Kerazyme Triggers the Production of Mosquitocidal Toxins

 

Suneetha V.

Instrumental and Food Analysis Laboratory, School of Biosciences and Technology VIT University,

Vellore-632 014, Tamil Nadu, India

 

ABSTRACT:

Recently various biopesticides have been produced by culturing keratinolytic bacteria to synthesize mosquitocidal toxins to minimize or decrease the risk of mosquito-borne diseases by controlling the mosquito population. Certain species of bacilli (mainly Bacillus spharicus and Bacillus thuringiensis) and Clostridium produce different form of toxins having different molecular weight those help to kill mosquito larvae at the concentrations in the picomolar range. Although, there are so many different genes those encode mosquitocidal toxins, which vary in their properties like potency, mode of action and species specificity. Mosquitocidal bacilli strains are safe for animals and the environment and unlike chemical insecticide they do not affect the non-target insects. These Mosquitocidal bacteria are very effective against Anopheles, Aedes and Culex mosquitoes, but generally they rapidly settle down from larval feeding zone and their narrow host range and UV-light sensitivity properties have hampered their development. These limitations could be overcome by the new genetic engineering approaches that allow stable expression of broad host range combinations of toxins in UV resistant and buoyant recombinant bacteria.

 

 

INTRODUCTION:

Mosquitoes, the most important vectors widely present in all over world are responsible to transmit the various deadly diseases. There are lots of  Mosquito-borne diseases those  involve the transmission of viruses and parasites from one animal to another animal, animal to person, or person-to-person, without affecting the insect vectors with symptoms of disease. Mosquito-borne diseases form a major component of communicable diseases like, dengue, malaria, filariasis, Japanese encephalitis, yellow fever, Rift Valley Fever, Eastern equine encephalitis, St. Louis encephalitis, La crosse encephalitis and Western equine encephalitis in India and in other Asian countries.[1] 

 

These diseases chiefly cause indisposition and death and disease-endemic countries face major economic burden attributable to these deadly diseases. Every year, around the world approx. 300 million people are affected by malaria, a fatal malady.[2,3]

 

Malaria could be a risk to 2,400 million individuals that is 40% of world population.

 

Uncontrolled urbanization and formation of conditions that are suited to dipteron development are the key reason for increment in mosquito-borne diseases.[3].

 

There are many methods those are adopted to manage the various mosquito-borne diseases. Synthetic insecticides are effectively used throughout the past many decades to manage these dipterious insect pests. However, the use of these chemical insecticides has become problematic, due to multiplicity of things as well as physiological resistance within the vectors, environmental pollution leading to bioamplification of food chain contamination and harmful effects on useful insects. Hence, an interest has been raised in recent years in the use of biological vector control agents.

Recently various biopesticides have been produced to replace these harmful chemicals.[4,5].

 

There are many mosquitocidal bacteria present in the environment those can be used to synthesize mosquitocidal toxins. Some keratinolytic bacteria may be used for this purpose can be isolated from poultry waste .Using these toxins the mosquito population can be reduced to minimize or decrease the risk of mosquito-borne diseases.

 

Mosquitocidal Bacteria and their mosquitocidal activity There are some bacteria present in the environment containing mosquitocidal activity has the ability to produce mosquitocidal toxins. [6,7,8] Keratinolytic bacteria like Bacillus sphaericus (Bs) and Bacillus thuringiensis serovar israelensis (Bti) are two main bacilli strains, degrades the chicken feather keratin,[9] those are helpful in the production of endotoxins to kill the mosquito larvae (Figure 1). Chicken feather waste is generated in a huge quantity all over the world per year. About 18.5 million thousand tons of chicken feather waste are generated all over the world.[10]

 

This may be the cheapest bio-organic waste, which can be used as a substrate for culturing different mosquitocidal bacteria producing mosquitocidal toxins in the laboratories.

