WO2012105824A1

WO2012105824A1 - Bioinsecticide and method for producing thereof

Info

Publication number: WO2012105824A1
Application number: PCT/MY2012/000005
Authority: WO
Inventors: Alan Erwin BRANDT; Robert Rose
Original assignee: Entogenex Industries Sdn Bhd
Priority date: 2011-01-31
Filing date: 2012-01-19
Publication date: 2012-08-09
Also published as: MY178859A

Abstract

A bioinsecticide active against Diptera insect larvae comprising at least two active ingredients, characterized in that the active ingredients comprise a regulatory hormone of enzyme biosynthesis, and a bacteria capable of releasing toxins. In preferred embodiments, the regulatory hormone is Trypsin Modulating Oostatic Factor (TMOF) and the bacteria are a Bacillus species that can release crystal toxins (in particular; Bacillus thuringiensis israelensis). The bioinsecticide can be produced in floating formats by applying the bioinsecticide to carriers such as rice hulls, or produced in a water dispersible form as a soluble free flowing powder.

Description

BIOINSECTICIDE AND METHOD FOR PRODUCING THEREOF

TECHNICAL FIELD OF THE INVENTION

This invention is related to a bioinsecticide and a method for producing thereof. More particularly, it relates to a bioinsecticide, which is active against Diptera insect larvae, and a method for producing thereof.

BACKGROUND OF THE INVENTION

Mosquito control is an ongoing worldwide challenge. Up till now, even with such technology advancement, more particularly, in biotechnology, the control of mosquito populations and diseases vectored by mosquitoes is still not accomplished. Reduction of mosquito population throughout the world, especially in the tropical regions, is imperative as they pose immense health problems to the human kind. This is because mosquitoes are able to spread deadly diseases such as malaria, dengue fever, yellow fever, West Nile disease, St. Louis encephalitis, and others to humans. They are able to do so as they serve as important vectors for viruses and/or parasites of such diseases, and transmit the same upon feeding on humans. Till today, these diseases have caused millions of death worldwide^-pei^ ear. This concerning death figure in millions is the main reason that mosquito populations must be managed accordingly. Therefore, numerous solutions have been provided to decrease the mosquito population, if not eradicate. According to the surrounding and circumstances, source reduction, biocontrol, larviciding, and adulticiding can be used for this purpose. Many of the products used for larviciding and adulticiding are neurotoxic synthetic chemicals, which are broad spectrum and have wide non-target effects. Not only do these chemicals exterminate mosquitoes, but also bring tremendous harm to other non-target organisms, including humans. For example, dichlorodiphenyltrichloroethane (DDT) was introduced since 1939 for controlling malaria during World War II. Although it was highly effective during then in combating mosquitoes, it is toxic to a wide range of animals, including marine animals. It has been known that DDT is a reproductive toxicant and the main culprit for the disturbing number decline in some bird species such as bald eagle and brown pelican. To humans, it is also suspected to be a reproductive toxicant in addition to being carcinogenic. This chemical has already been banned in United States of America for nearly 40 years due to the reasons above. Further, effectiveness of this chemical is vastly reduced as mosquitoes have developed resistance against the same. As such, there is a need to develop new insecticides for controlling mosquitoes.

In view of the above, several biocompatible solutions have been offered in the market. One of these environmental friendly solutions is the introduction of Bacillus thuringiensis israelensis, a strain of Bacillus thuringiensis, which is highly effective in killing mosquito larvae. Bti, as it is more commonly known, works by being consumed by mosquito larvae and producing toxins, which are crystal proteins, that bind to specific mid gut cell receptors on the lumen side of the gut and punch holes in the cells, causing cell lysis and eventually larval death.

