WO1996018295A1

WO1996018295A1 - Pesticide composition and method for controlling the mediterranean fruit fly and the oriental fruit fly

Info

Publication number: WO1996018295A1
Application number: PCT/US1995/015962
Authority: WO
Inventors: Nicanor J. Liquido; Roy T. Cunningham; James R. Heintz; John P. Spencer
Original assignee: The United States Of America, Represented By The Secretary, Department Of Agriculture; Photodye International, Inc.
Priority date: 1994-12-12
Filing date: 1995-12-12
Publication date: 1996-06-20
Also published as: MX9704337A; IL116343A0; BR9510021A; EP0797385A1; IL116343A; EP0797385A4; AU705515B2; AU4596496A

Abstract

A composition and method for eradicating or suppressing a population of Mediterranean fruit flies or oriental fruit flies which introduces substantially minimal safety risks to humans, agriculture and nontargeted living creatures includes: providing an insecticide composition which includes at least one photoactive dye which is preferably a mixture of Phloxine B and uranine; causing the composition to be ingested by the targeted fruit fly population by spraying or providing the insecticide at a location where the fruit flies feed; and whereby ingestion of the photoactive dye component of the composition by a Mediterranean fruit fly or oriental fruit fly causes a toxic quantity of photons to penetrate within the body of the fly.

Description

PESTICIDE COMPOSITION AND METHOD

FOR CONTROLLING THE MEDITERRANEAN FRUIT FLY

AND THE ORIENTAL FRUIT FLY

This is a continuation-in-part of application Serial No. 08/353,726

filed December 12, 1994.

Background of the Invention

The fruit flies of the family Tephritidae include several species tha

are major pests of agriculture throughout the world and that represent

serious threat to U.S. agriculture. The U.S. Department of Agricultur

(USDA), Animal and Plant Health Inspection Service (APHIS), i

cooperation with other Federal and State organizations, has conducted

number of programs to eradicate some species of fruit flies when thes

insects have been introduced. These programs generally have employe

an integrated pest management approach to eradication. Many recen

programs have involved application of malathion bait spray to effectivel

lower fly populations in the infested area followed by release of steril

flies. Aerial applications of the bait spray over populated areas to contr

infestations of fruit flies have been controversial. Concerns about advers

health effects from exposure to malathion bait spray have been raised b

residents of treated neighborhoods. The currently used malathion bai system for control of Mediterranean fruit flies contains as the main

ingredients, Nu-Lure (acid hydrolyzed protein) as an attractant food bait

and the contact insecticide, malathion. This system has a bad public

perception, damages paint finishes on cars, and, because of the high

concentration of insecticides (10-20%) in the bait to ensure temporal

stability, this insecticide is extremely detrimental to beneficial insects that

may contact the bait surface or be exposed to volatile fumes after

application. The United States government has mandated that certain

currently listed pesticides, such as malathion, should have a more

restricted use pattern. The Environmental Protection Agency has

specifically requested that safer pesticides be developed for use in the

agricultural sector.

Summary of the Invention

In the quest for these aforementioned safer pesticides, it has been

unexpectedly discovered that dye-sensitized photoactive substances can

be effectively utilized as the active ingredient in insecticides for use in a

bait station or as bait spray component which target Mediterranean fruit

flies and many other tephritid fruit flies. The preferred photoactive dye

for these purposes is a halogenated xanthene such as phloxine B or a mixture of a halogenated xanthene and uranine. Other known xanthen

dyes which could be used include erythrosin B and rose bengal. Thes

dyes are more fully described in the U.S. patents to Crounse and Heitz

U.S. Patent No. 4,647,578 and U.S. Patent No. 4,320,140, the subjec

matter of which is incorporated herein by reference. When employin

halogenated xanthene dyes, light activated toxicity is especially marke

with respect to Mediterranean fruit flies and oriental fruit flies. Th

photoactive dye ingredient of the present invention is effective due to th

release of light induced free radical molecules within the body of th

insect which has ingested the composition containing the dye. Due to it

small proportionate body mass, the dose of light is fatal to the insect.

