Parasitol Res (2012) 110:1841–1847

DOI 10.1007/s00436-011-2708-6

ORIGINAL PAPER

Insecticidal potency of bacterial species Bacillus thuringiensis

SV2 and Serratia nematodiphila SV6 against larvae
of mosquito species Aedes aegypti, Anopheles stephensi,
and Culex quinquefasciatus
Chandrashekhar D. Patil & Satish V. Patil &
Bipinchandra K. Salunke & Rahul B. Salunkhe

Received: 22 July 2011 / Accepted: 20 October 2011 / Published online: 9 November 2011
# Springer-Verlag 2011

Abstract The tremendous worldwide efforts to isolate mosquito species. Genetic relatedness of the strains was
novel mosquito larvicidal bacteria with improved efficacy confirmed on the basis of phylogenetic reconstructions
present significant promise to control vector-borne diseases based on alignment of 16S rRNA gene sequences which
of public health importance. In the present study, two native indicated a strong clustering of the strain SV2 with B.
bacterial isolates, Bacillus thuringiensis (Bt SV2) and thuringiensis and the strain SV6 with Serratia nematodi-
Serratia species (SV6) were evaluated for mosquito phila. In conclusion, the native isolate B. thuringiensis SV2
larvicidal potential against the early fourth instar larvae of showed significant toxicity while Serratia SV6 showed less
Aedes aegypti, Anopheles stephensi, and Culex quinque- and delayed toxicity against several mosquito species
fasciatus with reference to B. thuringiensis subsp. israel- compared with BtiH14. They may be used as novel
ensis (Bti) H 14. The native Gram-positive, spore-forming bacterial insecticidal agents in mosquito vector-borne
Bt SV2 isolate showed 100% mortality against early fourth disease control. To our knowledge, this is the first report
instars of Aedes aegypti, Anopheles stephensi, and Culex on mosquito larvicidal potential of Serratia species.
quinquefasciatus, in parallel to Bti H14 strain. After 24 h,
Bt SV2 showed 98%, 89%, and 80.67%, and Bti H14
showed 92%, 98.33%, and 60% mortality against Aedes Introduction
aegypti, Anopheles stephensi, and Culex quinquefasciatus,
respectively. Serratia SV6 showed highest activity against Several mosquito species belonging to genera Anopheles,
Culex quinquefasciatus (100%) followed by Anopheles Culex, and Aedes are vectors for the pathogens of various
stephensi (95%) and Aedes aegypti (91%) after 48 h of diseases like malaria, filariasis, Japanese encephalitis,
exposure. The Gram-negative Serratia SV6 showed dengue, yellow fever, chikungunya, etc. (Redwane et al.
delayed toxicity compared to Bti H14 and Bt SV2 against 2002). Recently, concerns increased with respect to public
early fourth instars of Aedes aegypti, Anopheles stephensi, health and environmental security requiring detection of
and Culex quinquefasciatus. The relative mortality of all natural products that may be used against insect pests
treatments after 12-h exposures showed the varied toxicity (Amer and Mehlhorn 2006a). It is known that larvicides
with respect to exposure time, bacterial treatment, and play a vital role in controlling mosquitoes in their
breeding sites as mosquitoes breed in water, and thus,
it is easy to deal with them in this habitat (Amer and
C. D. Patil : S. V. Patil : B. K. Salunke : R. B. Salunkhe
Mehlhorn 2006a, b). The common mosquito larvicides,
School of Life Sciences, North Maharashtra University,
Post Box 80, Jalgaon 425001( Maharashtra, India nowadays, include an organophosphate temephos and
methoprene, but these have caused their own problems,
S. V. Patil (*) : B. K. Salunke such as adverse effects on the environment and the
North Maharashtra Microbial Culture Collection Centre (NMCC),
buildup of pesticide resistance in some mosquitoes (Su
North Maharashtra University,
Post Box 80, Jalgaon 425001( Maharashtra, India and Mulla 1998). Several insecticides have been with-
e-mail: satish.patil7@gmail.com drawn for economic or regulatory reasons, resulting in
1842 Parasitol Res (2012) 110:1841–1847

