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 novel mosquito larvicidal bacteria with improved efficacy present significant promise to control vector-borne diseases of public health importance. In the present study, two native bacterial isolates, Bacillus thuringiensis (Bt SV2) and Serratia species (SV6) were evaluated for mosquito larvicidal potential against the early fourth instar larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus with reference to B. thuringiensis subsp. israelensis (Bti) H 14. The native Gram-positive, spore-forming Bt SV2 isolate showed 100% mortality against early fourth instars of Aedes aegypti, Anopheles stephensi, and Culex 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 aegypti, Anopheles stephensi, and Culex quinquefasciatus, respectively. Serratia SV6 showed highest activity against Culex quinquefasciatus (100%) followed by Anopheles stephensi (95%) and Aedes aegypti (91%) after 48 h of exposure. The Gram-negative Serratia SV6 showed delayed toxicity compared to Bti H14 and Bt SV2 against early fourth instars of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. The relative mortality of all treatments after 12-h exposures showed the varied toxicity with respect to exposure time, bacterial treatment, and C. D. Patil : S. V. Patil : B. K. Salunke : R. B. Salunkhe School of Life Sciences, North Maharashtra University, Post Box 80, Jalgaon 425001( Maharashtra, India S. V. Patil (*) : B. K. Salunke North Maharashtra Microbial Culture Collection Centre (NMCC), North Maharashtra University, Post Box 80, Jalgaon 425001( Maharashtra, India e-mail:
[email protected]
mosquito species. Genetic relatedness of the strains was confirmed on the basis of phylogenetic reconstructions based on alignment of 16S rRNA gene sequences which indicated a strong clustering of the strain SV2 with B. thuringiensis and the strain SV6 with Serratia nematodiphila. In conclusion, the native isolate B. thuringiensis SV2 showed significant toxicity while Serratia SV6 showed less and delayed toxicity against several mosquito species compared with BtiH14. They may be used as novel bacterial insecticidal agents in mosquito vector-borne disease control. To our knowledge, this is the first report on mosquito larvicidal potential of Serratia species.
Introduction Several mosquito species belonging to genera Anopheles, Culex, and Aedes are vectors for the pathogens of various diseases like malaria, filariasis, Japanese encephalitis, dengue, yellow fever, chikungunya, etc. (Redwane et al. 2002). Recently, concerns increased with respect to public health and environmental security requiring detection of natural products that may be used against insect pests (Amer and Mehlhorn 2006a). It is known that larvicides 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 Mehlhorn 2006a, b). The common mosquito larvicides, nowadays, include an organophosphate temephos and methoprene, but these have caused their own problems, such as adverse effects on the environment and the buildup of pesticide resistance in some mosquitoes (Su and Mulla 1998). Several insecticides have been withdrawn for economic or regulatory reasons, resulting in
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greater selection pressure and more rapid resistance development to the remaining materials (Kaufman et al. 2011). These problems stimulated a search for safer and effective alternative bioactive larvicidal material. Although various biocontrol measures are in vogue, their effective control of larval mosquitoes has not been hitherto highlighted. Microorganisms and microbial product with potential insecticidal activity can play an important role in controlling diseases by interrupting transmission mechanism by killing insect vectors at community level (Patil et al. 2011a). Worldwide efforts to screen effective entomopathogenic microorganisms for control of agriculturally and medically important insect pests have yielded many Bacillus thuringiensis (Bt) isolates with various insecticidal properties (Feitelson et al. 1992). In the last 20 years, there has been a continuous worldwide search for natural bacterial isolates active against economically and medically important target insects (Martin and Travers 1989; Bernhard et al. 1997). Today, several tens of thousands of isolates, probably more than 50,000 obtained from numerous screening procedures are distributed among various private and public collections, and are considered