Citation
The behavior of the egg parasitoid Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) in response to kairomones produced by its host, the southern green stink bug Nezara viridula (L.) (Hemiptera: Pentatomidae)

Material Information

Title:
The behavior of the egg parasitoid Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) in response to kairomones produced by its host, the southern green stink bug Nezara viridula (L.) (Hemiptera: Pentatomidae)
Creator:
Sales, Fernando João Montenegro de, 1944-
Publication Date:
Language:
English
Physical Description:
xiv, 141 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Eggs ( jstor )
Female animals ( jstor )
Hemolymph ( jstor )
Insects ( jstor )
Palpation ( jstor )
Parasite hosts ( jstor )
Parasitoids ( jstor )
Solvents ( jstor )
Stimulants ( jstor )
Stink bugs ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Insects -- Parasites ( lcsh )
Nezara viridula -- Biological control ( lcsh )
Trissolcus basalis -- Behavior ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 132-139.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Fernando João Montenegro de Sales.

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THE BEHAVIOR OF THE EGG PARASITOID TRISSOLCUS BASALIS
(WOLLASTON) (HYMENOPTERA: SCELIONIDAE) IN RESPONSE TO KAIROMONES PRODUCED BY ITS HOST, THE SOUTHERN GREEN STINK BUG NEZARA VIRIDULA (L.) (HEMIPTERA: PENTATOMIDAE)











By

FERNANDO JOAO MONTENEGRO DE SALES


A DISSERTATION PRESENTED TC
THE UNIVERSITY IN PARTIAL FULFILLMENT OF THE
OF DOCTOR OF


THE GRADUATE COUNCIL OF OF FLORIDA REQUIREMENTS FOR THE DEGREE PHILOSOPHY


UNIVERSITY OF FLORIDA


1978
















ACKNOWLEDGMENTS


The author is grateful to Dr. G.E. Allen for his advice,

encouragement and guidance during the experimental work and preparation of this dissertation. Appreciation is extended to the staff members of his laboratory for help and understanding.

Recognition and gratitude is given to Dr. R.I. Sailer, Dr. J.H. Tumlinson, Dr. J.R. McLaughlin and Dr. F.W. Zettler for serving on my graduate committee. Special thanks are extended to Dr. D.L. Chambers for allowing me to use the facilities of the USDA-ARS, Insect Attractants, Behavior and Basic Biology Research Laboratory.

Special thanks are extended to the chairman, Dr. Fowden Maxwell, as well as the staff and graduate students of the Department of Entomology and Nematology for their unselfish service, instruction, and encouragement.

Gratitude is also expressed to the Brazilian colleagues at the University of Florida for their support and encouraging words.

I am also indebted to Mrs. Maria I. Cruz, Campus Coordinator of the AID program for her cooperation, assistance and concern.

The author was supported by funds from the United States Agency for International Development (USAID), the Federal University of CearA, Brazil, to whom sincere appreciation is expressed.



















TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.................................................. ** ii

LIST OF TABLES ....................................................vii

LIST OF ILLUSTRATIONS ............................................. x

ABSTRACT ..........................................................********* xiii

INTRODUCTION ...................................................... 1

LITERATURE REVIEW ................................................. 4

Current Status of the Host Southern Green Stink Bug,
Nezara viridula (L.) and Its Parasitoid Trissolcus basalis
(Wollaston) .................................................. 4

The Southern Green Stink Bug, Nezara viridula (L.)....... 4

Origin ............................................. 4

Distribution ....................................... 4

Host plants ........................................ 5

Life history ....................................... 6

Trissolcus basalis (Wollaston) .......................... 7

Origin ............................................. 7

Distribution and host insects ...................... 8

Life history ....................................... 8

Interspecific Communication .................................. 9

METHODS AND MATERIALS ............................................. 11

Rearing of N. viridula (L.) .................................. 11

Rearing of T. basalis (Wollaston) ............................ 11

Experiment 1: Response of the Female T. basalis to the
Eggs of the Host, N. viridula ................................ 12


iii










Page

Experiment 2: Orientation of the Female T. basalis Inside a "Y" Type Olfactometer................................ 16

Experiment 3: Temporal Analysis of the Ovipositional Behavior of the Female T. basalis ............................ 19

Experiment 4: Cues Useful in Location of the Host, N. viridula, by the Parasitoid T. basalis ....................... 20

Scent of Male N. viridula ............................... 20

Scent of Female N. viridula. ............................. 23

Male and Female N. viridula Hemolymph................... 23

N. viridula Eggs of Different Ages. ...................... 24

Experiment 5: Reactions of the Male and Female T. basalis to the Eggs of the Host, N. viridula at Different Degrees of Parasitism......................................... 24

Experiment 6: Reactions of the Female T. basalis to the Kairomonal Solutions Prepared with Different Solvents........ 25

Dichloromethane Washes Required for Removal of the
Kairomone from Eggs of N. viridula. ...................... 26

Reaction of the Female T. basalis to Kairomonal
Solutions Prepared by Different Methods................. 27

Experiment 7: Behavior Patterns of the Female T. basalis When Stimulated by Different Concentrations of the Crude Kairomonal Extract from Eggs of the Host, N. viridula........ 27 Experiment 8: Effects of the Crude Kairomonal Extract from Eggs of the Host, N. viridula in the Orientation of the Female T. basalis. ............................................ 29

Experiment 9: Enhancement of Host Location by Scent Combinations.................................................. 30

Experiment 10: Normality Studies with the Crude Kairomonal Solution from Eggs of the Host, N. viridula.................. 31

Response to the Kairomonal Solution on the Filter Paper. 31

Evaluation of Parasitism in Areas Treated with the
Crude Kairomonal Solution................................ 32

Responses of Female T. basalis to Egg Shells and 12Hour-Old Eggs of the Host N. viridula ................... 33










P

Antennal Palpation Previous to Oviposition..............

RESULTS ...........................................................

Experiment 1: Response of the Female T. basalis to the
Eggs of the Host, N. viridula ................................

Experiment 2: Orientation of the Female T. basalis Inside
a "Y" Type Olfactometer ......................................

Experiment 3: Temporal Analysis of the Ovipositional
Behavior of the Female T. basalis ............................

Experiment 4: Cues Useful in Location of the Host, N.
viridula by the Parasitoid T. basalis ........................

Scent of Male N. viridula ...............................

Scent of Female N. viridula .............................

Scent of Male N. viridula Hemolymph .....................

Scent of Female N. viridula Hemolymph...................

N. viridula Eggs of Different Ages ......................

Experiment 5: Reactions of the Male and Female T. basalis
to the Eggs of the Host, N. viridula at Different
Degrees of Parasitism ........................................

Experiment 6: Reactions of the Female T. basalis to the
Kairomonal Solutions Prepared with Different Solvents........

Number of Dichloromethane Washes Required for Removal
of the Kairomonal Extract from Eggs of N. viridula......

Reaction of the Female T. basalis to Kairomonal
Solutions Prepared by Different Methods.................

Experiment 7: Behavior Patterns of the Female T. basalis When Stimulated by Different Concentrations of the Crude
Kairomonal Extract from Eggs of the Host, N. viridula........

The Concept of Stimulant Concentration..................

The Concept of Stimulant Dose ...........................

The Concept of Stimulant Time ...........................

Experiment 8: Effects of the Crude Kairomonal Extract from
Eggs of the Host, N. viridula in the Orientation of the
Female T. basalis. ............................................


age 34 35 35


35


38 49 49 49 52 53 53




58 63 64


64




71 75 82 83










Page


Experiment 9: Enhancement of Host Location by Scent
Combinations ................................................. 84

Experiment 10: Normality Studies with the Crude Kairomonal
Solution from Eggs of the Host, N. viridula................... 87

Response to the Kairomonal Solution on the Filter Paper. 88

Evaluation of Parasitism in Areas Treated with the Crude
Kairomonal Solution..................................... ** 88

Responses of the Female T. basalis to the Egg Shells
and 12-Hour-Old Eggs of the Host, N. viridula. ........... 88

Antennal Palpation Previous to Oviposition. .............. 89

DISCUSSION ........................................................ 91

SUMMARY AND CONCLUSIONS ...........................................102

APPENDIX: SUMMARY OF THE EXPERIMENTAL DATA .......................106

REFERENCES CITED ..................................................132

BIOGRAPHICAL SKETCH ...............................................140
















LIST OF TABLES


Table Page

1. Analysis of variance for the simple effects between the number of female Trissolcus basalis (Wollaston)
orienting within a single tube olfactometer containing
eggs of the host, Nezara viridula (L.) at 10 levels of time..107

2. Percentage of female Trissolcus basalis (Wollaston) found within different sections of a "Y"-type olfactometer
containing 12-hour-old host eggs (B), egg shells (B'),
and no eggs of the host, Nezara viridula (L.) at different
time intervals ...............................................108

3. Analysis of variance for Trissolcus basalis (Wollaston) orienting within a single tube olfactometer with the scent
of 0, 1, and 3 male Nezara viridula (L.) .....................109

4. Analysis of variance for the number of Trissolcus basalis (Wollaston) orienting within a single tube olfactometer with
the scent of 0, 1, and 3 male Nezara viridula (L.)........... 110

5. Analysis of variance for the number of Trissolcus basalis (Wollaston) orienting within a single tube olfactometer
with the scent of 0 and 3x10-3 cm3 of male Nezara viridula
(L.) hemolymph ...............................................111

6. Total number of Trissolcus basalis (Wollaston) orienting within a single tube olfactometer with different levels of the
male hemolymph of Nezara viridula (L.), averaged over 10
equally spaced time interval .................................112

7. Analysis of variance for the number of Trissolcus basalis (Wollaston) orienting within a single tube olfactometer to
the scent of 0 and 3x10-3 cm3 of female Nezara viridula
(L.) hemolymph ...............................................113

8. Total number of Trissolcus basalis (Wollaston) orienting within a single tube olfactometer with different levels of the hemolymph of female Nezara viridula (L.), averaged over 10 equally
spaced time interval. ........................................114

9. Analysis of variance for the simple effects between the number of female Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer containing 50 eggs of the
host, Nezara viridula (L.) at different host age levels......115





viii


Table Page

10. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orientina within a single tube olfactometer to eggs of the host, Nezara viridula (L.) at different levels
of parasitism ................................................116

11. Analysis of variance for the simple effects between Trissolcus
basalis (Wollaston) orienting within a single tube olfactometer
containing 50 eggs of the host, Nezara viridula (L.) at
different levels of parasitism, averaged over 10 equally
spaced time interval. ........................................117

12. Treatment totals for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing 50 egg equivalents of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.) removed
by different solvents ........................................118

13. Treatment means for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing crude kairomonal extract from eggs of the host,
Nezara viridula (L.), removed by 4 solvents, after 4 minutes
of exposure. ..................................................119

14. Analysis of variance for the number of female Trissolcus
basalis (Wollaston) orienting within a single tube olfactometer
containing crude kairomonal extract from fifty 12-hour-old
eggs of the host, Nezara viridula (L.), extracted with
dichloromethane by different methods .........................120

15. Treatment totals for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing 50 egg equivalent solutions obtained from 12-hourold eggs of the host, Nezara viridula (L.) with dichloromethane by different methods .................................121

16. Percentage of female Trissolcus basalis (Wollaston) exhibiting
different types of behavior patterns on filter paper when stimulated by different concentrations of crude kairomonal
extract from eggs of the host, Nezara viridula (L.).......... 122

17. Parameters for the behavior patterns of the female parasitoid,
Trissolcus basalis (Wollaston) when stimulated by four equally
spaced concentrations, ranging from 10-4 to 10-1 egg
equivalents/ul of crude kairomonal extract from eggs of the
host Nezara viridula (L.) ....................................123

18. Percentage of the female parasitoid Trissolcus basalis
(Wollaston) exhibiting antennal palpation on a treated filter
paper spot, when stimulated by 10-2 egg equivalents/ul of
the crude kairomonal extract of the eggs of the host, Nezara
viridula (L.) at different time intervals.................... 124










Table Paqe

19. Parameters for the stimulant timeofor the female parasitoid
Trissolcus basalis (Wollaston) exhibiting antennal palpation,
when stimulated by 10-2 egg equivalent/ul of the crude
kairomonal extract from the eggs of the host, Nezara
viridula (L.). ................................................125

20. Average time in seconds spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper area with
different concentrations of the crude kairomonal extract from
eggs of the host, Nezara viridula (L.) .......................126

21. Average velocity in cm/s of the female Trissolcus basalis
(Wollaston) in orienting within a single tube olfactometer
containing scent combinations from the host, Nezara
viridula (L.). ................................................127

22. Percentage of female Trissolcus basalis (Wollaston) exhibiting
antennal palpation on filter paper treated with 103 cm3 of
solutions containing different concentrations of crude
kairomonal extract from eggs of the host Nezara viridula (L.)128

23. Percentage of eggs of Nezara viridula (L.) parasitized by
Trissolcus basalis (Wollaston) when placed on filter paper
=-2 3
treated with 5xlO2 cm3 of a 10-2 egg equivalent solution in
dichloromethane ..............................................129

24. Percentage of female Trissolcus basalis (Wollaston) exhibiting
ovipositional behavior when stimulated by 6 12-hour-old eggs
and egg shells of the host, Nezara viridula (L.).............130

25. Time required by the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation previous to a successful oviposition in the eggs and egg shells of the host Nezara
viridula (L.). ................................................131

















LIST OF ILLUSTRATIONS


Figure Page

1. A single glass tube olfactometer is pictured................. 14

2. A "Y" type olfactometer is pictured. .......................... 18

3. The glass chamber is shown connected to a single glass tube olfactometer ............................................ 22

4. Relationship between percentage of female Trissolcus basalis (Wollaston) responding within a single tube olfactometer
versus different levels of eggs of the host southern green
stink bug, Nezara viridula (L.), is shown.................... 37

5a and b. Female Trissolcus basalis (Wollaston) exhibits preovipositional behavior in an egg mass of its host, Nezara
viridula (L.). Antennal palpation of (a) marginal egg and
(b) central egg is shown. ..................................... 41

5c and d. Female Trissolcus basalis (Wollaston) exhibits ovipositional behavior on an egg mass of its host, Nezara viridula
(L.). Shown are (c) drilling the chorium for ovipositing and
(d) ovipositor thrust and marking the egg after oviposition.. 43

5e and f. Female Trissolcus basalis (Wollaston) exhibits ovipositional behavior on an egg mass of its host, Nezara
viridula (L.). Oviposition of the central eggs by drilling
the chorium, (e) the lateral wall, and (f) the operculum
is shown...................................................... 46

6. Ovipositional ethogram of the female parasitoid Trissolcus basalis (Wollaston) when stimulated by six 12-hour-old eggs
of the host, Nezara viridula (L.), is shown.................. 48

7. Number of male and female Trissolcus basalis (Wollaston) reacting to (a) scent of 0, 1, and 3 male Nezara viridula
(L.) and (b) 0, 1, and 3 female N. viridula, averaged over
10 equally spaced time intervals is shown.................... 51

8. Number of male and female Trissolcus basalis (Wollaston) reacting to combined levels of (a) hemolymph of male Nezara
viridula (L.) and (b) hemolymph of female N. viridula at
different time intervals is shown. ............................ 55










Figure Page

9. Number of female Trissolcus basalis (Wollaston) reacting to different egg ages of the host, Nezara viridula (L.),
at different time intervals is shown. ......................... 57

10. Number of male Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown............. 60

11. Number of female Trissolcus basalis (Wollaston) reacting
to the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown............. 62

12a. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), after soaking
in dichloromethane for different periods of time is shown.... 66

12b. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), after soaking
in dichloromethane for different periods of time is shown.... 68

13. Number of female Trissolcus basalis (Wollaston) reacting to
crude kairomonal extract from eggs of the host, Nezara
viridula (L.), removed with dichloromethane by different
methods at certain time intervals is shown................... 70

14. The behavior patterns of the female Trissolcus basalis
(Wollaston) on a filter paper treated with crude kairomonal
extract from eggs of the host, southern green stink bug,
Nezara viridula (L.), arediagrammed. Shown are parasitoid: (a) in direct movement to the treated spot; (a') in random
movement to the spot; (a") after antennal palpation, leaving
the spot and starting to search; (b) after searching,
returning to the treated area for reinforcement; and (b')
leaving the spot for new cycle of searching. Either a
or a' will occur for every single trial ...................... 73

15a and b. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.), is shown:
(a) random movement (possibly kinesis), (b) chemotaxis....... 77

15c and d. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract from eggs of the host, Nezara viridula (L.), is shown: (c) antennal palpation, (d) searching. ................................. 79

15e. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract





xii




Figure Page

from eggs of the host, Nezara viridula (L.), is shown:
(e) reinforcement. ............................................ 81

15f. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation when stimulated by 10-2 egg equivalent solution per microliter from eggs of the host,
Nezara viridula (L.), versus time, is shown.................. 81

16. Percent of time spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper spot with
different concentrations of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.), is shown........ 86










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



THE BEHAVIOR OF THE EGG PARASITOID TRISSOLCUS BASALIS
(WOLLASTON) (HYMENOPTERA: SCELIONIDAE) IN RESPONSE
TO KAIROMONES PRODUCED BY ITS HOST, THE SOUTHERN GREEN
STINK BUG NEZARA VIRIDULA (L.) (HEMIPTERA: PENTATOMIDAE) By

Fernando Joao Montenegro de Sales March, 1978

Chairman: Dr. George E. Allen
Major Department: Entomology and Nematology

The mediators of behavior patterns among insects have been a focal point of research in recent years as potential tools for management of insect pest populations. Laboratory studies were conducted to reveal the factors involved in the interspecific communication between the egg parasitoid, Trissolcus basalis (Wollaston) and its host, the southern green stink bug, Nezara viridula (L.). Temporal analysis of the female parasitoid ovipositional behavior showed that location of the egg masses of the host takes place by random movement (kinesis), chemotaxis, and combination of both. It was demonstrated that the ovipositional behavior is divided in two distinct steps: the appetitive behavior and the most stereotyped consummatory behavior.

Olfactometer tests indicated that orientation toward the host eggs is in great extent purposeful rather than random, and that the male and female stink bug scents as well as their hemolymph act as cues in eliciting orientation of T. basalis toward the eggs. The data implied that the host egg age is a factor in stimulating the quest for eggs, and the parasitoid mating behavior is possibly mediated by a pheromone.


xiii





xiv


A crude kairomonal extract was isolated by soaking host eggs in dichloromethane and the results of bioassays with this extract showed that visual clues are not as important as chemical clues in increasing the parasitoid velocity, consequently reducing the orientation time and enhancing host location. Assays on filter paper proved that the kairomonal solution induced 5 behavior patterns; i.e., random movement (kinesis), chemotaxis, antennal palpation, searching, and reinforcement. Mathematical equations were developed to describe these patterns. The concepts of Stimulant Concentration (SC50), Stimulant Dose (SD50), and Stimulant Time (ST50) were introduced as measurements of the potency of chemicals involved in interspecific and intraspecific communication among insects.

The overall performance of the female T. basalis was improved in terms of orientation, velocity, oviposition, and parasitism by the kairomonal solution, and a very stereotyped and precisely timed behavior pattern indicated that action of the isolated releaser is within the limits of normality.

















INTRODUCTION


The ability of insects to compete with man for the products of agriculture is a continuing challenge to entomologists. In recent years increased scientific and public awareness of the environment has greatly increased the complexity of the problem. To meet the challenge entomologists have resorted to a variety of new methods designed to solve or at least alleviate this problem of controlling insects with minimum detrimental effects to the environment. One of these new methods involves research on chemical substances that influence inter and intraspecific behavior of insects.

The southern green stink bug, Nezara viridula (L.) is a cosmopolitan insect pest destructive to many crops of economic importance throughout the world. Until now most of the control measures have relied on chemical insecticides however with the demand for a cleaner environment, new opportunities have been opened to alternatives that could assure an acceptable injury level and meet the new standards set for the agroecosystems common to this insect.

The scelionid parasitoid Trissolcus basalis (Wollaston) has been utilized as one of those alternatives, and in fact, the specialized biocontrol literature includes it as one of the major tools in balancing the populations of many stink bug species, in the United States and other countries. However there is a dearth of quantitative information regarding the potential of this parasitoid as a major control agent.










With the growing research on the inter and intraspecific communication among insects (Kennedy, 1956; Johnston, Jr. et al., 1970; Wilson, 1962, 1977), the chemical, physical, and biological releasers have been studied more accurately in order to find out new ways to manage insect pests with minimum detrimental effects to the environment (Metcalf and Luckmann, 1975).

The only published study involving chemical interspecific communication between N. viridula and its parasites and predators is the work of Mitchell and Mau (1971) with the dipterous parasitoid Trichopoda pennipes.

Since experimental works have suggested that kairomones can be used as valuable tools in management of insect pests (Lewis et al., 1971, 1975, 1976) the aim of this research was to assess the importance of the releaser that triggers all sequences involved in the ovipositional behavior of T. basalis. A series of bioassays were conducted to determine: (1) threshold for host-seeking stimulation of the female T. basalis by the eggs of the host; (2) ovipositional ethogram of the female T. basalis with temporal analysis; (3) cues that enhance host location; (4) discriminatory behavior of the male and female T. basalis to parasitized and non-parasitized host eggs; (5) solvents and number of washes required to remove the kairomone from the eggs and solution with the best potential activity; (6) effect of host-eggs and kairomonal solution on orientation of the parasite; (7) behavior patterns mediated by the kairomonal solution and relationship between those patterns and concentration; (8) Stimulant Concentration (SC50), Stimulant Dose (SD50), and Stimulant Time (ST50) as measurements of the potency of the kairomonal extract utilized in this work. (These are units that should

be useful in evaluations of chemicals involved in both inter and intra-





3



specific communication among insects.) (9) The normality of the extract obtained through standard and original procedures.

















LITERATURE REVIEW


Current Status of the Host Southern Green Stink Bug,
Nezara viridula (L.) and Its Parasitoid Trissolcus basalis (Wollaston).


The Southern Green Stink Bug, Nezara viridula (L.)


Origin

According to Van Duzee (1917), Nezara viridula (L.) was first

described by Linnaeus in 1758 under the scientific name Cimex viridulus. Linnaeus' description was based on specimens collected in India. The first new world record for the species is from the West Indies, and since then the species has been redescribed by various authors under numerous other scientific names.

Freeman (1940) placed the species into the genus Nezara of Amyot

and Serville, 1843 and Drake (1920) indicated that three color varieties are recognized as: smaragdula (Fabricius), torquata (Fabricius) and hepatica Horvath.


Distribution

The southern green stink bug, N. viridula (L.) is widely distributed throughout the world. It is found in Europe, Asia, Africa, and Americas (DeWitt and Godfrey, 1972). Van Duzee (1917) and Jones (1918) have pointed out that as many other insect pests in this Country, N. viridula was introduced from West Indies and is established in Virginia, Florida, Louisiana, South Carolina, Georgia, Alabama, Mississippi,










Texas, New Mexico, Arizona, California. Davis and Krauss (1963) have reported its recent importation to Hawaii.


Host plants

Nezara viridula (L.) is a phytophagous insect with a broad range of host plants. Hoffman (1935) indicates that this insect attacks both monocotyledons and dicotyledons. Among the former he pointed out that Graminae are the most important, and within the dicotyledons he stated that 29 families are injured and ranked the following in order of importance: Leguminosae having 27 species damaged, Cruciferae with

8 and Solanaceae with 6 species. Drake (1920), Gallo et al. (1970), Todd (1973), Issa (1973), Turnipseed and Kogan (1976) have reported this insect as feeding on radish, mustard, turnip, collard, cauliflower, cabbage, okra, peas, beans, peanut, tomato, potato, cotton, tobacco, pepper, eggplant, sunflower, sugar cane, corn, orange, lime, peach, pecan, rise, snap bean, squash, cucumber, soybean, lemon, and grapefruit.

Drake (1920) also reports a number of weeds that serve as host to the bug as: pokeweed, Phytolacca decandra; lamb's quarters, Chenopodium spp.; nut grass, Cypepus esculentus L.; spiny amaranth, Amaranthus spinosus; beggarweed, Desmodium spp.; crotolaris, Crotolaris spp.; wild grape, Vitis spp.; castor bean, Ricinus communis L.; maypops, Passiflora incarinata L.; and wild plum, Prunus spp.

In spite of the broad spectrum of host plants, N. viridula does

not breed in all those plants and only occassionally feeds on a number of them. Drake (1920) indicates that the southern green stink bug has

a remarkable preference for the legumes, with the greatest degree of preference when those plants are in the stage of fruit formation.










Life history

Life history and behavior of the southern green stink bug has been intensively studied by Japanese researchers [Kariya (1961), Kiritani (1963, 1965), Kiritani and Hokyo (1965), Kiritani, Hokyo, and Iwao (1966), Kiritani, Hokyo, and Kimura (1966), Kiritani, Hokyo, Kimura, and Nakasuji (1965), Kiritani and Kimura (1965), and Kobayashi (1959)]. In general, life cycle and generation time are much the same and variations are usually correlated to temperature fluctuation at a given location.

In the United States, the basic information in this area comes from the classical works of Drake (1920) and Jones (1918). They indicate that this insect, like most other pentatomids, hibernates in the adult stage under litter, bark, and other objects which offers protection. Drake (1920) points out that mating begins almost immediately upon emergence from hibernation. The female and male usually remain attached to one another by the tips of their abdomens and with their heads facing in opposite directions. Under natural conditions copulation is repeated a number of times before and after the eggs have been deposited. Drake (1920) also reported that after feeding a few days, newly emerged adults reach sexual maturity, and this period was found to vary from 3 to 6 weeks. The eggs are generally laid in regularly shaped compact clusters in which the individual eggs are arranged in very regular rows and firmly glued together. The incubation period is about 6 days in summer, but during early spring and late fall the period is often extended to 2 or 3 weeks.

The southern green stink bug has 5 nymphal instars and during the first instar, the nymphs normally cluster together near or on the egg-









shells. Drake (1920) indicated that no individuals have been observed to feed while clustered; but just before or subsequent to molting, the nymphs become active, scatter more or less and begin to feed. The nymphs like the adults, are usually found upon those portions of the plant on which they prefer to feed--the tender growing shoot and specially the developing fruit. Jones (1918) and Drake (1920) reported that during the summer, the period from egg to adult is about 35 days with temperature conditions having an important effect.


Trissolcus basalis (Wollaston)


Regulation of southern green stink bug is attributed to biotic

and abiotic factors. A lot of work has been done in Japan in relation to population dispersion and control. Kiritani (1965) suggests that mortality factors work in a stage-specific way, i.e. parasite against eggs, weather factors against the first instar and predators against the second. He also indicates that the complex age structure during the breeding seasons increases the population plasticity against a specified mortality factor.

The specialized literature lists 12 parasites of the southern

green stink bug and T. basalis stands out as one of the most important biocontrol agents. Since that time, it has been recorded from such widely separated locations as the island of Saint Vincent, Florida, and Egypt (Priesner, 1931).


Origin

This parasite was first described by Wollaston in 1858 from specimens collected on the Ilha da Madeira.












Masner (1971) pointed out that Trissolcus basalis (Wollaston) has the following synonyms: Telenomus basalis Wollaston, 1858; Telenomous maderensis Wollaston, 1858; Telenomus magacephalus Ashmead, 1894; and Telenomus piceipes Dodd, 1919.


Distribution and host insects

Trissolcus basalis has been reported as a polyphagous parasitoid with broad range of dispersion throughout the world. It has been reported in Europe, Asia, Africa, and North America (Cumber, 1964; Davis, 1964; Hokyo and Kiritani, 1965; Kamal, 1937; Wilson, 1961). Trissolcus basalis parasitizes N. viridula, Acrosternum hilare (Say), A. marginatum (Palisot de Beauvois), Euchistus servus (Say), E. variolatus (Palisot de Beauvois) and Cumber (1964) reported that this parasitoid also develops on eggs of the following pentatomids: Antestia orbona Kirk., Dictyotus caenosus (Westw.), Cermatulus nasalis (Westw.), Glaucias amyoti (A. White) and Cuspicona simplex Walk.


Life history

Wilson (1961) indicated that T. basalis is a solitary arrhenotokous parasite that develops from egg to adult within the host egg. He reports that this parasite passes through a number of generations each year and that the development is correlated to the temperature, i.e., at 270C, the males lived for 4 to 5 days and the females for 4 to 15 days. Sailer (1976) has indicated and the Author has confirmed that at 60 + 5% RH and 27 C, male T. basalis have a life span varying from 3 to 5 weeks or months while the females last from 4 to 15 weeks, and he also recorded that females are able to live as long as 10 months with a honey and










water supply.

Thomas, Jr. (1972) indicates that the polyphagous behavior of T.

basalis is a positive factor in its utilization as a released biological regulator, since other stink bug species can serve as alternate hosts for maintenance and increase of the parasitoid population. He also points out that field releases of T. basalis at rates of 5000 and 8000 adults per 1/10 acre increased the rate of parasitism substantially and releases of at least 50,000 adults per acre would be required for minimal effective suppression in a large scale augmentative release program.


Interspecific Communication


Investigations involving interspecific communication were reported by Laing (1937) who pointed out that the parasitoid Trichogramma evanescens (Westwood) perceives an odor left by adult moth as a cue that helps in locating host eggs. Thorpe and Jones (1937) also indicated that odor plays an important role in a parasitoid/host relationship. It was Brown, Jr. et al. (1970) that coined the term kairomone to define the mediators involved in those processes of communication. Since then, researches dealing with either behavioral studies, isolation, identification, synthesis, and proof of effectiveness, or combination with one or more of those aspects mediated by kairomones have been reported by Corbet (1973), Greany and Oatman (1972), Gross et al. (1975), Hays and Vinson (1971), Hendry et al. (1973, 1976), Jones et al. (1973), Leonard et al. (1975), Lewis et al. (1971, 1975, 1976), Nettles and Burks (1975), Nordlund et al. (1974), Nordlund and Lewis (1976), Tucker and Leonard (1977), Vinson (1975, 1976), Vinson et al. (1975, 1976) and










Weseloh (1974, 1976).

Interspecific chemical communication between Trissolcus basalis

(Wollaston) and its host, the southern green stink bug, Nezara viridula

(L.) has not been reported, however Russian researchers have conducted a series of investigations involving associations between many species of scelionids, particularly Trissolcus spp., and pentatomid species (Buleza, 1973; Meier, 1970; Zatyamina et al., 1976; Gennadiev et al., 1976). Viktorov et al. (1975) found Trissolcus grandis ability to encounter egg masses was significantly increased by extracts of adults of two pentatomid hosts.

















METHODS AND MATERIALS


Rearing of N. viridula (L.)


Southern green stink bugs were reared in wide-mouth Mason-type

jars measuring 8.0 cm and 6.5 cm bottom and top diameter, respectively, and 17.5 cm in height. The top portion was covered with white paper towel to confine and prevent the escape of nymphs and adults. Colonies were established by placing 5 male and 5 female adults per jar with 3 to 5 string beans and occasionally either peanuts, carrots, and corn as the food source. Oviposition sites were provided by a circle of paper towel measuring 8 cm in diameter at the bottom of the jar and one strip of the same paper measuring 3x20 cm with one end stuck to the top of the container.


Rearing of T. basalis (Wollaston)



The Floridian strain of the parasitoid was reared in large wooden cages with Plexi-glass top, with the front wall measuring 37.5 x 40.5 cm, back wall 37.5 x 45.5 cm and 42.5 cm in length. The front wall had an 18 cm hole and was provided with a cotton cloth sleeve to prevent escape and allow manual work inside the cage. Honey and water were available to all parasitoids and they were exposed to a photophase of
+0 +
14 hours day per 24 hr. cycle, a temperature of 25+1 C and 65-5% relative humidity. The host, N. viridula (L.), was reared under the same conditions of light, temperature, and humidity.









To reduce circadian and any other endogenous physiological

variability, the tests were conducted between 11:00 and 14:00 hour with 3-day old T. basalis without any previous ovipositional experience and host eggs 12-hour old or less.


Experiment 1: Response of the Female T. basalis to the Eggs of the Host, N. viridula.


Responsiveness of the female parasitoid to the eggs of the host was tested by means of a glass single tube olfactometer as shown in Fig. 1. The dispenser consisted of a small tube measuring 7.5 cm in length and

0.35 cm in diameter. One extreme of this tube had its internal diameter reduced to 0.20 cm and held a cotton filter. The dispenser was connected to the main tube with a hollowed rubber stopper.

The main tube measured 30 cm in length and 0.60 cm in internal diameter with one end linked to the dispenser and the other with its

internal diameter reduced to 0.30 cm retained a cotton plug which functioned as a filter and prevented movement of the female parasitoids beyond that point. This end of the tube was connected to a plastic hose, which linked to a vacuum source and an air flow meter. Air flow was adjusted to 3.3 cm3/s throughout the experiment.

Chilled female T. basalis were placed on a white filter paper cut out to a trapezoidal shape with bases measuring 0.5 and 5 cm and 4.5 cm. The immobilized parasitoids were transferred to the main tube by removing the rubber stopper and dispenser and placing the small base of the trapezoidal paper inside the tube and raising it to a 200 angle and gently tapping the paper. Then, the dispenser with the rubber stopper was returned to the original situation.




































Fig. 1. A single glass tube olfactometer is pictured.









__ __ Plastic tube
*.



Cotton plug












Main tube


Air flow direction







0.6Cm




Filter



Rubber stopper




0.35cm



Disoenser tube


30cm


7.5Cm









The olfactometer was placed on a white horizontal surface and ca. 109 cm away from 2 fluorescent lamps (Sylvania F40 cwx Lifeline) and the apparatus was properly positioned to allow uniform dispersion of the female parasitoids inside it.

Each experimental unit consisted of 10 healthy female T. basalis within the main tube. Previous to any treatment, they were allowed to complete recovery from the process of immobilization and run inside the olfactometer for no less than 5 minutes, and after every single treatment they were discarded.

Treatments were prepared in a spare dispenser. Host eggs were

introduced with soft forceps then they were covered by a thin cotton layer. This dispenser containing the treatment was then used to replace the blank one in a very quick stroke to avoid parasitoid escape.

Responsiveness of the female T. basalis was measured in terms of the number of parasitoids that concentrated within 10 cm of the tip of the dispenser. The readings were performed at 1 minute intervals for a period of 10 minutes following insertion of the treated dispenser.

Experiment 1 consisted of 13 treatments (numbers of eggs) with 4 replicates applied to every experimental unit. The levels of those treatments consisted of 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, and 300 eggs of the host, N. viridula. Reliability of the readings were compared with a blank with no eggs applied to the testing parasitoids.

Regression analysis was carried out with the treatments transformed to logarithm and multiplied by 10. The response of the female T. basalis was measured as the percentage of individuals orienting to the scent source after 3 minutes of exposure.

A test for lack of fit was conducted to check the adequacy of the









model in describing the relationship between the percentage of female parasitoids orienting to the kairomonal source and the treatments applied.

To determine the threshold for stimulation of the female T. basalis the same experimental procedure was used; however the treatments applied to the experimental units consisted of 0, 1, 2, 3, 4, and 5 eggs of the southern green stink bug.

A completely randomized 6 x 10 factorial experiment with 4 replications was developed for testing the response of the female parasitoid when stimulated by different levels of the eggs of N. viridula in order to find the threshold of stimulation.


Experiment 2: Orientation of the Female T. basalis Inside a "Y" Type Olfactometer.


Orientation of the female parasitoid was determined by the "Y"

type olfactometer shown in Fig. 2. Connections of the parallel tubes, to vacuum, and air flow meter were with plastic tubing. The parasitoid releasing tube and the filter tube were linked to the "Y" connections through hollowed rubber stoppers. Organdi screen at one end of the parallel tubes prevented movement of the parasitoids to the section "C", the "Y" connection that is linked to the air flow meter.

The experimental unit consisted of 100 healthy female T. basalis

transferred to the parasitoid releasing tube by the method of Experiment 1. Twenty females were tested in each of five olfactometers of the same type, under the same experimental conditions.

The air current passing through the filter tube of the olfactometer was 6.6 cm3/s, and the apparatus was positioned properly on a white horizontal surface and a blank test was run to detect any tendentious




































Fig. 2. A "Y" type olfactometer is pictured.

























to vaccum







I
A=13Cm





























8:B' 15Cm










-=0.35Cm


C=13Cm


iastic tube itton plug ibber stopper


arasitold releasing tube plastic tube kir flow direction )roandi screen


liter


Connection to air flow meter









response.

A treatment consisted of 50 eggs of the host introduced into the parallel tube (B) and dispersed 7.5 cm away from the organdi screen. Fifty egg shells of N. viridula were similarly placed inside the other tube (B'). The shells were thoroughly washed in a solution of 1 part
R
of liquid non-phosphorous soap, 1 part of Clorox and 100 parts of water, and then thoroughly rinsed in running water. The shells were dried under lab conditions and then washed with dichloromethane. After complete evaporation of the solvent, they were used in the experiment.

The test began after the parasitoids were fully recovered inside the release tube which was then connected to the olfactometer.

Responsiveness was measured as the percentage of female T. basalis orienting to tube (B), (B'), or remaining in the non-choice area (A). Readings were performed at 5-minute intervals for a 30-minute period.

Test for non-preference orientation to each of those sites (A, B, or B') was done with the chi-square test for multinomial experiments (Snedcor and Cochran, 1973).


Experiment 3: Temporal Analysis of the Ovipositional Behavior of the Female T. basalis


Experiments were conducted within a 5.0 cm Petri dish (lid 5.6 cm)
3
with a volume of 28.46 cm The lid of the dish was lined with a 5.5 cm diameter circle of WhatmanR No. 1 qualitative filter paper.

The experimental unit consisted of 1 healthy 3-day-old female T. basalis with no prior ovipositional experience. The parasitoid was chilled and transferred to a white surface and covered with the bottom of the Petri dish. A piece of paper supporting an egg mass was pinned to the center of the filter paper with a dissecting pin. Six eggs were









used per mass because this is in the range of the threshold for stimulation. To reach that number, large egg masses were broken apart and 3 rows made up of 1, 2, and 3 eggs were left as the best physical support upon which the female T. basalis could oviposit.

Completely recovered female T. basalis were exposed to the egg

masses when bottom and cover were put together. The container with the parasitoid was held vertically and 25 cm away from a fluorescent light source (cool white Sylvania F 15T8-CW), with the cover toward the light.

The experiment was repeated 20 times, and both eggs and parasitoid were discarded after each test. All ovipositional behavior was observed and every step was timed.


Experiment 4: Cues Useful in Location of the Host,
'N. viridula, by the Parasitoid T. basalis Scent of Male N. viridula


Responsiveness of the male and female T. basalis to the scent of

the male host was determined using the single tube olfactometer described

in Experiment 1. The dispenser tube was joined to another glass chamber measuring 10.0 cm in length and 1.3 cm in diameter (Fig. 3). All other experimental procedures were similar to those described for Experiment 1, except for the use of both male and female parasitoids for the treatment combinations.

Treatment combinations consisted of 2 levels of the parasitoid, male or female; 3 host levels of 0, 1, or 3 male N. viridula; and 10 time intervals obtained by 1 minute readings recorded every 10 minutes.

The glass chamber was used to confine the host insects during the experiment. When using 0 male host, the chamber was kept empty with a




































Fig. 3. The glass chamber is shown connected to a single glass
tube olfactometer.




















Ifactometer main tube :otton plug


lubber stopper Chamber Air flow direction


75cm


10Cm









current of air (3.3 cm3/ Is) passing through it and reaching the main

tube which contained 10 healthy 3-day old parasitoids (either male or female); readings were recorded at every 1-minute interval for 10 minutes. Responsiveness was reported when the parasitoid stayed around

the tip of the dispenser tube or within 10 cm of it. For the remaining treatments (2nd and 3rd levels of the male host) 1 and 3 15-day old male N. viridula were placed inside the glass chamber and the reactions of the parasitoid recorded as already described.

Analysis of the data was performed by a three-way analysis of

variance for completely randomized 2x3x10 factorial experiment with 4 replicates. Interactions of significance for the experiment were studied by a thorough analysis of the simple effects.


Scent of Female N. viridula


The methodology for the response of the parasitoid to the female host was identical to that described above for the male N. viridula.


Male and Female N. viridula Hemolymph


The response of male or female parasitoids to 3ul of male or female hemolymph or to blank controls was evaluated using the apparatus described in Experiment 1.

The hemolymph was obtained from healthy 15-day-old virgin males

and the liquid was drawn from the prothorax at the point of insertion of the forewing. The wing was removed and the exuded hemolymph collected with a micropipette, and transferred to a thin piece of cotton measuring

0.5 x 0.6 cm. After drying the cotton was transferred to a spare dispenser tube which later replaced the blank one from the olfactometer.









Evaluation of the reactions were similar to those described for

Experiment 1, and the readings were taken during each 1-minute interval for a 10-minute period.
22
The experimental procedure was arranged in a 2 x 10 factorial

design for each hemolymph type with 4 replicates. Analysis of variance was carried out and the interactions of importance were investigated through the simple effects.


m. ziiu Eggs of Different Ages


Response of the female T. basalis to N. viridula eggs of different ages was measured by means of a single tube olfactometer as described in Experiment 1.

Treatments were prepared by placing 0, 50 12-hour-, 50 24-hour-, or 50 48-hour-old eggs of the host in the dispenser tube and proceeding as previously described. All treatments were replicated 4 times and readings made at 1-minute intervals for a 10-minute period.

A two-way analysis of variance was computed for the 4 x 10 factorial design, and simple effects were investigated for the interaction between egg age and time required for female parasitoid reaction.


Experiment 5: Reactions of the Male and Female
T. basalis to the Eggs of the Host,
N. viridula at Different Degrees of Parasitism


Treatments were set up to test parasitoid response to nonparasitized and parasitized host eggs as well as eggs from which parasitoids had already emerged, i.e., 0 N. viridula eggs, 50 12-hour, 50 parasitized eggs from which adult T. basalis were ready to emerge, and 50 host egg shells 4 days after emergence of the parasitoids.









Except for the statistical analysis and utilization of both male and female parasitoids as experimental units, the methods used were similar to that described for Experiment 1.

The levels of the factors utilized during the test were: male and female T. basalis, 4 different situations of host eggs, and 10 1-minute readings recorded during a 10 minute period.

The experimental design was a 2 x 4 x 10 factorial with 4 replicates. F-test values were determined for main effects, interactions and simple effects at 0.01 and 0.05 levels of significance.


Experiment 6: Reactions of the Female T. basalis
to the Kairomonal Solutions Prepared with Different Solvents


The solutions were prepared by soaking 12-hour-old (or less) eggs of N. viridula in 4 different solvents, i.e., water, dichloromethane, ethanol, and hexane, for 1 hour. The resulting suspension was filtered through a WhatmanR No. 1 qualitative filter paper. The ratio of egg and solvent was 1 egg:l ul of solvent.

Treatments were set up by drawing a 50 ul aliquot from the

solutions and applying it to different pieces of a thin piece of cotton measuring 0.5 x 0.6 cm and allowing enough time for solvent evaporation. The treated pieces of cotton were transferred to spare dispenser tubes by means of a forceps and pushed in to the narrowed end with a microsyringe plunger. These dispensers were utilized to replace the blank ones in the olfactometer. The experimental procedure and methods of recording responsiveness of the female T. basalis to the stimuli were identical to Experiment 1.

The experimental procedure was a completely randomized design with









5 replications. Selection of the solvent that removed most of the active ingredient(s) was done by contrasting the treatment means at the 4th minute interval, utilizing Duncan's test (Duncan, 1955).