Numerous formulations of Bti for water application have been introduced. However, Bti has the problem of having a high density, which causes it to consequently sink to the bottom of water column and quickly become unavailable for the consumption of mosquito larvae. Therefore, re-application of Bti has to be done regularly and within a very short interval, e.g. 2 weeks, in order to sustain its effectiveness against mosquito larvae. Apart from that, Bti is also not effective against the mosquito larvae when other food sources are available. This is due to the reason that the other food sources prevent the activity of toxic crystal proteins of Bti in the mid gut of mosquito larvae. Beside that, Bti is easily degraded under sunlight, making it less persistent. Some of the formulations of Bti available nowadays in the market only persist for less than a week.

Another known biocompatible solution available in the market is methoprene. It is an insect juvenile hormone compound that can be used as insecticide to control insects, mostly mosquitoes. This compound is able to interfere with normal maturation process of insects by mimicking their growth regulation hormone, which is essential for the process of metamorphosis. In other words, treated larvae will pupate but adults do not emerge from the pupal stage. Therefore, methoprene does not kill adult insects directly; rather it breaks their biological life cycle, preventing these insects from reaching maturity or reproducing stage.

Nonetheless, usage of methoprene to control insect populations has a few disadvantages. Some of the animals, such as mice and rats, are prone to liver toxicity when they are exposed to methoprene for long-term. This finding suggests that methoprene might cause liver toxicity to other organisms as well, especially mammals. Beside, to assure optimum effectiveness of methoprene, its use requires professional skill and knowledge, as this compound needs to be administered at the proper stage of the life cycle of target insects.

In light of the above, there is a need to provide a novel bioinsecticide that is able to overcome all aforesaid technical problems.

SUMMARY OF THE PRESENT INVENTION

It is an object of this present invention to provide a bioinsecticide, which is highly effective in exterminating Diptera insect larvae. In one of the embodiments, the bioinsecticide is used to kill mosquito larvae, fly larvae, gnat larvae, and midge larvae. Preferably, the bioinsecticide is used to kill mosquito larvae, such as Diptera culicidae. It is another object of this present invention to provide a bioinsecticide comprising at least two active ingredients, wherein each active ingredient has a different mechanism in reacting and exterminating target organisms. One of the active ingredients is a regulatory hormone capable of down regulating or inhibiting enzyme biosynthesis, whereas the other active ingredient is a bacteria capable of releasing toxins. This combination of active ingredients increases the efficiency of the bioinsecticide.

In another embodiment of the present invention, the regulatory hormone is capable of down regulating or inhibiting serine protease biosynthesis by gut epithelial cells of Diptera insect larvae, including the biosynthesis of the enzyme trypsin and other serine proteases. In another preferred embodiment of the present invention, the regulatory hormone is Trypsin Modulating Oostatic Factor (hereinafter is referred to as TMOF).

In a further embodiment of the present invention, the bacteria, which is capable of releasing toxins, is selected from any subspecies of Bacillus thuringiensis, Bacillus sphaericus, or a combination thereof. In a further preferred embodiment of the present invention, the bacteria is a strain of Bacillus thuringiensis, namely Bacillus thuringiensis israelensis (hereinafter is referred to as Bti). The toxins produced and released by Bti are crystal proteins, namely Cry4, Cry10, Cry11 , Cyt1 , and Cyt2, which work by lysing gut epithelial cells of Diptera insect larvae. It is also an object of this present invention to provide a bioinsecticide that is capable to float on water surface and control the release of the active ingredients to surroundings by further comprising a floatable carrier and an adhesive material. The floatable carrier plays the role as a carrier of the active ingredients for easy storage and dispersal. Beside that, the floatable carrier is able to float on the water surface and consequently allows the release of active ingredients into the feeding zone of Diptera insect larvae. The adhesive material is responsible for adhering the active ingredients to the floatable carrier. Additionally, the adhesive material controls the release of the active ingredients to the surroundings, thus increasing the persistence of the bioinsecticide according to the present invention. In still another embodiment of the present invention, the floatable carrier is a rice husk, whereas the adhesive material is a mineral oil.

If desired, the bioinsecticide, which is capable to float on water surface and control the release of the active ingredients to the surrounding, further comprises a food colouring and a preservative agent. The food colouring is added to give a more visually appealing and acceptable product. The preservative agent is added for extending the shelf life.

