Title: Effects of insect growth regulators on the bionomics and control of the horn fly, Haematobia irritans (Linnaeus) /
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Title: Effects of insect growth regulators on the bionomics and control of the horn fly, Haematobia irritans (Linnaeus) /
Physical Description: x, 104 leaves : ; 28 cm.
Language: English
Creator: Greer, Norman Ivan, 1945-
Publication Date: 1975
Copyright Date: 1975
 Subjects
Subject: Horn fly -- Control   ( lcsh )
Insect hormones   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida, 1975.
Bibliography: Includes bibliographical references (leaves 97-103).
Statement of Responsibility: by Norman Ivan Greer.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098931
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000413960
oclc - 38046310
notis - ACG1102

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EFFECTS OF INSECT GROWTH REGULATORS ON ITH BTIOOMICS
AND CONTROL OF THE HORN FLY, Haeaatobia irritangs (LI-NXNEUS)











By

MORMIAN IVAi GREER


A DISSERTATION PRESE ':TE) TO THE C-GADU.ATE COUNCIL OF
TP}H. UNIVERSITY 3O :E.JD3 .
IN PARTIAL FULFil LtMi.'-N" 01'I T;E i C-r!Q:'i-,"P' EX:;iTS FOR Ti E
DEGRl:A OF DOCTOR OF FLIOSc 0Y




UNIV ,,STY OF FLORIDA


1975

















ACKNOWLEDGEMENTS


I wish to express sincere gratitude to those persons who have made

this study possible.

I am extremely grateful to Dr. Jerry F. Butler, Chairman of the

Supervisory Committee for his invaluable assistance, advice, and guid-

ance during the course of graduate study. Dr. Butler unselfishly extended

on many occasions knowledge much needed for the success of this study.

I thank Dr. F. S. Blanton for serving on the Supervisory Committee,

and sharing his knowledge of medical and veterinary entomology.

Much appreciation is extended to Dr. P. B. Morgan, USDA, Gainesville,

for serving as a member of the Supervisory Committee. Dr. Morgan will-

ingly gave encouragement and invaluable assistance on the laboratory

testing of insect growth regulators on flies.

I also extend appreciation to Dr. J. A. Himes who served as a mcm-

ber of the Supervisory Committee and also assisted and advised ne on tcx-

icity and methods of application of insect growth regulators to cattle.

I also wish to express appreciation to Dr. W. G. Eden, Chai-r.a'i of

the Department of tntomology and NErmaology for awarding a graduate re-

search assistantshLp during the terry of graduate study.

I also w;sh to express my sincere approciatiion to my wife, Susan

for her uendning support, understanding, and invaluable assistance that

she so willingly gave during the entire period of graduate study,
















TABLE OF CONTENTS


PAGE

ACKNOWLEDGEMENTS ii

LIST OF TABLES v

ABSTRACT ix

INTRODUCTION 1

REVIEW OF LITERATURE 3
History and Economic Importance of the Horn Fly 3
Biology of the Horn Fly 3
Diapause P
Mass Rearing of Horn Flies b
Parasitism and Fredation 11
Feed Additive Fly Control 13
Insect Growth Regulators 13
Effects of Juvenile Hormone on Eggs 16
Effect of Juvenile Hormone on Larvae 17
Effect of Juvenile Hormone on Pupae 18
Effect of Juvenile Hormone on Lice 1.
Effect of Juvenile Hormone on Adults 18
Effects of Juvenile Hormone on Diptera 19
Crossresistance and Selectivity of IGR 22

METHODS AND MATERIALS 23
Establishment of the Florida Strain of Horn Fly 23
Rearing Chambers for the Colony 23
Care of the Colony 24
Laboratory Screening Tests 26
ICR Active Comoounds 27
Cattlr Feeding Trials 30

RFSULTS AND D-ISCUSqION 33
Lab.'orttory Studies of Larval Medium Treated w.it h Insect 33
Greoth Reg'..) ateor
Lab;ra.tory St-'Ces with Eggs Treated Topically with IGR 50
Laboratory Studies- o Pupai TreaLed Topically with Insect 54

L'ab"oiraLcr h.d.-ie s rf AdulLs Treated Topic:illy with IGR 60
Lal-crutory ;..-s with Manurc FromT Cattle ted Yethoprone 69
in a Fe<- ST pleJ rnt
Large S.za. Field Studies dS










PAGE

SUMMARY 92
Laboratory Tests with IGR 92
Feeding Methoprene to Cattle 93

BIBLIOGRAPHY 97

BIOGRAPHICAL SKETCH 104
















LIST OF TABLES


TABLE PAGE

1. Production of Horn Fly Pupae, Adults, and Eggs From 34
MGK-264-Treated Medium (100 eggs/rep).

2. Sex Ratios of Horn Flies Reared From MGK-264-Treated 36
Medium (100 eggs/rep).

3. Production of Horn Fly Pupae, Adults, and Eggs From 37
R-20458-Treated Medium (100 eggs/rep).

4. Sex Ratios of Horn Flies Reared From R-20458-Treated 40
Medium (100 eggs/rep).

5. Production of Horn Fly Pupae, Adults, and Eggs From 41
Piperonyl Butoxide-Treated Medium (100 eggs/rep).

6. Sex Ratios of Horn Flies Reared From Piperonyl 43
Butoxide-Treated Medium (100 eggs/rep),

7. Production of Horn Fly Pupae, Adults, and Eggs From 45
MGK-264-Treated Medium (100 eggs/rep).

8. Sex Ratios of Horn Flies Reared From CRD-9499-Treated 46
Medium (100 eggs/rep).

9. Production of Horn Fly Pupae, Adults, and Eggs From 48
Methoprene-Treated Medium (100 eggs/rep).

10. Sex Ratios of Horn Flies Reared From Methoprene- 49
Treated Medium (100 eggs/rep).

11. Percent Hatch of 25 Horn Fly Eggs per Replication When 51
Group Treated with 5 pl of Acetone or MGK-264 in Acetone.

12. Percent Hatch of 25 Horn Fly Eggs per Replication When 51
Group Treated with 5 1l of Acetone or Piperonyl Butoxide
in Acetone.

13. Percent Hatch of 25 Horn Fly Eggs per Replication When 52
Group Treated with 5 pl of Acetone or CR0-9499 in Acetone.

14. Percent Hatch of 25 Horn Fly Eggs per Replication Wnhen 52
Group Treated with 5 ul of Acetone or Methoprene in
Acetone.











15. Percent Hatch of 25 Horn Fly Eggs per Replication When 53
Group Treated with 5 p1 of Acetone or R-20458 in Acetone.

16. Production of Horn Fly Adults and Eggs From Pupae 55
Treated Topically with HGK-264 (10 pupae/rep).

17. Production of Horn Fly Adults and Eggs From Pupae 56
Treated Topically with Piperonyl Butoxide (10 pupae/rep).

18. Production of Horn Fly Adults and Eggs From Pupae 58
Treated Topically with R-20458 (10 pupae/rep).

19. Production of Horn Fly Adults and Eggs From Pupae 59
Treated Topically with CRD-9499 (10 pupae/rep).

20. Production of Horn Fly Adults and Eggs From Pupae 61
Treated Topically with Methoprene (10 pupae/rep).

21. Survival and Egg Hatch of Horn Fly Adults Treated 62
Topically with CRD-9499 (10 adults/rep).

22. Survival and Egg Hatch of Horn Fly Adults Treated 64
Topically with Piperonyl Eutoxide (10 adults/rep).

23. Survival and Egg Hatch of Horn Fly Adults Treated 65
Topically with MGK-264 (10 adults/rep).

24. Survival and Egg Hatch of Horn-Fly Adults Treated 67
Topically with R-20458 (10 adults/rep).

25. Survival and Egg Hatch of Horn Fly Adults Treaced 68
Topically with Methoprene (10 adults/rep).

26. Laboratory Production of Horn Fly Pupae and Adults in 69
Manure From Animals Fed Methoprene at 48 or 480 pg/kg/day
(100 eggs/rep).

27. Laboratory Production of Horn Fly Pupae and Adults in 70
Manure From Animals Fed Metboprene at 24 or 48 pg/kg/day
(100 eggs/rep).

28. Laboratory Production of Horn Fly Pupae, Adults, and Eggs 71
in Manure From Animals Fed Methoprene at 2.4 or 24
vg/h1:/day (100 eggs/rep).

29. Sex Ratios of Horn Fly Adults Produced in the Laboratory 72
in Manure From Animals Fed Mlethoprene at 2.4 and 24
pg/kg/day (00 eggs/rep).

30. Laboratory Production of Horn Fly Pupae, Adults, and Eggs 73
in Manure prom Animals Fed Methoprene Under Field Conditions
at 24 pg/kg/day (100 eggs/rep).


TABLE


PAGE









TABLE PAGE

31. Sex Ratios of Horn Fly Adults Proouced in the Laboratory 74
in Manure From Animals Fed Methoprene Under Field
Conditions at 24 pg/kg/day (100 eggs/rep).

32. Field Emergence of Insects From the Manure of Animals 75
Fed 1% Methoprene Granular at the Rate of 24 pg/kg/day.

33. Laboratory Production of Horn Fly Pupae, Adults, and Eggs 77
in Manure From Animals Fed Methoprene in Field Studies at
12 pg/kg/day (100 eggs/rep).

34. Sex Ratios of Horn Fly Adults Produced in the Laboratory 77
in Manure From Animals Fed Methoprene in Field Studies at
12 pg/kg/day (100 eggs/rep).

35. Field Emergence of Insects From the Manure of Animals 73
Fed Methoprene Granular at the Rate of 12 pg/kg/day.

36. Laboratory Production of Horn Fly Pupae, Adults and Eggs 80
in Manure From Animals Fed Methoprene in Field Studies at
6 pg/kg/day (100 eggs/rep).

37. Sex Ratios of Horn Fly Adults Produced in the Laboratory 80
in Manure From Animals Fed Mlethoprene in Field Studies at
6 pg/kg/day (100 eggs/rep).

38. Field Emergence of Insects From Manure of Animals 81
Fed 1% Methoprene at the Rate of 6 ug/kg/day/head.

39. Laboratory Production of Horn Fly Pupae, Adults, and Eggs 83
in Manure From Animals Fed Methoprene in Field Studies at
3 pg/kg/day (100 eggs/rep).

40. Sex Ratios of Horn Fly Adults Produced in the Laboratory 83
in Manure From Animals Fed Methoprene in Field Studies at
3 pg/kg/day (100 eggs/rep).

41. Field Emergence of Insects From Manure of Animals 84
Fed 1% Methcprene at the Rate of 3 pg/kg/day/hcad.

42. The Percent Parasitism of Horn Fly Pupae Recovered From 85
Field Samples of Manure From Animals Treated with
Methopreno at the Rate of 24 pg/kg/day.

43. The Percent Parasitism of Horn Fly Pupae Recovered From 87
Field Samples of Manure From Animals Treated with
Methoprene at the Rate of 12 pg/kg/day.

44. The Percent Parasitism of Horn Fly Pupae Recovered From 87
Field Samples of Manure From Animals Treated with
Metboprone at the Rate of 6 pg/kg/day.









TABLE


PAGE


45. The Percent Parasitism of Horn Fly Pupae Recovered From 87
Field Samples of Manure From Animals Treated with
Methoprene at the Rate of 3 pg/kg/day.

46. Laboratory Production cf.Horn Fly Pupae, Adults, and Eggs 88
in Manure From Beef Cattle Fed Methoprene.

47. Sex Ratios of Horn Fly Adults Produced in the Laboratory 89
in Manure From Beef Animals Fed Methoprene.

48. Field Emergence of Insects From Manure of Beef Cattle 91
Fed Methoprene at the Rate of 24 igkgg/day/head.
















Abstract of Dissertation Presented to the
Graduate Council of the University of Florida
in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy



EFFECTS OF INSECT GROWTH REGULATORS ON THE BIONOMICS
AND CONTROL OF THE HORN FLY, Haemazobia irritans (LINNAEUS)

By

Norman Ivan Greer

March, 1975

Chairman: J. F. Butler
Major Department: Entomology and Nematology

insect Growth Regulators (ICR) were tested against the horn fly

Haematobia Irritans (Linnaeus) to determine their effect on development.

Laboratory tests with IGR in horn fly larval medium showed that reduc--

tion of emergence with MGK-264, [N-(2-ethylhexyl)-5-nor=bornene-2,3-

dicarboximide], occurred at 1000 ppm. Piperonyl butoxide, {a[2-(2-

butoxyethoxy)=ethcxy]-4,5-methylenedioxy-2-propyltoluene), R-20458, [1-

(4'ethylphenoxy)-6,7-epoxy-3,7-dimethyl-2-octene], and CRD-9499, [ELT

70284] reduced emergence at 150 ppm and merhoprene, (is'opropyl 11-methoxy-

3,7,11-trimethyldodeca-2,4-dienoate], reduced emergence at 0.05 ppm.

Topical application to eggs showed that R-20458 reduced egg hatch at 2 pg

per egg. Topical application to pupae showed that R--20458 reduced emer-

ger(i: at 0.1 pg per pupa and CRD-9499 reduced emergence at 1 pg-per pupa.

Methoprene was the most effective, being active at 0.01 pg per pupa.

Topical application to adults showed that CRD-9499, 1G%-264, and R-20458

caused a reduction i. adult survival at 10 u, per di:it. uhile p*ipercnyl









butoxide produced a reduction cf survival at 1 pg per adult. Reduction

of hatching of eggs from treated adults was found with piperonyl butoxide,

MGK-264, and P-20458. Feeding nrethoprene.to cattle for control of horn

flies breeding in manure showed that 12 pg per kg per day produced 100%

reduction in horn fly emergence in laboratory bioassays. A dosage of

24 yg per kg per day was required to reduce fly emergence 100% in manure

under field conditions. Field manure samples from cattle fed 24 or 12

pgper kg per day of methoprene showed a significant increase in para-

sitism of horn fly pupae. Sepsidae were the only other diptera signifi-

cantly affected. A herd of cattle fed methoprene at the average dosage

of 24 pg per kg per day showed a reduction of horn fly adults emerging

from sampled field patties but no reduction of the adult population on

the animals was observed.





( `ChI--ran
Chairman
















INTRODUCTION


The horn fly, haematobia irritans (Linnaeus), is one of the major

cattle pests of the United States. This fly has been called the cattle

fly, cattle horn fly, cow horn fly, stockyard fly, Texac fly, Texas horn

fly, and the third party fly. The adult horn fly is a cosmopolitan obli-

gate bloodsucking ectoparasice of cattle which will occasionally attack

goats, mules, horses, and dogs. It will rarely attack man. Infestations

of 4,000 flies per animal are commonly observed cn Texas cattle, and pop-

ulation densities may reach 10,000 flies per animal. Damage to cattle

results from annoyance, blood loss, reduction of weight gain, reduction

in milk production, severe dermal lesions, and the possibility of dis-

ease transmission. Dermal lesions are susceptible to infcctiodn with

screwworms, Cochitomyia hominivorax (Coquerel), or other wound infesting

parasites. The ability of the horn fly to reproduce throughout the year

in the Southeastern United States, especially in Florida, increases the

necessity of developing more effective control measures. Since the lar-

val stages of the horn fly develop i. fresh cattle manure, the possibil-

ity of controlling the horn fly by us- of cattle feed izaditive- seemora

worth studying. The use of feed additives for control of flies breeding

in ding has been investigated for rany years. Kniplinl in 1.938 and

Eruce nr 1939 tested phenothiazine for control of the b orn f.-.. Other

workers: continued alongp these lines uting v''riLus :nsec'ticideC

Becatus of th'- i creasinlg eorv1,": rcinoita po1ltit1 n prc' le'-m ] i peir--

sist;: p.hsticl2s. frd t.he ii .ear inr d'evelopn't or insacu res;staa.e-









to insecticides, the need for alternate horn fly control measures is be-

coming increasingly important. New chemicals are required which provide

effective and specific control on target insects with little or no damage

to plants and animals. Some alternatives the entomologists are developing

for insect pest management are biological control, integrated control,

pheromones, and the use of insect hormones which are commonly referred to

as insect growth regulators or simply IGR. Insect growth regulators

sach as most juvenile hormones closely fit these requirements.

