Fate of Bacillus thuringiensis subsp.

israelensis in the Field: Evidence

for Spore Recycling and Differential Persistence of Toxins in Leaf
Litter
Guillaume Tetreau, Mattia Alessi, Sylvie Veyrenc, Sophie Périgon, Jean-Philippe David, Stéphane Reynaud, and Laurence Després
Laboratoire d’Ecologie Alpine, LECA-UMR 5553, Université de Grenoble 1, Grenoble, France

Bacillus thuringiensis subsp. israelensis is a bioinsecticide increasingly used worldwide for mosquito control. Despite its appar-
ent low level of persistence in the field due to the rapid loss of its insecticidal activity, an increasing number of studies suggested
that the recycling of B. thuringiensis subsp. israelensis can occur under specific, unknown conditions. Decaying leaf litters sam-
pled in mosquito breeding sites in the French Rhône-Alpes region several months after a treatment were shown to exhibit a high

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level of larval toxicity and contained large amounts of spores. In the present article, we show that the high concentration of tox-
ins found in these litters is consistent with spore recycling in the field, which gave rise to the production of new crystal toxins.
Furthermore, in these toxic leaf litter samples, Cry4Aa and Cry4Ba toxins became the major toxins instead of Cyt1Aa in the com-
mercial mixture. In a microcosm experiment performed in the laboratory, we also demonstrated that the toxins, when added in
their crystal form to nontoxic leaf litter, exhibited patterns of differential persistence consistent with the proportions of toxins
observed in the field-collected toxic leaf litter samples (Cry4 > Cry11 > Cyt). These results give strong evidence that B. thurin-
giensis subsp. israelensis recycled in specific breeding sites containing leaf litters, and one would be justified in asking whether
mosquitoes can become resistant when exposed to field-persistent B. thuringiensis subsp. israelensis for several generations.

B acillus thuringiensis-based insecticides are often considered

environmentally safe and efficient alternatives to chemical in-
secticides for the control of pest populations, including lepi-
dopteran, coleopteran, and mosquito larvae (36, 37). The modes
of action of some B. thuringiensis subspecies together with their
fate in the environment have been largely studied (6, 30, 45). B.
thuringiensis is a natural larval pathogen (bacterium) that pro-
duces a toxic crystal during its sporulation (34), and its fate in the
field can be monitored by using three different approaches. First,
its insecticidal activity can be tested by performing bioassays using
environmental samples (i.e., water, soil, or leaf litter samples, etc.)
(9, 38, 44). Unfortunately, the sensitivity of this approach is often
too low to detect slight modifications of toxicity, as B. thuringiensis
usually does not exhibit a high level of residual activity (36). Sec-
ond, spores, which usually persist longer than the larvicidal activ-
ity, can be detected in field samples by culturing them on petri
dishes (18, 46) or by PCR and quantitative PCR, allowing the
detection of potential recycling events (12, 17). Finally, the fate of
Cry toxins, which are mainly responsible for the toxicity of B.
thuringiensis, has often been studied by using enzyme-linked im-
munosorbent assays (ELISAs) (1, 29). Although it is widely used,
such an approach does not guarantee that the native full protein is
present, because antibodies react only with a specific part of the
target protein. Nevertheless, focusing on toxins is the only ap-
proach that can confirm if the recycling of spores, detected by
quantitative PCR or by plating, is combined with crystal produc-
tion or not. Only the combination of these three approaches al-
lows the precise monitoring of the persistence of B. thuringiensis in
the field.
Bacillus thuringiensis subsp. israelensis, whose crystal is consti-
tuted of four main toxins (Cry4Aa, Cry4Ba, Cry11Aa, and
Cyt1Aa), is being increasingly used worldwide for mosquito con-
trol due to its high specificity in targeting dipteran insects and due
to the absence of firm cases of resistance detected in the field so far
(24). As its larvicidal activity often quickly decreases after spray-
ing, B. thuringiensis subsp. israelensis is often said to be low-level
persistent in the field (24, 38). Even though spores have already
been described to germinate and proliferate in mosquito cadavers
(2, 23), they are not believed to be able to proliferate in the envi-
ronment and to produce a large enough amount of crystals to kill
mosquitoes (4). Nevertheless, an increasing number of studies
suggested that B. thuringiensis subsp. israelensis can persist and
even recycle (i.e., germination, vegetative growth, sporulation,
and the production of toxins) under specific conditions in the
environment (4, 11). Moreover, decaying leaf litter sampled in
mosquito breeding sites several months after a treatment exhib-
ited a high level of toxicity against mosquito larvae (7, 8) and
contained large amounts of spores (43), suggesting a higher level
of persistence than expected and a potential recycling of spores,
leading to an unexpected high level of toxicity. The selection of an
Aedes aegypti laboratory strain for 30 generations with these litter
samples led to moderate resistance to B. thuringiensis subsp. israel-
ensis (3.5-fold) but higher levels of resistance to separate Cry tox-
ins (up to 60-fold) (41). This strain provides evidence that mos-
quitoes can become resistant when exposed to field-persistent B.
thuringiensis subsp. israelensis. It is therefore essential to charac-
terize the behavior of B. thuringiensis subsp. israelensis toxins in
the field and to precisely quantify the concentrations of these tox-
ins in toxic leaf litter.
Only a few studies have investigated the fate of toxins of B.
Received 2 July 2012 Accepted 16 September 2012
Published ahead of print 21 September 2012
Address correspondence to Guillaume Tetreau, guillaume.tetreau@gmail.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.02088-12

