Biocontrol Potential of Bacillus thuringiensis Isolated From Soil Samples Against Mosquito Larvae
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BIOCONTROL POTENTIAL OF Bacillus thuringiensis ISOLATED FROM SOIL SAMPLES AGAINST MOSQUITO LARVAE.
1Najibullah B. A., 1Abba H. A. and *1Gashua I. B.
1Department of Science Laboratory Technology, School of Science and Technology, Federal Polytechnic Damaturu.
*Corresponding Email: bgashua@gmail.com
(+234)8164313012
ABSTRACT
Mosquitoes as vectors have been transmitting several etiological agents of important human diseases, including malaria, causing millions of deaths every year. Overcoming insecticide resistance becomes a lingering challenge for recording a successful Mosquito control. In this research work, Larvicidal potential (LP) of Bacillus thuringiensis isolated from different soil samples were tested as a control strategy of Mosquitoes and monitoring of larvae susceptibility. Mosquito larvae were assessed by isolation from different soil habitats in Yobe State. Confirmation of the isolated organisms to be Bacillus thuringiensis was based on biochemical characterization and microscopic observation. Two out of the five isolates of Bacillus thuringiensis obtained from the soil samples labeled P1 and Ga2 were used for carrying the study. At the intervals of 4, 12 and 24 hours, the Larvicidal activity (LA) on the mosquito larvae (which were measured by mortality rate and change in morphology of the larvae) were observed and recorded at 10-1, 10-2, 10-3, 10-4, and 10-5 dilution factors. The isolates of Bacillus thuringiensis showed a slight level of variation in their LA. Both isolates P1 and Ga2 caused 100% mortality of the larvae at the highest concentration of 10-1 at 4 hours while 100% mortality was recorded in other dilution factors at 12 hours. From this study, it is concluded that Bacillus thuringiensis is a very potent biolarvicide that brings about mortality of mosquito larvae at a short duration of time.
Key words: Mosquitoes, Bacillus thuringiensis, Malaria, Mortality, Biolarvicide.
INTRODUCTION
Worldwide, mosquito vectors are transmitting several etiological agents of important human diseases, including malaria, causing millions of deaths every year (El-Kersh et al., 2016). Mosquito borne diseases such as Malaria, Filariasis, Yellow fever and Dengue cause extensive morbidity and mortality and are a major economic burden within disease-endemic countries (Sachs and Malaney, 2002; Boutayeb, 2006). Every year, about 300 million people are estimated to be affected by Malaria, a major killer disease, which threatens 2,400 million (about 40%) of the world’s population (Sharma, 1999; Snow et al., 2005). About 20 million people are infected every year by dengue viruses transmitted by Aedes mosquitoes with about 24,000deaths. The incidence of mosquito-borne diseases is increasing due to uncontrolled urbanization, creating mosquito-genic conditions for the vector mosquito populations. Therefore, mosquito control forms an essential component for the control of mosquito borne diseases.
However, of all the microbial agents, Bacillus thuringiensis has been successfully used as a bio-control agent. This bacterium is widely distributed in the environment and can be isolated from soil, insects, agricultural environments and leaves of certain deciduous and coniferous trees (Jara et al., 2006). B. thuringiensis is a spore-forming, gram-positive bacterium which could be distinguished by production of one or more proteinaceous parasporal crystals (δ-endotoxin) during sporulation (Lacey et al., 2001; Kumar, 2002). In certain strains, the delta-endotoxin proteins are toxic to members of specific insect genera and this has led to commercial development and use of some strains as microbial insecticides. Morphological and biochemical techniques have been used to differentiate newly isolated B. thuringiensis strains obtained from a variety of sources (Schnepf et al., 2005).
Due to their high specificity and their safety to most non-target organisms and to the environment in general, Bacillus thuringiensis crystal proteins are preferred and widely used as an alternative to chemical pesticides in pest management strategies against insect pests of agricultural crops (Roh et al., 2007; van Frankenhuyzen, 2009).
