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Sex and aggregation pheromone bioassays and mating observations of the Caribbean fruit fly, Anastrepha suspensa (Loew), under field conditions

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Title:
Sex and aggregation pheromone bioassays and mating observations of the Caribbean fruit fly, Anastrepha suspensa (Loew), under field conditions
Creator:
Perdomo, Alberto Javier, 1943-
Publication Date:
Language:
English
Physical Description:
xi, 127 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Bioassay ( jstor )
Boll weevils ( jstor )
Female animals ( jstor )
Fruit flies ( jstor )
Insects ( jstor )
Mating behavior ( jstor )
Pheromones ( jstor )
Sex attractants ( jstor )
Species ( jstor )
Virgin females ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Fruit-flies ( lcsh )
Key Biscayne ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1974.
Bibliography:
Includes bibliographical references (leaves 115-125).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Alberto Javier Perdomo.

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University of Florida
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SEX AND AGGREGATION PHEROMONE BIOASSAYS AND
MATING OBSERVATIONS OF THE CARIBBEAN FRUIT FLY,
Anastrepha suspensa (LOEW), UNDER
FIELD CONDITIONS










By

ALBERTO JAVIER PERDOMO


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY








UNIVERSITY OF FLORIDA


1974













ACKNOWLEDGMENTS


I am deeply indebted to Dr. James L. Nation and Dr. Richard M. Baranowski, who directed the present research. Special thanks are extended to Dr. J. L. Nation for his criticism, assistance, and enthusiastic encouragement in the preparation of this dissertation. I thank Dr. R. M. Baranowski for his liberal support, and for providing the necessary supplies, equipment, and direction during my stay in Homestead, Florida.

Grateful acknowledgments is given to Dr. H. L. Cromroy, Dr. D. L. Chambers, and Dr. F. W. Zettler for their critical review and suggestions in the preparation of this dissertation and for serving as members of the Supervisory Committee.

I extend thanks to Dr. and Mrs. D. 0. Wolfenbarger, and Dr. R. A. Conover for providing housing and various other services, and also to the personnel of the Agricultural Research and Education Center, for their friendship during my stay at Homestead, Florida.

I am indebted to the International Atomic Energy Agency, and to Dr. W. G. Eden, Chairman of the Department of Entomology and Nematology, for their financial support during the period of graduate study. Special thanks are extended to the National Academy of Sciences for their administrative assistance. Finally thanks go to my wife Esther, my children Tito and Malena, and to my family who provided moral support.

ii














TABLE OF CONTENTS


ACKNOWLEDGMENTS. . . . * . . . . . . LIST OF TABLES . . . . . . . . , . . LIST OF FIGURES. . . . . . . . . . .

ABSTRACT . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . .

LITERATURE REVIEW. . . . . . . . . .

Insect Sex Pheromones . . . . .

Utilization of Sex Pheromones in
Management . . . . . . . . .

Pheromones as Survey Tools Potential Control with the
Technique . . . . a . .

Potential Control with the
Technique . . . . . . .


Page



. . . * . * . . . , vi . . . . . . . . . viii

. . . . . . . . ix

. . . . . . . . . . 1

. . . * . . 5 . . 5 3

. . . . . . . . . . 3


Pest
. . .S


. .


. . 0 . . Mass Trapp a a . . , Confusion
. 0 . . .


. . . . - 5

. . . . . 7

ing
. . . . . 11


. . . . . 13


Integration of Sex Pheromones with other
Control Agents. . . . . . . . . . . . . .

Sex and Aggregating Pheromones in the Tephritidae

Female-produced Pheromones . . . . . . . .

Male-produced Pheromones , . . . . . . .

Aggregating Pheromones . . . . . . . . .

Courtship and Mating Behavior in the Tephritidae. Courtship and Mating Behavior in other Diptera. Economic Importance of the Tephritidae. . . . . Taxonomic Relationships . . . . . . . . . .


iii


. 14 . 16 . 16 . 16 . 17

* 19

* 25 . 27 . 30










MATERIALS AND METHODS. . . . . . . . . . . . *

Experimental Insects. . . . . . . . .

Field Bioassays . . . . . . . . ..

Field Observations. . . . . . . . . .

Screen-mesh House Bioassays to Test Sex
Pheromone. . . . . . . . . . . . # . .

Laboratory Bioassays to Test Aggregating
Pheromone. . . . . . . . . . . . .


Page
- - - - - 33

. . - - . 33

. 9 - - - 35 . . *. . -43


* * - - - 43 . . . . . 44


RESULTS. . . . . . . . # . . . . e a - * @ . s @ @

Recapture of Virgin Females with Male-baited
Traps in the Screen House. . . . . . . .

Field Bioassays . . . . . . . . . . . ..

Recapture of Virgin Females with Malebaited Traps. . . . . . ,. . . . . . .

Recapture of Virgin Females and Males with
Male-baited Traps . . . . . . . . . . .

Recapture of Virgin Females and Males with
Female-baited Traps . . . . . . . . . a

Effect of Age and Virgin or Mated Status
Upon Female Attraction to Malebaited Traps. . . . . . . . . . . .

Recapture of Virgin Females with Pheromone
Extract-baited Traps. . . . . . . . . .

Laboratory Bioassays to Test Aggregating
Pheromone. . . . . . . . * . . . . . . . . .

Ability of Laboratory-reared Males to Attract
Wild Flies . . . . . . . . . . . . . . a .

Courtship and Mating Behavior of A. suspensa
in Nature. . . . . . . . . . . ,. . . . . .

Some Environmental Factors Associated with
Mating . . . . . . . . . . . . . . . . a . .


iv


- - 47 ..47

. . 48 . . 48


. 56


60 63


. . 71 . . 74


75


. . 81 . . 89


.








Page

DISCUSSION. . . . . . . , , . . , . . . . . , . . . . . 98

The Experimental Procedures. . . . . . . . . . . . 98

The Field Bioassays. . , , . . . . . . . . . . . 100

Courtship and Mating Behavior of A. suspensa
in Nature . . . . . . # V . . a . . * . . 107

The Future Application of This Research. . . . . 110 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . 115

BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . 126


V













LIST OF TABLES


Table Page
1 Recapture of released A. suspensa adult virgin
females with male-baited sticky traps. . . . . . . 49

2 Recapture of released A. suspensa adult virgin
females with sticky traps baited with 10, 20, and
40 live males as compared with McPhail traps . . . 51

3 Recapture of released A. suspensa adult virgin
females with boll weevil sticky traps baited with
40 live males. . . . . . . . . . . . . . . . . . . 55

4 Recapture of released A. suspensa adult virgin
females and males with sticky traps baited with
20 and 40 live males as compared with McPhail
traps. . . . . . . . .1 . . - - - - - - a . & . . a 57

5 Recapture of released A. suspensa adult virgin
females and males with sticky traps baited with
food only and 40 live males as compared with
McPhail traps. . . . . . . . . . . . . - - . a & - 59

6 Recapture of released A. suspense adult virgin
males with sticky traps baited with food only
and 40 live males as compared with McPhail traps . 61

7 Recapture of released A. suspense adult virgin
females and males with sticky traps baited with
food only, 5, and 10 live females as compared
with McPhail traps . . . . . . . . . . . . . . . . 62

8 Recapture of released A. suspensa adult virgin
and non-virgin females-of different age with
sticky traps baited with 40 live males . . . . . . 65

9 Recapture of released A. suspense adult virgin
and non-virgin females of different age with
sticky traps baited with 40 live males . . . . . . 68

10 Recapture of released A. suspensa adult virgin and non-virgin females of different age with
sticky traps baited with 40 live males . . . . . . 70


vi











11 Recapture of released A. suspensa adult virgin
females with triangular-sticky-traps baited with 0 and 5 live males, and 100 pl of male
pheromone extract. . . . . . . . . . . . . . . - & 73

12 Wild A. suspensa adult flies per trap per day
captured during the summer of 1972 with 40-malebaited sticky traps compared with McPhail and
control traps at AREC. . . . . . . . . . . . . . . 78

13 Wild A. suspense adult flies per trap per day
captured during the summer of 1973 with 40-malebaited sticky traps compared with McPhail traps
at AREC . . . . . . . . . . . . . . o . . . o 80

14 Wild A. suspensa adult flies per trap per day
captured during the summer of 1973 with 40-malebaited sticky traps compared with McPhail traps
at Key Biscayne. . . . . . . . . e , . . . . . . . 82

15 Number of mating pairs of wild Caribbean fruit
flies observed in daylight hours during the
summer of 1972 at AREC . . . . . . . . . . . . . . 91

16 Mating pairs of wild A. suspensa flies observed
during the summer of 1972 at AREC. . . . . . . . . 92

17 Total number of pair matings and courting males
observed among 6 to 10 days old A. suspense
adults when exposed to wind currents of7T and
16 kph compared with flies tested in still air
under laboratory conditions. . . . . . . . . . . . 97

18 Recapture of released sterile and non-irradiated
A. suspensa adult virgin females and males with
sticky traps baited with 40 sterile males, and
40 non-irradiated males as compared with control
traps. . . . . . . . v . . . . . v . . . a a . * .114


vii


Page


Table













LIST OF FIGURES


Figure Page
1 Cages used to maintain adult A. suspensa in
the laboratory. . . . . . . . . . . . . . . . . 34

2 Adult A. suspensa females emerging from a
release cylinder in the field . . . . . . . . . 36

3 Partial view of the avocado grove showing the
grid pattern distribution of trees. . . . . . . 37

4 Diagram of a section of the avocado grove
showing the distribution of traps, release
points, and tree orientation. . . . . . . . . . 38

5 Cotton boll weevil trap, also known as wing
trap, used in field bioassays to test sex and
aggregation pheromone of A. suspensa. . . . . . 40

6 Invaginated glass or McPhail trap used in
field bioassays . . . . . . . . . . . . . . . . 41

7 Triangular sticky trap baited with males
and food. . . . . . . . , . . . . . . . . . . . 42

8 Diagram of the Plexiglas plastic chamber
(45 X 45 X 45 cm) used as a bioassay test
apparatus in the laboratory . . . . . . . . . . 45

9 Total recapture of A. suspensa adult virgin
females at intervals after release May 26, 1972, at 1545 hours with four sticky traps
baited with 5 males each and four sticky traps
baited with 10 males each . . . . . . . . . . . 52

10 Recapture of virgin female A. suspensa adults
48 hours after release, and final recovery for
observation period with sticky traps baited
with males. . . . . . . . . . . . . . . . . . . 54


viii









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


SEX AND AGGREGATION PHEROMONE BIOASSAYS AND
MATING OBSERVATIONS OF THE CARIBBEAN FRUIT FLY,
Anastrepha suspensa (Loew), UNDER FIELD CONDITIONS


By

ALBERTO JAVIER PERDOMO

June, 1974

Chairman: Dr. J. L. Nation
Major Department: Entomology and Nematology

A bioassay for the sex pheromone produced by male Caribbean fruit flies, Anastrepha suspensa (Loew), was developed in field studies at Homestead, Florida. Cotton boll weevil traps coated with Stickem and baited with live male flies attracted and recaptured released female flies. Experiments were conducted in an avocado grove, and wild flies were not present in the immediate grove area. Males used as bait and females released were from a laboratory stock colony. Sexes were segregated after adult emergence.

Sticky traps baited with 10 or 40 live males consistently recaptured more released females than did McPhail traps baited with yeast hydrolysate-borax-water mixture. A maximum recapture of 48% of released females occurred in one experiment in which all of 9 traps used were baited with 40 live males. Numbers of females recaptured tended to be greater in the late afternoon hours, and during the first 48 hours folix










lowing release.

Male-baited and McPhail traps also attracted male flies when males were released with females or alone. Male-baited traps caught significantly more males than did McPhail traps. When males were released with females in a 1 : 1 ratio, the male-baited traps recaptured a 1 : 1 ratio. The effect of varying the ratio released was not tested. The attraction of males to male-baited traps was interpreted as evidence of an aggregation phenomenon, and possibly aggregation to a pheromone. Female-baited sticky traps were not as effective as McPhail traps in attracting released males and females.

Recapture of mated and virgin females varying from 1 to 19 days of age, marked with fluorescent dye, showed that virgin females were more attracted to male-baited traps than were mated flies. Little attraction of flies occurred before 6 days of age, and a peak of attraction occurred at 9-12 days of age. Older mated females were very poorly attracted. At

9 days of age the attraction of mated females as compared with virgin females was about 50% reduced.

Sticky traps baited with sex pheromone extracted from the cloth and cage surfaces of male holding cages attracted released females. These results and earlier demonstrations of female attraction to male-baited traps were interpreted as indicating attraction to a male sex pheromone.

In the summers of 1972 and 1973 traps baited with live

lab-reared males attracted wild females and males at Homestead

x










and Key Biscayne, Florida. In total, McPhail traps at the same locations captured more wild flies than male-baited traps, but during some periods and in some locations, malebaited traps caught more wild flies than McPhail traps. Male-baited traps consistently detected a greater proportion of males in wild populations than did McPhail traps.

Males court females and mate on the underside of leaves of host trees. Aggregation of males and females seems to occur in certain trees. Males exhibit a "calling" behavior involving distention of lateral abdominal and anal pouches, slow wing waving, and intermittent bursts of rapid wing fanning. Females move toward males a short distance or several branches away, but they do not usually fly to the male by a direct path. Mating occurred during windless afternoons, but no mating was seen during morning hours. Twenty-six mating pairs were observed.









Chairman L


xi













INTRODUCT ION


The Caribbean fruit fly, Anastrepha suspensa (Loew),

known informally as the caribfly, is one of several species of fruit flies indigenous to the West Indies. It invaded Florida on at least three occasions, the last in Miami Springs (Dade county) in April 1965 (Weems 1965, 1966). Since then it has spread over 21 counties throughout the southern and central part of the state. In south Florida it infests mainly dooryard tropical and subtropical fruits, including Surinam and Barbados cherry, common guava, cattley guava, loquat, rose apple, tropical almond, peach, and Citrus species. More than 80 species of fruit have been recorded as hosts of caribflies (Swanson and Baranowski 1972). For this reason it is considered by many scientists and c9Wizens to be an urban pest rather than a commercial pest.

However, some individuals believe A. suspensa represents a potential threat to the citrus industry of the state of Florida and of other parts of the country (Arizona, California, Texas) and to the peach industry in Florida and Georgia. In Florida this insect occasionally attacks several important citrus crops, such as grapefruits and oranges. A strain of A. suspensa in Puerto Rico attacks grapefruits, sour oranges, sweet oranges and other tropical and subtropical fruits

(Weems 1965).


1







2

Laws of California, Arizona, and Texas require that citrus shipments from Florida be fumigated. The cost to the citrus industry for treating citrus fruits is estimated at approximately $35,000 per year. Three fumigation chambers were built in 1971 at Gainesville, Florida, at a cost to the citrus industry of $72,000. The fumigation treatments are conducted by the Division of Plant Industry (H. A. Denmark and R. Brown, personal communications).

Research with A. suspensa is being conducted by the University of Florida, the U. S. Department of Agriculture, and the Florida State Division of Plant Industry. One phase of this research is the investigation of improved methods of detection involving the determination of a lure or lures which might replace the present food-baited McPhail traps.

Nation (1972) demonstrated that males of A. suspensa

release one or more volatile pheromone compounds that attract females under laboratory conditions. Insects utilize infinitesimally small quantities of such chemical compounds to communicate with each other.

Since the practical application of this basic information needs to be ultimately tested in nature, I decided to study the sexual attraction and behavior of the caribfly under field conditions. The objectives of this work were (1) to develop a field bioassay for the sex pheromone, and determine certain parameters of female response to it, such as the influence of age and mated status, and (2) to observe courtship and mating behavior in the field.













LITERATURE REVIEW


Insect Sex Pheromones


Although the name "pheromone" was proposed only recently (Karlson and Butenandt 1959) to describe chemicals that, produced by one animal, influenced the behavior of others of the same species, many examples had been observed over the past 100 years.

In 1837 Carl von Siebold, a German zoologist, suggested that insect odors were probably attractants and aphrodisiacs for members of the same species. Apparently, his view was not taken seriously by most other scientists, for many early authors believed that male insects were provided with acute vision that enabled them to see females at great distances (cited by Grosser 1971).

Lefebvre (cited by Jacobson 1972) initiated research in the field of insect olfaction. Weismann (cited by Jacobson 1972) suggested that odors from scent glands on the wings of certain male lepidopterous insects function as an aphrodisiac for females. He reported that numerous males of the eyed hawk moth, Smerinthus ocellata (L.), were attracted to a single virgin caged female.

Around 1890, Jean Henri Fabre, a French naturalist, found that male moths of Saturnia pyri were attracted in

3







4

large numbers, even in the dark, to caged females (cited by Grosser 1971). Urbahn (cited by Jacobson 1972) reported numerous findings of sex pheromones from laboratory and field experiments in the order Lepidoptera, and described the abdominal scent glands of many female lepidopterous insects.

Law and Regnier (1971) proposed the name "semiochemicals" for those substances that convey chemical messages between insects and they divided semiochemicals into two broad classes: those conveying signals between individuals of the same species (intraspecific) and those conveying signals between different species (interspecific). They also proposed the names "allomones" and "kairomones" for those interspecific semiochemicals whose release favors the producer or receiver, respectively.

On a functional basis pheromones are divided into releasers or primers (Wilson and Bossert 1963). Releaser pheromones stimulate an immediate behavioral response, while primers change the physiology of the receptor animal, reprogramming it for an altered response pattern that usually appears later. Some pheromones act as both primers and releasers (Grosser 1971). Sex attractants are releaser pheromones.

Although noting that the same chemical may have multiple effects upon behavior, Dethier et al. (1960) erected the following classification in terms of the behavioral responses that chemicals elicit upon insects: (1) Arrestants - chemicals that cause insects to aggregate, (2) Locomotor stimulants -







5


chemicals that cause insects to disperse from a region more rapidly than if the area did not contain the chemicals,

(3) Attractants - chemicals which cause insects to make oriented movements towards the source, (4) Repellents - chemicals which cause oriented movements away from the source,

(5) Feeding, mating, or ovipositional stimulants - chemicals which elicit feeding, mating, or oviposition, and (6) Deterrents - chemicals that inhibit feeding or oviposition when present in a place where insects would normally feed or oviposit.

A sex pheromone is usually emitted by one sex to attract the opposite sex (and sometimes both sexes) for mating (Beroza 1972). There is evidence that hundreds of insects utilize sex attractants to find each other. Most of these sex pheromones are chemically unidentified.


Utilization of Sex Pheromones in Pest Management


It is strongly felt by many that pheromones will be the key to truly integrated pest management for many crops (Antognini 1972). The potential use of pheromones in pest management for survey, mass trapping, and mate confusion programs is being explored (Marx 1973). Survey or monitoring the population of an insect pest helps to determine proper timing of sprays, or whether a pesticide spray is actually needed. Sex pheromone-baited traps assist in locating infestations, and are especially useful when these







6

are not easy to find by visual inspection. Pheromone traps also help detect new pest introductions and proper control measures can be instituted before the infestation becomes so large that it is uncontrollable. Monitoring quality control of sterile insects by means of sex pheromone or live insectbaited traps has been useful.

Mass trapping and mate confusion techniques are related to control or prevention of reproduction. Mass trapping techniques may be directed toward preventing an insect from expanding its range, or help to exclude an insect from a particular crop. Usually a combination of the sex pheromone (or aggregating pheromone) with a sticky trap or other suitable trapping device is used (Tette 1972). One procedure has been to drop from an airplane large numbers of safe and inexpensive mass-produced traps (such as small tubes with an adhesive inside) containing attractant to capture one or both sexes before they can find each other and mate. If the ratio of traps to the target insects is high enough, the population of lightly infested areas might be eradicated. This overflooding approach is similar to the one devised by Knipling (1960) for use in the sterile-male technique.

The mate confusion technique is known by several names

such as "male confusion", "male inhibition", "disruptive communication", and "misdirection" (Grosser 1971). This new approach to control consists of permeating the atmosphere with the sex pheromone, or attractant, so that the males







7

will not be able to orient to the females, or vice'versa. Their odor receptors may simply become fatigued, insensitive, or saturated, thereby preventing them from responding. The advantages offered by this method are that it may be applicable to both lightly and heavily infested areas (Beroza 1960, 1971, 1972).

Pheromones as Survey Tools

A variety of commercially produced pheromone-baited

sticky traps are available which serve to monitor effectively in the field more than a dozen important economic pests. Included among these are methods for two fruit flies, the apple maggot fly, Rhagoletis pomonella (Walsh), and the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Two types of traps are in use for these fruit pests. The wing-type trap of paperboard construction has a sticky adhesive on the upper surface of the bottom section. Another type is constructed of a pre-folded, heavy gauge cardboard with a tacky adhesive coating on the inside. When the trap is opened, it takes the form of a rectangle, resembling a pagoda-like shape. Insects entering these traps are captured and firmly held in the sticky substance.

In 1972 Madsen and Vakenti (1972) compared female-baited traps against traps baited with Codlemone, a synthetic sex pheromone found by Roelofs et al. (1971) for the codling moth, Laspeyresia pomonella (L.) (Lepidoptera). The results indicated that both types of traps can be used to estimate pop-







8

ulation levels and to determine the need for chemical treatment. In the next year, Madsen and Vakenti (1973) reported that sprays were applied only if Codlemone-baited traps within the orchard captured two or more moths per trap per week and if infested apples were found. In this way, only one or two sprays were required in different orchards, instead of the three sprays usually applied.

In the case of the California red scale, Aonidiella

aurantii (Maskell), routine inspection of groves represents a difficult, tedious, and costly task. Many trees have to be checked in the area, identification of the insect can be confused, and infestations may go unnoticed because scales are usually in cryptic places. A citrus scale trap was developed by Shaw et al. (1971) in which the sex pheromone produced by the female (Tashiro and Chambers 1967) attracts males, which are then caught on a sticky card. In California and Arizona, citrus growers are starting to use these traps in their normal operations (Anonymous 1972). In California alone, A. aurantii causes an estimated $5 million loss annually in damage from loss of trees and diminished yields. The cost of spraying once or twice a year is about $45 per acre. An inspector searching visually for scale infestations covers about 5 acres a day and costs $20. In contrast, each baited sticky trap monitors from 1 to 5 acres, costs about $2, and detects infestations not readily apparent to an inspector. Tests show that such traps capture







9

4,000 to 7,000 male scales per day in areas where the pest is well established (Anonymous 1972).

Sex pheromone traps are used in surveying infestations of the pink bollwor Pectinophora gossypiella (Saunders). State and federal agencies rely on traps to monitor uninfested and lightly infested cotton areas for spread of this insect.

Although the traps are baited with a synthetic sex attractant (hexalure or cis-7-hexadecenyl acetate) (Sharma et al. 1973), specific for males, the true sex pheromone has now been identified (Hummel et al. 1973) and can also be used in traps. McLaughlin et al. (1973) demonstrated that living females were more effective in locating low population levels of males than hexalure-baited traps.

The USDA uses disparlure, the sex pheromone of the gypsy moth, Porthetria dispar (L.), to bait its 60,000 survey traps in the United States. Gypsy moths have been detected with these traps in many places where they were not previously known to exist (Beroza 1971).

Grandlure, an aggregating and sex pheromone of the boll weevil, Anthonomus grandis Boheman, has been used by the USDA in cotton areas of the United States to bait traps which attract and destroy weevils emerging from hibernation during spring. This monitoring plan provided for intensification of sampling effort in and around detected "hot-spots" of weevil infestations (Eden et al. 1973).

Fruit fly detection programs have been continuously run








10

since 1956 by the Division of Plant Industry and the USDA throughout the state of Florida. Two types of traps are used. The combination (Steiner) trap is baited with cue-lure, methyl eugenol, and trimedlure. Cue-lure attracts melon and Queensland fruit flies; methyl eugenol attracts Oriental fruit flies; and trimedlure attracts Mediterranean and Natal fruit flies. The other type of trap is the wet or McPhail

trap and is baited primarily to attract Mexican and Caribbean fruit flies. The wet trap is baited with cottonseed protein (ENT-44,014-X) which contains one per cent technical borax. This mixture is made into pellets, two of which are dropped into each trap with the required amount of water (300 ml). The traps are cleaned and rebaited once a week (Poucher 1970).

Experiments are being conducted to determine the optimum proportion of the sex pheromone and activating acids from C. capitata males for practical application (Anonymous 1973). Investigations with A. suspensa are conducted by different agencies to determine which lure or lures might be used to replace the present one, which is not considered completely satisfactory. Until a good lure is available for A. suspensa any attempt to study the effect of chemicals, sterile release, or population densities will be hindered.

Quality control of sterile insects has been monitored by many researchers using different methods. Fletcher and Giannakakis (1973b) used a sex pheromone bioassay to evaluate








11

the effect of irradiation upon sexual behavior of Dacus tryoni (Frogg.). A pheromone field bioassay, consisting of hanging baited traps containing females in trees in a grid pattern, was used by Moffitt and Hathaway (1973) to monitor the quality of adult codling moth, L. pomonella.

Olfactometers provide an additional tool to compare the effectiveness of the attractant from insects of different strains and ages, and the effect of administering different diets or radiation treatments. The comparative ability of sterile and wild individuals to elicit a response from virgin wild ones can also be studied (Steiner 1969). Potential Control with the Mass Trapping Technique

Tette (1972) reviewed mass trapping with the redbanded

leafroller, Argyrotaenia velutinana (Walker), the gypsy moth, P. dispar, bark beetles, Dendroctonus spp., and the codling moth, L. pomonella.

In an experiment that lasted 4 years, Roelofs and his colleagues (cited by Marx 1973) achieved 99 percent control of A. velutinana in a lightly infested area by using traps baited with sex pheromones. However, in a heavily infested area, mass trapping did not produce a satisfactory control. It is possible that when populations are high, vision may be as important as pheromones for locating a mate (Marx 1973).

In 1972, tests were conducted in areas with gypsy moth populations below detectable levels by means other than pheromone traps. Glassine-liquid cardboard tube traps coated







12

inside with Tack-Trap containing disparlure were distributed by aircraft over large test areas. The results showed that apparently mating was not inhibited in the treated plots and was actually higher than in the controls (Cameron 1973). Some evidence (not published) was later obtained that the sticky Tack-Trap was perhaps too good a keeper for the lure, retaining rather than releasing it. A continuing study of gypsy moth populations with pheromone traps is in progress.

Southern pine beetles, Dendroctonus frontalis Zimmerman, concentrated their attacks on highly immune longleaf pine trees, Pinus palustris, when these trees were baited with the aggregation pheromone of the beetles. As a result they were completely decimated by the resins from the trees (cited by Grosser 1971).

Hardee et al. (1970) showed that in areas with low populations of boll weevils, wing traps baited with males were efficient in suppressing the spring infestation until migrant weevils infested the field in late summer. A large-scale experiment was conducted on a core area of roughly 25 miles radius around Columbia, Mississippi to determine if various population suppression techniques, when used in concert, were adequate to eradicate a boll weevil population from a prescribed area (Eden et al. 1973). One of the population suppression techniques used was traps baited with the sex pheromone, grandlure. The results showed that pheromone traps were effective in monitoring the population and also








13

in detecting "hot-spots" of weevil infestation, although effectiveness declined as cotton reached the squaring stage. The committee that reviewed this pilot experiment (Eden et al. 1973) strongly recommended continued research on the pheromone "grandlure" as a population regulation tool. Potential Control with the Confusion Technique

Promising experiments on male confusion or inhibition were conducted by Shorey et al. (1971) at the University of California at Riverside with the cabbage looper moth, Trichoplusia ni (Hubner). The synthetic sex pheromone, continuously evaporated at a rate of 1 mg per ha per night, almost completely prevented males from locating pheromone-releasing females. The cost of the program was $0.01 per ha per night. The only remaining problem, according to Tette (1972), will be to prevent gravid females from entering or migrating into the test area.

Several tests conducted in 1971 and 1972 with the gypsy moth, P. dispar, suggested that it might be possible to confuse male gypsy moths and prevent them from finding females (Stevens and Beroza 1972, Cameron 1973). In 1971, pieces of disparlure-treated paper were uniformly distributed by aircraft over 40-acre-plots. VMales released periodically in these plots were unable to find traps containing unmated females or disparlure for 6 days. In contrast, males were readily captured by such traps in untreated plots. However, by the 21st day the attraction of the treated papers appar-







14

ently had declined sufficiently to allow some males to locate traps containing females or disparlure (Beroza and Knipling 1972).

Cameron (1971) tested granular cork impregnated with

disparlure and a microencapsulated formulation of disparlure containing a sticker to adhere the capsules to the foliage and achieve vertical stratification of the lure. These microcapsules released the pheromone gradually so that it was expected to be effective for 6 to 8 weeks. The results of spreading granular cork impregnated with disparlure in areas with a natural population indicated a reduction in mating of about 46% compared with the control. The results with microcapsules in areas where laboratory-reared insects were previously released were very encouraging, and no successful mating was detected in the two treated plots. Integration of Sex Pheromones with other Control Agents

Many sterile-release programs have been less successful than hoped for because the irradiated insects did not compete well with wild ones. Irradiation in many cases (Steiner and Christenson 1956, LaBrecque and Keller 1965, Holbrook and Fujimoto 1970, Hooper and Katiyar 1971, Ohinata et al. 1971) reduces the vigor of insemination, mating competitiveness, and sex pheromone production by the sterile insects. It would be desirable then to counter with an alternative method which would enhance the sterile insects, even if their condition is near normal or completely normal, so that the ste-







15

rility principle itself can be raised to a higher level of efficiency. Chambers et al. (1972) suggested that treating tephritids with attractants or a sex pheromone might attract native partners to sterile mates, allow them to compete more effectively with wild males for wild females, or make it possible to use the sterile flies as disease or toxicant carriers. They were able to prove that untreated flies of the Oriental fruit fly, Dacus dorsalis Hendel, and of the melon fly, Dacus cucurbitae Coquillet, were attracted to flies treated with synthetic attractants.

The male annihilation technique was used successfully

by Steiner et al. (1965) and by Steiner et al. (1970) in the Mariana islands to eradicate the Oriental fruit fly through aerial distribution of fiberboard blocks containing synthetic male lure, methyl eugenol, and an insecticide, naled. In a more recent experiment, Cunningham and Steiner (1972) reduced the populations of male D. cucurbitae more than 99% through the distribution of fiberboard blocks soaked with cue-lure and a solution of 5% naled (wt/wt) in a 2-squaremile plot in Hawaii. Cunningham et al. (1970) and Cunningham et al. (1972) reduced substantially the populations of D. dorsalis, D. cucurbitae and C. capitata for periods of up to two weeks by spraying the plot areas with a formulation of cue-lure and methyl eugenol mixed with 5% technical naled.








16


Sex and Aggregating Pheromones in the Tephritidae


Female-produced Pheromones

Literature reviewed by Jacobson (1972), Law and Regnier (1971), and Beroza (1972) indicate that no species of tephritid is known in which the female attracts the male. However, Prokopy and Bush (1972) presented evidence that mature females of R. pomonella produce and deposit an unidentified marking pheromone on fruit. The marking pheromone serves both to act as a male arrestant and to regulate numbers of eggs deposited per fruit. Male-produced Pheromones

Among the cases of male-produced pheromones reported by Jacobson (1972) seven species of tephritid flies were included: the Mediterranean fruit flyC. capitata, the solanum fruit fly,Dacus cacuminatus (Hering), the melon fly,D. cucurbitae, the Oriental fruit fly,D. dorsalis, the olive fruit fly,Dacus oleae (Gmelin.), the Queensland fruit fly,D. tryoni, and the island fruit fly,Rioxa pornia (Walker).

Jacobson et al. (1973) identified two components of the

sex pheromone of male medflies as a combination of methyl (E)6-nonenoate and (E)-6-nonen-1-ol. Fletcher (1968, 1969) described the rectal gland complex responsible for the production and storage of the sex pheromone of D. tryoni males.