Dichloromethane Washes Required for Removal of the Kairomone from Eggs of N. viridula


From the 4 solvents tested, dichloromethane was selected for

utilization throughout this assay, based on results presented later in this text.

Treatments in this experiment consisted in soaking 12-hour-old

(or less) N. viridula eggs in dichloromethane for different periods of time. For every single wash fresh aliquots of dichloromethane were used. No re-use or recycling was permitted through the bioassay.

After the soaking process the washed eggs were spread on a WhatmanR No. 1 qualitative filter paper to allow solvent evaporation. The ratio of host eggs per milliliter of dichloromethane during the washing (=soaking) process was 0.025/1.

After a given washing cycle, and complete solvent evaporation, the eggs were placed in a spare dispenser tube, and later used to replace the blank tube in the olfactometer with the female parasitoid. The testing procedure was similar to that described for Experiment 1, except for the analysis of the data.

During this experiment, 10 treatments including a blank with no

eggs, were applied to the experimental unitS. The treatments consisted of 50 N. viridula eggs treated as follows: host eggs soaked 1 time for

3 minutes, soaked 1 time for 5 minutes, soaked 2 times for 5 minutes, 3 times for five minutes, soaked 1, 2, 3, and 4 times for 1 hour, and









finally, host eggs soaked 1 time for 4 hours.

The data were analyzed as a 102 factorial experiment with 4

replicates, then the F-test values were determined for the statistical parameters of relevant interest to the experiment. Reaction of the Female T. basalis to Kairomonal Solutions Prepared by Different Methods


Activity of the solutions was measured by the percentage of female T. basalis orienting to the tip of the dispenser or within 10.0 cm of it (refer to Experiment 1). The solutions were obtained by soaking host eggs 1 time in dichloromethane for 4 hours, grinding the host eggs with the same solvent, and by soaking host eggs 4 times at 1 hour intervals. Suspensions obtained were filtered with a WhatmanR No. 1 qualitative filter paper, and concentrations were set, such that 100 ul of the solution would contain 50 egg equivalents.

Fifty-egg equivalents of each solution were applied to a thin piece of absorbent cotton (0.5 x 0.6 cm). After solvent evaporation the treated cotton was introduced into the glass dispenser with forceps and a microsyringe plunger. Controls were dichloromethane-treated pieces of cotton.

A completely randomized 4 x 10 factorial experiment with 4

replications was developed for testing the responses of the female T. basalis to the different extraction procedures. Analysis of variance was performed and F-test values obtained for the inference-making process.


Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the
Crude Kairomonal Extract from Eggs of the Host, N. viridula


The kairomonal solution was prepared from N. viridula eggs 12 hours









old or less. The eggs were ground with dichloromethane and the resulting suspension filtered through a WhatmanR No. 1 qualitative filter paper. The ratio of egg and solvent was 1 egg:l ul of dichloromethane. Dilutions were then prepared to provide the concentrations used during the experiment, such that 1 ul of the prepared dilutions would contain
4 -3 2 -l1
10-4, 10-3, 10-2, and 10- egg equivalents.

The experiments were conducted within 5 cm Petri dishes with lids
R
lined with Whatman filter paper as described in Experiment 3. The experimental unit consisted of 30 healthy 3-day-old female T. basalis with no prior ovipositional experience. The females were chilled and transferred to a plain white surface then individually covered with the bottom of a Petri dish. One microliter of the test solution was dropped + 2
on the center of a filter paper covering an area of (0.17-0.008) cm The solvent was allowed to evaporate and the paper then transferred to the inside of a Petri dish cover. Fully recovered female parasitoids were exposed to the crude extract when bottom and cover were put together. The container with the parasitoid was held vertically and 25 cm away from a fluorescent light source (cool white Sylvania F15T8-CW), with the cover toward the light.

Preliminary assays without solution were conducted to eliminate

any bias due to the light or handling during the experimental procedure. The activity of the parasitoids was measured in terms of their ability to locate the treated spot and their subsequent reactions when exposed to different concentrations of the crude extract.

Responsiveness to the different stimuli were recorded in terms of

percentage of females exhibiting kinesis, chemotaxis, antennal palpation, searching and reinforcement.









Data obtained through the experiment were submitted to probit

analysis for quantal response experiments (Finney, 1971). Subsequently, regression equations were established for all behavioral patterns exhibited by the female parasitoids then Stimulant Concentration (SC50 and Stimulant Dose (SD50) values were determined for those patterns.
-2
The Stimulant Time (ST50) for a 102 egg equivalent solution was calculated for antennal palpation; however, the procedure is applicable to any behavior pattern.


Experiment 8: Effects of the Crude Kairomonal Extract
from Eggs of the Host, N. vid in the
Orientation of the Female T. basalis


The methodology and the experimental material utilized in this test was identical to those used in Experiment 7, except that the experimental unit consisted of 10 healthy 3-day-old female T. basalis, and a different statistical design.

Treatments were four equally spaced concentrations of the crude

kairomonal extract, ranging from 10-4 to 10-1 egg equivalents/ul, plus a check which consisted of 1 ul of dichloromethane dropped on the center of the filter paper and after evaporation, exposed to the experimental units.

Effects of the treatments were observed by tracking the time spent by the female parasitoids in orienting themselves to the treated spot for the first antennal contact.

A completely randomized design with 3 replications was developed for checking the effect of the kairomonal concentrations on the orientation time of the female parasitoid. The F-test was determined and contrast of the means was done by Duncan's technique (Duncan, 1955).










Experiment 9: Enhancement of Host Location by Scent Combinations


Responsiveness of the female T. basalis to combination of scents of the kairomonal solution, host eggs, and solvent was tested with the single tube olfactometer and the olfactometer-glass chamber tube as shown in Figs. 1 and 3 respectively.

For every single treatment 5 female T. basalis under the same

biological conditions of the previous experiments were introduced into the single tube olfactometer as described in Experiment 1 and submitted to 7 different scent combinations.

The first treatment consisted of 3 healthy 15-day-old virgin female N. viridula in the glass chamber. Treatment 2 consisted of 10 12-hourold eggs of the host combined with 3 female N. viridula offered simultaneously. The eggs were placed inside the olfactometer and approximately 0.5 cm away from the tip of the dispenser. The adult female hosts were introduced in the glass tube chamber.

The third treatment consisted in applying 25 ul of dichloromethane to a thin piece of cotton measuring 0.5 x 0.6 cm which after evaporation was introduced into a spare dispenser tube.

The next treatment was 25 ul of the solution containing 12.5 egg equivalents of the crude kairomonal solution in dichloromethane was dispensed on a thin cotton piece. After solvent evaporation the treated cotton was placed inside the dispenser tube as in the previous test.

Treatment 5 consisted of introducing 10 12-hour-old eggs of N. viridula into the olfactometer and 0.5 cm away from the tip of the dispenser. Treatments 6 and 7 were identical to 5 except that a thin piece of cotton treated with 25 ul of dichloromethane and 25 ul of the









kairomonal solution with 12.5 egg equivalents was placed in the dispenser, respectively. Reactions of the female T. basalis to the scent combinations was determined when one of the 5 parasitoids made the first physical contact with the egg mass or the tip of the dispenser.

The velocity of the parasitoid toward the scent source was

determined for the first female T. basalis scored in each of the situations. For evaluation purposes, it was assumed that the movement toward the source of stimuli was preformed in a linear and uniform fashion.

The experiment was set up as a completely randomized design with 7 treatments and 5 replications. Contrast of the treatments and means were performed by the appropriate statistical procedures already mentioned.


Experiment 10: Normality Studies with the Crude
Kairomonal Solution from Eggs of the Host, N. viridula


Reactions to exaggerated releasers by higher animals and insects have been reported in the specialized literature (Tinbergen, 1951; Marler and Hamilton, 1967; Wallace, 1973; Hogan et al., 1974; Magnus, 1958; and Staddon, 1975).

To observe if female T. basalis were reacting to a normal stimulus a series of tests were performed for certain behavior patterns.


Response to the Kairomonal Solution on the Filter Paper


Bioassays were conducted in Petri dishes described in Experiment 3. The experimental unit was 10 healthy 3-day-old female T. basalis without any ovipositional experience. The female parasitoids were exposed to









-i
1 ul of a 10 egg equivalent solution in dichloromethane or to dichloromethane controls using the procedure described in Experiment 7.

Responsiveness of the parasitoids to the solvent and to the solution were recorded in terms of the percentage of the testing insects reaching the treated spot and drumming the area with the antennal flagellum. The data obtained were analyzed by a paired-difference t-test.


Evaluation of Parasitism in Areas Treated with the Crude Kairomonal Solution


The method utilized was a modification of the procedure of Jones et al. (1973). A circle of WhatmanR No. 1 qualitative filter paper measuring 12.5 cm in diameter was cut in 4 quadrants and placed in a Petri dish with bottom and cover measuring 14 cm and 15 cm of internal diameter respectively. The quadrants were positioned 1.2 cm apart to reduce the chance that the parasitoid would search in adjacent areas not treated with the kairomone. An aliquot of 50 ul of a 10-2 egg equivalent solution was applied to each of two opposite quadrants, and 50 ul of dichloromethane was applied to the remaining quadrants. After the solvent evaporation 1 egg mass made up of 10 12-hour-old eggs of N. viridula was placed on each quadrant. Two immobilized (by chilling) female T. basalis were placed on the center of the dish and after recovery the dish was covered and the parasitoid allowed to search and oviposit for 45 minutes. Parasitism was determined 12 or more days after the treatment by counting the darkened eggs typical of T. basalis close to emergence.

The data obtained were analyzed by paired-difference t-test and the ratio between parasitized eggs on solution and solvent treated










area computed.


Responses of Female T. basalis to Egg Shells and 12-Hour-Old Eggs of the Host N. viridula


The consummatory phase of the ovipositional behavior was tested with egg shells treated with the crude kairomonal solution, with the solvent, and with 12-hour-old eggs of N. viridula.

Egg shells with the operculi were obtained 15 days after hatching of the host insect. They were thoroughly washed in a 1:1:100 nonR
phosphorous dish wash detergent, CloroxR, and water, then extensively rinsed in tap water and finally washed in distilled water. The shell remained glued through the washing process to the paper towel ovipositional site. They were allowed to dry on filter paper under laboratory conditions and later rinsed 3 times in dichloromethane. After solvent evaporation, they were ready for the experimental procedure.

The experimental unit was made up of 10 healthy 3-day-old female T. basalis without any prior ovipositional experience. Except for the treatments assigned to the female parasitoids, all bioassay procedures were the same as those for Experiment 3.

The treatments were 6 12-hour-old eggs of N. viridula, 6 egg shells treated with 1 ul of dichloromethane, and 6 treated with 1 ul of 10-1 egg equivalent solution of the kairomonal extract.

Response of the parasitoids to the treatments were recorded as the percentage of female T. basalis assuming the ovipositional posture after exploring the treated egg masses and bringing the ovipositor into contact with the chorial surface.










Datawere analyzed for a completely randomized design with 3 treatments and 4 replications. Contrast of the means was done by Duncan's test (Duncan, 1955).


Antennal Palpation Previous to Oviposition


Egg shells treated with 1 ul of a 10-1 egg equivalent solution of the crude kairomonal extract and 12-hour-old eggs of N. viridula were utilized to assess the degree of normality of the isolated releaser. The time spent by the female T. basalis palpating the eggs and treated egg shells previous to the contact of the ovipositor with the chorium was recorded as the measurement of evaluation.

The bioassay consisted in exposing 1 healthy 3-day-old female parasitoid without ovipositional experience to the treatments. The procedure was replicated 30 times and the amount of time spent by T. basalis exhibiting antennal palpation was recorded. The methodology was similar to that of the previous section except for the points already mentioned and analysis of the data, which consisted of applying a paired-difference t-test to the results.
















RESULTS


Experiment 1: Response of the Female T. basalis
to the Eggs of the Host, N. viridula


Responsiveness of the parasitoid inside the single tube olfactometer (Fig. 1) was recorded after 3 minutes of exposure. The results are shown in Fig. 4. There is a strong linear relationship (r=0.97) between the percentage of female T. basalis stimulated to move toward the scent source and the log number of eggs of N. viridula. The adequacy of the linear model was contrasted at 0.05 level of significance, and the F-test (=1.29) indicated that the relationship already studied is properly represented by the model.

Analysis of the main effects, i.e., egg and exposure time revealed F values equal to 14.12 and 4.13 respectively. They were highly significant ata=0.05, but do not interact. Their effects are additive to the population mean of the number of female parasitoid orienting to the scent source.

The threshold for stimulation was 5 12-hour-old eggs of the southern green stink bug. The analysis of variance for the simple effects is shown in Table 1, of the Appendix. An F-test for all time levels within the 5-egg level was highly significant, indicating that the parasitoids started to react to the host presence at that point.


Experiment 2: Orientation of the Female
T. basalis Inside a "Y" Type Olfactometer

































Fig. 4. Relationship between percentage of female Trissolcus basalis (Wollaston) responding
within a single tube olfactometer versus different levels of eggs of the host
southern green stink bug, Nezara viridula (L.), is shown.










100


o
0 o
A
80 Y = 22.15 + 32.76 log x

r = 0.97
IM 0
F =1.29
S60
'........ Threshold for stimulation
0
x =1.70

U) 0
40

0 0


E
a 20 o

0




I I I 1 2 3 log of the number of eggs of N. viridula x 10










When introduced into the olfactometer and after recovery the

parasitoids began to move to the different sections of the apparatus. During the first 15 minutes of observation the parasitoids kept moving within the region A of the olfactometer, running either on a straight or circular path into the Y part of the olfactometer. Sometimes the females returned to the releasing tube and remained there or resumed movement to the other parts of the apparatus. After 30 minutes, 63 percent of the female parasitoids were found in the section B palpating host eggs with the antennal flagellum, ovipositing, or running inside the tube. Five percent of the tested insects were observed in section B' performing the same behavior except ovipositing. Finally 32 percent of the sample tested stayed in Section A either running in the Y part of the olfactometer or remaining in a resting position inside the releasing tube.

It is evident that the parasitoids showed a highly significant

preference for the side of the olfactometer containing the 12-hour-Old eggs of the host, N. viridula. These data are presented in Table 2, of the Appendix. The chi-square value was 50.54.


Experiment 3: Temporal Analysis of the
Ovipositional Behavior of the Female T. basalis


When the female T. basalis is introduced into the Petri dish she starts to either run on the margins of the filter paper exhibiting a random movement, possibly kinesis, or moves straight to the egg mass, after spending a certain amount of time in this kinetic pattern, the female parasitoid may purposefully move toward the source of stimulus. During the orientation process the parasitoids required an average of









+
103.65 20.40 seconds (a=0.05) to encounter the egg mass. During this period considerable antennal and body grooming took place, sometimes the female moved straight to the eggs, then suddenly changed direction and moved away and later returned. It is evident that the source of stimulus is reached either by random movement, chemotaxis, or a combination of both.

When a female T. basalis makes first contact with the egg mass, she begins to explore it by drumming the lateral wall of the marginal eggs or the crevices of the central eggs with the antennal flagellum (Fig.

+
5 a,b). The parasitoid spends 116.55 18.56 seconds with 95% of the fiducial limit, in this exploratory behavior, then prepares for oviposition.

The female T. basalis finishes the exploratory phase when it turns its head away from the point chosen to insert the ovipositor. Drilling of the chorium begins when the parasitoid slightly bends the terminal portion of the abdomen and inserts its ovipositor through the egg wall. When insertion is performed in the marginal eggs, the female T. basalis may take three positions: (a) the parasitoid has the ventral portion of the body parallel to the substratum that supports the egg mass and the tip of the wings reaching the eggs to be parasitized; (b) the female parasitoid stands sideways in relation to the substratum and the wings may touch the egg or stay away from it (Fig. Sc). In both situations, drilling of the chorium occurs in the bottom third of the egg. Oviposition in the central eggs is performed in two distinct postures: (a) female T. basalis flexes its abdomen and introduces the terminal portion between the crevices then inserts the ovipositor 1/3 below the operculum (Fig. Se); (b) the parasitoid perforates the egg cap, by introducing



































Fig. 5a and b. Female Trissolcus basalis (Wollaston) exhibits
pre-ovipositional behavior in an egg mass of its
host, Nezara viridula (L.). Antennal palpation of
(a) marginal egg and (b) central egg is shown.






















































I


i


1~


































Fig. 5c and d. Female Trissolcus basalis (Wollaston) exhibits
ovipositional behavior on an egg mass of its host, Nezara viridula (L.). Shown are (c) drilling the chorium for ovipositing and (d) ovipositor thrust
and marking the egg after oviposition.





43
























9.



(c)















f.s N9


L- .. :7 o .....


(d)










the ovipositor through the operculum and beneath the point of junction with the surrounding walls. In this posture the female T. basalis stands with the sternal portion of the thorax approximately in a straight angle with the horizontal plane of the opercular surface (Fig. 5f). Deposition of the egg is characterized by a rocking movement similar to those observed by Wilson (1961) and Hokyo and Kiritani (1965) in other scelionid parasitoids. All legs serve as support to the female T. basalis, however; the meso and metathoracic pairs act more remarkably to this function. It was observed that the prothoracic legs and forewings are used very effectively in agressive behavior, especially when the egg density drops to 0.5 egg per female parasitoid or less, and more than one parasitoid is looking for ovipositional site.

The process of oviposition which starts with the preparation of the parasitoid to drill into the egg shell, ends wheh the female T. basalis withdraws the ovipositor from the chorium. This behavioral step
+
requires 117.97 5.86 seconds, with a fiducial limit of 95%.

After successful oviposition the female parasitoid begins the egg
+
marking process (Askew, 1971; Safavi, 1968). It took 19.15 1.02 seconds for T. basalis to complete this behavior. Marginal eggs are marked when the female drags the tip of the ovipositor from the point of insertion up to the margins of the operculum in a sinuous pattern (Fig. 5d). Central eggs are marked in the same manner, starting from the point of drilling and covering the opercular margins and going down ca. 1/3 of the upper portion of the surrounding egg wall.

An ethogram of the ovipositional behavior of T. basalis is shown in Fig. 6. It could be seen that the orientation to the egg mass is accomplished by either random movement or chemotaxis or combination of


































Fig. 5e and f. Female Trissolcus basalis (Wollaston) exhibits
ovipositional behavior on an egg mass of its
host, Nezara viridula (L.). Oviposition of the
central eggs by drilling the chorium, (e) the lateral wall, and (f) the operculum is shown.






46




. viy*


(f)


- - - - - .. 11 111- jil -



































Fig. 6. Ovipositional ethogram of the female parasitoid Trissolcus
basalis (Wollaston) when stimulated by six 12-hour-old
eggs of the host, Nezara viridula (L.), is shown.










OVIPOSITIONAL BEHAVIOR OF Trissolcus basalis (Wollaston)


ORIENTAT ION (103.65 20.40) s


RANDOM MOVEMENT -


I CHEMOTAXIS


L


I


ANTENNAL PALPATION (116.55 18.56) s


OV I PO S I TI ON (117.971 5.86) s

t


MAR K I N G 19.15 1.02 ) s



I
S-- AT- NATION










both. After the physical contact, antennal palpation, oviposition, and marking follows in a very defined sequence. Those behavioral steps were observed when the number of eggs per mass is within the threshold limit, that is, 6 eggs of N. viridula per mass.

The broken lines of the Fig. 6 indicate repetition of a series of behavioral steps that ultimately leads the parasitoid to satiation. Tracking of time during this cycle was omitted.


Experiment 4: Cues Useful in Location
of the Host, N. viridula by the Parasitoid T. bassalis


Scent of Male N. viridula


Responsiveness of the male and female T. basalis to the scent of

different levels of the male N. viridula in the single tube olfactometer was determined by the analysis of variance of the treatment combinations. As shown in Table 3 of the Appendix, parasitoid and host factors interact. There is evidence that host levels and parasitoid levels do not act independently, that is, a change in the levels of the host correspond to a response by the male and female parasitoid which is different in direction and intensity. Analysis of the simple effects by combination of 10 levels of time is shown in Fig. 7a. Both male and female T. basalis reacted to the scent of the male host levels combined, F.values were 6.20** and 13.87** respectively. The differences in variability was assigned to 1 and 3 male N. viridula, since no significant response was observed at 0 level (F=0.80). Response of the female parasitoid sharply increased when 3 male hosts were placed in the olfactometer.


Scent of Female H. viridula



































Fig. 7. Number of male and female Trissolcus basalis (Wollaston)
reacting to (a) scent of 0, 1, and 3 male Nezara viridula (L.)
and (b) 0, 1, and 3 female N. viridula, averaged over
10 equally spaced time intervals is shown.













-0 ..... o o 0 0-o Male +- female, F=1.67

Male, F = 6.20**


Female, F=13.87**




-o_


0 1 2
Number of male N. viridula









o o
(a)











o- F =45.42**

F =3.18**


.... F = 17.96 **,


- ... *-


0 1 2 3

Number of female N. viridula

(b)


-- 20(




150




- 100
0


E
z


3







o


250


S20 a,

150




I 100


E
z


4i I I I I


so 00 Ov


-


0









Orientation of the parasitoid to the scent source was analyzed and the results are presented in Table 4 of the Appendix. A highly significant interaction indicated that the responsiveness of the parasitoids vary with the levels of the female host. Response of the male parasitoid was lower than the response exhibited by the female (Fig. 7b), and the F-test was significant for both parasitoid sexes, at 0.05 levL.

Responsiveness of male and female T. basalis combined over 10 time intervals were not significantly different (F=1.67) for three male host levels utilized at 0.05 level of significance. When female hosts were offered to the male and female parasitoids, the combined response was highly significant, F=45.42**. Most of this is accounted by the female parasitoid reaction.


Scent of Male N. viridula Hemolymph


It was observed that male and female T. basalis responded differently to the different levels of the male host hemolymph. As shown in Table 5, the three-factor interaction implies that the male host hemolymph versus T. basalis differs with the levels of time.

Data for the treatment totals are presented in Table 6 of the

Appendix. There is an indication that the male parasitoid did react poorly to the male hemolymph levels when averaged over 10 levels of time. Analysis of the simple effects confirmed that assumption as the F-test was not significant at 0.05 level of significance (F=0.45). On the other hand the female T. basalis exhibited a response to the male N. viridula hemolymph, which was highly significant, F=83.60**.

The two-factor interaction observed for male host hemolymph versus T. basalis suggests that the responses of the male and female parasitoid










are different in magnitude and direction when the two levels of the male host hemolymph are available to both sexes (Table 5 of the Appendix).

Reactions of the male and female parasitoids to combined levels of the male N. viridula hemolymph in relation to time are shown in Fig. 8a. Male parasitoid reaction was not statistically significant. Female T. basalis reaction was highly significant. It was observed that after 2 minutes responsiveness of the female parasitoid reached its highest value. After this, reactivity decreased and returned to the level of the control at the 10th minute of observation.


Scent of Female N. viridula Hemolymph


Reactions of the male and female T. basalis to the female host

hemolymph were analyzed and the results are summarized in Tables 7 and

8 of the Appendix. Responsiveness to combined levels of the female hemolymph at different time intervals is presented in Fig. 8b.

Male parasitoids were poorly stimulated by the female host hemolymph, there was no significant difference between the two levels of hemolymph applied when averaged over 10 time levels (F=0.05). Female parasitoids responded positively to the female N. viridula hemolymph (F=156.80**). The remaining inferences about responsiveness of the parasitoid sexes to the female host hemolymph are identical to those already discussed for the male host hemolymph.


R. xi idl& Eggs of Different Ages


Treatment responses shown in Fig. 9 implies that the female T., basalis responded better to 12 and 24-hour-old host eggs.

Analysis of variance for the simple effects was carried out and the



































Fig. 8. Number of male and female Trissolcus basalis (Wollaston)
reacting to combined levels of (a) hemolymph of male
Nezara viridula (L.) and (b) hemolymph of female N. viridula
at different time intervals is shown.










Number of T. basalis reacting
-0 a o


Number of T. basalis reacting


) '4
/ -;. I
.- I


I

I f
~1
Oi
2


/
/
/
/
/


I f

-n
3


oc


/ /'

(


CnI-



































Fig. 9. Number of female Trissolcus basalis (Wollaston) reacting
to different egg ages of the host, Nezara viridula (L.),
at different time intervals is shown.



















30



en
S 25



U)
\ 20O/



= is..........


E

.... 12-hour-old host eggs 10
....N 24 -hour-old host eggs

a
.E 0 egg z 5
-.... 48-hour-old host eggs


o 0 I I I

1 2 3 4 5 Minutes









results are presented in Table 9 of the Appendix.

F values for variation between time within 12-hour- and 24-hour-old eggs were 8.44 and 5.19 respectively. They were highly significant and suggested that the female T. basalis were stimulated by volatile chemicals carried by the air stream that passed through the eggs inside the dispenser tube. There is also indication that such chemicals decrease in concentration as the eggs get older. The F value for the response of females to 48-hour-old eggs was not significantly different from the blank control at the 0.05 level.

The female parasitoid reactions are shown in Fig. 9. The maximum reaction occurred at the third minute of exposure. Following that, a decline in response took place and after the 5th minute the reactions were identical for all treatments applied. The decline after the 5th minute could be a result of either habituation, decline in concentration of the volatile chemicals or a combination of both.


Experiment 5: Reactions of the Male and Female T. basalis to the
Eggs of the Host, N. viridula at Different Degrees of Parasitism


Responsiveness of the male and female T. basalis to the treatments are summarized in Table 10 of the Appendix. Significant interactions were found for egg levels versus time, egg versus male and female parasitoid, time versus male and female T. basalis. There is evidence that the stimulation of the parasitoids by the scent source inside the main tube of the olfactometer depended upon the levels of parasitism of the eggs of N. viridula and upon levels of time.

The reactions of male and female T. basalis to the different

conditions of the host eggs are presented in Figs. 10 and 11 respectively.

Treatment totals revealed that again the maximum reaction of male



































Fig. 10. Number of male Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown.




60




















70 o
0 o

o
O

60




50-o- Treatment totals
50
c Parasitized host eggs
U
0M 12-hour-old host eggs
40
.......... Egg shells from parasitized eggs cc
cc 0 host eggs 30



E
o20
0


E
Z 10
.**


1 2 3 4 5
Minutes


































Fig. 11. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), at different
levels of parasitism and time intervals-is shown.






















100


0

90

~00


80~

0) 0
80
Co

oo
70 -o Treatment totals
0
"0 host egg
50 12.hour-old host eggs
.0.. 50 parasitized host eggs
60 ............ 50 egg shells from parasitized host eggs
L.
IIB
30

E


20


.:,.. :

z 10




I I I I I 1 2 3 4 5
Mi n utes









and female parasitoids happened at the 2nd or 3rd time interval.

Analysis of variance for the simple effects are shown in Table 11 of the Appendix. It can be observed that the response of the male parasitoid to the blank tube without host eggs and to 50 12-hour-old host eggs were statistically identical. The response of male parasitoids to the 12-hour-old eggs was not high enough to rule out chance factor. On the other hand, 50 parasitized host eggs containing T. basalis ready to emerge, and 50 egg shells from parasitized eggs 4 days after emergence, triggered a highly significant response in the male parasitoids.

Female parasitoid response was statistically high for the different situations of the egg host except for the blank, when no eggs were available (Table 11 of the Appendix).


Experiment 6: Reactions of the Female T. basalis to the
Kairomonal Solutions Prepared with Different Solvents


Since it was demonstrated that female T. basalis exhibit a

noticeable preference for sites containing eggs of its host N. viridula, the next step was to extract the chemical(s) from eggs of the host. Four solvents were used and the activities of the crude kairomonal extract obtained tested in the olfactometer.

Results of the evaluation process are shown in Table 12 of the

Appendix. Treatment totals indicated that the maximum response of the female parasitoid tolthe crude kairomonal extract obtained with the solvents was at the 4th minute of observation. Data from this time interval were submitted to analysis of variance and the F-test (=12.14**) was highly significant.

Contrast of the means was done by the Duncan's procedure (Duncan, 1955) and the results are presented in Table 13 of the Appendix. The










data implied that dichloromethane was the best solvent in removing the active ingredient(s) from the host eggs. Activity of hexane was similar to the ethanol but different from water. Water had the lowest activity, which was statistically identical to ethanol.


Number of Dichloromethane Washes Required for Removal of the Kairomonal Extract from Eggs of N. viridula


Evaluation of the methods utilized consisted in offering the eggs

of N. viridula to the female parasitoids after soaking in dichloromethane. Responsiveness of the female T. basalis was measured inside the olfactometer and the results are shown in Table 14 of the Appendix. Methods and time do not interact; they are independent of one another (F=1.49).

Treatment responses are shown in Figures 12 a and b. Removal of the kairomone from eggs of the host was partial for all methods tested, except for the procedures of soaking eggs 1 time for 1 and 4 hours. Analysis of the simple effects indicated that the activity present on the eggs treated by those methods elicited responses from the female parasitoids not significant at 0.05 level. Reaction of the Female 1. basalis to Kairomonal Solutions Prepared by Different Methods


Responsiveness of the female parasitoids to the crude kairomonal extract obtained by 3 techniques was compared when host eggs were: (a) soaked 1 time for 4 hours in the solvent; (b) ground with the solvent;

(c) soaked 4 times at 1-hour intervals.

A summary of the female parasitoid reactions in the single tube olfactometer is shown in Fig. 13. Solutions with best activity were



































Fig. 12a. Number of female Trissolcus basalis (Wollaston) reacting
to the eggs of the host, Nezara viridula (L.), after
soaking in dichloromethane for different periods of time
is shown.




























4
N%~~ :~


0
o
.1I


Sp 4.


I


F=O.55 -F=7.55** F=1.93**
Fa .9 ....'....
F.2.19** F=2.29** F=1.25


4.
4.
4.


0 host egg host eggs host eggs host eggs host eggs host eggs


soaked for 3minutes soaked for 5 minutes soaked 2 times for 5 minutes Soaked 3 times for 5 minutes soaked 1 time for 1hour


I I I I 1 2 3 4 5
Minu t es
(a)


24 L


r 20




16
(V


































Fig. 12b. Number of female Trissolcus basalis (Wollaston) reacting
to the eggs of the host, Nezara viridula (L.), after
soaking in dichloromethane for different periods of time
is shown.





































-4~
q
\~


V
V
V
V
V


+
.9 -

S


SF0.55 F. 2.59** F= 2 03** SF= 2.48**. F= 0.64 o


0 host egg host eggs host eggs host eggs host eggs


soaked 2 times soaked 3 times soaked 4 times soaked 1 time


Minutes
(b)


/


for for for for


hour hour hour hours



































Fig. 13. Number of female Trissolcus basalis (Wollaston) reacting
to crude kairomonal extract from eggs of the host, Nezara
viridula (L.), removed with dichloromethane by different
methods at certain time intervals is shown.






























Treatment totals
F=4.43"*_ solution from eggs soaked
1 time for 4 hours
F=3.49"......_ solution from eggs ground
with solvent
F=4.10* solution from eggs soaked
4 times for 1 hour F=0.84 ...... solvent


100 90


Minutes









those when the 12-hour-old eggs of N. viridula were immersed 1 time for 4 hours in the solvent, and those when the eggs were ground in dichloromethane. The results are summarized in Table 15 of the Appendix. From the treatment totals it is evident that the solution with the highest activity was prepared by grinding eggs of N. viridula. However analysis of the simple effects failed to reveal any statistically significant difference between the methods under investigation. F values are presented in Fig. 13.


Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the Crude
Kairomonal Extract from Eggs of the Host, g. viridula


Table 16 of the Appendix shows the percentage of each type of

behavior exhibited when different concentrations of the crude kairomonal solution were used. It can be seen that all behavior steps become more stereotyped when the concentration increases, except for the initial random movement which has a reverse trend.

When introduced into the Petri dish, the female T. basalis starts to run either on the margins of the treated filter paper or lateral walls of the bottom of the container.

Sometimes the parasitoid stops, grooms its antennae, wings, and

abdomen then continues to move. This random movement is caused by some sort of stimulus which has not been determined. It could be light intensity or neurophysiological factors of the female T. basalis. This behavior is also observed when no treatment has been applied to the filter paper (Fig. 14). Kinesis occurs when as a result of random movement the parasitoid encounters the treated spot and remains there for a period of time. On the other hand, the percentage of parasitoids


































Fig. 14. The behavior patterns of the female Trissolcus basalis
(Wollaston) on a filter paper treated with crude
kairomonal extract from eggs of the host, southern green
stink bug, Nezara viridula (L.), are diagrammed. Shown are parasitoid: (a) in direct movement to the treated
spot; (a') in random movement to the spot; (a") after
antennal palpation, leaving the spot and starting to search;
(b) after searching, returning to the treated area for
reinforcement; and (b') leaving the spot for new cycle of
searching. Either a or a' will occur for every single
trial.


























































Legends:
0 Treated spot.
SFemale T. basalis.
Random (kinesis),
chemotaxis,
searching,
.reinforcement, and
searching paths.










locating the treated spot randomly, decreases as the concentration of the crude extract increases.

Chemotaxis is observed when the female parasitoid after random

movements inside the Petri dish and from a certain point moves directly to the treated area and is arrested by the stimulus. Such orientation type is directly proportional to the concentration of the crude extract.

Orientation of the female T. basalis to the treated spot can occur by random movement possibly kinesis or chemotaxis (Fraenkel and Gunn, 1961). In either event the following subsequent behavior patterns occur: antennal palpation happens while the female parasitoid explores the treated area and by means of the antennal flagellum "drums" the surface of the area. After this phase the female T. basalis leaves the spot, increases its velocity, and for a period of time searches the adjacent area then returns to the spot where she repeats antennal palpation. This cycle of behavior may be repeated many times.

There is evidence that the crude kairomonal extract under test acted as a reinforcer stimulus, and the repetition of the parasitoid adaptive behavior was a result of reinforcement (Fig. 14).

These behavioral activities are completed in a time period that is highly correlated with the concentration of the solution under test.

Parameters of the described behavior patterns are presented in

Table 17. Concentration values were converted to logarithm and multiplied
5
by 10 so when, solving equations for "x" for any behavioral step the "y" values should be entered as the probit corresponding to a certain concentration value. The final "x" obtained is then transformed to egg equivalents/ul by finding the antilogarithm and multiplying it by
-5
10 When solving for "y" the "x" original values have to be converted










as previously described and the "y" found is expressed in probits. These values may be converted to percentage with a probit table (Fisher and Yates, 1963).

By Table 17 of the Appendix, it can be seen that the regression equation for random movement has a negative slope, indicating that for every unit of increase in concentration of egg equivalents/ul there is a decrease in 0.75 units for random movement. The equations for chemotaxis, antennal palpation, searching, and reinforcement had positive slopes; consequently there is an increase in response when the concentration increases. Correlation coefficient values were close to 1, indicating a strong linear relationship between log concentrations of the crude kairomonal extract and the behavioral responses of the female parasitoid. The equations are graphically represented in Fig. 15.


The Concept of Stimulant Concentration


Stimulant Concentration (SC) can be defined as a term to express the concentration of a chemical stimulant involved in inter and intraspecific communication among insects, required to stimulate the innate releasing mechanism of the insects tested resulting in a percentage of a characteristic behavioral response in a natural or artificial environment at a certain time interval. Concentration can be represented by conventional metric units and its sub-multiples when the situation requires, and it will depend upon the type of relationship involved, or through other usual means as parts per million in a certain volume or mass unit, or those measures already common to the chemo-entomological field of transspecific chemical messengers as: egg equivalent/ul, body equivalent/ul, abdomen equivalent/ml, etc.


































Fig. 15a and b.


Relationship of the female Trissolcus basalis (Wollaston) exhibiting certain behavior patterns when stimulated by different concentrations of the crude kairomonal extract from eggs of the host, Nezara viridula (L.), is shown: (a) random movement (possibly kinesis), (b) chemotaxis.













Y= 6.58 0.75x


Log concentration in egg eq./pl=105
(a)




Y= 3.38 + 0.76x


4

eq./lllOS


Log concentration in egg
(b)


MI
ml


E XE
.0 S

0 m
X
E~x


0
,.0-


Co
- E

0 EU



































Fig. 15c and d.


Relationship of the female Trissolcus basalis (Wollaston) exhibiting certain behavior patterns when stimulated by different concentrations of the crude kairomonal extract from eggs of the host, Nezara viridula (L.), is shown: (c) antennal palpation, (d) searching.
















Y= 3.96 + 0.69x


1 2 3 4 Log concentration in egg eq./px 105

(c)






Y = 3.50 + 0.67x


Log concentration in egg eq./pIl105

(d)


0

-.



EU
m






.0
0


IL 0 o C= o,.C




















Relationship of the female Trissolcus basalis (Wollaston) exhibiting certain behavior patterns when stimulated by different concentrations of the crude kairomonal extract from eggs of the host, Nezara viridula (L.), is shown:
(e) reinforcement.























Relationship of the female Trissolcus basalis (Wollaston) exhibiting antennal palpation when stimulated by 10-2 egg equivalent solution per microliter from eggs of the host, Nezara viridula (L.), versus time, is shown.


Fig. 15e.




























Fig. 15f.














Y= 3.38 + 0.71x


1 2 3 4 Log concentration in egg eq. /l.105
(e)


Y = 6.34 0.98x


1 2 3 Log timroe in seconds 10
(f)










Stimulant Concentration values for five behavior patterns of the female parasitoid are shown in Table 17, with 95% confidence limits.
-3
The SC for random movement (possibly kinesis) was 1.23x10 egg
50
equivalents; this represents the concentration of the stimulant that caused 50% of the female T. basalis to orient themselves to the treated spot in approximately 3 minutes. Stimulant Concentration values for the other behavior patterns can be interpreted the same way.


The Concept of Stimulant Dose


Stimulant Dose (SD) can be defined as a term used to express the amount of a chemical stimulant involved in inter and intraspecific communication among insects, required to stimulate the innate releasing mechanism of the insects tested in a natural or artificial environment, resulting in a percentage of responsiveness characteristic of the species under study at a given time. Stimulant Dose units should represent the exact amount of the stimulant utilized in any sort of bioassay, either by direct application of the chemical on the animal tested or by impregnation of a given surface or volume. Units of mass, volume, and area of multiples and sub-multiples of the metric system can be used for measurements of the doses.

Under the conditions of the actual experiment, all concentrations were delivered with a constant amount of solution, that is, 1 ul which 2 3 covered an area of (0.17 0.008) cm within a Petri dish of 28.46 cm of internal volume. It was found that 1 N. viridula egg weighs (533.96 15.75) ug a correction factor was determined to transform egg equivalents in nanogram equivalents per square millimeter per cubic centimeter. Any concentration can be expressed in terms of SD when










mnultiplying it by the factor "1066," consequently the SC values could be transformed to SD in terms of Ng equivalents/mm2/cm3

Equations of Table 17 of the Appendix, represent the Stimulant Concentration and Stimulant Dose. They are statistically sound for determinations of any SC or SD values within the range of the established lines.

Stimulant Time (ST) data are presented in Tables 18 and 19 of the Appendix. Stimulant Time was determined for antennal palpation when
-2
1 ul of 10 egg equivalent solution of the kairomonal crude extract was applied on the filter paper. According to Table 18, the class limits of time were converted to logarithm and since the logarithm of time for a given dose is normally distributed (Hastings and Peacock, 1975), the class limits were transformed to mid-point of log intervals. The quantal response technique was utilized to determine the ST because 50
the number of female T. basalis palpating the treated spot at a given time are unrelated in terms of responsiveness to the stimulus (Modification of the procedure used by Bliss, 1937).

The correlation coefficient for ST was minus 0.945, and chi-square

7.54. The percentage of antennal palpation transformed to probits is highly correlated with the mid-point of log intervals in seconds, and the regression line appropriately describes this relationship (ata=0.05). The Concept of Stimulant Time


Stimulant Time can be defined as a term to express the time required for a defined dose of a chemical stimulant involved in inter and intraspecific communication among insects required to stimulate the innate releasing mechanism of the insects tested in a natural or artificial










environment, resulting in a percentage of response characteristic of the species under study.


Experiment 8: Effects of the Crude Kairomonal Extract from Eggs of the Host, _. viridula in
the Orientation of the Female T. basalis


When introduced in the Petri dish the females T. basalis start to orient toward the treated spot. Location of the area results either from random movement or chemotaxis. During the orientation process, the parasitoid stops, grooms the body and resumes searching. The
-4
grooming behavior is intensified when the concentration reached 10 egg equivalent. As the concentration increases the time required for location of the treated area decreases. This trend can be seen in Fig. 16. When the solvent was offered to the female T. basalis, she required 27.8% of the time for the treatment totals to encounter the spot,
-4
and 19.9%, 21.0%, 16.1% and 16.0%, when the concentrations are 10 ,
-3 -2 -1
10 10 and 10 egg equivalents/ul respectively. The F-test (= 5.78) at 0.05 level disclosed that the solutions tested affected the orientation process of the parasitoid. It was found that the
-1
highest concentration, i.e., 10 egg equivalents/ul reduced the orientation time 42.58% in comparison to the control (Table 20 of the Appendix).


Experiment 9: Enhancement of Host Location by Scent Combinations


Responsiveness of the female T. basalis to the scent combinations in the olfactometer is summarized in Table 21 of the Appendix. Duncan's test (Duncan, 1955) disclosed that the best velocity toward the scent source was achieved when the crude kairomonal solution from eggs of the



































Fig. 16. Percent of time spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper spot
with different concentrations of the crude kairomonal
extract from eggs of the host, Nezara viridula (L.), is
shown.