 

Figure 1. A prototype for the production of endotoxins from Bacillus to kill the Culex larvae by using chicken feather waste

 

These mosquitocidal bacteria (eg. Bs and Bti) [6,7,8] can also be grouped under the parasites of vector mosquitoes and pathogens. Some of the Bs and Bt strains generally produce mosquitocidal protein crystals (inactive protoxin) during their sporulation.

 

They form depositions alongside the spores. These crystals are extremely toxic to the vulnerable species that ingest them with the spores as food. The alkaline pH midgut milieu of larva solubilizes the protoxins, which get activated for proteolysis with proteases and bind to the specific glycoproteins cell receptors present on midgut epithelial cells of insects in target. This leads to pore formation in the cells, which allows the water inflow rapidly. Due to this the osmoregulatory mechanism of cell membrane gets disturbed. Hence, the cells swell and lyse, thus larva starves to death.

 

Bacillus sphaericus was reassigned to the genus Lysinibacillus on the basis of both physiology and phylogenetic analysis, Some of the strains of Lysinibacillus sphaericus have toxic effects on mosquito larvae. They are used as a biological control method for the vectors of mosquito-born diseases like malaria, dengue fever and West Nile fever. [8,9]

Lysinibacillus sphaericus is a genetically heterogeneous species which is divided into five DNA homology groups. Subgroup IIA constitutes the strains pathogenic to mosquitoes, but some of the non-pathogenic isolates are also a part of this homology group. The subgroup IIB has been allocated to the species Lysinibacillus fusiformis.[10] L. sphaericus sensu lato was classified into seven similarity groups using 16S rDNA sequence. In agreement with whole-cell fatty acid profiles, four of the phylogenetic groups correspond to the DNA hybridization groups. In a study it was revealed that 16s rRNA gene sequences and phylogenetic analysis clustered 84% of the metal-tolerant strains in the L. sphaericus group 1, which shows the mosquitocidal property. The larvicidal activity of sporulated and vegetative cells and its high tolerance to arsenate, hexavalent chromium and lead indicates that L. sphaericus  OT4b.2b strain has strong potential for biological control of mosquito population in waters contaminated with metals. Here L. sphaericus metal (loid) tolerant strains are classified according to their toxicity towards Culex quinquefacsiatus larvae (Table 1).

 

Due to some of the reports on development of resistance to Bs (4), a number of workers have started looking for alternate microbial agents or their metabolites, in order to control mosquito. In Malaysia and Japan, the mangrove swamps and mangrove sediments, have been reported as a habitat for highly potent mosquitocidal microorganisms like Clostridium bifermentans and B. thuringiensis subsp. israelensis/ tochigiensis .Using the standard microbiological methods, a mosquitocial bacterium was isolated from the Andaman and Nicobar Islands of India and then identified as Bacillus amyloliquefaciens using classical biochemical tests and rpoB gene sequences. The extracellular metabolite(s) of this mosquitocidal bacilli exhibit larvicidal and pupicidal activities. The larvicidal and pupicidal efficacy was determined in terms of LC50 and LC90, after separating it from the culture supernatant solution of the bacterium (Figure 2.). 

 

Figure 2. Pupicidal activity of B. amyloliquefaciens from Mangrove forest

 

Table 1. Mosquitocidal bacteria, their toxins (types and/or molecular mass) and mosquito host range

Bacterial strain	Toxins (Molecular mass of protoxin (s) in kDa and/ or types)	Mosquito host range of bacterium
Bacillus sphaericus	32(Mtx 2), 36(Mtx 3), 42(BIN A), 51(BIN B), 100(Mtx 1)	Culex > Anopheles >> Aedes aegypti
Bacillus thuringiensis subsp. israelensis (Bti)	27, 72, 78, 128, 134	Aedes aegypti, Culex > Anopheles
Bacillus thuringiensis subsp. jegathesan (Btj)	16,26, 37, 65, 70-72,81	Anopheles stephensi > Culex pipiens > Aedes aegypti
Clostridium bifermentans ser. Malaysia	16, 18, 66	Anophelese ≥ Adese detritus, Adese caspius > Aedes aegypti, Culex
Bacillus subtilis	Surfactin	Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti
Bacillus thuringiensis subsp. Kurstaki	65	Aedes aegypti
Bacillus amyloliquefaciens	Lipopeptide biosurfactant	Anopheles stephensi > Culex quinquefasciatus > Aedes aegypti

 

Another bacterial strain, isolated from mangrove forests of Andaman and Nicobar islands and identified as a strain of B. subtilis subsp. subtilis (B471), is a gram-positive, aerobic, spore forming bacterium.

