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Ormia depleta

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Ormia depleta laboratory maintenance, strain identification, and evaluation of Aphis neriil as a banker species
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Welch, Craig H
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ix, 77 leaves : ill. ; 29 cm.

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Female animals ( jstor )
Gravid females ( jstor )
Honeydew ( jstor )
Humidity ( jstor )
Larvae ( jstor )
Mortality ( jstor )
Parasite hosts ( jstor )
Parasitoids ( jstor )
Photoperiod ( jstor )
Pupae ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF ( lcsh )
Entomology and Nematology thesis, Ph. D ( lcsh )
Flies ( lcsh )
Insect rearing ( lcsh )
Tachinidae ( lcsh )
City of Bradenton ( local )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 2004.
Bibliography:
Includes bibliographical references.
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Printout.
General Note:
Vita.
Statement of Responsibility:
by Craig H. Welch.

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University of Florida
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Ormia depleta:
LABORATORY MAINTENANCE, STRAIN IDENTIFICATION,
AND EVALUATION OF Aphis nerii AS A BANKER SPECIES














By

CRAIG H. WELCH


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

UNIVERSITY OF FLORIDA















ACKNOWLEDGMENTS

I would like to thank Dr. J. Howard Frank for the help, support, and patience he

has offered me over the past years and my committee for the advice and guidance offered

in the development of this dissertation. I would like to thank Dr. Robert Hemenway for

his countless hours of work in maintaining fly and cricket colonies, without which none

of this research would have been possible. I would like to thank the many people at the

Gulf Coast Research and Education Center in Bradenton, Florida, for their help in

running the sound traps and collecting flies. I would like to thank the Green Section of

the US Golf Association for their financial support of this research. Their support is what

made it possible to import the Os6rio strain of Ormia depleta, which was crucial to this

project. I would also like to thank Dr. Ken Portier for his help in statistical analysis.

Finally, I want to express my love and appreciation to my wife, Celeste, who for the past

seven years of my time in graduate school has shown me great love and patience, has

cooked dinners, done five million loads of laundry, managed our household, paid our

bills, and given birth to four daughters, who are the joy of our lives.















TABLE OF CONTENTS
Page

ACKNOWLEDGMENTS................................... ........... .............................ii

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

LIST O F FIG U R E S .................................................................. ....................... ......vi

A B ST R A C T ................................................................................ ............................. viii

CHAPTER

1 REVIEW OF LITERATURE .................................................... .......... .............. 1

2 VARIATION OF PARAMETERS FOR REARING ORMIA DEPLETA (DIPTERA:
TACHINIDAE) AND INDUCING DIAPAUSE...................... ......... .............. 7

Introduction ................................................................................ ......................7
M materials and M methods ......................................... ................. ............................ 10
Environmental Chamber Rearing Parameters................................................ 10
M elezitose and Fecundity ..................... ......... .................... ... 12
R esu lts ................................................................................... ............................... 13
Environmental Chamber Rearing Parameters............................. ............ 13
Melezitose and Fecundity ....................................................... 15
D iscu ssio n .................................................................. ....................... .................. 16


3 INTRASPECIFIC COMPETITION FOR RESOURCES BY ORMIA DEPLETA
(DIPTERA: TACHINIDAE) LARVAE ................................. ............................20

Introdu action ............................................................................ .............................. 20
M materials and M methods ............................................................. ...................... 22
R results ..................... ................................................................................................ 23
D iscu ssio n .................................................................................... ........................2 8


4 REARING ORMIA DEPLETA (DIPTERA: TACHINIDAE), A PARASITOID OF
MOLE CRICKETS (ORTHOPTERA: GRYLLOTALPIDAE), ON A FACTITIOUS
HOST, ACHETA DOMESTICUS (ORTHOPTERA: GRYLLIDAE)........................33

Introduction .............................................................................. ............................ 33


iii









M materials and M methods ................................................................ ............................. 36
Larval Development of Ormia depleta in Acheta domesticus .........................36
Larval Behavior within host............................................................................. 37
R results ......................................................................................... ................... 37
Larval Development of Ormia depleta in Acheta domesticus .........................37
Larval Behavior within host................... ................................38
Scapteriscus abbreviatus- .........................................................38
Acheta domesticus-.................................. ............. ............44
D iscu ssion .............................................................................. ..............................4 9


5 IDENTIFICATION OF TWO SOUTH AMERICAN GEOGRAPHICAL ISOLATES
OF ORMIA DEPLETA BY ANALYSIS OF CUTICULAR HYDROCARBONS.... 53