A method to produce the bioinsecticide that is capable to float on water surface and control the release of the active ingredients to the surrounding is described herein. Initially the floatable carrier, regulatory hormone, and bacteria are mixed together. Subsequently, the adhesive material is sprayed onto the mixture whilst the mixing process is ongoing. The mixing and spraying processes are stopped when the regulatory hormone and bacteria are adhered to the floatable carrier. If the additions of food colouring and preservative agent are desired, the method further comprises dissolving the food colouring in minimal amount of water and spraying the same onto the mixture whilst the mixing process is ongoing. Thereafter, the preservative agent is dissolve in acetone and spraying the same onto the mixture whilst the mixing process in ongoing. The mixing process is stopped when the acetone is completely evaporated.

It is yet another object of this present invention to provide a bioinsecticide that is water dispersible by further comprising a water soluble carrier. The water soluble carrier plays the role as a carrier of the active ingredients for easy storage and dispersal. Upon direct contact with water, the water soluble carrier is dissolved resulting in the release of the active ingredients. In yet another embodiment of the present invention, the water soluble carrier is maltodextran. If desired, the bioinsecticide, which is dispersed in water, further comprises a preservative agent. The preservative agent is added for extending the shelf life.

A method to produce the bioinsecticide, which is water dispersible, is described herein. The water soluble carrier, regulatory hormone, and bacteria are combined and mixed well until a flowing dry powder is produced. If the addition of preservative agent is desired, the method further comprises mixing the preservative agent with the water soluble carrier, regulatory hormone, and bacteria until a flowing dry powder is produced.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the results of a simulated field trial of bioinsecticide according to present invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The above mentioned and other features and objects of this invention will become more apparent and better understood by reference to the following detailed description. It should be understood that the detailed description made known below is not intended to be exhaustive or limit the invention to the precise form disclosed. On the contrary, the detailed description covers all the relevant modifications and alterations made to the present invention, unless the claims expressly state otherwise.

The present invention is related to a bioinsecticide with high biological activity against Diptera insect larvae, wherein said bioinsecticide comprises at least two active ingredients.

The bioinsecticide in accordance with the present invention can be used to exterminate a wide range of Diptera insect larvae. Particularly, it is highly effective in eradicating mosquito larvae, fly larvae, gnat larvae, and midge larvae. More particularly, it is mainly used to control mosquito population by killing mosquito larvae in an aquatic area, preventing the emergence of adult mosquitoes. The active ingredients present in the bioinsecticide are natural, biocompatible, and biodegradable. The Diptera insect larvae must consume them in order to take effect. Therefore, the bioinsecticide as well as the active ingredients are employed as food sources for Diptera insect larvae. Each active ingredient has a different mechanism on reacting and killing Diptera insect larvae. As such, the existence of other additional food sources will not hamper the efficiency of the bioinsecticide. Further, this feature also tremendously increases the efficiency and biological activity of the bioinsecticide against Diptera insect larvae. In accordance with the present invention, one of the active ingredients is a regulatory hormone capable of down regulating or inhibiting enzymes biosynthesis. Generally, enzymes are proteins that are able to catalyse chemical reactions and they are essential to every living organism, as almost all processes in a biological cell need enzymes to occur at significant rates. By down regulating or preventing the biosynthesis of enzymes of Diptera insect larvae, the same would not be able to survive. Preferably, the regulatory hormone is capable of down regulating or preventing the synthesis of serine proteases by gut epithelial cells of Diptera insect larvae. The serine proteases are digestive enzymes vital for catalysing the break down of proteins into simpler form in order for easy absorption of nutrients in the digestive system. One of the important types of serine protease in the digestive system of Diptera insect larvae is trypsin. As such, in a preferred mode, Trypsin Modulating Oostatic Factor (TMOF) is used as the active ingredient of regulatory hormone as it terminates the serine protease biosynthesis, including trypsin biosynthesis by the gut epithelial cells of Diptera insect larvae. After consumption of TMOF, the Diptera insect larvae would die of starvation as their digestive system has malfunctioned. TMOF can be obtained via expression by microorganism. Preferably, yeast is used to express TMOF, as it is a preferred food source for Diptera insect larvae, especially mosquito larvae. For example, TMOF is engineered into Pichia pastoris yeast strain. Thereafter, the transformed yeast is grown by fermentation. Subsequently, the yeast is killed and dried to yield TMOF, which can be used as the regulatory hormone according to the present invention.