8362 aem.asm.org Applied and Environmental Microbiology p. 8362– 8367 December 2012 Volume 78 Number 23

thuringiensis subsp. israelensis in the field (4, 13). Contrary to
other B. thuringiensis subspecies, for which toxins are studied
mainly in the context of genetically modified crops, which pro-
duce large amounts of a unique soluble Cry toxin (20, 29, 40), B.
thuringiensis subsp. israelensis is sprayed as a suspension of spores
and crystals. The crystals are constituted of a mixture of four tox-
ins often found at very low concentrations in the environment
even a few hours after a treatment, rendering them difficult to
detect (24). This may explain why, while numerous ELISAs exist
for a large range of different Cry toxins, no efficient tool is avail-
able so far to extract and detect B. thuringiensis subsp. israelensis
toxins in field samples. Furthermore, most previous studies inves-
tigated the interaction between Cry toxins and soil, but there have
been few studies on the behavior of Cry and Cyt toxins in leaf litter
under aquatic conditions (42), which is one of the main constitu-
ents of several mosquito breeding sites and the main food source
for larvae.
In the present article, we used a recently developed and
patented extraction protocol followed by detection using an
ELISA targeting each toxin of B. thuringiensis subsp. israelensis to
determine the concentrations of the toxins and their relative pro-
portions in several field-collected leaf litter samples (42). We then
applied this test to leaf litter samples contaminated in the labora-
tory with different amounts of toxins in their crystal form to better
understand how long toxins can persist in leaf litter and if they
have different behaviors. We also tested whether the drying/
watering episodes, often experienced by the mosquito breeding
sites, have an impact on the persistence of toxins. All these results
are discussed in regard to B. thuringiensis subsp. israelensis recy-
cling in the field, treatment strategies, and resistance management
in the environment.
MATERIALS AND METHODS
Leaf litter sampling. All the leaf litter samples were taken from mosquito
breeding sites in the French Rhône-Alpes region. Toxic leaf litter samples
were collected in 2008 at four different sites 4 months after a treatment
with B. thuringiensis subsp. israelensis (Vectobac WG) (3,500 interna-
tional toxic units [ITU] · mg⫺1) at the beginning of spring (43). Two
samples (samples P23 and P26) were constituted mainly of Populus nigra
leaves, and two other samples (samples Aul1 and Aul2) were constituted
mainly of Alnus glutinosa leaves. Litter samples were left to dry at room
temperature, powdered, and stored at ⫺20°C until the extraction of tox-
ins and ELISA quantification were performed.
Leaf litter samples used for further contaminations with B. thuringien-
sis subsp. israelensis toxins were collected in 2011 in mosquito breeding
sites never treated with the bioinsecticide. These samples were constituted
mainly of Quercus sp. leaves and some Alnus glutinosa leaves (42). Litter
samples were left to dry and were stored at room temperature until the
toxins were added for the experiments. The samples were tested by ELISAs
prior to contamination, and no signal was recorded, indicating that no
indigenous B. thuringiensis subsp. israelensis was present.
Production of individual B. thuringiensis subsp. israelensis toxins.
A crystal-negative strain of B. thuringiensis subsp. israelensis (4Q2-81)
transformed with plasmids pHT606, pHT618, pWF53, and pWF45 for
Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, respectively, was used to produce
each protoxin separately (10, 32, 49). Spores and crystal suspensions were
produced as previously described (41, 42) and conserved in distilled water
at ⫺20°C until use.
Contamination of leaf litter samples and parameters tested. Ten
grams of leaf litter samples from untreated sites was put into crystallizing
glass dishes (capacity, 300 ml) containing 60 ml of tap water. Litter sam-
ples were contaminated with two different concentrations of one of the
December 2012 Volume 78 Number 23
Spore Recycling and Toxin Persistence in Leaf Litter
four toxins, a low dose (LD) and a 4-fold-higher concentrated dose (high
dose [HD]). For each dose and each toxin, two conditions of watering/
drying were also tested: either the water level was maintained by adding 4