Introduction ............................................................................. ............................. 53
M materials and M methods ............................................................ ....................... 53
R results ..................... .................................................................................................. 55
D discussion ............................................................................... ....................... 59


6 SURVIVAL OF ORMIA DEPLETA WHEN CAGED WITH HONEYDEW-
PRODUCING APHIS NERII........................... ..............................60

Introdu action ............................................................................ .............................. 60
M materials and M methods ............................................................ ....................... 62
R esu lts .................................................................................... .............................. 6 3
D iscu ssio n .............................................................................. .............................. 6 3


APPENDIX

A THE EFFECT OF PUPAL SIZE ON EMERGENCE........................ .................66

B ATTEMPTED INDUCTION OF DIAPAUSE BY SHORT DAY PHOTOPERIOD IN
OSORIO STRAIN ORML4 DEPLETA .......................................... ......................67

C GAS CHROMATOGRAPHIC ANALYSIS OF THE CUTICULAR
HYDROCARBONS OF ORMIA DEPLETA TO IDENTIFY STRAINS .............69

LIST OF REFERENCES.......................................................................................71

BIOGRAPHICAL SKETCH............................. ..... ...... ....................77















LIST OF TABLES


Table page


1 Levels of parameters tested and treatment numbers assigned....................................... 12

2 Percentage of females gravid arcsine square root transformation F-values and p-
values for the main factors and interactions........................ ........ ............. 14

3 Percentage of females becoming gravid within each treatment combination...............14

4 The effect if temperature, humidity, and photoperiod on the percentage of mortality of
0 depleta ............................................... ...... ................................ 15

5 Mortality F-values and p-values for the main factors and interactions....................... 15

6 Mean number and length of 0. depleta larvae found in S. abbreviatus and A.
domesticus hosts.................. ............................................................... 38

7 Retention times of GC peaks for the cuticular hydrocarbons of 0. depleta..................56

8 The percent emergence of O. depleta pupae of varying sizes .....................................66















LIST OF FIGURES


Figure page

1 Old style rearing cages for 0. depleta................................... ........................... 11

2 Experimental style of rearing cage for 0. depleta....................... ............................ 11

3 The effect of planidia density used to inoculate mole crickets on the number of pupae
produced (error bars indicate standard deviation, significantly different means
indicated by letters over bars as determined by Duncan's procedure, a = 0.05).....24

4 The effect of number of planidia used to inoculate mole crickets on the number of
pupae produced regression analysis with 95% confidence bands.......................25

5 The effect of number of planidia used to inoculate mole crickets on the survival rate of
the larvae to the pupal stage (error bars indicate standard deviation, significantly
different means indicated by letters over bars as determined by Duncan's
procedure, a = 0.05) ..................................................... ... ................................... 25

6 The effect of number of planidia used to inoculate mole crickets on the survival rate of
the larvae to the pupal stage -regression analysis with 95% confidence bands .....26

7 The effect of number of planidia used to inoculate mole crickets on the mean weight of
the pupae produced (error bars indicate standard deviation, significantly different
means indicated by letters over bars as determined by Duncan's procedure, a =
0 .0 5 ) ............................................................ .................... ...............................2 6

8 The effect of number of planidia used to inoculate mole crickets on the mean weight of
the pupae produced regression analysis with 95% confidence bands...................27

9 The effect of host cricket weight on mean pupal weight (error bars indicate standard
deviation, significantly different means indicated by letters over bars as determined
by Duncan's procedure, a = 0.05) .................................................28

10 The effect of host cricket weight class on the mean weight of the pupae produced -
regression analysis with 95% confidence bands.................................... ........... 28

11 Larva of 0. depleta in S abbreviatus host one day after inoculation.......................40

12 Larva of 0. depleta in S. abbreviatus host two days after inoculation......................40









13 Larva of 0. depleta in S. abbreviatus host three days after inoculation..................... 41

14 Larva of 0. depleta in S. abbreviatus host four days after inoculation.....................41

15 Scapteriscus abbreviatus host five days after inoculation showing the external
evidence of the attachment point of 0. depleta larva ............................................. 42

16 Larvae of 0. depleta in S. abbreviatus host five days after inoculation....................42

17 Larvae of 0. depleta in S abbreviatus host six days after inoculation.....................43

18 Two 0. depleta larvae in S. abbreviatus host six days after inoculation showing detail
of the larval attachment to the host................................... ......................43

