You are on page 1of 9

Biocontrol Science, 2015, Vol. 20, No.

2, 115123

Original

Novel Toxicity of Bacillus thuringiensis Strains against the


Melon Fruit Fly, Bactrocera cucurbitae
Diptera: Tephritidae
MD. ASADUZZAMAN SHISHIR1, ASMA AKTER1, MD. BODIUZZAMAN1,
M. AFTAB HOSSAIN2, MD. MUSFIQUL ALAM1, SHAKIL AHMED KHAN2,
SHAKILA NARGIS KHAN1 AND M. MOZAMMEL HOQ1
1

Department of Microbiology, University of Dhaka, Dhaka- 1000, Bangladesh


2
Insect Biotechnology Division, Institute of Food and Radiation Biology,
Atomic Energy Research Establishment, Savar- 1349, Bangladesh.
Received 23 September, 2014/Accepted 25 December, 2014

Bactrocera cucurbitaemelon fruit yis one of the most detrimental vegetable-damaging


pests in Bangladesh. The toxicity of Bacillus thuringiensisBthas been reported against a few
genera of Bactrocera in addition to numerous other insect species. Bt strains, harbouring
cry1A-type genes were, therefore, assayed in vivo against the 3rd instar larvae of B. cucurbitae
in this study. The biotype-based prevalence of cry1 and cry1A genes was calculated to be 30.8%
and 11.16%, respectively, of the test strainsn=224while their prevalence was greatest in
biotype kurstaki. Though three indigenous Bt strains from biotype kurstaki with close genetic
relationship exhibited higher toxicity, maximum mortalities were recorded for Btk HD-7396%
and the indigenous Bt JSc193%. LC50 and LC99 values were determined to be 6.81 and 8.32
for Bt JSc1, 7.30 and 7.92 for Bt SSc2, and 6.99 and 7.67 for Btk HD-73, respectively. The cause
of toxicity and its variation among the strains was found to be correlated with the synergistic
toxic effects of cry1, cry2, cry3 and cry9 gene products, i.e. relevant Cry proteins. The novel
toxicity of the B. thuringiensis strains against B. cucurbitae revealed in the present study thus
will help in developing efcient and eco-friendly control measures such as Bt biopesticides and
transgenic Bt cucurbits.
Key words Pest management / Bacillus thuringiensisBt, / Cry genes / Synergistic toxicity / Bactrocera
cucurbitae.

INTRODUCTION
B. thuringiensisBtbiopesticides, for their highly
specific mode of actions, are the key components of
Integrated Pest ManagementIPMstrategies aimed at
preserving natural enemies of pests and managing
insect resistanceKumar et al., 2008. Bt biopesticides
are eco-friendly as they are free of recalcitrant residues
which, upon bioaccumulation and biomagnification,
might become carcinogenic, mutagenic, teratogenic or
allergenicZahm et al., 1997. Hence, Bt biopesticides
have served as valuable alternatives to synthetic chem

Corresponding author. Tel: +88-01717083673, E-mail :


mmzhoqa
gmail.com

ical pesticides in agriculture, forestry and mosquito


control for last many decadesMohan and Gujar,
2000.
B. thuringiensis, a gram-positive spore forming
bacterium, synthesizes proteinaceous insecticidal crystals or - endotoxins during sporulation which can
specically kill the insects belonging to the Lepidoptera,
Coleoptera, Diptera, Hymenoptera, Hemiptera, and
Mallophaga as well as some invertebrates at their larval
stageBen-Dov et al., 1997; Schnepf et al., 1998;
Feitelson et al., 1999.
Bangladesh, a country of subtropical climate,
produces various vegetable crops covering an area of
ca. 498,073 acres. However, the yield per unit area is
low as 25% annual yield losses occur due to the pests

116

H. M. MOZAMMEL ET AL.

aloneRahman, 2000. More than 200 major species


of insects and mites from different eld crops, fruit trees,
and stored products have been recorded in Bangladesh
Rahman, 2000. The melon fruit fly, B. cucurbitae
Diptera: Tephritidae, is one of the widely distributed
and detrimental pests that damages about 81 host
plantsHollingsworth and Allwood, 2000mainly from
cucurbitaceous cropsDhillon et al., 2005, and it
causes signicant losses in different cucurbitsincluding
cucumber, melon, watermelon, squash, pumpkin,
gourds, etc.in Bangladesh also. The female melon y
is capable of destroying large numbers of fruits in its
lifespan of 10 months to a year as one may deposit up
to 1,000 eggs in soft tender fruit tissues by piercing
them with the ovipositorMohan and Gujar, 2000.
Maggots feed inside the fruits, owers and stems, and
young larvae cause necrotic regions, often introducing
various pathogens and hastening fruit decomposition
Dhillon et al., 2005.
A lot of control measures have been reported against
the melon fruit y such as the bagging of fruit, eld sanitation, monitoring and control with parapheromone
lures/cue-lure traps, increasing the host plant resistance, chemical control, wide area management, malesterile technique, transgene based embryo-specific
lethality system, quarantine and also biological control
Dhillon et al., 2005
. In Bangladesh, with no exception,
chemical insecticides are the major and most widely
administered control measure against the melon fruit y.
Continuous and indiscriminate use of these insecticides
due to their long residual action and toxicity against a
wide spectrum of insects has polluted the environment
largely by bioaccumulation and biomagnications, and
simultaneously led to the development of resistance in
agricultural pests and the spread of human diseases
Marrone and MacIntosh, 1993. As a result, ecofriendly pest management or organic farming is an
undeniable necessity in agricultureFrankenhuyzen
1993; Margalit et al., 1995; Salehi et al., 2005. The Bt
biopesticide, therefore, should be the most appropriate
choice for insect control.
Although a few species from genus Bactrocera have
been reported to be killed by the - endotoxins of Bt,
no such development has been found against B. cucurbitaeAnsari et al., 2012. Worldwide continuous
screening programs for new strains with different
combinations of crystal proteins as well as the discovery
of new toxins have broadened the activity spectrum of
Bt toxins. The present study was therefore designed by
combining in vitro and in vivo molecular techniques with
a view to nding potential Bt strains that are toxic to B.
cucurbitaemelon fruit y
.