17

Similar glands have been found in the males of Dacus neohumeralis Birch, Dacus kraussi Hardy and Afrodacus jarvisi (Tryon) (Fletcher 1969), and in D. dorsalis, D. cucurbitae and in D. oleae (Schultz and Boush 1971, Economopoulos et al. 1971).

Evidence for a sex pheromone produced by the males of R. pornia and A. suspensa was given by Pritchard (1967) and Nation (1972), respectively. Nation (personal communication) identified the major components of the male-produced pheromone of A. suspensa as two lactone esters and two alcohols. These compounds, when fractionated and tested individually in the laboratory showed reduced or no biological activity, although when recombined, they strongly attracted mature females.

Aggregating Pheromones

Insects aggregate for various purposes including mutual protection, hibernation, aestivation, mating, and oviposition (Butler 1967). Aggregations may be temporary or persistent (Butler 1967). Temporary aggregations are those that occur during part of the life cycle and are intended usually for only one biological activity (hibernating Coccinellidae, mating swarms of Ephemeroptera, etc.). Persistent aggregations occur only in social insects living in colonies in which the well-being of each individual depends on its companions. Usually, one or more pheromones are involved, together with several other biological and physical factors of the environment in the promotion and maintenance of aggregation (Butler 1967).







18

Jacobson (1972) cited 42 cases in which assembling or

aggregating pheromones lure both sexes, including 32 species of Coleoptera, 9 species of Orthoptera, and only one species of Hemiptera. Some of these aggregating compounds are produced by females and some by males, but they attract both sexes.

Although no chemical has been identified from tephritids that causes aggregation, several authors have suggested that aggregation occurs in the field in response to host odors or other factors. Cunningham and Steiner (1972) called localized pockets of high populations of males of D. cucurbitae "hot-spots". Males of D. tryoni are attracted to their own pheromone, and apparently feed upon the secretions from the rectal glands of other males (Fletcher 1968). Fletcher suggested that the pheromone may play a specific part in the sexual behavior of males, and cited some evidence that males congregate together when stridulating. Bateman (1972) observed mating swarms of highly active males of D. tryoni aggregating at dusk in an orchard containing a dense population. He suggested that tropical species of fruit flies tend to congregate in locations which provide shelter and food and that these locations are usually restricted to patches of evergreen foliage such as citrus, banana, or other "favorable" plants. He further suggested that adults feed during the warmer hours of the days, but return to the same sheltered foliage when temperatures fall.








19
Males and females of Eutreta sp. fed upon froth masses

secreted by other males from the proboscis, indicating a possible attraction of both sexes to the same chemical (Stoltzfus and Foote 1965). Feron (1962) reported that males of the medfly are attracted to the "sexual odor" of other males.

In one well-studied fruit fly, R. pomonella, neither

male nor female seems to produce an attractant (Prokopy and Bush 1972). Instead of attraction to a pheromone odor, both sexes are attracted to fruit odors, and thus the sexes come into close contact. Females also deposit a marking pheromone onto the fruit following oviposition. The marking pheromone acts as a male arrestant. Thus the biological purpose of retaining males at a site where females have been and are likely to be is served. Prokopy and Bush (1972) referred to these effects as aggregation of flies.


Courtship and Mating Behavior in the Tephritidae


Three fundamental aspects of the sexual behavior of any

species are courtship, mating, and coitus (Guhl 1968). Courtship was defined by Oliver (1955) as the behavior preceding mating that stimulates one or both individuals and initiates the mating performance. It is considered separately from the act of mating, but it may be difficult to designate the point at which courtship ends and mating behavior begins.- According to Bastock (1967) in a typical case of courtship, individuals of one sex display to the opposite sex immediately before


I







20

mating. This implies communication between displayer and audience.

Prokopy and Bush (1973) gave a detailed and extensive description of the courtship behavior of R. pomonella. Mature flies gather at the host fruit, and periodically move from fruit to fruit as if in search of potential mates. Flies spend as much as 89% of their time on fruits, while only 10% is spent elsewhere in the trees. The predominant activity of females on the fruit is oviposition-type activity, while only a very small percentage of females (or males) engage in feeding-type activity. Either sex may initiate the flight which brings it to the particular fruit occupied by a member of the opposite sex. Prokopy and Bush (1972) concluded that olfactory and acoustical stimuli emanating from other apple maggot flies did not play a role in eliciting any response from males or females several cm or more away.

Many species of Dacus initiate mating activity at dusk. In laboratory cages D. tryoni and D. cacuminatus usually mate on the dark ceiling of the cage (Myers 1952), and in outdoor cages mating was restricted to a period of about 30 minutes at dusk (Tychsen and Fletcher 1971). For many other species of Tephritidae, falling illumination at dusk acts as a stimulus for the initiation of sexual activity. These include D. dorsalis (Roan et al. 1954, Chambers et al. 1972); D. tryoni (Barton 1957, Myers 1952); Dacus ciliatus Lw.







21

(Syed 1969); Euleia fratria (Loew) (Tauber and Toschi 1965a); Dacus zonatus (Saund.) (Syed et al. 1970); Dacus scutellaris Bezzi, Dacus diversus Coq. (Syed 1970); D. cucurbitae (Back and Pemberton 1914, Schroeder et al. 1973); D. cacuminatus (Myers 1952); D. oleae (Economopoulos et al. 1971); and Anastrepha ludens (Loew) (Baker et al. 1944). In contrast to most of the Dacinae, D. neohumeralis mates during the day at high light intensities (Gee 1969).

A "call" produced by immobile males of D. tryoni and D. oleae when vibrating the wings rapidly was described by Myers (1952) and Martelli (1908), respectively. The "call" is clearly heard as rather high, flute-like notes emitted in series, each note varying in duration from a half to two seconds and rarely longer (Myers 1952). Monro (1953) described a similar mating call in D. tryoni, but the sound consisted of a stridulation produced by drawing the wings across a pair of combs formed from large bristles on the third abdominal tergite. The sound, audible to the human ear, consisted of a high pitched buzz. Sound production by males has been observed in D. cacuminatus (Myers 1952), which produces an audible higher pitch than that of D. tryoni; in D. dorsalis (Roan et al. 1954); in D. oleae (Fe'ron 1960); in D. cucurbitae (Back and Pemberton 1917); and, in D. passiflorae Frogg. (Simmonds 1936).

The sexual behavior of the medfly C. capitata was extensively treated by Fe'on (1962). The basic behavior was







22

the same in nature as in the laboratory. Attraction of the mate pair from a certain distance occurs in response to a chemical signal; and the sexual "parade" that occurs after the pair gets closer is in response to other signals (mainly visual). The sequence of activities involved, according to Fe'ron (1962), is as follows: (1) the sexually excited male stays motionless, and the female is attracted to the male. The abdomen of the male is strongly retracted longitudinally and expanded laterally into pouches, in a similar way as in the picture of males of the asparagus fly, Platyparea poeciloptera Schrank (Se'guy 1951), the island fruit fly, R. pornia (Pritchard 1967), and the Caribbean fruit flyA. suspensa (Nation 1972). The male of C. capitata also exhibits an anal "ampoule", apparently inflated and shiny like a liquid droplet. This posture is related to the release by sexually mature medfly males of an odorous pheromone that attracts mature virgin females (Fe'ron 1959, Lhoste and Roche 1960);

(2) a female fly arrives a few centimeters away from the male, who in turn vibrates both wings rapidly; (3) the female slowly orients toward the male in a face to face position; (4) when the flies are closer, the excitation of the male increases, characterized by movements of the wings. Fe'ron (1962) believed that the rapid wing vibration served to emit a sonorous signal, to produce an air current, and to release a chemical; (5) the last ,step is the leap of the male onto the

female, and mating ensuns.







23

Holbrook and Fujimoto (1970) determined that the peak mating response in C. capitata occurs at about 0600 hours. Myburgh (1962) reported the lowest temperature for mating of medflies to be 190 C, and the most favorable zone was between 220 and 300 C. However, Steiner (1967) mentioned that other workers had observed mating in C. capitata early in the morning at temperatures of 170 C and above, and also at 40.50 C under high humidity. The range of relative humidities at which mating occurred in C. capitata was between 30 and 60% according to Myburgh (1962). He considered that light intensity was the environmental factor which had the greatest bearing on differences in mating habits observed in medflies.

Oldroyd (1964) reported that males of an African species, Afrocnerus mundus (Loew), use small balls of foam as mating lures. Similar behavior was reported by Stoltzfus and Foote (1965) in Eutreta sparsa (Wiedemann). Pritchard (1967) described extensively and in detail the mating behavior of the. island fruit fly, R. pornia, whose male also produces a mound of foam when courting the female. During courtship behavior, R. pornia males distend the pleural regions of abdominal segments 3, 4, and 5. Wing waving is also common. When a female approaches the male's pleural regions deflate, and the pair make wing movements. A mound of white foam is produced and the female feeds upon the foam while the male attempts copulation. The foam apparently serves to keep the female







24

quiescent (Pritchard 1967). Copulation occurs in the late afternoon (at dusk) and is usually broken after dark.

The courtship behavior of the Caribbean fruit fly, A. suspensa, was described by Nation (1972). The mature male distends pouches from the pleural regions of the abdomen. The males also distend a thin membranous pouch of cuticle surrounding the anal area, similar to the anal pouch in C. capitata (Fe'ron 1959). While exhibiting the courtship or "puffing" behavior, the male remains relatively stationary for long periods of time. Wing waving movements are interrupted by bursts of rapid wing vibration. The metathoracic legs are frequently rubbed over the abdomen and the puff areas, and then over the wings. Nation (1972) relates this cleaning behavior to the spread of a sex pheromone over the body and wings, providing a greater surface from which it can evaporate. When a female approaches, the male ceases wing fanning and puffing, crouches with the body near the surface, and appears to touch the surface with extended proboscis. According to Nation (personal communication) deposition of chemicals may take place when males extend the proboscis to touch the substrate, and these chemicals could serve to keep the female quiescent. When the female advances and contacts the male, he leaps upon her and attempts to copulate.

Many tephritid males will court individuals of their

own sex as well as females (Boyce 1934, Economopoulos et al.







25

1971, Fel'on 1962, Nation 1972, Prokopy and Bush 1973, Tauber and Toschi 1965a, 1965b).

Wing waving and stridulation seem to be important in the sexual behavior of most tephritids as many authors have reported. Some (Fletcher 1968, Fletcher and Giannakakis 1973a, Feron 1962, Nation 1972, Pritchard 1967, Tychsen and Fletcher 1971, Tauber and Toschi 1965a, 1965b) believe wing movements facilitate evaporation of a pheromone by production of air currents, as well as subserve a visual function; others (Back and Pemberton 1917, Fe'ron 1960, Fe'ron 1962, Fletcher 1968, Fletcher and Giannakakis 1973a, Monro 1953, Myers 1952, Roan et al. 1954, Simmonds 1936) believe the sounds produced

during waving and stridulation of the wings enable the female to find the precise location of the male.

In summary the sexual behavior of Tephritidae involves auditory, visual, gustatory, olfactory and tactile stimuli. Bateman (1972) considers smell and hearing as the two most important sensory stimuli for mating response in the tephritids.


Courtship and Mating Behavior in other Diptera

The sexual behavior of some other dipterous species closely resembles the behavior of tephritid species.

Courtship behavior in the Hawaiian Drosophilidae does not occur at the feeding sites. Males tend to assemble in considerable numbers at specific localized sites in the







26

vegetation surrounding the feeding and oviposition sites. Such breeding sites retain their attractiveness over prolonged periods of time. Each male "patrols and defends" a small and limited area (i.e. a single leaf) and simultaneously "advertises" his presence by body movements and especially wing movements. Spieth (1968) called this behavior "true lek behavior" and considered it an adaptation to avoid predators. Males of some Drosophila species repeatedly drag the tip of their abdomen over the substrate and in doing so deposit a thin film of liquid, while others assume a ritualized posture and extrude and retract a bubble of liquid from their anal papillae. Spieth (1968) suggests that scents from these fluids attract females into the immediate vicinity of the male.

Carlson and Beroza (1973) found that (Z)-9-tricosene,

the sex pheromone of the house fly, Musca domestica L., acted as an aggregation pheromone in the field, rather than just as a sex attractant for males as in the laboratory bioassays. Willson and Mulla (1973) observed distinct zones of activity in poultry ranches in which mating females were concentrated, while recently emerged and gravid females were fairly evenly dispersed along the cage rows of the poultry house. Such behavior suggested to Willson and Mulla (1973) the existence of specific zones of mating activity.






27


Economic Importance of the Tephritidae


The distribution of the family Tephritidae is virtually worldwide but it is largely restricted to the temperate, subtropical, and tropical parts of the world. About 4,000 species have been described. They are usually referred to as fruit flies because some are among the most destructive pests of fruit known.

In the typical developmental cycle, gravid females insert their eggs into plant hosts with an eversible, sclerotized ovipositor. The developing larva sheds its skin twice as it feeds and grows. An inactive fourth-instar larval stage within the puparium precedes the formation of a pupa. Pupation may take place within or on the host plant, but most often it occurs in the soil. Diapause is inherent in many temperate-zone tephritids but has been inadequately studied (Foote and Blanc 1963).

Miller (1970) considered the total economic losses due to fruit flies to include the following:

(1) Direct loss of fruits and vegetables.

(2) Cost of control treatments and extra labor.

(3) Reduction in market value of the produce.
(4) Drawback to horticultural development and/or prospective markets.

(5) Indirect cost of numerous stringent quarantine measures.

The amount of damage which could result through the in-







28


troduction of fruit flies into fly-free areas is so great that elaborate efforts are usually taken to prevent establishment in new territory. As an illustrative example, the second medfly introduction into the state of Florida in 1956 covered 28 counties and required treatment of more than
6 million acres with chemical poisons and bait sprays. Throughout the state 46,499 traps were intensively monitored. The peak work load involved 701 state personnel (excluding personnel contracted to do aerial spraying and federal personnel). Fumigation chambers were established in 264 sites and numerous road blocks resulted in inspection of 4,672,901 vehicles. The state and federal funds expended amounted to almost 10 million dollars (Oberbacher and Denmark 1957).

More than 200 varieties of fruits and vegetables are hosts of the medfly (Christenson and Foote 1960). Other species of fruit flies that attack close to 100 or more plant varieties are the melon fly, D. cucurbitae, and the Oriental fruit fly, D. dorsalis, both present in Africa, Indonesia and many Pacific islands including Hawaii; the Queensland fruit fly, D. tryoni, present in Australia (Christenson and Foote 1960); and the Caribbean fruit fly, A. suspensa (Swanson and Baranowski 1972).

A. ludens, the Mexican fruit fly, breeds in wild and

cultivated citrus in north-eastern Mexico and each year migrates into the Rio Grande Valley of southern Texas. It has spread also into the cultivated citrus sections of the west








29

coast of Mexico and northward toward Arizona and California, resulting in continual detection, survey, quarantine measures, and eradication campaigns (Weems 1963). Of less importance are Anastrepha interrupta Stone, Anastrepha serpentina Wiedemann,and Anastrepha mombinpraeoptans Seln (Weems 1967, 1969, 1970). All have been found in parts of the United States (southern tip of Florida and/or Rio Grande Valley of Texas), and the latter two are distributed along Central America and part of South America. An economically important species not present in the United States is t.he South American fruit fly, Anastrepha fraterculus (Wiedemann),which is distributed from Texas to Argentine and in the Caribbean islands of Trinidad and Tobago.

Other species of fruit flies of economic importance in the continental United States are the apple maggot fly, R. pomonella, attacking apples, pears, plums,and other deciduous fruits in northeast U. S. and southeast Canada, and the walnut husk fly, Rhagoletis completa Cresson, that attacks all Juglans spp., peaches,and other fruits in the-western U. S. (Christenson and Foote 1960). Rhagoletis cingulata (Loew) and' Rhagoletis indifferens Curran (eastern and western cherry fruit fly, respectively) damage sweet and tart cherries (Bush 1969).

A. suspensa is one of several species of fruit flies

which are indigenous to the West Indies. It invaded Florida on three occasions, the last in April 1965 at Miami Springs








30
(Dade county) (Weems 1965, 1966). Since then it has spread over 21 counties throughout the southern and central part of the state. Within its normal range of distribution the economic damage caused by this species has been relatively small, although more than 80 species of plants, including tomatoes and bell pepper, are hosts of the Caribbean fruit fly. Preferred hosts include common guava, Psidium guajava L., cattley guava, Psidium cattleianum Sabine, Surinam cherry, Eugenia uniflora L., tropical almond, Terminalia catappa L., loquat, Eriobotrya japonica (Thunb.) Lindl., rose apple, Syzygium jambos (L.) Alst., Barbados cherry, Malpighia glabra L., and calamondin, Citrus mitis Blanco (Swanson and Baranowski 1972). A strain of A. suspensa found in Puerto Rico attacks a number of tropical and subtropical fruits, including grapefruit, Citrus paradisi Macf., sour orange, Citrus aurantium L., and sweet orange, Citrus sinensis Osbeck (Weems 1965). There is no assurance that A. suspensa could not become a major pest of citrus or other crops such as peaches and apples, found in Florida or neighboring states. Thus far A. suspensa has been collected only in Florida, Puerto Rico, Dominican Republic, Haiti, Jamaica and Cuba (Weems 1965, Anonymous 1970).


Taxonomic Relationships

The first taxonomic summary of North American Tephritidae was presented by Loew (1862), who placed all the known species







31

in the genus Trypeta. In 1868 Schiner described the genus Anastrepha. In 1873 Loew again reviewed the family, but in much greater detail, assigning the species to various genera. He erected the genus Acrotoxa for Anastrepha. Bezzi (1909) placed the species back in Anastrepha and included only 19 species. Hendel (1914) in his revision of the genus treated 34 species, including a number of new ones. The genus then remained untouched until Costa Lima (1934) described 62 species, based almost entirely on Brazilian material, of which 22 species and 1 variety were new. His paper was the first that used extensively the ovipositor as a character. Greene in 1934 reviewed the genus Anastrepha, and based on a study of the wings and on the length of the ovipositor sheath included 54 species, of which 16 were new. Further work by Stone (1942), based on the wing patterns and on the female terminalia, removed several species and added new ones to the genus, ending with 114 species, of which 49 were new species.

Host races are common and these may be rather rapidly

formed within a species. It is generally assumed that physiological differences enable a population formerly using only one species of plant as a host to maintain itself on another with apparent ease. These populations are often beyond the eyes of the museum taxonomist if the differences are of relatively recent origin and they are among the most difficult of taxonomic problems. Some species are morphologically separable only by extremely obscure characters and satisfac-








32

tory identifications can be made only when such supporting data as host, locality, and date are supplied with the specimen (Foote and Blanc 1963).

Unfortunately, most of the keys are designed for females only. Greene (1934) devised a key for males. There remains great need for biological work, since hosts are not known for many species.

The following names and synonyms have been used in the past for A. suspensa:

Trypeta suspensa Loew

(Trypeta) Acrotoxa suspensa (Loew)

Anastrepha unipuncta Sein

Anastrepha longimacula Greene

Crosses of A. suspensa females with A. mombinpraeoptans males in the laboratory have been obtained on two occasions. The offspring show characters of both parents, but it is rather doubtful that such crossing takes place in nature, according to Stone (1942), who never saw any specimens collected in the field that agreed with these hybrid specimens.












:ATERIALS AND METHODS


Experimental Insects


All flies used in these experiments were reared at the University of Florida, Agricultural Research and Education Center (AREC) in Homestead, Florida. Flies were sexed and separated when between 1-3 days of age. The sexes were kept in separate rooms in large screened cages except when mated females were needed (Fig. 1). Sugar and water (as 1% agar) was placed at the top of the cage and yeast hydrolysate paste on one of the screen mesh wire sides. Food was replaced or added when necessary. To obtain mated females a ratio of

1 : 2 ( was maintained in the same cage. An aspirator consisting of a bag sleeve on one end tied to a plastic tube 38 cm long and 1.3 cm diameter and connected to a vacuum pump was used to collect flies. About 500 flies could be collected each time without any apparent adverse effect on the flies' vigor. Flies were never handled with the hands nor were they anesthetized when segregating sexes or transferring from cage to cage.

Flies to be released for recapture bioassays were transferred on the day of the release to screen mesh cylinders 16 cm long and 6 cm diameter with an aspirator after knocking the flies down in a freezer for approximately 3 minutes.

33







34


'UP


Figure 1.-


Cages used to maintain adult A. suspensa in the laboratory. Dry sucrose, yeast hydrolysate paste, and water as 1% agar was provided at the top of the cage and was replaced or added when necessary.







35

Flies were always- allowed to emerge from the cylinders in the field by themselves (Fig. 2).


Field Bioassays


Field bioassays utilizing release and recapture of labreared flies tested the sexual attraction of caribflies. Bioassays were conducted at the Homestead AREC in approximately 1 ha of an isolated avocado grove (Fig. 3). Avocados are not a host of the caribfly, and the nearest host trees were guavas approximately 0.7 km away. No wild flies were observed in the avocado grove, and only plowed ground occurred between the avocado and the guava groves. Survey traps were placed in the avocado grove before and after most of the experiments and these traps consistently indicated an absence of wild flies. In tests with flies marked with fluorescent powders, all flies captured were marked, further confirming the absence of wild flies.

Avocado trees were spaced 6 m apart in a square design. Traps were spaced 18 m or 30 m apart and 2 or 4 trees, respectively, occurred between traps (Fig. 4). Lab-reared flies for recapture data were released at points 13 to 21 m equidistant from any trap at the intersection points of 4 traps arranged in a square.

Three different traps were used in the bioassays:

(1) Live males or females were caged in 16 cm long and 6 cm diameter screen mesh cylinfers which were attached to stickum-








36


Figure 2.- Adult A. suspensa females emerging from a release
cylinder in the field.








37


(A. ~. 4 4, AT*. -


Figure 3.- Partial view of the avocado grove showing the
grid pattern distribution of trees.





0


0


of
0
0


0


a
Cl


0


0

0
0
C


H - i


0
0
0
C
0
0


C
C


C
0


00
00


C
0


C
C


1 30m


Figure 4.- Diagram of a section of the avocado grove showing the distribution of traps,
release points, and tree orientation when traps were spaced 18 m and 30 m
apart. , avocado trees; 4 , trap trees; *.I~ # release points


00
*C


C

0
0
0


C

0
C
U


11-j


-i







39
coated cotton boll weevil traps (Cross et al. 1969). The traps, also called wing traps, consisted of four yellow crossbaffles 15.2 cm X 25.4 cm height with a top of 23 X 23 cm (Fig. 5). The cylinder was placed at the center of the trap at the bottom. Flies in cylinders had access to dry sucrose, yeast hydrolysate paste, and water. Control Stickem-coated traps contained food only.

(2) Glass invaginated McPhail traps, currently used in the field trapping program in Florida (Poucher 1970) to study the population of Anastrepha spp., were used in several experiments. They were originally used and described by McPhail (1939). In most of the experiments, 300 ml of aqueous suspension of yeast hydrolysate were used per trap, which were always prepared 3 days in advance of the experiment date. However, in one bioassay the yeast hydrolysate was replaced with distilled water containing 3 drops of an emulsifier (Pergosperse L-9) (Fig. 6). The emulsifier was added to prevent flies from crawling outside of the liquid and/or the trap by reducing the intersurface tension.

(3) Triangular sticky board traps (Gutie'rrez et al. 1970), currently used in the field trapping program in Florida to detect the presence of the Mediterranean fruit fly C. capitata were used in two bioassays (Fig. 7). Dental cotton wicks 3.8 cm long X 1.0 cm diameter covered with Parafilm in the middle were hung from the center of the traps with the same wire used to hang the trap from a tree branch. A cardboard









40


U
*


b V


~\ ~
~ j~b ~ '-I
.. ~ 7~ .,
4 4
"
*'.A
- '4' h. 4
Is,
~ -.

~


Cotton boll weevil trap, also known as wing trap, used in field bioassays to test sex and aggregation pheromone of A. suspensa. Adult flies in the cylinder had access to dry sucrose, yeast hydrolysate paste, and water.


Figure 5.-







41


Figure 6.- Invaginated glass or McPhail trap used in field
bioassays. The McPhail trap is normally baited
with 2 pellets of a mixture of torula yeast hydrolysate-borax added to 300 ml of water.



























I,



A'

-~..,,.


~ ~-~-:*'A






-K S


Figure 7.- Triangular sticky trap baited with males and food.







43

inset 15.9 cm long X 9.5 cm wide was almost entirely coated with Stickem and placed on the bottom surface of the triangular-shaped trap.



Field Observations


Field observations of the activities and behavior, especially courtship and mating behavior, of wild caribflies were made during the summers of 1972 and 1973.

Sex ratios were counted at various points in host groves, and detailed observations over long periods of time were made at those sites or trees where more flies than usual were found. Courtship and mating behavior were observed. Other data recorded included the host trees and grove, height of flies location, and wind movement.

Meteorological data on temperature, relative humidity, wind movement and type of general weather were obtained from records at the AREC weather station located about 0.8 km distant from the observation sites.


Screen-mesh House Bioassays to Test Sex Pheromone


Three early experiments were conducted in a greenhouse frame covered with screen-mesh of 10 X 5 m located in block 1 of the AREC grounds. Four male-baited and 4 control wing traps, similar to the boll weevil traps (Cross et al. 1969), were suspended from the ceiling of the screen house. The 8







44
traps were arranged in two lines and spaced 3 m apart. Each male-baited trap was baited with 10 caged males in the first and second experiment and with 25 males in the third experiment. Control and male-baited traps always contained food

and water.


Laboratory Bioassays to Test Aggregating Pheromone


Attempts were made in the laboratory to demonstrate the male to male attraction found in the field. A square Plexiglas cage (Fig. 8) of 0.09 cubic meter routinely used to bioassay fractions during pheromone isolation was used in two bioassays. Six Tygon plastic cylinders were connected from the outside to holes in the only wooden side of the cage. In the center of the opposite side, a screened hole was connected through a wind sleeve to an electric fan. The fan pulled air through the cylinders at a rate of less than 8 kph. This trap design has been used by Nation (unpublished data) to test the sex pheromone.

Males to be tested for pheromone production were placed inside small screened vials containing food (Fig. 8). The vials were inserted at the air intake side of the cylinders and on the opposite side a conical screen trap was positioned. Control vials always contained food only.

Attraction was measured by determining the number of

flies moving past the conical trap from the cage to the cylinders. Tests were run with continuous light and wind move-










B


A


I


D E


.4r


Figure 8.- Diagram of the Plexiglas plastic chamber (45 X 45 X 45 cm) used as a bioassay
test apparatus in the laboratory. A, air intake; B, position of male or
control traps; C, release area; D, air exit; E, electric fan; F, cage with
screened ends for holding males and/or food; G, trap space; H, conical screen
funnel.


C


F G H








46


ment for 24 hours.

Three bioassays were conducted with the test apparatus and by the procedure reported by Nation (1972) for showing female to male attraction. Air flow from the caged males to the free males was provided by means of a compressed air tank and tubing connections to each cylinder. Males to be tested were placed inside small screened vials and these were inserted at one end of the cylinder. Males were released at the opposite end and attraction to the caged males was measured by determining the number of flies moving past a conical screen trap placed between them. Control cylinders with food only were always used. Food was provided to both sides of the cylinders (caged and free males). Data were collected periodically over a period of 2 to 4 days. A third technique consisted of placing 4 to 6 traps baited with males inside of an indoor nylon cage of 6 cubic m.













RESULTS


Recapture of Virgin Females with Male-baited Traps in the Screen House


Virgin females 8, 9, and 10 days of age were released inside the screen house in successive weeks and recapture data were obtained periodically for a period of 1 to 5 days

after the release date.

In the first experiment metal boll weevil traps with

two yellow baffles and two unpainted baffles were used, while in the second and third experiments red boll weevil plastic traps were used. Also in the third experiment a mixture of 100 ml tap water, 70 g sucrose, and 40 g hydrolyzed yeast was sprayed periodically in the screen house on small citrus trees and other plants to provide food for the released flies.

The results showed that male-baited traps were no more

attractive than control traps. In the first experiment traps baited with 10 males recaptured 38% of 500 females released and the control traps recaptured 41%. Of the females recapture&with both traps, 85% were caught in the yellow baffles. This high percentage indicated a strong attraction to the yellow color instead of to the males and/or food under screen house conditions. In the second and third experiments with red traps, the male-baited traps (10 and 25 males per trap, 47







48

respectively) recaptured 33% of 1425 females released and the control traps caught 21%.


Field Bioassays


Recapture of Virgin Females with Male-baited Traps

The attraction of females to food only, 1, 5, and 10

live caged males was compared in the first experiment. All flies were 8 days old when first put into the field. Captures were recorded over a period of 2-6 days. The experiment was replicated 4 times in successive weeks with a total release of 6,250 females. Flies were released between 1500 and 1630 hours.
Traps baited with food only captured 36 females, while traps baited with 1 male attracted 85 females (Table 1). Traps baited with 5 males attracted 227 females, and traps baited with 10 males attracted 695 females. The total percentage recovery of released females varied from 6.5 to 28% between replicates. Traps baited with 10 males attracted significantly more females than traps baited with food, 1 or

5 males (P ! 0.05), and traps baited with 5 males attracted more than those containing food only. The difference between traps baited with food and 1 male was not significant. Variation in percentages of recovery among experiments may have been caused by weather conditions prevailing when experiments were performed. In the test started on 3 May 1972 the low total recovery of 6.45% was possibly related to the fact that








Table 1.- Recapture of released A. suspensa adult virgin females with male-baited sticky
traps from tests in a non-host avocado grove at Homestead, Florida. A Latin
square 4 X 4 design was used in each test date. Flies were 8 days old when
first put into the field and captures were recorded over a period of 2-6 days.


Source Total Final %
Release date (1972)
of females recapture
5-3 5-12 5-16 5-26
attractant recaptured per trap


Food only 11 3 9 13 36 0.14 a
1 Male 16 13 30 26 85 0-34 ab

5 Males 21 51 84 71 227 0.91 b
10 Males 81 204 170 240 695 2.78 c



Total recapture 129 271 293 350 1043

Total released 2000 1000 2000 1250 6250
Final % recovery 6.45 27.10 14.65 28.00 16.69


*


by the same letter are not significantly different at 0.05 level.


Values followed







50

too many females were held in much smaller cages prior to release than in subsequent experiments. These experiments indicated that males attracted virgin females under field conditions.

A second experiment was designed to compare the attraction of the food-baited McPhail trap against 10, 20, and 40 live males. Males and females were again 8 days old when first put into the field. The experiment was replicated 4 times in successive weeks with a total release of 8,650 females. Flies were released between 1530 and 1700 hours. Captures were recorded over a period of 3-6 days.
Traps baited with 40 males attracted significantly more females than McPhail traps or traps baited with 10 or 20 males (Table 2). Traps baited with 20 males also attracted significantly more females than McPhail traps; but traps baited with 10 males were not significantly different from the McPhail traps. In 2 replicates, traps baited with 10 males captured fewer females than McPhail traps.

Recapture of females at intervals after release was recorded for the test of 26 May 1972 (Fig. 9). Most of the females recaptured were caught during the first 48 hours and during the daylight hours. From 1945 hours on 26 May to 0615 hours on 27 May only 24 flies were captured, representing only 0.29 flies per trap per hour, but during the daylight hours on 27 May'substantially more females were captured, though not as many as during the first 24 hours. These data







Table 2.- Recapture of released A. suspensa adult virgin females with sticky traps baited
with 10, 20, and 40 live males as compared with McPhail traps from tests in a non-host avocado grove at Homestead, Florida. A Latin square 4 X 4 design was
used in each test date. Flies were 8 days old when first put into the field
and captures were recorded over a period of 3-6 days.


Source Total Final %
Release date (1972)
of females recapture
5-30 6-7 6-13 6-20
attractant recaptured per trap


McPhail 97 71 26 31 225 0.65 a
10 Males 75 52 57 95 279 0.80 ab
20 Males 155 108 63 186 512 1.48 b
40 Males 356 170 93 349 968 2.80 c



Total recapture 683 401 239 661 1984
Total released 2900 2000 2000 1750 8650
Final % recovery 23.55 20.00 11.95 37.77 22.94


*


Values followed by the same letter are not significantly different at 0.05 level.