19 Larva of 0 depleta in S. abbreviatus host seven days after inoculation ....................44

20 Larva of 0. depleta in A. domesticus host one day after inoculation........................45

21 Larva of 0. depleta in A. domesticus host two days after inoculation......................46

22 Larva of 0. depleta in A. domesticus host three days after inoculation ....................46

23 Larva of 0. depleta in A. domesticus host four days after inoculation......................47

24 Larvae of 0. depleta in A. domesticus host five days after inoculation ....................47

25 Larvae of 0. depleta in A. domesticus host six days after inoculation......................48

26 Larvae of 0. depleta in A. domesticus host seven days after inoculation ...................48

27 Seven day old larvae of 0. depleta from S. abbreviatus (top) and A. domesticus
(bottom ).................................................... .................................................... 49

28 The growth of 0. depleta larvae in alternate hosts: S. abbreviatus and A. domesticus49

29 The mean percentage of total peak area for the gas chromatograms of the cuticular
hydrocarbons of female Piracicaba strain Ormia depleta.............................. ..56

30 The mean percentage of total peak area for the gas chromatograms of the cuticular
hydrocarbons of female Os6rio strain Ormia depleta.............................................57

31 The mean percentage of total peak area for the gas chromatograms of the cuticular
hydrocarbons of male Piracicaba strain Ormia depleta...................................57

32 The mean percentage of total peak area for the gas chromatograms of the cuticular
hydrocarbons of male Os6rio strain Ormia depleta............................................ 58

33 Scatter plot representation of the values for peaks F and I for male 0. depleta of the
Piracicaba and Os6rio strains........................... .... ........................58















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

ORMIA DEPLETA:
LABORATORY MAINTENANCE, STRAIN IDENTIFICATION,
AND EVALUATION OF APHIS NERII AS A BANKER SPECIES

By

Craig H. Welch

August, 2004

Chair: J. Howard Frank
Major Department: Entomology and Nematology

Ormia depleta (Wiedemann) is a tachinid fly used in the biological control of

Scapteriscus spp. mole crickets. Several changes to the current method of rearing 0.

depleta in the laboratory were attempted. Manipulations of temperature, humidity, and

photoperiod during the pupal and adult stages showed that the percentage of gravid

females was higher at 230C than at 270C and at 70% relative humidity than at 85%, but

photoperiod did not have a significant effect. A diet of melezitose did not increase the

fecundity of flies compared to a melezitose-free diet. Increasing the number of planidia

used to inoculate host mole crickets increased the number of pupae produced, but reduced

the survivability of those planidia and the mean weights of the pupae produced. Larger

host mole crickets were also found to produce larger pupae. Acheta domesticus was tried

as a factitious host and found to be unsuitable. Although pupae were produced, they were

small and few in number. Dissections of hosts with developing larvae showed that the








larvae in Acheta developed slowly and apparently had trouble emerging from the host.

Identification of two Brazilian strains of O depleta was performed by gas

chromatographic analysis of cuticular hydrocarbons. The results indicated that, using the

model used in this experiment, the strains could be identified with 80% confidence. An

evaluation of the honeydew of Aphis nerii on its milkweed host indicated that it is an

adequate carbohydrate source for 0. depleta, but further study is needed in this area

before any recommendations can be made.













CHAPTER 1
REVIEW OF LITERATURE

Four species of mole crickets are currently established in Florida. Three of these

are immigrant pests of the genus Scapteriscus: S. vicinus Scudder (the tawny mole

cricket), S. abbreviatus Scudder (the short-winged mole cricket), and S. borellii Giglio-

Tos (the southern mole cricket). These three species can be differentiated by several

features including dactyl spacing and the characteristics of the male song (Nickle and

Castner 1984). All three Scapteriscus spp. mole crickets are believed to have arrived

from South America around the year 1900 (Walker and Nickle 1981). S. vicinus and S.

borellii are thought to have initially arrived in the United States through Brunswick,

Georgia, in 1899 and 1904, respectively (Walker and Nickle 1981, Nickle and Castner

1984). Scapteriscus abbreviatus apparently arrived several times between 1899 and 1923

into several ports in Florida and southernmost coastal Georgia (Walker and Nickle 1981).

Since then, Scapteriscus mole crickets have become major agricultural pests throughout

Florida and into Georgia, Mississippi, South Carolina, and other states (Frank et al.

