AU744876B2 - Methods and compositions to control tephritidae fruit flies - Google Patents

Description

WO 99/13724 PCT/US97/16475 1

DESCRIPTION

METHODS AND COMPOSITIONS TO CONTROL TEPHRITIDAE FRUIT FLIES Field of the Invention The present invention relates to compositions and methods utilizing borax toxicants, such as borax, to control fruit fly populations of the Tephritidae family.

Background Roaches, ants, termites, house flies and fruit files are common pests that have plagued mankind for ages. House flies are serious nuisance pests to the poultry and livestock industries, whereas fruit flies are a serious problem to, for example, citrus fruit, and have continued to plague agricultural industry for decades. Extensive efforts have been made heretofore to exterminate these difficult and sometimes disease-bearing and/or fruit infecting insects.

Boric acid is known as a killing agent in roach, fruit fly and ant-killing compositions.

For example, Australian patent 22,579 (Fenwicke, 1935) teaches the use of boric acid as a "germicidal antiseptic" in combination with castor oil and turpentine as "cleaning agents" to be applied to sheep for killing maggots. Japanese patents J5-8052-205 (Nakamoto, 1981), J6-1030- 506-A (Watkayama, 1984) and J6-1078-705-A (Amachir, 1984) teach the use of boric acid as the killing agent in various complex compositions for killing roaches (Nakamoto and Amachir) and white ants (Wakayama). All three Japanese patents are dried and used in a pellet, tablet or ball form. Enkerlin, W. et al.: Use of a Mixture of Boric Acid, Borax, Hydrolyzed Protein, and Water to Control Anastrepha Fruit Flies, Fruit Flies: Biology and Management, ed. Alua, P. and Liedo, Springer-Verlag, NY, Inc., pp. 353-358 (1993) discusses the use of a toxic bait consisting of boric acid, hydrolyzed protein (PIB.7) and water to kill, for instance adult Anastrepha Ludens (Loew) and adult Ceratitis capitaia (Wied).

French patent 2,491,296 (Lagache, 1982) shows a 50/50 by weight composition of boric acid or one of its salts plus sweetened condensed milk which was placed, without spreading, in a ship's hold to control cockroaches. Japanese document JA-72-23198-R (Sankyo Co. Ltd. 47- 23198) shows a toxic roach bait comprising insecticidal compositions, e.g. dieldrin, BHC (Lindane), DDT, Sumithic, and boric acid mixed with more than 4 weight percent glycerol in carriers, such as cereal, fish meal, rice bran, starch paste, sugar, maltose, fatty acids, faulty acid esters and fatty alcohols. Japanese patent J5-4017-120 (Sakamoto) shows a cockroach bait of WO 99/13724 PCT/US97/16475 2 1.5-10 weight percent boric acid, 10-50 weight percent starch and an extract of fish or animal bones prepared by boiling the bones in water for not over 2 hours.

While boric acid has been used previously, the art teaches that it must be kept dry, as wet boric acid will not work; Wellness Letter, University of Calif. at Berkeley, September 1991, page 7. Thus, use of boric acid with aqueous liquefiers, such as water, is not expected to be effective.

U.S. Patent No. 4,205,066 (Hennant et al.) discloses a bait composition for anthropophilic flies which utilizes boric acid, for example, as the insecticidal material in such bait compositions.

U.S. Patent No. 4,440,746 (Maglio) is concerned with a granular pesticide composition which relies upon borax as a source of borate ions to effect gelation of polyvinyl alcohol.

U.S. Patent No. 4,617,188 (Page) relates to natural insecticides employing borax and carob to control cockroaches.

Grace, J.K. et al.: J. Econ. Entomol, 84(6):1753-1757 (1991) is concerned with the response of certain subterranean termites to borate dust and soil treatments.

Enkerlin, W. et al.: Use of a Mixture of Boric Acid, Borax, Hydrolyzed Protein, and Water to Control Anastrepha Fruit Flies, Fruit Flies: Biology and Management, ed. Aluja, P. and Liedo, Springer-Verlag, NY, Inc., pp. 353-358 (1993) suggest that borate compounds may be used as insecticides against fruit flies and that a mixture of boric acid, borax, hydrolyzed protein and water may be used to control Anastrepha fruit flies.

Hogsette, J.A. et al.: J. Econ. Entomol., 85(4):1209-1212 (1992) compare toxicity of aqueous solutions of boric acid and polybor (disodium octaborate tetrahydrate) to house flies (Diptera: Muscidae).

Mullens, B.A. et al.: J. Econ. Entomol., 85(1):137-143 (1992) is concerned with the effects of disodium octaborate tetrahydrate (polybor) on the survival, behavior and egg viability of adult Muscoid flies (Diptera: Muscidae), house flies.

Lopez, F.D. et al.: J. Econ. Entomol., 61(1):316-317 (1968) disclose the use of pelletized lures formulated with borax and either PIB.7 (protein insect bait) or ENT-44, 014-X (enzyme hydrolyzed cottonseed protein) to trap and catch Mexican fruit flies.

Lopez, F.D. et al.: J. Econ. Entomol., 60(1):137-140 (1967) suggest that sodium borate inhibits decomposition of two protein hydrolysates attractive to the Mexican fruit fly.

Ken, A.J. et al.: Insect Pests Leaflets. Noll.-Fruit Flies, Gov't Printer, Dept. of Agriculture, N.S.W. Australia (1930) disclose the use of lures containing borax to trap Mediterranean and Queensland fruit flies.

WO 99/13724 PCT/US97/16475 3 Newman, L.J. et al.: Fruit Fly (Ceratitis capitata) Baiting and Trapping Experiments, leaflet No. 244, Gov't Printer, Dept. of Agriculture, Western Australia disclose the use of arsenate of soda as a rapid killer of certain fruit flies and that trapping or luring methods utilizing arsenate of soda appear to be somewhat more effective than baiting methods.

The prior art also shows the high degree of specificity of attractants in different insecticide compositions. For example, U.S. Patent No. 4,049,460 (Broadbent, 1977), teaches a composition of brown sugar, a binder material (paraffin or wax), dry dog food, maltose ad Dursban (a commercially available insecticide) in pellet form. Roaches are attracted to the dog food, maltose and sugar mixture. The pellets are coated with paraffin or wax to protect them from disintegrating upon exposure to environmental factors. The Dursban is ingested by the roaches, along with the attractant. Japanese patent J53091-140 (Kao Soap KK) teaches the use of pure concentrated sesame oil, preferably mixed with an extract of cockroaches faeces as an attractant for cockroaches.

U.S. Patent No. 4,332,792 (Kohn et al., 1982) teaches a process for preparing a pyrolyzate solution of corn syrup and N-methylnicotinic acid for attracting insects, particularly roaches.

U.S. Patent No. 4,369,176 (Ott, 1983) teaches a sugar, bacteria and carrier material (such as ground comrncobs, sawdust or sand) for use as an insect attractant. The sugar is degraded by the bacteria, causing fermentation by-products which are the attractant. The attractant is combined with an insecticide to kill insects. The insects ingest the insecticide along with the composition.

U.S. Patent No. 4627,981 (Shimano et al., 1986) discloses the use of various alcohols dissolved in an organic solvent and impregnated on a carrier (such as cardboard or cloth) for use in attracting and killing insects in pellet form.

Notwithstanding the above, there is still a need in the art for improved toxicants that are effective against fruit flies of the family Tephritidae and that are specific and powerful attractants, that have features for direct and easy application, and that are not environmental pollutants or potential carcinogens.

Summary of the Invention In brief, the present invention overcomes and alleviates certain of the above-mentioned drawbacks and shortcomings of the prior art and is directed to novel methods and compositions for attracting and either killing or controlling reproduction of fruit flies of the family Tephritidae.

