# Isolation ofBacillus sphaericus from Lombok Island, Indonesia, and Their Toxicity againstAnopheles aconitus

Authors: Bambang Fajar Suryadi; Bagyo Yanuwiadi; Tri Ardyati; Suharjono
Journal: International Journal of Microbiology
 (2015)

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## Abstract

Malaria is endemic to Lombok Island, Indonesia. One approach to suppress malaria spread is to eliminate anopheline larvae in their habitat and the environmentally safe agent is bacteria, that is,Bacillus sphaericus. However, there is no information regarding local isolate ofB. sphaericus that is toxic to mosquito larvae from Lombok. The aims of the study were to isolateB. sphaericus from soil in areas close to beach surrounding Lombok Island and to test their toxicity against 3rd instarAnopheles aconitus larvae. Soil samples were collected from 20 different sampling locations from Lombok Island and homogenized with sterile physiological salt solution. Suspension was heat-shocked at 80°C for 30 minutes and then spread onto antibiotic-supplemented NYSM solid medium. Colonies grown were characterized and subjected to initial toxicity test against anopheline larvae. Isolates with more than 50% killing percentage were subjected to bioassay testing against anopheline larvae. From 20 locations, 1 isolate showed mild toxicity (namely, isolate MNT) and 2 isolates showed high toxicity (namely, isolates SLG and TJL2) againstAn. aconitus. Those 3 isolates were potentially useful isolates, as they killed almost all larvae in 24 hours. The discovery of toxic indigenous isolates ofB. sphaericus from Lombok Island opens opportunity to develop a biopesticide from local resources.


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## Body

## 1. Introduction

Lombok Island is one island in West Nusa Tenggara Province of Indonesia. One of common infectious diseases on Lombok is malaria. It is predicted that at minimum 13,000 people are suffering from the disease [1]. Malaria is caused by a protozoa calledPlasmodium and spreads among humans by bites from anopheline mosquitoes. One species that has been identified as a malarial vector on Lombok isAnopheles aconitus. The species lives at locations ranging from sea level to 600–800 m above sea level. The larvae ofAn. aconitus can be found on rice fields (planted and unplanted), various shallow pools (rock, stream, and flood), and slow moving streams with grassy margins [2].Mosquito control is the primary method used to suppress the spread of malaria. This is commonly done in 3 ways: mosquito larvae control (using larvicide), adult mosquito control (using adulticide), and breeding habitat modification [3]. The most effective approach is mosquito larvae control and this can be accomplished in several ways. One safe agent for controlling anopheline larvae is bacteria calledBacillus sphaericus [4].The use of indigenousB. sphaericus is highly desirable as it would build a local capability to produce a biopesticide in developing countries. The capability would suppress dependency on imported product and accelerate biopesticide production [5]. However, there is no information on prevalence of environmentally relevantB. sphaericus on Lombok nor its potential to be used as a biopesticide. Therefore, studies to reveal indigenous isolate ofB. sphaericus are important to the island from both a public health and economic perspectives.In this study, isolates ofB. sphaericus were taken from some areas close to beach area and villages known to be endemic to malaria. The isolates were tested againstAn. aconitus larvae that is widely found on the island.


## 2. Material and Methods

### 2.1. Soil Collection

Collection was done at 20 different locations close to beach area surrounding Lombok Island, West Nusa Tenggara, Indonesia. Five hundred grams of soil was collected from each chosen point compositely and stored in sterile screw-capped container. The chosen areas were close to village and/or river opening/estuaries presumably an appropriate location for anopheline breeding habitat.


### 2.2. Bacterial Isolation

Soil samples were homogenized with sterile physiological salt solution forming 10% w/v suspension. The suspension was heated to 80°C for 30 minutes and then serially diluted with sterile physiological saline solution (in 10−1 to 10−5 dilutions). Diluted suspension was spread on NYSM (nutrient agar enriched with 0.5 g/L yeast extract, 0.2 g/L MgCl2, 0.01 g/L MnCl2, and 0.1 g/L CaCl2) plating medium supplemented with 100 μg/mL streptomycin to avoid unwanted bacteria growth [6]. Incubation was done at 30°C for 2 × 24 hours. Colonies that showed Gram positive rod with bulging endospore on the terminal end were purified for detailed characterization and toxicity testing. PutativeB. sphaericus isolates were further characterized using key biochemical/physiological tests such as catalase, oxidase, nitrate reduction, urease, sugar utilization, starch hydrolysis, and antibiotic sensitivity test [7].