The regulatory hormone is added to the bioinsecticide to a final concentration of 0.1 % to 25% by weight.

In accordance with the present invention, the other active ingredient is a bacteria capable of releasing toxins, which are effective in killing Diptera insect larvae. Preferably, the toxins react and kill Diptera insect larvae by lysing their gut epithelial cells. As such, any subspecies of Bacillus thuringiensis, Bacillus sphaehcus, or a combination thereof can be used for this purpose. In a preferred mode, the subspecies of Bacillus thuringiensis is Bacillus thuringiensis israelensis {Bti). Upon consumption of Bti by Diptera insect larvae, Bti is able to produce toxins in the form of crystal proteins, namely Cry4, Cry10, Cry11 , Cyt1 , and Cyt2. These toxins subsequently lyse the gut epithelial cells by forming pores on their membranes.

The bacteria is added to the bioinsecticide to a final concentration of 0.1% to 25% by weight. In one of the aspects of the present invention, the bioinsecticide is formulated and invented in such a way that it is able to float on water surface and control the release of the active ingredients into the surroundings. In this case, the bioinsecticide further comprises a floatable carrier and an adhesive material, in addition to the aforementioned regulatory hormone and bacteria.

The floatable carrier mainly functions as a carrier for the active ingredients for easy storage and dispersal. Moreover, this floatable carrier allows the bioinsecticide to float on the water surface after application. This is important as the Diptera insect larvae linger and feed near the water surface. As some of the active ingredients have higher density than water, those active ingredients will sink to the bottom of the aquatic area and thus not available for consumption by the Diptera insect larvae, resulting in lower persistence and bioavailability of bioinsecticide. Therefore, it is imperative to have such floatable carrier to ensure that the bioinsecticide remains near the water surface and release the active ingredients into the feeding region of Diptera insect larvae. In a preferred mode of the present invention, rice husk is used as the floatable carrier.

The adhesive material is mainly responsible for the adhesion of the active ingredients to the floatable carrier. Furthermore, its other functions also include controlling the release of active ingredients into the surrounding in order to increase the persistence and bioavailability of bioinsecticide. As mentioned above, some of the active ingredients with density higher than water will sink to the bottom of the aquatic area and consequently become unavailable for consumption by Diptera insect larvae. The adhesive material will ensure that the active ingredients are released in such a manner that active ingredients are always available as food sources for Diptera insect larvae. In another words, active ingredients which have sunk will be replaced by newly released active ingredients. In a preferred mode of the present invention, mineral oil is used as the adhesive material.

According to the above-described bioinsecticide that is able to float on the water surface and control the release of the active ingredients to surrounding, the regulatory hormone is preferably added to the bioinsecticide to a final concentration of 0.1% to 8% by weight; the bacteria is preferably added to the bioinsecticide to a final concentration of 0.1% to 8% by weight; the floatable carrier is added to the bioinsecticide to a final concentration of 81% to 98% by weight; and the adhesive material is added to the bioinsecticide to a final concentration of 1 % to 11 % by weight. If desired, any types of food colouring that is available in the market can be added to the bioinsecticide to provide a more visually appealing and acceptable product. For examples, FD&C Blue Nos. 1 and 2, FD&C Green No. 3, FD&C Red Nos. 3 and 40, FD&C Yellow Nos. 5 and 6, Orange B, Citrus Red No. 2, annatto extract, beta-carotene, grape skin extract, cochineal extract, carmine, paprika oleoresin, caramel colour, fruit and vegetable juices, and saffron can be used for this purpose.