Although being extremely toxic with respect to the targete

Mediterranean fruit fly or oriental fruit fly, the preferred dyes of th

instant invention are non-toxic to humans, most mammals, an

nontargeted insects. This is a substantial advance in the industry, a

prior art pesticides which were adapted for the purpose of controlling frui

fly populations, namely malathion, have posed health risks to humans

Because malathion is highly penetrating and invades cells very quickl

upon contact, malathion is highly potent as a poison, both to insects an

nontargeted species. The present invention, on the other hand, i directed to an active ingredient which is toxic to the targeted

Mediterranean or oriental fruit fly, not due to cell penetration, but rather

due to exposure to a quantity of light which is fatal to the fly. The effect

of the same quantity of exposure by a human or other mammal would be

negligible. In this regard, the compound phloxine B

(2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein) has been

consumed by humans for decades. It is registered as D & C Red No. 28

as a drug and cosmetic additive and has been included into such

commercially available products as Pepto Bismol. In fact, the intrinsic

toxicity of malathion is 62.5 times greater than that of phloxine B as

measured by acute LD₅₀ in rats.

Additionally, unlike malathion which kills any insect merely upon

contact, even nontargeted species such as honeybees, the photoactive

dye ingredient of the present invention is not harmful unless ingested.

Indeed, the skin penetrability of phloxine B has been calculated to be

about 87 times lower than that of malathion by octanol/water partition

coefficient analysis conducted at the USDA.

Furthermore, because the active xanthene dye component is

light-activated, a much smaller quantity is necessary to provide the

desired effect. Moreover, contrary to the prior art broad spectrum pesticides which have a half-life on the order of months or even years

the xanthene based ingredient will naturally photodegrade in th

environment within hours or, at the most, days. Thus, not only provid

an extremely high kill rate when introduced in the targeted Mediterranea

or oriental fruit fly populations, but additionally pose little or no risk fo

use in the agriculture industry.

An additional benefit which is offered by the use of xanthene base

dyes as the active ingredient in pesticide compositions, resides in the fac

that, after ingestion, the dye does not kill the insect immediately. Thi

allows the insect which has ingested the pesticide to effectively transfe

some quantity of the pesticide among other insects in the population

Therefore, substantially more insects will be eliminated due to the transfe

of the toxic material between flies.

Another aspect of the invention is use of phloxine B in combinatio

with uranine. Uranine is registered as D & C Yellow Dye No. 8 for use a

a color additive in drugs and cosmetics.

The present insecticidal composition further contemplates th

inclusion of an attractant bait which has the following properties: i

attracts the target fruit flies to the location of the bait, and it stimulate

the target fruit flies to feed on the bait. Attractant baits include (1 ) carbohydrate sugar source, for example, sugars such as sucrose or

fructose, or sugar substrates such as molasses or honey; (2) a

proteinaceous food bait such as hydrolyzed protein (either by acid

hydrolysis or enzymatic action), or (3) a combination of carbohydrate

sugar source and hydrolyzed protein bait. The composition may

optionally include anti-foaming agents and other adjuvants to increase the

permeability of the ingested dye within the gut of the insect.

It is therefore an important object of the present invention to provide

a pesticide that is toxic only when ingested by particular species of

insects and in particular the Mediterranean or oriental fruit fly and other

tephritid fruit flies.

It is a further object to provide a pesticide that has a delayed

toxicity to the insect.

A still further object lies in the provisions of a pesticide which has

minimal detrimental effect on the behavior of the insect between the

period of ingestion and the onset of toxicity.

A still further object of the present invention lies in the provision of

an insecticidal composition which is highly attractive to both sexes of the

Mediterranean and oriental fruit fly.

A yet further object of the present invention resides in the provision of an insecticidal composition which contains no feeding inhibitors th

may limit feeding or may induce a learned behavior to avoid the bait.

Lastly, it is an object of the present invention to provide a

insecticidal composition which incorporates a bait that stimulates feedin

and induces engorgement by the insects.

These together with other objects of the invention, are pointed o

with particularity in the following detailed description and claims annexe

hereto and forming part of this disclosure.

Brief Description of the Drawings

Figure 1 shows percent mortality of adult Mediterranean fruit flie

as a function of varying concentrations of 1 :1 molar mixture of phloxin

B and uranine and time after feeding.

Figure 2 shows the functional relationship between concentratio

of dyes in 10% molasses and mortality of Mediterranean fruit fly adult

Figure 3 shows the functional relationship between concentratio

of dyes in 1 % Nu-Lure and mortality of Mediterranean fruit fly adults.

Figure 4 shows the functional relationship between concentratio

(M) of 1 :1 molar mixture of phloxine B and uranine in 1 % Nu-Lure an

mortality of oriental fruit fly adults. Figures 5A and 5B show percent mortality of adult oriental fruit fly

males and females, respectively, as a function of varying concentrations

of 1 :1 molar mixture of phloxine B and uranine and time after feeding.

