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NATIVE MOSQUITOCIDAL BACTERIA AS BIOCONTROL AGENTS
AGAINST THE MOSQUITO VECTOR, CULEX PIPIENS
1
Ashraf M. Ahmed*1, 2, Ahmed A. M. Abdel Megeed3 Talat A. El-kersh4 and Hosain M. Al-Qahtaney1
Zoology Department, Collage of Science, King Saud University, PO. Box 2455, Riyadh 11451, Saudi Arabia.
2
Zoology Department, Faculty of Science, Minia University, El-Minia, Egypt.
3
Botany and Microbiology Department., College of Science - King Saud University, P.O. BOX 2455
Riyadh 1145, Saudi Arabia.
1
Department of Clinical Laboratory, College of Applied Medical Sciences,
King Saud University, Riyadh, Saudi Arabia.
[email protected], [email protected]
ABSTRACT
Samples of the Bacillus thuringiensis (Bt) and Pseudomonas frederiksbergensis (Pf), both proved to be safe to living
organisms and environment, were isolated from rural soil of Saudi Arabia, and evaluated for entomopathogenicity
against 3rd larval stage of Culex pipiens. Spore-crystal mixture of the Bt isolate showed a mosquitocidal activity
similar to that of the reference B. t. israelensis H14 (Bti-H14). However, toxin extract of Pf, identified as glycolipid
biosurfactants, showed 14 times higher toxicity than that of the Bti-H14. This significant difference in toxicity could
be attributed to the difference in the bacterial product used as it was the purified crude toxins of pf compared to
spore-crystal mixture of Bt isolate or Bti-H14. Light and electron microscopy showed destructive effects for the Pf
toxin extract on the midgut epithelial layer of treated larvae. Epithelial cells were first appeared with cytoplasmic
extensions followed by cellular and nuclear degradation. Additionally, Pf toxin extract has a devastating effect on
the peritrophic membrane that may have resulted in septicemia. In addition, scanning electron microscopy showed
body shrinkage in Pf toxin-treated larvae which was clearly noted compared to untreated control ones. This could be
attributed to the surface activity of characteristic to this bacterial extract. Molecular characterization of the current
Bt isolate, investigating the synergistic effect of combining both the Bt spore-crystal mixture (or δ-endotoxins) and
the Pf toxin on mosquito larvae, as well as isolating more native Bt isolates from the Saudi environment, are
currently under investigation. These data may be of a great help for stepping up an effective control measure against
mosquito vectors in Saudi Arabia
Keywords: Bacillus thuringiensis, Pseudomonas frederiksbergensis, biocontrol, Culex pepiens, histological effects,
morphological alterations
INTRODUCTION
Haematophagous mosquito vectors (Diptera, Culicidae: Nematocera) are responsible for the transmission of a
number of viral and parasitic human pathogens. In fact, mosquitoes are the primary, worldwide arthropod vectors
for fevers and parasitic diseases (Nasci and Miller, 1996). These vectors affect two-thirds of the world’s population
and kill millions annually (Gubler, 1998). In this context, different types of mosquito vectors are spread all over
Saudi Arabia (Al-Khreji, 2005 and Ahmed et al., 2011) and transmit common mosquito-borne diseases including
dengue fever (Khan et al., 2008), filaria (Hawking 1973), malaria (Al-Hazmi et al., 2003; Balkhy and Memish, 2003
and Madani, 2005). Thus, the Saudi Ministry of Health developed and implemented strict plans to prevent the
appearance of mosquito-borne diseases especially in Hajj (pilgrimage) time. Hence, and in support of these
governmental plans, this study here was proposed as a support of utilizing eco-friendly bio-agents for controlling
mosquito population in the country.