Full Text
Fig. 1. A single glass tube olfactometer is pictured.
V


117
Table 11. Analysis of variance for the simple effects between Trissolcus
basalis (Wollaston) orienting within a single tube olfacto
meter containing 50 eggs of the host, Nezara viridula (L.) at
different levels of parasitism, averaged over 10 equally
spaced time interval.
Degrees of
Source of Variation Freedom
Sum of
Squares
Mean
Square
F
Between time within no host
egg for male T. basalis
9
5.00
0.55'
0.56
Between time within 12-hour
old N. viridula eggs for
male T. basalis
9
8.90
0.98
. 1.01
Between time within parasi
tized eggs of N. viridula
for male T. basalis
9
48.02
5.33
5.49**
Between time within egg
sheels of parasitized eggs
for male T. basalis
9
29.00
3.22
3.31**
Between time within no host
egg for female T basalis
9
15.60
1.73
1.78
Between time within 12-hour
old N. viridula eggs for
female T. basalis
9
31.12
3.45
3.55**
Between time within parasi
tized eggs of N. viridula
for female T. basalis
9
106.12
11.79
12.15**
Between time within egg
shells of parasitized eggs
for female T. basalis
9
40.22
4.46
4.59**
Error
240
232.50
0.97


active chemicals will result in comparing materials that will
certainly require different concentrations for an optimal activity.
It may also be that two or more compounds will happen to have the
same SD^ for inducing a key behavioral response. In these situations
the selection must fall on that one which has the smallest variance
(Snedcor and Cochran, 1973) An immediate application of this proce
dure of selection by SC^, SD or STso' '*'n synt^es;*-s f the
natural compounds. There is a minimal margin for error in electing
a chemical for synthesis, when such chemical had SC, SD, and ST
values determined for an important behavioral response.
As the concentration increases the time spent during appetitive
behavior decreases. This is an obvious consequence of the
decreased time required for the female T. basalis to encounter the
treated area when the solution reaches its highest concentration level
This phenomenon can be observed by examining data from Experiment 8,
where it is evident that the time of location of the area with 10 ^
egg equivalent solution, by the female T. basalis is reduced 1.74
times when compared to the control (Dichloromethane).
The crude kairomonal solution also increases the parasitoid
velocity during the orientation phase 1.89 times when compared to the
control, and 1.49 and 1.34 times when contrasted with the 12-hour-old
eggs and three female hosts, respectively. However, when the crude
kairomonal solution was combined with scent of three female hosts and
when matched with ten 12-hour-old eggs, the speed of the female T.
basalis was reduced to the control level. This may have been caused
by a conflict situation resulting from the concentration of the
scents which induced that behavior and consequently reduced the


134
Gennadiev, V. G., M. P. Suntsova, and N. G. Mel' Nikov. 1976.
Comparative evaluation of various freezing profile effects on
eggs of Graphosoma itlica L. at low temperatures. (in Russian,
English summary). Zh. Obshch. Biol. 37(4):615-619.
Gerling, D., and A. Schwartz. 1974. Host selection by Telenomus
remus, a parasite of Spodoptera litoralis eggs. Ent. Exp. Appl.
17:391-396.
Greany, P. D., and E. R. Oatman. 1972. Analysis of host discrimination
in the parasite Orgilus lepidus (Hymenoptera: Braconidae). Ann.
Ent. Soc. Am. 65:377-383.
Gross, H.R., Jr., W. J. Lewis, R. L. Jones and D. A. Nordlund. 1975.
Kairomones and their use for management of entomophagous insects.
III. Stimulation of Trichogramma achaeae, T. pretiosum, and Micro-
plitis croceips with host-seeking stimuli at time of release to
improve their efficiency. J. Chem. Ecol. 1(4):431-438.
Hastings, N. A. J., and J. B. Peacock. 1975. Statistical distributions.
Butterworths, London. 130 pp.
Hays, D. B., and S. B. Vinson, 1971. Acceptance of Heliothis virescens
(F.) (Lepidoptera: Noctuidae) as a host by the parasite Cardiochiles
nigriceps Vicreck (Hymenoptera: Braconidae) Anim. Behav. 19:344-352.
Hendry, L. B., P. D. Greany, and R. J. Gill. 1973. Kairomone mediated
host-finding behavior in the parasitic wasp, Orgilus lepidus. Ent.
Expt. Appl. 16:471-477.
Hendry, L. B., J. K. Wichmann, D. M. Hindenlang, K. M. Weaver, and S.
H. Korzeniowski. 1976. Plants: The origin of kairomone
utilized by parasitoids of phytophagous insects? J. Chem. Ecol.
2(3):271-283.
Hess, E. H. 1962. Ethology: an approach toward the complete analysis
of behavior. In New Directions in Psychology. Holt, Rinehart and
Winston, New York. 159-266.
Hinton, H. E. 1969. Respiratory systems of insect egg shells. Ann.
Rev. Entomol. 14:343-368.
Hoffman, W. E. 1935. The foodplants of Nezara viridula Linn. (Hem,
Pent). Proc. VI. Int. Congr. Entomol. Madrid. 6:811-816.
Hogan, J. A., J. p. Kruijt, and J. H. Frijlink. 1975. "Super
normality" in a learning situation. Z. Tierpsychol. 38:212-218.
Hokyo, N. and K. Kiritani. 1965. Oviposition behaviour of two
egg parasites, Asolcus mitsukurii Ashmead and Telenomus
nakagawai Watanabe (Hym., Proctotrupoidea, Scelionidae).
Wakayama Agrie. Expt. Sta. Assoc., Japan. 191-201.


Probit of female T. basalis
6.34 0.98x
Pro bit of female T. basalis
exhibiting reinforcement
oo


12
To reduce circadian and any other endogenous physiological
variability, the tests were conducted between 11:00 and 14:00 hour
with 3-day old T. basalis without any previous ovipositional experience
and host eggs 12-hour old or less.
Experiment 1: Response of the Female T. basalis
to the Eggs of the Host, N. viridula.
Responsiveness of the female parasitoid to the eggs of the host was
tested by means of a glass single tube olfactometer as shown in Fig. 1.
The dispenser consisted of a small tube measuring 7.5 cm in length and
0.35 cm in diameter. One extreme of this tube had its internal diameter
reduced to 0.20 cm and held a cotton filter. The dispenser was connected
to the main tube with a hollowed rubber stopper.
The main tube measured 30 cm in length and 0.60 cm in internal
diameter with one end linked to the dispenser and the other with its
internal diameter reduced to 0.30 cm retained a cotton plug which
functioned as a filter and prevented movement of the female parasitoids
beyond that point. This end of the tube was connected to a plastic hose,
which linked to a vacuum source and an air flow meter. Air flow was
adjusted to 3.3 cm'Vs throughout the experiment.
Chilled female Th basalis were placed on a white filter paper cut
out to a trapezoidal shape with bases measuring 0.5 and 5 cm and 4.5 cm.
The immobilized parasitoids were transferred to the main tube by removing
the rubber stopper and dispenser and placing the small base of the
trapezoidal paper inside the tube and raising it to a 20 angle and
gently tapping the paper. Then, the dispenser with the rubber stopper
was returned to the original situation.


138
Staddon, J. E. R. 1975. A note on the evolutionary significance
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Sta. Univ. MS Thesis. 100 pp.
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London. 558 pp.
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Tinbergen, N. 1951. The study of instinct. Oxford Univ. Press,
London. 228 pp.
Todd, J. W. 1973. Effects of damage by the southern green stink
bug, Nezara viridula (L.) on yield and quality of soybeans.
Clemson Univ. Ph.D. Dissertation. 56 pp.
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host recognition and host acceptance behavior of the parasite,
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North of Mexico. Univ. of Calif. Publ. Tech. Bull. 2:902 pp.
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1975. The search for the host in Trissolcus grandis and
and Telenomus chloropus, egg-parasites of Eurygaster integriceps
(Hym., Scelionidae; Het., Scutelleridae)(in Russian, English
summary). Zool. Zh. 54(6)922-927.
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Heliothis virescens (Lep.: Noctuidae) which initiates host
searching by the egg-larval parasitoid, Chelonus texanus (Hym.:
Braconidae). Ann. Entomol. Soc. Am. 68:381-384.
Vinson, S. B. 1976. Host selection by insect parasitoids. Ann.
Rev. Entomol. 21:109-132.
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1975. Isolation, identification, and synthesis, of host-seeking
stimulants for Cardiochiles nigriceps, a parasitoid of tobacco
budworm. Ent. Exp. Appl. 18:443-450.
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behavior of Bracon mellitor Say (Hymenoptera: Braconidae) a para
sitoid of boll weevil, Anthonomus grandis Boh. I. Isolation and


METHODS AND MATERIALS
Rearing of N. viridula (L.)
Southern green stink bugs were reared in wide-mouth Mason-type
jars measuring 8.0 cm and 6.5 cm bottom and top diameter, respectively,
and 17.5 cm in height. The top portion was covered with white paper
towel to confine and prevent the escape of nymphs and adults. Colonies
were established by placing 5 male and 5 female adults per jar with 3
to 5 string beans and occasionally either peanuts, carrots, and corn as
the food source. Oviposition sites were provided by a circle of paper
towel measuring 8 cm in diameter at the bottom of the jar and one strip
of the same paper measuring 3x20 cm with one end stuck to the top of
the container.
Rearing of T. basalis (Wollaston)
The Floridian strain of the parasitoid was reared in large wooden
cages with Plexi-glass top, with the front wall measuring 37.5 x 40.5
cm, back wall 37.5 x 45.5 cm and 42.5 cm in length. The front wall had
an 18 cm hole and was provided with a cotton cloth sleeve to prevent
escape and allow manual work inside the cage. Honey and water were
available to all parasitoids and they were exposed to a photophase of
14 hours day per 24 hr. cycle, a temperature of 25lC and 65^5%
relative humidity. The host, N. viridula (L.), was reared under the
same conditions of light, temperature, and humidity.
11


25
Except for the statistical analysis and utilization of both male
and female parasitoids as experimental units, the methods used were
similar to that described for Experiment 1.
The levels of the factors utilized during the test were: male and
female T. basalis, 4 different situations of host eggs, and 10 1-minute
readings recorded during a 10 minute period.
The experimental design was a 2 x 4 x 10 factorial with 4 replicates
F-test values were determined for main effects, interactions and simple
effects at 0.01 and 0.05 levels of significance.
Experiment 6: Reactions of the Female T. basalis
to the Kairomonal Solutions Prepared
with Different Solvents
The solutions were prepared by soaking 12-hour-old (or less) eggs
of N. viridula in 4 different solvents, i.e., water, dichloromethane,
ethanol, and hexane, for 1 hour. The resulting suspension was filtered
through a Whatman No. 1 qualitative filter paper. The ratio of egg
and solvent was 1 egg:l ul of solvent.
Treatments were set up by drawing a 50 ul aliquot from the
solutions and applying it to different pieces of a thin piece of cotton
measuring 0.5 x 0.6 cm and allowing enough time for solvent evaporation.
The treated pieces of cotton were transferred to spare dispenser tubes
by means of a forceps and pushed in to the narrowed end with a micro
syringe plunger. These dispensers were utilized to replace the blank
ones in the olfactometer. The experimental procedure and methods of
recording responsiveness of the female T. basalis to the stimuli were
identical to Experiment 1.
The experimental procedure was a completely randomized design with


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.
March, 1978
Dean, Graduate School


LIST OF TABLES
Table Pa9e
1.Analysis of variance for the simple effects between
the number of female Trissolcus basalis (Wollaston)
orienting within a single tube olfactometer containing
eggs of the host, Nezara viridula (L.) at 10 levels of time..107
2. Percentage of female Trissolcus basalis (Wollaston) found
within different sections of a "Y"-type olfactometer
containing 12-hour-old host eggs (B), egg shells (B'),
and no eggs of the host, Nezara viridula (L.) at different
time intervals 108
3. Analysis of variance for Trissolcus basalis (Wollaston)
orienting within a single tube olfactometer with the scent
of 0, 1, and 3 male Nezara viridula (L.) 109
4.Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer with
the scent of 0, 1, and 3 male Nezara viridula (L.) 110
5. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
with the scent of 0 and 3xl0-3 cm3 of male Nezara viridula
(L.) hemolymph Ill
6. Total number of Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer with different levels of the
male hemolymph of Nezara viridula (L.), averaged over 10
equally spaced time interval 112
7. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer to
the scent of 0 and 3xl0-3 cm3 of female Nezara viridula
(L.) hemolymph 113
8. Total number of Trissolcus basalis (Wollaston) orienting within
a single tube olfactometer with different levels of the hemo
lymph of female Nezara viridula (L.), averaged over 10 equally
spaced time interval 114
9. Analysis of variance for the simple effects between the
number of female Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer containing 50 eggs of the
host, Nezara viridula (L.) at different host age levels 115
vi 1


he has tenure, to continue his teaching and research in Entomology.
He is married to the
Ceara, Brazil. They have
former Elane Garcia de
one daughter, Cynthia,
Arruda of Fortaleza,
and one son, Elano.
141


Number of male T. b a s a I is reacting
Minutes


75
as previously described and the "y" found is expressed in probits.
These values may be converted to percentage with a probit table (Fisher
and Yates, 1963).
By Table 17 of the Appendix, it can be seen that the regression
equation for random movement has a negative slope, indicating that for
every unit of increase in concentration of egg equivalents/ul there is
a decrease in 0.75 units for random movement. The equations for chemo-
taxis, antennal palpation, searching, and reinforcement had positive
slopes; consequently there is an increase in response when the concentra
tion increases. Correlation coefficient values were close to 1,
indicating a strong linear relationship between log concentrations of
the crude kairomonal extract and the behavioral responses of the female
parasitoid. The equations are graphically represented in Fig. 15.
The Concept of Stimulant Concentration
Stimulant Concentration (SC) can be defined as a term to express
the concentration of a chemical stimulant involved in inter and
intraspecific communication among insects, required to stimulate the
innate releasing mechanism of the insects tested resulting in a percentage
of a characteristic behavioral response in a natural or artificial
environment at a certain time interval. Concentration can be represented
by conventional metric units and its sub-multiples when the situation
requires, and it will depend upon the type of relationship involved, or
through other usual means as parts per million in a certain volume or
mass unit, or those measures already common to the chemo-entomological
field of transspecific chemical messengers as: egg equivalent/ul, body
equivalent/ul, abdomen equivalent/ml, etc.


DISCUSSION
Trissolcus basalis (Wollaston) is a member of the Scelionidae
Family, Order Hymenoptera. This family includes many species that
have specialized as parasites of eggs of many insect groups, especially
Heteroptera and Orthoptera (Wilson, 1961; Davis and Krauss, 1963;
Davis, 1964; Cumber; 1964).
The mechanisms of N. viridula location by T. basalis have not been
completely determined; however, it is evident that this parasitoid
follows the basic steps in the process of parasitism as reported by
Salt (1937), Flanders (1939) and Doutt (1959), that is: host habitat
location, host location, host acceptance, and host suitability.
The aim of this investigation was to determine the factors
involved in the sequences of host location and host acceptance and the
characteristics of the crude kairomonal extract that mediate those
reactions.
A linear relationship was found between orientation of the
female parasitoid toward the scent source and the number of eggs
inside the dispenser tube. When the egcs are introduced and the scent
stimulates the female parasitoid, they start to move in a zig-zag
fashion and some parasitoids move down wind and later turn to the
source of stimulus by moving up wind. As the number of eggs increases
from ca. 20, some female |T. basalis start to "jump" when initially
stimulated by the crude kairomonal extract. This jumping act increases
91


46
(f)


Fig. 12a. Number of female Trissolcus basalis (Wollaston) reactina
to the eggs of the host, Nezara viridula (L.), after
soaking in dichloromethane for different periods of time
is shown.


18
tube


27
finally, host eggs soaked 1 time for 4 hours.
2
The data were analyzed as a 10 factorial experiment with 4
replicates, then the F-test values were determined for the statistical
parameters of relevant interest to the experiment.
Reaction of the Female T. basalis to Kairomonal Solutions Prepared by
Different Methods
Activity of the solutions was measured by the percentage of female
T. basalis orienting to the tip of the dispenser or within 10.0 cm of it
(refer to Experiment 1). The solutions were obtained by soaking host
eggs 1 time in dichloromethane for 4 hours, grinding the host eggs with
the same solvent, and by soaking host eggs 4 times at 1 hour intervals.
Suspensions obtained were filtered with a Whatman No. 1 qualitative
filter paper, and concentrations were set, such that 100 ul of the
solution would contain 50 egg equivalents.
Fifty-egg equivalents of each solution were applied to a thin piece
of absorbent cotton (0.5 x 0.6 cm). After solvent evaporation the
treated cotton was introduced into the glass dispenser with forceps and
a microsyringe plunger. Controls were dichloromethane-treated pieces of
cotton.
A completely randomized 4 x 10 factorial experiment with 4
replications was developed for testing the responses of the female T.
basalis to the different extraction procedures. Analysis of variance
was performed and F-test values obtained for the inference-making process.
Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the
Crude Kairomonal Extract from Eggs of the Host, N. viridula
The kairomonal solution was prepared from N. viridula eggs 12 hours


136
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Lewis, W. J., and R. L. Jones, 1971. Substance that stimulates host
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site of Heliothis Species. Ann. Entomol. Soc. Am. 64:471-473.
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Farm, Ottawa, Ontario, Canada. (Cited by J. W. Thomas, Jr. 1972
[q.v.].)
Mayer, M. S. 1973. Attraction studies of male Trichoplusia ni
(Lepidoptera: Noctuidae) with new combination of olfactometer
and pheromone dispenser. Ann. Ent. Soc. Am. 66:1191-1196.


2
With the growing research on the inter and intraspecific communica
tion among insects (Kennedy, 1956; Johnston, Jr. et al., 1970; Wilson,
1962, 1977), the chemical, physical, and biological releasers have been
studied more accurately in order to find out new ways to manage insect
pests with minimum detrimental effects to the environment (Metcalf and
Luckmann, 1975).
The only published study involving chemical interspecific communica
tion between N. viridula and its parasites and predators is the work of
Mitchell and Mau (1971) with the dipterous parasitoid Trichopoda pennipes.
Since experimental works have suggested that kairomones can be used
as valuable tools in management of insect pests (Lewis et al., 1971,
1975, 1976) the aim of this research was to assess the importance of the
releaser that triggers all sequences involved in the ovipositional
behavior of T. basalis. A series of bioassays were conducted to
determine: (1) threshold for host-seeking stimulation of the female
T. basalis by the eggs of the host; (2) ovipositional ethogram of the
female T. basalis with temporal analysis; (3) cues that enhance host
location; (4) discriminatory behavior of the male and female T. basalis
to parasitized and non-parasitized host eggs; (5) solvents and number of
washes required to remove the kairomone from the eggs and solution with
the best potential activity; (6) effect of host-eggs and kairomonal
solution on orientation of the parasite; (7) behavior patterns mediated
by the kairomonal solution and relationship between those patterns and
concentration; (8) Stimulant Concentration (SC ) Stimulant Dose (SDrri) ,
50 50
and Stimulant Time (ST^g) as measurements of the potency of the
kairomonal extract utilized in this work. (These are units that should
be useful in evaluations of chemicals involved in both inter and intra-


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.
Dr-f George. Allen, Chairman
Dr
^^rofessor of Entomology
I certify that 1 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.
LL
Ic
Dr. Reece I. Sailer
Graduate Research Professor of
Entomology
1 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 a. .dj-sep^t-ation for the
degree of Doctor of Philosophy.
Dr. James H. Tumlinson
Associate 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
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 qualit^w aw a dissertation for the
degree of Doctor of Philosophy.
Dr. Frncis w.1Zettler
Associate Professor of Plant
Pathology


105
be manipulated.
Experiments in progress will provide more information about how
closed the program controlling this interspecific system is;
consequently, more tangible data will be gathered and analyzed in
detail in order to obtain a kairomone with risk of habituation
reduced to a minimum.


122
Table 16. Percentage of female Trissolcus basalis (Wollaston) exhibiting
different types of behavior patterns on filter paper when sti
mulated by different concentrations of crude kairomonal extract
from eggs of the host, Nezara viridula (L.).
Dose in
Egg Equivalents
per Microliter
Percent of Behavior Patterns
Kinesis
(*)
Chemotaxis
Antennal
Palpation
Searching
Reinforcement
1J
o
1
-p
83.33
16.67
33.33
20.00
16.67
lo"3
53.33
46.67
66.67
43.33
40.00
10-2
13.33
86.67
90.00
73.33
73.33
11
1
O
r\
13.33
86.67
93.33
86.67
86.67
(*) Random movement, possibly kinesis.


71
those when the 12-hour-old eggs of N. viridula were immersed 1 time for
4 hours in the solvent, and those when the eggs were ground in dichloro-
methane. The results are summarized in Table 15 of the Appendix. From
the treatment totals it is evident that the solution with the highest
activity was prepared by grinding eggs of N. viridula However
analysis of the simple effects failed to reveal any statistically
significant difference between the methods under investigation. F values
are presented in Fig. 13.
Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the Crude
Kairomonal Extract from Eggs of the Host, N. viridula
Table 16 of the Appendix shows the percentage of each type of
behavior exhibited when different concentrations of the crude kairomonal
solution were used. It can be seen that all behavior steps become more
stereotyped when the concentration increases, except for the initial
random movement which has a reverse trend.
When introduced into the Petri dish, the female T. basalis starts
to run either on the margins of the treated filter paper or lateral
walls of the bottom of the container.
Sometimes the parasitoid stops, grooms its antennae, wings, and
abdomen then continues to move. This random movement is caused by some
sort of stimulus which has not been determined. It could be light
intensity or neurophysiological factors of the female T. basalis. This
behavior is also observed when no treatment has been applied to the
filter paper (Fig. 14). Kinesis occurs when as a result of random
movement the parasitoid encounters the treated spot and remains there
for a period of time. On the other hand, the percentage of parasitoids


90
of the crude kairomonal solution.
The t-test revealed no evidence of supernormality, that is, there
is insufficient evidence to reject the hypothesis under test (t=1.02)<,
The crude kairomonal extract is a highly specific releaser which
triggered the same behavior event on the same temporal basis of that
observed for 12-hour-old eggs subjected to oviposition by the female
T. basalis.


89
44.70** confirmed that the parasitoid responses were affected by the
treatments assigned. Contrast of the means indicated that the crude
kairomonal extract was better than the other two treatments in stimu
lating oviposition of the female parasitoids.
Antennal Palpation Previous to Oviposition
All experimental data thus far obtained have shown that exposure
to crude kairomonal extract improves performance of female T. basalis
in many steps of the ovipositional behavior. At the present time there
is no indication that the isolated is other than a chemical cue acting
as an exaggerated releaser (Magnus, 1958; Staddon, 1975). It also is
not known what kind of interaction a supernormal releaser (Tinbergen,
1951) would have in long or short-term on the process of interspecific
behavior among insects.
It was observed that the crude kairomonal extract obtained, acted
upon the innate releasing mechanism system of the female T. basalis and
induced a series of fixed action patterns characteristic of the ovi
positional behavior of this parasitoid (refer to Fig. 6) It is
evident that the species-specific behavior of the female T. basalis
is part of a behavioral pattern (Wallace, 1973; Baerends, 1976) that
remains relatively constant throughout life.
Considering all behavior steps preceding oviposition, antennal
palpation at first contact with the host egg mass is the most
stereotyped and precisely timed behavior event. This pattern was
analyzed and the results are presented in Table 25 of the Appendix.
The null hypothesis was that of no difference on time, when the female
parasitoids drum the eggs and egg shells treated with 0.1 egg equivalent


Fig. 11. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), at different
levels of parasitism and time intervals-is shown.


Fig. 5c and d. Female Trissolcus basalis (Wollaston) exhibits
ovipositional behavior on an egg mass of its host,
Nezara viridula (L.). Shown are (c) drilling the
chorium for ovipositing and (d) ovipositor thrust
and marking the egg after oviposition.


33
area computed.
Responses of Female T, basalis to Egg Shells and 12-Hour-Old Eggs of the
Host N. viridula
The consummatory phase of the ovipositional behavior was tested
with egg shells treated with the crude kairomonal solution, with the
solvent, and with 12-hour-old eggs of N. viridula.
Egg shells with the operculi were obtained 15 days after hatching
of the host insect. They were thoroughly washed in a 1:1:100 non-
phosphorous dish wash detergent, Clorox and water, then extensively
rinsed in tap water and finally washed in distilled water. The shell
remained glued through the washing process to the paper towel
ovipositional site. They were allowed to dry on filter paper under
laboratory conditions and later rinsed 3 times in dichloromethane.
After solvent evaporation, they were ready for the experimental
procedure.
The experimental unit was made up of 10 healthy 3-day-old female
T. basalis without any prior ovipositional experience. Except for the
treatments assigned to the female parasitoids, all bioassay procedures
were the same as those for Experiment 3.
The treatments were 6 12-hour-old eggs of N_. viridula, 6 egg shells
treated with 1 ul of dichloromethane, and 6 treated with 1 ul of 10 ^
egg equivalent solution of the kairomonal extract.
Response of the parasitoids to the treatments were recorded as the
percentage of female T. basalis assuming the ovipositional posture after
exploring the treated egg masses and bringing the ovipositor into
contact with the chorial surface.


44
the ovipositor through the operculum and beneath the point of junction
with the surrounding walls. In this posture the female T. basalis
stands with the sternal portion of the thorax approximately in a
straight angle with the horizontal plane of the opercular surface (Fig.
5f). Deposition of the egg is characterized by a rocking movement
similar to those observed by Wilson (1961) and Hokyo and Kiritani
(1965) in other scelionid parasitoids. All legs serve as support to
the female T. basalis, however; the meso and metathoracic pairs act
more remarkably to this function. It was observed that the prothoracic
legs and forewings are used very effectively in agressive behavior,
especially when the egg density drops to 0.5 egg per female parasitoid
or less, and more than one parasitoid is looking for ovipositional site.
The process of oviposition which starts with the preparation of the
parasitoid to drill into the egg shell, ends when the female T. basalis
withdraws the ovipositor from the chorium. This behavioral step
requires 117.97 5.86 seconds, with a fiducial limit of 95%.
After successful oviposition the female parasitoid begins the egg
marking process (Askew, 1971; Safavi, 1968). It took 19.15 1.02
seconds for T. basalis to complete this behavior. Marginal eggs are
marked when the female drags the tip of the ovipositor from the point of
insertion up to the margins of the operculum in a sinuous pattern (Fig.
5d) Central eggs are marked in the same manner, starting from the point
of drilling and covering the opercular margins and going down ca. 1/3
of the upper portion of the surrounding egg wall.
An ethogram of the ovipositional behavior of T. basalis is shown
in Fig. 6. It could be seen that the orientation to the egg mass is
accomplished by either random movement or chemotaxis or combination of


137
Mayr, E. 1974. Behavior programs and evolutionary strategies.
Amer. Sci. 62:650-659.
Meier, N. F. 1970. Parasites cultured in USSR in 1938-1939 on
the eggs of corn-bug (Eurygaster integricipes, Osch.)(in Russian,
English translation). Vestnik Zashchity Rastenii. 3:79-82.
Metcalf, R. L., and W. Luckmann. 1975. Introduction to insect pest
management. J. Wiley and Sons, New York. 587 pp.
Mitchell, W. C., and R. F. L. Mau. 1971. Response of the female
southern green stink bug and its parasite, Trichopoda pennipes,
to male stink bug pheromones. J. Econ. Entomol. 64:856-859.
Nettles, W. C., Jr., and M. L. Burks. 1975. A substance from
Heliothis virescens (Lep.: Noctuidae) larvae stimulating
larviposition by females of the tachinid, Archytal marmoratus
(Dipt.). J. Insect Physiol. 21:965-978.
Nordlund, D. A., and W. J. Lewis. 1976. Terminology of chemical
releasing stimuli in intraspecific and interspecific inter
actions. J. Chem. Ecol. 2(2) :211-220.
Nordlund, D. A., W. J. Lewis, H. R. Gross, Jr., and E. A. Harrel.
1974. Description and evaluation of a method for field
application of Heliothis zea (Lep., Noctuidae) eggs and
kairomones for Trichogramma (Hym., Trichogrammatidae).
Environ. Entomol. 3:981-984.
Priesner, H. 1931. Notes on hymenopterous egg parasites of Nezara
viridula (L.). Bull. Entomol. Egypt. 15:137-139.
Safavi, M. 1968. Etude biologique et e'cologique des hymenopteres
parasites des oeufs des punaises des cereales. Entomophaga
13:381-495.
Sailer, R. I. 1976. Department of Entomology and Nematology, Univ.
of Fla., personal communication.
Salt, G. 1937. Experimental studies in insect parasitism. V. The
sense used by Trichogramma to distinguish between parasitized
and unparasitized host. Proc. Roy. Soc. London, Ser. B 122:57-76.
Shorey, H. H. 1973. Behavioral responses to insect pheromones.
Ann. Rev. Entomol. 18:349-379.
Shorey, H. H., and L. K. Gaston. 1964. Sex pheromone of noctuid
moths. III. Inhibition of male responses to the sex pheromone in
Trichoplusia ni (Lepidoptera: Noctuidae). Ann. Entomol. Soc.
Am. 57:775-779.
Snedecor, G. W., and W. G. Cochran. 1973. Statistical methods. The
Iowa State Univ. Press. 593 pp.


Fig. 16. Percent of time spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper spot
with different concentrations of the crude kairomonal
extract from eggs of the host, Nezara viridula (L.), is
shown.


31
kairomonal solution with 12.5 egg equivalents was placed in the
dispenser, respectively. Reactions of the female T. basalis to the
scent combinations was determined when one of the 5 parasitoids made
the first physical contact with the egg mass or the tip of the dispenser.
The velocity of the parasitoid toward the scent source was
determined for the first female T. basalis scored in each of the
situations. For evaluation purposes, it was assumed that the movement
toward the source of stimuli was preformed in a linear and uniform
fashion.
The experiment was set up as a completely randomized design with 7
treatments and 5 replications. Contrast of the treatments and means
were performed by the appropriate statistical procedures already
mentioned.
Experiment 10: Normality Studies with the Crude
Kairomonal Solution from Eggs of the Host,
N. viridula
Reactions to exaggerated releasers by higher animals and insects
have been reported in the specialized literature (Tinbergen, 1951;
Marler and Hamilton, 1967; Wallace, 1973; Hogan et al., 1974; Magnus,
1958; and Staddon, 1975).
To observe if female T. basalis were reacting to a normal stimulus
a series of tests were performed for certain behavior patterns.
Response to the Kairomonal Solution on the Filter Paper
Bioassays were conducted in Petri dishes described in Experiment 3.
The experimental unit was 10 healthy 3-day-old female T. basalis without
any ovipositional experience. The female parasitoids were exposed to


101
kairomonal extract by drumming the antennal flagella against the
area treated with 1 ul of 10 ^ egg equivalent solution.
Parasitism in areas treated with the kairomonal solution was
69.60% higher than in areas where no kairomonal extract was available.
So, there is evidence that the performance of the female T. basalis
is considerably improved by the presence of the extract obtained.
There is also indication that the crude kairomonal extract act
ed as an oviposition stimulant in the presence of a physical clue
(host egg-shells) Host egg-shells treated with a 10 ^ egg equivalent
induced the female parasitoid to bring the tip of the ovipositor in
contact with the egg-shell and assume the characteristic ovipositional
posture, and exhibited the rocking movement.
Unquestionably the crude kairomonal extract improved the effec
tiveness of the female T. basalis to an overoptimal level s already
discussed. The quality of the isolated releaser was assessed by
comparing female response to the normal rigidly fixed stereotyped
behavior pattern that occurs prior to a successful oviposition, i.e.,
antennal palpation. Statistically there was no difference between the
time spent in palpating intact 12-hour-old host eggs or host egg
shells treated with the kairomonal solution.


94
potency and normality of the releaser(s) involved in interspecific
interactions. It also provides valuable information that could be
helpful in behavior manipulation when improvement of the parasitoid
performance is under consideration.
Responsiveness of the female T. basalis to male and female N.
viridula scent was 1.00 and 1.13 times better than that exhibited by
the male parasitoid, respectively. Combined parasitoid reactions to
male N. viridula were not different at 0.05. However, reactions of
the male and female T. basalis to the female host was highly signif
icant. The data support the rationale that the female southern green
stink bug provides more clues for its location by both male and female
parasitoids. It is possible that volatization of one or more specific
chemical(s) from the cement and wax layers of the epicuticle (Locke,
1974) will enhance host location in the natural environment.
When host male and female hemolymph was offered to both male and
female T. basalis, the males reacted poorly to the scent; however, the
females were highly stimulated and oriented to the tip of the dispenser
tube. Their reaction to the male and female southern green stink bug
hemolymph was 1.23 and 1.33 times higher than that shown by the male
parasitoids, respectively. The data corroborate the evidence that the
female T^. basalis has possibly a better chemosensory mechanism for
host location.
It has not been determined how certain specific chemicals present
in the N. viridula hemolymph, i.e., volatile fatty acids, glycerol,
hydrocarbons, amino acids, proteins, etc. (Florkin and Jeuniaux, 1974)
could reach the external environment and trigger a sequence of
behavior steps that might lead the parasitoid to the host. Actually,


135
Issa, E. 1973. Soja-problemas fitopatologico na safra 1972/73.
0 Biolgico. 39(7):174-177.
Jones, R. L., W. J. Lewis, M. Beroza, B. A. Bierl, and A. N. Sparks.
1973. Host-seeking stimulants (kairomones) for the egg parasite,
Trichogramma evanescens (Hymenoptera: Trichogrammatidae).
2:593-596.
Jones, T. H. 1918. The southern green plant bug. N. S. Dept. Agr.
Bull. 689. 27 pp.
Johnston, J. W., Jr., D. G. Moulton, and A. Turk. 1970. Chemical
communication. Appleton-Century-Crofts, New York. 412 pp.
Kamal, M. 1937. The cotton green bug, Nezara viridula, L. and its
important egg-parasite, Microphanurus megacephalus (Ashmead)
(Hymenoptera: Proctotrupidae). Bull. Soc. Roy. Entomol. Egypte.
21:176-207.
Kariya, H. 1961. Effect of temperature on the development and
mortality of the southern green stink bug, Nezara viridula
and the oriental green stink bug, N. antennata. Jap. J.
Appl. Entomol. Zool. 5:191-196.
Kennedy, J. S. 1956. The experimental analysis of aphid behaviour and
its bearing on current theories of instinct. Proc. 10th Int.
Congr. Entomol. 2:397-404.
Kiritani, K. 1963. The change in reproductive system of the southern
green stink bug, Nezara viridula, and its application to fore
casting of the seasonal history. Jap. J. Appl. Entomol. Zool.
7:327-337.
Kiritani, K. 1965. The natural regulation of the population of the
southern green stink bug, Nezara viridula (L.). Proc. XII Int.
Congr. Entomol., London. 12:375.
Kiritani, K., and N. Hokyo. 1965. Variation of egg mass size in
relation to the oviposition pattern in Pentatomidae. Kontyu,
33:427-432.
Kiritani, K., N. Hokyo, and K. Kimura. 1966. Factors affecting the
winter mortality in the southern green stink bug, Nezara viridula
(L.). Ann. Soc. Entomol. Fr. 2:199-207.
Kiritani, K., N. Hokyo, K. Kimura, and F. Nakasuji. 1965. Imaginal
dispersal of the southern green stink bug, Nezara viridula (L.),
in relation to feeding and oviposition. Jap. J. Appl. Entomol.
Zool. 9:291-297.
Kiritani, K., and S. Iwao. 1966. Population behavior of the southern
green stink bug, Nezara viridula with special reference to the
developmental stages of early planted paddy. Res. Pop. Ecol.
8:133-146.


V
Page
Antennal Palpation Previous to Oviposition 34
RESULTS 35
Experiment 1: Response of the Female [T. basalis to the
Eggs of the Host, N. viridula 35
Experiment 2: Orientation of the Female T. basalis Inside
a "Y" Type Olfactometer 35
Experiment 3: Temporal Analysis of the Ovipositional
Behavior of the Female T. basalis 38
Experiment 4: Cues Useful in Location of the Host, N.
viridula by the Parasitoid T. basalis 49
Scent of Male N. viridula 49
Scent of Female N. viridula 49
Scent of Male N. viridula Hemolymph 52
Scent of Female KL viridula Hemolymph 53
N. viridula Eggs of Different Ages 53
Experiment 5: Reactions of the Male and Female T. basalis
to the Eggs of the Host, N. viridula at Different
Degrees of Parasitism 58
Experiment 6: Reactions of the Female TL basalis to the
Kairomonal Solutions Prepared with Different Solvents 63
Number of Dichloromethane Washes Required for Removal
of the Kairomonal Extract from Eggs of N. viridula...... 64
Reaction of the Female T. basalis to Kairomonal
Solutions Prepared by Different Methods 64
Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the Crude
Kairomonal Extract from Eggs of the Host, N. viridula 71
The Concept of Stimulant Concentration 75
The Concept of Stimulant Dose 82
The Concept of Stimulant Time 83
Experiment 8: Effects of the Crude Kairomonal Extract from
Eggs of the Host, N. viridula in the Orientation of the
Female T. basalis 84


Xll
Figure Page
from eggs of the host, Nezara viridula (L.), is shown:
(e) reinforcement 81
15f. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation when stimulated by 102 egg
equivalent solution per microliter from eggs of the host,
Nezara viridula (L.), versus time, is shown 81
16. Percent of time spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper spot with
different concentrations of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.), is shown 86


24
Evaluation of the reactions were similar to those described for
Experiment 1, and the readings were taken during each 1-minute interval
for a 10-minute period.
2
The experimental procedure was arranged in a 2 x 10 factorial
design for each hemolymph type with 4 replicates. Analysis of variance
was carried out and the interactions of importance were investigated
through the simple effects.
H. viridula Eggs of Different Ages
Response of the female T^. basalis to N. viridula eggs of different
ages was measured by means of a single tube olfactometer as described
in Experiment 1.
Treatments were prepared by placing 0, 50 12-hour-, 50 24-hour-, or
50 48-hour-old eggs of the host in the dispenser tube and proceeding as
previously described. All treatments were replicated 4 times and
readings made at 1-minute intervals for a 10-minute period.
A two-way analysis of variance was computed for the 4 x 10 factorial
design, and simple effects were investigated for the interaction between
egg age and time required for female parasitoid reaction.
Experiment 5: Reactions of the Male and Female
T. basalis to the Eggs of the Host,
N. viridula at Different Degrees of Parasitism
Treatments were set up to test parasitoid response to non-
parasitized and parasitized host eggs as well as eggs from which
parasitoids had already emerged, i.e., 0 N. viridula eggs, 50 12-hour,
50 parasitized eggs from which adult T?. basalis were ready to emerge,
and 50 host egg shells 4 days after emergence of the parasitoids.


129
Table 23. Percentage of eggs of Nezara viridula (L.) parasitized by
Trissolcus basalis (Wollaston) w^en placed on filter paper
treated with 5x10^ cin of a 10 egg equivalent solution
in dichloromethane.
No. of Eggs
per Replicate
5x10 2cm^ of a 10 2 Egg
Equivalent Solution
5x10 2cm^ of
Dichloromethane
No. of Eggs
Parasitized
% Parasitism
No. of Eggs
Parasitized
% Parasitism
10
1.00
10.00
6.00
60.00
10
8.00
80.00
5.00
50.00
10
2.00
20.00
9.00
90.00
10
2.00
20.00
10.00
100.00
10
10.00
100.00
6.00
60.00
10
0.00
0.00
3.00
30.00
E
23.00
230.00
39.00
390.00
X
3.83
38.33
6.50
65.00
Ratio = 1.69


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132


Number of T. basalis reacting
Number of T.basalis reacting
Ln
ui


14
Plastic tube
<
30cm


Fig. 7. Number of male and female Trissolcus basalis (Wollaston)
reacting to (a) scent of 0, 1, and 3 male Nezara viridula (L.)
and (b) 0, 1, and 3 female N. viridula, averaged over
10 equally spaced time intervals is shown.


125
Table 19. Parameters for the Stimulant Time for the female parasitoid
Trissolcus basalis (Wollaston) exhibiting antennal palpation,
when stimulated by 10 egg equivalent/pl of the crude kairo-
monal extract from eggs of the host, Nezara viridula (L.).
Behavior
Parameters
Pattern
Correlation
95% Confidence
Equation
Coefficient
Limits for ST- *
dU
Antennal
Palpation
y=6.34-0.98x
-0.94
(2.34+0.19)
^Values expressed in seconds.


88
Response to the Kairomonal Solution on the Filter Paper
The mean number of female T basalis exhibiting antennal palpation
is response to the crude kairomonal extract from eggs of N. viridula
is shown in Table 22 of the Appendix. Ninty-two percent of the popula
tion tested responded to the stimulus and no reaction was observed for
1/2
the solvent treatment. The results were transformed to Arcsin x.
i
(Bartlett, 1947) and the paired-difference t-test revealed the signifi
cant difference between the treatments.
Evaluation of Parasitism in Areas Treated with the Crude Kairomonal
Solution
The activity of the parasitoid was measured by the number of
host eggs (%) parasitized, when the filter paper was treated with
dichloromethane and with the crude kairomonal solution. The results
are presented in Table 23 of the Appendix. The t-test revealed a highly
significant difference between the treatments. The performance of the
female T^. basalis in terms of parasitism was improved by 1.69 times by
the addition of the egg extract to the egg masses.
Responses of the Female T. basalis to the Egg Shells and 12-Hour-Old Eggs
of the Host, N. viridula
Assessment of the crude kairomonal extract in triggering oviposition
of the female parasitoids were compared to the natural situation, when
egg masses of the southern green stink bug, Nc viridula were offered
to the female T. basalis under test. Responsiveness of the parasitoids
are shown, in Table 24 of the Appendix. Oviposition induced by the crude
kairomonal solution was 35.48% better than that observed for 6 12-hour-
old host eggs. Analysis of variance was conducted and the F-test =


Fig. 3. The glass chamber is shown connected to a single glass
tube olfactometer.


48
l
I
l
SATIATION I
l


VIH
T?.ble Paqe
10. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orientina within a single tube olfactometer to
eggs of the host, Nezara viridula (L.) at different levels
of parasitism 116
11. Analysis of variance for the simple effects between Trissolcus
basalis (Wollaston) orienting within a single tube olfactometer
containing 50 eggs of the host, Nezara viridula (L.) at
different levels of parasitism, averaged over 10 equally
spaced time interval 117
12. Treatment totals for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing 50 egg equivalents of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.) removed
by different solvents 118
13. Treatment means for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing crude kairomonal extract from eggs of the host,
Nezara viridula (L.), removed by 4 solvents, after 4 minutes
of exposure 119
14. Analysis of variance for the number of female Trissolcus
basalis (Wollaston) orienting within a single tube olfactometer
containing crude kairomonal extract from fifty 12-hour-old
eggs of the host, Nezara viridula (L.), extracted with
dichloromethane by different methods 120
15. Treatment totals for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing 50 egg equivalent solutions obtained from 12-hour-
old eggs of the host, Nezara viridula (L.) with dichloro
methane by different methods 121
16. Percentage of female Trissolcus basalis (Wollaston) exhibiting
different types of behavior patterns on filter paper when
stimulated by different concentrations of crude kairomonal
extract from eggs of the host, Nezara viridula (L.) 122
17. Parameters for the behavior patterns of the female parasitoid,
Trissolcus basalis (Wollaston) when stimulated by four equally
spaced concentrations, ranging from 10^ to lO-^ egg
equivalents/ul of crude kairomonal extract from eggs of the
host Nezara viridula (L.) 123
Percentage of the female parasitoid Trissolcus basalis
(Wollaston) exhibiting antennal palpation on a treated filter
paper spot, when stimulated by 10~2 egg equivalents/ul of
the crude kairomonal extract of the eggs of the host, Nezara
viridula (L.) at different time intervals 124
18.


StT
M&Kft
mmmzrz
:..' £ ?F-tT*$3i$S**r' i
;a;t^i¡iiilifti

Fig. 5a and b. Female Trissolcus basalis (Wollaston) exhibits
pre-ovipositional behavior in an egg mass of its
host, Nezara viridula (L.). Antennal palpation of
(a) marginal egg and (b) central egg is shown.


107
Table 1. Analysis of variance for the simple effects between the number
of female Trissolcus basalis (Wollaston) orienting within a
single tube olfactometer containing eggs of the host, Nezara
viridula (L.) at 10 levels of time.
Source
of Variation
Degrees of
Freedom
Sum of
Squares
Mean
Square
F
Between
time within
0
egg
of N.
viridula
9
10.53
1.17
0.79
Between
time within
1
egg
of N.
viridula
9
9.73
1.08
0.73
Between
time within
2
eggs
of N.
viridula
9
5.10
0.56
0.38
Between
time within
3
eggs
of N.
viridula
9
8.90
0.98
0.66
Between
time within
4
eggs
of N.
viridula
9
12.03
1.33
0.90
Between
time within
5
eggs
of N.
viridula
9
34.72
3.86
2.62**
Error
180
265.50
1.47
'Highly significant at 0.05 level.
t


Fig. 15e. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.), is shown:
(e) reinforcement.
Fig. 15f. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation when stimulated by 10^ egg
equivalent solution per microliter from eggs of the host,
Nezara viridula (L.), versus time, is shown.


of female T. basalis reacting
68
28
24
. F=0.55
0 host egg
F=e2.59**
host
eggs
soaked
2
times
fo r
1
F_ 2.0 3 *__ _
host
eggs
soaked
3
times
fo r
1
_ F- 2.48**
host
eggs
s o a k ed
4
times
for
1
F- 0.64
host
eggs
soaked
1
time
f o r
4
1
JL
3
Minutes
(b)
hour
hour
hour
hours
1
2
4
5


130
Table 24. Percentage of female Trissolcus basalis (Wollaston) exhibiting
ovipositional behavior when stimulated by 6 12-hour-old eggs
and egg shells of the host, Nezara viridula (L.).
Replicates
-3 3
10 cm of Dichloro-
Methane on Egg Shells
of the Host
6 12-Hour-Old
Eggs of the
Host
10 cm of a 0.1
Egg Equivalent
Solution on Egg
Shells of the
Host
1
0.0
40.0
90.0
2
10.0
60.0
70.0
3
30.0
70.0
80.0
4
10.0
70.0
80.0
5
20.0
70.0
100.0
l
70.0
310.0
420.0
X
14.0a
62.0b
84.0C
3. ]d C
5 Means were significantly different at 0.05 level (Duncan, 1955).