WO 99/13724 PCT/US97/16475 4 Generally speaking, the present invention is premised upon the realization that borax or borate compounds in effective amounts will not only attract fruit flies, but will either kill fruit flies or cause female fruit flies to stop producing eggs for at least about seven days, depending upon the amount formulated into the bait or lure and ingested by the fruit flies. While the preferred toxicant contemplated by the present invention is borax (sodium borate mol Na 2 ,B,4010H 2 0 or sodium borate pentahydrate-5 mol Na 2

B

4 O5H 2 other suitable borate compounds may be utilized in effective amounts as substitutes for borax or may be utilized in effective amounts in combination with borax or one another. Exemplary of boraxtype compounds envisioned by the present invention include anhydrous borax Na 2 BO0,, ammonium tetraborate (NH 4 2

B

4 0 7 ,4HO, ammonium pentaborate (NHH4)2BioO 1 6 8H 2 0, potassium pentaborate K 2 B3 0 0 16 8H 2 O, potassium tetraborate K 2

B

4 0,4 O, sodium metaborate (8 mol) Na 2 B20 4 .8HO, sodium metaborate (4 mol) Na 2 B20O 4 4H 2 0, disodium tetraborate decahydrate NazB 4

O

7 1OH 2 0, disodium tetraborate pentahydrate Na 2

B

4 0 7 ,O5HO and disodium octaborate tetrahydrate Na 2

B

8

O,,

3 4H 2 0. Thus, the term "borax toxicant(s)" is used herein broadly and includes collectively and/or individually such borax and any other suitable borax type compounds.

Also in accordance with the present invention, the borax toxicants may be utilized alone or in combination with baits, insecticides, other toxicants, agars, liquefiers, sweeteners, carriers and the like. Moreover, the borax toxicants may be utilized in the.anhydrous and hydrous forms; however, when the anhydrous forms are selected, it should be appreciated by those versed in this art that such compounds are typically more expensive and will generally convert to a hydrated form in water or moisture environments. It should also be appreciated by those versed in this art that mixtures of borax toxicants may be utilized, as long as the borax toxicant available in such mixtures achieves the proper molarity to ensure that the objectives of the present invention are not defeated.

In accordance with the present invention, an effective molarity for a borax toxicant is believed to be in the range of between about 0.02 M and about 0.12 M or higher. When the end result to be accomplished is to kill fruit flies of the Tephritidae family, the amount of a borax toxicant that is believed that should be ingested by a fruit fly is between about 5 micromoles and about 10 micromoles during about a 24 hour period of time. However, if the objective is to prevent or stop the female fruit flies from laying eggs for about seven days or more, the borax toxicant concentrate may be adjusted, so that the amount ingested by a female fruit fly is believe to be between about 2.5 micromoles and about 5.0 micromoles during about a 24 hour period of time. It should be appreciated by those of skill in this art that the fruit flies of the Tephritidae WO 99/13724 PCT/US97/16475 family, as contemplated by the present invention, include the Caribbean fruit flies, the Mediterranean fruit flies, the Mexican fruit flies, the Oriental fruit flies and any other fruit flies which may be controlled by the methods and compositions of the present invention.

In accordance with the present invention, the methods and compositions are believed to be safe and effective and, therefore, can be used on any surface or at any location, such as paper, cardboard, concrete, plastic, metal, glass, plants, in kitchens of homes and restaurants, trees, utility poles, fences, signs, etc. In addition, the compositions of the present invention can be easily applied directly to areas of infestation and will remain active for extended periods of time. Therefore, the borax toxicants of the present invention may be used in residential preparations, commercial crop production, eradication programs and suppression programs for Tephritid fruit fly control.

In accordance with the present invention, the compositions include a mixture of a borax toxicant in an effective amount and, for example, protein hydrolysate bait or any synthetic bait to generate a bait or lure in the form of a patty, heavy cream, pellet, gel, foam, paste, liquid or spray. An example of a patty in accordance with the present invention includes borax in an amount of between about 0.01M and about 0.1M or more, agar, yeast hydrosylate, sugar and water. Notwithstanding, the bait or lure may be in the free form or, alternatively, in a form, such as granules or tablets, agglomerated with or without the aid of a binder. Moreover, the bait or lure can be fixed or impregnated on a support or absorbed therein, and this support may include, for instance, agar, paper, cardboard, plastic such as polystyrene, polyvinyl chloride, polyvinyl acetate and cellulose acetate, glass, pumice, crushed marble, silica or silica minerals.

Optionally, other toxicants, such as Malathion, Dibrom® and Naled®, attractants, such as Male Lure I 1®and methyl eugenol, sweeteners, carriers and/or liquefiers may be used as indicated hereinbefore. The bait or lure may then be placed in selected locations such that the fruit flies are likely to encounter and ingest the borax toxicant to assure the desired effect, but preferably out of the way of normal human or animal traffic.

It should therefore now be understood by those versed in this art that the novel methods and compositions of the present invention provide a simple, yet unique solution to controlling fruit fly populations by providing an attractant and a food arrestant for fruit flies. More particularly, it has been surprisingly discovered that the methods and compositions of the present invention uniquely attract the fruit flies and cause the fruit flies to stay and engorge, so that the objectives of the present invention are accomplished, controlling the population of fruit flies by either killing the fruit flies or preventing the female fruit flies from producing eggs for at least about a seven day period.

WO 99/13724 PCT/US97/16475 6 The above features and advantages of the present invention will be better understood with reference to the detailed description and examples set out hereinafter. It will also be understood that the methods and compositions of this invention are exemplary only and are not to be regarded as limitations of this invention.

Detailed Description By way of illustrating and providing a more complete appreciation of the present invention and many of the attendant advantages thereof, the following detailed description is provided covering the novel methods and compositions which utilize borax toxicants to eradicate or control fruit flies. Moreover, the following detailed description illustrates the invention by way of example, not by way of limitation of the principles of the invention. This detailed description should therefore enable one skilled in the art to make and use the inventions and describes several embodiments, adaptations, variations, alternatives and uses of the invention, including what is presently believed to be the best mode of carrying out the invention.

One objective of the present invention is in wide-area suppression and eradication programs. Currently, hard pesticides, such as malathion, are formulated with a protein hydrolysate bait, such as Miller's Nu-Lure®. The protein hydrolysates are usually corn-based.

Instead of the hard pesticide, one of the borates detailed above, and especially borax, may be substituted in one of the concentrations previously described. This substitution results in a pH change from about 5.0 to 8.5. A precipitate will form. This is filtered to prevent clogging the spray nozzles of either ground or air application equipment. The protein hydrolysate may be used full-strength or diluted to about 10% with water before the borate compound is added. The final proteinaceous bait spray may be used over wide inhabited areas, as the borates described above are exempt from tolerances for fire ant control (Fed. Reg. 58(124):34972 (June 30, 1993)).

A second innovative method for use in accordance with this invention is to formulate the borax toxicant with an extender or gel, such as Min-U-Gel®, Thixcin Myverol® and CAB-O-SIL®. These are commercially available. In this case, the precipitate is believed not to be important as gels are sprayed in a solid stream to adhere to tree trunks, telephones poles, buildings and so forth. The gels are formulated with synthetic bait and/or natural proteinaceous baits. This method of application reduces worker and public inconvenience of aerial spraying of large areas. For the homeowner, either the gel formulation or the liquid formulation may be applied to individual host trees for Tephritid fly control.

For lethal concentrations to reduce a Tephritidae fly population below economic threshold at a targeted area, a borax toxicant of the present invention should be applied once per WO 99/13724 W PCT/US97/16475 7 week for at least six weeks (7 days per week). For lower concentrations which suppress egg production, a weekly application schedule for six weeks (7 days per week) is also believed to be necessary. These schedules are based on a life-span of about 40 days for Tephritidae fruit flies in the field and egg production suppression of at least 7 days. In a limited geographical area, these schedules are believed to be capable of suppressing fly populations below economic levels. With wide geographical use, it is also believed that Tephritidae fruit flies will be eradicated.