150 140 40


2000


0800


24-HOUR


" MAY 26, 1972 " MAY 27, 1972


A 27r,
Z
;00




X0


0
w


C-)

Cl)
w
-j

w
Li-


o/,/


U' N)


1200 TIME


1600


2000


Figure 9.- Total recapture of A. suspensa adult virgin females at intervals after release
May 26, 1972, at 1515 hours with four sticky traps baited with 5 males each and four sticky traps baited with 10 males each in a non-host avocado grove
at Homestead, Florida.


7


302010-


/4


C


p- -- ~ -- i-


1600


w
L.~ ~ * r~ g* i I.







53

suggest that flies were not particularly active or sexually attracted to the traps at night, and indicate a tendency to recapture more flies in the late afternoon, as happened on 27 May between 1800 and 1945 hours in which 33 flies were captured. Full darkness during late May occurred approximately at 2000 hours.

Recapture percentages at the end of 48 hours and the

total recapture percentage for 6 replicates are shown in Fig. 10. Data from 2 replicates are not included because no observations were made at 48 hours. Almost all the flies recaptured were caught during the first 48 hours following release.

An additional experiment was conducted releasing virgin females 8 days old and using boll weevil traps, each baited with 40 males, to check the maximum possible attraction that could be obtained when all the traps were baited with males. A Latin square 4 X 4 design was used on 6-28-72, 7-3-72, and 7-10-72 with traps spaced at 18 m. A Latin square 3 X 3 design was used on 7-18-72 with traps spaced at 30 m. Virgin female flies were released between 1500 and 1830 hours at the intersecting points of every 4 traps arranged in a square pattern. Captures were recorded over a period of 2-6 days. The results, presented in Table 3, showed that substantial numbers of released females were attracted. The final percent recapture varied between 22 and 48%.







48 Hours after the release = Final recovery


(6)

MI


wj W
D~ C4 0i
W


---


(4)


(2)


- I


(4)


5-16


RELEASE


5-26 6-7 DATE (1972)


Figure 10.- Recapture of virgin female A. suspense adults 48 hours after release, and
final recovery for observatTon period with sticky traps baited with males
in a non-host avocado grove at Homestead, Florida. The numerals above the
bars refer to the number of days observations were continued.


5040-


3020-


/
/
/


/


(6)


10


0


(5)


5-3


5-12


I - Em


-z


6-20








55


Table 3.-


Recapture of released A. suspensa adult virgin females with boll weevil sticky traps baited with 40 live males from tests in a non-host avocado grove at Homestead, Florida. A Latin square 4 X 4 design was used on 6-28-72, 7-3-72, and 7-10-72. A Latin square 3 X 3 design was used on 7-18-72. Flies were 8 days old when first put into the field and captures were recorded over a period of 2-6 days.


Release Total females Final % Final % recapdate released recaptured recapture ture per trap


6-28-72 1028 222 21.6 1.3

7- 3-72 1175 378 32.2 2.0

7-10-72 1300 519 39.9 2.5

7-18-72 1220 581 47.6 5.3







56


Recapture of Virgin Females and Males with Male-baited Traps

In previous experiments only females were released.

Since I did not know if females would be attracted in the presence of free males, I decided to release females and males in a ratio of 1 : 1. Females and males were released from separate cartridges at the intersecting release points of each 4 traps arranged in a square. McPhail traps, control traps containing only food and water, and male-baited traps were used. Males serving as bait and released flies were 8 days old when first put into the field.

In the first release attraction to 20 and 40 males in

traps and to McPhail traps was tested (Table 4). McPhail traps captured 36 females, while 20 and 40 males captured 135 and 222 females, respectively. Unexpectedly, males were captured by male-baited traps in approximately the same ratio as females. McPhail traps captured 26 males, while the 20 and 40 males traps captured 168 and 216 males, respectively. McPhail traps captured slightly more females than males. The final percentage recovery of released females was 29% and that of released males was 30%. Traps baited with 20 and 40 males attracted significantly more flies than McPhail traps (P ! 0.05). However, the interaction of treatments by sex was not significantly different at the 0.05 level of confidence. This experiment confirmed earlier results that males attracted virgin females under field conditions. No previous work with caribflies had suggested that males would







Table 4.- Recapture of released A. suspense adult virgin females and males with sticky
traps baited with 20 and 40 live males as compared with McPhail traps from
tests in a non-host avocado grove at Homestead, Florida. A completely randomized block design with six replicates was used on the 7-24-72. Flies were 8
days old when first put into the field and captures were recorded over a
period of 7 days.



Source*
Flies recaptured Final % recapture per trap Ratio
of
oftotal mean
attractant


McPhail 36 26 62 0.44 0.32 0.38 a 1.4:1

20 Males 135 168 303 1.65 2.05 1.85 b 1:1.2

40 Males 222 216 438 2.71 2.64 2.67 b 1:1



Total recapture 393 410 803

Total released 1365 1365 2730

Final % recovery 28.8 30.0 29.4


*


by the same letter are not significantly different at 0.05 level.


KJ-t


Values followed








58

be captured by male-baited traps, although males are routinely captured by the food-baited McPhail traps. Thus, the results were very surprising and indicated an aggregation phenomenon among males not previously known in this species.

In order to confirm these results, two additional replicates were 'made limiting the traps to McPhail traps, foodbaited boll weevil traps, and boll weevil traps baited with 40 males. Virgin males and females in a 1 : 1 ratio were again released, and the recapture results are presented in Table 5. The food-baited control traps captured 55 females, while McPhail traps and traps baited with 40 males attracted 238 and 491 females, respectively. Food-baited traps captured 38 males, while McPhail traps and 40-male-baited traps captured 128 and 482 males, respectively. As in the first demonstration with male recapture, the sex ratio of recaptured flies was 1 : 1 in male-baited traps, while the foodbaited control boll weevil trap and the food-baited McPhail traps captured more females than males. The final percentage recovery of released females was 30% and of released males was 25%. Traps baited with 40 males attracted significantly more flies than McPhail and control traps (P e 0.05). The interaction of treatments by sex was again not significantly different at the 0.05 level of confidence.

It seemed possible that males were attracted to the females which became stuck to Stickem-covered boll weevil

traps, rather than being attracted to the males acting as







Table 5.- Recapture of released A. suspense adult virgin females and males with sticky
traps baited with food only and 40 live males as compared with McPhail traps from tests in a non-host avocado grove at Homestead, Florida. A completely
randomized block design with six replicates was used in each test date. Flies were 8 days old when first put into the field and captures were recorded over
a period of 3-7 days.


Source Release date (1972) Flies Final % recap* Ratio
of 7-31 8-7 recaptured ture per trap
attractant total mean


Food only 26 13 29 25 55 38 93 0.3 0.2 0.3 a 1.4:1
McPhail 178 82 60 46 238 128 366 1.5 0.8 1.2 a 1.8:1
40 Males 194 191 297 291 491 482 973 3.1 3.1 3.1 b 1:1



Total recapture 398 286 386 362 784 648 1432 Total released 1300 1300 1300 1300 2600 2600 5200 Final % recovery 30.6 22.0 29.7 27.8 30.1 24.9 27.5


*


Values followed by the same letter are not significantly different at 0.05 level.




7


60

bait. In order to study this possibility, two replicates were conducted in which only males were released. Foodbaited McPhail traps, food-baited boll weevil traps, and boll weevil traps baited with 40 males were again used, and results are shown in Table 6. Control traps captured 55 males, while McPhail and 40-male-baited traps attracted 178 and 346 males, respectively. The final percentage recovery varied from 21% in one replicate to 27% in the other. These percentage recoveries are in agreement with those obtained in the previous experiments when both males and females were released. Traps baited with 40 males attracted significantly more males than McPhail and control traps (P ! 0.05); and McPhail traps attracted significantly more males than foodbaited boll weevil traps (P ! 0.05). These experiments confirm earlier experiments that males are indeed attracted to other males under field conditions. Recapture of Virgin Females and Males with Female-baited Traps

The results from tests of attraction of flies to food

only, 5, and 10 live females caged with food and water, serving as bait in Stickem-coated boll weevil traps and to McPhail traps are presented in Table 7. All flies were 8 days old when first put into the field. The experiment was replicated three times in successive weeks with a total release of 3,000 flies of each sex. Releases were made between 1700 and 1830 hours. Captures were recorded over a period of 5 days.

Traps baited with food, 5, and 10 females captured 117,







Table 6.- Recapture of released A. suspensa adult virgin males With sticky traps baited
with food only and 40 live males as compared with McPhail traps from tests in a non-host avocado grove at Homestead, Florida. A completely randomized block
design with six replicates was used in each test date. Flies were 8 days old when first put into the field and captures were recorded over a period of 311 days.



Source Total Final %
Release date (1972)
of males recapture
8-3 8-15
attractant recaptured per trap


Food only 26 29 55 0.38 a

McPhail 132 46 178 1.24 b
40 Males 170 176 346 2.40 c



Total recapture 328 251 579

Total released 1200 1200 2400

Final % recovery 27.3 20.9 24.1


*


Values followed by the same letter are not significantly different at 0.05 level.


ON







Table 7.- Recapture of released A. suspensa adult virgin females and males with sticky
traps baited with food only, 5, and 10 live females as compared with McPhail traps from tests in a non-host avocado grove at Homestead, Florida. A Latin
square 4 X 4 design was used in each test date. Flies were 8 days old when
first put into the field and captures were recorded over a period of 5-9 days.


Source Release date (1973) Flies Final % recapRatio
of 6-26 7-2 8-2 recaptured ture per trap
attractant d'' CI' I total 0 ('mean

Food only 38 50 20 23 28 44 86 117 203 0.7 1.0 0.8 1:1.4

5 Females 56 93 52 48 18 27 126 168 294 1.0 1.4 1.2 1:1.3
10 Females 82 106 54 70 13 20 149 196 345 1.2 1.6 1.4 1:1.3
McPhail 48 43 124 137 60 60 232 240 472 1.9 2.0 2.0 1:1


Recaptured 224 292 250 278 119 151 593 721 1314 Released 1000 1000 1000 1000 1000 1000 3000 3000 6000
Recovery (%) 22.4 29.2 25.0 27.8 11.9 15.1 19.8 24.0 21.9







63

168 and 196 males, respectively, while McPhail traps baited with protein hydrolysate attracted 240 males. The final percentage recovery of released males in replicates of the experiment varied from 15 to 29%.

Traps baited with food, 5, and 10 females captured 86, 126 and 149 females, respectively, while McPhail traps attracted 232 females. The final percentage recovery of released females varied from 12 to 25% between replicates.

McPhail traps attracted more virgin males and females

than traps baited with food, 5, or 10 females, although they were not significantly different at 0.05 level of confidence. In contrast to all previous experiments, this is the first time in which McPhail traps captured more flies than traps baited with live flies. The results indicate that virgin

females do not attract virgin males or females under field conditions as well as McPhail traps. The differences between traps baited with food, 5, or 10 females were not significantly different at the 0.05 level of confidence. Effect of Age and Virgin or Mated Status Upon Female Attraction to Male-baited Traps

These experiments were conducted in 1973. Stickemcoated boll weevil traps were baited with 40 live males, 9 days old at the beginning of each experiment. Traps were spaced 30 m from each other in a square pattern. All females were released at six intersecting release sites of each 4 traps arranged in a square and each age group was marked with a fluorescent powder applied to the pupae at the









64

rate of 4 g/liter (Steiner 1965). The emerging flies became marked as they crawled up through the dye.

Mated females were obtained by keeping both sexes together after adult eclosion. Dissection of the female reproductive system in samples from each group indicated that 100% of the females mated. The virgin female groups were kept in a separate room isolated from male contact and/or odors.

Three experiments were conducted with release of different age groups. In the first experiment 2-, 6-, and 9-day virgin and 9-, 11-, and 15-day mated females were released. For the second experiment 1-, 7-, and 9-day virgin and 9-, 12-, and 15-day mated females were released, and in the third experiment 4-, 11-, and 13-day virgin and 13- and 16- or 19day mated females were tested.

Females were the age indicated upon the day they were

released. A total of 1,000 females per age group was released in the first experiment except in the 2-day virgin group in which 600 flies were released. In the second and third experiments 400 flies per age group were released. Male-baited traps were prepared and put into the field during the morning and female flies were released between 1600 and 1900 hours of the same day. Captures were recorded approximately 24, 48, and 96 or 114 hours after the release. Recaptured flies were identified under a UV lamp in the laboratory.

The results of the first experiment, presented in Table 8, show that more virgin flies were recaptured than mated







Table 8.- Recapture of released A. suspense adult virgin and non-virgin females of different age with sticky traps baited with 40 live males from tests in a non-host avocado grove at Homestead, Florida. A completely randomized block design with twelve replicates was used on 8-19-73. Caged males were 9 days old when first
put into the field.


Age (days)/mated Days after release Total Final %
females recapture
status 1 2 6 recaptured per trap*

2 Virgin* 24 53 25 102 1.42 b
6 " 34 72 69 175 1.46 b

9 " 189 49 17 255 2.12 c
9 Non-virgin 92 47 12 151 1.26 b

11 87 15 8 110 0.92 a

15 " 75 15 1 91 0.76 a

Total recapture 501 251 132 884
Final % recovery 8.9 4.5 2.4 15.8


600 virgin females were released at 2 days and


1,000 females released in all other


age groups.

Values followed by the same letter are not significantly different at 0.05 level.


*








66


flies. During the first 24 hours following the initial release of flies, the age group showing the greatest recapture was 10-day virgin females. It is noteworthy that the greatest total recapture of flies occurred in the first 24 hours, confirming earlier findings presented in Fig. 9.

There was good recapture of all three age groups of

virgin flies. Although 2- and 6-day virgin females were not recaptured well during the first 24 hours, the recapture numbers increased sharply by the time these flies had become

4 to 8 days old. Total recapture data suggest that the young 2-day and 6-day females were staying in the avocado grove in large numbers for up to 6 days, whereas most females released at 9 days of age or older were not recaptured after 2 days and had presumably left the avocado grove. These data also support similar recapture data from virgin females released at 8 days of age in previous bioassays reported in Tables 1 and 2.

Failure to recapture very many 2-day virgin females in the first 24 hours in contrast to the final good recapture indicates that very young females are not highly attracted to males in the field, but are more attracted as they age.

The greatest total recapture (25.5% of those released) occurred in the group of flies released at 9 days of age, and here the great majority of those recaptured were caught by the time the flies were 10 days old. The results strongly suggest that an optimum age for attraction of females is near








67


10 days of age, and although mated females near this optimum age can still be attracted in substantial numbers, older mated females are poorly attracted. However, the initial response of mated females released at 9 days of age was reduced about 50% compared with virgin females of the same age. Although the recapture of mated flies 11 and 15 days old was the lowest, a substantial percentage (352/3000 X 100) of mated flies were recaptured, which is of importance to future possible use of pheromone-baited traps.

The results from the second experiment, shown in Table 9, indicate as in the previous experiment that about twice as many virgin females were recaptured as mated females. The importance of the first 24 hour recapture was not clearly demonstrated in this experiment. Results again show the largest first day recapture of virgin flies that were 10 days old. The recapture of flies released at 7 days of age parallels that in the previous experiment with the 6-day flies, namely, that substantial numbers of flies were caught only after they had become 9 days old. Very few virgin flies released on the day they emerged (1-day-group) were recaptured. These data again suggest an optimum recapture age or response to the male pheromone.

It is a measure of the consistency of the results from

these experiments that a total recapture of 23 to 25% (93/400 X 100 and 255/1000 X 100, respectively) of flies 9 days old at release occurred in the first and second experiments, and







Table 9.- Recapture of released A. suspensa adult virgin and non-virgin females of different age with sticky traps baited with 40 live males from tests in a non-host avocado grove at Homestead, Florida. A completely randomized block design with twelve replicates was used on 9-17-73, and 400 females of each group were released. Caged males were 9 days old when first put into the field.


Age (days)/mated Days after release Total Final %
status 1 2 4 females recapture
recaptured per trap

1 Virgin 9 5 2 16 0.33 a

7 " 16 57 27 100 2.08 d
9 " 53 34 6 93 1.94 d
9 Non-virgin 17 21 10 48 1.00 c
12 " 15 15 6 36 0.75 bc

15 " 10 11 7 28 0.58 ab


Total recapture 120 143 58 321

Final % recovery 5.0 6.0 2.4 13.4


*


by the same letter are not significantly different at 0.05 level.


co







69

from 17 to 25% (175/1000 X 100 and 100/400 X 100, respectively) of flies released at 6 to 7 days were recovered.
The results of the third experiment, summarized in Table 10, are consistent with those from the two previous ones. Although equal numbers of virgin and mated females were not released, a greater percentage of virgin flies than mated ones were recaptured. The 24 hour recapture was greater than for any following period, and young females (4 days old at release) were caught in increasing numbers as they aged. A total of 20.5% (82/400 X 100) of the virgin 11-day females were recaptured over the 4 day observation period, suggesting that 11-12 days is still near a broad peak of maximum attraction to males. In this experiment as in the two previous ones, older mated flies were very poorly attracted.

The combined results from these three experiments suggest that virgin females 9-12 days old are maximally attracted to males in field tests. This includes the same range found in laboratory tests (Nation 1972). Both virgin and mated females near or younger than 9-12 days can also be attracted in substantial numbers. Young virgin females may have a tendency to be less active in moving out of a non-host area than older virgin females. Possibly the pressure to oviposit, even if unmated, may drive mature females to seek suitable host fruit for egg laying.

The ability to attract young virgin flies and younger mated flies is of great importance to the potential use of







Table 10.- Recapture of released A. suspensa adult virgin and non-virgin females of different age with sticky traps baited with 40 live males from tests in a non-host avocado grove at Homestead, Florida. A completely randomized block design with twelve replicates was used on 9-21-73, and 400 females of each group were released. Caged males were between 7 and 13 days old (8.5 days old on the average) when first put into the field.


Age (days)/mated Days after release Total Final %
females recapture
status 1 2 4 recaptured per trap

4 Virgin 13 17 20 50 1.04 b

11 " 37 19 26 82 1.71 c

13 " 25 11 9 45 0.94 b
13 non-virgin 21 6 2 29 0.60 a

16-19 " 13 3 3 19 0.39 a


Total recapture 109 56 60 225

Final % recovery 5.4 2.8 3.0 11.2


**


Values followed


Two hundred virgin females were released at 16 days and 200 at 19 days of age.


0


by the same letter are not significantly different at 0.05 level.







71

pheromone-baited traps in survey and detection of caribfly

infestations.

Recapture of Virgin Females with Pheromone Extract-baited Traps

The attraction of females to chemical extracts, obtained from washing 82 male cages with water followed by hexane extraction of the pheromone from the wash water was compared using McPhail and triangular sticky board traps.

The first experiment was done oily with McPhail traps, each one containing 300 ml of water instead of the usual borax-yeast hydrolysate-water food mixture. Ten of the traps were baited with 100 pl of the pheromone extract and ten traps served as a check and were baited with 100 pl of the solvent. Three drops of an emulsifier (Pergosperse L-9) was

added to the water of half of the traps baited with extract and to half of the control traps. The pheromone extract and the solvent were placed on the trap wick (hanging from the center of the stopper) in the laboratory 1/2 hour before releasing the flies (1645 hours). A completely randomized design was used with traps spaced 18 m from each other in a square pattern. McPhail traps baited with 100 pl of the pheromone extract captured only one male out of 1,000 flies of each sex (10 days old) released. Control McPhail traps did not catch any flies.

The next experiment was done with triangular sticky

board traps. In this experiment the chemicals were applied to wicks hanging from the center of the traps. Six of the







72

traps were treated with 100 p1 of the pheromone extract obtained from the same source as the one used in the previous experiment. Another six traps were treated with 100 )l of the solvent and the remaining six traps consisted of 5 tenday-old males per trap. The males were placed in screen-mesh cages of about 5 X 3 X 3 cm with food and these were suspended from the center of the traps.

In a final experiment with triangular sticky board traps, the extract or solvent was mixed with the Stickem and spread over the entire surface of traps 1/2 hour before releasing the flies (1600-1700 hours). In contrast with the first test, in which both sexes were released, 1,000 virgin females (10 days old) were released in the second and third experiments. A completely randomized block design with 6 replicates was used with traps spaced 18 m apart in a square pattern. Females were released at 10 release sites each equidistant from any four traps arranged in a square. Captures were recorded periodically for 4 days. Statistical analyses were made individually for each test date and data were not combined as in all previous experiments.

Traps baited with 100 pl of the pheromone extract on the wick captured 72 females while 5-male-baited traps attracted 55 and control traps none (Table 11). The pheromone-baited and male-baited traps attracted similar numbers of females and were significantly different from the control traps (P : 0.05), but not from each other.







Table 11.-


Recapture of released A. suspensa adult virgin females with triangular-stickytraps baited with 0 and 5 live males, and 100 pl of male pheromone extract from tests in a non-host avocado grove at Homestead, Florida. A completely randomized block design with six replicates was used in each test date. Flies were 10 days old when first put into the field and captures were recorded over a period of 3 days.


Source Release date (1973) Total Final %

of females recapture
10-11 10-18
attractant recaptured per trap


0 Males 0 a 0 a 0 0

5 Males 55 b 6 a 61 .51

100 ul extract 72 b 74 b 146 1.22



Total recapture 127 80 207

Total released 1000 1000 2000

Final % recovery 12.7 8.0 10.35


*
Values in each column followed by the same letter are not significantly different at
0.01 level of confidence.


_Q2







74

Traps baited with 100 pl of the pheromone extract mixed with Stickem captured 74 females while 5-male-baited traps attracted only 6 and control traps none (Table 11). The pheromone-baited trap attracted significantly more females than the male-baited and the control trap.

The attraction of virgin 10-day females to chemical compounds obtained from secretions of virgin 10-day males showed that chemical attraction between the sexes is very important.


Laboratory Bioassays to Test Aggregating Pheromone

Bioassays with the Plexiglas cage (Fig. 8) showed that

baits of 10, 20, and 30 males attracted 11, 20, and 13 males, respectively, while cylinders baited with 20 females, male pheromone extract, and with food attracted 9, 8, and 6 males, respectively. Another bioassay showed that baits of 50 males and food only attracted 26 and 45 males, respectively, out of 200 released males in the cage. All males were 8 days old when first used.

Bioassays using Plexiglas plastic cylinders as described by Nation (1972) showed that baits of 20 males (9 days old) and food attracted 20 and 14 males (4 days old), respectively, out of a total of 80 free males~released at the opposite end of the cylinders. In another bioassay, baits of 10 males (9 days old) and food attracted 37 and 36 males (4 days old), respectively, out of a total of 100 free males. In a last







75

test with cylinders, baits of 10 males and food attracted 40 and 32 males, respectively. All males were 5 days old when first used and the maximum attraction to both males and food was obtained when males were 9 days old.

These bioassays failed to show that males were any more attractive than food or control traps for free males. The next experiments made use of a nylon cage of 6 cubic meters set up inside the laboratory. Fluorescent lights provided light. In the first bioassay 250 males and 10 females were released in the cage. Two traps baited with 10 and 20 males caught 55 and 26 males, respectively, while two control traps caught 65 males. All males and females were 7 days old when first used and the maximum attraction to both males and food was obtained when males were 9 and 10 days old. In another bioassay in the nylon cage two traps baited with 10 males caught 78 males and two control traps caught 92 males out of 250 males and 10 females released when they were 7 days old. Maximum attraction to both males and food was obtained when flies were 8 and 9 days old.

These laboratory experiments conducted with three different techniques did not prove to be an adequate laboratory bioassay for the aggregating phenomenon of male flies demonstrated in field bioassays.


Ability of Laboratory-reared Males to Attract Wild Flies

The attraction of wild flies to live male-baited and







76


McPhail traps was compared at Homestead during the summers of 1972 and 1973, and at Key Biscayne during the summer of 1973.
From June to August of 1972 a boll weevil trap baited

with 50 virgin males (5 days old on the average) was hung in a guava tree near a McPhail trap (Trap No. 9 AREC) on AREC grounds to study attraction of wild flies. A control Stickem-coated trap was baited with food but no males. The male-baited trap was rotated with the control trap once every two weeks and sometimes weekly, but the McPhail trap was not moved from its place, since it was part of a long term trapping survey. Caged males in the trap were replaced once a week.

In July and August, 1972, a boll weevil trap baited with 50 virgin males (6 days old on the average) was hung in a sapodilla tree at the AREC. A McPhail and a control trap were placed in the two nearest sapodilla trees. These traps were rebaited and rotated counterclockwise once a week.

During August, 1972, a male-baited and a control trap were hung from cattley guava tree branches at the AREC. Traps were rebaited and rotated once a week. The average male age was 8 days when first put into the field. Results were compared with the capture of a nearby McPhail trap (Trap No. 4 AREC).

In 1972 mortality of caged males was high and most males were dying within the first three days after they were set up in the field.







77

The results in Table 12 show that McPhail traps captured four times more flies than male-baited traps, and male-baited traps captured almost as twice as many as control traps. During June, 1972, male-baited traps captured almost five times more flies than McPhail and eleven times more than control traps. As the population increased substantially in July and August, male-baited traps became relatively inefficient. McPhail traps always attracted more females than males (a ratio of 5 : 1), while male-baited traps detected a relatively higher number of males by capturing a ratio of

2.3 : 1.

In 1973 several modifications were made to the cylinders to help males survive a longer period of time. Water was provided in small vials containing dental cotton wicks, instead of using a block of 1% agar or water in a sponge as had been done in 1972. The aluminum caps positioned on the lower part of the cartridges were perforated and a circular screen mesh wire was fitted into the cap. In this way any excess water from rainfall drained outside the cylinder and less drowning of flies occurred. Eventually two paper towels were added inside the cartridges to provide more surface to the flies for resting, and for adsorption of the pheromone. Mortality of caged males during 1973 was reduced substantially and in some cases males survived for a week or more.

From June to October, 1973, sixteen boll weevil traps, each baited with 40 virgin males (7 days old on the average)







Table 12.- Wild A. suspensa adult flies per trap per day captured during the summer of
1972 with 40-male-baited sticky traps compared with McPhail and control traps
at AREC, Homestead, Florida for 3 months and at 3 different locations.


Dates Type of trap
and Food only 40 Males McPhail

Locations C total Y total e total


.66 .05


.03


.71 .36


.73 1.74


6.04 1.96 8.00


.56 .55


.13


.69


.51 1.06


1.41 0.31 1.72 4.44 0.18 4.62 8.86 2.19 11.05


Guava grove Sapodilla grove

Cattley guava grove Total capture Trapping days Flies/trap/day Ratio :Cl


1.48 .72 2.20 2.60 .99 3.56 7.05 .95 8.00

.05 .01 .06 .23 .02 .25 4.09 .59 4.68
.27 .50 .77 .35 .73 1.08 9.78 3.88 13.66

128 72 200 248 106 354 1215 249 1464

171 171 171 170 170 170 189 189 189

.75 .42 1.17 1.46 .62 2.08 6.43 1.32 7.75


1.77:1 2.34:1


June July


August


.33
1.01


--j


4.88: 1







79

were used. Six were located at the AREC and ten were located at Key Biscayne areas. All were located near continuously operated McPhail traps. Male-baited traps were usually located in a tree near the tree containing the McPhail trap; but sometimes it was necessary to hang both traps in the same tree, in which case they were separated by about 6 m. Male-baited and McPhail traps were rebaited and rotated once a week. At the beginning of the comparison McPhail bait was mixed 3 days before using it, but after several weeks, each trap was rebaited in the field by adding two pellets of torula yeast-hydrolysate-borax to 300 ml of water. No gross differences in attraction response were noted between the two ways of baiting the traps, but this was not checked

statistically.

The results in Table 13 show that McPhail traps captured 1,997 flies, which represented 3.58 flies per trap per day and was five times higher than male-baited traps. This is consistent with the 1972 data, although populations were lower in 1973. During June and July, 1973, caged males- captured slightly more flies than McPhail traps, but as the population increased in August, the caged males became relatively-inefficient. These results parallel those of 1972; namely, that caged males apparently do not attract flies consistently when the population increases. Again McPhail traps attracted more females than males in a ratio of about 3 : 1, while male-baited traps attracted fewer females per






80


Table 13.-


Wild A. suspensa adult flies per trap per day captured during the summer of 1973 with 40-malebaited sticky traps compared with McPhail traps at AREC, Homestead, Florida. Six traps of each type were used for 5 months (93 trapping days) at 6 locations.


Dates Type of trap

and 40 Males McPhail

Locations total 0 total


June July


August


September


October


1.17


.54

.38 .61

.11


.25 1.42 1.58


.20

.17


.74


.23


.55 1.91


.08 1.66


.01


.24


.45 2.36


.50 1.11 4.95 2.06 7.01


.08


.19 2.12


.67 2.79


1 (Loquat)


2 (Loquat)

3 (Surinam cherry)


4 (Guava) 5 (Guava) 6 (Guava)


Total capture Flies/trap/day



Ratio g:0


.09

.14 .05 .69 .99 .69


.03

.12 .01


.12 .26

.06


.06 .06 .o4


0


.01

0


.06

.07

.04


.35 1.04 4.01 1.71 5.72 .78 1.77 7.41 2.65 10.06


.22


.91 4.38 1.13 5.51


246 142 388 1485 512 1997 .44 .25 .69 2.66 .92 3.58


1.73 : 1


2.90 : 1







81


male with a ratio of 1.7 : 1.

It is noteworthy to mention here that male-baited traps, although showing lower overall capture than McPhail traps, detected larger numbers of females and males in three locations (locations 1, 2, and 3) where McPhail traps caught

very few (Table 13).

The results in Table 14 from Key Biscayne show that

McPhail traps in four months captured 387 flies, while malebaited traps caught 358 flies. The much lower population in Key Biscayne, 0.46 flies per trap per day in Key Biscayne against 3.58 flies per trap per day in AREC with McPhail traps, was probably due to spraying against mosquitoes.

The data confirm the results of previous experiments

wherein caged males attracted flies when the population was low. Almost twice the number of males were attracted with

male-baited traps as were attracted with McPhail traps (Table 14)'. Females and males were detected in male-baited traps in larger numbers than detected in McPhail traps in five locations (Nos. 3, 5, 7, 8, and 10).


Courtship and Mating Behavior of A. suspensa in Nature

Male calling behavior started when a male arrived at the

substrate and spread both wings outward from the body at an angle of 900 with the costal vein parallel to the substrate. The male frequently made short running movements to one side and then the other, usually returning to about the same place





82


Table 14.- Wild A. suspensa adult flies per trap per day
captured during the summer of 1973 with 40-malebaited sticky traps compared with McPhail traps
at Key Biscayne, Florida. Ten traps of each type
were used for 4 months (84 trapping days) at 10
different locations.


Dates Type of trap
and 40 Males McPhail
Locations I total I total

July .66 .45 1.11 .96 .17 1.13
August .17 .05 .22 .16 .03 .19
September .23 .12 .35 .41 .11 .52
October .15 .16 .31 .16 .07 .23


1 (Mango) .09 .04 .13 .27 .07 .34
2 (Clusia rosae) .30 .25 .55 .65 .13 .78

3 (Guava & Lime) .24 .18 .42 .21 .13 .34
4 (Grapefruit) .05 .03 .08 -07 .11 .18

5 (Surinam cherry &
Tropical almond) .32 .17 .49 .21 .05 .26
6 (Surinam cherry &
Sapodilla) .10 .07 .17 .51 .04 .55

7 (Kumquat & Mango) .10 .05 .15 .07 0 .07
8 (Rose apple) .83 .60 1.43 .32 .02 .34
9 (Tropical almond) .41 .11 .52 1.25 .28 1.53 10 (Tropical almond) .23 .09 .32 .15 .04 .19







83


Table 14 - continued


Type of trap


40 Males

04


and

Locations


total


McPhail

( $ tot


Total capture, Flies/trap/day Ratio Y:04


225 .27


133

.16


358

.43


314 .37


73

.09


1.69 : 1


Dates


387

.46


4.30 : 1







84


where he started. These lateral movements did not extend more than one-half cm to either side. After several of the above movements, the male stopped moving and started flicking and waving the wings. The costal vein was held parallel to the substrate at all times during these wing movements.