According to the present invention, the food colouring is added to the bioinsecticide to a final concentration of 0.03% to 0.07% by weight.

If desired, any types of preservative agent that is available in the market can be added to the bioinsecticide to prolong its shelf life. Preservative agents that are commonly used for preventing spoilage of food through fungal or bacterial growth can be used for this purpose. For examples, methyl paraben, ascorbic acid, citric acid, sodium benzoate, calcium propionate, sodium erythorbate, sodium nitrite, calcium sorbate, potassium sorbate, butylated hydroxylanisole (BHA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), and tocopherols (vitamin E) can be used for this purpose. In a preferred mode, methyl paraben is used as the preservative agent.

According to the present invention, the preservative agent is added to the bioinsecticide to a final concentration of 0.1% to 0.3% by weight. The present invention also provides a method to produce the bioinsecticide as described above. Initially, the floatable carrier, regulatory hormone, and bacteria are evenly mixed together. During the mixing process, the adhesive material is sprayed onto the mixture, preferably in a spurting manner. The mixing and spraying processes are stopped when the active ingredients are adhered to the floatation carrier.

If the additions of food colouring and preservative agent are desired, the mixing process described above is continued even after the regulatory hormone and bacteria are adhered to the floatable carrier. The food colouring is dissolved in a minimal amount of water before it is sprayed in a spurting manner onto the mixture whilst the mixing process is ongoing. Subsequently, the preservative agent is dissolve in acetone before it is sprayed in a spurting manner onto the mixture whilst the mixing process is ongoing. The mixing process continues until the acetone is being fully evaporated.

Having described the above bioinsecticide that encompasses the features of being able to float on water surface and control the release of active ingredients to surrounding, the present invention will now disclose another aspect of the invention, which is a bioinsecticide that is water dispersible. In this case, the bioinsecticide further comprises a water soluble carrier, in addition to the above-mentioned regulatory hormone and bacteria. The water soluble carrier mainly functions as a earner for the active ingredients for easy storage and dispersal. Upon direct contact with water, the water soluble carrier is dissolved, thus causing the roleata of the active ingredients into the surroundings. In a preferred mode of the present invention, maltodextran is used as the water teftu ftt carrier.

According to the above-described bioinsecticide that is water dispersible, the regulatory hormone is preferably added to the bioinsecticide to a final concentration of 5% to 25% by weight; the bacteria is preferably added to the bioinsecticide to a final concentration of 5% to 25% by weight; and the water soluble carrier is added to the bioinsecticide to a final concentration of 50% to 70% by weight.

According to the present invention, the preservative agent is added bioinsecticide to a final concentration of 0.1 % to 0.5% by weight.

The present invention further provides a method to produce the bioinsecticide as described above, wherein the method comprises at the least the step of combining and mixing the water soluble carrier, regulatory hormone, and bacteria until a flowing dry powder is produced.

If the addition of preservative agent is desired, the method further comprises at least the step of mixing the preservative agent with the water soluble carrier, regulatory hormone, and bacteria until a flowing dry powder is produced.

The present invention will now be further explained by the following examples. The examples are intended for assisting in understanding the core aspects of the present invention, including its embodiments. The present invention is not in any way limited by these examples.

Example 1

Bioinsecticide formulation:

a) 92 g dry rice husk (82.73% by weight)

b) 4 g Bti powder (3.60% by weight)

c) 4 g TMOF (3.60% by weight)

d) 11.2 g mineral oil (10.07% by weight)

Place the rice husk, Bti and TMOF in a drum mixer and mix thoroughly. Spray the mineral oil into the rotating drum in spurts to assure complete coverage of the material. Halt mineral oil application when all of the Bti and TMOF are adhered to the rice husk. The end product, which is the bioinsecticide in accordance with the present invention, is ready for packaging.

