Citation
Aspects of the reproductive biology of Pediobius foveolatus (Crawford) (Eulophidae: Hymenoptera)

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Title:
Aspects of the reproductive biology of Pediobius foveolatus (Crawford) (Eulophidae: Hymenoptera) parasite of Epilachna spp. (Coccinellidae: Coleoptera)
Uncontrolled:
Epilachna
Creator:
Limhuot Nong, 1939-
Publication Date:
Language:
English
Physical Description:
xvi, 193 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Beetles ( jstor )
Female animals ( jstor )
Larvae ( jstor )
Mating behavior ( jstor )
Mummies ( jstor )
Oviposition ( jstor )
Parasite hosts ( jstor )
Parasites ( jstor )
Sex ratio ( jstor )
Spermatozoa ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Eulophidae ( lcsh )
Mexican bean beetle -- Biological control ( lcsh )
Pediobius foveolatus ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1982.
Bibliography:
Bibliography: leaves 176-184.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Limhuot Nong.

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University of Florida
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ASPECTS OF THE REPRODUCTIVE BIOLOGY OF
PEDIOBIUS FOVEOLATUS (CRAWFORD) (EULOPHIDAE: HYMENOPTERA),
PARASITE OF EPILACHNA SPP. (COCCINELLIDAE: COLEOPTERA)




By

LIMHUOT NONG


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


1982
















Copyright 1982

by

LIMHUOT NONG
















To my father-in-law Mr. Yip Nguon Ung















ACKNOWLEDGEMENTS


The author wishes to express his deep gratitude and appreciation to Dr. Reece I. Sailer, supervisory committee chairman, for his invaluable guidance and assistance, continuous understanding and morale support during the whole period of this study and the preparation of this dissertation, and for making the overall involved efforts a very pleasant experience.

He owes a deep debt of gratitude to Dr. Vernon G. Perry who, together with Dr. Sailer, gave him a rare opportunity to pursue and make this graduate study possible; Dr. Perry's helpful advice, continuous encouragement and thoughtful consideration are here profoundly appreciated.

Special appreciation is directed to Dr. Jerry L. Stimac for his advice, assistance, and constructive criticism whenever these were called for.

Deep appreciation is due to Dr. Daniel A. Roberts for his unfailed advice and helpful suggestions during the whole period of this study and preparation of this manuscript.

He also wishes to direct a deep appreciation to Dr. John A.

Cornell for his very useful advice and untiring assistance in statistical analyses of the research data and the review of this manuscript.

Heartfelt thanks are specially directed to Mrs. May Morita

Buckingham for her dedicated and most wholehearted help on the typing


iv










work of this manuscript from the very beginning to the end of its preparation. Special appreciation is directed to her husband, Dr. Gary R. Buckingham for being very considerate and thoughtful and for making his laboratory and office facilities always available for use during this research and the preparation of this manuscript.

To his wife Bicheng, not only for her love and care, but also for her assistance in some laboratory work that necessitated help, he would like to sincerely say thanks.


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TABLE OF CONTENTS



Page

ACKNOWLEDGEMENTS ................................................ iv

LIST OF TABLES ................................................... ix

LIST OF FIGURES ................................................. xii

ABSTRACT ......................................................... xv

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

CHAPTER 1: LITERATURE REVIEW .................................. 3

CHAPTER 2: GENERAL MATERIALS AND METHODS USED TO STUDY
THE BIOLOGY OF PEDIOBIUS FOVEOLATUS ............. 15

CHAPTER 3: MODE OF REPRODUCTION ............................... 26

Introduction ....................................... 26
Materials and Methods ............................. 27
Results and Discussion .......................... 28

CHAPTER 4: SEXUAL BEHAVIOR .................................... 32

Introduction ....................................... 32
Materials and Methods ............................. 33
Results and Discussion ............................ 36

CHAPTER 5: PREEMERGENCE MATING ................................ 53

Introduction ....................................... 53
Materials and Methods ............................. 54
Results and Discussion .......................... 55

CHAPTER 6: SEX RATIOS RESULTING FROM SEQUENTIALLY MATED
MALES .... ......................................... 63

Introduction ....................................... 63
Materials and Methods ............................. 67
Results and Discussion ............................ 68


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Page

CHAPTER 7: FECUNDITY ........................................... 91

Introduction ......................................... 91
Materials and Methods ............................... 93
Results and Discussion ............................... 94

CHAPTER 8: INFLUENCE OF AGE ON MATING CAPABILITY OF FEMALES
AND MALES ... ........................................ 111

Introduction ......................................... 111
Materials and Methods ............................... 112
Results and.Discussion ............................... 113

CHAPTER 9: INFLUENCE OF AGE ON MATING CAPABILITY OF FEMALES .. 119

Introduction ......................................... 119
Materials and Methods ............................... 119
Results and Discussion ............................... 120

CHAPTER 10: MULTIPLE MATINGS OF FEMALES ....................... 128

Introduction ......................................... 128
Materials and Methods ............................... 129
Results and Discussion ............................... 131

CHAPTER 11: OVIPOSITION PREFERENCE WITH REFERENCE TO HOST
INSTARS ... .......................................... 149

Introduction ... ..................................... 149
Materials and Methods ............................... 150
Results and Discussion ............................... 152

CHAPTER 12: SUMMARY AND CONCLUSIONS ............................. 162

Summary of Results ................................... 162
Conclusions . --...................................... 167

REFERENCES CITED .............---..-.................................. 176

APPENDICES

5-1. Per-mummy numbers of P. foveolatus progeny (females/
males) produced through 3 consecutive host exposures
by individual parent females isolated immediately
after emergence from host mummies ....................... 185

6-2. Per-host-mummy numbers of P. foveolatus progeny
(females/males) produced through 6 consecutive host exposures by parent females sequentially mated with
single males ..................................--......... 186

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Page

6-3. Per-host-mummy percentages of P. foveolatus female
progeny produced through 6 consecutive host exposures by parent females sequentially mated with
single males ... ......................................... 187

7-4. Per-mummy numbers of P. foveolatus progeny (females/
males) produced by individual females with respect
to host exposures ... .................................... 188

7-5. Per-mummy numbers of P. foveolatus progeny produced
through single ovipositions by 38 individual parent
females .................................................. 189

8-6. Per-mummy numbers of P. foveolatus progeny (females/
males) produced through 3 consecutive host exposures
by 3 pairing combinations of young and old parents .... 190

9-7. Per-mummy numbers of P. foveolatus progeny (females/
males) produced through 3 consecutive host exposures
by 1-, 5-, 10-, 15-, 20-, and 25-day old females
paired with 1-day old males ............................. 191

BIOGRAPHICAL SKETCH .............................................. 192


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LIST OF TABLES


Table Page

3-1. Live adult progeny (females/males) produced
through 3 separate host exposures by
Pediobius foveolatus virgin females .............. 29

5-2. Progeny of P. foveolatus produced through 3
consecutive host exposures by females isolated immediately after emergence from the
host mummies ...... .................................. . 57

6-3. Average percentages of female progeny of
P. foveolatus produced by each quintet of parent
females with respect to individual males and
host exposures ........................................ 69

6-4. Significant difference between rates of sperm
depletion in increasing sequence of female
quintets ...... ....................................... 76

6-5. Significant difference between rates of sperm
depletion in increasing sequence of host
exposures ...... ...................................... 76

7-6. Fecundity, longevity, and numbers of 4thinstar host larvae successfully parasitized
by individual female P. foveolatus ............... 95

7-7. Sex ratios of P. foveolatus progeny computed
on the basis of 9 range categories of 5-individual increments ................................... 102

7-8. Numbers of successfully parasitized host
larvae, total numbers of parasite progeny
(females + males), average numbers of parasite
progeny per host mummy, total numbers of
parasite progeny (females/males), and parasite sex ratios, all corresponding to each exposure
of the host to the 32 parasite parent females ..... 105


ix










Table Page

7-9. Percentages of female progeny produced by
P. foveolatus individual parent females
with respect to host exposures ................... 108

8-10. Numbers of mated parasite females resulting from 3 pairing combinations of young and
old adults and 3 separate host exposures ......... 114

8-11. Results of Z-tests between pairing combinations of young and old P. foveolatus adults of both sexes, based on numbers of successfully mated
parent females of Table 8-10 ..................... 114

8-12. Numbers of females failing to produce progeny throughout 3 separate host exposures ............. 115

8-13. Results of Z-tests between pairing combinations of young and old P. foveolatus adults,
based on data in Table 8-12 involving numbers
of parent females that failed to produce
progeny ..... ......................................... 116

9-14. Effect of age on mating capability of P. foveolatus females ............................... 121

9-15. Results of X2 tests showing difference in mating capability between P. foveolatus
females of different age ......................... 123

10-16. Occurrence of single and multiple matings, and
dubious occurrence of multiple matings revealed
by P. foveolatus females after introduction of
second males ...................................... 132

10-17. Numbers of P. foveolatus parent females falling
in each of the 11 categories described in
materials and methods .............................. 134

10-18. per-host-exposure numbers of P. foveolatus
progeny (females/males) and corresponding
female to male ratios of progeny produced by
the first 10 parent females sequentially
mated with single males (replicated 4 times)
in the study on multiple matings and the study
on sperm depletion (Chapter 6) ..................... 136


x










Table Page

10-19. Significant difference between the slope
of equation Eq. 10.24 (linear regression
between P. foveolatus female to male ratios and host exposures in the study on multiple
matings) and the slopes of equation Eq. 10.25 (quadratic polynomial regression between the
parasite progeny sex ratios and host exposures
in the study on sperm depletion in Chapter 6)
at each level of host exposure ................... 139

10-20. Per-host-exposure numbers of P. foveolatus
progeny (females/males) and corresponding
female to male ratios produced by all females
in the study on multiple matings and the study
on sperm depletion .................................. 140

10-21. Significant difference between the slope of
equation Eq. 10.26 (quadratic polynomial regression between P. foveolatus female to male
progeny ratios and host exposures in the study on multiple matings) and the slope of equation
Eq. 10.28 (quadratic polynomial regression
between the parasite progeny sex ratios and
host exposures in the study on sperm depletion
in Chapter 6) at each level of host exposure ...... 145

10-22. Significant difference between the slope of
equation Eq. 10.27 (quadratic polynomial regression between P. foveolatus female to male
progeny ratios and host exposures in the study
on multiple matings where female progeny produced by the 6 obvious multimated females being converted into males) and the slope of equation Eq. 10.28 (quadratic polynomial regression between the parasite progeny sex ratios and host exposures in the
study on sperm depletion in Chapter 6) at each
level of host exposure .............................. 146

11-23. Numbers and percentages of parasitized hosts
corresponding to each of the 3 larval instars used
in the test for oviposition preference by
P. foveolatus females .................. ............ 153

11-24. Numbers and percentages of dead parasitized hosts
corresponding to each of the 3 larval instars
used in the test for oviposition preference by
P. foveolatus females ................................ 155


xi















LIST OF FIGURES



Figure Page

2-1. Mexican bean beetle, Epilachna varivestis
Mulsant ...... ....................................... 17

2-2. Pediobius foveolatus (crawford), parasite of
Epilachna spp. ...................................... 17

2-3. Containers and tools for Mexican bean beetle
rearing ..... ........................................ 19

2-4. Young greenhouse-grown lima bean plants ......... 19

2-5. Beggarweed, Desmodium tortuosum (SW) D. C.
at fruiting stage .................................... 23

2-6. Containers and tools for rearing and handling
of parasite adults ................................... 23

2-7. Hood for handling of parasite adults ............ 24

4-8. Observation arena specially designed for study
on parasite sexual behavior ..................... 34

4-9. Female-searching pattern of a male P. foveolatus
in a 30 cm-diameter cardboard arena from the time of emergence from the host mummy to the
end of mating activities ........................... 43

4-10. Schematic representation (transposed from photographs) showing gradual outward movement
of P. foveolatus adults (females and males) undergoing postemergence mating activities
around the host mummy ................................ 44

4-11. Schematic representation (transposed from
photographs) showing gradual outward movement of P. foveolatus adult males (host mummy contained only males) undergoing postemergence
searching for female mates ...................... 45




xii










Figure Page

5-12. Positions of P. foveolatus females and males in 4 series of emergence from their respective
host mummies ....... ................................. 56

6-13. Simple linear regression lines representing the relationship between the percentages of
female progeny of P. foveolatus and the
quintets of parent females sequentially mated
with single males ..................................... 71

6-14. Simple linear regression lines representing the relationship between the percentages of
female progeny of P. foveolatus and the
exposures of host larvae to the parasite
parent females sequentially mated with single
males ....... ........................................ 74

6-15. Tridimensional representation illustrating the general pattern of male sperm depletion, expressed in terms of percentages of female
progeny produced by P. foveolatus parent
females sequentially mated with single males .... 77

6-16. Quadratic polynomial regression curves representing the relationship between male
sperm depletion (expressed in terms of percentages of female progeny produced by P. foveolatus
parent females sequentially mated with single males) and the quintets of parent females in
the 1st and 2nd host exposures .................. 80

6-17. Relationship between male sperm depletion (expressed in terms of percentages of female
progeny produced by parent females sequentially mated with single males) and the host exposures
in the 1st and 2nd quintets of parent females,
showing a trend toward quadratic polynomial
regression ....... ................................... 82

7-18. Distribution of P. foveolatus females with respect to range categories of 20-progeny
increments ....... ................................... 96

7-19. Longevity vs. fecundity of female P. foveolatus . 98

7-20. Simple linear relationship between sex ratios and numbers of P. foveolatus adults produced through host mummies corresponding to range
categories of 5-progeny increments .............. 104


xiii










Figure Page

7-21. Linear relationship between sex ratios and per-host-mummy average numbers of progeny.
produced through individual host exposures
by 32 P. foveolatus parent females .............. 106

7-22. Quadratic polynomial regression curve representing the relationship between the
percentages of P. foveolatus female progeny
and the host exposures ............................. 110

8-23. Number of successfully mated females and number of females failing to produce progeny
with respect to 3 female-male pairing combinations of young and old P. foveolatus adults,
based on 3 consecutive host exposures ........... 117

9-24. Percentage of mated P. foveolatus females with respect to age ..................................... 122

10-25. Relationship between female to male ratios of
P. foveolatus progeny produced by the first 10 parent females mated sequentially with initial
single males (replicated 4 times), and host
exposures ..... ...................................... 137

10-26. Quadratic polynomial regression curves representing relationships between female to male ratios
of P. foveolatus progeny and host exposures ..... 143

11-27. Screened cage for confinement of host larvae
and parasite adults for the study on parasite
oviposition preference vs. host instars ......... 151

11-28. Percentage of parasitization of 2nd-, 3rd-, and
4th-instar host larvae, and percentage of dead
parasitized 2nd-, 3rd-, and 4th-instar host
larvae as based on percentage of parasitization... 156

11-29. Simple linear relationship between percentage of
parasitization and host instars, representing
preference vis-a-vis host instars by
P. foveolatus ........................................ 157

11-30. Simple linear relationship between percentage of
dead parasitized host larvae and host instars ... 157


xiv














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


ASPECTS OF THE REPRODUCTIVE BIOLOGY OF
PEDIOBIUS FOVEOLATUS (CRAWFORD) (EULOPHIDAE: HYMENOPTERA),
PARASITE OF EPILACHNA SPP. (COCCINELLIDAE: COLEOPTERA) By
LIMHUOT NONG

May 1982

Chairman: Reece I. Sailer
Major Department: Entomology and Nematology


Nine aspects of the reproductive biology of Pediobius foveolatus

are subjected to experimental study. These include (1) mode of reproduction, (2) sexual behavior, (3) preemergence mating, (4) sex ratios resulting from sequentially mated males, (5) fecundity, (6) influence of age on mating capability of females and males, (7) influence of age on mating capability of females, (8) multiple matings of females, and

(9) oviposition preference with reference to host instars.

In common with most Hymenoptera, P. foveolatus is arrhenotokous. Sex ratio is female-biased. Sibling adults mate in close proximity to their host mummy immediately following emergence. Mating activities following emergence last about 30 minutes and rarely exceed 1 hour. Once mated, females move away from the host mummy and thus afford more opportunity for the remaining virgin females to mate. Male behavior is characterized by a remarkably energetic search for and readiness to mate with receptive females. The male's mate-searching area expands gradually as adult emergence proceeds. Termination of mating activities xv










is marked by disorderly movement and flight of both sexes. Within the local mating area, mate encountering success results from innate postemergence behavior of both.sexes, and appears to involve a female sex pheromone. Males are unable to discriminate between virgin and mated females or between young and old females. Male wing vibration during courtship appears to attract outside males for participation in the local mating activities. Females seldom mate more than once. Subsequent matings are unrelated to success or failure of sperm transfer from the first mating. Receptivity of females to mating is substantially reduced after 2 weeks, but 15-day old males retain the ability to mate successfully.

When given a choice females preferentially oviposit in large host larvae. The total number of adult progeny produced by individual females from 4th-instar host larvae may range from 0 to 199 with an average of 126. Fecundity is inversely correlated with female lifespan.

For a given host instar, female to male ratio varies from one

host larva to another and increases with increasing numbers of parasite adults per host mummy. Sperm regulation by females, expressed in percentage of female progeny produced through successive ovipositions, follows a mathematical quadratic polynomial model and features in succession the so-called "maturation," "optimum," and "degradation" biological phases. A similar pattern is exhibited by single males undergoing successive matings with a series of virgin females. This is especially evident from analysis of the distribution of percentage of female progeny produced in the first 2 ovipositions by the sequentially mated females.


xvi















INTRODUCTION


This study of the reproductive biology of Pediobius foveolatus (Crawford) was undertaken with two primary objectives in mind.

First, P. foveolatus, a eulophid introduced into the USA from India in 1966 (Angalet et al., 1968), is being used as a parasite to control the Mexican bean beetle, Epilachna varivestis Mulsant, a severe pest of common beans and soybeans. Although the parasite does not overwinter successfully in the United States, it exhibits such a remarkably high host-searching ability and reproductive potential that effective control has been obtained through annual inoculative releases of comparatively small numbers early in the growing season. This implies that, in order to be available when needed, the parasite must be maintained in culture. A better understanding of its reproductive biology would not only enhance maintenance and quality of stock cultures but would also provide information needed to determine when and how many parasite individuals should be released under a given set of field conditions.

Second, P. foveolatus is an arrhenotokous species (females and males produced, respectively, from fertilized and unfertilized eggs). Other than thelytoky (females produced by uniparental females, males lost or rare), arrhenotoky is the only other widespread major mode of reproduction that departs from the common pattern of diplodiploid sexuality occurring among animals; it has received much less attention


1







2


even though it may occur in as many as one-quarter of all arthropod species and in some rotifers (Borgia, 1980). It is hoped that the present investigation will constitute a contribution to the basic knowledge relating to this mode of animal reproduction.

In addition to literature review, the present study encompasses

9 research aspects: (1) mode of reproduction, (2) sexual behavior,

(3) preemergence mating, (4) sex ratios resulting from sequentiallymated males, (5) fecundity, (6) influence of age on mating capability of females and males, (7) influence of age on mating capability of females, (8) multiple matings of females, and (9) oviposition preference with reference to host instars. Results are presented under 12 chapters. Chapter 1 deals entirely with pertinent literature concerning E. varivestis and P. foveolatus. Chapters 2-11 treat methodology and the above-mentioned research aspects, including the scope and literature specifically relevant to each. Chapter 12 is reserved for the general conclusions.
















CHAPTER 1

LITERATURE REVIEW



This chapter treats the literature on Mexican bean beetle, Epilachna varivestis Mulsant, a new host of the parasite Pediobius foveolatus (Crawford), and the parasite itself. It is intended to provide salient features in regard to the status of biological and applied knowledge about these insect species.



The Host Insect, E. varivestis


Taxonomy

The Mexican bean beetle, E. varivestis, also earlier known as ladybird, bean beetle, bean bug, and spotted beetle (Chittenden and Marsh, 1920) belongs to the Epilachna varivestis group, tribe Epilachni, subfamily Epilachninae, family Epilachnidae, and order Coleoptera (Gordon, 1975). First described by Mulsant in 1850, it was also once known under the name Epilachna corrupta Mulsant (Chapin, 1936).


Distribution

The original home of the beetle was southern North America, where it occurred in many parts of Mexico and Guatemala (Howard and English, 1924). According to the Commonwealth Institute of Entomology


3







4


(1954), it was known to inhabit southern Canada, the United States of America, Mexico, and Guatemala. Gordon (1975) added El-Salvador, Honduras, Nicaragua, and Costa Rica to the list. In Canada, the beetle was first reported from southwestern Quebec in 1943 (Auclair, 1959). In Mexico, it has a wide range of distribution, from 3 to 8,845 feet elevations within areas delimited by the 200C isotherm (Landis and Plummer, 1935).

In the United States, distribution of the beetle includes

Alabama, Arizona, Arkansas, Colorado, Connecticut, Delaware, Florida, Georgia, Idaho, Illinois, Iowa, Kansas, Kentucky, Louisiana, Maine, Maryland, Massachusetts, Michigan, Mississippi, Missouri, Nebraska, New Hampshire, New Jersey, New Mexico, New York, North Carolina, Ohio, Pennsylvania, Rhode Island, South Carolina, South Dakota, Tennessee, Texas, Utah, Vermont, Virginia, West Virginia, Wisconsin, and Wyoming (Nichols and Kogan, 1972). Within the United States, the beetle was first discovered at Watrous, New Mexico,in 1849 (Chittenden, 1924), but it was not until 1883 that anything concerning its habits appeared in publication (Chittenden, 1898). In Utah, it was officially recorded only in 1922 (List, 1922). By 1930, the beetle had been discovered in all but seven of the states east of the Mississippi River (Tissot, 1943). Howard and English (1924) presumed that it had reached Alabama at least as early as 1918 through shipments of alfalfa hay from Utah. They also reported that the beetle was present in Georgia, Tennessee, Kentucky, South Carolina, and Virginia by 1921. In Pennsylvania, it was found for the first time in 1921 and its entry was probably through West Virginia and Ohio (Guyton and Knull, 1925).







5


Watson (1942) reported that, since its introduction into Alabama, the beetle had spread rapidly to the north and east, but much more slowly to the south. He also reported that the first beetles captured in Florida were discovered at Monticello by Fred Walker in 1930. The second definite record of a Florida infestation was made in 1938 (Tissot, 1943). In 1942, they were found at three localities in Alachua County: Gainesville, Hawthorne, and Island Grove, when the nearest known infestation was at Havana in Gadsden County. According to Nichols and Kolgan (1972), the lowest borderline of Florida infestation was delimited by Levy, Marion, Putnam, and Flagler Counties. Since then, the beetle has spread southward to Citrus, Sumter, Hernando, Pasco, and Hillsborough Counties.

In the west, an isolated infestation was found in Ventura

County, California, in 1946 and another in Twin Falls County, Idaho, in 1954. The insect was eradicated from both regions (Entomology Research Division, ARS, 1958).


Life History, Economic Role, and Damage

The complete life history of the beetle was studied by Thomas (1924). Bernhardt and Shepard (1978) reported that the adult beetle survived the winter in heavy accumulation of pine litter, field litter, or sweet gum litter.

Both larval and adult stages are destructive to host plants. Howard (1922) stated that the beetle had demonstrated its importance not only in actual monetary loss, but also through its capacity for destruction wherever it became established, and by its tremendous






6


capacity for rapid spread. In 1924, he reported that the pest quickly destroyed practically all the beans over an area of about 4,500 square miles in northern Alabama. In Maryland, Stevens et- al. (1975a) pointed out that the beetle had inflicted serious economic damage to soybean, and that with the increasing cash value of this crop, the problem has steadily worsened, due to the greater tendency for the growers to use chemicals for control of the pest. Chittenden and Marsh (1920) reported that injuries caused by the beetle were practically confined to beans, and no variety seemed to be exempt from injurious attack. Various forms of the kidney bean, Phaseolus vulgaris L. et al., including string, pole, navy, and tepary or Mexican, and lima bean, P. lunatus L., were affected, and on one occasion, the soybean, Soja hispida Moench (now Glycine max (L.) Merr.) was also attacked. They estimated that annual damage in New Mexico varied from 5 to 100% of the crop, the average loss being set conservatively at 10%. Howard and English (1924) reported that wherever it occurred, the Mexican bean beetle was a more serious pest than the Colorado potato beetle. Hinds (1920b) gave the following assessments. Loss to common snap beans in Alabama was likely to be complete, except for a partial yield from the earliest planted beans. This was almost equally true for pole beans and shell beans. Lima beans made a partial crop, but certainly less than a half crop. California blackeyed peas were destroyed. Soybeans also suffered heavily in some fields, but the infestation was not as general as on the other food plants. Kudzu was not attacked noticeably in the field, but complete development of the insect was obtained upon that plant. No wild food plants were found, and there was no field attack of velvet beans, although slight feeding occurred







7


in confinement. He also reported that food plants or other fresh materials most likely to aid in disseminating the pest included all soybeans, fresh beans, and cowpeas of any kind, but did not include English peas and velvet beans. Cowpea was first reported to be a host for the insect by Hinds (1920a).

Sherman and Todd (1939) listed snap beans (bush), snap beans

(pole), lima beans, soybeans, velvet beans, crotalaria, alfalfa, peanut, beggarweed, and kudzu as food plants in decreased order of preference by the Mexican bean beetle. The Entomology Research Division of the USDA (1958) included common beans, such as snap (green or string), kidney, pinto, navy, and lima beans as primary food plants, while also stating that the beetle can reproduce successfully on cowpeas and soybeans and reported that injury to soybeans had become more common in parts of the south. It was also noted that the beetle's second choice of food was beggarweed (Desmodium tortuosum (SW) D. C.) or beggartick, which grows wild throughout the southeastern states.

Lockwood and Rabb (1979) found that the beetle adults consistently lived longer and produced more eggs when feeding on reproductive as opposed to vegetative soybeans and when feeding on lima beans as compared to reproductive soybeans. Whitfield and Ellis (1976), from their survey on insect pests of soybeans and white beans in 1975 and 1976 found no Mexican bean beetle on soybeans but only on white beans. Turner (1932) reported serious injury of rye caused by the beetle. Sunzenauer et al. (1980) found that the mean daily soybean leaf area consumption rates by the beetle were 4.65 cm2 for population consisting of second-generation adults, 4.87 cm2 for population consisting of adults






8


that had overwintered in the field, and 3.80 cm2 for populations consisting of adults that had overwintered in the laboratory.

McAvoy and Smith (1979) reported that the laboratory daily consumption of soybean foliage was 2.70 and 3.40 cm2 at 200C and 3.70 and 3.80 cm2 at 260C for male and female adult beetles, and that the development times of larvae were 5.30, 4.00, 4.80, 7.90, and 7.30 days at 200C for the 1st, 2nd, 3rd, and 4th instars and pupa, respectively; the figures were slightly smaller at 260C. Bernhardt and Shepard (1979) reported that the beetle adults that were given Phaseolus lunatus and had fed on P. vulgaris as larvae had higher fecundity, male and female longevity, as well as shorter preoviposition period and number of days between ovipositions than adults with any other combination of diets, i.e., P. vulgaris - soybeans, soybeans - soybeans, and soybeans P. lunatus.

Besides the damage directly inflicted on the host plants, -the beetle is also reported to transmit cowpea mosaic virus (Jansen and Staples, 1970), and the blackgram (mungo bean) mottle virus (Scott and Phatak, 1979).


Control

Early in the century, as far as chemical control is concerned, only rotenone gave satisfactory protection of beans from injury by the Mexican bean beetle (Howard et al., 1948; Entomology Research Division, ARS, 1958; Ditman and Bickley, 1951). Carbophenothion, disulfoton (granules), diazinon, malathion, methoxychlor, parathion- carbaryl,







9


and rotenone were later listed by Cantelo (1977) as pesticides for controlling the beetle.

Walker and Bowers (1970) found that synthetic juvenile hormones, Methylenedioxyphenoxyterpenoid ethers, prevented hatch of the beetle eggs. They concluded that these ovicidal properties warranted further investigation toward practical application for control of the beetle eggs.

An eradication program primarily composed of (a) winter survey and treatment of backyard gardens, (b) elimination of winter hibernation quarters, (c) planting of trap crops, (d) chemical treatment of bean plants within one mile radius of known infestations, (e) intensive survey of all bean plantings in the infested area, (f) establishment of quarantine line, and (g) development of effective commodity treatments, was successful in Ventura County, California where the beetle had been established since 1946 (Armitage, 1956).

Resurgence of the beetle population following the treatment of soybean with methyl parathion and methomyl was reported by Shepard et al. (1977) at Clemson University Edisto Experiment Station. These investigators concluded that removal of natural biotic agents by the chemical insecticides was probably the major reason for such resurgence.

Cultural practices have also been used to control the beetle. Immediate plowing under bean vines at the completion of harvest was encouraged in areas of occurrence to reduce the number of beetles entering hibernation (Chapman and Gould, 1930; Cantelo, 1977). Turner and Friend (1933) recommended that beans be planted 4 inches apart in







10


areas where the beetle was a serious pest, since the beetle preferred closely planted beans for oviposition. Turner (1935) later found that injuries caused by the beetle was 37% and 67.5% when plants were spaced, respectively, 8 and 2 inches apart. Variations in soybean cropping practices were found to affect significantly the abundance of the beetle (Sloderbeck and Edwards, 1979). The beetle adults and larvae were more abundant in tilled soybeans than in non-tilled soybeans, and was limited to one larval generation on double-crop soybeans compared to 2 generations on early planted crops. Destruction of the beetle generation on snap beans planted at the border of soybeans as trap crops has been demonstrated to result in the protection of the adjacent soybean fields (Rust, 1977).

Sources of plant resistance through screening of world collections of soybean were found in 3 cultivars (Van Duyn et al., 1971). Forced feeding tests showed that these lines were unsatisfactory as food even when no alternate food was available. Further studies by the same investigators showed reduction in longevity and fecundity in beetle adults, and weight loss and high mortality in larvae (Van Duyn et al., 1972). Field cage studies revealed that soybean cultivar "Shore" suffered no loss in yield at initial infestation rates of 1 and 2 adult beetles per linear foot of row as compared to susceptible cultivar "York" (Elden and Paz, 1977). Analyses for contents of total nitrogen, carbohydrates, organic acids, and sterols of leaf samples at different growth stages revealed that susceptible cultivars accumulated more total nitrogen at faster rate than did the resistant plant introductions (Tester, 1977).







11


Drought periods accompanied by dry winds decreased soil moisture and desiccated the plants. As a consequence, bean leaves turned upright, and the beetle eggs and young larvae exposed to the sun dried and collapsed (Douglass, 1933).

Despite the considerable list of natural enemies they had accumulated, Howard and Landis (1936) stated that the Mexican bean beetle had been practically unimpeded by parasites or predators in its spread throughout the intensively cultivated areas of the United States. The Entomology Reserach Division of the USDA Agricultural Research Services reported no record of internal parasites until 1922, when 2 native flies, the tachinid Euphorocera claripennis (Macq.), formerly known under the name Phorocera claripennis Macq., and the sarcophagid Helicobia rapax (Walker) were found to parasitize the insect in rare instances in northern Alabama; these parasites never became abundant enough to be of any value (Entomology Research Division, ARS, USDA, 1958). Paradexodes epilachnae, described by Aldrich in 1923 and now known as Aplomyiopsis epilachnae (Aldrich), was introduced into the United States from Mexico, and was bred and reared. The parasite was not recovered in any locality the year following liberation (Landis and Howard, 1940). Many species of predators of the families Pentatomidae, Reduviidae, and Cleridae were observed to feed on Mexican bean beetle by Plummer and Landis (1932). Stiretrus anchorago (F.) and Podisus maculiventris (Say), both feeding on eggs and larvae of the beetle, were found to respond to increasing prey density with negatively accelerating rises in the curves to an asymptote (Waddill and Shepard, 1975). A mite, Coccipolipus epilachnae Smiley, found in







12


Central America has been observed to cause reduction in egg production of the Mexican bean beetle (Smiley, 1974). Host specificity tests

conducted by Schroder (1979) revealed that hosts were limited to the members of the subfamily Epilachninae, all of which are phytophagous.

On soybeans, Stevens et al. (1975a) observed that egg predation by predaceous coccinellids appeared to be most common, and that, from several hundred thousand field-collected larvae, only 2 were found parasitized. Neither quantitative data on egg predation nor the identity of those 2 parasites were given.

Quattlebaum and Garner (1980) reported Mexican bean beetle

adults infected by a fungus different from Beauvaria. He pointed out that the beetle larvae were more susceptible to the fungus than any of the lepidopterous larvae tested, i.e., corn earworm (Heliothis zea (Boddie)), tobacco budworm (H. virescens (Fabricius)), cabbage looper (Trichoplusia ni (Hubner)), soybean looper (Pseudoplusia includens (Walker)), and velvet bean caterpillar (Anticarsia gemmatalis Hubner).

As indicated by the rapid dispersal of the Mexican bean beetle and continued importance as a pest, native biological agents and early efforts to introduce the parasite A. epilachnae (Landis and Howard, 1940) failed to provide control at a satisfactory level. However, the results of inoculative releases of Pediobius foveolatus (Crawford) made in Maryland in 1972 to 1974 (Stevens et al., 1975a) and in Florida in 1974 to 1977 (Reece I. Sailer, unpublished report) have proved to be an effective means for control of the beetle.







13


The Parasite, P. foveolatus


History, Taxonomy

This parasite was first recorded in India by Ayyar (Angalet

et al., 1968). In his survey made to discover beneficial insects in India in 1956, Angalet (Angalet et al., 1968) recovered several larvae of Henosepilachna sparsa (Herbst) parasitized by P. foveolatus. This recovery was of particular interest to him because of the possibility it might accept a new host, the Mexican bean beetle, E. varivestis, a species that does not occur in India. From the stock reared by the Commonwealth Institute of Biological Control in India on H. sparsa, a pest of potato and eggplant, the parasite was introduced into the United States by the Insect Identification and Parasite Introduction Research Branch at Moorestown, New Jersey, in 1966 and was found to develop readily on the Mexican bean beetle. In 1967, more than.25,000 P. foveolatus were released in the eastern states from New Jersey to Alabama (Clausen, 1978). The first quantitative account of its use through annual inoculative releases on soybeans was reported by Stevens et al. (1975a).

P. foveolatus belongs to the family Eulophidae, superfamily

Chalcidoidea, and order Hymenoptera. It was once placed in the genus Pleurotropis Foerster by Crawford (1912). Pleurotropis was later made a synonym of Pediobius Walker by Ferriere (1953). Role as Biological Agent, Biology, Natural Enemy

The role of P. foveolatus in biological control in the United States was already mentioned under the control of the Mexican bean







14


beetle. In India, Lall (1961) reported field maximum and minimum percentages of parasitization on Epilachna spp. as being 37.8 and 9.03, respectively, during April. He also cited the paper of Appana, and Usman and Thontadarya, in which Epilachna 28-punctata (Fabricius) and Henosepilachna sparsa (Herbst) were reported as other hosts of the parasite.

Little has been worked out on the details of the basic biology of this gregarious parasite. In his study designed to determine the efficiency of P. foveolatus as a parasite of phytophagous Epilachna spp. in India, Lall (1961) also worked on some aspects of its biology, namely mating, oviposition, larval growth, and adult emergence. Laboratory rearing of the beetle and the parasite, as well as studies on the parasite developmental rate, fecundity, sex ratio, effect of the parasite age on fecundity, and effect of parasite-host ratio on parasite production were published by Stevens et al. (1975b). Superparasitism of E. varivestis by the parasite and parasite-host ratios in regard to temperatures were reported by Shepard and Gale (1977). Gale and Shepard (1978) also studied the response of the parasite to temperature and time of exposure to the host. The parasite failed to overwinter, due to lack of diapause capability and/or available host material (Reece I. Sailer, unpublished report).

So far as natural enemies are concerned, Eupteromalus viridescens (Walsh), a native pteromalid, was found in Maryland to be associated with the Mexican bean beetle larvae parasitized by P. foveolatus, the first and only reported instance that appeared to Zungoli (1979) to represent hyperparasitization.















CHAPTER 2

GENERAL MATERIALS AND METHODS USED TO STUDY THE BIOLOGY OF PEDIOBIUS FOVEOLATUS



Unless otherwise mentioned, the following methodology regarding the rearing of the host insect, Epilachna varivestis Mulsant (Fig. 2-1) and the parasite Pediobius foveolatus (Crawford) (Fig. 2-2) was applied to all studies undertaken.

All experiments were conducted at the room temperatures (22-240C) of the Biological Control Laboratory of the Division of Plant Industry, Department of Agriculture and Consumer Services, Gainesville, Florida.



Rearing of Adults of Epilachna varivestis


Clear plastic containers (Fig. 2-3), 31 cm x 22.9 cm x 10.8 cm, were used to rear adults of the Mexican bean beetle, E. varivestis. Ventilation, to avoid water condensation, was assured by circular holes (4.4 cm diameter), usually 2 at either end or 2-4 in the cover of each container, all screened with 80-mesh brass strainer. Bean (snap or lima) (Fig. 2-4) or beggarweed (Desmodium tortuosum (SW) D. C., Fig. 2-5) leaves served as food for the beetle. Plant leaves were kept fresh, particularly during periods of low humidity, by inserting their petioles through holes in the cap of a small container partially filled with water. Excess of humidity inside the rearing containers was


15

















FIGURE 2-1. Mexican bean beetle, Epilachna varivestis Mulsant. Extreme left vertical row: 4 adult beetles; upper middle: 6 2ndinstar host mummies; upper right: 6 3rd-instar host mummies; bottom horizontal row: 4 4th-instar host mummies.




























FIGURE 2-2. Pediobius foveolatus (Crawford), parasite of Epilachna spp. (approximately 60 X).







17


I

, t



a,...






iI.Ie~et.aIa3ompsIegss3SpSSBIsIaIgIuI.gesmIgssIlIIIIp~I~I1I~Ispt
7 1 A. a Y



~'"' 4,



l1~1~S~ ~ '~
kit


- II
//
I













4





















FIGURE 2-3. Containers and tools for Mexican bean beetle rearing. Left: clear plastic container for rearing of adult beetles; middle: plastic container and petri dishes for rearing of beetle larvae; lower right: camel hair brush and locally-made bamboo tweezers.


Young greenhouse-grown lima bean plants.


FIGURE 2-4.













-4






ii








0"-4














I.


p


N







20


minimized by using containers with fewer vent holes or by placing leaves directly on the towelling paper covering the bottom of the containers. In all instances, towelling paper was used because it reduced soilage. Ten to 12 female-male pairs of adult beetle were reared in each container. Food was renewed daily. Egg masses, collected daily at the time of each food renewal, were incubated in petri dishes until the eggs hatched. Rearing containers were changed at 3 to 4 day intervals, depending on soilage. Beetle adults were discarded when egg production declined, usually when they were about 6 weeks old.



Rearing of E. varivestis Larvae


The newly hatched larvae, aggregated on the empty shells of the egg masses, were transferred to larger plastic petri dishes (14 cm diameter, Fig. 2-3). Larvae from 2 to 3 egg masses (about 100 larvae) were then reared in these dishes through the 2nd instar. Clear plastic containers (Fig. 2-3) having dimensions of 18.4 cm x 13.3 cm x 8.9 cm, and 31 cm x 22.9 cm x 10.8 cm were then used to rear about 100 3rd- and 4th-instar larvae, respectively. Each end of all rearing units contained a circular screen vent. Bean and beggarweed leaves were the food. A towel paper sheet of appropriate size was placed underneath the food material to reduce soilage. Fresh leaves were added daily and rearing units were changed when needed. Adult beetles for production of host larvae and maintenance of the beetle culture were produced in this way.







21


Rearing of P. foveolatus


Of the 3 stocks denoted "Ocala," "Perry," and "NF S-121" permanently maintained in the laboratory, only the last was used for all studies conducted here.

Clear plastic containers with dimensions 12 cm x 9.3 cm x 7 cm (Fig. 2-6) were used for rearing adults. To assure air circulation, rearing units were provided with 3 circular vents, one on either lateral side and one on the cover of the rearing unit; all were screened with nylon cloth. Another circular hole, normally plugged with a cork or rubber stopper, was located at one end of the unit to facilitate exposure of the host larvae to the parasite and the removal of the empty host mummies left after emergence of the adult parasites. To prevent the adults of the parasite from being caught between the wall and the lid rim of the rearing unit, the inner rim of the container body was lined with tape. Honey, applied in streaks on the bottom of the rearing unit and covered with a moistened sheet of towelling paper, served as food for adults. Water was supplied in a glass test tube of 1 cm diameter and 10 cm tall. The test tube was loosely plugged with cotton and, placed inside the rearing unit in a slightly inclined position to avoid water flow. A plastic vial (snap-cap type) lid having a diameter of 3.5 cm placed in the unit near the access hole was used to hold the host mummies, thereby preventing them from sticking to the honey-impregnated paper towelling. Only 4th-instar larvae of E. varivestis were used for all experiments, except the one dealing with oviposition preference study. Exposure of the host larvae to the
























FIGURE 2-5. Beggarweed plants, Desmodium tortuosum (SW) D. C., at fruiting stage.






















FIGURE 2-6. Containers and tools for rearing and handling of parasite adults. Upper left: clear plastic container for culture maintenance of parasite adults; upper middle (in glass petri dish cover): small glass tubes for isolation of parasite pupae until emergence and sexing of the virgin adults; upper right: clear plastic container topped with acetate sheet, used as mating chamber for study on sex ratios resulting from sequentially mated males (Chapter 6) and study on multiple matings of females (Chapter 10); lower left: aspirator for transfer and isolation of mated females from container at upper right; lower middle: plastic tubes for isolation and confinement of single females or femalemale separate pairs; lower right: standard aspirator for collection of parasite adults.







23


77 a..-.





d 6






24


ii


r






-H


-FIGURE 2-7. Hood for handling of parasite adults (designed bi Dr. Reece I. Sailer).







25


parasite was made in the hood (Fig. 2-7) with the light on. Host larvae, collected beforehand in a large petri dish (14 cm diameter) were transferred quickly one by one by means of a pair of locally made bamboo tweezers of appropriate length (Fig. 2-3) into the rearing unit through the open access hole. The transfer operation was interrupted from time to time by the removal of those host larvae that were in the process of being stung (oviposition) by the parasite females. Stung host larvae, once removed from the exposure unit, were placed in a separate dish until termination of parasite oviposition. They were then transferred to the rearing containers, large petri dishes or more roomy containers, depending on the numbers of host larvae needed. Only one parasitic female was allowed to parasitize one host larva. Upon termination of oviposition, parasitic females were collected with an aspirator and returned to the rearing unit only at the end of each host exposure operation. Exposed host larvae were reared on bean or beggarweed leaves until cessation of feeding. For the stock culture, host mummies were transferred to the rearing unit of the parasites 2 to 3 days prior to the parasite adult emergence. Parasite colony was renewed at 3- to 4-week intervals.