Meanwhile, if a female was located in a nearby branch

several ft distant, she flew short distances from one branch to the other, usually not directly towards the male, until eventually she landed on the same leaf or one a short distance from the male. Sometimes the female apparently arrived from another tree or perhaps from a more distant branch. However, I observed that in most cases the female flew short distances until arriving at the same leaf with the male. These flight movements usually took long periods of time to occur, sometimes more than an hour and many times the female did not reach the male at all, especially when disturbed by breezes or another insect.

During the time that the female took to land near the male, the male continued waving his wings and small pouches were distended in the pleuro-ventral region of the abdomen. After a few seconds an anal pouch was everted. After the anal area was everted the male touched the substrate with the tip of the abdomen. The male then moved both wings laterally, simultaneously vibrating them rapidly. The vibration movements were brief and intermittent.

Between fanning movements males turned, touched the







85


anal pouch to the substrate at intervals, and followed a circular pattern as though marking a circular territory of about one-half cm in diameter. During intervals between wing vibrations males started movements involving scratching of the ventral surface of the abdomen with the metathoracic legs, and cleaning-like movements with the tarsus of the forelegs. The forelegs were also moved upward and downward touching the frontal area of the antennae. Both forelegs were used in this procedure without a definite alternation. One more cleaning-like movement consisted of folding one wing downward, oriented in such a way that the 3rd leg on the same side held the wing from the upper side, while the male rubbed the underside of the wing against the ventral side of the abdomen several times. There was an alternation of both wings in this rubbing movement that lasted several seconds and was repeated between intervals of wing vibrations.

After the abdomen was rubbed several times with the hindlegs and the wings, the pouches from the abdomen grew disproportionally larger until two large white areas appeared which could be seen from almost any angle from which the male was observed. This together with the anal eversion and the rubbing movements was termed by Nation (1972) as the puffing behavior of A. suspensa.

A female was never observed to come directly to a

puffing male, but rather made several flights from one leaf to another, not overtly approaching the male. This type of







86


orientation in which the insect makes successive comparison of the intensities of stimulation from two sides is known as klino-taxis. Once the female was a short distance from the male, but usually separated by a few leaves or the next branch, the male stopped rubbing movements and spent more time vibrating the wings and touching the substrate with the anal pouch. If the arriving female was disturbed, she usually ceased movement toward the male and flew away. The male sometimes returned to the rubbing movements and wing vibration, and sometimes flew to another location.

If the sequence was not interrupted, a female might fly to the same leaf, but stayed 2 to 3 cm from the male. As a female started moving forward, the male spent less time in vibrating the wings. Movements of a female toward a male were slow and several minutes passed before the two were within a range of 1 cm. Then the male stopped all movement and kept the wings spread at 900 while facing the female. For the first time, the male started moving from its area toward the female. This movement of the male was almost imperceptible and slower than movement of the female.

When the flies were very close, they faced each other and made contact by touching each other with the frontal area of the head. The male jumped to the female and turned his body 180 0, landing on top of the female and facing the same direction as the female. The male grasped the female at the leading edge of the wings with the forelegs, extended







87


his proboscis to the vertex of the head of the female, and bent his abdomen downward and forward in order to contact the ovipositor of the female. The male on top was positioned slightly behind the female with the head aligned with the prothorax of the female. The female could bend the ovipositor forward at an angle so that the male claspers were incapable of finding the female genitalia. Females might also vibrate

their wings and walk short distances with the male.

Males trying to copulate with females in the field were easily detected at this point because there was rapid wing vibration produced by both flies that was easily heard by human observers when the wind was calm. Sometimes the female intercepted the male in the jump with a quick movement of one wing and the male never reached the top of the female. Contact in this way lasted a few seconds, but in case of successful jumping, the male could stay on top of the female for many seconds without copulating and would try through several interruptions to successfully mate with the female.

Males were frequently observed to jump onto the back of

females and attempt intromission. Unfortunately no successful copulation was observed from inception, but 26 pairs were observed already in copula. The male on top of the female remained with the wings spread outward at 900 from the body while the female wings were maintained outward at 450 from the body. No wing movements occurred. The ovipositor of the female was pointed upward and was held by the claspers of the







88


male. The male frequently extended the proboscis and touched the frontal area of the head of the female during copulation. The lateral pouches were not distended during copulation. On some occasions another male or female flew directly to the

mating pair and touched the genitalia with their mouthparts. On most instances the female walked a short distance with the male on top and only on a few occasions was copulation interrupted. Just before copulation ended the female started walking, moving the wings, and kicking the male with the hindlegs and the wings. Separation of the aedeagus from the vagina did not take place immediately. Usually the male got down from the female and turned 1800 to the opposite direction, with the aedeagus still inserted in the vagina. Sometimes the female was observed pulling the male with the aedeagus still inserted. After complete separation the flies stayed a few cm apart for a few seconds while making rubbing movements with the forelegs and the head. Males never used the same spot to start calling more females, but these spots were commonly visited, immediately afterwards, by males who walked in the same area most of the time and made frequent proboscis extensions to touch the substrate.

Males courting and/or attempting copulation with other males were commonly observed, but this behavior did not involve a similar sequence of courtship behavior as in the case of a female approaching a male. What usually happened was that a very excited male was approached directly by







89


another male, the first leaping upon the other almost immediately and attempting to copulate. The male beneath combined fluterring with kicking and moving the forelegs and the wings to thwart the copulation attempt in most cases after only a few seconds, but sometimes the male remained on top for several seconds.

At least four senses seemed to be involved in the courtship and mating behavior of A. suspensa. At a long distance the olfactory sense was probably the most important in bringing the sexes together to a suitable area (i.e. several trees with dense canopies). Males probably were also attracted to the same area by detecting an aggregating pheromone. The auditory sense might be important at a shorter distance after the female reached the area (i.e. a tree). The visual sense could be important also, especially when the flies were in the same branch or close branches in the same tree. The tactile sense was involved upon actual contact. The gustatory sense might be important, too, but it was not carefully observed to determine its order of importance.


Some Environmental Factors Associated with Mating

More than 55 hours were spent in the field observing

caribflies during the summers of 1972 and 1973. Observation periods covered most of the daylight hours starting from 0700 hours in the morning and ranging to, late evening (2100




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SEX AND AGGREGATION PHEROMONE BIOASSAYS AND MATING OBSERVATIONS OF THE CARIBBEAN FRUIT FLY, Anastrepha suspensa (LOEW), UNDER FIELD CONDITIONS By ALBERTO JAVIER PERDOMO A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 197^

PAGE 2

ACKNOWLEDGMENTS I am deeply indebted to Dr. James L. Nation and Dr. Richard M. Baranowski, who directed the present research. Special thanks are extended to Dr. J. L. Nation for his criticism, assistance, and enthusiastic encouragement in the preparation of this dissertation. I thank Dr. R. M. Baranowski for his liberal support, and for providing the necessary supplies, equipment, and direction during my stay in Homestead, Florida. Grateful acknowledgments is given to Dr. H. L. Cromroy, Dr. D. L. Chambers, and Dr. F. W. Zettler for their critical review and suggestions in the preparation of this dissertation and for serving as members of the Supervisory Committee. I extend thanks to Dr. and Mrs. D. 0. Wolfenbarger, and Dr. R. A. Conover for providing housing and various other services, and also to the personnel of the Agricultural Research and Education Center, for their friendship during my stay at Homestead, Florida. I am indebted to the International Atomic Energy Agency, and to Dr. W. G. Eden, Chairman of the Department of Entomology and Nematology, for their financial support during the period of graduate study. Special thanks are extended to the National Academy of Sciences for their administrative assistance. Finally thanks go to my wife Esther, my children Tito and Malena, and to my family who provided moral support. ii

PAGE 3

TABLE OF CONTENTS Pa ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES V ' ABSTRACT INTRODUCTION LITERATURE REVIEW Insect Sex Pheromones Utilization of Sex Pheromones in Pest Management . . Pheromones as Survey Tools Potential Control with the Mass Trapping Technique ...... Potential Control with the Confusion Technique Integration of Sex Pheromones with other Control Agents Sex and Aggregating Pheromones in the Tephritidae . Female-produced Pheromones • Male-produced Pheromones ... Aggregating Pheromones Courtship and Mating Behavior in the Tephritidae. . Courtship and Mating Behavior in other Diptera. . . Economic Importance of the Tephritidae Taxonomic Relationships

PAGE 4

Page MATERIALS AND METHODS 33 Experimental Insects 33 Field Bioassays 35 Field Observations ....... 43 Screen-mesh House Bioassays to Test Sex Pheromone ....... 43 Laboratory Bioassays to Test Aggregating Pheromone 44 RESULTS 47 Recapture of Virgin Females with Male-baited Traps in the Screen House 47 Field Bioassays ......... 48 Recapture of Virgin Females with Malebaited Traps 48 Recapture of Virgin Females and Males with Male -baited Traps ............. 56 Recapture of Virgin Females and Males with Female-baited Traps 60 Effect of Age and Virgin or Mated Status Upon Female Attraction to Malebaited Traps 63 Recapture of Virgin Females with Pheromone Extract-baited Traps 71 Laboratory Bioassays to Test Aggregating Pheromone 74 Ability of Laboratory-reared Males to Attract Wild Flies 75 Courtship and Mating Behavior of A. suspensa in Nature 81 Some Environmental Factors Associated with Mating 89 iv

PAGE 5

Page DISCUSSION 98 The Experimental Procedures. .... 98 The Field Bioassays. 100 Courtship and Mating Behavior of A. suspensa in Nature 10 ? The Future Application of This Research. .... 110 BIBLIOGRAPHY • 11 5 BIOGRAPHICAL SKETCH 126 v

PAGE 6

LIST OF TABLES Table Pa S e 1 Recapture of released A. suspensa adult virgin females with male-baited sticky traps 49 2 Recapture of released A. suspensa adult virgin females with sticky traps baited with 10, 20, and 40 live males as compared with McPhail traps ... 51 3 Recapture of released A. suspensa adult virgin females with boll weevil sticky traps baited with 40 live males 55 4 Recapture of released A. suspensa adult virgin females and males with sticky traps baited with 20 and 40 live males as compared with McPhail traps • • 57 5 Recapture of released A. suspensa adult virgin females and males with sticky traps baited with food only and 40 live males as compared with McPhail traps 59 6 Recapture of released A. suspensa adult virgin males with sticky traps baited with food only and 40 live males as compared with McPhail traps . 61 7 Recapture of released A. suspensa adult virgin females and males with sticky traps baited with food only, 5, and 10 live females as compared with McPhail traps ....... 62 8 Recapture of released A. suspensa adult virgin and non-virgin females of different age with sticky traps baited with 40 live males 65 9 Recapture of released A. suspensa adult virgin and non-virgin females of different age with sticky traps baited with 40 live males .68 10 Recapture of released A. suspensa adult virgin and non-virgin females of different age with sticky traps baited with 40 live males 70 vi

PAGE 7

Table Pa g e 11 Recapture of released A. suspensa adult virgin females with triangular-sticky-traps baited with 0 and 5 live males, and 100 ul of male pheromone extract • • • • 73 12 Wild A. suspensa adult flies per trap per day captured during the summer of 1972 with ^0-malebaited sticky traps compared with McPhail and control traps at AREC .78 13 Wild A. suspensa adult flies per trap per day captured during the summer of 1973 with ^0-malebaited sticky traps compared with McPhail traps at AREC 80 Ik Wild A. suspensa adult flies per trap per day captured during the summer of 1973 with ^-0-malebaited sticky traps compared with McPhail traps at Key Biscayne 82 15 Number of mating pairs of wild Caribbean fruit flies observed in daylight hours during the summer of 1972 at AREC 91 16 Mating pairs of wild A. suspensa flies observed during the summer of 1972 at AREC 92 17 Total number of pair matings and courting males observed among 6 to 10 days old A. suspensa adults when exposed to wind currents of 8 and 16 kph compared with flies tested in still air under laboratory conditions 97 18 Recapture of released sterile and non-irradiated A. suspensa adult virgin females and males with sticky traps baited with 40 sterile males, and if-0 non-irradiated males as compared with control traps .114 vii

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LIST OF FIGURES Figure Page 1 Cages used to maintain adult A. suspensa in the laboratory. ......... 3^ 2 Adult A. suspensa females emerging from a release cylinder in the field 36 3 Partial view of the avocado grove showing the grid pattern distribution of trees. ...... 37 4 Diagram of a section of the avocado grove showing the distribution of traps, release points, and tree orientation. . 38 5 Cotton boll weevil trap, also known as wing trap, used in field bioassays to test sex and aggregation pheromone of A. suspensa 40 6 Invaginated glass or McPhail trap used in field bioassays 41 7 Triangular sticky trap baited with males and food. . • 42 8 Diagram of the Plexiglas plastic chamber (45 X 45 X 45 cm) used as a bioassay test apparatus in the laboratory 45 9 Total recapture of A. suspensa adult virgin females at intervals after release May 26, 1972, at 1545 hours with four sticky traps baited with 5 males each and four sticky traps baited with 10 males each . 52 10 Recapture of virgin female k_. suspensa adults 48 hours after release, and final recovery for observation period with sticky traps baited with males 54 • • • Vlll

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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 SEX AND AGGREGATION PHEROMONE BIOASSAYS AND MATING OBSERVATIONS OF THE CARIBBEAN FRUIT FLY, Anastrepha suspensa (Loew) , UNDER FIELD CONDITIONS By ALBERTO JAVIER PERDOMO June, 197^ Chairman: Dr. J. L. Nation Major Department! Entomology and Nematology A bioassay for the sex pheromone produced by male Caribbean fruit flies, Anastrepha suspensa (Loew), was developed in field studies at Homestead, Florida. Cotton boll weevil traps coated with Stickem and baited with live male flies attracted and recaptured released female flies. Experiments were conducted in an avocado grove, and wild flies were not present in the immediate grove area. Males used as bait and females released were from a laboratory stock colony. Sexes were segregated after adult emergence. Sticky traps baited with 10 or 40 live males consistently recaptured more released females than did McPhail traps baited with yeast hydrolysate-borax-water mixture. A maximum recapture of ^8% of released females occurred in one experiment in which all of 9 traps used were baited with kO live males. Numbers of females recaptured tended to be greater in the late afternoon hours, and during the first 48 hours folix

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lowing release. Male-baited and McPhail traps also attracted male flies when males were released with females or alone. Male-baited traps caught significantly more males than did McPhail traps. When males were released with females in a 1 t 1 ratio, the male-baited traps recaptured a 1 : 1 ratio. The effect of varying the ratio released was not tested. The attraction of males to male-baited traps was interpreted as evidence of an aggregation phenomenon, and possibly aggregation to a pheromone. Female-baited sticky traps were not as effective as McPhail traps in attracting released males and females. Recapture of mated and virgin females varying from 1 to 19 days of age, marked with fluorescent dye, showed that virgin females were more attracted to male-baited traps than were mated flies. Little attraction of flies occurred before 6 days of age, and a peak of attraction occurred at 9-12 days of age. Older mated females were very poorly attracted. At 9 days of age the attraction of mated females as compared with virgin females was about 50% reduced. Sticky traps baited with sex pheromone extracted from the cloth and cage surfaces of male holding cages attracted released females. These results and earlier demonstrations of female attraction to male-baited traps were interpreted as indicating attraction to a male sex pheromone. In the summers of 1972 and 1973 traps baited with live lab-reared males attracted wild females and males at Homestead x

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and Key Biscayne, Florida. In total, McPhail traps at the same locations captured more wild flies than male-baited traps, but during some periods and in some locations, malebaited traps caught more wild flies than McPhail traps. Male-baited traps consistently detected a greater proportion of males in wild populations than did McPhail traps. Males court females and mate on the underside of leaves of host trees. Aggregation of males and females seems to occur in certain trees. Males exhibit a "calling" behavior involving distention of lateral abdominal and anal pouches, slow wing waving, and intermittent bursts of rapid wing fanning. Females move toward males a short distance or several branches away, but they do not usually fly to the male by a direct path. Mating occurred during windless afternoons, but no mating was seen during morning hours. Twenty-six mating pairs were observed. xi

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INTRODUCTION The Caribbean fruit fly, Anastrepha suspensa (Loew) , known informally as the caribfly, is one of several species of fruit flies indigenous to the West Indies. It invaded Florida on at least three occasions, the last in Miami Springs (Dade county) in April 1965 (Weems 1965i 1966). Since then it has spread over 21 counties throughout the southern and central part of the state. In south Florida it infests mainly dooryard tropical and subtropical fruits, including Surinam and Barbados cherry, common guava, cattley guava, loquat, rose apple, tropical almond, peach, and Citrus species. More than 80 species of fruit have been recorded as hosts of caribflies (Swanson and Baranowski 1972) . For this reason it is considered by many scientists and citizens to be an urban pest rather than a commercial pest. However, some individuals believe A. suspensa represents a potential threat to the citrus industry of the state of Florida and of other parts of the country (Arizona, California, Texas) and to the peach industry in Florida and Georgia. In Florida this insect occasionally attacks several important citrus crops, such as grapefruits and oranges. A strain of A. suspensa in Puerto Rico attacks grapefruits, sour oranges, sweet oranges and other tropical and subtropical fruits (Weems 1965) . 1

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Laws of California, Arizona, and Texas require that citrus shipments from Florida be fumigated. The cost to the citrus industry for treating citrus fruits is estimated at approximately $35,000 per year. Three fumigation chambers were built in 1971 at Gainesville, Florida, at a cost to the citrus industry of $72,000. The fumigation treatments are conducted by the Division of Plant Industry (H. A. Denmark and R. Brown, personal communications). Research with A. suspensa is being conducted by the University of Florida, the U. S. Department of Agriculture, and the Florida State Division of Plant Industry. One phase of this research is the investigation of improved methods of detection involving the determination of a lure or lures which might replace the present food-baited McPhail traps. Nation (1972) demonstrated that males of A. suspensa release one or more volatile pheromone compounds that attract females under laboratory conditions. Insects utilize infinitesimally small quantities of such chemical compounds to communicate with each other. Since the practical application of this basic information needs to be ultimately tested in nature, I decided to study the sexual attraction and behavior of the caribfly under field conditions. The objectives of this work were (1) to develop a field bioassay for the sex pheromone, and determine certain parameters of female response to it, such as the influence of age and mated status, and (2) to observe courtship and mating behavior in the field.

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LITERATURE REVIEW Insect Sex Pheromones Although the name "pheromone" was proposed only recently (Karlson and Butenandt 1959) to describe chemicals that, produced by one animal, influenced the behavior of others of the same species, many examples had been observed over the past 100 years. In 1837 Carl von Siebold, a German zoologist, suggested that insect odors were probably attractants and aphrodisiacs for members of the same species. Apparently, his view was not taken seriously by most other scientists, for many early authors believed that male insects were provided with acute vision that enabled them to see females at great distances (cited by Grosser 1971). Lefebvre (cited by Jacobson 1972) initiated research in the field of insect olfaction. Weismann (cited by Jacobson 1972) suggested that odors from scent glands on the wings of certain male lepidopterous insects function as an aphrodisiac for females. He reported that numerous males of the eyed hawk moth, Smerinthus ocellata (L.), were attracted to a single virgin caged female. Around 1890, Jean Henri Fabre, a French naturalist, found that male moths of Saturnia pyri were attracted in

PAGE 15

large numbers, even in the dark, to caged females (cited byGrosser 1971). Urbahn (cited by Jacobson 1972) reported numerous findings of sex pheromones from laboratory and field experiments in the order Lepidoptera, and described the abdominal scent glands of many female lepidopterous insects. Law and Regnier (1971) proposed the name "semiochemicals for those substances that convey chemical messages between insects and they divided semiochemicals into two broad classes: those conveying signals between individuals of the same species (intraspecif ic) and those conveying signals between different species (interspecific). They also proposed the names "allomones" and "kairomones" for those interspecific semiochemicals whose release favors the producer or receiver, respectively. On a functional basis pheromones are divided into releasers or primers (Wilson and Bossert 1963) . Releaser pheromones stimulate an immediate behavioral response, while primers change the physiology of the receptor animal, reprogramming it for an altered response pattern that usually appears later. Some pheromones act as both primers and releasers (Grosser 1971). Sex attractants are releaser pheromones. Although noting that the same chemical may have multiple effects upon behavior, Dethier et al. (i960) erected the following classification in terms of the behavioral responses that chemicals elicit upon insects: (1) Arrestants chemical that cause insects to aggregate, (2) Locomotor stimulants -

PAGE 16

chemicals that cause insects to disperse from a region more rapidly than if the area did not contain the chemicals, (3) Attractants chemicals which cause insects to make oriented movements towards the source, (k) Repellents chemicals which cause oriented movements away from the source, (5) Feeding, mating, or ovipositional stimulants chemicals which elicit feeding, mating, or oviposition, and (6) Deterrents chemicals that inhibit feeding or oviposition when present in a place where insects would normally feed or oviposit . A sex pheromone is usually emitted by one sex to attract the opposite sex (and sometimes both sexes) for mating (Beroza 1972). There is evidence that hundreds of insects utilize sex attractants to find each other. Most of these sex pheromones are chemically unidentified. Utilization of Sex Pheromones in Pest Management It is strongly felt by many that pheromones will be the key to truly integrated pest management for many crops (Antognini 1972). The potential use of pheromones in pest management for survey, mass trapping, and mate confusion programs is being explored (Marx 1973)* Survey or monitoring the population of an insect pest helps to determine proper timing of sprays, or whether a pesticide spray is actually needed. Sex pheromone-baited traps assist in locating infestations, and are especially useful when these

PAGE 17

are not easy to find by visual inspection. Pheromone traps also help detect new pest introductions and proper control measures can be instituted before the infestation becomes so large that it is uncontrollable. Monitoring quality control of sterile insects by means of sex pheromone or live insectbaited traps has been useful. Mass trapping and mate confusion techniques are related to control or prevention of reproduction. Mass trapping techniques may be directed toward preventing an insect from expanding its range, or help to exclude an insect from a particular crop. Usually a combination of the sex pheromone (or aggregating pheromone) with a sticky trap or other suitable trapping device is used (Tette 1972). One procedure has been to drop from an airplane large numbers of safe and inexpensive mass-produced traps (such as small tubes with an adhesive inside) containing attract ant to capture one or both sexes before they can find each, other and mate. If the ratio of traps to the target insects is high enough, the population of lightly infested areas might be eradicated. This overflooding approach is similar to the one devised by Knipling (i960) for use in the sterile-male technique. The mate confusion technique is known by several names such as "male confusion", "male inhibition", "disruptive communication", and "misdirection" (Grosser 1971). This new approach to control consists of permeating the atmosphere with the sex pheromone, or attractant, so that the males

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7 will not be able to orient to the females, or viceversa. Their odor receptors may simply become fatigued, insensitive, or saturated, thereby preventing them from responding. The advantages offered by this method are that it may be applicable to both lightly and heavily infested areas (Beroza I960, 1971, 1972). Pheromones as Survey Tools A variety of commercially produced pheromone-baited sticky traps are available which serve to monitor effectively in the field more than a dozen important economic pests. Included among these are methods for two fruit flies, the apple maggot fly, Rhagoletis pomonella (Walsh), and the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Two types of traps are in use for these fruit pests. The wing-type trap of paperboard construction has a sticky adhesive on the upper surface of the bottom section. Another type is constructed of a pre-folded, heavy gauge cardboard with a tacky adhesive coating on the inside. When the trap is opened, it takes the form of a rectangle, resembling a pagoda-like shape. Insects entering these traps are captured and firmly held in the sticky substance. In 1972 Madsen and Vakenti (1972) compared female-baited traps against traps baited with Codlemone, a synthetic sex pheromone found by Roelofs e_t al. (1971) for the codling moth, Laspeyresia pomonella (L.) (Lepidoptera) . The results indicated that both types of traps can be used to estimate pop-

PAGE 19

8 ulation levels and to determine the need for chemical treatment. In the next year, Madsen and Vakenti (1973) reported that sprays were applied only if Codlemone-baited traps within the orchard captured two or more moths per trap per week and if infested apples were found. In this way, only one or two sprays were required in different orchards, instead of the three sprays usually applied. In the case of the California red scale, Aonidiella aurantii (Maskell), routine inspection of groves represents a difficult, tedious, and costly task. Many trees have to he checked in the area, identification of the insect can be confused, and infestations may go unnoticed because scales are usually in cryptic places. A citrus scale trap was developed by Shaw et al. (1971) in which the sex pheromone produced by the female (Tashiro and Chambers 1967) attracts males, which are then caught on a sticky card. In California and Arizona, citrus growers are starting to use these traps in their normal operations (Anonymous 1972) . In California alone, A. aurantii causes an estimated $5 million loss annually in damage from loss of trees and diminished yields. The cost of spraying once or twice a year is about $^5 per acre. An inspector searching visually for scale infestations covers about 5 acres a day and costs $20. In contrast, each baited sticky trap monitors from 1 to 5 acres, costs about $2, and detects infestations not readily apparent to an inspector. Tests show that such traps capture

PAGE 20

9 4,000 to 7,000 male scales per day in areas where the pest is well established (Anonymous 1972) . Sex pheromone traps are used in surveying infestations of the pink bollworm, Pectinophora gossypiella (Saunders). State and federal agencies rely on traps to monitor uninfested and lightly infested cotton areas for spread of this insect. Although the traps are baited with a synthetic sex attractant (hexalure or cis-7-hexadecenyl acetate) (Sharma et al. 1973) t specific for males, the true sex pheromone has now been identified (Hummel et al. 1973) and can also be used in traps. McLaughlin et al. (1973) demonstrated that living females were more effective in locating low population levels of males than hexalure-baited traps. The USDA uses disparlure, the sex pheromone of the gypsy moth, Porthetria dispar (L.), to bait its 60,000 survey traps in the United States. Gypsy moths have been detected with these traps in many places where they were not previously known to exist (Beroza 1971)' Grandlure, an aggregating and sex pheromone of the boll weevilt Anthonomus grandis Boheman, has been used by the USDA in cotton areas of the United States to bait traps which attract and destroy weevils emerging from hibernation during spring. This monitoring plan provided for intensification of sampling effort in and around detected "hot-spots" of weevil infestations (Eden et al. 1973) • Fruit fly detection programs have been continuously run

PAGE 21

10 since 1956 by the Division of Plant Industry and the USDA throughout the state of Florida. Two types of traps are used. The combination (Steiner) trap is baited with cue-lure, methyl eugenol, and trimedlure. Cue-lure attracts melon and Queensland fruit flies; methyl eugenol attracts Oriental fruit flies; and trimedlure attracts Mediterranean and Natal fruit flies. The other type of trap is the wet or McPhail trap and is baited primarily to attract Mexican and Caribbean fruit flies. The wet trap is baited with cottonseed protein (ENT-44, 014-X) which contains one per cent technical borax. This mixture is made into pellets, two of which are dropped into each trap with the required amount of water (300 ml). The traps are cleaned and rebaited once a week (Poucher 1970) . Experiments are being conducted to determine the optimum proportion of the sex pheromone and activating acids from C. capitata males for practical application (Anonymous 1973) • Investigations with A. suspensa are conducted by different agencies to determine which lure or lures might be used to replace the present one, which is not considered completely satisfactory. Until a good lure is available for A. suspensa any attempt to study the effect of chemicals, sterile release, or population densities will be hindered. Quality control of sterile insects has been monitored by many researchers using different methods. Fletcher and Giannakakis (1973b) used a sex pheromone bioassay to evaluate

PAGE 22

the effect of irradiation upon sexual behavior of Dacus tryoni (Frogg.). A pheromone field bioassay, consisting of hanging baited traps containing females in trees in a grid pattern, was used by Moffitt and Hathaway (1973) "to monitor the quality of adult codling moth, L. pomonella . Olfactometers provide an additional tool to compare the effectiveness of the attractant from insects of different strains and ages, and the effect of administering different diets or radiation treatments. The comparative ability of sterile and wild individuals to elicit a response from virgin wild ones can also be studied (Steiner 1969). Potential Control with the Mass Trapping Technique Tette (1972) reviewed mass trapping with the redbanded leafroller, Argyrotaenia velutinana (Walker), the gypsy moth, P. dispar , bark beetles, Dendroctonus spp. , and the codling moth, L. pomonella . In an experiment that lasted k years, Roelofs and his colleagues (cited by Marx 1973) achieved 99 percent control of A. velut in ana in a lightly infested area by using traps baited with sex pheromones. However, in a heavily infested area, mass trapping did not produce a satisfactory control. It is possible that when populations are high, vision may be as important as pheromones for locating a mate (Marx 1973). In 1972, tests were conducted in areas with gypsy moth populations below detectable levels by means other than pheromone traps. Glassine-liquid cardboard tube traps coated

PAGE 23

inside with Tack-Trap containing disparlure were distributed by aircraft over large test areas. The results showed that apparently mating was not inhibited in the treated plots and was actually higher than in the controls (Cameron 1973). Some evidence (not published) was later obtained that the sticky Tack-Trap was perhaps too good a keeper for the lure , retaining rather than releasing it. A continuing study of gypsy moth populations with pheromone traps is in progress. Southern pine beetles, Dendroctonus frontalis Zimmerman, concentrated their attacks on highly immune longleaf pine trees, Pinus palustris , when these trees were baited with the aggregation pheromone of the beetles. As a result they were completely decimated by the resins from the trees (cited by Grosser 1971) . Hardee et al. (1970) showed that in areas with low populations of boll weevils, wing traps baited with males were efficient in suppressing the spring infestation until migrant weevils infested the field in late summer. A large-scale experiment was conducted on a core area of roughly 25 miles radius around Columbia, Mississippi to determine if various population suppression techniques, when used in concert, were adequate to eradicate a boll weevil population from a prescribed area (Eden et al. 1973) • One of the population suppression techniques used was traps baited with the sex pheromone, grandlure. The results showed that pheromone traps were effective in monitoring the population and also

PAGE 24

in detecting "hot-spots" of weevil infestation, although effectiveness declined as cotton reached the squaring stage. The committee that reviewed this pilot experiment (Eden et al. 1973) strongly recommended continued research on the pheromone "grandlure" as a population regulation tool. Potential Control with the Confusion Technique Promising experiments on male confusion or inhibition were conducted by Shorey et al. (1971) at the University of California at Riverside with the cabbage looper moth, Tricho plusia ni (Hubner) . The synthetic sex pheromone, continuously evaporated at a rate of 1 mg per ha per night, almost completely prevented males from locating pheromone-releasing females. The cost of the program was $0.01 per ha per night. The only remaining problem, according to Tette (1972), will be to prevent gravid females from entering or migrating into the test area. Several tests conducted in 1971 and 1972 with the gypsy moth, P. dispar , suggested that it might be possible to confuse male gypsy moths and prevent them from finding females (Stevens and Beroza 1972, Cameron 1973). In 1971, pieces of dispar lure-treated paper were uniformly distributed by aircraft over ^-0-acre-plots. Males released periodically in these plots were unable to find traps containing unmated females or disparlure for 6 days. In contrast, males were readily captured by such traps in untreated plots. However, by the 21st day the attraction of the treated papers appar-

PAGE 25

lif ently had declined sufficiently to allow some males to locate traps containing females or disparlure (Beroza and Knipling 1972). Cameron (1971) tested granular cork impregnated with disparlure and a microencapsulated formulation of disparlure containing a sticker to adhere the capsules to the foliage and achieve vertical stratification of the lure. These microcapsules released the pheromone gradually so that it was expected to be effective for 6 to 8 weeks. The results of spreading granular cork impregnated with disparlure in areas with a natural population indicated a reduction in mating of about k6% compared with the control. The results with microcapsules in areas where laboratory-reared insects were previously released were very encouraging, and no successful mating was detected in the two treated plots. Integration of Sex Pheromones with other Control Agents Many sterile-release programs have been less successful than hoped for because the irradiated insects did not compete well with wild ones. Irradiation in many cases (Steiner and Christenson 1956, LaBrecque and Keller 1965 t Holbrook and Fujimoto 1970, Hooper and Katiyar 1971, Ohinata et al. 1971) reduces the vigor of insemination, mating competitiveness, and sex pheromone production by the sterile insects. It would be desirable then to counter with an alternative method which would enhance the sterile insects, even if their condition is near normal or completely normal, so that the ste-

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15 rility principle itself can be raised to a higher level of efficiency. Chambers et al. (1972) suggested that treating tephritids with attractants or a sex pheromone might attract native partners to sterile mates, allow them to compete more effectively with wild males for wild females, or make it possible to use the sterile flies as disease or toxicant carriers. They were able to prove that untreated flies of the Oriental fruit fly, Dacus dorsalis Hendel, and of the melon fly, Dacus cucurbitae Coquillet, were attracted to flies treated with synthetic attractants. The male annihilation technique was used successfully by Steiner et al. (1965) and by Steiner et al. (1970) in the Mariana islands to eradicate the Oriental fruit fly through aerial distribution of fiberboard blocks containing synthetic male lure, methyl eugenol, and an insecticide, naled. In a more recent experiment, Cunningham and Steiner (1972) reduced the populations of male D. cucurbitae more than 99?S through the distribution of fiberboard blocks soaked with cue-lure and a solution of naled (wt/wt) in a 2-squaremile plot in Hawaii. Cunningham e_t al. (1970) and Cunningham et al . (1972) reduced substantially the populations of D. dorsalis , D. cucurbitae and C. capitata for periods of up to two weeks by spraying the plot areas with a formulation of cue-lure and methyl eugenol mixed with 5% technical naled.