ml of water every day, or the litter was left to dry until it was totally dried
after 17 days. For each condition, each dose, and each toxin, the litter was
sampled after six durations of exposure: immediately after toxin addition
(T0), 72 h, 7 days, 14 days, 1 month, and 2 months. For each duration of
exposure, a negative control not exposed to toxins was also sampled. After
sampling, litter samples were left to dry overnight in order to powder
them efficiently, a necessary step for the subsequent reproducible toxin
extraction, and were stored at ⫺20°C until use. All the conditions tested
were performed in triplicate. Litter samples were left at 27°C with 80%
relative humidity with a 14 h–10 h light/dark period.
Toxin extraction from leaf litter samples and detection by ELISA.
For the quantification of toxins extracted from leaf litter samples, calibra-
tion curves were performed by using purified toxins. Crystal toxins were
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solubilized by using an alkaline buffer (pH 10.8) containing Na2CO3 (50
mM) and dithiothreitol (DTT) (10 mM) and were incubated at 60°C for 1
h. Solubilized toxins were then purified by using a HiPrep 16/10 DEAE FF
ion-exchange column (Amersham Biosciences) and desalted by using a
HiPrep 26/10 desalting column (Amersham Biosciences). Toxins were
lyophilized and stored at ⫺20°C until use. For each ELISA, lyophilized
toxins were resuspended in distilled water, and their concentrations were
determined by a Bradford assay, using bovine serum albumin (BSA) as a
standard (5). Two aliquots of each toxin were then used in duplicate as
standards for each ELISA at concentrations ranging from 2 to 500 ng/ml.
Toxins were extracted from leaf litter samples by using a previously
described extraction procedure (42), adapted and modified from refer-
ences 19, 20, and 29. The procedure is described in pending French patent
no. FR1160365. This protocol is highly reproducible for each toxin and
with a large range of concentrations, suggesting that the extraction pro-
cedure in itself has no impact on the integrity of the toxins (42). All the
extractions were performed in duplicate. After extraction, toxins were
detected by sandwich ELISAs using anti-Cry4 (Cry4Aa and Cry4Ba), anti-
Cry11 (Cry11Aa), and anti-Cyt (Cyt1Aa) antibodies, as previously de-
scribed (28, 42). Each extracted sample was tested in triplicate by an
ELISA. For each toxin and each dose, the time needed to lose 50% of the
ELISA signal detected at T0 (T1/2) was calculated by performing an expo-
nential decay analysis using XLSTAT v.2009.4.06 software (Addinsoft).
For the toxic leaf litter samples, the concentration of each toxin was
compared to the concentration obtained by testing a commercial B. thu-
ringiensis subsp. israelensis strain at the operational dose (on the basis of 1
kg of formulated product/hectare of B. thuringiensis subsp. israelensis Vec-
tobac WG). Moreover, the proportion of each toxin among all the toxins
and among Cry toxins was also calculated. These values were compared to
the proportion obtained by testing a solubilized commercial B. thurin-
giensis subsp. israelensis strain (“B. thuringiensis subsp. israelensis solubi-
lized”) directly and by testing B. thuringiensis subsp. israelensis added to
leaf litter for 3 h and extracted according to the protocol described above
(“B. thuringiensis subsp. israelensis extracted”) (42). For each toxin or
group of toxins, the statistical differences in their proportions between the
different conditions/litters were tested by using a Fisher exact test per-
formed by using R 2.8.1 software (33).
RESULTS
The proportions of the different toxins calculated after extraction
and quantification by ELISAs were conserved among the four
toxic leaf litter samples tested regardless of their leaf compositions
(Table 1). In the toxic leaf litter samples, the proportion of the
Cyt1Aa toxin decreased by about 75% compared to the propor-
tion of solubilized B. thuringiensis subsp. israelensis tested by
ELISAs (Table 1). While a brief exposure of B. thuringiensis subsp.
israelensis to leaf litter (“B. thuringiensis subsp. israelensis ex-
tracted”) did not induce modifications of the proportions of Cry
toxins among them, Cry4 toxins were overrepresented among the
aem.asm.org 8363
Tetreau et al.