CHAPTER 3

MODE OF REPRODUCTION



Introduction


Of haplodiploid sexuality, Borgia (1980) wrote "among arthropods, haplodiploidy is the exclusive mode of reproduction in the orders Hymenotera and Thysanoptera, and also occurs in Homoptera, Coleoptera, and mites and ticks (order Acarina)" (p. 104).

Flanders (1939) stated that "sex control in the Hymenoptera

under normal conditions results in preponderance of females. Extreme variability in the proportion of sexes, however, is often evident. This condition apparently results from the fact that in the majority of species the males are usually derived from unfertilized eggs and the females from fertilized eggs" (p. 12). This clearly describes a haplodiploid system also referred as arrhenotoky. Stevens et al. (1975b) investigated reproduction of the eulophid Pediobius foveolatus and found that unmated females oviposited, but their progeny were all males, thus confirming the arrhenotokous nature of this species. They did not pursue the study of the reproductive biology of this species beyond this point.

The objective of the present investigation was to provide qualitative and quantitative evidence relative to arrhenotoky as the mode of reproduction of P. foveolatus and to gain insight to factors that


26







27


influence sex ratios and reproductive success of this species.



Materials and Methods


To obtain virgin females, pupae were separated from the host mummies through dissection, and confined individually in small glass tubes of about 0.5 cm diameter and 3 cm tall. The tubes were then plugged with absorbent tissue. The separation of the pupae was made 2 days prior to the normal emergence of the adults from the host mummies. Sexing was made soon after emergence from the pupae., Thirty-eight virgin females, picked at random from among those issued from 30 4thinstar host mummies (approximately 300 females), were reared separately in penicillin vials provided with honey and water impregnated in small circles of paper towelling. Three 4th-instar host larvae were separately exposed to each virgin parasite female within 3 consecutive days (one host exposure/day) beginning on the 3rd day after the adult emergence from the host mummies. They were then reared until cessation of feeding in separate petri dishes (8.9 cm diameter) labeled according to the given numbers of the parasite female and their exposure sequence. Two days prior to the day of normal emergence of the progeny, the host mummies were confined in separate gelatin capsules (no. 000) with corresponding labels. Sexing and counting were made only after the death of all emerged progeny adults.







28


Results and Discussion


Out of the total 38 tested virgin parasite females, 36 produced only live male progeny, whereas 2 (females no. 13 and 28) completely failed to produce offspring throughout the 3 host exposures (Table 3-1). No live female progeny was recorded. However, among those offspring that were not able to emerge from either the host mummies or their own pupae and those that failed to develop to adulthood (sexunidentifiable individuals), only one female (female no. 27) with incomplete development was recorded following dissection of all host mummies and the parasite pupae. The recognizable feature of this single female was the obvious presence of ovipositor. Before these convincing results, it is concluded that arrhenotoky is the type of reproduction of P. foveolatus.

In connection with the occurrence of the above-recorded single female that failed to fully develop, Suomalanien (1962) stated that unfertilized eggs of many bisexual insect species begin to develop, although the development, as a rule, comes to a standstill sooner or later (rudimentary parthenogenesis). He further stated that such unfertilized eggs occasionally develop quite far, even to adult stages in cases of accidental or tychoparthenogenesis, a process that may be regarded by many authors as the primitive type from which normal thelytoky has developed. Whether the occurrence of that particular P. foveolatus female should be called rudimentary parthenogenesis or tychoparthenogenesis or accidental parthenogenesis, the situation remains conditional.







29


TABLE 3-1

Live adult progeny (females/males) produced through 3 separate
host exposures by P. foveolatus virgin females


Parasite parent female no. host


1
2 3
4 5 6 7 8 9 10 11
12 13
14 15 16 17 18 19
20 21 22 23
24 25 26 27 28 29 30 31 32 33
34 35 36 37 38
* Host larva pupated;
developed female was


Parasite progeny (females/males) 1st 2nd 3rd
exposure host exposure host expo
0/17 0/18 0/22
* 0/17 0/22
0/20 0/15 **
* ** 0/37
0/20 0/18 0/25
0/14 0/16 0/23
* ** 0/33
0/17 0/20 0/12
* 0/17 0/18
0/23 0/19 0/27
* * 0/20
0/20 ** **
* * **
0/21 * **
0/23 0/23 **
** 0/16 *
** 0/18 0/34
0/19 0/15 0/12
0/22 0/18
0/16 0/23 **
* 0/15 0/18
0/17 0/17 **
0/25 0/17 **
* 0/11 0/32
* * 0/24
0/18 0/15 **
0/15 (*) ** **
* * **
0/18 0/37 0/23
0/19 0/21 **
0/16 0/21 **
0/21 0/14 **
* 0/18 **
0/14 0/19 **
0/17 0/16 **
* 0/26 **
0/8 0/17 **
0/16 0/27 **
** host larva dea; (*) 1 incomple observed


sure


tely






30


Based on the results of the sexual behavior study (Chapter 4),

P. foveolatus reproduction is characterized by a high level of inbreeding. All investigators who have studied the species have reported female-biased sex ratios. The association of inbreeding with haplodiploidy has been noted by many authors notably Ghiselin (1975) and Borgia (1980). Entwistle (1964) working on Xyleborus compactus (Eichh.) reported that unmated females on coffee stems produced only male progeny with which they subsequently mated to produce daughters. He attributed this high degree of inbreeding to the low mobility of the females living in "closed galleries". He concluded that the inhibiting effect on gene flow between populations combined with close inbreeding resulted in low individual variation within a local population but high interpopulation variation.

Brown (1964) noted that the cost of initiating a viable haploid male to produce a haplodiploid species would be reduced by prior close inbreeding resulting in a high level of homozygosity. Thus haploid males would not suffer the consequences of the numerous deleterious recessive alleles that are normally present in an outbreeding population. Supported by extensive data, Hamilton (1967) showed that sex ratios are biased when close inbreeding is common. He also pointed out that, in many instances, severe reduction of male size further enhances diversion of parental investment into daughters. In addition to the factor of size noted by Hamilton, P. foveolatus males have shorter life expectancy than females, a feature that further strengthens Hamilton's conclusion regarding diversion of parental investment into daughters. From an evolutionary standpoint, Borgia (1980) concluded that






31


haplodiploidy seems most likely to evolve in an inbreeding situation. He also noted that the transition from diplodiploidy to haplodiploidy through evolutionary time has occurred much less frequently than transitions to thelytoky. However, the haplodiploid transitions established the evolutionary origin of major systematic groups of organisms while the more numerous thelytokous transitions have normally been of little evolutionary consequence beyond the species level.

While it is beyond the scope of the present study to speculate on the pathway through which arrhenotokous species have evolved, the results of the studies on mating behavior of P. foveolatus support the view that inbreeding mating systems tend to be a common characteristic of haplodiploid species. The one dead, incompletely developed female encountered among the male progeny of the 38 virgin females would appear to be an example of the phenomenon noted by Speicher and Speicher (1938). They reported the appearance of an occasional uniparental female in Bracon hebetor Say and speculated that they resulted from patches of tetraploid tissue in an otherwise diploid ovary.















CHAPTER 4

SEXUAL BEHAVIOR



Introduction


Arrhenotoky, as already noted in Chapter 3, is the mode of

reproduction of P. foveolatus. Borgia (1980), working with models for inbred and outbred systems in the evolution of haplodiploidy, noted that the inbreeding context seems the most likely situation for the evolution of haplodiploidy. Hamilton (1967) pointed out that under inbreeding or under the effects of more severe competition between brothers than between nonrelatives, parents should produce investment ratios that favor females. Correlation between inbreeding and male haploidy has also been noticed by M. T. Ghiselin who was quoted by Borgia (1980) as saying "the adaptive significance of male haploidy may have something to do with controlling the sexuality of offspring" (p. 111). Borgia (1980) also concluded that "even though inbreeding models seem to provide the best explanations for the evolution of male haploidy, in specific instances pathways to male haploidy specified by outbreeding models may be important" (p. 125). Among haplodiploid species of parasite Hymenoptera, inbred or outbred, sexual behavior is expressed in different ways (Borgia (1980), Boush and Baerwald (1967), Ghiselin (1975), Hamilton (1967), King et al. (1969), Leonard and Ringo (1978), Miller and Tsao (1974), Schlinger and Hall (1960, 1961), Simser


32






33


and Coppel (1980a, 1980b), Van den Assem and Povel (1973), Vinson (1972), Werren (1980), and Yoshida (1978)). Little is known of the sexual behavior of P. foveolatus despite the important role of the species in biological control.

The purpose of the present study was to gather information

regarding sexual activities of males and females following emergence from their host mummy until their eventual dispersal.




Materials and Methods


The study was conducted under laboratory (22-24 C) and summer field conditions.


Laboratory Study

Gelatin capsules (no. 000) and a locally made cardboard arena (Fig. 4-8) were used to contain the parasite adults for observations on their sexual behavior.


Use of gelatin capsules

A number of host mummies were separately confined in gelatin

capsules (one host mummy per gelatin capsule), and held under constant observation. Mating behavior of adult female and male parasites was observed soon after they emerged from the host mummies. This set-up permitted close observations whenever needed, even under microscope. Use of the cardboard arena

Gelatin capsules did not allow observations on how the parasite females and males behaved within a wider space in accordance with time.







34


FIGURE 4-8. Observation arena specially designed for study on parasite sexual behavior. The arena floor, made of white cardboard and marked with a series of circles distant from each other by 1 cm, is of 30 cm diameter. The clear acetate wall is 3 cm tall. A circular glass plate of 40 cm diameter is placed on the top to prevent escape of the parasite adults from the arena and also enable tracking male mate-searching.







35


To get information in this regard, a locally made cylindrical arena of 30 cm diameter and 3 cm deep was used (Fig. 4-8). The wall of the arena was made of clear acetate. The floor, made of white cardboard, was provided with a series of pencil-drawn circles distant from each other by 1 cm. A clear circular glass plate of 40 cm diameter served not only as a cover to prevent escape of the parasites, but also minimized disturbances. It also provided a surface on which male matesearching paths could be drawn with a marking pen.

A single host mummy was placed at the center of the innermost circle of the arena floor and kept under continuous observation. Following emergence of the first male parasite, its movement was tracked until termination of mating activities, which was regularly followed by the active flight and dispersal of all individuals within the arena. In the course of track tracing, time lapses (in minutes) were also recorded whenever convenient. The inked male mate-searching pattern was then copied by means of Xerox.

Information on the distribution of the adult parasites within the arena during the active mating period was obtained through photographs taken at half- or one-minute intervals, depending on the intensity of mating activities. The first picture was made when the first emerging adult appeared. The last picture was taken when mating activities ceased and individuals began to take flight and disperse.

At no time did observational data or photographs involve adult parasites from more than a single host mummy.







36


Study under Field Conditions

A number of 4th-instar host larvae, following exposure to

P. foveolatus females in the laboratory, were released on garden-grown lima bean plants (University of Florida Organic Gardens area) and caged to prevent escape or predation by natural enemies prior to the day parasite adults were expected to emerge. Probable period of emergence was established by dissection of one or 2 host mummies daily during the last 3 days prior to the day of normal laboratory emergence; the presence of adults in the process of emergence from pupae was an indication that actual emergence of the adults from the host mummies would occur within the next 24 hours. Close watch was maintained during daylight hours of this period for emergence of adult parasites. All observable behavioral activity of emerged adults, both females and males was recorded.



Results and Discussion


Laboratory Study


Emergence of the parasite adults

Emergence from pupae. From the hundreds of pupae separated

from the host mummies 2 days prior to the day of normal emergence and individually confined in small glass tubes (to obtain virgin females and males for use in the studies of sex ratios resulting from sequentially mated males, Chapter 6, and multiple matings, Chapter 10), the process of emergence of individual P. foveolatus adults was observed to last from a few to several hours. The overall emergence terminated






37


within 2 days. More than half of the total number of adults emerged during the first day. Males commonly emerged earlier than females.

Emergence from the host mummies. During a period of over 5

years of continuous culture, emergence of the parasite adults from host mummies of 20-25 simultaneously exposed 4th instars (usual numbers used for each stock renewal) took place over a period of about 2 days. Within this range, observed difference of emergence from individual mummies appeared to correlate with the number of the parasite eggs laid in relation to the quantity of food available in each host larva. An observed difference in rate of mummification among the parasitized host larvae appeared to be an early indication of rate of food use by the parasite larvae.

The process of ecdysis to the adult stage while still inside the host mummy and preparation of an emergence hole is normally completed within no more than 24 hours. The emergence hole is circular in shape, single and generally found in the abdominal region of the host mummy, and are made by both females and males. Emergence of adults from any host mummy took place only when all individual females and males are ready. This process, followed by the relatively short period of mating activities appears to ensure that a maximum number of females will be mated by their brothers before dispersal. The comparatively small size of males, the variable female-biased sex ratios, and the remarkably high level of male sexual activity (described elsewhere in the present chapter) appears to explain the adaptive advantage of simultaneous emergence of both sexes and subsequent mating activity normally observed within a reduced area around the host mummy, here called local mating area. Among females and males emerging from







38


any single host mummy, there is no specific sequence of sexes. Behavior of females

Compared to males, sexual behavior of females is simple. Following emergence, unless disturbed by more than one male attempting to mate with her, a female normally waits for a male by stationing herself in the vicinity of the host mummy from which she emerged. This behavior, consistently exhibited by all females, not only reduced the search area to be covered by sibling males but also allowed opportunity for grooming and establishing their composure. During this period, females were routinely observed to extend their legs, and through rubbing motions used them to clean their wings, body, and antennae. Except for a slow up-and-down movement of their antennae, a female responded to male courtship by remaining immobile. Acceptance of copulation by a male was manifested by an abrupt change in the posture of the female, with the female standing high on her legs to allow copulation. Upon termination of copulation which lasted from 5 to 14 seconds, she resumed her initial rest position, occasionally remaining there for some period of time, but more often moved away for some distance following a subsequent mating attempt by the same or other males. This movement was directed toward the outside away from the host mummy (Fig. 4-10). The number of stops on her way out depended primarily on subsequent male mating attempts. Such behavior, in addition to making room available for her newly emerged virgin sisters and thereby enhancing their mating prospects, would minimize wastage of male energy in courting already mated females. On the way out,







39


particularly at the perimeter of the observation arena (large petri dish) and prior to the eventual dispersal, some females in rare occasions were seen to accept a second mating. Occasionally, some females were approached by males just as they emerged from the host mummy. These females would continue to move until they were a short distance from host mummy before assuming courtship stance. Competition for individual females, even briefly, was rarely observed among brothers. Not all virgin females were mated on the first attempt by males. Generally, males moved away in search of new females when the female was not receptive after a 40- to 70-second male precopulatory attempt. The erratic movement of females increased with time. Meanwhile, males also gradually expanded their mate-searching area. In other words, the whole process proceeded smoothly in conjunction with progress of emergence. Mating activity diminished and eventually ceased not long after all parasite adults had emerged from the host mummy. Concurrently with diminished mating activity, there was increasing intensity of overall ground movement followed by increasing flight activity and eventually by general flight movement of all individuals inside the arena. The whole process of mating activities around single host mummies was observed to last from 30 minutes to about one hour. Behavior of males

Males, in contrast to females, were usually very active. No matter what sequential position they occupied in any series of emergence, their behavioral pattern remained similar. Upon emergence from the host mummy, they lost no time searching for females, patrolling






40


around or nearby, and frequently returning to the host mummy. The behavior of one male was especially revealing. This male happened to be the first adult to emerge from the host mummy. After patrolling the vicinity of the mummy for 9 minutes, this male returned and stationed himself at the exit hole from which he had emerged. After 4 minutes, a second adult emerged and proved to be a female with which he immediately mated.

When a male encountered a female, especially after making his

presence known, he always positioned himself -at one side of the female's body. Meanwhile, the male while standing on his meso- and metathoracic legs, grasped the upper thoracic portion of the female with one of his prothoracic legs while keeping the other loose without any contact. The female rested low on her legs. Male courtship then proceeded to the next phase. This was manifested by strong vibration of the male's wings in upright position, regularly interrupted by brief periods during which the male stroked the female's head or thorax with his antennae; during

each stroking episode, the male body always moved in rhythmical synchrony with his antennae. Except for a slow up-and-down movement of her antennae, the female remained motionless during this period. As mentioned earlier under the study of female behavior, unsuccessful courtship was observed to last between 40 to 70 seconds after which the male moved away in search for other females and the female would remain in the same position. When a female was ready for copulation, she stood high on her legs. The male, while holding the female, then bent his abdomen toward the base of the female ovipositor, and copulation followed. The duration of copulation varied from 5 to







41


14 seconds after which the male resumed the precopulatory posture, whereas the female lowered herself to the normal resting position. In no case did a male move away from his mating partner immediately after copulation. Rather he maintained the precopulatory posture and exhibited an act (postcopulatory behavior) similar to the precopulatory courtship for a short while before leaving the female in search for others. In some instances, as when confined to a gelatin capsule where courtship arena was very small, it was not unusual to record a higher number of females mated by a single male within a short period of time.

In case of a moving female, the male may approach her from any direction and must make his presence known by contact. If the female continues to move, the male circles her closely moving very rapidly. Whereas mated females more often kept moving away in a generally outward direction from the host mummy particularly upon male courtship attempts, newly-emerged virgin females normally stopped and allowed male courtship.

Encounters between moving males never led to courtship. However, on rare occasions, courtship may be exhibited by a moving male upon contact with another male at rest.

Males did not have the ability' to discriminate mated from virgin females. Such behavior might theoretically be disastrous for the system particularly if all mated females accepted subsequent matings. Males would have to expend more time and energy for regeneration of sperm supply and courtship activities. Since both sperm supply and time are needed in order to cover a maximum number of newly emerged virgin females within a relatively short period of time before






42


dispersal from the local mating area, such a costly investment would tax the capacity of the species comparatively small-sized and very active males. However, the system has evolved in such a way that the lack of discriminating ability of males vis-a-vis the mated females is compensated for by the low percentage of mated females undergoing multiple matings (Chapter 10) because of the refusal of most females to respond to subsequent courtship attempts by males. In fact, only on some rare occasions were mated females observed to be receptive to subsequent matings during the postemergence period. This occurred only in the later part of the local mating period when mated females had already reached the periphery of the observation arena.

Expansion of the searching area by males and the outward movement of mated females in the arena was a coordinated and gradual process (Fig. 4-9 and 4-10).

When host mummies contained only adult males, mate-finding behavior of males was similar to that exhibited by males issued from mummies containing adults of both sexes. The only difference was that the activities were less intense and of shorter duration, lasting only 15 to 20 minutes (Fig. 4-11) compared to 30 minutes to one hour when both sexes were present.


Field Study

Despite the small area of lima bean leaflets, observation on sexual behavior of adult siblings made on 3 separate host mummies under field conditions did not reveal a noticeable difference from that recorded under laboratory conditions. This includes the mate-searching,








43


cm is- O
$4f 17min.

1 3 2 0 m n .


11

10






7

6

9

-






C






7min. 14 min.

















FIGURE 4-9. Female-searching pattern of a male P. foveolatts in a 30 cm-diameter cardboard arena from the time of emergence from the host mummy to the end of mating activities. C is the center of the arena where the host mummy was placed. Numbers 1, 2, ..., 14, and 15 represent the distances in cm from the center of the arena. Arrows indicate the direction of male's movement. Male's travel time is given in minutes (min.).







44


0








5'

















[24'3'



nee


6'30' 13' 28El


2'








8' 15' 333


FIGURE 4-10. Schematic representation (transposed from photographs) showing gradual outward movement of P. foveolatus adults (females and males) undergoing postemergence mating activities around the host mummy. Each black spot at the center of the innermost circle represents the host mummy from which the parasite adults emerged; each black speck represents 1 adult parasite individual (male or female). Each ensemble of circles represents the 30 cm-diameter floor of the observation arena. Elapsed times (in minutes and seconds) at which photographs were made are shown at the left corner of each square; the first was made when the first parasite adult emerging from the host mummy appeared; time zero (0) was given to this; the last (last square of the 4th row) was made when general flight (dispersal) of the parasite was observed.


3'30 9'30 20-3 39,1'







45


0

















9'


2'








5'30' 114'3


3'













1'3 1'3


4' 8'













2 3p ,,


FIGURE 4-11. Schematic representation (transposed from photographs) showing gradual outward movement of P. foveolatus adult males (host mummy contained only males) undergoing postemergence searching for female mates. Each black speck represents 1 adult parasite male; the larger and elongated black spot at the center of the innermost circle represents the host mummy. Circles in the top row represent the innermost 7 cm of the 30 cm-diameter observation arena while each ensemble of circles of the 2nd, 3rd, and 4th rows represent the entire floor area of the arena. Distance between circles in the first row represents 1 cm and those of the remaining rows 2 cm of the arena floor. Elapsed times (in minutes and seconds) at which photographs were taken are shown at the left corner of each square. The first photograph was made when the first parasite male emerging from the host mummy appeared; time zero (0) was given to this. The last photograph (last square of the 4th row) was made when general flight (dispersal) of the parasite within the arena was observed.







46


mate-approaching, pre- and postcopulatory behavior, expansion of the searching area of the males, and the postemergence behavior of females and their response to mating attempts by males.

The following describes observed sexual behavior of males and females in two successive occasions. In the first, a single male emerged following emergence of 2 females. Contrary to usual laboratory and field behavior, this male moved 2 cm from the host mummy and assumed a grooming position for some minutes before starting to court either of the 2 already emerged females. His attempt to mate failed. He resumed a resting position again 2 cm from the host mummy. During the next 15 minutes, 3 more virgin females emerged, at which time he resumed activity. At this time, 7 males were observed actively competing for 2 females about 30 cm distant from the mummy already under observation. These appeared to have emerged about 2 hours earlier. All males displayed wing vibrations while closely following the females. The females were obliged to move, but movement was nondirectional. At different times, these females approached within 2 cm of the host mummy

from which virgins had continued to emerge. Despite the proximity of the newly emerged virgin females,' the males continued to pursue the other females until they were lost from view in the bean foliage. No successful matings were observed during the period of intense competition among these outsider-males. Meanwhile, at the local mating area of the mummy under active parasite emergence, the lone male became active and began courting a virgin female. Within about 30 seconds, 2 males from an unknown source lighted in the mating area and proceeded to search for females in a manner similar to sibling males. This







47


phenomenon was observed on a second occasion, when a strange male lighted in the mating area almost immediately after courtship behavior was observed among males and females that had just emerged from a host mummy. The fact that these males did not appear until local males began to exhibit precopulatory vibration of their wings suggests that nearby males were attracted to the mating area by vibratory sounds produced by the courting males. The similarity in mate-searching behavior exhibited by the outsider males once they were in the active local mating area and while local males were still pursuing their courtship wing vibration would suggest that the vibratory sound might be superseded by the effects of female sex-attractant. In fact, no competition for females by local or outsider males has been observed in the local mating area.

The above observations appear to indicate that successful

mating of P. foveolatus is basically accomplished through a concurrence of innate male and female behavior as influenced by sex pheromone, tactile stimuli, and perhaps auditory effect of local male wing vibration.

Innate behavior was basically expressed through (a) the patrolling of males in search for females around the host mummy even though females are totally absent, (b) the exhibition of pre- and postcopulatory behavior of males, and (c) the waiting-for-male posture and the gradual male-assisted dispersal of mated females from the mating area. Werren (1980), working with Nasonia vitripennis (Wlkr), a haplodiploid and gregarious parasite of cyclorraphous fly pupae, reported that males emerged from the host puparium first and waited for females, a






48


situation that appears to reflect the inherent behavior of this species. King et al. (1969), working with the same species also noted that males tend to emerge before females from a single host puparium.

An indication of the presence of a female sex-attractant was observed on several occasions. First, as already mentioned in the beginning paragraph of the description of male sexual behavior, the presence of such sex-attractant was indicated by the return of the male (the first individual adult to emerge from the host mummy) to the host mummy after a 9-minute patrolling tour followed by a 4-minute period of waiting until the emergence of the first female with whom he later mated. Later, in a replicated test, 3 host mummies on the verge of adult parasite emergence were placed side by side in a triangular figure within a large petri dish (one mummy containing parasite adults of both sexes, emergence hole present but not yet large enough for the parasite adults to pass through; one mummy containing only male parasite adults with no emergence hole; and one mummy containing parasite adults of both sexes with no emergence hole). Thirty newly emerged virgin males were introduced to each of the 2 petri dishes. At the end of 30-minute observation, 6 and 8 males, respectively, in the first and second petri dish were found to be attracted only to the host mummies with emergence hole and adults of both sexes. These males were present on and in the immediate vicinity of the host mummies. This behavior was also occasionally observed throughout the period of the present investigation.

Pheromones have been reported to be involved in mating activities of other hymenopterous parasites. Schlinger and Hall (1960, 1961)







49


reported that male Trioxys utilis Muesebeck and Praon palitans Muesebeck, parasites of spotted alfalfa aphid, apparently detected virgin females by odor, not by sight. Boush and Baerwald (1967) noted a strong indication of a female-secreted attractant in Opius alloeus Muesebeck, the parasite of the apple maggot. Leonard and Ringo (1978) reported a pheromone role in mate-finding of Brachymeria intermedia (Nees), a parasite of the gypsy moth and other lepidopteran pupae. Simser and Coppel (!980b) working on B. intermedia (Nees) and B. lasus (Walker) found that a femaleproduced sex pheromone serves to aid mate recognition by male. These investigators pointed out that male response to the pheromone remains constant with increasing male age, but pheromone activity declines with age in females. They further noted that activity of the pheromone did not elicit male response at distances greater than 3 cm. Beyond this distance, males exhibited only random movement. Some similarity in regard to the sex pheromone stimulus reported by Simser and Coppel (1980b) has been observed on P. foveolatus. It appears that the pheromone produced by female P. foveolatus serves as a short-range cue that leads males to the female. Its effective radius depends on concentration. The behavior of the males in returning to the host mummy with decreasing frequence as females emerged, mated and dispersed from the mating area may be the result of the gradual reduction of pheromone emanating from the host mummy following departure of the females. This would further imply that (a) intact (without emergence hole) mummy is the source of maximum pheromone concentration, (b) the pheromone begins to disperse when emergence hole is opened; its gradient decreases with the distance from the host mummy, and (c) it gradually loses its






50


strength as emergence of the adult parasite thins out. Therefore, it is expected that the pattern of mating activities may not be obvious or typical. This is indicated by the evidence that the somewhat disarrayed pattern of mating activities occasionally observed in the course of this study, usually involved host mummies where emergence holes had been opened for a relatively long period before the emergence of the parasite adults, thus allowing time for dissipation of pheromone. Attraction of males to isolated virgin females from only a short distance may also be attributed to the low concentration of pheromone produced by a single female as opposed to an aggregate of females within a mummy. The decline of pheromone activity through female aging

was not investigated. However, older females (about 20 days or older) were observed to be generally nonreceptive to mating when presented with a male. They either walked away or did not allow copulation, although precopulatory courtship was sometimes observed. The attraction of males to the host mummies with emergence holes but still containing unemerged adults of both sexes and the return of males to their host mummies after female-searching tours in the case of mummies containing only male parasite adults suggests that females sex pheromone does not act alone; a concurrence of male and female innate behavior is required in bringing the sexes together.

Display of wing vibration prior to mating have also been reported for other parasites. The ichneumonid Campoletis sonorensis (Cameron) male, when exposed to a female, displayed wing vibration which persisted until mounting (Vinson, 1972). Van den Assem and Povel (1973) suggested that amplitude, speed and duration of wing






51


vibrations may function as reproductive isolating mechanism in species of Muscidifurax. The effect of wing vibration of male Nasonia vitripennis (Walker) on mating receptivity was reported by Miller and

Tsao (1974); less than 20% of reproducing females successfully mated by wingless (wing-removed) males, whereas 78% successful mating was recorded for females confined with winged males. These investigators further noted that absence of wings in males resulted in production of only male offspring by most females. By means of an oscilloscopic analysis, Leonard and Ringo (1978) recorded a courtship song consisted of 3 distinct and orderly auditory displays, i.e., rock, wing quiver, and buzz, frequently repeated by male B. intermedia (Nees). Yoshida (1978) reported that secretion of sex pheromone by female pteromalid Anisopteromalus calandrae (Howard) released wing vibration of males.

As described earlier, males of P. foveolatus display wing.

vibration, only and always, upon contact with females (precopulatory wing vibration display) and immediately after copulation (postcopulatory wing vibration display). Such displays may be interpreted as follows: the precopulatory display, triggered by contact with female, triggered female receptivity to coitus; the postcopulatory display would somehow appear to be involved in the facilitation of sperm storage in the female spermatheca. Since attraction of outsider males to local mating area-was coincidentally observed to occur during the courtship of local males, wing vibration in this instance appears to have a recruiting role with a relatively long-range effect. The shortrange means of communication between both sexes is apparently assured by female sex pheromone. The various mate-finding mechanisms imply







52


evolution of a system that ensures maximum opportunity for impregnation of females before their dispersal from the immediate area of the host mummy from which they emerge while allowing opportunity for a degree of outbreeding as influenced by host and parasite densities. In this context, the mating system of P. foveolatus is basically of the inbreeding type. The conclusion is supported by evidence of diminished mating capability of both males and females (Chapters 8 and 9) and associated reduction in production of female progeny when mating is delayed for a prolonged period.
















CHAPTER 5

PREEMERGENCE MATING



Introduction


Pediobius foveolatus (Crawford) is a gregarious parasite with haplodiploid mode of reproduction and female-biased sex ratios. In Maryland, according to Stevens et al. (1977), the numbers of parasite adults per host mummy ranged from 1 to 100. In Florida, the following accounts are obtained from an unrelated laboratory study and fieldcollected parasitized Mexican bean beetle larvae. The average numbers of live adult progeny per 4th-instar host larva subjected to laboratory-controlled parasitization by 1, 2, 3-4 parent females were, respectively, 17.5 (average of 38 host mummies), 28.83 (average of 28 host mummies), and 56.77 (average of 22 host mummies). The respective ranges of numbers of parasite individuals per host mummy were 6 to 43, 11 to 53, and 33 to 94. The average numbers of live adult progeny produced from 2nd-, 3rd, and 4th-instar parasitized host larvae collected in the field from different places on different dates were, respectively, 4.45 (average of 68 2nd-instar host larvae), 8.25 (average of 24 3rd-instar host larvae), and 18.27 (average of 30 4thinstar host larvae). The ranges of numbers of parasite adults per host mummy with each respective host instar were 1-9, 1-14, and 5-45.


53







54


So far, no studies have been made in regard to where (inside or outside the host mummies) mating of this parasite occurs. Stevens et al.(1975b) stated that "although we assumed that mating takes place within the mummified host larva prior to emergence, we have observed mating within a few minutes to several hours after emergence" (p. 955). However, observed postemergence sexual behavior exhibited by the parasite, both male and female, raised a strong doubt about the possibility that mating takes place within the host mummies before the parasite emergence.

The objective of this study was to confirm or refute the possibility of preemergence mating.



Materials and Methods


Among the 30 4th-instar host mummies placed under continuous observation for emergence of the parasite adults, only 4 were picked for study. Selection of the mummies was determined by order of adult emergence and work convenience. Parasite individuals were isolated immediately on emergence from the host mummies and confined individually in separate gelatin capsules (no. 000). Order of emergence was recorded and sex established as soon as emergence ceased. Individual females were reared in separate snap-cap vials (2.6 cm inner mouth diameter, 5.2 cm tall) labeled according to their host mummies and order of emergence. Each rearing vial was provided with a small drop of honey impregnated in a moistened circle of towelling paper of suitable size. Three 4th-instar host larvae were exposed to each parasite







55


female on 3 consecutive days, starting from the 2nd day after the parasite emergence. The exposed host larvae were reared in separate petri dishes (14 cm diameter) labeled according to mummy number, order of emergence, and order of host exposure. Individual mummies (parasitized host larvae) were then confined in gelatin capsules (no. 000) and appropriately labeled. Emerging adult parasites were sexed and counted when all were dead. Tests to establish fertility of males were undertaken in the one instance where only one -male emerged from a host mummy. This male was confined with 3 young virgin females for 24 hours. Three 4th-instar host laravae were then separately exposed to each female. The presence of female progeny produced by the tested females was proof of the male's fertility.



Results and Discussion


Sequential positions occupied by P. foveolatus males and females in their respective series of emergence'from each of the 4 host mummies are shown in Figure 5-12. The per-mummy numbers of females and males, ovipositing females producing only male progeny, and females failing to produce progeny are presented in Table 5-2. Details regarding the number of progeny produced by each of the above-mentioned parent females with respect to each initial host mummy and each of the 3 consecutive host exposures are given in Appendix 5-1.

The results show that there was no particular or orderly sequence or trend of emergence of males and females from the host mummies (Fig. 5-12), and that, among the total of 84 parent females, 60



























Lu


Mummy Order of parasite emergence from mummies
No. T 1| 4 | | 81 9 1 11 12 13 14 15 16 17 |118 19 20 1 21 22 1 23 24 25 126 127









FIGURE 5-12. Positions of P. foveolatus females and males in 4 series of emergence from their respective host mummies.


















TABLE 5-2


Progeny of P. foveolatus produced through 3 consecutive host exposures by females isolated immediately after emergence from the host mummies



No. of parasite No. of females No. of females failing
Host adults/host No. of ovipositing No. of females failing com- to produce progeny
mum mummy females producing failing to pletely to but dead host larvae
no. (females/males) only male progeny produce progeny ._/ oviposit b/ were recorded

1 25/1 16 9 8 1

2 16/2 13 3 2 1

3 26/1 17 8 6 2

4 18/2 14 4 4 0


Based on presence of pupating and/or dead host larvae through 3 consecutive host exposures Based on pupating host larvae through 3 consecutive host exposures only


Ul


a/ b/







58


produced only male progeny, and 24 failed to produce any offspring (Table 5-2).

The disorderly sequence of male and female emergence was also

observed repeatedly not only in this study and a preceding preliminary test, but also during the study on parasite sexual behavior (Chapter 4). This behavior of P. foveolatus, when compared to that of the chalcidoid Nasonia vitripennis (Wlkr), a parasite of cyclorraphous fly pupae, presents certain differences, although the 2 species exhibit similar properties such as gregarious parasitism, haplodiploid mode of reproduction, and female-biased sex ratios. King et al. (1969) reported that male N. vitripennis tend to emerge before females from a single host puparium and about 3% of emerged females were already mated. Werren (1980) went a step further and reported that male N. vitripennis emerge first from the host puparium, wait for females at the proximity of the exit hole, and no mating occurred within the puparium. For P. foveolatus, sequential position occupied by males in any series of emergence seems of little consequence. When females happen to emerge first, they wait for males. The reverse holds true for males (Chapter 4).

The production of only male progeny by 60 parent females, and no female progeny by any of the 84 total number of parent females (Table 5-2) clearly demonstrated that mating of P. foveolatus must take place outside host mummies. Postemergence sexual behavior of males and females (Chapter 4) explains why mating should not occur within the host mummies. Pre- and postcopulatory behaviors and postures of females and males during copulation require considerable space, time,







59


and convenience. There is no possibility that host mummies irrespective of size could accommodate these requirements. The number of the parasite adults, the pupal skins they left behind, the process of emergence from the pupae, the opening of the emergence hole, and the behavior of the emerged adults are among the primary limitations. Fertility of the only male emerging from host mummy no. I was proved when the 3 young virgin females produced female progeny, following a 24-hour confinement with the male in question.

Table 5-2 also shows that 20 of the 24 parent females (column

4) completely failed to produce progeny as evidenced by the development to adults of all host larvae subjected to the 3 consecutive exposures to the parasite within the 3-day period. Although sterile females were observed in the preliminary test on "Ocala" laboratory stock, the high level of sterility was not expected and is the more remarkable when compared with the results obtained from other studies such as those on the mode of reproduction (Chapter 3), influence of age on mating capability of females (Chapter 9), and fecundity (Chapter 7) of the parasite. In the mode of reproduction study, only 2 out of 38 females failed to produce progeny from one or more of the 3 consecutive host exposures. Of the 6 hosts exposed to the 2 "sterile" females, 4 pupated and 2 died as larvae (Table 3-1). In the study of influence of age on mating capability of females (Chapter 9), no single case of progeny production failure was recorded in any of the 190 females of age ranging from 1 to 25 days, to which 3 consecutive host exposures were made within a 4-day period (Appendix 9-6). In the fecundity study (Chapter 7), only 1 female among the total of 33 failed to







60


produce progeny yielding only dead and pupating exposed host larvae. Inbreeding due to laboratory culture maintenance should not be a factor, since the gap between the generations of the parasite used in the above-referred studies were very close to each other, namely generation 31 to generation 33. Besides, P. foveolatus is essentially an inbreeding species and no inbreeding depression of the kind commonly

encountered in small cultures of outbreeding species has been experienced. The most likely explanation for the high level of apparent "sterility" in this experiment is the manner in which the females were handled. In the mode of reproduction study, the parasite males and females were separated from each other while they were still in the pupal stage (about 2 days prior to their emergence), and remained isolated during the entire period of study. In the study on influence of age on mating capability of females, separation of sexes was also made at the pupal stage; each female was then paired with each male and allowed to remain together throughout the period of host exposures; exposures of the host larvae to the parasite were made only 24 hours after the pairing. In the study on fecundity, all individual males and females were allowed to emerge and mate freely within the rearing unit for a period of about 36 hours, after which the 33 females were selected randomly from the unit. The relatively high success of progeny production suggests that the poor performance of the females in this experiment may be attributed to disruption of the normal mating process resulting from the separation of individual parasite adults immediately after their emergence from the host mummies. Their earlier association with males while inside the host mummy may provide a






61


stimulus of male presence that inhibits oviposition by the subsequently

unmated females. Since similar disturbances may occur under natural conditions, this behavior would serve to avoid wastage of eggs prior to mating. Although males would be produced, the normally femalebiased sex ratio and tendency toward an inbreeding mating system suggest that production of female progeny is adaptively advantageous to the individual female parent. Any overproduction of males that would result in excessive competition for females would reduce the reproductive fitness of female parent and in the extreme result in total collapse of some populations. For P. foveolatus, a mechanism that inhibits.oviposition by unmated females would thus serve to maximize reproductive fitness of individual females and evidently increase probability of population continuity under certain conditions adverse to the species. The existence of such a mechanism in P. foveolatus would seem to conform to views expressed by Borgia (1980) in discussion of his models for evolution of haplodiploidy where, in regard to mother-son matings, he states that:

An increase in the fraction of females a mother produces
can be achieved by reducing the rate at which eggs are
laid before she is fertilized. Presumably, eggs laid
early reduce the female's subsequent egg production.
Since producing only a few males should guarantee her
fertilization, the greatest output of females might be
achieved by reducing the rate of oviposition until after
she is fertilized.(pp. 113-114)

As supporting evidence, he mentions studies of J. P. Gutierrez working with Tetranychus neocaledonicus Andre and J. L. Nickel, who worked with Tetranychus desertorum (Banks). In each instance, the authors found that the unmated spider mite females produced considerably fewer







62


eggs and lived longer than mated females. Similar behavior was observed by Browne (1922) in studies involving the chalcid wasp Melitobia acasia (Walker). Thus, while effected through a somewhat different mechanism, these admittedly preliminary observations of behavior of P. foveolatus provide additional support for evidence of behavioral and physiological responses associated with female fertilization that tend to maximize reproductive fitness of haplodiploid species.















CHAPTER 6

SEX RATIOS RESULTING FROM SEQUENTIALLY MATED MALES



Introduction


Among authors who have propounded theories relating to evolutionary aspects of sex ratio in bisexual-species, the foremost are Fisher (1930) and Hamilton (1967). Darwin's theory speaks of the struggle among individuals for reproductive success, i.e., maximal reproductive capacity, and contains no statement relevant to success of the population, the species, or the ecosystem (Gould, 1980). The Darwin argument as framed by R. A. Fisher (Gould, 1980) contended that selection causes parents to produce the sexes in numbers such that the ratio of parent expenditure in the sexes is equalized over the population. Excellent agreement with Fisher's prediction of 1:1 ratio of parental investment (Fisher's parental expenditure) in each sex was found by Metcalf (1980) who worked on Polistes metricus Say and Polistes variatus Cress (synonym of Polistes fuscatus fuscatus (F.)). Hamilton's theory deals with local mate competition and predicts a female-biased sex ratio in species where sons of a parent compete with each other for mates (Werren, 1980). Gould (1980) wrote that "exclusive sibmating destroys the major premises of Fisher's argument for 1:1 sex ratio" (p. 206).


63







64

Sex ratios of haplodiploid insects have been the subject of many investigations. Several factors, extrinsic or intrinsic, have been found or conceived to have influenced the sex ratios.

King (1962) suggested that increase in percentage of males

Nasonia vitripennis (Walker) is related to increase in number of eggs undergoing resorption in the female ovarioles at the time of oviposition, particularly under the conditions where host puparia are intermittently available. Werren (1980) reported that N. vitripennis females adjust the sex ratio of their broods according to whether they are first or second wasp to parasitize a host; progeny of the first wasp show a strong daughter bias, the second adjusts the production of sons to the relative level of local mate competition.

Colgan and Taylor (1981), while establishing a model of sex ratio based on the reproductive pattern of the aphelinid Coccophagus scutellaris Dalman, stated that "in some haplodiploid species a certain coarse control of offspring sex ratio is obtained from timing of mating: eggs laid before mating are haploid, after mating are diploid" (p. 564).