1998). They are the most damaging pests of turf and pasture grasses in Florida and are

also pests of vegetable and field crops (Reinert and Short 1980). Bahiagrass, Paspalum

notatum Fluegge, is apparently the preferred, and therefore most damaged, plant species

(Reinert and Short 1980). Damage is typically caused by either the tunneling of the mole

crickets, which exposes the roots to desiccation, or by direct feeding on the roots (Reinert

and Short 1980). Traditionally, these pests have been controlled through the use of

chemical pesticides such as carbaryl, chlorpyrifos, acephate, bifenthrin, fipronil,








imidacloprid, and others (Buss et al. 2002). Pesticide use is only a short-term solution

because most of Florida's over three million hectares of bahiagrass are in low-

maintenance areas such as pasture and roadside (Short and Reinert 1982), providing an

unlimited supply of mole crickets during flight periods in September-October and April-

May (Walker et al. 1992a). However, because multiple treatments are required to achieve

adequate levels of control (Parkman et al. 1996) and because of other concerns raised by

pesticide use, alternative methods of control have been sought.

At least four biocontrol agents have been considered for control of immigrant mole

crickets, three of which have already been released in the field. The entomopathogenic

nematode Steinernema scapterisci Nguyen and Smart has been effective against mole

cricket populations, with infected individuals being found up to eight years in pastures

after initial treatment and twelve years on golf courses after initial treatment (Frank et al.

1999, Frank et al. 2002). Steinernema scapterisci shows a much higher specificity toward

its mole cricket hosts than do other Steinernema species (Nguyen and Smart 1990) and

has had a measurable effect on mole cricket populations in treated areas (Parkman et al.

1994). The sphecid wasp Larra bicolor F. has been released and established in Florida,

and its population is spreading in northern Florida. However, little research has been

done on its effectiveness to date. The bombardier beetle Pheropsophus aequinoctialis L.,

a predator of mole cricket eggs, has not yet been released in Florida, but may be released

in the future (personal communication from Dr. J.H. Frank, University of Florida).

The tachinid fly Ormia depleta (Wiedemann) is an obligate parasitoid of

Scapteriscus mole crickets. Female flies are phonotactic to the call of the male

Scapteriscus spp. crickets (Fowler 1987, Fowler and Garcia 1987, Walker et al. 1996).









Phonotaxis is an uncommon host-locating method known in only one other dipteran,

Colcondamyia auditrix Shewell, a sarcophagid parasitoid of cicadas (Soper et al. 1976).

Ormia spp. are nocturnal and crepuscular, and collection can only be done reliably with

sound emitter traps that mimic the call of the host. Ormia depleta was originally brought

from Piracicaba, Brazil, to be used as a biocontrol agent against Scapteriscus spp. mole

crickets and was first released in 1988 (Frank et al. 1996). Since then, it has become

established in 38 counties in Florida, and in some it has been demonstrated to suppress

mole cricket populations (Parkman et al. 1996). This original strain seemed to have a

northern limitation to its establishment around 280 N latitude (Walker et al. 1996).

Because the strain was collected near Piracicaba, Estado de Slo Paulo, around 230 S

latitude (Piracicaba strain), it was speculated that flies collected from more temperate

latitudes would be more cold-hardy and able to establish farther north in the United

States. Dr. Howard Frank returned to Brazil in 1999, and collected a new strain of flies

near Os6rio in Estado do Rio Grande do Sul, approximately 300 S latitude (Os6rio strain)

(Frank 2002). The Os6rio strain was laboratory-reared and in 2000-2001, several releases

were made in North Carolina, Georgia, Texas, and Louisiana. Adequate trapping and

monitoring of flies in these locations have not yet occurred to determine whether the

Os6rio strain has established (Frank 2002).

Flies in the family Tachinidae are all parasitoids of other arthropods, almost

exclusively other insects (Cantrell and Crosskey 1989, Andersen 1996). In the more

primitive tachinid species, eggs are deposited directly on the host, whereas other species

deposit microtype eggs on their host's food plant, inject eggs into the host's body wall, or

drop newly hatched larvae on or near the host (Cantrell and Crosskey 1989). The most








common hosts are the larvae of Lepidoptera and Coleoptera, but Hymenoptera,

Hemiptera, Orthoptera, Dermaptera, other Diptera and, in some species, centipedes and

isopods are hosts (Cantrell and Crosskey 1989, Andersen 1996). Tachinids are among the

most important natural regulators of insect populations (Oesterbroek 1998). Few

tachinids are known to be species-specific in their choice of host, with some species

attacking hosts from three orders of insects (Cantrell and Crosskey 1989). Their success

as biocontrol agents has been mixed, with many attempts failing (Cantrell and Crosskey

1989). Adult tachinid flies, in general, are known to feed at flowers and do not require

any significant source of protein (Andersen 1996). They are very active and require a

daily source of sugar from plant nectar, homopteran honeydew, or sap from tree wounds

(Oesterbroek 1998).

Wineriter and Walker (1990) developed the original rearing protocol for 0. depleta.