MATERIALS AND METHODS

Bacterial strains and growth conditions


Indigenous Bt strains obtained from different locations
of Bangladesh were studied in terms of their abundance
and diversityShishir et al., 2014. These Bt strains are
preserved in the Bt Collection Centre, Bangladesh, in
the Department of Microbiology, University of Dhaka.
Two hundred and twenty four Bt strains from this collection were used in this study. The biotype kurstakiBtk
strain HD-73 and the biotype sottoBtsstrain T84A1
were kindly provided from Bt stock collection of
Okayama University, Japan and used as reference
strains. LB agarper litre: tryptone 10 g, yeast extract 5
g, NaCl 10 g, agar 15 gwas used for culture maintenance and T3- brothper litre: Tryptone 3.0 g, tryptose
2.0 g, yeast extract 1.5 g, MnCl2 0.005 g, phosphate
buffer 50 mM
Travers et al., 1987was used to
promote sporulation. Phase contrast microscopy was
used to observe sporulation as well as endotoxin
synthesisFig. 1. Incubation temperature was maintained at 30 for all types of culture conditions.
Total DNA preparation
Total DNA was prepared from the indigenous Bt
isolates streaked on LB agar mediumBravo et al.,
1998. After 12 hours of incubation at 30, a single
colony was transferred into 100 l sterile de-ionized
water in a microfuge tube, vortexed and kept at -70
for 20 min. It was then incubated in boiling water for 10
min to lyse the cells and briey centrifuged for 20 s at
12,000g. The upper aqueous phase transferred into
sterile microfuge tubes was used as a template and
preserved at 20 for further use. 50-100 ng of DNA
from this suspension was used as a template in PCR.
PCR detection of cry1 and cry1A genes
Bt strains were characterized on the basis of cry1
and cry1A genes by PCR detection with respective
p r i m e r s i . e . c r y 1 , f w d 5 CATGATTCATGCGGCAGATAAAC-3, rev 5TTGTGACACTTCTGCTTCCCATT-3; cry1A, fwd5C C G G T G C T G G AT T T G T G T TA - 3 , r e v 5 AATCCCGTATTGTACCAGCG-3
Carozzi et al.,
1991; Ben-Dov et al., 1997. PCR detection of the
genes were performed in 25 l reaction mixture
[containing 10 mM Tris, 50 mM KCl, 1.5 mM MgCl 2,
200 M dNTPs, 0.5 M of each primer, 50-100 ng of
the template and 0.5 U of Taq DNA polymerase
Promega, USA] in a thermal cyclerMJ mini,
Bio-Rad, USAfor 35 cycles95 for 50 s, 53 for 45
s, 72 for 60 swith an initial denaturation step at 95
for 4 min and a nal extension step at 72 for 10 min
Ben-Dov et al., 1997. PCR products were analysed
by electrophoresis in 1.5% w/v agarose gel
submerged in 1 TBE buffer89 mM TrispH 7.6
, 89