Figure 6 shows percent mortality of wild oriental fruit fly adults as

a function of methyl eugenol alone, methyl eugenol plus a 1 :1 molar

mixture of phloxine B and uranine, and methyl eugenol plus naled, and

time after exposure to light.

Figure 7 shows the increase in mortality over time of wild oriental

fruit fly adults exposed to methyl eugenol alone and methyl eugenol plus

a 1 :1 molar mixture of phloxine B and uranine at two concentrations.

Figure 8 shows the increase in mortality over time of wild oriental

fruit fly adults exposed to methyl eugenol alone and methyl eugenol plus

phloxine B at two concentrations.

Detailed Description of the Invention

Toxicity of several substituted xanthene dyes to many species o

insects has been documented in the laboratory and field (Heitz, Chapte

1 , Lioht Activated Pesticides. J. R. Heitz and K. R. Downum (eds.),

American Chemical Society Symposium Series 339, American Chemica

Society, Washington, D.C., 1987). Among these are the dye-sensitize

photoactive toxicity of phloxine B to adult housefly, Musca domestica L

(Fondren and Heitz, Environmental Entomology 8:432-436, 1979), fac

fly, M. autumnalis De Geer (Fondren and Heitz, Environmenta

Entomology 7:843-846, 1978), boll weevil, Anthonomus orandi

Boheman (Callaham et al., Environmental Entomology 4:837-841 , 1975)

black cutworm, Aorotis ipsilon (Clement et al., Journal of Economi

Entomology 73:390-392, 1980), and imported fire ant, Solenoosis richter

(Forel) (Callaham et al., Comp. Biochem. Phvsiol. 51 :123-128, 1975

Broome et al., Environmental Entomology 4:883-886, 1975). Ligh

activated toxicity has also been reported in uranine-fed adult house fl

(Fondren and Heitz, 1979, supra), face fly (Fondren and Heitz, 1978

supra), and imported fire ant (Callaham et al., 1975, supra). Althoug

dye-sensitized photooxidative toxicity from uranine is considerably les

than phloxine B, uraπ' e has been found to cause synergistic effect when combined with other xanthene dyes. Phloxine B

(2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein) is registered for

use as a color additive in drugs and cosmetics. Uranine, chemically

known as fluorescein 3',6'-dihydroxyspiro[isobenzofuran-1 (3H,9'-

[9H]xanthen]-3-one, disodium salt, is registered for use as a color additive

in cosmetics as D & C Yellow No. 8.

The toxicity of phloxine B combined with uranine is of importance

in finding suitable alternatives to insecticides that are mixed with food

baits in sprays that are used in eradication or suppressing many tephritid

fruit fly populations. Thus, in an effort to find an environmentally

acceptable alternative to malathion, we determined the toxicity of a

mixture of phloxine B and uranine to Mediterranean fruit fly and oriental

fruit fly adults. Data presented here are fundamental information in

developing bait formulations, containing phloxine B and uranine for

controlling Mediterranean fruit fly and other tephritid fruit fly pests.

As discussed above, it is contemplated that the present insecticidal

composition includes an attractant bait which attracts the target insect

and stimulates it to feed on the bait. Attractant baits include 1 ) a

carbohydrate sugar source, for example, sugars such as sucrose or

fructose or complex sugar substrates such as molasses or honey; 2) a proteinaceous food bait such as hydrolyzed protein (either by aci

hydrolysis or enzymatic action); 3) a combination of sugar source an

hydrolyzed protein bait or 4) methyl eugenol.

Examples of hydrolyzed protein sources are the commerciall

available preparation, Nu-Lure™ (44% corn gluten meal, hydrolyzed, an

56% inert ingredients, Miller Chemical and Fertilizer, Hanover, PA

Mazoferm™ (condensed fermented corn extractives, E802, Corn Product

Summit-Argo, II.); hydrolyzed torula yeast, and hydrolyzed brewers yeas

The dye and bait formulation can be used in dispensers set out i

feeding bait stations or used in a sprayable formulation for ground or a

applications. The formulation may be applied by various types

equipment from hand held sprayers to high pressure ground sprayers t

helicopters to fixed-wing aircraft.