Example 2

Bioinsecticide formulation:

a) 90.75 kg dry rice husk (90.75% by weight)

b) 4.0 kg Bti powder (4% by weight)

c) 4.0 kg TMOF (4% by weight)

d) 1.0 kg mineral oil (1% by weight)

e) 0.2 kg methyl paraben (0.2% by weight)

f) 0.05 kg FD&C yellow #6 food colouring (0.05% by weight)

Place the rice husk, 6f/^' and TMOF in a drum mixer and mix thoroughly. Spray the mineral oil into the rotating drum in spurts to assure complete coverage of the material. Halt mineral oil application when all of the Bti and TMOF are adhered to the rice husk. Dissolve the FD&C yellow #6 food colouring in a minimal amount of water and spray in spurts into the rotating mixture to assure uniform coverage of the product. Dissolve the methyl paraben in 500 ml of acetone. Spray in spurts into the rotating mixture to assure uniform coverage. Continue mixing until acetone is completely evaporated. The end product, which is the bioinsecticide in accordance with the present invention, is ready for packaging.

Example 3

Bioinsecticide formulation:

a) 10 kg Bti powder (20% by weight)

b) 10 kg TMOF (20% by weight)

c) 29.8 kg maltodextran (59.6% by weight)

d) 0.2 kg methyl paraben (0.4% by weight)

The maltodextran, Bti, TMOF, and methyl paraben are added and mixed thoroughly in a drum mixer until an evenly flowing dry powder is produced. The end product, which is the bioinsecticide in accordance with the present invention, is ready for packaging.

Simulated field trial

Materials used in this trial were buckets, mosquito netting, crushed dry leaf powder as additional food source, Aedes aegypti larvae, and the bioinsecticide comprising 4% TMOF and 4% Bti as described in Example 2.

Four buckets containing 4 liters of water were prepared. 20 first instar larvae of Aedes aegypti were put into each bucket. The bioinsecticide in following weights (10 mg, 25 mg, 50 mg and 100 mg) were distributed into each bucket, respectively. Crushed dry leaf powder was supplied as additional food source for the larvae. All buckets were covered by mosquito netting. Larval mortality was recorded after 24 hours and weekly. A new batch of 20 first instar larvae of Aedes aegypti was introduced into each bucket weekly.

The results of this simulated field trial is presented in Figure 1. Referring to Figure 1 now, it can be observed that the bioinsecticide is indeed very effective in controlling mosquito population by exterminating the mosquito larvae. The efficiency of the bioinsecticide is increased with the higher amount of bioinsecticide added. The additional food source does not hamper the biological activity of the bioinsecticide.

Field trial

The field trial was carried out in residential area of Gombak Phase 9 of Selangor, Malaysia. The area was selected as it had the most dengue cases reported weekly, as stated in Table 1 below. The reported dengue cases as in Table 1 were recorded on a weekly basis by the local Vector Control Agency, which is under the supervision of the Malaysia Ministry of Health.

The bioinsecticide used in this field trial was as described in Example 2. The measure employed to gauge the success of product application was the local incidence of reported dengue cases following application as compared to reported dengue cases before application in Gombak Phase 9, nearby areas (Gombak Phase 7 and 8 of Selangor, Malaysia), and unconnected areas (Crystal Heights, Pinggiran 21 , 22, and 23 of Selangor, Malaysia).

Table 1 : Weekly dengue incidence in selected areas of Selangor, Malaysia¹

Weekly Dengue Weekly Dengue Site Designation Incidence Before Incidence After

Treatment² Treatment²

Gombak Phase 9 Treatment 2.1 ± 0.32 0.2 ± 0.13 Gombak Phase 7 Control 1.3 ± 0.36 0.1 ± 0.10

Gombak Phase 8 Control 0.1 ± 0.13 0.3 ± 0.15

Crystal Heights Control 0.1 ± 0.10 Pinggiran 21 0.1 ± 0.09 0.1 ± 0.10 Pinggiran 22 Control 0.2 ± 0.11 0.3 ± 0.0.15 Pinggiran 23 Control 0.7 ± 0.21 0.3 ± 0.15

1) Data provided by Selangor Vector Control Agency

2) Weekly average ± SEM.