In addition, Bt. strains produce a wide variety of insecticidal proteins active against larvae of very diverse insect orders as well as, in some cases, against species from other phyla. This has led Bt-based products to become the best-selling biological insecticides to date (Roh et al., 2007). Since the genes encoding insecticidal proteins have been successfully used in novel insecticidal formulations and in the construction of transgenic crops (Sanchis, 2011).
Malaria vector control relies mostly on the use of an effective insecticide, which is commonly used through indoor residual spraying (IRS) or community-based deployment of insecticide impregnated/treated bed nets (ITN).
Chemical insecticides provide many benefits to food production and human health and have proven very effective at increasing agriculture and forestry productivities. However, they also pose some hazards as contamination of water and food sources, poisoning of non-target fauna and flora, concentration in the food chain and selection of insect pest populations resistant to the chemical insecticides (Wojciech and Korsten 2002). However, uncontrolled use of chemical insecticides has resulted in irreparable damage to environment (El-Kersh et al., 2012).
Radhika et al. (2011) reported that repetitive use of man-made insecticides for mosquito control disrupts natural ecosystems leading to reemergence of, and increase in mosquito populations. In their studies, Das et al. (2007) and Zhang et al. (2011) also pointed out that the continuous use of chemical-based insecticides has resulted in the development of resistance, detrimental effects on non-target organisms and human health problems. Consequently, they suggested the need for alternative control measures which leaves biological control as a viable alternative to chemical control. Microbial based insecticides are especially valuable because their toxicity to non-target animals and humans is extremely low and a crucial part of integrated pest management (Aramideh et al., 2010; El-kersh et al., 2012). Compared to other commonly used insecticides, they are safe for both the pesticide user and consumers of treated crops.
Interestingly, Bacillus thuringiensis is an important insect pathogen which is highly toxic to mosquito larvae and related dipterans (Poopathi and Abidha, 2010; Zulfaidah, et al., 2013). Bacillus thuringiensis is selectively active on pests and less likely to cause resistance hence it is considered beneficial to humans, animals and plants and also as a suitable replacement to chemical pesticides in many countries.
Thus, it is obvious that Bacillus thuringiensis is widespread in nature. However, the normal habitat of the organism is soil. The organism grows naturally as a saprophyte, feeding on dead organic matter. Therefore, the spores of Bacillus thuringiensis persist in soil and its vegetative growth occurs when there is nutrient available. Moreover Bacillus thuringiensis has recently been isolated from marine environments (Maeda et al., 2000).
The microbial insecticides most widely used in the world are preparation of Bacillus thuringiensis (Bt). Its insecticidal activity is due to the protein parasporal inclusions that are produced during sporulation. Insecticides based on the proteinaceous ᵹ-endotoxin of Bt. constitute part of a more ecologically rational pest control strategy. Bacillus thuringiensis sub sp. Isrealensis (Bt.) or serotype H-14, exhibit acute toxicity towards dipteran insects such as larval mosquitoes and black flies and is currently used in mosquito control programs worldwide (Mario and Montserrat, 2012).
Bacillus theringiensis is effective against the early stages of mosquito larvae and has not been reported to affect mosquito eggs, mature larvae, pupae or adults. Mosquito larvae must eat the Bt. formulated product containing dormant spores. Crystals which are known as insecticidal crystals proteins (ICPs) or delta – endotoxin produced during Bacillus thuringiensis sporulation. The mosquito larvae stop feeding and die when these proteins are converted into toxins that work by damaging the gut wall of mosquitoes (Lacey and Merrit, 2003).
The main aim of this research work is to isolate Bacillus thuringiensis from soil in different locations of Yobe state, and to test its potential as a Biolarvicide. From the literature search, this work can be considered as the first its kind in Yobe state, Nigeria.
METHODOLOGY
Study Location
This study was carried out in Yobe State, Nigeria between April and September 2022 (Rainy season).
Soil samples were collected from four locations: Potiskum, Gujba, Gashua and Fika local government areas of Yobe State.