PAGE 27

16 Sex and Aggregating Pheromones in the Tephritidae Female -produced Pheromones Literature reviewed by Jacobson (1972), Law and Regnier (1971), and Beroza (1972) indicate that no species of tephritid is known in which the female attracts the male. However, Prokopy and Bush (1972) presented evidence that mature females of R. pomonella produce and deposit an unidentified marking pheromone on fruit. The marking pheromone serves both to act as a male arrestant and to regulate numbers of eggs deposited per fruit. Male -produced Pheromones Among the cases of male-produced pheromones reported by Jacobson (1972) seven species of tephritid flies were included t the Mediterranean fruit fly,C. capitata , the solanum fruit fly, Dacus cacuminatus (Hering), the melon fly, D. cucur bit ae , the Oriental fruit fly,D. dorsalis , the olive fruit fly, Dacus oleae (Gmelin.), the Queensland fruit fly, D . tryoni and the island fruit fly, Rioxa pornia (Walker). Jacobson et al. (1973) identified two components of the sex pheromone of male medflies as a combination of methyl (E) 6-nonenoate and (E) -6-nonen-l-ol. Fletcher (1968, 1969) described the rectal gland complex responsible for the production and storage of the sex pheromone of D. tryoni males.

PAGE 28

17 Similar glands have been found in the males of Dacus neohume ralis Birch, Dacus kraussi Hardy and Afrodacus .jarvisi (Tryon) (Fletcher 1969), and in D. dorsalis , D. cucurbitae and in D. oleae (Schultz and Boush 1971 1 Economopoulos et al. 1971). Evidence for a sex pheromone produced by the males of R. pornia and A. suspensa was given by Pritchard (1967) and Nation (1972), respectively. Nation (personal communication) identified the major components of the male-produced pheromone of A. suspensa as two lactone esters and two alcohols. These compounds, when fractionated and tested individually in the laboratory showed reduced or no biological activity, although when recombined, they strongly attracted mature females. Aggregating Pheromone s Insects aggregate for various purposes including mutual protection, hibernation, aestivation, mating, and oviposition (Butler 1967) . Aggregations may be temporary or persistent (Butler 1967). Temporary aggregations are those that occur during part of the life cycle and are intended usually for only one biological activity (hibernating Coccinellidae , mating swarms of Ephemeroptera, etc.). Persistent aggregations occur only in social insects living in colonies in which the well-being of each individual depends on its companions. Usually, one or more pheromones are involved, together with several other biological and physical factors of the environment in the promotion and maintenance of aggregation (Butler 1967).

PAGE 29

18 Jacobson (1972) cited 42 cases in which assembling or aggregating pheromones lure both sexes, including 32 species of Coleoptera, 9 species of Orthoptera, and only one species of Hemiptera. Some of these aggregating compounds are produced by females and some by males, but they attract both sexes . Although no chemical has been identified from tephritids that causes aggregation, several authors have suggested that aggregation occurs in the field in response to host odors or other factors. Cunningham and Steiner (1972) called localized pockets of high populations of males of D. cucurbitae "hot-spots". Males of D. tryoni are attracted to their own pheromone, and apparently feed upon the secretions from the rectal glands of other males (Fletcher 1968) . Fletcher suggested that the pheromone may play a specific part in the sexual behavior of males, and cited some evidence that males congregate together when stridulating. Bateman (1972) observed mating swarms of highly active males of D. tryoni aggregating at dusk in an orchard containing a dense population. He suggested that tropical species of fruit flies tend to congregate in locations which provide shelter and food and that these locations are usually restricted to patches of evergreen foliage such as citrus, banana, or other "favorable" plants. He further suggested that adults feed during the warmer hours of the days, but return to the same sheltered foliage when temperatures fall.

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19 Males and females of Eutreta sp. fed upon froth masses secreted by other males from the proboscis, indicating a possible attraction of both sexes to the same chemical (Stoltzfus and Foote 1965). Fe'ron (1962) reported that males of the medfly are attracted to the "sexual odor" of other males. In one well-studied fruit fly, R. pomonella , neither male nor female seems to produce an attract ant (Prokopy and Bush 1972). Instead of attraction to a pheromone odor, both sexes are attracted to fruit odors, and thus the sexes come into close contact. Females also deposit a marking pheromone onto the fruit following oviposition. The marking pheromone acts as a male arrestant. Thus the biological purpose of retaining males at a site where females have been and are likely to be is served. Prokopy and Bush (1972) referred to these effects as aggregation of flies. Courtship and Mating Behavior in the Tephritidae Three fundamental aspects of the sexual behavior of any species are courtship, mating, and coitus (Guhl 1968). Courtship was defined by Oliver (1955) as the behavior preceding mating that stimulates one or both individuals and initiates the mating performance. It is considered separately from the act of mating, but it may be difficult to designate the point at which courtship ends and mating behavior begins.According to Bastock (1967) in a typical case of courtship, individuals of one sex display to the opposite sex immediately before

PAGE 31

20 mating. This implies communication between displayer and audience . Prokopy and Bush (1973) gave a detailed and extensive description of the courtship behavior of R. pomonella . Mature flies gather at the host fruit, and periodically move from fruit to fruit as if in search of potential mates. Flies spend as much as 89% of their time on fruits, while only 10% is spent elsewhere in the trees. The predominant activity of females on the fruit is oviposition-type activity while only a very small percentage of females (or males) engage in feeding-type activity. Either sex may initiate the flight which brings it to the particular fruit occupied by a member of the opposite sex. Prokopy and Bush (1972) concluded that olfactory and acoustical stimuli emanating from other apple maggot flies did not play a role in eliciting any response from males or females several cm or more away. Many species of Dacus initiate mating activity at dusk. In laboratory cages D. tryoni and D. cacuminatus usually mate on the dark ceiling of the cage (Myers 1952), and in outdoor cages mating was restricted to a period of about JO minutes at dusk (Tychsen and Fletcher 1971) • For many other species of Tephritidae, falling illumination at dusk acts as a stimulus for the initiation of sexual activity. These include D. dorsalis (Roan et al. 195^» Chambers et al. 1972); D. tryoni (Barton 1957i Myers 1952); Dacus ciliatus Lw.

PAGE 32

21 (Syed 1969); Euleia fratria (Loew) (Tauber and Toschi 1965a) } Dacus zonatus (Saund.) (Syed et al. 1970); Dacus scutellaris Bezzi, Dacus diversus Coq. (Syed 1970); D. cucurbitae (Back and Pemberton 1914, Schroeder et al. 1973) 5 D. cacuminatus (Myers 1952); D. oleae (Economopoulos et al. 1971); and Anastrepha ludens (Loew) (Baker et al. 19^4). In contrast to most of the Dacinae, D. neohumeralis mates during the day at high light intensities (Gee 1969) . A "call" produced by immobile males of D. tryoni and D. oleae when vibrating the wings rapidly was described by Myers (1952) and Martelli (1908), respectively. The "call" is clearly heard as rather high, flute-like notes emitted in series, each note varying in duration from a half to two seconds and rarely longer (Myers 1952). Monro (1953) described a similar mating call in D. tryoni , but the sound consisted of a stridulation produced by drawing the wings across a pair of combs formed from large bristles on the third abdominal tergite. The sound, audible to the human ear, consisted of a high pitched buzz. Sound production by males has been observed in D. cacuminatus (Myers 1952), which produces an audible higher pitch than that of D. tryoni ; in D. dorsalis (Roan et al. 195^); in D. oleae (Feron I960); in Q* cucurbitae (Back and Pemberton 1917); and, in D. passi florae Frogg. (Simmonds 1936) . The sexual behavior of the medfly C. capitata was extensively treated by Feron (1962). The basic behavior was

PAGE 33

22 the same in nature as in the laboratory. Attraction of the mate pair from a certain distance occurs in response to a chemical signal; and the sexual "parade" that occurs after the pair gets closer is in response to other signals (mainly visual). The sequence of activities involved, according to Feron (1962), is as follows! (1) the sexually excited male stays motionless, and the female is attracted to the male. The abdomen of the male is strongly retracted longitudinally and expanded laterally into pouches, in a similar way as in the picture of males of the asparagus fly, Platyparea poeci loptera Schrank (Se'guy 1951) t "the island fruit fly, R. pornia (Pritchard 1967), and the Caribbean fruit fly, A. suspensa (Nation 1972). The male of C. capitata also exhibits an anal "ampoule", apparently inflated and shiny like a liquid droplet. This posture is related to the release by sexually mature medfly males of an odorous pheromone that attracts mature virgin females (Feron 1959i Lhoste and Roche I960); (2) a female fly arrives a few centimeters away from the male, who in turn vibrates both wings rapidly; (3) the female slowly orients toward the male in a face to face position; (4) when the flies are closer, the excitation of the male increases, characterized by movements of the wings. Fe'ron (1962) believed that the rapid wing vibration served to emit a sonorous signal, to produce an air current, and to release a chemical; (5) the last step is the leap of the male onto the female, and mating ensue.s. N

PAGE 34

23 Holbrook and Fujimoto (1970) determined that the peak mating response in C. capitata occurs at about 0600 hours. Myburgh (1962) reported the lowest temperature for mating of medflies to be 19° C, and the most favorable zone was between 22° and 30° C. However, Steiner (1967) mentioned that other workers had observed mating in C. capitata early in the morning at temperatures of 17° C and above, and also at 40.5° C under high humidity. The range of relative humidities at which mating occurred in £. capitata was between 30 and 60% according to Myburgh (1962). He considered that light intensity was the environmental factor which had the greatest bearing on differences in mating habits observed in medflies. Oldroyd (196*0 reported that males of an African species, Afrocnerus mundus (Loew), use small balls of foam as mating lures. Similar behavior was reported by Stoltzfus and Foote (I965) in Eutreta sparsa (Wiedemann). Pritchard (I967) described extensively and in detail the mating behavior of the . island fruit fly, R. pornia , whose male also produces a mound of foam when courting the female. During courtship behavior, Pornia males distend the pleural regions of abdominal segments 3» ^1 and 5. Wing waving is also common. When a female approaches the male's pleural regions deflate, and the pair make wing movements. A mound of white foam is produced and the female feeds upon the foam while the male attempts copulation. The foam apparently serves to keep the female

PAGE 35

2^ quiescent (Pritchard 1967) . Copulation occurs in the late afternoon (at dusk) and is usually broken after dark. The courtship behavior of the Caribbean fruit fly, A. suspensa , was described by Nation (1972). The mature male distends pouches from the pleural regions of the abdomen. The males also distend a thin membranous pouch of cuticle surrounding the anal area, similar to the anal pouch in C. capitata (Fe'ron 1959). While exhibiting the courtship or "puffing" behavior, the male remains relatively stationary for long periods of time. V/ing waving movements are interrupted by bursts of rapid wing vibration. The metathoracic legs are frequently rubbed over the abdomen and the puff areas, and then over the wings. Nation (1972) relates this cleaning behavior to the spread of a sex pheromone over the body and wings, providing a greater surface from which it can evaporate. When a female approaches, the male ceases wing fanning and puffing, crouches with the body near the surface, and appears to touch the surface with extended proboscis. According to Nation (personal communication) deposition of chemicals may take place when males extend the proboscis to touch the substrate, and these chemicals could serve to keep the female quiescent. When the female advances and contacts the male, he leaps upon her and attempts to copulate. Many tephritid males will court individuals of their own sex as well as females (Boyce 1934, Economopoulos et al.

PAGE 36

25 1971. Feron 1962, Nation 1972, Prokopy and Bush 1973t Tauber and Toschi 1965a, 1965b). Wing waving and stridulation seem to be important in the sexual behavior of most tephritids as many authors have reported. Some (Fletcher 1968, Fletcher and Giannakakis 1973a, Feron 1962, Nation 1972, Pritchard 1967, Tychsen and Fletcher 1971, Tauber and Toschi 1965a, 1965b) believe wing movements facilitate evaporation of a pheromone by production of air currents, as well as subserve a visual function; others (Back and Pemberton 1917. Feron i960, Feron 1962, Fletcher 1968, Fletcher and Giannakakis 1973a, Monro 1953i Myers 1952, Roan et_ al. 1954, Simmonds 1936) believe the sounds produced during waving and stridulation of the wings enable the female to find the precise location of the male. In summary the sexual behavior of Tephritidae involves auditory, visual, gustatory, olfactory and tactile stimuli. Bateman (1972) considers smell and hearing as the two most important sensory stimuli for mating response in the tephritids . Courtship and Mating Behavior in other Diptera The sexual behavior of some other dipterous species closely resembles the behavior of tephritid species. Courtship behavior in the Hawaiian Drosophilidae does not occur at the feeding sites. Males tend to assemble in considerable numbers at specific localized sites in the

PAGE 37

26 vegetation surrounding the feeding and oviposition sites. Such breeding sites retain their attractiveness over prolonged periods of time. Each male "patrols and defends" a small and limited area (i.e. a single leaf) and simultaneously "advertises" his presence by body movements and especially wing movements. Spieth (1968) called this behavior "true lek behavior" and considered it an adaptation to avoid predators. Males of some Drosophila species repeatedly drag the tip of their abdomen over the substrate and in doing so deposit a thin film of liquid, while others assume a ritualized posture and extrude and retract a bubble of liquid from their anal papillae. Spieth (1968) suggests that scents from these fluids attract females into the immediate vicinity of the male. Carlson and Beroza (1973) found that (Z) -9-tricosene, the sex pheromone of the house fly, Muse a domestica L. , acted as an aggregation pheromone in the field, rather than just as a sex attractant for males as in the laboratory bioassays. Willson and Mulla (1973) observed distinct zones of activity in poultry ranches in which mating females were concentrated, while recently emerged and gravid females were fairly evenly dispersed along the cage rows of the poultry house. Such behavior suggested to Willson and Mulla (1973) the existence of specific zones of mating activity.

PAGE 38

27 Economic Importance of the Tephritidae The distribution of the family Tephritidae is virtually worldwide but it is largely restricted to the temperate, subtropical, and tropical parts of the world. About ^,000 species have been described. They are usually referred to as fruit flies because some are among the most destructive pests of fruit known. In the typical developmental cycle, gravid females insert their eggs into plant hosts with an eversible, sclerotized ovipositor. The developing larva sheds its skin twice as it feeds and grows. An inactive f ourth-instar larval stage within the puparium precedes the formation of a pupa. Pupation may take place within or on the host plant, but most often it occurs in the soil. Diapause is inherent in many temperate-zone tephritids but has been inadequately studied (Foote and Blanc 1963) . Miller (1970) considered the total economic losses due to fruit flies to include the following! (1) Direct loss of fruits and vegetables. (2) Cost of control treatments and extra labor. (3) Reduction in market value of the produce. (4) Drawback to horticultural development and/or prospective markets. (5) Indirect cost of numerous stringent quarantine measures . The amount of damage which could result through the in-

PAGE 39

28 troduction of fruit flies into fly-free areas is so great that elaborate efforts are usually taken to prevent establishment in new territory. As an illustrative example, the second medfly introduction into the state of Florida in 1956 covered 28 counties and required treatment of more than 6 million acres with chemical poisons and bait sprays. Throughout the state ^6,^99 traps were intensively monitored. The peak work load involved 701 state personnel (excluding personnel contracted to do aerial spraying and federal personnel) . Fumigation chambers were established in 26k sites and numerous road blocks resulted in inspection of 4-, 672, 901 vehicles. The state and federal funds expended amounted to almost 10 million dollars (Oberbacher and Denmark 1957). More than 200 varieties of fruits and vegetables are hosts of the medfly (Christenson and Foote i960). Other species of fruit flies that attack close to 100 or more plant varieties are the melon fly, D. cucurbit ae , and the Oriental fruit fly, D. dorsalis , both present in Africa, Indonesia and many Pacific islands including Hawaii; the Queensland fruit fly, D. tryoni , present in Australia (Christenson and Foote i960) ; and the Caribbean fruit fly, A. suspensa (Swanson and Baranowski 1972). A. ludens , the Mexican fruit fly, breeds in wild and cultivated citrus in north-eastern Mexico and each year migrates into the Rio Grande Valley of southern Texas. It has spread also into the cultivated citrus sections of the west

PAGE 40

29 coast of Mexico and northward toward Arizona and California, resulting in continual detection, survey, quarantine measures, and eradication campaigns (Weems 1963) . Of less importance are Anastrepha interrupta Stone, Anastrepha serpentina Wiedemann, and Anastrepha momb inpr ae o pt ans Sein (Weems 1967, 1969, 1970). All have been found in parts of the United States (southern tip of Florida and/or Rio Grande Valley of Texas), and the latter two are distributed along Central America and part of South America. An economically important species not present in the United States is the South American fruit fly, Anastrepha fraterculus (Wiedemann), which is distributed from Texas to Argentine and in the Caribbean islands of Trinidad and Tobago. Other species of fruit flies of economic importance in the continental United States are the apple maggot fly, R. pomonella , attacking apples, pears, plums, and other deciduous fruits in northeast U. S. and southeast Canada, and the walnut husk fly, Rhagoletis completa Cresson, that attacks all Juglans spp., peaches* and other fruits in the western U. S, (Christenson and Foote i960). Rhagoletis cingulata (Loew) and Rhagoletis indif f erens Curran (eastern and western cherry fruit fly, respectively) damage sweet and tart cherries (Bush 1969) . A. suspensa is one of several species of fruit flies which are indigenous to the West Indies. It invaded Florida on three occasions, the last in April I965 at Miami Springs

PAGE 41

30 (Dade county) (Weems 1965i 1966). Since then it has spread over 21 counties throughout the southern and central part of the state. Within its normal range of distribution the economic damage caused by this species has been relatively small, although more than 80 species of plants, including tomatoes and bell pepper, are hosts of the Caribbean fruit fly. Preferred hosts include common guava, Psidium guajava L. , cattley guava, Psidium cattleianum Sabine, Surinam cherry, Eugenia unif lora L. , tropical almond, Terminalia catappa L. , loquat, Eriobotrya japonica (Thunb.) Lindl., rose apple, Syzygium jambos (L.) Alst., Barbados cherry, Malpighia glabra L. , and calamondin, Citrus mitis Blanco (Swanson and Baranowski 1972) . A strain of A. suspensa found in Puerto Rico attacks a number of tropical and subtropical fruits, including grapefruit, Citrus par ad is i Macf., sour orange, Citrus aurantium L. , and sweet orange, Citrus sinensis Osbeck (Weems I965) . There is no assurance that A. suspensa could not become a major pest of citrus or other crops such as peaches and apples, found in Florida or neighboring states. Thus far A. suspensa has been collected only in Florida, Puerto Rico, Dominican Republic, Haiti, Jamaica and Cuba (Weems 1965, Anonymous 1970) . Taxonomic Relationships The first taxonomic summary of North American Tephritidae was presented by Loew (1862), who placed all the known species

PAGE 42

31 in the genus Trypeta . In 1868 Schiner described the genus Anastrepha . In I873 Loew again reviewed the family, but in much greater detail, assigning the species to various genera. He erected the genus Acrotoxa for Anastrepha . Eezzi (1909) placed the species back in Anastrepha and included only 19 species. Hendel (191*0 in his revision of the genus treated 3^ species, including a number of new ones. The genus then remained untouched until Costa Lima (193^) described 62 species, based almost entirely on Brazilian material, of which 22 species and 1 variety were new. His paper was the first that used extensively the ovipositor as a character. Greene in 193^ reviewed the genus Anastrepha , and based on a study of the wings and on the length of the ovipositor sheath included 5^ species, of which 16 were new. Further work by Stone (19^2), based on the wing patterns and on the female terminalia, removed several species and added new ones to the genus, ending with 114 species, of which 49 were new species. Host races are common and these may be rather rapidly formed within a species. It is generally assumed that physiological differences enable a population formerly using only one species of plant as a host to maintain itself on another with apparent ease. These populations are often beyond the eyes of the museum taxonomist if the differences are of relatively recent origin and they are among the most difficult of taxonomic problems. Some species are morphologically separable only by extremely obscure characters and satisfac-

PAGE 43

32 tory identifications can be made only when such supporting data as host, locality, and date are supplied with the specimen (Foote and Blanc 1963) • Unfortunately, most of the keys are designed for females only. Greene (193^) devised a key for males. There remains great need for biological work, since hosts are not known for many species. The following names and synonyms have been used in the past for A. suspensa t Trypeta suspensa Loew ( Trypeta ) Acrotoxa suspensa (Loew) Anastrepha unipuncta Sein Anastrepha longimacula Greene Crosses of A. suspensa females with A. momb inpr ae opt ans males in the laboratory have been obtained on two occasions. The offspring show characters of both parents, but it is rather doubtful that such crossing takes place in nature, according to Stone (19^2), who never saw any specimens collected in the field that agreed with these hybrid specimens .

PAGE 44

MATERIALS AND METHODS Experimental Insects All flies used in these experiments were reared at the University of Florida, Agricultural Research and Education Center (AREC) in Homestead, Florida. Flies were sexed and separated when between 1-3 days of age. The sexes were kept in separate rooms in large screened cages except when mated females were needed (Fig. 1). Sugar and water (as 1% agar) was placed at the top of the cage and yeast hydrolysate paste on one of the screen mesh wire sides. Food was replaced or added when necessary. To obtain mated females a ratio of 1 Q_ « 2 0*was maintained in the same cage. An aspirator consisting of a bag sleeve on one end tied to a plastic tube 38 cm long and 1.3 cm diameter and connected to a vacuum pump was used to collect flies. About 500 flies could be collected each time without any apparent adverse effect on the flies' vigor. Flies were never handled with the hands nor were they anesthetized when segregating sexes or transferring from cage to cage. Flies to be released for recapture bioassays were transferred on the day of the release to screen mesh cylinders 16 cm long and 6 cm diameter with an aspirator after knocking the flies down in a freezer for approximately 3 minutes. 33

PAGE 45

34 Figure 1.Cages used to maintain adult A. suspensa in the laboratory. Dry sucrose, yeast hydrolysate paste, and water as 1% agar was provided at the top of the cage and was replaced or added when necessary.

PAGE 46

35 Flies were always allowed to emerge from the cylinders in the field by themselves (Fig. 2). Field Bioassays Field "bioassays utilizing release and recapture of labreared flies tested the sexual attraction of caribflies. Bioassays were conducted at the Homestead AREC in approximately 1 ha of an isolated avocado grove (Fig. 3) • Avocados are not a host of the caribfly, and the nearest host trees were guavas approximately 0.7 km away. No wild flies were observed in the avocado grove, and only plowed ground occurred between the avocado and the guava groves. Survey traps were placed in the avocado grove before and after most of the experiments and these traps consistently indicated an absence of wild flies. In tests with flies marked with fluorescent powders, all flies captured were marked, further confirming the absence of wild flies. Avocado trees were spaced 6 m apart in a square design. Traps were spaced 18 m or 30 m apart and 2 or k trees, respectively, occurred between traps (Fig. k) . Lab-reared flies for recapture data were released at points 13 to 21 m equidistant from any trap at the intersection points of 4 traps arranged in a square . Three different traps were used in the bioassays: (1) Live males or females were caged in 16 cm long and 6 cm diameter screen mesh cylinders which were attached to stickum-

PAGE 47

36

PAGE 48

37 Figure 3.Partial view of the avocado grove showing the grid pattern distribution of trees.

PAGE 49

38 oo o o ooo oo o o o qp o o o o cvo o o oooooo oo o o e o to oo 00,00 O OO o 00 e CO

PAGE 50

39 coated cotton boll weevil traps (Cross e_t al. 1969). The traps, also called wing traps, consisted of four yellow crossbaffles 15.2 cm X 25. k cm height with a top of 23 X 23 cm (Fig. 5) • The cylinder was placed at the center of the trap at the bottom. Flies in cylinders had access to dry sucrose, yeast hydrolysate paste, and water. Control Stickem-coated traps contained food only. (2) Glass invaginated McPhail traps, currently used in the field trapping program in Florida (Poucher 1970) to study the population of Anastrepha spp. , were used in several experiments. They were originally used and described by McPhail (1939). In most of the experiments, 300 ml of aqueous suspension of yeast hydrolysate were used per trap, which were always prepared 3 days in advance of the experiment date. However, in one bioassay the yeast hydrolysate was replaced with distilled water containing 3 drops of an emulsifier (Pergosperse L-9) (Fig. 6). The emulsifier was added to prevent flies from crawling outside of the liquid and/or the trap by reducing the intersurface tension. (3) Triangular sticky board traps (Gutie'rrez et al. 1970), currently used in the field trapping program in Florida to detect the presence of the Mediterranean fruit fly C. capitata were used in two bioassays (Fig. 7). Dental cotton wicks 3.8 cm long X 1.0 cm diameter covered with Parafilm in the middle were hung from the center of the traps with the same wire used to hang the trap from a tree branch. A cardboard

PAGE 51

40 Figure 5«Cotton boll weevil trap, also known as wing trap, used in field bioassays to test sex and aggregation pheromone of A. suspensa . Adult flies in the cylinder had access to dry sucrose, yeast hydrolysate paste, and water.

PAGE 52

Figure 6.Invaginated glass or McPhail trap used in field bioassays. The McPhail trap is normally baited with 2 pellets of a mixture of torula yeast hydrolysate-borax added to 300 ml of water.

PAGE 53

42 Figure 7.Triangular sticky trap baited with males and food.

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^3 inset 15.9 cm long X 9.5 cm wide was almost entirely coated with Stickem and placed on the bottom surface of the triangular-shaped trap. Field Observations Field observations of the activities and behavior, especially courtship and mating behavior, of wild caribflies were made during the summers of 1972 and 1973. Sex ratios were counted at various points in host groves and detailed observations over long periods of time were made at those sites or trees where more flies than usual were found. Courtship and mating behavior were observed. Other data recorded included the host trees and grove, height of flies location, and wind movement. Meteorological data on temperature, relative humidity, wind movement and type of general weather were obtained from records at the AREC weather station located about 0.8 km distant from the observation sites. Screen-mesh House Bioassays to Test Sex Pheromone Three early experiments were conducted in a greenhouse frame covered with screen-mesh of 10 X 5 m located in block 1 of the AREC grounds. Four male-baited and b control wing traps, similar to the boll weevil traps (Cross et al. I969) , were suspended from the ceiling of the screen house. The 8

PAGE 55

traps were arranged in two lines and spaced 3 ro apart. Each male-baited trap was baited with 10 caged males in the first and second experiment and with 25 males in the third experiment. Control and male-baited traps alv/ays contained food and water. Laboratory Bioassays to Test Aggregating Pheromone Attempts were made in the laboratory to demonstrate the male to male attraction found in the field. A square Plexiglas cage (Fig. 8) of 0.09 cubic meter routinely used to bioassay fractions during pheromone isolation was used in two bioassays. Six Tygon plastic cylinders were connected from the outside to holes in the only wooden side of the cage. In the center of the opposite side, a screened hole was connected through a wind sleeve to an electric fan. The fan pulled air through the cylinders at a rate of less than 8 kph. This trap design has been used by Nation (unpublished data) to test the sex pheromone. Males to be tested for pheromone production were placed inside small screened vials containing food (Fig. 8). The vials were inserted at the air intake side of the cylinders and on the opposite side a conical screen trap was positioned. Control vials always contained food only. Attraction was measured by determining the number of flies moving past the conical trap from the cage to the cylinders. Tests were run with continuous light and wind move-

PAGE 56

^5 UJ 10 CO o id CO a) o o C k C O 3 CQ «H 00
PAGE 57

k6 merit for 24 hours. Three bioassays were conducted with the test apparatus and by the procedure reported by Nation (1972) for showing female to male attraction. Air flow from the caged males to the free males was provided by means of a compressed air tank and tubing connections to each cylinder. Males to be tested were placed inside small screened vials and these were inserted at one end of the cylinder. Males were released at the opposite end and attraction to the caged males was measured by determining the number of flies moving past a conical screen trap placed between them. Control cylinders with food only were always used. Food was provided to both sides of the cylinders (caged and free males) . Data were collected periodically over a period of 2 to 4 days. A third technique consisted of placing 4 to 6 traps baited with males inside of an indoor nylon cage of 6 cubic m.