NOVEL TOXICITY OF BT AGAINST B. CUCURBITAE


mM boric acid, 2 mM EDTAand visualized against UV
after Ethidium BromideEtBrstaining in a Gel Doc
systemAlpha Imager Mini, USA.
Insect rearing
Larvae were maintained on a locally developed semi
liquid articial diet. Adult melon ies were stocked in a
stainless steel framed cage12012090 cm
covered with a stainless steel net. The insects were
usually supplied with a laboratory dietyeast extract:
casein: sugar- 1: 1: 2and water soaked cotton. In
general, 2000-2500 adult fruit ies were maintained in a
stock cage. Temperature and relative humidityRHof
the rearing room were maintained at 282 and
70-80%, respectively. To collect huge numbers of eggs
the matured ies in the cage were provided with a piece
of sweet gourd for oviposition. The piece of sweet
gourd was removed after 2 hours from the adult cage
and placed in a plastic bowl with sawdust for further
larval development.
Bioassay
The toxicity of the Bt isolates was analysed in vivo by
bioassay against the 3rd instar larvae of melon fruit y, B.
cucurbitae. Spore-Cry protein mixtures were prepared
with cry1A gene positive isolatesObeidat et al., 2004
and mixed with 10g of boiled and smashed sweet
gourd paste on which 20 larvae were kept and fed at
252 and 7010% RH, with a photoperiod of 16:8
L: D
. The mortality was then scoredFig 4A and 4B
for the Bt strains along with a parallel control prepared
with sterile distilled water, and it was used to correct
test mortality using Abbots formulaDaffonchio et al.,
1998
. The concentration of spores in the suspension,
determined by spore count technique, was the basis for
estimating LC50 and LC99 values for the strains causing
more than 50% mortality. Bioassays were performed in
triplicate in all cases and LC 50 and LC99 values were
determined by Probit analysis using Statplus 2009 software for Windows.
16S rRNA gene sequence analysis
16S rRNA genes from indigenous Bt strains were
amplified by PCR with universal primers for Bacillus
spp.: fwd20F; 5'-GAGTTTGATCCTGGCTCAG-3'
p o s i t i o n 9 - 2 7 , a n d r e v 1 5 0 0 R ;
5'-GTTACCTTGTTACGACTT-3'position 1492-1509
Souane and Cote, 2009. The PCR was conducted
in a thermal cycler by performing 35 cycles96 for 50
s, 50 for 45 s, 72 for 2 minwith an initial denaturation step at 96 for 10 min and a nal extension step
at 72 for 10 min in 25 l reaction mixturefwd and
rev primers 0.5 M each, 50-100 ng of template, 0.5 U
of Taq DNA polymerase, 200 M dNTPs, 10 mM Tris,

117

50 mM KCl and 1.5 mM MgCl2.


5 l of the PCR product was electrophoresed on
1.5%w/vagarose gelPromega, USAsubmerged
in 1 TBE buffer at 60 V for 1 hour and stained with
0.5 g/mL EtBrSigma, USAfor visualization against
a UV light in a gel documentation systemAlpha imager
mini, USA. Purified PCR productsWizard SV Gel
and PCR Clean-Up System, Promega, USAwere
sequenced at CARSABI PRISM 310 Genetic
Analyzer, University of Dhaka, Bangladesh, and the
sequences were submitted to the NCBI database
GenBank KF741358- KF741360 and GenBank
KF812552- KF812557.Based on the sequences,
multiple alignments and phylogenetic analysis were
carried out to compare the Bt strains among each other
and to determine the evolutionary relatedness as well as
the genetic distance.
Identication of putative cry genes responsible for
toxicity
The amplicons for the cry1 gene from the potential
strains were purified and sequenced, and the
sequences were aligned using MEGA 5.2 software to
see if any differences persisted. Moreover, we evaluated
whether or not the variation in toxicity of the strains
against B. cucurbitae was due to the expression of
some other cry genes. This was done by analyzing their
cry genes prole. PCR was performed to detect cry2,
cry3, cry4, cry8, cry9, cry10 and cry11 genesShishir
et al., 2014
, and the PCR products were then analyzed
by agarose1.5%gel electrophoresis following
staining and destaining in EtBr. The image was then
captured against UV by the gel doc system
Alphaimager mini, USA. The molecular weight of the
amplicons was determined from the images with the aid
of Alphaview SA softwareversion 3.4.0.0. Amplicons
of certain sizes were considered to be the desired PCR
products, and the strains were assumed to harbour
those particular genes.
Cry protein prole analysis
The Cry proteins were partially purified from the Bt
strainsztrk et al., 2009harbouring cry1A genes
and were analysed by SDS-PAGE in a 10% separating
gelSambrook et al., 1989. Colonies formed on
T3-agar medium upon incubation at 30 for 7 days
were scraped off and resuspended in cold sterile
de-ionized water. Washing was performed twice with
cold sterile de-ionized water to remove exotoxins, once
with 1.0 M NaCl containing 5.0 mM EDTA and finally
with 5.0 mM EDTA alone. The pellet, after being resuspended in 1Laemmli buffer lacking Bromophenol blue
50 mM Tris-HClpH 6.8, 10%v/vglycerol, 2%w/
vSDS and 100 mM dithiothreitol, was incubated at

118

H. M. MOZAMMEL ET AL.

boiling temperature for 5 min and the supernatant was


collected by centrifugation for 5min at 10,000 rpm
Eppendorf centrifuge, 5415D. Protein concentration
in the supernatant was estimated by the Bradford
methodBradford, 1976prior to SDS-PAGE analysis
to ensure that an equal amount of proteins was loaded
in each lane. Upon the completion of electrophoresis,
the gel was stained in a staining solution0.02%
Coomassie Brilliant Blue- G250 in 2%w/vphosphoric
acid, 5% aluminum sulfate and 10% ethanol
Kang et
al. 2002for 2 hours. The molecular weight of the
proteins was determined with the help of Alphaview SA
software.