Considering the importance of these diseases and their vectors, control strategies, depend primarily on insecticides
to reduce vector populations and drugs to kill the parasites for long time. Unfortunately, both vectors and parasites
of some mosquito-borne diseases (like for example malaria) have eventually developed resistance against many
commonly used pesticides and drugs respectively. Moreover, the use of broad spectrum chemical insecticides in the
battle against insect pests had left both the soil and ground water contaminated with hazard chemicals. Moreover,
Chemical insecticides are further main causes of some human diseases, which vary from mild allergy to severe cases
of cancer. Thus, the urgent need for a clean and safe environment has forced the majority of scientists to focus on
the utilization of environmentally safe biocontrol agents for keeping vectors numbers down the threading level, and
in the same time, keeping the environment safe.
Biocontrol measures, using entomopathogenic agents is the simplest and perhaps still the most effective and readily
deployable technique being used to control harmful insects belonging to a variety of different orders. For instance,
fungi, and bacteria, from Genus Bacillus, are widely used to control mosquitoes (Becker and Margalit, 1993;
Margalith and Ben-Dov, 2000 and Kay, et al., 2002). Thus, in the current study, two different types of locally
*
Corresponding author.
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isolated soil bacteria were isolated and screened against the larval stages of the Saudi filaria vector, Cx. pipiens in
the lab as a first step towards utilization in the biocontrol measures in a larger scale. One of these is Bacillus
thuringiensis (Bt) a well-known Gram-positive spore forming saprophyte bacterium belongs to Genus Bacillus and
known with its mosquito larvicidal activity (Goldberg and Margalit, 1977). The other is Pseudomonas
frederiksbergensis (Pf) (Andersen et al., 2000) a Gram-negative bacterium belongs to Genus Pseudomonas, known
as a decomposer of organic matter in soil, water and food products, is used as a biological control of fungal diseases
in plants for example (Nielsen et al., 1998). Abdel-Megeed et al. (2006) found that the surface active agents
produced by these bacteria could have biotechnological application for insect control. Both bacteria were tested
against 3rd larval stages of Cx. pipiens mosquito in the current study. Once an effective novel bacterial product is
ensured, it will be of a great help for stepping up an effective control measure against mosquito vectors in Saudi
Arabia, specially, in the areas endemic with infectious diseases like Dengue fever in Eastern region for example.
MATERIALS AND METHODS
Mosquito Rearing
The Saudi filaria vector, Cx. pepiens, used in the current study were originally collected from the Eastern region of
Saudi Arabia (Ahmed et al., 2011), and purified from the cyclic colony (lab-reared for more than 25 generation), in
the mosquito research lab, Zoology Dept. Collage of Science, King Saud University. Mosquitoes were reared under
standardized insectarium conditions (26ºC, 12h: 12h light/dark period and 80–82% humidity). Larvae were reared in
tap water in the insectary of Zoology Department, College of Science, King Saud University, as previously detailed
in Ahmed et al. (1999). Adults emerging within a 24h period were maintained in rearing cages (30 × 30 × 30 cm
each) with continuous access to a 10% glucose solution (w/v). After adult emergence, mosquitoes of the same age
were used for the relevant experiments in this study. To maintain a stock of mosquito colony, they were kept
accessing 10% glucose until being allowed to feed on CD mouse blood for triggering vitellogenesis as detailed in
Ahmed et al. (1999). Resulting larvae were used for the experimental purposes.
Bacterial Isolation and Preparation
Bothe bacterial types used in this study were isolated from contaminated soil in Saudi Arabia and prepared for
experimental purposes.
B. Thuringiensis (Bt)
The bacterium Bt was isolated from contaminated soil in borders of Al-Madinah Al-Monawara city of the Western
region of Saudi Arabia (24° 28′ 0″ N, 39° 36′ 0″ E) according to Hong et al., (2009) and El-Kersh et al., (2012).