Collection of Soil Samples
The soil samples were taken two to five (5– 10cm) below the surface, after scrapping of the surface material with sterile spatula. Collected samples were placed in a sterile polyethene bag, transported to the laboratory and stored at 4°C until processed for Bacillus thuringiensis isolation. Ten soil samples each were collected from four locations in Yobe State. Two samples each from Potiskum, Gujba, Gashua and Fika local government areas of Yobe State respectively.
Culturing and Isolation of B. thuringiensis from Soil Samples
Culturing and isolation of B. thuringiensis were carried out following the procedure described by Elkersh et al., (2014) with slight modification, in which1g of fine grinded soil, was added to 2.0 ml of sterile distilled water and suspended vigorously using a Vortex Mixer, of these specimens, 2 ml aliquots were mixed with 2 ml absolute ethanol to obtain 50% ethanol concentration, vortexed for 1 min and then incubated at 30°C for 45 min with regular shaking. At the end of this time, 0.5 ml of each soil suspension was drawn into test-tube and pasteurized in a water bath at 80oC for 10 minutes to kill vegetative cells and non-spore forming bacteria. After cooling at room temperature, the mixture was then serially diluted with sterile distilled water of 4.5 ml in five folds. A volume of 0.1 ml of each dilution was streaked on nutrient agar medium. Plates were incubated at 37oC for 24hours.
Purification and Preservation of Typical B. thuringiensis Isolate
The morphological appearance of the B. thuringiensis colonies over the plate were creamy-white, rough and spread out. These colonies were picked and streaked again on nutrient agar and incubated for 48 hours at 37oC to obtain axenic culture. Isolated organisms that were suspected to be B. thuringiensis were then kept in nutrient broth medium at 4oC for further tests.
Identification of isolates
The isolates were identified using morphological characterization and conventional biochemical procedures according to Claus and Berkeley (1986) and Barrow and Feltham (1993). Gram staining and spore staining procedures were performed. Biochemical tests conducted include catalase, indole, coagulase, and amylase activity.
Detection of Endospore, Crystal Proteins and Morphology
Bacillus species do not stain, and they may be seen as unstained bodies within bacterial cells stained with methylene blue. Smears of Bacillus isolates were prepared and they were fixed by heat. The bacterial smears were then flooded with methylene blue. Staining lasted for 5 minutes.
Finally, de-staining was performed by washing under slow running tap water. The stained bacterial colonies were observed under oil immersion objective for endospore position, crystal production and morphology. The isolates having visible parasporal crystals next to the spore in the sporangium cells were identified as Bt. Isolates having ellipsoidal and sub terminal spores in un-swollen bacterial cells. The colonies showing morphology and crystal shape were scraped off from the plates and transferred into sterile vials containing 1 ml of nutrient broth. After vortex mixing, they were stored at 4oC as stock.
Breeding of Mosquito Larvae
A procedure described by Thomas et al., (2014) was followed to breed the mosquito larvae, water containers were left to stand in an open space at ambient temperature of about 30oC for one week to facilitate laying of eggs by the mosquito. The water containers were monitored daily to observe the emergence of the larvae. The larvae of the female anopheles mosquitoes were harvested using sieve and placed in a moistened cotton-wool to preserve them before exposure to Bacillus thuringiensis.
Bioassay and Activity of B. thuringiensis against Mosquito Larvae
From the laboratory screening carried out, two Bacillus thuringiensis isolates having different crystal shapes were selected and tested against larvae of mosquito. A similar procedure used by Adeyemo et al., (2018) was followed with slight modification. Were five mosquito larvae were introduced into each test tube with labeled dilution factor 10-1, 10-2, 10-3, 10-4 and 10-5 respectively of the Bacillus thuringiensis suspension. The Bacillus thuringiensis from broth slants were diluted with sterile distilled water by diluting 1ml of the suspension in 9 ml of sterile distilled water to obtain the various dilutions. The test tubes were kept at 30oC; where mortality rate was checked at 4, 12 and 24 hours for each dilution factor and larval mortality was recorded. A control test was also carried out using distilled water and a pond water.
RESULT
From the soil samples collected around Yobe state, B. thuringiensis was isolated from five of the eight samples as shown in Table 1.
