Recombinant materials for production of a protein toxic to...

Details Recombinant materials for production of a protein toxic to... Recombinant materials for production of a protein toxic to...

2000-10-24

2003-04-22

Sisson, B. L. (Department: 1634)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using a micro-organism to make a protein or polypeptide

C435S006120, C435S069100, C435S091100, C435S270000, C435S471000, C435S485000, C435S252300, C435S252310, C435S320100, C530S324000, C530S350000, C536S023100, C536S023710

Reexamination Certificate

active

06551800

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention relates to methods and compositions for controlling populations of Hymenopteran insect pests in the Formicidae (ant) family using a novel
Bacillus thuringiensis
(“PT”) toxin and preparation. In particular, the invention relates to effective methods of controlling populations of various fire ants of the family Solenopsis using a BT toxin effective in killing fire ants, and a novel strain of BT producing such toxin.
BACKGROUND OF THE INVENTION
Without limiting the scope of the invention, its background is described in connection with uses of
Bacillus thuringiensis
toxins as fire ant biocidal agents as an example. The imported fire ant,
Solenopsis invicta,
is an introduced species that arrived in Mobile, Ala. from South America around the 1930s. The imported fire ant has spread rapidly across the southern United States and continues to expand into areas of North America with mild climates and adequate moisture and food.
The imported fire ant is an agriculturally and medically important pest harmful to domestic animals, wildlife and humans. Fire ants particularly threaten obligate ground dwelling species and young animals of all species. The imported fire ant is thought to be responsible for the decline of several native species. Proliferation of the imported fire ant has been largely unchecked due to the absence of predators, pathogens and parasites that control its numbers in its native environment. The fire ant typically causes painful stings in humans and more severe reactions may occur in allergic individuals. High densities of fire ants have been responsible for damage to roads, pastures, and electrical and mechanical equipment. The recent appearance of multiple queen colonies has made control of fire ant populations even more difficult. An effective compound and method for control of fire ant populations that is safe to users and consumers is urgently needed.
Past attempts at fire ant control have involved the highly toxic chlorinated hydrocarbons heptachlor, dieldrin, and Mirex. The EPA has outlawed the use of these highly toxic albeit relatively effective compounds for all but exceptional applications. The most commonly used modern control methods used include the chemicals hydramethylnon, avermectin, and the synthetic insect growth regulator fenoxycarb. These compounds must be regularly reapplied and have not been able to significantly impact severe fire ant infestations or to control further spread. The search for control methods is now being conducted in the areas of sterile insect release and the introduction of natural enemies, both of which are only potential solutions with uncertain outcomes.
Bacillus thuringiensis
(“BT”) is the genus and species of a large number of strains of gram-positive, spore-forming bacteria which, under certain conditions, form a parasporal crystal comprised of insecticidal protein toxin (Bulla, et al.,
Crit. Rev. Microbiol.,
8:147-204, (1980); Höfte and Whitely,
Microbiol Rev
(1989) 53:242, (1989). The toxin itself is a glycoprotein product of cry genes (“Cry” is used to denote the protein; “cry” is used to denote the gene) as described by Höfte (Id.). Because the effects of the various Cry proteins are mediated by binding to unique receptors, the species specificity of a given BT toxin is typically quite limited as exemplified by the original classification proposed by Höfte (Id.): CryI (Lepidopteran specific); CryII (Lepidopteran and Dipteran specific); CryIII (Coleoptera specific); and CryIV (Diptera specific). The BT toxins function in the brush border of insect midgut epithelial cells and, although highly insecticidal to certain insects, are non-toxic to other organisms lacking toxin binding receptors (Gill, S. S. et al.
Ann. Rev. Entomol
(1992) 37:615).
Keen interest in BT toxins over the last 30 years has resulted in the isolation of more than 100 different BT crystal protein genes and the development of bioinsecticides for the control of insect species in the orders Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera and Mallophaga and against nematodes, mites and protozoa (Schnepf et al.,
Microbiol. Mol. Biol. Rev.
(1998) 62(3):775). BT toxin in various forms now accounts for 90% of the world sales of non-chemical insecticides.
U.S. Pat. Nos. 5,260,058, 5,268,297, 5,596,071 and 5,824,792, disclose processes and compositions for controlling pharoah ants (
Monomorium pharaonis
) using toxin containing bacterial cells of various BT strains. Although these toxins are alleged to be effective against all Hymenoptera and all ants, no testing beyond the pharoah ant was performed. Further, the protoxins of this invention were much larger (120-140 kD) than that described herein.
SUMMARY OF THE INVENTION
Prior to this invention, no BT toxins have been reported that are known to be effective in controlling fire ants, nor are any BT toxins commercially available for this indication. What is needed is a biocidal composition that is effective in controlling populations of the fire ant, yet is not toxic to non-insect organisms. Such a biocide would have broad applicability including the agricultural, domestic, environmental and biomedical arenas. For this reason a BT toxin effective in killing fire ants would be particularly desirable.
Fire ants are omnivorous, although a large portion of their diet comprises invertebrates which the fire ants sting and kill. They also feed on dead animal and plant tissues, seeds, developing and ripe fruits, and are attracted to honeydew and sap flows. They are attracted to sugars, certain amino acids, ions in solution, and to some oils containing polyunsaturated fatty acids in these food sources.
Worker ants can only consume liquid foods, and nearly half of the resources that are returned to the nest are in the form of liquids. Liquids consumed and stored by the foraging workers are fed to other workers through trophallaxis. Once the worker arrives back at the colony, the oils are slowly transferred to nurses and from the nurses to larvae. Soluble sugars in addition to some soluble protein and amino acid mixtures are strongly attractive and encourage trophallaxis among workers. This both dilutes the solution and reduces the speed of movement of these nutrients to larvae.
Undissolved solids greater than 0.88 microns are screened from the liquid in the pharynx of the worker fire ant and cannot be ingested by the worker ants. The solids accumulate in the buccal region as pellets and are later expelled to feed fourth-instar stage larvae, which are able to consume particles as large as 45 microns. Because solid food may be used by the mature larvae but not the workers, solids move from the field to these larvae more quickly and directly than liquid foods. After processing by the fourth instar larvae, previously solid foods can be utilized by the queen and young larvae through trophallaxis. As it is desirable that worker ants transport poisons back to the colony and distribute the poison throughout the colony, consideration to the favored foods of fire ants together with the physical size of the poison may influence the efficacy of treatment modalities.
The present invention provides a BT protein that is active against members of the Formicidae family, in particular the imported fire ant,
Solenopsis invicta,
and related species. Generally, the invention provides BT preparations that are effective in reducing populations of fire ants and related species. The invention further provides novel
B. thuringiensis
strains that produce toxins effective against fire ants and related species. Effectiveness is defined as the ability to reduce the numbers of ants within a local fire ant population, typically a mound-type community, by killing immature and/or mature individuals within the population who are exposed to the toxin. Exposure may take the form of ingestion of the subject toxin either directly in a bait formulation or as expressed in a food source or by trophallaxis.
This invention provides biologically pure cultures of an isolated BT bac

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