RESULTS
Identication of cry genes in B. thuringiensis
Glowing spores and juxtaposed dark crystal shaped
proteins were observed with phase contrast microscopy
upon sporulation of the Bt strainsFig. 1. As for the

FIG. 3Prevalence and distribution of cry1 and cry1A genes


in different biotypes of indigenous Bt from Bangladesh.thu=
thuringiensis, kur= kurstaki, ind= indiana, gal= galleriae, sot=
sotto, den= dendrolimus, mor= morrisoni, dar=
darmstadiensis, ost= ostriniae, isr= israelensis, 9, 10, 11, 13,
15, 16= undened biotypes
n= number of isolates available
in each biotype, tested for the presence of cry1 and cry1A
genes
.

presence of cry1 and cry1A genes, 30.8% and 11.16%


n=224were detected, respectivelyFig. 2A & 2B
. Bt
strains producing 277 bp and 490 bp amplicons were
considered positive for the cry1 and cry1A gene,
respectively.
Biotype-based prevalence study of these genes
revealed that 78% and 42% of the strainsn=42of Bt
biotype kurstaki harboured the cry1 and cry1A gene,
respectivelyFig. 3. Significant prevalence was also
observed with the biotype thuringiensis28% and 10%
for cry1 and cry1A genes, respectively.
FIG. 1Spores and crystal proteins of indigenous B.
thuringiensis strains are distinguished under phase contrast
microscopy. Glowing parts are spores and juxtaposed dark
parts are crystal proteins.Bar= 2m

FIG. 2ADetection of the cry1 gene in the Bt strains as


amplicons of about 270 bp were visualized by agarose gel
electrophoresis.Marker= 100 bp DNA ladder, Bioneer,
Korea. BDetection of the cry1A gene with 490 bp
ampliconsM= 100 bp DNA ladder, TaKaRa, Japan
. Names
of the strains have been written over each lane.

Bioassay
Six Bt strains out of twenty five harbouring cry1A
gene were found to exhibit significant toxicity against
the 3 rd instar larvae of B. cucurbitaeFig. 4C.
Mortalities were recordedFig. 4A and 4Bas JSc193%, SSc2- 80%, SSe2- 63% and JaS8- 56% among
the indigenous Bt strains while reference strain, Btk
HD-73 exhibited 96% mortality and Bts T84A1 only
16%Fig. 4C. Hence, the bioassay was repeated with
the Bt strains causing more than 50% mortality for
further determination of LC50 and LC99 values.
The LC50 and LC99 values varied from 6.81 to 7.61
and from 7.67 to 9.13, respectivelyTable 1. The
lowest LC 50 value was observed for the indigenous Bt
strain JSc1LC50- 6.81whereas for reference strain
Btk HD-73, it was slightly higherLC50- 6.99. On the
other hand, Bt strain SSc2LC99- 7.92demonstrated
.
a comparable LC99 value with Btk HD-73LC99- 7.67

NOVEL TOXICITY OF BT AGAINST B. CUCURBITAE

FIG. 4Bioassay performed with 3 rd instar larvae of B.


cucurbitae. Fate of the larvae that were fed with the diet
supplemented with ASterile distilled water, or BSpore- Cry
protein mixture of Bt. CEfficiency of the indigenous and
reference Bt strains in causing death to the test larvae.
TABLE 1
LC50 and LC99 values estimated for indigenous and
reference Bt strains
Strains

LC50

LC99

X2

Df

Bt SSc2
Bt SSe2
Bt JSc1
Bt JaS8
Btk HD-73

7.30
7.42
6.81
7.61
6.99

7.92
8.81
8.32
9.13
7.67

0.852
3.005
0.278
1.453
0.364

1
1
1
1
1

0.355
0.222
0.597
0.228
0.545

LC50 and LC99: logspore concentration ml-1. X2: Chi-square

16S rRNA gene sequence analysis


Sequences of amplicons obtained for the 16S rRNA
gene from the indigenous Bt strains were aligned to
analyse the phylogenetic relationship between them.
Following the bootstrap neighbor joining method, a
phylogenetic tree was constructed with 19 indigenous
Bt strains and reference Btk HD-73Fig. 5. The tree
was observed to contain 2 major distinct phylogenetic
groups consisting of clusters A and B. Cluster A, the
largest one, contained 16 native Bt strains and 1 reference strain whereas cluster B contained the other 3.
Nine of the indigenous Bt strains from biotype kurstaki
and reference Btk HD-73 remained in the same subcluster A1 among which were strains JSc1, SSc2 and
SSe2 that exhibited toxicity against B. cucurbitae.

119

FIG. 5Neighbor-joining tree showing the phylogenetic


relationship between indigenous Bacillus thuringiensis strains
and reference strain Btk HD-73 based on the 16S rRNA gene
sequence analysis. This is an un-rooted tree reconstructed
with 1000 bootstrap replicates based on maximum
composite likelihood using tree construction software MEGA
version 5.22.

Causes of toxicity and its variation based on the


gene and protein prole
It was observed from the alignment of sequences of
the cry1 conserved region, obtained from the indigenous Bt strains JSc1, SSc2, SSe2 and two reference
strains Btk HD-73 and Bts T84A1, that the indigenous
strains were more similar to Bts T84A1. Mismatches
were observed at 24 and 231 base positions with Btk
HD-73Fig. 6A.
Upon agarose gel electrophoresis of the PCR products, the presence of cry1, cry2, cry3 and cry9 genes in
the indigenous Bt strains was revealed whereas reference Btk HD-73cry1Acand Bts T84A1cry1Aa
were positive for only the cry1 geneTable 2. Bt JSc1
was found to harbour cry1, cry2 and cry9 genesFig.
6Bwhereas Bt SSc2 and SSe2 harboured cry1 and
cry3 genesTable 2. The correlation between the cry
gene profiles and LC50 values of these Bt strains was
observed in this studyTable 2. The presence of cry2
and cry9 genes had a positive effect on the toxicity as
the LC50 value for Bt JSc1 was found to be the lowest
among all the cry1 gene-harbouring Bt strains. The
effect of the cry3 gene was also observed since the
toxicity was found to be more intense in strains SSc2
and SSe2 than JaS8 that lacked the genes. The toxicity
of Bt strains against B. cucurbitae can therefore be
concluded as the synergistic effects of cry1, cry2, cry3
and cry9 gene products.