To accommodate for the different spray volume delivery rate

varying amounts of water is used as a diluent to the active ingredients

the spray formulation. An exemplary formulation is:

photodye from 0.1 to 1 % of total formulation hydrolyzed protein from 35.0 to 99.0% of total formulatio sugar source from 0.0 to 20% of total formulation added water from 0.0 to 70% of total formulation O 96/18295 PCI7US95/ 15962

The total formulation should be applied so as to obtain good spray

coverage with from 0.5 to 2.0 pounds of hydrolyzed protein per treated

acre.

Another exemplary formulation is:

photodye from 0.1 to 1 % of total formulation sugar source from 5 to 20% in water

A dye plus bait formulation using methyl eugenol as the attractant

bait is suitable for control of oriental fruit flies in male annihilation

eradication programs. Male annihilation treatments are most often applied

as spot applications of paste-like globs (generally about 600 spot

applications per square mile) or as solutions adsorbed into solid carriers

such as cigarette filter tips or 5 cm square fiberboard blocks hung on

trees at rates of 500 to several thousand per square mile.

As known to those in the art, any specific methyl eugenol

formulation and use pattern is chosen based on various factors including

accessibility and/or whether it is applied in an inhabited area.

An exemplary formulation is:

photodye from 0.5 to 5.0% of total formulation by wt. methyl eugenol from 79.5 to 70.0% of total formulation

Min-U-Gel 400 from 20 to 25 % of total formulation (attapugite clay)

The total formulation is applied as about 10 to 1 5 gram globs on tree trunks, telephone poles, etc. at a rate of about 600 spots per squar

mile about every two weeks. It can also be applied as a thickened spra

by aircraft at a rate of about 20 lbs per square mile every 1 to 2 week

Another exemplary formulation is:

photodye from 0.5 to 10.0% of total formulation by wt. methyl eugenol from 99.5 to 90.0% of total formulation

This liquid formulation is adsorbed into various solid carriers such a

cotton strings or wicks, cigarette filter tips, or cellulosic fiber wallboar

cut into pieces of various sizes. These saturated solid carrier pieces ca

be distributed by aircraft or hung on foliage at a rate so that the tot

amount of liquid formulation is from about 20 to 100 lbs per square mil

Generally, applications are repeated every 2 to 8 weeks.

This liquid formulation can also be sprayed without any thickener

solid carrier at rates of 20 lbs per square mile in aerial applications a

about weekly intervals.

The following examples describe the various innovative aspects o

the present invention.

Materials and Methods

Insects:

Mediterranean fruit fly pupae and Oriental fruit fly pupae wer

obtained from the mass rearing facility of the USDA-ARS, Honolulu

Pupae were kept in an insectary at 75 + 5 degrees C, 60 - 75% relativ

humidity, and a 12:1 2 h (L.D.) photoperiod. Test adults were held under th

same temperature, relative humidity, and photoperiod as the pupae, an

were fed with water (in agar, consisting of 9.975 part water and 0.025

part agar (Gelcarin™) and diet consisting of 3 parts sucrose, 1 par

protein yeast hydrolysate and 0.5 part Torula yeast. Five-d old adult

were used in the succeeding experiments.

Example 1 :

Determining Variation in Medite rranean Fruit Fly Adul

Mortality due to Concentration of Dyes, Sex, and Time afte

Feeding. Phloxine B and Uranine Mixed in 1 0% Molasses. Fiv

dilutions (percentage by weight) consisting of 0.001 %, 0.01 %, 0.1 %, 1 .0 and 10.0% of 1 :1 molar mixture of phloxine B (829 g per mole) and uranin