It, of course, should be understood by those versed in this art that the compositions of the present invention may be applied by any suitable means, such as by pressurized applications, hydraulic oil squirt cans and aerial sprays. Moreover, it should be appreciated by those skilled in this art that, whenever the compositions of the present invention are to be applied, weather conditions should be taken into account. For instance, treatment or retreatment should not be considered if weather reports indicate a 50% or greater chance of precipitation within 48 hours.

Overall, the more telling effects of use of the present invention are suppression of egg production which breaks the life cycle of the Tephritidae fruit flies, the lack of environmental effects of the treatment, and worker safety. Moreover, the present invention is adaptable to integrated pest management programs, and it is believed that the components and methods of the present invention do not attract honeybees.

The present invention will now be further illustrated, without limitation, with reference to the following examples.

EXAMPLE I Agar is made as indicated below. Some difficulty, however, is encountered with about 0.25N and saturated agars. The agar makes a thick scum layer which rises to the top, but the agar is heated and stirred repeatedly until this layer seems to melt and be dispersed. Upon pouring these 2 agars, there appears to be some precipitation of either the agar or borax or both, but none can be seen upon gelling, unless the precipitate rises to the top with the foam. Each cage is set up with food and one cage is supplied with a "water only" agar patty and an agar patty with borax (control had 2 "water only" agar patties). Each cage contained 50? and The about 0.25N and saturated agars did not gel due to extreme pH. These two borax concentrations are not included and duplicate cages for control, about 0.01N and about 0.05N borax, are set up to run instead. Cages are set by about 4:45pm on the first day, and the results are recorded in Table I below.

WO 99/13724 WO 9913724PCTIUS97/1 6475 Borax* Toxicity Experiment with Borax Agar Molarily of I Amount of I Amount of Amount of Borax j Borax Water Agar 0.005M 0.572gmn 300m] 3gmn 0.025M 2.861 gm 300m1 3gm 0.12M 14.303gmn 300m1 3gm saturated 11 5.0gmn 500m1 3gmn 0.0 0.0 300m1 3gmn Borax (sodium tetraborate) NaIB 4 0.IOH2O F.W. 381.37. About 1 16 ml] water 164M).

g dissolves in about TABLE I 1 Results of three day experiment.* [72 Hr.

___Day 3 1Cage [Borax Coni Dead Dead INo.

Day Time #I centration d Age of I. J Eggs Day 1 10:30 A 0.0-control 0 0 9 days 2553 Dayl1 10:30 B 0.02N 0 0 9 days 1072 Dayl1 10:39 C 0.10N 2- 13 9 days 18 Day 1 10:30 D 0.0-control 0 0 9 days 1064 Dayl1 10:40 E 0.02N 1 3 9 days 2165 Dayl1 10:45 F 0J .1ON 5 21 9 days 113 Presumptive starting numbers, 50e, 50? in eac h cage EXAMPLE II The agars, as prepared below, are used in the following five Mortality and Egg Production Studies.

Measure about 30m1 of the agar described below into a graduated cylinder and pour into the bottom of a petri dish. This agar should be made daily. Weigh the agar patty on a sheet of WO 99/13724 PCT/US97/16475 9 glassine paper and leave this paper on the patty so it can be weighed later. Agar is placed on cages for about 24 hrs. then removed and weighed. Regular food is then placed on cages for the duration of the experiment.

Borax (Sodium Tetraborate) Solutions for Toxicity Study LCso Normality Molarity (borax) Weight Volume pH Wt/Vol (borax) 0.02N 0.954g 500ml 9.19 0.1908% 0.005 0.1 ON 4.768g 500ml 9.26 0.9536% 0.0250 0.20N 9.535g 500ml 9.22 1.9% 0.05 0.40N 19.070g 500ml 9.32 3.8140% 0.10 0.04N 1.908g 500ml 9.04 0.3816% 0.01 0.14N 6.678g 500ml 9.17 1.3356% 0.035 about 0.05g Agar about 2.0g Yeast Hydrolysate about 55ml distilled Water about 10.0g Sugar combine ingredients and heat until the agar is melted (about 1 min.) after the above is heated properly, add the sugar and stir until dissolved do not heat WO 99/13724 WO 9913724PCTJUS97/1 6475 l.a. Mortality and Egg Production Study AegeCageg Conc. of B(N) Time Agar Dead f~~d lOd I 0.ON-control 3:50 pm Day 1 0 1 2 0.02N (0.005M) 3:55 pm Day 1 I 1 3 0.02N (0.25M) 4:00 pm Day 1 1 0 4 0.20N (0.05M) 4:05 pm Day 1 21 5 0.40N 1OM) 4:10 pm Day 1 24 10 1 ld 6 0.0N-control 11:11 am DaylI 1 0 ld 7 0.02N 11:17 am Dayl1 0 0 lid 8 0.I1ON 11:25 am Dayl1 0 0 ld 9 0.20N 11:34 am Dayl1 8 1 ld 10 0.40N 11:38 am Dayl1 19 2 12d 11 I 0.ON-control 9:35 am Day 1 0 0 12d 12 0.02N 9:3 8am Dayl1 1 0 12d 13 M.ON 9:41 am Dayl1 0 0 12d 14 0.20N 9:45 am Day 1 3 0 12d 15 0.40N 10:30 am Dayl1 12 4 2d la 0.ON-control 8:30 am Day 1 0 0 2d 2a 0.02N 8:35 am Day 1 0 0 2d 3a M.ON 8:40 am Day 1 0 0 2d 4a 0.20N 8:45 am Day 1- 0 1 2d 5a 0.40N 8:50 am Day 1 0 0 3d 6a 0.ON-control 10:45 am Day 1 0 0 3d 7a 0.02N 10:47 am Day 1 0 0 3d 8a MO1N 10:49 am Dayl1 0 0 3d 9a 0.20N 10:51 am Day 1 0 0 3d 10a 0.40N 10:53 am Dayl1 4 1 1Lb. Mortality and Egg Production Study #1 Cage 1Total No.f Dead] Dead No. of J Dead I Dead fTotal No.1 of Eggs 48 hr e J Eggs J72hr I of Eggs 1 844 Day 2 0 0 615 Day 3 0 0 541 2 787 Day 2 0 0 472 Day 3 0 2 98 3 0 Day 2 13 0 1 Day 3 6 0 13 4 3 Day 2 4 17 2 Day 3 0 2 0 1 Day 2 1 15 4 Day 3 6 982 Day 2 3 1 764 Day 3 1 0 844 7 986 Day 2 0 0 903 Day 3 0 1 465 8 542 Day 2 7 0 20 Day 3 4 1 41 9 7 Day 2 16 3 4 Day 3 1 1 0 24 Day 2 6 13 0 Day 3 0 1 1 11 491 Day 2 1 1 965 -Day 3 0 2 686 12 1330 Day 2 0 0 902 Day 3 0 0 668 13 287 Day 2 9 0 41 Day 3 5 2 14 121 Day 2 12 6 0 Day 3 10 8 1078 Day 2* 13 12 0 Day 3 1 4 0 *9:45am only 6 total flies alive in cage 15 on Day 2 1 a 0 Day 2 0 0 0 Day 3 0 0 0 2a 0 Day 2 0 0 0 Day 3 0 0 0 3a 0 Day 2 0 0 0 Day 3 0 0 0 4a 0 Day 2 4 0 0 Day 3 5 0 0 0 Day 2 19 5 1 0 Day 3 6 7 0 6a 0 Day 2 1 0 0 Day 3 0 0 0 7a 0 Day 2 0 0 1 Day 3 0 0 0 8a 0 Day 2 0 0 1 Day 3 2 0* 0 IQan 0 Day 2 1 5 1 0 1Day 3 1 6 1 3 1 0 l 1a 10 1 Day 2 1 15 'L 1 Day 3 1 5 1 6 0 one escapee, sex not noted 0 '0