Table 1: Habitats and Locations of Bacillus thuringiensis Isolates
S/No. Habitat of Isolate Location Isolate code
1 Clay soil Potiskum P1
2 Loamy soil Fika F1
3 Loamy soil Potiskum P2
4 Loamy soil Gashu’a Ga1
5 Sandy soil Gashu’a Ga2
Table 2: Morphological Characteristics of Bacillus thuringiensis Isolates
| Characteristic | P1 | F1 | P2 | Isolate Code. Ga1 | Ga2 |
| Gram staining | + | + | + | + | + |
| Formation of spore | + | + | + | + | + |
| Presence of crystal | + | + | + | + | + |
| Shape of spore | O | O | O | O | O |
Key: + Present/Positive and O Ovoid
Table 3: Biochemical Reactions of Bacillus thuringiensis Isolates
Biochemical Isolate code
Test P1 F1P2 Ga1 Ga2
Catalase test + + + + +
Indole test – – – – –
Coagulase + + + + +
Amylase activity + + + + +
Table 4: Larvicidal activity of Bacillus thuringiensis isolates P1 and Ga2 against Mosquito Larvae at the intervals of 4 hours, 12 hours and 24 hours.
| 4 hours | 12 hours | 24 hours | ||||||||||
| P1 | Ga2 | P1 | Ga2 | P1 | Ga2 | |||||||
| Dilution Factor | No.of live larvae | No.of dead larvae | No.of live larvae | No.of dead larvae | No.of live larvae | No.of dead larvae | No.of live larvae | No.of dead larvae | No.of live larvae | No.of dead larvae | No.of live larvae | No.of dead larvae |
| 10-1 | 2 | 3 | 1 | 5 | 1 | 4 | 0 | 5 | 0 | 5 | 0 | 5 |
| 10-2 | 1 | 4 | 2 | 3 | 1 | 5 | 1 | 5 | 0 | 5 | 0 | 5 |
| 10-3 | 3 | 1 | 3 | 1 | 1 | 5 | 0 | 5 | 0 | 5 | 0 | 5 |
| 10-4 | 3 | 0 | 4 | 1 | 0 | 5 | 1 | 5 | 0 | 5 | 0 | 5 |
| 10-5 | 5 | 0 | 3 | 0 | 0 | 5 | 0 | 5 | 0 | 5 | 0 | 5 |
Statistical Analysis
Data for mortality rate of the mosquito larvae were generated by the use of the two B. thuringiensis specie with two distinct morphological characteristics and quantitative measurement determined by counting. Duncan multiple range tests were employed to analyze the similarities and differences in the mean values of the quantitative characters.
DISCUSSION
This research was carried out to find a collection of native B. thuringiensis isolates that can potentially be used for developing bio-control tools to help fight mosquito-borne diseases. Under the assumption that sampling from different places might uncover novel genetic diversity and toxic potentials, B. thuringiensis isolates were obtained from a variety of soil samples collected in different locations around Yobe State as place like Gashu’a has extensive irrigation systems which creates an abundance of suitable mosquito breeding sites. Adeyemo et al., (2018) isolated six Bacillus thuringiensis from eight soil samples, this shows that not in all the samples collected produce the isolates as similar to the isolates obtained in this research.
CONCLUSION
Following series of laboratory analysis, the results obtained in this study clearly shows efficient the Bacillus thuringiensis is in the control of mosquito larvae. The use of Bacillus thuringiensis as a bio-control agent against mosquito larva is preferred as it is environmentally friendly and does not deplete the ozone layer unlike the regular pesticides used in killing mosquitoes in most communities.
It is however very significant to search for more microbial toxins to control insects’ orders which have the ability to develop resistance against selected insecticides. Screening of soil samples from different sources and habitats may be useful to obtain Bacillus thuringiensis strain with broader host ranges and novel crystal proteins.
Funding/Sponsorship
This project was sponsored/funded by the Tertiary Education Trust Fund (TETFUND), in form of Institutional Based Research (IBR).
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