133
Smaragdula (Fabricius) in Hawaii (Heteroptera: Pentatomidae)
Proc. Haw. Ent. Soc. 18(3):369-375.
Davis, C. J., and L. H. Krauss. 1963. Recent introductions for
biological control in Hawaii. VIII. Proc. Haw. Ent. Soc.
18(2):245-249.
De Bach, P. 1964. Biological control of insect pests and weeds.
Reinhold, New York. 844 pp.
DeWitt, N. B., and G. L. Godfrey. 1972. The literature of arthropods
associated with soybeans. II. A bibliography of the southern
green stink bug, Nezara viridula (Linnaeus)(Hemiptera: Pentatomidae)
Illinois Nat. Hist. Surv. Biol. Notes No. 78:1-23.
Doutt, R. L. 1959. The biology of parasitic Hymenoptera. Ann. Rev.
Entomol. 4:161-182.
Drake, C. J. 1920. The southern green stink bug in Florida. The
Quart. Bull., State Plant Board of Fla., 4(3):1-95.
Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics.
11:1-42.
Ebeling, W. 1974. Permeability of insect cuticle. Pages 271-243 in
M. Rockstein, ed. The physiology of Insecta, vol. 6. Academic
Press, New York.
Finney, D. J. 1971. Probit analysis. Cambridge Univ. Press, New
York. 333 pp.
Fisher, R. A., and F. Yates. 1963. Statistical tables for biological,
agricultural and medical research. Hafner Publ. Co., New York.
146 pp.
Flanders, S. E. 1939. Environmental control of sex in hymenopterous
insects. Ann. Entomol. Soc. Am. 32:11-26.
Florkin, M., and C. Jeuniaux. 1974. Hemolymph: composition. Pages
255-307 iii M. Rockstein, ed. The physiology of Insecta, vol. 5.
Academic Press, New York.
Fraenkel, G. S., and D. L. Gunn. 1961. The orientation of animals,
taxes and compass reactions. Dover Publications, Inc. New York.
376 pp.
Freeman, P. 1940. A contribution to the study of the genus Nezara Amyot
and Serville (Hemiptera, Pentatomidae). Trans. R. Ent. Soc. Lond.
90(12):351-374.
Gallo, D., O. Nakano, F. M. Wiendl, s. S. Neto, R. P. L. Carvalho. 1970.
Manual de entomologa, pragas das plantas e seu contrle. Editora
Agronmica Ceres, S. Paulo. 858 pp.


103
in assessing the potential characteristics cf chemical(s) involved
in interspecific communication.
Evaluation of cues that could act as "helpers" in location of
the host eggs revealed that presence of both male and female N.
viridula enhance host finding, and the latter elicited stronger
reactions than the former. Hemolymph of the southern green stink bug
was also tested, and the results showed that the males T. basalis
were not stimulated by the scent; however, the female parasitoids
were highly oriented to the source of stimulus. It was not determined
if or how chemicals from the hemolymph could be available to the
parasitoids in the external environment and act as a clue to T.
basalis.
Investigations with 4-day-old egg-shells from parasitized host
eggs and parasitized host eggs containing adult parasitoids ready
for emergence indicated that they were highly attractive to both
male and female T. basalis. It is possible that pheromone present in
the female parasitoid or in the metabolic excrements act as a mediator
to the mating behavior of this species.
The kairomonal extract from the eggs of N. viridula was obtained
with dichloromethane, which proved to be the most effective of three
methods of extraction in removing the active compound(s) with the
highest level of activity without interfering with the normal behavior
of the parasitoid.
Visual clues were not found to be as critical as chemical clue
in location of host eggs. The crude kairomonal extract induced five
behavioral patterns, i.e., random movement (possibly kinesis), chemo-
taxis, antennal palpation, searching and reinforcement, which are


THE BEHAVIOR OF THE EGG PARASITOID TRISSOLCUS BASALIS
(WOLLASTON) (HYMENOPTERA: SCELIONIDAE) IN RESPONSE
TO KAIROMONES PRODUCED BY ITS HOST, THE SOUTHERN GREEN
STINK BUG NEZARA VIRIDULA (L.) (HEMIPTERA: PENTATOMIDAE)
By
FERNANDO JOO MONTENEGRO DE SALES
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
1978

ACKNOWLEDGMENTS
The author is grateful to Dr. G.E. Allen for his advice,
encouragement and guidance during the experimental work and preparation
of this dissertation. Appreciation is extended to the staff members
of his laboratory for help and understanding.
Recognition and gratitude is given to Dr. R.I. Sailer, Dr. J.H.
Tumlinson, Dr. J.R. McLaughlin and Dr. F.W. Zettler for serving on my
graduate committee. Special thanks are extended to Dr. D.L. Chambers
for allowing me to use the facilities of the USDA-ARS, Insect Attractants,
Behavior and Basic Biology Research Laboratory.
Special thanks are extended to the chairman, Dr. Fowden Maxwell,
as well as the staff and graduate students of the Department of
Entomology and Nematology for their unselfish service, instruction,
and encouragement.
Gratitude is also expressed to the Brazilian colleagues at the
University of Florida for their support and encouraging words.
I am also indebted to Mrs. Maria I. Cruz, Campus Coordinator of
the AID program for her cooperation, assistance and concern.
The author was supported by funds from the United States Agency
for International Development (USAID), the Federal University of
Cear, Brazil, to whom sincere appreciation is expressed.
n

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES vii
LIST OF ILLUSTRATIONS X
ABSTRACT xiii
INTRODUCTION 1
LITERATURE REVIEW 4
Current Status of the Host Southern Green Stink Bug,
Nezara viridula (L.) and Its Parasitoid Trissolcus basalis
(Wollaston) 4
The Southern Green Stink Bug, Nezara viridula (L.) 4
Origin 4
Distribution 4
Host plants 5
Life history 6
Trissolcus basalis (Wollaston) 7
Origin 7
Distribution and host insects 8
Life history 8
Interspecific Communication 9
METHODS AND MATERIALS 11
Rearing of N. viridula (L.) 11
Rearing of T. basalis (Wollaston) 11
Experiment 1: Response of the Female T. basalis to the
Eggs of the Host, N. viridula 12
iii

IV
Page
Experiment 2: Orientation of the Female T. basalis
Inside a "Y" Type Olfactometer 16
Experiment 3: Temporal Analysis of the Ovipositional
Behavior of the Female Th basalis 19
Experiment 4: Cues Useful in Location of the Host, N.
viridula, by the Parasitoid T. basalis 20
Scent of Male N. viridula 20
Scent of Female N. viridula 23
Male and Female N. viridula Hemolymph 23
N. viridula Eggs of Different Ages 24
Experiment 5: Reactions of the Male and Female T. basalis
to the Eggs of the Host, N. viridula at Different
Degrees of Parasitism 24
Experiment 6: Reactions of the Female T. basalis to the
Kairomonal Solutions Prepared with Different Solvents 25
Dichloromethane Washes Required for Removal of the
Kairomone from Eggs of N. viridula 26
Reaction of the Female T. basalis to Kairomonal
Solutions Prepared by Different Methods 27
Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the Crude
' Kairomonal Extract from Eggs of the Host, N. viridula 27
Experiment 8: Effects of the Crude Kairomonal Extract from
Eggs of the Host, N. viridula in the Orientation of the
Female T. basalis 29
Experiment 9: Enhancement of Host Location by Scent
Combinations 30
Experiment 10: Normality Studies with the Crude Kairomonal
Solution from Eggs of the Host, N. viridula 31
Response to the Kairomonal Solution on the Filter Paper. 31
Evaluation of Parasitism in Areas Treated with the
Crude Kairomonal Solution 32
Responses of Female T. basalis to Egg Shells and 12-
Hour-Old Eggs of the Host N. viridula 33

V
Page
Antennal Palpation Previous to Oviposition 34
RESULTS 35
Experiment 1: Response of the Female [T. basalis to the
Eggs of the Host, N. viridula 35
Experiment 2: Orientation of the Female T. basalis Inside
a "Y" Type Olfactometer 35
Experiment 3: Temporal Analysis of the Ovipositional
Behavior of the Female T. basalis 38
Experiment 4: Cues Useful in Location of the Host, N.
viridula by the Parasitoid T. basalis 49
Scent of Male N. viridula 49
Scent of Female N. viridula 49
Scent of Male N. viridula Hemolymph 52
Scent of Female KL viridula Hemolymph 53
N. viridula Eggs of Different Ages 53
Experiment 5: Reactions of the Male and Female T. basalis
to the Eggs of the Host, N. viridula at Different
Degrees of Parasitism 58
Experiment 6: Reactions of the Female TL basalis to the
Kairomonal Solutions Prepared with Different Solvents 63
Number of Dichloromethane Washes Required for Removal
of the Kairomonal Extract from Eggs of N. viridula...... 64
Reaction of the Female T. basalis to Kairomonal
Solutions Prepared by Different Methods 64
Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the Crude
Kairomonal Extract from Eggs of the Host, N. viridula 71
The Concept of Stimulant Concentration 75
The Concept of Stimulant Dose 82
The Concept of Stimulant Time 83
Experiment 8: Effects of the Crude Kairomonal Extract from
Eggs of the Host, N. viridula in the Orientation of the
Female T. basalis 84

VI
Page
Experiment 9: Enhancement of Host Location by Scent
Combinations 84
Experiment 10: Normality Studies with the Crude Kairomonal
Solution from Eggs of the Host, N. viridula 87
Response to the Kairomonal Solution on the Filter Paper. 88
Evaluation of Parasitism in Areas Treated with the Crude
Kairomonal Solution 88
Responses of the Female T. basalis to the Egg Shells
and 12-Hour-Old Eggs of the Host, N. viridula 88
Antennal Palpation Previous to Oviposition 89
DISCUSSION 91
SUMMARY AND CONCLUSIONS 102
APPENDIX: SUMMARY OF THE EXPERIMENTAL DATA 106
REFERENCES CITED 132
BIOGRAPHICAL SKETCH 140

LIST OF TABLES
Table Pa9e
1.Analysis of variance for the simple effects between
the number of female Trissolcus basalis (Wollaston)
orienting within a single tube olfactometer containing
eggs of the host, Nezara viridula (L.) at 10 levels of time..107
2. Percentage of female Trissolcus basalis (Wollaston) found
within different sections of a "Y"-type olfactometer
containing 12-hour-old host eggs (B), egg shells (B'),
and no eggs of the host, Nezara viridula (L.) at different
time intervals 108
3. Analysis of variance for Trissolcus basalis (Wollaston)
orienting within a single tube olfactometer with the scent
of 0, 1, and 3 male Nezara viridula (L.) 109
4.Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer with
the scent of 0, 1, and 3 male Nezara viridula (L.) 110
5. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
with the scent of 0 and 3xl0-3 cm3 of male Nezara viridula
(L.) hemolymph Ill
6. Total number of Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer with different levels of the
male hemolymph of Nezara viridula (L.), averaged over 10
equally spaced time interval 112
7. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer to
the scent of 0 and 3xl0-3 cm3 of female Nezara viridula
(L.) hemolymph 113
8. Total number of Trissolcus basalis (Wollaston) orienting within
a single tube olfactometer with different levels of the hemo
lymph of female Nezara viridula (L.), averaged over 10 equally
spaced time interval 114
9. Analysis of variance for the simple effects between the
number of female Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer containing 50 eggs of the
host, Nezara viridula (L.) at different host age levels 115
vi 1

VIH
T?.ble Paqe
10. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orientina within a single tube olfactometer to
eggs of the host, Nezara viridula (L.) at different levels
of parasitism 116
11. Analysis of variance for the simple effects between Trissolcus
basalis (Wollaston) orienting within a single tube olfactometer
containing 50 eggs of the host, Nezara viridula (L.) at
different levels of parasitism, averaged over 10 equally
spaced time interval 117
12. Treatment totals for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing 50 egg equivalents of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.) removed
by different solvents 118
13. Treatment means for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing crude kairomonal extract from eggs of the host,
Nezara viridula (L.), removed by 4 solvents, after 4 minutes
of exposure 119
14. Analysis of variance for the number of female Trissolcus
basalis (Wollaston) orienting within a single tube olfactometer
containing crude kairomonal extract from fifty 12-hour-old
eggs of the host, Nezara viridula (L.), extracted with
dichloromethane by different methods 120
15. Treatment totals for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
containing 50 egg equivalent solutions obtained from 12-hour-
old eggs of the host, Nezara viridula (L.) with dichloro
methane by different methods 121
16. Percentage of female Trissolcus basalis (Wollaston) exhibiting
different types of behavior patterns on filter paper when
stimulated by different concentrations of crude kairomonal
extract from eggs of the host, Nezara viridula (L.) 122
17. Parameters for the behavior patterns of the female parasitoid,
Trissolcus basalis (Wollaston) when stimulated by four equally
spaced concentrations, ranging from 10^ to lO-^ egg
equivalents/ul of crude kairomonal extract from eggs of the
host Nezara viridula (L.) 123
Percentage of the female parasitoid Trissolcus basalis
(Wollaston) exhibiting antennal palpation on a treated filter
paper spot, when stimulated by 10~2 egg equivalents/ul of
the crude kairomonal extract of the eggs of the host, Nezara
viridula (L.) at different time intervals 124
18.

IX
Table
Page
19.Parameters for the stimulant timetfor the: female parsitoid
Trissolcus basalis (Wollaston) exhibiting antennal palpation,
when stimulated by 10^ egg equivalent/ul of the crude
kairomonal extract from the eggs of the host, Nezara
viridula (L.)
20. Average time in seconds spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper area with
different concentrations of the crude kairomonal extract from
eggs of the host, Nezara viridula (L.) y
21. Average velocity in cm/s of the female Trissolcus basalis
(Wollaston) in orienting within a single tube olfactometer
containing scent combinations from the host, Nezara
viridula (L.) -i
22. Percentage of female Trissolcus basalis (Wollaston) exhibiting
antennal palpation on filter paper treated with 10 3 cm3 of
solutions containing different concentrations of crude
kairomonal extract from eggs of the host Nezara viridula (L.)^28
23. Percentage of eggs of Nezara viridula (L.) parasitized by
Trissolcus basalis (Wollaston) when placed on filter paper
treated with 5xl0~^ cm3 of a 102 egg equivalent solution in
dichloromethane ^29
24. Percentage of female Trissolcus basalis (Wollaston) exhibiting
ovipositional behavior when stimulated by 6 12-hour-old eggs
and egg shells of the host, Nezara viridula (L.) 130
25.Time required by the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation previous to a successful ovi-
position in the eggs and egg shells of the host Nezara
viridula (L.)
.131

LIST OF ILLUSTRATIONS
Figure Page
1. A single glass tube olfactometer is pictured 14
2. A "Y" type olfactometer is pictured 18
3. The glass chamber is shown connected to a single glass
tube olfactometer 22
4.Relationship between percentage of female Trissolcus basalis
(Wollaston) responding within a single tube olfactometer
versus different levels of eggs of the host southern green
stink bug, Nezara viridula (L.) is shown 37
5a and b. Female Trissolcus basalis (Wollaston) exhibits pre-
ovipositional behavior in an egg mass of its host, Nezara
viridula (L.). Antennal palpation of (a) marginal egg and
(b) central egg is shown 41
5c and d. Female Trissolcus basalis (Wollaston) exhibits ovi-
positional behavior on an egg mass of its host, Nezara viridula
(L.). Shown are (c) drilling the chorium for ovipositing and
(d) ovipositor thrust and marking the egg after oviposition.. 43
5e and f. Female Trissolcus basalis (Wollaston) exhibits ovi-
positional behavior on an egg mass of its host, Nezara
viridula (L.). Oviposition of the central eggs by drilling
the chorium, (e) the lateral wall, and (f) the operculum
is shown 46
6. Ovipositional ethogram of the female parasitoid Trissolcus
basalis (Wollaston) when stimulated by six 12-hour-old eggs
of the host, Nezara viridula (L.) is shown 48
7. Number of male and female Trissolcus basalis (Wollaston)
reacting to (a) scent of 0, 1, and 3 male Nezara viridula
(L.) and (b) 0, 1, and 3 female N. viridula, averaged over
10 equally spaced time intervals is shown 51
8. Number of male and female Trissolcus basalis (Wollaston)
reacting to combined levels of (a) hemolymph of male Nezara
viridula (L.) and (b) hemolymph of female N. viridula at
different time intervals is shown 55
x

XI
Figure Page
9.Number of female Trissolcus basalis (Wollaston) reacting
to different egg ages of the host, Nezara viridula (L.),
at different time intervals is shown 57
10. Number of male Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown 60
11. Number of female Trissolcus basalis (Wollaston) reacting
to the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown 62
12a. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), after soaking
in dichloromethane for different periods of time is shown.... 66
12b. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), after soaking
in dichloromethane for different periods of time is shown.... 68
13. Number of female Trissolcus basalis (Wollaston) reacting to
crude kairomonal extract from eggs of the host, Nezara
viridula (L.), removed with dichloromethane by different
methods at certain time intervals is shown 70
14. The behavior patterns of the female Trissolcus basalis
(Wollaston) on a filter paper treated with crude kairomonal
extract from eggs of the host, southern green stink bug,
Nezara viridula (L.), are digrammed. Shown are parasitoid:
(a) in direct movement to the treated spot; (a') in random
movement to the spot; (a") after antennal palpation, leaving
the spot and starting to search; (b) after searching,
returning to the treated area for reinforcement; and (b')
leaving the spot for new cycle of searching. Either a
or a' will occur for every single trial 73
15a and b. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.), is shown:
(a) random movement (possibly kinesis), (b) chemotaxis 77
15c and d. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract from
eggs of the host, Nezara viridula (L.), is shown: (c) anten
nal palpation, (d) searching 7g
15e. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract

Xll
Figure Page
from eggs of the host, Nezara viridula (L.), is shown:
(e) reinforcement 81
15f. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation when stimulated by 102 egg
equivalent solution per microliter from eggs of the host,
Nezara viridula (L.), versus time, is shown 81
16. Percent of time spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper spot with
different concentrations of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.), is shown 86

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
THE BEHAVIOR OF THE EGG PARASITOID TRISSOLCUS BASALTS
(WOLLASTON) (HYMENOPTERA: SCELIONIDAE) IN RESPONSE
TO KAIROMONES PRODUCED BY ITS HOST, THE SOUTHERN GREEN
STINK BUG NEZARA VIRIDULA (L.) (HEMIPTERA: PENTATOMIDAE)
By
Fernando Joo Montenegro de Sales
March, 1978
Chairman: Dr. George E. Allen
Major Department: Entomology and Nematology
The mediators of behavior patterns among insects have been a focal
point of research in recent years as potential tools for management of
insect pest populations. Laboratory studies were conducted to reveal
the factors involved in the interspecific communication between the egg
parasitoid, Trissolcus basalis (Wollaston) and its host, the southern
green stink bug, Nezara viridula (L.). Temporal analysis of the female
parasitoid ovipositional behavior showed that location of the egg
masses of the host takes place by random movement (kinesis), chemotaxis,
and combination of both. It was demonstrated that the ovipositional
behavior is divided in two distinct steps: the appetitive behavior and
the most stereotyped consummatory behavior.
Olfactometer tests indicated that orientation toward the host eggs
is in great extent purposeful rather than random, and that the male and
female stink bug scents as well as their hemolymph act as cues in
eliciting orientation of T. basalis toward the eggs. The data implied
that the host egg age is a factor in stimulating the quest for eggs,
and the parasitoid mating behavior is possibly mediated by a pheromone.
xiii

XIV
A crude kairomonal extract was isolated by soaking host eggs in
dichloromethane and the results of bioassays with this extract showed
that visual clues are not as important as chemical clues in increasing
the parasitoid velocity? consequently reducing the orientation time
and enhancing host location. Assays on filter paper proved that the
kairomonal solution induced 5 behavior patterns; i.e., random movement
(kinesis), chemotaxis, antennal palpation, searching, and reinforcement.
Mathematical equations were developed to describe these patterns. The
concepts of Stimulant Concentration (SC ) Stimulant Dose (SD5q) and
Stimulant Time (ST^q) were introduced as measurements of the potency of
chemicals involved in interspecific and intraspecific communication
among insects.
The overall performance of the female T. basalis was improved in
terms of orientation, velocity, oviposition, and parasitism by the
kairomonal solution, and a very stereotyped and precisely timed behavior
pattern indicated that action of the isolated releaser is within the
limits of normality.

INTRODUCTION
The ability of insects to compete with man for the products of
agriculture is a continuing challenge to entomologists. In recent
years increased scientific and public awareness of the environment has
greatly increased the complexity of the problem. To meet the challenge
entomologists have resorted to a variety of new methods designed to
solve or at least alleviate this problem of controlling insects with
minimum detrimental effects to the environment. One of these new
methods involves research on chemical substances that influence inter
and intraspecific behavior of insects.
The southern green stink bug, Nezara viridula (L.) is a cosmopolitan
insect pest destructive to many crops of economic importance throughout
the world. Until now most of the control measures have relied on chemical
insecticides however with the demand for a cleaner environment, new
opportunities have been opened to alternatives that could assure an
acceptable injury level and meet the new standards set for the agroeco
systems common to this insect.
The scelionid parasitoid Trissolcus basalis (Wollaston) has been
utilized as one of those alternatives, and in fact, the specialized
biocontrol literature includes it as one of the major tools in balancing
the populations of many stink bug species, in the United States and
other countries. However there is a dearth of quantitative information
regarding the potential of this parasitoid as a major control agent.
1

2
With the growing research on the inter and intraspecific communica
tion among insects (Kennedy, 1956; Johnston, Jr. et al., 1970; Wilson,
1962, 1977), the chemical, physical, and biological releasers have been
studied more accurately in order to find out new ways to manage insect
pests with minimum detrimental effects to the environment (Metcalf and
Luckmann, 1975).
The only published study involving chemical interspecific communica
tion between N. viridula and its parasites and predators is the work of
Mitchell and Mau (1971) with the dipterous parasitoid Trichopoda pennipes.
Since experimental works have suggested that kairomones can be used
as valuable tools in management of insect pests (Lewis et al., 1971,
1975, 1976) the aim of this research was to assess the importance of the
releaser that triggers all sequences involved in the ovipositional
behavior of T. basalis. A series of bioassays were conducted to
determine: (1) threshold for host-seeking stimulation of the female
T. basalis by the eggs of the host; (2) ovipositional ethogram of the
female T. basalis with temporal analysis; (3) cues that enhance host
location; (4) discriminatory behavior of the male and female T. basalis
to parasitized and non-parasitized host eggs; (5) solvents and number of
washes required to remove the kairomone from the eggs and solution with
the best potential activity; (6) effect of host-eggs and kairomonal
solution on orientation of the parasite; (7) behavior patterns mediated
by the kairomonal solution and relationship between those patterns and
concentration; (8) Stimulant Concentration (SC ) Stimulant Dose (SDrri) ,
50 50
and Stimulant Time (ST^g) as measurements of the potency of the
kairomonal extract utilized in this work. (These are units that should
be useful in evaluations of chemicals involved in both inter and intra-

3
specific communication among insects.) (9) The normality of the extract
obtained through standard and original procedures.

LITERATURE REVIEW
Current Status of the Host Southern Green Stink Bug,
Nezara viridula (L.) and Its Parasitoid
Trissolcus basalis (Wollaston).
The Southern Green Stink Bug. Nezara viridula (L.)
Origin
According to Van Duzee (1917) Nezaara viridula (L.) was first
described by Linnaeus in 1758 under the scientific name Cimex viridulus.
Linnaeus' description was based on specimens collected in India. The
first new world record for the species is from the West Indies, and
since then the species has been redescribed by various authors under
numerous other scientific names.
Freeman (1940) placed the species into the genus Nezara of Amyot
and Serville, 1843 and Drake (1920) indicated that three color varieties
are recognized as: smaragdula (Fabricius), torguata (Fabricius) and
heptica Horvath.
Distribution
The southern green stink bug, N. viridula (L.) is widely distributed
throughout the world. It is found in Europe, Asia, Africa, and Americas
(DeWitt and Godfrey, 1972). Van Duzee (1917) and Jones (1918) have
pointed out that as many other insect pests in this Country, N.
viridula was introduced from West Indies and is established in Virginia,
Florida, Louisiana, South Carolina, Georgia, Alabama, Mississippi,
4

5
Texas, New Mexico, Arizona, California. Davis and Krauss (1963) have
reported its recent importation to Hawaii.
Host plants
Nezara viridula (L.) is a phytophagous insect with a broad range
of host plants. Hoffman (1935) indicates that this insect attacks
both monocotyledons and dicotyledons. Among the former he pointed out
that Graminae are the most important, and within the dicotyledons he
stated that 29 families are injured and ranked the following in order
of importance: Leguminosae having 27 species damaged, Cruciferae with
8 and Solanaceae with 6 species. Drake (1920), Gallo et al. (1970),
Todd (1973), Issa (1973), Turnipseed and Kogan (1976) have reported
this insect as feeding on radish, mustard, turnip, collard, cauliflower,
cabbage, okra, peas, beans, peanut, tomato, potato, cotton, tobacco,
pepper, eggplant, sunflower, sugar cane, corn, orange, lime, peach,
pecan, rise, snap bean, squash, cucumber, soybean, lemon, and grapefruit.
Drake (1920) also reports a number of weeds that serve as host to the
bug as: pokeweed, Phytolacca decandra; lamb's quarters, Chenopodium
spp.; nut grass, Cypepus esculentus L.; spiny amaranth, Amaranthus
spinosus; beggarweed, Desmodium spp.; crotolaris, Crotolaris spp.; wild
grape, Vitis spp.; castor bean, Ricinus communis L.; maypops, Passiflora
incarinata L.; and wild plum, Prunus spp.
In spite of the broad spectrum of host plants, N. viridula does
not breed in all those plants and only occassionally feeds on a number
of them. Drake (1920) indicates that the southern green stink bug has
a remarkable preference for the legumes, with the greatest degree of
preference when those plants are in the stage of fruit formation.

6
Life history
Life history and behavior of the southern green stink bug has been
intensively studied by Japanese researchers [Kariya (1961), Kiritani
(1963, 1965), Kiritani and Hokyo (1965) Kiritani, Hokyo, and Iwao
(1966), Kiritani, Hokyo, and Kimura (1966) Kiritani, Hokyo, Kimura,
and Nakasuji (1965), Kiritani and Kimura (1965), and Kobayashi (1959)].
In general, life cycle and generation time are much the same and
variations are usually correlated to temperature fluctuation at a
given location.
In the United States, the basic information in this area comes from
the classical works of Drake (1920) and Jones (1918). They indicate that
this insect, like most other pentatomids, hibernates in the adult stage
under litter, bark, and other objects which offers protection. Drake
(1920) points out that mating begins almost immediately upon emergence
from hibernation. The female and male usually remain attached to one
another by the tips of their abdomens and with their heads facing in
opposite directions. Under natural conditions copulation is repeated
a number of times before and after the eggs have been deposited. Drake
(1920) also reported that after feeding a few days, newly emerged adults
reach sexual maturity, and this period was found to vary from 3 to 6
weeks. The eggs are generally laid in regularly shaped compact clusters
in which the individual eggs are arranged in very regular rows and firmly
glued together. The incubation period is about 6 days in summer, but
during early spring and late fall the period is often extended to 2 or 3
weeks.
The southern green stink bug has 5 nymphal instars and during the
first instar, the nymphs normally cluster together near or on the egg-

7
shells. Drake (1920) indicated that no individuals have been observed
to feed while clustered; but just before or subsequent to molting, the
nymphs become active, scatter more or less and begin to feed. The
nymphs like the adults, are usually found upon those portions of the
plant on which they prefer to feedthe tender growing shoot and
specially the developing fruit. Jones (1918) and Drake (1920) reported
that during the summer, the period from egg to adult is about 35 days
with temperature conditions having an important effect.
Trissolcus basalis (Wollaston)
Regulation of southern green stink bug is attributed to biotic
and abiotic factors. A lot of work has been done in Japan in relation
to population dispersion and control. Kiritani (1965) suggests that
mortality factors work in a stage-specific way, i.e. parasite against
eggs, weather factors against the first instar and predators against
the second. He also indicates that the complex age structure during
the breeding seasons increases the population plasticity against a
specified mortality factor.
The specialized literature lists 12 parasites of the southern
green stink bug and T. basalis stands out as one of the most important
biocontrol agents. Since that time, it has been recorded from such
widely separated locations as the island of Saint Vincent, Florida,
and Egypt (Priesner, 1931).
Origin
This parasite was first described by Wollaston in 1858 from
specimens collected on the Ilha da Madeira.

8
Masner (1971) pointed out that Trissolcus basalis (Wollaston) has
the following synonyms: Telenomus basalis Wollaston, 1858; Telenomous
maderensis Wollaston, 1858; Telenomus magacephalus Ashmead, 1894; and
Telenomus piceipes Dodd, 1919.
Distribution and host insects
Trissolcus basalis has been reported as a polyphagous parasitoid
with broad range of dispersion throughout the world. It has been
reported in Europe, Asia, Africa, and North America (Cumber, 1964;
Davis, 1964; Hokyo and Kiritani, 1965; Kamal, 1937; Wilson, 1961).
Trissolcus basalis parasitizes N. viridula, Acrosternum hilare (Say),
A. marginatum (Palisot de Beauvois), Euchistus servus (Say), E.
variolatus (Palisot de Beauvois) and Cumber (1964) reported that this
parasitoid also develops on eggs of the following pentatomids: Antestia
orbona Kirk., Dictyotus caenosus (Westw.), Cermatulus nasalis (Westw.),
Glaucias amyoti (A. White) and Cuspicona simplex Walk.
Life history
Wilson (1961) indicated that T. basalis is a solitary arrhenotokous
parasite that develops from egg to adult within the host egg. He reports
that this parasite passes through a number of generations each year and
that the development is correlated to the temperature, i.e., at 27C,
the males lived for 4 to 5 days and the females for 4 to 15 days. Sailer
(1976) has indicated and the Author has confirmed that at 60 5% RH
o
and 27 C, male TL basalis have a life span varying from 3 to 5 weeks or
months while the females last from 4 to 15 weeks, and he also recorded
that females are able to live as long as 10 months with a honey and

9
water supply.
Thomas, Jr. (1972) indicates that the polyphagous behavior of T.
basalis is a positive factor in its utilization as a released biological
regulator, since other stink bug species can serve as alternate hosts
for maintenance and increase of the parasitoid population. He also
points out that field releases of T. basalis at rates of 5000 and 8000
adults per 1/10 acre increased the rate of parasitism substantially
and releases of at least 50,000 adults per acre would be required for
minimal effective suppression in a large scale augmentative release
program.
Interspecific Communication
Investigations involving interspecific communication were reported
by Laing (1937) who pointed out that the parasitoid Trichogramma
evanescens (Westwood) perceives an odor left by adult moth as a cue
that helps in locating host eggs. Thorpe and Jones (1937) also indicated
that odor plays an important role in a parasitoid/host relationship. It
was Brown, Jr. et al. (1970) that coined the term kairomone to define
the mediators involved in those processes of communication. Since then,
researches dealing with either behavioral studies, isolation, identi
fication, synthesis, and proof of effectiveness, or combination with
one or more of those aspects mediated by kairomones have been reported
by Corbet (1973), Greany and Oatman (1972), Gross et al. (1975), Hays
and Vinson (1971), Hendry et al. (1973, 1976), Jones et al. (1973),
Leonard et al. (1975), Lewis et al. (1971, 1975, 1976), Nettles and
Burks (1975), Nordlund et al. (1974), Nordlund and Lewis (1976), Tucker
and Leonard (1977), Vinson (1975, 1976), Vinson et al. (1975, 1976) and

10
Weseloh (1974, 1976).
Interspecific chemical communication between Trissolcus basalis
(Wollaston) and its host, the southern green stink bug, Nezara viridula
(L.) has not been reported, however Russian researchers have conducted
a series of investigations involving associations between many species
of scelionids, particularly Trissolcus spp., and pentatomid species
(Buleza, 1973; Meier, 1970; Zatyamina et al., 1976; Gennadiev et al.,
1976). Viktorov et al. (1975) found Trissolcus grandis ability to
encounter egg masses was significantly increased by extracts of adults
of two pentatomid hosts.

METHODS AND MATERIALS
Rearing of N. viridula (L.)
Southern green stink bugs were reared in wide-mouth Mason-type
jars measuring 8.0 cm and 6.5 cm bottom and top diameter, respectively,
and 17.5 cm in height. The top portion was covered with white paper
towel to confine and prevent the escape of nymphs and adults. Colonies
were established by placing 5 male and 5 female adults per jar with 3
to 5 string beans and occasionally either peanuts, carrots, and corn as
the food source. Oviposition sites were provided by a circle of paper
towel measuring 8 cm in diameter at the bottom of the jar and one strip
of the same paper measuring 3x20 cm with one end stuck to the top of
the container.
Rearing of T. basalis (Wollaston)
The Floridian strain of the parasitoid was reared in large wooden
cages with Plexi-glass top, with the front wall measuring 37.5 x 40.5
cm, back wall 37.5 x 45.5 cm and 42.5 cm in length. The front wall had
an 18 cm hole and was provided with a cotton cloth sleeve to prevent
escape and allow manual work inside the cage. Honey and water were
available to all parasitoids and they were exposed to a photophase of
14 hours day per 24 hr. cycle, a temperature of 25lC and 65^5%
relative humidity. The host, N. viridula (L.), was reared under the
same conditions of light, temperature, and humidity.
11

12
To reduce circadian and any other endogenous physiological
variability, the tests were conducted between 11:00 and 14:00 hour
with 3-day old T. basalis without any previous ovipositional experience
and host eggs 12-hour old or less.
Experiment 1: Response of the Female T. basalis
to the Eggs of the Host, N. viridula.
Responsiveness of the female parasitoid to the eggs of the host was
tested by means of a glass single tube olfactometer as shown in Fig. 1.
The dispenser consisted of a small tube measuring 7.5 cm in length and
0.35 cm in diameter. One extreme of this tube had its internal diameter
reduced to 0.20 cm and held a cotton filter. The dispenser was connected
to the main tube with a hollowed rubber stopper.
The main tube measured 30 cm in length and 0.60 cm in internal
diameter with one end linked to the dispenser and the other with its
internal diameter reduced to 0.30 cm retained a cotton plug which
functioned as a filter and prevented movement of the female parasitoids
beyond that point. This end of the tube was connected to a plastic hose,
which linked to a vacuum source and an air flow meter. Air flow was
adjusted to 3.3 cm'Vs throughout the experiment.
Chilled female Th basalis were placed on a white filter paper cut
out to a trapezoidal shape with bases measuring 0.5 and 5 cm and 4.5 cm.
The immobilized parasitoids were transferred to the main tube by removing
the rubber stopper and dispenser and placing the small base of the
trapezoidal paper inside the tube and raising it to a 20 angle and
gently tapping the paper. Then, the dispenser with the rubber stopper
was returned to the original situation.

Fig. 1. A single glass tube olfactometer is pictured.
V

14
Plastic tube
<
30cm

15
The olfactometer was placed on a white horizontal surface and ca.
109 cm away from 2 fluorescent lamps (Sylvania F40 cwx Lifeline) and
the apparatus was properly positioned to allow uniform dispersion of
the female parasitoids inside it.
Each experimental unit consisted of 10 healthy female T. basalis
within the main tube. Previous to any treatment, they were allowed to
complete recovery from the process of immobilization and run inside
the olfactometer for no less than 5 minutes, and after every single
treatment they were discarded.
Treatments were prepared in a spare dispenser. Host eggs were
introduced with soft forceps then they were covered by a thin cotton
layer. This dispenser containing the treatment was then used to replace
the blank one in a very quick stroke to avoid parasitoid escape.
Responsiveness of the female T. basalis was measured in terms of the
number of parasitoids that concentrated within 10 cm of the tip of the
dispenser. The readings were performed at 1 minute intervals for a
period of 10 minutes following insertion of the treated dispenser.
Experiment 1 consisted of 13 treatments (numbers of eggs) with 4
replicates applied to every experimental unit. The levels of those
treatments consisted of 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, and
300 eggs of the host, N. viridula. Reliability of the readings were
compared with a blank with no eggs applied to the testing parasitoids.
Regression analysis was carried out with the treatments transformed
to logarithm and multiplied by 10. The response of the female T. basalis
was measured as the percentage of individuals orienting to the scent
source after 3 minutes of exposure.
A test for lack of fit was conducted to check the adequacy of the

16
model in describing the relationship between the percentage of female
parasitoids orienting to the kairomonal source and the treatments
applied.
To determine the threshold for stimulation of the female T. basalis
the same experimental procedure was used; however the treatments applied
to the experimental units consisted of 0, 1, 2, 3, 4, and 5 eggs of the
southern green stink bug.
A completely randomized 6 x 10 factorial experiment with 4
replications was developed for testing the response of the female
parasitoid when stimulated by different levels of the eggs of N. viridula
in order to find the threshold of stimulation.
Experiment 2: Orientation of the Female T. basalis
Inside a "Y" Type Olfactometer.
Orientation of the female parasitoid was determined by the "Y"
type olfactometer shown in Fig. 2. Connections of the parallel tubes,
to vacuum, and air flow meter were with plastic tubing. The parasitoid
releasing tube and the filter tube were linked to the "Y" connections
through hollowed rubber stoppers. Organdi screen at one end of the
parallel tubes prevented movement of the parasitoids to the section "C",
the "Y" connection that is linked to the air flow meter.
The experimental unit consisted of 100 healthy female T. basalis
transferred to the parasitoid releasing tube by the method of Experiment
1. Twenty females were tested in each of five olfactometers of the same
type, under the same experimental conditions.
The air current passing through the filter tube of the olfactometer
was 6.6 cm^/s, and the apparatus was positioned properly on a white
horizontal surface and a blank test was run to detect any tendentious

Fig. 2.
A "Y" type olfactometer is pictured.

18
tube

19
response.
A treatment consisted of 50 eggs of the host introduced into the
parallel tube (B) and dispersed 7.5 cm away from the organdi screen.
Fifty egg shells of N. viridula were similarly placed inside the other
tube (B1). The shells were thoroughly washed in a solution of 1 part
of liquid non-phosphorous soap, 1 part of Clorox and 100 parts of
water, and then thoroughly rinsed in running water. The shells were
dried under lab conditions and then washed with dichloromethane. After
complete evaporation of the solvent, they were used in the experiment.
The test began after the parasitoids were fully recovered inside
the release tube which was then connected to the olfactometer.
Responsiveness was measured as the percentage of female T. basalis
orienting to tube (B), (B'), or remaining in the non-choice area (A).
Readings were performed at 5-minute intervals for a 30-minute period.
Test for non-preference orientation to each of those sites (A, B,
or B') was done with the chi-square test for multinomial experiments
(Snedcor and Cochran, 1973).
Experiment 3: Temporal Analysis of the Ovipositional
Behavior of the Female T. basalis
Experiments were conducted within a 5.0 cm Petri dish (lid 5.6 cm)
3
with a volume of 28.46 cm The lid of the dish was lined with a 5.5 cm
diameter circle of Whatman No. 1 qualitative filter paper.
The experimental unit consisted of 1 healthy 3-day-old female T.
basalis with no prior ovipositional experience. The parasitoid was
chilled and transferred to a white surface and covered with the bottom
of the Petri dish. A piece of paper supporting an egg mass was pinned
to the center of the filter paper with a dissecting pin. Six eggs were

20
used per mass because this is in the range of the threshold for
stimulation. To reach that number, large egg masses were broken apart
and 3 rows made up of 1, 2, and 3 eggs were left as the best physical
support upon which the female T. basalis could oviposit.
Completely recovered female T. basalis were exposed to the egg
masses when bottom and cover were put together. The container with the
parasitoid was held vertically and 25 cm away from a fluorescent light
source (cool white Sylvania F 15T8-CW), with the cover toward the light.
The experiment was repeated 20 times, and both eggs and parasitoid
were discarded after each test. All ovipositional behavior was
observed and every step was timed.
Experiment 4: Cues Useful in Location of the Host,
N. viridula, by the Parasitoid T. basalis
Scent of Male N. viridula
Responsiveness of the male and female T. basalis to the scent of
the male host was determined using the single tube olfactometer described
in Experiment 1. The dispenser tube was joined to another glass chamber
measuring 10.0 cm in length and 1.3 cm in diameter (Fig. 3). All other
experimental procedures were similar to those described for Experiment
1, except for the use of both male and female parasitoids for the
treatment combinations.
Treatment combinations consisted of 2 levels of the parasitoid,
male or female; 3 host levels of 0, 1, or 3 male N. viridula; and 10
time intervals obtained by 1 minute readings recorded every 10 minutes.
The glass chamber was used to confine the host insects during the
experiment. When using 0 male host, the chamber was kept empty with a

Fig. 3. The glass chamber is shown connected to a single glass
tube olfactometer.

22

23
current of air (3.3 cm /s) passing through it and reaching Fhe main
tube which contained 10 healthy 3-day old parasitoids (either male or
female) ; readings were recorded at every 1-minute interval for 10
minutes. Responsiveness was reported when the parasitoid stayed around
the tip of the dispenser tube or within 10 cm of it. For the remaining
treatments (2nd and 3rd levels of the male host) 1 and 3 15-day old
male N. viridula were placed inside the glass chamber and the reactions
of the parasitoid recorded as already described.
Analysis of the data was performed by a three-way analysis of
variance for completely randomized 2x3x10 factorial experiment with 4
replicates. Interactions of significance for the experiment were
studied by a thorough analysis of the simple effects.
Scent of Female N. viridula
The methodology for the response of the parasitoid to the female
host was identical to that described above for the male N. viridula.
Male and Female N. viridula Hemolymph
The response of male or female parasitoids to 3ul of male or female
hemolynph or to blank controls was evaluated using the apparatus
described in Experiment 1.
The hemolymph was obtained from healthy 15-day-old virgin males
and the liquid was drawn from the prothorax at the point of insertion of
the forewing. The wing was removed and the exuded hemolymph collected
with a micropipette, and transferred to a thin piece of cotton measuring
0.5 x 0.6 cm. After drying the cotton was transferred to a spare
dispenser tube which later replaced the blank one from the olfactometer.

24
Evaluation of the reactions were similar to those described for
Experiment 1, and the readings were taken during each 1-minute interval
for a 10-minute period.
2
The experimental procedure was arranged in a 2 x 10 factorial
design for each hemolymph type with 4 replicates. Analysis of variance
was carried out and the interactions of importance were investigated
through the simple effects.
H. viridula Eggs of Different Ages
Response of the female T^. basalis to N. viridula eggs of different
ages was measured by means of a single tube olfactometer as described
in Experiment 1.
Treatments were prepared by placing 0, 50 12-hour-, 50 24-hour-, or
50 48-hour-old eggs of the host in the dispenser tube and proceeding as
previously described. All treatments were replicated 4 times and
readings made at 1-minute intervals for a 10-minute period.
A two-way analysis of variance was computed for the 4 x 10 factorial
design, and simple effects were investigated for the interaction between
egg age and time required for female parasitoid reaction.
Experiment 5: Reactions of the Male and Female
T. basalis to the Eggs of the Host,
N. viridula at Different Degrees of Parasitism
Treatments were set up to test parasitoid response to non-
parasitized and parasitized host eggs as well as eggs from which
parasitoids had already emerged, i.e., 0 N. viridula eggs, 50 12-hour,
50 parasitized eggs from which adult T?. basalis were ready to emerge,
and 50 host egg shells 4 days after emergence of the parasitoids.