Briefly, one gram of fine grinded soil was added to 2.0 ml of sterile distilled water and suspended vigorously. Two
ml aliquots were then mixed with 2 ml absolute Ethanol to obtain 50% Ethanol concentration and then incubated at
30°C for 45min with shaking from time to time. After ethanol-treatment, 1/10 serial dilutions in sterile distilled
water were prepared, then appropriate dilutions were spread on nutrient agar medium supplemented with 0.2 %
yeast extract (Sisco research laboratories, Mumbai, India); and 0.0005% of manganese chloride (MnCl 2), and
incubated at 30°C for 2 to 3 days depending on spore maturation. The spores-crystals mixture on nutrient agar
medium plates were scrapped into 10 ml of sterile water, to give an average count of 109 cfu/ml. the crystal-spore
mixture was used in parallel with that of the reference B.t. israelensis H14 (Bti-H14) strain. The resulting sporescrystals mixture was then used for carrying out the Bioassay against mosquito larvae for determining LC50 & LC90.
P. Frederiksbergensis (Pf)
The biosurfactants extraction of the bacterium Pf was used in the current study. The Pf was first isolated from
Riyadh, the capital of Saudi Arabia, as detailed in Abdel Megeed and Mueller, (2009). Then, extraction and
purification of the P. frederiksbergiensis biosurfactants is carried out according to Abdel-Megeed and Majhdi
(2009). Briefly, 200 ml liquid bacterial culture was centrifuged at 10,000 rpm for 10 min at 4°C. The floating
materials resulting from the treatment of the supernatant by 40% ammonium sulphate were collected by
centrifugation and then dissolved in a small fraction of water. Chilled acetone is added to the solution to remove the
protein and acetone-insoluble materials. This step is critical in the purification procedure because the biosurfactant is
soluble in acetone at high concentration. Any decade that may have remained in the acetone fraction is removed by
extracting it three times with hexane. For sugar fatty acid esters separation, the mixture is subjected to extraction
with warm hexane (50°C) and filtration. This step resulted in fatty acids in the organic phase and sugar esters,
enzymes and sugars onto the filter.
Determination of the Hydrophilic moiety was carried out according to dinitrosalicylic assay that depends mainly on
reducing ends of carbohydrates. Reagent composition is 1 % of 3,5-dinitrosalicylic acid (DNSA), 30 % of sodium
potassium tartrate, and 0.4 M NaOH. Equal volumes of the sample and the previously mentioned reagent were
mixed and heated in a boiling water bath for 10 min. After rapid cooling to RT and diluting with 10 volumes of
water, the absorbance at 570 nm will be measured. Sucrose was used as a reference for the calibration curve. For
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further identification of the sugar moiety, the analysis was carried out by HPLC. Methyl esters of fatty acids were
prepared as previously described by Morrison (1964). Equal amounts from the sample and 1M of methanolic NaOH
were mixed well in a glass vial. The mixture was incubated in a water bath at 60°C for 20 min in order to cleave off
fatty acids. Equal amount of BF3/methanol was then added after cooling in ice for 1 min in order to create methyl
esters. After incubation at 60°C for 10 min, 200 µl of saturated NaCl was added and mixed well. The sample was
transferred to a 1.5 ml Eppendorf tube. Then 300 µl hexane was added and finally centrifuged at 13,000 rpm for 10
min after which the hexane layer was separated. The extract is dried over anhydrous Na 2SO4 before analysis with
GC-MS. The supernatant was transferred to a fresh tube. The water layer was extracted for second time and the
hexane layer was combined with the previous extract, and then subjected to GC-MS.