120

H. M. MOZAMMEL ET AL.
TABLE 2Comparison of the cry gene proles of Bt strains tested in bioassay
Strains

cry1

cry2

cry3

cry4A/4B

cry8

cry9

cry10

cry11

LC50

Bt SSc2

7.30

Bt SSe2

7.42

Bt JSc1

6.81

Bt JaS8

7.61

Btk HD-73

6.99

Bts T84A1

Cry protein prole of the potential Bt strains


SDS-PAGE analysisFig. 7of the partially purified
Cry proteins revealed that diverse Cry proteins are
synthesized by the indigenous Bt strains at different
expression levels. Based on the molecular weight of the
proteinsTable 3, Cry1, Cry2, Cry3 and Cry9 proteins
could be presumed to be expressed. From the analysis,
common 23, 45, 50 kDa protein bands were observed
in all Bt strains which were not considered as Cry
proteins. Another 67 kDa common band was observed
in all Bt strains except strain JaS8 which might be the

FIG. 7
SDS-PAGE analysis of partially puried Cry proteins of
Bt strains. Lanes have been labelled with the names of the
strains.HD73: Reference strain Btk HD-73
M: Precision
plus protein standards; All blue; Bio-Rad, USA
.

FIG. 6AThe sequences obtained from the conserved


regions of cry1 genes of indigenous and reference Bt strains
were aligned by ClustalW. Mismatches at 24 and 231 base
positions of Btk HD-73 are visible with the sequences of other
strains. BInvestigation of causative cry genes in Bt JSc1
rendering toxicity against the melon fruit fly. Amplicons of
approximately 490, 639 and 492 bp were observed in
detecting cry1A, cry2 and cry9 genes as indicated with
arrows whereas spurious amplicons of about 564 and 365 for
cry4A/4B, 667 for cry9, 480, 700 bp for cry10 and 200, 270
for cry11 genes were observed.

TABLE 3Determination of MW of the partially purified Cry


proteins
Strain
Bt SSc2
Bt SSe2
Bt JSc1
Bt JaS8
Btk HD-73

MW of the visible protein


bandskDa

Presumptive Cry
proteins

23, 45, 50, 67, 73, 90, 109,


204
23, 45, 50, 67, 73, 90, 109,
204
23, 45, 50, 67, 73, 90, 109,
134, 204
23, 45, 56, 109
23, 45, 50, 66, 134

Cry1, Cry3
Cry1, Cry3
Cry1, Cry2, Cry9
Cry1
Cry1Ac

NOVEL TOXICITY OF BT AGAINST B. CUCURBITAE


degraded product of Cry1 proteinArmengol et al.,
2006. An unusual but prominent band at 109 kDa was
common in all indigenous strains. A faint band in the
range of 130-140 kDa was observed in Bt JSc1 which
could be either the Cry1 and Cry9 protein whose
expression level seems to be low. The molecular weight
of Cry1Ac protein of reference Btk HD-73 was determined at 133 kDa.

DISCUSSION
B. thuringiensis, the entomo-pathogenic bacterium,
has gained considerable attention since 1960 and is
widely preferred over chemical insecticides for ecofriendly pest management as it is environmentally
benignSezen et al. 2010
. Discovery of novel potential
Bt strains is necessary to solve the problem of resistance that has been reported in many pests against
many Bt biopesticide formulations and transgenic Bt
cropsShishir et al., 2014. As a result, the screening
for potential Bt strains that are highly toxic against
different insect orders including resistant species is a
rudimentary and continuous process all over the world,
and many such novel Bt strains have been recovered
from numerous sources such as soil, grain dust,
diseased insect larvae, animal feed mills and aquatic
environmentsSezen, et al. 2010.
Bt is widely distributed and approximately 70% of soil
samples from all continents have been found to
produce this bacteria, and are especially plentiful in
Asian samples. Because of this, the particular interest
of this research work was to isolate and identify potential Bt strains with novel toxicity against the melon fruit
flyB. cucurbitae, an important vegetable-damaging
pest in Bangladesh.
Bt strains used in this study were observed with the
phase contrast microscope upon sporulationFig. 1.
Crystal proteins and the spores were distinguished from
each other as the lights of different phases were passed
through the specimens that revealed glowing spores
and dark crystal proteins.
Bt has also been reported to produce parasporin,
another type of parasporal crystal protein which has
anti-cancer cell activity. It was shown that parasporal
proteins from non-hemolytic Bt strains are mainly noninsecticidal but may have anti-cancer cell activity
Mizuki et al. 1999. That is why, hemolytic strains were
mainly used in cry1 and cry1A gene detection.
As all of the biotypes describing different subspecies
were found to consist of both hemolytic and non-hemolytic Bt strainsShishir et al., 2014, all of the 16
biotypes were included in the process of detecting cry
genes. Initially, the cry1 gene was detected in the strains
producing amplicons of ca. 277 bp, and the positive