### 2.3. Larvae Preparation

Anopheline eggs came from mosquitoes reared intensively in rearing facility at IVRCRD (Institute for Vector and Reservoir Control Research and Development), Salatiga, Central Java, Indonesia. Anopheline eggs were submerged into well water (nontreated water) to hatch them. Larvae resulting from hatched eggs were reared for 6 days to reach 3rd instar stadium (3-4 mm in length).


### 2.4. Initial Toxicity Testing

This testing is done to observe toxicity potential of allB. sphaericus isolates. The procedure was described by Dulmage et al. [8]. TheB. sphaericus isolates were grown in NYSM liquid medium at 30°C for 72 hours with 170 rpm shaking. Sixty anopheline larvae (60 larvae in 3 containers) were put into 10% v/v isolatedB. sphaericus grown on the NYSM liquid medium. Larva death on each test replication was observed and mean value of larva death was calculated. The test was also done withB. sphaericus 2362 for comparison.


### 2.5. Bioassay

In order to obtain LC (lethal concentration) value, bioassay was done on isolates that showed more than 50% toxicity on initial toxicity testing. As mention by Dulmage et al. [8], seven concentrations (in 10-fold concentration differences with 3 replications) of chosen isolatedB. sphaericus grown in 3 × 24-hour NYSM liquid medium were prepared (totally there were 21 testing containers). Four hundred and twentyAn. aconitus larvae were distributed evenly in the container (20 larvae for each testing container). SixtyAn. aconitus larvae in 3 containers (20 larvae per testing container) were mixed with 10% v/v culture medium (without bacteria) as negative control and other 60An. aconitus larvae were mixed with culture ofB. sphaericus 2362 as positive control.Mortality rate of anopheline larvae was calculated using this formula:(1)
Mortality  rate
=
number  of  dead larvae
number  of  total  larvae
×
100
%
.If in the negative control group 5–20% dead larva are found, Abbott’s correction formula [9] is used to obtain corrected mortality rate:(2)
Corrected  mortality  rate
=
Mortality  rate  of  test  group
-
mortality  rate  of  control  group
100
%
-
Mortality  rate  of  control  group
×
100
%
.Lethal concentration (LC) values in 24 and 48 hours were calculated using Probit analysis [10] applying software Minitab V16 for Windows.


## 2.1. Soil Collection

Collection was done at 20 different locations close to beach area surrounding Lombok Island, West Nusa Tenggara, Indonesia. Five hundred grams of soil was collected from each chosen point compositely and stored in sterile screw-capped container. The chosen areas were close to village and/or river opening/estuaries presumably an appropriate location for anopheline breeding habitat.


## 2.2. Bacterial Isolation

Soil samples were homogenized with sterile physiological salt solution forming 10% w/v suspension. The suspension was heated to 80°C for 30 minutes and then serially diluted with sterile physiological saline solution (in 10−1 to 10−5 dilutions). Diluted suspension was spread on NYSM (nutrient agar enriched with 0.5 g/L yeast extract, 0.2 g/L MgCl2, 0.01 g/L MnCl2, and 0.1 g/L CaCl2) plating medium supplemented with 100 μg/mL streptomycin to avoid unwanted bacteria growth [6]. Incubation was done at 30°C for 2 × 24 hours. Colonies that showed Gram positive rod with bulging endospore on the terminal end were purified for detailed characterization and toxicity testing. PutativeB. sphaericus isolates were further characterized using key biochemical/physiological tests such as catalase, oxidase, nitrate reduction, urease, sugar utilization, starch hydrolysis, and antibiotic sensitivity test [7].


## 2.3. Larvae Preparation

Anopheline eggs came from mosquitoes reared intensively in rearing facility at IVRCRD (Institute for Vector and Reservoir Control Research and Development), Salatiga, Central Java, Indonesia. Anopheline eggs were submerged into well water (nontreated water) to hatch them. Larvae resulting from hatched eggs were reared for 6 days to reach 3rd instar stadium (3-4 mm in length).


## 2.4. Initial Toxicity Testing

This testing is done to observe toxicity potential of allB. sphaericus isolates. The procedure was described by Dulmage et al. [8]. TheB. sphaericus isolates were grown in NYSM liquid medium at 30°C for 72 hours with 170 rpm shaking. Sixty anopheline larvae (60 larvae in 3 containers) were put into 10% v/v isolatedB. sphaericus grown on the NYSM liquid medium. Larva death on each test replication was observed and mean value of larva death was calculated. The test was also done withB. sphaericus 2362 for comparison.