PAGE 58

RESULTS Recapture of Virgin Females with Male -baited Traps in the Screen House Virgin females 8, 9» and 10 days of age were released inside the screen house in successive weeks and recapture data were obtained periodically for a period of 1 to 5 days after the release date. In the first experiment metal boll weevil traps with two yellow baffles and two unpainted baffles were used, while in the second and third experiments red boll weevil plastic traps were used. Also in the third experiment a mixture of 100 ml tap water, 70 g sucrose, and kO g hydrolyzed yeast was sprayed periodically in the screen house on small citrus trees and other plants to provide food for the released flies. The results showed that male-baited traps were no more attractive than control traps. In the first experiment traps baited with 10 males recaptured 38% of 500 females released and the control traps recaptured kVfo, Of the females recaptured, with both traps, 85% were caught in the yellow baffles. This high percentage indicated a strong attraction to the yellow color instead of to the males and/or food under screen house conditions. In the second and third experiments with red traps, the male-baited traps (10 and 25 males per trap, **7

PAGE 59

48 respectively) recaptured 33$ of 1^25 females released and the control traps caught 21%. Field Bioassays Recapture of Virgin Females with Male -baited Traps The attraction of females to food only, 1, 5, and 10 live caged males was compared in the first experiment. All flies were 8 days old when first put into the field. Captures were recorded over a period of 2-6 days. The experiment was replicated k times in successive weeks with a total release of 6,250 females. Flies were released between 1500 and 1630 hours. Traps baited with food only captured 36 females, while traps baited with 1 male attracted 85 females (Table 1). Traps baited with 5 males attracted 227 females, and traps baited with 10 males attracted 695 females. The total percentage recovery of released females varied from 6.5 to 28$ between replicates. Traps baited with 10 males attracted significantly more females than traps baited with food, 1 or 5 males (P 0.05), and traps baited with 5 males attracted more than those containing food only. The difference between traps baited with food and 1 male was not significant. Variation in percentages of recovery among experiments may have been caused by weather conditions prevailing when experiments were performed. In the test started on 3 May 1972 the low total recovery of 6.k5fo was possibly related to the fact that

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50 too many females were held in much smaller cages prior to release than in subsequent experiments. These experiments indicated that males attracted virgin females under field conditions . A second experiment was designed to compare the attraction of the food-baited McPhail trap against 10, 20, and 40 live males. Males and females were again 8 days old when first put into the field. The experiment was replicated k times in successive weeks with a total release of 8,650 females. Flies were released between 1530 and 1700 hours. Captures were recorded over a period of 3-6 days. Traps baited with ^0 males attracted significantly more females than McPhail traps or traps baited with 10 or 20 males (Table 2). Traps baited with 20 males also attracted significantly more females than McPhail traps; but traps baited with 10 males were not significantly different from the McPhail traps. In 2 replicates, traps baited with 10 males captured fewer females than McPhail traps. Recapture of females at intervals after release was recorded for the test of 26 May 1972 (Fig. 9). Most of the females recaptured were caught during the first ^8 hours and during the daylight hours. From 19^5 hours on 26 May to 0615 hours on 27 May only 2k flies were captured, represent in only 0.29 flies per trap per hour, but during the daylight hours on 27 May substantially more females were captured, though not as many as during the first 2k hours. These data

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51 1} CD to p cd cd •H Cd •H C O • GQ rH A+> CO CD ed CO CD CD > CD TJ Xl CD p p p rH J* vo >> g o CD * cd o X -p \& U ft cd o O u b d 3 cd • •H > CD •rH O t-1 CD cd CO Jh X) CD S X: TJ rH CD 3 i Xl VO p cd ft CM OtH VO co vo ON H C P T) 1 o g cd CM CM vo ON ON vO • P •r-l •H • rH CO EH CD O r-i co CM d MS O 'H CD CM cd Sh o •H 73 «H CO O •H > CD £h >> ^ o cd t3 «H •H P cd H T) o o d H ft ft »H CM vo VO ON o CNa g CO ^ CO o\ 00 NO VO •H O CD co vO o• CO cd O T5 CD ft cd ^ tH IN CO P cd CO CD CD Cd On! O to cd -p £ Od C to s-i On CO cu CO CD CO CD t-H VO CN CO co ON o vo 0 p. CD g CD > 1 CM vo VO ON co o ON $H to r-H O'H O vO CM o • cd 3 cd K H CD CM tH to g ft T3 4-> CD P cd i-H h CD • CD cd TJ Tj P <\> ' U P •H CD CD O CD rH CM CO o r-t o o CD T> -p o CO VO o oo o o rH cu O Cd CD cd \o -3o • CO O k T) r-l CD CM o CD cd-3W) i— 1 CM g CD +3 CD CD cd rl t) O U ^ CO CD C -0 CD CD o $h cd cd -p £ co oVO VO vo co o VO CD o 1 Ov ts. vo vo CO o VO » o xi to VO tH CO vo ON • O O > O CD CM co cm cd cd $-i CM >> CD CD 3 ,D «P -P 3 o co d ft T) -P i-H O "H Cd T5 >> CD ft „c o Q) CD $h >. cd x: i tj 5h CO CD o cd > rH CD •H O CO C p CD O rH tm 1 P to CO to cap rel rec o * CD H CD 0 CD 0) CO CM O P •H rH rH rH Jh Of CD f-l 4H O cd cd cd cd CD 3 o cd S S s H rH rH rH rH o u ft cd cd cd cd CO p o o o o p p d > cd p tH CM o O •H cd Eh En Ph *

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52 CM CM s CM N 0> CM >• < 2 O O o CM O O CO LU o o 00 o *8 o CM o" "o "o o o o ifi ^ t fO OJ ~ Q3Un±dVD3U S3"lVVM3d 31 tr O X I CM CD n co 0 H a> U U CD P «H CO I— I Cd X t'H 0) £ -p •H Q) -P •H cc 13 to cd H id e CD > O u Q o T3 cd o o SS p to o 43 i C o C cd •H -P •H 3 cd to C CD to C at p o a! CD n cO-h •c-* U U P o P H d) CO >3pL4 3 • O • -P CM -H TJ PnO--P CO Cd O CO 0> O ,-1 -P SO O g rH CV! Cm O cO X P >sT3 o cd c "P Eh £ 3 cd •H

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53 suggest that flies were not particularly active or sexually attracted to the traps at night, and indicate a tendency to recapture more flies in the late afternoon, as happened on 27 May between 1800 and 1945 hours in which 33 flies were captured. Full darkness during late May occurred approximately at 2000 hours. Recapture percentages at the end of 48 hours and the total recapture percentage for 6 replicates are shown in Fig. 10. Data from 2 replicates are not included because no observations were made at 48 hours. Almost all the flies recaptured were caught during the first 48 hours following release . An additional experiment was conducted releasing virgin females 8 days old and using boll weevil traps, each baited with 40 males, to check the maximum possible attraction that could be obtained when all the traps were baited with males. A Latin square 4X4 design was used on 6-28-72, 7-3-72, and 7-10-72 with traps spaced at 18 m. A Latin square 3X3 design was used on 7-18-72 with traps spaced at 30 m. Virgin female flies were released between 1500 and I83O hours at the intersecting points of every 4 traps arranged in a square pattern. Captures were recorded over a period of 2-6 days. The results, presented in Table 3, showed that substantial numbers of released females were attracted. The final percent recapture varied between 22 and 48%.

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5^ in o % c3 D 0) 0) CD x: o w i_ o X 00 ii CvJ UJ <£ CO > o o 0) o c IT (X) CVJ ro i in o o o K) o CM 3dnidV03d % It! 0) Cap «J H Cti 0) o to x: x> cti p cti CD *H £ CO H •O CCj CD P 0) U CD P s cd O «H •H T3 CD C •H P c o o CD U CD 5 Cti CO C CD to O •H «P T3 Cti Cti > CD P CO CD S CD O p CD to o CO ti T3 H cti 6 CD cu "in > O o W CD M o o CD u p ftrH Cti Cti o C CD -H K
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55 Table 3.Recapture of released A. suspensa adult virgin females with boll weevil sticky traps baited with ^-0 live males from tests in a non-host avocado grove at Homestead, Florida. A Latin square k X k design was used on 6-28-72, 7-3-72, and 7-10-72. A Latin square 3X3 design was used on 7-18-72. Flies were 8 days old when first put into the field and captures were recorded over a period of 2-6 days. Release Total females Final % Final % recapdate released recaptured recapture ture per trap 6-28-72 1028 222 21.6 1.3 73-72 1175 378 32.2 2.0 7-10-72 1300 519 39.9 2.5 7-18-72 1220 581 1*7.6 5.3

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Recapture of Virgin Females and Males with Male-bait e d Traps In previous experiments only females were released. Since I did not know if females would be attracted in the presence of free males, I decided to release females and males in a ratio of 1 z 1. Females and males were released from separate cartridges at the intersecting release points of each ktraps arranged in a square. McPhail traps, control traps containing only food and water, and male-baited traps were used. Males serving as bait and released flies were 8 days old when first put into the field. In the first release attraction to 20 and 40 males in traps and to McPhail traps was tested (Table h) . McPhail traps captured 36 females, while 20 and males captured 135 and 222 females, respectively. Unexpectedly, males were captured by male-baited traps in approximately the same ratio as females. McPhail traps captured 26 males, while the 20 and males traps captured 168 and 216 males, respectively. McPhail traps captured slightly more females than males. The final percentage recovery of released females was 29fo and that of released males was 30%. Traps baited with 20 and ^0 males attracted significantly more flies than McPhail traps (P 0.05). However, the interaction of treatments by sex was not significantly different at the 0.05 level of confidence. This experiment confirmed earlier results that males attracted virgin females under field conditions. No previous work with caribflies had suggested that males would

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57 M o e •H O P 5-4 tO 3 Cd cd to 5^ CD CO H ft Q) u CO CD •H O Q) H H P H ftlH to 'd CD O 10 CM cd CD X r-H -p CD iH 5H £ O 0) CD > O U fclfl CD in o cd »H o to O cd -p tO rH CD CD P -H Cd > 5h cd 0 -O ft o •H P cd Or ft cd U P 5h 0 ft 0 5-. h P ft cd o CD 5-i cd •H 0 5-i P ft cd o 0 5-i to 0 •H rH fx, 0 a 5-i o to 0 g rH rd P o P O CVi tH TH • H J" tH rH 0 h • > 0 rH VPl O o Q o CO U l L^t i CjCJ MJ cd o , — | CM p> 0 u 0 CH £h •H * /— \ -V •CJ t3 O VO • >> CM CM ntl ica { H •H -3rH P HNO •H t — i OJ to P o © Cd CO rr~\ o u o fr\ o cd c~\ HCO Ov CM CM 0 P> 0 NO CO VO o VN o rH C\i tH tH VO ^i CM o 0 rr\ CO 0 vO to CM CO x: L r cn o^ CM On vo • p> i CM 0^1 CO , — i CM rD X) i>. 0 m u c , M Lv 0 o >-< i — ' 0 •> r rH -1-^ CO o rH ft cd o O J_> cd 0 0 •H rH rH U 0 o cd cd cd cd x: E rH H rH H f-i ft cd cd cd cd -P o o o P > P CM -3o o •H cd rH Eh *

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58 be captured by male-baited traps, although males are routinely captured by the food-baited McPhail traps. Thus, the results were very surprising and indicated an aggregation phenomenon among males not previously known in this species. In order to confirm these results, two additional replicates were made limiting the traps to McPhail traps, foodbaited boll weevil traps, and boll weevil traps baited with ^0 males. Virgin males and females in a 1 t 1 ratio were again released, and the recapture results are presented in Table 5. The food-baited control traps captured 55 females, while McPhail traps and traps baited with 4-0 males attracted 238 and 491 females, respectively. Food-baited traps captured 38 males, while McPhail traps and ^0-male-baited traps captured 128 and ^82 males, respectively. As in the first demonstration with male recapture, the sex ratio of recaptured flies was 1 : 1 in male-baited traps, while the foodbaited control boll weevil trap and the food-baited McPhail traps captured more females than males. The final percentage recovery of released females was 30$ and of released males was 25$. Traps baited with ^0 males attracted significantly more flies than McPhail and control traps (P 0.05). The interaction of treatments by sex was again not significantly different at the 0.05 level of confidence. It seemed possible that males were attracted to the females which became stuck to Stickem-covered boll weevil traps, rather than being attracted to the males acting as

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59 >J CO » ft >> rt H $H cd p to CD o •H P> P P CO 0 H H P •H rt xi Pn o CO cd rH ^ rt P g »H rt cd o U i— l BJ (in ft e O T) O Cti CU P CO cu e M CD O U H X •H rt > e T3 • CD cd -a P $-4 CO CD rH rt e CD •H a) > p rH o rtUt crj P to a> p & o cd • H CD CO CO Ki CO CD P rt o •r-1 rH ft CD u o o CD u CD CD CO CD 5h P ft o CO C CD ft] rt CO CO o d ts qrt o rt rH CD •H > X rt £ -a O T3 O rH £ H O H ,Q CO tH >> O rt •H O o CD f-i CD 5 CD rH r-l CD ft rt rt Eh P rt « of CD CJ O CO Ch o 1 Mh Ml CU O f . rH CD P M _ M CD ft rH rt CD C rH •H pH P> CD $h CO -J CD P •H ft rH rt Pn o CD rH CM Or>On i tH 00 CD P> rt -a CD CO rt cm • • CD rH VM rt rt o O • CM C\l o • • • v — ' at CM CO rH p § <-> 0^ f. M rt* UJ CM tH f, , *ri • r_i *H o en i • ri rW Tj VO CM O r-l Q\ \o o_ o • -P 0V CM oc T-l CM S o •ri CO CD CM co o O Ci-\ C\J 00 o •H -V vO VO & CM CM (/} ir\ CO ^-j o rH xr\ p"\ Q\ 00 o • -P CM o_ VO o O CM CM C CD *o VO CM o CO t . C\i -V — -i Ov VO o CO CM C^v o_ CM M CD P o\ o £SVO o -P CM VO Ov 00 o • CD CM Ov iH T-l CM CD • — CM r-| VO o o rH 00 ON co o • CO r-l CM CM CM r-l CM CD -p VO CO — -J CO o VO CO Ov Ov Q • >> ^ 1 C*-\ X5 ^ 1 CM TJ CD a) > Sh CD O 2' CD > H P> CO O tH ft rt o O >> rt CD rH CO o rH Sh rH CD CD CD to o •H rH r-l r-l CD rt rt r d rH rH rH rH o Ph rt rt rt rt o o o P P > S o o •H EH EH Ph *

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60 bait. In order to study this possibility, two replicates were conducted in which only males were released. Foodbaited McPhail traps, food-baited boll weevil traps, and boll weevil traps baited with 40 males were again used, and results are shown in Table 6. Control traps captured 55 males, while McPhail and ^0-male-baited traps attracted 178 and 3^6 males, respectively. The final percentage recovery varied from 21% in one replicate to 27$ in the other. These percentage recoveries are in agreement with those obtained in the previous experiments when both males and females were released. Traps baited with 40 males attracted significantly more males than McPhail and control traps (P ^ 0.05); and McPhail traps attracted significantly more males than foodbaited boll weevil traps (P ^ 0.05). These experiments confirm earlier experiments that males are indeed attracted to other males under field conditions. Recapture of Virgin Females and Males with Female-baited Traps The results from tests of attraction of flies to food only, 5» and 10 live females caged with food and water, serving as bait in Stickem-coated boll weevil traps and to McPhail traps are presented in Table 7. All flies were 8 days old when first put into the field. The experiment was replicated three times in successive weeks with a total release of 3,000 flies of each sex. Releases were made between 1700 and I83O hours. Captures were recorded over a period of 5 days. Traps baited with food, 5, and 10 females captured 117,

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61 0> P •H cd C O T3 •H O H CO ,Q -P CO T) Q) CD CO p ft a s ^ o p u ft >5 H O -a Kl ft >> O cd Xi X) o H a) ft to H goo O to U 5 cd o ft H cd >> to k P U H CD CD CO P CD -H > -P H O -P •H rl •H cd ft o ft O CD H CO O O P O d)S cd o rH ajT) a> cd k E P -P •H • CO CD £ Cd CD ^ •l-l T) -P CD in cd u x: f-t O O 10 > cd H cd cd ftft CD k p e 3 H O -CP O *H ft cd cd cd CO CD 13 CJ cd p CD ft • •H CD -P cd a o o£ 13 O H 'H -p CD -3tjflH CO ft o cd ti o cd p CD H cd cd -h CD o X H > tO 3 ft C cd ft O O £ -p -p -p CD T) CO -H 10 O O £ >H • 3 to >J ft cd cd ,C O -H £ xi O -P C CO Q) o •H CD ,C *H ft £ Cd TJ £ rH NO CD i — I EH CD * cd ft o cd H P CO o cd ft P 0"N. CM cd • •H o U o CM ft re pe Xi CD rH CO cd CD P CO P ft itn. o cd cd Eh S o CD U C\i ON 00 r-l CD P cd CD to cd CD H CD CA « 1 CO O NO CM -3" NO NO CM CM CA O o T-l •rH tv o • xi C\J CM >5 CM O O CM 00 CM CN O O CM o CM CNCM >J CD CD CO CD cd > P CD O P, H o p >> CO cd CD CD § H ft CD o (4 u ce p on ai al CI. A re ft o x: o cd ft ft H o U o o o cd cd cd m P> o p p C ft o o •H Id EH Eh ft

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62 O • H H DO P H 5 -p tO CD T3 0) rt B s 09 CD H rt s CD cd U O P rH CO CO (in ft e O T5 O rt CD to -p o5 CO CO to 6 0) o to >> P rt CD TJ > Os I H O "in O to >>-d rt o P cu •H Ch P CO •H > > «H rH +» rH O 3 t-H T3 rt T» cd (0 C 0.) p to 3 to CO CO > T3 O P -P bfl to CO o -p CO X o o O rt > CO rH CD CVS P CD > O T5 0 t3 P O O CD p CD u CD CO CD U 3 P ft o to * O tJ'ti H a • o <\ TJ O CO O tO rH o * V Sj S. Si -> C*S Q ^ — j SO CM i. CD vn ^ — ' o tH SO Os CM o • I T | -V— ~i CM On CM c*^ 00 CD (V Os o • T 1 CM \rs V — Os i — l — r v. /~> ^ — { CM VO o • rH T-H 0\ Q Os CM so O * ^ | 1 — 1 , l r-H tH pr\ CO r~s » — ' oo Q CO CM oo • CM T-l cs; Q CM — i Q Q Q cm CM o • CM US t-H CSJ Q CS] Q CM O -dOs o • T-l CM o On ' — 1 m CSJ CO — j 4. — j pr\ l 1 — j CM i\> * — f ^ ( f\) v \J CO CO rrH rH CD 0) rH CD P rH rH •H P T5 O rt rt rt P CD p e jp -P to 0 CD CO Ph ft rt > o o rt 0 o o S o rH O Ph o CD 0 0 K PS OS

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63 168 and 196 males, respectively, while McPhail traps baited with protein hydrolysate attracted 2^0 males. The final percentage recovery of released males in replicates of the experiment varied from 15 to 29^. Traps baited with food, 5t and 10 females captured 86, 126 and 149 females, respectively, while McPhail traps attracted 232 females. The final percentage recovery of released females varied from 12 to 25% between replicates. McPhail traps attracted more virgin males and females than traps baited with food, 5, or 10 females, although they were not significantly different at 0.05 level of confidence. In contrast to all previous experiments, this is the first time in which McPhail traps captured more flies than traps baited with live flies. The results indicate that virgin females do not attract virgin males or females under field conditions as well as McPhail traps. The differences between traps baited with food, 5i or 10 females were not significantly different at the 0.05 level of confidence. Effect of Age and Virgin or Mated Status Upon Female Attrac tion to Male-baited Traps These experiments were conducted in 1973* Stickemcoated boll weevil traps were baited with kO live males, 9 days old at the beginning of each experiment. Traps were spaced 30 m from each other in a square pattern. All females were released at six intersecting release sites of each 4 traps arranged in a square and each age group was marked with a fluorescent powder applied to the pupae at the

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64 rate of 4 g/liter (Steiner 1965). The emerging flies became marked as they crawled up through the dye. Mated females were obtained by keeping both sexes together after adult eclosion. Dissection of the female reproductive system in samples from each group indicated that 100% of the females mated. The virgin female groups were kept in a separate room isolated from male contact and/or odors. Three experiments were conducted with release of different age groups. In the first experiment 2-, 6-, and 9-day virgin and 9-, 11-, and 15-day mated females were released. For the second experiment 1-, 7-, and 9-day virgin and 9-t 12-, and 15-day mated females were released, and in the third experiment 4-, 11-, and 13-day virgin and 13and 16or 19day mated females were tested. Females were the age indicated upon the day they were released. A total of 1,000 females per age group was released in the first experiment except in the 2-day virgin group in which 600 flies were released. In the second and third experiments 400 flies per age group were released. Male-baited traps were prepared and put into the field during the morning and female flies were released between 1600 and 1900 hours of the same day. Captures were recorded approximately 24, 48, and 96 or 114 hours after the release. Recaptured flies were identified under a UV lamp in the laboratory. The results of the first experiment, presented in Table 8, show that more virgin flies were recaptured than mated

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65 i i u > H •do? I o P to •H tH o co CD cd s 0 i o o o rH H m CO H >> C -P rQ cd H CO W) CD T3 ^P (DON H E « o g M o «H tsl o CO cu g •H M U •H iH > O H 3 X, P Cd «H cd CO a 0 0) fH CD > cd co U CD H >> cd H 6 0 CO -P 'O > 0 0 H W) ft cd g o o CJ < cn oI • ON cd th id t3 I 0 -H 00 P U PfH O £ O cd H X> CO 0 • ft* in <<|cd cd 3 5-i 0 t3 -p -p 0 co co >» 0 cd g 0 o o H'H K P CO -p cd tH X, o 0 0 ?H P CO cd £ • CO rH 0 0 -P cd tH o • H CD tH X ft-P £ b 0 w O 0 M P W) 0 c ft cd O > »H Cd TJ rH o -p cd 0 0 C o 5 en 0 o p I 00 0 rH cd EH p rH cd q •H * 0 * k ft 3 p cd ft p> cd p o tH O 0 CO 0 H cd g 0 0 ft 13 0 5-i P ft nJ o 0 0 CO cd 0 rH 0 u 0 p Ch cd co >i cd Q NO CM 0 p cd < CO * CD -P cd • H P W) CO M •H > 0 ttfl CM •<; X) X) o ,Q cd cd CO NO CM NO CM NO ^3CM C\ T-H CM t-H o o CM UN O O UA H CM CM On NO OA u^ CM CM On CN) CO On 3On CO CM On Oj UN NO ON tlO > I o ON M 0 • x: H -p 0 o > 0 • i — t | 1 1 H cd UN, o • •H o id -p 0 cd CO cd p 0 rH 0 0 u 0 tH CO r +H /l^ U> • n CO • H T5 00 cd T-l e >j 0 H tH P 1 f -^ o o sJ o •H 1 tH tH •H c id & •H CM c CO OA CM CO P> O cd t3 0 cnj H cd p cd 0 T3 p rH 0 P UN • CO 0 CM cd rH 0 rH 0 0 ( , H cl CU CO 0 u 0 0 x: ? p tH ON CO >a O • 0 xi vn CO rH cd •o g 0 0 :< tH o 0 U H u 0 H > •H O p o tH Pi o u • cd 0 •H CO [fl o M > ft 0 Q) U O O rH O M cd H H NO W) > cd cd P 0 O • H * EH cd *

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66 flies. During the first 2k hours following the initial release of flies, the age group showing the greatest recapture was 10-day virgin females. It is noteworthy that the greatest total recapture of flies occurred in the first 2k hours, confirming earlier findings presented in Fig. 9. There was good recapture of all three age groups of virgin flies. Although 2and 6-day virgin females were not recaptured well during the first 2k hours, the recapture numbers increased sharply by the time these flies had become if to 8 days old. Total recapture data suggest that the young 2-day and 6-day females were staying in the avocado grove in large numbers for up to 6 days, whereas most females released at 9 days of age or older were not recaptured after 2 days and had presumably left the avocado grove. These data also support similar recapture data from virgin females released at 8 days of age in previous bioassays reported in Tables 1 and 2. Failure to recapture very many 2-day virgin females in the first 24 hours in contrast to the final good recapture indicates that very young females are not highly attracted to males in the field, but are more attracted as they age. The greatest total recapture (25. 5$ of those released) occurred in the group of flies released at 9 days of age, and here the great majority of those recaptured were caught by the time the flies were 10 days old. The results strongly suggest that an optimum age for attraction of females is near

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67 10 days of age, and although mated females near this optimum age can still be attracted in substantial numbers, older mated females are poorly attracted. However, the initial response of mated females released at 9 days of age was reduced about 50?o compared with virgin females of the same age. Although the recapture of mated flies 11 and 15 days old was the lowest, a substantial percentage (352/3000 X 100) of mated flies were recaptured, which is of importance to future possible use of pheromone-baited traps. The results from the second experiment, shown in Table 9t indicate as in the previous experiment that about twice as many virgin females were recaptured as mated females. The importance of the first 2k hour recapture was not clearly demonstrated in this experiment. Results again show the largest first day recapture of virgin flies that were 10 days old. The recapture of flies released at 7 days of age parallels that in the previous experiment with the 6-day flies, namely, that substantial numbers of flies were caught only after they had become 9 days old. Very few virgin flies released on the day they emerged (1 -day-group) were recaptured. These data again suggest an optimum recapture age or response to the male pheromone. It is a measure of the consistency of the results from these experiments that a total recapture of 23 to 25% (93/^00 X 100 and 255/1000 X 100, respectively) of flies 9 days old at release occurred in the first and second experiments, and

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68 i i u Z 0 cd H C co cd t3 •H H O O CO H -P ,Q CQ t(fl CD T3 k -P H > e r o e f~, o I 0 Sh 0 IH 0) ft 2 • O T3 H W) CD •H o cd QJ o CD tsl O CD P O -P •H CO e •H CD M > •H H > O P^t H TJ -P cd -H 5 CD >>j CD cd m e moo rH «H CD -P J-J to CO CD £ en I H oo I to cd o ON TCi CD CD CO U 3 CD to a) 5 CO CD X cd b O -P •H CD =J 3 p cd CU CQ 0 •P Cd O CD •H faD rH CO fto CD U 0 > o u bJ3 CD Td ft cd o > o CO rH CO o -p cd 0 cd 0 C o 5 0 « 0 O -P rH i O 0 H cd 0* o *-l ft cd X) O & cd 3 cd rH -P U cn CO -3o 00 cd ft -P o ON o IN cd * • * • •H o U o rH o o fin 0 0 k ft 13 0 CO H 0 cd H p> p CvJ ft o B cd EH 0 o re 0 CO 0 rH 0 u u 0 J> cd P eg SO o o ON CO NO CO CM Cvj CM vo o NO IN CM ON NO 0 p cd < to •H W) CO -p •H cd •H > : cd -P 1 to No 0 W) »— 1 IN ON On CNJ CU , 1 w-\ *J l ) CO p C 0 rH 0 r-t CN) •H C^l 13 >> rH O •H iif nn UJ M •H \ r\ u i to p> O 0 u cd 0 -P — T -P x — t 0 H 0 § to 0 4: P o O CnJ rH £> t3 0 IV M O 0 rH r-T( > rH o O p. o cd 0 o to 0 0 rH rH rH cd cd cd > p c o EH *

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69 . from 17 to 25% (175/1000 X 100 and 100/400 X 100, respectively) of flies released at 6 to 7 days were recovered. The results of the third experiment, summarized in Table 10, are consistent with those from the two previous ones. Although equal numbers of virgin and mated females were not released, a greater percentage of virgin flies than mated ones were recaptured. The Zk hour recapture was greater than for any following period, and young females (4 days old at release) were caught in increasing numbers as they aged. A total of 20.5% (82/400 X 100) of the virgin 11 -day females were recaptured over the 4 day observation period, suggesting that 11-12 days is still near a broad peak of maximum attraction to males. In this experiment as in the two previous ones, older mated flies were very poorly attracted. The combined results from these three experiments suggest that virgin females 9-12 days old are maximally attracted to males in field tests. This includes the same range found in laboratory tests (Nation 1972). Both virgin and mated females near or younger than 9-12 days can also be attracted in substantial numbers. Young virgin females may have a tendency to be less active in moving out of a non-host area than older virgin females. Possibly the pressure to oviposit, even if unmated, may drive mature females to seek suitable host fruit for egg laying. The ability to attract young virgin flies and younger mated flies is of great importance to the potential use of

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71 pheromone -baited traps in survey and detection of caribfly infestations. Recapture of Virgin Females with Pheromone Extract-baited Traps The attraction of females to chemical extracts, obtained from washing 82 male cages with water followed by hexane extraction of the pheromone from the wash water v/as compared using McPhail and triangular sticky board traps. The first experiment was done only with McPhail traps, each one containing 300 ml of water instead of the usual borax-yeast hydrolysate-water food mixture. Ten of the traps were baited with 100 fil of the pheromone extract and ten traps served as a check and were baited with 100 ul of the solvent. Three drops of an emulsifier (Pergosperse L-9) was added to the water of half of the traps baited with extract and to half of the control traps. The pheromone extract and the solvent were placed on the trap wick (hanging from the center of the stopper) in the laboratory 1/2 hour before releasing the flies (16^5 hours). A completely randomized design was used with traps spaced 18 m from each other in a square pattern. McPhail traps baited with 100 jul of the pheromone extract captured only one male out of 1,000 flies of each sex (10 days old) released. Control McPhail traps did not catch any flies. The next experiment was done with triangular sticky board traps. In this experiment the chemicals were applied to wicks hanging from the center of the traps. Six of the

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72 traps were treated with 100 jul of the pheromone extract obtained from the same source as the one used in the previous experiment. Another six traps were treated with 100 jil of the solvent and the remaining six traps consisted of 5 tenday-old males per trap. The males were placed in screen-mesh cages of about 5 X 3 X 3 cm with food and these were suspended from the center of the traps. In a final experiment with triangular sticky board traps, the extract or solvent was mixed with the Stickem and spread over the entire surface of traps 1/2 hour before releasing the flies (1600-1700 hours). In contrast with the first test, in which both sexes were released, 1,000 virgin females (10 days old) were released in the second and third experiments. A completely randomized block design with 6 replicates was used with traps spaced 18 m apart in a square pattern. Females were released at 10 release sites each equidistant from any four traps arranged in a square. Captures were recorded periodically for ^ days. Statistical analyses were made individually for each test date and data were not combined as in all previous experiments. Traps baited with 100 ul of the pheromone extract on the wick captured 72 females while 5-male-baited traps attracted 55 and control traps none (Table 11). The pheromone -baited and male-baited traps attracted similar numbers of females and were significantly different from the control traps (P 0.05), but not from each other.

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7k Traps baited with 100 jul of the pheromone extract mixed with Stickem captured 7k females while 5-male-baited traps attracted only 6 and control traps none (Table 11). The pheromone -baited trap attracted significantly more females than the male-baited and the control trap. The attraction of virgin 10-day females to chemical compounds obtained from secretions of virgin 10-day males showed that chemical attraction between the sexes is very important . Laboratory Bioassays to Test Aggregating Pheromone Bioassays with the Plexiglas cage (Fig. 8) showed that baits of 10, 20, and 30 males attracted 11, 20, and 13 males, respectively, while cylinders baited with 20 females, male pheromone extract, and with food attracted 9t 8, and 6 males, respectively. Another bioassay showed that baits of 50 males and food only attracted 26 and 45 males, respectively, out of 200 released males in the cage. All males were 8 days old when first used. Bioassays using Plexiglas plastic cylinders as described by Nation (1972) showed that baits of 20 males (9 days old) and food attracted 20 and lk males (k days old), respectively, out of a total of 80 free males , released at the opposite end of the cylinders. In another bioassay, baits of 10 males (9 days old) and food attracted 37 and 36 males (k days old), respectively, out of a total of 100 free males. In a last

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75 test with cylinders, baits of 10 males and food attracted ^0 and 32 males, respectively. All males were 5 days old when first used and the maximum attraction to both males and food was obtained when males were 9 days old. These bioassays failed to show that males were any more attractive than food or control traps for free males. The next experiments made use of a nylon cage of 6 cubic meters set up inside the laboratory. Fluorescent lights provided light. In the first bioassay 250 males and 10 females were released in the cage. Two traps baited with 10 and 20 males caught 55 and 26 males, respectively, while two control traps caught 65 males. All males and females were 7 days old when first used and the maximum attraction to both males and food was obtained when males were 9 and 10 days old. In another bioassay in the nylon cage two traps baited with 10 males caught 78 males and two control traps caught 92 males out of 250 males and 10 females released when they were 7 days old. Maximum attraction to both males and food v/as obtained when flies were 8 and 9 days old. These laboratory experiments conducted with three different techniques did not prove to be an adequate laboratory bioassay for the aggregating phenomenon of male flies demonstrated in field bioassays. Ability of Laboratory-reared Males to Attract Wild Flies The attraction of wild flies to live male-baited and

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76 McPhail traps was compared at Homestead during the summers of 1972 and 1973, and at Key Biscayne during the summer of 1973. From June to August of 1972 a boll weevil trap baited with 50 virgin males (5 days old on the average) was hung in a guava tree near a McPhail trap (Trap No. 9 AREC) on AREC grounds to study attraction of wild flies. A control Stickem-coated trap was baited with food but no males. The male-baited trap was rotated with the control trap once every two weeks and sometimes weekly, but the McPhail trap was not moved from its place, since it was part of a long term trapping survey. Caged males in the trap were replaced once a week. In July and August, 1972, a boll weevil trap baited with 50 virgin males (6 days old on the average) was hung in a sapodilla tree at the AREC. A McPhail and a control trap were placed in the two nearest sapodilla trees. These traps were rebaited and rotated counterclockwise once a week. During August, 1972, a male -baited and a control trap were hung from cattley guava tree branches at the AREC. Traps were rebaited and rotated once a week. The average male age was 8 days when first put into the field. Results were compared with the capture of a nearby McPhail trap (Trap No. k AREC) . In 1972 mortality of caged males was high and most males were dying within the first three days after they were set up in the field.