to be potential reservoirs for novel toxins (Lecadet et al. 1999). The larvicidal bacilli commonly being used for this purpose are B. thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Balaraman 1995). It has long been believed that the occurrence of B. thuringiensis was closely related with insect-breeding environments. However, more recent studies have shown that it is an indigenous bacterium in many ecosystems and is distributed worldwide (Schnepf et al. 1998; Forsyth and Logan 2000). Strains have been isolated from many habitats, including soil (Hossain et al. 1997), stored grains (Chaufaux et al. 1997), insect cadavers (Carozzi et al. 1991), and the phylloplane (Smith and Couche 1991; Damgaard et al. 1998). The bacterium is probably best described as an opportunistic pathogen in insect habitats (Chaufaux et al. 1997; Schnepf et al. 1998). However, most of the commercially available Bt products 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 attributes such as greater field persistence at high temperatures (Brownbridge and Onyango 1992). Currently, several insect pests have developed resistance to Bt, which makes it difficult to control these insect pests with Bt only. Bt tolerance is associated with loss or modification of Bt receptors (Ferré and Van Rie 2002; Gahan et al. 2001), altered proteolysis of Bt toxin(s) (Oppert et al. 1997), and recovery of damaged midgut epithelial cells (Forcada et al. 1999). In previous study, Kim et al. (2009) reported that the Serratia sp. isolate significantly elevated Bt pathogenicity against insect pest Spodoptera exigua. This enhanced
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potency of Bt by the Serratia sp. would be applicable to improve Bt availability to control vectors responsible for diseases of public health importance. Serratia species are a rod-shaped, Gram-negative, facultative bacterium belonging to the Enterobacteriaceae family. Several Serratia strains have been shown to be lethal to insect pests when ingested in high doses; however, in some cases, the strains can be highly virulent at low doses, killing the larvae in 2–3 days with symptoms similar to a virus infection (Steinhaus 1959; Lysyk et al. 2002). Especially, red-colored Serratia biotypes are more often associated with insects than non-pigmented biotypes (Grimont et al. 1977). In our previous study treatment of Aedes aegypti and Anopheles stephensi mosquito larvae with red-colored pigment prodigiosin extracted from Serratia marcescens NMCC46 was produced sublethal to lethal effects, such as reduced adult longevity, larval mortality (Patil et al. 2011b). Potential insecticidal activity of Serratia sp. EML-SE1 was found against diamondback moth (Jeong et al. 2010) Strains of S. marcescens have been reported to be recovered from other tephritids such as Ceratitis capitata Weidermann and Dacus (Bactrocera) dorsalis hendel flies (Grimont et al. 1977), and these bacteria may possess some utility as insect control agents. Also, entomopathogenic strains of Serratia entomophila have been used to control various insect genera including Anomala, Costelytra, and Phyllophaga (Nunez-Valdez et al. 2008). In addition, strains of S. entomophila and S. proteamaculans were shown to kill grass grub, Costelytra zealandica (Jackson et al. 2001; Sikorowski et al. 2001; Nunez-Valdez et al. 2008). These combined results demonstrate that this species acts as a bioinsecticidal agent. In continuation of our previous studies (Patil et al. 2010, 2011a, b; Salunkhe et al. 2011) on searching of insecticidal agents for mosquito vector-borne disease control, this study was to isolate insecticidal B. thuringiensis and Serratia species against the early fourth instar larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus with reference to B. thuringiensis subsp. israelensis H 14.
Material and methods Sample collection Sediment samples were collected from the sites of Jalgaon district as described by Park et al. (2008), with slight modification. The upper crust of sediment in mosquito habitats was chosen as the source of mosquitocidal bacteria because moribund larvae were difficult to obtain from the field. Sediment samples were collected in presterilized containers from the bottom of only stagnant water where the immature stages of mosquitoes were prevalent. Collect-
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ed samples were labeled, brought in the laboratory, and stored at 4°C until used.