## 2.5. Bioassay

In order to obtain LC (lethal concentration) value, bioassay was done on isolates that showed more than 50% toxicity on initial toxicity testing. As mention by Dulmage et al. [8], seven concentrations (in 10-fold concentration differences with 3 replications) of chosen isolatedB. sphaericus grown in 3 × 24-hour NYSM liquid medium were prepared (totally there were 21 testing containers). Four hundred and twentyAn. aconitus larvae were distributed evenly in the container (20 larvae for each testing container). SixtyAn. aconitus larvae in 3 containers (20 larvae per testing container) were mixed with 10% v/v culture medium (without bacteria) as negative control and other 60An. aconitus larvae were mixed with culture ofB. sphaericus 2362 as positive control.Mortality rate of anopheline larvae was calculated using this formula:(1)
Mortality  rate
=
number  of  dead larvae
number  of  total  larvae
×
100
%
.If in the negative control group 5–20% dead larva are found, Abbott’s correction formula [9] is used to obtain corrected mortality rate:(2)
Corrected  mortality  rate
=
Mortality  rate  of  test  group
-
mortality  rate  of  control  group
100
%
-
Mortality  rate  of  control  group
×
100
%
.Lethal concentration (LC) values in 24 and 48 hours were calculated using Probit analysis [10] applying software Minitab V16 for Windows.