(376 g per mole) in a stock of 10% molasses were prepared. Test adul

flies confined in feeding chambers were provided with dye-molasse

preparations saturated in cotton wicks; control flies were provided wit

10% molasses. Each feeding chamber had either 20 males or 20 females

Adult feeding with dye-molasses mixtures commenced at 06.00 h an

terminated at 08:00 h, providing a 2-h feeding duration. From the onset o

feeding, each feeding chamber was kept 10 cm below two high intensity

cool fluorescent lights yielding surface intensity of 1 8,000 LUX; the sam

lighting condition was maintained throughout the 1 2-h light phase of th

experiment duration. Following removal of wicks containing the dyes

flies were provided with water and a diet consisting of sucrose, protei

hydrolysate, and torula yeast. Mortality counts were made every hour fo

the first 4 hours following feeding, at 1 8:00 h when lights wer

automatically turned off, and during each succeeding morning at 8:00 h fo

5 d. The experiment followed a randomized complete block design with

replicates. Adults used in the experiments were provided with only wate

24 h (i.e., 1 2-h dark and 12-h lights periods) prior to their use in th

experiment. Results

Fig. 1 shows the Variation in Mediterranean fruit fly adul

mortality due to concentration of dyes, sex and time afte

feeding. Phloxine B and Uranine were mixed in 10% Molasses

Mortality of Mediterranean fruit fly adults fed with variou

concentrations (by weight) of 1:1 molar mixture of phloxine B and uranin

differed significantly with dose, sex, and number of hours afte

termination of feeding. Mortality of both males and females increase

with the increase in concentration of dyes from 0.001% to 0.1%; however

at 1% to 10% concentrations, adult mortality was lower than tha

observed at 0.1%. Five days after feeding molasses with phloxine B an

uranine, the trend in mortality by dose for males was 0.1% > 0.01% = 1%

10% > 0.001% = 0%; for females, the mortality trend was 0.1% > 0.01%

1% > 10% > 0.001% > 0%. The cumulative mortality of females wad highe

than that of males for all concentrations, except at 10%. There was n

significant increase in mortality 24 h after feeding on molasses wit

dyes for females and after 48 hours for males. Example 2

Determining Functional Relationship Between Concentratio

of Dyes and Mortality of Mediterranean Fruit Fly Adults

Phloxine B and Uranium Mixed in 10% Molasses. Based on result

obtained in study 1, concentration-mortality relationship was determine

using the following molarity (M) concentrations of 1:1 molar mixture o

phloxine B and uranine in 10% molasses: 2.5 x 10^"5, 5 x 10^{' 5}, 1

10-⁴, 2 x 10^"4, 4x 10^"4, 8x l0-⁴, 1.6x l0^"3, 3.2x l0^"3, 6.4X 10^{' 3}

and 1.28 x 10"². The assay procedure was similar to the methodolog

described above. Control flies were fed with 10% molasses. Based o

results of study 1, mortality counts were done only up to 2 d. Twent

groups of 20 flies (by sex) were tested for each dye concentration. Adult

used in the test were not starved (i.e., 24 h prior to their use in th

experiment, adults had sugar-protein-water diet during the 12 h ligh

phase and were provided with water only during the (12 h dark phase).

Results

Figure 2 shows the functional relationship betwee

concentration of dyes and mortality. Phloxine B and uranin

were in 10% molasses. Mortality of both males and females increase with the increase in concentration of dyes from 2.5 x 1 0' ⁵M to 1 .6

10^{" 3}M; however, at 3.2 x 1 0^{* 3}M to 1.28 x 1 0^{" 2}M, adult mortality wa

lower that that observed at 1 .6 x 1 0" ²M. The LC₅₀ was 1 .7 x 1 0" ³M fo

males and 1.3 x 1 0^{' 3}M for females. Both sexes were deterred fro

feeding at concentrations greater than 1 .6 x 10^{" 3}M. The LT₅₀ at 1 .6

1 0^{" 3}M was about 7 hours for both sexes.

Example 3

Phloxine B and Uranine Mixed in 1 % NuLure. Various dilution

of dyes were prepared in a stock of 1 % NuLure (a commercial hydrolyze

protein bait preparation) 44% corn gluten meal, hydrolyzed and 56% iner

ingredients, Miller. Concentration mortality relationship was determine

using the following molarity (M) concentrations: 2.5 x 1 0^{" 5}, 1 x 10^{" 4}

4 x 1 0" ⁴, 8 x 1 0^{" 4}, 1.6 x 1 0^{" 3}. Control flies were fed with 1 % NuLure

The assay procedure was similar to the method described above, excep

that fourteen groups of 20 females were tested for each dy

concentration. Test adults were not starved (i.e. 24 h prior to their use i

the experiment, adults had sugar-protein-water diet during the 1 2 h ligh

phase and were provided with water only during the 1 2 h dark phase). R es u lts

Figure 3 shows that when the food carrier for dye was 1 % NuLur

(i.e., with relatively much lower feeding stimulant than molasses), ther

was no significant increase in mortality from 2.5 x 10" ⁵ to 4 x 10"

significant increase in mortality was observed from 4 x 10" ⁴ to 1.6

1 0" ³. The LC50 was 1.6 x 10^{_ 3}M for. females. However, the LT₅₀ at 1.6

10^{" 3}M was about 35 h. Combined results shown in Figs. 2 & 3 emphasiz

the importance of food carrier in the adult feeding consumption an

consequently the level of mortality due to the light-activated dy

toxicity.