Flanders (1939) pointed out that, in arrhenotokous species of Hymenoptera, the spermatheca is a sex-controlling mechanism, able to modify sex ratio in response to stimuli of inconstant environmental factors. He later theorized that the small spermathecal gland of a chalcid parasite may not be able to keep pace with egg deposition and as a result more unfertilized eggs would be produced. This rate of oviposition could be a factor influencing sex ratio (Flanders, 1956). Flanders noted that his concept was supported by the fatigue theory proposed by Marchal who suggested that an increase in the male progeny of a mated female parasite which oviposited rapidly would indicate







65


fatigue of spermatheca. Abdelrahman (1974b) has since reported that sex ratio of the chalcid Aphytis melinus DeBach, a parasite of the red scale Aonidiella aurantii (Mask.) is influenced by both the number of eggs laid per host and by the density of the parasite population relative to hosts. He also listed the size and quality of the host, small size of spermathecal gland, differential mortality, temperature, and age of mothers as other factors influencing the final sex ratios of A. melinus. Wilkes (1965) studied the structure and function of male and female reproductive systems of the eulophid Dahlbominus fuscipennis (Zett.) in an effort to explain the apparent ability of this parasite to control the sex ratio of its offspring. He pointed out that the production of male- and female-producing eggs during oviposition is not influenced by the environment except under the extremely unfavorable environment, nor by the host, and that discontinuity in the release of sperm is unlikely the regulating mechanism involved in the production of haploid males. He assumed that "for most of the inseminated females, if the effect of the female sperm storage organ of the passage of all eggs from the ovarioles to vagina is constant, fertilization must be constant; if not, apart from the possibility of dimorphism of the sperm or egg, the intervention of male production must be externally induced" (pp. 647-648). He also noted that external stimuli operating through spermathecal gland as theorized by Flanders (1939) for regulating sexes has never been adequately supported by observation. In apparent contradiction to Wilkes' concept, Gordh and DeBach (1976), working with the aphelinid Aphytis lingnanensis Compere, stated that "it is difficult to imagine any factor which would tend to suppress female progenies production







66


with subsequent matings other than the reduction in the absolute amount of sperm being deposited" (p. 588).

Sekhar (1957) reported that the sex ratio of the progeny of Aphidius testaceipes (Cresson) and Praon aguti Smith shifted to male bias with females later in the mating sequence.

Schlinger and Hall (1960) found that mated females of the

braconid aphid parasite Praon palitans Muesebeck produce 1:1 sex ratio, but multimated males supply so little sperm at each mating that the sex ratio may go as high as 58:1 in favor of males. Similar findings were reported for the braconid Trioxys utilis Muesebeck by Schlinger (1961), in which mated females produced progeny of both sexes in a 1:1 ratio; however, virgin females mated to males having a previous history of multiple matings cause a shift toward higher ratios of males and in the case of those males having the highest number of previous matings the ratio male:female progeny was as high as 24:1. This was interpreted as the result of sperm depletion. Male inseminative potential of the aphelinid A. melinus was studied by Gordh and DeBach (1976) to determine the number of females a single male could inseminate during the course of a lifetime and the number of progeny resulting from these copulations. They found that males courted and copulated with females in rapid succession, resulting in a decreasing percentage of F, females with successive matings. They concluded that females inseminated by a male that had experienced coitus with several females earlier tended to produce more males, thus suggesting that fewer spermatozoa were transferred during later copulations.

Male to female sex ratio of P. foveolatus was reported by Lall (1961) from India (the native country of the parasite) as being 1:2.







67


Seasonal changes in sex ratio of this parasite was studied by Stevens et al. (1977) who observed no apparent relationship between sex ratios of the parasite adults and collection dates. These investigators also reported that male to female ratio declined as the number of parasite developing within the host larva increased.

The objective of the present investigation was to determine the effect of sequential matings of P. foveolatus by single males with a series of virgin females on the sex ratios of their progeny.



Materials and Methods


The method used in the study of mode of reproduction (Chapter 3) was applied here to obtain virgin parasite adults of both sexes. All females and males subjected to this study were picked at random from among those issued from 30 host mummies. Each of the 4 virgin males used were allowed to mate sequentially with 50 virgin females. This operation was carried out under the hood (Fig. 2-7) with the light on. A parasite rearing unit, with the cover replaced by a sheet of clear acetate of appropriate size (Fig. 2-6), was used to contain the parasite adults. Sixty-five virgin females were introduced into the container. Thirty minutes were allowed to them to regain their composure. A virgin male was then introduced into the container. Male activities were watched continuously until the 50th virgin female was mated. Each mated female was removed by means of an aspirator (Fig. 2-6) and released into a rearing plastic vial (snap-cap type, 2.6 cm inner mouth diameter, 5.2 cm tall) (Fig. 2-6) prepared and labeled beforehand in accordance with male number and order of mating. On the second day







68

after mating, one 4th-instar host larva was exposed to each parasite female on each of six successive days. The exposed host larvae were reared in separate petri dishes (8.9cm diameter). The mummies were then individually confined in gelatin capsules (No. 000) also labeled in accordance with male number, order of mating of female, and order of host exposure. Sexing and counting were made only when all emerging individual died.



Results and Discussion


Raw data representing the numbers of adult parasite progeny of both sexes produced from individual host mummies resulting from 6 separate host exposures of all parent females sequentially mated by single males are given in Appendix 6-2. The corresponding percentages of female progeny are presented in Appendix 6-3.

Table 6-3 represents the average percentages of the parasite female progeny produced by parent females arbitrarily grouped in 10 successive quintets under each of the 4 sequentially mated males and each of the 6 separate host exposures; the grouping was made for statistical analysis convenience.

Statistical analysis based on data presented in Table 6-3 showed that male sperm depletion, expressed in percentage of female progeny, did occur both in the direction of increasing order of quintets of parent females and direction of increasing order of host exposures. Sperm Depletion in Increasing Order of Parent Female Quintets

Based on the average percentages of female progeny corresponding to individual single males of each host exposure (Table 6-3), depletion


























TABLE 6-3


Average percentages of female progeny of P. foveolatus produced by each quintet
of parent females with respect to individual males and host exposures


oupaenst Ist host exposure 2nd host exposure 3rd host exposure 4th host exposure 5th host exposure 6th host exposure
females o'i o12 o'13 orol4 o'll e 1I2 o3 o'14 ell o'2 o13 o'r4 e'l r'12 o13 ol4 oell o02 o'I3 o14 cr1I o' 2 o"3 o'4

1 88.26 75.79 79.91 83.33 87.72 84.12 91.25 85.37 84.74 91.85 92.79 89.86 80.65 88.18 88.04 87.44 85.45 56.14 88.65 91.84 94.73 54.90 93.30 84.57
(81.82) (87.11) (89.81) * (86.08) . (80.52) (85.87)
2 81.67 64.12 85.18 87.08 84.82 69.05 89.93 89.81 81.72 71.89 89.54 90.49 89.58 71.93 89.93 89.39 87.35 70.57 82.35 91.03 92.46 67.86 84.26 90.19
(79.51) (83.40) (83.41) (85.21) (82.82) (83.69)
3 76.35 69.58 84.88 79.45 68.09 85.90 85.60 89.29 69.54 90.49 77.32 88.04 51.17 35.82 82.02 66.28 58.67 28.57 65.95 50.67 33.98 44.73 35.37 42.89
(77.56) (82.22) (81.35) (58.82) (50.96) (39.24)
4 84.58 44.34 85.35 78.33 89.84 58.09 76.99 68.30 89.23 42.92 56.90 65.98 83.45 27.59 42.85 89.29 89.07 33.33 54.89 61.11 89.83 17.14 67.43 46.87
(73.15) (73.30) (63.76) (60.79) (59.6n) (55.32)
5 82.85 79.61 86.60 42.50 71.64 89.60 65.81 43.10 89.56 70.78 54.74 0 80.27 64.65 60.48 0 50.05 42.27 65.59 0 45.28 53.32 31.50 0
(72.89) (67.54) (53.77) 451.35) (39.48) (32.52)
6 63.76 79.98 70.02 70.22 88.10 89.39 40.67 69.21 69.69 61.35 32.16 27.83 61.10 64.83 0 17.60 61.26 47.73 0 0 57.22 43.05 0 17.89
(70.99) (71.84) (46.76) a (35.88) (27.25) (29.54)
7 40.38 31.08 65.22 79.06 58.14 23.75 52.34 26.19 56.09 45.31 20.73 18.75 30.30 31.67 1.04 1.31 27.59 0 0 0 29.41 23.07 0 7.41
(53.93) (40.10) (35.22) (16.08) (6.90) (14.97)
8 34.13 88.72 78.46 51.67 18.71 47.72 30.93 2.46 2.78 45.24 21.43 0 0 0.95 33.33 0 0 0 5.26 0 0 0 0 0
(63.24) (24.95) (17.36) (8.57) (1.31) (0)
9 50.00 27.71 75.00 1.85 0 34.94 86.97 0 0 1.67 82.35 0 0 0 28.57 0 0 0 0 0 0 0 31.82 0
(38.64) (30.48) (21.00) (7.14) (0) (7.95)
10 0 2.70 24.89 1.00 0 9.52 2.86 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0
(7.15) (3.09) (0) (0) (0) (0)
Note: Figures in parentheses represent per-host-exposure average percentages of female progeny produced by each 4-time
replicated (4 single males) female quintet.


C7'







70


of male sperm determined by sented by the icant at 0.01


in increasing order of quintets of parent females as order of sequential matings by single males is reprefollowing simple linear regression equations, all signiflevel, and illustrated in Figure 6-13.


Y(exp 1) = 98.20 - 6.60 Quintet

(coefficient of correlation


Y(exp 2) = 106.68 - 9.14 Quintet
(coefficient of correlation


(Eq. 6.1)


r = - 0.70)


(Eq. 6.2)


r = - 0.81)


= 104.57 - 10.04 Quintet (coefficient of correlation


(Eq. 6.3)


r = - 0.83)


= 98.39 - 10.44 Quintet Y(exp 4)
(coefficient of correlation


= 92.32 - 10.44 Quintet Y(exp 5)
(coefficient of correlation


i(exp 6) = 87.42 - 9.62 Quintet

(coefficient of correlation


(Eq. 6.4)


r = - 0.84)


(Eq. 6.5)


r = - 0.86)


(Eq. 6.6)


r = - 0.84)


where: Y(exp 1), Y(exp 2), ..., Y(exp 6) respectively represent the

estimated percentages of female progeny for the 1st, 2nd, ...,

6th host exposures, and

Quintet stands for quintet of parent females. Sperm Depletion in Increasing Order of Host Exposures

Aside from the first 2 quintets (first 2 rows of Table 6-3;

equations Eq. 6.7 and Eq. 6.8) which showed no significant slopes (rate


Y(exp 3)







71


1st exposure 2nd exposure 3rd exposure


100 90 80 70 60
C
aD
C) 50


30 02



1
40

E a) 30


20

10 0

-10

-20


1 2 3 4 5 6 7 8 9 10
Quintets of female parents


FIGURE 6-13. Simple linear regression lines representing the relationships between the percentages of female progeny of P. foveolatus and the quintets of parent females sequentially mated with single males.


4th exposure 5th exposure .. 6th exposure



.



\ \\










I \ . \





.


......-.







72


of sperm depletion), simple linear relationships between the percentages of female progeny and the host exposures for the next 7 female quintets, i.e., 3rd to 9th quintets, are all significant at 0.01 level (equations Eq. 6.9 to Eq. 6.15), whereas that of the 10th quintet (equation Eq. 6.16) is significant only at 0.05 level. The statistical analyses here involved were based on the per-host-exposure average percentages of female progeny produced by each quintet of parent females (figures in parentheses, Table 6-3). All equations are represented below according to the sequence of parent female quintets and illustrated in Figure 6-14.


Y(Q 1) = 86.86 - 0.66 E
(coefficient of correlation r = - 0.34)


Y(Q.2) = 80.96 - 0.56 E

(coefficient of correlation r = - 0.59)


Y(Q.3) = 95.82 - 8.80 E

(coefficient of correlation r = - 0.91)


Y(Q.4)


= 77.64 - 3.81 E (coefficient of correlation r = - 0.96)


Y(Q.5) = 81.77 - 8.24 E

(coefficient of correlation r = - 0.99)


Y(Q.6) = 82.23 - 10.05 E
(coefficient of correaltion r = - 0.94)


(Eq. 6.7) (Eq. 6.8) (Eq. 6.9) (Eq. 6.10) (Eq. 6.11) (Eq. 6.12)


I







73


Y 7) = 59.22 - 8.96 E (Eq. 6.13)

(coefficient of correlation r = - 0.93)


Y(Q.8) = 58.83 - 11.31 E (Eq. 6.14)

(coefficient of correlation r = - 0.90)


YQ9) = 43.41 - 7.39 E (Eq. 6.15)

(coefficient of correlation r = - 0.92)


Y(Q.1O) = 6.21 - 1.29 E (Eq. 6.16)

(coefficient of correlation r = - 0.82)


where: Y(Q.1), Y(Q.2), - Y(Q.10) respectively represent the

estimated percentages of female progeny for the 1st, 2nd, ...,

10th quintets of parent females, and

E stands for host exposure.


Difference in Rates of Sperm Depletion and Biological Significance

The results of analysis of variance primarily aimed at determining the difference among the rates of sperm depletion, i.e., the difference between the slopes of the simple regression lines of male sperm depletion in increasing sequence of matings (equations Eq. 6.1 to Eq. 6.6; Fig. 6-13) and in increasing sequence of host exposures (equations Eq. 6.7 to Eq. 6.16; Fig. 6-14) are respectively presented in Tables 6-4 and 6-5.

A comparative view on the general behavior of the regression

lines illustrated in Figure 6-13 and Figure 6-14 appears to indicate that sperm depletion occurred in a more regular pattern in the









74


100


90 80 70

60


50

40


30

20


10


0


-10


-20


-94


~5 N. -~--S- 96
............. ' 9





...........
- - - - - - -*





10

Q8


1st


2nd


3rd


4th


5th


6th


Host exposures


FIGURE 6-14. Simple linear regression lines representing the relationships between the percentages of female progeny of P. foveolatus and the exposures of host larvae to the parasite parent females sequentially mated with single males; Q Q ' N''., 10 represent regression lines of equations Eq. 6.7, Eq. 6.8,*..., q. 6.16 respectively.


0
Q
a

CO
E a)


I


I


I


I







75


direction of increasing number of matings than in the direction of increasing number of host exposures. The F-test for equality of slopes of all simple regression equations, namely equations Eq. 6.1 to Eq. 6.6, showed no significant difference, and suggests the closeness among these slopes. The difference between the slopes of the combined equations (Eq. 6.1 + Eq. 6.2 + Eq. 6.3) and that of the combined equations (Eq. 6.4 + Eq. 6.5 + Eq. 6.6), being significant only at

0.10 level (Table 6-4) further suggests such closeness. The varying degrees of steepness among the slopes (rates of sperm depletion) of the simple regression lines of Figure 6-14 (sperm depletion in increasing sequence of host exposures) suggest irregularity in the amount of sperm deposited by the males during successive copulations, while going toward complete depletion. In this study where females were readily accessible to each male (65 virgin females vs. on virgin male at the onset of the serial matings, all aggregated about the upper portion of the mating container near the light source of the hood), time appeared to be a critical factor for the process of sperm . replenishment in the males. In the field, such extreme accessibility of virgin females would rarely be encountered; however, as females normally outnumber males, there are no doubt occasions when males have opportunity to mate in rapid succession. This should tend to reduce the normally observed sex ratio bias in favor of females and may in part explain the variations in sex ratio encountered in the field.

Figure 6-15 features a general pattern of sperm depletion, both

in increasing sequence of matings (female quintets) and host exposures.







76


TABLE 6-4

Significant difference between rates of sperm depletion
in increasing sequence of female quintets


Slopes tested Level of significance
not significant 0.10 0.05

Those of equations Eq.6.1, Eq.6.2, Eq.6.3, Eq.6.4, Eq.6.5, Eq.6.6 not significant

Those of combined equations (Eq.6.1 + Eq.6.2 + Eq.6.3), (eq.6.4 + Eq.6.5 + Eq.6.6) significant

Those of combined equations (Eq.6.1 + Eq.6.2), (Eq.6.3 + Eq.6.4), (Eq.6.5 + Eq.6.6) significant




TABLE 6-5

Significant difference between rates of sperm depletion
in increasing sequence of host exposures


Slopes tested Level of significance
not significant 0.05 0.01

Those of equations Eq.6.7, Eq.6.8 not significant

Those of equations Eq.6.7, Eq.6.8, Eq.6.9 significant

Those of equations Eq.6.9, Eq.6.10, Eq.6.11, Eq.6.12, Eq.6.13, Eq.6.14, Eq. 6.15 significant

Those of equations Eq.6.15, Eq.6.16 significant






77


//



5 Quintets of
6 parent females

Host /
exposures 4




FIGURE 6-15. Tridimensional representation illustrating the general pattern of male sperm depletion, expressed in terms of percentages of female progeny produced by parasite parent females sequentially mated with single males (based on data of Table 6-3).


.100 90 80
70 > 60 50 Q.

40 30
20 10







78


Table 6-3, illustrated in Figures 6-14 and 6-15, also show that higher doses of male sperm were received by the first 2 female quintets, as evidenced by the insignificant slopes of equations Eq. 6.7 and Eq. 6.8, in contrast to the 8 subsequent female quintets respectively represented by equations Eq. 6.9 to Eq. 6.16 which show significant slopes. However, evidence of sperm depletion did appear as early in a sequence as the 2nd mating (Appendix 6-2, case of male no. 2), and unsuccessful mating, though most likely rare, may also occur (Appendix 6-2, case of male no.2, 6th mating).

In a general sense, the results indicate that a single male is capable of effectively impregnating about 10 virgin females in rapid succession before sperm depletion can be noticed. The per-mummy average of 83.43% female progeny produced by the first 2 parent quintets (Table 6-3), i.e., 40 females, from 174 parasitized host larvae for the 6 host exposures, appears to confirm the results of statistical analyses above-described.


Further Relationships and Biological Implications

In addition to the above-determined simple linear relationships, male sperm depletion was, in some instances, found to fit quadratic polynomial regression or show an apparent trend toward similar curvilinear relationship.

In increasing sequence of matings (increasing sequence of quintets of parent females, Table 6-3), curvilinear relationships represented by equations Eq. 6.17 and Eq. 6.18 and illustrated in Figure 6-16 were revealed in the 1st and 2nd host exposures; the level of significance







79


for the test on the magnitude of the quadratic coefficient estimate was 0.01 for equation Eq. 6.17, and 0.05 for equation Eq. 6.18. The analyses were based on the average percentages of female progeny produced by each quintet of parent females with respect to each host exposure and each of the 4 single males (Table 6-3).

2
Y (sd.Q)l= 70.91 + 7.04 Q - 1.24 Q (Eq. 6.17)

(coefficient of determination R = 0.60)

Y sd.Q)2 = 87.02 + 0.69 Q - 0.89 Q2 (Eq. 6.18)

(coefficient of determination-R = 0.70)

where: Y and Y respectively represent the estimated
(sd.Q)l (sd.Q)2
-percentages of female progeny for the 1st and 2nd host

exposures, and

Q stands for parent female quintet.

In increasing sequence of host exposures, the trend of male sperm depletion based on the per-host-exposure average percentages of female progeny produced by each quintet of parent females (figures in parentheses, Table 6-3) appeared to incline toward the curvilinear relationship. Such tendency is represented by equations Eq. 6.19 and Eq. 6.20 which are close to being significant at 0.05 level (t0.05, 31 d.f. =-2.353 4 tcalc. =-2.24 for equation Eq. 6.20). The

2 equations are illustrated in Figure 6-17.

A 2
Y (sd.E) 78.07 + 5.93 E -7 0.94 E (Eq. 6.19)

(coefficient of determination R2 = 0.60)







80


100

90 Y(sd.Q)1 = 70.91 + 7.04 Q - 1.24 Q


800

70

C 60
(D
04
S50

Ca 40 0
E

* 30

20

10 Y= 87.02 + 0.69 Q - 0.89 Q2
(s d. 2p

0
1 2 3 4 5 6 7 8 9 10

Female quintets


FIGURE 6-16. Quadratic polynomial regression curves representing the relationship between male sperm depletion (expressed in terms of percentages of parasite female progeny produced by parent females sequentially mated with single males) and the quintets of parasite parent females in the 1st and 2nd host exposures.







81


Y(sd.E)2 = 76.79 + 3.69 E - 0.44 E2 (Eq. 6.20)

(coefficient of determination R2= 0.76)


where: Y(sd.E)1, (sd.E)2 respectively represent the estimated

percentages of female progeny for the 1st and 2nd quintets of

parent female, and

E stands for host exposure.


In light of the above statistical analyses, the behavior of the regression lines (those representing the relationship between the percentages of female progeny and host exposures, and those representing the relationship between the percentages of female progeny and quintets of parent females) appear to follow a similar pattern: a gradual shift from quadratic polynomial (curvilinear) to simple linear (straight line) regressions. This transitional state which appears to be basically dependent on the intensity of sperm depletion may be explained as follows.

In the direction of increasing order of host exposures, the

early quintets of parent females, particularly the 1st and 2nd, seem to be inclined toward a curvilinear relationship (quadratic polynomial regression), if more than 6 host exposures are available. This interpretation is essentially based on supportive evidence provided by the study on the fecundity (Chapter 7). In that study, despite the wide range of fecundity among parent females, it was found that the distribution of the percentages of female progeny over 10 out of 11 exposures of the host larvae to the 32 free-mated parasite parent females is well represented by the quadratic polynomial model (Chapter 7, equation







82


Y(sd.E)1

100 CL--- Y(sd.E)2



Y(sd.E)1 = 78.07 + 5.93 E - 0.94 E


90

e0

0
0
80

0)0




10 . (sd.E)2 = 76.79 + 3.69 E - 0.44 E






0 I I I I I
1 2 3 4 5 6

Host exposures


FIGURE 6-17. Relationship between male sperm depletion (expressed in terms of percentages of parasite female progeny produced by parent females sequentially mated with single males) and the host exposures in the 1st and 2nd quintets of parent females, showing trend toward quadratic polynomial regression.






83


Eq. 7.23). Reasons for the exclusion of the average value (percentage of female progeny) of the l1th host exposure from the 10 preceding values used to fit the quadratic polynomial model rested on the grounds that (a) only 3 out of the total 32 parasite parent females produced progeny in this host exposure, thus putting their validity in doubt, (b) the percentage of female progeny produced in this host exposure appeared to be very erratic, most probably due to the loss of sperm regulating ability by the exhausted parasite parent females, and

(c) ignoring this excluded value (average percentage of female progeny obtained from only 3 host mummies resulting from the parasitization by the 3 parasite parent females mentioned in (a)) permitted a very good fit of the data to the quadratic polynomial model. The change from curvilinear to simple linear relationship is basically dependent on the amount of sperm received by parasite parent females during copulations, and in this case (sperm depletion in increasing sequence of host exposures) appears to go from the low through higher level of significance of fit before ending up with a good fit to simple linear regression. This apparent trend is indicated by equation Eq. 6.19 (1st quintet of parent females) and equation Eq. 6.20 (2nd quintet of parent females), whose respective calculated t-values are

- 1.93 and - 2.24, both almost significant at 0.05 level (t0.05, 3 d.f. = - 2.353; coefficient of determination R2 is 0.60 for equation Eq. 6.19, and 0.76 for equation Eq. 6.20). As sperm depletion becomes more and more intense in the subsequent female quintets, the percentage of female progeny decreases linearly with increasing numbers of host exposures.







84


The change in behavior of regression lines as above-described also holds true for the distribution of the percentages of parasite progeny in relation to the sequence of matings (sequence of quintets of parasite parent females). Here the situation concerns the amount of sperm delivered by single males during each subsequent copulation. In the first 2 host exposures where a relatively large amount of sperm was received by parasite parent females, the data (Table 6-3) showed significant fit by the quadratic polynomial model. A straight line relationship becomes apparent when sperm depletion becomes more pronounced, i.e., in subsequent host exposures.

The curvilinear relationship represented by equations Eq. 6.17 and Eq.-6.18, and equations Eq. 6.19 and Eq. 6.20, respectively illustrated in Figures 6-16 and 6-17 , appears to represent a mathematical translation of a fundamental biological feature and brings out

3 apparent phases in succession, i.e., (a) the "maturation phase" whereby males perfect their sperm delivery during successive copulations, and females perfect their successive ovipositions, in order to reach their optimal performance (left portion of each curve); (b) the "optimum phase" representing the stage where perfection in sperm delivery by males and ovipositions by females are optimal (portion about the maximum of each curve); and (c) the "degradation phase" where sperm delivery by males and ovipositions by females gradually decline, most likely due to sperm depletion in males and general fatigue in females (right portion of each curve). The 3 phases disappear or become imperceptible in simple linear regression, most probably beacause of the superseding effect of sperm depletion.




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ASPECTS OF THE REPRODUCTIVE BIOLOGY OF PEDIOBIUS FOVEOLATUS (CRAWFORD) (EULOPHIDAE: HYMENOPTERA) , PARASITE OF EPILACHNA SPP. (COCCINELLIDAE: COLEOPTERA) By LIMHUOT NONG 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 1982

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Copyright 1982 by LIMHUOT NONG

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To my father-in-law Mr. Yip Nguon Ung

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ACKNOWLEDGEMENTS The author wishes to express his deep gratitude and appreciation to Dr. Reece I. Sailer, supervisory committee chairman, for his invaluable guidance and assistance, continuous understanding and morale support during the whole period of this study and the preparation of this dissertation, and for making the overall involved efforts a very pleasant experience. He owes a deep debt of gratitude to Dr. Vernon G. Perry who, together with Dr. Sailer, gave him a rare opportunity to pursue and make this graduate study possible; Dr. Perry's helpful advice, continuous encouragement and thoughtful consideration are here profoundly appreciated. Special appreciation is directed to Dr. Jerry L. Stimac for his advice, assistance, and constructive criticism whenever these were called for. Deep appreciation is due to Dr. Daniel A. Roberts for his unfailed advice and helpful suggestions during the whole period of this study and preparation of this manuscript. He also wishes to direct a deep appreciation to Dr. John A. Cornell for his very useful advice and untiring assistance in statistical analyses of the research data and the review of this manuscript. Heartfelt thanks are specially directed to Mrs. May Morita Buckingham for her dedicated and most wholehearted help on the typing iv

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work of this manuscript from the very beginning to the end of its preparation. Special appreciation is directed to her husband. Dr. Gary R. Buckingham for being very considerate and thoughtful and for making his laboratory and office facilities always available for use during this research and the preparation of this manuscript. To his wife Bicheng, not only for her love and care, but also for her assistance in some laboratory work that necessitated help, he would like to sincerely say thanks. V

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TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv LIST OF TABLES Ix LIST OF FIGURES xii ABSTRACT xv INTRODUCTION 1 CI^APTER 1: LITERATURE REVIEW 3 CHAPTER 2: GENERAL MATERIALS AND METHODS USED TO STUDY THE BIOLOGY OF PEDIOBIUS FOVEOLATUS 15 CHAPTER 3: MODE OF REPRODUCTION 26 Introduction . 26 Materials and Methods 27 Results and Discussion 28 CHAPTER 4: SEXUAL BEHAVIOR 32 Introduction 32 Materials and Methods 33 Results and Discussion 36 CHAPTER 5 : PREEMERGENCE MATING 53 Introduction 53 Materials and Methods 54 Results and Discussion 55 CHAPTER 6: SEX RATIOS RESULTING FROM SEQUENTIALLY MATED MALES 63 Introduction 63 Materials and Methods 67 Results and Discussion 68 vi

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Page CHAPTER 7: FECUNDITY 91 Introduction 91 Materials and Methods 93 Results and Discussion 94 CHAPTER 8: INFLUENCE OF AGE ON MATING CAPABILITY OF FEMALES AND MALES Ill Introduction Ill Materials and Methods 112 Results and. Discussion 113 CHAPTER 9: INFLUENCE OF AGE ON MATING CAPABILITY OF FEMALES .. 119 Introduction 119 Materials and Methods 119 Results and Discussion 120 CHAPTER 10: MULTIPLE MATINGS OF FEMALES 128 Introduction 128 Materials and Methods 129 Results and Discussion 131 CHAPTER 11: OVIPOSITION PREFERENCE WITH REFERENCE TO HOST INSTARS 149 Introduction 149 Materials and Methods 150 Results and Discussion 152 CHAPTER 12: SUMMARY AND CONCLUSIONS 162 Summary of Results 162 Conclusions 167 REFERENCES CITED 176 APPENDICES 51. Per-mummy numbers of P^. foveolatus progeny (females/ males) produced through 3 consecutive host exposures by individual parent females isolated immediately after emergence from host mummies 185 62. Per-host-mummy numbers of P^. foveolatus progeny (females /males) produced through 6 consecutive host exposures by parent females sequentially mated with single males jgg vii

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Page 63. Per-host-munnny percentages of P^. foveolatus female progeny produced through 6 consecutive host exposures by parent females sequentially mated with single males 187 74. Per-muramy numbers of P. foveolatus progeny (females/ males) produced by individual females with respect to host exposures 188 75. Per-mummy numbers of P^. foveolatus progeny produced through single oviposit ions by 38 individual parent females 189 86. Per-mummy numbers of P^. foveolatus progeny (females/ males) produced through 3 consecutive host exposures by 3 pairing combinations of young and old parents .... 190 97. Per-mummy numbers of P^. foveolatus progeny (females/ males) produced through 3 consecutive host exposures by 1-, 5-, 10-, 15-, 20-, and 25-day old females paired with 1-day old males 191 BIOGRAPHICAL SKETCH 192 viii

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LIST OF TABLES Table ^^ge 3-1. Live adult progeny (females /males) produced through 3 separate host exposures by Pedlobius foveolatus virgin females 29 52, Progeny of P^. foveolatus produced through 3 consecutive host exposures by females isolated immediately after emergence from the host mummies 57 63. Average percentages of female progeny of P. foveolatus produced by each quintet of parent females with respect to individual males and host exposures 69 6-4. Significant difference between rates of sperm depletion in increasing sequence of female quintets 6-5. Significant difference between rates of sperm depletion in increasing sequence of host exposures 76 7-6. Fecundity, longevity, and numbers of 4thinstar host larvae successfully parasitized by individual female P^. foveolatus 95 7-7. Sex ratios of P^. foveolatus progeny computed on the basis of 9 range categories of 5-individual increments 102 7-8. Numbers of successfully parasitized host larvae, total nxombers of parasite progeny (females + males) , average numbers of parasite progeny per host mummy, total numbers of parasite progeny (females/males) , and parasite sex ratios, all corresponding to each exposure of the host to the 32 parasite parent females 105 ix

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-nr-'j • Table Page 7-9. Percentages of female progeny produced by P^. f oveolatus individual parent females with respect to host exposures 108 8-10. Numbers of mated parasite females resulting from 3 pairing combinations of young and old adults and 3 separate host exposures 8-11. Results of Z-tests between pairing combinations of young and old P^. f oveolatus adults of both sexes, based on numbers of successfully mated parent females of Table 8-10 11^ 8-12. Numbers of females failing to produce progeny throughout 3 separate host exposures 115 813. Results of Z-tests between pairing combinations of young and old P^. f oveolatus adults, based on data in Table 8—12 involving numbers of parent females that failed to produce -progeny 116 914. Effect of age on mating capability of P^. f oveolatus females 121 9-15. Results of X tests showing difference in mating capability between P^. foveolatus females of different age 123 10-16. Occurrence of single and multiple matings, and dubious occurrence of multiple matings revealed by P^. foveolatus females after introduction of second males 132 10-17. Numbers of P. foveolatus parent females falling in each of the 11 categories described in materials and methods 134 10-18. per-host-exposure numbers of P^. foveolatus progeny (females /males) and corresponding female to male ratios of progeny produced by the first 10 parent females sequentially mated with single males (replicated 4 times) in the study on multiple matings and the study on sperm depletion (Chapter 6) 136 X

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Table Page 10-19. Significant difference between the slope of equation Eq. 10.24 (linear regression ,. between P^. f oveolatus female to male ratios and host exposures in the study on multiple matings) and the slopes of equation Eq. 10.25 (quadratic polynomial regression between the parasite progeny sex ratios and host exposures in the study on sperm depletion in Chapter 6) at each level of host exposure 139 10-20. Per-host-exposure numbers of P^. f oveolatus progeny (females/males) and corresponding female to male ratios produced by all females in the study on multiple matings and the study on sperm depletion 140 10-21. Significant difference between the slope of equation Eq. 10.26 (quadratic polynomial regression between P^. f oveolatus female to male progeny ratios and host exposures in the study on multiple matings) and the slope of equation Eq. 10.28 (quadratic polynomial regression between the parasite progeny sex ratios and host exposures in the study on sperm depletion in Chapter 6) at each level of host exposure 145 1022. Significant difference between the slope of equation Eq. 10.27 (quadratic polynomial regression between P^. f oveolatus female to male progeny ratios and host exposures in the study on multiple matings where female progeny produced by the 6 obvious multimated females being converted into males) and the slope of equation Eq. 10.28 (quadratic polynomial regression between the parasite progeny sex ratios and host exposures in the study on sperm depletion in Chapter 6) at each level of host exposure 146 1123. Numbers and percentages of parasitized hosts corresponding to each of the 3 larval instars used in the test for oviposition preference by P^. f oveolatus females 153 11-24. Numbers and percentages of dead parasitized hosts corresponding to each of the 3 larval instars used in the test for oviposition preference by P^. foveolatus females I55 xi

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LIST OF FIGURES Figure Page 2-1. Mexican bean beetle, Epilachna varivestis Mulsant 17 2-2, Pediobius foveolatus (crawford), parasite of Epilachna spp 17 2-3. Containers and tools for Mexican bean beetle rearing 19 2-4. Young greenhouse-grown lima bean plants 19 2-5. Beggarweed, Desmodium tortuosum (SW) D. C. at fruiting stage 23 2-6. Containers and tools for rearing and handling of parasite adults 23 2-7. Hood for handling of parasite adults 24 4-8. Observation arena specially designed for study on parasite sexual behavior 34 4-9. Female-searching pattern of a male P. foveolatus in a 30 cm-diameter cardboard arena from the time of emergence from the host mummy to the end of mating activities 43 4-10. Schematic representation (transposed from photographs) showing gradual outward movement of P^. foveolatus adults (females and males) undergoing postemergence mating activities around the host mummy 44 4-11. Schematic representation (transposed from photographs) showing gradual outward movement of P^. foveolatus adult males (host mummy contained only males) undergoing postemergence searching for female mates 45 xii

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Figure Page 5-12. Positions of P, foveolatus females and males in 4 series of emergence from their respective host mummies 56 6-13. Simple linear regression lines representing the relationship between the percentages of female progeny of P. foveolatus and the quintets of parent females sequentially mated with single males 71 6-14. Simple linear regression lines representing the relationship between the percentages of female progeny of P^. foveolatus and the exposures of host larvae to the parasite parent females sequentially mated with single males 74 6-15. Tridimensional representation illustrating the general pattern of male sperm depletion, expressed in terms of percentages of female -progeny produced by P^. foveolatus parent females sequentially mated with single males 77 6-16. Quadratic polynomial regression curves representing the relationship between male sperm depletion (expressed in terms of percentages of female progeny produced by P^. foveolatus parent females sequentially mated with single males) and the quintets of parent females in the 1st and 2nd host exposures 80 6-17. Relationship between male sperm depletion (expressed in terms of percentages of female progeny produced by parent females sequentially mated with single males) and the host exposures in the 1st and 2nd quintets of parent females, showing a trend toward quadratic polynomial regression 32 7-18. Distribution of P. foveolatus females with respect to range categories of 20-progeny increments 95 7-19. Longevity vs. fecundity of female P^. foveolatus . 98 7-20. Simple linear relationship between sex ratios and numbers of P. foveolatus adults produced through host mummies corresponding to range categories of 5-progeny increments 104 xiii

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Figure Page 7-21. Linear relationship between sex ratios and per-host-mummy average numbers of progeny . produced through individual host exposures by 32 P^. f oveolatus parent females 106 722. Quadratic polynomial regression curve representing the relationship between the percentages of P^. f oveolatus female progeny and the host exposures 110 823. Number of successfully mated females and number of females failing to produce progeny with respect to 3 female-male pairing combinations of young and old P^. f oveolatus adults, based on 3 consecutive host exposures 117 924. Percentage of mated P^. f oveolatus females with respect to age 122 10-25. Relationship between female to male ratios of p. f oveolatus progeny produced by the first 10 parent females mated sequentially with initial single males (replicated 4 times) , and host exposures 137 1026. Quadratic polynomial regression curves representing relationships between female to male ratios of P^. f oveolatus progeny and host exposures 143 1127. Screened cage for confinement of host larvae and parasite adults for the study on parasite oviposition preference vs. host instars 151 11-28. Percentage of parasitization of 2nd-, 3rd-, and 4th-instar host larvae, and percentage of dead parasitized 2nd-, 3rd-, and 4th-instar host larvae as based on percentage of parasitization. . . 156 11-29. Simple linear relationship between percentage of parasitization and host instars, representing preference vis-a-vis host instars by P^. f oveolatus 157 11-30. Simple linear relationship between percentage of dead parasitized host larvae and host instars . . . 157 xiv

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Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Reqiurements for the Degree of Doctor of Philosophy ASPECTS OF THE REPRODUCTIVE BIOLOGY OF PEDIOBIUS FOVEOLATUS (CRAWFORD) (EULOPHIDAE: HYMENOPTERA) , PARASITE OF EPILACHNA SPP. (COCCINELLIDAE: COLEOPTERA) By LIMHUOT NONG May 1982 Chairman: Reece I. Sailer ; Major Department: Entomology and Hematology Nine aspects of the reproductive biology of Pediobius f oveolatus are subjected to experimental study. These include (1) mode of reproduction, (2) sexual behavior, (3) preemergence mating, (4) sex ratios resulting from sequentially mated males, (5) fecundity, (6) influence of age on mating capability of females and males, (7) influence of age on mating capability of females, (8) multiple matings of females, and (9) oviposition preference with reference to host instars. In common with most Hymenoptera, P. f oveolatus is arrhenotokous. Sex ratio is female-biased. Sibling adults mate in close proximity to their host mummy immediately following emergence. Mating activities following emergence last about 30 minutes and rarely exceed 1 hour. Once mated, females move away from the host mummy and thus afford more opportunity for the remaining virgin females to mate. Male behavior is characterized by a remarkably energetic search for and readiness to mate with receptive females. The male's mate-searching area expands gradually as adult emergence proceeds. Termination of mating activities XV

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is marked by disorderly movement and flight of both sexes. Within the local mating area, mate encountering success results from innate postemergence behavior of both sexes, and appears to involve a female sex pheromone. Males are unable to discriminate between virgin and mated females or between young and old females. Male wing vibration during courtship appears to attract outside males for participation in the local mating activities. Females seldom mate more than once. Subsequent matings are unrelated to success or failure of sperm transfer from the first mating. Receptivity of females to mating is substantially reduced after 2 weeks, but 15-day old males retain the ability to mate successfully. When given a choice females preferentially oviposit in large host larvae. The total number of adult progeny produced by individual females from 4th-instar host larvae may range from 0 to 199 with an average of 126. Fecundity is inversely correlated with female lifespan. For a given host instar, female to male ratio varies from one host larva to another and increases with increasing numbers of parasite adults per host mummy. Sperm regulation by females, expressed in percentage of female progeny produced through successive ovipositions , follows a mathematical quadratic polynomial model and features in succession the so-called "maturation," "optimum," and "degradation" biological phases. A similar pattern is exhibited by single males undergoing successive matings with a series of virgin females. This is especially evident from analysis of the distribution of percentage of female progeny produced in the first 2 ovipositions by the sequentially mated females. xvi

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INTRODUCTION This study of the reproductive biology of Pediobius foveolatus (Crawford) was undertaken with two primary objectives in mind. First, P. foveolatus , a eulophid introduced into the USA from India in 1966 (Angalet et al., 1968), is being used as a parasite to control the Mexican bean beetle, Epllachna varivestis Mulsant, a severe pest of common beans and soybeans. Although the parasite does not overwinter successfully in the United States, it exhibits such a remarkably high host-searching ability and reproductive potential that effective control has been obtained through annual inoculative releases of comparatively small numbers early in the growing season. This implies that, in order to be available when needed, the parasite must be maintained in culture, A better understanding of its reproductive biology would not only enhance maintenance and quality of stock cultures but would also provide information needed to determine when and how many parasite individuals should be released under a given set of field conditions. Second, P^. foveolatus is an arrhenotokous species (females and males produced, respectively, from fertilized and unfertilized eggs). Other than thelytoky (females produced by uniparental females, males lost or rare), arrhenotoky is the only other widespread major mode of reproduction that departs from the common pattern of diplodiploid sexuality occurring among animals; it has received much less attention

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2 even though it may occur in as many as one-quarter of all arthropod species and in some rotifers (Borgia, 1980). It is hoped that the present investigation will constitute a contribution to the basic knowledge relating to this mode of animal reproduction. In addition to literature review, the present study encompasses 9 research aspects: (1) mode of reproduction, (2) sexual behavior, (3) preemergence mating, (4) sex ratios resulting from sequentially' mated males, (5) fecundity, (6) influence of age on mating capability of females and males, (7) influence of age on mating capability of females, (8) multiple matings of females, and (9) oviposition preference with reference to host instars. Results are presented under 12 chapters. Chapter 1 deals entirely with pertinent literature concerning E. varivestis and P. foveolatus . Chapters 2-11 treat methodology and the above-mentioned research aspects, including the scope and literature specifically relevant to each. Chapter 12 is reserved for the general conclusions .

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CHAPTER 1 LITERATURE REVIEW This chapter treats the literature on Mexican bean beetle, Epilachna varivestis Mulsant, a new host of the parasite Pediobius foveolatus (Crawford), and the parasite itself. It is intended to provide salient features in regard to the status of biological and applied knowledge about these insect species. The Host Insect, E^. varivestis Taxonomy The Mexican bean beetle, E, varivestis , also earlier known as ladybird, bean beetle, bean bug, and spotted beetle (Chittenden and Marsh, 1920) belongs to the Epilachna varivestis group, tribe Epilachni, subfamily Epilachninae, family Epilachnidae, and order Coleoptera (Gordon, 1975). First described by Mulsant in 1850, it was also once known under the name Epilachna corrupta Mulsant (Chapin, 1936). Distribution The original home of the beetle was southern North America, where it occurred in many parts of Mexico and Guatemala (Howard and English, 1924). According to the Commonwealth Institute of Entomology

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4 (1954), it was known to inhabit southern Canada, the United States of America, Mexico, and Guatemala. Gordon (1975) added El-Salvador, Honduras, Nicaragua, and Costa Rica to the list. In Canada, the beetle was first reported from southwestern Quebec in 1943 (Auclair, 1959). In Mexico, it has a wide range of distribution, from 3 to 8,845 feet elevations within areas delimited by the 20°C isotherm (Landis and Plummer, 1935). In the United States, distribution of the beetle includes Alabama, Arizona, Arkansas, Colorado, Connecticut, Delaware, Florida, Georgia, Idaho, Illinois, Iowa, Kansas, Kentucky, Louisiana, Maine, Maryland, Massachusetts, Michigan, Mississippi, Missouri, Nebraska, New Hampshire, New Jersey, New Mexico, New York, North Carolina, Ohio, Pennsylvania, Rhode Island, South Carolina, South Dakota, Tennessee, Texas, Utah, Vermont, Virginia, West Virginia, Wisconsin, and Wyoming (Nichols and Kogan, 1972). Within the United States, the beetle was first discovered at Watrous, New Mexico, in 1849 (Chittenden, 1924) , but it was not until 1883 that anything concerning its habits appeared in publication (Chittenden, 1898). In Utah, it was officially recorded only in 1922 (List, 1922). By 1930, the beetle had been discovered in all but seven of the states east of the Mississippi River (Tissot, 1943). Howard and English (1924) presumed that it had reached Alabama at least as early as 1918 through shipments of alfalfa hay from Utah. They also reported that the beetle was present in Georgia, Tennessee, Kentucky, South Carolina, and Virginia by 1921. In Pennsylvania, it was found for the first time in 1921 and its entry was probably through West Virginia and Ohio (Guyton and Knull, 1925).