16S rRNA identification and phylogenetic affiliation of bacterial strains
Isolation of bacteria
The bacterial cultures were grown overnight on Luria Agar (LA) plates, and genomic DNA was extracted from colonies for each bacteria using phenol–chloroform method (Sambrook et al. 1989). Each DNA sample was PCR amplified (Applied Biosystems) using universal bacterial 16S rRNA gene specific primers 27F/1525R. The amplicon product (approximately 1,500 bp) was verified by agarose gel electrophoresis and purified using PEG–NaCl method (Sambrook et al. 1989). Aliquots of the PCR purified products were distributed to separate sequencing reactions, each containing a primer specific for a region of the 16S rRNA gene in forward or backward direction, to cover the entire gene and sequenced bidirectionally using BigDye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems). Sequences were obtained using an automatic DNA sequencer (3730 DNA analyzer, ABI) and were deposited in the GenBank database. To construct phylogenetic trees, gene sequences generated in this study were aligned with homologous sequences deposited in GenBank for closely related bacteria using ClustalX (version 2.0.9) (Larkin et al. 2007). All sequences were manually edited using DAMBE (Xia and Xie 2001). All uninformative sites were removed from further analysis. The phylogenetic trees were constructed by neighbor-joining method for nucleotide sequences of each dataset using Molecular Evolutionary Genetic Analysis (MEGA) 4.1 (Tamura et al. 2007) with 1,000 bootstrap replications and Kimura 2-parameter as a model of nucleotide substitution.
Two separate isolation strategies were followed for the isolation of Bacillus and Serratia species, respectively. Pasteurized samples were plated on nutrient agar (HiMedia, Mumbai) for isolation of spore-forming Bacillus species. Single colonies were subjected to microscopic observation of bacterial cells including Gram staining and spore staining, isolated, and then cultured for further studies. The medium used for isolation of Serratia sp. was mannitol containing nutrient agar (Patil et al. 2011b).The single red pigmented colonies were preliminarily identified with Gram staining. Isolate was maintained on an agar slant and Petri plate at 4°C. Reference bacterial strain The commercial B. thuringiensis spore-crystal formulation, Vectobac, was used for the isolation of reference strain. B. thuringiensis subsp. israelensis H 14 density was estimated by a standard pour plate method on Nutrient agar (HiMedia, Mumbai). Colonies showing typical colony appearance were maintained on an agar slant and Petri plate at 4°C. Insect rearing and bioassay Mosquito larvae were maintained as per Patil et al. (2011b). For the laboratory trial, identified early fourth instar larvae of Aedes Aegypti, Anopheles stephensi, and Culex quinquefasciatus were obtained from District Malaria Control Department, Jalgaon (21°2′54″ N, 76°32′3″ E; elevation, 209 m). The larvae were kept in plastic enamel trays containing dechlorinated tap water. They were maintained, and all the experiments were carried out at 28±2°C and 75– 85% relative humidity under 14:10 light and dark cycles. Larvae were fed with a diet of finely ground brewer’s yeast and dog biscuits (3:1). To test for potential toxicity of individual isolates, cultures of bacterial isolates were assayed on early fourth instar larvae of Aedes Aegypti, Anopheles stephensi, and Culex quinquefasciatus. Bioassays were performed using 10 ml of 6×105 CFU/ml bacterial cultures for 20 mosquito species in 100 ml of sterile distilled water for checking larval mortality after 24 h of incubation. Each treatment was performed in three replicates each. In all cases, the mortality of control larvae, reared on a bacterial cell free diet (or water medium) and under the same environmental conditions as the experimental larvae, was recorded and calculated by Abbott (1925) formula.