This is the protocol used throughout this dissertation unless specified otherwise. This

protocol requires that gravid female flies be sacrificed and the oviducts removed. The

oviducts are then placed on moist filter paper and gently torn open, releasing the planidia.

Three fully developed planidia are then placed behind the posterior margin of the

pronotum of the host. The hosts (usually S. abbreviatus) are then placed in individual

vials of moist sand while the larvae develop. Both male and female mole crickets are

used. Twelve days after inoculation, the vials are emptied and the pupae retrieved from

the sand and placed in emergence boxes. Each box contains -2 cm moist sand and 100

pupae are placed on the sand and then covered with another 5 mm layer of moist sand.

Each emergence box is then placed in a large rearing cage consisting of a clear acetate

tube covered on both ends with bucket lids. These cages are placed near a window that








receives indirect sunlight and the flies are allowed to emerge into the cage. Food and

water are offered in Petri dishes wicked with cotton balls. After approximately four

weeks in the rearing cage, gravid females are removed and sacrificed to produce the next

generation. For the colonies kept at the University of Florida mole cricket rearing

laboratory, two rearing cages are maintained under normal circumstances. Each cage is

started with approximately 100 pupae. To obtain this number of pupae, approximately

200 S. abbreviatus are inoculated with three planidia each, with the expectation of

obtaining two viable pupae per host (personal communication with Robert Hemenway,

University of Florida).

Although this rearing protocol has been fairly successful in maintaining laboratory

colonies of 0. depleta, a new protocol was desired that would reduce the unpredictable

variability in the percentage of gravid flies produced in each generation as well as

allowing for smaller colonies to be maintained. These smaller colonies could be reared in

environmental chambers to control the factors of photoperiod, temperature, and humidity.

The number of planidia per host could be manipulated to determine the optimal use of

this limiting factor in colony growth.

Because the Os6rio strain has been released but not yet recaptured, a method was

needed to differentiate between the two strains to identify the strain of individuals

recaptured in the future. This is especially important in identifying flies from areas that

lie between the established range of the Piracicaba strain and the new release sites of the

Os6rio strain.

In addition, there is some demand for a method to locally augment populations of

0. depleta in areas where a greater level of mole cricket control is desired, such as golf






6

courses and bahiagrass pastures. The use of a plant/aphid banker system for the

production of honeydew and subsequent attraction of 0. depleta was investigated to

determine whether the honeydew produced by this system is an adequate food source for

the flies.














CHAPTER 2
VARIATION OF PARAMETERS FOR REARING ORMIA DEPLETA (DIPTERA:
TACHINIDAE) AND INDUCING DIAPAUSE


Introduction

The protocol for rearing 0. depleta set by Wineriter and Walker (1990) has been

successful in keeping colonies at the University of Florida's mole cricket rearing

laboratory almost continuously since 1988. The greatest limitation of this method is

primarily in the large number of flies required per cage in order to produce gravid

females. Many advantages would be gained if a method for rearing 0. depleta in smaller

colonies were to be devised.

Although the Piracicaba strain of 0. depleta has become well established

throughout southern Florida and somewhat established in central Florida, its range falls

far short of that of its host (Frank et al. 1996). The releases of the Os6rio strain may

increase the fly's range, but this has not yet been demonstrated (Frank 2002).

Augmentative releases may be a possible solution, but 0. depleta is a very difficult

organism to reliably rear under the protocol set by Wineriter and Walker (1990). The

populations show a great deal of instability and reproductive success is unpredictable. In

an attempt to control some of the unpredictability of laboratory colonies, a revised

protocol was tested to rear flies under the controlled conditions of an environmental

chamber. The effects that had the greatest probability of affecting the outcome of the

colonies (humidity, temperature, and photoperiod) were tested at two levels each. Natural

sunlight, one of the factors long thought to be necessary for 0. depleta to mate








successfully (Wineriter and Walker 1990), was not investigated. Dependence on natural

sunlight introduces another uncontrollable variable and other research has proved that

Ormia depleta can reproduce successfully in the absence of natural light (Welch 2000).

Perhaps the most compelling reason for a new rearing method is to maintain smaller

colonies. The current method requires at least 100 pupae to be used in each large (30 cm

diameter x 60 cm tall, approximately 35 liter) cage (Figure 1). Maintaining fewer flies in

smaller cages would facilitate experimentation and reduce the risk to the entire laboratory

colony, should one colony fail. This experiment was designed to determine the levels of

temperature, humidity, and photoperiod that would be most conducive to rearing O.

depleta in small colonies in environmental chambers.