-J

1 Mortality and Egg Production Study #1 IBegn. Cage jConcentration] Dead jDead ITotal# ofl Dead Dead~ Total No.1 Age of Borax 96 hr Jd Eggs L120 hr e_ J of Eggs 1 0.0N-control Day 4 0 0 632 Day 5 0 0 882 2 0.02N Day 4 1 1 206 Day 5 0 1 512 3 0.10N Day4 1 3 32 Day 5 0 9 31 4 0.20N Day 4 0 1 0 Day5 5 0.40N Day 4 Day5 1ld 6 0.ON-control Day 4 0 0 754 Day 5 0 1 1336 1ld 7 0.02N Day 4 0 0 759 Day 5 4 0 699 I1d 8 0.1ON Day 4 4 0 90 Day 5 0 0 41 lid 9 0.20N Day 4 0 7 0 Day 5 0 1 32 1ld 10 0.40N Day 4 0 3 0 Day 5 0 3 3 12d 11 0.ON-control Day 4 2 0 1126 Day 5 1 1 436 12d 12 0.02N Day 4 1 1 1495 Day 5 1 1 573 12d 13 0.1ON Day 4 3 1 209 Day 5 1 3 42 12d 14** 0.20N Day 4 0 6 6 Day 5 1 3 0 12d 15"* 0.40N Day 4 0 2 11 Day 5 0 0 0 **Cage #14 destroyed on Day 5. Only ld 2 was left alive/*Cage #15 destroyed on Day 5. Only 1 left alive 2d Ia control Day 4 0 0 0 Day 5 0 0 0 2d 2a 0.02N Day 4 0 0 0 Day 5 0 0 0 2d 3a 0.10N Day 4 0 0 0 Day 5 0 0 0 2d 4a 0.20N Day 4 1 1 0 Day 5 2 2 0 2d 5a 0.40N Day 4 2 6 0 Day 5 1 6 0 3d 6a control Day 4 0 0 135 Day 5 0 0 555 3d 7a 0.02N Day 4 0 0 32 Day 5 0 0 506 3d 8a 0.10N Day 4 3 0 0 Day 5 1 0 0 3d 9a 0.20N Day 4 8 3 0 Day 5 5 8 0 3d 10a 0.40N Dav 4 1 6 0 Day 5 1 0 1 Mortality and Egg Production Study #1 Begn. Cage Boron Dead Dead Dead Dead Total Age J C144 hr J Eggs 168 hr d of Eggs 1 0.0 control Day 6 2 0 943 Day 7 0 0 1283 2 0.02 Day 6 1 0 847 Day 7 0 1 582 3 0.10 Day 6 0 3 22 Day 7 0 1 0 lOd 4 terminated Day 6 5 terminated Day 6 lid 6 0.0 control Day 6 0 1 956 Day 7 0 0 988 lid 7 0.02 Day 6 0 1 815 Day 7 1 0 516 U.1U Day 0 Dav 7 ~Da 7 v lid 9 0.20 Day 6 0 2 57 Day 7 0 2 5 F nal Live Count lid 10 0.40 Day 6 0 0 0 Day 7 0 3 0* Coun 12d 11 0.0 control Day 6 0 0 1214 Day 7 0 1 989* o"19/920 12d 12 0.02 Day 6 0 0 777 Day 7 0 0 678* c?22/?23 12d 13 0.10 Day 6 0 3 18 Day 7 0 0 o"12/916 12d 14 0.20 Day 6 term'd Day 5- 12d 15 0.40 Day 6 term'd Day 5 2d la ___Day 6 0 0 1505 Day 7 0 0 955 de25/?25 2d 2a 6 0 0 75 Day 7 0 0 774 o25/?25 2d 3a Day 6 1 0 16 Day 7 0 0 119 d24/?25 2d 4a Day 6 0 2 0 Day 7 3 0 30 cl 1/?18 2d 5a Day 6 3d 6a Day 6 2 0 642 Day 7 0 0 1001 d'22/?25 3d 7a Day 6 0 1 668 Day 7 0 0 c&26/$24 3d 8a Day 6 1 2 20 Day 7 0 0 202 c18/?24 3d 9a 6 3 2 0 Day 7 4 0 do0/?2 3d l 10a Day 6 *cages 10, 11, 12, 13 terminated as of the end of day 7 iLe. Mortality and Egg Production Study #1 Live Live Fia Final Total No.Tta Cae# Cn.Brx 216 hr Deadd Dead$ Total oa of Eggs I control Day 9 0024 21 710* 2 0.02N Day 9 0 0 20 21 562* 3 O.ION Day 9 I 1 0* 4 192 hr 6 control Day 8 0 0 19 20 494*' 7 0.02N Day 8 0 0 21 22 8 M.ON Day 8 2 0 7 22 279** 9 0.20N Day 8 0 0 0 7 *Terminated Day 9 Terminated Day 8 2.a. Mortality and Egg Production Study #2 Beginning 11Concen. TimeAgar J24 hr Total Wt. Total No Age JCage I Borax IPut on Cagej 24 hr d'J~ J Paper Agar of Eggs 4d I1I control 9:16 am Dayl1 0 0 16.5g 2 4d 12 0.OlN 9:18 am Dayl1 0 1 17.7g 2 4d 13 0.05N 9:20 am Day 1 0 0 19.8g 11I 4d 14 0.10N 9:22 am DaylI 1 0 2'1.7g 3 4d 15 0.20N 9:26 am Day 1 7 13 2 1I.Og 7 16 control 11:05 am Dayl1 0 0 21.9g 17 0.OIN 11:11 am Dayl1 0 0' 21.7g 77 18 0.05N 11:13 am Dayl1 0 0 22.7g 23 19 0.10N 11:15 am Dayl1 0 0 22.lg 20 0.20N 11: 17 am Dayl1 3 0 21.5g 21 6d 21 control 10:00 am Dayl1 0 0 15.6g 2008 6d 22 0.O1N 10:02 am Dayl1 0 0 15.7g 770 6d 23 0.05N 10:04 am Day 1 0 0 18.7g 630 6d 24 0.LON 10:10 am Dayl1 2 0 19.lg 263 6d 25 0.20N 10:45 am Day 1 10 0 25.7g 150 7d 26 control 10:24 am Dayl1 0 0 19.13g 697 7d 27 0.02N 10:26 am Day 1 1 10 19.83g 820 7d28 M.ON 10:28 am Dayl1 0 0 18.78g 433 7d 29 0.20N 10:30 am Day 1 7 1 18.97g 477 7d 30 -0.20N *10:32 am Day 1 1 3 4 1 19.90g 0 2.b. Mortality and Egg Production Study #2 IfDead Dead Total No. jDeadJDead [Total No.