Larvicidal Activity Bioassay
Bioassay for mosquito-larvicidal activity was performed using different concentrations of Bt spores-crystals mixture
(El-Kersh et al., 2012) or Pf biosurfactants extract (Prabakaran et al., 2003). In case of Pf extract, fractions were
dissolved in hexane and made up to different concentrations in serial dilutions from the stock solution of the extracts
in hexane. In case of Bt spores-crystals mixture, serial concentrations were prepared from the stack mixture using
distilled water. Preliminary bioassays of each bacterial product were performed using a wide range of nine
ascending concentrations (data not shown). Then, a narrower range of the 4 lethal concentrations, in five replicates
each (n = 5), were used for carrying out the bioassay experiments in plastic cups according to WHO (2005). Briefly,
entomopathogenicity of each was assessed by adding 200μl of a bacterial product, to 300 ml plastic cups containing
100 mL of de-chlorinated water and 50 third-instar Cx. pipiens larvae. In case of Bt, negative controls received 200
μl of de-chlorinated water instead of spore suspension and the positive controls received 200μl of Bti-H14 reference
strain that known to be highly entomopathogenic to dipterans (Fillinger et al., 2003), at the same spore
concentration. However, in case of Pf, negative controls received 200 μl of hexane, the solvent of the bacterial
extract. Mortality in both cases was evaluated 24h post-treatment. Larval food was added as normal, so that larvae
were feeding normally until feeding cessation was noticed starting from 8-12h post-treatment. Larvae were
monitored each 3h for observing feeding cessation and death until 24h had occurred. The number of larvae surviving
was counted at 24h. Death or lack of reaction to gentle prodding with a glass pipette was the measured mortality
according to (Brown et al., 1998). Larval mortality percentages were calculated at 24h post-treatment in 5 replicates
for each concentration (n = 5) for each concentration using the Abbott’s formula (Abbott, 1925) as following:
Mortality (%) = [(X – Y)/X] 100
Where X = % survival in control,
and Y = % survival in treated mosquitoes.
Based on the resulting mortality %, the suitable concentrations used in the main toxicity experiment of the current
study were assessed in the 4 ascending lethal concentrations in each case. Fifty percent and ninety five percent lethal
concentrations (LC50 and LC95, respectively) were determined using Probit analysis.
Cytological Alterations Of Larval Midgut Epithelium
This part of study has been performed in Pf-treated larvae only as the histological impact of Bt infection is wellknown. The 3rd instar larvae of Cx. pipiens were treated with LC50 dose of Pf toxin (0.31µl/ml). Then, alive, but
sluggish, larvae were used for investigating histological alterations at 24h post-treatment under light microscopy
according to Ahmed et al. (2010) and Villalon et al., (2003). Briefly, the midgut sections were fixed overnight in
cold 2.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.2) and post-fixed for 1 h at room temperature in 1%
OsO4 in the 100 mM phosphate buffer. The tissues then were dehydrated through an ethanol series, treated with
propylene oxide and embedded in Poly/Bed 812 (Polysciences Inc., Warrington, PA). Thin 10 μm sections were
mounted on slides, stained with hematoxylin and eosin (Sigma-Aldrich) and mounted with Paramount (Fisher) and
examined by light microscopy using a Zeiss Axioskop 50 compound microscope (Carl Zeiss, Inc., Thornwood, NY).
Resulting images from control or treated larval preparations were imported into Adobe Illustrator Cs, 2003 Software
and adjusted for contrast and suitable qualities.
Ultrastructural Alterations Of Larval Midgut Epithelium
The impact of Pf extract on the ultrastructure of larval gut epithelial cells was investigated at 24h post-treatment
with the LC50 dose of Pf toxin (0.31µl/ml) using transmission electron microscopy. Control or treated midguts (from
alive, but sluggish, 3rd instar larvae) were dissected at 24 h post-treatment. Dissected midguts were fixed overnight
in cold 0.8% glutaraldehyde, 4% Paraformaldehyde in 0.1 M sodium cacodylate, (pH 7.0) and post-fixed for 4 h at
4°C in 1% OsO4 in 0.1 M sodium cacodylate (pH 7.0). Midguts were then dehydrated through ascending ethanol
series, treated with propylene oxide and embedded in Epon-Araldite resin (1:1). Ultrathin 4 μm sections were
mounted on slides, stained with 2% uranyl acetate for 30 min and then incubated in lead citrate for 10 min. Samples
were then analyzed with a transmission electron microscope (JEOL Ltd., model JEM-100CX II) at 80 kV.