Effects of juvenile hormone mimics (IGR) on flies in the family

Muscidae have been studied. These studies include the stable fly Stonoxys

calcitrans, and the house fly Musca domestic. Insecticides when used as

feed additives in cattle rations have proven to be successful in control

of larvae of dung breeding flies. Research was required to dete:,rne if

the horn fly could be effectively controlled through the use of ICR, thus

limiting treatment to the developmental site of the horn fly and minimizing

environmental contamination, since the IGR would be isolated in the manure

or the soil near the manure.

The major impetus for this study is to utilize the success of feeding

insecticides to cattle for horn fly larvae control by developing a similar

technique for feeding insect growth regulators (IGR, juvenile hormones)

to control the horn fly.

The objectives of this study were: 1.. to determine through labo-

ratory tesarng rhe effectiveness of some IGR compounds on all stag-s of

the horn fly life cycle; 2. to determine the practicality of using the

most active conipou-d as a feed additive to cattle rations for horn fly

control: and 3. to determine if the most active compound adversely

affected the ecology of p-rariti;n, predation, species interaction, and

the survival of born fl!ts ii the amluure ecosystem.
















REVIEW OF LITERATURE


History and Economic Importance of the Horn Fly

The horn fly is a cosmopolitan species (McLintock and Depner, 195Z).

It invaded the United States during the late nineteenth certiury, the

apparent port of entry_.being in the vicinity of Philadelphia, and by the

turn of the century, the horn fly had invaded most of the United States.

Puerto Rico, and Canada (Hargett and Goulding, 1962).

The horn fly has been a very serious economic cattle pest through-

out America (Dorsey et al., 1962). Animals showed weight gains of 30 to

70 pounds more per head when treated with insecticide for fly control

(Laake, 1946). Cutkomp and Harvey (1958) in a two-year study again ob-

served a significant increase in weight gains in beef cattle treated

with insecticide for control of horn flies and stable flies and con-

cluded that fly control would be profitable.

Granett and Hansens (1957) and Bruce (1940) studied the effect of

high populations of biting flies on dairy cattle in Salem County, New

Jersey. Thase studies showed a decrease in milk production in unpro-

tected dairy herds of one-fourth to one-half by severe parasitism of

biting flies.

Large populations of horn flies may produce dermal lesions cau'ied

directly by injury from the constant probing of the biting flies or daU-

age may be indirect from self-inflicted injury by ciials attempting to

escape the biting pcsts (Bruce, 1964). Animals will run through tall

brush, or tub on 1rets, fence po.-ts wire, or other objects trying to

escape the flies.









The amount of damage to the parasitized animals due to blood loss

has been studied in efforts to determine the economic importance resulting

from debilitation and anemia of the host. Daily blood loss due to a horn

fly infestation has been found to average 2.19 mg per mature female fly;

therefore, an infestation of 288,000 flies would-consume about 600 ml of"

blood per day (Bruce, 1964).

The horn fly, like all blood-sucking insects, is believed to be a

vector of disease. It may be a carrier..of Bacillus anthracis (Morris,

1918) and Trypansoma americana (Glaser, 1922).


Biology of the Horn Fly

Bruce (1964) conducted extensive studies on the biology of the horn

fly. Oviposition under natural conditions occurs only in fresh cattle

manure. Gravid females deposit eggs immediately after the manure is

voided and generally do not deposit eggs on the manure after ten minutes.

Flies remain on the manure from 1 to 10 minutes and have been observed

to deposit from 1 to 14 eggs. An entire batch is not necessarily de-

posited at one time. Eggs develop at the rate of 1 per ovariole, with

maximum batches of 24 in the ovaries. The female may produce 15 batches

or approximately 360 eggs in her lifetime.

Approximately 75 per cent of the eggs are deposited on the underside

edge of the manure, some on the lower sides and a few in crevices on the

sides and upper surfaces. Frequently eggs are deposited on grass or straw

beneath the manure. Sggs are rarely deposited on the smooth upper sur-

face of thc manure.

The eggs of the horn fly develop rapidly after being deposited. The

inctbati n period .is less than 24 hours even at room temperature. JI









manure, eggs are exposed to a very high humidity which is necessary to

prevent desiccation (McLintock and Depner, 1954). The egg'of the horn

fly varies from a light straw color at deposition to a dark brown at

hatching. The average dimensions of horn fly eggs are 1.2 mm long and

approximately 0.32 mm wide (Bruce, 1964).

The larvae pass through three instars. Those of the first instar

are slender, widest at the posterior end and taper gradually to a nar-

row cephalic segment. Anterior spiracles are apparently absent in this

instar. The second instar larvae are shaped the same but are larger.

Anterior spiracles are present in the second instar and have four to

six finger-like branches. The general external characteristics of the

third instar larvae are similar to those of the first two instars; how-

ever, there is increased pigmentation as the larvae mature. Also, the

older larvae are more sluggish than the earlier instars particularly

immediately prior to pupation.

Immediately after hatching, the first instar larvae burrow into a

crack or crevice in the manure to seek food and shelter. During devel-

opment in their isolated -micro-climate, the larvae move to the moist

parts of the manure as the outer surfaces become dry (Bruce, 1964).

The time that is spent in each of the three larval instars is quite

different. Characteristically, the first larval instar is the shortest,

the second- instar taking about one-and one-half times as long for devel-

opment. The third instar takes longer than either the first or second,

being six Limes the duration of the first larval instar. At a constant

temperature of 30C, the average duration of the first instar was 10,25

hours, 18.25 hours for the second instar, and 63.75 hours Eor te third

instar. The average time required for de.velopn't-.t of the larvae from

hatching to pupatic. vas 92.25 hours (Bruce, 1964).









The third instar larvae seek a drier environment immediately before

pupation. Factors influencing the selection of the pupation site of

horn fly larvae are dependent on the type of soil, moisture content of

the manure (McLintock and Depner, 1954), and the moisture content of

soil beneath the manure (Bruce, 1964). If the manure is dry in relation

to the soil, the third instar larvae will migrate to the soil where pupa-

tion takes place. If the soil is excessively dry, all pupae will be

found in the moist areas in the manure. The pupation site may be as

deep as one and a half inches in the soil beneath the manure. The dura-

tion of the pupal stage under natural field conditions is about 5.5 days

(Bruce, 1964). The pupae are brown and appear seed-like. After flies -

emerge, they seek out and begin to feed on cattle within two or three

hours (Bruce, 1940).

The adult is about 4 mm long or about one-half the size of the

stable fly with palps about two thirds as long as its conspicious beak

(Metcalf et al., 1962). Usually, the flies cluster around the shoulders

and sides of the animal, but during extremely hot or rainy weather they

congregate on the underside of the belly.

The fact that the horn fly larval stages are obligated to develop in

cow manure as a growth medium has been assumed for many years. Studies

conducted by Greer and Butler (1973) showed that in the laboratory horn

fly eggs reared in manure of cattle bison, sheep or horses produced fer-

tile adults; however, adults were not produced in hog manure. Field ob-

servations showed that there was no natural horn fly development in horse

manure. Clearly some factor or factors under natural field conditions

prevent the female fly from depositing eggs in the manure of these other

hosts since mature adult horn flies are found on these animals.









The horn fly is extremely sensitive to changes in temperature. At

20C the adult fly becomes extremely sluggish and at 4.4C it becomes

inactive. The optimum temperature range fcr normal horn fly activity

falls in a very narrow temperature range of 270C to 32C. -The incuba-

tion period of the horn fly eggs was found to average 14 hours 25 ain-

utes and 238 hours 34 minutes was the average duration frch oviposition

to emergence. An incubation period of 18 hours at 30C was reported by

Depner (1961).

The horn fly has a marked host color preference. They are more

numerous on black and other dark colored cattle than on lighter colored

animals (Bruce, 1940). Using dark colored cloth backgrounds to provide

areas of contrast, Hargett and Goulding (1952) found that more horn

flies congregated on dark than clear cr white test surfaces. Franks et

al. (1964) found that horn flies populated black heifer cattle more than

red cattle while fewer flies were found on white animals.

Bruce (1964) stated that the horn fly mates as early as the second

day after emergence. Mating occurs on castle generally but copulating

pairs have been observed on vegetation in pastures. Harris et al. (1968)

found that mating of horn flies occured as early as the first day after

emergence when the flies were on the host. In the laboratory, mating

took place on the second day after emergence. Also, it was found that

one male may mate up to 8 females but the females appeared to be monog-

amous. Harris and Frazar (1970) found in laboratory studies that the

total daily consumption of blood by 500 horn flies would be approxi-

mately 7 ml/day.

Tungwell et al. (1966) studied the flight behavior of the horn fly.

The flies 'ere released in dylIighL or dark to test the response to CO,










baited sticky traps.- More fly activity was found during the-morning. A

larger number of females were trapped 600 meters from the release point

after 4 hours.

Hoelscher et al. (1968) found that horn flies dispersed in excess

of 400 yards in 48 hours. The maximum rate of dispersal was at night.

Kinzer and Reeves (1974)'found marked horn flies on cattle 7.3 miles from

the'point of releasewithin 10 hours.


Diapause

Hoelscher et al. (1967) found that horn flies in Northeastern

Mississippi overwinter as diapausing pupae. Sufficient numbers of pupae

are found in dung and the soil immediately beneath dung to produce a

spring reproducing generation. Pupae collected in late winter (January,

February) and stored at 1.670C and 70% RH had 60% emergence after 8

months.

In Mississippi, Hoelscher and Combs (1971) found that the first

emergence of diapausing pupae occurred in March and early April. The

middle of November was the last time during the year suitable for fall

adult emergence.


Mass Rearing of Horn Flies

McLintock and Depner (1957) tried to establish a colony of the horn

flies on a diet of defibrinated and citrated beef looid. The wild flies

stopped laying after 2 or 3 days and no eggs were laid by laboratory

reared flies. Dissection showed that ovari-an maturation was not occurring

in laboratory reared flies. The spermathecae of laboratory reared flies

contained ro sppermato7oa.








Harris (1962) established the first successful procedure for con-

tinuous rearing of horn flies under laboratory conditions. Before this

time, colonies were reared by keeping flies on cattle in fly cages or by

confining -flies under lamp chimneys or screen cages attached to the skin

of stanchioned cattle (McLintock and Depner, 1957; Lewis and Eddy, 1961;

Depner, 1962; Hargett and Goulding, 1962). Horn flies have been main-

tained in colony since 1947-on caged steers at the USDA laboratory,

Kerrville, Texas. However, until 1961 the colony could be maintained

only by periodically adding wild field-collected flies. Adult horn

flies were fed sterile bovine blood containing a saline extract of beef

muscle, acid-citrate dextrose solution (ACD) and certain antibiotics.

These flies mated and females produced viable eggs when confined in small

cylindrical plastic cages (Harris, 1962).

Harris (1962) postulated that the reason horn flies never produced

viable eggs in the laboratory was that because of microorganisms in cit-

rated bovine blood, it did not-contain the essential nutrients for horn

fly reproduction. Through his experimentation, he found that an adult

diet of 1 part citrated bovine blood, 1 part beef juice, and a standard

antibiotic mixture containing 1 mg streptormcin, 1,000 units penicillin

and 250 units of mycostatin per ml produced sufficient production of

viable eggs to maintain a colony away from a host.

Harris et al. (1967) reported the use of an artificial medium for

rearing horn flies. The ingredients were 264 g ground sugarcane pulp,

48 g whole wheat flour, 12 g dehydrated bovine plasma, 6 g sodium bicar-

bonate, and 1100 ml of distil.led water. This medium proved successful

in rearing horn fly larvLa. without dasnure. Rate of pupation, adult emer-

gence and egg hacch ere satisfactnry.









Morgan (1966) found that horn fly adults reared in continuous-light

with Glo-luxTM fluorescent lamps laid 50.% more eggs than flies exposed.

to cool-white fluorescent lamps. He used gauze-covered cotton pads

soaked with liquified-extract-from bovine manure as a substrate for ovi-

position. These pads were inverted in fresh cow manure in pans and cov-

ere6 with Saran WrapTM and incubated at a temperature of 27 20C for 8

days.

Morgan and Grahman (1966) determined that prairie hay manure pro-

duces smaller pupae than alfalfa and sorghum-hay manure. Sorghum hay

manure produced the heaviest pupae.

A concentrated effort began in 1965 at Kerrville, Texas- to develop

a laboratory mass-rearing procedure for the horn fly (Schmidt et al.,

1967). The studies of Harris (1962) and Morgan and Schmidt (1966), and

Harris et al. (1967) were the foundation for the following mass-rearing

technique. Blood for the adults was obtained nonaseptically once or

twice weekly from a slaughterhouse. Standard acid citrate dextrose beef

anticoagulent was added to the whole blood at the rate of 360 ml/1400 ml.

Also, 250 units of nystatin and 1/3 mg ChloromycetinTM (Chloramphenicol)/

ml -ere added to the blood just prior to refrigeration at 4.5C. The

flies were fed at 0800 and 1600 hour daily by dipping 6.5 x 6.5 x 1.0 cm

cotton pads in the blood and placing the wet pads on 7.5 cm squares of

unbleached cotton rmuslin on top of the cages. -One pad of -this-size- was

sufficient for 1,500 fli-s. A 10 x 1.2 cm plastic petri dish was in-

vr"ted over each pad to prevent desicaticon

Schmidt i t al. (1957) report J thie-fol.loinig modification in the

ndult Lorn fly iiet. Thecy found that sodium;c -erate could be substtiLtLc

.pr :cid Cit c xL dextrose and re"le recently a substj utionr o0 2. g of









potassium oxalate dissolved in 20 ml of distilled water/178-ml blood.

The use of 1000 units of potassium penicillin G per ml of blood reduced

spoilage. Also, nonsterile ChloromycetinT was found to be just as sat-

isfactory as the expensive medicinal product.

Size of the blood pad was reduced to a 15 cm square sandwiched between

2 muslin strips. Each.pad was .sufficient for 5000 flies. AluminumP pie

pans 20 cm in diameter were placed over the pads to prevent desiccation.

Schmidt et al. (1967) reported the larval-starting diet is prepared

by blending 2 parts steer manure with 3 parts distilled water and then

freezing the mixture in 35 ml plastic cup. One unit of the frozen diet

was removed from the freezer and placed on an egging pad and allowed to

thaw.

The current larval diet is similar to that described by Harris et al.

