability to adapt to environmental changes and the
robustness of populations, which assures fast recovery
after intervention. An important event opened new
perspectives for the control of mosquitoes and blackfly
populations: the discovery of the mosquiticidal action
of some bacterial strains from the genus Bacillus. For
example, Bacillus sphaericus1 (Bs) and Bacillus
thuringiensis serovar. israelensis2 (Bti) act as per os
larvicides. These aerobic, Gram-positive, widespread
sporulating bacteria have a major advantage over
synthetic insecticides: selectivity owing to their
specific mode of action. Bs is toxic to some species of
the Culicidae, whereas Bti is also highly toxic to
Simuliidae. At the operational level, Bs shows good
persistence in the organically polluted water habitats
typical of Culex species and good efficacy against
some Anopheles species, whereas Bti is mainly used
for controlling Simulium and Aedes species.
Mode of action
Both Bti and Bs produce, during sporulation, a very
large amount of protein (10–30% dry weight of the
Lêda Regis* bacteria) that accumulates in the bacterial
Maria Helena Silva-Filha
Fundação Oswaldo Cruz-
sporangium as parasporal inclusions3 (Fig. 1). These
Fiocruz, CPqAM, Dept crystals, which contain the protoxins, are released in
Entomology, Av Moraes the medium when sporulation is completed. For Bti,
Rego s/n 50670.420
the crystals contain four major polypeptides of
Recife, PE, Brazil.
*e-mail: 123 kDa, 135 kDa, 72 kDa and 28 kDa, called Cry4A,
leda@cpqam.fiocruz.br Cry4B, Cry11A and CytA, respectively4. For Bs, the
Christina Nielsen-LeRoux
crystals contain a toxin (Bin) made of two
Jean-François Charles polypeptides of 42 kDa and 51 kDa, called BinA and
Institut Pasteur, BinB, respectively. For purified crystals from both Bti
Laboratoire des Bactéries
and Bs, the concentration that can kill 50% of a
Entomopathogènes, 25
rue du Dr Roux, 75015 mosquito larval population within 48 h (LC50) is 1–5
Paris, France. (ng protein) ml−1.
http://parasites.trends.com 1471-4922/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)01953-5
377
The general mode of action of these insecticidal
proteins involves the following steps (Fig. 2).
Aquatic larvae of mosquitoes and blackflies ingest
crystals and spores in suspension in water. Inside
the midgut lumen, under the combined action of
alkaline pH and intestinal proteinases, protoxins
contained in the crystals are solubilized and
activated5. Released toxins bind to apical microvilli
of midgut cells6,7 and then cytopathological
alterations are observed in midgut cells8,9, leading to
the death of larvae.
For Bti, the four toxins act in synergy10. The most
drastic cytopathological changes consist of the
swelling of midgut epithelium cells11. The colloid-
osmotic lysis hypothesis has been proposed to explain
the cellular damage12. After binding of toxin
molecules to specific membrane receptors, or to
membrane phospholipids (for Cyt proteins13), a part
of the toxin inserts into the membrane and forms a
transmembrane channel. Entry of water into the cells
and exit of ions and other, larger components from the
cells could lead to the cell swelling and lysis. The
receptors for the Cry4A and Cry11 toxins have not yet
been identified, despite a report that unactivated
Cry11A could bind to a 148 kDa protein in Anopheles
stephensi larvae14.
For Bs, the BinA and BinB components must both
be present in an equimolar ratio for the full
expression of toxicity15. Although BinB alone is not
toxic, high doses of BinA alone might be larvicidal16.
The most important damages in Culex pipiens midgut
cells are the appearance of large vacuoles and
mitochondrial swelling9. The posterior stomach and
the gastric caecae are the most severely damaged by
the Bin toxin, but late damage to neural tissue and
skeletal muscles has also been reported17.
Ultrastructural effects have been reported in
cultured cells of C. pipiens within a few minutes of
treatment with soluble and activated Bs Bin toxin8,
suggesting that the toxin exerts its effects at the cell
membrane itself18. The electrophysiological effects of
the Bin toxin has been investigated in cultured
C. pipiens cells using the patch clamp technique19.
This showed a reduction in whole-cell membrane
resistance, suggesting that the toxin could create
pores or channels in the cell membrane.