Cage 48 hr d, I I of Eggs 72k hr_ of Eggs I1I Day 2 0 0 0 Day 3 1 0 29 12 Day 2 0 0 0 Day 3 0 0 13 13 Day 2 0 0 0 Day 3 2 0 0 14 Day 2 10 4 0 Day 3 8 3 0 Day 2 14 12 0 Day 3 2 9 0 16 Day 2 0 0 813 Day 3 0 0 719 17 Day 2 0 0 1010 Day 3 0 0 827 18 Day 2 0 0 252 Day 3 0 0 7 19 Day 2 7 1 5 Day 3 7 0 3 Day 2 16 8 0 Day 3 3 6 0 21 Day 2 0 0 1099 Day 3 0 0 1185 22 Day 2 0 0 963 Day 3 0 0 847 23 Day 2 2 0 91 Day 3 4 0 3 24 Day 2 17 1 113 Day 3 2 5 9 Day 2 12 5 45 Day 3 3 7 26 Day 2 0 0 876 Day 3 0 1 1528 27 Day 2 0 0 702 Day 3 1 1 1442 28 Day 2 2 0 103 Day 3 9 0 9 29 Day 2 14 6 55 Day 3 4 9 4 30 1 Day 2 11 _L 14 1 0 Day 3 4 9 2.c. Mortality and Egg Production Study #2 ~Beginning Concen. JDead Dead Total No. JDead JDa oa o Age I Cage I Borax 96 hr I j ofgg 120 hr d Eggs 4d 11 control Day 4 0 0 606 Day 5 0 0 1437 4d 12 0.OIN Day 4 0 0 229 Day 5 1 0 490 4d 13 0.05N Day 4 4 0 2 Day 5 3 0 8 4d 14 0.10N Day 4 5 6 0 Day 5 0 7 0 4d 15 0.20N Day 4 1 0 2 DayS 2 0 16 control Day 4 0 0 803 Day 5 0 1 993 17 0.0IN Day 4 0 1 1143 DayS5 1 0 1236 18 0.05N Day 4 3 1 49 DayS5 1 0 128 19 0.1ON Day 4 3 7 9 DayS5 5 1 20 0.20N Day 4 3 6 0 Day 5 3 7 6d 21 control Day 4 0 0 1289 DayS5 0 0 2538 6d22 0.O1N Day 4 0 0 587 Day 5 0 0 1587 6d 23 0.5N Day 4 6 0 6 DayS5 1 0 6d 24 0.1ON Day 4 2 4 10 DayS5 1 2 4 6d 25 0.20N Day 4 0 4 32 Day 5 4 3 7d 26 control Day 4 0 0 1 1573 DayS5 0 0 1054 7d 27 0.O1N Day 4 0 1 1130 DayS5 0 0 1103 7d 28 0.05N Day 4 3 1 0 Day 5 4 6 4 7d 29 0.1ON Day 4 0 4 1 1 DayS5 2 6 7d 30 AO.4N Day 4 3 5 1 Day 5 2.d. Mortality and Egg Production Study #2 Cage I 14r Dead Dead Total No. DrDead ed Ttlo1 Final 1Fnl Cage_ 14 hr ofgs 168 ej6 of Eggs jLive? Live?~ I1I Day 6 0 0 1992 Day 7 0 0 2044 24 12 Day 6 0 0 998 Day 7 0 1 1546 24 23 13 Day 6 2 1 0 Day 7 1 0 18 12 24 14 Day 6 1 5 0 Day 7 Day 6 16 Day 6 0 0 1556 Day 7 0 0 735 25 24 17 Day 6 0 1 1374 Day 7 0 0 1007 24 23 18 Day 6 1 2 310 Day 7 0 0 376 21 22 19 Day 6 1 7 1 Day 7 1 5 2 0 4 Day 6 0 1 0 Day 7 1 0 0 0 21 Day 6 0 0 2506 Day 7 0 0 1964 25 22 Day 6 0 0 2502 Day 7 0 0 1610 25 24 23Day 6 0 0 34 Day 7 0 99 7 23 24 Day 6 0 2 13 Day 7 1 7 0 11 Day 6 4 0 Day 7 2 0 0 0 26 Day 6 0 0 Day 7 0 0 956 25 24 27 Day 6 0 0 Day 7 0 0 912 23 23 28 Day 6 0 0 Day 7 1 2 0 6 16 29 Day 6 3 Day 7 Day 6 I Dayv7 1 I E WO 99/13724 19 3.a. Mortality and Egg Production Study #3 PCTfUS97/16475 Eggs Start Age Concen. Dead Dead Total Per in Days Cage Borax 24 hr 6'No.

Jof Eggs 4d I control Dayl1 0 0 66 3 4d 2 0.02N Dayl1 0 0 8 0 4d 3 0.05N Dayl1 0 0 1 0 4d 4 0.07N Day 1 0 0 0 0 4d 5 O.lON Dayl1 0 2 0 0 4d 11I control Dayl1 0 0 0 0 4d 12 0.02N Dayl1 0 0 0 0 4d 13 0.05N Dayl1 0 0 5 0 4d 14 0.07N DaylI 1 0 0 0 4d 1 15 0.ION Dayl1 2 1 0 0 4d la control Dayl1 0 0 0 0 4d 2a 0.02N Dayl1 0 0 0 0 4d 3a 0.05N Dayl1 0 0 0 0 4d 4a 0.07N Dayl1 0 0 0 0 4d 5a 0.1ON Dayl1 0 0 0 0 4d Ila control Dayl1 0 0 0 0 4d 12a 0.02N Dayl1 0 0 0 0 4d 13a 0.05N Dayl1 0 0 0 0 4d 14a 0.07N ,Dayl1 0 0 0 0 4d 15a 0.ION Dpayl 01 0 0 0 3.b. Mortality and Egg Production Study #3 JJ1Dead Dead JTotal No. IEggs Dead Dead Total No. Eggs Cage L 8 hr I I 1ofEggs J Per? 1 72 hr I e' I I of Eggs -~Per IDay 2 0 0 677 27 Day 3 0 0 822 33 2 Day 2 0 1 188 7 Day 3 0* 0 134 3 Day 2 1 0 52 2 Day 3 3 0 1 0 4 Day 2 0 0 15 1 Day 3 11 1 2 0 Day 2 10 3 2 0 Day 3 11 9 0 0 11 Day 2 0 0 0 0 Day 3 0 0 3 0 12 Day 2 0 0 0 0 Day 3 0 0 3 0 13 Day 2 0 0 0 0 Day 3 2 0 0 0 14 Day 2 2 0* 0 0 Day 3 -2 1 0 0 Day 2 2 2 0 0 Day 3 9 1 0 0 la Day 2 0 0 0 0 Day 3 0 0 5 0 2a Day 2 0 0 0 0 Day 3 0 0 0 0 3a Day 2 1 0 0 0 Day 3 1 0 0 0 4a Day 2 1 0 0 0 -Day 3 0 0 0 0 Day 2 4 2 0 0 Day 3 14 5 0 0 Ila Day 2 0 0 0 0 Day 3 0 0 954 12a Day 2 0 0 0 0 Day 3 0 0 0 0 13a Day 2 0 0 0 0 Day 3 0 0 0 0 14a Day 2 0 0 0 0 Day 3 1 0 1 0 0 Day 2 0 1 0 0 Day 3 3 2 1 0 0 0 *1I escapee 3.c. Mortality and Egg Production Study #3 IfTotal Eggs Total Eggs Cager Dead_ JDead_ No. of Per Dea Dead_ No. of Per D6hr ead Dea Eggs D 2 r ea Dea Eggs I Day 4 1 0 669 27 Day 5 0 0 827 33 2 Day 4 1 1 235 9 Day 5 1 0 522 22 3 Day 4 3 1 0 0 DayS5 4 0 0 0 4 Day 4 5 3 0 0 Day 5 8 5 0 0 Day 4 3 9 0 0 Day 5 2 0 1 0 11 Day 4 0 0 511 20 DayS5 1 0 1070 43 12 Day 4 0 0 0 0 Day 5 0 0 43 2 13 Day 4 1 0 0 0 Day 5 3 0 0 0 14 Day 4 8 1 1 0 0 Day 5 6 3 0 0 Day 4 7 8 0 0 Day 5 3 8 0 0 la Day 4 0 0 338 14 Day 5 0 0 682 27 2a Day 4 0 0 15 1 Day 5 0 0 97 4 3a Day 4 1 1 0 0 1DayS5 4 1 0 0 4a Day 4 7 3 0 0 DayS5 8 4 0 0 Day 4 4 4 0 0 DayS5 1 6 0 0 Ila Day 4 0 0 527 21 Day 5 0 0 1082 43 12a -Day 4 0 0 0 0 DayS5 0 0 0 1 0 13a Day 4 1 1 0 0 Day 5 1 0 0 0 14a Day 4 7 0 0 0 DayS5 11 3 0 0 Dayv4 7 3 1 0 1 0 1 Day 5 6 L 10 0 0 3.d. Mortality and Egg Production Study #3 Total Eggs Total Eggs Dead Dead No. of Per Dead Dead No. of Per Live Live Begn.