Results The native Gram-positive, spore-forming Bt SV2 isolate showed 100% mortality against early fourth instars of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, in parallel to Bti H14 strain (Table 1). The relative mortality of all treatments after 12 h exposure (Figs. 1, 2, and 3) showed the extent of toxicity with respect to exposure time. After 24 h, Bt SV2 showed 98%, 89%, and 80.67% and Bti H14 showed 92%, 98.33%, and 60% mortality against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, respectively. Interestingly, after 12 h of exposure, Bt SV2 (80.67%) showed significantly higher mortality than Bti H14 (60%) against Culex quinquefasciatus (Fig. 3). The Gram-negative Serratia SV6 showed lower (43– 49%) mortality against tested mosquito species. The toxicity was delayed with Serratia SV6 treatment compared to Bti H14 and Bt SV2 against early fourth instars of Aedes
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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
46.33 (35.99–56.68) 58.33 (47.99–68.68)
92 (73.25–110.8) 98.33 (91.16–105.5)
48 h
Bti H14
Aedes aegypti Anopheles stephensi Culex quinquefasciatus
43.67 (34.26–53.07)
60 (41.25–78.25)
Bt SV2
Aedes aegypti Anopheles stephensi
61.33 (51.29–71.37) 55.67 (47.94–65.39)
98 (91.43–104.6) 89 (79.06–98.94)
Culex quinquefasciatus
67.67 (60.08–75.26)
80.67 (76.87–84.46)
99.33 (96.46–102.2)
100
29 (20.04–37.96) 28 (20.55–35.45)
49 (31.09–66.91) 45.67 (37.68–53.65)
82.67 (72.63–92.71) 69.33 (58.13–80.53)
91 (82.04–99.96) 95 (82.58–107.4)
Serratia SV6
Aedes aegypti Anopheles stephensi Culex quinquefasciatus
24.33 (19.16–29.5)
100 100
100 100
97.33 (91.08–103.6) 100 100
43 (36.43–49.57)
100 100 100
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
100 90 80 70 60 50 40 30 20 10 0
to different S. marcescens and other Serratia strains. The sequences generated during this study have been deposited in the GenBank database and accession numbers are as follows: B. thuringiensis species (JN315886) and Serratia nematodiphila (JN315887) (Fig. 4).
Discussion As a part of our search for the biodiversity resource available in India for natural products with utilizable bioactivity, we have isolated potential mosquito larvicidal bacterial species. Isolates of B. thuringiensis subsp. israelensis (H14) are well known, since 1977 (Goldberg and Margalit 1977), for their activity against mosquitoes. It has been reported that the mortality occurs in mosquito larvae within 12–24 h when treated with vegetative cells of Bti H14 (Walther et al. 1986). However, mortality occurred in Aedes aegypti and Culex quinquefasciatus larvae within early 6 h of exposure of Bt SV2 (Table 1). This may be due to high toxicity and
Mortality (%)
Mortality (%)
aegypti, Anopheles stephensi, and Culex quinquefasciatus which caused 91–100% mortality within 48 h of exposure (Table 1). The comparison of the generated bacterial 16S rRNA gene sequences from this study was analyzed using the standard nucleotide-nucleotide Basic Local Alignment Search Tool (BLAST) program (National Center for Biotechnology Information). BLASTn analysis of the strains from our studies showed 100% and 99% sequence similarities with B. thuringiensis species (HM854748) and Serratia nematodiphila (FJ662869) from GenBank, respectively. Genetic relatedness of the strains was confirmed on the basis of phylogenetic reconstructions based on alignment of gene sequences which indicated a strong clustering of the strain SV2 with B. thuringiensis and the strain SV6 with Serratia nematodiphila (Fig. 4). The isolate SV2 clustered together in the group representing B. thuringiensis (HM854748) and formed sister cluster with B. thuringiensis (EU240371) divergent alongside other B. thuringiensis strains (FJ772071 and EU168410). The strain SV6 clustered together with Serratia nematodiphila (FJ662869) divergent
Bti H14
Bt SV2
Serratia SV6
Fig. 1 The relative mortality of Aedes aegypti larvae after 12 h of Bti H14, Bt SV2, and Serratia SV6 treatment
100 90 80 70 60 50 40 30 20 10 0 Bti H14
Bt SV2
Serratia SV6
Fig. 2 The relative mortality of Anopheles stephensi larvae after 12 h of Bti H14, Bt SV2, and Serratia SV6 treatment
Mortality (%)
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100 90 80 70 60 50 40 30 20 10 0 Bti H14
Bt SV2
Serratia SV6
Fig. 3 The relative mortality of Culex quinquefasciatus larvae after 12 h of Bti H14, Bt SV2, and Serratia SV6 treatment