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5 Watson (1942) reported that, since its introduction into Alabama, the beetle had spread rapidly to the north and east, but much more slowly to the south. He also reported that the first beetles captured in Florida were discovered at Monticello by Fred Walker in 1930. The second definite record of a Florida infestation was made in 1938 (Tissot, 1943). In 1942, they were found at three localities in Alachua County: Gainesville, Hawthorne, and Island Grove, when the nearest known infestation was at Havana in Gadsden County. According to Nichols and Kolgan (1972), the lowest borderline of Florida infestation was delimited by Levy, Marion, Putnam, and Flagler Counties. Since then, the beetle has spread southward to Citrus, Sumter, Hernando, Pasco, and Hillsborough Counties. In the west, an isolated infestation was found in Ventura County, California, in 1946 and another in Twin Falls County, Idaho, in 1954. The insect was eradicated from both regions (Entomology Research Division, ARS, 1958). Life History, Economic Role, and Damage ' The complete life history of the beetle was studied by Thomas (1924). Bernhardt and Shepard (1978) reported that the adult beetle survived the winter in heavy accumulation of pine litter, field litter, or sweet gum litter. Both larval and adult stages are destructive to host plants. Howard (1922) stated that the beetle had demonstrated its importance not only in actual monetary loss, but also through its capacity for destruction wherever it became established, and by its tremendous

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capacity for rapid spread. In 1924, he reported that the pest quickly destroyed practically all the beans over an area of about 4,500 square miles in northern Alabama. In Maryland, Stevens et al. (1975a) pointed out that the beetle had inflicted serious economic damage to soybean, and that with the increasing cash value of this crop, the problem has steadily worsened, due to the greater tendency for the growers to use chemicals for control of the pest. Chittenden and Marsh (1920) reported that injuries caused by the beetle were practically confined to beans, and no variety seemed to be exempt from injurious attack. Various forms of the kidney bean, Phaseolus vulgaris L. et al., including string, pole, navy, and tepary or Mexican, and lima bean, P^. lunatus L., were affected, and on one occasion, the soybean, Soja hispida Moench (now Glycine max (L.) Merr.) was also attacked. They estimated that annual damage in New Mexico varied from 5 to 100% of the crop, the average loss being set conservatively at 10%. Howard and English (1924) reported that wherever it occurred, the Mexican bean beetle was a more serious pest than the Colorado potato beetle. Hinds (1920b) gave the following assessments. Loss to common snap beans in Alabama was likely to be complete, except for a partial yield from the earliest planted beans. This was almost equally true for pole beans and shell beans. Lima beans made a partial crop, but certainly less than a half crop. California blackeyed peas were destroyed. . Soybeans also suffered heavily in some fields, but the infestation was not as general as on the other food plants. Kudzu was not attacked noticeably in the field, but complete development of the insect was obtained upon that plant. No wild food plants were found, and there was no field attack of velvet beans, although slight feeding occurred

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in confinement. He also reported that food plants or other fresh materials most likely to aid in disseminating the pest included all soybeans, fresh beans, and cowpeas of any kind, but did not include English peas and velvet beans. Cowpea was first reported to be a host for the insect by Hinds (1920a). Sherman and Todd (1939) listed snap beans (bush), snap beans (pole), lima beans, soybeans, velvet beans, crotalaria, alfalfa, peanut, beggarweed, and kudzu as food plants in decreased order of preference by the Mexican bean beetle. The Entomology Research Division of the USDA (1958) included common beans, such as snap (green or string), kidney, pinto, navy, and lima beans as primary food plants, while also stating that the beetle can reproduce successfully on cowpeas and soybeans and reported that injury to soybeans had become more common in parts of the south. It was also noted that the beetle's second choice of food was beggarweed ( Desmodium tortuosum (SW) D. C.) or beggartick, which grows wild throughout the southeastern states. ' ,. Lockwood and Rabb (1979) found that the beetle adults consistently lived longer and produced more eggs when feeding on reproductive as opposed to vegetative soybeans and when feeding on lima beans as compared to reproductive soybeans. Whitfield and Ellis (1976), from their survey on insect pests of soybeans and white beans in 1975 and 1976 found no Mexican bean beetle on soybeans but only on white beans. Turner (1932) reported serious injury of rye caused by the beetle. Sunzenauer et al. (1980) found that the mean dally soybean leaf area consumption rates by the beetle were 4.65 cm^ for population consisting of second-generation adults, 4.87 cm2 for population consisting of adults

PAGE 24

that had overwintered in the field, and 3.80 cm^ for populations consisting of adults that had overwintered in the laboratory. McAvoy and Smith (1979) reported that the laboratory daily consumption of soybean foliage was 2.70 and 3.40 cm^ at 20°C and 3.70 and 3.80 cm at 26°C for male and female adult beetles, and that the development times of larvae were 5.30, 4.00, 4.80, 7.90, and 7.30 days at 20°C for the 1st, 2nd, 3rd, and 4th instars and pupa, respectively; the figures were slightly smaller at 2b°C. Bernhardt and Shepard (1979) reported that the beetle adults that were given Phaseolus lunatus and had fed on P. vulgaris as larvae had higher fecundity, male and female longevity, as well as shorter preoviposition period and number of days between ovipositions than adults with any other combination of diets. I.e., P. vulgaris soybeans, soybeans soybeans, and soybeans JP. lunatus . Besides the damage directly inflicted on the host plants, the beetle is also reported to transmit cowpea mosaic virus (Jansen and Staples, 1970), and the blackgram (mungo bean) mottle virus (Scott and Phatak, 1979). ( Control Early in the century, as far as chemical control is concerned, only rotenone gave satisfactory protection of beans from injury by the Mexican bean beetle (Howard et al., 1948; Entomology Research Division. ARS, 1958; Ditman and Bickley, 1951). Carbophenothion, disulfoton (granules), diazinon, malathlon, methoxychlor , parathioncarbaryl.

PAGE 25

9 and rotenone were later listed by Cantelo (1977) as pesticides for controlling the beetle. Walker and Bowers (1970) found that synthetic juvenile hormones, Methylenedioxyphenoxyterpenoid ethers, prevented hatch of the beetle eggs. They concluded that these ovicidal properties warranted further investigation toward practical application for control of the beetle eggs. ' An eradication program primarily composed of (a) winter survey and treatment of backyard gardens, (b) elimination of winter hibernation quarters, (c) planting of trap crops, (d) chemical treatment of bean plants within one mile radius of known infestations, (e) intensive survey of all bean plantings in the infested area, (f) establishment of quarantine line, and (g) development of effective commodity treatments, was successful in Ventura County, California where the beetle had been established since 1946 (Armitage, 1956). Resurgence of the beetle population following the treatment of soybean with methyl parathion and methomyl was reported by Shepard et al. (1977) at Clemson University Edisto Experiment Station. These investigators concluded that removal of natural biotic agents by the chemical insecticides was probably the major reason for such resurgence. Cultural practices have also been used to control the beetle. Immediate plowing under bean vines at the completion of harvest was encouraged in areas of occurrence to reduce the number of beetles entering hibernation (Chapman and Gould, 1930; Cantelo, 1977). Turner and Friend (1933) recommended that beans be planted 4 inches apart in

PAGE 26

10 areas where the beetle was a serious pest, since the beetle preferred closely planted beans for oviposition. Turner (1935) later found that injuries caused by the beetle was 37% and 67.5% when plants were spaced, respectively, 8 and 2 inches apart. Variations in soybean cropping practices were found to affect significantly the abundance of the beetle (Sloderbeck and Edwards, 1979). The beetle adults and larvae were more abundant in tilled soybeans than in non-tilled soybeans, and was limited to one larval generation on double-crop soybeans compared to 2 generations on early planted crops. Destruction of the beetle generation on snap beans planted at the border of soybeans as trap crops has been demonstrated to result in the protection of the adjacent soybean fields (Rust, 1977). Sources of plant resistance through screening of world collections of soybean were found in 3 cultivars (Van Duyn et al., 1971). Forced feeding tests showed that these lines were unsatisfactory as food even when no alternate food was available. Further studies by . the same investigators showed reduction in longevity and fecundity in beetle adults, and weight loss and high mortality in larvae (Van Duyn et al., 1972). Field cage studies revealed that soybean cultlvar "Shore" suffered no loss in yield at initial infestation rates of 1 and 2 adult beetles per linear foot of row as compared to susceptible cultivar "York" (Elden and Paz, 1977). Analyses for contents of total nitrogen, carbohydrates, organic acids, and sterols of leaf samples at different growth stages revealed that susceptible cultivars accumulated more total nitrogen at faster rate than did the resistant plant introductions (Tester, 1977).

PAGE 27

11 Drought periods accompanied by dry winds decreased soil moisture and desiccated the plants. As a consequence, bean leaves turned upright, and the beetle eggs and young larvae exposed to the sun dried and collapsed (Douglass, 1933). Despite the considerable list of natural enemies they had accumulated, Howard and Landis (1936) stated that the Mexican bean beetle had been practically unimpeded by parasites or predators in its spread throughout the intensively cultivated areas of the United States. The Entomology Reserach Division of the USDA Agricultural Research Services reported no record of internal parasites until 1922, when 2 native flies, the tachinid Euphorocera claripennis (Macq.), formerly known under the name Phorocera claripennis Macq . , and the sarcophagid Helicobia rapax (Walker) were found to parasitize the insect in rare instances in northern Alabama; these parasites never became abundant enough to be of any value (Entomology Research Division, ARS, USDA, 1958) . Paradexodes epilachnae , described by Aldrich in 1923 and now known as Aplomyiopsis epilachnae (Aldrich) , was introduced into the United States from Mexico, and was bred and reared. The parasite was not recovered in any locality the year following liberation (Landis and Howard, 1940). Many species of predators of the families Pentatomidae, Reduviidae, and Cleridae were observed to feed on Mexican bean beetle by Plummer and Landis (1932). Stiretrus anchorago (F.) and Podisus maculiventris (Say) , both feeding on eggs and larvae of the beetle, were found to respond to increasing prey density with negatively accelerating rises in the curves to an asymptote (Waddill and Shepard, 1975). A mite, Coccipolipus epilachnae Smiley, found in

PAGE 28

12 Central America has been observed to cause reduction in egg production of the Mexican bean beetle (Smiley, 1974). Host specificity tests conducted by Schroder (1979) revealed that hosts were limited to the members of the subfamily Epilachninae, all of which are phytophagous . On soybeans, Stevens et al. (1975a) observed that egg predation by predaceous coccinellids appeared to be most common, and that, from several hundred thousand field-collected larvae, only 2 were found parasitized. Neither quantitative data on egg predation nor the identity of those 2 parasites were given. Quattlebaum and Garner (1980) reported Mexican bean beetle adults infected by a fungus different from Beauvaria . He pointed out that the beetle larvae were more susceptible to the fungus than any of the lepidopterous larvae tested, i.e., corn earworm ( Heliothis zea (Boddie)), tobacco budworm (H. vires cens (Fabricius) ) , cabbage looper (Trichoplusia nl (Hubner)), soybean looper ( Pseudoplusia includens (Walker)), and velvet bean caterpillar ( Anticarsia gemmatalis Hubner). As indicated by the rapid dispersal of the Mexican bean beetle and continued importance as a pest, native biological agents and early efforts to introduce the parasite A. epilachnae (Landis and Howard, 1940) failed to provide control at a satisfactory level. However, the results of inoculative releases of Pediobius foveolatus (Crawford) made in Maryland in 1972 to 1974 (Stevens et al. , 1975a) and in Florida in 1974 to 1977 (Reece I. Sailer, unpublished report) have proved to be an effective means for control of the beetle.

PAGE 29

13 The Parasite, P. f oveolatus History, Taxonomy This parasite was first recorded in India by Ayyar (Angalet et al., 1968). In his survey made to discover beneficial insects in India in 1956, Angalet (Angalet et al., 1968) recovered several larvae of Henosepilachna sparsa (Herbst) parasitized by P^. f oveolatus . This recovery was of particular interest to him because of the possibility it might accept a new host, the Mexican bean beetle, E^. varivestis , a species that does not occur in India. From the stock reared by the Commonwealth Institute of Biological Control in India on H. sparsa , a pest of potato and eggplant, the parasite was introduced into the United States by the Insect Identification and Parasite Introduction Research Branch at Moorestown, New Jersey, in 1966 and was found to develop readily on the Mexican bean beetle. In 1967, more than25,000 P^. f oveolatus were released in the eastern states from New Jersey to Alabama (Clausen, 1978). The first quantitative account of its use through annual inoculative releases on soybeans was reported by Stevens et al. (1975a). ' P^. f oveolatus belongs to the family Eulophidae, superfamily Chalcidoidea, and order Hymenoptera. It was once placed in the genus Pleurotropis Foerster by Crawford (1912) . Pleurotropis was later made a synonym of Pediobius Walker by Ferriere (1953) . Role as Biological Agent, Biology, Natural Enemy The role of foveolatus in biological control in the United States was already mentioned under the control of the Mexican bean

PAGE 30

14 beetle. In India, Lall (1961) reported field maximum and minimum percentages of parasitization on Epilachna spp. as being 37.8 and 9.03, respectively, during April. He also cited the paper of Appana, and Usman and Thontadarya, in which Epilachna 28-punctata (Fabricius) and Henosepilachna sparsa (Herbst) were reported as other hosts of the parasite. Little has been worked out on the details of the basic biology of this gregarious parasite. In his study designed to determine the efficiency of foveolatus as a parasite of phytophagous Epilachna spp. in India, Lall (1961) also worked on some aspects of its biology, namely mating, oviposition, larval growth, and adult emergence. Laboratory rearing of the beetle and the parasite, as well as studies on the parasite developmental rate, fecundity, sex ratio, effect of the parasite age on fecundity, and effect of parasite-host ratio on parasite production were published by Stevens et al. (1975b). Superparasitism of E^. varivestis by the parasite and parasite-host ratios in regard to temperatures were reported by Shepard and Gale (1977). Gale and Shepard (1978) also studied the response of the parasite to temperature and time of exposure to the host. The parasite failed to overwinter, due to lack of diapause capability and/or available host material (Reece I. Sailer, unpublished report). So far as natural enemies are concerned, Eupteromalus viridescens (Walsh), a native pteromalid, was found in Maryland to be associated with the Mexican bean beetle larvae parasitized by P^. foveolatus , the first and only reported instance that appeared to Zungoli (1979) to represent hjrperparasitization.

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CHAPTER 2 GENERAL MATERIALS AND METHODS USED TO STUDY THE BIOLOGY OF PEDIOBIUS FOVEOLATUS Unless otherwise mentioned, the following methodology regarding the rearing of the host insect, Epilachna varivestis Mulsant (Fig. 2-1) and the parasite Pediobius foveolatus (Crawford) (Fig. 2-2) was applied to all studies undertaken. All experiments were conducted at the room temperatures (22-24°C) of the Biological Control Laboratory of the Division of Plant Industry, Department of Agriculture and Consumer Services, Gainesville, Florida. Rearing of Adults of Epilachna varivestis Clear plastic containers (Fig. 2-3), 31 cm x 22.9 cm x 10.8 cm, were used to rear adults of the Mexican bean beetle, E. varivestis . Ventilation, to avoid water condensation, was assured by circular holes (4.4 cm diameter), usually 2 at either end or 2-4 in the cover of each container, all screened with 80-mesh brass strainer. Bean (snap or lima) (Fig. 2-4) or beggarweed ( Desmodium tortuosum (SW) D, C. , Fig. 2-5) leaves served as food for the beetle. Plant leaves were kept fresh, particularly during periods of low humidity, by inserting their petioles through holes in the cap of a small container partially filled with water. Excess of humidity inside the rearing containers was 15

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FIGURE 2-1. Mexican bean beetle, Epilachna varivestls Mulsant. Extreme left vertical row: 4 adult beetles; upper middle: 6 2ndinstar host mummies; upper right: 6 3rd-instar host mummies; bottom horizontal row: 4 4th-instar host miimmies. FIGURE 2-2. Pediobius foveolatus (Crawford), parasite of Epilachna spp . (approximately 60 X) .

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17 1 1 1 iii|iiiiptii|iiii|»i»i|iiii|iiii|iMi|in»|ini)i»ii|iiii{M II tifti|M i 9 % A. Ml A

PAGE 34

FIGURE 2-3. Containers and tools for Mexican bean beetle rearing. Left: clear plastic container for rearing of adult beetles; middle plastic container and petri dishes for rearing of beetle larvae; lower right: camel hair brush and locally-made bamboo tweezers. FIGURE 2-4. Young greenhouse-grown lima bean plants.

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20 minimized by using containers with fewer vent holes or by placing leaves directly on the towelling paper covering the bottom of the containers. In all instances, towelling paper was used because it reduced soilage. Ten to 12 female-male pairs of adult beetle were reared in each container. Food was renewed daily. Egg masses, collected daily at the time of each food renewal, were incubated in petri dishes until the eggs hatched. Rearing containers were changed at 3 to 4 day intervals, depending on soilage. Beetle adults were discarded when egg production declined, usually when they were about 6 weeks old. Rearing of E^. varivestis Larvae The newly hatched larvae, aggregated on the empty shells of the egg masses, were transferred to larger plastic petri dishes (lA cm diameter, Fig. 2-3). Larvae from 2 to 3 egg masses (about 100 larvae) were then reared in these dishes through the 2nd instar. Clear plastic containers (Fig. 2-3) having dimensions of 18.4 cm x 13.3 cm x 8.9 cm, and 31 cm x 22.9 cm x 10.8 cm were then used to rear about 100 3rdand 4th-instar larvae, respectively. Each end of all rearing units contained a circular screen vent. Bean and beggarweed leaves were the food. A towel paper sheet of appropriate size was placed underneath the food material to reduce soilage. Fresh leaves were added daily and rearing units were changed when needed. Adult beetles for production of host larvae and maintenance of the beetle culture were produced in this way.

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21 Rearing of P^. foveolatus Of the 3 stocks denoted "Ocala," "Perry," and "NF S-121" permanently maintained in the laboratory, only the last was used for all studies conducted here. Clear plastic containers with dimensions 12 cm x 9 . 3 cm x 7 cm (Fig. 2-6) were used for rearing adults. To assure air circulation, rearing units were provided with 3 circular vents, one on either lateral side and one on the cover of the rearing unit; all were screened with nylon cloth. Another circular hole, normally plugged with a cork or rubber stopper, was located at one end of the unit to facilitate exposure of the host larvae to the parasite and the removal of the empty host mummies left after emergence of the adult parasites. To prevent the adults of the parasite from being caught between the wall and the lid rim of the rearing unit, the inner rim of the container body was lined with tape. Honey, applied in streaks on the bottom of the rearing unit and covered with a moistened sheet of towelling paper, served as food for adults. Water was supplied in a glass test tube of 1 cm diameter and 10 cm tall. The test tube was loosely plugged with cotton and placed inside the rearing unit in a slightly inclined position to avoid water flow. A plastic vial (snap-cap type) lid having a diameter of 3.5 cm placed in the unit near the access hole was used to hold the host mummies, thereby preventing them from sticking to the honeyimpregnated paper towelling. Only 4th-instar larvae of E. varivestis were used for all experiments, except the one dealing with oviposition preference study. Exposure of the host larvae to the

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FIGURE 2-5. Beggarweed plants, Desmodium tortuosum (SW) D. C. , at fruiting stage. FIGURE 2-6. Containers and tools for rearing and handling of parasite adults. Upper left: clear plastic container for culture maintenance of parasite adults; upper middle (in glass petri dish cover): small glass tubes for isolation of parasite pupae until emergence and sexing of the virgin adults; upper right: clear plastic container topped with acetate sheet, used as mating chamber for study on sex ratios resulting from sequentially mated males (Chapter 6) and study on multiple matings of females (Chapter 10); lower left: aspirator for transfer and isolation of mated females from container at upper right; lower middle: plastic tubes for isolation and confinement of single females or femalemale separate pairs; lower right: standard aspirator for collection of parasite adults.

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FIGURE 2-7. Hood for handling of parasite adults (designed hy Dr. Reece I. Sailer).

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25 parasite was made in the hood (Fig. 2-7) with the light on. Host larvae, collected beforehand in a large petri dish (14 cm diameter) were transferred quickly one by one by means of a pair of locally made bamboo tweezers of appropriate length (Fig. 2-3) into the rearing unit through the open access hole. The transfer operation was interrupted from time to time by the removal of those host larvae that were in the process of being stung (oviposition) by the parasite females. Stung host larvae, once removed from the exposure unit, were placed in a separate dish until termination of parasite oviposition. They were then transferred to the rearing containers, large petri dishes or more roomy containers, depending on the numbers of host larvae needed. Only one parasitic female was allowed to parasitize one host larva. Upon termination of oviposition, parasitic females were collected with an aspirator and returned to the rearing unit only at the end of each host exposure operation. Exposed host larvae were reared on bean or beggarweed leaves until cessation of feeding. For the stock culture, host mummies were transferred to the rearing unit of the parasites 2 to 3 days prior to the parasite adult emergence. Parasite colony was renewed at 3to 4-week intervals.

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CHAPTER 3 MODE OF REPRODUCTION Introduction Of haplodiploid sexuality, Borgia (1980) wrote "among arthropods, haplodiploidy is the exclusive mode of reproduction in the orders Hymenotera and Thysanoptera, and also occurs in Homoptera, Coleoptera, and mites and ticks (order Acarina)" (p. 104). Flanders (1939) stated that "sex control in the Hymenoptera under normal conditions results in preponderance of females. Extreme variability in the proportion of sexes, however, is often evident. This condition apparently results from the fact that in the majority of species the males are usually derived from unfertilized eggs and the females from fertilized eggs" (p. 12). This clearly describes a haplodiploid system also referred as arrhenotoky. Stevens et al. (1975b) investigated reproduction of the eulophid Pediobius foveolatus and found that unmated females oviposited, but their progeny were all males, thus confirming the arrhenotokous nature of this species. They did not pursue the study of the reproductive biology of this species beyond this point. The objective of the present investigation was to provide qualitative and quantitative evidence relative to arrhenotoky as the mode of reproduction of P. foveolatus and to gain insight to factors that 26

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27 influence sex ratios and reproductive success of this species. Materials and Methods To obtain virgin females, pupae were separated from the host mummies through dissection, and confined individually in small glass tubes of about 0.5 cm diameter and 3 cm tall. The tubes were then plugged with absorbent tissue. The separation of the pupae was made 2 days prior to the normal emergence of the adults from the host mummies. Sexing was made soon after emergence from the pupae. Thirty-eight virgin females, picked at random from among those issued from 30 4thinstar host mummies (approximately 300 females) , were reared separately in penicillin vials provided with honey and water impregnated in small circles of paper towelling. Three 4th-instar host larvae were separately exposed to each virgin parasite female within 3 consecutive days (one host exposure/day) beginning on the 3rd day after the adult emergence from the host mummies. They were then reared until cessation of feeding in separate petri dishes (8.9 cm diameter) labeled according to the given numbers of the parasite female and their exposure sequence. Two days prior to the day of normal emergence of the progeny, the host mummies were confined in separate gelatin capsules (no. 000) with corresponding labels. Sexing and counting were made only after the death of all emerged progeny adults.

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28 Results and Discussion Out of the total 38 tested virgin parasite females, 36 produced only live male progeny, whereas 2 (females no. 13 and 28) completely failed to produce offspring throughout the 3 host exposures (Table 3-1). No live female progeny was recorded. However, among those offspring that were not able to emerge from either the host mummies or their own pupae and those that failed to develop to adulthood (sexunidentifiable individuals), only one female (female no. 27) with incomplete development was recorded following dissection of all host mummies and the parasite pupae. The recognizable feature of this single female was the obvious presence of ovipositor. Before these convincing results, it is concluded that arrhenotoky is the type of reproduction of P^. foveolatus . In connection with the occurrence of the above-recorded single female that failed to fully develop, Suomalanien (1962) stated that unfertilized eggs of many bisexual insect species begin to develop, although the development, as a rule, comes to a standstill sooner or later (rudimentary parthenogenesis) . He further stated that such unfertilized eggs occasionally develop quite far, even to adult stages in cases of accidental or tychoparthenogenesis , a process that may be regarded by many authors as the primitive type from which normal thelytoky has developed. Whether the occurrence of that particular P. foveolatus female should be called rudimentary parthenogenesis or tychoparthenogenesis or accidental parthenogenesis, the situation remains conditional.

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29 TABLE 3-1 Live adult progeny (females /males) produced through 3 separate host exposures by P^. foveolatus virgin females Parasite Parasite progeny (females /males) parent female no. 2nd 3rd host exposure host exposure host exposure 1 0/17 0/18 0/22 2 * 3 0/20 0/15 4 * 16 17 ** 21 * 24 25 * 0/17 0/22 22 0/17 0/17 23 0/25 0/17 * 26 0/18 0/15 27 0/15 (*) ** 28 * 29 0/18 0/37 30 0/19 0/21 31 0/16 0/21 32 0/21 0/14 33 * 34 0/14 6/19 35 0/17 0/16 36 * 37 0/8 Q/Tl 3« 0/16 0/27 0/37 5 0/20 0/18 0/25 6 0/14 0/16 0/23 7 . * ** 0/33 8 0/17 0/20 0/12 ,1 * 0/17 0/18 10 0/23 0/19 0/27 11 * * 0/20 12 0/20 ** A* 13 * * ** 1* 0/21 * 15 0/23 0/23 ** 0/16 * 0/18 0/34 18 0/19 0/15 0/12 19 0/22 0/18 ** 20 0/16 0/23 *A 0/15 0/18 AA AA 0/11 0/32 0/24 AA *A * AA . 0/23 AA AA *A 0/18 *A AA AA 0/26 ** ** H^f /^T/"^f ** ^^^^^ (*) 1 incompletely developed female was observed

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30 Based on the results of the sexual behavior study (Chapter 4), P. foveolatus reproduction is characterized by a high level of inbreeding. All investigators who have studied the species have reported female-biased sex ratios. The association of inbreeding with haplodiploidy has been noted by many authors notably Ghiselin (1975) and Borgia (1980). Entwistle (1964) working on Xyleborus compactus (Eichh.) reported that unmated females on coffee stems produced only male progeny with which they subsequently mated to produce daughters. He attributed this high degree of inbreeding to the low mobility of the females living in "closed galleries". He concluded that the inhibiting effect on gene flow between populations combined with close inbreeding resulted in low individual variation within a local population but high interpopulation variation. Brown (1964) noted that the cost of initiating a viable haploid male to produce a haplodiploid species would be reduced by prior close inbreeding resulting in a high level of homozygosity. Thus haploid -. males would not suffer the consequences of the numerous deleterious recessive alleles that are normally present in an outbreeding population. Supported by extensive data, Hamilton (1967) showed that sex ratios are biased when close inbreeding is common. He also pointed out that, in many instances, severe reduction of male size further enhances diversion of parental investment into daughters. In addition to the factor of size noted by Hamilton, P. foveolatus males have shorter life expectancy than females, a feature that further strengthens Hamilton's conclusion regarding diversion of parental investment into daughters. From an evolutionary standpoint. Borgia (1980) concluded that

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31 haplodiploldy seems most likely to evolve in an inbreeding situation. He also noted that the transition from diplodiploidy to haplodiploldy through evolutionary time has occurred much less frequently than transitions to thelytoky. However, the haplodiploid transitions established the evolutionary origin of major systematic groups of organisms while the more numerous thelytokous transitions have normally been of little evolutionary consequence beyond the species level. While it is beyond the scope of the present study to speculate on the pathway through which arrhenotokous species have evolved, the results of the studies on mating behavior of P. foveolatus support the view that inbreeding mating systems tend to be a common characteristic of haplodiploid species. The one dead, incompletely developed female encountered among the male progeny of the 38 virgin females would appear to be an example of the phenomenon noted by Speicher and Speicher (1938). They reported the appearance of an occasional uniparental female in Bracon hebetor Say and speculated that they resulted from patches of tetraploid tissue in an otherwise diploid ovary.

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CHAPTER 4 SEXUAL BEHAVIOR Introduction Arrhenotoky, as already noted in Chapter 3, is the mode of reproduction of P. foveolatus . Borgia (1980), working with models for inbred and outbred systems in the evolution of haplodiploidy , noted that the inbreeding context seems the most likely situation for the evolution of haplodiploidy. Hamilton (1967) pointed out that under inbreeding or under the effects of more severe competition between brothers than between nonrelatives , parents should produce investment ratios that favor females. Correlation between inbreeding and male haploidy has also been noticed by M. T. Ghiselin who was quoted by Borgia (1980) as saying "the adaptive significance of male haploidy may have something to do with controlling the sexuality of offspring" (p. 111). Borgia (1980) also concluded that "even though inbreeding models seem to provide the best explanations for the evolution of male haploidy, in specific instances pathways to male haploidy specified by outbreeding models may be important" (p. 125). Among haplodiploid species of parasite Hymenoptera, inbred or outbred, sexual behavior is expressed in different ways (Borgia (1980), Boush and Baerwald (1967), Ghiselin (1975). Hamilton (1967), King et al. (1969), Leonard and Ringo (1978), Miller and Tsao (1974), Schlinger and Hall (I960, 1961), Simser 32

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33 and Coppel (1980a, 1980b), Van den Assem and Povel (1973), Vinson (1972), Werren (1980), and Yoshida (197g)). Little is known of the sexual behavior of P. foveolatus despite the important role of the species in biological control. The purpose of the present study was to gather information regarding sexual activities of males and females following emergence from their host mummy until their eventual dispersal. Materials and Methods . The study was conducted under laboratory (22-24°C) and summer field conditions. Laboratory Study Gelatin capsules (no. 000) and a locally made cardboard arena (Fig. 4-8) were used to contain the parasite adults for observations on their sexual behavior. Use of gelatin capsules A number of host mummies were separately confined in gelatin capsules (one host mummy per gelatin capsule), and held under constant observation. Mating behavior of adult female and male parasites was observed soon after they emerged from the host mummies. This set-up permitted close observations whenever needed, even under microscope. Use of the cardboard arena Gelatin capsules did not allow observations on how the parasite females and males behaved within a wider space in accordance with time

PAGE 50

34 FIGURE 4-8. Observation arena specially designed for study on parasite sexual behavior. The arena floor, made of white cardboard and marked with a series of circles distant from each other by 1 cm, is of 30 cm diameter. The clear acetate wall is 3 cm tall. A circular glass plate of 40 cm diameter is placed on the top to prevent escape of the parasite adults from the arena and also enable tracking male matesearching.

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35 To get information in this regard, a locally made cylindrical arena of 30 cm diameter and 3 cm deep was used (Fig. 4-8). The wall of the arena was made of clear acetate. The floor, made of white cardboard, was provided with a series of pencil-drawn circles distant from each other by 1 cm. A clear circular glass plate of 40 cm diameter served not only as a cover to prevent escape of the parasites, but also minimized disturbances. It also provided a surface on which male matesearching paths could be drawn with a marking pen. A single host mummy was placed at the center of the innermost circle of the arena floor and kept under continuous observation. Following emergence of the first male parasite, its movement was tracked until termination of mating activities, which was regularly followed by the active flight and dispersal of all individuals within the arena. In the course of track tracing, time lapses (in minutes) were also recorded whenever convenient. The inked male mate-searching pattern was then copied by means of Xerox. Information on the distribution of the adult parasites within the arena during the active mating period was obtained through photographs taken at halfor one-minute intervals, depending on the intensity of mating activities. The first picture was made when the first emerging adult appeared. The last picture was taken when mating activities ceased and individuals began to take flight and disperse. At no time did observational data or photographs involve adult parasites from more than a single host mummy.

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36 Study under Field Conditions A number of 4th-instar host larvae, following exposure to P. foveolatus females in the laboratory, were released on garden-grown lima bean plants (University of Florida Organic Gardens area) and caged to prevent escape or predation by natural enemies prior to the day parasite adults were expected to emerge. Probable period of emergence was established by dissection of one or 2 host mummies daily during the last 3 days prior to the day of normal laboratory emergence the presence of adults in the process of emergence from pupae was an Indication that actual emergence of the adults from the host mummies would occur within the next 24 hours. Close watch was maintained during daylight hours of this period for emergence of adult parasites. All observable behavioral activity of emerged adults, both females and males was recorded. Results and Discussion Laboratory Study Emergence of the parasite adults Emergence from pupae. From the hundreds of pupae separated from the host mummies 2 days prior to the day of normal emergence and individually confined in small glass tubes (to obtain virgin females and males for use in the studies of sex ratios resulting from sequentially mated males. Chapter 6, and multiple matings. Chapter 10), the process of emergence of individual P. foveolatus adults was observed to last from a few to several hours. The overall emergence terminated

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37 within 2 days. More than half of the total number of adults emerged during the first day. Males commonly emerged earlier than females. Emergence from the host mummies . During a period of over 5 years of continuous culture, emergence of the parasite adults from host mummies of 20-25 simultaneously exposed 4th Instars (usual numbers used for each stock renewal) took place over a period of about 2 days. Within this range, observed difference of emergence from individual mummies appeared to correlate with the number of the parasite eggs laid in relation to the quantity of food available in each host larva. An observed difference in rate of mummification among the parasitized host larvae appeared to be an early Indication of rate of food use by the parasite larvae. The process of ecdysis to the adult stage while still inside the host mummy and preparation of an emergence hole is normally completed within no more than 24 hours. The emergence hole is circular in shape, single and generally found in the abdominal region of the host mummy, and are made by both females and males. Emergence of adults from any host mummy took place only when all individual females and males are ready. This process, followed by the relatively short period of mating activities appears to ensure that a maximum number of females will be mated by their brothers before dispersal. The comparatively small size of males, the variable female-biased sex ratios, and the remarkably high level of male sexual activity (described elsewhere in the present chapter) appears to explain the adaptive advantage of simultaneous emergence of both sexes and subsequent mating activity normally observed within a reduced area around the host mummy, here called local mating area. Among females and males emerging from

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38 any single host mummy, there is no specific sequence of sexes. Behavior of females Compared to males, sexual behavior of females is simple. Following emergence, unless disturbed by more than one male attempting to mate with her, a female normally waits for a male by stationing herself in the vicinity of the host mummy from which she emerged. This behavior, consistently exhibited by all females, not only reduced the search area to be covered by sibling males but also allowed opportunity for grooming and establishing their composure. During this period, females were routinely observed to extend their legs, and through rubbing motions used them to clean their wings, body, and antennae. Except for a slow up-and-down movement of their antennae, a female responded to male courtship by remaining immobile. Acceptance of copulation by a male was manifested by an abrupt change in the posture of the female, with the female standing high on her legs to allow copulation. Upon termination of copulation which lasted from 5 to 14 seconds, she resumed her initial rest position, occasionally remaining there for some period of time, but more often moved away for some distance following a subsequent mating attempt by the same or other males. This movement was directed toward the outside away from the host mummy (Fig. 4-10). The number of stops on her way out depended primarily on subsequent male mating attempts. Such behavior, in addition to making room available for her newly emerged virgin sisters and thereby enhancing their mating prospects, would minimize wastage of male energy in courting already mated females. On the way out.

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39 particularly at the perimeter of the observation arena (large petri dish) and prior to the eventual dispersal, some females in rare occasions were seen to accept a second mating. Occasionally, some females were approached by males just as they emerged from the host mummy. These females would continue to move until they were a short distance from host mummy before assuming courtship stance. Competition for individual females, even briefly, was rarely observed among brothers. Not all virgin females were mated on the first attempt by males. Generally, males moved away in search of new females when the female was not receptive after a 40to 70-second male precopulatory attempt. The erratic movement of females increased with time. Meanwhile, males also gradually expanded their mate-searching area. In other words, the whole process proceeded smoothly in conjunction with progress of emergence. Mating activity diminished and eventually ceased not long after all parasite adults had emerged from the host mummy. Concurrently with diminished mating activity, there was increasing intensity of overall ground movement followed by increasing flight activity and eventually by general flight movement of all individuals inside the arena. The whole process of mating activities around single host mummies was observed to last from 30 minutes to about one hour. Behavior of males Males, in contrast to females, were usually very active. No matter what sequential position they occupied in any series of emergence, their behavioral pattern remained similar. Upon emergence from the host mummy, they lost no time searching for females, patrolling

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40 around or nearby, and frequently returning to the host mummy. The behavior of one male was especially revealing. This male happened to be the first adult to emerge from the host mummy. After patrolling the vicinity of the mummy for 9 minutes, this male returned and stationed himself at the exit hole from which he had emerged. After 4 minutes, a second adult emerged and proved to be a female with which he immediately mated. When a male encountered a female, especially after making his presence known, he always positioned himself -at one side of the female's body. Meanwhile, the male while standing on his mesoand metathoracic legs, grasped the upper thoracic portion of the female with one of his prothoracic legs while keeping the other loose without any contact. The female rested low on her legs. Male courtship then proceeded to the next phase. This was manifested by strong vibration of the male's wings in upright position, regularly interrupted by brief periods during which the male stroked the female's head or thorax with his antennae; during each stroking episode, the male body always moved in rhythmical synchrony with his antennae. Except for a slow up-and-down movement of her antennae, the female remained motionless during this '"'''^ period. As mentioned earlier under the study of female behavior, unsuccessful courtship was observed to last between 40 to 70 seconds after which the male moved away in search for other females and the female would remain in the same position. When a female was ready for copulation, she stood high on her legs. The male, while holding the female, then bent his abdomen toward the base of the female ovipositor, and copulation followed. The duration of copulation varied from 5 to

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41 14 seconds after which the male resumed the precopulatory posture, whereas the female lowered herself to the normal resting position. In no case did a male move away from his mating partner immediately after copulation. Rather he maintained the precopulatory posture and exhibited an act (postcopulatory behavior) similar to the precopulatory courtship for a short while before leaving the female in search for others. In some instances, as when confined to a gelatin capsule where courtship arena was very small, it was not unusual to record a higher number of females mated by a single male within a short period of time. In case of a moving female, the male may approach her from any direction and must make his presence known by contact. If the female continues to move, the male circles her closely moving very rapidly. Whereas mated females more often kept moving away in a generally outward direction from the host mummy particularly upon male courtship attempts, newly-emerged virgin females normally stopped and allowed male courtship. Encounters between moving males never led to courtship. However, on rare occasions, courtship may be exhibited by a moving male upon contact with another male at rest. Males did not have the ability to discriminate mated from virgin females. Such behavior might theoretically be disastrous for the system particularly if all mated females accepted subsequent matings. Males would have to expend more time and energy for regeneration of sperm supply and courtship activities. Since both sperm supply and time are needed in order to cover a maximum number of newly emerged virgin females within a relatively short period of time before

PAGE 58

42 dispersal from the local mating area, such a costly investment would tax the capacity of the species comparatively small-sized and very active males. However, the system has evolved in such a way that the lack of discriminating ability of males vis-a-vis the mated females is compensated for by the low percentage of mated females undergoing multiple matings (Chapter 10) because of the refusal of most females to respond to subsequent courtship attempts by males. In fact, only on some rare occasions were mated females observed to be receptive to subsequent matings during the postemergence period. This occurred only in the later part of the local mating period when mated females had already reached the periphery of the observation arena. Expansion of the searching area by males and the outward movement of mated females in the arena was a coordinated and gradual process (Fig. 4-9 and 4-10). When host mummies contained only adult males, mate-finding behavior of males was similar to that exhibited by males issued from mummies containing adults of both sexes. The only difference was that the activities were less intense and of shorter duration, lasting only 15 to 20 minutes (Fig. 4-11) compared to 30 minutes to one hour when both sexes were present. Field Study Despite the small area of lima bean leaflets, observation on sexual behavior of adult siblings made on 3 separate host mummies under field conditions did not reveal a noticeable difference from that recorded under laboratory conditions. This includes the mate-searching.

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43 FIGURE 4-9. Female-searching pattern of a male P. foveolatus in a 30 cm-diameter cardboard arena from the time of eme rgence from the host mummy to the end of mating activities. C is th! center of the arena where the host mummy was placed. Numbers 1. 2, U and 15 represent the distances in cm from the center ^f ^he ;;ena' Arrows indicate the direction of male's movement. Mlle'rtra;el time IS given in minutes (min.). "-ravei

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44 FIGURE 4-10. Schematic representation (transposed from photographs) showing gradual outward movement of P. foveolatus adults (females and males) undergoing postemergence mating activities around the host mummy. Each black spot at the center of the innermost circle represents the host mummy from which the parasite adults emergedeach black speck represents 1 adult parasite individual (male or' female). Each ensemble of circles represents the 30 cm-diameter floor of the observation arena. Elapsed times (in minutes and seconds) at which photographs were made are shown at the left corner of each square; the first was made when the first parasite adult emerging from the host mummy appeared; time zero (0) was given to this; the last (last square of the 4th row) was made when general tlight (dispersal) of the parasite was observed.

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45 FIGURE 4-11. Schematic representation (transposed from photographs) showing gradual outward movement of P. foveolatus adult males (host mummy contained only males) undergoing postemergence searching for female mates. Each black speck represents 1 adult parasite male; the larger and elongated black spot at the center of the innermost circle represents the host mummy. Circles in the top row represent the innermost 7 cm of the 30 cm-diameter observation arena while each ensemble of circles of the 2nd, 3rd, and 4th rows represent the entire floor area of the arena. Distance between circles in the first row represents 1 cm and those of the remaining rows 2 cm of the arena floor. Elapsed times (in minutes and seconds) at which photographs were taken are shown at the left corner of each square. The first photograph was made when the first parasite male emerging from the host mummy appeared; time zero (0) was given to this. The last photograph (last square of the 4th row) was made when general flight (dispersal) of the parasite within the arena was observed.