121

strains were further searched for the presence of cry1A


genes. The targeted amplicons of about 490 bp were
observed in 25 out of 69 cry1 positive Bt strains. It was
found from the previous studies that the cry1 gene was
present mainly in the biotypes of kurstaki, thuringiensis,
sotto, dendrolimus, morrisoni, galleriae and darmstadi. As exceptions, in our study
ensisCarozzi et al., 1991
we detected the cry1 gene in the biotypes indiana and
israelensis and in 11 undescribed biotypes at percentages of 31%, 18% and 21%, respectively, and the
absence of the cry1 gene in the biotype galleriae.
Bt toxin was reported to cause mortalityat more
than 65-80%to the olive fruit fly, Bactrocera oleae
Ansari et al., 2012. However, there has been no
report of Bt toxicity against the melon y, B. cucurbitae,
nor is it listed in the toxin specificity data summary.
Though Dipteran insect orders have been found to be
susceptible to Bt subsp. israelensis and mostly to Cry4,
Cry10 and Cry11 proteins, Cry1Ab and Cry1Ac proteins
were also found to exert toxicity against themThe
Canadian Forest Service: http://cfs.nrcan.gc.ca/
projects/119/6. In this connection, the toxicity of Bt
strains harbouring cry1A-type gene was tested against
the 3rd instar larvae of B. cucurbitae in this study.
The larvae were fed on sweet gourd paste in which a
Bt spore-Cry protein suspension was mixed. The larvae
were observed up to 7 days as the unaffected larvae
grew up into pupae and nally matured into y. On the
other hand, the effect of Cry toxins over the larvae was
evidenced as their movement and feeding gradually
stopped, and nally they turned black and diedFig. 4A
and 4B. The result of the bioassay performed in our
experiment revealed that the indigenous Bt JSc1 and
reference Btk HD-73 were highly toxic to the melon y
larvaeFig. 4C.
The experiment was repeated thrice, and each time it
was done in triplicate to evaluate the results statistically.
Bt strains causing more than 50% mortality were
studied to determine the lethal concentrations. Thus,
four indigenous Bt strains and one reference strain, Btk
HD-73, were used. The logarithmic value of the concentration of spores was the basis for LC50 and LC99 determination instead of proteins because actual amount of
the active proteins should be conrmed for the strains
expressing more than one protein. From this analysis,
the LC50 value of Bt JSc1 and the LC99 value of Bt SSc2
were found to be highly comparable to those of Btk
HD-73Table 1.
16S rRNA gene sequence analysis has been used as
a molecular identication tool for Bt and the claims of its
ability to discriminate Bt in different H-serotypes also
was reportedJoung and Cote, 2002; Soufiane and
Cote, 2009; Poornima et al., 2010. In the present
study, 16S rRNA gene analysis was performed with 19

122

H. M. MOZAMMEL ET AL.

Bt strains16 harbouring the cry1 gene, 10 harbouring


the cry1A gene, 3 without the cry1 geneand one
reference strain Btk HD-73. Two main clustersA and
Bwere generated from the dendrogram analysisFig.
5with two sub-clusters in cluster A. Sub-cluster A1
was the largest with 12 strains in which Bt DSf3,
harbouring no cry1 gene, was present and 7 Bt strains
harbouring the cry1A gene were found. In cluster A2,
two Bt strains were found without any cry1 gene and
the rest of them did not harbour the cry1A gene as well.
In cluster B, all three Bt strains were found to harbour
both cry1 and cry1A genes. Interestingly, all the Bt
strains that were found to be highly toxic against the
larvae of melon fruit y were observed in the same subcluster A1Fig. 5.
The alignment of sequences of the cry1 gene from
the potential strains obtained from bioassay revealed
that the sequences are more similar to that of cry1Aa of
Bts T84A1 rather than Btk HD-73. Hence, the variation
in toxicity is very likely. However, the toxicity of Bts
T84A1 was very low compared to the indigenous strains
and reference Btk HD-73Fig. 4C. That is why, some
other gene products, i.e. Cry proteins, might be involved
in the toxicity against B. cucurbitae. To get an insight
into this, potential strains were investigated for the
genes cry2, cry3, cry4, cry8, cry9, cry10 and cry11 as
most of the lepidopteran and dipteran vegetabledamaging pests were reported to be susceptible to the
proteins encoded by these genes. Interestingly, the
indigenous strains were found to possesssome of the
genes searched for in this regard. Besides the cry1
gene, Bt JSc1 was positive for cry2 and cry9 genes and
Bt SSc2 as well as SSe2 were positive for cry3 genes.
The PCR products of expected sizes were the only
basis for the presumption of the presence of a gene,
and non-specic products were disregardedShishir et
al., 2014
. From this analysis, the toxicity was found to
be correlated with the cry gene proles of the Bt strains
Table 2. The lowest LC50 value was obtained for Bt
JSc1 which was detected with cry1, cry2 and cry9
genes whereas it was slightly higher for reference Btk
HD-73 that harboured only the cry1Ac gene. The LC50
values of Bt SSc2 and SSe2 were higher than that of Bt
JSc1 and Btk HD-73 as they lacked cry2 and cry9
genes, and the cry1 gene in them was not similar to
cry1Ac. The presence of the cry3 gene enhanced their
toxicity more than Bt JaS8 harbouring only the cry1
gene.
The Cry protein proles also support the idea as the
correlation between Cry proteins and cry genes was
observed, and the presence of a PCR band alone was
not sufficient to guarantee its expression. Cryptic cry
genes can arise by frequent recombination events
among the genes present in the Bt strains. The insecti-