## 3. Results and Discussion

The use ofB. sphaericus as a biocontrol agent to suppress anopheline larvae has been done intensively in some countries, such as United States, some European countries, and China. It is primarily used at standing waters, swamps/marshland, paddy fields, and lake shores that are known as primary mosquito breeding habitats [11–13].In this study 20 isolates ofB. sphaericus (and their toxicity attributes) were found at 20 locations close to beach area around Lombok Island Indonesia as presented in Figure 1.Figure 1
Sampling location ofB. sphaericus around Lombok Island (Indonesia) and their toxicity attributes ((−): nontoxic; (+): toxic).Morphology characteristics ofB. sphaericus isolated from Lombok Island are presented in Figure 2 and their characteristics are presented on Table 1.Table 1
Characteristics ofB. sphaericus isolated from Lombok Island.
Characteristics
Results/isolated
Standard
Cell
Form
Rod
Rod
Gram reaction
Positive
Positive
Size (L
×
W)
3.0–5.0 × 0.5–0.75μm
1.5–5.0 × 0.6–1.0μm
Endospore
Positive
Positive
Endospore position
Terminal
Terminal/subterminal
Bulging endosporangium
Positive
Positive
Morphology
Form
Round
Round
Margin
Entire
Entire
Surface
Flat and smooth
Flat and smooth
Color
White-cream
Opaque (grown on nutrient agar)
Biochemical and physiological
Catalase
Positive
Positive
Starch hydrolysis
Negative
Negative
Acid production from sugar
Negative
Negative
Nitrate reduction
Negative
Negative
Urease
Positive
Positive
Oxidase
Positive
Positive
Aerobicity
Aerobe
Aerobe
Sensitivity to streptomycin
Resistant
Resistant
Sensitivity to chloramphenicol
Sensitive
Sensitive
Sensitivity to penicillin
Sensitive
Sensitive
Sensitivity to tetracycline
Sensitive
Sensitive
Sensitivity to amoxicillin
Sensitive
Sensitive
Sensitivity to vancomycin
Sensitive
Sensitive
Sensitivity to erythromycin
Sensitive
Sensitive
Sensitivity to gentamicin
Sensitive
Sensitive
Sensitivity to ciprofloxacin
Sensitive
SensitiveFigure 2
Morphology of colony (a) and cell (b) ofB. sphaericus from Lombok Island in NYSM agar medium on 3 × 24-hour incubation.
(a)
(b)The characteristics of putativeB. sphaericus isolates were in agreement with standard characteristics mentioned inBergey’s Manual of Determinative Bacteriology [7].We have found that all these isolates could be not collected from area exposed to sea water directly (in form of rip-tide or salt dam/pool).B. sphaericus isolates were isolated from sand/soil covered/shaded with leaves and rich of organic matter (grass, fallen leaves, branches, etc.). Some locations formed small puddles, while other locations were dry and/or moist soil.From 20 locations explored, medium and highly toxicB. sphaericus was obtained only from 3 locations. Compared to other locations, these 3 locations were areas that formed small puddles that were rich in organic matter, shaded from the sun, undisturbed by human activity, and inhabited by mosquito larvae, whereas other locations were dry or moist areas (and no mosquito larvae was found). However, the latter were also rich in organic matter, shaded, and untouched. These locations were in accordance with the first discovery ofB. thuringiensis in Israel Nagev Dessert [14] and discovery ofB. sphaericus in the United States [15]. Those reports had similarities that those entomopathogenic bacteria were isolated from small puddle inhabited by mosquito larvae.Soil is potential habitat forBacillus, as soil provides nutrients and growing factors for the bacteria. However, in this study richness in organic matter did not make a given area a suitable habitat for toxicB. sphaericus. It was seen that, from 20 locations explored, there were 10 locations that gave nontoxicB. sphaericus isolates, 7 locations that gave lowly toxicB. sphaericus isolates, and only 3 locations that gave very toxicB. sphaericus isolates. We suggest that richness in organic matter is not main factor for obtaining such toxicB. sphaericus isolates. Contact with mosquito larvae should be taken into consideration and it was shown in some report in early discovery of entomopathogenic bacteria [14, 15].LC50 and LC90 values in 24 and 48 hours of threeB. sphaericus isolates based on cell concentration (cell/mL) are shown in Table 2. Isolate MNT showed higher LC values compared toB. sphaericus 2362 as standard. Isolates SLG and TJL2 showed LC values that were close to those ofB. sphaericus 2362. LC (lethal concentration) value informs us how low the concentration or the dilution of certain microbe or ingredient able to kill targeted organism is. From these values it can be concluded thatB. sphaericus isolate MNT was of lower toxicity thanB. sphaericus 2362, whereasB. sphaericus isolates SLG and TJL2 had LC values that were almost similar to toxicity ofB. sphaericus 2362.Table 2
LC value ofB. sphaericus isolated from Lombok Island against An. aconitus and its comparison with B. sphaericus  2362.
Isolates
LC values (cell/mL)
LC50
-
24
h
r
s
LC90
-
24
h
r
s
LC50
-
48
h
r
s
LC90
-
48
h
r
s
B. sphaericus isolate MNT
1.28 × 108
1.76 × 107
1.98 × 108
4.57 × 107
B. sphaericusisolate SLG
1.51 × 107
3.69 × 106
2.54 × 105
5.45 × 105
B. sphaericus isolate TJL2
1.12 × 106
4.33 × 106
1.01 × 105
4.25 × 105
B. sphaericus 2362 (standard)
1.52 × 106
1.34 × 105
5.64 × 106
5.88 × 105TheseB. sphaericus isolates were the first toxicB. sphaericus isolated from Lombok Island, Indonesia. Other entomopathogenic bacterium that was isolated and tested wasB. thuringiensis that came from some areas in Indonesia.B. thuringiensis’ susceptible targets are larvaeAedes andCulex.Anopheles is the least susceptible to this bacterium. In contrast,B. sphaericus’ susceptible targets areCulex andAnopheles, whereasAedes is the least susceptible. The toxicity and LC value of this new isolatedB. sphaericus suggest that it would be good candidate for local biocontrol agent on Lombok Island.B. sphaericus can kill mosquito larvae because of toxin activities it harbors. There are 2 kinds of toxins: binary toxins/Bin (51 and 42 kDa) are produced on sporulating stage and mosquitocidal toxins/Btx (100, 32 and 36 kDa) are produced on vegetative stage [16, 17]. The binary toxins which are the most potent toxins can interact with receptor along larvae midgut specifically, whereas the mosquitocidal toxins are weaker toxins that will kill the larvae in longer period or will not kill at all (just weaken the larvae) [18]. The activities of the toxins cause nervous and muscle system collapse of the larvae. The larvae will lose its ability to move and consequently undergo asphyxia by drowning [19]. The existence of the toxins varies. Some strains may have both toxins; others may have only one or none. That explains varied killing capability among strains ofB. sphaericus worldwide [20]. From its low toxicity and higher LC values (compared toB. sphaericus 2362), we predict that isolate MNT may only have binary toxins, while other 2 isolates may have binary toxin and mosquitocidal toxin altogether.Compared to other biocontrol bacteria such asB. thuringiensis,B. sphaericus will last longer in environment (some study reported 20–30 days after application) [21]. Also,B. sphaericus is still effective in killing mosquito larvae on polluted waters [22]. These reasons makeB. sphaericus a popular biocontrol agent in some countries.Even though manyB. sphaericus strains from any places in the world have been collected, the existence of indigenous isolates is still important to study, as it will open opportunity to develop local-strain-based biopesticide production in developing countries such as Indonesia. This capability will suppress cost used for importing commercial biopesticide from other countries and also promote local biopesticide industry as well.


## 4. Conclusion

Twenty local isolates ofB. sphaericus were found from 20 locations close to beach area on Lombok Island with varied toxicity against anopheline larvae. Isolate MNT was mildly toxic againstAn. aconitus larvae, while isolates SLG and TJL2 were highly toxic againstAn. aconitus.



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*Source: 854709-2015-12-14.xml*