25
Except for the statistical analysis and utilization of both male
and female parasitoids as experimental units, the methods used were
similar to that described for Experiment 1.
The levels of the factors utilized during the test were: male and
female T. basalis, 4 different situations of host eggs, and 10 1-minute
readings recorded during a 10 minute period.
The experimental design was a 2 x 4 x 10 factorial with 4 replicates
F-test values were determined for main effects, interactions and simple
effects at 0.01 and 0.05 levels of significance.
Experiment 6: Reactions of the Female T. basalis
to the Kairomonal Solutions Prepared
with Different Solvents
The solutions were prepared by soaking 12-hour-old (or less) eggs
of N. viridula in 4 different solvents, i.e., water, dichloromethane,
ethanol, and hexane, for 1 hour. The resulting suspension was filtered
through a Whatman No. 1 qualitative filter paper. The ratio of egg
and solvent was 1 egg:l ul of solvent.
Treatments were set up by drawing a 50 ul aliquot from the
solutions and applying it to different pieces of a thin piece of cotton
measuring 0.5 x 0.6 cm and allowing enough time for solvent evaporation.
The treated pieces of cotton were transferred to spare dispenser tubes
by means of a forceps and pushed in to the narrowed end with a micro
syringe plunger. These dispensers were utilized to replace the blank
ones in the olfactometer. The experimental procedure and methods of
recording responsiveness of the female T. basalis to the stimuli were
identical to Experiment 1.
The experimental procedure was a completely randomized design with

26
5 replications. Selection of the solvent that removed most of the
active ingredient(s) was done by contrasting the treatment means at
the 4th minute interval, utilizing Duncan's test (Duncan, 1955).
Dichloromethane Washes Required for Removal of the Kairomone from Eggs
of N. viridula
From the 4 solvents tested, dichloromethane was selected for
utilization throughout this assay, based on results presented later in
this text.
Treatments in this experiment consisted in soaking 12-hour-old
(or less) N. viridula eggs in dichloromethane for different periods of
time. For every single wash fresh aliquots of dichloromethane were used.
No re-use or recycling was permitted through the bioassay.
After the soaking process the washed eggs were spread on a Whatman
No. 1 qualitative filter paper to allow solvent evaporation. The ratio
of host eggs per milliliter of dichloromethane during the washing
(=soaking) process was 0.025/1.
After a given washing cycle, and complete solvent evaporation, the
eggs were placed in a spare dispenser tube, and later used to replace
the blank tube in the olfactometer with the female parasitoid. The
testing procedure was similar to that described for Experiment 1, except
for the analysis of the data.
During this experiment, 10 treatments including a blank with no
eggs, were applied to the experimental units.. The treatments consisted
of 50 N. viridula eggs treated as follows: host eggs soaked 1 time for
3 minutes, soaked 1 time for 5 minutes, soaked 2 times for 5 minutes,
3 times for five minutes, soaked 1, 2, 3, and 4 times for 1 hour, and

27
finally, host eggs soaked 1 time for 4 hours.
2
The data were analyzed as a 10 factorial experiment with 4
replicates, then the F-test values were determined for the statistical
parameters of relevant interest to the experiment.
Reaction of the Female T. basalis to Kairomonal Solutions Prepared by
Different Methods
Activity of the solutions was measured by the percentage of female
T. basalis orienting to the tip of the dispenser or within 10.0 cm of it
(refer to Experiment 1). The solutions were obtained by soaking host
eggs 1 time in dichloromethane for 4 hours, grinding the host eggs with
the same solvent, and by soaking host eggs 4 times at 1 hour intervals.
Suspensions obtained were filtered with a Whatman No. 1 qualitative
filter paper, and concentrations were set, such that 100 ul of the
solution would contain 50 egg equivalents.
Fifty-egg equivalents of each solution were applied to a thin piece
of absorbent cotton (0.5 x 0.6 cm). After solvent evaporation the
treated cotton was introduced into the glass dispenser with forceps and
a microsyringe plunger. Controls were dichloromethane-treated pieces of
cotton.
A completely randomized 4 x 10 factorial experiment with 4
replications was developed for testing the responses of the female T.
basalis to the different extraction procedures. Analysis of variance
was performed and F-test values obtained for the inference-making process.
Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the
Crude Kairomonal Extract from Eggs of the Host, N. viridula
The kairomonal solution was prepared from N. viridula eggs 12 hours

28
old or less. The eggs were ground with dichloromethane and the resulting
suspension filtered through a Whatman No. 1 qualitative filter paper.
The ratio of egg and solvent was 1 egg:l ul of dichloromethane.
Dilutions were then prepared to provide the concentrations used during
the experiment, such that 1 ul of the prepared dilutions would contain
-4 -3 -2 -1
10 ,10 ,10 and 10 egg equivalents.
The experiments were conducted within 5 cm Petri dishes with lids
lined with Whatman filter paper as described in Experiment 3. The
experimental unit consisted of 30 healthy 3-day-old female T. basalis
with no prior ovipositional experience. The females were chilled and
transferred to a plain white surface then individually covered with the
bottom of a Petri dish. One microliter of the test solution was dropped
+ 2
on the center of a filter paper covering an area of (0.17-0.008) cm .
The solvent was allowed to evaporate and the paper then transferred to
the inside of a Petri dish cover. Fully recovered female parasitoids
were exposed to the crude extract when bottom and cover were put together.
The container with the parasitoid was held vertically and 25 cm away
from a fluorescent light source (cool white Sylvania F15T8-CW), with the
cover toward the light.
Preliminary assays without solution were conducted to eliminate
any bias due to the light or handling during the experimental procedure.
The activity of the parasitoids was measured in terms of their ability
to locate the treated spot and their subsequent reactions when ejqposed
to different concentrations of the crude extract.
Responsiveness to the different stimuli were recorded in terms of
percentage of females exhibiting kinesis, chemotaxis, antennal palpation,
searching and reinforcement.

29
Data obtained through the experiment were submitted to probit
analysis for quantal response experiments (Finney, 1971). Subsequently,
regression equations were established for all behavioral patterns
exhibited by the female parasitoids then Stimulant Concentration (SC^)
and Stimulant Dose (SD^) values were determined for those patterns.
-2
The Stimulant Time for a 10 egg equivalent solution was
calculated for antennal palpation; however, the procedure is applicable
to any behavior pattern.
Experiment 8: Effects of the Crude Kairomonal Extract
from Eggs of the Host, N. viridula in the
Orientation of the Female T. basalis
The methodology and the experimental material utilized in this test
was identical to those used in Experiment 7, except that the experimental
unit consisted of 10 healthy 3-day-old female T. basalis, and a different
statistical design.
Treatments were four equally spaced concentrations of the crude
-4 -1
kairomonal extract, ranging from 10 to 10 egg equivalents/ul, plus
a check which consisted of 1 ul of dichloromethane dropped on the center
of the filter paper and after evaporation, exposed to the experimental
units.
Effects of the treatments were observed by tracking the time spent
by the female parasitoids in orienting themselves to the treated spot
for the first antennal contact.
A completely randomized design with 3 replications was developed
for checking the effect of the kairomonal concentrations on the
orientation time of the female parasitoid. The F-test was determined
and contrast of the means was done by Duncan's technique (Duncan, 1955).

30
Experiment 9: Enhancement of Host Location
by Scent Combinations
Responsiveness of the female T. basalis to combination of scents of
the kairomonal solution, host eggs, and solvent was tested with the
single tube olfactometer and the olfactometer-glass chamber tube as
shown in Figs. 1 and 3 respectively.
For every single treatment 5 female T. basalis under the same
biological conditions of the previous experiments were introduced into
the single tube olfactometer as described in Experiment 1 and submitted
to 7 different scent combinations.
The first treatment consisted of 3 healthy 15-day-old virgin female
N. viridula in the glass chamber. Treatment 2 consisted of 10 12-hour-
old eggs of the host combined with 3 female N. viridula offered
simultaneously. The eggs were placed inside the olfactometer and
approximately 0.5 cm away from the tip of the dispenser. The adult
female hosts were introduced in the glass tube chamber.
The third treatment consisted in applying 25 ul of dichloromethane
to a thin piece of cotton measuring 0.5 x 0.6 cm which after evaporation
was introduced into a spare dispenser tube.
The next treatment was 25 ul of the solution containing 12.5 egg
equivalents of the crude kairomonal solution in dichloromethane was
dispensed on a thin cotton piece. After solvent evaporation the treated
cotton was placed inside the dispenser tube as in the previous test.
Treatment 5 consisted of introducing 10 12-hour-old eggs of N.
viridula into the olfactometer and 0.5 cm away from the tip of the
dispenser. Treatments 6 and 7 were identical to 5 except that a thin
piece of cotton treated with 25 ul of dichloromethane and 25 ul of the

31
kairomonal solution with 12.5 egg equivalents was placed in the
dispenser, respectively. Reactions of the female T. basalis to the
scent combinations was determined when one of the 5 parasitoids made
the first physical contact with the egg mass or the tip of the dispenser.
The velocity of the parasitoid toward the scent source was
determined for the first female T. basalis scored in each of the
situations. For evaluation purposes, it was assumed that the movement
toward the source of stimuli was preformed in a linear and uniform
fashion.
The experiment was set up as a completely randomized design with 7
treatments and 5 replications. Contrast of the treatments and means
were performed by the appropriate statistical procedures already
mentioned.
Experiment 10: Normality Studies with the Crude
Kairomonal Solution from Eggs of the Host,
N. viridula
Reactions to exaggerated releasers by higher animals and insects
have been reported in the specialized literature (Tinbergen, 1951;
Marler and Hamilton, 1967; Wallace, 1973; Hogan et al., 1974; Magnus,
1958; and Staddon, 1975).
To observe if female T. basalis were reacting to a normal stimulus
a series of tests were performed for certain behavior patterns.
Response to the Kairomonal Solution on the Filter Paper
Bioassays were conducted in Petri dishes described in Experiment 3.
The experimental unit was 10 healthy 3-day-old female T. basalis without
any ovipositional experience. The female parasitoids were exposed to

32
1 ul of a 10 ^ egg equivalent solution in dichloromethane or to
dichloromethane controls using the procedure described in Experiment 7.
Responsiveness of the parasitoids to the solvent and to the solution
were recorded in terms of the percentage of the testing insects reaching
the treated spot and drumming the area with the antennal flagellum.
The data obtained were analyzed by a paired-difference t-test.
Evaluation of Parasitism in Areas Treated with the Crude Kairomonal
Solution
The method utilized was a modification of the procedure of Jones
et al. (1973). A circle of Whatman No. 1 qualitative filter paper
measuring 12.5 cm in diameter was cut in 4 quadrants and placed in a
Petri dish with bottom and cover measuring 14 cm and 15 cm of internal
diameter respectively. The quadrants were positioned 1.2 cm apart to
reduce the chance that the parasitoid would search in adjacent areas not
-2
treated with the kairomone. An aliquot of 50 ul of a 10 egg equivalent
solution was applied to each of two opposite quadrants, and 50 ul of
dichloromethane was applied to the remaining quadrants. After the
solvent evaporation 1 egg mass made up of 10 12-hour-old eggs of N.
viridula was placed on each quadrant. Two immobilized (by chilling)
female T. basalis were placed on the center of the dish and after
recovery the dish was covered and the parasitoid allowed to search and
oviposit for 45 minutes. Parasitism was determined 12 or more days after
the treatment by counting the darkened eggs typical of T. basalis close
to emergence.
The data obtained were analyzed by paired-difference t-test and
the ratio between parasitized eggs on solution and solvent treated

33
area computed.
Responses of Female T, basalis to Egg Shells and 12-Hour-Old Eggs of the
Host N. viridula
The consummatory phase of the ovipositional behavior was tested
with egg shells treated with the crude kairomonal solution, with the
solvent, and with 12-hour-old eggs of N. viridula.
Egg shells with the operculi were obtained 15 days after hatching
of the host insect. They were thoroughly washed in a 1:1:100 non-
phosphorous dish wash detergent, Clorox and water, then extensively
rinsed in tap water and finally washed in distilled water. The shell
remained glued through the washing process to the paper towel
ovipositional site. They were allowed to dry on filter paper under
laboratory conditions and later rinsed 3 times in dichloromethane.
After solvent evaporation, they were ready for the experimental
procedure.
The experimental unit was made up of 10 healthy 3-day-old female
T. basalis without any prior ovipositional experience. Except for the
treatments assigned to the female parasitoids, all bioassay procedures
were the same as those for Experiment 3.
The treatments were 6 12-hour-old eggs of N_. viridula, 6 egg shells
treated with 1 ul of dichloromethane, and 6 treated with 1 ul of 10 ^
egg equivalent solution of the kairomonal extract.
Response of the parasitoids to the treatments were recorded as the
percentage of female T. basalis assuming the ovipositional posture after
exploring the treated egg masses and bringing the ovipositor into
contact with the chorial surface.

34
Data were analyzed for a completely randomized design with 3
treatments and 4 replications. Contrast of the means was done by
Duncan's test (Duncan, 1955).
Antennal Palpation Previous to Oviposition
Egg shells treated with 1 ul of a 10 1 egg equivalent solution
of the crude kairomonal extract and 12-hour-old eggs of N. viridula
were utilized to assess the degree of normality of the isolated releaser.
The time spent by the female T. basalis palpating the eggs and treated
egg shells previous to the contact of the ovipositor with the chorium
was recorded as the measurement of evaluation.
The bioassay consisted in exposing 1 healthy 3-day-old female
parasitoid without ovipositional experience to the treatments. The
procedure was replicated 30 times and the amount of time spent by T.
basalis exhibiting antennal palpation was recorded. The methodology was
similar to that of the previous section except for the points already
mentioned and analysis of the data, which consisted of applying a
paired-difference t-test to the results.

RESULTS
Experiment 1: Response of the Female T. basalis
to the Eggs of the Host, N. viridula
Responsiveness of the parasitoid inside the single tube olfactometer
(Fig. 1) was recorded after 3 minutes of exposure. The results are
shown in Fig. 4. There is a strong linear relationship (r=0.97) between
the percentage of female T. basalis stimulated to move toward the scent
source and the log number of eggs of N. viridula. The adequacy of the
linear model was contrasted at 0.05 level of significance, and the
F-test (=1.29) indicated that the relationship already studied is
properly represented by the model.
Analysis of the main effects, i.e., egg and exposure time revealed
F values equal to 14.12 and 4.13 respectively. They were highly
significant ata=0.05, but do not interact. Their effects are additive
to the population mean of the number of female parasitoid orienting to
the scent source.
The threshold for stimulation was 5 12-hour-old eggs of the
southern green stink bug. The analysis of variance for the simple
effects is shown in Table 1, of the Appendix. An F-test for all time
levels within the 5-egg level was highly significant, indicating that
the parasitoids started to react to the host presence at that point.
Experiment 2: Orientation of the Female
T. basalis Inside a "Y" Type Olfactometer
35

Fig. 4. Relationship between percentage of female Trissolcus basalis (Wollaston) responding
within a single tube olfactometer versus different levels of eggs of the host
southern green stink bug, Nezara viridula (L.), is shown.

80
60
40
20
i
1
1
2
3
log of the number of eggs
of N. viridula
10
u>

38
When introduced into the olfactometer and after recovery the
parasitoids began to move to the different sections of the apparatus.
During the first 15 minutes of observation the parasitoids kept
moving within the region A of the olfactometer, running either on a
straight or circular path into the Y part of the olfactometer. Sometimes
the females returned to the releasing tube and remained there or
resumed movement to the other parts of the apparatus. After 30 minutes,
63 percent of the female parasitoids were found in the section B
palpating host eggs with the antennal flagellum, ovipositing, or running
inside the tube. Five percent of the tested insects were observed in
section B' performing the same behavior except ovipositing. Finally
32 percent of the sample tested stayed in Section A either running in
the Y part of the olfactometer or remaining in a resting position inside
the releasing tube.
It is evident that the parasitoids showed a highly significant
preference for the side of the olfactometer containing the 12-hour-Old
eggs of the host, N. viridula. These data are presented in Table 2, of
the Appendix. The chi-square value was 50.54.
Experiment 3: Temporal Analysis of the
Ovipositional Behavior of the Female T. basalis
When the female T. basalis is introduced into the Petri dish she
starts to either run on the margins of the filter paper exhibiting a
random movement, possibly kinesis, or moves straight to the egg mass,
after spending a certain amount of time in this kinetic pattern, the
female parasitoid may purposefully move toward the source of stimulus.
During the orientation process the parasitoids required an average of

39
103.65 20.40 seconds (a=0.05) to encounter the egg mass. During
this period considerable antennal and body grooming took place, sometimes
the female moved straight to the eggs, then suddenly changed direction
and moved away and later returned. It is evident that the source of
stimulus is reached either by random movement, chemotaxis, or a
combination of both.
When a female T_. basalis makes first contact with the egg mass, she
begins to explore it by drumming the lateral wall of the marginal eggs
or the crevices of the central eggs with the antennal flagellum (Fig.
5 a,b). The parasitoid spends 116.55 18.56 seconds with 95% of the
fiducial limit, in this exploratory behavior, then prepares for
oviposition.
The female T. basalis finishes the exploratory phase when it turns
its head away from the point chosen to insert the ovipositor. Drilling
of the chorium begins when the parasitoid slightly bends the terminal
portion of the abdomen and inserts its ovipositor through the egg wall.
When insertion is performed in the marginal eggs, the female T. basalis
may take three positions: (a) the parasitoid has the ventral portion of
the body parallel to the substratum that supports the egg mass and the
tip of the wings reaching the eggs to be parasitized; (b) the female
parasitoid stands sideways in relation to the substratum and the wings
may touch the egg or stay away from it (Fig. 5c). In both situations,
drilling of the chorium occurs in the bottom third of the egg. Ovi
position in the central eggs is performed in two distinct postures: (a)
female |T. basalis flexes its abdomen and introduces the terminal portion
between the crevices then inserts the ovipositor 1/3 below the operculum
(Fig. 5e); (b) the parasitoid perforates the egg cap, by introducing

Fig. 5a and b. Female Trissolcus basalis (Wollaston) exhibits
pre-ovipositional behavior in an egg mass of its
host, Nezara viridula (L.). Antennal palpation of
(a) marginal egg and (b) central egg is shown.

41
3
(a)

Fig. 5c and d. Female Trissolcus basalis (Wollaston) exhibits
ovipositional behavior on an egg mass of its host,
Nezara viridula (L.). Shown are (c) drilling the
chorium for ovipositing and (d) ovipositor thrust
and marking the egg after oviposition.

StT
M&Kft
mmmzrz
:..' £ ?F-tT*$3i$S**r' i
;a;t^i¡iiilifti
44
the ovipositor through the operculum and beneath the point of junction
with the surrounding walls. In this posture the female T. basalis
stands with the sternal portion of the thorax approximately in a
straight angle with the horizontal plane of the opercular surface (Fig.
5f). Deposition of the egg is characterized by a rocking movement
similar to those observed by Wilson (1961) and Hokyo and Kiritani
(1965) in other scelionid parasitoids. All legs serve as support to
the female T. basalis, however; the meso and metathoracic pairs act
more remarkably to this function. It was observed that the prothoracic
legs and forewings are used very effectively in agressive behavior,
especially when the egg density drops to 0.5 egg per female parasitoid
or less, and more than one parasitoid is looking for ovipositional site.
The process of oviposition which starts with the preparation of the
parasitoid to drill into the egg shell, ends when the female T. basalis
withdraws the ovipositor from the chorium. This behavioral step
requires 117.97 5.86 seconds, with a fiducial limit of 95%.
After successful oviposition the female parasitoid begins the egg
marking process (Askew, 1971; Safavi, 1968). It took 19.15 1.02
seconds for T. basalis to complete this behavior. Marginal eggs are
marked when the female drags the tip of the ovipositor from the point of
insertion up to the margins of the operculum in a sinuous pattern (Fig.
5d) Central eggs are marked in the same manner, starting from the point
of drilling and covering the opercular margins and going down ca. 1/3
of the upper portion of the surrounding egg wall.
An ethogram of the ovipositional behavior of T. basalis is shown
in Fig. 6. It could be seen that the orientation to the egg mass is
accomplished by either random movement or chemotaxis or combination of

Fig. 5e and f. Female Trissolcus basalis (Wollaston) exhibits
ovipositional behavior on an egg mass of its
host, Nezara viridula (L.). Oviposition of the
central eggs by drilling the chorium, (e) the
lateral wall, and (f) the operculum is shown.

46
(f)

Fig. 6. Ovipositional ethogram of the female parasitoid Trissolcus
basalis (Wollaston) when stimulated by six 12-hour-old
eggs of the host, Nezara viridula (L.), is shown.

48
l
I
l
SATIATION I
l

49
both. After the physical contact, antennal palpation, oviposition, and
marking follows in a very defined sequence. Those behavioral steps
were observed when the number of eggs per mass is within the threshold
limit, that is, 6 eggs of N. viridula per mass.
The broken lines of the Fig. 6 indicate repetition of a series of
behavioral steps that ultimately leads the parasitoid to satiation.
Tracking of time during this cycle was omitted.
Experiment 4: Cues Useful in Location
of the Host. N. viridula by the Parasitoid T. bassalis
Scent of Male N. viridula
Responsiveness of the male and female T. basalis to the scent of
different levels of the male N. viridula in the single tube olfactometer
was determined by the analysis of variance of the treatment combinations.
As shown in Table 3 of the Appendix, parasitoid and host factors interact.
There is evidence that host levels and parasitoid levels do not act
independently, that is, a change in the levels of the host correspond to
a response by the male and female parasitoid which is different in
direction and intensity. Analysis of the simple effects by combination
of 10 levels of time is shown in Fig. 7a. Both male and female T.
basalis reacted to the scent of the male host levels combined, F.values
were 6.20** and 13.87** respectively. The differences in variability
was assigned to 1 and 3 male N. viridula, since no significant response
was observed at 0 level (F=0.80). Response of the female parasitoid
sharply increased when 3 male hosts were placed in the olfactometer.
Scent of Female N,. viridula

Fig. 7. Number of male and female Trissolcus basalis (Wollaston)
reacting to (a) scent of 0, 1, and 3 male Nezara viridula (L.)
and (b) 0, 1, and 3 female N. viridula, averaged over
10 equally spaced time intervals is shown.

51
250
O)
c
o
03
0)
C/>
03
(/>
03
JO
h- I
O
0)
-Q
E
3
2
200
150
Male 4. female, F = 1.67
Male F = 6.20**
Female, F =13-87**
4iL
_1 o
i
1
Number of male
(a)
I
2
N. v ¡ r ¡ d u I a
-L
3
(b)
3

52
Orientation of the parasitoid to the scent source was analyzed and
the results are presented in Table 4 of the Appendix. A highly signifi
cant interaction indicated that the responsiveness of the parasitoids
vary with the levels of the female host. Response of the male parasitoid
was lower than the response exhibited by the female (Fig. 7b) and the
F-test was significant for both parasitoid sexes, at 0.05 levl.
Responsiveness of male and female T. basalis combined over 10 time
intervals were not significantly different (F=1.67) for three male host
levels utilized at 0.05 level of significance. When female hosts were
offered to the male and female parasitoids, the combined response was
highly significant, F=45.42**. Most of this is accounted by the female
parasitoid reaction.
Scent of Male N. viridula Hemolymph
It was observed that male and female T. basalis responded differently
to the different levels of the male host hemolymph. As shown in Table 5,
the three-factor interaction implies that the male host hemolymph
versus T. basalis differs with the levels of time.
Data for the treatment totals are presented in Table 6 of the
Appendix. There is an indication that the male parasitoid did react
poorly to the male hemolymph levels when averaged over 10 levels of time.
Analysis of the simple effects confirmed that assumption as the F-test
was not significant at 0.05 level of significance (F=0.45). On the other
hand the female "T. basalis exhibited a response to the male N. viridula
hemolymph, which was highly significant, F=83.60**.
The two-factor interaction observed for male host hemolymph versus
T. basalis suggests that the responses of the male and female parasitoid

53
are different in magnitude and direction when the two levels of the male
host hemolymph are available to both sexes (Table 5 of the Appendix).
Reactions of the male and female parasitoids to combined levels of
the male N. viridula hemolymph in relation to time are shown in Fig. 8a.
Male parasitoid reaction was not statistically significant. Female T.
basalis reaction was highly significant. It was observed that after 2
minutes responsiveness of the female parasitoid reached its highest
value. After this, reactivity decreased and returned to the level of
the control at the 10th minute of observation.
Scent of Female N. viridula Hemolymph
Reactions of the male and female Th basalis to the female host
hemolymph were analyzed and the results are summarized in Tables 7 and
8 of the Appendix. Responsiveness to combined levels of the female
hemolymph at different time intervals is presented in Fig. 8b.
Male parasitoids were poorly stimulated by the female host hemolymph,
there was no significant difference between the two levels of hemolymph
applied when averaged over 10 time levels (F=0.05). Female parasitoids
responded positively to the female N^. viridula hemolymph (F=156.80**) .
The remaining inferences about responsiveness of the parasitoid sexes to
the female host hemolymph are identical to those already discussed for
the male host hemolymph.
N. viridula Eggs of Different Ages
Treatment responses shown in Fig. 9 implies that the female .
basalis responded better to 12 and 24-hour-old host eggs.
Analysis of variance for the simple effects was carried out and the

Fig. 8. Number of male and female Trissolcus basalis (Wollaston)
reacting to combined levels of (a) hemolymph of male
Nezara viridula (L.) and (b) hemolymph of female N. viridula
at different time intervals is shown.

Number of T. basalis reacting
Number of T.basalis reacting
Ln
ui

Fig. 9. Number of female Trissolcus basalis (Wollaston) reacting
to different egg ages of the host, Nezara viridula (L.),
at different time intervals is shown.

Number of female T. basalis reacting
57

58
results are presented in Table 9 of the Appendix.
F values for variation between time within 12-hour- and 24-hour-old
eggs were 8.44 and 5.19 respectively. They were highly significant and
suggested that the female T. basalis were stimulated by volatile chemicals
carried by the air stream that passed through the eggs inside the dispens
er tube. There is also indication that such chemicals decrease in concen
tration as the eggs get older. The F value for the response of females to
48-hour-old eggs was not significantly different from the blank control
at the 0.05 level.
The female parasitoid reactions are shown in Fig. 9. The maximum
reaction occurred at the third minute of exposure. Following that, a
decline in response took place and after the 5th minute the reactions were
identical for all treatments applied. The decline after the 5th minute
could be a result of either habituation, decline in concentration of the
volatile chemicals or a combination of both.
Experiment 5: Reactions of the Male and Female T. basalis to the
Eggs of the Host, N. viridula at Different Degrees of Parasitism
Responsiveness of the male and female T. basalis to the treatments
are summarized in Table 10 of the Appendix. Significant interactions
were found for egg levels versus time, egg versus male and female parasit
oid, time versus male and female T. basalis. There is evidence that the
stimulation of the parasitoids by the scent source inside the main tube
of the olfactometer depended upon the levels of parasitism of the eggs
of N. viridula and upon levels of time.
The reactions of male and female T. basalis to the different
conditions of the host eggs are presented in Figs. 10 and 11 respectively.
Treatment totals revealed that again the maximum reaction of male

Fig. 10. Number of male Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown.

Number of male T. b a s a I is reacting
Minutes

Fig. 11. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), at different
levels of parasitism and time intervals-is shown.

Number of female T. basal is reacting
62
100
90
80
70
60
30
20
10
\
\
Treatment totals
o
0 host egg
50 12-hour-old host eggs
50 parasitized host eggs
50 egg shells from parasitized
host eggs
i 1!I L
3
Minutes
1
2
4
5

63
and female parasito ids happened at the 2nd or 3rd time interval.
Analysis of variance for the simple effects are shown in Table 11
of the Appendix. It can be observed that the response of the male
parasitoid to the blank tube without host eggs and to 50 12-hour-old
host eggs were statistically identical. The response of male parasitoids
to the 12-hour-old eggs was not high enough to rule out chance factor.
On the other hand, 50 parasitized host eggs containing T. basalis
ready to emerge, and 50 egg shells from parasitized eggs 4 days after
emergence, triggered a highly significant response in the male parasitoids.
Female parasitoid response was statistically high for the different
situations of the egg host except for the blank, when no eggs were
available (Table 11 of the Appendix).
Experiment 6: Reactions of the Female T. basalis to the
Kairomonal Solutions Prepared with Different Solvents
Since it was demonstrated that female T. basalis exhibit a
noticeable preference for sites containing eggs of its host N. viridula,
the next step was to extract the chemical (s) from eggs of the host.
Four solvents were used and the activities of the crude kairomonal
extract obtained tested in the olfactometer.
Results of the evaluation process are shown in Table 12 of the
Appendix. Treatment totals indicated that the maximum response of the
female parasitoid to:the crude kairomonal extract obtained with the
solvents was at the 4th minute of observation. Data from this time
interval were submitted to analysis of variance and the F-test (=12.14**)
was highly significant.
Contrast of the means was done by the Duncan's procedure (Duncan,
1955) and the results are presented in Table 13 of the Appendix. The

64
data implied that dichloromethane was the best solvent in removing the
active ingredient(s) from the host eggs. Activity of hexane was
similar to the ethanol but different from water. Water had the lowest
activity, which was statistically identical to ethanol.
Number of Dichloromethane Washes Required for Removal of the Kairomonal
Extract from Eggs of N. viridula
Evaluation of the: methods utilized consisted in offering the eggs
of N. viridula to the female parasitoids after soaking in dichloromethane.
Responsiveness of the female T. basalis was measured inside the
olfactometer and the results are shown in Table 14 of the Appendix.
Methods and time do not interact; they are independent of one another
(F=l.49).
Treatment responses are shown in Figures 12 a and b. Removal of
the kairomone from eggs of the host was partial for all methods tested,
except for the procedures of soaking eggs 1 time for 1 and 4 hours.
Analysis of the simple effects indicated that the activity present
on the eggs treated by those methods elicited responses from the female
parasitoids not significant at 0.05 level.
Reaction of the Female T. basalis to Kairomonal Solutions Prepared by
Different Methods
Responsiveness of the female parasitoids to the crude kairomonal
extract obtained by 3 techniques was compared when host eggs were: (a)
soaked 1 time for 4 hours in the solvent; (b) ground with the solvent;
(c) soaked 4 times at 1-hour intervals.
A summary of the female parasitoid reactions in the single tube
olfactometer is shown in Fig. 13. Solutions with best activity were

Fig. 12a. Number of female Trissolcus basalis (Wollaston) reactina
to the eggs of the host, Nezara viridula (L.), after
soaking in dichloromethane for different periods of time
is shown.

Number of female T. basalis reacting
66
F=0.55 0 host egg
F=7.55** host eggs soaked for 3m¡nutes
F=1.93** host eggs soaked for 5 minutes
4 _Fc2.19** host eggs soaked 2 times for 5 minutes
F=2.29** host eggs soaked 3 times for 5 minutes
F=1.2 5 o host eggs soaked 1 time for 1 hour
i 1 1|L
3
Minutes
(a)
1
2
4
5

Fig. 12b. Number of female Trissolcus basalis (Wollaston) reacting
to the eggs of the host, Nezara viridula (L.), after
soaking in dichloromethane for different periods of time
is shown.

of female T. basalis reacting
68
28
24
. F=0.55
0 host egg
F=e2.59**
host
eggs
soaked
2
times
fo r
1
F_ 2.0 3 *__ _
host
eggs
soaked
3
times
fo r
1
_ F- 2.48**
host
eggs
s o a k ed
4
times
for
1
F- 0.64
host
eggs
soaked
1
time
f o r
4
1
JL
3
Minutes
(b)
hour
hour
hour
hours
1
2
4
5

Fig. 13. Number of female Trissolcus basalis (Wollaston) reacting
to crude kairomonal extract from eggs of the host, Nezara
viridula (L.), removed with dichloromethane by different
methods at certain time intervals is shown.

Number of female T. basalis reacting
70
100
90
80
T reatment totals
F=4.43tJL. solution from eggs soaked
1 time for 4 hours
F = 3.49** solution from eggs ground
with solvent
F = 4.10tJ! solution from eggs soaked
4 times for 1 hour
F= 0.84 solvent
Mi utes

71
those when the 12-hour-old eggs of N. viridula were immersed 1 time for
4 hours in the solvent, and those when the eggs were ground in dichloro-
methane. The results are summarized in Table 15 of the Appendix. From
the treatment totals it is evident that the solution with the highest
activity was prepared by grinding eggs of N. viridula However
analysis of the simple effects failed to reveal any statistically
significant difference between the methods under investigation. F values
are presented in Fig. 13.
Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the Crude
Kairomonal Extract from Eggs of the Host, N. viridula
Table 16 of the Appendix shows the percentage of each type of
behavior exhibited when different concentrations of the crude kairomonal
solution were used. It can be seen that all behavior steps become more
stereotyped when the concentration increases, except for the initial
random movement which has a reverse trend.
When introduced into the Petri dish, the female T. basalis starts
to run either on the margins of the treated filter paper or lateral
walls of the bottom of the container.
Sometimes the parasitoid stops, grooms its antennae, wings, and
abdomen then continues to move. This random movement is caused by some
sort of stimulus which has not been determined. It could be light
intensity or neurophysiological factors of the female T. basalis. This
behavior is also observed when no treatment has been applied to the
filter paper (Fig. 14). Kinesis occurs when as a result of random
movement the parasitoid encounters the treated spot and remains there
for a period of time. On the other hand, the percentage of parasitoids

Fig. 14. The behavior patterns of the female Trissolcus basalis
(Wollaston) on a filter paper treated with crude
kairomonal extract from eggs of the host, southern green
stink bug, Nezara viridula (L.), are diagrammed. Shown
are parasitoid: (a) in direct movement to the treated
spot; (a') in random movement to the spot; (a") after
antennal palpation, leaving the spot and starting to search;
(b) after searching, returning to the treated area for
reinforcement; and (b') leaving the spot for new cycle of
searching. Either a or a' will occur for every single
trial.

73
O T reated spot.
Female T. basalis.
Random (kinesis),
. chemotaxis,
searching,
reinforcement, and
^searching paths.

74
locating the treated spot randomly, decreases as the concentration of
the crude extract increases.
Chemotaxis is observed when the female parasitoid after random
movements inside the Petri dish and from a certain point moves directly
to the treated area and is arrested by the stimulus. Such oriehtation
type is directly proportional to the concentration of the crude extract.
Orientation of the female T basalis to the treated spot can occur
by random movement possibly kinesis or chemotaxis (Fraenkel and Gunn,
1961). In either event the following subsequent behavior patterns occur:
antennal palpation happens while the female parasitoid explores the
treated area and by means of the antennal flagellum "drums" the surface
of the area. After this phase the female T. basalis leaves the spot,
increases its velocity, and for a period of time searches the adjacent
area then returns to the spot where she repeats antennal palpation.
This cycle of behavior may be repeated many times.
There is evidence that the crude kairomonal extract under test
acted as a reinforcer stimulus, and the repetition of the parasitoid
adaptive behavior was a result of reinforcement (Fig. 14).
These behavioral activities are completed in a time period that is
highly correlated with the concentration of the solution under test.
Parameters of the described behavior patterns are presented in
Table 17. Concentration values were converted to logarithm and multiplied
5
by 10 so when, solving equations for "x" for any behavioral step the
"y" values should be entered as the probit corresponding to a certain
concentration value. The final "x" obtained is then transformed to
egg equivalents/ul by finding the antilogarithm and multiplying it by
-5
10 When solving for "y" the "x" original values have to be converted

75
as previously described and the "y" found is expressed in probits.
These values may be converted to percentage with a probit table (Fisher
and Yates, 1963).
By Table 17 of the Appendix, it can be seen that the regression
equation for random movement has a negative slope, indicating that for
every unit of increase in concentration of egg equivalents/ul there is
a decrease in 0.75 units for random movement. The equations for chemo-
taxis, antennal palpation, searching, and reinforcement had positive
slopes; consequently there is an increase in response when the concentra
tion increases. Correlation coefficient values were close to 1,
indicating a strong linear relationship between log concentrations of
the crude kairomonal extract and the behavioral responses of the female
parasitoid. The equations are graphically represented in Fig. 15.
The Concept of Stimulant Concentration
Stimulant Concentration (SC) can be defined as a term to express
the concentration of a chemical stimulant involved in inter and
intraspecific communication among insects, required to stimulate the
innate releasing mechanism of the insects tested resulting in a percentage
of a characteristic behavioral response in a natural or artificial
environment at a certain time interval. Concentration can be represented
by conventional metric units and its sub-multiples when the situation
requires, and it will depend upon the type of relationship involved, or
through other usual means as parts per million in a certain volume or
mass unit, or those measures already common to the chemo-entomological
field of transspecific chemical messengers as: egg equivalent/ul, body
equivalent/ul, abdomen equivalent/ml, etc.

Fig. 15a and b. Relationship of the female Trissolcus basalis
(Wollaston) exhibiting certain behavior patterns
when stimulated by different concentrations of
the crude kairomonal extract from eggs of the
host, Nezara viridula (L.), is shown: (a) random
movement (possibly kinesis) (b) chemotaxis.

Probit of female T. basalis
exhibiting chemotaxis
00
t
-<
II
3.38 + 0.7 6 x
Log concentration in egg eq.//ilx10
Probit of female T. basalis
exhibiting kinesis
ro
Fig. 15c and d. Relationship of the female Trissolcus basalis
(Wollaston) exhibiting certain behavior patterns
when stimulated by different concentrations of
the crude kairomonal extract from eggs of the
host, Nezara viridula (L.), is shown: (c) antennal
palpation, (d) searching.

Probit of female T. basa lis
exhibiting searching
03
T
<
II
3.50 + 0.6 7 x
Probit of female T. basalis
exhibiting antennal palpation

Fig. 15e. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.), is shown:
(e) reinforcement.
Fig. 15f. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation when stimulated by 10^ egg
equivalent solution per microliter from eggs of the host,
Nezara viridula (L.), versus time, is shown.

Probit of female T. basalis
6.34 0.98x
Pro bit of female T. basalis
exhibiting reinforcement
oo

82
Stimulant Concentration values for five behavior patterns of the
female parasitoid are shown in Table 17, with 95% confidence limits.
-3
The SC for random movement (possibly kinesis) was 1.23x10 egg
50
equivalents; this represents the concentration of the stimulant that
caused 50% of the female T. basalis to orient themselves to the treated
spot in approximately 3 minutes. Stimulant Concentration values for
the other behavior patterns can be interpreted the same way.
The Concept of Stimulant Dose
Stimulant Dose (SD) can be defined as a term used to express the
amount of a chemical stimulant involved in inter and intraspecific
communication among insects, required to stimulate the innate releasing
mechanism of the insects tested in a natural or artificial environment,
resulting in a percentage of responsiveness characteristic of the species
under study at a given time. Stimulant Dose units should represent the
exact amount of the stimulant utilized in any sort of bioassay,
either by direct application of the chemical on the animal tested or by
impregnation of a given surface or volume. Units of mass, volume, and
area of multiples and sub-multiples of the metric system can be used for
measurements of the doses.
Under the conditions of the actual experiment, all concentrations
were delivered with a constant amount of solution, that is, 1 ul which
+ 2 3
covered an area of (0.17 0.008) cm within a Petri dish of 28.46 cm
of internal volume. It was found that 1 N. viridula egg weighs
(533.96 15.75) ug a correction factor was determined to transform egg
equivalents in nanogram equivalents per square millimeter per cubic
centimeter. Any concentration can be expressed in terms of SD when

83
multiplying it by the factor "1066," consequently the SC values could
2 3
be transformed to SD in terms of Ng equivalents/mm /cm .
Equations of Table 17 of the Appendix, represent the Stimulant
Concentration and Stimulant Dose. They are statistically sound for
determinations of any SC or SD values within the range of the established
lines.
Stimulant Time (ST) data are presented in Tables 18 and 19 of the
Appendix. Stimulant Time was determined for antennal palpation when
-2
1 ul of 10 egg equivalent solution of the kairomonal crude extract was
applied on the filter paper. According to Table 18, the class limits of
time were converted to logarithm and since the logarithm of time for a
given dose is normally distributed (Hastings and Peacock, 1975), the
class limits were transformed to mid-point of log intervals. The
quantal response technique was utilized to determine the ST because
50
the number of female T. basalis palpating the treated spot at a given
time are unrelated in terms of responsiveness to the stimulus (Modifica
tion of the procedure used by Bliss, 1937) .
The correlation coefficient for ST was minus 0.945, and chi-square
7.54. The percentage of antennal palpation transformed to probits is
highly correlated with the mid-point of log intervals in seconds, and
the regression line appropriately describes this relationship (ata =0.05).
The Concept of Stimulant Time
Stimulant Time can be defined as a term to express the time required
for a defined dose of a chemical stimulant involved in inter and intra
specific communication among insects required to stimulate the innate
releasing mechanism of the insects tested in a natural or artificial

84
environment, resulting in a percentage of response characteristic of the
species under study.
Experiment 8: Effects of the Crude Kairomonal
Extract from Eggs of the Host, N. viridula in
the Orientation of the Female T. basalis
When introduced in the Petri dish the females T. basalis start to
orient toward the treated spot. Location of the area results either
from random movement or chemotaxis. During the orientation process,
the parasitoid stops, grooms the body and resumes searching. The
-4
grooming behavior is intensified when the concentration reached 10
egg equivalent. As the concentration increases the time required for
location of the treated area decreases. This trend can be seen in
Fig. 16. When the solvent was offered to the female T. basalis, she
required 27.8% of the time for the treatment totals to encounter the spot,
-4
and 19.9%, 21.0%, 16.1% and 16.0%, when the concentrations are 10 ,
-3 -2 -1
10 ,10 and 10 egg equivalents/ul respectively. The F-test
(= 5.78) at 0.05 level disclosed that the solutions tested affected
the orientation process of the parasitoid. It was found that the
-1
highest concentration, i.e., 10 egg equivalents/ul reduced the orienta
tion time 42.58% in comparison to the control (Table 20 of the Appendix).
Experiment 9: Enhancement of Host Location
by Scent Combinations
Responsiveness of the female TP. basalis to the scent combinations
in the olfactometer is summarized in Table 21 of the Appendix. Duncan's
test (Duncan, 1955) disclosed that the best velocity toward the scent
source was achieved when the crude kairomonal solution from eggs of the

Fig. 16. Percent of time spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper spot
with different concentrations of the crude kairomonal
extract from eggs of the host, Nezara viridula (L.), is
shown.

86

87
host was introduced into the dispenser tube. When compared to the
treatment with dichloromethane, the solution with 12.5 egg equivalent
caused a 47.05% increase in the speed of the female parasitoid in
relation to the control. The reactions induced by the remaining scent
combinations, including the control, were statistically equivalent at
0.05 level of significance.
When the crude kairomonal solution was offered to the parasitoids,
they tended to move down wind in a zig-zag pattern for a few seconds,
then change direction and in the same fashion move up wind toward the
scent source. The same pattern is observed for the scent-combination
treatments, however when the parasitoids are approximately 6 cm away
from the stimulant source, they turn back and later resume movement
toward the stimulus. Actually it is assumed that the concentration of
the volatile chemical(s) emanated from the treatment combinations
applied could be responsible for a situation of conflict (Marler and
Hamilton, 1967), consequently resulting in a decline in the female T.
basalis velocity.
Experiment 10: Normality Studies with the Crude
Kairomonal Solution from Eggs of the Host,
N. viridula
Previous experiments have indicated that orientation and velocity
of the female T. basalis toward the crude kairomonal extract from eggs
of the host southern green stink bug, N. viridula, were improved by
42.58 and 47.05% in relation to the control respectively. Evaluation of
the following results will indicate whether the behavior patterns
exhibited by the female parasitoid were triggered by a normal releaser.