cidal potential of a Bt strain can be more appropriately


ascertained by detection of cry genes present followed
by analysis of crystal proteins produced by that strain
Sarvjeet, 2006. This method can supplement the
PCR strategy to determine which genes are actually
expressed. Thus, it was observed from the protein
prole of the indigenous Bt strains that multiple protein
bands ranging from 23 kDa to more than 200 kDa were
present. Proteins bands with molecular weights similar
to those of the reported Cry proteins were considered
as Cry proteins, and thus the presence of Cry proteins
in both protoxin and toxin forms were observed. The
Cry protein prole of the indigenous Bt strains revealed
the presence of Cry1, Cry2, Cry3 and Cry9 proteins
Table 3. Thus the Cry protein prole of Bt JSc1 with
Cry1, Cry2 and Cry9, strain JaS8 with Cry1 and strain
SSc2 as well as SSe2 with Cry1 and Cry3 comply with
the cry gene proles.
It can, therefore, be concluded that the synergistic
effects of Cry proteins encoded by cry1, cry2, cry3 and
cry9 are the causes of novel toxicity of the indigenous
Bt strains against B. cucurbitae and that the Cry1Ac
protein is the toxic agent in Btk HD-73.

CONCLUSION
The report of the novel toxicity of the Bt strains
against the larvae of melon y, B. cucurbitae, will help to
develop efficient and eco-friendly control measures
which in turn will prevent bioaccumulation and biomagnification of toxic substances through the food chain
and enhance the food safety and security.

ACKNOWLEDGEMENT
This work was supported by a Grant-in-Aid from the
USDA as a project entitled
Production of Bacillus
thuringiensis biopesticides by biotechnological
approach for the control of vegetable pests in
Bangladesh.
We thank Okayama University, Japan for
providing the reference strains. We also thank CARS,
University of Dhaka, Dhaka-1000, Bangladesh, for
providing the sequencing facility.
REFERENCES
Ansari, M. S., Hasan, F., and Ahmed, N.2012Threats to
fruit and vegetable crops: Fruit iesTephritidae- Ecology,
behaviour, and management. J. Crop. Sci. Biotech. 15,
169-188.
Armengol, G., Escobar, M. C., Maldonado, M. E., and Orduz,
S.2006Diversity of Colombian strains of Bacillus
thuringiensis insecticidal activity against dipteran and lepidopteran insects. J. Appl. Microbiol. 102, 7788.
Ben-Dov, E., Zaritsky, A., Dahan, E., Barak, Z., Sinai, R.,

NOVEL TOXICITY OF BT AGAINST B. CUCURBITAE


Manasherob, R., Khamraev, A., Troitskaya, E., Dubitsky, A.,
Berezina, N., and Margalith, Y.1997Extended screening
by PCR for seven cry-group genes by eld-collected strains
of Bacillus thuringiensis. Appl. Environ. Microbiol. 63,
4883-4890.
Bradford, M. M.1976A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Analyt. Biochem. 72,
248-254.
Bravo, A., Sarabia, S., Lopez, L., Ontiveros, H., Abarca, C.,
Ortiz, A., Ortiz, M., Lina, L., Villalobos, V., Pena, G., NunezValdez M., Soberon, M., and Quintero, R.1998
Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl. Environ. Microbiol. 64,
4965-4972.
Carozzi, N. B., Kramer, V. C., Warren, G. W., Evola, S., and
Koziel, M. G.1991Prediction of insecticidal activity of
Bacillus thuringiensis strains by polymerase chain reaction
product proles. Appl. Environ. Microbiol. 57, 3057-3061.
Daffonchio, D., Borin, S., Frova, G., Manachini, P. L., and
Sorlini, C.1998PCR fingerprinting of whole genomes:
the spacers between the 16S and 23S rRNA genes and of
intergenic tRNA gene regions reveal a different intraspecic
genomic variability of Bacillus cereus and Bacillus licheniformis. Int. J. Syst. Bacteriol. 48, 107-116.
Dhillon, M. K., Singh, R., Naresh, J. S., and Sharma, H. C.
2005The melon fruit y, Bactrocera cucurbitae: A review
of its biology and management. J. Insec. Sci. 5, 40-55.
Feitelson, J. S., Payne, J., and Kim, L.1999Bacillus
thuringiensis: insects and beyond. Biotechnology. 10,
271-275.
Frankenhuyzen, K. V.1993The challenge of Bacillus
thuringiensis. In Bacillus thuringiensis, An Environmental
Biopesticide: Theory and PracticeEntwistle, P. F., Cory, J.
S., Bailey, M. J., and Higgs, S. R., ed., pp. 1-35, Wiley,
Chichester.
Hollingsworth, R., and Allwood, A.J.2002Melon y. In SPC
Pest Advisory Leaets, pp. 1-2.
Joung, K. B., and Cote, J. C.2002A single phylogenetic
analysis of Bacillus thuringiensis strains and bacilli species
inferred from 16S rRNA gene restriction fragment length
polymorphism is congruent with two independent phylogenetic analyses. J. Appl. Microbiol. 93, 1075-1082.
Kang, C., Kang, D., Gho, Y. S., Suh, M.2002Highly
Sensitive and Fast Protein Detection with Coomassie
Brilliant Blue in Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis. Bull. Kor. Chem. Soc. 23, 1511-1512.
Kumar, S., Chandra, A., and Pandey, K. C.2008Bacillus
thuringiensisBttransgenic crop: An environment friendly
insect-pest management strategy. J. Environ. Biol. 29,
641-653.
Margalit, J., Becker, N., Back, C., and Zaritsky, A.1995
Bacillus thuringiensis subsp. israelensis as a biological
control agent of mosquitoes and black flies. In Bacillus
thuringiensis: Biotechnology and Environmental Benefits
Feng, T. Y., Chak, K. F., Smith, R. A., Yamamoto, T.,
Margalit, J., Chilcott, C., and Rose, R. I., ed., pp.
521-556, Hua Shiang Yuan Publishing Co, Taipei.
Marrone, P. G., MacIntosh, S. C.1993Resistance to
Bacillus thuringiensis and resistance management. In
Bacillus thuringiensis, An Environmental Biopesticide:

123

Theory and PracticeEntwistle, P. F., Cory, J. S., Bailey, M.


J., and Higgs, S. R., ed.
, pp. 221-235, Wiley, Chichester.
Mizuki, E., Ohba, M., Akao, T., Yamashita, S., Saitoh, H., and
Park, Y. S.1999Unique activity associated with noninsecticidal Bacillus thuringiensis parasporal inclusions: in
vitro cell-killing action on human cancer cells. J. Appl.
Microbiol. 86, 477-486.
Mohan, M., and Gujar, G. T.2000Susceptibility pattern
and development of resistance in the diamondback moth,
Plutella xylostella L, to Bacillus thuringiensis Berl var
kurstaki in India. P. Manag. Sci. 56, 189-194.
Obeidat, M., Hassawi, D., and Ghabeish, I. 2004
Characterization of Bacillus thuringiensis strains from
Jordan and their toxicity to the Lepidoptera, Ephestia
kuehniella Zeller. Afri. J. Biotech. 3, 622-626.
ztrk, F., Ak, L., Ayvaz, A., Bozdoan, B., Suludere, Z.
2009Isolation and characterization of native Bacillus
thuringiensis strains from soil and testing the bioactivity of
isolates Against Ephestia Kuehniella ZellerLepidoptera:
Pyralidaelarvae. Turk. J. Biochem. 33, 202-208.
Poornima, K., Selvanayagam, P., and Shenbagarathai, R.
2010Identication of native Bacillus thuringiensis strain
from South India having specic cytocidal activity against
cancer cells. J. Appl. Microbiol. 109, 348-354.
Rahman, M. M.2000Pesticides: their uses and problems
in context of Bangladesh. IFRB, AERE, Savar, 15-19
October.
Salehi, J. G. R., Komakhin, R. A., and Piruzian, E. S.2005
Comparative study of the expression of the native modied
and hybrid cry3a genes of Bacillus thuringiensis in prokaryotic and eukaryotic cells. Russ. J. Genet. 41, 116-121.
Sambrook, J., and Russel D. W.2001SDS-poly acrylamide
gel electrophoresis of proteins. In Molecular Cloning: A
Laboratory Manual 3, Cold Spring Harbor Laboratory
Press, NY. ISBN 0-87969-577-3.
Sarvjeet, K.2006Molecular approaches for identification
and construction of novel insecticidal genes for crop
protection. W. J. Microbiol. Biotech. 22, 233-253.
Schnepf, E., Crickmore, N., Van, R. J., Lereclus, D., Baum, J.,
Feitelson, J., Zeigler, D. R., and Dean, D. H.1998
Bacillus thuringiensis and its pesticidal crystal proteins.
Microbiol. Mol. Biol. Rev. 62, 775-806.
Sezen, K., Kati, H., Muratoglua, H., and Demirbaga, Z.
2010Characterisation and toxicity of Bacillus thuringiensis strains from hazelnut pests and elds. P. Manag. Sci.
66, 543548.
Shishir, A., Roy, A., Islam, N., Rahman, A., Khan, S. N., and
Hoq, M. M.2014Abundance and diversity of Bacillus
thuringiensis in Bangladesh and their cry genes profile.
Front. Environ. Sci. 2, 20. doi: 10.3389/fenvs.2014.00020
Souane, B., and Cote, J. C.2009Discrimination among
Bacillus thuringiensis H serotypes, serovars and strains
based on 16S rRNA, gyrB and aroE gene sequence analyses. Anton. V. Leeuwen. 95, 33-45.
Travers, R. S., Martin, P. A. W., and Reichelderfer, C. F.,
1987Selective process for efficient isolation of soil
Bacillus spp. Appl. Environ. Microbiol. 56, 1263-1266.
Zahm, S. H., Ward, M. H., and Blair, A.1997Pesticides
and Cancer. In Occupational Medicine: State of the Art
ReviewsKeifer, M., ed., pp. 269-289, Hanley and Belfus
Inc., Philadelphia.