(1967) as follows:

Part by wt. (g)
Finely ground-sugar cane pulp (Poultry litter) 246
Wheat flour (Baking) 48
Fish meal (60% protein) 360
Sodium Bicarbonate (baking soda) 6
Distilled water 1300
Manure (alfalfa fed cattle) -545

The dry ingredients are mixed together and the manure and.water is

blended together. The manure and water is then thoroughly hand mixed

with the dry ingredients. This mixture provides enough medium for 5,000 -

6,000 larvae. Our modifications of-this diet are reported- in the-methods

and materials section of this paper.


Parasitism and Predation

There were early reports of hymcmoptcrous parasatolds of the horn

fly. Marlatt (1910) reported that Sp,_-Larmia hirta and Spajr.aa









lanaiensis were important enemies of the horn fly. Later, Pinkus (1913)

observed Spalangia muscidarum Richardson being parasitic on horn fly

pupae.

Lindquist (1936) studied parasites of the horn fly. He found that

Spalangia muscidarum attacked the horn-fly. Spalangia drosophilae Ashmead

generally parasitizes small dung-breeding diptera, but it will also attack

the larger horn fly. Out of 67 cattle droppings examined, 38.2% of the

horn fly pupae in the sand were parasitized, whereas, 64.3% of the pupae

in the manure were parasitized-by Spalangia. Over 95% of these were

Spalangia muscidarum stomoxysiae.

Depner 41968) reported two parasites in the genu Spalangia para-

sitizing horn fly pupae. They were Spalangia drosophilae and Spalangia

haematobiae Ashmead.

Combs and Hoelscher (1969) also studied the hymenopterous pupal

parasitoids of -the horn fly. -They discovered that the population of the

parasitoids peaked during the fall season.

Blume et al. (1970) found that cattle manure exposed in thefield

for a longer period before it was covered with emergence traps had f,:wer

horn flies and more of the other types of insects than when the manure

was covered much sooner. This implies that specific competition with

horn flies develops later in the manure.

Bourne and Hays (1968) studied larvae of the beetle predator

Sphaeridi u scarabaenieos (Linnaeus). iNo predation was found at 4,40C;

while lirval activity -of horn fly larvae was noted at this temperature,

the larv-v of the pr-edator .ere inactive. At 26.70C, 95% predation was

found and at 32.2'C, toe many horn fly larvae pupated before the predator

could succes:sfully ieed on them. Peasites and predators of horn fly









larvae may well be one of tha most important limiting factors in reducing -

the severity of summer population outbreaks.

Burns and Chapin (1969) determined the insect fauna used as food for

the cattle egret, Bubulcus ibis (Linnaeus). Grasshoppers were a large

portion of the-diet, a few tabanid-s were eaten but too few horn flies

-were consumed to effect control. Therefore, this bird is not considered

important in horn fly control.


Feed Additive Fly Control

Drummond (1963) found that insecticides fed to Eolstein cattle

could control horn fly and house fly larvae developing in cattle manure.

House fly control using coumaphos as a feed additive was reported by

Skaptason and Pitts (1962) and Miller et al. (1970a). Anthony et al.

(1961) found that feeding coumaphos to Holstein cattle at 1-lg/kg!day

produced larval mortality in the house fly and the face fly. Feed addi-

tives for face fly control were also reported by Treace (1962, 1964),

and Ode and Matthysse (1964). Miller et al. (1970b) found that GardonaTM

fed to dairy cattle successfully controlled house fly larvae. Butler

and Greer (1973) found that Rabon and vapona fed to cattle would destroy

horn fly larvae breeding in cattle manure. Miller and Gordon (1972)

found that when feeding RabonTM, encapsulated Rabon was present in the

feces at higher levels than when unencapsulated formulations- wre fed.


Insect Growth Regulators

The Brain, Prothoracic Gland, and Corpora Allata

The processes of growth and molting of immature insects is coordi-

nated by three groups of hormones. The first group, called the brain

hor.irne, i., secreted by neurosecretory cells located in the protocerebrum.









This secretion stimulates the activity-of the prothoracic glands. The

prothoracic glands when activated secrete the second group of hormones,

the ecdysones or some substance necessary for its production such as an

.ecdysone glycoside (Willig et al., 1971). After the prothoracic glands

are activated, ecdysone appears in the insect's hemolymph-and their pre-

sence is correlated with the insect molt.

A third group of hormones secreted from the corpora allata are

called the juvenile hormones, .which control the type of cuticle.secreted

by the epidermal cells. Larvae with a high concentration of juvenile

hormone and stimulated with ecdysone molt into larvae. With a low con-

centration-or the absence of -juvenile-hormone they molt into pupae or

adults.

Brain Hormone

Using the gypsy moth Porthetria dispar (Linnaeus)-in a group of de-

cisive experiments, Kopec (1922)-discovered that the insect brain was

an organ of internal secretion. The neurosecretory cells of the brain

which control molting were found to be located in the pars intercere-

bralis of several insects (Wigglesworth, 1940; van der Kloot, 1961;

Girardie,-1964).

Prothoracic Gland Hormone

Fukuda (1940) demonstrated the importance of the prothoracic glands.

He ligatured silkworm larvae behind the prothorax and- found. that-they

would molt only when the prothoracic glands were implanted posterior to

the ligature.

It was Williams (1947) who found the interaction between the brain

and the prothoracic glands. Isolated pupal abdomens of the Cecropia silk-

worn did not molt if either active brains or inactive prothoracic glands









were implanted. However, the abdomens methamorphosed if both active

brains and inactive prothoracic glands were implanted.

Juvenile Hormone

The function of the corpora allata secretion in maintaining larval

development was discovered by Wigglesworth (1935, 1936, 1940) who named

the secretion juvenile hormone. In the Cecropia silkworm, -he corpora

allata-were-active during the third and fourth larval instars but inac-

tive in the pupal stage (Williams, 1961). Gilbert and Schneiderman (1961)

found that the actual juvenile hormone titer in a Saturniid moth was

high during larval life and low in the pupae. Meyer et al. (1965) pre-

pared a highly purified extract of juvenile hormone from the male Cecropia

silkworm. They used the Galleria wax moth test and the A. 2olyphemus in-

jection test to determine the hormone activity. The extract was found

by gas chromatography to be about 90% pure and the analysis revealed that

there were two biologically highly active portions present in the purified

extract.

The structure of juvenile hormone was successfully elucidated by

Roller et al. (1967). Since then, synthesis of juvenile hormone has-

been achieved and many compounds with juvenile hormone activity have been

found. Some examples found were farnesol in Tenebrio feces
1961), and the "paper factor" from Balsam fir (Slama and Williams, 1965)

which was later identified as the methyl ester 6f todomatuic acid and

named juvabione (Bowers et al., 3966).

Two closely related juvenile hormones have been isolated and iden-

tified from the Cecropia silkworm. The first was called JH 1 (methyl

10, 11, -epoxy- 7 -ethyl- 3, 11 -dimethyl- 2, 6, -tridecadienoate, [Meyer

et al'., 1968; Heyer et al., 1970]). The second was called JH 2 .(methyl









10, 11 -epoxy- 3, 7, 11 -trimethyl- 2, 6 -tridecadienoate). The action

of juvenile hormone that is most easily demonstrated is the kind of cut-

icle secreted by epidermal cells stimulated by ecdysones. Not only does

juvenile hormone block metamorphosis of larvae to the adult, but it also

interferes with metamorphosis of the embryo to the larva. White (1971)

studied the relationship of juvenile hormone with polymorphism in aphids.

He found that the activity of the corpora allata in female aphids influ-

enced the development of wing buds and therefore, controlled polymorphism.

Juvenile hormone had a gonadatrophic influence which was seen through the

control of yolk protein synthesis by fat body and this resulted in the

accumulation of protein by the developing oocyte.

Engelmann et al. (1971) found that in.Sarcophaga bullata Parkar that

a female specific protein is synthesized in some but not all allatecto-

mized flies. After extirpation of the neurosecretory cells little yolk

was deposited in the oocytes even though female specific protein was pre-

sent in the hemolymph.


Effects of Juvenile Hormone on Eggs

Riddiford et al. (1967) found that development of embryos can be

blocked in the silkworm eggs as early as the blastoderm stage by exposing

unfertilized eggs to juvenile hormone or its analogues by treating the

adult female moth prior to oviposition. If treated after fertilization

and-oviposition, embryo development cannot be blocked but the first instar

larvae commonly fail to hatch. Larvae that hatch from treated eggs often

have anatomical Cefects as well as poor viability and various abnormali-

ties in postenbryonic development. Riddiford (1970) found that metamor-

phosis -is blocked in the bugs Pvrrhocoris apt rus (Linnaeus) and

Oncoelcus fasctatus by application of juvenile hormone analogues to eggs.









There are one or more supernumerary larval molts forming'giant larvae

-which usually die at the fifth larval stage. By removal -of the corpora

allata at the beginning of the fifth larval stage, supernumerary molts

disappear and the insects undergo normal metamorphosis. From this it is

concluded that there is a continuation of endogenous juvenile hormone

secretion in mature larvae by the corpora allata-due to the treatment

of eggs from which the larvae arose. Eggs of Epilachna varivestis Mulsant,

the Mexican bean beetle, were sensitive to the synthetic hormones during

the first half of the egg stage (Walker and Bowers,-1970). They used

3 methylene-dioxyphenoxy-terpenoid ethers. They also showed that

methylete-dioxyphenoxy--terpenoid ethers prevented-egg-hatch when applied

to.the Mexican bean beetle, Epilachna varivestis and cigarette beetle

Lasioderma serricorne (Fabricius). Riddiford and Truman (1972) showed

that juvenile hormone can somehow interfere with the programming of the

embryonic corpus allatum. The gland fails to cease secretion at the on-

set of the last larval instar.


Effect of Juvenile Hormone on-Larvae

Sehnal et al. (1968). using a purified extract of juvenile hormone

obtained from the Cecropia silkmoth found that transformation of larva

to pupa in Galleria was prevented. Theextract, which was injected,

acted independently of the insect's own corpoca allata. The morphogenic

response occurred when juvenile hormone was present in the insect at the

time the cells were sensitive to the juvenile hormone. The amount of the

effect depended on the age at which the larvae uer. injected. Maximum

effect, was produced when the extract was provided not later than the

first one-third of the last instar.










Effect of Juvenile Hormone on Pupae

Reddy and Krishnakunaran (1972) studied the relationship between

natural cecropia juvenile hormone and some of its synthetic analogues on

Tenebrio pupae. They found through parabiosis that the natural hormone

but not the analogue was netabolized-during the 5 day period. -In the

last instar Galleria larvae, injections showed the same effect. This

longer half life is probably associated with a greater metabolic stabil-

ity of these synthetic analogues. Herzog and Monrce (1972)-reported an

-inhibitor of synthetic juvenile hormone in the house fly. 'They found

that citric acid applied separately to hormone-treated pupae inhibited

the action of the hormone. -


Effect of Juvenile Hormone on Lice

Hopkins et al. (1970) tested topical application of synthetic juven-

ile hormone on the Angora-goat biting louse Bovicola limbata (Gervais).

They found that some lice molted prematurely, some molted a 4th and 5th

time. Nymphal characteristics were retained and sexually nonfunctional

pseudoadults developed. Chamberlain and Hopkins (1970), experimenting

with the same louse, found that lice fed on a diet containing synthetic

juvenile hormone at 50 ppm-remained nymphs and incapable of-depositing

eggs or successfully mating,. .


Effect of Juvenile Hormone on Adults

Master et al. (1970) applied trans dihydro-dichloro-farnesenic acid

methyl ester and trans-dihydro-dichloro-farnesenic acid ethyl ester to

males of Pyrrhocoris apterus (Linnaeus). -Transmission of the material

during matin i was sufficient so that the resulting eggs ceased embryonic

development.









Metwally et al. (1972) applied juvenile hormone mimics to the khapra

beetle, Trogoderma granarium Everts and found that it resulted in severe

defects in the ovaries and reduced fecundity of the adults. Eggs were

often morphologically abnormal, hatchability was low and sometimes reduced

to zero. -Defects in the ovaries included cell death in the gernarium,

resorption of the oocytes in the previtellarium and vitellarium, forma-

tion of compound egg chambers and proliferation of follicular cells re-

sulting sometimes in malformation of the whole ovary.


Effects of Juvenile Hormone on Diptera

Mosquitoes

Juvenile hormone application has been tested in mosquitoes of the

genera Anopheles (Jakob and Schoof,-1971, 1972), Aedes (Spielman and

Williams, 1966;-Jakob and Schoof,_1971, 1972), and Culex (Jakob and

Schoof, 1971, 1972; Wheeler and Thebault, 1971).

Spielman and Williams (1966) used a crude synthetic material pro-

duced by treatment of ethanolic solutions of farnesoic acid with hydrogen

chloride. Adult emergence of A. aegypti was prevented at a concentra-

tion of 10 ppm. Metamorphosis was stopped at stages from pupae to fully

foLmed pharate adults unable to emerge. The most sensitive stage was

late fourth instar larvae. Also, the material inhibited hatching of eggs.

Patterson (1971) tested the sterilizing effects of juvenile hormone

mimics on female Aedes aegypti. The greatest effect on fertility was

about halfway through the gonadotrophic cycle, 32-36 hours after feeding.

The data indicated that juvenile hormone may be broken down or excreted

rapidly by the mosquito. The mosquiLtoes laid large numbers of abnormally

formed eggs which did not darken when exposed to air.









Steelman and Schilling (1972) found that when a juvenile hormone

mimic was used in the field to control Psoro.hora confinnis (Linnaeus)

it reduced Dytiscidae larvae and possibly Hydrophilidae larvae.

House Flies

-Adams-and Nelson (1969) investigated the effects of the corpus

allacum on fat body content. Topical application of synthetic juvenile

hormone on mature female house flies caused an increase in adult fat

body volume. Adult fat body was not affected in allatectomized flies or

flies that were both allatectomized and ovariectomized. There was in-

creased adult fat body in flies that were ovariectomized. Ovariectomy

-probably removed inhibition of the corpus allatum and resulted in an in-

crease in juvenile hormone titer which caused an increase in adult fat

body volume.

Adams (1970) found that ovariectomy and oviposition resulted in a

reduction of area of the corpus allatum of the house fly. He concluded

that a small corpus allatum released juvenile hormone whereas a large

gland was involved in the process of storage. He found that an injection

of an extract containing oostatic hormone into ovariectomized flies

caused an increase in area of the corpus allatum. A humorally mediated

inhibitory feedback mechanism may exist between the ovaries and the cor-

pus allatum.

Morgan and LaBrecque (1971) found that some hormone-li'ke substances

produced sterility when applied to house fly adults or larvae. Compounds

that possessed sterilant activity when used to treat pupae caused high

mortality and the sterility produced was variable from-test to test.

Stable Flies

gri.ght (1972) tested 3 juvenile hormone analogs on stable flies in

both laboratory and outdoor tests. He found that in both tests-emergence









was prevented and the resulting prevention of eclosion was caused by the

formation of pupal-adult intermediates within the puparia.

Wright and Schwarz (1972) tested 62 compounds for morphogenetic

activity on the stable fly. Six compounds were highly active at a dose

of 10 mg. Five of these were epoxides of arylterpenoids with a 9-carbon

backbone and one was an aryl carbamate n-substituted by an 8-carbon

epoxide.