88
Response to the Kairomonal Solution on the Filter Paper
The mean number of female T basalis exhibiting antennal palpation
is response to the crude kairomonal extract from eggs of N. viridula
is shown in Table 22 of the Appendix. Ninty-two percent of the popula
tion tested responded to the stimulus and no reaction was observed for
1/2
the solvent treatment. The results were transformed to Arcsin x.
i
(Bartlett, 1947) and the paired-difference t-test revealed the signifi
cant difference between the treatments.
Evaluation of Parasitism in Areas Treated with the Crude Kairomonal
Solution
The activity of the parasitoid was measured by the number of
host eggs (%) parasitized, when the filter paper was treated with
dichloromethane and with the crude kairomonal solution. The results
are presented in Table 23 of the Appendix. The t-test revealed a highly
significant difference between the treatments. The performance of the
female T^. basalis in terms of parasitism was improved by 1.69 times by
the addition of the egg extract to the egg masses.
Responses of the Female T. basalis to the Egg Shells and 12-Hour-Old Eggs
of the Host, N. viridula
Assessment of the crude kairomonal extract in triggering oviposition
of the female parasitoids were compared to the natural situation, when
egg masses of the southern green stink bug, Nc viridula were offered
to the female T. basalis under test. Responsiveness of the parasitoids
are shown, in Table 24 of the Appendix. Oviposition induced by the crude
kairomonal solution was 35.48% better than that observed for 6 12-hour-
old host eggs. Analysis of variance was conducted and the F-test =

89
44.70** confirmed that the parasitoid responses were affected by the
treatments assigned. Contrast of the means indicated that the crude
kairomonal extract was better than the other two treatments in stimu
lating oviposition of the female parasitoids.
Antennal Palpation Previous to Oviposition
All experimental data thus far obtained have shown that exposure
to crude kairomonal extract improves performance of female T. basalis
in many steps of the ovipositional behavior. At the present time there
is no indication that the isolated is other than a chemical cue acting
as an exaggerated releaser (Magnus, 1958; Staddon, 1975). It also is
not known what kind of interaction a supernormal releaser (Tinbergen,
1951) would have in long or short-term on the process of interspecific
behavior among insects.
It was observed that the crude kairomonal extract obtained, acted
upon the innate releasing mechanism system of the female T. basalis and
induced a series of fixed action patterns characteristic of the ovi
positional behavior of this parasitoid (refer to Fig. 6) It is
evident that the species-specific behavior of the female T. basalis
is part of a behavioral pattern (Wallace, 1973; Baerends, 1976) that
remains relatively constant throughout life.
Considering all behavior steps preceding oviposition, antennal
palpation at first contact with the host egg mass is the most
stereotyped and precisely timed behavior event. This pattern was
analyzed and the results are presented in Table 25 of the Appendix.
The null hypothesis was that of no difference on time, when the female
parasitoids drum the eggs and egg shells treated with 0.1 egg equivalent

90
of the crude kairomonal solution.
The t-test revealed no evidence of supernormality, that is, there
is insufficient evidence to reject the hypothesis under test (t=1.02)<,
The crude kairomonal extract is a highly specific releaser which
triggered the same behavior event on the same temporal basis of that
observed for 12-hour-old eggs subjected to oviposition by the female
T. basalis.

DISCUSSION
Trissolcus basalis (Wollaston) is a member of the Scelionidae
Family, Order Hymenoptera. This family includes many species that
have specialized as parasites of eggs of many insect groups, especially
Heteroptera and Orthoptera (Wilson, 1961; Davis and Krauss, 1963;
Davis, 1964; Cumber; 1964).
The mechanisms of N. viridula location by T. basalis have not been
completely determined; however, it is evident that this parasitoid
follows the basic steps in the process of parasitism as reported by
Salt (1937), Flanders (1939) and Doutt (1959), that is: host habitat
location, host location, host acceptance, and host suitability.
The aim of this investigation was to determine the factors
involved in the sequences of host location and host acceptance and the
characteristics of the crude kairomonal extract that mediate those
reactions.
A linear relationship was found between orientation of the
female parasitoid toward the scent source and the number of eggs
inside the dispenser tube. When the egcs are introduced and the scent
stimulates the female parasitoid, they start to move in a zig-zag
fashion and some parasitoids move down wind and later turn to the
source of stimulus by moving up wind. As the number of eggs increases
from ca. 20, some female |T. basalis start to "jump" when initially
stimulated by the crude kairomonal extract. This jumping act increases
91

92
in intensity as the number of eggs offered increases. Such response
has been observed in some other insects and it has been described as
part of the "activation stage" in the process of attraction (Mayer,
1973).
It is evident that the air stream that passes through the eggs of
the host southern green stink bug, N. viridula, acts as a carrier of
some volatile chemical(s) that could be present in the air spaces of
the exochorium and endochorium (Hinton, 1969; Chapman, 1971). It is
also possible that some chemical constituent of the chorium could
volatilize and reach the parasitoids.
Analysis of the simple effects revealed that the threshold in the
olfactometer for stimulation of the innate releasing mechanism of
the insect was five eggs.
The results of the Experiment 2 showed that orientation of the
female parasitoid toward eggs of the host is purposeful (Thorpe,
1963) even though the specialized literature (Clausen, 1940; De Bach,
1964; Gerling and Schwartz, 1974) suggests that discovery of a host by
entomophagous species is a random event.
Data from Experiment 3 support the view that both random movement
(possibly kinesis) and chemotaxis commonly occur in the process of
host location, although the latter appears to be the more important.
The act of orienting and finding the eggs of the host, N. viridula,
is usually interrupted by the parasitoid's selfgrooming behavior.
Such action is not fully understood even though Corbet (1973) ascribes
this pattern to a receptor-clearing process. However, it is our
understanding that either a conflict situation or an act of ritual-
ization may also be possible explanations for this phenomenon.

93
When the female parasitoid encounters the egg mass, she starts
to explore the chorial surface by drumming the antennal flagellum
against the egg walls, and occasionally moving from marginal eggs to
central eggs and vice-versa.
It is clear that the parasitoid reaches the source of stimulus
by a well defined sequence of locomotory actions which are character
istic of appetitive behavior (Tinbergen, 1951; Hess, 1962). This phase
ends when the female T. basalis stops antennal palpation and starts to
drill the egg-shell with her ovipositor. The appetitive phase is
accomplished in 220.20 + 33.17 seconds.
The consummatory behavior starts when the female parasitoid thrusts
its ovipositor against the exochorion initiating the process of ovi
posit ion. After laying the egg in the host substratum, T. basalis
withdraws its ovipositor from the host egg and begins the marking phase.
Locomotion is considerably reduced during the consummatory behavior,
and the parasitoid spends 137.10 + 5.79 seconds during this event.
It is axiomatic that the consummatory behavior is more stereo- >
typed than the appetitive behavior. This could be seen by comparing
the time spent in those two phases, and also by the variance of the
time parameter during these two behavioral cycles, i.e., it was
determined that the appetitive behavior had a variance 32.77 larger
than that of the consummatory phase.
The female T. basalis requires 359.25 + 64.55 seconds to
accomplish these two distinct phases of its ovipositional behavior.
This is the first report of these two behavior cycles among insects
which interact interspecifically.
Determination of these phases is extremely useful in assessing

94
potency and normality of the releaser(s) involved in interspecific
interactions. It also provides valuable information that could be
helpful in behavior manipulation when improvement of the parasitoid
performance is under consideration.
Responsiveness of the female T. basalis to male and female N.
viridula scent was 1.00 and 1.13 times better than that exhibited by
the male parasitoid, respectively. Combined parasitoid reactions to
male N. viridula were not different at 0.05. However, reactions of
the male and female T. basalis to the female host was highly signif
icant. The data support the rationale that the female southern green
stink bug provides more clues for its location by both male and female
parasitoids. It is possible that volatization of one or more specific
chemical(s) from the cement and wax layers of the epicuticle (Locke,
1974) will enhance host location in the natural environment.
When host male and female hemolymph was offered to both male and
female T. basalis, the males reacted poorly to the scent; however, the
females were highly stimulated and oriented to the tip of the dispenser
tube. Their reaction to the male and female southern green stink bug
hemolymph was 1.23 and 1.33 times higher than that shown by the male
parasitoids, respectively. The data corroborate the evidence that the
female T^. basalis has possibly a better chemosensory mechanism for
host location.
It has not been determined how certain specific chemicals present
in the N. viridula hemolymph, i.e., volatile fatty acids, glycerol,
hydrocarbons, amino acids, proteins, etc. (Florkin and Jeuniaux, 1974)
could reach the external environment and trigger a sequence of
behavior steps that might lead the parasitoid to the host. Actually,

95
it is assumed that those compounds could be eliminated during the
process of metabolism and gain the external environment through wax
canals (Ebeling, 1974) or pass to the eggs of the host.
Egg age is also a factor in stimulation of the innate releasing
mechanism of the females T. basalis. They are highly stimulated by
12- and 24-hour-old N. viridula eggs.
Mating behavior of T. basalis is performed by males that emerge
in advance of the females and the dominant male takes possession of
the egg mass and copulates with the emerging females (Wilson, 1961) .
Olfactometer tests revealed that a possible pheromone, attractive
to both male and female T. basalis, is present in parasitized egg
masses of the host N. viridula, as well as in egg-shells from host eggs
that have been parasitized. It was also observed that this attractant
has a considerable chemical stability; it was noticed that after 4
days under 25 + 1 C, 65 + 5% RH, and a photophase of 14 hours, the
activity of the "pheromone" remained equivalent to that of parasitized
eggs with adult T. basalis ready for emergence. At the present time
there is indication that such material could be present in the emerging
adult female parasitoid, or could be in the excrement inside the egg
shells as a result of the metabolism of the parasitoid. Males T.
basalis are not activated by non-parasitized host eggs.
Among the solvents used to remove the crude kairomonal extract,
dichloromethane emerged as the best in removing the active ingredient(s)
from the eggs of the host. Twelve-hour-old eggs have their kairomonal
activity removed when soaked in dichloromethane by the following
procedures: (a) soaking eggs one time for 4 hours; (b) soaking them
four times at 1 hour intervals; or (c) grinding them with the solvent.

96
In any of those cases the resulting suspension should be filtered
through a filter paper.
From results of Experiment 7 it isclear that visual clues are
not critical in the location of the N^. viridula eggs by the female
T. basalis. Orientation toward the area treated with the crude
kairomonal solution can happen either by random movement or chemotaxis.
In either situation some degree of grooming takes place, and is
-4
substantially increased when the concentration reaches 10 egg
equivalent/ul. However, grooming has been reported in the absence
of any applied treatment or presence of host eggs on the threshold
attraction limit.
This grooming behavior remains to be studied in more detail
to determine its real significance. Actually it is assumed that a
conflict situation is taking place, probably generated by chemical(s)
in vapor phase stimulating the innate releasing mechanism of the
parasitoid or an act of ritualization without any significance to the
performance of T. basalis.
Antennal palpation takes place when the parasitoid drums the
treated spot with its antennal flagellum. There is an indication
that the parasitoid responds to a contact chemical stimulant(s) that
will help in locating the host. Such rationale is corroborated by
the subsequent behavioral steps, i.e., searching and reinforcement.
Searching is characterized by an increase in velocity when
female T. basalis scans the area adjacent to the spot and is motivated
by the crude extract which acts as a reinforcer, that is, it induces
the parasitoid to repeat a behavioral cycleantennal palpation and
searching.

97
The data support the reasoning that the female T. basalis when
sensing the odor given off by possible volatile chemicals from the
treated region moves in that direction (chemotaxis). Such behavior
is adaptively advantageous since it enhances the parasitoids1 prospects
of finding hosts and producing progeny. Since the crude kairomonal
extract was isolated from the eggs of the host, and considering the
fact that T. basalis is strictly an egg parasitoid, it becomes evident
that the systems of sensory input of the female parasitoid is adapted
to seek eggs of its host in order to maximize fitness through success
ful occupation of its particular ecological niche and assuring the
success of the species through reproduction. The hierarchy of N.
viridula location by T. basalis has not been completely determined, but
it has been proven that the crude kairomonal extract from the eggs of
the southern green stink bug has no indication of supernormality.
As the concentration of the extract increases, the repetitive
frequency of the behavior patterns exhibited by T. basalis population
increases; thiff is particularly noticeable during reinforcement. Further
experiments are under development to determine whether such behavior
is an act of learning (Thorpe, 1963) or whether it is part of the
parasitoid's innate reaction to the kairomone of the host.
It is evident that the crude kairomonal extract evoked a
frequently repeated pattern of movement that is part of a behavioral
plan (Wallace, 1973; Baerends, 1976) which is relatively constant
throughout animal species. The occurrence of this plan was confirmed
by the highly significant correlation between the behavior patterns
and the concentration of the releaser. Chi-square values at <*=0.05
for random movement, chemotaxis, antennal palpation, searching, and

98
reinforcement were 3.76, 0.153, 0.046, 0.015, and 0.006, respectively.
There is evidence that the linear regression lines calculated are
representing adequately the relationship already discussed.
The concepts of Stimulant Concentration, Stimulant Dose, and
Stimulant Time and their median values, i.e., SC^, SD,_0, an<^ ST50'
were introduced here to assess the potency of the crude kairomonal
extract as a trigger for the behavioral responses studied. The
applicability of the concepts are extended to intraspecific communica
tion systems since the literature reports situations where behavioral
responses are related to pheromonal concentrations (Mayer, 1973;
Shorey and Gaston, 1964; Bartell and Lawrence, 1973: Shorey, 1973).
By the dose-response lines obtained, there is possibility that
female T. basalis populations can be manipulated genetically for
selection of individuals at the lower, median, or upper limits of the
lines in order to increase the number of desirable genetic combinations
resulting in an improvement of the efficiency of the parasitoid.
Research is under development to determine the nature of the compound(s)
in the crude extract tested.
This technique also can provide ways to detect the major tissue
or organ responsible for synthesis of transspecific chemical messengers
by comparisons of the SC,.,., SDrrt, and ST,.. of the crude extract or
dU dU oU
already identified chemicals from different sources of the insect body.
It can also furnish more accurate results when two or more active
chemicals in inter- or intraspecific communication are compared for
effectiveness. In many cases, this is done by comparing equal amounts
of the chemicals and the responses obtained are registered and evaluated.
Such a procedure is fallacious because use of the same amount of various

active chemicals will result in comparing materials that will
certainly require different concentrations for an optimal activity.
It may also be that two or more compounds will happen to have the
same SD^ for inducing a key behavioral response. In these situations
the selection must fall on that one which has the smallest variance
(Snedcor and Cochran, 1973) An immediate application of this proce
dure of selection by SC^, SD or STso' '*'n synt^es;*-s f the
natural compounds. There is a minimal margin for error in electing
a chemical for synthesis, when such chemical had SC, SD, and ST
values determined for an important behavioral response.
As the concentration increases the time spent during appetitive
behavior decreases. This is an obvious consequence of the
decreased time required for the female T. basalis to encounter the
treated area when the solution reaches its highest concentration level
This phenomenon can be observed by examining data from Experiment 8,
where it is evident that the time of location of the area with 10 ^
egg equivalent solution, by the female T. basalis is reduced 1.74
times when compared to the control (Dichloromethane).
The crude kairomonal solution also increases the parasitoid
velocity during the orientation phase 1.89 times when compared to the
control, and 1.49 and 1.34 times when contrasted with the 12-hour-old
eggs and three female hosts, respectively. However, when the crude
kairomonal solution was combined with scent of three female hosts and
when matched with ten 12-hour-old eggs, the speed of the female T.
basalis was reduced to the control level. This may have been caused
by a conflict situation resulting from the concentration of the
scents which induced that behavior and consequently reduced the

100
velocity of the female T. basalis.
From the evolutionary standpoint it is possible that the presence
of the adult hosts close to their eggs could act in some degree as
an inhibitor to the parasitoid activity, since it is during this stage
of development that the southern green stink bug, N. viridula, is highly
vulnerable to the attack of parasitoid species. This hypothesis is
under investigation, to disclose the interactions involved between
N. viridula and T. basalis, in order to assure a correct manipulation
of the parasitoid behavior via kairomone.
Studies in the area of supernormality have been extensively
reported as related to higher animals (Alcock, 1975; Hogan et al., 1975;
Staddon, 1975) These authors have suggested that physical factors
play a major role in triggering abnormal responses.
The results of the assays conducted and described throughout this
study imply that the crude kairomonal extract obtained from the eggs
of the host southern green stink bug, N. viridula, mediated behavioral
responses in the female parasitoid that could be regarded as within the
range of over-optimal (Magnus, 1958). Since the behavioral program
of the parasitoid is very closed (Mayr, 1974) experiments have been
undertaken to rule out any possibility of response-preference due to
interference of an extraneous supernormal releaser (Tinbergen, 1951;
Marler and Hamilton, 1967).
It is evident that dichloromethane did not interfere in the
performance of T. basalis. This is evident from data resulting from
the filter paper bioassay where no antennal palpation was recorded
for the treatment involving only this solvent. On the other hand,
92% of the females T. basalis tested responded to the crude

101
kairomonal extract by drumming the antennal flagella against the
area treated with 1 ul of 10 ^ egg equivalent solution.
Parasitism in areas treated with the kairomonal solution was
69.60% higher than in areas where no kairomonal extract was available.
So, there is evidence that the performance of the female T. basalis
is considerably improved by the presence of the extract obtained.
There is also indication that the crude kairomonal extract act
ed as an oviposition stimulant in the presence of a physical clue
(host egg-shells) Host egg-shells treated with a 10 ^ egg equivalent
induced the female parasitoid to bring the tip of the ovipositor in
contact with the egg-shell and assume the characteristic ovipositional
posture, and exhibited the rocking movement.
Unquestionably the crude kairomonal extract improved the effec
tiveness of the female T. basalis to an overoptimal level s already
discussed. The quality of the isolated releaser was assessed by
comparing female response to the normal rigidly fixed stereotyped
behavior pattern that occurs prior to a successful oviposition, i.e.,
antennal palpation. Statistically there was no difference between the
time spent in palpating intact 12-hour-old host eggs or host egg
shells treated with the kairomonal solution.

SUMMARY AND CONCLUSIONS
Interspecific interactions between the egg parasitoid, Trissolcus
basalis (Wollaston) and its host southern green stink bug, Nezara
viridula (L.), were studied as relating to host location and host
acceptance.
Laboratory studies indicated that the female parasitoids have a
remarkable ability to orient toward and find host eggs, especially
those not more than 24 hours old. A highly significant linear rela
tionship was found between the number of parasitoids attracted and number
of host eggs. The threshold for the female parasitoid stimulation was
determined to be five 12-hour-old host eggs. The data support the
reasoning that the "glue" that binds the host eggs, the exo- and
endochorion, and the internal contents of the egg, i.e., cytoplasm
and nucleus, are possibly the major reservoir of the kairomone.
Olfactometer assays indicated that the orientation of the
parasitoid to the eggs is in great extent purposeful rather than
random, as was thought by earlier researchers.
Temporal analysis of the ovipositional behavior revealed that
the female T. basalis encounters the host eggs through random movement,
chemotaxis, or combination of both. There are two distinct patterns
involved in the parasitoid ovipositional behavior: the appetitive
behavior and the consummatory behavior, with the latter being more
stereotyped than the former. Such distinction is of great importance
102

103
in assessing the potential characteristics cf chemical(s) involved
in interspecific communication.
Evaluation of cues that could act as "helpers" in location of
the host eggs revealed that presence of both male and female N.
viridula enhance host finding, and the latter elicited stronger
reactions than the former. Hemolymph of the southern green stink bug
was also tested, and the results showed that the males T. basalis
were not stimulated by the scent; however, the female parasitoids
were highly oriented to the source of stimulus. It was not determined
if or how chemicals from the hemolymph could be available to the
parasitoids in the external environment and act as a clue to T.
basalis.
Investigations with 4-day-old egg-shells from parasitized host
eggs and parasitized host eggs containing adult parasitoids ready
for emergence indicated that they were highly attractive to both
male and female T. basalis. It is possible that pheromone present in
the female parasitoid or in the metabolic excrements act as a mediator
to the mating behavior of this species.
The kairomonal extract from the eggs of N. viridula was obtained
with dichloromethane, which proved to be the most effective of three
methods of extraction in removing the active compound(s) with the
highest level of activity without interfering with the normal behavior
of the parasitoid.
Visual clues were not found to be as critical as chemical clue
in location of host eggs. The crude kairomonal extract induced five
behavioral patterns, i.e., random movement (possibly kinesis), chemo-
taxis, antennal palpation, searching and reinforcement, which are

104
highly linear related to the log concentration of the active material(s).
The data support the rationale that these reactions are triggered
by contact chemical(s) and compound(s) in vapor phase.
Mathematical models were established for every behavior pattern
elicited by the crude kairomonal extract and the concepts of Stimulant
Concentration, Stimulant Dose, Stimulant Time with their respective
median values were introduced as standard measures of the potency of
chemicals involved in interspecific and intraspecific communication
among insects.
It was demonstrated that the velocity of the parasitoid is
substantially increased by the kairomonal solution and it was found
that the time required during the orientation is reduced to 42% in
relation to the control.
Analysis of the experimental data proved that the performance
of the egg parasitoid T. basalis was improved by the crude kairomonal
extract, i.e. oviposition, time for orientation, velocity, parasitism,
etc. Since the behavior programs of parasitoid insects is rigidly
fixed, a very stereotyped behavior pattern was used to check the
normality of the releaser obtained. The results supported the
claim that the crude kairomonal solution when applied to egg-shells
of the host insect, N. viridula, elicited the same reactions as
host eggs when offered to the female T. basalis.
Identification of the chemical(s) from this crude kairomonal
extract will make it possible to better assess the potential usefulness
of the kairomone in controlling economic populations of the southern
green stink bug. Under laboratory conditions the isolated kairomonal
extract demonstrated that the behavior of the female T. basalis can

105
be manipulated.
Experiments in progress will provide more information about how
closed the program controlling this interspecific system is;
consequently, more tangible data will be gathered and analyzed in
detail in order to obtain a kairomone with risk of habituation
reduced to a minimum.

APPENDIX
SUMMARY OF THE EXPERIMENTAL DATA

107
Table 1. Analysis of variance for the simple effects between the number
of female Trissolcus basalis (Wollaston) orienting within a
single tube olfactometer containing eggs of the host, Nezara
viridula (L.) at 10 levels of time.
Source
of Variation
Degrees of
Freedom
Sum of
Squares
Mean
Square
F
Between
time within
0
egg
of N.
viridula
9
10.53
1.17
0.79
Between
time within
1
egg
of N.
viridula
9
9.73
1.08
0.73
Between
time within
2
eggs
of N.
viridula
9
5.10
0.56
0.38
Between
time within
3
eggs
of N.
viridula
9
8.90
0.98
0.66
Between
time within
4
eggs
of N.
viridula
9
12.03
1.33
0.90
Between
time within
5
eggs
of N.
viridula
9
34.72
3.86
2.62**
Error
180
265.50
1.47
'Highly significant at 0.05 level.
t

108
Table 2. Percentage of female Trissolcus basalis (Wollaston) found
within different sections of a "Y"-type olfactometer con
taining 12-hour-old host eggs (B), egg shells (B'), and
no eggs of the host, Nezara viridula (L.) at different
time intervals.
Treatments
Esposure time in minute
0 5 10 15 20 25 30
Section with
50 eggs (B)
Section with
egg shells (B)
Section with
no eggs (A)
0 9 16
0 3 4
100 88 80
31
5
64
44
8
48
52 63
6 5
42 32

109
Table 3. Analysis of variance for Trissoicus basalis (Wollaston)
orienting within a single tube olfactometer with the scent
of 0, 1, and 3 male Nezara viridula (L.).
Degrees of
Source of Variation Freedom
Sum of
Squares
Mean
Square
F
Male N. viridula
2
13.41
6.70
1.24
Time
9
16.79
1.86
0.35
Male and female T. basalis
1
7.00
7.00
1.30
N. viridula vs. time
18
111.17
6.18
1.15
N. viridula vs. T. basalis
2
2477.11
1238.55
230.13**
T. basalis vs. time
9
75.12
8.35
1.55
N. viridula x time x T.
basalis
18
1075.64
59.76
11.10**
Error
180
966.75
5.38
Total
239
4744.99

110
Table 4. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer with
the scent of 0, 1
and 3
female Nezara
viridula
(L.).
Degrees of
Sum of
Mean
Source of Variation
Freedom
Squares
Square
F
Female N. viridula
2
21.32
10.66
9.52**
Time
9
16.73
1.85
1.66
Male and female T. basalis
1
8.07
8.06
7.20**
N. viridula vs. time
18
23.59
1.31
1.17
N. viridula vs. T. basalis
2
22.85
11.43
10.21**
T. vasalis vs. time
9
29.43
3.27
2.92**
N. viridula x time x T.
basalis
18
31.89
1.77
1.58
Error
180
201.50
1.12
Total
239
355.40

Ill
Table 5. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within_a single tube olfactometer
with the scent of 0 and 3x10 cm of male Nezara viridula
(L.) hemolymph.
Degrees of
Source of Variation Freedom
Sum of
Squares
Mean
Square
F
Male N. viridula hemolymph
1
37.06
37.06
35.95**
Time
9
34.28
9.36
9.09**
Male and female T. basalis
1
20.31
20.31
19.69**
Hemolymph vs. time
9
44.63
4.96
4.81**
Hemolymph vs. T. basalis
1
49.51
49.51
48.00**
Time vs. T. basalis
9
13.63
1.51
1.47
Hemolymph x time x T.
basalis
9
25.43
2.82
2.74**
Error
120
123.75
1.03
Total
159
338.59

112
Table 6. Total number of Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer with different levels of
the male hemolymph of Nezara viridula (L.), averaged over
10 equally spaced time interval.
T. basalis sex
No. of Parasitoids Reacting to
Different Amounts of Male Host Hemolymph
Total
0 Cm3
3x10 3 Cm3
Male
125
119
244
Female
109
192
301
Total
234
311
545

113
Table 7. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting wi|hin3a single tube olfactometer to
the scent of 0 and 3x10 Cm of female Nezara viridula (L.)
hemolymph.
Source of Variation
Degrees of
Freedom
Sum of
Squares
Mean
Square
F
Female N. viridula
hemolymph
1
75.62
75.62
105.52**
Time
9
74.35
8.26
11.53**
Male and female T. basalis 1
42.02
42.02
58.64**
Hemolymph vs. time
9
35.50
3.94
5.50**
Hemolymph vs. T. basalis
1
81.22
81.22
113.34**
Time vs. T. basalis
9
12.10
1.34
1.87
Hemolymph x time x T.
basalis
9
17.15
1.90
2.66**
Error
120
86.00
0.72
Total
159
423.97

114
Table 8. Total number of Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer with different levels of
the hemolymph of female Nezara viridula (L.), averaged over
10 equally spaced time interval.
T. basalis sex
No. of Parasitoids Reacting to
Different Amounts of Male Host Hemolymph
Total
0 .Cm3
3xl0~3 Cm3
Male
125
123
248
Female
109
221
330
Total
234
344
578

115
Table 9. Analysis of variance for the simple effects between the number
of female Trissolcus basalis (Wollaston) orienting within a
single tube olfactometer containing 50 eggs of the host,
Nezara viridula (L.) at different host age levels.
Source of Variation
Degrees of
i Freedom
Sum of
Squares
Mean
Square
F
Between time
within
no egg
9
2.52
0.28
0.28
Between time
within
12-hour
old eggs
9
107.00
11.89
8.44**
Between time
within
24-hour
old eggs
9
65.73
7.30
5.18**
Between time
within
48-hour
old eggs
9
15.02
1.67
1.18
Error
120
169.00
1.41

116
Table 10. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
to eggs of the host, Nezara viridula (L.) at different
levels of parasitism.
Source
of Variation
Degrees of
Freedom
Sum of
Squares
Mean
Square
F
Eggs of
N. viridula
3
184.42
61.47
63.46**
Time
9
126.74
14.08
14.54**
Male and female T. basalis 1
46.51
46.51
48.01**
Eggs vs
. time
27
54.39
2.01
2.08**
Eggs vs
. T. basalis
3
158.36
52.79
54.49**
Time vs
. T. basalis
9
62.92
6.99
7.22**
Eggs x
time x T. basalis
27
39.95
1.48
1.53
Error
240
232.50
0.97
Total
319
905.80

117
Table 11. Analysis of variance for the simple effects between Trissolcus
basalis (Wollaston) orienting within a single tube olfacto
meter containing 50 eggs of the host, Nezara viridula (L.) at
different levels of parasitism, averaged over 10 equally
spaced time interval.
Degrees of
Source of Variation Freedom
Sum of
Squares
Mean
Square
F
Between time within no host
egg for male T. basalis
9
5.00
0.55'
0.56
Between time within 12-hour
old N. viridula eggs for
male T. basalis
9
8.90
0.98
. 1.01
Between time within parasi
tized eggs of N. viridula
for male T. basalis
9
48.02
5.33
5.49**
Between time within egg
sheels of parasitized eggs
for male T. basalis
9
29.00
3.22
3.31**
Between time within no host
egg for female T basalis
9
15.60
1.73
1.78
Between time within 12-hour
old N. viridula eggs for
female T. basalis
9
31.12
3.45
3.55**
Between time within parasi
tized eggs of N. viridula
for female T. basalis
9
106.12
11.79
12.15**
Between time within egg
shells of parasitized eggs
for female T. basalis
9
40.22
4.46
4.59**
Error
240
232.50
0.97

118
Table 12. Treatment totals for the number of female Trissolcus
basalis (Wollaston) orienting within a single tube
olfactometer containing 50 egg equivalents of the crude
kairomonal extract from eggs of the host, Nezara
viridula (L.) removed by different solvents.
1
2
3
4
5
6
7
8
9
10
Water
19
24
16
24
18
19
16
13
13
12
Ethanol
27
32
31
29
25
25
19
19
16
15
Hexane
30
33
36
33
24
22
20
15
15
15
Dichloromethane
31
32
35
41
21
19
15
14
16
11
Total
107
121
118
127
88
85
70
61
60
53

119
Table 13. Treatment means for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer con
taining crude kairomonal extract from eggs of the host,
Nezara viridula (L.), removed by 4 solvents, after 4 minutes
of exposure.
Water
Ethanol
Hexane
Dichloromethane
4.8a
5.8ab
6.6b
00
NO
O
a,b,CMeans with
ferent at
the same exponential letter
0.05 level (Duncan, 1955).
are not significantly dif-

120
Table 14. Analysis of variance for the number of female Trissolcus
basalis (Wollaston) orienting within a single tube
olfactometer containing curde kairomonal extract from
fifty 12-hour-old eggs of the host, Nezara viridula (L.),
extracted with dichloromethane by different methods.
Source of Variation
Degrees of
Freedom
Sum of
Squares
Mean
Square
F
Methods
9
383.29
42.59
24.33**
Time
9
131.34
14.59
8.34**
Method vs. time
81
210.76
2.60
1.49
Error
300
525.00
1.75
Total
399
1250.39

121
Table 15. Treatment totals for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer con
taining 50 egg equivalent solutions obtained from 12-hour-old
eggs of the host, Nezara viridula (L.) with dichloromethane
by different methods.
Removal Method
with
Dichloromethane
Time
in
minute
Total
1
2
3
4
5
6
7
8
9
10
Solvent only
14
10
10
8
14
11
15
10
9
10
111
Eggs washed 1 time
for 4 hrs.
24
33
34
27
24
23
23
22
19
17
246
Eggs ground with
the solvent
17
31
33
34
32
33
29
29
30
29
297
Eggs washed 4 times
for 1 hour 18
17
28
31
29
27
23
21
19
18
231
Total
73
91
105
100
99
94
90
82
77
74
885

122
Table 16. Percentage of female Trissolcus basalis (Wollaston) exhibiting
different types of behavior patterns on filter paper when sti
mulated by different concentrations of crude kairomonal extract
from eggs of the host, Nezara viridula (L.).
Dose in
Egg Equivalents
per Microliter
Percent of Behavior Patterns
Kinesis
(*)
Chemotaxis
Antennal
Palpation
Searching
Reinforcement
1J
o
1
-p
83.33
16.67
33.33
20.00
16.67
lo"3
53.33
46.67
66.67
43.33
40.00
10-2
13.33
86.67
90.00
73.33
73.33
11
1
O
r\
13.33
86.67
93.33
86.67
86.67
(*) Random movement, possibly kinesis.

123
Table 17. Parameters for the behavior patterns of the female parasitoid,
Trissolcus basalis (Wollaston) when stimulated lj>y four equally
spaced concentrations, ranging from 10 to 10 egg equiva-
lents/yl of crude kairomonal extract from eggs of the host
Nezara viridula (L.).
Behavior
Pattern
Parameters
Equation
Correlation
Coefficient
95% Confidence
Limits for
SC50*
95% Confidence
Limits for
SD50**
Kinesis
y=6.58-0.75x
-0.94
(1.23x10 3+0.02x10~3)
(1.31+0.21)
Chemotaxis
y=3.38+0.76x
0.95
(1.26x10_3+0.02x10_3)
(1.34+0.21)
Antennal
palpation
y=3.96+0.69x
0.97
(0.31x10_3+2.6x10_3)
(0.33+2.77)
Searching
y=3.50+0.67x
0.99
(1.67x10-3+1.1x10_3)
(1.78+1.17)
Reinforce
ment
y=3.38+0.71x
0.99
(1.87xl0_3+0.87xl0~3)
(1.99+0.92)
Values expressed in egg equivalent/pl
, 2
'"Values expressed in Ng eg. /mm .

124
Table 18. Percentage of the female parasitoid Trissolcus basalis
(Wollaston) exhibiting antennal palpation on a treated fil
ter paper spot, when stimulated by 10 egg equivalents/yl
of the crude kairomonal extract of the eggs of the host,
Nezara viridula (L.) at different time intervals.
Time Interval
in Seconds
Log of Time
Interval x 10
Mid-point of
Log Intervals
Antennal
Palpation (%)
1-15
1.000 -
2.176
1.588
39
16 30
2.204 -
2.477
2.340
21
31 45
2.491 -
2.653
2.572
14
46 60
2.662 -
2.778
2.720
9
61 75
2.785 -
2.875
2.830
7
76 90
2.880 -
2.954
2.917
9
91 -105
2.959 -
3.021
2.990
1

125
Table 19. Parameters for the Stimulant Time for the female parasitoid
Trissolcus basalis (Wollaston) exhibiting antennal palpation,
when stimulated by 10 egg equivalent/pl of the crude kairo-
monal extract from eggs of the host, Nezara viridula (L.).
Behavior
Parameters
Pattern
Correlation
95% Confidence
Equation
Coefficient
Limits for ST- *
dU
Antennal
Palpation
y=6.34-0.98x
-0.94
(2.34+0.19)
^Values expressed in seconds.

126
Table 20. Average time in seconds spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper area with
different concentrations of the crude kairomonal extract from
eggs of the host, Nezara viridula (L.).
1 pi of
Dichloro-
10~4 Egg
10 3 Egg
10"2 Egg
10 1 Egg
methane/pl
Equivalent/pl
Equivalent/pl
Equivalent/pl
Equivalent/pl
195.93a
134.40b
147.70b
113.00b
112.57b
means with the same exponential letter were not significantly dif
ferent at a=0.05 (Duncan, 1955).

127
Table 21. Average velocity in cm/s of the female Trissolcus basalis
(Wollaston) in orienting within a single tube olfactometer
containing scent combinations from the host, Nezara
viridula (L.).
10 12-Hr-
10 12-Hr-
10 12-Hr-
10 12-Hr-
3
12.5
Old N.
Old Eggs
Old Eggs
Old Eggs
Female
Egg
Dichloro-
viridula
Plus 3
Plus Di-
of N.
N.
Equi-
methane
Eggs Plus
12.5 Egg
Eq. So
lution
Female
N.
viridula
Chloro-
methane
viridula
viridula
valent
Solu
tion
0.63a
o
o
CD
0.70a
0.80ab
0.83ab
0.89ab
1.19b
Means with the same exponential letter were not significantly dif
ferent at a=0.05 (Duncan, 1955).

128
Table 22. Percentage of female Trissolcus basalis (Wollaston)3exh^biting
antennal palpation on filter paper treated with 10 cm of
solutions containing different concentrations of crude kairo-
monal extract from eggs of the host Nezara viridula (L.).
Antennal Palpation
Replicates
0.1 Egg Equivalent
Dichloromethane
%
Arcsin X.1^
i
%
A V V2
Arcsin X.
i
1
90.00
71.57
0.00
2.85
2
80.00
63.43
0.00
2.85
3
100.00
87.15
0.00
2.85
4
90.00
71.57
0.00
2.85
5
100.00
87.15
0.00
2.85
I
460.00
380.87
0.00
14.25
X
92.00
76.17
0.00
2.86

129
Table 23. Percentage of eggs of Nezara viridula (L.) parasitized by
Trissolcus basalis (Wollaston) w^en placed on filter paper
treated with 5x10^ cin of a 10 egg equivalent solution
in dichloromethane.
No. of Eggs
per Replicate
5x10 2cm^ of a 10 2 Egg
Equivalent Solution
5x10 2cm^ of
Dichloromethane
No. of Eggs
Parasitized
% Parasitism
No. of Eggs
Parasitized
% Parasitism
10
1.00
10.00
6.00
60.00
10
8.00
80.00
5.00
50.00
10
2.00
20.00
9.00
90.00
10
2.00
20.00
10.00
100.00
10
10.00
100.00
6.00
60.00
10
0.00
0.00
3.00
30.00
E
23.00
230.00
39.00
390.00
X
3.83
38.33
6.50
65.00
Ratio = 1.69

130
Table 24. Percentage of female Trissolcus basalis (Wollaston) exhibiting
ovipositional behavior when stimulated by 6 12-hour-old eggs
and egg shells of the host, Nezara viridula (L.).
Replicates
-3 3
10 cm of Dichloro-
Methane on Egg Shells
of the Host
6 12-Hour-Old
Eggs of the
Host
10 cm of a 0.1
Egg Equivalent
Solution on Egg
Shells of the
Host
1
0.0
40.0
90.0
2
10.0
60.0
70.0
3
30.0
70.0
80.0
4
10.0
70.0
80.0
5
20.0
70.0
100.0
l
70.0
310.0
420.0
X
14.0a
62.0b
84.0C
3. ]d C
5 Means were significantly different at 0.05 level (Duncan, 1955).

131
Table 25. Time required by the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation previous to a successful
oviposition in the eggs and egg shells of the host Nezara
viridula (L.).
Time in Seconds
Egg Shells Treated
with 10 Cm of a
0.1 Egg Equivalent
Replicates
6-12-Hour-Old Eggs
Solution
1
210
96
2
158
118
3
67
90
4
136
102
5
97
99
6
69
56
7
286
60
8
31
75
9
104
103
10
54
106
11
131
90
12
58
180
13
59
101
14
111
75
15
106
70
16
98
65
17
88
87
18
112
125
19
125
60
20
55
77
21
58
96
22
239
103
23
34
202
24
158
96
25
37
56
26
56
120
27
45
93
28
88
50
29
63
53
30
53
307
£
2986
3011
X
99.53
100.36
t = 1.02

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139
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BIOGRAPHICAL SKETCH
Fernando Joo Montenegro de Sales was born in Fortaleza,
Ceara', Brazil, on March 18, 1944. He attended Cole'gio Castelo
Branco, Colegio S. Joo, and was graduated from Colegio Estadual
do Ceara' (Liceu) in 1961. After graduation he attended Curso
Picaneo, a pre-university course in Fortaleza, Ceara' for one year.
He obtained his undergraduate education at Escola de
Agronomia da Universidade Federal do Ceara", Fortaleza, Ceara"",
Brazil, where he received his Bachelor of Science degree in Agronomy,
in 1966.
In 1967 he was hired to serve as a faculty member at the
Escola de Agronomia da Universidade Federal do Ceara (= Department
of Plant Science of the College of Agriculture, Federal University
of Ceara) Since then, he has been teaching and working in ento
mological projects and published papers in his major field.
In 1970 he was awarded a United States Agency for International
Development (USAID) scholarship to work toward the degree of Master
of Science at the University of Arizona. He granted his MS degree
in February, 1972, before returning to Brazil.
In 1974 he was awarded a new USAID scholarship to work toward
the Doctor of Philosophy degree. From 1974 to 1978 he was a graduate
student in the Department of Entomology and Nematology, University of
Florida. He now returns to the Federal University of Ceara', where
140

he has tenure, to continue his teaching and research in Entomology.
He is married to the
Ceara, Brazil. They have
former Elane Garcia de
one daughter, Cynthia,
Arruda of Fortaleza,
and one son, Elano.
141

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.
Dr-f George. Allen, Chairman
Dr
^^rofessor of Entomology
I certify that 1 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.
LL
Ic
Dr. Reece I. Sailer
Graduate Research Professor of
Entomology
1 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 a. .dj-sep^t-ation for the
degree of Doctor of Philosophy.
Dr. James H. Tumlinson
Associate 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
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 qualit^w aw a dissertation for the
degree of Doctor of Philosophy.
Dr. Frncis w.1Zettler
Associate Professor of Plant
Pathology

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.
March, 1978
Dean, Graduate School



84
environment, resulting in a percentage of response characteristic of the
species under study.
Experiment 8: Effects of the Crude Kairomonal
Extract from Eggs of the Host, N. viridula in
the Orientation of the Female T. basalis
When introduced in the Petri dish the females T. basalis start to
orient toward the treated spot. Location of the area results either
from random movement or chemotaxis. During the orientation process,
the parasitoid stops, grooms the body and resumes searching. The
-4
grooming behavior is intensified when the concentration reached 10
egg equivalent. As the concentration increases the time required for
location of the treated area decreases. This trend can be seen in
Fig. 16. When the solvent was offered to the female T. basalis, she
required 27.8% of the time for the treatment totals to encounter the spot,
-4
and 19.9%, 21.0%, 16.1% and 16.0%, when the concentrations are 10 ,
-3 -2 -1
10 ,10 and 10 egg equivalents/ul respectively. The F-test
(= 5.78) at 0.05 level disclosed that the solutions tested affected
the orientation process of the parasitoid. It was found that the
-1
highest concentration, i.e., 10 egg equivalents/ul reduced the orienta
tion time 42.58% in comparison to the control (Table 20 of the Appendix).
Experiment 9: Enhancement of Host Location
by Scent Combinations
Responsiveness of the female TP. basalis to the scent combinations
in the olfactometer is summarized in Table 21 of the Appendix. Duncan's
test (Duncan, 1955) disclosed that the best velocity toward the scent
source was achieved when the crude kairomonal solution from eggs of the


20
used per mass because this is in the range of the threshold for
stimulation. To reach that number, large egg masses were broken apart
and 3 rows made up of 1, 2, and 3 eggs were left as the best physical
support upon which the female T. basalis could oviposit.
Completely recovered female T. basalis were exposed to the egg
masses when bottom and cover were put together. The container with the
parasitoid was held vertically and 25 cm away from a fluorescent light
source (cool white Sylvania F 15T8-CW), with the cover toward the light.
The experiment was repeated 20 times, and both eggs and parasitoid
were discarded after each test. All ovipositional behavior was
observed and every step was timed.
Experiment 4: Cues Useful in Location of the Host,
N. viridula, by the Parasitoid T. basalis
Scent of Male N. viridula
Responsiveness of the male and female T. basalis to the scent of
the male host was determined using the single tube olfactometer described
in Experiment 1. The dispenser tube was joined to another glass chamber
measuring 10.0 cm in length and 1.3 cm in diameter (Fig. 3). All other
experimental procedures were similar to those described for Experiment
1, except for the use of both male and female parasitoids for the
treatment combinations.
Treatment combinations consisted of 2 levels of the parasitoid,
male or female; 3 host levels of 0, 1, or 3 male N. viridula; and 10
time intervals obtained by 1 minute readings recorded every 10 minutes.
The glass chamber was used to confine the host insects during the
experiment. When using 0 male host, the chamber was kept empty with a


93
When the female parasitoid encounters the egg mass, she starts
to explore the chorial surface by drumming the antennal flagellum
against the egg walls, and occasionally moving from marginal eggs to
central eggs and vice-versa.
It is clear that the parasitoid reaches the source of stimulus
by a well defined sequence of locomotory actions which are character
istic of appetitive behavior (Tinbergen, 1951; Hess, 1962). This phase
ends when the female T. basalis stops antennal palpation and starts to
drill the egg-shell with her ovipositor. The appetitive phase is
accomplished in 220.20 + 33.17 seconds.
The consummatory behavior starts when the female parasitoid thrusts
its ovipositor against the exochorion initiating the process of ovi
posit ion. After laying the egg in the host substratum, T. basalis
withdraws its ovipositor from the host egg and begins the marking phase.
Locomotion is considerably reduced during the consummatory behavior,
and the parasitoid spends 137.10 + 5.79 seconds during this event.
It is axiomatic that the consummatory behavior is more stereo- >
typed than the appetitive behavior. This could be seen by comparing
the time spent in those two phases, and also by the variance of the
time parameter during these two behavioral cycles, i.e., it was
determined that the appetitive behavior had a variance 32.77 larger
than that of the consummatory phase.
The female T. basalis requires 359.25 + 64.55 seconds to
accomplish these two distinct phases of its ovipositional behavior.
This is the first report of these two behavior cycles among insects
which interact interspecifically.
Determination of these phases is extremely useful in assessing


34
Data were analyzed for a completely randomized design with 3
treatments and 4 replications. Contrast of the means was done by
Duncan's test (Duncan, 1955).
Antennal Palpation Previous to Oviposition
Egg shells treated with 1 ul of a 10 1 egg equivalent solution
of the crude kairomonal extract and 12-hour-old eggs of N. viridula
were utilized to assess the degree of normality of the isolated releaser.
The time spent by the female T. basalis palpating the eggs and treated
egg shells previous to the contact of the ovipositor with the chorium
was recorded as the measurement of evaluation.
The bioassay consisted in exposing 1 healthy 3-day-old female
parasitoid without ovipositional experience to the treatments. The
procedure was replicated 30 times and the amount of time spent by T.
basalis exhibiting antennal palpation was recorded. The methodology was
similar to that of the previous section except for the points already
mentioned and analysis of the data, which consisted of applying a
paired-difference t-test to the results.