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Morphological Alterations of Larval Bodies
Third instar larvae of control or treated (with LC50 of Pf) larvae (alive, but sluggish), were routinely prepared, at 24h
post-treatment of bacterial product, for investigating the morphological alterations on larval body under scanning
electron microscope according to the instructions of the Electron Microscopy Unit in Zoology Department, King
Saud University. Briefly, control or treated larvae were immediately fixed in 2.5% glutaraldehyde in phosphate
buffer. Following a minimum of 24h of fixation, larvae were rinsed with 0.1 M PBS three times at 10 min intervals
then fixed in 2% osmium tetroxide in distilled water. Larvae were then rinsed three times in 0.1 M PBS, dehydrated
in ethanol series (30%, 50%, 70%, 95%) for 10 min each, then rinsed three times in 100% ethanol. This was
followed by treatments with 50% ethanol: 50% acetone, 100% acetone, and 50% acetone: 50%
hexamethyldisilazane (HMDS), each for 15 min. Finally, larvae were rinsed twice in100% HMDS for 15 min and
allowed to air dry overnight. Larvae were then gold coated using a sputter machine (SPI Module Sputter Coater,
USA) and observed under the SEM (JSM-6380 LA, Japan). Resulting images from control or treated larval
preparations were imported into Adobe photoshop Cs, 2003 Software and adjusted for contrast and suitable qualities
Statistical Analysis
Mean % mortalities were calculated using MINITAB software (MINITAB, State College, PA, v: 13.1, 2001). LC50,
LC95, Slope and confidence limits were calculated using Probit analysis. Five replicates (five different mosquito
groups: n = 5) were carried out for better statistical analysis.
RESULTS
Mosquito-Larvicidal Bioassay
This part of study was conducted to determine the LC50 & LC95 of Bt spore-crystal and Pf toxin extract against the
3rd larval instar of Cx. pipiens mosquito at 24h post-treatment. Larvae were treated with wide range of ascending
concentrations preliminary study (data not shown). Thus, ascending lethal concentrations were assessed for
determining LC50 and LC95 at 24h post treatment (Table 1). The mean larval mortality percentage (calculated by
Abbott’s formula) was increased by increasing concentrations. Furthermore, cessation of feeding was noticed in
treated larvae starting from 12h post-treatment. LC50 and LC90 were calculated by Probit analysis which showed
LC50 of the native Bt similar to that of the reference bacteria Bti H-14 (4.8 µ/ml; CL: 4.1-5.6 v 4.88 µ/ml; CL: 4.25.4), and LC95 of the native Bt also similar to that of the reference bacteria Bti H-14 (33.9 µ/ml; CL: 21-54.5 v 21.93
µ/ml; CL: 17.3-28.03). On the other hand, Pf toxin extract showed LC50 and LC90 of 0.35 µ/ml (CL: 031-0.39) and
0.94 µ/ml (0.73-1.2) respectively (slope: 3.84 ± 0.143). The LC50 of Pf toxin extract (0.35µ/ml) was used for the
subsequent experimental purposes of the current study.
Histological Impact on Larval Midgut Tissues
Results from light microscopy revealed destructive effect of LC50 (0.35µ/ml) of Pf toxin extract on the internal
structure of the midgut epithelium of Cx. pipiens 3rd larval stage compared to the untreated midgut control (Figure
1). This effect was appeared as numerous cytoplasmic extensions in epithelial cells, followed by cellular and nuclear
degradation at 24h post-treatment. Although gut was still filled with nutritional contents, the peritrophic membrane
and the microvilli were disappeared, whereas those of the control retained their structural integrity (Figure 1).