Gombak Phase 9 prior to application of the bioinsecticide was considered a 'hotspot' for dengue with an average reported incidence of 2.1 cases per week with a range of 1 - 4 cases per week. Gombak Phase 9 has historically had a high incidence of dengue, which had been very difficult to control. The data prior to application was accumulated over the first 15 weeks of 2010. Application of the bioinsecticide occurred on 18 April 2010. The post- application data covered 10 weeks, concluding on 19 June 2010.

There were a total of 31 reported cases of dengue in Gombak Phase 9 in the 15 weeks prior to application and only 2 reported cases in the 10 weeks after application. One of these post-application cases was reported during the first week of post-application and very likely could have resulted from mosquito vectored transmission prior to application, as it takes 3 days for the symptoms of dengue to appear and another day for laboratory confirmation. Consequently, confirmed reported cases of dengue take at least 5 days to be recorded. The other post-application reported dengue case in Gombak Phase 9 occurred 4 weeks after application with no reported dengue cases for the remainder of the report period, i.e. 6 weeks. This case appeared to be an "isolated island case" and could have arisen by the individual being infected in an outside area with it being reported as a Gombak Phase 9 case as the individual resided there.

The pre-treatment weekly dengue incidence of 2.1 cases per week is 7 - 10 times the weekly average for other areas for which information has been provided and qualified Gombak Phase 9 for designation as a 'hotspot'. The neighbouring district of Gombak Phase 7, which shares a large stationary water feature also has a higher than normal weekly incidence of dengue. The weekly incidence of dengue for both Gombak Phase 9 and 7 was reduced to a similar incidence level compared to outside districts, thus permitting removal of the dengue "hotspot" list.

Claims

l/WE CLAIM

1. A bioinsecticide active against Diptera insect larvae comprising at least two active ingredients, characterized in that the active ingredients comprise a regulatory hormone of enzyme biosynthesis, and a bacteria capable of releasing toxins.

2. A bioinsecticide in accordance with claim 1 , wherein the regulatory hormone is added to the bioinsecticide to a final concentration of 0.1 % to 25% by weight.

3. A bioinsecticide in accordance with any one of claims 1 and 2, wherein the regulatory hormone is capable of down regulating or inhibiting serine proteases biosynthesis by gut epithelial cells of Diptera insect larvae.

4. A bioinsecticide in accordance wMh cttm 3, wherein the serine protease is trypsin.

5. A bioinsecticide in accordance with any one of claims 1 , 2 and 3, wherein the regulatory hormone is Trypsin Modulating Oostatic Factor (TMOF).

6. A bioinsecticide in accordance with claim 1 , wherein the bacteria is added to the bioinsecticide to a final concentration of 0.1% to 25% by weight.

7. A bioinsecticide in accordance with any one of claims 1 and 6, wherein the bacteria is any subspecies of Bacillus thuringiensis, Bacillus sphaericus, or a combination thereof.

8. A bioinsecticide in accordance with claim 7, wherein the bacteria is Bacillus thuringiensis israelensis.

9. A bioinsecticide in accordance with claim 1, wherein the toxins are crystal proteins.

10. A bioinsecticide in accordance with any one of claims 1 and 9, wherein the toxins are capable of lysing gut epithelial cells of Diptera insect larvae.

11. A bioinsecticide in accordance with any one of claims 1 , 9 and 10, wherein the toxins are any one or a combination of Cry4, Cry10, Cry11 , Cyt1 , and Cyt2.

12. A bioinsecticide in accordance with claim 1 , wherein the Dipteral insect larvae comprise any one or a combination of mosquito larvae, fly larvae, gnat larvae, and midge larvae.