127
Table 21. Average velocity in cm/s of the female Trissolcus basalis
(Wollaston) in orienting within a single tube olfactometer
containing scent combinations from the host, Nezara
viridula (L.).
10 12-Hr-
10 12-Hr-
10 12-Hr-
10 12-Hr-
3
12.5
Old N.
Old Eggs
Old Eggs
Old Eggs
Female
Egg
Dichloro-
viridula
Plus 3
Plus Di-
of N.
N.
Equi-
methane
Eggs Plus
12.5 Egg
Eq. So
lution
Female
N.
viridula
Chloro-
methane
viridula
viridula
valent
Solu
tion
0.63a
o
o
CD
0.70a
0.80ab
0.83ab
0.89ab
1.19b
Means with the same exponential letter were not significantly dif
ferent at a=0.05 (Duncan, 1955).


Fig. 2.
A "Y" type olfactometer is pictured.


97
The data support the reasoning that the female T. basalis when
sensing the odor given off by possible volatile chemicals from the
treated region moves in that direction (chemotaxis). Such behavior
is adaptively advantageous since it enhances the parasitoids1 prospects
of finding hosts and producing progeny. Since the crude kairomonal
extract was isolated from the eggs of the host, and considering the
fact that T. basalis is strictly an egg parasitoid, it becomes evident
that the systems of sensory input of the female parasitoid is adapted
to seek eggs of its host in order to maximize fitness through success
ful occupation of its particular ecological niche and assuring the
success of the species through reproduction. The hierarchy of N.
viridula location by T. basalis has not been completely determined, but
it has been proven that the crude kairomonal extract from the eggs of
the southern green stink bug has no indication of supernormality.
As the concentration of the extract increases, the repetitive
frequency of the behavior patterns exhibited by T. basalis population
increases; thiff is particularly noticeable during reinforcement. Further
experiments are under development to determine whether such behavior
is an act of learning (Thorpe, 1963) or whether it is part of the
parasitoid's innate reaction to the kairomone of the host.
It is evident that the crude kairomonal extract evoked a
frequently repeated pattern of movement that is part of a behavioral
plan (Wallace, 1973; Baerends, 1976) which is relatively constant
throughout animal species. The occurrence of this plan was confirmed
by the highly significant correlation between the behavior patterns
and the concentration of the releaser. Chi-square values at <*=0.05
for random movement, chemotaxis, antennal palpation, searching, and


74
locating the treated spot randomly, decreases as the concentration of
the crude extract increases.
Chemotaxis is observed when the female parasitoid after random
movements inside the Petri dish and from a certain point moves directly
to the treated area and is arrested by the stimulus. Such oriehtation
type is directly proportional to the concentration of the crude extract.
Orientation of the female T basalis to the treated spot can occur
by random movement possibly kinesis or chemotaxis (Fraenkel and Gunn,
1961). In either event the following subsequent behavior patterns occur:
antennal palpation happens while the female parasitoid explores the
treated area and by means of the antennal flagellum "drums" the surface
of the area. After this phase the female T. basalis leaves the spot,
increases its velocity, and for a period of time searches the adjacent
area then returns to the spot where she repeats antennal palpation.
This cycle of behavior may be repeated many times.
There is evidence that the crude kairomonal extract under test
acted as a reinforcer stimulus, and the repetition of the parasitoid
adaptive behavior was a result of reinforcement (Fig. 14).
These behavioral activities are completed in a time period that is
highly correlated with the concentration of the solution under test.
Parameters of the described behavior patterns are presented in
Table 17. Concentration values were converted to logarithm and multiplied
5
by 10 so when, solving equations for "x" for any behavioral step the
"y" values should be entered as the probit corresponding to a certain
concentration value. The final "x" obtained is then transformed to
egg equivalents/ul by finding the antilogarithm and multiplying it by
-5
10 When solving for "y" the "x" original values have to be converted


Ill
Table 5. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within_a single tube olfactometer
with the scent of 0 and 3x10 cm of male Nezara viridula
(L.) hemolymph.
Degrees of
Source of Variation Freedom
Sum of
Squares
Mean
Square
F
Male N. viridula hemolymph
1
37.06
37.06
35.95**
Time
9
34.28
9.36
9.09**
Male and female T. basalis
1
20.31
20.31
19.69**
Hemolymph vs. time
9
44.63
4.96
4.81**
Hemolymph vs. T. basalis
1
49.51
49.51
48.00**
Time vs. T. basalis
9
13.63
1.51
1.47
Hemolymph x time x T.
basalis
9
25.43
2.82
2.74**
Error
120
123.75
1.03
Total
159
338.59


120
Table 14. Analysis of variance for the number of female Trissolcus
basalis (Wollaston) orienting within a single tube
olfactometer containing curde kairomonal extract from
fifty 12-hour-old eggs of the host, Nezara viridula (L.),
extracted with dichloromethane by different methods.
Source of Variation
Degrees of
Freedom
Sum of
Squares
Mean
Square
F
Methods
9
383.29
42.59
24.33**
Time
9
131.34
14.59
8.34**
Method vs. time
81
210.76
2.60
1.49
Error
300
525.00
1.75
Total
399
1250.39


116
Table 10. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer
to eggs of the host, Nezara viridula (L.) at different
levels of parasitism.
Source
of Variation
Degrees of
Freedom
Sum of
Squares
Mean
Square
F
Eggs of
N. viridula
3
184.42
61.47
63.46**
Time
9
126.74
14.08
14.54**
Male and female T. basalis 1
46.51
46.51
48.01**
Eggs vs
. time
27
54.39
2.01
2.08**
Eggs vs
. T. basalis
3
158.36
52.79
54.49**
Time vs
. T. basalis
9
62.92
6.99
7.22**
Eggs x
time x T. basalis
27
39.95
1.48
1.53
Error
240
232.50
0.97
Total
319
905.80


63
and female parasito ids happened at the 2nd or 3rd time interval.
Analysis of variance for the simple effects are shown in Table 11
of the Appendix. It can be observed that the response of the male
parasitoid to the blank tube without host eggs and to 50 12-hour-old
host eggs were statistically identical. The response of male parasitoids
to the 12-hour-old eggs was not high enough to rule out chance factor.
On the other hand, 50 parasitized host eggs containing T. basalis
ready to emerge, and 50 egg shells from parasitized eggs 4 days after
emergence, triggered a highly significant response in the male parasitoids.
Female parasitoid response was statistically high for the different
situations of the egg host except for the blank, when no eggs were
available (Table 11 of the Appendix).
Experiment 6: Reactions of the Female T. basalis to the
Kairomonal Solutions Prepared with Different Solvents
Since it was demonstrated that female T. basalis exhibit a
noticeable preference for sites containing eggs of its host N. viridula,
the next step was to extract the chemical (s) from eggs of the host.
Four solvents were used and the activities of the crude kairomonal
extract obtained tested in the olfactometer.
Results of the evaluation process are shown in Table 12 of the
Appendix. Treatment totals indicated that the maximum response of the
female parasitoid to:the crude kairomonal extract obtained with the
solvents was at the 4th minute of observation. Data from this time
interval were submitted to analysis of variance and the F-test (=12.14**)
was highly significant.
Contrast of the means was done by the Duncan's procedure (Duncan,
1955) and the results are presented in Table 13 of the Appendix. The


15
The olfactometer was placed on a white horizontal surface and ca.
109 cm away from 2 fluorescent lamps (Sylvania F40 cwx Lifeline) and
the apparatus was properly positioned to allow uniform dispersion of
the female parasitoids inside it.
Each experimental unit consisted of 10 healthy female T. basalis
within the main tube. Previous to any treatment, they were allowed to
complete recovery from the process of immobilization and run inside
the olfactometer for no less than 5 minutes, and after every single
treatment they were discarded.
Treatments were prepared in a spare dispenser. Host eggs were
introduced with soft forceps then they were covered by a thin cotton
layer. This dispenser containing the treatment was then used to replace
the blank one in a very quick stroke to avoid parasitoid escape.
Responsiveness of the female T. basalis was measured in terms of the
number of parasitoids that concentrated within 10 cm of the tip of the
dispenser. The readings were performed at 1 minute intervals for a
period of 10 minutes following insertion of the treated dispenser.
Experiment 1 consisted of 13 treatments (numbers of eggs) with 4
replicates applied to every experimental unit. The levels of those
treatments consisted of 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, and
300 eggs of the host, N. viridula. Reliability of the readings were
compared with a blank with no eggs applied to the testing parasitoids.
Regression analysis was carried out with the treatments transformed
to logarithm and multiplied by 10. The response of the female T. basalis
was measured as the percentage of individuals orienting to the scent
source after 3 minutes of exposure.
A test for lack of fit was conducted to check the adequacy of the


Probit of female T. basalis
exhibiting chemotaxis
00
t
-<
II
3.38 + 0.7 6 x
Log concentration in egg eq.//ilx10
Probit of female T. basalis
exhibiting kinesis
ro

115
Table 9. Analysis of variance for the simple effects between the number
of female Trissolcus basalis (Wollaston) orienting within a
single tube olfactometer containing 50 eggs of the host,
Nezara viridula (L.) at different host age levels.
Source of Variation
Degrees of
i Freedom
Sum of
Squares
Mean
Square
F
Between time
within
no egg
9
2.52
0.28
0.28
Between time
within
12-hour
old eggs
9
107.00
11.89
8.44**
Between time
within
24-hour
old eggs
9
65.73
7.30
5.18**
Between time
within
48-hour
old eggs
9
15.02
1.67
1.18
Error
120
169.00
1.41


Fig. 8. Number of male and female Trissolcus basalis (Wollaston)
reacting to combined levels of (a) hemolymph of male
Nezara viridula (L.) and (b) hemolymph of female N. viridula
at different time intervals is shown.


28
old or less. The eggs were ground with dichloromethane and the resulting
suspension filtered through a Whatman No. 1 qualitative filter paper.
The ratio of egg and solvent was 1 egg:l ul of dichloromethane.
Dilutions were then prepared to provide the concentrations used during
the experiment, such that 1 ul of the prepared dilutions would contain
-4 -3 -2 -1
10 ,10 ,10 and 10 egg equivalents.
The experiments were conducted within 5 cm Petri dishes with lids
lined with Whatman filter paper as described in Experiment 3. The
experimental unit consisted of 30 healthy 3-day-old female T. basalis
with no prior ovipositional experience. The females were chilled and
transferred to a plain white surface then individually covered with the
bottom of a Petri dish. One microliter of the test solution was dropped
+ 2
on the center of a filter paper covering an area of (0.17-0.008) cm .
The solvent was allowed to evaporate and the paper then transferred to
the inside of a Petri dish cover. Fully recovered female parasitoids
were exposed to the crude extract when bottom and cover were put together.
The container with the parasitoid was held vertically and 25 cm away
from a fluorescent light source (cool white Sylvania F15T8-CW), with the
cover toward the light.
Preliminary assays without solution were conducted to eliminate
any bias due to the light or handling during the experimental procedure.
The activity of the parasitoids was measured in terms of their ability
to locate the treated spot and their subsequent reactions when ejqposed
to different concentrations of the crude extract.
Responsiveness to the different stimuli were recorded in terms of
percentage of females exhibiting kinesis, chemotaxis, antennal palpation,
searching and reinforcement.


110
Table 4. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer with
the scent of 0, 1
and 3
female Nezara
viridula
(L.).
Degrees of
Sum of
Mean
Source of Variation
Freedom
Squares
Square
F
Female N. viridula
2
21.32
10.66
9.52**
Time
9
16.73
1.85
1.66
Male and female T. basalis
1
8.07
8.06
7.20**
N. viridula vs. time
18
23.59
1.31
1.17
N. viridula vs. T. basalis
2
22.85
11.43
10.21**
T. vasalis vs. time
9
29.43
3.27
2.92**
N. viridula x time x T.
basalis
18
31.89
1.77
1.58
Error
180
201.50
1.12
Total
239
355.40


Fig. 15c and d. Relationship of the female Trissolcus basalis
(Wollaston) exhibiting certain behavior patterns
when stimulated by different concentrations of
the crude kairomonal extract from eggs of the
host, Nezara viridula (L.), is shown: (c) antennal
palpation, (d) searching.


SUMMARY AND CONCLUSIONS
Interspecific interactions between the egg parasitoid, Trissolcus
basalis (Wollaston) and its host southern green stink bug, Nezara
viridula (L.), were studied as relating to host location and host
acceptance.
Laboratory studies indicated that the female parasitoids have a
remarkable ability to orient toward and find host eggs, especially
those not more than 24 hours old. A highly significant linear rela
tionship was found between the number of parasitoids attracted and number
of host eggs. The threshold for the female parasitoid stimulation was
determined to be five 12-hour-old host eggs. The data support the
reasoning that the "glue" that binds the host eggs, the exo- and
endochorion, and the internal contents of the egg, i.e., cytoplasm
and nucleus, are possibly the major reservoir of the kairomone.
Olfactometer assays indicated that the orientation of the
parasitoid to the eggs is in great extent purposeful rather than
random, as was thought by earlier researchers.
Temporal analysis of the ovipositional behavior revealed that
the female T. basalis encounters the host eggs through random movement,
chemotaxis, or combination of both. There are two distinct patterns
involved in the parasitoid ovipositional behavior: the appetitive
behavior and the consummatory behavior, with the latter being more
stereotyped than the former. Such distinction is of great importance
102


26
5 replications. Selection of the solvent that removed most of the
active ingredient(s) was done by contrasting the treatment means at
the 4th minute interval, utilizing Duncan's test (Duncan, 1955).
Dichloromethane Washes Required for Removal of the Kairomone from Eggs
of N. viridula
From the 4 solvents tested, dichloromethane was selected for
utilization throughout this assay, based on results presented later in
this text.
Treatments in this experiment consisted in soaking 12-hour-old
(or less) N. viridula eggs in dichloromethane for different periods of
time. For every single wash fresh aliquots of dichloromethane were used.
No re-use or recycling was permitted through the bioassay.
After the soaking process the washed eggs were spread on a Whatman
No. 1 qualitative filter paper to allow solvent evaporation. The ratio
of host eggs per milliliter of dichloromethane during the washing
(=soaking) process was 0.025/1.
After a given washing cycle, and complete solvent evaporation, the
eggs were placed in a spare dispenser tube, and later used to replace
the blank tube in the olfactometer with the female parasitoid. The
testing procedure was similar to that described for Experiment 1, except
for the analysis of the data.
During this experiment, 10 treatments including a blank with no
eggs, were applied to the experimental units.. The treatments consisted
of 50 N. viridula eggs treated as follows: host eggs soaked 1 time for
3 minutes, soaked 1 time for 5 minutes, soaked 2 times for 5 minutes,
3 times for five minutes, soaked 1, 2, 3, and 4 times for 1 hour, and


121
Table 15. Treatment totals for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer con
taining 50 egg equivalent solutions obtained from 12-hour-old
eggs of the host, Nezara viridula (L.) with dichloromethane
by different methods.
Removal Method
with
Dichloromethane
Time
in
minute
Total
1
2
3
4
5
6
7
8
9
10
Solvent only
14
10
10
8
14
11
15
10
9
10
111
Eggs washed 1 time
for 4 hrs.
24
33
34
27
24
23
23
22
19
17
246
Eggs ground with
the solvent
17
31
33
34
32
33
29
29
30
29
297
Eggs washed 4 times
for 1 hour 18
17
28
31
29
27
23
21
19
18
231
Total
73
91
105
100
99
94
90
82
77
74
885


123
Table 17. Parameters for the behavior patterns of the female parasitoid,
Trissolcus basalis (Wollaston) when stimulated lj>y four equally
spaced concentrations, ranging from 10 to 10 egg equiva-
lents/yl of crude kairomonal extract from eggs of the host
Nezara viridula (L.).
Behavior
Pattern
Parameters
Equation
Correlation
Coefficient
95% Confidence
Limits for
SC50*
95% Confidence
Limits for
SD50**
Kinesis
y=6.58-0.75x
-0.94
(1.23x10 3+0.02x10~3)
(1.31+0.21)
Chemotaxis
y=3.38+0.76x
0.95
(1.26x10_3+0.02x10_3)
(1.34+0.21)
Antennal
palpation
y=3.96+0.69x
0.97
(0.31x10_3+2.6x10_3)
(0.33+2.77)
Searching
y=3.50+0.67x
0.99
(1.67x10-3+1.1x10_3)
(1.78+1.17)
Reinforce
ment
y=3.38+0.71x
0.99
(1.87xl0_3+0.87xl0~3)
(1.99+0.92)
Values expressed in egg equivalent/pl
, 2
'"Values expressed in Ng eg. /mm .


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
THE BEHAVIOR OF THE EGG PARASITOID TRISSOLCUS BASALTS
(WOLLASTON) (HYMENOPTERA: SCELIONIDAE) IN RESPONSE
TO KAIROMONES PRODUCED BY ITS HOST, THE SOUTHERN GREEN
STINK BUG NEZARA VIRIDULA (L.) (HEMIPTERA: PENTATOMIDAE)
By
Fernando Joo Montenegro de Sales
March, 1978
Chairman: Dr. George E. Allen
Major Department: Entomology and Nematology
The mediators of behavior patterns among insects have been a focal
point of research in recent years as potential tools for management of
insect pest populations. Laboratory studies were conducted to reveal
the factors involved in the interspecific communication between the egg
parasitoid, Trissolcus basalis (Wollaston) and its host, the southern
green stink bug, Nezara viridula (L.). Temporal analysis of the female
parasitoid ovipositional behavior showed that location of the egg
masses of the host takes place by random movement (kinesis), chemotaxis,
and combination of both. It was demonstrated that the ovipositional
behavior is divided in two distinct steps: the appetitive behavior and
the most stereotyped consummatory behavior.
Olfactometer tests indicated that orientation toward the host eggs
is in great extent purposeful rather than random, and that the male and
female stink bug scents as well as their hemolymph act as cues in
eliciting orientation of T. basalis toward the eggs. The data implied
that the host egg age is a factor in stimulating the quest for eggs,
and the parasitoid mating behavior is possibly mediated by a pheromone.
xiii


49
both. After the physical contact, antennal palpation, oviposition, and
marking follows in a very defined sequence. Those behavioral steps
were observed when the number of eggs per mass is within the threshold
limit, that is, 6 eggs of N. viridula per mass.
The broken lines of the Fig. 6 indicate repetition of a series of
behavioral steps that ultimately leads the parasitoid to satiation.
Tracking of time during this cycle was omitted.
Experiment 4: Cues Useful in Location
of the Host. N. viridula by the Parasitoid T. bassalis
Scent of Male N. viridula
Responsiveness of the male and female T. basalis to the scent of
different levels of the male N. viridula in the single tube olfactometer
was determined by the analysis of variance of the treatment combinations.
As shown in Table 3 of the Appendix, parasitoid and host factors interact.
There is evidence that host levels and parasitoid levels do not act
independently, that is, a change in the levels of the host correspond to
a response by the male and female parasitoid which is different in
direction and intensity. Analysis of the simple effects by combination
of 10 levels of time is shown in Fig. 7a. Both male and female T.
basalis reacted to the scent of the male host levels combined, F.values
were 6.20** and 13.87** respectively. The differences in variability
was assigned to 1 and 3 male N. viridula, since no significant response
was observed at 0 level (F=0.80). Response of the female parasitoid
sharply increased when 3 male hosts were placed in the olfactometer.
Scent of Female N,. viridula


126
Table 20. Average time in seconds spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper area with
different concentrations of the crude kairomonal extract from
eggs of the host, Nezara viridula (L.).
1 pi of
Dichloro-
10~4 Egg
10 3 Egg
10"2 Egg
10 1 Egg
methane/pl
Equivalent/pl
Equivalent/pl
Equivalent/pl
Equivalent/pl
195.93a
134.40b
147.70b
113.00b
112.57b
means with the same exponential letter were not significantly dif
ferent at a=0.05 (Duncan, 1955).


INTRODUCTION
The ability of insects to compete with man for the products of
agriculture is a continuing challenge to entomologists. In recent
years increased scientific and public awareness of the environment has
greatly increased the complexity of the problem. To meet the challenge
entomologists have resorted to a variety of new methods designed to
solve or at least alleviate this problem of controlling insects with
minimum detrimental effects to the environment. One of these new
methods involves research on chemical substances that influence inter
and intraspecific behavior of insects.
The southern green stink bug, Nezara viridula (L.) is a cosmopolitan
insect pest destructive to many crops of economic importance throughout
the world. Until now most of the control measures have relied on chemical
insecticides however with the demand for a cleaner environment, new
opportunities have been opened to alternatives that could assure an
acceptable injury level and meet the new standards set for the agroeco
systems common to this insect.
The scelionid parasitoid Trissolcus basalis (Wollaston) has been
utilized as one of those alternatives, and in fact, the specialized
biocontrol literature includes it as one of the major tools in balancing
the populations of many stink bug species, in the United States and
other countries. However there is a dearth of quantitative information
regarding the potential of this parasitoid as a major control agent.
1


131
Table 25. Time required by the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation previous to a successful
oviposition in the eggs and egg shells of the host Nezara
viridula (L.).
Time in Seconds
Egg Shells Treated
with 10 Cm of a
0.1 Egg Equivalent
Replicates
6-12-Hour-Old Eggs
Solution
1
210
96
2
158
118
3
67
90
4
136
102
5
97
99
6
69
56
7
286
60
8
31
75
9
104
103
10
54
106
11
131
90
12
58
180
13
59
101
14
111
75
15
106
70
16
98
65
17
88
87
18
112
125
19
125
60
20
55
77
21
58
96
22
239
103
23
34
202
24
158
96
25
37
56
26
56
120
27
45
93
28
88
50
29
63
53
30
53
307
£
2986
3011
X
99.53
100.36
t = 1.02


112
Table 6. Total number of Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer with different levels of
the male hemolymph of Nezara viridula (L.), averaged over
10 equally spaced time interval.
T. basalis sex
No. of Parasitoids Reacting to
Different Amounts of Male Host Hemolymph
Total
0 Cm3
3x10 3 Cm3
Male
125
119
244
Female
109
192
301
Total
234
311
545


Fig. 6. Ovipositional ethogram of the female parasitoid Trissolcus
basalis (Wollaston) when stimulated by six 12-hour-old
eggs of the host, Nezara viridula (L.), is shown.


64
data implied that dichloromethane was the best solvent in removing the
active ingredient(s) from the host eggs. Activity of hexane was
similar to the ethanol but different from water. Water had the lowest
activity, which was statistically identical to ethanol.
Number of Dichloromethane Washes Required for Removal of the Kairomonal
Extract from Eggs of N. viridula
Evaluation of the: methods utilized consisted in offering the eggs
of N. viridula to the female parasitoids after soaking in dichloromethane.
Responsiveness of the female T. basalis was measured inside the
olfactometer and the results are shown in Table 14 of the Appendix.
Methods and time do not interact; they are independent of one another
(F=l.49).
Treatment responses are shown in Figures 12 a and b. Removal of
the kairomone from eggs of the host was partial for all methods tested,
except for the procedures of soaking eggs 1 time for 1 and 4 hours.
Analysis of the simple effects indicated that the activity present
on the eggs treated by those methods elicited responses from the female
parasitoids not significant at 0.05 level.
Reaction of the Female T. basalis to Kairomonal Solutions Prepared by
Different Methods
Responsiveness of the female parasitoids to the crude kairomonal
extract obtained by 3 techniques was compared when host eggs were: (a)
soaked 1 time for 4 hours in the solvent; (b) ground with the solvent;
(c) soaked 4 times at 1-hour intervals.
A summary of the female parasitoid reactions in the single tube
olfactometer is shown in Fig. 13. Solutions with best activity were


92
in intensity as the number of eggs offered increases. Such response
has been observed in some other insects and it has been described as
part of the "activation stage" in the process of attraction (Mayer,
1973).
It is evident that the air stream that passes through the eggs of
the host southern green stink bug, N. viridula, acts as a carrier of
some volatile chemical(s) that could be present in the air spaces of
the exochorium and endochorium (Hinton, 1969; Chapman, 1971). It is
also possible that some chemical constituent of the chorium could
volatilize and reach the parasitoids.
Analysis of the simple effects revealed that the threshold in the
olfactometer for stimulation of the innate releasing mechanism of
the insect was five eggs.
The results of the Experiment 2 showed that orientation of the
female parasitoid toward eggs of the host is purposeful (Thorpe,
1963) even though the specialized literature (Clausen, 1940; De Bach,
1964; Gerling and Schwartz, 1974) suggests that discovery of a host by
entomophagous species is a random event.
Data from Experiment 3 support the view that both random movement
(possibly kinesis) and chemotaxis commonly occur in the process of
host location, although the latter appears to be the more important.
The act of orienting and finding the eggs of the host, N. viridula,
is usually interrupted by the parasitoid's selfgrooming behavior.
Such action is not fully understood even though Corbet (1973) ascribes
this pattern to a receptor-clearing process. However, it is our
understanding that either a conflict situation or an act of ritual-
ization may also be possible explanations for this phenomenon.


Fig. 15a and b. Relationship of the female Trissolcus basalis
(Wollaston) exhibiting certain behavior patterns
when stimulated by different concentrations of
the crude kairomonal extract from eggs of the
host, Nezara viridula (L.), is shown: (a) random
movement (possibly kinesis) (b) chemotaxis.


16
model in describing the relationship between the percentage of female
parasitoids orienting to the kairomonal source and the treatments
applied.
To determine the threshold for stimulation of the female T. basalis
the same experimental procedure was used; however the treatments applied
to the experimental units consisted of 0, 1, 2, 3, 4, and 5 eggs of the
southern green stink bug.
A completely randomized 6 x 10 factorial experiment with 4
replications was developed for testing the response of the female
parasitoid when stimulated by different levels of the eggs of N. viridula
in order to find the threshold of stimulation.
Experiment 2: Orientation of the Female T. basalis
Inside a "Y" Type Olfactometer.
Orientation of the female parasitoid was determined by the "Y"
type olfactometer shown in Fig. 2. Connections of the parallel tubes,
to vacuum, and air flow meter were with plastic tubing. The parasitoid
releasing tube and the filter tube were linked to the "Y" connections
through hollowed rubber stoppers. Organdi screen at one end of the
parallel tubes prevented movement of the parasitoids to the section "C",
the "Y" connection that is linked to the air flow meter.
The experimental unit consisted of 100 healthy female T. basalis
transferred to the parasitoid releasing tube by the method of Experiment
1. Twenty females were tested in each of five olfactometers of the same
type, under the same experimental conditions.
The air current passing through the filter tube of the olfactometer
was 6.6 cm^/s, and the apparatus was positioned properly on a white
horizontal surface and a blank test was run to detect any tendentious


52
Orientation of the parasitoid to the scent source was analyzed and
the results are presented in Table 4 of the Appendix. A highly signifi
cant interaction indicated that the responsiveness of the parasitoids
vary with the levels of the female host. Response of the male parasitoid
was lower than the response exhibited by the female (Fig. 7b) and the
F-test was significant for both parasitoid sexes, at 0.05 levl.
Responsiveness of male and female T. basalis combined over 10 time
intervals were not significantly different (F=1.67) for three male host
levels utilized at 0.05 level of significance. When female hosts were
offered to the male and female parasitoids, the combined response was
highly significant, F=45.42**. Most of this is accounted by the female
parasitoid reaction.
Scent of Male N. viridula Hemolymph
It was observed that male and female T. basalis responded differently
to the different levels of the male host hemolymph. As shown in Table 5,
the three-factor interaction implies that the male host hemolymph
versus T. basalis differs with the levels of time.
Data for the treatment totals are presented in Table 6 of the
Appendix. There is an indication that the male parasitoid did react
poorly to the male hemolymph levels when averaged over 10 levels of time.
Analysis of the simple effects confirmed that assumption as the F-test
was not significant at 0.05 level of significance (F=0.45). On the other
hand the female "T. basalis exhibited a response to the male N. viridula
hemolymph, which was highly significant, F=83.60**.
The two-factor interaction observed for male host hemolymph versus
T. basalis suggests that the responses of the male and female parasitoid


LIST OF ILLUSTRATIONS
Figure Page
1. A single glass tube olfactometer is pictured 14
2. A "Y" type olfactometer is pictured 18
3. The glass chamber is shown connected to a single glass
tube olfactometer 22
4.Relationship between percentage of female Trissolcus basalis
(Wollaston) responding within a single tube olfactometer
versus different levels of eggs of the host southern green
stink bug, Nezara viridula (L.) is shown 37
5a and b. Female Trissolcus basalis (Wollaston) exhibits pre-
ovipositional behavior in an egg mass of its host, Nezara
viridula (L.). Antennal palpation of (a) marginal egg and
(b) central egg is shown 41
5c and d. Female Trissolcus basalis (Wollaston) exhibits ovi-
positional behavior on an egg mass of its host, Nezara viridula
(L.). Shown are (c) drilling the chorium for ovipositing and
(d) ovipositor thrust and marking the egg after oviposition.. 43
5e and f. Female Trissolcus basalis (Wollaston) exhibits ovi-
positional behavior on an egg mass of its host, Nezara
viridula (L.). Oviposition of the central eggs by drilling
the chorium, (e) the lateral wall, and (f) the operculum
is shown 46
6. Ovipositional ethogram of the female parasitoid Trissolcus
basalis (Wollaston) when stimulated by six 12-hour-old eggs
of the host, Nezara viridula (L.) is shown 48
7. Number of male and female Trissolcus basalis (Wollaston)
reacting to (a) scent of 0, 1, and 3 male Nezara viridula
(L.) and (b) 0, 1, and 3 female N. viridula, averaged over
10 equally spaced time intervals is shown 51
8. Number of male and female Trissolcus basalis (Wollaston)
reacting to combined levels of (a) hemolymph of male Nezara
viridula (L.) and (b) hemolymph of female N. viridula at
different time intervals is shown 55
x


Number of female T. basalis reacting
66
F=0.55 0 host egg
F=7.55** host eggs soaked for 3m¡nutes
F=1.93** host eggs soaked for 5 minutes
4 _Fc2.19** host eggs soaked 2 times for 5 minutes
F=2.29** host eggs soaked 3 times for 5 minutes
F=1.2 5 o host eggs soaked 1 time for 1 hour
i 1 1|L
3
Minutes
(a)
1
2
4
5


95
it is assumed that those compounds could be eliminated during the
process of metabolism and gain the external environment through wax
canals (Ebeling, 1974) or pass to the eggs of the host.
Egg age is also a factor in stimulation of the innate releasing
mechanism of the females T. basalis. They are highly stimulated by
12- and 24-hour-old N. viridula eggs.
Mating behavior of T. basalis is performed by males that emerge
in advance of the females and the dominant male takes possession of
the egg mass and copulates with the emerging females (Wilson, 1961) .
Olfactometer tests revealed that a possible pheromone, attractive
to both male and female T. basalis, is present in parasitized egg
masses of the host N. viridula, as well as in egg-shells from host eggs
that have been parasitized. It was also observed that this attractant
has a considerable chemical stability; it was noticed that after 4
days under 25 + 1 C, 65 + 5% RH, and a photophase of 14 hours, the
activity of the "pheromone" remained equivalent to that of parasitized
eggs with adult T. basalis ready for emergence. At the present time
there is indication that such material could be present in the emerging
adult female parasitoid, or could be in the excrement inside the egg
shells as a result of the metabolism of the parasitoid. Males T.
basalis are not activated by non-parasitized host eggs.
Among the solvents used to remove the crude kairomonal extract,
dichloromethane emerged as the best in removing the active ingredient(s)
from the eggs of the host. Twelve-hour-old eggs have their kairomonal
activity removed when soaked in dichloromethane by the following
procedures: (a) soaking eggs one time for 4 hours; (b) soaking them
four times at 1 hour intervals; or (c) grinding them with the solvent.


108
Table 2. Percentage of female Trissolcus basalis (Wollaston) found
within different sections of a "Y"-type olfactometer con
taining 12-hour-old host eggs (B), egg shells (B'), and
no eggs of the host, Nezara viridula (L.) at different
time intervals.
Treatments
Esposure time in minute
0 5 10 15 20 25 30
Section with
50 eggs (B)
Section with
egg shells (B)
Section with
no eggs (A)
0 9 16
0 3 4
100 88 80
31
5
64
44
8
48
52 63
6 5
42 32


LITERATURE REVIEW
Current Status of the Host Southern Green Stink Bug,
Nezara viridula (L.) and Its Parasitoid
Trissolcus basalis (Wollaston).
The Southern Green Stink Bug. Nezara viridula (L.)
Origin
According to Van Duzee (1917) Nezaara viridula (L.) was first
described by Linnaeus in 1758 under the scientific name Cimex viridulus.
Linnaeus' description was based on specimens collected in India. The
first new world record for the species is from the West Indies, and
since then the species has been redescribed by various authors under
numerous other scientific names.
Freeman (1940) placed the species into the genus Nezara of Amyot
and Serville, 1843 and Drake (1920) indicated that three color varieties
are recognized as: smaragdula (Fabricius), torguata (Fabricius) and
heptica Horvath.
Distribution
The southern green stink bug, N. viridula (L.) is widely distributed
throughout the world. It is found in Europe, Asia, Africa, and Americas
(DeWitt and Godfrey, 1972). Van Duzee (1917) and Jones (1918) have
pointed out that as many other insect pests in this Country, N.
viridula was introduced from West Indies and is established in Virginia,
Florida, Louisiana, South Carolina, Georgia, Alabama, Mississippi,
4


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30
Experiment 9: Enhancement of Host Location
by Scent Combinations
Responsiveness of the female T. basalis to combination of scents of
the kairomonal solution, host eggs, and solvent was tested with the
single tube olfactometer and the olfactometer-glass chamber tube as
shown in Figs. 1 and 3 respectively.
For every single treatment 5 female T. basalis under the same
biological conditions of the previous experiments were introduced into
the single tube olfactometer as described in Experiment 1 and submitted
to 7 different scent combinations.
The first treatment consisted of 3 healthy 15-day-old virgin female
N. viridula in the glass chamber. Treatment 2 consisted of 10 12-hour-
old eggs of the host combined with 3 female N. viridula offered
simultaneously. The eggs were placed inside the olfactometer and
approximately 0.5 cm away from the tip of the dispenser. The adult
female hosts were introduced in the glass tube chamber.
The third treatment consisted in applying 25 ul of dichloromethane
to a thin piece of cotton measuring 0.5 x 0.6 cm which after evaporation
was introduced into a spare dispenser tube.
The next treatment was 25 ul of the solution containing 12.5 egg
equivalents of the crude kairomonal solution in dichloromethane was
dispensed on a thin cotton piece. After solvent evaporation the treated
cotton was placed inside the dispenser tube as in the previous test.
Treatment 5 consisted of introducing 10 12-hour-old eggs of N.
viridula into the olfactometer and 0.5 cm away from the tip of the
dispenser. Treatments 6 and 7 were identical to 5 except that a thin
piece of cotton treated with 25 ul of dichloromethane and 25 ul of the


5
Texas, New Mexico, Arizona, California. Davis and Krauss (1963) have
reported its recent importation to Hawaii.
Host plants
Nezara viridula (L.) is a phytophagous insect with a broad range
of host plants. Hoffman (1935) indicates that this insect attacks
both monocotyledons and dicotyledons. Among the former he pointed out
that Graminae are the most important, and within the dicotyledons he
stated that 29 families are injured and ranked the following in order
of importance: Leguminosae having 27 species damaged, Cruciferae with
8 and Solanaceae with 6 species. Drake (1920), Gallo et al. (1970),
Todd (1973), Issa (1973), Turnipseed and Kogan (1976) have reported
this insect as feeding on radish, mustard, turnip, collard, cauliflower,
cabbage, okra, peas, beans, peanut, tomato, potato, cotton, tobacco,
pepper, eggplant, sunflower, sugar cane, corn, orange, lime, peach,
pecan, rise, snap bean, squash, cucumber, soybean, lemon, and grapefruit.
Drake (1920) also reports a number of weeds that serve as host to the
bug as: pokeweed, Phytolacca decandra; lamb's quarters, Chenopodium
spp.; nut grass, Cypepus esculentus L.; spiny amaranth, Amaranthus
spinosus; beggarweed, Desmodium spp.; crotolaris, Crotolaris spp.; wild
grape, Vitis spp.; castor bean, Ricinus communis L.; maypops, Passiflora
incarinata L.; and wild plum, Prunus spp.
In spite of the broad spectrum of host plants, N. viridula does
not breed in all those plants and only occassionally feeds on a number
of them. Drake (1920) indicates that the southern green stink bug has
a remarkable preference for the legumes, with the greatest degree of
preference when those plants are in the stage of fruit formation.


86


29
Data obtained through the experiment were submitted to probit
analysis for quantal response experiments (Finney, 1971). Subsequently,
regression equations were established for all behavioral patterns
exhibited by the female parasitoids then Stimulant Concentration (SC^)
and Stimulant Dose (SD^) values were determined for those patterns.
-2
The Stimulant Time for a 10 egg equivalent solution was
calculated for antennal palpation; however, the procedure is applicable
to any behavior pattern.
Experiment 8: Effects of the Crude Kairomonal Extract
from Eggs of the Host, N. viridula in the
Orientation of the Female T. basalis
The methodology and the experimental material utilized in this test
was identical to those used in Experiment 7, except that the experimental
unit consisted of 10 healthy 3-day-old female T. basalis, and a different
statistical design.
Treatments were four equally spaced concentrations of the crude
-4 -1
kairomonal extract, ranging from 10 to 10 egg equivalents/ul, plus
a check which consisted of 1 ul of dichloromethane dropped on the center
of the filter paper and after evaporation, exposed to the experimental
units.
Effects of the treatments were observed by tracking the time spent
by the female parasitoids in orienting themselves to the treated spot
for the first antennal contact.
A completely randomized design with 3 replications was developed
for checking the effect of the kairomonal concentrations on the
orientation time of the female parasitoid. The F-test was determined
and contrast of the means was done by Duncan's technique (Duncan, 1955).


128
Table 22. Percentage of female Trissolcus basalis (Wollaston)3exh^biting
antennal palpation on filter paper treated with 10 cm of
solutions containing different concentrations of crude kairo-
monal extract from eggs of the host Nezara viridula (L.).
Antennal Palpation
Replicates
0.1 Egg Equivalent
Dichloromethane
%
Arcsin X.1^
i
%
A V V2
Arcsin X.
i
1
90.00
71.57
0.00
2.85
2
80.00
63.43
0.00
2.85
3
100.00
87.15
0.00
2.85
4
90.00
71.57
0.00
2.85
5
100.00
87.15
0.00
2.85
I
460.00
380.87
0.00
14.25
X
92.00
76.17
0.00
2.86


Fig. 10. Number of male Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown.


7
shells. Drake (1920) indicated that no individuals have been observed
to feed while clustered; but just before or subsequent to molting, the
nymphs become active, scatter more or less and begin to feed. The
nymphs like the adults, are usually found upon those portions of the
plant on which they prefer to feedthe tender growing shoot and
specially the developing fruit. Jones (1918) and Drake (1920) reported
that during the summer, the period from egg to adult is about 35 days
with temperature conditions having an important effect.
Trissolcus basalis (Wollaston)
Regulation of southern green stink bug is attributed to biotic
and abiotic factors. A lot of work has been done in Japan in relation
to population dispersion and control. Kiritani (1965) suggests that
mortality factors work in a stage-specific way, i.e. parasite against
eggs, weather factors against the first instar and predators against
the second. He also indicates that the complex age structure during
the breeding seasons increases the population plasticity against a
specified mortality factor.
The specialized literature lists 12 parasites of the southern
green stink bug and T. basalis stands out as one of the most important
biocontrol agents. Since that time, it has been recorded from such
widely separated locations as the island of Saint Vincent, Florida,
and Egypt (Priesner, 1931).
Origin
This parasite was first described by Wollaston in 1858 from
specimens collected on the Ilha da Madeira.