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77 The results in Table 12 show that McPhail traps captured four times more flies than male-baited traps, and male-baited traps captured almost as twice as many as control traps. During June, 1972, male-baited traps captured almost five times more flies than McPhail and eleven times more than control traps. As the population increased substantially in July and August, male -baited traps became relatively inefficient. McPhail traps always attracted more females than males (a ratio of 5 : 1), while male-baited traps detected a relatively higher number of males by capturing a ratio of 2.3 i 1. In 1973 several modifications were made to the cylinders to help males survive a longer period of time. Water was provided in small vials containing dental cotton wicks, instead of using a block of lfo agar or water in a sponge as had been done in 1972. The aluminum caps positioned on the lower part of the cartridges were perforated and a circular screen mesh wire was fitted into the cap. In this way any excess water from rainfall drained outside the cylinder and less drowning of flies occurred. Eventually two paper towels were added inside the cartridges to provide more surface to the flies for resting, and for adsorption of the pheromone. Mortality of caged males during 1973 was reduced substantially and in some cases males survived for a week or more. From June to October, 1973 » sixteen boll weevil traps, each baited with ^0 virgin males (7 days old on the average)

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78 CO >> Sh ft** O O <+H to •H CD -P Cd H W T3 H H cd cd rH a> Ul Cd -P e w a> 1 CD P o 6 w 4o en -p P cd •p o 03 P >j EH DO 03 P Cd Q cd Oh O CO CO H cd o >> H sJ o T3 o o rH cd CM CM -P VO O -P tH *b Of rH •P o -p *b Of cd f 3 o P ^b Of ca Pi o «H P cd o o t-H rH O o ^3o no co no CO CO rO vo o th co o\ CO tH CM 00 to On no NO O 00 VO CO t-I ON tH VO no >o VO vo NO -j" oco t>vo co co o o o oo no O NO NO CO o CD > o U CO M > cd -P O W U rH rH rH Ml •H r-3 cd T3 O P. cd O to CO vo On 00 On VO OO CO VO ON 00 O O OOn no vo 00 vo CM O CO On CM CO O O OCO vo CM CO CM O NO Cn» CM O OCM CM tH O o vo co vo o O CM CO > o Cd 5 qC >> a) rH -P -P cd o On VO NO CO £>rH rH OtH On 0\ CM co CO CO CM t-H • oo rH • -3" VO On CO tH 00 CM T-H tH NO ^ o 00 VO oo CO i-H • CM T-H NO o CM o NO T-H t-H CO • CM 00 o NO H; .eh CM T-l • t-H O OO ot-I CM rH • T-H T-H CM t — 1 CM IN t-H • o• t-H 00 *H VO CM oOt-H «-H >> W cd u % -d J \ pt ft cd Xb cd M CJ in -p \ Of rH p CO O cd p 03 • H -p cd •H -P o U rH cd En Eh P>H OS

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79 were used. Six were located at the AREC and ten were located at Key Biscayne areas. All were located near continuously operated McPhail traps. Male-baited traps were usually located in a tree near the tree containing the McPhail trap; but sometimes it was necessary to hang both traps in the same tree, in which case they were separated by about 6 m. Male-baited and McPhail traps were rebaited and rotated once a week. At the beginning of the comparison McPhail bait was mixed 3 days before using it, but after several weeks, each trap was rebaited in the field by adding two pellets of torula ye ast-hydrolysate -borax to 300 ml of water. No gross differences in attraction response were noted between the two ways of baiting the traps, but this was not checked statistically. The results in Table 13 show that McPhail traps captured 1,997 flies, which represented 3. 58 flies per trap per day and was five times higher than male-baited traps. This is consistent with the 1972 data, although populations were lower in 1973During June and July, 1973. caged males captured slightly more flies than McPhail traps, but as the population increased in August, the caged males became relatively-inefficient. These results parallel those of 1972; namely, that caged males apparently do not attract flies consistently when the population increases. Again McPhail traps attracted more females than males in a ratio of about 3:1, while male-baited traps attracted fewer females per

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80 Table 13.Wild A. suspensa adult flies per trap per day captured during the summer of 1973 with 40-malebaited sticky traps compared with McPhail traps at AREC, Homestead, Florida. Six traps of each type were used for 5 months (93 trapping days) at 6 locations. Dates Type of trap and 40 Males McPhail Locations $ total $ (y total June 1.17 .25 1.42 1.58 .08 1.66 July • 54 .20 .74 .23 .01 .24 August .38 .17 .55 1.91 .45 2.36 September .61 .50 1.11 4.95 2.06 7.01 October .11 .08 .19 2.12 .67 2.79 1 (Loquat) .09 .03 . 12 .06 0 .06 2 (Loquat) .14 .12 .26 .06 .01 .07 3 (Surinam cherry) .05 .01 .06 .04 0 .04 4 (Guava) .69 • 35 1.04 4.01 1.71 5.72 5 (Guava) .99 .78 1.77 7.41 2.65 10.06 6 (Guava) .69 .22 .91 4.38 1.13 5.51 Total capture 246 142 388 1485 512 1997 Flies/trap/day .44 .25 .69 2.66 .92 3.58 Ratio
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81 male with a ratio of 1.7 : 1. It is noteworthy to mention here that male-baited traps, although showing lower overall capture than McPhail traps, detected larger numbers of females and males in three locations (locations 1, 2, and 3) where McPhail traps caught very few (Table 13) . The results in Table lk> from Key Biscayne show that McPhail traps in four months captured 387 flies, while malebaited traps caught 358 flies. The much lower population in Key Biscayne, flies per trap per day in Key Biscayne against 3.58 flies per trap per day in AREC with McPhail traps, was probably due to spraying against mosquitoes. The data confirm the results of previous experiments wherein caged males attracted flies when the population was low. Almost twice the number of males were attracted with male-baited traps as were attracted with McPhail traps (Table 1^-)'. Females and males were detected in male-baited traps in larger numbers than detected in McPhail traps in five locations (Nos. 3, 5, 7, 8, and 10). Courtship and Mating Behavior of A. suspensa in Nature Male calling behavior started when a male arrived at the substrate and spread both wings outward from the body at an angle of 90° with the costal vein parallel to the substrate. The male frequently made short running movements to one side and then the other, usually returning to about the same place

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82 Table 14.Wild A. suspensa adult flies per trap per day captured during the summer of 1973 with 40-malebaited sticky traps compared with McPhail traps at Key Biscayne, Florida. Ten traps of each type were used for 4 months (84 trapping days) at 10 different locations. Dates Type of trap and 40 Males Locations 0 total 0 T tota! July .66 .45 1.11 .96 .17 1.13 August .1? .05 .22 .16 .03 .19 September .23 .12 .35 .41 .11 .52 October .15 .16 .31 .16 .07 .23 1 (Mango) .09 nil . 04 • 13 .27 .07 .34 2 (Clusia rosae) • 30 .25 •55 .05 .13 no •70 3 (Guava & Lime) .24 -1 O . 18 .42 . 21 • 13 .34 4 (Grapefruit) .05 .03 . 08 .07 .11 .18 5 (Surinam cherry & Tropical almond) .32 .17 .49 .21 .05 .26 6 (Surinam cherry & Sapodilla) .10 .07 .17 .51 .04 .55 7 (Kumquat & Mango) .10 .05 .15 .07 0 .07 8 (Rose apple) .83 .60 1.43 .32 .02 .34 9 (Tropical almond) .41 .11 • 52 1.25 .28 1.53 1C (Tropical almond) .23 .09 .32 .15 .04 .19

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83 Table 14 continued Dates Type of trap and 40 Males McPhail Locations 0. & total 0. (f total Total capture 225 133 358 314 73 387 Flies/trap/day .27 .16 .43 .37 .09 .46 Ratio 1 4.30 : 1

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8k where he started. These lateral movements did not extend more than one-half cm to either side. After several of the above movements, the male stopped moving and started flicking and waving the wings. The costal vein was held parallel to the substrate at all times during these wing movements. Meanwhile, if a female was located in a nearby branch several ft distant, she flew short distances from one branch to the other, usually not directly towards the male, until eventually she landed on the same leaf or one a short distance from the male. Sometimes the female apparently arrived from another tree or perhaps from a more distant branch. However, I observed that in most cases the female flew short distances until arriving at the same leaf with the male. These flight movements usually took long periods of time to occur, sometimes more than an hour and many times the female did not reach the male at all, especially when disturbed by breezes or another insect. During the time that the female took to land near the male, the male continued waving his wings and small pouches were distended in the pleuro-ventral region of the abdomen. After a few seconds an anal pouch was everted. After the anal area was everted the male touched the substrate with the tip of the abdomen. The male then moved both wings laterally, simultaneously vibrating them rapidly. The vibration movements were brief and intermittent. Between fanning movements males turned, touched the

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85 anal pouch to the substrate at intervals, and followed a circular pattern as though marking a circular territory of about one-half cm in diameter. During intervals between wing vibrations males started movements involving scratching of the ventral surface of the abdomen with the metathoracic legs, and cleaning-like movements with the tarsus of the forelegs. The forelegs were also moved upward and downward touching the frontal area of the antennae. Both forelegs were used in this procedure without a definite alternation. One more cleaning-like movement consisted of folding one wing downward, oriented in such a way that the 3rd leg on the same side held the wing from the upper side, while the male rubbed the underside of the wing against the ventral side of the abdomen several times. There was an alternation of both wings in this rubbing movement that lasted several seconds and was repeated between intervals of wing vibrations. After the abdomen was rubbed several times with the hindlegs and the wings, the pouches from the abdomen grew disproportionally larger until two large white areas appeared which could be seen from almost any angle from which the male was observed. This together with the anal eversion and the rubbing movements was termed by Nation (1972) as the puffing behavior of A. suspensa . A female was never observed to come directly to a puffing male, but rather made several flights from one leaf to another, not overtly approaching the male. This type of

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86 orientation in which the insect makes successive comparison of the intensities of stimulation fr'om two sides is known as klino-taxis. Once the female was a short distance from the male, but usually separated by a few leaves or the next branch, the male stopped rubbing movements and spent more time vibrating the wings and touching the substrate with the anal pouch. If the arriving female was disturbed, she usually ceased movement toward the male and flew away. The male sometimes returned to the rubbing movements and wing vibration, and sometimes flew to another location. If the sequence was not interrupted, a female might fly to the same leaf, but stayed 2 to 3 cm from the male. As a female started moving forward, the male spent less time in vibrating the wings. Movements of a female toward a male were slow and several minutes passed before the two were within a range of 1 cm. Then the male stopped all movement and kept the wings spread at 90° while facing the female. For the first time, the male started moving from its area toward the female. This movement of the male was almost imperceptible and slower than movement of the female. When the flies were very close, they faced each other and made contact by touching each other with the frontal area of the head. The male jumped to the female and turned his body 180°, landing on top of the female and facing the same direction as the female. The male grasped the female at the leading edge of the wings with the forelegs, extended

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87 his proboscis to the vertex of the head of the female, and bent his abdomen downward and forward in order to contact the ovipositor of the female. The male on top was positioned slightly behind the female with the head aligned with the prothorax of the female. The female could bend the ovipositor forward at an angle so that the male claspers were incapable of finding the female genitalia. Females might also vibrate their wings and walk short distances with the male. Males trying to copulate with females in the field were easily detected at this point because there was rapid wing vibration produced by both flies that was easily heard by human observers when the wind was calm. Sometimes the female intercepted the male in the jump with a quick movement of one wing and the male never reached the top of the female. Contact in this way lasted a few seconds, but in case of successful jumping, the male could stay on top of the female for many seconds without copulating and would try through several interruptions to successfully mate with the female. Males were frequently observed to jump onto the back of females and attempt intromission. Unfortunately no successful copulation was observed from inception, but 26 pairs were observed already in copula. The male on top of the female remained with the wings spread outward at 90° from the body while the female wings were maintained outward at 45° from the body. No wing movements occurred. The ovipositor of the female was pointed upward and was held by the claspers of the

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88 male. The male frequently extended the proboscis and touched the frontal area of the head of the female during copulation. The lateral pouches were not distended during copulation. On some occasions another male or female flew directly to the mating pair and touched the genitalia with their mouthparts. On most instances the female walked a short distance with the male on top and only on a few occasions was copulation interrupted. Just before copulation ended the female started walking, moving the wings, and kicking the male with the hindlegs and the wings. Separation of the aedeagus from the vagina did not take place immediately. Usually the male got down from the female and turned 180° to the opposite direction, with the aedeagus still inserted in the vagina. Sometimes the female was observed pulling the male with the aedeagus still inserted. After complete separation the flies stayed a few cm apart for a few seconds while making rubbing movements with the forelegs and the head. Males never used the same spot to start calling more females, but these spots were commonly visited, immediately afterwards, by males who walked in the same area most of the time and made frequent proboscis extensions to touch the substrate. Males courting and/or attempting copulation with other males were commonly observed, but this behavior did not involve a similar sequence of courtship behavior as in the case of a female approaching a male. What usually happened was that a very excited male was approached directly by

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89 another male, the first leaping upon the other almost immediately and attempting to copulate. The male beneath combined fluterring with kicking and moving the forelegs and the wings to thwart the copulation attempt in most cases after only a few seconds, but sometimes the male remained on top for several seconds. At least four senses seemed to be involved in the courtship and mating behavior of A. suspensa . At a long distance the olfactory sense was probably the most important in bringing the sexes together to a suitable area (i.e. several trees with dense canopies). Males probably were also attracted to the same area by detecting an aggregating pheromone. The auditory sense might be important at a shorter distance after the female reached the area (i.e. a tree). The visual sense could be important also, especially when the flies were in the same branch or close branches in the same tree. The tactile sense was involved upon actual contact. The gustatory sense might be important, too, but it was not carefully observed to determine its order of importance . Some Environmental Factors Associated with Mating More than 55 hours were spent in the field observing caribflies during the summers of 1972 and 1973 • Observation periods covered most of the daylight hours starting from 0700 hours in the morning and ranging to late evening (2100

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90 hours) (Table 15) with an average of 237 minutes spent in observation each daylight interval. A total of 26 pairs of flies were observed copulating between 1325 hours to 1955 hours. No mating pairs were observed during the period 0700 to 1300 hours (Table 15). Copulation was observed always on the underside of leaves, in cattley guava, sapodilla and guava trees at an average height of 2.3 m and with a mean temperature and relative humidity of 29.4° C and 70%, respectively (Table 16) . An exact estimate of the duration of copulation was not possible, since no pair was observed just as copulation was initiated. The longest duration of copulation observed was 24 minutes. Twenty-four mating pairs were observed in 2 to 3 neighboring trees, but on the surrounding trees it was not possible to find mating, except for two isolated cases that occurred in guava trees, although considerable time was spent in observing flies on the surrounding trees. On 1 August, 1972, I counted 57 females and 37 males in two cattley guava trees, and observed two matings, while only 26 females and 3 males were counted in several surrounding cattley guava trees. On 30 August, 1972, 22 females and 22 males were counted in these same two trees, while only 15 females and 2 males were counted in several surrounding trees. Exactly the same time was spent in both cases counting flies in the two trees and in the surrounding area. In the sapodilla

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91 Table 15.Number of mating pairs of wild Caribbean fruit flies observed in daylight hours during the summer of 1972 at AREC, Homestead, Florida. * Total minutes Number of mating Weather conditions Time period 0 observation pairs observed Temp. (-C) RH (%) 0700 0800 220 0 22.8 100 0800 0900 240 0 24.0 100 0900 1000 175 0 26.8 87.2 1000 1100 285 0 28.9 72.1 1100 1200 340 0 29.4 70.0 1200 1300 215 0 29.4 64.0 1300 1400 250 2 29.7 64.0. 1400 1500 300 4 30.1 60.6 1500 1600 125 0 30.4 53.0 1600 1700 309 4 29.9 59.0 1700 1800 348 11 29.3 65.5 1800 1900 235 2 27.9 76.7 1900 2000 245 3 26.1 84.5 2000 2100 . 40 0 26.1 87.5 Total 3327 minutes 26 pairs Mean logical average station data calculated from records at AREC, Homestead, Florida. of the meteoro-

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92 Table 16.Mating pairs of wild A. suspens a flies observed during the summer of 1972 at AREC, Homestead, Florida, including data on dates, duration in copula, host tree, height and general weather conditions. Duration # Mating Host Height Weather Date Time in copula No. tree (meters) -C RH Wind (minutes) 1 J. 6-??_7? J-™ 7 2 Sapodilla 2.1 31.1 57 6-11 9 c 6-?7-7? 1Q16 j Guava 2.1 30 .0 66 2-5 J 6-?R-7? 1 717 6 Sapodilla 2.3 32.8 k8 M 77-7? 1 71 6 Cattley guaval.9 7? 11 J 77-7? 1 7/i? < »» 1.9 10 0 68 |l f. u 7— 7-7? 1 7/j.Q 1.6 TO 0 67 II n f (-1 }( tL 1DUO Q 7 2.5 ?R Q CO % 7 f £ IT 8 1656 2 " 1.7 28.9 72 ft 9 7-^3-72 1703 7 11 2.6 28.9 71 II 10 7-13-72 1707 19 11 2.2 28.9 71 fff 11 7-13-72 1723 1.7 28.9 71 12 7-13-72 1725 9 2.2 28.9 71 13 7-13-72 1728 6 11 2.1 28.9 71 14 7-13-72 1729 16 2.1 28.9 71 15 7-13-72 1735 16 11 2.1 28.9 71 16 7-19-72 1955 8 11 2. ^ 23.9 9k 17 81-72 16^9 10 2.1 30.0 6k 6-11 18 81-72 I651 19 11 2.5 30.0 65

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93 Table 16 continued Duration * Mating Host Height Weather Date Time in copula No. tree (meters) -C RH Wind (minutes) 19 8-23-72 1443 7 Sapodilla 2.0 31.I 62 2-5 20 8-23-72 1452 5 " 2.9 31.1 62 21 91-72 1325 2 " 3.2 30.0 65 22 91-72 1358 2 " 3.1 31.1 63 23 91-72 1428 3 " 2.4 30.0 70 24 91-72 1817 24 " 2.2 27.8 82 25 91-72 1841 5 " 3.4 28.3 80 26 91-72 1918 5 " 2.9 27.8 81 Mean 1711 8min l4sec 2.3 29.4 69.5 Calm itRelative humidity is expressed in % and wind was estimated using the "Rule of Thumb" system in kilometers per hour.

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94 grove where mostly unripe fruits were found in very small numbers during the summer season, 9 mating pairs were observed in three neighboring trees. It is noteworthy that I never observed females ovipositing in the few ripe sapodilla fruits that occasionally were found in the trees. In contrast to the above observations, most females in the guava and cattley guava grove were observed laying eggs in the fruits, although some grouping of flies always occurred in a few guava trees. For example, on 30 August, 1972, in two guava trees, 19 females and 29 males were counted. Ten of the males were very actively courting females. Males were frequently found aggregated at the tip of tree branches. I counted 9 males and 1 female on only one branch of a sapodilla tree. At the beginning of the field observations in May 1972, large numbers of females were counted among the guava grove while males were scarce. In this guava grove the mean ratio of females to males counted was about h.2 i 1 (12 May 12 June 1972). When my observations progressed (after mid June 1972) I found nearly a 1 : 1 ratio in those areas where mating was observed. In the sapodilla trees this ratio was 1.1 females to 1 male, and in the cattley guava trees the ratio was 1.3 females to 1 male. The mean ratio in places where mating was observed averaged 1.03 females to 1 male, indicating that sex ratio might be an important factor in successful mating. The percentage of courting males observed in the groves

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95 varied from 6% in the morning to G\% in the afternoon. Most courting in the morning was observed close to noon, with a few exceptions. In the cattley guava grove, more than 85?? of the males observed exhibited courting behavior during the afternoon, while only about lfo of the males observed in the morning exhibited this behavior. In sapodilla trees about 70% of the males were courting in the afternoon and 11% in the late morning, near noon. In the guava grove courting males were less common, and were estimated at 38% in the afternoon and 6% in the morning. These data agree significantly with the mating observations. Apparently males start courting females near noon and this characteristic behavior becomes more common during the afternoon and late afternoon. When flies were observed in copulation, wind movement was almost imperceptible, even in the form of gentle breezes. According to the "Rule of Thumb" system in judging the velocity of wind (devised by USDA Weather Bureau, Official Record, Nov. 20, 1930) breezes were rarely more than 5 km an hour when copulations were observed. On only three occasions was wind velocity considered to be 6 to 11 km an hour. Whenever windy afternoons occurred no mating was observed. In laboratory bioassays with flies exposed to different wind currents, mating and courtship behavior were studied. Flies unexposed to wind current were more active with males leaping upon females, while flies exposed to 16

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96 kph were calm and only occasionally attempted to copulate. Forty-six mating pairs out of 80 pairs were counted when flies were unexposed to wind current, while 16 and 6 pairs mated out of a total of 80 pairs when flies were exposed to 8 and 16 kph, respectively (Table 17). Fewer puffing males were observed with wind currents of 8 and 16 kph. As soon as the experiments were ended, many flies among those previously exposed to 8 and 16 kph started copulating.

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DISCUSSION The Experimental Procedures The bioassay test area, an avocado grove, was chosen to study the attraction of caribflies because of absence of wild flies, relative isolation from host groves, and uniform distribution of trees. Trapping indicated that no wild flies were present and experiments with marked flies showed that 100% of the recaptured flies were marked. In view of the considerable reduction demonstrated in 1973 in recapture of mated flies (on the average h2% fewer than virgin ones of the same age), the decision made at the very beginning of this study to segregate the sexes upon emergence was an important one to the success of the work, and it is recommended for any future work with this fruit fly. It would certainly be prudent to segregate the sexes in similar pheromone research with any other tephritid flies. Working entirely with wild populations would have created many difficulties especially related to variation in relative population density, age, sex ratio, and competition from fruit and/or oviposition odors. It would not have been possible to obtain recapture percentages as was done in this work, since no precise measures are presently available to estimate the wild population of caribflies in even a limited 98

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99 area. No effort was made in this study to test parameters such as trap shape, size, color, or design, and these might well be the subject of fruitful investigations in the future. The boll weevil traps were readily available, relatively easy to use, and in preliminary tests proved to be suitable. A more suitable trap for use with pheromone will undoubtedly have to be devised. The boll weevil trap is bulky, requires large space for storage, and requires periodic cleaning. The McPhail and triangular sticky board traps were selected because these are the conventional traps actually used by state and federal agencies for survey and detection of fruit flies in the continental United States and in many other parts of the world. The triangular sticky board traps offer the added attraction of being relatively inexpensive and disposable . An important factor contributing to the success of these experiments was the release of flies in a uniform grid pattern, at the same distance from each trap, which provided for a more uniform distribution of flies over the grove than release from a single site would have. The field bioassays developed in this way were highly precise and accurate and similar recapture data were obtained between replicates of the experiments and among the treatments. It may be especially useful in future field bioassays to know that most of the recapture of sexually mature females occurred within

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100 ^8 hours after release as shown in Figs. 9 and 10. Future experiments can be conducted in successive weeks, provided that laboratory-reared insects are available. A similar field bioassay was developed by AliNiazee and Stafford (1973) to evaluate the sex pheromone of the grape leaffolder, Desmia funeralis (Hubner), which indicates that the general field design may be useful for other insects in addition to tephritid species. This work is the first development of a field bioassay for the sex pheromone produced by male caribflies. The ultimate test of a pheromone must be in the field, and collectively these experiments constitute a workable and recommended field bioassay for further sex pheromone or aggregation pheromone studies. I believe that future field mating observations in A. suspensa need be carried out only in the afternoon, approximately starting at noon time and continued until late afternoon. Special attention should be paid to finding specific areas where matings occur. Future mating observations may help in planning a more effective comparison of traps, or trapping program, by using a more precise criterion when distributing the traps. The Field Bioassays Preliminary experiments in a screened enclosure showed that live virgin males caged in boll weevil sticky-covered

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101 traps were not any more attractive than McPhail traps to virgin females of the same age. Consequently, subsequent experiments were conducted in an avocado grove. Recapture of released virgin females showed for the first time that virgin female flies were attracted under field conditions to virgin males. A progressive increase in the recapture of virgin females was obtained when the number of caged males in traps was increased, thus, providing more evidence of male attractiveness . In all experiments with comparison of recapture of released virgin females, male-baited traps always recaptured more flies than McPhail traps. The population density in the avocado grove was controlled and was always low (maximum number of flies released at any one time in the 1 ha of avocado grove was 2,900). Pheromone traps are not usually as effective in competition with wild mates under high density conditions as under low population densities (Beroza 1971. Beroza and Knipling 1972, Hardee et al. 1969 i Hardee et al. 1970, Lloyd et al. 1972, Marx 1973. McLaughlin et al. 1973). Recapture percentages of released females generally averaged better than 20fo with male-baited traps and larger recapture percentages occurred in some cases. For example, kQfo of the released virgin females were recaptured in one case. These recoveries of released virgin females suggest

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102 that substantial population reduction might be possible with pheromone -baited traps. The use of pheromone-baited traps in addition to the McPhail traps or the use of both in combination might be very useful for future survey of caribfly populations. In the first and second experiments only females were released; in nature wild males are present to compete with male-baited traps. Nakagawa e_t al. (1970) showed with medflies that when males were scarce or absent, sexually mature virgin females were attracted by synthetic lures, but when sexually mature males were introduced into field populations, virgin females ceased their response to lures. Thus, bioassays were designed to see if virgin males released at the same time as virgin females would reduce the recapture percentage of females. Unexpectedly, the recapture of females was not reduced, and more surprising, males were also attracted to male -baited traps, and in the same numbers as females (Tables 4, 5) • Indeed the percentage recovery of females and males was 29 and JOfo respectively, which was slightly higher than that obtained in previous experiments when only females were released. Because I was not sure whether males were attracted to the females which became stuck to St ickem-covered boll weevil traps, rather than being attracted to the males acting as bait, I conducted two further experiments in which males were released in the absence of any released females. The results (Table 6) showed

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103 unequivocally that males were attracted to males. These experiments provide the first documentation by direct means that male to male attraction occurs in the Tephritidae and that there may be both aggregation and sex pheromones, or the sex pheromone may also act as an aggregation pheromone. Other authors (Bateman 1972, Cunningham and Steiner 1972, Feron 1962, Fletcher 1968, Prokopy and Bush 1972, Stoltzfus and Foote 1965) have suggested that aggregation of fruit flies may occur. Virgin females caged in boll weevil sticky traps did not attract released males or females as well as McPhail traps (Table 7). This was not unexpected, since there is no evidence from laboratory or field studies that female caribflies, or females of any other tephritid species, produce an attract ant pheromone. McPhail traps attracted more flies than boll weevil sticky traps baited with live females. A sex ratio near 1 was recaptured with female-baited traps. No significant differences were found between McPhail traps and boll weevil traps baited with food, 5» or 10 live virgin females. In order to keep the caged females serving as trapbait alive, food and water had to be included with the females. Much of the attraction that occurred to the femalebaited traps may have been response to the food. In contrast to these results, Table 1 showed a significant difference between boll weevil traps baited with 10 males and those baited with food, 1 or 5 males and also showed that traps

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104 with 5 males as bait were significantly better than traps baited only with food. It would be most desirable in using a sex pheromone trap if a wide range of ages of flies were attracted, and especial ly if flies could be attracted before mating occurs. Baranowski (1968) and Nation (1972) have shown that female caribflies begin mating and laying eggs in the lab at 5-7 days but no data are available on the age of mating or sexual maturity in nature. The question may be asked "What is the likelihood that a female will mate within 7 days in nature?" Marked virgin females of different ages ranging from 1 to 16 days (Tables 8, 9» and 10) were only slightly attracted at 1, 2, or 4 days, so one would expect very few females of these ages to be mated. Virgin females 6 and 7 days old were attracted somewhat, so that chances for mating at 6 or 7 days go up substantially. Virgin females 9 to 12 days old were maximally attracted to virgin males, and the probability of mating within these ages must be very high indeed. The data suggest a tentative answer to the question asked. Since there is little apparent attraction between the sexes prior to about 6 days, there is probably little mating before 6 days. At 6 to 7 days probably less than $0% are mated. Saturation of an area with pheromone traps could very possibly trap many females before they found a wild mate and thus before they laid fertile eggs. Mated females were about hjfo less responsive at 9 days

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105 of age than virgin females of the same age to male-baited traps. Older mated females were much less attracted. This ability to attract young virgin females (5i 6, or 7 days old) and younger mated females is of great importance to the potential use of pheromone-baited traps in survey and detection of car ibf lies. Attraction possibly due to visual and auditory orientation to caged males was eliminated by using triangular sticky board traps baited with 100 jdl of male pheromone extract obtained by washing male holding cages from males maintained in incubators for 9 or 10 days. The results demonstrated for the first time that virgin females responded under field conditions to natural lures obtained from secretions of mature males, and thus confirm that attraction to the male pheromone definitely occurs in the field. The aggregation phenomenon demonstrated in the field did not work under any laboratory conditions tested. Similarly, muscalure and grandlure attracted both sexes of M. domestica and A. grand is , respectively, in the field, but not in laboratory bioassays (Carlson and Beroza 1973, Hardee et al. 1972). This work has established that laboratory-reared males will attract wild flies, a finding of considerable importance to subsequent pheromone isolation studies with lab-reared males. Comparison of the overall capture during 1972 and 1973 for each month (Tables 12, 13, and Ik) shows that McPhail traps caught on the average three and a half times more wild

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106 flies than male-baited traps. However, during June of 1972 and 1973, AREC male-baited traps caught almost three times more flies than McPhail traps on the average. During July of 1973 at Key Biscayne and at AREC, male -baited traps caught slightly more flies than McPhail traps on the average. It seems possible that male-baited or pheromone traps might attract more flies during certain periods of the year and further testing should be continued throughout the year to observe these possible differences. Comparing the overall captures during 1973 by locations (Table 13 and 14), McPhail traps caught larger numbers of wild flies in about half of the locations. At locations 1, 2, and 3 in AREC and at locations 3» 5$ 1% 8, and 10 in Key Biscayne, male-baited traps caught more wild flies than McPhail traps in 1973» In many other locations (1, 2, and 3 in AREC and 2, 3, 5, 6, 7, 8, and 10 in Key Biscayne), malebaited traps greater numbers of wild males in the population than McPhail traps, which caught very few or none. These data strongly suggest that pheromone traps could be more valuable than McPhail traps in detecting low level infestations during population surveys. Pheromone traps may also give information about the sex ratio in populations, which McPhail traps probably cannot do. The sex ratio of trapped wild females to wild males was quite variable with McPhail traps, ranging from 2.3 0. s 1 (fin October 1973 (Key Biscayne) to 25 $ i 1 6* in July 1972 (AREC) with a final av-

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107 erage of 3.6 § : 1 0^. The total capture in Key Biscayne and AREC in 1972 and 1973 was 3,014 females and 834 males. Malebaited traps showed more consistency throughout the comparison ranging from 0.9 $ » 1 (? in October 1973 (Key Biscayne) to 4.7 0. t 1 (f in June 1973 (AREC) with a final average of 1.9 (j) s 1 0^ The total capture in Key Biscayne and AREC in 1972 and 1973 was 719 females and 381 males. Courtship and Mating Behavior of A. suspensa in Nature Courting males in nature evert lateral and anal pouches, and it is during this behavior that the pheromone is probably released in largest quantities. A "calling" male tends to remain in a small area on the underside of a leaf alternating slow wing waving with bursts of rapid wing fanning. The wing fanning produces audible sounds that can be heard by human observers several feet away on quiet calm afternoons. Webb (1973) recorded and analyzed the sounds, and is investigating their biological significance. Possibly female caribflies may use the sound for orientation. Visual stimuli are probably important at close range, when the female is presented with the broad wing expanse and the banded pattern. When a male extends the proboscis, deposition of chemicals for gustatory as well as olfactory stimulation of females may occur. Tactile as well as chemosensory mechanisms probably are important at the very last when face to face physical contact between male and female occurs and when the male leaps onto