The variation in susceptibility between mosquito
species seems to be due to differences at the cellular
level. Indeed, a number of studies report the binding
of Bin toxin to the gastric caecae and the posterior
378 Review TRENDS in Parasitology Vol.17 No.8 August 2001
Bacillus thuringiensis serovar. israelensis
Spore Crystal
Bacillus sphaericus
Spore Crystal
TRENDS in Parasitology
Fig. 1. Sporangia of Bacillus thuringiensis serovar. israelensis and
Bacillus sphaericus before the end of the sporulation process as seen
by transmission electron microscopy. The parasporal inclusions
(crystals), which contain the entomopathogenic toxins, are visible close
to the spore. Scale bar = 0.5 µm.
stomach only in susceptible Culex species20. For
Anopheles, no regionalized binding is observed and no
binding is observed for Aedes aegypti21. Furthermore,
in C. pipiens, only BinB binds with high affinity to the
caecum and the posterior stomach, the binding of
BinA being conditioned by the binding of the BinB
(Refs 22,23). However, for Anopheles gambiae,
binding studies showed that both BinA and BinB are
essential for full receptor interaction23. The receptor
of the Bin toxin in C. pipiens has been recently
identified as a 60 kDa α-glucosidase, anchored in the
apical midgut membrane via a glycosyl-
phosphatidylinositol anchor24,25.
Field application
The development of Bacillus-based products for
vector control took place relatively soon after the
discovery of Bti. The first field trials were carried out
in the early 1980s for controlling mosquitoes and
blackflies in temperate and tropical countries
to reduce nuisance or to interrupt disease
transmission. Bti was the first bacterium adopted in a
long-term public health program, to control larvae
belonging to the Simulium damnosum complex,
which are vectors of Onchocerca volvulus, as part
of the Onchocerciasis Control Program (OCP).
This involved 11 countries in West Africa. The OCP
started in 1975 using organophosphates and Bti was
introduced in 1983, to manage insecticide resistance.
After 16 years of this program running,
onchocerciasis was no longer a public health problem
in the original area26.
The Simulium Control Program (Programa de
Controle de Simulídeos; PCS) conducted in the
State of Rio Grande do Sul in Brazil involved
pioneering work in the regular use of
entomopathogenic bacteria in South America. Bti
http://parasites.trends.com
was introduced to replace the organophosphorate
temephos, owing to the resistance recorded in
Simulium pertinax populations27. The successful
results obtained have stimulated the use of Bti by
other programs in South America28–30.
In temperate countries, a successful model has
been conducted in Germany to control Aedes vexans,
C. pipiens and other mosquito species in large
areas of the Rhine Valley, while conserving
biodiversity. The German Mosquito Control
Association (KABS) has conducted a programme for
more than 15 years using Bti and Bs on a 600 km2
breeding area, reducing nuisance to a tolerable
level31. Bti-based formulations are also used in
control programmes in France, Spain, Hungary,
Switzerland, Russia, Italy, Slovenia and Yugoslavia31.
In the USA, Bti has became one of the leading
larvicides used in mosquito control31,32.
The operational use of Bs in control programmes
was limited by the late availability of commercial
formulations. A Bs-based commercial product was
first used in 1989 in France, where the
Interdepartmental Service of Mosquito Control
(EID) (http://www.eid-med.org/) has gradually
introduced Bs to replace chemicals to control
C. pipiens, which is a seasonal nuisance. Bs is now
being used in combination with other larvicides to
prevent resistance.
Formulations of Bs have been used against Culex
and Anopheles species (which are vectors of
lymphatic filariasis, malaria and Japanese
encephalitis) in several areas of Asia, Africa and
America. In subtropical region of China, several
tons of Bs have been locally produced and
successfully used to reduce populations of Culex and
Anopheles species33. A sharp decline in vector
populations has been achieved in Goa, Pondicherry,
Chennai and small villages using Bs-based
formulations developed in India34–36. Large-scale Bs
trials in Africa conducted in two cities in Cameroon,
Yaoundé and Maroua, showed that Bs can be as
effective as chemical insecticides to control Culex
quinquefasciatus in urban areas37. In South America,
large-scale trials of Bs have also been conducted in
Recife, Brazil, a city with endemic Bancroftian
filariasis. This had a drastic effect on
C. quinquefasciatus population and a decrease in
filariasis transmission38,39.
Data from the operational use of microbial
larvicides in mosquito and blackfly control in several
countries in the past two decades have confirmed the
effectiveness of Bti and Bs, and their safety to non-
target species. Some problems have been pointed out,
such as the short-lasting larvicidal activity in certain
larval habitats, increasing the costs of programmes.
This limitation especially concerns Bti, because Bs
provides greater residual activity lasting 1–3 months.
This feature has been attributed to the ability of Bs to
persist in organically rich water and to its recycling in
mosquito larva cadavers40,41.