13. A bioinsecticide in accordance with any one of claims 1 to 12, wherein the bioinsecticide further comprises:

a) a floatable carrier acting as a carrier of the active ingredients and permits the bioinsecticide to float on water surface;

b) an adhesive material for adhering the active ingredients to the floatable carrier, and controlling the release of the active ingredients to the surrounding.

14. A bioinsecticide in accordance with claim 13, wherein the regulatory hormone is preferably added to the bioinsecticide to a final concentration of 0.1 % to 8% by weight.

15. A bioinsecticide in accordance with claim 13, wherein the bacteria is preferably added to the bioinsecticide to a final concentration of 0. % to 8% by weight.

16. A bioinsecticide in accordance with claim 13, wherein the floatable carrier is added to the bioinsecticide to a final concentration of 81 % to 98% by weight.

17. A bioinsecticide in accordance with claim 13, wherein the adhesive material is added to the bioinsecticide to a final concentration of 1% to 11% by weight.

18. A bioinsecticide in accordance with any one of claims 13 and 16, wherein the floatable carrier is rice husk.

19. A bioinsecticide in accordance with any one of claims 13 and 17, wherein the adhesive material is mineral oil.

20. A bioinsecticide in accordance with any one of claims 1 to 19, wherein the bioinsecticide further comprises:

a) a food colouring; and

b) a preservative agent.

21. A bioinsecticide in accordance with claim 20, wherein the food colouring is added to the bioinsecticide to a final concentration of 0.03% to 0.07% by weight.

22. A bioinsecticide in accordance with claim 20, wherein the preservative agent is added to the bioinsecticide to a final concentration of 0.1% to 0.3% by weight.

23. A bioinsecticide in accordance with any one of claims 20 and 22, wherein the preservative agent is methyl paraben.

24. A method for producing a bioinsecticide in accordance with any one of claims 1 to 23, comprising at least the steps of:

a) mixing of the floatable carrier, regulatory hormone, and bacteria; b) spraying the adhesive material into the mixing process; and c) stop the processes of mixing and spraying when the regulatory hormone and bacteria are adhered to the floatable carrier.

25. A method in accordance with claim 24, further comprising at least the steps of:

a) dissolving the food colouring in minimal amount of water;

b) spraying the dissolved food colouring into the mixing process;

c) dissolving the preservative agent in acetone;

d) spraying the dissolved preservative agent into the mixing process; and

e) continue the mixing process until acetone is being evaporated.

26. A bioinsecticide in accordance with any one of claims 1 to 12, wherein the bioinsecticide further comprises a water soluble carrier as a carrier of the active ingredients.

27. A bioinsecticide in accordance with claim 26, wherein the regulatory hormone is preferably added to the bioinsecticide to a final concentration of

5% to 25% by weight.

28. A bioinsecticide in accordance with claim 26, wherein the bacteria is preferably added to the bioinsecticide to a final concentration of 5% to 25% by weight.

29. A bioinsecticide in accordance with claim 26, wherein the water soluble carrier is added to the bioinsecticide to a final concentration of 50% to 70% by weight.

30. A bioinsecticide in accordance with any one of claims 26 and 29, wherein the water soluble carrier is maltodextran.

31. A bioinsecticide in accordance with any one of claims 1 to 12 and claims 26 to 30, wherein the bioinsecticide further comprises a preservative agent.

32. A bioinsecticide in accordance with claim 31 , wherein the preservative agent is added to the bioinsecticide to a final concentration of 0.1 % to 0.5% by weight.

33. A bioinsecticide in accordance with any one of claims 31 and 32, wherein the preservative agent is methyl paraben.

34. A method for producing a bioinsecticide in accordance with any one of claims 1 to 12 and claims 26 to 33, comprising at least the step of mixing the water soluble carrier, regulatory hormone, and bacteria to produce a flowing dry powder.

35. A method in accordance with claim 34, further comprising at least the step of mixing the preservative agent with the water soluble carrier, regulatory hormone, and bacteria to produce a flowing dry powder.