 

The pupicidal and larvicidal activities were found in the supernatant of the culture of this bacterium. Different biochemical parameters like, gyrA gene sequencing and 16S ribosomal DNA were used to identify it.[7,8]

 

In the control programs, it could be a promising tool, as there are only few biocontrol agents or insecticides which are effective against mosquito pupae. Table1.

 

Mosquitocidal bacteria, their toxins (types and/or molecular mass) and mosquito host range Bacterial strain Toxins (Molecular mass of protoxin (s) in kDa and/or types) Mosquito host range of bacterium Bacillus sphaericus 32(Mtx 2), 36(Mtx 3), 42(BIN A), 51(BIN B), 100(Mtx 1) Culex > Anopheles >> Aedes aegypti Bacillus thuringiensis subsp. israelensis (Bti) 27, 72, 78, 128, 134 Aedes aegypti, Culex > Anopheles Bacillus thuringiensis subsp. jegathesan (Btj) 16,26, 37, 65, 70-72,81 Anopheles stephensi > Culex pipiens > Aedes aegypti Clostridium bifermentans ser. Malaysia 16, 18, 66 Anophelese ≥ Adese detritus, Adese caspius > Aedes aegypti, Culex Bacillus subtilis Surfactin Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti Bacillus thuringiensis subsp. Kurstaki 65 Aedes aegypti Bacillus amyloliquefaciens Lipopeptide biosurfactant Anopheles stephensi > Culex quinquefasciatus > Aedes aegypti Another group of toxins known as Mtx toxins, are generally synthesized during the exponential phase of growth and degraded during the stationary phase. Highly toxic strains synthesize both Mtx and Bin toxin and low toxic strains synthesize only Mtx. Three types of Mtx toxins, such as, Mtx1, Mtx2 and Mtx3 with molecular mass of 100, 31.8 and 35.8 kDa, respectively, have been reported.[2,4] During vegetative growth phase, the Bs toxins (of 100 and 32 kDa) are synthesized having less toxin potency. The 100 kDa toxin, synthesized by the natural strain SSII-1, is unstable but when derived from recombinant E. coli, an engineered and purified 97 kDa version of this toxin (lacking some N-terminal sequences) is as toxic to Culex mosquitoes as the binary toxin(LC50, ~15 ng/ml).[4,1] The 100 kDa toxin is deliberately toxic to A. aegypti (120-290 ng/ml) unlike most of the binary toxins, which are less toxic to these mosquitoes.[7] The 32 kDa toxin is toxic to Culex but is otherwise poorly characterized and shows limited but significant homology to - (1) the Clostridium perfringense toxin and (2) the Pseudomonas aeruginosa cytotoxin, both of which are specific for the mammalian cells, and are  believed to form pores on the cell surface. The 100 kDa toxin appears to kill cells by  inactivation of certain cellular proteins via ADP-ribosylation.

 

Another bacilli strain i.e. Bacillus thuringiensis serovar israelensis (Bti), available to date as most effective microbial control agent is also active against mosquitoes] Intracellular crystal inclusions are synthesized by this strain containing multiple components of protein having molecular weight of 134, 125, 67 and 27 kDa.[4,5] These proteins have also been cloned individually and are shown as toxic to mosquito larvae.[4] Although, a combination of these protein components shows much higher toxicity than the individual components. Individually, none of the crystal proteins of mosquitocidal Bt strains are as toxic as the spore-crystal complex, suggesting that synergy between toxins (or between toxins and unknown spore components) is an important factor in toxicity.