Many homopterans, including aphids, feed on the liquids they extract from plants

by piercing the phloem tissues with their stylets (Owen 1978). The result is a diet very

high in sugars, and excess sugars are excreted as honeydew, which is used as a food

source for many organisms such as fungi, bacteria, bees, moths, ants and flies, including

0. depleta (Owen 1978, Welch 2000). Most of the sugars found in homopteran honeydew

are also occur in the saps of the plants on which they feed, such as fructose, glucose and

sucrose (Percival 1965). Melezitose (a-D-glucose-[1 --3]-P-D-fructose-[2-+ 1]- a-D-

glucose) is a trisaccharide found in homopteran honeydew, but not in plants (Petelle

1980). The enzyme which makes melezitose has been isolated from the midgut of aphids

and shown to produce melezitose, fructose, and glucose from sucrose (Petelle 1980).

It is not fully understood why homopteran insects produce melezitose. Owen

(1978) hypothesized that aphids produce honeydew because it offers them an

evolutionary advantage. His theory has two requirements. The first requirement is that an








aphid and all her parthenogenically-produced offspring be considered a single

evolutionary individual (Janzen 1977) such that any benefit provided to offspring can be

considered benefit to self. The second, and most crucial to his argument, is that

melezitose must be a sugar source which is not as widely usable to other organisms as

other sugars, but highly usable to nitrogen-fixing bacteria. Aphids that produced

melezitose-heavy honeydew would be benefiting their host plants by providing a nitrate

source and, therefore, benefiting their offspring and, from the evolutionary individual

standpoint, themselves. This theory was refuted by Petelle (1980), who found that

fructose was nine times better than melezitose as a carbohydrate source for nitrogen-

fixing bacteria. Instead, he postulated that, because the enzymatic action which produces

melezitose prevents sucrose from breaking down into fructose and glucose and in the

process ties up two molecules of glucose and one of fructose, the production of

melezitose reduces the concentration of monosaccharides in the midgut of the insect. This

reduction in concentration would be beneficial to the insect in that it would reduce the

uptake of these sugars. Too much sugar uptake would increase the osmotic pressure and

require the insect to ingest a large amount of water.

The question of how melezitose influences the longevity of 0. depleta has been

investigated (Welch 2000). It was found to be a suitable diet, indicating that 0. depleta

has the necessary enzymes to hydrolyze melezitose, but it did not perform better than a

diet of hummingbird feeder nectar containing no melezitose. Although this may provide

some insight into the importance of melezitose in the diet of 0. depleta, for the purpose

of maintaining laboratory colonies, longevity is not as important as the ability to produce








offspring. An experiment was designed to determine the influence of melezitose on the

fecundity of mated female 0. depleta.

Materials and Methods

Environmental Chamber Rearing Parameters

Scapteriscus abbreviatus were obtained from the mole cricket rearing laboratory

at the University of Florida. This colony was originally collected from Broward County,

Florida, and was inoculated with planidia from Os6rio strain 0. depleta. Each mole

cricket was inoculated by placing four planidia behind the posterior margin of the

pronotum and then placing it in a vial of moist sand. After 12 days, pupae were collected

from the mole cricket vials. Four small cups (approximately 10 cm diameter, 4 cm deep)

were filled with approximately 2 cm damp sand, and 35 0. depleta pupae were placed in

each cup on top of the sand with the spiracles pointing upward. The pupae were then

covered with a thin layer (0.5-1.0 cm) of moist sand and each cup was placed in a small

plastic rearing box (10 x 15 x 20 cm) lined with paper towel and covered with a lid

(Figure 2). The lid was ventilated with a 10-cm-diameter hole covered with a fine screen

mesh. A nutrient solution containing a 1:1:1 solution of sucrose, fructose, and melezitose

(40g each /L) was kept in a small plastic Petri dish wicked with cotton and placed in each

rearing box. Each rearing box was also provided with water in a small plastic Petri dish

wicked with cotton. The nutrient solution was colored green for identification with

approximately 251pL/L of food coloring (McCormick, Hunt Valley, MD). Both the

nutrient solution and water were refilled as needed and replaced if mold appeared. Each

box with pupae was then placed in a separate Florida Reach-In environmental chamber

(Walker et al. 1993)



























Figure 1: Old style rearing cages for 0. depleta


Figure 2: Experimental style of rearing cage for 0 depleta


set to a specific temperature, humidity and photoperiod. Each of these three variables had

two levels determined by preliminary experimentation (Table 1) and was set up according

to a balanced incomplete block design (a=8, b=14, k=4, r=7, k=3). This design was

necessary to accommodate all eight treatment combinations. A fully randomized model








would have been preferred, but this would require eight environmental chambers, which

were not available, and enough pupae to set up eight experimental units, which were a

greater number than our laboratory colony could reliably produce. The low humidity of