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46 mate-approaching, preand postcopulatory behavior, expansion of the searching area of the males, and the postemergence behavior of females and their response to mating attempts by males. The following describes observed sexual behavior of males and females in two successive occasions. In the first, a single male emerged following emergence of 2 females. Contrary to usual laboratory and field behavior, this male moved 2 cm from the host mummy and assumed a grooming position for some minutes before starting to court either of the 2 already emerged females. His attempt to mate failed. He resumed a resting position again 2 cm from the host mummy. During the next 15 minutes, 3 more virgin females emerged, at which time he resumed activity. At this time, 7 males were observed actively competing for 2 females about 30 cm distant from the mummy already under observation. These appeared to have emerged about 2 hours earlier. All males displayed wing vibrations while closely following the females. The females were obliged to move, but movement was nondirectional. At different times, these females approached within 2 cm of the host mummy from which virgins had continued to emerge. Despite the proximity of the newly emerged virgin females,' the males continued to pursue the other females until they were lost from view in the bean foliage. No successful matings were observed during the period of intense competition among these outsider-males. Meanwhile, at the local mating area of the mummy under active parasite emergence, the lone male became active and began courting a virgin female. Within about 30 seconds, 2 males from an unknown source lighted in the mating area and proceeded to search for females in a manner similar to sibling males. This

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47 phenomenon was observed on a second occasion, when a strange male lighted in the mating area almost immediately after courtship behavior was observed among males and females that had just emerged from a host mummy. The fact that these males did not appear until local males began to exhibit precopulatory vibration of their wings suggests that nearby males were attracted to the mating area by vibratory sounds produced by the courting males. The similarity in mate-searching behavior exhibited by the outsider males once they were in the active local mating area and while local males were still pursuing their courtship wing vibration would suggest that the vibratory sound might be superseded by the effects of female sex-attractant . In fact, no competition for females by local or outsider males has been observed in the local mating area. The above observations appear to indicate that successful mating of P. foveolatus is basically accomplished through a concurrence of innate male and female behavior as influenced by sex pheromone, tactile stimuli, and perhaps auditory effect of local male wing vibration. Innate behavior was basically expressed through (a) the patrolling of males in search for females around the host mummy even though females are totally absent, (b) the exhibition of preand postcopulatory behavior of males, and (c) the waiting-f or-male posture and the gradual male-assisted dispersal of mated females from the mating area. Werren (1980) , working with Nasonia vitripennis (Wlkr) , a haplodiploid and gregarious parasite of cyclorraphous fly pupae, reported that males emerged from the host puparium first and waited for females, a

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48 situation that appears to reflect the inherent behavior of this species, King et al. (1969), working with the same species also noted that males tend to emerge before females from a single host puparium. An indication of the presence of a female sex-attractant was observed on several occasions. First, as already mentioned in the beginning paragraph of the description of male sexual behavior, the presence of such sex-attractant was indicated by the return of the male (the first individual adult to emerge from the host mummy) to the host mummy after a 9-minute patrolling tour followed by a 4-minute period of waiting until the emergence of the first female with whom he later mated. Later, in a replicated test, 3 host mummies on the verge of adult parasite emergence were placed side by side in a triangular figure within a large petri dish (one mummy containing parasite adults of both sexes, emergence hole present but not yet large enough for the parasite adults to pass through; one mummy containing only male parasite adults with no emergence hole; and one mummy containing parasite adults of both sexes with no emergence hole) . Thirty newly emerged virgin males were introduced to each of the 2 petri dishes. At the end of 30-minute observation, 6 and 8 males, respectively, in the first and second petri dish were found to be attracted only to the host mummies with emergence hole and adults of both sexes. These males were ' present on and in the immediate vicinity of the host mummies. This behavior was also occasionally observed throughout the period of the present investigation. Pheromones have been reported to be involved in mating activities of other hymenopterous parasites. Schlinger and Hall (I960, 1961)

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49 reported that male Trioxys utllls Muesebeck and Praon palitans Muesebeck, parasites of spotted alfalfa aphid, apparently detected virgin females by odor, not by sight. Boush and Baerwald (1967) noted a strong indication of a female-secreted attractant in Opius alloeus Muesebeck, the parasite of the apple maggot. Leonard and Ringo (1978) reported a pheromone role in mate-finding of Brachymeria intermedia (Nees) , a parasite of the gypsy moth and other lepidopteran pupae. Simser and Coppel (!980b) working on B. intermedia (Nees) and B. las us (Walker) found that a femaleproduced sex pheromone serves to aid mate recognition by male. These investigators pointed out that male response to the pheromone remains constant with increasing male age, but pheromone activity declines with age in females. They further noted that activity of the pheromone did not elicit male response at distances greater than 3 cm. Beyond this distance, males exhibited only random movement. Some similarity in regard to the sex pheromone stimulus reported by Simser and Coppel (1980b) has been observed on P. f oveolatus . It appears that the pheromone produced by female P. foveolatus serves as a short-range cue that leads males to the female. Its effective radius depends on concentration. The behavior of the males in returning to the host mummy with decreasing frequence as females emerged, mated and dispersed from the mating area may be the result of the gradual reduction of pheromone emanating from the host mummy following departure of the females. This would further imply that (a) intact (without emergence hole) mummy is the source of maximum pheromone concentration, (b) the pheromone begins to disperse when emergence hole is opened; its gradient decreases with the distance from the host mummy, and (c) it gradually loses its

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50 strength as emergence of the adult parasite thins out. Therefore, it is expected that the pattern of mating activities may not be obvious or typical. This is indicated by the evidence that the somewhat disarrayed pattern of mating activities occasionally observed in the course of this study, usually involved host mummies where emergence holes had been opened for a relatively long period before the emergence of the parasite adults, thus allowing time for dissipation of pheromone. Attraction of males to isolated virgin females from only a short distance may also be attributed to the low concentration of pheromone produced by a single female as opposed to an aggregate of females within a mummy. The decline of pheromone activity through female aging was not investigated. However, older females (about 20 days or older) were observed to be generally nonreceptive to mating when presented with a male. They either walked away or did not allow copulation, although precopulatory courtship was sometimes observed. The attraction of males to the host mummies with emergence holes but still containing unemerged adults of both sexes and the return of males to their host mummies after female-searching tours in the case of mummies containing only male parasite adults suggests that females sex pheromone does not act alone; a concurrence of male and female innate behavior is required in bringing the sexes together. Display of wing vibration prior to mating have also been reported for other parasites. The ichneumonid Campgletis sonorensis (Cameron) male, when exposed to a female, displayed wing vibration which persisted until mounting (Vinson, 1972). Van den Assem and Povel (1973) suggested that amplitude, speed and duration of wing

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51 vibrations may function as reproductive isolating mechanism in species of Muscidifurax. The effect of wing vibration of male Nasonia vitripennls (Walker) on mating receptivity was reported by Miller and Tsao (1974); less than 20% of reproducing females successfully mated by wingless (wing-removed) males, whereas 78% successful mating was recorded for females confined with winged males. These investigators further noted that absence of wings in males resulted in production of only male offspring by most females. By means of an oscilloscopic analysis, Leonard and Ringo (1978) recorded a courtship song consisted of 3 distinct and orderly auditory displays, i.e., rock, wing quiver, and buzz, frequently repeated by male B. intermedia (Nees) . Yoshida (1978) reported that secretion of sex pheromone by female pteromalid Anisopteromalus calandrae (Howard) released wing vibration of males. As described earlier, males of P. foveolatus display wing, vibration, only and always, upon contact with females (precopulatory wing vibration display) and Immediately after copulation (postcopulatory wing vibration display). Such displays may be interpreted as follows: the precopulatory display, triggered by contact with female, triggered female receptivity to coitus; the postcopulatory display would somehow appear to be involved in the facilitation of sperm storage in the female spermatheca. Since attraction of outsider males to local mating area. was coincidentally observed to occur during the courtship of local males, wing vibration in this instance appears to have a recruiting role with a relatively long-range effect. The shortrange means of communication between both sexes is apparently assured by female sex pheromone. The various mate-finding mechanisms imply

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52 evolution of a system that ensures maximum opportunity for impregnation of females before their dispersal from the immediate area of the host mummy from which they emerge while allowing opportunity for a degree of outbreeding as influenced by host and parasite densities. In this context, the mating system of P. foveolatus is basically of the inbreeding type. The conclusion is supported by evidence of diminished mating capability of both males and females (Chapters 8 and 9) and associated reduction in production of female progeny when mating is delayed for a prolonged period.

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CHAPTER 5 PREEMERGENCE MATING Introduction Pediobius foveolatus (Crawford) is a gregarious parasite with haplodiploid mode of reproduction and female-biased sex ratios. In Maryland, according to Stevens et al. (1977). the numbers of parasite adults per host mummy ranged from 1 to 100. In Florida, the following accounts are obtained from an unrelated laboratory study and fieldcollected parasitized Mexican bean beetle larvae. The average numbers of live adult progeny per Ath-instar host larva subjected to laboratory-controlled parasitization by 1, 2, 3-4 parent females were, respectively, 17.5 (average of 38 host mummies), 28.83 (average of 28 host mummies), and 56.77 (average of 22 host mummies). The respective ranges of numbers of parasite individuals per host mummy were 6 to 43, 11 to 53. and 33 to 94. The average numbers of live adult progeny produced from 2nd-, 3rd, and 4th-instar parasitized host larvae collected in the field from different places on different dates were, respectively. 4.45 (average of 68 2nd-instar host larvae). 8.25 (average of 24 3rd-lnstar host larvae), and 18.27 (average of 30 4thinstar host larvae). The ranges of numbers of parasite adults per host mummy with each respective host instar were 1-9, 1-14, and 5-45. 53

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54 So far, no studies have been made in regard to where (inside or outside the host mummies) mating of this parasite occurs. Stevens et al. (1975b) stated that "although we assumed that mating takes place within the mummified host larva prior to emergence, we have observed mating within a few minutes to several hours after emergence" (p. 955). However, observed postemergence sexual behavior exhibited by the parasite, both male and female, raised a strong doubt about the possibility that mating takes place within the host mummies before the parasite I emergence. |The objective of this study was to confirm or refute the possibility of preemergence mating. Materials and Methods Among the 30 4th-instar host mummies placed under continuous observation for emergence of the parasite adults, only 4 were picked for study. Selection of the mummies was determined by order of adult emergence and work convenience. Parasite individuals were isolated immediately on emergence from the host mummies and confined individually in separate gelatin capsules (no. 000). Order of emergence was recorded and sex established as soon as emergence ceased. Individual females were reared in separate snap-cap vials (2.6 cm inner mouth diameter, 5.2 cm tall) labeled according to their host mummies and order of emergence. Each rearing vial was provided with a small drop of honey impregnated in a moistened circle of towelling paper of suitable size. Three 4th-instar host larvae were exposed to each parasite

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55 female on 3 consecutive days, starting from the 2nd day after the parasite emergence. The exposed host larvae were reared in separate petri dishes (14 cm diameter) labeled according to mummy number, order of emergence, and order of host exposure. Individual mtmimies (parasitized host larvae) were then confined in gelatin capsules (no. 000) and appropriately labeled. Emerging adult parasites were sexed and counted when all were dead. Tests to establish fertility of males were undertaken in the one instance where only one male emerged from a host mummy. This male was confined with 3 young virgin females for 24 hours. Three 4th-instar host laravae were then separately exposed to each female. The presence of female progeny produced by the tested females was proof of the male's fertility. Results and Discussion Sequential positions occupied by P. foveolatus males and females in their respective series of emergence from each of the 4 host mummies are shown in Figure 5-12. The per-mummy numbers of females and males, ovipositing females producing only male progeny, and females failing to produce progeny are presented in Table 5-2. Details regarding the number of progeny produced by each of the above-mentioned parent females with respect to each initial host mummy and each of the 3 consecutive host exposures are given in Appendix 5-1. The results show that there was no particular or orderly sequence or trend of emergence of males and females from the host mummies (Fig. 5-12), and that, among the total of 84 parent females, 60

PAGE 72

56

PAGE 73

57 CM I m n 0) iH n) B 0) <4-( 1 CO CO 0) •H e U o 3 u O >w 0) CO (U C a o C o (U 60 m ^ ,c: 6 3 O M (U ^ 4-) U-l 0) a iH 3 0) •T3 4-1 O CO •H 0) to 3 •u •H cS iH -a O (U 0) u > cfl o iH l(-J O W •H o c 60 o i-l 60 C •H >, H C •H (U Cd 60 M-l O to o, CO > U T3 CO 0) 4J O CO O CU T) m o u t3 (U CO U 0) QJ 13 3 o 2 0) 4J 3 ,J2 O ^ >^ -H IH 60 iH CO O a< O rH (1) •H iH > • CO O O 4-1 CO 0) col to 4-1 o u c (X OJ O rH CD CO m Q) 6 O T-\ • B rH O (U c Z MH O QJ ^ 4-1 CO •H 4J Q) CO CO rH CO O CO u >^ B CO ^ B ^ & to g CO 4-13 0) MH rH B rH O 3 CO • CO (1) O IH CO o B o 00 00 u-1 CM CM CM CM 00 «) 3 cn o 0) 4J a o > c o CO 0) !-i 3 CO c: o 60 (X CU 3 o !-i X 4J 4J CO O Q) Xi CO > (U ^1 > CO -rl H 4J 3 4-1 O CO (U T3 o CO 0) ro 13 x: U 60 O 3 O 13 ^-1 C J2 CO 4-J 60 CO CO O. rH 3 IX 4J CO 14H O O X! OJ 60 O C C -H 0) 4J CO CO (U (X V4 3 (X 13 T3 CU (U CO CO CO CO PQ rt C0|X3|

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58 produced only male progeny, and 24 failed to produce any offspring (Table 5-2). The disorderly sequence of male and female emergence was also observed repeatedly not only in this study and a preceding preliminary test, but also during the study on parasite sexual behavior (Chapter 4) . This behavior of P. foveolatus , when compared to that of the chalcidoid Nasonia vitripennis (Wlkr) , a parasite of cyclorraphous fly pupae, presents certain differences, although the 2 species exhibit similar properties such as gregarious parasitism, haplodiploid mode of reproduction, and female-biased sex ratios. King et al. (1969) reported that male N. vitripennis tend to emerge before females from a single host puparium and about 3% of emerged females were already mated. Werren (1980) went a step further and reported that male N. vitripennis emerge first from the host puparium, wait for females at the proximity of the exit hole, and no mating occurred within the puparium. For P. foveolatus . sequential position occupied by males in any series of emergence seems of little consequence. When females happen to emerge first, they wait for males. The reverse holds true for males (Chapter 4). The production of only male progeny by 60 parent females, and no female progeny by any of the 84 total number of parent females (Table 5-2) clearly demonstrated that mating of P. foveolatus must take place outside host mummies. Postemergence sexual behavior of males and females (Chapter 4) explains why mating should not occur within the host mummies. Preand postcopulatory behaviors and postures of females and males during copulation require considerable space, time,

PAGE 75

59 and convenience. There is no possibility that host mummies irrespective of size could accommodate these requirements. The number of the parasite adults, the pupal skins they left behind, the process of emergence from the pupae, the opening of the emergence hole, and the behavior of the emerged adults are among the primary limitations. Fertility of the only male emerging from host mummy no. 1 was proved when the 3 young virgin females produced female progeny, following a 24-hour confinement with the male in question. Table 5-2 also shows that 20 of the 24 parent females (column 4) completely failed to produce progeny as evidenced by the development to adults of all host larvae subjected to the 3 consecutive exposures to the parasite within the 3-day period. Although sterile females were observed in the preliminary test on "Ocala" laboratory stock, the high level of sterility was not expected and is the more remarkable when compared with the results obtained from other studies such as those on the mode of reproduction (Chapter 3) , influence of age on mating capability of females (Chapter 9), and fecundity (Chapter 7) of the parasite. In the mode of reproduction study, only 2 out of 38 females failed to produce progeny from one or more of the 3 consecutive host exposures. Of the 6 hosts exposed to the 2 "sterile" females, 4 pupated and 2 died as larvae (Table 3-1) . In the study of influence of age on mating capability of females (Chapter 9) , no single case of progeny production failure was recorded in any of the 190 females of age ranging from 1 to 25 days, to which 3 consecutive host exposures were made within a 4-day period (Appendix 9-6) . In the fecundity study (Chapter 7) , only 1 female among the total of 33 failed to

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60 produce progeny yielding only dead and pupating exposed host larvae. Inbreeding due to laboratory culture maintenance should not be a factor, since the gap between the generations of the parasite used in the above-referred studies were very close to each other, namely generation 31 to generation 33. Besides, P. foveolatus is essentially an inbreeding species and no inbreeding depression of the kind commonly encountered in small cultures of outbreeding species has been experienced. The most likely explanation for the high level of apparent "sterility" in this experiment is the manner in which the females were handled. In the mode of reproduction study, the parasite males and females were separated from each other while they were still in the pupal stage (about 2 days prior to their emergence) , and remained isolated during the entire period of study. In the study on influence of age on mating capability of females, separation of sexes was also made at the pupal stage; each female was then paired with each male and allowed to remain together throughout the period of host exposures; exposures of the host larvae to the parasite were made only 24 hours after the pairing. In the study on fecundity, all individual males and females were allowed to emerge and mate freely within the rearing unit for a period of about 36 hours, after which the 33 females were selected randomly from the unit. The relatively high success of progeny production suggests that the poor performance of the females in this experiment may be attributed to disruption of the normal mating process resulting from the separation of individual parasite adults immediately after their emergence from the host mummies. Their earlier association with males while inside the host mummy may provide a

PAGE 77

stimulus of male presence that inhibits oviposition by the subsequently unmated females. Since similar disturbances may occur under natural conditions, this behavior would serve to avoid wastage of eggs prior to mating. Although males would be produced, the normally femalebiased sex ratio and tendency toward an inbreeding mating system suggest that production of female progeny is adaptively advantageous to the individual female parent. Any overproduction of males that would result in excessive competition for females would reduce the reproductive fitness of female parent and in the extreme result in total collapse of some populations. For P. foveolatus , a mechanism that inhibits .oviposition by unmated females would thus serve to maximize reproductive fitness of individual females and evidently increase probability of population continuity under certain conditions adverse to the species. The existence of such a mechanism in P. foveolatus would seem to conform to views expressed by Borgia (1980) in discussion of his models for evolution of haplodiploidy where, in regard to mother-son matings, he states that: An increase in the fraction of females a mother produces can be achieved by reducing the rate at which eggs are laid before she is fertilized. Presumably, eggs laid early reduce the female's subsequent egg production. Since producing only a few males should guarantee her fertilization, the greatest output of females might be achieved by reducing the rate of oviposition until after she is fertilized. (pp. 113-114) As supporting evidence, he mentions studies of J. P. Gutierrez working with Tetr anychus neocaledonicus Andre and J. L. Nickel, who worked with Tetranychus desertorum (Banks). In each instance, the authors found that the unmated spider mite females produced considerably fewer

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62 eggs and lived longer than mated females. Similar behavior was observed by Browne (1922) in studies involving the chalcid wasp Melitobia acasia (Walker), Thus, while effected through a somewhat different mechanism, these admittedly preliminary observations of behavior of P^. foveolatus provide additional support for evidence of behavioral and physiological responses associated with female fertilization that tend to maximize reproductive fitness of haplodiploid species.

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CHAPTER 6 SEX RATIOS RESULTING FROM SEQUENTIALLY MATED MALES Introduction Among authors who have propounded theories relating to evolutionary aspects of sex ratio in bisexualspecies , the foremost are Fisher (1930) and Hamilton (1967). Darwin's theory speaks of the struggle among individuals for reproductive success, i.e., maximal reproductive capacity, and contains no statement relevant to success of the population, the species, or the ecosystem (Gould, 1980). The Darwin argument as framed by R. A. Fisher (Gould, 1980) contended that selection causes parents to produce the sexes in numbers such that the ratio of parent expenditure in the sexes is equalized over the population. Excellent agreement with Fisher's prediction of 1:1 ratio of parental investment (Fisher's parental expenditure) in each sex was found by Metcalf (1980) who worked on Polistes metricus Say and Polistes variatus Cress (synonym of Polistes fuscatus fuscatus (F.)) . Hamilton's theory deals with local mate competition and predicts a female-biased sex ratio in species where sons of a parent compete with each other for .ates (Werren, 1980). Gould (1980) wrote that "exclusive sibmating destroys the major premises of Fisher's argument for 1:1 sex ratio" (p. 206). 63

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64 Sex ratios of haplodiploid insects have been the subject of many investigations. Several factors, extrinsic or intrinsic, have been found or conceived to have influenced the sex ratios. King (1962) suggested that increase in percentage of males Nasonia vitripennis (Walker) is related to increase in number of eggs undergoing resorption in the female ovarioles at the time of oviposition, particularly under the conditions where host puparia are intermittently available. Werren (1980) reported that N. vitripennis females adjust the sex ratio of their broods according to whether they are first or second wasp to parasitize a host; progeny of the first wasp show a strong daughter bias, the second adjusts the production of sons to the relative level of local mate competition. Colgan and Taylor (1981), while establishing a model of sex ratio based on the reproductive pattern of the aphelinid Coccophagus scutellaris Dalman, stated that "in some haplodiploid species a certain coarse control of offspring sex ratio is obtained from timing of mating: eggs laid before mating are haploid, after mating are diploid" (p. 564). Flanders (1939) pointed out that, in arrhenotokous species of Hymenoptera, the spermatheca is a sex-controlling mechanism, able to modify sex ratio in response to stimuli of inconstant environmental factors. He later theorized that the small spermathecal gland of a chalcid parasite may not be able to keep pace with egg deposition and as a result more unfertilized eggs would be produced. This rate of oviposition could be a factor influencing sex ratio (Flanders, 1956). Flanders noted that his concept was supported by the fatigue theory proposed by Marchal who suggested that an increase in the male progeny of a mated female parasite which oviposited rapidly would indicate

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65 fatigue of spermatheca. Abdelrahman (1974b) has since reported that sex ratio of the chalcid Aphytis melinus DeBach, a parasite of the red scale Aonidiella aurantii (Mask.) is influenced by both the number of eggs laid per host and by the density of the parasite population relative to hosts. He also listed the size and quality of the host, small size of spermathecal gland, differential mortality, temperature, and age of mothers as other factors influencing the final sex ratios of A. melinus . Wilkes (1965) studied the structure and function of male and female reproductive systems of the eulophid Dahlbomlnus fusclpennls (Zett.) in an effort to explain the apparent ability of this parasite to control the sex ratio of its offspring. He pointed out that the production of maleand female-producing eggs during oviposition is not influenced by the environment except under the extremely unfavorable environment, nor by the host, and that discontinuity in the release of sperm is unlikely the regulating mechanism involved in the production of haplold males. He assumed that "for most of the inseminated females, if the effect of the female sperm storage organ of the passage of all eggs from the ovarloles to vagina is constant, fertilization must be constant; if not, apart from the possibility of dimorphism of the sperm or egg, the Intervention of male production must be externally induced" (pp. 647-648). He also noted that external stimuli operating through spermathecal gland as theorized by Flanders (1939) for regulating sexes has never been adequately supported by observation. In apparent contradiction to Wilkesconcept, Gordh and DeBach (1976), working with the aphe linid AEh^ lingnanensis Compere, stated that "it is difficult to imagine any factor which would tend to suppress female progenies production

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66 with subsequent matings other than the reduction in the absolute amount of sperm being deposited" (p. 588). Sekhar (1957) reported that the sex ratio of the progeny of Aphidius testaceipes (Cresson) and Praon aguti Smith shifted to male bias with females later in the mating sequence. Schlinger and Hall (1960) found that mated females of the braconid aphid parasite Praon palitans Muesebeck produce 1:1 sex ratio, but multimated males supply so little sperm at each mating that the sex ratio may go as high as 58:1 in favor of males. Similar findings were reported for the braconid Trioxys utilis Muesebeck by Schlinger (1961), in which mated females produced progeny of both sexes in a 1:1 ratio; however, virgin females mated to males having a previous history of multiple matings cause a shift toward higher ratios of males and in the case of those males having the highest number of previous matings the ratio male: female progeny was as high as 24:1. This was interpreted as the result of sperm depletion. Male inseminative potential of the aphelinid A. melinus was studied by Gordh and DeBach (1976) to determine the number of females a single male could inseminate during the course of a lifetime and the number of progeny resulting from these copulations. They found that males courted and copulated with females in rapid succession, resulting in a decreasing percentage of females with successive matings. They concluded that females inseminated by a male that had experienced coitus with several females earlier tended to produce more males, thus suggesting that fewer spermatozoa were transferred during later copulations. Male to female sex ratio of P. foveolatus was reported by Lall (1961) from India (the native country of the parasite) as being 1:2.

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67 Seasonal changes in sex ratio of this parasite was studied by Stevens et al. (1977) who observed no apparent relationship between sex ratios of the parasite adults and collection dates. These investigators also reported that male to female ratio declined as the number of parasite developing within the host larva increased. The objective of the present investigation was to determine the effect of sequential matings of P. foveolatus by single males with a series of virgin females on the sex ratios of their progeny. Materials and Methods The method used in the study of mode of reproduction (Chapter 3) was applied here to obtain virgin parasite adults of both sexes. All females and males subjected to this study were picked at random from among those issued from 30 host mummies. Each of the 4 virgin males used were allowed to mate sequentially with 50 virgin females. This operation was carried out under the hood (Fig. 2-7) with the light on. A parasite rearing unit, with the cover replaced by a sheet of clear acetate of appropriate size (Fig. 2-6), was used to contain the parasite adults. Sixty-five virgin females were introduced into the container. Thirty minutes were allowed to them to regain their composure. A virgin male was then introduced into the container. Male activities were watched continuously until the 50th virgin female was mated. Each mated female was removed by means of an aspirator (Fig. 2-6) and released into a rearing plastic vial (snap-cap type, 2.6 cm inner mouth diameter, 5.2 cm tall) (Fig. 2-6) prepared and labeled beforehand in accordance with male number and order of mating. On the second day

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68 after mating, one 4th-instar host larva was exposed to each parasite female on each of six successive days. The exposed host larvae were reared in separate petri dishes (8.9cm diameter). The mummies were then individually confined in gelatin capsules (No. 000) also labeled in accordance with male number, order of mating of female, and order of host exposure. Sexing and counting were made only when all emerging individual died. Results and Discussion Raw data representing the numbers of adult parasite progeny of both sexes produced from individual host mummies resulting from 6 separate host exposures of all parent females sequentially mated by single males are given in Appendix 6-2. The corresponding percentages of female progeny are presented in Appendix 6-3. Table 6-3 represents the average percentages of the parasite female progeny produced by parent females arbitrarily grouped in 10 • successive quintets under each of the 4 sequentially mated males and each of the 6 separate host exposures; the grouping was made for statistical analysis convenience. Statistical analysis based on data presented in Table 6-3 showed that male sperm depletion, expressed in percentage of female progeny, did occur both in the direction of increasing order of quintets of parent females and direction of increasing order of host exposures. Sperm Dep letion in Increasing Order of Parent Female Quintets Based on the average percentages of female progeny corresponding to individual single males of each host exposure (Table 6-3), depletion

PAGE 85

69 I 4J 0) U (S •H a CO 3 CO o CO o S"-" s-» s-^ 2 2 n => <^ w«) w— C*^ Co C^o w w V— o — SJ O »^ 0» CO ^ CM fM o so ^ C7» <0 CTl o to a> \o to r» o o o rsj in CO o o ffi V o *-» CO ^ cy< JB.CM . , fsj O rx CO r~ tn f-» CO O f^CM « •rt — • \0'^ tn*M «»Ot o"* — *-) s c s 5S r* *^ "i-^ CO r^. n CM « to n o o o ir> CX3 cn to U1 M ^ CM o o en Q ot — « fn o o m o o o V ^ 0^ V CO CO O GO lA W O CD irt O 00 OS CO lO CM CO o o o 0% *W C\j o A CM CM tMCM so CO o Ot V «NI * CO to (-(CM ->tO tM *— ^ ^ to o r«. (-1 ^ CM — • V -J*-". TNI cr> m ic-» V 00 CM ^ «n CD f>. (MOO— CO«oCOk-,CD<7« « in Ml — » — CM Ot —« *^ M »n XT oi 0 i-F. ^ ! T _ * ^ * u er c^ « eg ttf Of tn *«* a. vt at •* 1. — -2 •t »— £ C7» c —

PAGE 86

70 of male sperm in Increasing order of quintets of parent females as determined by order of sequential matings by single males is represented by the following simple linear regression equations, all significant at 0.01 level, and illustrated in Figure 6-13. ^(exp 1) = 98.20 6.60 Quintet (Eq. 6.1) (coefficient of correlation r = 0.70) A '^(exp 2) = 106.68 9.14 Quintet (Eq. 6.2) (coefficient of correlation r = 0.81) ^(exp 3) = 104.57 10.04 Quintet (Eq. 6.3) (coefficient of correlation r = 0.83) ^(exp 4) = 10.44 Quintet (Eq. 6.4) (coefficient of correlation r = 0.84) ^(exp 5) = 92.32 10.44 Quintet (Eq, 6.5) (coefficient of correlation r = 0.86) ^(exp 6) = 87.42 9.62 Quintet (Eq. 6.6) (coefficient of correlation r = 0.84) where: Y (^^^^ , ^(^^^p . . . , ^ (e^p 6) respectively represent the estimated percentages of female progeny for the 1st, 2nd, 6th host exposures, and Quintet stands for quintet of parent females. Sperm Depletion in Inc reasing Order of Host Exposures Aside from the first 2 quintets (first 2 rows of Table 6-3; equations Eq. 6.7 and Eq. 6.8) which showed no significant slopes (rate

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71 100 90 80 70 60 c 0) O) 50 o I— Q. ^ 40 CO E S 30 20 10 0 -10 -20 1st exposure 2nd exposure 3rd exposure 4th exposure 5th exposure 6th exposure J. -» ' ' 1 2 3 4 5 6 7 Quintets of female parents 9 I 10 rp?^M I t^""^ ^^""^^^ regression lines representing the relationships between the percentages of female progeny of |2Z£2lStH£ and the quintets of parent females sequentially mated with single males. . '^"'-La-L-Ly

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72 of sperm depletion), simple linear relationships between the percentages of female progeny and the host exposures for the next 7 female quintets, i.e., 3rd to 9th quintets, are all significant at 0.01 level (equations Eq. 6.9 to Eq. 6.15), whereas that of the 10th quintet (equation Eq. 6.16) is significant only at 0.05 level. The statistical analyses here involved were based on the per-host-exposure average percentages of female progeny produced by each quintet of parent females (figures in parentheses. Table 6-3). All equations are represented below according to the sequence of parent female quintets and illustrated in Figure 6-14. ^(Q.l) = 86.86 0.66 E (Eq. 6.7) (coefficient of correlation r = 0.34) Y(Q.2) = 80.96 0.56 E (Eq. 5.8) (coefficient of correlation r = 0.59) , Y(Q.3) = 95.82 8.80 E (Eq. g.gj (coefficient of correlation r = 0.91) ^(Q.4) = 77.64 3.81 E (Eq. 6.10) (coefficient of correlation r = 0.96) Y(Q.5) = 81.77 8.24 E (Eq. 6.11) (coefficient of correlation r = 0.99) Y(Q,6) = 82.23 10.05 E (Eq. 6.12) (coefficient of correaltion r = 0.94)

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73 ^(Q.7) = 59.22 8.96 E (Eq. 6.13) (coefficient of correlation r = 0.93) Y(Q 8) = 58.83 11.31 E (Eq. 6.14) (coefficient of correlation r = 0.90) ^(Q.9) = 7.39 E (Eq. 6.15) (coefficient of correlation r = 0.92) ^(Q.IO) = 6.21 1.29 E (Eq. 6.16) (coefficient of correlation r = 0.82) where: Y(q ^(Q.2)» ^(q ^q) respectively represent the estimated percentages of female progeny for the 1st, 2nd, 10th quintets of parent females, and E stands for host exposure. Difference in Rat es of S p er m Depletion and Biological Si^ni f in.... The results of analysis of variance primarily aimed at determining the difference among the rates of sperm depletion, i.e., the difference between the slopes of the simple regression lines of male sperm depletion in increasing sequence of matings (equations Eq. 6.1 to Eq. 6.6; Fig. 6-13) and in increasing sequence of host exposures (equations Eq. 6.7 to Eq. 6.16; Fig. 6-14) are respectively presented in Tables 6-4 and 6-5. A comparative view on the general behavior of the regression lines illustrated in Figure 6-13 and Figure 6-14 appears to indicate that sperm depletion occurred in a more regular pattern in the

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74 4 A A 100 90 80 70 60 50 c o a 40 © CO 30 E o >S a* 10 0 10 20 1st 2nd 3rd 4th Host exposures J 5th 6th FIGURE 6-14. Simple linear regression lines representing the relationships between the percentages of female progeny of P. foveolatus and the exposures of host larvae to the parasite parent~f e^iiHii sequentially mated with single males; Q Q q represent regression lines of equations Eq. 6.7, Eq. 5.8, ... % 6 16 respectively. ' V'

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75 direction of increasing number of matings than in the direction of increasing number of host exposures. The F-test for equality of slopes of all simple regression equations, namely equations Eq. 6.1 to Eq. 6.6, showed no significant difference, and suggests the closeness among these slopes. The difference between the slopes of the combined equations (Eq. 6.1 + Eq. 6.2 + Eq. 6.3) and that of the combined equations (Eq. 6.A + Eq. 6.5 + Eq. 6.6), being significant only at 0.10 level (Table 6-4) further suggests such closeness. The varying degrees of steepness among the slopes (rates of sperm depletion) of the simple regression lines of Figure 6-14 (sperm depletion in increasing sequence of host exposures) suggest irregularity in the amount of sperm deposited by the males during successive copulations, while going toward complete depletion. In this study where females were readily accessible to each male (65 virgin females vs. on virgin male at the onset of the serial matings, all aggregated about the upper portion of the mating container near the light source of the hood) , time appeared to be a critical factor for the process of sperm replenishment in the males. In the field, such extreme accessibility of virgin females would rarely be encountered; however, as females normally outnumber males, there are no doubt occasions when males have opportunity to mate in rapid succession. This should tend to reduce the normally observed sex ratio bias in favor of females and may in part explain the variations in sex ratio encountered in the field. Figure 6-15 features a general pattern of sperm depletion, both in increasing sequence of matings (female quintets) and host exposures.

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76 TABLE 6-4 Significant difference between rates of sperm depletion in increasing sequence of female quintets Slopes tested Level of significance not significant 0. 10 0.05 Those of equations Eq.6.1, Eq.6.2, Eq.6.3, Eq.6.4, Eq.6.5, Eq.6.6 Those of combined equations (Eq.6. 1 + Eq.6.2 + Eq.6.3) , (eq.6.4 + Eq.6.5 + Eq.6.6) Those of combined equations (Eq.6.1 + Eq.6.2), (Eq.6.3 + Eq.6.4), (Eq.6.5 + Eq.6.6) not significant significant significant TABLE 6-5 Significant difference between rates of sperm depletion in increasing sequence of host exposures Slopes tested Level of significance not significant 0.05 0.01 Those of equations Eq,6.7, Eq.6. 8 Those of equations Eq.6. 7, Eq.6. 8, Eq.6. 9 Those of equations Eq.6. 9, Eq.6. 10, Eq.6. 11, Eq.6. 12, Eq.6. 13, Eq.6. 14, Eq. 6.15 Those of equations Eq.6. 15, Eq.6. 16 not significant significant significant significant

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77 100 . 90 . 80 ^1 >. c (U . 60 o . 50 a. . 40 mal 0) . 30 . 20 . 10 _0 /I ^ Quintets of y 6 parent females 4 •: FIGURE 6-15. Tridimensional representation illustrating the general pattern of male sperm depletion, expressed in terms of percentages of female progeny produced by parasite parent females sequentially mated with single males (based on data of Table 6-3),

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78 Table 6-3, illustrated in Figures 6-14 and 6-15, also show that higher doses of male sperm were received by the first 2 female quintets, as evidenced by the insignificant slopes of equations Eq. 6.7 and Eq. 6.8, in contrast to the 8 subsequent female quintets respectively represented by equations Eq. 6.9 to Eq. 6.16 which show significant slopes. However, evidence of sperm depletion did appear as early in a sequence as the 2nd mating (Appendix 6-2, case of male no. 2), and unsuccessful mating, though most likely rare, may also occur (Appendix ^6-2, case of male no. 2, 6th mating). In a general sense, the results indicate that a single male is capable of effectively impregnating about 10 virgin females in rapid succession before sperm depletion can be noticed. The per-mummy average of 83.43% female progeny produced by the first 2 parent quintets (Table 6-3), i.e., 40 females, from 174 parasitized host larvae for the 6 host exposures, appears to confirm the results of statistical analyses above-described. Further Relationships and Biological Implications In addition to the above-determined simple linear relationships, male sperm depletion was, in some instances, found to fit quadratic polynomial regression or show an apparent trend toward similar curvilinear relationship. In increasing sequence of matings (increasing sequence of quintets of parent females. Table 6-3), curvilinear relationships represented by equations Eq. 6.17 and Eq. 6.18 and illustrated in Figure 6-16 were revealed in the 1st and 2nd host exposures; the level of significance

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79 for the test on the magnitude of the quadratic coefficient estimate was 0.01 for equation Eq. 6.17, and 0.05 for equation Eq. 6.18. The analyses were based on the average percentages of female progeny produced by each quintet of parent females with respect to each host exposure and each of the 4 single males (Table 6-3) . ^(sd.Q)l = 70-91 + 7.04 Q .1.24 (Eq. 6.17) (coefficient of determination = 0.60) ^(sd.Q)2 = ^^-O^ + 0.69 Q 0.89 (Eq. 6.18) (coefficient of determination = 0.70) where: Y^^^ and Y^^^^^^^ respectively represent the estimated -percentages of female progeny for the 1st and 2nd host exposures, and Q stands for parent female quintet. , In increasing sequence of host exposures, the trend of male sperm depletion based on the per-host-exposure average percentages of female progeny produced by each quintet of parent females (figures in parentheses. Table 6-3) appeared to incline toward the curvilinear relationship. Such tendency is represented by equations Eq. 6.19 and Eq. 6.20 which are close to being significant at 0.05 level. ^^0.05, 31 d.f. =-2.353 <^ t^alc. ="2-24 for equation Eq. 6.20). The 2 equations are illustrated in Figure 6-17. ^(sd.E)l = 78.07 + 5.93 E 0.94 E^ (Eq. 6.19) (coefficient of determination r2 = 0.60)

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80 100 90 80 70 60 50 I 40 30 20 10 0 ^(sd.Q)l = 70.91 + 7.04 Q 1.2A '(sd.Q)2= ^^-^^ + 0.69 Q 0.89 Q 123456789 10 Female quintets FIGURE 6-16. Quadratic polynomial regression curves representing the relationship between male sperm depletion (expressed in terms of percentages of parasite female progeny produced by parent females sequentially mated with single males) and the quintets of parasite parent females in the 1st and 2nd host exposures .

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81 ^(gd^E)2 = 76.79 + 3.69 E 0.44 E^ (Eq. 6.20) (coefficient of determination = 0.76) where: Y^^^ g^j^^ Y(sd.E)2 respectively represent the estimated percentages of female progeny for the 1st and 2nd quintets of parent female, and E stands for host exposure. In light of the above statistical analyses, the behavior of the regression lines (those representing the relationship between the percentages of female progeny and host exposures, and those representing the relationship between the percentages of female progeny and quintets of parent females) appear to follow a similar pattern: a gradual shift from quadratic polynomial (curvilinear) to simple linear (straight line) regressions. This transitional state which appears to be basically dependent on the intensity of sperm depletion may be explained as follows. In the direction of increasing order of host exposures, the early quintets of parent females, particularly the 1st and 2nd, seem to be inclined toward a curvilinear relationship (quadratic polynomial regression), if more than 6 host exposures are available. This interpretation is essentially based on supportive evidence provided by the study on the fecundity (Chapter 7). In that study, despite the wide range of fecundity among parent females, it was found that the distribution of the percentages of female progeny over 10 out of 11 exposures of the host larvae to the 32 free-mated parasite parent females is well represented by the quadratic polynomial model (Chapter 7, equation

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82 100 90 • ''(sd.E)l °^(sd.E)2 ''^(sd.E)l = 78.07 + 5.93 E 0.94 E' 80 _ 6 1/ 10 '(sd.E)2 = 76.79 + 3.69 E 0.44 E^ J_ 3 4 Host exposures FIGURE 6-17. Relationship between male sperm depletion (expressed in terms of percentages of parasite female progeny produced by parent females sequentially mated with single males) and the host exposures in the 1st and 2nd quintets of parent females, showing trend toward quadratic polynomial regression.

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83 Eq. 7.23). Reasons for the exclusion of the average value (percentage of female progeny) of the 11th host exposure from the 10 preceding values used to fit the quadratic polynomial model rested on the grounds that (a) only 3 out of the total 32 parasite parent females produced progeny in this host exposure, thus putting their validity in doubt, (b) the percentage of female progeny produced in this host exposure appeared to be very erratic, most probably due to the loss of sperm regulating ability by the exhausted parasite parent females, and (c) ignoring this excluded value (average percentage of female progeny obtained from only 3 host mummies resulting from the parasitization by the 3 parasite parent females mentioned in (a)) permitted a very good fit of the data to the quadratic polynomial model. The change from curvilinear to simple linear relationship is basically dependent on the amount of sperm received by parasite parent females during copulations, and in this case (sperm depletion in increasing sequence of host exposures) appears to go from the low through higher level of significance of fit before ending up with a good fit to simple linear regression. This apparent trend is indicated by equation Eq. 6.19 (1st quintet of parent females) and equation Eq. 6.20 (2nd quintet of parent females) , whose respective calculated t-values are 1.93 and 2.24, both almost significant at 0.05 level (tr> nr -> ^ U.05, 3 d.f . = 2.353; coefficient of determination is 0.60 for equation Eq. 6.19, and 0.76 for equation Eq. 6.20). As sperm depletion becomes more and more intense in the subsequent female quintets, the percentage of female progeny decreases linearly with increasing numbers of host exposures.

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d4 The change in behavior of regression lines as above-described also holds true for the distribution of the percentages of parasite progeny in relation to the sequence of matings (sequence of quintets of parasite parent females) . Here the situation concerns the amount of sperm delivered by single males during each subsequent copulation. In the first 2 host exposures where a relatively large amount of sperm was received by parasite parent females, the data (Table 6-3) showed significant fit by the quadratic polynomial model. A straight line relationship becomes apparent when sperm depletion becomes more pronounced, i.e., in subsequent host exposures. The curvilinear relationship represented by equations Eq. 6.17 and Eq. 6.18, and equations Eq. 6.19 and Eq. 6.20, respectively illustrated in Figures 6-16 and 6-17 , appears to represent a mathematical translation of a fundamental biological feature and brings out 3 apparent phases in succession, i.e., (a) the "maturation phase" whereby males perfect their sperm delivery during successive copulations, and females perfect their successive ovipositions , in order to reach their optimal performance (left portion of each curve) ; (b) the "optimum phase" representing the stage where perfection in sperm delivery by males and ovipositions by females are optimal (portion about the maximum of each curve) ; and (c) the "degradation phase" where sperm delivery by males and ovipositions by females gradually decline, most likely due to sperm depletion in males and general fatigue in females (right portion of each curve). The 3 phases disappear or become imperceptible in simple linear regression, most probably beacause of the superseding effect of sperm depletion.