Hence, this may indicate a high toxicity of Pf toxin extract on both external and internal morphology of treated
larvae.
In order to examine the impact of the LC50 (0.35µ/ml) of the larvicidal bacterial extract on sub-cellular organelles,
TEM revealed that the treatment with this dose of larvicidal bacterial extract is associated with severe damage in
microvilli (Fig. 2B). This effect was also noticed at the nuclear level as a clear degradation of the nuclear contents
(Fig. 2D). Moreover, degradation was noticed at the mitochondrial level (Fig. 3F). The observed mitochondrial
degradation, visualized as white spots or degraded mitochondria as compared to those of the control (Fig. 2F).
Morphological Impact on Larval Body
Both control and Pf extract-treated larvae were observed under SEM for investigating its impact on the whole body
of treated larvae (in terms of head and thorax widths) alterations at 24h post-treatment. SEM investigation revealed
that the size of thoracic region was considered as an indication of the body size alteration in this study. As shown in
Fig. 3, an obvious shrinkage in the treated larval body was clear compared to that of control one (Fig. 2A & B).
Thorax and abdomen of Pf-treated larvae showed a significant shrinkage in size compared to those of the control
one (Fig. 3C & D). These data clearly show a significant shrinkage in the body size of Pf extract-treated larvae.
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Table 1. Toxicity of Bt spores-crystals suspension and Pf purified crude toxin against 3rd larval stage of Cx. pipiens
at 24h post-treatment. Larvae were treated with ascending lethal concentrations of Bt or Pf product (v/v) in sterile
water, and mortality was monitored at 24h post-treatment for each concentrations of each product.
Parameter
B. thuringiensis
(Suspension)
4.80
(4.1 – 5.6)
33.9
(21.8 – 54.5)
1.94 ± 0.044
LC50 (µl/ml)
(95% CL)
LC95 (µl/ml)
(95% CL)
Slope
× 105
4.3 ± 0.7
(CFU/µg)*
Replicates (n) 5
* CFU: No. of bacterial cell; CL: Confidence Limit
Reference
(Bti-H14)
4.88
(4.2-5.4)
21.93
(17.3-28.03)
2.52 ± 0.044)
P. frederiksbergiensis
(Toxin extract)
0.35
(0.31-0.39)
0.94
(0.73-1.2)
3.84 ± 0.143)
4.5 ± 0.3
-
5
5
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4
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DISCUSSION
Mosquito-borne diseases are major causes of death and morbidity worldwide (Knight and Stone, 1977; Tolle, 2009).
The current further problematic issue is the absence of effective vaccines for the majority of mosquito-borne
diseases (Pierson et al., 2008 and Perera et al., 2008), the harmful effects of chemical insecticides on non-target
populations and the ever growing resistance by mosquito vectors to these chemicals have induced scientists to
explore alternative and eco-friend sustainable methods of mosquito control. One of the main alternatives is primarily
employing non-chemical based control measures, which includes the use of larvicidal bacteria such as Bacillus spp.
(Federici et al., 2007). To this end, the primary objective of the current study was to shed some light on new novel
locally isolated mosquito larvicidal bacteria from contaminated soil that may be proposed as new biopesticides in
the battle against the Saudi filaria mosquito vector Cx. pipiens (Omar, 1996).
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Figure 1. Histopathological impact of P. frederiksbergiensis extract in midgut epithelial tissue of Cx. pipiens larvae
18h post-treatment. A & B show cross-sections (at 200 & 600× magnifications respectively) of untreated control
larvae showing normal gut epithelial layer (EL), peritrophic membrane (PM) and microvellii (MV), and nutritional
gut contents (GC) filling the gut lumen. C & D: show gut epithelia of treated larvae (at 400 and 1000×
magnifications respectively) with clear cytoplasmic extensions (CE) in the epithelial cells, and devastated epithelial
cells (DEC), nuclei (DN) and microvellii (DMV).