greater selection pressure and more rapid resistance potency of Bt by the Serratia sp. would be applicable to
development to the remaining materials (Kaufman et al. improve Bt availability to control vectors responsible for
2011). diseases of public health importance.
These problems stimulated a search for safer and Serratia species are a rod-shaped, Gram-negative,
effective alternative bioactive larvicidal material. Although facultative bacterium belonging to the Enterobacteriaceae
various biocontrol measures are in vogue, their effective family. Several Serratia strains have been shown to be
control of larval mosquitoes has not been hitherto high- lethal to insect pests when ingested in high doses; however,
lighted. Microorganisms and microbial product with poten- in some cases, the strains can be highly virulent at low
tial insecticidal activity can play an important role in doses, killing the larvae in 2–3 days with symptoms similar
controlling diseases by interrupting transmission mecha- to a virus infection (Steinhaus 1959; Lysyk et al. 2002).
nism by killing insect vectors at community level (Patil et Especially, red-colored Serratia biotypes are more often
al. 2011a). Worldwide efforts to screen effective entomo- associated with insects than non-pigmented biotypes
pathogenic microorganisms for control of agriculturally and (Grimont et al. 1977). In our previous study treatment of
medically important insect pests have yielded many Aedes aegypti and Anopheles stephensi mosquito larvae
Bacillus thuringiensis (Bt) isolates with various insecticidal with red-colored pigment prodigiosin extracted from Ser-
properties (Feitelson et al. 1992). In the last 20 years, there ratia marcescens NMCC46 was produced sublethal to
has been a continuous worldwide search for natural lethal effects, such as reduced adult longevity, larval
bacterial isolates active against economically and medically mortality (Patil et al. 2011b). Potential insecticidal activity
important target insects (Martin and Travers 1989; of Serratia sp. EML-SE1 was found against diamondback
Bernhard et al. 1997). Today, several tens of thousands of moth (Jeong et al. 2010) Strains of S. marcescens have been
isolates, probably more than 50,000 obtained from numer- reported to be recovered from other tephritids such as
ous screening procedures are distributed among various Ceratitis capitata Weidermann and Dacus (Bactrocera)
private and public collections, and are considered to be dorsalis hendel flies (Grimont et al. 1977), and these
potential reservoirs for novel toxins (Lecadet et al. 1999). bacteria may possess some utility as insect control agents.
The larvicidal bacilli commonly being used for this purpose Also, entomopathogenic strains of Serratia entomophila
are B. thuringiensis subsp. israelensis (Bti) and Bacillus have been used to control various insect genera including
sphaericus (Balaraman 1995). It has long been believed Anomala, Costelytra, and Phyllophaga (Nunez-Valdez et al.
that the occurrence of B. thuringiensis was closely related 2008). In addition, strains of S. entomophila and S.
with insect-breeding environments. However, more recent proteamaculans were shown to kill grass grub, Costelytra
studies have shown that it is an indigenous bacterium in zealandica (Jackson et al. 2001; Sikorowski et al. 2001;
many ecosystems and is distributed worldwide (Schnepf et Nunez-Valdez et al. 2008). These combined results dem-
al. 1998; Forsyth and Logan 2000). Strains have been onstrate that this species acts as a bioinsecticidal agent.
isolated from many habitats, including soil (Hossain et al. In continuation of our previous studies (Patil et al. 2010,
1997), stored grains (Chaufaux et al. 1997), insect cadavers 2011a, b; Salunkhe et al. 2011) on searching of insecticidal
(Carozzi et al. 1991), and the phylloplane (Smith and agents for mosquito vector-borne disease control, this study
Couche 1991; Damgaard et al. 1998). The bacterium is was to isolate insecticidal B. thuringiensis and Serratia
probably best described as an opportunistic pathogen in species against the early fourth instar larvae of Aedes
insect habitats (Chaufaux et al. 1997; Schnepf et al. 1998). aegypti, Anopheles stephensi, and Culex quinquefasciatus
However, most of the commercially available Bt products with reference to B. thuringiensis subsp. israelensis H 14.
have their strains from temperate zones and their success in
the tropics has not been as expected. This has necessitated
search for effective local isolates that possess useful Material and methods
attributes such as greater field persistence at high temper-
atures (Brownbridge and Onyango 1992). Currently, several Sample collection
insect pests have developed resistance to Bt, which makes it
difficult to control these insect pests with Bt only. Bt Sediment samples were collected from the sites of Jalgaon
tolerance is associated with loss or modification of Bt district as described by Park et al. (2008), with slight
receptors (Ferré and Van Rie 2002; Gahan et al. 2001), modification. The upper crust of sediment in mosquito
altered proteolysis of Bt toxin(s) (Oppert et al. 1997), and habitats was chosen as the source of mosquitocidal bacteria
recovery of damaged midgut epithelial cells (Forcada et al. because moribund larvae were difficult to obtain from the
1999). In previous study, Kim et al. (2009) reported that the field. Sediment samples were collected in presterilized
Serratia sp. isolate significantly elevated Bt pathogenicity containers from the bottom of only stagnant water where
against insect pest Spodoptera exigua. This enhanced the immature stages of mosquitoes were prevalent. Collect-
Parasitol Res (2012) 110:1841–1847 1843

ed samples were labeled, brought in the laboratory, and 16S rRNA identification and phylogenetic affiliation
stored at 4°C until used. of bacterial strains