Our data attest to the insecticidal activity of Phloxine B and Uranin

mixture to Mediterranean fruit fly adults and provide fundamenta

information for developing phloxine B + uranine + bait formulations fo

large-scale, area-scale, area-wide spray applications.

The ratio and absolute amounts of the photodye, attractant bait i

commercial formulations may vary and may be readily predetermined b

the practitioner skilled in the art by routine testing. It will be recognize

that the photodye should be employed in an amount effective to result in

significant growth inhibition or mortality rate of a test group of th target insect as compared to an untreated group. Furthermore, the amoun

of attractant bait employed should be effective to attract the targe

insect and stimulate it to feed on the bait. The actual effective amount

of the photodye and attractant bait may vary with environment

conditions such as temperature, humidity and wind, the type of vehicle

carrier employed, application protocol, and stage of target inse

development.

Example 4

Determining Functional Relationship Betwee

Concentration of Dyes and Mortality of Oriental Fruit Fly Adults

Phloxine B and Uranium Mixed i/i 1% NuLure; Adult Provided Wit

A Feeding Choice. Concentration-mortality relationship wa

determined using the following molarity (M) concentrations of 1:1 mola

mixture B and uranine in 1% NuLure: 4 x 10^{" 4}, 8 x 10^"4, 1.6 x 10" ³. Th

assay procedure was similar to the methodology described abov

However, the test adults were provided with a feeding choice of tw

types of food: 2 wicks with only 1% NuLure and another 2 wicks with 1

NuLure mixed with dyes. Control flies were fed with 1% NuLure. Mortalit

counts were done up to 2 d. Three groups of 20 flies (by sex) were teste for each dye concentration. Adults used in the test were not starved (i.e

24 h prior to their use in the experiment, adults had sugar-protein-wat

diet during the 1 2-h light phase and were provided with water only durin

the 12-h dark phase).

Res u lts

(Figure 4) show the high toxicity (>60%) of 1 :1 molar mixture B an

uranine to Oriental fruit fly adults, even in the presence of a choice

food that do not contain the dyes.

Example 5

Hydrolyzed Protein Bait Sprays Containing Phloxine B an

Uranine for Controlling Mediterranean Fruit Fly and Orient

Fruit Fly (Diptera: Tephritidae) Infestations. Insecticidal efficac

of Phloxine B and Uranine mixed in hydrolyzed protein bait sprays wa

assessed against an established, high density Mediterranean fruit fl

population in coffee fields on Kauai, Hawaii. An experimental use perm

from the State of Hawaii allowed treatment of 10 acres amidst thousand

of acres of productive coffee fields. A mixture of 2.2 oz of a 1 :1 mola

mixture of Phloxine B and Uranine, 1 6.23 fl oz of hydrolyzed protein ba (NuLure), and 1 5.5 gal of water was ground-sprayed for 20 application

over a 3 month period on 1 0 acres of coffee, while there was no sprayin

on an adjacent (control) 1 0 acres. Densities of Mediterranean fruit fly an

oriental fruit fly adults were reduced by 50% in the treated field after th

completion of the spraying program, with corresponding suppression o

their level densities in coffee berries collected from the treated field

These results were significant because the thousand of acres surroundin

the 10-acre treatment plot produced hundreds of thousands of adults frui

flies that were constantly immigrating into the treatment plot.

Example 6

Determining Variation in Oriental Fruit Fly Adult Mortality due to Concentration of Dyes, Sex, and Time after Feeding. Phloxine B and Uranine

Mixed in Aqueous Solution containing 20% Yeast Hydrolysate and 20% Fructose. Eleven concentrations, consisting of 1.25 x 10^"5 M, 2.5 x 10^*δ M, 5.0 x 10^"5 M, 1.0 x 1CT*

, 2.0 x 10-⁴ M, 4.0 x 10-* M, 8.0 x 10^"4 M, 1.6 x 10^"3 M, 3.2 x 10^"3 M, 6.4 x 10^'3 M, and