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Figure 2. Transmission electron microscopic micrographs showing cytological effects of P. frederiksbergiensis toxin
extract on the ultrastructure of midgut epithelium of Cx. pipiens 3rd larval stage at 18 h post treatment. A: normal
microvilli (Mv) in control larvae (Scale bar = 2μm). B: degenerated microvilli (DMv) with bubbling and stretching
in treated larvae (Scale bar = 1μm). C: normal nucleus with clear normal nucleolus (n) and chromatin contents in
control epithelial cell (Scale bar = 2μm). D: degenerating nucleus and contents with clear degenerated nucleolus
(Dn) of an epithelial cell of treated gut (Scale bar = 2μm). E: normal mitochondrial structures in control larvae (M)
(Scale bar = 0.5μm). F: degraded mitochondria (DM) in treated midgut cells showing deformation and loss of cristae
and matrix (Scale bar = 0.5μm). These ultrathin 4μm sections were analyzed with transmission electron microscope
model JEOL JEM-100CX II at 80 kV.
One of the targeted bacteria in the current study was the native Bt rod-shaped Gram-positive bacteria belongs to
genus Bacillus. This based on the fact that this genus is characterized by its ability to produce robust endospores as
a survival mechanism in response to adverse environmental conditions (Gibson and Gordon, 1974), and hence,
produces crystalline prasporal inclusions called ‘‘δ-endotoxin proteins” during sporulation. These crystalline
inclusions, along with the spores, have a great potential to control a number of insect pests of different orders. Thus,
it is being utilized in the production of biopesticides as it is considered the safest insecticides and constitute safe and
environmentally friendly substitute of the harmful chemically synthesized insecticides. Thus, spore-crysta mixture
was used in the current study, and proved that the locally isolated Bt isolate has a mosquitocidal activity similar to
that of the reference Bti-H14 [4.80(4.1-5.6) v 4.88(4.2-5.4)]. This reference bacterium was used as a reference to our
local Bt isolate as it is being used extensively in the field for the control of mosquito vectors since 25 years ago
(Goldberg and Margalit 1977). We assume that this is a promising step that encourages us to isolate more local Bt
isolates which may lead to the discovery of an even more toxic mosquito larvicidal Bt isolate than Bti-H14 does.
The molecular identification and characterization of this native Bt isolate is currently being under investigation.
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Figure 3. Scanning electron microscopy of body regions of Cx. pipiens 3rd larval stage at 24h post-treatment with P.
frederiksbergiensis extract, or untreated larvae. A & B: whole mounts of control and infected larvae respectively at
low magnifications showing clear shrinkage in thorax and abdomen of treated larva immediately after death. C & D:
Heads and thoraces of control & treated larvae respectively, at higher magnifications showing shrinkage in heads
and thoracic regions in treated larva comparing to untreated one.
The other targeted bacterium in the current study was Pf, a rod-shaped Gram-negative bacterium belongs to Genus
Pseudomonas that contains members function as important decomposers of organic matter in soil, water and food
products (Palleroni, 1993). It was first isolated from a coal gasification site in Frederiksberg, Copenhagen, Denmark
(Anderson et al., 2000) and from Riyadh, Saudi Arabia by Abdel Megeed and Mueller (2009). It has been proved as
safe and eco-friend, and thus is used as biocontrol agent against plant pathogens (Haas and Defago, 2005), and
preliminarily tested against some aedine and anopheline mosquito showing reasonable and promising effects
(Prabakaran et al., 2003). Further, its biotechnological importance is partly due to its application in biological
control of fungal diseases in plants (Nielsen et al., 1998). Thus, targeting of Pf by the current study was based on
those studies and the assumption that its toxins could have biotechnological application for insect control. Hence,
the crude toxic surface-active extract from this bacterium was tested against 3rd larval stage of Cx. pipiens which
showed significantly 14 times higher toxicity compared to that of the Bti-H14 [Probit Analysis: LC50: 0.35(0.310.39) v 4.88(4.2-5.4) µl/ml respectively]. This difference in toxicity could be attributed to the difference in the
bacterial product used as it was crude toxins of pf compared to spore-crystal mixture of Bti-H14. Therefore,
extraction of the local Bt isolate and Bti-H14 toxins are currently being taking place for use in the future toxicity
tests. These data may indicate that Pf product may be considered as an effective candidate in the battle against Cx.
pipiens in Saudi Arabia.