70% was used because, in preliminary trials, flies kept much below that range suffered

high mortality. The high humidity of 85% was chosen because the Florida Reach In units

do not do a good job of maintaining relative humidity higher than that amount. After 30

days in the chambers, all flies were removed and checked for gravidity. Mature planidia

are easily visible through the abdomen of gravid females. The numbers of dead males,

dead females, live males, live females, and gravid females were recorded. Gravid females

were then sacrificed and their planidia used to inoculate mole crickets for the next

generation. For statistical analysis, the percentage of gravid females was transformed

using the arcsine square root transformation (sinl x). Both the arcsine square root of the

percentage of gravid females and the mortality of the flies were analyzed by ANOVA

(SAS Institute 2001).

Table 1: Levels of parameters tested and treatment numbers assigned

Treatment # 1 2 3 4 5 6 7 8

Humidity 70% 85% 85% 70% 70% 85% 70% 85%

Temperature 23 C 23 C 27C 27 C 23 C 23 C 27 C 27C

Photoperiod 10hrs 14hrs l0hrs 10hrs 14hrs 10obrs l4hrs 14 hrs


Melezitose and Fecundity

Colonies of Os6rio strain 0. depleta were obtained from the mole cricket rearing

laboratory at the University of Florida. The colonies were reared on S. abbreviatus and








maintained following the protocols outlined previously, with the exception of the food

provided. Colonies were provided a diet of either hummingbird nectar (Perky-Pet

Denver, Colorado, Instant Nectar, containing: unspecified proportions of sucrose,

glucose, tartaric acid, sodium benzoate, artificial color and flavor), which had long been

used as a sugar source for laboratory rearing at the University of Florida mole cricket

rearing laboratory, or a solution of melezitose only (100 g/L). Both diets were provided

ad lib and replaced if signs of mold appeared. When gravid females appeared

(approximately 6 weeks after mole crickets were inoculated), females were sacrificed and

their oviducts removed by dissection of the abdomen. The planidia were removed and

used to inoculate mole crickets for the next generation. During inoculation, the number of

mature planidia was counted and recorded. Only dark, well-sclerotized larvae were

counted. Immature, light colored planidia and eggs were not counted. This procedure was

repeated over several generations of 0. depleta during the course of maintaining the

laboratory colonies.

Results

Environmental Chamber Rearing Parameters

Determining the statistical significance of the variables temperature, humidity and

photoperiod was not possible using the variable "ratio gravid" (number of gravid females

/ number of live females). Only 18 out of the 56 trials produced gravid females. The large

number of zeros shifted the distribution so that parametric statistics were not meaningful.

By changing the unit of measurement from treatments to individual flies, it was possible

to produce a binomial distribution of female flies that survived the duration of the

treatment and indicate whether or not each one became gravid. Using the arcsine square

root transformation, the ANOVA results indicated that the lower temperature (230C)








produced a higher percentage of gravid females (F = 9.40, P = 0.004) and that humidity

was marginally significant (F = 4.05, P = 0.052), with the lower humidity (70% RH)

producing more gravid females. Photoperiod was not significant (P > 0.05) nor were any

of the two-way or three-way interactions (Table 2). The analysis of these data remains

somewhat suspect, however, due to the fact that blocks 3, 10 and 12 failed to produce any

gravid females. Because of this, the statistical software was unable to converge on a

likelihood confidence interval, so some observations were eliminated from the analysis

(SAS Institute 2001). Both the lower temperature and the lower humidity produced a

larger percentage of gravid females (Table 3).

Table 2: Percentage of females gravid arcsine square root transformation F-values and
p-values for the main factors and interactions

Block 0.54 0.884

Temperature 9.40 0.004

Humidity 4.05 0.052

Photoperiod 2.56 0.119

Temperature x Humidity 1.60 0.214

Temperature x Photoperiod 0.75 0.394

Humidity x Photoperiod 0.01 0.994

Temperature x Humidity xPhotoperiod 1.01 0.323


Table 3: Percentage of females becoming gravid within each treatment combination.
23 C 27 C
10 Hours 14 Hours 10 Hours 14 Hours
70% RH 15.98% 17.92% 3.90% 3.57%

85% RH 18.79% 1.10% 0.00% 0.00%








An ANOVA analysis of the mortality of the flies showed the treatment effect to be

highly significant (F = 3.67, P < 0.003). The percentage mortality for the eight treatments

ranged from 30.4% to 72.6% (Table 4). The higher temperature and longer photoperiod

produced higher mortality, whereas humidity and all two and three way interactions were

not significant (Table 5). The block effect was not significant (F= 1.51, P > 0.15).