1.28 x 10^*2 M of 1:1 molar mixture of phloxine B (829 g per mole) and uranine (376 g per mole) wera-prepared in a stock of 20% yeast hydrolysate and 20% fructose. Six-d old adult flies confined in feeding chambers were provided with the treatment solutions

saturated in cotton wicks; control flies were provided with the stock solution. Each

feeding chamber had either 20 males or 20 females. Adult feeding with dye-stock mixtures commenced at 08:00 h and terminated at 12:00 h, providing a 4-h feeding duration. At the onset of feeding each feeding chamber was kept 10 cm below two hig

intensity, cool fluorescent lights covered with an 80% shade cloth yielding surface light

intensity of 600 lux. Following the feeding period, the shade cloth was removed and

two additional fluorescent tubes were turned on, resulting in a light intensity of 18,000

lux which was then maintained throughout the remaining 12-h light phase of the experiment Upon removal of the wicks containing the treatment solutions, flies were

provided with water and a diet consisting of sucrose, protein hydrolysate, and torula yeast. Mortality counts were made at the time wicks were removed and 2 h, 4 h, 6 h,

24 h, and 48 h later. The experiment followed a randomized complete block design

with 7 replicates per sex-treatment combination.

Results

Figures 5 A and 5B show the variation in oriental fruit fly adult mortality due to

concentration of dyes and time after feeding for males and females, respectively.

Mortality of oriental fruit fly adults differed with dose and number of hours after termination of feeding, but was similar for males and females. Rate of kill increased with increasing concentration, with 100% mortality achieved after 2 h of light exposure

at high dye concentrations (6.4 x 10^*3 M and 1.28 x 10^"2M). Concentrations less than equal to 1.0 x 10"* failed to increase mortality beyond control levels. Example 7

Determining Relative Mortality of Oriental Fruit Flies when Exposed to D

Mixed with Methyl Eugenol as compared to Methyl Eugenol alone or Methyl

Eugenol + dog collar (i.e., source of the insecticide Naled). Containers with 4 - 2.

cm diameter openings on the sides and holding a 2.5 cm long cotton wick holding 1.0

ml of either methyl eugenol alone (treatments 1 and 2) or a 1.28 x 10^"2 M concentrati

of Phloxine B + Uranine in methyl eugenol (treatment 3) were set out in an orchard,

supplemental release of irradiated oriental fruit flies. Treatment 2, additionally, had a

strip of dog collar added to the interior of the container as a knock-down toxicant. On and one-half hours after the fly release, containers were collected with screen added

the openings to trap all contained flies. Containers were then exposed to full dayligh

and mortality counts were taken every half hour for the next two hours, after which th

total number of flies was counted in each chamber. The experiment followed a

randomized complete block design with 5 replicates per treatment.

Results

Figure 6 shows the average mortality in the three treatments over time. Ther was a high initial rate of mortality in the treatment including the dog collar strip. Two hours after exposure to full sunlight, there was greater than 98% mortality in contain

to which dye had been added to the methyl eugenol wicks, compared to 100% morta

in containers which had a strip of dog collar and less than 25% mortality in chambers

with only methyl eugenol contained in the wicks. Example 8

Determining Whether Increase of Dye Concentration in Methyl Eugenol

above 1.28 x 10^"2 M Improves the Rate of Kill of Oriental Fruit Flies. Containers

with 4 - 2.5 cm diameter openings on the sides and holding a 1.8 cm long cotton wick

holding 1.0 ml of either methyl eugenol alone (treatments 1 and 2) or a 1.28 x 10'² M

(treatment 3) or 4.29 x 10^"2 M (treatment 4) concentration of Phloxine B + Uranine in

methyl eugenol were set out in a citrus orchard known to have a wild population of

oriental fruit flies. After two hours traps were recovered with screens added to the hole io entrap any contained flies. Wicks In treatment numbers 2 - 4 were replaced will ι

water saturated wicks while wicks in treatment number 1 were not exchanged or

removed. Containers were moved to full daylight and mortality counts were taken

periodically over the next two hours, after which the total number of flies was counted i

each container, and the number of live flies with reddened abdomens in the container

which had included dye in their initial wicks. The experiment followed a randomized

complete block design with 3 replicates per treatment.

Results

Figure 7 shows the increase in mortality overtime in the 4 treatments. By 30 minutes after the exposure of the chambers to full daylight, over 66% of the flies in th

4.29 x 10^*2 M dye treatment containers had died compared to less than 10% of those i

the 1.28 x 10^"2 M dye treatment containers. By 2 hours these percentages had

increased to 100% of the flies in the 4.29 x 10^"2 dye treatment containers and less

than 50% of those in the 1.28 x 10^"2 M dye treatment containers. None of the flies in the 1.28 x 10^"2 dye treatment containers which continued to survive at the end of th experiment had reddened abdomens. Average mortalities in the two methyl eugeno

only treatments were less than 8% and less than 20% at these two time periods,

respectively.