73
O T reated spot.
Female T. basalis.
Random (kinesis),
. chemotaxis,
searching,
reinforcement, and
^searching paths.


98
reinforcement were 3.76, 0.153, 0.046, 0.015, and 0.006, respectively.
There is evidence that the linear regression lines calculated are
representing adequately the relationship already discussed.
The concepts of Stimulant Concentration, Stimulant Dose, and
Stimulant Time and their median values, i.e., SC^, SD,_0, an<^ ST50'
were introduced here to assess the potency of the crude kairomonal
extract as a trigger for the behavioral responses studied. The
applicability of the concepts are extended to intraspecific communica
tion systems since the literature reports situations where behavioral
responses are related to pheromonal concentrations (Mayer, 1973;
Shorey and Gaston, 1964; Bartell and Lawrence, 1973: Shorey, 1973).
By the dose-response lines obtained, there is possibility that
female T. basalis populations can be manipulated genetically for
selection of individuals at the lower, median, or upper limits of the
lines in order to increase the number of desirable genetic combinations
resulting in an improvement of the efficiency of the parasitoid.
Research is under development to determine the nature of the compound(s)
in the crude extract tested.
This technique also can provide ways to detect the major tissue
or organ responsible for synthesis of transspecific chemical messengers
by comparisons of the SC,.,., SDrrt, and ST,.. of the crude extract or
dU dU oU
already identified chemicals from different sources of the insect body.
It can also furnish more accurate results when two or more active
chemicals in inter- or intraspecific communication are compared for
effectiveness. In many cases, this is done by comparing equal amounts
of the chemicals and the responses obtained are registered and evaluated.
Such a procedure is fallacious because use of the same amount of various


139
identification of a synthetic releaser of ovipositor probing.
J. Chem. Ecol. 2(4):431-440.
Wallace, R. A. 1973. The ecology and evolution of animal behavior.
Goodyear Publ. Col, Inc., California. 348 pp.
Weseloh, R. M. 1974. Host recognition by the gypsy moth (Porthetria
dispar: Lep. Lymantriidae) larval parasitoid, Apanteles melanoscelus
(Hym.: Braconidae). Ann. Entomol. Soc. Am. 67:583-587.
Weseloh, R. M. 1976. Behavioral responses of the parasite, Apanteles
melanoscelus, to gypsy moth silk. Environ. Entomol. 5:1128-1132.
Wilson, E. 0. 1962. Chemical communication among workers of the fire
ant, Solenopsis saevissima (Fr. Smith). Anim. Behav. 10:134-147.
Wilson, E. 0. 1977. Sociobiology: The new synthesis. The Belknap
Press, Cambridge. 697 pp.
Wilson, F. 1961. Adult reproductive behaviour in Asolcus basalis
(Hymenoptera: Scelionidae). Aust. J. Zool. 9(5):737-751.
Wollaston, V. T. 1858. Brief diagnostic characters of undescribed
Madeiran insects. Ann. Mag. Nat. Hist. 3:18-28. (Cited by
J. W. Thomas, Jr., 1972 [q.v.].)
Zatyamina, V. V., F. R. Klechkoviski, V. I. Burakova. 1976. The
ecology of egg parasites of shield bugs in the Voronezh
district (in Russian, English summary). Zool. Zh. 55(7):1001-1004.


VI
Page
Experiment 9: Enhancement of Host Location by Scent
Combinations 84
Experiment 10: Normality Studies with the Crude Kairomonal
Solution from Eggs of the Host, N. viridula 87
Response to the Kairomonal Solution on the Filter Paper. 88
Evaluation of Parasitism in Areas Treated with the Crude
Kairomonal Solution 88
Responses of the Female T. basalis to the Egg Shells
and 12-Hour-Old Eggs of the Host, N. viridula 88
Antennal Palpation Previous to Oviposition 89
DISCUSSION 91
SUMMARY AND CONCLUSIONS 102
APPENDIX: SUMMARY OF THE EXPERIMENTAL DATA 106
REFERENCES CITED 132
BIOGRAPHICAL SKETCH 140


100
velocity of the female T. basalis.
From the evolutionary standpoint it is possible that the presence
of the adult hosts close to their eggs could act in some degree as
an inhibitor to the parasitoid activity, since it is during this stage
of development that the southern green stink bug, N. viridula, is highly
vulnerable to the attack of parasitoid species. This hypothesis is
under investigation, to disclose the interactions involved between
N. viridula and T. basalis, in order to assure a correct manipulation
of the parasitoid behavior via kairomone.
Studies in the area of supernormality have been extensively
reported as related to higher animals (Alcock, 1975; Hogan et al., 1975;
Staddon, 1975) These authors have suggested that physical factors
play a major role in triggering abnormal responses.
The results of the assays conducted and described throughout this
study imply that the crude kairomonal extract obtained from the eggs
of the host southern green stink bug, N. viridula, mediated behavioral
responses in the female parasitoid that could be regarded as within the
range of over-optimal (Magnus, 1958). Since the behavioral program
of the parasitoid is very closed (Mayr, 1974) experiments have been
undertaken to rule out any possibility of response-preference due to
interference of an extraneous supernormal releaser (Tinbergen, 1951;
Marler and Hamilton, 1967).
It is evident that dichloromethane did not interfere in the
performance of T. basalis. This is evident from data resulting from
the filter paper bioassay where no antennal palpation was recorded
for the treatment involving only this solvent. On the other hand,
92% of the females T. basalis tested responded to the crude


Number of female T. basal is reacting
62
100
90
80
70
60
30
20
10
\
\
Treatment totals
o
0 host egg
50 12-hour-old host eggs
50 parasitized host eggs
50 egg shells from parasitized
host eggs
i 1!I L
3
Minutes
1
2
4
5


113
Table 7. Analysis of variance for the number of Trissolcus basalis
(Wollaston) orienting wi|hin3a single tube olfactometer to
the scent of 0 and 3x10 Cm of female Nezara viridula (L.)
hemolymph.
Source of Variation
Degrees of
Freedom
Sum of
Squares
Mean
Square
F
Female N. viridula
hemolymph
1
75.62
75.62
105.52**
Time
9
74.35
8.26
11.53**
Male and female T. basalis 1
42.02
42.02
58.64**
Hemolymph vs. time
9
35.50
3.94
5.50**
Hemolymph vs. T. basalis
1
81.22
81.22
113.34**
Time vs. T. basalis
9
12.10
1.34
1.87
Hemolymph x time x T.
basalis
9
17.15
1.90
2.66**
Error
120
86.00
0.72
Total
159
423.97


41
3
(a)


3
specific communication among insects.) (9) The normality of the extract
obtained through standard and original procedures.


Fig. 5e and f. Female Trissolcus basalis (Wollaston) exhibits
ovipositional behavior on an egg mass of its
host, Nezara viridula (L.). Oviposition of the
central eggs by drilling the chorium, (e) the
lateral wall, and (f) the operculum is shown.


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES vii
LIST OF ILLUSTRATIONS X
ABSTRACT xiii
INTRODUCTION 1
LITERATURE REVIEW 4
Current Status of the Host Southern Green Stink Bug,
Nezara viridula (L.) and Its Parasitoid Trissolcus basalis
(Wollaston) 4
The Southern Green Stink Bug, Nezara viridula (L.) 4
Origin 4
Distribution 4
Host plants 5
Life history 6
Trissolcus basalis (Wollaston) 7
Origin 7
Distribution and host insects 8
Life history 8
Interspecific Communication 9
METHODS AND MATERIALS 11
Rearing of N. viridula (L.) 11
Rearing of T. basalis (Wollaston) 11
Experiment 1: Response of the Female T. basalis to the
Eggs of the Host, N. viridula 12
iii


19
response.
A treatment consisted of 50 eggs of the host introduced into the
parallel tube (B) and dispersed 7.5 cm away from the organdi screen.
Fifty egg shells of N. viridula were similarly placed inside the other
tube (B1). The shells were thoroughly washed in a solution of 1 part
of liquid non-phosphorous soap, 1 part of Clorox and 100 parts of
water, and then thoroughly rinsed in running water. The shells were
dried under lab conditions and then washed with dichloromethane. After
complete evaporation of the solvent, they were used in the experiment.
The test began after the parasitoids were fully recovered inside
the release tube which was then connected to the olfactometer.
Responsiveness was measured as the percentage of female T. basalis
orienting to tube (B), (B'), or remaining in the non-choice area (A).
Readings were performed at 5-minute intervals for a 30-minute period.
Test for non-preference orientation to each of those sites (A, B,
or B') was done with the chi-square test for multinomial experiments
(Snedcor and Cochran, 1973).
Experiment 3: Temporal Analysis of the Ovipositional
Behavior of the Female T. basalis
Experiments were conducted within a 5.0 cm Petri dish (lid 5.6 cm)
3
with a volume of 28.46 cm The lid of the dish was lined with a 5.5 cm
diameter circle of Whatman No. 1 qualitative filter paper.
The experimental unit consisted of 1 healthy 3-day-old female T.
basalis with no prior ovipositional experience. The parasitoid was
chilled and transferred to a white surface and covered with the bottom
of the Petri dish. A piece of paper supporting an egg mass was pinned
to the center of the filter paper with a dissecting pin. Six eggs were


Fig. 9. Number of female Trissolcus basalis (Wollaston) reacting
to different egg ages of the host, Nezara viridula (L.),
at different time intervals is shown.


XI
Figure Page
9.Number of female Trissolcus basalis (Wollaston) reacting
to different egg ages of the host, Nezara viridula (L.),
at different time intervals is shown 57
10. Number of male Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown 60
11. Number of female Trissolcus basalis (Wollaston) reacting
to the eggs of the host, Nezara viridula (L.) at different
levels of parasitism and time intervals is shown 62
12a. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), after soaking
in dichloromethane for different periods of time is shown.... 66
12b. Number of female Trissolcus basalis (Wollaston) reacting to
the eggs of the host, Nezara viridula (L.), after soaking
in dichloromethane for different periods of time is shown.... 68
13. Number of female Trissolcus basalis (Wollaston) reacting to
crude kairomonal extract from eggs of the host, Nezara
viridula (L.), removed with dichloromethane by different
methods at certain time intervals is shown 70
14. The behavior patterns of the female Trissolcus basalis
(Wollaston) on a filter paper treated with crude kairomonal
extract from eggs of the host, southern green stink bug,
Nezara viridula (L.), are digrammed. Shown are parasitoid:
(a) in direct movement to the treated spot; (a') in random
movement to the spot; (a") after antennal palpation, leaving
the spot and starting to search; (b) after searching,
returning to the treated area for reinforcement; and (b')
leaving the spot for new cycle of searching. Either a
or a' will occur for every single trial 73
15a and b. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract
from eggs of the host, Nezara viridula (L.), is shown:
(a) random movement (possibly kinesis), (b) chemotaxis 77
15c and d. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract from
eggs of the host, Nezara viridula (L.), is shown: (c) anten
nal palpation, (d) searching 7g
15e. Relationship of the female Trissolcus basalis (Wollaston)
exhibiting certain behavior patterns when stimulated by
different concentrations of the crude kairomonal extract


IX
Table
Page
19.Parameters for the stimulant timetfor the: female parsitoid
Trissolcus basalis (Wollaston) exhibiting antennal palpation,
when stimulated by 10^ egg equivalent/ul of the crude
kairomonal extract from the eggs of the host, Nezara
viridula (L.)
20. Average time in seconds spent by the female Trissolcus basalis
(Wollaston) in orienting to a treated filter paper area with
different concentrations of the crude kairomonal extract from
eggs of the host, Nezara viridula (L.) y
21. Average velocity in cm/s of the female Trissolcus basalis
(Wollaston) in orienting within a single tube olfactometer
containing scent combinations from the host, Nezara
viridula (L.) -i
22. Percentage of female Trissolcus basalis (Wollaston) exhibiting
antennal palpation on filter paper treated with 10 3 cm3 of
solutions containing different concentrations of crude
kairomonal extract from eggs of the host Nezara viridula (L.)^28
23. Percentage of eggs of Nezara viridula (L.) parasitized by
Trissolcus basalis (Wollaston) when placed on filter paper
treated with 5xl0~^ cm3 of a 102 egg equivalent solution in
dichloromethane ^29
24. Percentage of female Trissolcus basalis (Wollaston) exhibiting
ovipositional behavior when stimulated by 6 12-hour-old eggs
and egg shells of the host, Nezara viridula (L.) 130
25.Time required by the female Trissolcus basalis (Wollaston)
exhibiting antennal palpation previous to a successful ovi-
position in the eggs and egg shells of the host Nezara
viridula (L.)
.131


Fig. 4. Relationship between percentage of female Trissolcus basalis (Wollaston) responding
within a single tube olfactometer versus different levels of eggs of the host
southern green stink bug, Nezara viridula (L.), is shown.


53
are different in magnitude and direction when the two levels of the male
host hemolymph are available to both sexes (Table 5 of the Appendix).
Reactions of the male and female parasitoids to combined levels of
the male N. viridula hemolymph in relation to time are shown in Fig. 8a.
Male parasitoid reaction was not statistically significant. Female T.
basalis reaction was highly significant. It was observed that after 2
minutes responsiveness of the female parasitoid reached its highest
value. After this, reactivity decreased and returned to the level of
the control at the 10th minute of observation.
Scent of Female N. viridula Hemolymph
Reactions of the male and female Th basalis to the female host
hemolymph were analyzed and the results are summarized in Tables 7 and
8 of the Appendix. Responsiveness to combined levels of the female
hemolymph at different time intervals is presented in Fig. 8b.
Male parasitoids were poorly stimulated by the female host hemolymph,
there was no significant difference between the two levels of hemolymph
applied when averaged over 10 time levels (F=0.05). Female parasitoids
responded positively to the female N^. viridula hemolymph (F=156.80**) .
The remaining inferences about responsiveness of the parasitoid sexes to
the female host hemolymph are identical to those already discussed for
the male host hemolymph.
N. viridula Eggs of Different Ages
Treatment responses shown in Fig. 9 implies that the female .
basalis responded better to 12 and 24-hour-old host eggs.
Analysis of variance for the simple effects was carried out and the


58
results are presented in Table 9 of the Appendix.
F values for variation between time within 12-hour- and 24-hour-old
eggs were 8.44 and 5.19 respectively. They were highly significant and
suggested that the female T. basalis were stimulated by volatile chemicals
carried by the air stream that passed through the eggs inside the dispens
er tube. There is also indication that such chemicals decrease in concen
tration as the eggs get older. The F value for the response of females to
48-hour-old eggs was not significantly different from the blank control
at the 0.05 level.
The female parasitoid reactions are shown in Fig. 9. The maximum
reaction occurred at the third minute of exposure. Following that, a
decline in response took place and after the 5th minute the reactions were
identical for all treatments applied. The decline after the 5th minute
could be a result of either habituation, decline in concentration of the
volatile chemicals or a combination of both.
Experiment 5: Reactions of the Male and Female T. basalis to the
Eggs of the Host, N. viridula at Different Degrees of Parasitism
Responsiveness of the male and female T. basalis to the treatments
are summarized in Table 10 of the Appendix. Significant interactions
were found for egg levels versus time, egg versus male and female parasit
oid, time versus male and female T. basalis. There is evidence that the
stimulation of the parasitoids by the scent source inside the main tube
of the olfactometer depended upon the levels of parasitism of the eggs
of N. viridula and upon levels of time.
The reactions of male and female T. basalis to the different
conditions of the host eggs are presented in Figs. 10 and 11 respectively.
Treatment totals revealed that again the maximum reaction of male


51
250
O)
c
o
03
0)
C/>
03
(/>
03
JO
h- I
O
0)
-Q
E
3
2
200
150
Male 4. female, F = 1.67
Male F = 6.20**
Female, F =13-87**
4iL
_1 o
i
1
Number of male
(a)
I
2
N. v ¡ r ¡ d u I a
-L
3
(b)
3


Number of female T. basalis reacting
57


32
1 ul of a 10 ^ egg equivalent solution in dichloromethane or to
dichloromethane controls using the procedure described in Experiment 7.
Responsiveness of the parasitoids to the solvent and to the solution
were recorded in terms of the percentage of the testing insects reaching
the treated spot and drumming the area with the antennal flagellum.
The data obtained were analyzed by a paired-difference t-test.
Evaluation of Parasitism in Areas Treated with the Crude Kairomonal
Solution
The method utilized was a modification of the procedure of Jones
et al. (1973). A circle of Whatman No. 1 qualitative filter paper
measuring 12.5 cm in diameter was cut in 4 quadrants and placed in a
Petri dish with bottom and cover measuring 14 cm and 15 cm of internal
diameter respectively. The quadrants were positioned 1.2 cm apart to
reduce the chance that the parasitoid would search in adjacent areas not
-2
treated with the kairomone. An aliquot of 50 ul of a 10 egg equivalent
solution was applied to each of two opposite quadrants, and 50 ul of
dichloromethane was applied to the remaining quadrants. After the
solvent evaporation 1 egg mass made up of 10 12-hour-old eggs of N.
viridula was placed on each quadrant. Two immobilized (by chilling)
female T. basalis were placed on the center of the dish and after
recovery the dish was covered and the parasitoid allowed to search and
oviposit for 45 minutes. Parasitism was determined 12 or more days after
the treatment by counting the darkened eggs typical of T. basalis close
to emergence.
The data obtained were analyzed by paired-difference t-test and
the ratio between parasitized eggs on solution and solvent treated


Fig. 12b. Number of female Trissolcus basalis (Wollaston) reacting
to the eggs of the host, Nezara viridula (L.), after
soaking in dichloromethane for different periods of time
is shown.


119
Table 13. Treatment means for the number of female Trissolcus basalis
(Wollaston) orienting within a single tube olfactometer con
taining crude kairomonal extract from eggs of the host,
Nezara viridula (L.), removed by 4 solvents, after 4 minutes
of exposure.
Water
Ethanol
Hexane
Dichloromethane
4.8a
5.8ab
6.6b
00
NO
O
a,b,CMeans with
ferent at
the same exponential letter
0.05 level (Duncan, 1955).
are not significantly dif-


83
multiplying it by the factor "1066," consequently the SC values could
2 3
be transformed to SD in terms of Ng equivalents/mm /cm .
Equations of Table 17 of the Appendix, represent the Stimulant
Concentration and Stimulant Dose. They are statistically sound for
determinations of any SC or SD values within the range of the established
lines.
Stimulant Time (ST) data are presented in Tables 18 and 19 of the
Appendix. Stimulant Time was determined for antennal palpation when
-2
1 ul of 10 egg equivalent solution of the kairomonal crude extract was
applied on the filter paper. According to Table 18, the class limits of
time were converted to logarithm and since the logarithm of time for a
given dose is normally distributed (Hastings and Peacock, 1975), the
class limits were transformed to mid-point of log intervals. The
quantal response technique was utilized to determine the ST because
50
the number of female T. basalis palpating the treated spot at a given
time are unrelated in terms of responsiveness to the stimulus (Modifica
tion of the procedure used by Bliss, 1937) .
The correlation coefficient for ST was minus 0.945, and chi-square
7.54. The percentage of antennal palpation transformed to probits is
highly correlated with the mid-point of log intervals in seconds, and
the regression line appropriately describes this relationship (ata =0.05).
The Concept of Stimulant Time
Stimulant Time can be defined as a term to express the time required
for a defined dose of a chemical stimulant involved in inter and intra
specific communication among insects required to stimulate the innate
releasing mechanism of the insects tested in a natural or artificial


109
Table 3. Analysis of variance for Trissoicus basalis (Wollaston)
orienting within a single tube olfactometer with the scent
of 0, 1, and 3 male Nezara viridula (L.).
Degrees of
Source of Variation Freedom
Sum of
Squares
Mean
Square
F
Male N. viridula
2
13.41
6.70
1.24
Time
9
16.79
1.86
0.35
Male and female T. basalis
1
7.00
7.00
1.30
N. viridula vs. time
18
111.17
6.18
1.15
N. viridula vs. T. basalis
2
2477.11
1238.55
230.13**
T. basalis vs. time
9
75.12
8.35
1.55
N. viridula x time x T.
basalis
18
1075.64
59.76
11.10**
Error
180
966.75
5.38
Total
239
4744.99


104
highly linear related to the log concentration of the active material(s).
The data support the rationale that these reactions are triggered
by contact chemical(s) and compound(s) in vapor phase.
Mathematical models were established for every behavior pattern
elicited by the crude kairomonal extract and the concepts of Stimulant
Concentration, Stimulant Dose, Stimulant Time with their respective
median values were introduced as standard measures of the potency of
chemicals involved in interspecific and intraspecific communication
among insects.
It was demonstrated that the velocity of the parasitoid is
substantially increased by the kairomonal solution and it was found
that the time required during the orientation is reduced to 42% in
relation to the control.
Analysis of the experimental data proved that the performance
of the egg parasitoid T. basalis was improved by the crude kairomonal
extract, i.e. oviposition, time for orientation, velocity, parasitism,
etc. Since the behavior programs of parasitoid insects is rigidly
fixed, a very stereotyped behavior pattern was used to check the
normality of the releaser obtained. The results supported the
claim that the crude kairomonal solution when applied to egg-shells
of the host insect, N. viridula, elicited the same reactions as
host eggs when offered to the female T. basalis.
Identification of the chemical(s) from this crude kairomonal
extract will make it possible to better assess the potential usefulness
of the kairomone in controlling economic populations of the southern
green stink bug. Under laboratory conditions the isolated kairomonal
extract demonstrated that the behavior of the female T. basalis can


114
Table 8. Total number of Trissolcus basalis (Wollaston) orienting
within a single tube olfactometer with different levels of
the hemolymph of female Nezara viridula (L.), averaged over
10 equally spaced time interval.
T. basalis sex
No. of Parasitoids Reacting to
Different Amounts of Male Host Hemolymph
Total
0 .Cm3
3xl0~3 Cm3
Male
125
123
248
Female
109
221
330
Total
234
344
578


23
current of air (3.3 cm /s) passing through it and reaching Fhe main
tube which contained 10 healthy 3-day old parasitoids (either male or
female) ; readings were recorded at every 1-minute interval for 10
minutes. Responsiveness was reported when the parasitoid stayed around
the tip of the dispenser tube or within 10 cm of it. For the remaining
treatments (2nd and 3rd levels of the male host) 1 and 3 15-day old
male N. viridula were placed inside the glass chamber and the reactions
of the parasitoid recorded as already described.
Analysis of the data was performed by a three-way analysis of
variance for completely randomized 2x3x10 factorial experiment with 4
replicates. Interactions of significance for the experiment were
studied by a thorough analysis of the simple effects.
Scent of Female N. viridula
The methodology for the response of the parasitoid to the female
host was identical to that described above for the male N. viridula.
Male and Female N. viridula Hemolymph
The response of male or female parasitoids to 3ul of male or female
hemolynph or to blank controls was evaluated using the apparatus
described in Experiment 1.
The hemolymph was obtained from healthy 15-day-old virgin males
and the liquid was drawn from the prothorax at the point of insertion of
the forewing. The wing was removed and the exuded hemolymph collected
with a micropipette, and transferred to a thin piece of cotton measuring
0.5 x 0.6 cm. After drying the cotton was transferred to a spare
dispenser tube which later replaced the blank one from the olfactometer.


87
host was introduced into the dispenser tube. When compared to the
treatment with dichloromethane, the solution with 12.5 egg equivalent
caused a 47.05% increase in the speed of the female parasitoid in
relation to the control. The reactions induced by the remaining scent
combinations, including the control, were statistically equivalent at
0.05 level of significance.
When the crude kairomonal solution was offered to the parasitoids,
they tended to move down wind in a zig-zag pattern for a few seconds,
then change direction and in the same fashion move up wind toward the
scent source. The same pattern is observed for the scent-combination
treatments, however when the parasitoids are approximately 6 cm away
from the stimulant source, they turn back and later resume movement
toward the stimulus. Actually it is assumed that the concentration of
the volatile chemical(s) emanated from the treatment combinations
applied could be responsible for a situation of conflict (Marler and
Hamilton, 1967), consequently resulting in a decline in the female T.
basalis velocity.
Experiment 10: Normality Studies with the Crude
Kairomonal Solution from Eggs of the Host,
N. viridula
Previous experiments have indicated that orientation and velocity
of the female T. basalis toward the crude kairomonal extract from eggs
of the host southern green stink bug, N. viridula, were improved by
42.58 and 47.05% in relation to the control respectively. Evaluation of
the following results will indicate whether the behavior patterns
exhibited by the female parasitoid were triggered by a normal releaser.


8
Masner (1971) pointed out that Trissolcus basalis (Wollaston) has
the following synonyms: Telenomus basalis Wollaston, 1858; Telenomous
maderensis Wollaston, 1858; Telenomus magacephalus Ashmead, 1894; and
Telenomus piceipes Dodd, 1919.
Distribution and host insects
Trissolcus basalis has been reported as a polyphagous parasitoid
with broad range of dispersion throughout the world. It has been
reported in Europe, Asia, Africa, and North America (Cumber, 1964;
Davis, 1964; Hokyo and Kiritani, 1965; Kamal, 1937; Wilson, 1961).
Trissolcus basalis parasitizes N. viridula, Acrosternum hilare (Say),
A. marginatum (Palisot de Beauvois), Euchistus servus (Say), E.
variolatus (Palisot de Beauvois) and Cumber (1964) reported that this
parasitoid also develops on eggs of the following pentatomids: Antestia
orbona Kirk., Dictyotus caenosus (Westw.), Cermatulus nasalis (Westw.),
Glaucias amyoti (A. White) and Cuspicona simplex Walk.
Life history
Wilson (1961) indicated that T. basalis is a solitary arrhenotokous
parasite that develops from egg to adult within the host egg. He reports
that this parasite passes through a number of generations each year and
that the development is correlated to the temperature, i.e., at 27C,
the males lived for 4 to 5 days and the females for 4 to 15 days. Sailer
(1976) has indicated and the Author has confirmed that at 60 5% RH
o
and 27 C, male TL basalis have a life span varying from 3 to 5 weeks or
months while the females last from 4 to 15 weeks, and he also recorded
that females are able to live as long as 10 months with a honey and


39
103.65 20.40 seconds (a=0.05) to encounter the egg mass. During
this period considerable antennal and body grooming took place, sometimes
the female moved straight to the eggs, then suddenly changed direction
and moved away and later returned. It is evident that the source of
stimulus is reached either by random movement, chemotaxis, or a
combination of both.
When a female T_. basalis makes first contact with the egg mass, she
begins to explore it by drumming the lateral wall of the marginal eggs
or the crevices of the central eggs with the antennal flagellum (Fig.
5 a,b). The parasitoid spends 116.55 18.56 seconds with 95% of the
fiducial limit, in this exploratory behavior, then prepares for
oviposition.
The female T. basalis finishes the exploratory phase when it turns
its head away from the point chosen to insert the ovipositor. Drilling
of the chorium begins when the parasitoid slightly bends the terminal
portion of the abdomen and inserts its ovipositor through the egg wall.
When insertion is performed in the marginal eggs, the female T. basalis
may take three positions: (a) the parasitoid has the ventral portion of
the body parallel to the substratum that supports the egg mass and the
tip of the wings reaching the eggs to be parasitized; (b) the female
parasitoid stands sideways in relation to the substratum and the wings
may touch the egg or stay away from it (Fig. 5c). In both situations,
drilling of the chorium occurs in the bottom third of the egg. Ovi
position in the central eggs is performed in two distinct postures: (a)
female |T. basalis flexes its abdomen and introduces the terminal portion
between the crevices then inserts the ovipositor 1/3 below the operculum
(Fig. 5e); (b) the parasitoid perforates the egg cap, by introducing


Probit of female T. basa lis
exhibiting searching
03
T
<
II
3.50 + 0.6 7 x
Probit of female T. basalis
exhibiting antennal palpation


THE BEHAVIOR OF THE EGG PARASITOID TRISSOLCUS BASALIS
(WOLLASTON) (HYMENOPTERA: SCELIONIDAE) IN RESPONSE
TO KAIROMONES PRODUCED BY ITS HOST, THE SOUTHERN GREEN
STINK BUG NEZARA VIRIDULA (L.) (HEMIPTERA: PENTATOMIDAE)
By
FERNANDO JOO MONTENEGRO DE SALES
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
1978


38
When introduced into the olfactometer and after recovery the
parasitoids began to move to the different sections of the apparatus.
During the first 15 minutes of observation the parasitoids kept
moving within the region A of the olfactometer, running either on a
straight or circular path into the Y part of the olfactometer. Sometimes
the females returned to the releasing tube and remained there or
resumed movement to the other parts of the apparatus. After 30 minutes,
63 percent of the female parasitoids were found in the section B
palpating host eggs with the antennal flagellum, ovipositing, or running
inside the tube. Five percent of the tested insects were observed in
section B' performing the same behavior except ovipositing. Finally
32 percent of the sample tested stayed in Section A either running in
the Y part of the olfactometer or remaining in a resting position inside
the releasing tube.
It is evident that the parasitoids showed a highly significant
preference for the side of the olfactometer containing the 12-hour-Old
eggs of the host, N. viridula. These data are presented in Table 2, of
the Appendix. The chi-square value was 50.54.
Experiment 3: Temporal Analysis of the
Ovipositional Behavior of the Female T. basalis
When the female T. basalis is introduced into the Petri dish she
starts to either run on the margins of the filter paper exhibiting a
random movement, possibly kinesis, or moves straight to the egg mass,
after spending a certain amount of time in this kinetic pattern, the
female parasitoid may purposefully move toward the source of stimulus.
During the orientation process the parasitoids required an average of


96
In any of those cases the resulting suspension should be filtered
through a filter paper.
From results of Experiment 7 it isclear that visual clues are
not critical in the location of the N^. viridula eggs by the female
T. basalis. Orientation toward the area treated with the crude
kairomonal solution can happen either by random movement or chemotaxis.
In either situation some degree of grooming takes place, and is
-4
substantially increased when the concentration reaches 10 egg
equivalent/ul. However, grooming has been reported in the absence
of any applied treatment or presence of host eggs on the threshold
attraction limit.
This grooming behavior remains to be studied in more detail
to determine its real significance. Actually it is assumed that a
conflict situation is taking place, probably generated by chemical(s)
in vapor phase stimulating the innate releasing mechanism of the
parasitoid or an act of ritualization without any significance to the
performance of T. basalis.
Antennal palpation takes place when the parasitoid drums the
treated spot with its antennal flagellum. There is an indication
that the parasitoid responds to a contact chemical stimulant(s) that
will help in locating the host. Such rationale is corroborated by
the subsequent behavioral steps, i.e., searching and reinforcement.
Searching is characterized by an increase in velocity when
female T. basalis scans the area adjacent to the spot and is motivated
by the crude extract which acts as a reinforcer, that is, it induces
the parasitoid to repeat a behavioral cycleantennal palpation and
searching.


22


XIV
A crude kairomonal extract was isolated by soaking host eggs in
dichloromethane and the results of bioassays with this extract showed
that visual clues are not as important as chemical clues in increasing
the parasitoid velocity? consequently reducing the orientation time
and enhancing host location. Assays on filter paper proved that the
kairomonal solution induced 5 behavior patterns; i.e., random movement
(kinesis), chemotaxis, antennal palpation, searching, and reinforcement.
Mathematical equations were developed to describe these patterns. The
concepts of Stimulant Concentration (SC ) Stimulant Dose (SD5q) and
Stimulant Time (ST^q) were introduced as measurements of the potency of
chemicals involved in interspecific and intraspecific communication
among insects.
The overall performance of the female T. basalis was improved in
terms of orientation, velocity, oviposition, and parasitism by the
kairomonal solution, and a very stereotyped and precisely timed behavior
pattern indicated that action of the isolated releaser is within the
limits of normality.


ACKNOWLEDGMENTS
The author is grateful to Dr. G.E. Allen for his advice,
encouragement and guidance during the experimental work and preparation
of this dissertation. Appreciation is extended to the staff members
of his laboratory for help and understanding.
Recognition and gratitude is given to Dr. R.I. Sailer, Dr. J.H.
Tumlinson, Dr. J.R. McLaughlin and Dr. F.W. Zettler for serving on my
graduate committee. Special thanks are extended to Dr. D.L. Chambers
for allowing me to use the facilities of the USDA-ARS, Insect Attractants,
Behavior and Basic Biology Research Laboratory.
Special thanks are extended to the chairman, Dr. Fowden Maxwell,
as well as the staff and graduate students of the Department of
Entomology and Nematology for their unselfish service, instruction,
and encouragement.
Gratitude is also expressed to the Brazilian colleagues at the
University of Florida for their support and encouraging words.
I am also indebted to Mrs. Maria I. Cruz, Campus Coordinator of
the AID program for her cooperation, assistance and concern.
The author was supported by funds from the United States Agency
for International Development (USAID), the Federal University of
Cear, Brazil, to whom sincere appreciation is expressed.
n


118
Table 12. Treatment totals for the number of female Trissolcus
basalis (Wollaston) orienting within a single tube
olfactometer containing 50 egg equivalents of the crude
kairomonal extract from eggs of the host, Nezara
viridula (L.) removed by different solvents.
1
2
3
4
5
6
7
8
9
10
Water
19
24
16
24
18
19
16
13
13
12
Ethanol
27
32
31
29
25
25
19
19
16
15
Hexane
30
33
36
33
24
22
20
15
15
15
Dichloromethane
31
32
35
41
21
19
15
14
16
11
Total
107
121
118
127
88
85
70
61
60
53


IV
Page
Experiment 2: Orientation of the Female T. basalis
Inside a "Y" Type Olfactometer 16
Experiment 3: Temporal Analysis of the Ovipositional
Behavior of the Female Th basalis 19
Experiment 4: Cues Useful in Location of the Host, N.
viridula, by the Parasitoid T. basalis 20
Scent of Male N. viridula 20
Scent of Female N. viridula 23
Male and Female N. viridula Hemolymph 23
N. viridula Eggs of Different Ages 24
Experiment 5: Reactions of the Male and Female T. basalis
to the Eggs of the Host, N. viridula at Different
Degrees of Parasitism 24
Experiment 6: Reactions of the Female T. basalis to the
Kairomonal Solutions Prepared with Different Solvents 25
Dichloromethane Washes Required for Removal of the
Kairomone from Eggs of N. viridula 26
Reaction of the Female T. basalis to Kairomonal
Solutions Prepared by Different Methods 27
Experiment 7: Behavior Patterns of the Female T. basalis
When Stimulated by Different Concentrations of the Crude
' Kairomonal Extract from Eggs of the Host, N. viridula 27
Experiment 8: Effects of the Crude Kairomonal Extract from
Eggs of the Host, N. viridula in the Orientation of the
Female T. basalis 29
Experiment 9: Enhancement of Host Location by Scent
Combinations 30
Experiment 10: Normality Studies with the Crude Kairomonal
Solution from Eggs of the Host, N. viridula 31
Response to the Kairomonal Solution on the Filter Paper. 31
Evaluation of Parasitism in Areas Treated with the
Crude Kairomonal Solution 32
Responses of Female T. basalis to Egg Shells and 12-
Hour-Old Eggs of the Host N. viridula 33


Fig. 13. Number of female Trissolcus basalis (Wollaston) reacting
to crude kairomonal extract from eggs of the host, Nezara
viridula (L.), removed with dichloromethane by different
methods at certain time intervals is shown.


APPENDIX
SUMMARY OF THE EXPERIMENTAL DATA


RESULTS
Experiment 1: Response of the Female T. basalis
to the Eggs of the Host, N. viridula
Responsiveness of the parasitoid inside the single tube olfactometer
(Fig. 1) was recorded after 3 minutes of exposure. The results are
shown in Fig. 4. There is a strong linear relationship (r=0.97) between
the percentage of female T. basalis stimulated to move toward the scent
source and the log number of eggs of N. viridula. The adequacy of the
linear model was contrasted at 0.05 level of significance, and the
F-test (=1.29) indicated that the relationship already studied is
properly represented by the model.
Analysis of the main effects, i.e., egg and exposure time revealed
F values equal to 14.12 and 4.13 respectively. They were highly
significant ata=0.05, but do not interact. Their effects are additive
to the population mean of the number of female parasitoid orienting to
the scent source.
The threshold for stimulation was 5 12-hour-old eggs of the
southern green stink bug. The analysis of variance for the simple
effects is shown in Table 1, of the Appendix. An F-test for all time
levels within the 5-egg level was highly significant, indicating that
the parasitoids started to react to the host presence at that point.
Experiment 2: Orientation of the Female
T. basalis Inside a "Y" Type Olfactometer
35


BIOGRAPHICAL SKETCH
Fernando Joo Montenegro de Sales was born in Fortaleza,
Ceara', Brazil, on March 18, 1944. He attended Cole'gio Castelo
Branco, Colegio S. Joo, and was graduated from Colegio Estadual
do Ceara' (Liceu) in 1961. After graduation he attended Curso
Picaneo, a pre-university course in Fortaleza, Ceara' for one year.
He obtained his undergraduate education at Escola de
Agronomia da Universidade Federal do Ceara", Fortaleza, Ceara"",
Brazil, where he received his Bachelor of Science degree in Agronomy,
in 1966.
In 1967 he was hired to serve as a faculty member at the
Escola de Agronomia da Universidade Federal do Ceara (= Department
of Plant Science of the College of Agriculture, Federal University
of Ceara) Since then, he has been teaching and working in ento
mological projects and published papers in his major field.
In 1970 he was awarded a United States Agency for International
Development (USAID) scholarship to work toward the degree of Master
of Science at the University of Arizona. He granted his MS degree
in February, 1972, before returning to Brazil.
In 1974 he was awarded a new USAID scholarship to work toward
the Doctor of Philosophy degree. From 1974 to 1978 he was a graduate
student in the Department of Entomology and Nematology, University of
Florida. He now returns to the Federal University of Ceara', where
140


Fig. 14. The behavior patterns of the female Trissolcus basalis
(Wollaston) on a filter paper treated with crude
kairomonal extract from eggs of the host, southern green
stink bug, Nezara viridula (L.), are diagrammed. Shown
are parasitoid: (a) in direct movement to the treated
spot; (a') in random movement to the spot; (a") after
antennal palpation, leaving the spot and starting to search;
(b) after searching, returning to the treated area for
reinforcement; and (b') leaving the spot for new cycle of
searching. Either a or a' will occur for every single
trial.


9
water supply.
Thomas, Jr. (1972) indicates that the polyphagous behavior of T.
basalis is a positive factor in its utilization as a released biological
regulator, since other stink bug species can serve as alternate hosts
for maintenance and increase of the parasitoid population. He also
points out that field releases of T. basalis at rates of 5000 and 8000
adults per 1/10 acre increased the rate of parasitism substantially
and releases of at least 50,000 adults per acre would be required for
minimal effective suppression in a large scale augmentative release
program.
Interspecific Communication
Investigations involving interspecific communication were reported
by Laing (1937) who pointed out that the parasitoid Trichogramma
evanescens (Westwood) perceives an odor left by adult moth as a cue
that helps in locating host eggs. Thorpe and Jones (1937) also indicated
that odor plays an important role in a parasitoid/host relationship. It
was Brown, Jr. et al. (1970) that coined the term kairomone to define
the mediators involved in those processes of communication. Since then,
researches dealing with either behavioral studies, isolation, identi
fication, synthesis, and proof of effectiveness, or combination with
one or more of those aspects mediated by kairomones have been reported
by Corbet (1973), Greany and Oatman (1972), Gross et al. (1975), Hays
and Vinson (1971), Hendry et al. (1973, 1976), Jones et al. (1973),
Leonard et al. (1975), Lewis et al. (1971, 1975, 1976), Nettles and
Burks (1975), Nordlund et al. (1974), Nordlund and Lewis (1976), Tucker
and Leonard (1977), Vinson (1975, 1976), Vinson et al. (1975, 1976) and


80
60
40
20
i
1
1
2
3
log of the number of eggs
of N. viridula
10
u>


Number of female T. basalis reacting
70
100
90
80
T reatment totals
F=4.43tJL. solution from eggs soaked
1 time for 4 hours
F = 3.49** solution from eggs ground
with solvent
F = 4.10tJ! solution from eggs soaked
4 times for 1 hour
F= 0.84 solvent
Mi utes


10
Weseloh (1974, 1976).
Interspecific chemical communication between Trissolcus basalis
(Wollaston) and its host, the southern green stink bug, Nezara viridula
(L.) has not been reported, however Russian researchers have conducted
a series of investigations involving associations between many species
of scelionids, particularly Trissolcus spp., and pentatomid species
(Buleza, 1973; Meier, 1970; Zatyamina et al., 1976; Gennadiev et al.,
1976). Viktorov et al. (1975) found Trissolcus grandis ability to
encounter egg masses was significantly increased by extracts of adults
of two pentatomid hosts.


124
Table 18. Percentage of the female parasitoid Trissolcus basalis
(Wollaston) exhibiting antennal palpation on a treated fil
ter paper spot, when stimulated by 10 egg equivalents/yl
of the crude kairomonal extract of the eggs of the host,
Nezara viridula (L.) at different time intervals.
Time Interval
in Seconds
Log of Time
Interval x 10
Mid-point of
Log Intervals
Antennal
Palpation (%)
1-15
1.000 -
2.176
1.588
39
16 30
2.204 -
2.477
2.340
21
31 45
2.491 -
2.653
2.572
14
46 60
2.662 -
2.778
2.720
9
61 75
2.785 -
2.875
2.830
7
76 90
2.880 -
2.954
2.917
9
91 -105
2.959 -
3.021
2.990
1


6
Life history
Life history and behavior of the southern green stink bug has been
intensively studied by Japanese researchers [Kariya (1961), Kiritani
(1963, 1965), Kiritani and Hokyo (1965) Kiritani, Hokyo, and Iwao
(1966), Kiritani, Hokyo, and Kimura (1966) Kiritani, Hokyo, Kimura,
and Nakasuji (1965), Kiritani and Kimura (1965), and Kobayashi (1959)].
In general, life cycle and generation time are much the same and
variations are usually correlated to temperature fluctuation at a
given location.
In the United States, the basic information in this area comes from
the classical works of Drake (1920) and Jones (1918). They indicate that
this insect, like most other pentatomids, hibernates in the adult stage
under litter, bark, and other objects which offers protection. Drake
(1920) points out that mating begins almost immediately upon emergence
from hibernation. The female and male usually remain attached to one
another by the tips of their abdomens and with their heads facing in
opposite directions. Under natural conditions copulation is repeated
a number of times before and after the eggs have been deposited. Drake
(1920) also reported that after feeding a few days, newly emerged adults
reach sexual maturity, and this period was found to vary from 3 to 6
weeks. The eggs are generally laid in regularly shaped compact clusters
in which the individual eggs are arranged in very regular rows and firmly
glued together. The incubation period is about 6 days in summer, but
during early spring and late fall the period is often extended to 2 or 3
weeks.
The southern green stink bug has 5 nymphal instars and during the
first instar, the nymphs normally cluster together near or on the egg-


82
Stimulant Concentration values for five behavior patterns of the
female parasitoid are shown in Table 17, with 95% confidence limits.
-3
The SC for random movement (possibly kinesis) was 1.23x10 egg
50
equivalents; this represents the concentration of the stimulant that
caused 50% of the female T. basalis to orient themselves to the treated
spot in approximately 3 minutes. Stimulant Concentration values for
the other behavior patterns can be interpreted the same way.
The Concept of Stimulant Dose
Stimulant Dose (SD) can be defined as a term used to express the
amount of a chemical stimulant involved in inter and intraspecific
communication among insects, required to stimulate the innate releasing
mechanism of the insects tested in a natural or artificial environment,
resulting in a percentage of responsiveness characteristic of the species
under study at a given time. Stimulant Dose units should represent the
exact amount of the stimulant utilized in any sort of bioassay,
either by direct application of the chemical on the animal tested or by
impregnation of a given surface or volume. Units of mass, volume, and
area of multiples and sub-multiples of the metric system can be used for
measurements of the doses.
Under the conditions of the actual experiment, all concentrations
were delivered with a constant amount of solution, that is, 1 ul which
+ 2 3
covered an area of (0.17 0.008) cm within a Petri dish of 28.46 cm
of internal volume. It was found that 1 N. viridula egg weighs
(533.96 15.75) ug a correction factor was determined to transform egg
equivalents in nanogram equivalents per square millimeter per cubic
centimeter. Any concentration can be expressed in terms of SD when