Wright and Spates .(1972) tested 29 materials against stable flies.

Eleven were juvenile hormone-analogues, 9 potential chemosterilants, and

9 plant extracts. The chemosterilants and plant extracts had no morpho-.

genic effect against the stable fly.- The juvenile hormone analogues

affected larval, pupal, and adult stages but not the eggs.

Synergists

Fiperonyl butoxide, sesoxane, piperonyl farnesol ether, and piperonyl

farnesol ether epoxide when used as IGR were active against Oncopeltus

fasciatus (Dallas) whereas, sesoxane was more effective on Tenebrio

molitor (Linnaeus) (Rociler et al., 1967). Bowers (1968) discovered that

some insecticide synergists such as piperonyl butoxide and sesoxane not

only enhance the effectiveness of juvenile hormone mimics, but also have

juvenile hormone activity when used alone. Redfern et al. (1972) used

aziridines as potentiators of juvenile hormone on the yellow mealworm

and the large milkweed bug. They found that activity was maximized when

the synergist to juvenile hormone ratio was 2:1.

Fales et al. (1970) tested juvenile hormone analogues for character-

istics of synergism in combination with pyrethrins, .They found a marked

synergism but none of the analogues tested were as active as the pyrethrin-

piperonyl butoxide standard. McGovern et al. (1971) found that 5 of 19









acetals were as active as standard farnesyl methyl ether when tested

topically for juvenile hormone activity on Tenebrio molitor. Sesamex, a

synergist, showed a high activity. In bioassays, it was found that va-

pors of the active chemicals also were sufficient to induce juvenilization.


Crossresistance and Selectivity of ICR

Cerf and Georghiou (1972) found evidence of crossresistance to ju-

venile hormone analogue in some insecticide resistant strains of house

flies.

Selectivity of structure of juvenile hormone has been reported. The

juvenile hormone from cecropia seems to be effective against all insects

but the "paper factor" (Slama and Williams, 1965) has juvenile hormone

activity in certain bugs of the family Pyrrhoccoridae and does not affect

other insects. This fact has led to search for other substances that may

be selective against specific pests.
















METHODS AND MATERIALS


Establishment of the Florida Strain of Horn Fly

Much difficulty was encountered in development of a laboratory strain

of horn fly from Florida. jWhen wild flies were captured in the field,

returned to the laboratory and fed on the standard blood diet, the flies

would lay only one batch of eggs. No subsequent ovarian cycles were pro-

duced. Eggs from these wild flies produced larvae that successfully

pupated and emerged. The F1 adults however, did not mate or lay eggs.

Dissection of the females demonstrated the lack of-ovary development even

though males did produce sperm. Similar observations were recorded by

McLintock and Depner (1957). Egg laying was obtained by Harri. (1962)

by adding beef juice to the blood.

In our attempts to establish a Florida strain, three fly collections

were made at the Range Cattle Beef Station, Ona, Florida. During each

of these collections, 8-10,000 wild flies were returned to the laboratory

and all eggs obtained were reared to F1 flies. From the approximately

75,000 F1 flies, 15 viable eggs were obtained. After six months of care-

ful rearing, these horn flies were increased to a 1,000 fly colony, but

the colony was unstable with periodic reductions of tctal numbers of

adults. This colony stabilized after about one year of colonization and

the Florida strain could be used for experimentation.


Fj- n*l fi__ .l ( '' '_ .i'- J : .'

The adults were reared ini a walk-in chamber with constant I fght from

six 40w coou white fluorescent lights located on the ceiling. The 232 X

23









203 X 232 cm chamber contained three metal stands of five shelves, each

positioned along thestyrofoam-insulated walls.

Larvae were reared in a PercivalTM incubator, model UDC 2 (Percival

Refrigeration and Manufacturing-Company, Des Moines, Iowa). Two 40w cool

white fluorescent lights maintained constant light. Temperature was main-

tained at 26.7 3.33"C. Humidity averaged 73 percent and ranged from 54-

92 percent RH. Larvae normally began pupating on the third day and all

pupated by the sixth day.


Care of the Colony

Adults

Laboratory reared adult horn flies were held in cages made from

standard aluminum window screen framing (Mclveen-, 1972, Fig.-2). The

dimensions were 51 X 26 X 27 cm. The bottom of the cage was covered

with 18 mesh aluminum screen and the top, sides, and back were covered

with 32 mesh nylon screen. The front cage opening was covered with 15

cm orthopedic tubular stockinette. The screen and stockinette were held

in place by an aluminum screen gasket.

Adults were fed on a diet of preserved bovine blood obtained from a

-slaughterhouse (Swift and Company, Ocala, Florida). These animals were

injected with papain before slaughter to produce protein beef. The blood-

ras treated with an anticoagulant (sodium citrate, 7g/1800ml), and the

antibiotics kanamycin sulfare (Ig/1800ml), and mycostatin (500,000 units/

1800a2) as preservatives. Blood was stored in a refrigerator and utilized

within four weeks.

I1i'a" in tli oviposition cages were fed twice daily with 6 X 8 X 1

cm cel''u tton pads covered with gauze. Pads were saturated with blood.

These pads were placed one each on the top of the cages and covered with

polvethylene nlastii to prveint detiz-atia..









For feeding flies during weekends, a 9 X 16 cm double thickness of

cellucotton was covered with a light weight Diamond "B" brand gutta

percha membrane tissue (Bemis Associates. Inc.). The edges were sealed

with a hot.air dryer. These pads were filled through the top with 200

ml of preserved blood. A pad'was placed on each cage providing suffi-

cient blood for 48 hours. Reduced survival of flies fed on membrane

pads was noted.

Eggs

The adult flies began producing some eggs four days from emergence

with maximum egg production on the sixth day. Eggs were obtained from

caged horn flies by placing the cages over 51 X 38 cm oviposition trays

containing a moistened layer of cellucotton. The cellucotton was covered

with a layer of paper toweling as a substrate for egg deposition. Eggs

were washed from the tray twice daily into a white enamel pan. The con-

tents of the pan were rinsed with a.wash bottle through a 16 cm diameter

filter in a funnel supported by a ring stand. Eggs were kept moist

until measured for seeding on medium for colony maintenance or used for

experiments. The eggs collected in the morning were used for colony

maintenance, whereas eggs collected in the evening-were used for experi-

mencation. No difference in egg hatch was noted for different egg col-

lections.

Larvae

The basic larval rearing procedure developed by the USDA Entomology

Research Division, Kerrville, Texas (Schmidt et al., 1967) was used with

several modifications.

Larval rearing diet contained a mixture of 246 g sugar cane bagasse

pellets, 48 g wheat flour, 36 g fish meal, 6 g sodium bicarbonate, 20 g

alfalfa meal, 545 g bovine nanure, and 1300 nl tap rater. Fresh manure









was obtained from beef cattle on pasture. It was weighed in 545 g

batches, placed in plastic bags, sealed, and stored in a freezer for pe-

riods up to 2 months. Freezing destroyed any insect contamination pre-

sent at the time of collection. The manure was removed at 1400 hours

and used the next day at 0800. A dry mix was prepared from wheat four,

fish meal, alfalfa meal, and sodium bicarbonate. In our method, 1300 ml

of hot tap water was added to 492 g bagasse pellets and the pellets were

given about 10 minutes to saturate. The pellets were then hand-mixed

with 1090 g manure and 220 g dry mix. The medium was placed in a 40 cm

diameter X 10.5 cm deep polyethylene pan. One ml of eggs was added to

3102 g prepared medium.

Pupae

The larval medium was removed from the larval chamber on the sixth

day, saturated with tap water, and allowed to stand one hour. The pan

was inverted on a tray and allowed to drain overnight. The pupae were

separated from the medium on the seventh day by water floatation. The

pupae were skimmed from the water with an 18 mesh aluminum screen and

placed on a layer of cellucotton which was covered with a paper towel.

The pupae were air dryed, placed in an emergence cup in the oviposition

cage which was then placed in the adult rearing chamber.


Laboratory Screening Tests

Larval Test Chamber

Tha test chamber for larvae was 239 X 58 X 71 cm and maintained at

a temperature of 26.7 3.33C. The temperature was controlled by a ther-

ncstat connected to a small electric heater and a squirrel cage blower

(McLean Engineering, Princeton, New Jersey, Model 2E 408). Humidity was

maintained by evaporation of water from a 5] X 38 cm saturated cellucotton

pad.









Adult Test Chamber

The-test chamber for adults was 148 X 141 X 225 cm and was main-

tained at-a temperature of 32.2 1.11C and a RH of 60%. Temperature :

was controlled by a thermoscar connected to a model 262-AH oxygen tent

(Melico Oxygen Tent, Melchior, Armstrong, Dessau Co. of Del., Ridgefield,

N. J.),.and a small electric space heater. Humidity was maintained as

in the larval chamber.


IGR Active Compounds

IGR active compounds used in tests on the horn fly were piperonyl

butoxide, a{2-[2-butoxyethoxy]=ethoxy}-4,5-methylenedioxy-2propyltoluene.

(Niagara Chemical Division, FMC Corporation); MGK--264, N--[2-ethylehexyl]-

5-nor=bornene-2,3-dicarboximide, (McLaughlin, Gormley, King Company);

R-20458, 1-[4'-ethylphenoxy]-6,7-epoxy-3,7-dimethyl-2-octene, (Stauffer

Chemical Company); CRD-9499, ENT 70284, (Niagara Chemical Division, FMC

Corporation); and methoprene, isopropyl ll-methoxy-3,7,11-trimethyldodeca-

2,4-dienoate, (Zoecon Corporation). A 0.1, 1.0, and 10.0 percent stock

solution of each compound was prepared on the basis of weight of techni-

cal per volume of acetone diluent. Stock solutions were stored in 100

ml volumetric flasks. To prevent evaporation, each glass stopper was

coated with a thin film of nontoxic silicone stopcock grease (Dow

Corning Corporation). Stock solutions and technicals were stored in a

refrigerator.

Egg Tests

Eggs were treated topically with IGR dissolved in acetone. Treat-

nents included acetone treated-checks, and the IGR treatments -in concen-

trations of 0.1, 1.0, and 10.0 percent. Twenty-five eggs, three hours

old or less, were counted and placed on a seven r diameter filter paper









disc. Five ip of the IGR active compound was pipetted onto the group.of

eggs. The treatment would avarage-0.2 pg/egg for a 0.1%- solution, 2 pg/

egg for a 1.0% solution, and 20 ug/egg for a 10% solution. The discs

were placed on a moist collucotton pad and placed in the larval test cham-

ber (26.7 3.33C). Hatchability was determined at a 24 hour post-

exposure since hatching at this temperature occurs at about 18 hours.

Larval Tests

Standard larval medium was treated with IGR active compounds diluted

with acetone and pipetted to produce dosages-of 10, 100, or 1000 ppm.

These dosages were adjusted according to the activity of the compound.

One hundred grams of the medium was placed in nine oz plastic cold drink

cups and 100 eggs were added to each cup. The pupae were collected, air

dryed, counted, observed for abnormalities and placed in a small 2.1 cm3

plastic cup in 10.2 X 9 cm diameter cylindrical cages made from petri

dishes and 18 mesh aluminum screen.(Mcllveen, 1972, Fig. 6). The 18 mesh

screen was stapled together to form a 9 cm diameter by 10.2 cm cylinder.

The 9 cm plastic petri dishes had a 8.5 cm circle removed and a 9 cm

diameter circle of 18 mesh aluminum screen was welded in the plastic with

a small soldering iron and these formed the ends of the cages. The pupal

cup was placed in these test cages and the cages were placed in the adult

test chamber. The pupae were observed for adult emergence and adults

were fed with standard bovine blood on 1 cm2 single thickness cellucotton

pads covered with gauze. Observations were made for oviposition by

placing the cages on a water soaked cellucotton pad covered by paper

towels. Eggs were counted and placed on filter paper over wet soaked

cellucotton for hatchabilit.y studies. After hatchability studies were

completed, the flies were killed, sexed, and counted to determine adult

emergence and sex ratios. Pupae that did not emerge were dissecrei.









Pupal Tests

Newly formed pupae-were removed from the larval medium, washed with

tap water, air dryed and treated with 1 pl of a 0.1%, 1.0%, or 10% solu-

tion of IGR. One pi of a 0.1% solution applied 1 pg/pupae, a 1.0% solu-

tion applied 10 pg/pupae and a 10% solution applied 100 pg/pupae. The

pupae were placed in a 16 X 125 mm KimaxT culture tube with a cellu-

cotton plug at the bottom which had been saturated with 0.1 ml water to

provide a source of humidity. The tube was capped, placed in a test

tube rack, and maintained in the larval test chamber. The caps were re--

moved from the test tubes on the sixth day. On the seventh day, the

pupae were removed from the-test tubes and placed in 2.1 cm3 plastic

pupal cups in the adult test cages. The test cages were put in the adult

test chamber and observed for adult emergence. Adults were fed, allowed

to mature, and any resulting eggs were counted and tested for hatcha-

bility.

Adult Topical Tests

Newly emerged flies were sexed and placed in test cages as in the

pupal tests. Five female and five male horn flies were used in each

test. The flies were treated topically with 1 p~ of a 0.1%, 1.0%, or a

10% acetone solution of the IGR compound while they were restrained by

the use of a vacuum holding unit. The cage was placed over a 2.54 cm X

1.59 cm orifice cut in a plastic container that was connected to an in-

take of a vacuum cleaner. This restrained the flies on the aluminum

screen at the bottom of the cage. The top of the cage and the aluminum

screen cylinder was removed after th e flics became restrained at the bot-

tom of the cage (Mcilveen, 1972, Fig. 9). The flies were positioned dor-

sal side up using a paint brush and then treated topically with 1 ul of









the test compound with the microapplicator syringe. The cylinder and

the top of the cage was replaced after treatment and the vacuum cleaner

was turned off. The flies were placed in the adult test chamber and fed

immediately.


Cattle Feeding Trials

Preliminary Cattle Feeding Trials

The IGR compound with the best potential for control in field

studies was determined by the laboratory testing and used in a prelimi-

nary feeding trial. Methoprene (Altosid ZR-515, Zoecon Corporation)

was fed to cattle as a 1% granular formulation. Two Angus heifers

weighing an average of 373 kg were used in the test. Dosages tested

were 480 (257 ppm), 48 (25.7 ppm), 24 (12.85 ppm), and 2.4 (1.285 ppm)

mg/kg/day per head. Half of the dosage was administered in the morning

and the other half was administered in the evening mixed with 700 g of

a standard cattle ration (PurinaTM Cattle Chow'M Complete, Ralston

Purina Company, St. Louis, Mo.). The animals had free access to water

and pasture. Manure samples were collected by rectal samples before

treatment and post-treatment daily for seven days. These samples ware

returned to the laboratory for bioassay. Two hundred g of the manure

were placed in 12 oz drink cups with 20 g of water saturated bagasse pel-

lets (1:1 by volume) on the bottom of the cup and 20 g placed on the top

to cover the manure. One hundred eggs were added to each cup and the

cups were placed in the larval test chamber. Pupae were separated on

day seven and placed in adult test cages for emergence. Adults were fed

bovine blood and subsequent eggs were tested for hatchability.