 Review TRENDS in Parasitology Vol.17 No.8 August 2001
Culex pipiens
(d)
B. thuringiensis
serovar.
israelensis
(c)
Spore
Crystal
(b)
(a)
TRENDS in Parasitology
Fig. 2. The mode of action of crystal toxins from an entomopathogenic bacteria (Bacillus
thuringiensis serovar. israelensis, in this case). After ingestion of spores and crystals by aquatic larvae
(a), the crystal proteins are solubilized and activated by serine proteinases in the insect midgut (b).
Active toxins then bind to specific receptors on the brush-border membranes of midgut cells (c) and
the larvae then die as a result of significant damage to midgut cells (d). Photograph of Culex pipens:
Pascal Goetgheluck.
Resistance and its management
The mechanism of action of insecticidal Bacillus
endotoxins involves a number of steps, which was
considered to be an advantage over synthetic
insecticides. However, resistance under laboratory and
field conditions has been reported for Bs. Different
levels of resistance to Bs have been reported in C. pipiens
populations33,42,43,*. The level and stability of resistance,
and the time needed for its development, depend on
various factors, including genetic background, selection
pressure, the insect generation turnover, migration
level and, most importantly, the genetic dominance of
resistance. In all studied cases, resistance seems to be
due to a recessive trait44–46, which is consistent with the
reversion of resistance under field conditions33,42,47.
Resistance is due to one major sex-linked gene in the
field resistant C. pipiens colony in France45 and to one
major and probably autosomic gene in a laboratory-
selected resistant colony from California44,46.
Until now, only one mechanism of resistance has
been identified. However, in vitro toxin–receptor binding
experiments conducted with larval midgut isolated
from Bs-resistant colonies showed that, in some cases,
the receptor was not functional44 but that, in others,
this interaction was similar to a susceptible colony45.
The facts that there are at least two possible
mechanisms of resistance, arising from two different
mutations, and that either one is sufficient to reduce
the larvicidal efficacy might explain why resistance to
Bs is likely to occur.
*Sinègre, G. et al. (1994) First field occurrence of Culex pipiens
resistance to Bacillus sphaericus in southern France. In VIII
European Meeting of Society for Vector Ecology, 5–8 September
1994, Faculty of Biology, University of Barcelona, Spain.
http://parasites.trends.com
379
In contrast to Bs, no field resistance to Bti has
occurred so far and selection trials in the laboratory have
shown that the evolution of resistance is correlated
with the number of toxins present in the selection
material. Resistance can occur to individual Bti toxins
but, when the entire toxin complex is used, only a low
level of resistance was found48. This explains why no
field resistance to Bti has ever occurred, these toxins
acting in synergy and apparently with different
target molecules. Even though the two subunits of Bs
Bin toxin also act in synergy, they cannot be
considered to be individual toxins, as can those of Bti.
Precautions for the use of Bs should be the same as for
any other single-site-acting insecticide.
Because all commercialized Bs products are based
on strains 2362, 1593M and C3-41, expressing strictly
identical binary toxins49, and because these toxins
apparently use the same receptor, it is not surprising
that they display reciprocal cross-resistance.
Fortunately, all resistant colonies are susceptible to
Bti (Refs 42,43). Searches for other Bs strains with
activity on Bs-resistant colonies have been undertaken
and two strains in particular proved to be of interest:
strain IAB-59 (Refs 46,50) and LP1-G (Refs 33,50).
Other attempts to reduce the risk of appearance of
resistance have been made by creating recombinant
Bs bacteria that express the Bin toxin and one or
several Bt toxins as well. It was found that Cyt toxins
of Bt clearly reduce the level of Bs resistance51,52.
Perspective
Since the discovery of Bti and highly mosquitocidal
strains of Bs, a large amount of laboratory and field work
has provided a wealth of knowledge about these bacteria,
their biology and genetics, their toxins and mode of
action, and their performance as larvicides. Both Bti
and Bs kill larvae shortly after ingestion, causing drastic
larval death within 48 h, and can be safely used
simultaneously with other biocontrol agents. As well
as this, their operational success is also due largely to
their ability to be mass produced in inexpensive
artificial media. Moreover, the persistence in breeding
sites has been extended through the continued
improvement of formulations53. Liquid formulations,
which are inherently less stable, are being progressively
replaced by dry formulations with excellent stability,
giving a shelf life of more than 2 years53.
Because mosquito populations can recover quickly
after intervention, their control requires a strong and
sustained pressure to succeed. It is therefore essential
to highlight that, although the choice of effective and
safe larvicides is important, other factors for achieving
successful control of mosquitoes population include
designing appropriate strategies, mapping and
treating all breeding sources, and gathering
entomological data to evaluate the control strategy
continuously. Resistance management strategies
should be considered in programmes based on Bs as a
larvicide, including the use of Bti and/or other
biocontrol agents50.
380 Review
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