Isolation of bacteria The bacterial cultures were grown overnight on Luria

Agar (LA) plates, and genomic DNA was extracted from
Two separate isolation strategies were followed for the colonies for each bacteria using phenol–chloroform
isolation of Bacillus and Serratia species, respectively. method (Sambrook et al. 1989). Each DNA sample was
Pasteurized samples were plated on nutrient agar PCR amplified (Applied Biosystems) using universal
(HiMedia, Mumbai) for isolation of spore-forming Bacillus bacterial 16S rRNA gene specific primers 27F/1525R.
species. Single colonies were subjected to microscopic The amplicon product (approximately 1,500 bp) was
observation of bacterial cells including Gram staining and verified by agarose gel electrophoresis and purified using
spore staining, isolated, and then cultured for further PEG–NaCl method (Sambrook et al. 1989). Aliquots of
studies. the PCR purified products were distributed to separate
The medium used for isolation of Serratia sp. was sequencing reactions, each containing a primer specific for a
mannitol containing nutrient agar (Patil et al. 2011b).The region of the 16S rRNA gene in forward or backward
single red pigmented colonies were preliminarily identified direction, to cover the entire gene and sequenced bidirection-
with Gram staining. Isolate was maintained on an agar slant ally using BigDye Terminator Cycle Sequencing kit version
and Petri plate at 4°C. 3.1 (Applied Biosystems). Sequences were obtained using an
automatic DNA sequencer (3730 DNA analyzer, ABI) and
Reference bacterial strain were deposited in the GenBank database.
To construct phylogenetic trees, gene sequences
The commercial B. thuringiensis spore-crystal formulation, generated in this study were aligned with homologous
Vectobac, was used for the isolation of reference strain. B. sequences deposited in GenBank for closely related
thuringiensis subsp. israelensis H 14 density was estimated bacteria using ClustalX (version 2.0.9) (Larkin et al.
by a standard pour plate method on Nutrient agar (HiMedia, 2007). All sequences were manually edited using DAMBE
Mumbai). Colonies showing typical colony appearance (Xia and Xie 2001). All uninformative sites were removed
were maintained on an agar slant and Petri plate at 4°C. from further analysis. The phylogenetic trees were
constructed by neighbor-joining method for nucleotide
Insect rearing and bioassay sequences of each dataset using Molecular Evolutionary
Genetic Analysis (MEGA) 4.1 (Tamura et al. 2007) with
Mosquito larvae were maintained as per Patil et al. (2011b). 1,000 bootstrap replications and Kimura 2-parameter as a
For the laboratory trial, identified early fourth instar larvae model of nucleotide substitution.
of Aedes Aegypti, Anopheles stephensi, and Culex quinque-
fasciatus were obtained from District Malaria Control
Department, Jalgaon (21°2′54″ N, 76°32′3″ E; elevation, Results
209 m). The larvae were kept in plastic enamel trays
containing dechlorinated tap water. They were maintained, The native Gram-positive, spore-forming Bt SV2 isolate
and all the experiments were carried out at 28±2°C and 75– showed 100% mortality against early fourth instars of
85% relative humidity under 14:10 light and dark cycles. Aedes aegypti, Anopheles stephensi, and Culex quinque-
Larvae were fed with a diet of finely ground brewer’s yeast fasciatus, in parallel to Bti H14 strain (Table 1). The
and dog biscuits (3:1). relative mortality of all treatments after 12 h exposure
To test for potential toxicity of individual isolates, (Figs. 1, 2, and 3) showed the extent of toxicity with respect
cultures of bacterial isolates were assayed on early fourth to exposure time. After 24 h, Bt SV2 showed 98%, 89%,
instar larvae of Aedes Aegypti, Anopheles stephensi, and and 80.67% and Bti H14 showed 92%, 98.33%, and 60%
Culex quinquefasciatus. Bioassays were performed using mortality against Aedes aegypti, Anopheles stephensi, and
10 ml of 6×105 CFU/ml bacterial cultures for 20 mosquito Culex quinquefasciatus, respectively. Interestingly, after
species in 100 ml of sterile distilled water for checking 12 h of exposure, Bt SV2 (80.67%) showed significantly
larval mortality after 24 h of incubation. higher mortality than Bti H14 (60%) against Culex
Each treatment was performed in three replicates each. quinquefasciatus (Fig. 3).
In all cases, the mortality of control larvae, reared on a The Gram-negative Serratia SV6 showed lower (43–
bacterial cell free diet (or water medium) and under the 49%) mortality against tested mosquito species. The
same environmental conditions as the experimental larvae, toxicity was delayed with Serratia SV6 treatment compared
was recorded and calculated by Abbott (1925) formula. to Bti H14 and Bt SV2 against early fourth instars of Aedes
1844 Parasitol Res (2012) 110:1841–1847