Cattle Feeding Trials Under Semi-Field Conditions

Five Angus heifers were used in this study. Two animals were used

for untreated checks. The other three animals received 24 (12.85 ppm),

12 (6.425 ppm), 6 (1.071 ppm), or 3 (.5355 ppm) pg/kg/day of methoprene

fed individually once a day in 700 g of Purina cattle ration. Freshly

dropped manure from each group was allowed to be exposed to natural fly

breeding activity from animals averaging 430 flies per side. After 24

hours, the manure was removed by digging the grass and soil to a depth

of 2 inches and moving the unit to an isolated area to prevent distur-

bance by the cattle and immediately covered by a trap for emergence.

The emergence trap was constructed of a cylinder formed from galvanized

metal 31 cm in diameter and 14 cm in height. A cone was attached to the

top of this galvanized ring and was made from 32 mesh nylon screen. The

cone height was 30 cm with a 31 cm bottom diameter and a 5 cm top dia-

meter. A 12 oz plastic cup with the bottom removed was secured at the

top of the cone. The emergence trap cups were softened with heat to

form an inverted cone in the cup bottom. The center was opened to a

diameter of 4 mm. A top was secured to each cup. Emergence traps were

placed over the transferred manure to study field emergence patterns.

Two replicate samples per treatment were set up each week for a 4 week

period. The samples were allowed only natural fly breeding activity.

These samples were also replicated in the laboratory and were frozen

overnight to destroy any insects present. These laboratory samples were

tested in the same manner as the laboratory samples in the preliminary

cattle feeding trials. Adult horn flies that emerged were counted and

sexed. Where no emergence was found, the manure was separated to collect

the pupae present. Pupae were dissected to determine the stage of









development. Other insects were also counted and identified if possible.

Emergence trap cups were changed daily during the emergence period.

Cattle Feeding Trials Under Field Conditions

A herd of twenty-five cattle were used to test the effectiveness of

methoprene under field conditions. The feed containing methoprene at the

rate of 24 pg/kg/day was administered at a rate of 700 g per head per

day. Since all of the animals used the same feeding trough, the cattle

could eat more or less than the average of 700 g. The more aggressive

animals may have consumed more than the docile animals. Two manure

patties were removed from the field twice a week from both treated and

untreated cattle. Laboratory samples were also-obtained weekly-from.

both herds. The manure samples from the field were allowed 24 hours of

egg laying activity from the insects before they were removed to a test

area and covered by emergence traps as described in the previous test.

Adult horn flies emerging were counted and sexed. All other insects

were counted and identified if possible.

Statistical Analysis

Analysis conducted on the data were analysis of variance, Ddncan's

multiple range test, and Chi-square. Both analysis of variance and

Duncan's multiple range test were computer programmed using statistical

analysis system (Service, 1972). The Chi-square analysis was determined

using a Casio AL-2000 programable calculator. All significant differ-

ences in the data reported in this paper are at the 5% confidence level

unless otherwise reported.
















RESULTS AND DISCUSSION


Laboratory Studies of Larval Medium Treated
with Insect Growth Regulators

Prior to the use of any IGR compounds for feed additives to cattle,

studies of their effectiveness on larval development were studied in

laboratory tests. Horn fly eggs were placed on treated larval medium.

Evaluation of the effectiveness of insect growth regulators was based on

the number of pupae produced from eggs placed on treated larval medium,

the number of adults emerging from pupae formed when eggs were placed on

treated larval medium and fertility of adults reared in a larval medium.

The compounds MGK-264, R-20458, piperonyl butoxide, CRD-9499, and metho-

prene were used to treat the larval medium. These compounds are dis-

cussed in the following text in order from the least to the most active.

MGK-264

As seen from the data presented in Table 1, MGK-264 was effective

in reducing the survival of horn fly larvae to the pupal stage at the

dosage of 1000 ppm mixed with the larval medium. Only one larva was

able to form a puparium and the adult was unable to emerge from the pupa.

The reduction of survival of horn fly larvae to the pupal stage, emer-

gence of the adult stage and hatchability of the eggs produced were sig-

nificantly different only at a rate of 1000 ppm. The significant differ-

ences seen in the 1000 ppm treatment are due to the 100% reduction of

adult emergence resulting in a 100% reduction in egg production. At'this

dosage, MGK-264 acr.d a; a toxicant of the larval stage. An IGR active

















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compound would not be expected to prevent formation of pupae. The pupa-

tion of horn flies reared in untreated medium averaged 66.5% per replica-

tion. Pupation averaged 66.0% per replication in larval medium treated

at the rate of 10 ppm and an average of 71.0% per replication success-

fully pupated when the treatment was 100 ppm. There was no significant

reduction in number of- adults produced or the percent hatch of eggs from

these adults when comparing treatments of 10 ppm and 100 ppm with the

untreated medium. The adults emerging from the untreated medium, medium

treated at the rate of 10 ppm or medium treated at the rate of 100 ppm

were respectively 56.25%, 54.0%, and 64.75%. The percent emergence from

the pupae' for horn flies reared in the untreated medium averaged 84.15%

while medium treated with 10 ppm or 100 ppm MGK-264 averaged respectively

81.92% and 91.32%. These three values were not significantly different.

The rate of 1000 ppm had no emergence. The percent hatch of eggs laid

by horn fly adults reared in the larval medium was 76.75%, 79.50%, and

85.75% respectively for untreated, 10 ppm, and 100 ppm. There was no

significant reduction in the percent egg hatch from larvae reared in the

treated medium except-where there were no adults produced as in 1000 ppm.

Chi-square analysis showed no significant difference in the sex ratios

obtained on all treatments. The data on sex ratios of adult flies pro-

duced on this test are shown in Table 2. The ratio for untreated horn

flies averaged 1.05 males to 1 female. At a dosage of 10'ppm of MGK-264

the male to female ratio averaged 0.96 ro 1. At 100 ppm the ratio

averaged 1.06 to 1.

R-20458

The next most active compound in the larval medium of the horn fly

was R-20458. In Table 3; it is noted that .he effect of R-20458 in

















Table 2. Sex Ratios of Horn Flies Reared From
MGK-264-Treated Medium (100 eggs/rep).


Treatment Replication


Adult
Male Female Total


Untreated



Total



10 PPM


lotal


100 PPM


Total


150 PPM



Total


Ratio
Male/Female


1.16:1
.75:1
.97:1
1.27:1





1.23:1
.79:1
1.14-1
.79:1





1.50:1
1.03:1
..88:1
.91:1


- -~----


















. V0V 0 0 C,
.q o0


l o m r-







IM


p ) Vncono o c O o
m a-; cn -^ir cn m a% c
0 o-4c-1cO r
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0 LCr
r- o0 C m co mn 3
en r-q t n -I t c i C"i r
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0
















0 &
o






















Lm
(0 0
0to
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4 4
CO










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Ch
H
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E C
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prevention of pupation is negligible. In fact, the untreated medium and

medium treated at rates of 10 ppm, 100 ppm, and 150 ppm were not signifi-

cantly different from each other and respectively averaged 54.5%, 54.8%,

49.3% and 43.5% successfully pupating. This result would be expected in

a compound-showing IGR activity. If a significant toxicity is seen when

larvae pupate as was observed with MGK-264, the compound would have an

action similar to a toxic insecticide on larva and not the delayed mor-

phogenic response expected. Emergence from pupae was reduced by treat-

ment of the-larval medium with R-20458. Treatment rates of 100 ppm and

150 ppm in the larval medium significantly reduced emergence. However,

the rate of 10 ppm did not. -The untreated horn flies averaged 38.0

adults per replication while treatments of 10 ppm, 100 ppm, and 150 ppm

respectively averaged 32.0, 17.5, and 8.0 adults per replication. The

percent emergence of horn flies from pupae in untreated medium averaged

70.2% while treatments of 10 ppm, 100 ppm, and 150 ppm respectively

averaged 57.8%, 34.6%, and 18.4%. The untreated larval medium and medium

treated at the rate of 10 ppm were not significantly different in adult

emergence, but medium treated at the rate of 100 ppm and 150 ppm signifi-

cantly reduced adult emergence when compared to the percent pupae emerging

of horn flies reared in the untreated and 10 ppm medium. The data on the

percent egg hatch showed a reduction in egg hatch. However, analysis of

variance showed that there was no significant reduction in the percent

egg hatch caused by treatment of larval medium with R-20458. This is

because of-the tremendous variability in the egg hatch of the different

replications. The percent hatch of eggs obtained from adult horn flies

reared in untreated medium averaged 70.5%. The horn flies reared in lar-

val medium treated with R-20458 at 10 ppm, 100 ppm, and 150 ppm were









respectively 67.8%, 57.S%, and 33.5%. The sex ratio of adults in this

test was also studied for differences. Using a Chi-sqiare test, it was

found that there was no significant difference in the expected sex ratio

of 1 to 1. Sex ratios are shown in Table 4. The sex ratio of the un- *

treated horn flies averaged 1.41 male to 1 female. Horn flies reared

at the dosage of 10 ppm had a sex ratio average of 0.94 to 1. The dos-

age of 100 ppm produced horn flies with an average sex ratio of 1.12 to

1. The dosage of 150 ppm reared horn flies with an average sex ratio of

1.13 to 1.

Piperonyl butoxide

The results with piperonyl butoxide are shown in Table 5. In this

experiment, there was no significant reduction in the number of horn fly

pupae reared in the treated medium when compared to the untreated medium.

The average number of horn fly pupae produced by eggs in the untreated

medium was 48. Treated medium containing 10 ppm, 100 ppm, 150 ppm

piperonyl butoxide averaged respectively 55.5, 54.0, and 54.3 pupae per

replication. There were no significant differences in the number of

pupae produced when comparisons of all treatments were made. Piperonyl

butoxide added to larval medium significantly reduced adult emergence

when used at 150 ppm. The number of adults emerging from pupae reared

in the untreated medium averaged 43. The number of adults reared in me-

dium treated at rates of 10 ppm, 100 ppm, 150 ppm averaged respectively

51.3, 35.8, and 1.8 adults per replication. Medium containing piperonyl

butoxide at 150 ppm significantly reduced adult production when compared

with the medium containing 10 ppm or 100 ppm piperonyl butoxide. Medium

treated with piperonyl butoxide at 100 ppm significantly reduced the num-

ber of adult horn flies produced when compared to medium treated with
















Table 4. Sex Ratios of Horn Flies Reared From
R-20458-Treated Medium (100 eggs/rep).


Treatment Reolication


Adult
Male Female Total


Untreated



Total



10 PPM



Total



100 PPM



Total



150 PPM


Total


Ratio
Male/Female


.83:1
1.67:1
1.86:1
.74:1




.68:1
1.00:1
1.13:1
1.04:1





.44:1
1.67:1
1.08:1
1.67:1




1.33:1
6.00:1
.67:1
.60:1


I _~__




















rI-













cn ci 00 )0
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t) h cc mC








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rcn
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ae -
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10 ppm piperonyl butoxide. A medium treated with 150 ppm piperonyl

butoxide was the only treatment tested that significantly reduced adult

production when compared with untreated medium. The percent emergence

of adult horn flies from pupae reared in untreated medium averaged 88.4%

while medium treated with 10 ppm, 100 ppm, or 150 ppm respectively

averaged 92.8%, 66.5%, and 3.3%. Medium treated with 100 ppm or 150 ppm

significantly reduced emergence when compared to untreated medium and

medium treated with 10 ppm. A treatment of 150 ppm significantly re-

duced adult emergence when compared to 100 ppm. The 10 ppm dosages did

not produce a significant reduction when compared to untreated medium.

Larval medium treated with 100 ppm and 150 ppm of piperonyl butoxide

caused a significant reduction in egg hatch when compared with the 10

ppm group. There were no significant reductions of egg hatch between

the untreated and the 10 ppm group. The average percent hatch for the

untreated, 10 ppm, and 100 ppm were respectively 90%, 90%, and 73.3%.

No eggs were produced by flies reared from larval medium treated with 150

ppm of piperonyl butoxide. The sex ratios of flies in this test are

shown in Table 6. Using a Chi-square analysis it was found that there

was no significant deviation from the expected sex ratio of 1 to 1. The

sex ratio of adults reared in untreated medium averaged 0.85 males to 1

female. The sex ratio of adults reared in medium treated with 10 ppm

piperonyl butoxide averaged 1.16 to 1. The sex ratio of adults reared

in medium treated with 100 ppm averaged 1.42 to 1. The sex ratio of the

medium treated with 150 ppm averaged 2 to 1. In replication 1 of medium

treated with 150 ppn, only 1 female emerged and in replication 2, there

were only 2 males that emerged. In replication 4, there were no adult

flies. Replication 3 is the only replication that had both sexes pre-

sent, but no eggs were produced.
















Table 6. Sex Ratios of Horn Flies Reared From
Piperonyl Butoxide-Treated Medium
(100 eggs/rep).


Adult
Treatment Replication Male Female Total


Untreated



Total



10 PPM



Total



100 PPM



Total



150 PPM



Total

aAdults


15
15
25
24
79



21
31
30
28
110



19
20
25
20
84



0
2a
2a
0
4


were unable to expand wings.


Ratio
Mala/Female


1.07:1
.94:1
.81:1
.75:1




1.05:1
1.11:1
1.20:1
1.27:1




1.73:1
1.43:1
1.32:1
1.34:1




0.00:1

1.00:1









CRD-9499

The compound CRD-9499 also affected horn flies when applied to the

larval medium. Significant reduction of puparion was observed in larvae

reared in medium (Table 7) treated with 100 ppm or 150 ppm CRD-9499 when

compared to larvae reared in untreated medium. The larvae in untreated

medium yielded an average of 31.8 pupae per replication. Medium treated

with 10 ppm, 100 ppm, or 150 ppm CRD-9499 averaged respectively 24.8,

19.8, and 18.3 pupae per replication. Reduction in adult emergence was

also produced with treated medium containing CRD-9499. All treatments

significantly reduced the number of adults produced from eggs placed in

treated larval medium when compared to the untreated medium. The un-

treated medium averaged 19.5 adults per replication while medium treated

with_10 ppm, 100 ppm, 150 ppm CRD-9499 were respectively 10.8, 10.8 and

4.3 flies per replication. The percent emergence of horn fly pupae

reared in untreated larval medium averaged 60.7% while medium treated

10 ppm, 100 ppm or 150 ppm CRD-9499 respectively averaged 42.2%, 55.9%,

and 23.3%. Treatment with 150 ppm CRD-9499 significantly reduced emer-

gence from pupae. The percent egg hatch from flies reared on CRD-9499

treated larval medium was affected only at 150 ppm. This treatment had

a significantly reduced hatch when compared to eggs from flies reared in

untreated medium and medium containing the other treatments. Those

adult flies that survived when reared in larval medium treated with 150

ppm CRD-9499 did not lay any eggs. The average percent hatch for the

four replications of the untreated flies averaged 74% while flies reared

in larval medium containing 10- ppm and-100 ppm CRD-9499 averaged respec-

tively 45% and 63%. Sex ratio data are shown in Table 8. Chi-square

analysis showed no difference in the expected sex ratio of 1 to 1. The
















.0
co
U 0 O4Orl2 0-O1M 0l I II I l I
4)o r-4










0 M:


u H

0.









01
6IID






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c1)







noi
k0 0

0 0













as, cls p U ) o o

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00 t < C ) 10 -00
















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41) 0 0:4 0:4 0:4riiM c ^ ^c r s n <










0J T




-4 s 4) < -
















Table 8. Sex Ratios of Horn Flies Reared From
CRD-9499-Treated Medium (100 eggs/rep).