Cage# 144 hr e Eggs 168 hr De Eggs 1 Total 1 Day 6 0 2 802 32 Day 7 0 1 827 36 24 22 250e/25 2 Day 6 0 0 1092 46 Day 7 0 0 940 39 21 24 24e?@/26? 3 Day 6 0 1 0 0 Day 7 4 1 0 0 10 22 25 4 Day 6 1 1 0 0 Day 7 2 0 0 13 25c/25 Day 6 0 0 0 Day 7 0 0 0 I 240"/26 11 Day 6 0 0 1990 80 Day 7 0 0 msg msg 25 25 26c?/25 data data 12 Day 6 0 1 214 9 Day 7 0 1 msg msg 25 23 250 data data 13 Day 6 0 1 0 0 Day 7 0 0 0 0 19 25 25ce/26Y 14 Day 6 1 4 0 0 Day 7 2 7 0 0 3 7 25"@/24Y Day 6 1 3 0 0 Day 7 1 0 0 1 24o"/25$ la Day 6 0 0 835 33 Day 7 0 0 164 66 24 25 24cr/25 2a Day 6 0 0 590 23 Day 7 0 0 1373 53 29 26 29e'/26? 3a Day 6 2 1 0 0 Day 7 0 1 0 0 16 21 250 /25 4a Day 6 4 6 0 0 Day 7 1 1 0 0 4 11 25e1Y25 Day 6 1 8 0 0 Day 7 1 0 0 0 0 24dY26? Ila Day 6 0 0 1125 45 0 0 968 39 25 25 25eo/25 12a Day 6 0 0 64 3 Day 7 0 0 214 10 26 25 26c?/25? 13a Day 6 3 0 0 0 Day 7 0 0 4 0 20 23 25oe/24? 14a Day 6 1 4 0 0 Day 7 1 5 0 0 4 13 25 e/25 Day6 -7 1 0 0 Day 7 1 0 0 0 23?/25? "ani males aeao Day 6 @1 escapee WO 99/13724 23 4.a. Mortalit and Egg Production Study #4 PCT/US97/16475 Start I JTotal Eggs Age in Concen. Dead Dead No. of Per Days Cage Borax 24 hr Eggs 6 control Day 1 10 1002 lOd 7 0.02N Dayl1 0 0 590 24 8 0.05N DaylI 1 0 549 22 9 0.07N Dayl1 3 0 555 23 10 0.lON Day 1 4 1 345 14 16 control Dayl1 0 0 1187 46 17 0.02N Dayl1 0 0 1139 48 18 0.05N Day 1 3 0 538 22 19 0.07N Dayl1 7 0 597 24 20 0.10ON Dayl1 13 1 542 23 lOd 6a control D ay 1 0 1 1100 44 7a 0.02N Day 1 0 0 758 8a 0.05N Day 1 4 1 1412 54 9a 0.07N Day 1 2 2 901 38 10a 0. 1ON Dayl1 7 0 756 16a control Day 1 0 0 1334 53 17a 0.02N Day 1 0- 0 895 36 18a 0.05N DaylI 1 0 1169 47 19a 0.07N Dayl1 5 0 618 20a 0.1ON Day 1 8 1"0344 4.b. Mortality and Egg Production Study #4 Cae~ 4~ Dead Dead Total No. Eggs Dead Dea Total No. Eggs Cage_ 1I8IrI of Eggs I Per 72 hr e I' I of Eggs jPer~ 6 Day 2 0 0 843 34 Day 3 0 2 813 33 7 Day 2 1 1 591 24 Day 3 1 1 43 2 8 Day 2 6 1 76 3 Day 3 2 0 69 3 9 Day 2 11 0 105 4 Day 3 3 4 11 1 Day 2 9 1 90 4 Day 3 7 2 20 1 16 Day 2 0 0 751 29 Day 3 0 0 1253 48 17 Day 2 0 0 111 5 Day 3 0 0 10 0 18 Day 2 5 0 122 5 Day 3 0 1 18 1 19 Day 2 9 0 92 4 Day 3 6 3 23 1 Day 2 11 4 24 1 Day 3 1 1 2 0 6a Day 2 0 0 492 21 Day 3 0 0 959 7a Day 2 0 0 66 3 Day 3 1 0 60 2 8a Day 2 8 0 96 4 Day 3 3 0 63 3 9a Day 2 11 0 44 2 Day 3 3 0 68 3 Day 2 15 6 50 2 Day 3 3 1 20 1 16a Day 2 0 0 850 34 Day 3 0 0 838 34 17a Day 2 0 0 175 7 Day 3 0 0 69 3 18a Day 2 6 0 61 -j 2 Day 3 1 0 1B 1 19a Day 2 16 0 53 1-2 Day 3 3* 0 39 2 -Dav 2 1 17 7 52 1 2 -Day 3 0 6 413 *one escapee 4.c. Mortality and Egg Production Study #4 IDead Dead Total#1 Eggs fDead Dead] Total Eggs Cage J 961 hrJ of Eggs JPer 1201 hr j ofEggs j Per? 1 6 Day 4 10 840 37 DayS 1 0 863 38 7 Day 4 0 0 12 1 Day 5 1 0 52 3 8 Day 4 5 2 12 1 Day 5 2 4 7 0 9 Day 4 4 2 4 0 Day 5 1 0 0 0 Day 4 2 0 28 1 Day 5 2 6 6 0 16 Day 4 0 0 1023 39 Day 5 2 0 1349 52 17 Day 4 2 2 12 1 Day 5 1 0 0 0 18 Day 4 8 0 2 0 DayS5 4 0 0 0 19 Day 4 3 2 5 0 DayS 5 0 0 Day 4 6 7 0 Day 5 4 0 0 6a Day 4 1 0 860 36 Day 5 0 1 685 29 7a Day 4 1 0 8 0 Day 5 1 0 50 2 8a Day 4 3 0 9 0 Day 5 4 0 0 0 9a Day 4 2 0 0 0 DayS5 3 1 0 0 Day 4 4 0 0 DayS5 3 0 0 16a Day 4 0 0 676 27 Day 5 0 0 613 17a Day 4 2 0 17 1 Day 5 2 1 361 18a Day 4 2 1 0 0 DayS5 1 0 5 0 19a Day 4 0 3 0 0 Day 5 0 1 3 0 Day 4 10 0 .0 Day 5 4 0 0 4.d. Mortality and Egg Production Study #4 Cage 144 hr Dead [Dead