Table 1 Mosquito larvicidal activity of Bt SV2 and Serratia SV6 in comparison to B. thuringiensis subsp. israelensis H14

Bacterial species Mosquito species Mortalitya (%)

6h 12 h 24 h 48 h

Bti H14 Aedes aegypti 46.33 (35.99–56.68) 92 (73.25–110.8) 100 100

Anopheles stephensi 58.33 (47.99–68.68) 98.33 (91.16–105.5) 100 100
Culex quinquefasciatus 43.67 (34.26–53.07) 60 (41.25–78.25) 97.33 (91.08–103.6) 100
Bt SV2 Aedes aegypti 61.33 (51.29–71.37) 98 (91.43–104.6) 100 100
Anopheles stephensi 55.67 (47.94–65.39) 89 (79.06–98.94) 100 100
Culex quinquefasciatus 67.67 (60.08–75.26) 80.67 (76.87–84.46) 99.33 (96.46–102.2) 100
Serratia SV6 Aedes aegypti 29 (20.04–37.96) 49 (31.09–66.91) 82.67 (72.63–92.71) 91 (82.04–99.96)
Anopheles stephensi 28 (20.55–35.45) 45.67 (37.68–53.65) 69.33 (58.13–80.53) 95 (82.58–107.4)
Culex quinquefasciatus 24.33 (19.16–29.5) 43 (36.43–49.57) 62 (18.05–105.09) 100

A value in parenthesis shows lower confidence limit and upper confidence limit at 95% confidence limit. Control shows nil mortality
a
Mean of three replicates

aegypti, Anopheles stephensi, and Culex quinquefasciatus to different S. marcescens and other Serratia strains. The
which caused 91–100% mortality within 48 h of exposure sequences generated during this study have been deposited in
(Table 1). the GenBank database and accession numbers are as follows:
The comparison of the generated bacterial 16S rRNA B. thuringiensis species (JN315886) and Serratia nematodi-
gene sequences from this study was analyzed using the phila (JN315887) (Fig. 4).
standard nucleotide-nucleotide Basic Local Alignment
Search Tool (BLAST) program (National Center for
Biotechnology Information). BLASTn analysis of the strains Discussion
from our studies showed 100% and 99% sequence
similarities with B. thuringiensis species (HM854748) and As a part of our search for the biodiversity resource
Serratia nematodiphila (FJ662869) from GenBank, respec- available in India for natural products with utilizable
tively. Genetic relatedness of the strains was confirmed on bioactivity, we have isolated potential mosquito larvicidal
the basis of phylogenetic reconstructions based on align- bacterial species.
ment of gene sequences which indicated a strong clustering Isolates of B. thuringiensis subsp. israelensis (H14) are
of the strain SV2 with B. thuringiensis and the strain SV6 well known, since 1977 (Goldberg and Margalit 1977), for
with Serratia nematodiphila (Fig. 4). The isolate SV2 their activity against mosquitoes. It has been reported that
clustered together in the group representing B. thuringiensis the mortality occurs in mosquito larvae within 12–24 h
(HM854748) and formed sister cluster with B. thuringiensis when treated with vegetative cells of Bti H14 (Walther et al.
(EU240371) divergent alongside other B. thuringiensis 1986). However, mortality occurred in Aedes aegypti and
strains (FJ772071 and EU168410). The strain SV6 clustered Culex quinquefasciatus larvae within early 6 h of exposure
together with Serratia nematodiphila (FJ662869) divergent of Bt SV2 (Table 1). This may be due to high toxicity and