Treatment Replication


Untreated



Total



10 PPM



Total



100 PPM



Total



150 PPM


Adult
Male Female Total


4
14
9
15
42



1
6
4
7
18



8
6
6
2
22



3(1)a
1
0
3(3)
7


Total


aNumber of flies that were unable to expand wings are in parenthesis.


Ratio
Male/Female


1.00:1
.57:1
1.11:1
.93:1




3.00:1
1.33:1
2.00:1
.86:1




.50:1
.50:1
1.16:1
3.00:1




.66:1
1.00:1

.33:1









sex ratio of males to females in the untreated group averaged 0.86 to 1.

Treatment of 10 ppm to the larval medium produced a male to female ratio

average of 1.39 to 1. The sex ratio with a treatment of 100 ppm averaged

0.91 to 1. The treatment of 150 ppm produced a sex ratio average of 1.43

to 1. Those flies treated with 150 ppm had abnormalities in wing forma-

tion. One female in replication 1 and 3 females in replication 4 were

found abnormal. Replication 3 produced no females.

Methoprene

The compound methoprene was by far the most effective as a larval

medium treatment. As is to be expected with a good IGR, the pupation of

the.flies was not affected (Table 9).. The average number of pupae per

replication produced from horn fly eggs added to untreated larval medium,

or larval medium treated with .005 ppm, .01 ppm or .05 ppm were respec-

tively 62.5%, 64.0%, 64.25%. There were significant reductions in adult

emergence from treatment of the larval medium with methoprene at .01 ppm

or .05 ppm with an average respectively of 48 and 2 adults produced per

replication. A treatment of .05 ppm significantly reduced adult emer-

gence when compared to a treatment of .01 ppm. There was no significant

reduction in the average number of adults produced with a treatment of

larval medium at .005 ppm with 53.50 adult flies produced per replication

when compared to the untreated horn files with 60.75 adults per replica-

tion. Also, medium treated with .05 ppm methoprene significantly reduced

adult emergence more than a medium treated with .005 ppm. The percent

emergence of horn flies reared in untreated larval medium averaged 97.12%,

while medium treated with-.005 ppm, .03 ppm or .05 ppm methoprene respec-

tively averaged 83.92%, 74.50%, and 4.11%. There was complete reduction

of production of eggs from insects created in the larval stage with .05

ppm. The average percent egg hatch for the flies reared in untreated,




















a r- \D r C







SC























C4m o m m r
r oo r-. oo I' D




















C)
mcu N

SC-. 0












0

On-m o


0

















O
















1 n -- -* m






0 (3 C 1 C!







r- r- Ln 0n P-
V.


000
cn 0 'o V) a'
-I I r













C- CD mi

0L N -1-














0







4














rlNmW *


v 0 0 0 .-r

C0N 0,-4







S0 C) oI II






r-C
aOC







N








r o- N- C- I
\'













H1N CM -4
















Table 10. Sex Ratios of Horn Flies Reared From
Methoprene-Treated Medium (100 eggs/rep).


Adults
Treatment Replication Male Female- Total


Untreated



Total



.005 PPM


1
2
3
4




1
2
3
4


Total


.01 PPM



Total



.05 PPM


27
22
38
27
114



36
30
25-
29
120



21(2)a
35(2)
34(3)
28(2)
118



3(3)a
0
3(3)
0
6


Total


25
29
35
40
129



25
28
21
20
94



12(3)
16(2)
20(6)
26(6)
74



0
3(3)
2(2)
0
5


aNumber of flies that were unable to expand wings are in parenthesis.


Ratio
Male/Female


1.08:1
.76:1
1.09:1
.68:1




1.44:1
1.07:1
1.19:1
1.45:1




1.75:1
2.19:1
1.70:1
1.08:1






1.50 :1









.005 ppm or .01 ppm medium were respectively 72.0, 58.5, and 59.0. These

differences were not significant. The sex ratio of male to female for

the flies reared in untreated medium or medium treated with -.005 ppm,

.01 -ppm or .05 ppm were respectively 0.88:1, 1.28:1, 1.59:1 and 1.20:1.

The sex ratios were evaluated with a Chi-square test and it was found

that the ratio for horn flies reared in medium with .01 ppm was signifi-

cantly different from the expected 1:1. All adults produced on this

treatment were unable to expand their wings. In replication 4 there were

no adults produced, but in replication 1 all those that wore produced

were males (Table 10).


Laboratory Studies with Eggs Treated Topically with IGR

MGK-264

No significant reduction in egg hatch was noted when 25 horn fly

eggs were group treated with an average of 0.2 pl acetone per egg or

treated with 0.2 p1 of acetone solutions containing .1%, 1%, or 10% MGK-

264 (Table 11). The average percent hatch was 68% with eggs treated

with acetone, 69% with eggs treated with 0.1% MCK-264, 52% with-eggs

treated with 1% MGK-264, and 35% with 10% MGK-264. From these averages,

it seems as if the dosage of 10% MGK-264 on eggs does produce a redu:-

tion. However, analysis of variance showed that the variability within

replications (Table 11) was so great that significance was not proven.

Notice the variability in the percent hatch at the 10% dosage. There is

a low of 8% hatch and a high value of 80%. Here the value of 8% in re-

plication 3 and 12% in replication 4 is inconsistent with the 40% in re-

plication 1, end the 80% In replication 2. Such differences can only be

explained by the very sensitive timing of application of IGR to eggs.









Table 11. Percent Hatch of 25 Horn Fly Eggs per Replication When
Group Treated with 5 4l of Acetone or MGK-264 in Acetone.

Replication
Treatment 1 2 3 4 Means

Acetone 56 64 80 72 68

0.1% 64 80 52 80 69

1.0% 48 76 24 60 52

10.0% 40 80 8 12 35


Piperonyl Butoxide

No significant reduction in egg hatch was noted in eggs treated with

an average of 0.2 pi acetone per egg or 0.2 41 per egg with .1%, 1%, or

10% piperonyl butoxide (Table 12). The average percent hatch for the

acetone treated, and piperonyl butoxide treatments of .1%, 1.0% and 10%

were respectively 50%, 56%, 57%, and 38%. Similar to the data of MGK-264,

analysis of variance showed that the variation between replications caused

the data not to be significant even with the average of 38% hatch seen

when eggs were treated with 10% piperonyl butoxide..


Table 12. Percent Hatch of 25 Horn Fly Eggs per Replication When
Group Treated with 5 pl of Acetone or Piperonyl Butoxide
in Acetone.

Replication
Treatment 1 2 3 4 Means

Acetone 44 56 52 48 50

0.1% 64 56 44 60 56

1.0% 60 56 56 56 57

10.0% 32 60 40 20 38









CRD-9499

No significant reduction in egg hatch was noted with an average

treatment of 0.2 4l of acetone per egg or treatments with 0.2 pi per

egg with .01%, 0.1%, and 1.0% CRD-9499 (Table 13). The average percent

hatch for eggs treated with acetone or eggs treated with .01%, .1%, or

1.0% solutions of CRD-9499 were respectively 68%, 58%, 65%, and 50%.

There was no significant variation between replications in this test.


Table



Treatme

Acetone

0.01%

0.1%

1.0%


13. Percent Hatch of 25 Horn Fly Eggs per Replication When
Group Treated with 5 pl of Acetone or CRD-9499 in Acetone.

Replication_
nt 1 2 3 4 Means

60 52 88 72 68

72 28 60 72 58

52 72 56 80 65

44 60 56 40 50


Methoprene

No significant reduction in egg hatch was noted with a treatment of

horn fly eggs with a average of 0.2 pl acetone per egg or treated with

0.2 pv per egg of .01%, 0.1%, or 1.0% methoprene. The average percent

hatch for the acetone treated and methoprene treatments of .01%, .1%,

and 1.0% were respectively 66%, 70%, 68%, and 60% (Table 14).


Table 14. Percent Hatch of 25 Horn Fly Eggs per Replication When
Group Treated with 5 pl of Acetone or Methoprene in Acetone.

Replication
Treatment 1 2 3 4 Means


Acetone

.01%

0.1%

1.0%


66

70

68

60


~









R-20458

The compound R-20458 was the only compound tested that significantly

reduced egg hatch (Table 15). Two-tenths microliters per egg of a 0.1%

solution produced an average of 40% hatch. This reduction proved to be

significant. At a dcsage of 0.2 pl of 1% R-20458, there was a 100% re-

duction of hatch. This was the lowest concentration of any compound

tested on eggs to produce a total blockage of hatch. Using a solution

of .01% R-20458, there was a 60% hatch compared to a 70% hatch in ace-

tone treated eggs.


Table 15. Percent Hatch of 25 Horn Fly Eggs per Replication When
Group Treated with 5 pv of Acetone or R-20458 in Acetone.

Replication
Treatment 1 2 3 4 Means

Acetone 68 76 64 72 70

.01% 52 72 48 68 60

0.1% 52 28 44 36 40

1.0% 0 0 0 0 0


Laboratory treatment of newly laid horn fly eggs with IGR showed

that eggs were not very sensitive to the treatment with most compounds

tested. The sensitivity of horn fly eggs to these chemicals may be unim-

portant for the use of these chemicals tested as possible control meas-

ures. The timing of exposure is apparently so critical that the treat-

ments were delayed enough to misp the critical stage where the IGR have

their effect on morphogensis.









Laboratory Studies of Pupae Treated Topically
with Insect Growth Regulators

MGK-264

When MGK-264 (Table 16) was applied topically to pupae of the horn

fly it was found not to affect emergence. The percent emergence for

pupae treated with 1 pl of acetone or 1 11 of 0.1%, 1.0%, or 10% solu-

tion of MGK-264 were respectively 65%, 82.5%, 82.5%, and 75%. The adults

that closed in the treatments were not sterilized since there was no

significant reduction in the percent egg hatch. The percent hatch of

eggs from adults treated in the pupal stage with acetone or treated with

0.1%, 1.0%, and 10% solutions were respectively 78%, 90.5%, 68.5%, and

64.5%. There was much variability in replications with the 10% solution.

In replication 1, 100% hatch was observed while in replication 2, 0%

hatch was observed. Also, the value of 63% in replication 2 and 100% in

replication 4 in the acetone treated group show high variation. The same

type of variation was observed in replication 2 of 10% solution with 90%

compared to 35% in replication 3.

Piperonyl Butoxide

When piperonyl butoxide (Table 17) was applied topically to pupae of

the horn fly, it did not significantly affect emergence. The percent

emergence for pupae treated with 1 pl acetone or 1 pi of 0.1%, 1.0%, or

10% solution of piperonyl butoxide were respectively 65%, 90%, 67.5%, and

60%. The adults that closed were not sterilized since there was no sig-

nificant reduction in the percent hatch. The percent hatch for acetone

treated and dosages of 0.1%, 1.0%, 10% piperonyl butoxide averaged respec-

tively 78%, 54.25%, 37.5%, and 28.25%. These averages were not signifi-

cant, because analysis of variance showed that the replication variability

was too high.
















Table 16. Production of Horn Fly Adults and Eggs From Pupae
Treated Topically .ith MGK-264 (10 pupae/rep).


Percent
Treatment Replication No. Adults Egg Hatch

Acetone 1 8 78
2 7 63
3 6 71
4 5 100
Means 6.5 78.0



0.1% 1 9 85
2 9 92
3 9 100
4 6 85
Means 8.25 90.50



1.0% 1 8 82
2 6 90
3 10 35
4 9 67
Means 8.25 68.50



10.0% 1 7 !00
2 5 0
3 8 69
4 10 89
7.50 64.50















Table 17. Production of Horn Fly Adults and Eggs From Pupae
Treated Topically with Piperonyl Butoxide (10 pupae/rep).


Percent
Treatment Replication No. Adults Egg Hatch

Acetone 1 8 78
2 7 63
3 6 71
4 5 100
Means 6.50 78.0



0.1% 1 9 Z
2 10 93
3 10 44
4 7 80
Means 9.0 54.25



1.0% 1 2
2 8 100
3 10 50
4 7 0
Means 6.75 37.50



10.0% 1 9 25
2 5
3 4(3)a -
4 6(1) 88
Means 6.0 28.25

Oaumber of flies unable to expand their wings are in parentheses.









R-20458

Results are more impressive with R-20458 (Table 18) when it was used

as a topical treatment to horn fly pupae. A solution of .01% or 0.1% R-

20458 at the rate of 1 1p per pupae completely blocked adult emergence.

At the lower concentration of .001% there was no significant effect on

adult emergence with the percent emergence of 80% compared to 87.5% in

the acetone treated group. The eggs laid by the adults emerging from

pupae treated at .001% were significantly reduced in hatchability. Only

in replication 4 was there any hatch which was 50%. In replication 1,

2 eggs were laid, but in replication 2 and 3, no eggs were laid. The

acetone treated group averaged 66% hatch and the hatch of horn fly eggs

from adults treated as pupae with 1 pl of a .001% solution of R-20458

averaged 12.5%.

CRD-9499

The results with CRD-9499 treatment to horn fly pupae (Table 19)

were also encouraging. The effective dosages were in the range of .01%

and 0.1%, the former allowing 5 flies to emerge. These two treatments

significantly reduced emergence from the treatment of .001% CRD-9499 and

the acetone treated control. The percent emergence for the acetone

treated and treatments of .001%, .01%, or 0.1% were respectively 87.5%,

77.5%, 12.5% and 0%. More important was that all treatments with CRD-

9499 significantly reduced hatchability of eggs produced by adults emer-

ging when the compound was applied to pupae. The percent hatch for the

acerone treated and the treatment of .001% were respectively 66%, and

17.75%. A treatment of .01% inhibited egg production 100%. Since no

adults emerged with a 0.1% solution, no eggs were produced.
















Table 18. Production of Horn Fly Adults and Eggs From Pupae
Treated Topically with R-20458 (10 pupae/rep).


Treatment


Acetone


Means



:001%



Means


Replication


No. Adults


8
10
8
9
8.75



7(4)a
9(5)
7(4)
9(3)
8.0


Percent
Egg Hatch


80
80
44
60
66.0



0


50
12.50


Means


Means U

aNumber of flies unable to expand their wings are in parentheses.
















Table 19. Production of Horn Fly Adults and Eggs From Pupae
Treated Topically with CRD-9499 (10 pupae/rep).


Percent
Treatment Replication No. Adults Egg Hatch

Acetone 1 8 80
2 10 80
3 8 44
4 9 60
Means 8.75 66.0



.001% 1 9
2 8
3 6
4 8 71
Means 7.75 17.75



.01% 1 1(1)a
2 1
3 3(3)
4 0
Means 1.25



0.1% 1 0 -
2 0
3 0 -
4 0
Means 0

aNumber of flies unable to expand their wings are in parentheses.










Methoprene

The most active compound was methoprene. A dosage of 1 ul of .0001%

methoprene was sufficient to prevent emergence of all but 6 flies, while

dosages of 1 ul of either a .001% or .01% inhibited adult emergence by

100%. These dosages significantly reduced emergence when compared to the

acetone treated control. Also, 100% reduction in egg hatch was produced

on all treatments tested (Table 20). The acetone treated group averaged

78.25% egg hatch.