TABLE 1 Proportion of each toxin (Cry4Aa, Cry4Ba, Cry11Aa, and Cry toxins and became the main toxin in the field-collected toxic
Cyt1Aa) among all the toxins of B. thuringiensis subsp. israelensis or leaf litter samples (Table 1). When the concentrations of toxins in
among Cry toxinsa the toxic leaf litter samples were compared to those in leaf litter
Proportion (%) of each toxin among: samples where commercial B. thuringiensis subsp. israelensis had
All toxins Cry toxins been added, the amount of Cry4 toxins was 26% higher in the
field-collected toxic leaf litter samples (calculated as the means of
Cry4Aa Cry4Aa
the four toxic leaf litter samples) than in litter samples contami-
and and
Sample Cry4Ba Cry11Aa Cyt1Aa Cry4Ba Cry11Aa
nated in the laboratory with B. thuringiensis subsp. israelensis at
the operational dose (37.92 and 30.07 ng/ml, respectively). Con-
B. thuringiensis subsp. 1.0* 8.1* 90.9* 11.1* 88.9*
israelensis
versely, the proportions of Cry11Aa and Cyt1Aa were 18- and
solubilizedb 1,557-fold lower in the field-collected toxic leaf litter samples than
B. thuringiensis subsp. 12.6† 61.7† 26.7† 17.2* 82.8* in litter samples where B. thuringiensis subsp. israelensis had been
israelensis added, respectively (111.79 and 278.16 ng/ml for Cry11Aa and
extractedc 1.28 and 13,845.51 ng/ml for Cyt1Aa, respectively).
P26 extractedd