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85 Whatever the regressions may be, sex ratios produced by mated females is under the control of the female sperm regulating mechanism, providing that copulation takes place while females are still young (about 1 to 15 days old) (Chapter 9). Abdelrahman (1974b) reported that sex ratio of A. melinus is influenced by environmental factors such as size and quality of the host, density of the parasite population relative to hosts, and by inherent factors such as the small size of female spermathecal gland. Other included factors are differential mortality, temperature, and age of mothers. He concluded that results of his work conformed with both Flanders' concept of depletion of spermathecal gland fluid and the fatigue theory conceived by Marchal who suggested that an increase in the male progeny of a mated female parasite which oviposited rapidly would indicate fatigue of the spermatheca (Flanders, 1956). With P. foveolatus , age of mothers have been proved to have obvious influence on sex ratio of progeny (Chapter 9) ; older females (20 days or older) , when placed in the presence of males, appeared to lose the willingness to mate or when mated, the ability to regulate sperm received. The loss of willingness to mate is indicated by failure of a majority of the females to produce female progeny from 3 consecutive host exposures (all female-male individual pairs were confined in separate rearing vials 24 hours prior to and throughout the whole period of host exposures) . The loss of ability to regulate sperm during ovipositions was evidenced by the production of female progeny in one host exposure followed by the presence of only male progeny in the next immediate host exposure (Chapter 9, Appendix 9-6 ) . These observations show why it is adaptively advantageous for

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86 mating to occur immediately after emergence of parasite adults from their respective host mummies, and why inbreeding prevails in this gregarious species. The fact that high percentages of female progeny were occasionally produced by some mothers after a series of decline, i.e., after the degradation phase and shortly before the death of the involved mothers (Chapter 7) , suggests that spermathecal fatigue may not be the only factor responsible for such change in sex ratio; general exhaustion must be therein involved. Although egg resorption by females and the extent of viability of male sperm in females are not known, surviving 3-month old females of the parasite stock culture maintained under laboratory conditions were found to still be able to produce female progeny (observation made only on one host exposure) . Oviposition preference vis-a-vis host instars has been demonstrated (Chapter 11); the parasite females prefer late instar host larvae to earlier ones. Laboratory and field observations under Florida conditions appear to agree with Stevens et al. (1975b) who reported that the sex ratio of female to male in laboratory-reared P^. foveolatus is higher than that recorded from the field. In India (Bihar), female to male sex ratio of the parasite recorded on monthly basis from October to April was respectively 2.4, 2.6, 2.3, 2.3, 1.5, 1.8, and 2.4 (Lall, 1961). Stevens et al. (1977) reported that, under Maryland field conditions, sex ratio of V_. foveolatus changes during the season according to the ratio of the parasite to its host; at low ratio of parasite to host, sex ratio favors females; at high ratio of parasite to host, sex ratio favors males. These investigators also found that male to female sex ratio, based on data grouped according to the

PAGE 103

87 "numbers of adults emerging from each parasitized larva, decreased as the nimber of emerging adults per parasitized larva increased. The sex ratio (malerfemale) dropped from 1:3.23 to 1:0.48 for the range category of number of parasites emerging per host mummy of 1-5 and 41-100 respectively. They cited 2 factors that are likely responsible for such correlation. First, the environmental factors within the host larva, obviously the reduced availability of food as the numbers of developing parasites increase, could impose a differential determination and/or survival between sexes as the number of developing parasites increases (case of superparasitization in connection with host scarcity) . Second, ovipositing parasite females are able to control fertilization of eggs as they are laid in response to an external stimulus such as marking pheromone on the host. However, the results of the study on the parasite fecundity (Chapter 7) appears to prove otherwise. Whether one or 2 host larvae (4th-instar) made daily available to each mated female are considered to be abundant or scarce as far as host availability in relation to the parasite is concerned, the ratio of female to male in parasite adults emerging from individual parasitized host larvae increased as the nximbers of parasite adults per host mummy increased (Chapter 7) . A similar result was obtained from a small laboratory test purposedly designed to create single and multiple ovipositions (the test comprised 3 sets of 4th-instar host larvae; only 1 parasite female was allowed to parasitize each host larva of the 1st set; 2 parasite females were allowed to parasitize each host larva of the 2nd set; and 3 to 4 parasite females were allowed to parasitize each host larva of the

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88 3rd set; only one ovlposition per parasite female was permitted). No decrease in ratio of female to male resulting from superparasitization (parasitlzation by 2, and 3 to 4 parasite females) was revealed. In fact, the ratio of female to male appeared to increase as the number of ovipositions increased. The average ratios corresponding to 1, 2, and 3 to 4 ovipositions were respectively 4.68, 5.11, and 5.85 (the numbers of successfully parasitized host larvae over the numbers of dead host larvae corresponding to each oviposition category were 38/1, 30/5, and 22/10; the ranges of numbers of parasite progeny per host mummy for each respective oviposition category were 6-43, 11-53, and 33-94; the average numbers of parasite adults per host mummy were, respectively, 17.50, 28.83, and 56.77). Therefore, superparasitization does not appear to be a factor likely to be responsible for a decreasing female to male sex ratio. Viability of male sperm in mated females in this experiment does not appear to be a factor likely to influence sex ratio of progeny produced by older females. This is indicated by a test mentioned earlier in which an effort was made to establish the effect of age on female ability to oviposit and produce progeny. Since the females were 3 months old and had been isolated from contact with males for at least 2 months (after death of males) , the fact that females were present among their progeny is evidence of sperm viability for a minimum of 2 months. Unfortunately, this test was undertaken out of curiosity and no data on sex ratio was recorded. In a second test also of an incidental nature, 6 4th-instar host larvae were parasitized by 78-day old females that had had no contact with males for at least 48 days.

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89 In this test, the number of female/male progeny per host mimnny was recorded and found to include an unusually high number of males, i.e., 11/2, 4/2, 8/2, 7/2, 4/17, and 2/11 for an average female to male ratio of 1:1. Although the number is too small to establish significance, there is reason to suspect substantial loss of sperm. Alternatively, loss of sperm regulating ability due to female age may be responsible. At first sight, this is more puzzling in view of the high percentage of female progeny produced from the last ovipositions of most females used in the fecundity studies (Chapter 7, Table 7-9). However, in these fecundity studies, there is evidence that longevity is inversely correlated with fecundity and since no females lived longer than 48 days, they may have had no opportunity to exhibit loss of sperm control due to age. Under natural conditions, the change of host population from the state of abundance to scarcity through involvement of parasitism is a slow process and requires extensive period of time. Finding the host under conditions of host scarcity takes time. In other words, the longer time a parasite female needs to find host, the more energy she must spend. As a consequence, she becomes progressively weaker, physically and physiologically, as time passes, while the host may become progressively more scarce. Therefore, it is not to be expected that the reproductive performance of these females would equal that of young and strong females. Some effects of physical and physiological debilitation may take the form of decreased female to male ratio as reported by Stevens et al. (1977) and may be explained as due to substantial death of sperm and/or to the loss of female sperm

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90 regulating ability as earlier discussed. Since the parasite is imable to overwinter, due to prolonged absence of host laravae, the ability to project or predict the effect of sex ratio changes occurring at low host density level remains obscure. However, information obtained through the present study, as well as those of Stevens et al. (1977) and Lall (1961) appear to reflect strategies evolved by P. f oveolatus imder environmental conditions of its indigenous distributional ranges. While insuring survival under conditions of that environment, it is ill adapted to those of the United States as I evidenced by failure to overwinter. At the same time, characteristics favorable to survival in the home environment appear to be responsible for the species remarkable capacity to increase and disperse in the course of a single season. Among these characteristics, one of the most important is the highly skewed sex ratio resulting in a disproportionate production of females.

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CHAPTER 7 FECUNDITY Introduction The extreme range of fecundities characteristic among insect species has long been known to biologists. Price (1975) noted that R.A.F. de Reaumur, early in the century, was the first biologist to express wonder at such great diversity. In his discussion of reproductive strategies of parasitoids. Price wrote: "for parasitoids, knowledge of the selective factors acting on egg production and the results of natural selection are necessary for a full understanding of the coevolution of host and parasitoid, host and parasitoid population dynamics, parasitoid community ecology, and for the development of a predictive science in biological control using introduced parasitoids" (p. 135). In an effort to explain different reproductive strategies among the parasite wasps. Price (1973) related difference in potential fecundity of members of the family Ichneumonidae to the availability of the host and the probability of survival of the developing parasite. He found the ovarioles per ovary to be less numerous in a sequence of species attacking successively less abundant host stages, and that fewer ovarioles were found in parasites attacking well-concealed hosts than those attacking exposed hosts. Similarly, fecundity was found 91

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92 to be closely correlated with ovariole number in Tachinidae. From these observations, he postulated that "the major determinant of fecundity in parasitoids are the probability of finding hosts and the probability of survival once established on or in the host" (p. 100). Sorokina (1973) stated that "in order to investigate the reproductive potential of a parasitic Hymenoptera relative to that of their hosts, it is essential to accumulate as much data as possible on the structure of their reproductive system" (p. 310). Study of the structure and development of the ovaries of 7 species of aphelinid females (parasites of armored scale coccids) led him to conclude that the potential fecundity of aphelinids is as a rule lower than the fecundity of their hosts. Flanders (1956) noted that "in species of Microbracon and Trichogramma , the female may be less fecund after mating, possibly because the female then exercises greater discrimination in hos selection with the consequent greater amount of ovisorption" (p. 331). Fecundity of P^. foveolatus was reported by Lall (1961) from India, the native country of the parasite, to range from 10 to 50; the number of host larvae ( Epilachna spp.) successfully parasitized by a parasite female during her lifetime was 8. In the United States, the average production of live adult progeny was 88 per parasite female, and the average number of Mexican bean beetle ( Epilachna varivestis ) larvae parasitized by a parasite female was 20.3 (Stevens et al., 1975b). The purpose of the present investigation was to determine not only the number of live adult progeny produced by each parasite female and the number of successfully parasitized host larvae, but also to

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93 record the distribution patterns of females and males among the progeny obtained through successive host exposures. Materials and Methods Thirty-three parasite females were picked at random from a colony emerging from 30 4th-instar host mummies. All females had mated within the colony during the preceding 36 hours. The whole colony was kept in a rearing unit (Fig. 2-6) provided with honey and water as described early in Chapter 2. Plastic snap-cap vials (2.6 cm mouth inner diameter, 5.2 cm tall) described in Chapter 5 were used to contain individual parasite females. A number was assigned to each female. Only 4th-instar host larvae were used to produce parasite progeny. The scheme for host exposures, which were started on the second day after emergence of the females, was: 2 exposures daily for the first 2 days; one exposure daily until the death of the parasite females with the exception of the 6th, 10th, 13th, 19th, 23rd, 25th, 27th, 28th, 30th, 32nd, 36th, 37th, 41st, 42nd, 44th, and 47th days when no larvae were exposed. Exposed host larvae were reared in separate plastic petri dishes (8.9 cm diameter) until cessation of feeding. The dishes were labeled according to the numbers given to the parasite females, the dates and sequence of host exposures. The resulting host mummies were then individually confined in separate gelatin capsules (no. 000) with corresponding labels, about 2 days prior to the normal emergence of the parasite adults. The numbers of female and male progeny were recorded for each host exposure and each

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94 female, and the date of death of each parent female noted. Only those adult progeny able to emerge from the host mummies were counted. Results and Discussion The per-mummy number of female and male offspring, dead or pupating exposed host larvae, all recorded under corresponding dates of exposures of host larvae to the parasite parent females, and dates of death of the parasite parent females are given in Appendix 7-4. These data were variously processed and tabulated as needed for convenience of interpretation and statistical analyses of the results (Tables 7-6, 7-7, 7-8, and 7-9). Fecundity, Longevity, and Number of Successfully Parasitized Host Larvae Fecundity, expressed in terms of live adult progeny, i.e., those adult progeny capable of emerging from the host mummies, ranged from 0 to 199 with a mean of 126.30 and a standard deviation of 51.00 (Table 7-6). The results also showed that, out of the 33 parasite parent females studied, 26 produced more than 100 offspring, 7 less than 100, and 22 in the range of 120 to 179 (Fig. 7-18). Longevity, the period extending from the day of emergence of the parasite females from the host mummies to their death, ranged from 9 to 48 days with a mean of 19.42 days and a standard deviation of 12.08 (Table 7-6). The number of host larvae successfully parasitized (those giving rise to adult parasite progeny capable of emerging from the

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95 TABLE 7-6 Fecundity, longevity, and numbers of 4th-lnstar host larvae successfully parasitized by individual female P. foveolatus Parent female nos. Number of , Number of in decreasing progeny per /^f^'^r^ parasitized host order of fecundity parent female *.aays; larvae 1 199 1 s X^ 1 n xu 2 196 1 L lU 3 176 1 xo lU 4 176 1 /i XH o O 5 174 XH 10 6 167 o O 7 1 66 X U LI 9 8 1 69 XD 9 9 Q 7 10 1 / 8 11 J. J J 1j 8 12 X J J 8 13 1 4Q Xt ? 8 14 1 4R X ^ (J XD 9 15 XHX 7 16 141 15 a o 17 138 18 7 18 136 14 8 19 131 12 7 20 129 14 8 21 126 12 7 22 124 11 7 23 123 12 7 24 123 12 7 25 119 15 8 26 113 15 6 27 95 40 6 28 68 38 4 29 49 45 4 30 46 48 3 31 25 46 2 32 11 48 1 33 0 15 0 Mean Standard deviation 126.30 51.00 19.42 12.08 6.94 2.52

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96 10 9 8 CO E _ 0) 6 0 O) a> a> o O) 1 CO 1 m r~ 1 1 CO T" m To o o o o 1 1 1 1 CM u> CO o o o o o o CM CO Number of progeny FIGURE 7-18. Distribution of P. foveolatus parent females with respect to range categories of 20-progeny increment

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97 host mummies) by Individual females ranged from 0 to 10 with a mean of 6.94 and a standard deviation of 2.52 (Table 7-6). When fecundity is compared to longevity, two broad categories of parent females, namely those of high fecundity and those of low fecundity, the former characterized by short and the second by long life expectancy (Table 7-6, Fig. 7-19). The inverse relation of longevity to fecundity is indicated by the abrupt drop in fecundity that coincides with a sharp rise in longevity at about female no. 26 on the abscissa. In other words, there is a clear indication that the fewer the number of progeny produced, the longer the life expectancy. In relation to longevity of the parasite, it is of interest to note that on one occasion, some females that had lived for 93 days in standard culture unit were offered 4th-instar host larvae and subsequently produced progeny of both sexes. These females belonged to a colony comprised of approximately 300 individuals to which only one exposure of 25 4th-instar host larvae was made, a situation implying that few of females had an opportunity to lay an appreciable number of eggs. This suggests that the relatively shorter life expectancy observed in the present study may be attributed to the effect of successive ovipositions which allowed females to rapidly expend their complement of eggs , The results showed further that the last ovipositions of the low fecundity females did not extend beyond the 12th day after the parasite emergence from the host mummies, and only one day beyond the latest ovipositions of the high fecundity females. Ovipositions of

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99 the less fecund females, though noticeably aggregated around the early host exposures, were also observed to be more or less scattered within the oviposition period (Appendix 7-4). Reasons for the variation in fecundity of females and the inverse correlation with longevity are conjectural. The predominance of early ovipositions consistent for all females seems to indicate that all females took full advantage of the host availability at the early stage of their reproductive life, whereas the delayed ovipositions may represent an adaptive response to relative host-parasite numbers that ensures maximum survival of progeny, perhaps through the avoidance of superparasitization that might cause premature death of the host or incomplete development and death of parasite larvae. Under conditions of a high parasite population relative to the numbers of available hosts, the ability to delay oviposition should be selectively advantageous to the individual female. Although the cause of premature death (without producing any parasite progeny) of exposed host larvae was not investigated in the present study, such deaths were frequent among larvae after exposure to parasite females and seldom observed in their absence. Since such premature host death entails death of parasite progeny, selection pressure would favor development of the biological process featuring the so-called "maturation," "optimum," and "degradation" phases described earlier in Chapter 6 (study on sex ratios resulting from sequentially-mated males). Here, attention is called to the variability in successful host parasitization exhibited by the females used in this experiment. It will be noted that parent female no. 33

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100 was almost uniformly unsuccessful, whereas occurrence of dead host larvae scattered in the middle portion of the oviposition period was characteristic of a few other individual parent females such as the cases of female no. 19, 20, 21, 24, and 25 (Appendix 7-4). Complete sterility (total absence of oviposition) was observed in some females in a preliminary test. In that test, 5 out of the 6 total females of "Ocala" laboratory stock failed to produce progeny, with only pupating host larvae (4th instar) recorded after each host . exposure until death of the females. Meanwhile, 3 other laboratory stocks, namely "Augusta," "NF-S-121," and "Perry" showed no progeny production failure ("NF-S-121" was the only stock used in the present study) . This would therefore imply that the parasite fecundity can be as low as complete absence of progeny production and as high as 199 offspring per female. In India, fecundity of the parasite was found to be in the range of 10 to 50 progeny per female (Lall, 1961). In Maryland (USA), the average production of live progeny (adults) per parasite female was 88 (Stevens et al., 1975b). Whereas Lall (1961) gave no details in regard to the parasite fecundity, Stevens et al.(1975b) reported that the average number of host larvae parasitized by a female was 20.3 with a range of 1-81, and noted that approximately 57% of parasitized host larvae failed to produce live adult progeny. They also found that few of the host larvae parasitized by older females produced live adult progeny and attributed the higher mortality to the continued access of females, aged 13-180 days, to host larvae until death of each female ("The higher mortality in this study than

PAGE 117

101 obtained with only 1-12-day-old parasites (Table 1) is because each parasite was allowed to parasitize host larvae until she died" (p. 955)) If correctly interpreted, the longevity of females as reported by Stevens et al. (1975b) departs significantly from results obtained in the present study, in which females were offered 4th-instar host larvae 2 days after their emergence as adults, hosts being exposed at the rate of 2 per day for the first 2 days and thereafter one per day except as noted earlier in the section. Materials and Methods. Under these conditions, no female lived longer than 48 days. Also, the lower average number of adult progeny produced by a female (88/female) and the higher percentage of premature death of parasitized host larvae (57%) reported by Stevens et al. (1975b) than those observed in this study (average number of live adult progeny/parent female =126.30; dead host larvae presumed to be due to the effect of parasitization = 15.80%) seem unlikely to be the result of continuous access to host larvae during the life of each female. Comments relating Lall's findings, particularly in regard to fecundity differences, are hazardous, because the host insect used was not Epilachna varlvestis . These different results must be attributed to a combination of differences in inherent ability of individuals to successfully parasitize the host, as mentioned earlier, and other causes such as age or exhaustion of the females, continuous ovipositions , as well as the handling of the host and the parasite. Price (1975) found a close correlation between the fecundity and the number of ovarioles in Ichneumonidae and Tachinidae. For instance, in the tachinid species Leschenaultia exul Townsend

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102 (parasite of Malacosoma americana Fabrlcius and Malacosoma dlstria (Hubner), the number of ovarioles per ovary of 119, 230, and 252 corresponded to observed fecundities of 2,800, 4,000, and 5,500, respectively. No study of this kind was undertaken on P. foveolatus . However, dissection of a few females revealed only 3 ovarioles per ovary. Further more detailed study in this regard would be useful as a means of checking the reliability of Price's hypothesis. Per-Mummy Number of Progeny vs. Sex Ratios Table 7-7 shows the average sex ratios of the. parasite progeny produced through individual host mummies that are included in range categories of 5 adult-progeny increments. TABLE 7-7 Sex ratios of P. foveolatus progeny computed on the basis of 9 range categories of 5-individual increments Range categories Number of host of progeny emerging mummies per ratios from single host mummies range category (female :male) 1-5 6-10 11-15 16-20 21-25 32 3 5.00 22 7.09 *3 5.37 101 8.60 26-30 31-35 36-40 41-45 6.18 9 7.55 13 10.08 5 7.71 1 10.00

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103 Statistical analysis based on these data revealed a simple linear relationship between the parasite progeny sex ratios and the numbers of parasite progeny per host mummy. The model, represented by equation Eq. 7.21 and significant at 0.05 level (t = 2,990 > calc. *^0.05, 7 d.f. ^ 1-895), is illustrated in Figure 7-20. ^(sr) = ^-0^ + 0-50 Rep (Eq. 7.21) (coefficient of correlation r = 0.75) where: Rep stands for range category of parasite progeny Average N umbers of Progeny per Host Exposure vs. Sex Ratios Table 7-8 shows the per-mummy average numbers of the parasite progeny and the corresponding sex ratios (f emalermale) , computed on the basis of individual host exposures. When all sex ratio values were tested against their corresponding per-mummy average numbers of progeny, no simple linear relationship was revealed. However, when the value of the last host exposure (11th host exposure) was ignored, a highly significant (0.005 level) linear relationship was obtained ^'^calc. 6.615 > ^Q QQ^^ g = 3.355). The model is represented by equation Eq. 7.22 and illustrated in Figure 7-21. A Y. (sr) " ~ + 0.43 Prog (Eq. 7.22) (coefficient of correlation r = 0.90) where: Prog stands for per-mummy average number of parasite progeny of each host exposure.

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104 10 9 8 7 0) TO E 6 0) ma 5 0) u— « 4 g i_ 3 X 0) CO 2 1 (SR) 5.01 + 0.50Rcp 5 6 7 8 9 (1-5) (11-15) (21-25) (31-35) (41-45) (6-10) (16-20) (26-30) (36-40) Range categories of progeny FIGURE 7-20. Simple linear relationship between sex ratios and numbers of P. foveolatus adults produced through host mummies corresponding to range categories of 5-progenv increments t& ^

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105 00 I w J + ny to ^ to 3 CO u ^ cu CO X5 CU 3 CO IS 6 (U O r-t •H CO 4-) e CO u , CO c e cu \ 60 CO o CU CO P. to w B o cu O W CD • O o x; I OJ C 3 60 (U g CO 60 u o (U u > (X . ^1 o cu CO C 60 cu O rH H CO CO & g u CU CD o X HA o o
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106 ^(SR) = 0-28 + 0.43 Prog 10 12 14 16 18 20 22 24 Per-munmy average numbers of parasite progeny per host exposure FIGURE 7-21. Linear relationship between sex ratios and per-mummy average numbers of progeny produced through individual host exposures by 32 P. foveolatus parent females '

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107 The reasons for the , deletion of the value of the 11th host exposure (last host exposure) are the same as those given in Chapter 6 (p. 83) except that the present case deals with simple linear regression. Whatever effects the value of the last host exposure (which accounts only 1.31% of the total number of 229 host mummies, or 0.84Z of the total number of progeny) might later show for the host-parasite biological system under natural conditions, this linear relationship is nevertheless considered as evidence lending extra strength to the similar relationship represented by equation Eq. 7.21. This shows again that the female to male ratio of the parasite increased witH the increasing number of progeny per host mummy. Percentages of Female Progeny vs. Host Exposures Processed data presented in Table 7-9 allow an analysis on how male sperm was used by parasite females in the subsequent host exposures or ovipositions (based on the data given in Appendix 7-5 obtained through a closely observed single ovipositions in 4th-instar host larvae by single parasite females, the term "host exposure" is here equivalent to "oviposition") . Statistical analysis based on the average percentages of female progeny produced by the 32 parasite parent females (the 33th female produced no progeny) through 10 successive host exposures showed that the parasite females used sperm they received from the males to fertilize their eggs in accordance with the quadratic polynomial regression model represented by equation Eq. 7.23 illustrated in Figure 7-22. The level of significance for the test on the magnitude of the quadratic coefficient estimate was 0.001.

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108 I 1^ U to CO 3 4J 3 tC CO iH o O (U o •u to o P.* x; o 4-1 -a U QJ U a (U 3 a •T3 to O (L) ^1 &. c •H QJ 60 O to U ^1 H 0) T3 C H ^^^^^"^ ^ ^ ^ ^ V CM ^ t\j ^ r\jr\j^n^ C3 CO >n a to o •0-0-0-0-0-0-OT5-0-OT3-0-DX>-0-OTD-aT3-DT3-0-D-S-0-S-0-0-S ccccccccccccccccccccccccccccc ooooooooooooooooooooooooooooo X)-O-O-O-D-O-OX>-O-OT3T0-O-OT3T3T3-D-O-ClT3-O-D-OT3T3TJ-OTJ •DT3T5T3-0-aX)-0-D-O-O-0-0T3-DT3-CI-a-O-OXl-013-O-DT3-O-5-D o «c «o «3 *o to e c n E ECEEEEECEEEEEECEEEEEE6 CI Cf o O CI E 6 CI CI c c c c c ccccccccccccc ciaiajojojaiaiQiQiaiajc/c^QjcuoaiQioajci '-'-'-'-'-^'->-i->-i-i->-i-i-i-i-i-!-i-i-i-i.i-c~crccr'r' o O o o vo' ' o CO CO o m in ro o n cc CO CO o o O m o ro O O O r— CD oo oo o CM o (X3 O ^ O UD CO O UD VO CO o o Or— — OOCCOVOVOOOOOV ^Of^r^cT»oo^ococovr>u->oco oin^ooinootM«>ji-.'r~,'oco «7^COCT»crvOr^OO^Cr»COCOOCO rnvooo^vD^r^OCOr** rO'^ococoro'cTOr^^ *^voocorsJcoo>OF-^c7v COCOOCOCT%COCCr^r^CO «.r2~r?^°^o^«n.— COCO coro^' cnvocncncccocnoicncococncococD coiS '"^cooovoCTvi — rovnir»cT»^o\t >e\j^r-ir)vncr»co.— vor^r^co^cot JP^S; — '~-vDcovo«Tmroco«'cdi '^rsj'^ccccrofn.-r^OLnKiiominr^ cncoo%coo^cooocooocncocooorocoa;«CTi vo •— o o — CO o% o o o o o vr» 0% Ov CO Ov CO in f\j .— <7\ CO CNJ 1 • 1 CO CO VD • • OO CO CO CO CO CO o o^o^c^l0^coc^^cor*.oo o o o *— tn CM KO o r~ o tn o% o% CO c\JOo oi^.— ^CDcccOf^ r— , — co^roo o^ocooooco^c^»coo^a^o en O CO o CO CO cn cc O ^ "<5CO »— CO ^ m Ln ^ O CT> 0^ OvoO« — r-*00vD u^^incocNjr^ror^ CTicncnco^o cocnco 'f^'^voovo^vocNJcocD o o vn o n o f\j vr> CM r— CNjn^ir» vor^GOCT»Of— ^i^coo%o^rof0^i/m5r^coo%o^fNj g CM V CJ* *^ *^ .* 2f O * >» c CI CO C7» O ^ OC Oi JT ro O c \£> o CO e • C7I m O Oj c L. * — CO o «o u 00 i E 3 "O o a; o >— *j a. CO « c E a> Cf 1. GO a s. 4-* o *-» u-> u CO cx «a CO «a a. a. 3 CM CX in u *-» i. cr% *o o CO O Ct a JZ o *-» o •o c: o cu u 4-> cn c# o^ 1jC oj a; « E > OJ 1. rs. OJ CO > CO «-*«^ t. o J= n o o o o CO v> C u o«-* cx K C UJ o >1

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109 ^(% female) = 8^.03 + 2.57 E 0.31 (Eq. 7.23) (coefficient of determination = 0.93) where: E stands for host exposure. The reasons for deletion of the value of the 11th host exposure are the same as those given in Chapter 6 (p. 83). Biological explanations relative to the curve behavior of this regression are the same as those defined in Chapter 6 (p. 84), i.e., the "maturation," "optimum," and "degradation" phases that occur in succession within the period of parasite oviposition.

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110 100 90 80 7 10 ^(%feinale) = 85.03 + 2.57 E 0.31 _L _L 4 5 6 7 Host exposures 10 li^^ Quadratic polynomial regression curve representfei^ ^^l^t-°^«hip between the percentages of P. foveolatus female progeny and the host exposures. ~

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CHAPTER 8 INFLUENCE OF AGE ON MATING CAPABILITY OF FEMALES AND MALES Introduction The influence of age of parents on mating and the sex ratio of their progeny has been reported for some insect parasites. Flanders (1956) noted that aging either weakens the mating instinct of female of hymenopterous insects or results in the loss of their attractiveness to the males. Abdelrahman (1974a) reported differential mortality, temperature, and age of mothers as 3 factors influencing final sex ratio of Aphytis melinus DeBach (parasite of California red scale, Aonidiella auran^ (Mask.)), in addition to environmental factors such as size and quality of the host and density of the parasite population relative to hosts, as well as inherent factors such as the small size of the spermathecal gland. Gordh and DeBach (1976) studying the inseminative potential of the aphelinid AEhZtis lingnanensis Campere (parasite of California red scale) found that most females were inseminated by males on the first day of adult life and the number of copulations and inseminative ability decreased rapidly through day 4, with only a few matings occurring during days 5 to 9. These investigators also pointed out that production of female progeny appeared to decline with male aging, and that the number of 111

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112 females producing only males, presumably uninseminated, appeared to increase with successive days of male age. Barbosa and Frongillo (1979) reported that maternal aging appeared to increase the proportion of male progeny of Brachymeria intermedia (Nees) (pupal parasite of the gypsy moth, Lymantria dispar (Linneus)). Results of the study on sexual behavior of P. foveolatus (Chapter 4) provide evidence that this parasite is predominently of the inbreeding type, for mating took place actively in close proximity of the host mummies during a period of 30 minutes to about one hour immediately after emergence of the adults. In regard to this finding, one question immediately came to mind: what would happen if mating were delayed for some period of time? The present investigation was focused on the effect of age on mating capability of this parasite adult of both sexes. Materials and Methods Three female-male pairing combinations, young females x old males, old females x old males, and old females x young males, all virgin, were tested. Young females and males were those emerged from host mummies no longer than 48 hours, whereas old females and old males were 30 and 15 days old, respectively. All parasite adults were obtained through isolation of pupae in small glass tubes (about 0.5 cm diameter, 3 cm tall) in the same manner as described for study of mode of reproduction (Chapter 3). The work plan was managed in such a way that all the female-male 3 pairing combinations fell on the same date.

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113 Each female-male pair was confined in a clear snap-cap vial (2.6 cm diameter, 5.2 cm tall), and allowed to stay together for 24 hours. One 4th-instar host larva was then exposed to each parasite female on each of 3 consecutive days, beginning on the 3rd day after removal of the males. The exposed host larvae were reared in separate plastic petri dishes (8.9 cm diameter) labeled according to pairing combination, parent female number, and order of host exposures. Mummies were confined in separate gelatin capsules (no. 000). Sexing and counting was done only after death of all adult parasite progeny. Presence of female progeny was evidence of successful mating. Results and Discussion Detailed data regarding the number of P. foveolatus progeny (females/males) produced by each parent female, each parent pairing combination (young females x old males; old females x old males; and old females x young males) , and each host exposure are given in Appendix 8-5. The number of successfully mated parent females and number of parent females failing to produce progeny throughout 3 separate host exposures in each of the 3 pairing combinations are presented in Tables 8-10 and 8-12. Table 8-10 shows that the pairing of young females and old males gives the highest number of successfully mated parent females, followed by old females with old males. Poorest performance was that of old females x young males. Statistical analysis (Z-test) resulted in a

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114 highly significant difference (0.01 level, two-tail test) between the pairing young female x old male and the other 2 pairings, namely old female x old male, and old female x young male (Table 8-11). TABLE 8-10 Numbers of mated parasite females resulting from 3 pairing combinations of young and old adults and 3 separate host exposures Adult pairings Numbers of successfully mated females a/ Total numbers of females tested Young ^ $ X Old 25 32 Old ? $ X Old cycf 2 32 Old ? ? X Young o* 0* 0 32 a/ Based on the presence of female progeny in any or host exposures all 3 separate TABLE 8-11 Results of Z-tests between pairing combinations of young and old P. foveolatus adults of both rpxps^ hj,«*.H .,r, T,..n,|,^r5 of successfully mated parent females of Table 8-10 Parent pairings Z-values (calculated vs. critical) Significant difference vs . Old $ ? x Old d* Old ? $ X Old ^ vs. Old + ? X Young d* 'calc. = ^•82>Zo.01 = 2.58 ^calc. = l-^^<^0.05 = 1-96 highly significant not significant The inclusion of dead parent females in the category of parent females failing to produce progeny (Table 8-12) is based on the assumption that death was primarily caused by physical exhaustion resulting from their

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115 continuous disturbance by courting males; in fact, dead parent females were only found in old female x young male pairings, one at the 2nd host exposure and 3 others at the 3rd host exposure (Appendix 8-5). The results of statistical analysis (two-tail Z-test) are presented in Table 8-13. TABLE 8-12 Number of females failing to produce progeny throughout 3 separate host exposures Parent pairings Number of females failing Total number of to produce progeny a/ females tested Young $ ? X Old cf 1 Old $ ? X Old cT . 7 Old ^ $ X Young cT d" 14 32 32 32 a/ Based on absence of parasite progeny production, due either to death and/or pupation of exposed host larvae, or death of the parasite females This shows that mating capability of 30-day-old females was practically lost whereas that of 15-day-old males appeared to be less affected. The decreasing likelihood that females will mate as their age increases implies that the production of male progeny would also be increased (arrhenotokous mode of reproduction) , and the sex ratio of progeny would therefore become increasingly male-biased. The fact that successful mating depends primarily on the female response to male mating attempts (Chapter 4) and data from this study showing that the highest number of successfully mated females was obtained in the young female X old male pairings suggests that female mating instinct diminishes with age. Direct observation of behavior exhibited by old females in

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116 response to male's mating attempts also revealed the same; females often moved away from courting males (precopulatory behavior) and showed no positive response to male presence (positive response is characteristically manifested by the sudden shift from low to raised body position of female to allow copulation by male) . The loss of female mating instinct is not the only effect of aging, Oviposition failure and death of parent females were also observed (Fig. 8-23) . The number of parent females that failed to produce progeny (including dead parent females) throughout the 3 consecutive host exposures are shown in Table 8-12. TABLE 8-13 Results of Z-tests between pairing combinations of young and old P^. f oveolatus adults, based on data in Table 8-12 involving number of parent females that failed to produce progeny Parent pairings Z-values (calculated vs. critical) Significant difference Young ? $ X Old cT vs. Old ? ^ X Old (/' Old ? ^ X Old d* vs. Old ? ? X Young d* Old ^ $ X Young d* cf vs. Young ? ^ X Old cf I , = 2.28>Z. = 1.96 calc. 0.05 I , = 1.86 < Z= 1.96 calc. 0.05 ^ T = 3.26 > Z= 2.58 calc. 0.01 significant not significant highly significant The ability of 2 old females to mate with old males (Table 8-10) appeared to show that old females are more compatible with old than with young males. The complete absence of successful mating in old female X young male pairing (although statistically not significant when

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117 FIGURE 8-23. Number of successfully mated females and number of females failing to produce progeny with respect to 3 female x male pairing combinations of young and old parasite parents (based on 3 separate host exposures).

PAGE 134

118 tested against old female x old male pairing that showed only 2 successfully mated females) (Table 8-10) and the significant increase in numbers of females that failed completely to produce progeny, i.e., 7 in the old female x old male pairing, compared to 14 in old female x young male pairing (Table 8-12) , appear to be primarily attributed to the aggressiveness of the young males which, through their continuous mating attempts, had further reduced the energy reserves of the already aged and weak females. The present study did not, however, reveal any age limit at or about which mating remains totally effective for either sex of the parasite. Nevertheless, as will be seen in the following chapter, which covers the study of influence of age on mating capability of females, the problem is partly solved by the fact that the number of successfully mated females dropped sharply beyond the age of 15 days. Males , on the other hand, have a shorter life expectancy than females. This difference in longevity of the sexes appears to be related to the smaller size of the males and the energy costs of courtship and mating activity. In this respect and in the absence of better supporting data, it is of interest to note that in a laboratory-maintained colony of approximately 300 parasite individuals of both sexes, all males but only a few females died within a period of 25 days after emergence of parasite adults from the host mummies. The progressive loss of female mating instinct and male mating capability with increased age clearly points to the advantages afforded by the inbreeding mating system exhibited by P. foveolatus.

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CHAPTER 9 INFLUENCE OF AGE ON MATING CAPABILITY OF FEMALES Introduction Literature related to the influence of age of some insect parasites on their mating behavior and sex ratio of their progeny has already been given in the preceding chapter. Studies on the influence of age on mating capability of P^. foveolatus (Chapter 8) revealed that (a) after 30 days, virgin females had virtually lost their instinct to mate while 15-day old males retained a high degree of mating capability, (b) old females appeared to be more compatible with old males than old females with young males, and (c) the pairing of old females with young males resulted in the highest proportion of females that failed to produce progeny and highest female death rate. The objective of the present investigation was focused on the mating capability of P. foveolatus females when virgin females of different age were paired with young virgin males. Materials and Methods Six batches of virgin parasite females of 1, 5, 10, 15, 20, and 25 days old were used to test influence of age on mating capability. 119

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120 Virgin adults (females and males) were obtained through the same procedure used for the study of mode of reproduction of the parasite (Chapter 3). Each of the 32 virgin females comprising each batch was paired with one 1-day old male and allowed to remain together in a snap-cap plastic vial (2.6 cm diameter, 5.2 cm tall) during the period of study. A small disc of towelling paper on which a small drop of honey and 2 drops of water were placed was kept in each vial. One 4th-instar host larva was exposed to each of the parasite females daily for 3 consecutive days, 24, 48, and 72 hours, respectively, after female-male pairing. Exposed host larvae were reared in separate small plastic petri dishes (8.9 cm diameter) labeled according to the age of the females, their respective numbers, and order of exposure. Host mummies were confined in separate gelatin capsules (no. 000), with labels as mentioned above. Sexing and counting of parasite progeny was done only after all individuals died. Mated or nonmated status of females was established by the presence or absence of females among progeny produced through the 3 consecutive host exposures by the parasite parent females; emergence of one or more female progeny from a host mummy was accepted as evidence of successful mating; conversely, no mating was recorded if only male progeny emerged. Results and Discussion The numbers of P. foveolatus progeny of both sexes produced by parent females of different age through 3 separate host exposures are

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121 presented in Appendix 9-7. The numbers of mated, nonmated parent females as well as the percentage of mated parent females are given in Table 9-14. The results show that aging exerted influence on mating capability of the parasite females (Table 9-14, Fig. 9-24); younger females produced higher mating successes than the older ones. Based on 2 data in Table 9-14, statistical analysis using X test showed (a) no significant difference between females of 1, 5, 10, and 15 days old, (b) no significant difference between those of 20 and 25 days old, and (c) highly significant difference between those of 1, 5, 10, and 15 days old and those of 20 days old, and, by deduction, between those of 1, 5, 10, and 15 days old and those of 25 days old (Table 9-15). TABLE 9-14 Effects of age on mating capability of P. f oveolatus females Age of parasite females (days) No. of mated females a/ No. of non-mated J^otal females b/ females — tested % mated females 1 32 0 32 100 5 32 0 32 100 10 29 1 30 96.67 15 30 2 32 93.75 20 10 22 32 31.25 25 13 18 31 41.93 aj Based on presence of female progeny W Based on absence of female progeny

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122 100 90 80 70 $ 60 CO E a 50 TO 0) ^ 40 E 5^ 30 20 10 0 5 10 15 Age of females 20 25 days FIGURE 9-24. Percentage of mated females with respect to age.

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I w J 0) •H i-l •H (U rt 60 CJ M u C c •H (U 4J ma fe c if •H 4-l O o c 0) CO (U 05 o U 4-1 CO 4J XI tM O n u iH 9 a 0) CO o o c •H (U 4-1 H •H 0) d 4-1 60 4-1 CO 13 CJ X CJ CO > (U M CO r-t 3 C rH to CJ 4J C (U u o ff LO •H -a T3 te 4-1 u o O cu •r-i CN CO CO 0) -§ U lO rH CO CO rH cfl (U B •u fe ays o > 4-1 4-1 4-1 4H iH -rl •H rH •H J= C J= C C 60 60 4J 60 60 60 U 60 •H -H O •H •H -H O •H X: CO c CO J3 CO c CO in 00 ^ to o 13 H in CN m rH o rH o CM CM

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124 The sharp drop of the number of mated females beyond the age of 15 days implies that mating capability of the parasite females remained effective within a period of about 15 days after their emergence from the host mummies. Cases of unusual presence of higher numbers of males than normal that were observed among mated older females are extracted from Appendix 9-6 and presented below according to age of parent females, parent female number, sequence of host exposures, and female/male numbers . Age of parent females 15 days 20 days Parent female Sequence of Number of progeny 25 days no. host exposure (females /males) 5 2nd 8/6 9 3rd 21/15 22 2nd 11/19 27 2nd 21/18 1 3rd 7/18 13 1st 20/11 18 2nd 4/37 22 1st 29/16 23 2nd 13/11 29 2nd, 3rd 25/34, 26/36 1 1st 1/32 9 1st 14/10 16 2nd, 3rd 13/22, 7/24 17 2nd 14/22 28 2nd 10/17 31 3rd 1/11 Furthermore, some of the mated older females were irregular in the production of female progeny, particularly in regard to ovipositions (host exposures). These females, instead of producing progeny of both sexes in successive ovipositions as young mated females normally do, presented a broken oviposition and sex ratio sequence. The cases, extracted from Appendix 9-7 are presented below.

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125 Parent female Progeny (females/males) /host exposure age and no. 1st exposure 2nd exposure 3rd exposure 15 days, no. 5 0/12 8/6 0/31 15 days, no. 27 21/18 0/23 20 days, no. 18 0/20 4/37 0/22 20 days, no. 22 29/16 0/25 29/1 20 days, no. 23 0/39 13/11 0/25 25 days, no. 2 15/1 0/20 25 days, no. 9 14/10 0/28 0/30 25 days, no. 18 0/27 11/3 0/23 25 days, no. 27 0/31 16/0 0/18 25 days, no. 28 0/20 10/17 0/16 This shows that sperm regulating ability in females is also affected by aging. However, it is not known whether the effect of old age is expressed differently when females mated when young as opposed to females not mated until they are old. Death of sperm inside the mated females (case of early matings when males and females are young) is not considered as a significant factor since some females that survived a 78-day period in culture maintenance were found to still be able to produce female progeny, although the sex ratio of the progeny (through one host exposure) appeared to favor males (Chapter 6). The presence of female progeny in some ovipositions preceded by the presence of only male progeny in other ovipositions may be the indication that aged parent females did not mate immediately after being placed with males while consistent presence of female progeny of younger parent females (Appendix 9-6) is indicative of immediate mating. Females exhibiting delay in production of female progeny are presented below. The list also includes some parent females producing female progeny in some ovipositions which are immediately followed by ovipositions containing only male progeny (cases of females that appeared to show irregularity in sperm regulation as previously noted) .