Laboratory Studies of Adults Treated Topically with IGR

Topical application of IGR compounds were tested on adult horn

flies. Data was recorded on a 24 hour percent adult survival and a per-

cent egg hatch from adults that survived treatment.

CRD-9499

Topical application of CRD-9499 (Table 21) showed significant 24

hour adult mortality which averaged 57.5%. This mortality was seen when

CRD-9499 was used at the rate of 1 pI of a 1.0% solution in acetone. The

other concentrations of CRD-9499 were not significantly different from

the 1% group. Adults treated with 1 pj of a .01% and .1% had a 100%

survival which was not significantly different from the acetone treated

with a 92.5% survival. There was no significant reduction in egg hatch

in any of the test groups. The acetone treated adults and adults

treated with .01%, .1% or 1% CRD-9499 had percent egg hatches respec-

tively of 73.75%, 65.75%, 63.75%, and 36%. Even though the averages of

egg hatch seem different the replication variability was severe enough

to prevent significance in the data.
















Table 20. Production of Horn Fly Adults and Eggs From Pupae
Treated Topically with Methoprene (10 pupae/rep).


Percent
Treatment Replication No. Adults Egg Hatch

Acetone 1 6 60
2 7 89
3 7 91
4 8 73
Means 7.0 78.25



0.0001% 1 5(5)a 0
2 0
3 0
4 1(1) 0
Means 1.50 0



.001% 1 0
2 0
3 0
4 0
Means 0



0.01% 1 0
2 0
3 0
4 0
Means 0

aNumber of flies unable to expand their wings are in parentheses.
















Table 21. Survival and Egg Hatch of Horn Fly Adults Treated
Topically with CRD-9499 (10 adults/rep).


Treatment


ReDlication


Percent
Survival
24 Hours


100
90
100
80
92.50


100
100
100
100
100.00


100
100
100
100
100.00


50
70
30
80
57.50


Percent
Eeq Hatch


64
74
91
66
73.75


83
53
68
59
65.75


66
54
53
74
61.75


31
71
0
42
36.00


Acetone


Means


Means


Means


Means


Treatent epliatio









Piperonyl Butoxide

Topical application of piperonyl butoxide (Table 22) to adults

showed that treatment with 1 pl per adult of .1% or 1% solutions signi-

ficantly reduced adult survival within a 24 hour period. The 0.1% solu-

tion caused a 32.5% survival whereas 1 pl per adult of 1% piperonyl

butoxide solution caused 100% mortality after 24 hours. These two treat-

ments were significantly different from each other. The .01% solution

of piperonyl butoxide when applied topically at 1 pl per adult was sig-

nificantly different from the 0.1% and 1%, but not the acetone treated

group. Also, the percent hatch of eggs from these adults was affected

at .1%. The 1.0% group lacked data on hatchability because no flies

survived longer than 24 hours. These two levels significantly reduced

egg hatch when compared with the adults treated with .01% solution or

acetone treated check groups. Egg hatch of adult treated with the .01%

solution was nor significantly reduced from that of the acetone treated

adults. The percent egg hatch for adult horn flies treated topically

with acetone or dosages of piperonyl butoxide at .01% or .l%'were re-

spectively 68.5%, 46.5%, and 16.5%

MGK-264

A topical application of MGK-264 (Table 23) significantly reduced

adult survival at 1 Pl of a 1% solution per fly. This treatment lowered

adult survival more than any other of the treatments in this test. One

pl of acetone, 1 pl of .01%, or .1% solution in acetone of MGK-264 per

adult were not significantly different from each other and averaged re-

spectively 95%, 97.5%, and 92.5%. Treatment of adults with 1% solution

caused a 5% survival at 24 hours. The percent egg hatch was significantly

lower for adults treated with 0.1% or the 1% solutions when compared to

the acetone treated check. The .1% group was not significantly different





64










Table 22. Survival and Egg Hatch of Horn Fly Adults Treated
Topically with Piperonyl Butoxide (10 adults/rep).

Percent
Survival Percent
Treatment. Replication 24 Hours Egg Hatch

Acetone 1 100 65
2 100 74
3 100 80
4 100 55
Means 100.00 68.50


.01% 1 100 50
2 100 20
3 90 75
4 90 41
Means 95.00 46.50


.1% 1 0
2 10
3 80 66
4 40
Means 32.50 16.50



1.0% 1 0
2 0
3 0
4 0
Means 0.00
















Table 23. Survival and Egg Hatch of Horn Fly Adults Treated
Topically with MGK-264 (10 adults/rep).

Percent
Survival Percent
Treatment Replication 24 Hours Egg Hatch

Acetone 1 100 57
2 100 55
3 90 65
4 90 48
Means 95.00 56.25


.01% 1 90 18
2 100 30
3 100 23
4 100 26
Means 97.50 24.25



.1% 1 100 64
2 90 46
3 100 11
4 80 66
Means 92.50 46.75


I.C% 1 0
2 20 38
3 0
4 0
Means 5.00 9.50









from the acetone treated group. The percent hatch for the acetone treated

and dosages of .01%, .1%, and 1.0% were respectively 56.25%, 24.25%,

47.75%, and 9.5%.

R-20458

The compound R-20458 (Table 24) was tested by topical application

to adults. There was a significant 100% reduction in survival of adults

treated topically with 1 Vp of a 1.0% solution of R-20458. The other

treatments showed no reduction in survival during 24 hours. The percent

survival for horn fly adults treated with 1 P1 acetone or 1 pl of ace-

tone solutions containing R-20458 at the rate of .01% or .1% were respec-

tively 92.5%, 95%, and 95%. The eggs were tested for hatchability and

it was found that the .01% solution significantly reduced egg hatch when

compared to the acetone treated check and the .1% solution. The lower

hatch of the .01% group compared to the .1% solution is unexplainable at

this time. There were no eggs found in the 1% group, since all flies

had died within 24 hours. The percent hatch for horn flies treated with

1 1l acetone or treatments of 1 vl of acetone solutions of R-20458 at

the rate of .01%, or .1% were respectively 73.75%, 51.25%, and 71%.

Methoprene

The compound methoprene (Table 25) when applied topically to adults

showed that when 1 pl of a 1% solution was used, no flies survived after

24 hours. Treatments with 1 p1 of .01% or .1% solution of methoprene in

acetone showed no effect on survival. The percent survival for the ace-

tone treated and dosages of .01%, or .1% were all 100%. Eggs from these

adults were tested for hatchability. It was found that a 1% solution sig-

nificantly reduced egg hatch when compared to the acetone treated check

because no flies survived long enough to produce eggs. The .1% treatment
















Table 24. Survival and Egg Hatch of Horn Fly Adults Treated
Topically with R-20458 (10 adults/rep).

Percent
Survival Percent
Treatment Replication 24 Hours Egg Hatch

Acetone 1 100 64
2 90 74
3 100 91
4 80 66
Means 92.50 73.75


.01% 1 90 59
2 90 38
3 100 55
4 100 53
Means 95.00 51.25


.1% 1 90 63
2 100 73
3 100 88
4 90 60
95.00 71.00


1.0% 1 0 0
2 0 0
3 0 0
S4 0 0
Means 0.00 0.00















Table 25. Survival and Egg Hatch of Horn Fly Adults Treated
Topically with Methoprene (10 adults/rep).

Percent
Survival Percent
Treatment Replication 24 Hours Egg Hatch

Acetone 1 100 84
2 100 83
3 100 84
4 100 90
Means 100.00 85.25


.01% 1 100 94
2 100 74
3 100 50
4 100 41
Means 100.00 64.75


0.1% 1 100 55
2 100 42
3 100 53
4 100 56
Means 100.00 51.50


1.0% 1 0
2 0
3 0
S4 0
Means 0.00









significantly reduced egg hatch by 48.5% when compared to the acetone

created group and the .01% solution was not significantly different from

the acetone treated check because no flies survived long enough to pro-

duce eggs. The .1% treatment significantly reduced egg hatch by 48.5%

when compared to the acetone treated group and the .01% solution was not

significantly different from the acetone treated or flies treated with a

.1% solution. The percent hatch for eggs produced by adults treated

with 1 ul of acetone or 1 vl of a .01% or 0.1% solution of methoprene

were respectively 85.25%, 64.75%, and 51.5%.


Laboratory Tests with Manure From Cattle Fed
Methoprene in a Feed Supplement

Samples of manure from treated and untreated animals were bioassayed

by adding 100 horn fly eggs to 200 g manure in 12 oz cold drink cups.

The results of the first test are shown in Table 26. The two dosages fed

to cattle were 48 pg/kg/day, or 480 ug/kg/day. Both of these dosages con-

trolled emergence of adult horn flies from the pupae by 100% but had no

significant effect on the number of larvae successfully pupating. Dis-

section of pupae from treated samples showed that the pupae contained

fully formed adults. However, these adults were unable to eclose.


Table 26. Laboratory Production of Horn Fly Pupae and Adults in Manure
From Animals Fed Methoprene at 48 or 480 pg/kg/day (100 eggs/rep).

Replication
Treatment Stage 1 2 3 4 5 6 7

Untreated Pupae 49 66 60 54 51 27 29
Adult 22 40 46 24 18 11 25

48 Pg/kF/day Pupae 52 63 59 50 62 48 44
Adult 0 0 0 0 0 0 0

480 pg/kg/day Pupae 53 67 57 48 43 40 37
Adult 0 0 0 0 0 0









In the next test the treatments were 24 pg/kg/day and 48 pg/kg/day.

These dosages did not affect pupation of horn fly larvae when compared to

growth of flies in the manure of the untreated cattle (Table 27). How-

ever, both dosages significantly prevented eclosion of the horn fly adult.

Again dissection was performed on the pupae that did not eclose. It was

found that each pupa contained a fully formed adult that from external

characteristics seemed quite normal. No significant reduction in pupa-

tion was seen in the treated replications when compared to the untreated

replications.


Table 27. Laboratory Production of Horn Fly Pupae and Adults in Manure
From Animals Fed Methoprene at 24 or 48 pg/kg/day (100 eggs/rep).

Replication
Treatment Stage 1 2 3 4 5 6 7

Untreated Pupae 81 59 55 61 61 53 66
Adult 75 57 45 1 55 46 61

24 pg/kg/day Pupae 67 61 65 61 60 53 62
Adult 0 0 0 0 0 0 0

48 pg/kg/day Pupae 65 59 60 50 64 55 59
Adult 0 0 0 0 0 0 0


In the last test of this preliminary feeding series, dosages were

2.4 ug/kg/day of an alfalfa premix, 24 pg/kg/day of an alfalfa premix,

and 24 pg/kg/day of a granular premix. There was no significant reduc-

tion in formation of pupae in all treatments when compared to the untreated

group (Table 28). Both formulations at 24 pg/kg/day had 100% reduction in

adult eclosion from the pupae. There was no significant reduction in

adult eclosion at the dosages of 2.4 ug/kg/day of a .1% alfalfa premix.

This dosage however, significantly reduced percent hatch of eggs produced

by the adults. Of course, no eggs were produced on the other two treat-

rmn nts.









Table 28. Laboratory Production of Horn Fly Pupae, Adults, and Eggs in
Manure From Animals Fed Methoprene at 2.4 or 24 pg/kg/day
(100 eggs/rep).

Replication
Treatment Stage 1 2 3

Untreated Pupae 66 68 54
Adult 63 43 49
% hatch 81 90 84

2.4 pg/kg/day Pupae 76 69 70
alfalfa premix Adult 69 53 18
% hatch 70 40 '0

24 pg/kg/day Pupae 53 57 60
alfalfa premix Adult 0 0 0
% hatch

24 pg/kg/day Pupae 68 74 53-
granular Adult 0 0 0
% hatch


Sex ratios of adults produced on this test were evaluated with Chi-

square. There were significantly (p < .01) more males emerging in the

untreated replications with an average ratio of 1.54 males to ] female

(Table 29). McLintock and Depner (1954) found a sex ratio of 1 male to

1.13 female and Claser (1924) found a 1:1 ratio. There was no difference

in the sex ratio with a treatment of 2.4 pg/kg/day of an alfalfa premix.

Initial Field Test

In these studies, data on emergence of horn flies and other insects

in manure maintained under field conditions were studied to determine

the effect of methoprene on the manure ecosystem. Data were also col-

lected on manure samples using the same laboratory bioassay technique as

in previous tests. Data on parasitism of horn fly pupae was also col-

lected.

The first dosage tested was 24 lig/kg/day, the lowest dosage tested

previously in preliminary laboratory trials that produced 100% reduction
















Table 29. Sex Ratios of Horn Fly Adults Produced in the Laboratory in
Manure From Animals Fed Methoprene at 2.4 and 24 Ug/kg/day
(100 eggs/rep).


Retaliation


Adult
Male Female Total


Untreated


2.4 pg/kg/day
alfalfa premix


24 Pg/kg/day
alfalfa premix


24 pg/kg/day
granular


36
28(16)a
11(6)

0
0
0

0
0
0


33
25(15)
7(5)

0
0
0


Ratio
Male/Female


1.33:1
4.38:1
.88:1

1.09:1
1.12:1
1.57:1


0 0
0 0
0 0


aNumber of flies there were unable to expand wings are in parenthesis.


Treatment


Treatment Replicatio









in adult emergence. The data were used to compare laboratory conditions

with field conditions. Again, the laboratory test confirmed that a

dosage of 24 pg/kg/day completely prevented emergence from the puape

(Table 30). Sex ratios of adults produced on this test were evaluated

by Chi-square analysis (Table 31). There was no significant difference

in the sex ratios of males and females in the untreated insects. The

ratio of adults for the 6 replications averaged 1.03 males to 1 female.

No adults emerged with a treatment of 24 pg/kg/day. Emergence of insects

from manure pats maintained under field conditions and exposed to natural

fly populations are shown in Table 32. The dosage of 24 jg/kg/day pre-

vented adults of the horn fly from emerging from the manure pats exposed

to field conditions. The untreated manure pats averaged 7.43 horn flies

per pile, whereas no adult horn flies were produced in the manure pats

from treated cattle. This difference was significant at the 1% level.


Table 30. Laboratory Production of Horn Fly Pupae, Adults, and Eggs in
Manure From Animals Fed Methoprene Under Field Conditions at
24 jg/kg/day (100 eggs/rep).

Replication
Treatment Stage 1 2 3 4 5 6

Untreated Pupae 52 50 4 53 43 35
i Adult 41 47 45 49 35 32
% hatch 93 85 69 97 84 71

24 pg/kg/day Pupae 57 65 43 62 54 37
Adult 0 0 0 0 0 0
% hatch


Dipterans in the family Sepsidae are abundant in cattle droppings

during the spring and summer and are important in manure degradation.

Therefore, the reduction of this insect in treated manure may be con-

sidered harmful. In untreated pats this small fly averaged 285.43 flies

per pat (Table 32). In the treated manure pots, they averaged only

















Table 31. Sex Ratios of Horn Fly Adults Produced in the Laboratory in
Manure From Animals Fed Methoprene Under Field Conditi6ns at
24 yg/kg/day (100 eggs/rep).


Treatment Replication


Untreated


24 pg/kg/day


Adult
Male Female Total


.86:1
1.04:1
1.25:1
1.72:1
.84:1
.55:1


0 0
0 0
0 0
0 0
0 0
0 0


Ratio
Male/Female















|I



0
,-








-1 -1 -A|5 -4 r- 4 4








(O 000000
4-1I
0
0



o
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130.86 flies per pat. The reduction of these flies in the treated manure

pat was significant at the 1% level. Even though there was a significant

reduction of sepsids in the manure pats, the numbers of these flies re-

maining seemed sufficient to work efficiently in manure degradation

during the early stage.