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59.7‡ 28.3‡ 11.9‡ 67.8† 32.2† For each toxin, a rapid decline in the concentration of detect-
P23 extractedd 56.7‡ 27.4‡ 15.9‡ 67.4† 32.6† able toxin during the first 14 days of contact with the leaf litter
Aul1 extractedd 68.9‡ 19.1‡ 12.0‡ 78.3† 21.7†
followed by a slower decrease was observed (Fig. 1). The drying of
Aul2 extractedd 55.8‡ 24.7‡ 19.6‡ 69.3† 30.7†
leaf litter samples did not modify the detectability of the toxins,
a
For each toxin or group of toxins, statistical differences (P ⬍ 0.05 by Fisher’s exact
test) of toxin proportions among the conditions are indicated by different symbols
regardless of the dose and the toxin considered (Fig. 1). When
(*, †, and ‡). Data obtained for solubilized and extracted B. thuringiensis subsp. toxins were added at higher doses, the concentration of detectable
israelensis have been reproduced from reference 42. toxins became the same as the concentration detected for lower
b
The proportion of each toxin was determined by ELISAs using a solubilized doses after 7 days (Cry11Aa and Cyt1Aa), 14 days (Cry4Aa), or 1
commercial B. thuringiensis subsp. israelensis strain (Vectobac WG) (3,500 ITU ·
mg⫺1).
month (Cry4Ba) of contact with the litter (Fig. 1). Cry11Aa and
c
The proportion of each toxin was determined by ELISAs after toxin extraction from Cyt1Aa toxins exhibited the highest decrease of detectability, with
leaf litters where B. thuringiensis subsp. israelensis had been added. the T1/2 being 2.2- to 23-fold lower than those of Cry4Aa and
d
The proportion of each toxin was determined by ELISAs after toxin extraction from Cry4Ba (Table 2). Even after 2 months of contact, the Cry4Aa,
toxic leaf litter samples. Samples P23 and P26 correspond to toxic leaf litter samples
constituted of mainly Populus nigra leaves, and samples Aul1 and Aul2 correspond to
Cry4Ba, and Cry11Aa toxins remained detectable at a basal level
toxic leaf litter samples constituted of mainly Alnus glutinosa leaves. (23%, 30%, and 15% of the ELISA signal detected at T0 when
toxins were added to the litter samples at high doses and 34%,

FIG 1 Detectability of Cry4Aa (A), Cry4Ba (B), Cry11Aa (C), and Cyt1Aa (D) toxins for the duration of contact with leaf litter, determined by using an ELISA
after extraction. Each toxin was inoculated at a low dose (LD) or at a high dose (HD), and the leaf litter was left to dry (W⫺), or the water level was maintained
during the entire incubation period (W⫹). Standard errors are indicated.