100 100
90 90
80 80
Mortality (%)

70
Mortality (%)

70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
Bti H14 Bt SV2 Serratia SV6 Bti H14 Bt SV2 Serratia SV6

Fig. 1 The relative mortality of Aedes aegypti larvae after 12 h of Bti Fig. 2 The relative mortality of Anopheles stephensi larvae after 12 h
H14, Bt SV2, and Serratia SV6 treatment of Bti H14, Bt SV2, and Serratia SV6 treatment
Parasitol Res (2012) 110:1841–1847 1845

100
90
exhibited a strong larvicidal activity specific for mosqui-
80 toes. This trend of discovery and practical application of
Mortality (%)
70 bacterial agents against mosquitoes led to search native
60 bacterial isolates for the effective control of insect pest.
50
Several other researchers reported potential efficacy of
40
30 commercial microbial formulations containing B. sphaer-
20 icus (Bs), B. thuringiensis subsp. israelensis (Bti), and
10 combination (Bti/Bs) against larval instars of Aedes,
0 Anopheles, and Culex in laboratory as well as field
Bti H14 Bt SV2 Serratia SV6
conditions (Mwangangi et al. 2011; Chenniappan and
Fig. 3 The relative mortality of Culex quinquefasciatus larvae after Ayyadurai 2011; Singh and Prakash 2009). These studies
12 h of Bti H14, Bt SV2, and Serratia SV6 treatment demonstrate that each mosquito species responds differently
to the microbial agent and that mortalities were dependent
sporulating capacity within short time of our isolate than on dose, age of larvae, time of exposure, level of insecticide
the Bti H14. The localization of larvicidal potential within resistance, and combination of active material in the
cell and ability of vegetative cells to sporulate during the treatment. Kovendan et al. (2011a, b) recently reported the
bioassay results in to larval death (Walther et al. 1986). excellent potential of B. sphaericus for larvae of malarial
Goldberg and Margalit (1977) isolated a Bacillus strain vector, Anopheles stephensi (0.051%, 0.057%, 0.062%, and
extremely toxic to mosquito larvae from Israel. On the basis 0.066% for I–IV instar, respectively), and B. thuringiensis
of this isolate, de Barjac (1978) established B. thuringiensis israelensis against the first to fourth instar larvae of Culex
serovar israelensis (flagellar antigen H14). The discovery quinquefasciatus of values LC 50 = 9.332%, 9.832%,
has been followed by the isolation of several highly 10.212%, and 10.622%, and LC90 =15.225%, 15.508%,
mosquitocidal B. thuringiensis strains, belonging to other 15.887%, and 15.986% larvae.
H serotypes, from Tropic Asia (Padua et al. 1984; Seleena The various reports on insecticidal potential of Serratia
et al. 1995) and Central and South America (Lo’pez-Meza species against agriculture pest (Grimont and Grimont
et al. 1995; Orduz et al. 1992). Recently, Ragni et al. (1996) 1978; Nunez-Valdez et al. 2008; Kim et al.. 2009)
have reported that a strain belonging to the B. thuringiensis prompted us to investigate insecticidal potential of Serratia
serovar canadensis originated from the soil of Iraq and species against mosquito species. Jeong et al. (2010)

Fig. 4 Phylogenetic relationships

between our isolates (bold) a B.
thuringiensis SV2 and b Serratia
nematodiphila SP6 and those
retrieved from GenBank (NCBI
database), based on bacterial 16S
rRNA gene. Names are those of
the bacterial species. The phylo-
genetic trees were constructed by
neighbor-joining method using
Kimura 2-parameter distance
(1,000 bootstrap) in MEGA v4.1.
Accession numbers are shown
before each species name in
parenthesis
1846 Parasitol Res (2012) 110:1841–1847

observed the 88.3–91.7% mortality against the diamond- Aucken HM, Pitt TL (1998) Antibiotic resistance and putative
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Pauli S, Rodgers P, Burges HD (1997) Natural isolates of
extracellular factors are hydrolytic in nature, it is reasonable to Bacillus thuringiensis: worldwide distribution, characterization,
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contribute to insecticidal activity. Alternatively, an unidenti- Brownbridge M, Onyango T (1992) Screening of exotic and locally
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of S. marcescens NMCC46 pigment prodigiosin. However, Carozzi NB, Kramer VC, Warren GW, Evola S, Koziel MG (1991)
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