Coleoptera adults averaged 10.4 per manure pat in untreated samples,

and 54.57 per treated dung pile. These differences in beetles captured

were not significantly different. Diptera of the family sarcophagidae

were not significantly reduced in emergence in manure pats from treated

animals. The untreated pats averaged 1.71 flies per pat, whereas there

were 0.14 flies in the treated samples.

Flies of the family calliphoridae in untreated samples averaged 0.14

flies per manure pat and treated samples averaged 0.43 flies per manure

pat. There was no significant reduction in adult calliphorid emergence

because of treatment. Unidentified diptera in untreated samples and

treated samples were not significantly different. The averages were

respectively 83.71 and 64.71 flies per untreated and treated manure pats.

The dosage of 24 pg/kg/day methoprene fed to cattle was halved to

12 pg/kg/day. This dosage also significantly reduced the number of adult

horn flies emerging from pupae in laboratory tests (Table 33), but had no

significant effect on the number of pupae. With this dosage, no adults

were produced. Sex ratios of adult horn flies produced on this test were

evaluated by the Chi-square (Table 34) analysis. There was no significant

difference in the sex ratios of the untreated group which was 0.92 male

to 1 female. There were no adults produced in the replications of treated

manure.










Table 33. Laboratory Production of Horn Fly Pupae, Adults,-and Eggs in
Manure From Animals Fed Methoprene in Field Studies at 12
pg/kg/day (-100 eggs/rep).


Treatment

Untreated



12..pig/kg/day


Stage

Pupae
Adult
% hatch

Pupae
Adult
% hatch


Replication
3

56
51
71

58
0


Table 34. Sex Ratios of Horn Fly Adults Produced in the Laboratory in
Manure From Animals Fed Methoprene in Field Studies at 12
pg/kg/day (100 eggs/rep).


Replication

1
2
3
4
5

1
2
3
4
5


Adult
Male Female Total

30 43 73
30 27 57
27 24 51
35 28 63
17 29 46

0 0 0
0 0 0
0 0 0
0 0 0
0 0 0


Ratio
Male/Female

0.70:1
1.11:1
1.13:1
1.25:1
0.59:1


In field studies the dosage of 12 ug/kg/day significantly reduced

the production of adult horn flies. The untreated manure pats averaged

23.33 horn flies per dung pile (Table 35). The treatment of 12 pg/kg/day

allowed an average of 0.83 flies per dung pile. The same manure main-

tained under laboratory conditions for testing as previously discussed

(Table 31) allowed no production of adults. Environmental factors in

the field apparently reduced the effectiveness of methoprene held under

environmental conditions as well as dilution of dosage rate by the ani-

mals feeding on pasture. The field emergence from untreated and treated


Treatment

Untreated





12 pg/kg/day

















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manure pats showed no significant difference in numbers of Diptera in

the family Sepsidae. The untreated replications averaged 174.42 flies

per dung pile and the treated replications averaged 166.17 flies per

dung pile. The differences in emergence of the sepsids is much more

favorable than in the treatment of 24 pg/kg/day as seen in Table 32.

The number of trapped adult Coleoptera were significantly different

between the untreated and treated groups. The untreated replications

averaged 7.67 beetles per dung pile, while the treated replications

averaged 16.08 beetles per dung pile. Beetles are important contribu-

tors in degradation of manure and help it to dry out rapidly thus

limiting the-success of.horn fly breading. Diptera of the"family

Sarcophagidae were not significantly affected. The untreated group

averaged 0.67 flies per dung pile and the treated group 2.25 flies per

dung pile. There were more unidentified Diptera in the treated groups

than in the untreated groups, but this difference was not significant.

The untreated group averaged 98.75 flies per manure pat and the treated

groups averaged 142.08 flies per pat.

The dosage of 12 ug/kg/day was halved in the next test to 6 ug/kg/day.

In laboratory bioassays of samples obtained in the field, it was found

that this dosage was not completely effective in prevention of adult

emergence. Adult emergence (Table 36) averaged 1.75 flies per replica-

tion in laboratory samples of treated manure. There was still a signi-

ficant reduction due to treatment, with the untreated replications

averaging 6.05 flies. No eggs were produced by flies which had emerged

from manure from treated animals. In replication 2, there were 3 female

flies with unexpanded wings and 2 male flies: however, 3 females may have

been unable to mate. In replication 3, all flies were males. Sex ratios










of adults produced on this test are shown in Table 37. The ratio;of

untreated insects was 0.85 males to 1 female. The use of a Chi-square

test showed that there was no significant difference in the expected sex

ratio of 1 male to 1 female. The treated insects had a-ratio of 1.33

males to 1 female.


Table 36. Laboratory Production of Horn Fly Pupae, Adults and Eggs in
Manure From Animals Fed Methoprene in Field Studies at 6
pg/kg/day (100 eggs/rep).

Replication
Treatment Stage -1 2 3 4

Untreated Pupae 63 58 74 62
Adult 58 55 68 61
% hatch 66 58- 54 47

6 ug/kg/day Pupae 70 69 70 59
Adult 0 5 2 0
% hatch -- 0 0 -


Table 37. Sex Ratios of Horn Fly Adults Produced in the Laboratory in
Manure From Animals Fed Methoprene in Field Studies at
6 vg/kg/day (100 eggs/rep).

Adult Ratio
Treatment Replication Male Female Total Male/Female

Untreated 1 31 27 58 1.15:1
2 25 30 55 .83:1
3 35 33 68 1.06:1
4 4 20 41 61 .49:1

6 pg/kg/day 1 0 0 0
2 2 3 5 .67:1
3 2 0 2
4 0 0 0


In field emergence studies, untreated samples averaged 8.88 adult

horn flies per pat. A treatment of methoprene at the rate of 6 ug/kg/day

caused an average emergence of 0.63 horn flies per pat (Table 38). Flies

in thi family St'nsiLae in the untreated group averaged 100 flies per untreated

::nure pat. The treated manure produced 79.75 flies per manure pat and





















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was not significantly different from the untreated samples. This is en-

couraging since these insects are considered beneficial to the manure.

Adult Coleoptera averaged 4.5 beetles per manure'pat in untreated

samples and 12.13 beetles per pat in-the treated samples. The treatment

had significantly more beetles than the untreated groups.

Adult sarcophagidae from untreated animals averaged 2.88 flies per

manure pat and treated animals averaged 0.89 flies per pat. These two

groups were not significantly different.

Unidentified diptera in the untreated group averaged 108.13 flies

per manure pat. In the treated samples the average was 83.50 flies per

---manure-pat-. These differences were also not significant.

The last dosage tested in this series was 3 pg/kg/day. In labora-

tory tests on the samples of manure obtained in the field, there was an

average of 58 flies produced per replication in untreated samples (Table

39). The treated samples averaged 8 flies per replication. This is an

86% reduction in adult production. These differences were significant.

The percent egg hatch was significantly reduced in the treatment group.

Only in replication 2 were there any-eggs laid and their hatchability

was 38%. Sex ratios of adults produced in this test are shown in Table

40. The ratio of untreated insects averaged 1.3 males to 1 female with

no significant difference. The treatment of 3 pg/kg/day had a ratio of

2.6 males to 1 female and was not significantly-different from the un-

treated group.

Field emergence tests were conducted on samples of manure from un-

treated and treated animals (Table 41).' The emergence of horn fly adults

in untreated samples averaged 7.63 flies per manure pat and the samples

from cattle treated at the rate of 3 ug/kg/day averaged 1.63 flies per












Table 39. Laboratory Production of Horn Fly Pupae, Adults, and Eggs in
Manure From Animals Fed Methoprene in Field Studies at
3-pg/kg/day (100 eggs/rep).


Treatment

Untreated



3 Ug/kg/day


Stage


SReplication
1 2 3


4 5


Pupae
Adult
% hatch

Pupae
Adult
% hatch


Table 40. Sex Ratios of Horn Fly Adults Produced in the Laboratory in
Manure From Animals Fed Methoprene in Field Studies at
3 pg/kg/day (100 eggs/rep).


Adult
Treatment Replication Male Female Total


Untreated





3 pg/kg/day


47
20
34
30
33

6(5)a
9(6)
1 -
3
7(6)


Ratio
Male/Female


1.81:1
1.25:1
1.31:1
.88:1
1.50:1

1.50:1
4.50:1
1.00:1
3.00:1
3.50:1


aNumber of flies that were unable to expand wings are in parentheses.




















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manure pat. This treatment caused a 79% reduction in adult horn fly

emergence and this difference was significant.

Diptera in the family Sepsidae in untreated samples averaged 79

flies per manure pat. The treated samples averaged 46.63 flies per

manure pat. These differences were not significant. Adult Coleoptera

in untreated samples averaged 2.88 beetles per manure pat. The treated

groups averaged 10.38 beetles per dung pile. The treatment average was

significantly higher than the untreated average.

Diptera of the family Sarcophagidae in untreated manure averaged

.38 flies per dung pile and the treated group averaged 0.00 flies per

dung pile. Unidentified diptera in the untreated group averaged 59.88

flies per dung pile and the treated groups averaged 121.75 flies per

dung pile. These differences were significant.

Parasitism was common in pupae of the horn fly in both treated and

untreated samples. It was important to determine the effect of methoprene

on the rate of parasitism. In Table 42, the number of pupae recovered

and the percent parasitism of the pupae are shown, The data were analyzed

statistically using analysis of variance. The average number of pupae

in the untreated manure was 13.4 pupae per replication and manure from

animals treated with 24 pg/kg/day averaged 11.9. Analysis of these

values showed that there was no significant difference in the number of

pupae produced in the untreated and the treated samples.

Table 42. The Percent Parasitism of Horn Fly Pupae Recovered From Field
Samples of Manure From Animals Treated with Methoprene at the
Rate of 24 pg/kg/day.

Replication
Treatmnent _1 2 3- 4 5 6 7 Means

Untreated No. IPuae 32 0 7 1 12 11 31 13.4
2 parasiti7ed 19 0 43 0 67 9 39 25.3

24 : 'day No. Pupae 47. 0 5 3 5 5 18 11.9
% parasitized 11 0 60 67 100 40 67 49.3









The average percent.parasitism of the untreated samples were 25.3%

per replication. The manure from animals fed 24 pg/kg/day averaged 49.3%

parasitism.- This value represents a 1.95 times increase in parasitism in

manure samples from treated animals.

The number of pupae recovered in field samples of manure of untreated

animals and animals fed methoprene at the rate of 12 ug/kg/day are shown

in Table 43. The untreated group averaged 18.6 pupae per replication and

the treatment group averaged 7.3 pupae per replication. These differences

-were found to be significant at the 1% level through analysis of variance.

Parasitism was also significantly different at the 1% level. The un-

treated group averaged 26.2% parasitism of-the pupae recovered. The ani-

mals treated at the rate of 12 pg/kg/day averaged 68.7%. This is a 2.6

times increase in parasitism in the manure of the treated animals.

In Table 44, the number of-pupae recovered and the percent parasi-

tism of pupae separated from field samples of manure are recorded. There

was no significant differences between the untreated samples and samples

of animals treated with 6 pg/kg/day of methoprene for either number of

pupae or percent parasitism. The average number of pupae per replica-

tion in untreated samples was 9.6 pupae and a treatment of 6 ug/kg/day

had 4.9 pupae. The percent parasitism of the untreated pupae was 33.6%

and a treatment of 6 pg/kg/day was 58:9%.

The data on the number of pupae recovered and the percent parasitism

of the pupae in untreated manure or manure from animals treated at the

rate of 3 pg/kg/day are. shown in Table 45. The number of pupae recovered

in untreated- samples averaged 7.8 pupae per replication and 3.2 pupae per

replication in manure samples from treated animals. The differences in

the imber of pupae was significant at the 1% level. The percent
























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parasitism for the untreated groups averaged 16.9% and the percent para-

sitism in manure from animals treated with 3 Vg/kg/day averaged 50.1%.

These differences were not significantly different.


Large Scale Field Studies

Manure samples obtained in the field from the treated and untreated

cattle were returned to the laboratory and to each sample 100 horn fly

eggs were added. The results of this test are shown in Table 46. Anal-

ysis of variance of the data showed successful pupation following a treat-

ment of 24 pg/kg/day when compared to untreated samples. But there was

a significant reduction in emergence of adults from pupae due to treat-

ment. Only 6 flies emerged in treated samples. The percent hatch was

significantly different because of those replications which had flies

emerging; only replication 6 had a female and she laid no eggs. The sex

ratios were analyzed and shown in Table 47. The average sex ratio for

the untreated group was 1.16 males to 1 female and this difference was

not significant from the expected ratio of 1 to 1. The sex ratio of horn

flies produced in manure from treated animals was 5 males to 1 female;

however, the sample size was so small that there was no significant dif-

ference in this ratio.


Table 46. Laboratory Production of Horn Fly Pupae, Adults, and Eggs in
Manure From Beef Cattle Fed Methoprene.

Replication
Treatment Stage 1 2 3 4 5 6 7 8

Untreated Pupae 74 62 73 37 66 69 79 63
Adult 68 61 73 36 60 64 55 58
% hatch 54 47 57 69 77 86 69 57

24 pgi>'::day Pupae 73 70 65 61 68 68 72 71
Adult 0 1 1 0 0 4 0 0
/ hatch -- 0 0 -- -- 0 -

















Table 47. Sex Ratios of Horn Fly Adults Produced in the
Manure From Beef Animals Fed Methoprene.


Treatment Replication


Adult
Male Female Total


Laboratory in


Ratio
Male/Female


1.06:1
0.49:1
1.81:1
1.25:1
1.31:1
0.88:1
1.50:1
1.64:1


Untreated


24 ig/kg/day


Treatment el : atio










Field emergence of insects from untreated manure patties and patties

from animals fed an average of 24 ug/kg/day are shown in Table 48. A

treatment of 24 pg/lg/day significantly reduced the number of horn flies

emerging when compared to the untreated samples. The untreated patties

averaged 5.94 flies per patty, whereas the treated patties averaged 0.06

flies per patty. This field test definitely demonstrates the feasibility

of reducing the numbers of adult horn flies emerging from manure from

treated animals. Diptera of the family Sepsidae suffered a significant

reduction from a treatment of 24 ug/kg/day. The average production per

patty for the untreated patties was 69.06 flies. The treated patties

averaged 18.63 flies per patty. Adult Coleoptera showed no significant

difference In untreated and treated patties. The untreated patties

averaged 3.31 beetles per patty and the treated patties averaged 5.69

beetles per patty. Also, there was no significant reduction of flies

in the family Sarcophagidae. The untreated patties averaged 0.31 flies

per patty and the treated patties averaged 0.25 flies per patty. Uniden-

tified diptera were not significantly reduced from a treatment of 24 pg/

kg/day. The untreated patties averaged 72.19 flies per patty.

Adult horn fly populations on cattle were compared between treated

and untreated cattle. Untreated animals averaged 78 flies per side and

treated animals averaged 76 flies per side. There was no reduction of

the anult population on treated cattle even though emergence from manure

was reduced 99%. This difference is probably due to migration of flies

from other herds in the area.




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