8364 aem.asm.org Applied and Environmental Microbiology


TABLE 2 Time to loss of 50% of the ELISA signal detected at T0 for all
the toxins at a low dose or at a high dose in leaf litter samples, and R2
values of the corresponding exponential decay curves
Cry4Aa Cry4Ba Cry11Aa Cyt1Aa
Condition T1/2 (h) R 2
T1/2 (h) R 2
T1/2 (h) R 2
T1/2 (h) R2
Low dose 140 0.78 212 0.95 63 0.77 86
High dose 308 0.83 543 0.87 24 0.93 53
21%, and 29% when toxins were added at low doses, respectively),
while the Cyt1Aa toxin was undetectable after only 1 week of con-
tact (Fig. 1).
DISCUSSION
Even if B. thuringiensis subsp. israelensis is usually said to be low-
level persistent in the field, an increasing number of studies have
shown that it can persist for a long time and suggest that it can
recycle under specific conditions in the environment (4, 11). The
high toxicity and the large quantity of spores found in the toxic
leaf litter samples suggested that such recycling events occurred in
mosquito breeding sites (8, 43). Here we demonstrated that the
concentration of Cry4 toxins detected in the toxic leaf litter sam-
ples by ELISAs was higher than expected after a treatment. The
most obvious explanation for this is that a recycling of the spores
led to a large amount of spores and toxins in the litters, inducing a
high toxicity to mosquito larvae. Further experiments are needed
to characterize the specific conditions (physicochemical parame-
ters and the presence of larvae) that allowed B. thuringiensis subsp.
israelensis to recycle in the field. The lower concentrations of
Cry11Aa and Cyt1Aa toxins observed in toxic leaf litter samples
are consistent with the differential persistence of the toxins ob-
served under laboratory conditions, with the Cry4Aa and Cry4Ba
toxins being more persistent than Cry11Aa and Cyt1Aa. It has to
be noted that the larger the protein, the more it persists: Cry4
toxins (130 kDa) have the highest molecular mass and are more
persistent than Cry11Aa (70 kDa) and Cyt1Aa (28 kDa). This
could be due to differences in their solubilities in water and/or to
their degradability by organisms naturally present in the leaf lit-
ters. The parameters increasing or decreasing the persistence of
each toxin (pH, salinity, and temperature) will be investigated to
better understand their fate in the field.
Cry4 toxins became the main toxins among all the toxins pres-
ent in the toxic leaf litter samples, together with a dramatic de-
crease in the concentration of Cyt toxins, which are known to
delay the development of resistance in mosquitoes (16, 47, 48).
This may explain why the LiTOX strain, selected with the toxic leaf
litters, exhibited a high level of resistance to the Cry4Aa toxin (up
to 60-fold) compared to the Cry4Ba and Cry11Aa toxins (about
9-fold) (41). As no antibody yet exists to distinguish between the
Cry4Aa and Cry4Ba toxins due to their highly conserved amino
acid sequences, it is impossible to know whether only Cry4Aa was
overrepresented in the toxic leaf litter samples or if both Cry4Aa
and Cry4Ba were dominant among all the toxins. The develop-
ment of more specific antibodies is needed to characterize more
precisely the toxin proportions in toxic leaf litter and to better
understand the selective pressures exerted on mosquitoes in the
field.
When toxins were added to leaf litter samples, a nonlinear
decrease in the detectability of toxins was observed: all the toxins
December 2012 Volume 78 Number 23
Spore Recycling and Toxin Persistence in Leaf Litter
had a fast decrease in detectability in the first days, followed by a
slower decrease. This profile of decreased detectability of toxins is
congruent with previous studies which investigated the fate of
soluble Cry3 and Cry1 toxins in different soils (1, 19, 21, 30). It
seems that the heterogeneous structure of the leaf litters is close to
the structure of soils, generating the same toxin adsorption pro-
0.77
files (25). In the present work, we used toxins in their crystal form
0.95
instead of soluble toxins, and we obtained similar results. Interac-
tions between crystals and litters have to be investigated further to
better understand why the B. thuringiensis subsp. israelensis toxins
have different behaviors in leaf litters. The same experiments have
to be performed with other constituents of mosquito breeding
sites, like soils, to determine if crystal toxins exhibit the same pat-
tern of persistence.
Due to its synergistic effect on Cry toxins, Cyt toxins are con-
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sidered key proteins in the cocktail of toxins produced by B. thu-
ringiensis subsp. israelensis, allowing a delay in the development of
resistance (16, 48). Our results show that Cyt is the less persistent
toxin, as it was underrepresented in the field-collected toxic leaf
litter samples and was undetectable after only 1 week of contact
with leaf litter samples contaminated in the laboratory. A previous
study showed that contact with leaf litter induced a strong de-