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126 Parent female Progeny (females /males) /host exposure age and no. 1st exposure 2nd exposure 3rd exposure 15 days , no . 5 0/12 8/6 0/31 20 days , no . 1 0/20 0/19 7/18 20 days, no. 14 0/31 15/2 12/6 20 days, no. 18 0/20 4/37 0/22 20 days, no. 23 0/39 13/11 0/25 20 days , no . 30 0/24 23/3 20/2 25 days, no. 5 0/32 14/3 0/21 25 days, no. 7 0/33 16/3 25 days, no. 15 0/20 0/17 9/21 25 days, no. 16 0/20 13/22 lllk 25 days , no . 17 0/24 14/22 25 days, no. 18 0/27 11/3 0/23 25 days, no. 27 0/31 16/0 0/18 25 days, no. 28 0/20 10/17 0/16 25 days, no. 31 0/4 0/1 1/11 Also worthy of mention is the ntmiber of ovipositing females of all age groups tested in the present investigation that performed uniformly despite the effect of progressing age. All females were able to oviposit. This situation is notably different from that involving the studies of mode of reproduction (Chapter 3) , and preemergence mating (Chapter 5) already discussed in Chapter 5. The loss of mating instinct and sperm regulating ability of females through aging is evident. However, considering the effect of age on sex ratio when old females are mated, the short longevity, and the postemergence sexual behavior around the host mummies (Chapter 4) , late matings of old virgin females of this parasite species would not appear likely to be a common phenomenon in nature. This further emphasizes the advantageous nature of the inbreeding system that ensures fertilization of females almost immediately after they emerge as adults from their host mummy.

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127 The effect of parental aging on progeny sex ratio obtained through the present investigation and the study in Chapter 8 as well indicate that it would be unwise to use old adults for the parasite production.

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CHAPTER 10 MULTIPLE MATINGS OF FEMALES Introduction Matings, whether single, multiple, or mixed are characteristic of each species of parasitic insects. Females of Trioxys utilis Muesebeck (internal parasite of the spotted alfalfa aphid, Therioaphis maculata (Buckton) were found by Schlinger and Hall (1961) to mate only once during their lifetime; once mated, the females either ran away or curled their abdomen downward upon attempts by the males to copulate. Wilkes (1965) reported that females of the eulophid Dahlbominus fuscipennis (Zett.) (parasite of sawflies) rarely mated more than once. He added that, once mated, females usually resisted male attempts to copulate, but occasionally after an interval of egg laying, some females would remate. Females of Aphidius testaceipes (Cresson) and Praon aguti Smith (endoparasitfes of various species of aphids) were reported by Sekhar (1957) to mate only once in their life. Females of Aphytis melinus DeBach (parasite of California red scale) are also known to mate only once in their lifetime (Abdelrahman, 1974b). Flanders (1956) pointed out that the progeny of multimated ichneumonid females represented by Macrocentrus ancylivorus Roh., are usually entirely males, and the highest proportion of female progeny are obtained from the once-mated females. 128

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129 For P^. foveolatus , only in rare occasions in the course of the study on sexual behavior (Chapter 4) , were multiple matings of females observed. Otherwise no previous studies have been reported. The present investigation was aimed at determining the relative extent of occurrence of multiple matings of females and its effects on the parasite progeny sex ratio. Materials and Methods The design of this experiment was fundamentally the same as that described in the study on sex ratios.rresulting "from sequentially mated males (Chapter 6) . It was essentially based on evidence of sperm depletion when virgin males were allowed to mate in rapid succession. The difference was that 7 host exposures were carried out in this study instead of 6 in the study in Chapter 6, and that, before the 4th host exposure was made, a young virgin male was made available to each of the once-mated 8-day-old females and allowed to remain with her for 24 hours. Because of the number of females involved and limitations of time, it was not possible through direct observation to determine whether matings occurred following the introduction of the second males (new virgin males). Thus, it was necessary to seek evidence of a second mating through change in sex ratio among progeny of each female as they emerged from the seven successfully exposed hosts. These data were then categorized as follows: 1. Positive occurrence: (a) those females that produced no female progeny in the first 3 hosts but produced females in the last 4; (b) those females that showed a significantly higher female to

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130 male ratios in the last 4 hosts than in the first 4; 2. Negative occurrence: (a) those females that produced no female progeny in all 7 hosts; (b) those females that produced no progeny in the first 3 hosts and only males in the last 4; (c) those females that produced female progeny in the first 3 hosts but only males in the last 4; (d) those females that showed a convincing trend toward an increasing proportion of males in the 7-host series; 3. Dubious occurrence: (a) those females that produced both female and male progeny with no clear change in sex ratios throughout the 7-host series; (b) those females that produced no progeny throughout the 7-host series as due to premature death or pupation of the exposed hosts ; (c) those females that produced female and male progeny in the first 3 hosts and failed to produce in the last 4; (d) those females that produced no progeny in the first 3 hosts and showed female and/or male progeny in the last 4; and (e) those females that produced no female progeny in the first 3 hosts and failed to produce progeny in the last 4. Whereas females belonging to the first two major categories (positive and negative occurrence) recognized with some confidence, it is not possible to determine whether females in the dubious category did or did not mate a second time. This is especially true of categories 3(b), 3(c). 3(d), and 3(e) where no data was available.

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131 In the case of females in category 3(a), where sex ratios showed no clear change, a statistical analysis of data for the first 10 sequentially mated females (A replications) and data from the first 10 females of the sperm depletion study (Chapter 6) (also replicated 4 times) was undertaken to reveal any differences that might have resulted from multiple matings. The results of this analysis were assessed on the basis of the behavior of the regression curves representing the relationship between sex ratios (female :male) and host exposures of the two corresponding studies. Results and Discussion The per-host-exposre numbers of parasite progeny produced by each parent female are given in Table 10-16. Table 10-17 shows individual parent females falling in each of the 11 categories described in materials and methods in correspondence to their individual sequential mating numbers and their initial males. Of the 11 categories characterized in material and methods, only 6 parent females, all belonging to categories 1(a) and 1(b) , clearly showed an effect of subsequent mating. These represent 3.00% of the total 200 parent females included in this study, or 5.94% of the total 101 parent females of the combined positive-negative categories, namely categories 1(a), 1(b), 2(a), 2(b). 2(c), and 2(d). The remaining 99 parent females belong to categories of dubious multiple mating occurrence, due either to missing data (cases of categories 3(b), 3(c), 3(d), and 3(e)), or obscurity vis-a-vis the parasite progeny sex

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132 >> to c 4 o -a to ,o C Q) O u o O (U C CO u 0) CO •T3 0) O 0) c X 'H C O r-\ O ,Q U T\ 'O o 0) C 3 Po W Q) JJ E -H C ^ 4-1 CO ct) ^ M C CO C -H H CO o "O (U u 3 tH O 60 vH C ^ •H 3 •> CO T3 >^ fi .
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133 ratios (cases of category 3(a)), which barred them from being placed in either broad category of positive or negative occurrence of multiple matings. Whereas assessment of multiple matings for females belonging to categories 3(b), 3(c), 3(d), and 3(e) was impossible because of missing data, that of females of category 3(a) was in part obtained through statistical analysis of data given in Table 10-18. This analysis involved the test of the per-host-exposure sex ratios of progeny produced by the first 10 sequentially mated parent females (replicated 4 times) of the present study and those of the study on sperm depletion (Chapter 6) against their corresponding host exposures. The results showed that female to male ratios of progeny produced by the females of the present study increased linearly with increasing sequence of host exposures (equation Eq. 10.24), whereas the sex ratios produced by the females of the study on sperm depletion showed a curvilinear relationship with increasing sequence of host exposures (equation Eq. 10.25); the level of significance for the test on the magnitude of the respective linear and quadratic coefficient estimates (linear for equation Eq. 10.24, quadratic for equation Eq. 10.25) was 0.01. ^(SRmm)l = ^'860 + 0.581 E (Eq. 10.24) (coefficient of determination = 0.72) ^(SRsd)l = 2.360 + 2.089 E 0.320 E^ (Eq. 10.25) (coefficient of determination R = 0.87) where: ^^^^^^^ represents sex ratio of progeny produced by the

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134 TABLE 10-17 Numbers of foveolatus parent females falling in each of the 11 categories described in materials and methods Category Initial male Parent female Total females no, a_/ no. h_l no. cj per category d^/ 1(a) 1 35 3 31 4 18, 46, 48 5 Kb) 4 23 1 2(a) 1 45, 50 2 30, 48, 49 3 36, 46, 49 4 37, 38, 40, 41, 42, 43, 44, 45, 47, 49, 50 19 2(b) 2 20 3 18 4 33 3 2(c) 1 14, 17, 18, 22, 23, 25, 26, 27, 30, 32, 38, 39, 40, 42, 46, 49 2 19, 31, 33, 35, 36, 40, 41, 43, 44, 45, 47, 50 3 11, 37, 38, 39, 40, 41, 42, 43, 45, 47, 48 4 25, 28, 30, 32, 34, 35 45 2(d) 1 19, 20, 28, 29, 31, 33, 37, 41, 43, 44 2 16, 18, 22, 32, 37, 38, 39, 42 3 12, 13, 14, 15, 16, 19, 27, 33 4 22, 36 28 3(a) 1 1, 4, 5, 6, 7, 8, 10, 11, 12 13, 15, 21, 24, 36 2 1, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 21, 24, 25, 26, 27, 29 3 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 17, 21, 23, 24, 28, 32 4 3, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 24 66 3(b) 1 9 2 2, 17 3 22, 35, 50 4 7 7

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135 TABLE 10-17 continued Category Initial male Parent female Total females no. a./ no. W no. c/ per category d/ 3(c) 1 2, 3, 34, 48 2 5, 12, 28, 34 3 25, 26, 29, 30, 34 4 4, 26, 29 16 3(d) 1 47 2 23 3 20 4 1, 2, 27, 33 3(e) 2 46 3 44 4 39 Total 200 a/ Description of each category is given in materials and methods W 1, 2, 3, and 4 are conventional numbers given respectively to each of the 4 single initial males (first males) , each male was allowed to mate sequentially with 50 virgin females £/ 1> 2, 3,..., and 50 are conventional numbers given to each of the 50 parent females in accordance to their sequence of mating with the involved single male d/ These are numbers of parent females that belong to each category of females described in materials and methods first 10 sequentially mated females of the present study; ^(SRsd)l ^spresents sex ratio of progeny produced by the first 10 sequentially mated females of the study on sperm depletion (Chapter 6) ; and E stands for host exposure. The obvious difference between the two relationships represented above by equations Eq. 10.24 and Eq. 10.25 and illustrated in Figure 10-25 clearly reflects the effect of multiple matings and is here considered as positive evidence of subsequent mating by females that had already received an ample amount of sperm through their first copulation. This

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136 0) <-s ^ VO O tfl u B u OJ 0) 4-> iH iH a, n) 60 CO B C J= 14-1 to w MX C C -u o •H -tH -H T) & -U C 0) O TJ rH O. 01 P. CO -u > U a 0) M c CO CO (U CO (0 c o 0) cr -a 3 4J CO CO CO (U iH to . CO e e Qj T3 CO C CO >^ 01 bO a u c (U CO -rl M D, -U O CO u o e OJ 4J CO O. l-i -H •rl 4-1 3 0) 6 O cu > o c o -a 3 IM 0) 4J 0 o CO 3 to tu ^1 O XI vo o 00 rH m CM ro Cvl 4J -a 13 J3 CO c U u rH CM CO m x: 4-1 VO I o csl I VO c Po CO (U CO CO tu o cj cj •H -H H g rH c & e a 6 tu 3 tu 3 ^1 rH ^1 rH o o -vlo o >4H C ItH C O -H O -H to to CO CO tu (U tu (U r-l r-i ^ CO to tO to e 6 e B to to CO CO tu tu (U 0) H rH rH rH CO to to to B B B B (U (U tu CU 14H MH 14H iH 14H m MH 14H o o o o )H M >J tu tu 0) 0) -S -2 -2 6 B B B 3 3 3 3 3 C C C to CO CO CO 4-1 4-1 4J 4.1 o o o o 4J 4J ^ 4J c c c c o o o o 0) Id 13 TJ cu
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137 E _0 E 0) g 2 Q) CO 10 9 8 7 6 5 4 3 2 \sRBm,)f ^.860 + 0.581 E ^(SRsd)i" ^*^^° ^-^^^E 0.320 1st 2nd 3rd 4th 5th 6th 7th Host exposures FIGURE 10-25. Relationships between female to male ratios of P. foveolatus progeny produced by the first 10 parent females mated sequentially with initial single males (replicated 4 times), and host exposures: Y. for study on multiple matmgs (present studv> : Y. ^^^Ai o<-„^,, „ matings (present study); "^.'^'^6^ study on sex ratios Aated males (Chapter 6) where multiple suiting from sequentially matings were not allowed. re-

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138 conclusion is reenforced by the results of the test for equality between the slopes (rate of change in female to male ratio for one unit change in host exposure) of equations Eq. 10.24 and those of equation Eq. 10.25. Table 10-19 shows that significant differences were found between the slope of equation Eq. 10.24 and the slopes of equation Eq. 10.25 at the 4th, 5th, and 6th host exposures, but not at the 1st, 2nd, and 3rd host exposures, which preceded exposure to a second male. This evidence, although indirect as it is, further shows that multiple matings of P. foveolatus females exert no negative effect on production of female progeny. Instead of producing entirely male progeny as Flanders (1956) observed on multimated ichneumonid females, the proportion of female progeny produced by multimated P. foveolatus females increased with increasing amount of sperm supplied through multiple matings. However, the observed sex ratios produced by parent females after introduction of new males (Table 10-18, Fig. 10-25), particularly those produced in the 4th and 5th host exposures, behaved in the following peculiar trend: the number of female progeny dropped sharply at the 4th host exposure but returned to a relatively high number in the 5th. The significance of this anomaly as it might relate to effect of multiple matings can only be established through repeated experiments. If these should yield results consistent with those observed here, it would then be of interest to learn why a second mating temporarily inhibits a females ability to lay fertilized eggs. Table 10-20 contains female to male ratios corresponding to each host exposure of the present study and those of the study on sex ratios resulting from sequentially mated males (sperm depletion) in

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139 I o Id C (0 0) 1-1 d) CO C 4-1 J3 C O & •H ^ CO 4J CO i-l Q) 3 u 6 Q) C I-l O U e -w •H CO CO c (u bO -U O CO !-i O •u o •H CO r-l « (1) ^4 > A, r-l (U X 4J ca (U c 0) 4J 0) CO St IN w c o •H 4J CO 3 D* (U •H OJ OJ vO 4.) V4 C (U O 4J •H & CO CO CO CO -d 0) 0) u >-l M 3 W) C CO 0) -H O Vj D. C X iH O (U CO -H •H 4J 4J e cu CO O I-l oca, X) o (1) cu ^ cr -u C ^ 3 (U 4J M in CO O CM >-i • <4-l -H CU O •H c •H CO CU ^, a o c c (U (U CO v< 4-l 4J o <4-l (U iH •H ^ CO -i cro M W <)• to O ^ t4-l . rH O tX) cu rH U-| o • o iH cr^ cu ^4 4-1 3 to CO o o K & X u (U > (U 4-1 4J 4J C c c 4J to to CO to u o a •H •H •H 4J >4-l 4-1 M-l c •H •H •H to c c C CJ 60 00 60 •H •H •H •1-1 M-l to CO to •rl 4J 4J 4J C 60 o O O •H c c 3 CO (U o o •H C 60 •H CO 00 00 o o CS (J cu 13 rH M (U to CO 3 o CU CO MH 8* rH O O X o< 0) X 0) u rH (U rH > H • cu • D< rH cr M O U to C O c OJ o MH o •H •H 4-1 4-1 to 4-1 to to XJ to 3 c 3 -arH cr to cy C^l 00 H a & OJ o O 42 rH H > CO CO mh a\ o 00 o o •H 4-) to OJ )H 3 60 OJ 3 O E VO o • u o •H W MH U 3 CO o (X X OJ cfl| ^1 0|

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140 CO c •H •O (0 C OO o c cn 4J (U CO u B u o . 13 CO 3 o H-l ^£ o m CO u CO o H 1 4J CO c (U ST I-I O 00 CN in iH iH O O O o 00 I-I < saro CT> CO VD CTi ve vo vO vO NO I-I I-I I-I iH CO •H vO o m < 00 00 iH 00 o sr CM cr> \o CM o 00 iH iH iH iH I-I rH SJin CO CO O cn in CM CN iH o o O o O o\ CO o VO VD I-I CM O vo iH iH I-I iH i-H iH CO CO 00 O m CO r-. o iH CM I-I iH 0\ CTi 00 CO CN o CM CO m vo oc o o 00 in Ov rH CTi O CO 00 CVl O CO iH 00 VD m CM CM iH rH o O o o 0\ o vO VD a\ rH CM CO o vo m vo iH rH as <• rH o CJ\ rH rH rH rH rH CO CO CO 00 00 as m CO o 00 O iH CM rH rH rVD CD c CM U CO 4-1 VD x: 4-) 0) ex CB CO 0) iH CD E •a 0) 4J CO E C v 3 cr (U cn E o VI I4H 00 c 3 cn Q> u CO o •H 4J T3 CO 3 U u cn X 0) cn c o 00 0) C 0) CO 0) VJ >^ 3 0) 4-1 JZ CD o o CM vD I VO O X -H •a (U c a) ^ -Q D. vO CO o. H < M 0) C C 4-1 •H a. CO CO CO X AJ CJ CO CO •o -a c B C O O O -H 4J "O "O (1) CU 0) rH CO CO O. CO CO OJ PQ pq "O rH •o (9 (U E 4-1 fe > 4J c o 0) o M CO 0) -a. o > o CN CO CU CU rH (Ut X CO 4J E (U v,_,-^ UH 43 ^ rH C CO (U W Q) -H rH c i-H bc 4-1 CO (U nJ o c E 00 c >-i tH C ex o CU 4-) CU u OJ CO C X u V4
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141 Chapter 6 where multiple matings were not allowed. Included in this table are the numbers of females/males of the parasite progeny and the corresponding sex ratios when female progeny produced by the 6 multimated parent females (the 6 obvious cases showing multiple matings) were converted into males. All data therein contained were statistically analyzed to detect the pattern of change in sex ratios of progeny produced by parent females under conditions of multiple matings as well as that of the progei^y produced by females under the sperm depletion condition where multiple matings were not allowed (Chapter 6, Appendix 6-2), Statistical analyses showed that each of the 3 sets of sex ratios presented in Table 10-20 in columns 3, 5, and 7, when fitted to the corresponding host exposure, produced quadratic polynomial regression equations. These relationships are represented by equations Eq. 10.26 for the first set (per-host-exposure sex ratios of progeny produced by females under conditions of multiple matings) , Eq. 10.27 for the second set (per-host-exposure sex ratios of progeny produced by females under conditions of multiple matings, except that all female progeny produced by the 6 multimated parent females were converted into males), and Eq. 10.28 for the third set (per-hostexposure sex ratios of progeny produced by females under sperm depletion condition where multiple matings were not allowed) . ^(SRmm)2= 2-^27 0.608 E + 0.039 e2 (Eq. io.26) (coefficient of determination R = 0.99) ^(SRmm)3 " ^'^^^ " ^'^^^ E + 0.047 E^ (Eq. 10.27) (coefficient of determination R =0.98)

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142 ^(SRsd)2 " ^-"^^^ " ^ ^ (Eq. 10.28) (coefficient of determination R = 0.98) where: "^(sRjj„n)2 represents female to male ratio of progeny produced by females under conditions of multiple matings; ^(SRmm)3 represents female to male ratio of progeny produced by females under conditions of multiple matings, except that all female progeny produced by the 6 multimated parent females were converted into males; ^(SRsd)2 'Represents female to male ratio of progeny produced by females under sperm depletion conditions where multiple .matings were not allowed; and E stands for host exposure. The 3 models are illustrated in Figure 10-26. The level of significance for the test on the magnitude of the quadratic coefficient estimate was 0.01 for equations Eq. 10.26 and Eq. 10.27, and 0.025 for equation Eq. 10.28. The test for equality between the slopes of equations Eq. 10.26 and Eq. 10.27 at each level of host exposure showed no significant differences, although the curve of equation Eq. 10.27 dropped slightly below equation 10.26 at the later host exposures. It is expected that this drop would be greater if female progeny produced by the undetected multimated females previously revealed by statistical analyses of data in Table 10-18 could be singled out and converted into males. A puzzling feature of Figure 10-26 is the consistently higher female to male ratio of progeny from females of the multimated series. There was no evidence of host quality difference

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143 to E 0) E 0) a 1 CO l_ X 0) CO • j:(SRinm)2 O ^(SRmm)3 Y(SRsd)2 n2 47 ^SRsd)2= 2.422 0.681 E + 0.061 1st 2nd 3rd 4th 5th 6th 7th Host exposures FIGURE 10-26. Quadratic polynomial regression curves representing relationship between female to male ratios and host exposures ^(SRmm)2 study on multiple matings where female and male progeny produced by all multimated females were included; ^(SRmm)3 ^^"^ study, except that the numbers of female progeny produced by the 6 obviously multimated females were converted int30 corresponding numbers of males; and Y. for the study on sex ratios resulting from sequentially migel'^Mles (Chapter 6) (sperm depletion where multiple matings were not allowed.

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144 or difference in environmental factors such as temperature and light between the two studies. There was a two generation difference in the females used in the studies; however, any effect of progressive inbreeding would be expected to decrease the female to male ratio of progeny. This is contrary to the observed results in which the later generation produced the higher female to male ratios. Whatever the explanation for the higher proportion of female progeny produced by the multimated females, it would be expected (assuming that females of both series received approximately the same amount of sperm from their first copulation) that these females would exhibit the most rapid rate of sperm depletion. This would have resulted in rapid convergence of the curvilinear regression lines as shown in Figure 10-26 and should be most pronounced for the last 4 host exposures. The fact that the multimated series line does not show a more rapid convergence with that of the sequential mating series (Chapter 6) is, at least in theory, indicative of an increased store of sperm resulting from a second mating. When the slope of equation Eq. 10.26 was tested against the slope of equation Eq. 10.28 at each exposure, a significant difference was found at the 4th and 5th host exposures; at the 6th exposure, the difference was significant only at 0.06 level (Table 10-21). When the slope of equation Eq. 10.27 was tested against the slope of equation Eq. 10.28 at each exposure, a significant difference was found also at the 4th and 5th host exposures (Table 10—2) . The results of these last two tests imply that the slopes at the 1st, 2nd, and 3rd host exposures are not different with the 3 equations (equations Eq. 10.26, Eq. 10.27, and Eq. 10.28); but

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145 I o w 0) c o •H Cli /-V CO -w x JJ cd •H B o c 3 1-1 (U 4-> a cfl •H c o iH (1) CO -H O ^ a c 60 •H -H Q) U t-i CO CO ^1 Cfl 3 -H CO B cr o o J cfl -a cfl CD 3 o o" •H 4J Cfl <30 Vj CN f"^ d . M-l O CJ-I CU •3 (U •H > C 60 T3 o CO o u 4J — s DC CO Cfl cJ 60 j-i cu o u 3 r-l CO a, cu X -H (U JJ a CO cfl O (U JJ Id cfl cu o 3 cfl <-i o (U -H > iJ-l in CU O "H 3 60 •H CO (U a o c ' 3 cu CO cu 0) cu M ^ a cu JJ o <+-! ^ CO •H Q 00 U 00 \D 0 CN • 14-1 •• O o 01 ^ Q. o • w iH cr CN CO W I CO o ^1 vD VO O CN • cu JJ 3 cfl o •H CJ-I •H 3 60 •H CO JJ o 3 4J 3 cfl a •H M-l •H 3 60 •H CO o 3 3 cfl o •H M-l •H 3 60 •H CO o 3 3 cfl o •H M-l •H 3 60 •H CO > 0) m o 3 CO O •H M-l •rl 3 60 O JJ 3 CO O •H M-l •H 3 60 •H to CTi C3> cn i-H CN f— 1 u-l o o o O r— 1 r-l o o O o O O u-1 » — 1 I-H t-H a\ in m 00 i-H o o o o o O o o o CM \o 00 o in 1-4 in CO CN CN f— 1 o o o O o o •a 3 CN •T3 U JJ JJ JJ m (1) cd rn s Q J_l E o Q Q f'S M-l H ) Q) 4-J r»*-< CO 1 1 ] 1 \J r" rU (H 1-1 C^ M— 1 /-\ \J 0} CO ni cfl cd T—H CO CO CO 0) »> *> t> c P3 cu QJ CO J CO ITl o o p. & 00 cu 0) CM JJ u O CO CO o o ,3 fi O M-l M-l •H O o 4J cu CO rH O O. cu CJ CO cu JJ Cfl JJ o cfl X. M-l CM O O r-4 (U > • CU cr 1-4 M u 3 O O M-l •H JJ CO CO 3 3 cr cfl 3 cu 0) CO H O M-4 3 &° u '-4 • cu cr rH . JJ CO cfl 13 3 3 cr cfl (U )-4 W o 3 o iJ CO 3 cr CO cu W M-l o cu (U ^ cx cu O X. H > CO lU o M-4 00 MH f-H O O O 00 II VO vO 3 • 3 CU O O o o u •H •H 3 JJ 1 4J 1 60 Cfl cfl •H JJ W JJ w MH 3 00 3 CN O, CN B o B t-H a o • o CO u o u d w ^1 ^1

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146 CM I O c o •H (0 CO (U )^ 60 (U ^1 (U r-( 3 -n B 01 a u O (U > o 3 u 4J CO too C u o CO (U C J3 (U 4J 60 o >^ U Xi D. X) (U (U iH O CO 3 o O !-t > 3 CO o o J3 a CO <3i 4J CO dl (U a c c 14-1 CU o •H hJ sign CU o c CU (U (U (U CO (U m CU u 4J u a c 4J CO CO CO CO o O o •H •H •H u H-4 m •H •H CO C C c o 60 60 60 •H •H •H •H M-l CO CO CO •H u 4J u 3 60 o o o •H C 3 3 CO CU > CU u-1 O 3 CO o •H U-i •H 3 60 a CO a •H <4-l •H 3 60 •H CO O 3 o 00 o CO ITl 00 o o o I— 1 I— 1 I— 1 o o o o o o m fO —1 I— I m CO in in ro 1—1 o o o o O o o o in .— I CO CJ^ 00 CJ^ o o 1— ( •— 1 in CM 1— 1 o o o O O C •3 -p 3 >-l 4J CN CO U m 3 B o M-l CU ^ 4J 3 O •3 CU CO CO Xi CO CO (U ^1 3 CO o & CU 0) S CO CO u CO 00 CM 3 o M-l 0) 3 CU CO > O 0) 3 CU X a o CO 4J CO 0) CO (u o CO 4-1 CO 4H O 00 CM CO CU 3 i-i cr 3 (U CO O «4H g,o CU (U a 4-1 o CO rH o CO x: I MH O CM O 0) O CU O rH > r-H J> ,— I CU CU . rH • rH • cf cr o* U V4 U M U 3 MH 3 MH 3 O O O •H CO -H CO -H 4J -3 4J -3 4J 3 CO 3 3 CO CO tr 4J cr 4J (U CO 0) CO M-l W MH W MH o o CU
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147 they are significantly different at the 4th and 5th host exposures where the slope of equation Eq. 10.26 drops more steeply than that of equation Eq. 10.28 and the slope of equation Eq. 10,27 drops more steeply than the slope of equation Eq. 10.28 (Tables 10-21 and 10-22, Fig. 10-26). No significant difference between the slopes of equation Eq. 10.26 and equation Eq. 10.27 were found at any of the host exposures. The significant difference only at 0.06 level between the slopes of equation Eq. 10.26 and equation Eq. 10.28 at the 6th host exposure (Table 10-21) , and the lack of significant difference between slopes of equation Eq. 10.27 and equation Eq. 10.28 at the same host exposure (the 6th host exposure) (Table 10-22) , also suggest that multiple matings can modify the distribution of sex ratios of the parasite progeny in the series of successive host exposures. The curve illustrating equation Eq. 10.27 (Fig. 10-26) occupies a transitional position; its slope would become steeper if female progeny produced by the undetectable multimated females could be singled out, converted into males and incorporated into the involved set of data (columns 4 and 5, Table 10-20) as discussed earlier in the paragraph dealing with the analysis of sex ratios of progeny produced by the first 10 females mated in sequence with single males. The steeper drop of the curve illustrating equation Eq. 10.26 than that of the curve illustrating equation Eq. 10.28 is mainly due to the higher proportion of female progeny produced by parent females at the early host exposures and the overriding effect of sperm depletion relative to the low rate of multiple matings among parent females. This appears to adequately explain the configuration of the slopes for equation

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148 Eq. 10.26 where there is a significant difference beginning with the 4th and continuing through the 5th host exposures. In conclusion, the results of the study on sexual behavior (Chapter 4) and such characteristics of P. foveolatus as the species (a) female-biased sex ratio, (b) smaller size and shorter longevity of males, all point to a low probability that a female will mate more than once during her lifetime. However, results of the multimating study and comparison of these results with those of the male sequential mating study (Chapter 6) indicate that some of the once-mated females remain receptive to subsequent matings and these matings are adaptively advantageous at least in those instances where the first copulation of a female happens to be with a sperm-depleted male.

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CHAPTER 11 OVIPOSITION PREFERENCE WITH REFERENCE TO HOST INSTARS Introduction The power of host perception is one among the several important qualities composing the searching ability of a parasite; others are the power of locomotion, power of survival, and aggressiveness and persistence (DeBach, 1964). The power of perception may include the ability to select the most suitable host developmental stage(s), and ability to discriminate parasitized from the unparasitized hosts. For Aphidius testaceipes (Cresson) and Praon aguti Smith, halfgrown and unparasitized aphids are usually chosen for attack (Sekhar, 1957) . The braconid Praon palitans Muesebeck oviposits in aphids at any stage, but apparently prefers the 3rd and 4th instars (Schlinger and Hall, 1960). Schlinger and Hall (1961) reported that Trioxys utilis Muesebeck preferred to oviposit in the first 3 instars of aphids, although it oviposits in all stages. For Aphytis melinus DeBach, the most preferred stage of the red scale, Aonidiella aurantii (Mask.), was the young adult female, followed by the maturing 2nd instar and male pupae in decreasing order; the number of scales parasitized, total of eggs laid, number of eggs per scale, female to male ratio and size of the parasite also followed the same order (Abdelrahman, 1974a). Abdelrahman (1974b) also reported that the parasite A. melinus seemed to discriminate between recently parasitized and 149

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150 unparasitlzed scale through recognition of an odor that inhibits attack. Small pupae of the gypsy moth, Lymantria dispar L. parasitized by Brachymeria intermedia (Nees) produced a greater proportion of male progeny (Barbosa and Frongillo, 1979). Lfoveolatus , field observations made repeatedly after inoculative releases of the parasite for control of the Mexican bean beetle in infested areas in Florida indicated that the parasite preferred to attack 4th-instar larvae before attacking the younger larval stages. The objective of the present study was to prove that such a preference vis-a-vis host instars does exist in P. foveolatus . . . 1 s Materials and Methods Second-, 3rd, and 4th-instar host larvae with a total number of 150 (50 larvae for each instar) , were released on foliage of cut beggarweed plant inside a cage, dimensions 30.5 cm x 30.5 cm x 60.1 cm, screened with fine mesh nylon cloth (Fig. 11-27). To maintain turgidity, plant material was inserted into a mason jar half -filled with water, through a suitable hole on the lid; the hole was then closed with absorbent tissue paper. This prevented the host larvae from falling into the jar. The larvae were then allowed a period of 60 minutes to settle on the leaves and begin feeding. Earlier, 50 4-day old parasite females, picked at random from a colony issued from 30 4thinstar host mummies, were placed in a parasite rearing unit provided with honey and water as described in Chapter 2, and

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151 FIGURE 11-27. Screened cage for confinement of host larvae and parasite adults for the study on parasite oviposition preference vs. host ins tars.

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152 allowed to resettle themselves down for 5 to 6 hours. The unit was then placed underneath the plant foliage, and the parasite females were released by removing the lid of the rearing unit. A period of 90 minutes was allowed for the parasite female oviposition activities. The host larvae were then retrieved and reared in groups of separate instars until cessation of feeding. The condition of exposed host larvae was observed as frequently as possible. Dead larvae were dissected and observed under microscope for presence of parasite eggs or larvae. Mummies from which adults did not emerge were also checked for the parasite presence. The test was replicated 4 times. The numbers of nonparasitized, successfully parasitized, dead, and dead parasitized host larvae were recorded. Results and Discussion Host preference was revealed when the parasite females were afforded to have a choice between 2nd-, 3rd, and 4th-instar host larvae. This was based on two observed biological criteria. First, the number of parasitized host larvae increased with the increasing rank of host instars (Table 11-23) . Statistical analysis based on arcsin transformed data (Table 11-23, figures in parentheses) revealed a significant (0.01 level) linear relationship between the percentage of parasitization and the host instars. This relationship is represented by equation Eq. 11,24 and illustrated in Figures ll-28a and 11-29.

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153 TABLE 11-23 Numbers and percentages of parasitized hosts corresponding to each of the 3 larval instars used in the test for oviposition preference by P. foveolatus females Host larvae Replicates 2nd ins tar ; No. parasitized/ total no. tested % parasitization a/ 3rd ins tar ; No. parasitized/ total no. tested % parasitization a_/ 4th instar ; No parasitized/ total no. tested % parasitization a/ 3/50 6 (14.18) 14/50 28 (31.95) 18/50 36 (36.87) II 6/50 12 (20.27) 8/50 16 (23.58) 18/50 36 (36.87) III . 4/50 8 (16.43) 8/50 16 (23.58) 14/50 28 (31.95) IV 1/50 2 (8.13) 3/50 6 (14.18) 12/50 24 (29.33) oitand.'f P^'^^^^^^^^^ represent arcsin Vp transformed data where p stands for percentage of parasitization

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154 (p = % parasitization) Second, the rate of death of host larvae computed on the basis of percentage of parasitization increased with the decreasing rank of the host instars (Table 11-24, Fig. ll-28b) . Statistical analysis based on arcsin transformed data (Table 11-24, figures in parentheses) gave a highly significant linear relationship between the percentage of the dead parasitized host larvae and the host instars (equation Eq. 11.25, Fig. 11-30). ^(arcsin l/d?) = ^^^'^'^ " 34.68 Instar (Eq. 11.25) (dp = % dead parasitized host larvae) The higher rate of premature death of 2ndand 3rd-instar host larvae before normal mummification may be due to one or a combination of 2 or more of the following factors. 1. The efficient use of ovipositor during the oviposition process may largely depend on how firm a parasite female is able to settle herself on the body of the host larva in preparation for egg laying. Firm settlement requires large host scoli for the parasite female to hold with her tarsi, thus providing firm and stable support. The scoli of the larger host larvae best accommodate this need. Direct observations made under laboratory conditions revealed that the females required substantially more time to settle on and oviposit in a small host larva than when the host was a large larva. In addition, females often abandoned small host larvae without inserting her

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155 TABLE 11-24 Numbers and percentages of dead parasitized hosts corresponding to each of the 3 larval instars used in the test for oviposition preference by P. foveolatus females Host larvae Replicates II III IV 2nd instar ; No. dead/ no. parasitized 3/3 6/6 4/4 1/1 ^ ^^^^ 100 100 100 100 (90) (90) (90) (90) 3rd instar ; No. dead/ no. parasitized 10/14 6/8 4/8 2/3 '^''^^^'^^Z 71.43 75.00 50.00 66.67 (57.69) (60.00) (45.00) (54.74) 4th instar : No. dead/ no. parasitized 2/8 3/I8 2/14 I/12 ^'Je^da/ 11.11 16,67 14.29 8 33 (19.47) (24. 10) (22.21) (16.'77) Itr.Tri "^heses represent arcsin /d? transformed data where dp stands for percentage of dead parasitized host larvae

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156 o o o c 0) > 03 to O CO c o CO o o to o in o o o CM uoijBzijisBjed % uo paseq meap o/^ CO •H W J3 (0 c > o •a iH c •H CIJ 1 <-l 4J 4J < CO O •o J3 c CO U CO 4J 1 CO •o c •H CO 1 u 1 -aT3 C •a cs a o o a c CM 0) CO > CO CO O TO "55 c I 13 CO I C CN •H to CO CO o CO o CM C o (U Ph 00 CN I o M fin •H CO 4J CO -H !-i CO CO to PU to 73 PCO 0) M-l XI O 14-1 0) O 60 to C C
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157 o 90 M 70 . 60 SO (arcsin fp) = A. 56 + 9.50 Instar < 30 . 20 10 2nd L_ 3rd L_ 4th Host instars FIGURE 11-29 . Simple linear relationship between the percentage of parasitization and host instars, representing preference of host instars by P. foveolatus. 90 SO it o u < 60 SO K 30 20 10 A ^(arcsin Vd^) " 159.04 34.68 Instar 2"d 3rd Host instars 4th FIGURE 11-30. Simple linear relationship between the percentage of dead parasitized host larvae and host instars!

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158 ovipositor. This behavior was most obvious in the presence of the 1stinstar host larvae; the parasite females, even though young and generally eager to oviposit, showed almost no interest in these small host larvae; those that appeared to be interested in oviposition often moved away after spending some time in attempting to position themselves over a small host. Occasional successful ovipositions always resulted in the death of the 1st instar host larvae within a period of less than 24 hours. Furthermore, no successful parasitization of 1st instars has been recorded under field conditions. Deaths of 2ndand 3rd-instar host larvae that occurred within 2 days after parasitization seem best attributed to the poor suitability of these smaller instars. Death appeared to result from the inability of the female to position her ovipositor accurately, thereby causing injury to the vital organs of the small and delicate hosts. 2. Oviposition eagerness of the parasite females may also contribute to the cause of the death of the smaller host larvae. This factor may be more significant when the parasite females are still very young and carrying a full complement of eggs ready to be laid as was true of those used in the present study. Oviposition eagerness, if added to the poor support (small scoli and light body weight) provided by the younger and smaller host larvae may intensify inaccuracy in use of the ovipositor, and perhaps explains the death of all the parasitized 2nd-instar host larvae in this study. Successful para' sitization in 2nd-instar host larvae under field conditions may therefore be a contribution of the least eager females.

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159 3. A higher number of developing parasite larvae per host larva than the host can normally support may also be responsible for the death of the host before normal mummification. The rapid development of the excessive number of the parasite larvae may result in exhaustion of the host larva's fat body and nonvital energy reserves causing the parasite larvae to start consuming the vital tissues, thus causing premature death of both the host and the parasite larvae. 4. Superparasitization may also be a factor; however, under conditions of the present study, this would not be likely to occur. If some hosts were superparasitized, this would have probably been the result of female oviposition eagerness since an ample number of hosts were available. Of the 4 causal factors above-postulated, the last 2 seem least likely to be responsible for the high mortality of smaller host instars. Although counts of the dead parasite larvae were not made during the dissection of the host after its death, no particular instances appeared to show excessive numbers of parasite larvae. However, deterioration of the parasite larvae after death might also be a possibility since some dead host larvae were in very bad shape when they were dissected primarily for purpose of checking for presence of parasite eggs or larvae. Despite the fact that in the present study all parasitized 2ndinstar host larvae died prematurely before mummification, successful parasitization of this host instar is common. This may indicate that the parasite has the ability to control the number of eggs laid and normally lays only the number that can develop in a host of given

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160 size. The mechanism used by the parasite for control of the number of eggs to be laid is not known. However, based on the fact that the average numbers of parasite progeny per host larva were found to approximately double from one instar to the next higher one, and that, within each host instar, there is a wide range of per-munnny numbers of parasite progeny, the physiological and anatomical state of the host larvae appears to be closely related to the number of eggs deposited by a female in each oviposition. The average numbers of parasite progeny per host larva were 4.46, 8.25, and 18.27, respectively, for 2nd-, 3rd-, and 4th-instar host larvae (figures based on 68, 24, and 30 field-collected 2nd-, 3rd, and 4th-instar host larvae). Probable factors in determining the number of eggs to be laid during an oviposition are host size in terms of volume as determined by tactile senses of the female and body turgor as detected by sensors on the tip of the parasite ovipositor. The wide range in number of adult parasite progeny produced from individual larvae of each host instar may reflect the variation in size and degree of maturity normally associated with development of each instar. For instance, ranges of 1-9, 1-14, and 4-45 were respectively recorded for the 2nd-, 3rd, and 4thinstar field-collected host larvae (based on respective 68, 24, and 30 collected larvae as previously mentioned) . Under the laboratory conditions, from a test in which no superparasitization is thought to have occurred, a range of 6-43 parasite progeny was also obtained from 38 4th-instar host larvae. This would imply that the control of the number of eggs laid in each oviposition is determined not before but during the process of oviposition and is governed by the host quality

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161 factors detected by sensors on the ovipositor. By analogy, for closed plastic containers, the internal pressure would provide a measure of the quantity of enclosed material which together with a measure of surface area would determine the quantity of material in the containerin this case the amount of food available for the development of the parasite larvae. Evidence regarding the parasite preference vis-a-vis different host instars is obvious. However, some other aspects such as (a) the frequency of contact with different host instars by the parasite within a certain given period of time, whether leading to oviposition or not, and (b) cues, of whatever number and nature, that bring the parasite to the host, have not been investigated. Repeated contacts with a host, whether leading to oviposition or not, and high rates of death due to parasitization in the younger host larvae represent a waste of time and energy. An ability of the parasite to detect already parasitized hosts and to control the number of eggs to be laid as influenced by host quality would minimize such wastage and be selectively advantageous. Control of number of eggs laid, whether in single or multiple oviposltions appears to play an important role in the Mexican bean beetlePediobius host parasite system.