The mode of action of Bt effects on the larval midgut epithelium has already been intensively studied. Thus, the
current study was focused on partial investigation of the mode of action of Pf toxin. Therefore, histopathological and
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morphological results carried out via light as well as transmission and scanning electron microscopies. Data revealed
that Pf extract treatments brought about a massive destruction to the larval midgut epithelial tissues as compared to
control larvae. This destruction of epithelial tissue might be the main reason of observed cessation in feeding by 812h post-treatment, septicemia and finally death at 24h post-treatment. This may indicate that Pf exhibit a similar
action as that recorded in case of Bt (Gill et al., 1992). This is confirmed by the observed massive subcellular
damage in the gut epithelial cells shown as degraded mitochondria and microvellii compared to untreated controls.
On the other hand, the morphological alteration, in terms of severe shrinkage in the whole body was clearly shown
in the thorax and abdomen sizes in infected larvae as compared to that of the control ones. This, in fact, may be
attributed to the surface activity of Pf extract (Abdel Megeed et al., 2006), which may indicate that Pf toxic extract
targets and causes the destruction of the integument as well as the gut epithelium. It has been reported that
destruction of the epithelial cells lining the midgut of mosquito larvae is often associated with midgut paralysis and
cessation of feeding as previously recorded with Bt (Clark et al., 2005 and Bravo et al., 2007). The current study
recorded similar symptoms which, in fact, may indicate high toxicity of these bacteria against mosquitoes and being
an effective biocontrol agent in the field of mosquito control since long time ago (e.g. Goldberg and Margalit, 1977
and Goettel et al., 1982).
In conclusion, and taking all together, our results provide solid evidence that the locally isolated Bt and Pf isolates
could be suggested as potential candidates in the biocontrol measures against Cx. pipiens mosquito in Saudi Arabia.
This suggests the possibility of exploiting for the development of a commercial mosquito larvicide, as bacteria
naturally occurring in Saudi Arabian soil. Moreover, testing this bacterial product against other mosquito is
underway in order to determine the infection range of these two bacterial isolates amongst mosquito vectors of
Saudi Arabia. Fortunately, safety of Pf toxic extract to environment and beneficial livestock has already been
confirmed (Nielsen et al., 1998, Haas and Defago, 2005; Abdel-Megeed et al., 2006 and Abdel-Megeed and Majhdi,
2009), which supports our assumption of proposing it as a potential biocontrol agent against mosquitos. Finally, our
ongoing work is focusing on the molecular characterization of the current Bt isolate, investigating the synergistic
effect of combining both the Bt spore-crystal mixture (or δ-endotoxins) and the Pf toxic extract on mosquito larvae
as well as isolating more native Bt isolates from the Saudi environment. This may be of a great help for stepping up
an effective control measure against mosquito vectors in Saudi Arabia, specially, in the areas endemic with
infectious diseases (Hawking 1973; Al-Hazmi et al., 2003; Balkhy and Memish, 2003; Madani, 2005 and Khan et
al., 2008)
ACKNOWLEDGEMENT
This research was supported by King Saud University through a National Program for Science and Technology
grant (11-MED-1848-02) from the Kingdom of Saudi Arabia. The authors would also like to thank Prof. H.
Husain, Faculty of Food Sciences and Agriculture for his help with statistical analyses. Author would like also to
thank the anonymous referees for their helpful comments and suggestions.
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