crease of the synergistic effect of Cyt on Cry toxins, leading to a
drastic decrease of the toxicity of B. thuringiensis subsp. israelensis
(42). As an increasing number of recombinant B. thuringiensis
strains containing Cyt toxins are developed with the goal of ob-
taining a more efficient insecticidal product than B. thuringiensis
subsp. israelensis (14, 15), it is necessary to better characterize the
fate of Cyt toxins in the environment.
No effect of litter drying on the persistence of the toxins was
observed, whatever the dose and the toxin considered. These re-
sults indicate that the watering/drying events often experienced by
mosquito breeding sites are not responsible for the low level of
persistence of B. thuringiensis subsp. israelensis in the environ-
ment. Moreover, higher doses of toxins did not led to a higher
level of persistence of the toxins. These results are congruent with
data from previous studies, indicating that an increase of the
quantity of B. thuringiensis subsp. israelensis sprayed does not re-
sult in the increased persistence of the toxins and of the insecti-
cidal activity (27). Our results confirm the importance of respect-
ing the operational doses of insecticides for mosquito control for
economic and ecological purposes.
Toxic leaf litters represent a unique opportunity to better un-
derstand how B. thuringiensis subsp. israelensis can persist and
increase its toxicity in the field. Based on the results obtained in the
present article and in previous studies, we propose a hypothetical
scenario of the sequential events that led to the formation of toxic
leaf litters in the field (Fig. 2). B. thuringiensis subsp. israelensis is
sprayed into mosquito breeding sites as a suspension of spores and
crystals (Fig. 2, step 1). Crystals and spores quickly settle in the
bottom of the mosquito breeding site (24) (Fig. 2, step 2). This
settling is partly responsible for the rapid decrease of the toxicity of
B. thuringiensis subsp. israelensis, notably for surface-feeding mos-
quitoes like Anopheles (3). After contact with leaf litter, B. thurin-
giensis subsp. israelensis drastically loses its toxicity, also explain-
ing a part of the toxicity loss, essentially for bottom-feeding
mosquitoes like Aedes larvae (42) (Fig. 2, step 3). Spores can recy-
cle under specific, unknown conditions (this work), as suggested
by previous works (11, 43) (Fig. 2, step 4). Numerous environ-
mental factors (the presence of organic matter, pollution, UV
aem.asm.org 8365
Tetreau et al.
FIG 2 Hypothetical scenario of Bacillus thuringiensis subsp. israelensis recycling in mosquito breeding sites containing leaf litters.
light, temperature, and solubilization) are known to strongly in-
fluence the residual activity of B. thuringiensis subsp. israelensis;
therefore, their influence on the persistence of the toxins in the
field must be investigated (22, 26, 38, 39). Moreover, the status of
B. thuringiensis as an exclusive larval pathogen was recently con-
firmed, indicating that the presence of mosquito cadavers must be
essential for recycling to occur (2, 34, 35). After proliferation,
bacteria will sporulate, leading to a large amount of spores and to
the liberation of crystals, inducing a high level of toxicity for mos-
quito larvae (7, 8, 43) (Fig. 2, step 5). At this step, toxins will
exhibit the same proportions in this recycled B. thuringiensis
subsp. israelensis strain as those in the commercial product. Tox-
ins will then persist differentially, leading to modified proportions
of toxins in the toxic leaf litters compared to the proportions of
commercial B. thuringiensis subsp. israelensis (this work). Such an
example shows that under specific, unknown conditions, B. thu-
ringiensis subsp. israelensis can recycle and achieve a high level of
toxicity for mosquito larvae. Considering that mosquitoes can be-
come resistant when selected with these toxic leaf litters in the
laboratory (31, 41), it can be expected that mosquito larvae ex-
posed to field-persistent B. thuringiensis subsp. israelensis in some
breeding sites may already be developing resistance to at least
some toxins (Fig. 2, step 6), even if bioassays with B. thuringiensis
subsp. israelensis still have not given evidence of it, probably due to
their sensitivity being too low to be able to detect arising resistance
in field mosquitoes. An understanding of the conditions that led
to the recycling of B. thuringiensis subsp. israelensis and the mo-
lecular basis of mosquito resistance is therefore of high interest to
ensure the long-term use of B. thuringiensis subsp. israelensis, one
of the main larvicidals authorized at present for mosquito control.
ACKNOWLEDGMENTS
We thank Brian Federici for providing recombinant B. thuringiensis
strains. We also thank Rolland Douzet for the identification of the litter.
This work was funded by the French National Research Agency (ANR)
(project ANR-08-CES-006-01 DIBBECO). Guillaume Tetreau was sup-
ported by the French Ministry of Research.
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