Example 9

Determining Whether Phloxine B Alone (no Added Uranine; 4.29 x 10^*2 M

and 8.58 x 10^*2 M Concentrations) in Methyl Eugenol is Effective in Killing

Oriental Fruit Fly Adults and Whether Rate of Kill is Greater with 8.S8 x 10"² M

than 4.29 x 10^*2 M. Containers with 4 - 2.5 cm diameter openings on the sides and

holding a 1.8 cm long cotton wick holding 1.0 ml of either methyl eugenol alone

(treatments 1 and 2) or a 4.29 x 10^"2 M (treatment 3) or 8.58 x 10^"2 M (treatment 4)

concentration of Phloxine B (without the addition of Uranine) in methyl eugenol were

set out in a citrus orchard known to have a wild population of oriental fruit flies. After

two hours traps were recovered with screens added to the holes to entrap any

contained flies. Wicks in treatment numbers 2 - 4 were replaced with water saturated

wicks while wicks in treatment number 1 were not exchanged or removed. Container

were moved to full daylight and mortality counts were taken periodically over the next

two hours, after which the total number of flies was counted in each container; during

these 2-h period, the weather condition was overcast with intermittent rains. The

experiment followed a randomized complete block design with 6 replicates per

treatment. Results

Figure 8 shows the increase in mortality over time in the 4 treatments. After 30

minutes of exposure to full daylight conditions, over 50% of the flies had died in the 8.58 x 10"² M dye treatment while less than 25% had died in the 4.29 x 10"² M dye

treatment. Both control treatments averaged less than 3% mortality after 30 minutes. The difference in total mortality between the two dye treatments, however, became less

over time, both reaching 100% after 2 hours of exposure, at which point both control treatments averaged less than 21% mortality.

Claims

What is Claimed is:

1. An insecticidal composition comprising:

an effective amount of an attractant bait, and

an effective insecticidal amount of photoactive dye.

2. The composition of claim 1 wherein said photoactive dye is

selected from the group consisting of halogenated xanthene and a mixture

of halogenated xanthene and uranine.

3. The composition of claim 2 which contains between about 0.001 %

to about 1 0.0% by weight of said photoactive dye.

4. The composition of claim 3 further including at least one

anti-foaming agent.

5. The composition of claim 4 wherein said halogenated xanthene is

Phloxine B.

6. A method of eradicating or suppressing a population of

Mediterranean fruit flies or oriental fruit flies and which introduces

substantially minimal safety risks to humans, agriculture, and

nontargeted living creatures comprising the steps of: providing the composition of claim 1 ;

causing said composition to be ingested by said fruit fly populatio

by spraying or providing said composition at a location where said fruit

flies feed; and

whereby ingestion of said photoactive dye by a Mediterranean frui

fly or oriental fruit fly causes a quantity of photons to penetrate withi

the body of said fly, said quantity of photons being toxic to said fruit fly,

said quantity of photons further beings non-toxic to humans and said

nontargeted living creatures.

7. A method for eradicating or suppressing a population o

Mediterranean fruit flies or oriental fruit flies and which introduces

substantially minimal safety risks to humans, agriculture, and

nontargeted living creatures comprising the steps of:

providing an insecticide composition which includes at least one

photoactive dye;

causing said composition to be ingested by said fruit fly populatio

by spraying or providing said composition at a location where said frui

flies feed; and

whereby ingestion of said photoactive dye by a Mediterranean fruit fly or oriental fruit fly causes a quantity of photons to penetrate within

the body of said fly, said quantity of photons being toxic to said fruit fly,

said quantity of photons further being non-toxic to humans and said

nontarget living creatures.

8. The method of claim 8 wherein said photoactive dye is selecte

from the group consisting of halogenated xanthene and a mixture of

halogenated xanthene and uranine.

9. The method of claim 9 wherein said halogenated xanthene is

Phloxine B.

1 0. The method of claim 9 wherein said composition further

includes molasses as an attractant.

1 1 . The composition of claim 1 wherein said attractant bait is

molasses.

12. The composition of claim 1 wherein said attractant bait is

hydrolyzed protein.

13. The composition of claim 1 wherein said attractant bait is

methyl eugenol.

14. The method of claim 7 wherein said fruit flies are oriental fruit

flies and wherein said composition further includes methyl eugenol as an

attractant.