T

tal JEggsg P 1 Dead Dead Total #of ]Eggs Live Live I_ I_ I _g Per J6 hr_ e Eggs Per L 6 Day 6 0 0 564 25 Day 7 1 0 1057 46 21 23 25e1259 7 Day 6 0 0 141 6 Day 7 0 0 368 16 22 23 256/25? 8 Day 6 5 0 8 0 Day 7 0 1 7 0 4 17 25e"/25? 9 Day 6 1 2 0 0 Day 7 1 6 0 0 1 10 25c?/24? Day 6 3 0 0 Day 7 4 1 0 7 24?/24? 16 Day 6 0 0 1125 43 Day 7 0 0 771 30 22 26 24oe/26? 17 Day 6 1 1 0 0 Day 7 2 0 0 0 18 21 240"/24? 18 Day 6 0 2 0 0 Day 7 0 0 0 0 5 21 25oe/249 19 Day 6 6 6 0 Day 7 all o" 2 0 0 0 7 25'/25 dead Day 6 2 0 0 Day 7 all e 3 0 0 0 3 25dV24? dead 6a Day 6 0 0 327 14 Day 7 1 1 219 10 23 22 25e125? 7a Day 6 4 0 55 2 Day 7 0 0 141 6 18 25 25ce/25 8a Day 6 3 1 0 0 Day 7 3 1 0 0 5 23 33d'/26? 9a Day 6 3 1 0 0 Day 7 1 0 0 0 0 20 25c?/24? Day 6 8 0 0 Day 7 all e 3 0 0 0 0 25 dead 16a Day 6 0 1 missing Day 7 0 1 450 19 25 23 25 e/25 data 17a Day 6 1 1 missing Day 7 0 0 155 7 20 23 25el25? data 18a Day 6 0 0 missing Day 7 0 2 49 2 14 22 25o'/25? data 19a Day 6 1 3 0 0 Day 7 3 0 0 0 15 260?*/25 Day 6 all? 3 0 0 Day 7 1 0 0 0 2 26o?/24? dead *1 escapee WO 99/13724 WO 9913724PCT/US97/I 6475 Mortality and Egg Production Study Start IITotal Age in jCage Concen. jDead Dead No. of Eggs Days J jBorax 24 hr- e I" Eggs Per 4d 21 control Dayl1 0 0 0 0 4d 22 0.02N Dayl1 0 0 0 0 4d 23 0.05N Dayl1 0 1 0 0 4d 24 0.07N Dayl1 0 0 0 0 4d 25 0.ION DaylI 1 0 0 0 lOd 26 control Dayl1 0 0 380 16 27 0.02N DaylI 1 0 162 7 28 0.05N DaylI 1 0 231 9 29 0.07N Dayl1 3 0 164 7 30 M.ON Dayl1 9 0 211 9 4d I control DaylI 1 0 0 0 4d 2 0.02N Dayl1 0 0 0 0 4d 3 0.05N Dayl1 0 0 0 0 4d 4 0.07N DaylI 1 0 0 0 4d 1 5 0.1ON Dayl1 2 0 0 0 6 control Dayl1 0 0 1769 74 7 0.02N Dayl1 0 0 0 0 8 0.05N Dayl1 4 0 8 0 9 0.07N Dayl1 5 0 341 14 -10 0.20N Dayl1 16 100 Mortality and Egg Production Study #5 I Dead JDead ITotal No.1 Eggs Dead Dead Total No. Egg Cage# J48 hr J ed of Eggs JPer~ 721u hr je of Eggs -Per$ 21 Day 2 0 0 0 0 Day 3 0 1 64 3 22 Day 2 0 0 0 0 Day 3 0 1 0 0 23 Day 2 0 0 0 0 Day 3 3 0 0 0 24 Day 2 3 0 0 0 Day 3 7 1 0 0 Day 2 12 2 0 0 Day 3 10 5 0 0 26 Day 2 0 0 691 29 Day 3 1 1 460 19 27 Day 2 0 0 450 18 Day 3 1 0 127 28 Day 2 6 0 169 7 Day 3 5 1 9 0 29 Day 2 8 0 93 4 Day 3 8 2 36 2 Day 2 9 4 91 4 Day 3 3 2 39 2 1 Day 2 1 1 0 0 Day 3 1 0 236 2 Day 2 0 0 0 0 Day 3 2 0 0 0 3 Day 2 2 0 0 0 Day 3 8 0 0 0 4 Day 2 2 0 0 0 Day 3 5 2 0 0 Day 2 11 3 0 0 Day 3 11 5 0 0 6 Day 2 0 0 371 16 Day 3 0 0 563 24 7 Day 2 8 0 42 2 Day 3 5 0 120 8 Day 2 16 2 21 1 Day 3 3 1 12 1 9 Day 2 13 2 101 4 Day 3 2 1 46 2 Day 2 9 15 12 1 Day 3 all e dead 4 0 0 Day 2 Mortality and Egg Production Study #5 I Dead Dead jTotal No.* Eggs [Dead Dead ITotal No.1 Eggs Cage# J 96 hr I I of Eggs .1Per 120hr C? of Eggs jPer~ 21 Day 4 0 0 270 11 Day 5 0 0 420 18 22 Day 4 0 0 0 0 Day 5 0 0 0 0 23 Day 4 6 2 0 0 Day 5 6 1 0 0 24 Day 4 6 4 0 0 Day 5 5 6 0 0 Day 4 2 8 0 0 Day 5 5 0 0 26 Day 4 0 0 698 30 Day 5 1 0 1158 27 Day 4 0 0 107 4 Day 5 0 0 252 28 Day 4 5 0 0 0 Day 5 3 0 0 0 29 Day 4 5 0 9 0 Day 5 0 4 0 0 Day 4 2 1 10 1 Day 5 2 4 0 0 1 Day 4 0 0 394 16 DayS5 0 0 missing missing data data 2 Day 4 2 0 0 0 DayS5 0 0 8 0 3 Day 4 8 7 0 0 DayS5 7 10 12 1 4 Day 4 13 7 0 0 DayS5 3 6 2 0 Day 4 2 9 0 0 DayS5 allde'dead 1 0 0 4 6 Day 4 1 1 442 18 Day 5 0 0 617 27 7 Day 4 6 0 43 2 DayS5 4 1 8 0 8 Day 4 2 10 35 2 DayS5 3 19 2 9 Day 4 4 5 23 1 DayS5 1 6 0 0 IDayv4 1 3 0 0 Day 5 2 0 0 Mortality and Egg Production Study Cage 144 JDead- Dead Total#1 Eggs/J Dead Dead Total Eggs Live Live JTotal ]Total hr_ a" of Eggs 1 168bhr e J of Eggs jPer~ j 6I 21 Day 6 0 0 1017 42 Day 7 0 0 601 25 25 24 25 22 Day 6 1 0 4 0 Day 7 0 0 30 1 24 24 25 23 Day 6 7 3 0 0 Day 7 0 4 0 0 2 14 24 24 Day 6 4 7 0 0 Day 7 3 0 0 10 4 25 Day 6 4 0 0 Day 7 0 0 25 24 26 Day 6 0 0 780 34 Day 7 0 2 1427 62 23 21 25 24 27 Day 6 0 0 318 13 Day 7 0 0 1069 43 23- 25 25 28 Day 6 0 0 0 0 Day 7 2 1 0 0 2 23 24 29 Day 6 1 4 0 0 Day 7 0 1 0 0 0 13 25 24 Day 6- 0 0 0 Day 7 3 0 0 0 10 25 24 1 Day 6 0 0 702 29 Day 7 1 0 651 27 21 24 25 2 Day 6 2 0 31 1 Day 7 0 0 115 5 18 24 24 24 3 Day 6 3 0 0 Day 7 2 0 0 0 3 25 125 4 Day 6 0 2 0 0 Day 7 1 2 0 0 0 8 25 27 Day 6 2 0 0 Day 7 3 0 0 0 1 26 24 6 .Day 6 1 0 576 25 Day 7 1 0 1097 48 23 23 26 24 7 Day 6 0 2 0 0 Day 7 1 3 0 0 1 20 25 26 8 Day 6 7 0 0 Day 7 3 0 0 0 1 125 9 Day 6 6 0 0 Day 7 4 0 0 0 0 25 124 Dayv6. Dayv7 0 0 0 1 25 1 female left alive (1) @all ae dead Day 6 WO 99/13724 PCT/US97/16475 31 EXAMPLE III Examples of various formulations that may be utilized in accordance with the present invention include: 6% borax, 1% agar and 94% water, wherein the agar and water are combined, the 94% water and 1% agar, and brought to a boil. The mixture is removed from the heat and the borax is added. This results in a gelatinous mixture which will not dry completely and which is believed will adhere to trees, buildings, etc. to which it is applied. It may be applied by spraying via a pressurized application or an hydraulic oil squirt can to utility poles, trees, fences, etc.

6% borax, 1% agar and 93% proteinaceous bait (10% (Miller's Nu-Lure®), wherein the proteinaceous bait is formulated with 90 parts water and 10 parts proteinaceous concentrated bait. This formulation 2 is made as formulation 1 above and may be applied similarly; however, this formulation 2 is believed to be a better attractant than formulation 1; 1% borax, 76% proteinaceous bait (Miller's Nu-Lure®) and 23% Min-U-Gel® (Floridin wherein this formulation 3 is made following the steps to make formulation 1, except without heating. This formulation 3 may be applied like formulations 1 and 2, i.e., simply mix and spray.

6% borax, 70% proteinaceous bait (Miller's Nu-Lure®) and 23% Min-U-Gel® (Floridin wherein this formulation 4 is made following the steps to make formulation 1, except without heating. This formulation 4 may be applied like formulations 1 and 2, i.e., simply mix and spray.

1% borax and 99% Min-U-Gel® (Floridin Co.) or 6% borax and 94% Min-U- Gel® (Floridin wherein these formulations are made following the steps to make formulation 1, except without heating. These two formulations may be applied like formulations 1 and 2, simply mix and spray.