 

Studies conducted with recombinant bacteria expressing these polypeptides individually have shown that in the absence of 51 kDa protein the 42 kDa protein of Bs could be toxic at high dosage, but it is not possible for the 51 kDa protein alone. Although, the presence of both proteins results in high toxicity to the mosquito larvae in equimolar amounts, as they seem to act in synergy.[8]

 

In case of another mosquitocidal bacillus satrin B. amyloliquefaciens, mosquito larvicidal activity in terms of LC50 against Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti was respectively, 22.2 µg, 26.4 µg, and 20.5 µg/ml and its pupicidal activity was 8.2 µg, 4.4 µg, and 14.5 µg/ml respectively. Clostridium bifermentans ser. malaysia is the first anaerobic bacterium with significant mosquitocidal activity.[7] During sporulation, it synthesizes three major proteins, probably having mosquitocidal  toxicity, but the mode of action of the toxin(s) and the function separately or in a complex is unknown.

 

The receptors presence in the insect midgut may help to determine the mosquito host range of toxins (correlation between the distribution of receptors and susceptibility of the species to be killed by the toxin has been explained via few studies that have been carried out.[8] The mosquitoes those got resistance may lack receptors for one or more toxins,[8] thus these toxins will not be effective for those resistant mosquitoes.

 

As compared to the chemical insecticides, various shortcomings of the mosquitocidal bacilli must be overcome. One of the solutions to it is the development of genetically engineered toxin producing bacteria.

 

CONCLUSIONS:

The number and diversity of mosquitocidal bacteria and their toxins have increased gradually since their discovery, and this increase is accelerating. Currently, about 19 distinct mosquitocidal toxin genes are known responsible to express mosquitocidal toxins. There are some keratin degrading bacteria i.e. keratinolytic bacteria also present in the environment produces some mosquitocidal toxins. The emergence of pesticide-resistant mosquitoes and drug-resistant parasites act as a strong incentive to develop effective mosquitocidal bacteria. The toxins are generally vary in their mode of action and species specificity, making it likely treat particular combinations cloned in recombinant micoorganisms can be chosen so as to delay or prevent the development of resistance and to enlarge insect host range. The mosquitocidal toxins potential towards susceptible species is generally excellent, but the bacilli naturally present in environment have some drawbacks that have hampered their widespread use.

 

However, several novel genetic manipulation approaches like high-level expression of toxin combinations and chromosomal integration of toxin genes, coupled with existing formulation technology or the use of engineered vegetative bacteria which can exist in the upper layer of water, may overcome these problems.

 

ACKNOWLEDGEMENT:

The author wants to express her gratitude to Hon’ble Chancellor, Dr G. Viswanathan, VIT University and Mr. Sekar Viswanathan, Vice President for their constant encouragement and support to carry out this valuable research work.

 

REFERENCES:

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4.        Suneetha, V. Screening, Characterization and Optimization of Keratinolytic Bacteria Isolated from Poultry Waste. Lambert Academic Publishing GmbH & Co. KG, 2011.

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6.        Hofte, H.; Whiteley, H.R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 1989, 53(2), 242-255.

7.        Baumann, P.; Clark, M. A.; Broadwell, A.H. Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxin. Microbiol. Rev. 1991, 55(3), 425-436.

8.        Porter, A.G.; Davidson, E.W.; Liu, J.W. Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes. Microbiol. Rev. 1993, 57, 838-861.

9.        Suneetha, V.; Kumar, S.; Ramalingam, C. Bioremediation of Poultry Waste. Adv. Biotech. J. Biotechnol., 2010, 10 (1), 28-32.

10.     Suneetha, V.; Ritika, S.; Abhishek, G.; Rahul, G. An attempt and a brief research study to Produce Mosquitocidal toxin using Bacillus spp. (VITRARS), isolated from different soil samples (Vellore and Chittoor), by degradation of Chicken Feather Waste. Res. J. Pharm. Biol. Chem. Sci. 2012, 3(4), 40-48.

 

 

Received on 08.08.2014          Modified on 20.08.2014

Accepted on 05.09.2014          © RJPT All right reserved