Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-25
2002-06-04
Slobodyansky, Elizabeth (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S195000, C435S218000
Reexamination Certificate
active
06399759
ABSTRACT:
BACKGROUND
Of the species of ants present in the United States,
Solenopsis richteri
and
Solenopsis invicta,
commonly referred to as the imported black fire ant and imported red fire ant, respectively, are not indigenous to North America. Both of these species are indigenous to the riverbanks of the Paraguay and Guapore regions of South America.
S. geminata
is a fire ant that is indigenous to North America.
The imported red fire ant (
Solenopsis invicta
), a major pest in the Southern United States, has had a large medical and economic impact because of its effects on man, livestock, and agriculture in general. For example, a study at the University of Texas, on a 70-acre tract of land, showed that as
S. invicta
moved across the effect was on the whole insect fauna. The number of other ant species declined by 70%, while a number of arthropod species, such as spiders, ticks, and other insects, dropped by 40%. It has been noted that fire ants are able to damage the roots of various crops, girdle the stems of young citrus trees, as well as feed on the fruits and flowers of several plants, particularly okra.
The most noticeable concern imposed by
S. invicta
is its physical and medical impact on man. Fire ants are a major nuisance to humans and other animals due to their powerful, painful sting, from which the name was acquired. In
S. invicta,
95% of the venom consists primarily of alkyloids (2,6-disubstituted piperidines). Allergic reactions which can lead to death from anaphylactic shock occur in 1-2% of the population. A 1989 survey announced that 32 persons died from fire ant stings, most of which involved less than five stings. Due to the prevalence of
S. invicta,
man cannot help but encounter them in any outdoor activity, resulting in between 67,000-85,000 people per year seeking medical treatment for fire ant stings.
In 1957, when it became apparent that fire ants posed a serious problem, the federal government launched a massive campaign to eliminate this pest. In five years, the United States government spent 70 million dollars dispensing chlordane, dieldrin and heptachlor over large areas. While these three poisons did kill fire ants, they also had adverse effects to a variety of other organisms, including the native ant fauna. Also, due to their stability, these compounds were soon detected in the environment and were found to be responsible for the death of birds, fish and reptiles. In view of this, the use of these chemicals immediately ceased. A few years later in the mid-sixties, the compound mirex was developed. It proved effective against
S. invicta,
but unfortunately, it was also found to be capable of killing all species of ants. Nonetheless, since mirex appeared to have minimal environmental effects and eradication seemed economically feasible, this poison was broadly employed to control fire ant infestation. However, after several years of use, low levels of residues were detected in some non-target organisms. Perhaps more significantly, remnants of this poison were found in adipose tissue from humans and studies suggested that mirex might be a carcinogen. Therefore, in 1978, the Environmental Protection Agency revoked the use of mirex, thus, ending the widespread distribution of this poison. Since that decision there has never been a more intensive effort to control an unwanted pest in the United States.
When the above poisons were used to fight the fire ant infestation, the indigenous species of ants, which happen to be the best defense against the fire ant, were also killed. By eliminating the fire ant's competition, in combination with their aggressiveness and high rate of reproduction, man played to the strength of
S. invicta
by clearing the way for it to invade the North American habitat.
Currently, the most common methods of treating fire ant infestation include the use of toxicants (amidohydrozones), juvenile hormone analogs, or avermectin B
1
a. However, in order to eliminate the colony completely it is necessary to kill the queen, and with the emergence of polygyne (multiple queen) colonies this has become more difficult. Juvenile hormone analogs interfere with the development of the brood, but the entire colony must be reached or it will quickly rebound. It has been observed that sometimes the colony will relocate in response to physical disturbance or stress if members of the colony suddenly die. Therefore a subtler and less invasive means of control would be useful.
In the course of maturation, the fire ant undergoes eight stages of development: egg—1st, 2nd, 3rd and 4th instar larvae—prepupae—pupae—adult. The 4th instar larvae plays an important role in the survival of the colony in that it is totally responsible for the digestion of solid foods and the source of nutrients for the queen and adult workers. It has been proposed that proteinase inhibitors might be a method for controlling undesirable insects; however, the use of non-specific inhibitors that inhibit a large number of ant proteolytic enzymes may cause a colony to relocate. Travis et al. (
Acta Biochemica Polonica,
43, 411-417 (1996)) disclose in a review the existence of three proteolytic enzymes isolated from the 4th instar larvae of
S. invicta.
However, the purification steps are not disclosed in sufficient detail to allow purification of the proteolytic enzymes.
SUMMARY OF THE INVENTION
The present invention represents a potential advance in the control and/or eradication of fire ants by describing the purification and characterization of fire ant proteinases. The present invention provides isolated polypeptides, preferably isolated from an ant, particularly a
S. invicta
4th instar larvae, having amidolytic activity for cleavage of a peptide bond present in a target polypeptide. In particularly preferred embodiments, four proteinases have been successfully isolated from the 4th instar larvae of
S. invicta
and characterized. Based on substrate specificity, they appear to represent two chymotrypsin-like and two elastase-like proteinases. These are referred to as Soli C1, Soli C2, Soli E1, and Soli E2, respectively, and have molecular weights of 25, 28, 23, and 24 kDa, respectively, based on SDS-PAGE. All enzymes are inhibited by diisopropyl fluorophosphate, a general serine class inhibitor. Each enzyme has been characterized as to pH optimum, pH stability, isoelectrofocusing, and susceptibility to inhibition by a broad range of natural and synthetic proteinase inhibitors. Such compounds may prove useful for the development of insecticides to control fire ant infestation.
In preferred embodiments, the polypeptide can have an elastase-like amidolytic activity, and can cleave a target polypeptide of MeOSuc-Ala-Ala-Pro-Val-pNA (SEQ ID NO:8), where the polypeptide and target polypeptide are in about 0.05 M Tris-HCl, about 100 mM NaCl at about pH 7.4 and about 25° C. for about 10 minutes. In other preferred embodiments, the polypeptide can have a chymotrypsin-like amidolytic activity, and can cleave a target polypeptide of Suc-Ala-Ala-Pro-Phe-pNA (SEQ ID NO:7), where the polypeptide and target polypeptide are in about 0.05 M Tris-HCl, about 100 mM NaCl at about pH 7.4 and about 25° C. for about 10 minutes. The polypeptide can include an amino terminal amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.
The invention is also directed at an isolated polypeptide, an active analog or an active fragment thereof, where the polypeptide includes an amino acid sequence that has a percentage amino acid identity of greater than 39% with SEQ ID NO:2, or alternatively including the amino acid sequence of SEQ ID NO:2, an active analog or an active fragment thereof.
Another aspect of the invention is an isolated nucleic acid fragment encoding a polypeptide, where the nucleic acid fragment has a nucleotide sequence including nucleotides 519 to 1198 of SEQ ID NO:1, and a complement thereto. Alternatively, the nucleic acid fragment of the invention is a complement of the nucleic acid fragment that hybridizes to nucleotides 519 to 1198 of SEQ ID NO:1
Blum Murray S.
Travis James
Whitworth S. Troy
Mueting Raasch & Gebhardt, P.A.
Slobodyansky Elizabeth
University of Georgia Research Foundation Inc.
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