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CHAPTER 12 SUMMARY AND CONCT.USIONS Summary of Regults Arrhenotoky Is the mode of reproduction of P. foveolatus ; females receiving sperm through copulations produce both female and male progeny, whereas unmated females or those mated with completely spermdepleted males give rise to only male progeny. Emergence of the parasite adults from pupae from many different host mummies occurred within a 2-day period. Emergence from any individual host mummies did not take place until all the parasite adults had emerged from their pupal stage. The emergence hole is made by parasite adults of both sexes. More than one emergence hole per host mummy has rarely been observed; the most common presence of single emergence holes perhaps helps prevent the rapid dissipation of a parasite female sex pheromone from the host mummies, thereby facilitating mating activities such as mate-searching by the parasite males that appears responsive to and in some degree controlled by concentration of the female sex pheromone. Whereas the majority of adult males emerged earlier than females from the several hundreds of pupae that were individually isolated from the host mummies, no particular sequence of emergence of the parasite adults of both sexes from their 162

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163 host mummies was noticed; each individual adult may occupy any position in the series of emergence. Matings occur actively outside the host mummies during the course of emergence from the host mummies. Mating outside the host mummies also admits, to some degree, the participation of outsider males in local mating activities, particularly under conditions of high host density. This factor, when combined with a high level of parasitism, greatly increases the probability of non-sibmating in an otherwise inbreeding mating system. Outbreeding, at whatever level, would seem desirable in order to retain some degree of genetic variability. The wide range of fecundity observed among females could be a result of the inand outbreeding of the founding stocks inside the rearing units during the long period of laboratory culture in which each stock culture renewal was made through simultaneous parasitization of the host larvae and thus provided opportunity for outbreeding since the adults emerged more or less simultaneously from mummies that were in contact or separated by only a few millimeters. Outbreeding under low host density, on the other hand, is not to be expected, due primarily to the distance separating the parasitized host larvae and/or the asynchrony of the emergence of the parasite adults. This would also imply that outbreeding of P. foveolatus under open field conditions may occur only at a relatively low rate. Mating activities beginning immediately at the onset of the parasite emergence from the host mummies were observed to generally last for about 30 minutes, rarely exceeding one hour. Expansion of mating area proceeds gradually in an outward direction. Similar

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164 behavior was also observed among the parasite males emerging from the mummies containing only male adults, except that mate-searching activities terminated sooner because of absence of females. Male mating behavior is mainly characterized by very active search for, and readiness to mate with all receptive females. Males do not have the ability to discriminate mated from unmated females or young from old females. Mating success depends on the females; only when the female shows readiness (by standing high on her legs following male courtship) is copulation possible. Multiple matings of females do occur, but only at low rate; only on rare occasions were multiple matings of females directly observed. Wing vibration sounds of the courting male appear to play a role in recruiting outsider males into the local mating activities. The fact that outsider males did not orientate themselves toward the source of sound production (wing vibration of male during courtship) upon landing in the active loCal mating area but showed a similar mate-searching behavior to that of the local males, appears to suggest that the vibratory sound effects were superseded by those of female sex-attractant. This would therefore imply that female sexattractant may serve only as a short-range means of communication, whereas more remote communication may be through sound produced by wing vibration of the males during the courtship. Preemergence matings (matings that take place inside the host mummies prior to the emergence of the parasite adults) are impossible due to the fact that courtship behavior essential to successful mating requires an amount of space that cannot be provided within the crowded host mummies.

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165 Mating capability of the parasites also depends much on age of adults. Females began to significantly lose the mating instinct from the age of about 2 weeks, whereas 15-day old males (old in terms of male longevity) still managed substantially successful matings with young females (under laboratory conditions, some females were observed to survive over 3-month period whereas males were observed to live for a period not exceeding one month) . Young males consistently mated with a succession of females as rapidly as one mating was completed and the next female was contacted, but a rapid succession of copulations resulted in sperm depletion. Single males continued mating activities even after having successfully copulated with 50 virgin females. Sperm depletion occurred when males were allowed to mate in succession with a high number of females. Normally female-biased sex ratios, the relatively short period of local mating activities around the host mummies soon after emergence of adults, and the relatively small size and shorter lifespan of the males are all indicative of a species in which females mate but once and store sufficient sperm for fertilization of eggs laid during a normal lifespan. However, retention of ability to mate a second time serves as a "hedge" against the consequences of a first mating with a sperm-depleted male. Both unmated and mated females lay eggs. However, the presence of unmated females under field conditions must be very rare as no fieldparasitized host larvae have been observed to produce only male progeny. Successful parasitization of the smaller host larvae, namely those of 2nd and 3rd instars, gave the indication that the parasite has the

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166 ability to control the number of eggs to be laid in each oviposit ion. For a given host instar or size of the host, such control may hypothetically rely on the physical stimuli such as the internal pressure in relation to the space available around the points of ovipositor insertion; the parasite may sense these stimuli through its ovipositor. Certainly, there is no evidence of differential mortality of parasite larvae in hosts of different size that would influence either the number or sex ratio of emerging adults. When the parasite is given a choice, it prefers larger host larvae to the smaller ones for oviposition. Cues, whatever their nature, intensity and relative effects may be, which enable the parasite to locate the host, are not yet known. Furthermore, despite the fact that the parasite females were observed to move away from smaller host larvae after attempting to oviposit, presumably because of poor structural accommodation (small scoli and body) offered by the host, frequency of visits to individual host larvae of different size by a given number of parasite females for a given period of time has not been investigated The apparent ability to control the number of eggs laid in each oviposi tion, thus ensuring higher probability of progeny survival in small hosts, must be a mechanism that has evolved to reduce wastage of eggs through premature death of parasitized smaller host larvae. Under the laboratory conditions of these studies, the number of live parasite adults produced by a female from 4th-instar host larvse ranged from 0 to 199, with an average of 126.30. The longevity of ovipositing females was observed to range from 9 to 48 days, with longevity inversely related to fecundity.

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167 The female to male sex ratio to the parasite varies from one host larva to the other for a given host instar, but increases with the increased numbers of parasite adults per host mummy. For a group of females, sperm regulating ability, recorded through successive ovipositions and expressed in percentage of female progeny, followed the mathematical quadratic polynomial regression, and thereby appeared to encompass in succession 3 biological phases, the so-called "maturation," "optimum," and "degradation" phases. For single males, the distribution of sperm through successive copulations with different females was also observed to fit a similar mathematical model; such a trend is particularly visible through the 1st and 2nd ovipositions , where the sperm depletion effect was seldom observed. Conclusions Relating to Basic Biology The present studies relating to reproductive biology and sexual behavior of Pediobius foveolatus fully support the conclusions of earlier workers (Stevens et al., 1975b) regarding mode of reproduction. The species is clearly arrhenotokous with female progeny developing from fertilized eggs and males from unfertilized eggs. The system of reproduction is thus of the haplodiploid type typical of Hymenoptera. Studies of sexual behavior (Chapter 4) show P. foveolatus to be a species characterized by a high level of inbreeding with most females being fertilized by sibling males. This provides additional evidence to support the contention of Ghiselin (1975), Borgia (1980) and others that haplodiploidy is closely associated with inbreeding mating systems.

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168 Whereas the sexual behavior of P. foveolatus results in a high probability of sib-mating, it is not a closed inbreeding system. If non-sibmates are present in the vicinity of a host mummy from which adult P. foveolatus are emerging they will be attracted to and participate in the postemergence mating activities in the immediate area surrounding the host mummy. This system has obvious advantages in terms of population fitness. First, it ensures that under conditions of low populationdensity, females will be fertilized prior to dispersal and thus not subject to the hazard of failure to find a mate. Secondly, under conditions of higher population density the system admits a degree of outbreeding that insures maintenance of genetic variability and population vigor. All earlier authors (Lall, 1961; Stevens et al., 1975b) who have discussed P. foveolatus reproduction have noted the species female-biased sex ratio. The present study while corroborating results of earlier workers with respect to the female bias yielded data that do not agree in other respects, most notably data on the effect of superparasitism. Stevens et al. (1977) reported that the ratio of females to males emerging from mummies producing 1-5 adults was 3.23:1 while for those emerging from mummies producing 41-100 adults was 0.48:1. Thus superparasitism had the effect of increasing the proportion of males. It should be noted that these were field collected mummies. Data from my laboratory study of fecundity (Chapter 7) show an increasing female bias as the numbers of adults emerging from individual host mummies increase. This was supported by data from one experiment (pp. 87-88) in which individual larvae were exposed to 1, 2, and 3-4 female P. foveolatus

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169 Only one ovlposition per female was allowed and the resulting female to male sex ratios for the adult parasites emerging from the three groups of hosts were 4.68:1, 5.11:1 and 5.85:1, respectively. Why superparasitism under controlled laboratory conditions should yield results so different from those encountered in the field is puzzling and should be the subject of further study. Results of the studies involving effects of allowing males to mate sequentially to 50 females clearly indicate that no reproductive liability is attached to the female-biased sex ratio. It was found that males were able to mate with up to 10 females in quick succession without evidence of significant sperm depletion. Data from the fecundity study (Chapter 7) indicate a substantially higher number of adult progeny per female than earlier studies. Lall (1961) working in India found female fecundity to range from 10 to 50, while in Maryland Stevens et al. (1975b) reported that the average number of live adult progeny per female was 88. In the present study the mean number of adult progeny per female was 126. This was based on 33 females to which 4th-instar host larvae were exposed to each female at the rate of 2 each day for the first two days and 1 daily until death of the female. Assuming equal female longevity and equal access to hosts it seems likely average fecundity of females in the field is nearer to the number foimd in this study than to the numbers reported by the earlier workers (Stevens et al., 1975b). Data from the 33 females used in the fecundity study (Chapter 7) also indicate a very clear inverse relation between fecundity and longevity. This finding seemingly represents a trade-off that should

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170 operate to increase population fitness. Short-lived females are able to exploit situations of high host density while long-lived females serve as a "hedge" in situations of low host numbers. One experiment was conducted for the purpose of determining whether females might mate prior to exit from the host mummy. Stevens et al. (1975b) assumed that this occurred though they observed postemergence mating. Data obtained earlier from sexual behavior studies (Chapter A) indicated that preemergence mating was unlikely because of space limitations within the host mummy. This was confirmed by results of the study of preemergence mating (Chapter 5) in which females were isolated on emergence and then allowed to parasitize host larvae. Among the 2,421 progeny produced by the 84 females used in this test only 1 dead and not fully developed female was found. This single dead female was found by dissection of mummies following emergence of all live adults and seems best expalined as an example of accidental parthenogenesis resulting from a patch of tetraploid tissue in the otherwise diploid ovarian tissue of the female parent as hypothesized by Speicher and Speicher (1938) who encountered a similar phenomenon in their work with Bracon hebetor . Data obtained from the study relating to the effect of female multiple matings (Chapter 10) are of interest primarily because they confirm that P^. f oveolatus females may normally be expected to mate no more than once. However, should sperm fail to be transferred during a first mating, individual female fitness and presumably population fitness are increased by the ability of females to remate. There was no evidence that access to mates following an initial successful mating

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171 significantly influenced the sex ratio of progeny when compared with progeny of females known to have mated only once. Relating to Pest Management Despite repeated releases that resulted in rapidly increasing populations during the season of release (Angalet et al., 1968) and even larger inoculative releases made first in Maryland (Stevens et al., 1975a), later in Florida and more recently in several additional States, P^. foveolatuS populations have failed to survive the winter season. However, when introduced into high and evenly distributed, host populations P^. foveolatus has repeatedly demonstrated the ability to overtake and suppress production of Mexican bean beetle adults. If host density and dispersal factors are highly favorable, release of a few hundred P^. foveolatus will in 3 to 5 parasite generations (60-90 days) increase to a level such that no, or very few, host larvae escape parasitization. The suppressive effect is most pronounced in the area of the initial release but in diminishing degree extends many miles from the release site by the end of the season. Reece I. Sailer (personal communication) reported finding Mexican bean beetle larvae parasitized hy foveolatus near the end of October at Commerce, Georgia in 1975 following releases at locations in Florida, the nearest of which was 400 miles distant. Therefore, until such time as a natural enemy is found that is able to live from season to season and maintain effective control of the Mexican bean beetle, inoculative releases of ' P^. foveolatus is a feasible management practice and is being used to suppress beetle infestations.

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172 Use of P^. foveolatus for large area or regional control of Mexican bean beetle infestations requires, first of all, knowledge of the host population. In order to arrive at any reasonable estimate of the number of adult parasites needed for field release, some knowledge of population density of the infested area and of the spacial distribution of the beetle populations within the area is required. Time of appearance and plant hosts of the first seasonal generation of the beetle are critical information. With this information, estimates of the timing and number of parasites to be produced can be made and it is at this point that results of these studies on the reproductive biology of P^. foveolatus should be useful. Of the several studies and experiments undertaken, those concerning the host instar preferred for oviposition (Chapter 11), sexual behavior (Chapter 4), and factors influencing sex ratio (Chapters 5, 6, 8, 9, 10) seem of greatest relevance to use of P^. foveolatus in control of Mexican bean beetle infestations. Incidental to these studies, data were obtained on longevity and fecundity that should be useful in planning production of P^. foveolatus . Perhaps, of greatest interest from a pest management viewpoint is the information obtained from the studies that relates to why P^. foveolatus is an effective agent for inoculative release against its host. Such information should be useful in searching for biological agents that can be used effectively against other insect pests for which no self-maintaining effective natural enemy can be found. In production of P^. foveolatus the factor most likely to be limiting will be production of host larvae. Unless carefully scheduled

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173 and managed there will be occasions when insufficient 4th instars are available and there will be a temptation to use 2nd and 3rd instars for production of the number of adults needed for anticipated field releases. Results of the test (Chapter 11) in which 2nd, 3rd, and 4th instars were exposed in a free choice situation indicate that 2nd instars should never be used for production purposes. Use of 3rdinstars will reduce the reproductive efficiency of the foveolatus breeding stock and probably adversely affect the sex ratio since this is usually most favorable in stock emerging from mummies producing the largest number of emerged P^. foveolatus adults. The very high mortality of parasitized 2nd instars and higher production of males from 3rd instars would seriously reduce the production efficiency of the female parents. The results of the studies on sexual behavior (Chapter 4) suggest that special care should be taken to ensure that production conditions during the period when adult parasites are emerging from their host mummies will not interfere with mating activity. The studies on sexual behavior (Chapter 4) indicate that courtship and mating normally occurs in the immediate area of the parent mummy. A certain amount of space is required and excessive crowding of host mummies from which adult P^. foveolatus are emerging is very likely to have a disruptive effect on mating activities and result in release of unmated females. While such released females might subsequently encounter and mate with released males the female bias of the released stock combined with the effect of spacial dispersion of the released stock would greatly reduce the probability of such encounters. If a high proportion of

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174 the released females were not fertilized prior to release resulting progeny could be largely male and greatly reduce build up of the second and third parasite generations that normally are responsible for economic reduction of the host population. Data on the effects of age, mostly obtained incidentally from studies on other aspects of the reproductive biology of P^. foveolatus are important as they relate to production and also as they explain failure of the species to successfully overwinter in the United States. Insofar as production is concerned, increasing age probably acting, through sperm depletion results in decreasing female to male ratio of progeny. Also the study concerning fecundity (Chapter 7) revealed that fecundity is inversely related to longevity. Based on these data females employed in production of release stock should not be used longer than 10 days. Data on longevity as related to failure of winter survival in the United States was obtained from survival in maintenance cultures. These stock cultures of live generations were retained and given routine attention to insure that food and water were available. Under conditions of the laboratory some females that had lived as long as 90 days produced female progeny, but these represented less than 1 percent of the original population. How the females would respond to normal winter temperatures is not known but it is unlikely that any would survive from October when the last host larvae are available to early April of the following year when larvae are again available. In the absence of a diapause response to winter conditions and the absence of hosts during a five month period (Florida) there seems

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175 little prospect that P^. f oveolatus will ever become permanently established in the United States. No experimental studies directly concerned dispersal characteristics of P^. f oveolatus . However, the studies on sexual behavior (Chapter A) revealed adaptations of the kind that would be expected in species exhibiting the dispersal capabilities noted by workers in Maryland and Florida. The fact that females usually mate with sibling males immediately after emerging from their host mummy insures that dispersing females can establish viable populations at any place they find a host population. This characteristic combined with the ability to produce an average of 126 progeny with a sex ratio strongly biased in favor of females and ability to produce two generations in the time required for the host to produce one adequately explains why inoculative releases of relatively small numbers of P^. foveolatus have so successfully controlled Mexican bean beetle infestations in Maryland and Florida. Subject to some modifications due to different characteristics of host species, any search for enemy species with the view of using them in inoculative releases programs should be directed toward species having characteristics similar to those of P. foveolatus.

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REFERENCES CITED Abdelrahman, I. 1974a. Growth, development, and innate capacity to increase in Aphytis chrysomphali Mercet and Aphytis melinus DeBach, parasites of California red scale Aonidiella aurantii (Mask.) in relation to temperature. Aust. J. Zool. 22:213-230. Abdelrahman, I. 1974b. Studies in ovipositional behavior and control of sex in Aphytis melinus DeBach, a parasite of California red scale, Aonidiella aurantii (Mask.). Aust. J. Zool. 22:231-247. Aldrich, J. M. 1923. A new parasitic fly bred from the bean beetle. Proc. Entomol. Soc. Wash. 25:95-96. Amitage, H. B. 1956. Successful eradication of the Mexican bean beetle in California. Calif. Dep. Agric. Bull. 45 (3) : 238-248. Angalet, G. W. , L. W. Coles, and J. A. Stewart. 1968. Two potential parasites of the Mexican bean beetle from India. J. Econ. Entomol. 61:1073-1075. ; Auclair, J. L. 1959. Life history, effects of temperature and relative humidity, and distribution of the Mexican bean beetle, Ep ilachna varivestis Mulsant (Coleoptera: Coccinellidae) in Quebec, with a review of the pertinent literature in North America. Ann. Entomol. Soc. Que. 5:18-43. Augustine, M. G., F. W. Fisk, R. H. Davidson, J. B. LaPidus, and R. W. Cleary. 1964. Host paint selection by the Mexican bean beetle, Epilachna varivestis . Ann. Entomol. Soc. Amer. 57(1): 127-134. Barbosa,_P., and E. A. Frongillo, Jr. 1979. Host-parasitoid interactions affecting reproduction and oviposition by Brachymeria intermedia (Hymenopt era: Chalcidae). Entomophaga 24(2) : 139-144. Bernhardt, J. L., and M. Shepard. 1978. Overwintered Mexican bean beetles, E pilachna varivestis : emergence from overwintering sites ;wc^'^i^T' ^"'^ longevity, on snap beans. Ann. Entomol. Soc. Amer. /i(5) : 724-727. Bernhardt, J. L., and M. Shepard. 1979. Comparative development reproduction, and leaf area consumption by Mexican bean beetles (Epilachna varivestis) on Phaseolus spp. (kidney beans, lima beans) and soybeans. J. Ga. Entomol. Soc. 14(3) • 191-198 176

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177 Borgia, G. 1980. Evolution of haplodiploidy : models for inbred and outbred systems. Theor. Popul. Biol. 17:103-128. Boush, G. M., and R. A. Baerwald. 1967. Courtship and behavior and evidence for a sex pheromone in the apple maggot parasite, Opius alloeus (Hymenoptera: Braconidae) . Ann. Entomol. Soc. Amer. 60:865-866. Brown, S. W. 1964. Automatic frequency response in the evolution of male haploidy and other coccid chromosome systems. Genetics 49:797-817. Browne, F. B. 1922. On the life history of Melitobia acasia (Walker), a chalcid parasite of bees and wasps. Parasitology 14:349-370. Cantelo, W. W. 1977. Controlling the Mexican bean beetle. U. S. Dep. Agric. Leafl. 548. 7 pp. Chapin, E. A. 1936. Correct name for Mexican bean beetle. J. Econ Entomol. 29:214. Chapman, P. J., and G. E. Gould. 1930. Plowing as an aid in Mexican bean beetle control. J. Econ. Entomol. 23:149-154. Chittenden, F. H. 1898. Insects injurious to beans and peas. U. S Dep. Agric. Yb. Agric, pp. 233-260. 767 pp. Chittenden, F. H. 1924. Evidence that Mexican bean beetle was present in the United States as early as 1850. Proc. Entomol. Soc. Wash. 29:19. Chittenden, F. H. , and H. 0. Marsh. 1920. The bean ladybird. U S Dep. Agric. Bull. 843:1-21. Clausen, C. P. 1978. Coccinellidae. In: Introduced parasites and predators of arthropod pests and weeds: a world review (C. P Clausen, ed.), p. 258. U. S. Dep. Agric. Agric. Handb. no. ifoU. 545 pp. Colgan, P.. and P. Taylor. 1981. Sex ratio in autoparasltic Hymenoptera. Amer. Natur. 117:564-566. Commonwealth Institute of Entomology. 1954. Distribution maps of pests. Series A, map no. 46: Epilachna varivestis Muls. Crawford, J. C. 1912. Description of new Hymenoptera. Proc. U. S. Nat. Mus. 42:1-10. DeBach, P 1964. Biological control of insect pests and weeds. Remhold Publishing Corporation, New York. 844 pp.

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178 Ditman, L. P., and W. E. Bickley. 1951. On control of the Mexican bean beetle. J. Econ. Entomol. 44:325-328. Douglass, J. R. 1933. Habits, life history and control of the Mexican bean beetle in New Mexico. U. S. Dep. Agric. Tech. Bull 376. 45 pp. Elden, T. C, and P. E. Paz. 1977. Field cage studies to determine effects of Mexican bean beetle Epilachna varivestis resistance m soybeans. J. Econ. Entomol. 70:26-29. Entomology Research Division, Agricultural Research Service, USDA. 1958. The Mexican bean beetle in the East and its control. U. S. Dep. Agric. Farmers Bull. 1624. 17 pp. Entwistle, P. F. 1964. Inbreeding and arrhenotoky in ambrosia beetles (Xyle borus compactus (Eichh.)) (Coleoptera: Scolytidae) . Proc. Roy. Entomol. Soc. London Ser. A Gen. Entomol. 39:83-88. Ferriere C. 1953. Les parasites de Lithocollites platani en Italie. Boll. Entomol. Univ. Bologna B:395-404. Fisher R. A. 1930. The genetical theory of natural selection. Clarendon, Oxford. 291 pp. Flanders, S. E. 1939. Environmental control of sex in hymenopterous insects. Ann. Entomol. Soc. Amer. 32:11-26. Flanders S.E. 1946. Control of sex and sex-limited polymorphism m the Hymenoptera. Q. Rev. Bio. 21:135-144. Flanders, S.E. 1956. The mechanisms of sex ratio regulation in the (.parasitic) Hymenoptera. Insectes Soc. 3:325-334. Gale, G. T., and M. Shepard. 1978. Response of PedioMus foveolatus to temperature and time of exposure to host,liniihia"^i?i^i^, Ghiselin, M. T. 1975. The economy of nature and evolution of sex University of California Press, Berkely. Gordh G. and P. DeBach. 1976. Male inseminative potential in AE|Z|x|^^|nsnane^ (Hymenoptera: Aphelinidae) . Can. Entomol. Gordon. R. D. 1975. A revision of the Epilachninae of the western B:u!l:93.'':J9ir'^^= Coccinellidae). U. S. Dep. A.r.TZ:,. Gould,^S. J. 1980. The throwaway male. New Sci. (April), pp. 205-

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179 Guyton, T. L., and J. N. Knull. 1925. Mexican bean beetle in Pennsylvania. Pa. Dep. Agric. Bull. 417. Hamilton, W. D. 1967. Extraordinary sex ratios. Science 156:477488 . Hinds, W. E. 1920a. Bean ladybird. J. Econ. Entomol. 13:430-431. Hinds, W. E. 1920b. Mexican bean beetle situation. J. Econ. Entomol 13:486-488. Howard, N. F. 1922. The Mexican bean beetle in the southeastern United States. J. Econ. Entomol. 15:265-275. Howard, N. F. 1924. The Mexican bean beetle in the East. U. S. Dep Agric. Farmers Bull. 1407. 13 pp. Howard, N. F., L. W. Brannon, and H. C. Mason. 1948. The Mexican bean beetle in the East and its control. U. S. Dep. Agric Farmers Bull. 1624. 17 pp. f 6 • Howard, N F., and L. L. English. 1924. Studies of the Mexican bean beetle m the Southeast. U. S. Dep. Agric. Bull. 1243. Howard N. F., and B. J. Landis. 1936. Parasites and predators of the Mexxcan bean beetle in the United States. U. S. Dep. Agric. Circ. 418. 12 pp. Jansen. W. P., and R. Staples. 1970. Transmission of cowpea mosaic virus by the Mexican bean beetle. J. Econ. Entomol. 63:1719King, P. E 1962. The effect of resorbing eggs upon the sex ratio of the offspring in Nasonia vitripennis (Hymenoptera: Pteromalidae) . J. Exp. Biol. 39:161-165. King, P. E.,_P. E. Askew, and C. Singer. 1969. The detection of parasitized hosts by male Nasonia vitripennis (Walker) Sr^EnLTr some possible implications. Proc. Roy. Entomol. Soc. London Ser. A Gen. Entomol. 44:85-90. Kolman, W. A. I960. The mechanism of selection for sex ratio Amer. Natur. 94:373-377. ''^^''fLL.-H^^^'H of Pediobius foveolatus (Crawford) (Eulophidae: Hymenoptera). Indian J. Entomol. 23:268-273. ''""'tLMn?;' Paradexodes epila^hnae, T:ch^"Buir^72l" 3^ pt bea^rbiiil^U:V^Ig.ie.

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180 Landis, B. J., and C. C. Plunmer. 1935. The Mexican bean beetle in Mexico. J. Agrlc. Res. 50:989-1001. List, G. M. 1922. Mexican bean beetle. J. Econ. Entomol. 15:373. Leonard, S. H. , and J. M. Ringo. 1978. Analysis of male courtship patterns and mating behavior of Brachymeria intermedia . Ann. Entomol. Soc. Amer. 7:817-826. Lockwood, D. F., and R. L. Rabb. 1979. The effects of two host plant species (soybeans, lima beans) and phenology on three population parameters of adult Mexican bean beetle ( Epilachna varivestis ) in North Carolina. J. Ga. Entomol. Soc. 14(3) :220-229. McAvoy, T. J., and J. C. Smith. 1979. Feeding and developmental rates of the Mexican bean beetle ( Epilachna varivestis ) on soybeans. J. Econ. Entomol. 72(6) : 835-836 . McPorter, R. , and M. Shepard. 1977. Response of Mexican bean beetle Epilachna varivestis larvae and the parasitoid Pedioblus foveolatus to Dimilin. Fla. Entomol. 60(l):55-66. Metcalf, R. A, 1980. Sex ratio, parent-offspring conflict, and local competition for mates in the social wasps Polistes metricus and Polistes variatus . Amer. Natur. 116(5) :642-654. Miller, M. C., and C. H. Tsao. 1974. Significance of wing vibration in male Nasonia vitripennis (Pteromalidae) during courtship. Ann. Entomol. Soc. Amer. 67:772-774. Nichols, M. P., and M. Kogan. 1972. The literature of arthropods associated with soybeans. I. A bibliography of the Mexican bean beetle Epilachna varivestis Mulsant (Coleoptera: Coccinellidae) . Biol. Notes nat. Hist. Surv. Div. St. Ill no 77. 20 pp. Plummer, C. C, and B. J. Landis. 1932. Records of some insects predaceous on Epilachna corrupta Muls. in Mexico. Ann. Entomol. Soc. Amer. 25:695-708. Price, P. W. 1973. Reproductive strategies in parasitoid wasps. Amer. Natur. 107:684-693. Price, P. W. 1975. Reproductive strategies of parsitoids. In: Evolutionary strategies of parasitic insects and mites (P. W. Price, ed.), pp. 87-111. Plenum Press, New York, London. 224 pp. Quattlebaum, E. C, and G. R. Garner. 1980. A new fungal pathogen (tentatively identified as a species of Beauvaria ) of the 35(3)320^322^^^^^^' ^P^^^^^"^ varivestis . J. Invertebr. Pathol.

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181 Quednau, F. W. 1967. Note on mating behavior and oviposition of Chrysocharis laricinellae (Hymenoptera: Eulophidae) , a parasite of the larch casebearer ( Coleophora laricella ) . Can. Entomol. 99:326-331. Rust, R. W. 1977. Evaluation of trap crop procedures for control of Mexican bean beetle, Epilachna varivestis , in soybeans and lima beans. J. Econ. Entomol. 70(5) : 630-632. Schlinger, E. I., and J. C. Hall. 1960. The biology, behavior and morphology of Praon palitans Muesebeck, an internal parasite of the spotted alfalfa aphid, Therioaphis maculata (Buckton) (Hymenoptera: Braconidae, Aphidiinae) . Ann. Entomol. Soc. Amer. 53:144-160. Schlinger, E. I., and J. C. Hall. 1961. The biology, behavior and morphology of Trioxys ( Trioxys ) utilis , an internal parasite of the spotted alfalfa aphid, Therioaphis maculata (Hymenoptera: Braconidae, Aphidiinae). Ann. Entomol. Soc. Amer. 54:34-45. Schroder, R. F. W. 1979. Host specificity tests of Coccipolipus epilachnae , a mite parasitic on the Mexican bean beetle ( Epilachna varivestis , biological control). Environ. Entomol. 8(1) :46-47. Scott, H. A., and H. C. Phatak. 1979. Properties of blackgram (mungo bean) mottle virus (transmitted by the bean leaf beetle, Cerotoma trifurcata , anf the Mexican bean beetle, Epilachna varivestis ) . Phytopathology 69(4) : 346-348. Sekhar, P. S. 1957. Mating, oviposition, and discrimination of hosts by Aphidius testaceipes (Cresson) and Praon aguti Smith, primary parasite of aphids. Ann. Entomol. Soc. Amer. 50:370-375. Shaw, R. F., and J. D. Mohler. 1953. The selective significance of the sex ratio. Amer. Natur. 87:337-342. Sherman, F., and J. N. Todd. 1939. The Mexican bean beetle in South Carolina. S. C. Agric. Exp. Stn. Bull. 322. 24 pp. Shepard, M., G. R. Carner, and S. G. Turnipseed. 1977. Colonization and resurgence of insect pests of soybean in response to insecticides and field isolation. Environ. Entomol. 6(4):501506. Shepard, M. , and G. T. Gale. 1977. Superparasitism of Mexican bean beetle, Epilachna varivestis (Coleoptera: Coccinellidae) by P edioblus foveolatus (Hymenoptera: Eulophidae): influence of temperature and parasitoid-host ratio. Entomophaga 22(3) :31532 1 •

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182 Simser, D. H., and H. C. Coppel. 1980a. Courtship and mating behavior of Brachymeria lasus (Hymenoptera: Chalcidae) , an imported gypsy moth parasitoid. Entomophaga 25 (4) : 349-355. Simser, D. H., and H. C. Coppel. 1980b. Female-produced sex pheromone in Brachymeria lasus and Brachymeria intermedia (Hymenoptera: Chalcidae). Entomophaga 25(4) : 373-380. Sloderbeck, P. E., and C. R. Edwards. 1979. Effects of soybean cropping practices on Mexican bean beetle ( Epilachna varivestis ) and redlegged grasshopper ( Melanoplus femur rub rum ) populations . J. Econ. Entomol. 72 (6) : 850-853. Smiley, R. L. 1974. A new species of Coccipollpus parasitic on the Mexican bean beetle (Acarina: Podaolipidae) . J. Wash. Acad. Sci. 64(4):298-302. Sorokina, A. P. 1973. The structure and development of reproductive system in chalcids (Hymenoptera, Chalcidae) which are parasites of coccids (Homoptera, Coccidea) . Part I. Entomol. Rev. 52(3): 396-403. Speicher, K. G., and B. R. Speicher. 1938. Diploids from unfertilized egg in Habrobracon . Biol. Bull. 74:247-252. Stevens, L. M. , A. L. Steinhauer, and J. R. Coulson. 1975a. Suppression of Mexican bean beetle on soybeans with annual inoculative releases of Pediobius foveolatus . Environ. Entomol. 4(6):947952. Stevens, L. M. , A. L. Steinhauer, and T. C. Elden. 1975b. Laboratory rearing of the Mexican bean beetle and the parasite, Pediobius foveolatus , with emphasis on parasite longevity and host-parasite ratios. Environ. Entomol. 4(6) :953-957. Stevens, L. M. , J. U. McGuire, A. L. Steinhauer, and P. A. Zungoli. 1977. The observed sex ratio of Pediobius foveolatus (Hymenoptera: Eulophidae) in field populations of Epilachna varivestis (Coleoptera: Coccinellidae) . Entomophaga 22(2): 175Sunzenauer, I. M., T. C. Elden, and A. L. Steinhauer. 1980. Soybean foliage consumption by the adult Mexican bean beetle, Epilachna varivestis Mulsant (Coleoptera: Coccinellidae). J. N. Y. Entomol Soc. 88(1) :76. Suomalainen, E. 1962. Significance of parthenogenesis in the evolution of insects. Annu. Rev. Entomol. 11:349-366. Tester, C. F. 1977. Constituents of soybean cultivars differing in insect resistance, Epilachna varivesti s. Phytochemistrv 16(12)1899-1901. ~

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183 Thomas, F. L. 1924. Life history and control of the Mexican bean beetle. Ala. Agric. Exp. Stn. Bull. 221. Tissot, A. N. 1943. The Mexican bean beetle in Florida. Fla. Entomol. 26(1): 1-8. Trivers, R. L., and H. Hare. 1976. Haplodiploidy and the evolution of social insects. Science 191:249-263. Turner, N. 1932. Mexican bean beetle injuring rye. J. Econ. Entomol. 25:1241. Turner, N. 1935. Effect of Mexican bean beetle injury on crop yield. J. Econ. Entomol. 28:147-149. Turner, N., and R. B. Friend. 1933. Cultural practices in relation to Mexican bean beetle control. J. Econ. Entomol. 26:115-123. Van den Assem, J., and G. D. E. Povel. 1973. Courtship behavior of some Muscidifurax species (Hymenoptera: Pteromalidae) : a possible example of a recently evolved ethological isolating mechanism. Neth. J. Zool. 23:465-487. Van Duyn, J, W., S. G. Turnipseed, and J. D. Maxwell. 1971. Resistance in soybeans to the Mexican bean beetle. I. Sources of resistance. Crop Sci. 11:572-573. Van Duyn, J. W,, S. G. Turnipseed, and J, D. Maxwell. 1972. Resistance in soybeans to the Mexican bean beetle. II. Reactions of the beetle to resistant plants. Crop Sci. 12:561-562. Vinson, S. B. 1972. Courtship behavior and evidence for sex pheromone in the parasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae) . Environ. Entomol. 1:409-414. Waddill, v., and M. Shepard. 1975. A comparison of predation by the pentatomids, Podisus maculiventris (Say) and Stiretrus anchorago (F.), on the Mexican bean beetle, Epilachna varivestis Mulsant, pest of soybeans. Ann. Entomol. Soc. Amer. 68(6) : 1023-1027. Walker, W. F., and W. S. Bowers. 1970. Synthetic juvenile hormones as potential coleopteran ovicides. J. Econ. Entomol. 63:1231-1233, Watson, J. R. 1942. The spread of Mexican bean beetle. Fla. Entomol. 25 • 23 • Werren, J. H. 1980. Sex ratio adaptation to local mate competition in parasitic wasp. Science 208:1157-1159. Whitfield, G. H., and C. R. Ellis. 1976. The pest status of foliar xnsects on soybeans and white beans in Ontario. Proc. Entomol Soc. Ont. 107:47-55.

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184 Wilkes, A. 1965. Sperm transfer and utilization by the arrhenotokous wasps Dahlbominus fuscipennis (Zett.) (Hymenoptera: Eulophidae) . Can. Entomol. 97:647-657. Yoshida, S. 1978. Behaviour of males in relation to female sex pheromone in the parasitoid wasp, Anisopteromalus calandrae (Hymenoptera: Pteromalidae) . Entomol. Exp. Appl. 23:152-162. Zungoli, P. A. 1979. Eupteromalus viridescens (Hymenoptera: Pteromalidae) , a new parasite association for Pediobius foveolatus (Hymenoptera: Eulophidae) and Epilachna varivestis (Coleoptera: Coccinellidae) . Proc. Entomol. Soc. Wash. 81(4): 663-665.

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APPENDIX 7-5 Per-mummy numbers of P. foveolatus progeny produced through single ovipositions by 38 individual parent females Parasite parent No. of parasite progeny per oviposit ion female no. Female + Male Female M^Ii 34 18 35 23 36 12 37 38 1 14 12 2 13 11 3 21 16 4 27 23 5 24 20 6 43 7 40 8 21 9 8 10 14 11 6 12 28 13 31 14 11 15 23 16 10 17 14 18 11 19 14 20 17 21 19 22 10 23 13 24 19 25 10 26 23 27 9 28 9 29 15 30 14 31 32 33 35 2 2 5 4 4 37 6 36 4 19 2 7 1 12 ^ 2 5 1 24 4 26 5 10 1 20 3 8 2 12 2 9 2 9 5 14 3 14 5 7 3 11 2 16 3 8 2 17 6 8 1 1 8 13 2 11 3 19 17 2 11 11 0 28 7 16 2 18 5 11 1 7 4 3 9 7 2 Total A 548 117 ^^^^^g^ 17.5 14.42 3.08 189

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APPENDIX 8-6 Per-mummy numbers of P^. foveolatus progeny (females /males) produced through 3 consecutive host exposures by 3 pairing combinations of young and old parents Numbers of progeny (females/males) with respect to their parent Pai'ent pairing combinations and host exposures f ema 1 e no. Young ?? X Old Old 5? X Old Old 52 X Youna dcT Exp 1 Exp 2 Exp 3 Exp 1 Exp 2 Exp 3 Exp 1 Exp 2 Exp 3 1 11/1 8/2 ** 0/18 0/14 ** ** * •* 2 23/2 18/3 16/1 ** * * U/ 1 *+ ** ** 3 ** 12/1 ** ** 0/32 0/18 * * 4 ** 17/0 * 0/27 *• U/ t*f * 0/11 5 0/25 0/22 ** ** ** U/ I U 0/16 0/12 6 16/3 * 1/32 * * 0/2 * 7 0/23 0/41 0/28 0/13 * 0/29 U/ 1 3 0/15 ** 8 35/2 18/2 26/5 0/18 0/34 0/3 0/10 0/18 0/26 9 * 33/5 * 0/14 0/32 0/15 ** ** ** 10 33/3 28/2 27/4 ** * ** * ** n 20/1 22/1 30/2 0/15 0/15 0/6 0/15 ** *• 12 0/15 * 0/24 0/14 * 0/17 0/20 0/22 0/16 13 0/24 0/25 0/15 ** * 0/15 * 14 15/3 32/3 • ** 0/38 * 0/12 0/12 0/10 15 7/4 32/3 ** 14/0 16/2 25/5 * * ** 16 16/3 * 18/6 0/9 0/16 0/34 * •* *** 17 17/1 35/4 30/6 * * ** 0/16 0/19 0/19 18 * 14/2 13/3 0/15 0/14 0/1 -** * * 19 26/5 * 28/4 * ** ** ** ** 20 0/26 0/45 0/24 0/20 0/29 0/16 0/19 0/16 « 21 11/2 * 0/16 * ** ** * *** 22 17/2 * ** 0/20 0/19 0/17 0/10 * * 23 15/3 13/4 * 0/23 * 0/19 ** *** 24 11/2 ** * * ** ** ** * ** 25 * 0/24 0/11 0/9 ** 0/18 * 0/14 26 13/1 * ** 14/2 * ** ** * 27 * 24/3 33/5 0/14 0/19 0/18 0/23 0/19 0/19 28 ** 35/3 18/2 0/32 0/33 0/12 ** *• 29 33/3 * 14/1 * 0/13 0/12 0/30 0/23 * 30 18/2 32/5 14/4 0/22 0/15 0/16 0/18 ** ** 31 22/2 * * 0/16 0/14 0/12 0/15 ** ** 32 31/4 29/3 15/3 0/12 ** ** * * ** * Host larva dead ** Host larva pupated *** Parent parasite female dead 190

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BIOGRAPHICAL SKETCH Limhuot Nong was born in 1939 in Krauchmar, province of Kompong Cham, Cambodia. He attended Sisowath high school from 1953 to 1957 and the National School of Agriculture, Animal Husbandry, and Sylviculture from 1957 to 1960. In 1960, he was awarded a USAID (United States Agency for International Development) scholarship to attend the University of Florida where he received his Bachelor and Master of Science degrees respectively in 1962 and 1963. In Cambodia, from 1964 to 1968, he was appointed by the government to serve the Ministry of Agriculture as Director of the Khmero-Japanese Center for Rice Research in the province of Battambang to help establish and implement rice research program. In 1968, he received a scholarship from the Deutsche Stiftung fur Entwicklungslander for training in plant protection in West Germany for 16 months. Upon his return to Cambodia in 1970, he served the Ministry of Agriculture as head of the Plant Protection Division. In 1971, he was designated by the government to be chief of the secretariat for the "General Secretariat for the Organization of the 7th General Assembly and the 11th and 12th Council Meetings of the Asian Parliamentarians' Union" held in Phnom Penh in 1971-72. In 1973, he was appointed by the government to serve the Ministry of Information, first as Deputy Director-General, then as Director of the National Television. 192

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193 From 1973 to 1975, he pursued his doctoral study in pM.nt Hematology at the University of the Bhilippines at Los Banos. His study was unfortunately disrupted by the takeover of Cambodia by the communists, a political situation that forced him to emigrate to the United States of America. At the University of Florida, he has been employed by the Department of Entomology and Hematology as research assistant since 1976. He is member of the Florida Entomological Society, and the International Organization for Biological Control of Noxious Animals and Plants (lOBC) . He was also member of the American Phytopathological Society; associate-member of the Sigma Chi Society (Florida Chapter); and member of the Phi Sigma Society (Alpha Chi Chapter, University of the Philippines at Los Banos). In Cambodia, he was secretary-general and member of the Khmer Alumni Association; president of the Association of the Directorate of Agriculture personnel; member of the Executive Committee of the Khmer Management Association; and member of the Association of Khmer Ingenieurs. He is married to Bicheng Ung in 1965 and has two sons, Yudyn and Yuvora, and two daughters, Yuda and Lyvia. His daughter Yuda died at the age of 10 in the midst of the Cambodian tragedy under the communist regime.

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Reece I. Sailer, Chairman Graduate Research Professor of Entomology and Nematology 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. Vernon G. Perry Professor of Entomology alid Nematology 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. ?rry U. Stimac Assistant Professor of Entomology and Nematology 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. ^ Daniel A. Roberts Professor of Plant Pathology

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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. May 1982 curtui Dean, /tzo liege of Agricurture Dean, Graduate School