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malaria is caused by a protozoa called plasmodium and spreads among humans by bites from anopheline mosquitoes .
one species that has been identified as a malarial vector on lombok is anopheles aconitus .
the species lives at locations ranging from sea level to 600800  m above sea level .
aconitus can be found on rice fields ( planted and unplanted ) , various shallow pools ( rock , stream , and flood ) , and slow moving streams with grassy margins .
this is commonly done in 3 ways : mosquito larvae control ( using larvicide ) , adult mosquito control ( using adulticide ) , and breeding habitat modification . the most effective approach is mosquito larvae control and this can be accomplished in several ways .
the use of indigenous b. sphaericus is highly desirable as it would build a local capability to produce a biopesticide in developing countries .
however , there is no information on prevalence of environmentally relevant b. sphaericus on lombok nor its potential to be used as a biopesticide .
therefore , studies to reveal indigenous isolate of b. sphaericus are important to the island from both a public health and economic perspectives . in this study , isolates of b. sphaericus were taken from some areas close to beach area and villages known to be endemic to malaria .
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 .
soil samples were homogenized with sterile physiological salt solution forming 10%  w / v suspension .
the suspension was heated to 80c for 30 minutes and then serially diluted with sterile physiological saline solution ( in 10 to 10 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  g / ml streptomycin to avoid unwanted bacteria growth .
colonies that showed gram positive rod with bulging endospore on the terminal end were purified for detailed characterization and toxicity testing .
putative b. 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 .
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 .
larvae resulting from hatched eggs were reared for 6 days to reach 3rd instar stadium ( 3 - 4  mm in length ) .
the b. sphaericus isolates were grown in nysm liquid medium at 30c for 72 hours with 170  rpm shaking .
sixty anopheline larvae ( 60 larvae in 3 containers ) were put into 10%  v / v isolated b. 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 with b. sphaericus 2362 for comparison . 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 . , seven concentrations ( in 10-fold concentration differences with 3 replications ) of chosen isolated b. sphaericus grown in 3  24-hour nysm liquid medium were prepared ( totally there were 21 testing containers ) .
aconitus larvae were distributed evenly in the container ( 20 larvae for each testing container ) .
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 60 an .
mortality rate of anopheline larvae was calculated using this formula:(1)mortality  rate = number  of  dead larvaenumber  of  total  larvae100%.if in the negative control group 520% dead larva are found , abbott 's correction formula   is used to obtain corrected mortality rate:(2)corrected  mortality  rate = mortality  rate  of  test  groupmortality  rate  of  control  group100%mortality  rate  of  control  group100%.lethal concentration ( lc ) values in 24 and 48 hours were calculated using probit analysis   applying software minitab v16 for windows .
the use of b. 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 [ 1113 ] . in this study 20
isolates of b. sphaericus ( and their toxicity attributes ) were found at 20 locations close to beach area around lombok island indonesia as presented in figure 1 .
morphology characteristics of b. sphaericus isolated from lombok island are presented in figure 2 and their characteristics are presented on table 1 .
the characteristics of putative b. sphaericus isolates were in agreement with standard characteristics mentioned in bergey 's manual of determinative bacteriology .
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 toxic b. 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 of b. thuringiensis in israel nagev dessert   and discovery of b. sphaericus in the united states .
those reports had similarities that those entomopathogenic bacteria were isolated from small puddle inhabited by mosquito larvae .
soil is potential habitat for bacillus , 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 toxic b. sphaericus .
it was seen that , from 20 locations explored , there were 10 locations that gave nontoxic b. sphaericus isolates , 7 locations that gave lowly toxic b. sphaericus isolates , and only 3 locations that gave very toxic b. sphaericus isolates .
we suggest that richness in organic matter is not main factor for obtaining such toxic b. 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 three b. sphaericus isolates based on cell concentration ( cell / ml ) are shown in table 2 .
isolates slg and tjl2 showed lc values that were close to those of b. 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 that b. sphaericus isolate mnt was of lower toxicity than b. sphaericus 2362 , whereas b. sphaericus isolates slg and tjl2 had lc values that were almost similar to toxicity of b. sphaericus 2362 .
these b. sphaericus isolates were the first toxic b. sphaericus isolated from lombok island , indonesia .
other entomopathogenic bacterium that was isolated and tested was b. thuringiensis that came from some areas in indonesia .
in contrast , b. sphaericus ' susceptible targets are culex and anopheles , whereas aedes is the least susceptible . the toxicity and lc value of this new isolated b. sphaericus suggest that it would be good candidate for local biocontrol agent on lombok island . 

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 ) .
some strains may have both toxins ; others may have only one or none . that explains varied killing capability among strains of b. sphaericus worldwide . from its low toxicity and higher lc values ( compared to b. 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 as b. thuringiensis , b. sphaericus will last longer in environment ( some study reported 2030 days after application ) .
even though many b. 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 .
twenty local isolates of b. sphaericus were found from 20 locations close to beach area on lombok island with varied toxicity against anopheline larvae .
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 of b. sphaericus that is toxic to mosquito larvae from lombok . 
 the aims of the study were to isolate b. sphaericus from soil in areas close to beach surrounding lombok island and to test their toxicity against 3rd instar anopheles 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 80c 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 ) against an . 
 aconitus . 
 those 3 isolates were potentially useful isolates , as they killed almost all larvae in 24 hours . 
 the discovery of toxic indigenous isolates of b. sphaericus from lombok island opens opportunity to develop a biopesticide from local resources .