sporulating capacity within short time of our isolate than the Bti H14. The localization of larvicidal potential within cell and ability of vegetative cells to sporulate during the bioassay results in to larval death (Walther et al. 1986). Goldberg and Margalit (1977) isolated a Bacillus strain extremely toxic to mosquito larvae from Israel. On the basis of this isolate, de Barjac (1978) established B. thuringiensis serovar israelensis (flagellar antigen H14). The discovery has been followed by the isolation of several highly mosquitocidal B. thuringiensis strains, belonging to other H serotypes, from Tropic Asia (Padua et al. 1984; Seleena et al. 1995) and Central and South America (Lo’pez-Meza et al. 1995; Orduz et al. 1992). Recently, Ragni et al. (1996) have reported that a strain belonging to the B. thuringiensis serovar canadensis originated from the soil of Iraq and 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 phylogenetic 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
exhibited a strong larvicidal activity specific for mosquitoes. This trend of discovery and practical application of bacterial agents against mosquitoes led to search native bacterial isolates for the effective control of insect pest. Several other researchers reported potential efficacy of commercial microbial formulations containing B. sphaericus (Bs), B. thuringiensis subsp. israelensis (Bti), and combination (Bti/Bs) against larval instars of Aedes, Anopheles, and Culex in laboratory as well as field conditions (Mwangangi et al. 2011; Chenniappan and Ayyadurai 2011; Singh and Prakash 2009). These studies demonstrate that each mosquito species responds differently to the microbial agent and that mortalities were dependent on dose, age of larvae, time of exposure, level of insecticide resistance, and combination of active material in the treatment. Kovendan et al. (2011a, b) recently reported the excellent potential of B. sphaericus for larvae of malarial vector, Anopheles stephensi (0.051%, 0.057%, 0.062%, and 0.066% for I–IV instar, respectively), and B. thuringiensis israelensis against the first to fourth instar larvae of Culex quinquefasciatus of values LC 50 = 9.332%, 9.832%, 10.212%, and 10.622%, and LC90 =15.225%, 15.508%, 15.887%, and 15.986% larvae. The various reports on insecticidal potential of Serratia species against agriculture pest (Grimont and Grimont 1978; Nunez-Valdez et al. 2008; Kim et al.. 2009) prompted us to investigate insecticidal potential of Serratia species against mosquito species. Jeong et al. (2010)
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observed the 88.3–91.7% mortality against the diamondback moth larva with the treatment of Serratia sp. EMLSE1 after 72 h. Serratia species secretes many known extracellular proteins, including nuclease, phospholipase, hemolysin, siderophore, chitinase, protease, and lipase (Hines et al. 1988; Aucken and Pitt 1998). Because these extracellular factors are hydrolytic in nature, it is reasonable to hypothesize that one or more of the factors may directly contribute to insecticidal activity. Alternatively, an unidentified factor may be responsible for killing of mosquito larvae. Patil et al. (2011b) reported the mosquito larvicidal potential of S. marcescens NMCC46 pigment prodigiosin. However, in the present study, active pigmented culture is used for the bioassay. Although several Serratia spp. have been found to be human opportunistic pathogens with a broad host range (Kurz et al. 2003), they are also the most often reported insect pathogens. Therefore, in order to determine whether toxicity of Serratia comes from other unknown metabolite and/or compounds, further study is underway. The results of this study showed the possibilities of highly mosquito larvicidal native bacteria in mosquito larval habitats. However, as liquid culture of bacterial isolates were used for our preliminary bioassay, therefore advanced bioassay using lyophilized powder of bacterial culture along with other molecular biological analysis is under way to determine median lethal concentrations (LC50s) and other characteristics of these isolates. In addition to this, more studies on the isolation of the active compounds, a possible mechanism of effect, and studies on non-target organisms are necessary. In conclusion, the native isolates B. thuringiensis SV2 and Serratia nematodiphila SV6 both showed significant toxicity against several mosquito species compared with BtiH14. They may be used as novel bacterial insecticidal agents in mosquito vector-borne disease control. Acknowledgments Authors would like to express their deep gratitude to Dr. Yogesh S. Shouche, Scientist F, National Centre for Cell Sciences (NCCS), Pune, India for 16S rRNA identification of bacterial samples. Conflict of interest conflict of interest.
The authors declare that they do not have any
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