The proper viscosity of each formulation should be maintained, the surface of a spot application is thick enough to hold indentations, to avoid splashback, runoff and possible ineffective treatments on new porous surfaces. Generally, 23% of Min-U-Gel® (Floridin Co.) is believed to be sufficient to maintain appropriate viscosity, however, additional or lesser amounts may be necessary to achieve desired results.

To treat infested area via spot treatment: apply one or more of the above formulations, formulations 3-5, with a pressurized application or a hydraulic oil squirt can to, utility poles, trees, fences, signs, etc. At least about 600 evenly distributed bait spots per squire mile, WO 99/13724 PCT/US97/1 6475 32 or approximately 60 to 80 bait spots per city block. Apply treatment once per week for at least about six weeks. The area of coverage will extend about nine square miles around each fly find.

As opposed to spot treatment, the above formulations, formulations 3-5, may be squirted on tree trunks, fences, utility poles, signs, etc. in areas thereon which are out of reach of children at a rate of about 0.1 oz to about 0.2 oz (about 3 to about 5 ml) per station.

With respect to aerial treatment or retreatment of the above formulations, such as formulations 1 and 2 above, it should not be considered if weather reports indicate a 50% or greater chance of precipitation within 48 hours. Applications of full coverage of the bait sprays, such as formulations 1 and 2 above, should be scheduled approximately once per week for at least six weeks. It is believed that the bait formulations of the present invention, such as bait formulations 1 and 2 identified above in this Example, are effective as full coverage aerial bait sprays when applied in amounts on the order of between about 5 oz and about 128 oz or more per acre per full coverage application. It should, of course, be understood that the amount of bait formulation actually applied will depend upon the objectives to be accomplished and the size of the area to be covered. Moreover, the area of full coverage spray should extend a minimum of about 1.5 miles beyond known fruit fly infestation. It, of course, may be expanded to about miles from any kind if the infestation is heavy. Weather conditions may also dictate change in spray schedules. After an estimated two off generations of negative trapping, spray operations may be discontinued.

The present invention may, of course, be carried out in other specific ways than those herein set forth without departing from the spirit and essential characteristics of the invention.

The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive and all changes coming within the spirit and scope of the appended claims are intended to be embraced herein.

Claims (33)

1. A method of treating fruit flies of the Tephritidae family so that the fruit flies die prematurely, said method comprising: feeding to the fruit flies an effective amount of a borax toxicant to cause the fruit flies to die prematurely, so that following ingestion of the borax toxicant by the fruit flies, the fruit flies will die prematurely due to the ingestion of the borax toxicant.

2. A method of claim 1, the borax toxicant being borax.

3. A method of claim 2, said feeding comprises feeding the fruit flies between about 5mM and about 10mM of borax within about a 24 hour period.

4. A method of claim 2 wherein the effective amount is in the range 15 between about 0.01M and about 0.12M.

5. A method of claim 1, the borax toxicant being selected from the group consisting of borax, anhydrous borax, ammonium tetraborate, ammonium pentaborate, potassium pentaborate, potassium tetraborate, sodium metaborate, 20 disodium tetraborate decahydrate, disodium tetraborate pentahydrate and disodium octaborate tetrahydrate.

6. A method of claim 1 wherein the borax toxicant included in a bait.

7. A method of interfering with reproduction by female fruit flies of the Tephriditae family so that the female fruit flies will stop producing eggs for about 7 days or longer, said method comprising: feeding to the female fruit flies an effective amount of a borax toxicant to interfere with the reproduction by the female fruit flies, so that following ingestion of 34 the borax toxicant by the female fruit flies, the female fruit flies will stop producing eggs for a period of about 7 days or more due to the ingestion of the borax toxicant.

8. A method of claim 7, the borax toxicant being borax.

9. A method of claim 8, said feeding comprises feeding the fruit flies between about 2.5mM and about 5mM of borax within about a 24 hour period. S..1

10. A method of claim 8 wherein the effective amount is in the range S: 10 between about 0.01M and about 0.12M. S*

11. A method of claim 7, the borax toxicant being selected from the group consisting of borax, annhydrous borax, ammonium tetraborate, ammonium pentaborate, potassium pentaborate, potassium tetraborate, sodium metaborate, 15 disodium tetraborate decahydrate, disodium tetraborate pentahydrate and disodium •octaborate tetrahydrate.

12. A method of claim 7, wherein the borax toxicant is included in a bait. 20

13. A method of reducing the population of fruit flies of the Tephritidae family at a targeted area, said method comprising: applying to the targeted area an effective amount of a borax toxicant for ingestion by the fruit flies at the targeted area, so that following ingestion of the borax toxicant by the fruit flies, the population is reduced at the targeted area due to of the ingestion of the borax toxicant.

14. A method of claim 13, the borax toxicant being borax.

A method of claim 14 wherein the ingestion by the fruit flies is between about 2.5mM and about 10mM of borax within about a 24 hour period.

16. A method of claim 15, said application of the borax toxicant at the targeted area being done at least about once per week for at least about six weeks.

17. A method of claim 14 wherein the ingestion by the fruit flies is between about 5mM and about 10mM of borax within about a 24 hour period.

18. A method of claim 17, said application of the borax toxicant at the targeted area being done at least about once per week for at least about six weeks.

19. A method of claim 14 wherein the effective amount is in the range between about 0.01M and about 0.12M.

20. A method of claim 13, the borax toxicant being selected from the group consisting of borax, anhydrous borax, ammonium tetraborate, ammonium 15 pentaborate, potassium pentaborate, potassium tetraborate, sodium metaborate, disodium tetraborate decahydrate, disodium tetraborate pentahydrate and disodium octaborate tetrahydrate.

21. A method of claim 13, wherein the borax toxicant is included in a bait.

22. A bait or lure used to reduce the number of fruit flies of the Tephritidae family, said bait or lure consisting essentially of a borax toxicant, said borax toxicant being present in said bait or lure in an effective amount so that following ingestion of said borax toxicant in said bait or lure by the fruit flies, the number of fruit flies is reduced due to the consumption of said borax toxicant by the fruit flies.

23. A bait or lure of claim 22, said bait or lure being in a form selected from the group consisting of a patty, heavy cream, pellet, gel, foam, paste, liquid and spray. 36

24. A bait or lure of claim 22, said bait or lure further including a bait selected from the group consisting of a protein hydrolysate bait and a synthetic bait.

A bait or lure of claim 22, said bait or lure further including other ingredients selected from the group consisting of agar, insecticide, bait, liquefier, sweetener and carrier.

26. A bait or lure of claim 22, said borax toxicant being borax.

27. A bait or lure of claim 26 wherein the consumption by the fruit flies is between about 2.5mM and about 10mM of borax within about a 24 hour period.

28. A bait or lure of claim 26 wherein the effective amount is in the range between about 0.01 M and about 0.12M.

29. A bait or lure of claim 26, said bait or lure further including agar yeast hydrosylate, sugar and water.

30. A bait or lure of claim 22, the borax toxicant being selected from the 20 group consisting of borax, anhhydrous borax, ammonium tetraborate, ammonium pentaborate, potassium pentaborate, potassium tetraborate, sodium metaborate, :oo: disodium tetraborate decahydrate, disodium tetraborate pentahydrate and disodium octaborate tetrahydrate. *I

31. A bait or lure used to reduce numbers of fruit flies substantially as hereinbefore described with reference to the accompanying examples.

32. A method for reducing the population of fruit flies substantially as hereinbefore described with reference to the accompanying examples.

33. A method of interfering with reproduction by female fruit flies as hereinbefore described with reference to the accompanying examples. DATED THIS SIXTEENTH DAY OF JANUARY 2002 UNIVERSITY OF FLORIDA BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS eq. C we.. C C C. 0 C* *e C C .0 0 ~0 CC C C 00*0 C@ Seed C *COC 0 S C. C. C C S C C