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108 the back of the female. At a long distance probably the olfactory sense is the most important in bringing the sexes near each other. Males probably are attracted to the same area by detecting an aggregating pheromone, which might also be identical to the sex pheromone. The importance of the above stimuli has not been quantified experimentally, so they are only presumed to be necessary in the courting and mating sequence in nature. Their importance should be thoroughly investigated and their potential use for population control alone or in combination should be tested. Field observations suggest that wild males attract more females during the afternoon hours than earlier and this is in agreement with data from the early release-recapture experiments (Fig. 9). I observed 26 mating pairs in the afternoon hours but none in the morning hours, although I spent approximately the same amount of time in observations during both periods. A higher percentage of those males observed in the afternoon were exhibiting the characteristic courtship behavior, while only a very small percentage of males observed in the morning were sexually active. Tephritid species mate during the morning, as in C. capitata (My burgh 1962, Holbrook and Fujimoto 1970); during high light intensities, as in D. neohumeralis (Gee 1969) ; and during the night until next day, as in Pterandrus (= Ceratitis ) rosa (Ksh.) (Myburgh 1962). However, most Dacinae, E. fratria , A. ludens f

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109 A. suspensa , and R. pornia are reported to initiate activity at dusk, or at late afternoon (Back and Pemberton 191^. Baker et al. 19^4, Earton 1957 » Chambers e_t al. 1972, Economopoulos et al. 1971, Fletcher and Giannakakis 1973a, Myers 1952, Nation 1972, Pritchard 1967i Roan et_ al. 195^i Schroeder et al. 1973, Syed I969, Syed 1970, Syed et al. 1970, Tauber and Toschi 1965a) . Caribflies court and mate in the field on the underside of leaves. In contrast, the sexes of some Rhagoletis spp. assemble for mating on the host fruit, having been attracted there by the size, shape, color, and odor of the fruit. Upon copulation the pair usually flies to the surrounding vegetation (Prokopy and Bush 1973). One consequence of the attraction of both sexes to the fruit is the apparent lack of need for a sex attractant (Bush, personal communication). Unfortunately, very few published observations of field behavior and mating are available on fruit flies, and none have been previously published on caribflies. Mating and aggregation of caribflies may be linked. Aggregations of males possibly allow production of a stronger gradient of the sex pheromone. Males were frequently found aggregated at the tip of tree branches in certain trees and were usually sexually active. Mating was observed in host trees not bearing ripe fruits, and in non-host or less preferred host trees. These observations could influence the location of McPhail and pheromone traps, although Farias and

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110 Nakagawa (1970) suggested that traps should be placed in host trees of C. capitata whenever possible. However there are indications by Bess and Haramoto (1961), Nishida and Bess (1957), Sigwalt etal. (1968), and Syed et al. (1970) that D. dorsalis , D. cucurbitae , C. capitata , and D. z on at us, respectively, move back and forth to the surrounding vegetation. Such "nondispersive" movements are associated with the normal activities of feeding, ovipositing, and mating according to Bateman (1972). I believe pheromone traps should be carefully distributed in the field according to these movements of flies. A windless afternoon seems to be important to mating. Most mating pairs were observed when wind movement was less than 5 to 11 km an hour and in laboratory bioassays mating and courtship behavior were inhibited at 16 kph (Table 17). The Future Application of This Research The general procedures used here for demonstrating aggregation and sex attraction, and for field observations, might serve as a general model for similar work with other fruit flies. Much of the work of isolating and identifying the sex pheromone will need to be done for practical reasons in the laboratory. It is important to future studies that these field observations generally support earlier laboratory observations (Nation 1972). Thus, we know that courting males generally exhibit the same characteristic sexual behav-

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Ill ior in the field as in the lab, that males attract females, and that females are maximally attracted when they are 9 to 12 days old. These data lend strength to the validity of laboratory observations. Nevertheless, total reliance upon laboratory data can not be supported, and as much should be done or confirmed in the field as possible. The attraction of males to males was not suspected from any previous laboratory data, and indeed, even now can not be demonstrated in the laboratory. In general a field bioassay for a sex pheromone is to be preferred, and should be developed as early as possible in work with any other fruit flies. Parameters such as the best concentration of the pheromone, the optimum amount of each chemical component forming the pheromone, and the best combination of the pheromone with preservatives or Stickem need to be investigated. After a suitable concentration and effective combination is found, testing should proceed with the development of an effective trap design with a long-lasting attraction to wild flies in the field, powerful enough to override wild males. Preservative chemicals which might synergize the pheromone can be of great value. Certainly, the stability of the pheromone to weather conditions is of importance. In relation to trap design, it would be interesting, for example, to determine if trapping can be improved by combining the pheromone with the food in McPhail traps. Perhaps application of pheromone to wicks hanging from the stopper of

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112 McPhail traps can improve catches, detect low density populations, and provide a better estimate of sex ratio. Other factors which may be important are color, size, and placement of traps. The attraction of males to males may prove very useful in population suppression techniques. It is not known whether males respond to the same pheromone as females, and this should be determined with extracts of the sex pheromone or with the identified chemical components. It is important to determine what ages of males can be attracted to a baited trap. This information could be obtained with male-baited traps by releasing males of known age as in the experiments with females reported here in Tables 8, 9, and 10, If very young males can be attracted to traps, then possibly effective population suppression might be achieved by trapping young males before they are old enough to mate. A potential use of the field bioassay might be to study the loss of sexual vigor and/or mating competitiveness of sterile insects. The following data v/ere not replicated and consequently have not been included in the results section. They are given here merely as a suggestion for future work. In a field bioassay on 4-9-72 in the avocado grove five traps baited with H-0 sterile males (irradiated with 7 Kr when they were 2 days old) attracted 41 radiation-sterilized and 50 non-irradiated females } and 56 sterilized and 72 nonirradiated males. Five traps baited with 40 non-irradiated

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113 males attracted 101 sterilized and 123 non-irradiated females; and 99 sterilized and 155 non-irradiated males. All flies were 8-dayold virgins when first put into the field and 1,200 flies of each group were released. Traps were spaced 30 m apart in a square pattern. The results (Table 18) show that non-irradiated males attracted twice the number of females and males that sterile males could attract. These data, although not properly replicated, suggest that the technique may be helpful in the future, probably not only with A. suspensa , but with other species to monitor the quality control of sterile insects for release in a large scale program. This work provides background for a variety of future possibilities for population suppression, such as combination of male trapping, female trapping, placement of food and specific sex attractant in traps, and location of traps in aggregation areas. The evidence also strongly supports the use of pheromone traps in population surveys, especially to locate movement of flies into new regions of the United States or into the countries of Latin America.

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BIBLIOGRAPHY AliNiazee, M. T . , and E. M. Stafford. 1973. Sex pheromones of the grape leaffolder, Desmia funeral is (Lepidopterat Pyralidae)s Laboratory and field evaluation. Ann. Entomol. Soc. Amer. 66 j 909-911. Anonymous. 1970. The major pest and diseases of economic crops in the Caribbean . Food and Agric. Organiz. of the U. N. Office for the Caribbean area. P. 0. Box 1071, Port of Spain, Trinidad and Tobago. Anonymous. 1972. Key to less pesticide use citrus scale trap. U. S. Dept. Agric. Agric. Res. 20 1 5. Anonymous. 1973. Attracting the female medfly. U. S. Dept. Agric. Agric. Res. 21s 10. Antognini, J. 1972. Insect growth regulators and insect sex attractants in pest control. Invitational paper presented at the 56th annu. meet, of the Pac. Br., Entomol. Soc. Amer. at Victoria, B. C, June 20, 1972. Back, E. A., and C. E. Pemberton. 191^. Life history of the melon fly ( Bactrocera cucurbitae Coq.). U. S. Dept. Agric. Agric. Res. 3« 269-27*+. Back, E. A., and C. E. Pemberton. 1917. The melon fly in Hawaii. U. S. Dept. Agric. Bull. ^91: 1-64. Baker, A. C, W. E. Stone, C. C. Plummer, and M. McPhail. 19^4. A review of studies on the Mexican fruit fly and related mexican species. U. S. Dept. Agric. Misc. Publ. 531 1 1-155* Baranowski, R. M. 1968. Research on the Caribbean fruit fly at the subtropical station (Homestead, Florida). Nurserymen's Buyer's Guide and Bull. 13s 7-8. Barton B., L. 1957. The effect of light on the mating behaviour of the Queensland fruit fly Strumeta tryoni Frogg. Aust. J. Zool. 5« 1^5-158. Bastock, M. 1967. Courtships An ethological study . Aldine Publishing Co., Chicago. 220 pp. U5

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116 Bateman, M. A. 1972. The ecology of fruit flies. Annu. Rev. Entomol. 17 i 493-518. Beroza, M. i960. Insect attractants are taking hold. Agric Chem. 15: 37-40. Beroza, M. 1971. Insect sex attractants. Amer. Sci. 59 » 320-325. Beroza, M. 1972. Attractants and repellents for insect pest control. In Pest control. Strategies for the future (Nat. Acad. Sci., Wash., ed.). pp. 226-253Beroza, M. , and E. F. Knipling. 1972. Gypsy moth control with the sex attractant pheromone. Science. 177 » 19-27. Bess, H. A., and F. H. Haramoto. 1961. Contributions to the biology and ecology of the Oriental fruit fly, Dacus dorsalis in Hawaii. Hawaii Agric. Exp. Sta. Techn. Bull. 44: 1-30. Bezzi, M. 1909. Le specie dei generi Ceratitis , Anastrepha e Dacus . Biol. Lab. Zool. Gen. Agric. 3* 280-286. Boyce, A. M. 1934. Bionomics of the walnut husk fly, Rhagoletis completa . Hilgardia. 8: 363-579. Bush, G. L. 1969. Mating behavior, host specificity, and the ecological significance of sibling species in frugivorous flies of the genus Rhagoletis (Diptera: Tephritidae) . The Amer. Natural. 103: 669-672. Butler, C. G. 1967. Insect heromones. Biol. Rev. 42: 42-87. Cameron, E. A. 1971. Field tests of disparlure in Pennsylvania. Paper read at annu. meet., Entomol. Soc. Amer., Los Angeles, California. Cameron, E. A. 1973. Disparlure: potential tool for gypsy moth population manipulation. Bull. Entomol. Soc. Amer. 19» 15-19. Carlson, D. A., and M. Beroza. 1973. Field evaluation of (Z) -9-tricosene, a sex attractant pheromone of the house fly. Envir. Entomol. 2: 555-559 . Chambers, D. L. , K. Ohinata, M. Fujimoto, and S. Kashiwai. 1972. Treating tephritids with attractants to enhance their effectiveness in sterile-release programs. J. Econ. Entomol. 65: 279-282.

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117 Christenson, L. D., and R. H. Foote. I960. Biology of fruit flies. Annu. Rev. Entomol. 5* 171-192. Costa Lima, A. da. 1934. Moscas de frutas do genero Anastrepha Schiner (Diptera: Trypetidae). Inst. Oswaldo Cruz Mem. 28: 487-575. Cross, W. H., D. D. Hardee, F. Nichols, H. C. Mitchell, E. B. Mitchell, P. M. Huddleston, and J. H. Tumlinson. 1969. Attraction of female boll weevils to traps baited with males or extracts of males. J. Econ. Entomol. 62 t 154-161. Cunningham, R. T . , and L. F. Steiner. 1972. Field trial of cue-lure + naled on saturated fiberboard blocks for control of the melon fly by the male -annihilation technique. J. Econ. Entomol. 65: 505-507. Cunningham, R. T . , L. F. Steiner, K. Ohinata, and G. J. Farias. 1970. Mortality of male melon flies and male Mediterranean fruit flies treated with aerial sprays of lure and naled formulated with a monoglyceride or siliceous extender. J. Econ. Entomol. 63: 106-110. Cunningham, R. T . , L. F. Steiner, and K. Ohinata. 1972. Field tests of thickened sprays of methyl eugenol potentially useful in male-annihilation programs against Oriental fruit flies. J. Econ. Entomol. 65» 556-559. Dethier, V. G., L. Barton B., and C. N. Smith, i960. The designation of chemicals in terms of the responses they elicit from insects. J. Econ. Entomol. 53s 134-136. Economopoulos, A. P., A. Giannakakis, M. E. Tzanakakis, and A. V. Voyadjoglou. 1971. Reproductive behavior and physiology of the olive fruit fly. 1.Anatomy of the adult rectum and odors emitted by adults. Ann. Entomol. Soc. Amer. 64: 1112-1116. Eden, W. G., H. C. Chiang, E. H. Glass, D. L. Haynes, P. Oman, and H. T. Reynolds. 1973. The pilot boll weevil eradication experiment. Bull. Entomol. Soc. Amer. 19 « 218-221. Farias, G. J., and S. Nakagawa. 1970. Host vs. non-host plants as sites for baited traps for Mediterranean fruit flies. J. Econ. Entomol. 63* 662-663. Feron, M. 1959. Attraction chimique du m^le de Cerat itis capitata V/ied. (Diptera: Trypetidae) pour la femelle. Compt. Rend. Acad. Sci. 248: 2403-2404.

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118 Fe'ron, M. i960. L'appel sonore du irf&le dans le comport ement sexuel de Dacus oleae Gmel. Bull. Soc. Entomol. Fr. 65« 139-lWFe'ron, M. 1962. L' instinct de reproduction chez lamouche me'diterrane'enne des fruits Ceratitis capitata Wied. (Diptera: Trypetidae). Comportement sexuel-comportement de ponte. Rev. Pathol. Ve'g. Entomol. Agric. Fr. kl x 1-129. Fletcher, B. S. 1968. Storage and release of a sex pheromone by the Queensland fruit fly, Dacus tryoni (Dipteral Trypetidae). Nature (London). 219: 63I-632. Fletcher, B. S. 1969. The structure and function of the sex pheromone glands of the male Queensland fruit fly, Dacus tryoni . J. Insect Physiol. 15» 1309-1322. Fletcher, B. S., and A. Giannakakis. 1973a. Factors limiting the response of females of the Queensland fruit fly, Dacus tryoni , to the sex pheromone of the male. J. Insect Physiol. 19» 114-7-H55. Fletcher, B. S., and A. Giannakakis. 1973b. Sex pheromone production in irradiated males of Dacus (Strumeta) tryoni . J. Econ. Entomol. 66 1 62-64-. Foote, R. H., and F. L. Blanc. 1963. The fruit flies or Tephritidae of California. Bull. Calif. Insect Survey. 7t 1-117. Gee, J. H. 1969. Effect of daily synchronization of sexual activity on mating success in laboratory populations of two species of Dacus (Dipteral Tephritidae). Aust. J. Zool. 17: 619-624. Greene, C. T. 193^. A revision of the genus Anastrepha based on a study of wings and on the length of the ovipositor sheath (Diptera: Trypetidae). Proc. Entomol. Soc. Wash. 36: 127-179. Grosser, Ms_ 1971. The language of scent. In Annu. Rep. Zoecon Corp., Palo Alto, California, pp. 9-20. Guhl, A. M. 1968. A glossary of terms used in animal behavior. In Animal behavior in laboratory and field (A. W. Stokes, ed.). W. H. Freeman and Co., San Francisco. 10 pp. Gutierrez, J., W. Calderon, and R. H. Rhode. 1970. Evaluation of traps for Mediterranean fruit fly detection. In Technical reports eradication medfly Central America (OIRSA, ed.). P. 0-. Box 3628, San Jose, Costa Rica.

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119 Hardee, D. D., W. H. Cross, S. B. Mitchell, P. M. Huddleston, H. C. Mitchell, M. E. Merkl, and T . B . Davich. 1969. Biological factors influencing responses of the female boll weevil to the male sex pheromone in field and large-cage tests. J. Econ. Entomol. bZt 161-166. Hardee, D. D., W. H. Cross, P. M. Huddleston, and T. B. Davich. 1970. Survey and control of the boll weevil m west Texas with traps baited with males. J. Econ. Entomol. 63: 1041-1048. Hardee, D. D., G. H. McKibben, R. C. Gueldner, E. B. Mitchell, J. H. Tumlinson, and W. H. Cross. 1972. Boll weevils in nature respond to grandlure, a synthetic pheromone. J. Econ. Entomol. 65 1 97-100. Hendel, F. 1914. Die bohrfliegen. Suedamerikos . 14» 84. Holbrook, F. R . , and M. S. Fujimoto. 1970. Mating competitiveness of unirradiated and irradiated Mediterranean fruit flies. J. Econ. Entomol. 63: 1175-1176". Hooper, G. H. S., and K. P. Katiyar. 1971. Competitiveness of gamma-sterilized males of the Mediterranean fruit fly. J. Econ. Entomol. 64: 1068-1071. Hummel, H. E., L. K. Gaston, H. H. Shorey, R. S. Kaae, K. S. Byrne, and R. M. Silverstein. 1973. Clarification of the chemical status of the pink bollworm sex pheromone. Science. 181 t 873-875. Jacobson, M. 1972. Insect sex pheromones . Academic Press, Inc. , N. Y. 382 pp. Jacobson, M . , K. Ohinata, D. L. Chambers, W. A. Jones, and M. S. Fujimoto. 1973. Insect sex attractants. 13.isolation, identification, and synthesis of sex pheromones of the male Mediterranean fruit fly. J. Med. Chem. I61 248-251. Karlson, P., and A. Butenandt. 1959. Pheromones (ectohormones) in insects. Annu. Rev. Entomol. 4» 39-58. Knipling, E. F. i960. The eradication of the screw-worm fly. Scient. Amer. 203 t 54-61. LaBrecque, G. C, and J. C. Keller (ed.). 1965. Advances in insect population control by the sterile-male tech nique . Intern. Atom. Energy Agency Tech. Ser. 44 1 20-21.

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120 Law, J. H., and F. E. Regnier. 1971. Pheromones. Annu. Rev. Biochem. 40i 533-5^8. Lhoste. J., and A. Roche. I960. Organes odorife'rants des msiles de Ceratitis capitata . Bull. Soc. Entomol. Fr. 65» 206-210. Lloyd, E. P., M. E. Merkl, F. C. Tingle, W. P. Scott, D. D. Hardee, and T. B. Davich. 1972. Evaluation of malebaited traps for control of boll weevils following a reproduction-diapause program in Monroe County, Mississippi. J. Econ. Entomol. 65« 552-555. Loew, H. 1862. Monographs of the Diptera of North America . Smithsonian Misc. Coll. li 1-221. Loew, H. 1873. Review of the North American Trypetina. In Monographs of the Diptera of North America . Smithsonian Misc. Coll. 3» 211-351. Madsen, H. F., and J. M. Vakenti. 1972. Codling moths t Female-baited and synthetic pheromone traps as population indicators. Envir. Entomol. li 55^-557Madsen, H. F., and J. M. Vakenti. 1973. Codling moth: Use of Codlemone-baited traps and visual detection of entries to determine need of sprays. Envir. Entomol. 2: 677-679. Martelli, G. 1908. Notizie dietologiche sulla mosca delle olive ( Dacus oleae Gmel.). Boll. Lab. Zool. Portici. 2« 3-11. Marx, J. L. 1973 • Insect control (I)t Use of pheromones. Research News. Science. 181 1 736-737. McLaughlin, J. R., R. S. Kaae, H. H. Shorey, and L. K. Gaston. 1973. Sex pheromones of Lepidoptera. XXXIX. Comparison of virgin female moths and hexalure as attractants for male Pect in ophora gossypiella. Envir. Entomol. 2: 488489. McPhail, M. 1939. Protein lures for fruit flies. J. Econ. Entomol. 32 » 758-761. Miller, J. E. 1970. An investigation of the present and potential economic losses caused by the Mediterranean fruit fly ( Ceratitis capitata Wied.) in Central America. In Technical reports eradication medfly Central America (OIRSA, ed.). P. 0. Box 3628, San Jose, Costa Rica.

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121 Moffitt, H. R., and D. 0. Hathaway. 1973Effects of three systems of laboratory collection on the vigour of adult codling moths. J. Econ. Entomol. 66: 37^-376. Monro, J. 1953. Stridulation in the Queensland fruit fly Dacus ( Strumeta ) tryoni Frogg. Aust. J. Sci. l6s 60yburgh, A. C. 1962. Mating habits of the fruit flies Ceratitis capitata (Wied.) and Pterandrus rosa (Ksh.). S. Afr. J. Agric. Sci. 5: 457-464. Myers, K. 1952. Oviposit ion and mating behaviour of the Queensland fruit fly (Dacus ( Strumeta ) tryoni (Frogg. )J and the solanum fruit fly ( Dacus ( Strumeta ) cacuminatus (Hering)) . Aust. J. Sci. Res. Ser. B5s 264-281. Nakagawa, S., G . J. Farias, and L. F. Steiner. 1970. Response of female Mediterranean fruit flies to male lures in the relative absence of males. J. Econ. Entomol. 63: 227-229. Nation, J. L. 1972. Courtship behavior and evidence for a sex attract ant in the male Caribbean fruit fly, Anas trepha suspensa . Ami. Entomol. Soc. Amer. 65 » 13641367. Nishida, T . , and H. A. Eess. 1957. Studies on the ecology and control of the melon fly Dacus (Strumeta) cucurbitae . Hawaii Agric. Exp. St a. Techn. Bull. 34: 1-44. Oberbacher, M. F., and H. A. Denmark. 1957. Mediterranean fruit fly eradication program in Florida (1956-1957) * hronology and summary. State Plant Board of Fla. , Gainesville. 6 pp. Ohinata, K., D. L. Chambers, M. Fujimoto, S. Kashiwai, and R. Miyabara. 1971. Sterilization of the Mediterranean fruit fly by irradiation: omparative mating effectiveness of'treated pupae and adults. J. Econ. Entomol. 64: 781-784. Oldroyd, H. 1964. The natural history of flies . Weidenfeld and Nicolson, London. 324 pp. Oliver, J. A. 1955. The natural history of .orth merican amphibians and reptiles . Van Nostrand, N. Y. 218 pp. Poucher, C. 1970. Twenty-eight biennial report July 1, 1968-June 30, 1970 of the Bureau of pest eradication and control. Div. of Plant Industry, Fla. Dept. Agric. pp. 126-166.

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122 Pritchard, G. 196? , Laboratory observations on the mating behaviour of the island fruit fly Rioxa pornia (Diptera: Tephritidae) . J. Aust. Entomol. Soc. 6: 127-132. Prokopv, R. J. i and G. L. Bush. 1972. Mating behavior in Rhagoletis pomonella. III. Male aggregation m response to an arrestant. Can. Entomol. 10^i 275-283. Prokopy, R. J., and G. L. Bush. 1973Mating behavior of Rhagoletis pomonella (Diptera: Tephritidae). IV. Courtship. Can. Entomol. 105: 873-891. Roan, C. C, N . E. Flitters, and C. J. Davis. < 195^. Light intensity and temperature as factors limiting the mating of the Oriental fruit fly. Ann. Entomol. Soc. Amer. 4-7: 593-59*+. Roelofs, W. L., A. Comeau, A. Hill, and G. Milicevic. 1971. Sex attractants of the codling moth: haracterization with electroantennogram technique. Science. 297299Schiner, J. R. 1868. Diptera. 2: 263. In: Reise der osterreichischen fregatte novara. Zoologischer Theil. Vienna. 388 pp. Schroeder, W. J., D. L. Chambers, and R. Y. Miyabara. 1973* Reproduction of the melon fly: ating activity and mating compatibility of flies treated to function in sterile-release programs. J. Econ. Entomol. 66: 66l663. Schultz, G. A., and G. M. Eoush. 1971. Suspected sexpheromone glands in three economically important species of Dacus. J. Econ. Entomol. 64t 3^7-3^9. Se'guy, E. 1951. Ordre des Diptbres. Traite de Zoologie. Pierre-P. Crasse' (ed . ) . (Masson, Paris. 975 PP.). 10: Sharma, R. K. , A. J. Mueller, H., T. Reynolds, and N. Toscano. 1973. Techniques for trapping pink bollworm males. Calif. Agric. 27« 1^-15. Shaw, J. G., D. S. Moreno, and J. Fargerlund. 1971. New red scale detector. Citrograph. 56 : 1^9-150. Shorey, H. H., L. K. Gaston, and L. L. Sower. 1971. The male inhibition technique ... cabbage looper control by confusing sex pheromone communication. Calif. Agric. 25: 11.

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123 Sigwalt, B. r F. Soria, A. Yana, and C. Baldy. 1968. Deplacement diurne apparent d'une population de Ceratites sur une parcelle d'agrumes. Ann. Epiphyt. 19: 169171. Simmonds, H. W. 1936. Fruit fly investigations. Bull. Dept. Agric. Fiji. 19: 1-18. Spieth, H. T. 1968. Evolutionary implications of sexual behavior in Drosophila . Evolut. Biol. 2: 157-193 • Steiner, L. F. 1965. A rapid method for identifying dyemarked fruit flies. J. Econ. Entomol. 58: 37^-375. Steiner, L. F. 1967. The use of trimedlure-baited traps to support the sterile fly release program (Memo # 3 directed to W. E. Stone, Project Manager, Medfly Eradication Project, P. 0. Box 3628, San Jose, Costa Rica). December 16-17, 1967. Steiner, L. F. I969. Mediterranean fruit fly research in Hawaii for the sterile fly release program. In Insect ecology and the sterile-male technique (Intern. Atom. Energy Agency , ed . ) . Vienna, Austria, pp. 73-82. Steiner, L. P. , and L. D. Christenson. 1956. Potential usefulness of the sterile fly release method in fruit fly eradication programmes. Proc. Hawaiian Acad. Sci. 3: 17-18. Steiner, L. F., W. C. Mitchell, E. J. Harris, T. T. Kozuma, and M. S. Fujimoto. 1965. Oriental fruit fly eradication by male annihilation. J. Econ. Entomol. 58: 961-964. Steiner, L. F., W. G. Hart, E. J. Harris, R. T. Cunningham, K. Ohinata, and D. C. Kamakahi. 1970. Eradication of the Oriental fruit fly from the Mariana islands by the methods of male annihilation and sterile insect release. J. Econ. Entomol. 63: 131-135. Stevens, L. J., and M. Beroza. 1972. Mating-inhibition field tests using disparlure, the synthetic gypsy moth sex pheromone. J. Econ. Entomol. 65 « 1090-1095* Stoltzfus, W. B., and B. A. Foote. 1965. The use of froth masses in courtship of Eutreta . Proc. Entomol. Soc. Wash. 67: 263-264. Stone, A, 19^2. The fruit flies of the genus Anastrepha . U. S. Dept. Agric. Misc. Publ. ^39. 112 pp.

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124 Swanson, R. W., and R. M. Baranowski. 1972. Host range and infestation by the Caribbean fruit fly, Anastrepha suspensa (Dipteras Tephritidae) in south Florida. Proc. Fla. State Hort. Soc. 85s 271-27^. Syed, R. A. 1969. Studies on the ecology of some important species of fruit flies and their natural enemies in west Pakistan. Pakistan Comm. Inst. Biol. Control Sta. Rep. 12 pp. Syed, R. A. 1970. Studies on trypetids and their natural enemies in west Pakistan. Dacus species of lesser importance. Pakistan J. Zool. 2s 17-24. Syed, R. A., M. A. Ghani, and M. Murtaza. 1970. Studies on trypetids and their natural enemies in west Pakistan. III. Dacus ( Strumeta ) zonatus . Pakistan Comm. Inst. Biol. Control Sta. Techn. Bull. 13s 1-16. Tashiro, H., and D. L. Chambers. 1967. Reproduction in the California red scale Aonidiella aurantii (Homopteras Diaspididae) . I. Discovery and extraction of a female sex pheromone. Ann. Entomol. Soc. Amer. 60s 1166-1170. Tauber, M. J., and C. A. Toschi. 1965a. Bionomics of Euleia fratria (Loew) (Dipteras Tephritidae). I. Life history and mating behavior. Can. J. Zool. ^3' 369-379. Tauber, M. J., and C. A. Toschi. 1965b. Life history and mating behavior of Tephritis stigmatica (Coquillet) (Dipteras Tephritidae) . The Pan-Pac. Entomol. kit 73-79. Tette, J. P. 1972. Pheromoness Prospects for control. An invitational paper presented at the 1^-th Intern. Congr. of Entomol., Canberra city, Australia. Tychsen, P. H., and B. S. Fletcher. 1971. Studies on the rhythm of mating in the Queensland fruit fly, Dacus tryoni . J. Insect Physiol. 17s 2139-2156. Webb, J. C. 1973. Analysis and identification of specialized sounds possibly used by the Caribbean fruit fly, Anastre pha suspensa (Loew), for communication purposes. Ph. D. Dissertation. The University of Tennessee. Knoxville, Tennessee . Weems, H. V., Jr. 1963. Mexican fruit fly ( Anastrepha ludens (Loew)) (Dipteras Tephritidae). Div. of Plant Industry, Fla. Dept. Agric. Entomol. Circ. 16. 2 pp.

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125 Weems, H. V., Jr. 1965. Anastrepha suspensa (Loew) (Dipterat Tephritidae). Div. of Plant Industry, Fla. Dept. Agnc. Entomol. Circ. 38. ^ pp. Weems, H. V., Jr. 1966. The Caribbean fruit fly in Florida. Proc. Fla. State Hort. Soc. 79 • 4-01-403. Weems, H. V., Jr. 196? • Anastrepha interrupta Stone (Dipteral Tephritidae). Div. of Plant Industry, Fla. Dept. Agric. Entomol. Circ. 61. 2 pp. Weems, H. V., Jr. 1969. Anastrepha serpentina (Wiedemann) (Dipterat TephritidaeT Div. of Plant Industry, Fla. Dept. Agric. Entomol. Circ. 91. 2 pp. Weems, H. V., Jr. 1970. West Indian fruit fly Anastrepha mombinpraeoptans Sein (Diptera: Tephritidae )• Div. of Plant Industry, Fla. Dept. Agric. Entomol. Circ. 101. 2 PP. Willson, H. R., and M. S. Mulla. 1973. Attractants for synanthropic flies II. Response patterns of house flies to attractive baits on poultry ranches. Envir. Entomol. 2: 815-822. Wilson, E. 0., and W. H. Bossert. I963. Chemical communication among animals. Recent Progr. Horm. Res. 19: 673.

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BIOGRAPHICAL SKETCH Alberto Javier Perdomo Ehlers was born May 11, 19^3» at Colon, Republic of Panama. He graduated from "Colegio San Jose' (La Salle)" in January, i960. From February, i960, until December, 1966, Mr. Perdomo attended the "Instituto Tecnolo'gico y de Estudios Superiores de Monterrey" (Technological Institute of Monterrey, Mexico) where he received his "Ingeniero Agronomo" and Master of Science in agricultural parasitology. From February, 1967, until August, I968, Mr. Perdomo worked as an extension entomologist and as chief of the Department of Entomology of the Ministry of Agriculture of the Republic of Panama. In September, 1968, he was employed by the International Atomic Energy Agency to work in the Mediterranean Fruit Fly Project in Central America as a technical expert and was stationed for two and a half years in Nicaragua, Central America. In January, 1971, he was employed by the "Organismo Internacional Regional de Sanidad Agropecuaria" to continue working in the Medfly Project, but he was transferred to San Jose, Costa Rica, where he worked until August, 1971. In September, 1971* he started graduate studies at the University of Florida toward the degree of Doctor of Philosophy. He has been financially assisted by the IAEA, by the Department of Entomology of the University of Florida and by 126

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127 the government of the Republic of Panama. Mr. Perdomo is married to Ethelrrida Esther Lewis. They have two children, Alberto Jr. and Malena Esther. He is a member of the Florida Entomological Society and of the Entomological Society of America.

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. fames L. Nation, Chairman Professor of Entomology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Richard M. Baranowski, Co-Chairman Professor of Entomology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ft Harvey JA Cromroy Professfo^ of Entomology//

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Ztoctor of Philosophy. This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. June, 197^ Derrell L. Chambers Associate Professor of Entomology Francis W. Zettler Associate Professor of Plant Pathology Dean, College of Agriculture Dean, Graduate School