Table 4: The effect if temperature, humidity, and photoperiod on the percentage of
mortality of 0. depleta
23 C 27 C
10 Hours 14 Hours 10 Hours 14 Hours
70 % RH 39.1% 41.3% 44.7% 67.3%

85% RH 30.4% 44.0% 55.6% 72.6%


Table 5: Mortality F-values and p-values for the main factors and interactions

Source F-value P
Block 1.51 0.156

Temperature 13.87 0.0005

Humidity 0.65 0.424

Photoperiod 11.24 0.002

Temperature x Humidity 1.46 0.232

Temperature x Photoperiod 0.77 0.386

Humidity x Photoperiod 0.43 0.514

Temperature x Humidity x Photoperiod 2.85 0.098

Melezitose and Fecundity

The planidia of 11 melezitose-fed flies and 12 non-melezitose-fed flies were

counted. The mean number of planidia for melezitose-fed females was 248 + 73 (SD)

with a range of 121-392. For non-melezitose fed females, the mean was 232 + 71 (SD)








with a range of 117-341. A t-test showed no difference between these two treatments (P=

0.61).

Discussion

The rearing of 0. depleta inside environmental chambers was only somewhat

successful. For some generations, more flies became gravid in the four environmental

chambers than in the two large laboratory colonies. However, as previously stated, there

were several generations that did not produce any gravid females in the environmental

chambers. No tests were conducted to determine whether the colonies inside the

chambers would be more successful if maintained as large colonies versus the small

colonies used in this experiment.

Temperature was the factor of greatest significance. Although the flies can

develop normally at temperatures above 300 C (Cabrera 2000), mold appeared more often

in the chambers kept at 270 C versus those kept at 230 C. Food containers had to be

changed very frequently and fungi were visible growing on the paper towel lining the

bottom of the cage on several occasions. The increase in mortality from 230 C to 270 C

(38.7% vs. 60.1%) could have also been due in part to problems caused by fungal growth.

Fungi have been known to cause high mortality in laboratory colonies of Drosophila

hydei Sturtevant and D. melanogaster Meigen (Hodge and Mitchell 1997).

It was surprising that humidity was not a more significant factor in the successful

rearing of 0. depleta than the data show (P = 0.052). It may have been discounted

somewhat in this experiment due to the fairly narrow range of humidities used. The lower

humidity level most likely was more successful again because of reduced fungal growth.

There was a greater incidence of fungi in the high humidity chambers over the low









humidity chambers. The mortality between the two levels of humidity was essentially the

same. Humidity is often a major source of mortality in rearing insects (Tsitsipis 1980,

Leatemia et al. 1995); this further supports the hypothesis that the range of humidity

tested here was too narrow to show the real significance of this factor.

Photoperiod was tested due to the traditional thinking that sunlight was in some

way necessary for 0. depleta to mate successfully. The production of gravid females

within the environmental chambers showed that sunlight is not a necessity for 0. depleta

to reproduce; however, it would be irresponsible to discount that the regular laboratory

colonies did, for some generations, produce more gravid females than those in the

environmental chambers. Until specific testing has been conducted, no changes would be

suggested to that particular practice. It was unexpected to find such a drastic difference

between the mortality of the flies kept on the two levels of photoperiod. The higher

mortality caused by the longer photoperiod may have been due to these flies having to go

longer periods without eating, as the flies do not often appear to feed actively during the

daylight hours.

Because some generations did not produce any gravid females in the small colonies

in the environmental chambers, it cannot be recommended that this method be adopted

for regular laboratory use. Future research in this area may investigate how larger

colonies of flies, such as those currently used in laboratory rearing of 0. depleta, are able

to produce gravid females in environmental chambers. The most likely parameters to be

successful would be in the range of the lower temperature and lower humidity used in

this experiment.








Previous research has shown that melezitose does not increase or decrease the

survival of 0. depleta compared to other carbohydrate diets (Welch 2000). In the

ichneumonid wasp Bathyplectes curculionis Thomson, longevity was significantly

reduced when fed a diet of melezitose versus other sugars (Jacob and Evans 2004). This

indicates that 0. depleta does have some benefit from melezitose. There is, however, no

evidence that it is important for reproductive success. Comparable fecundities were

produced by both a diet of melezitose and a non-melezitose diet supports the hypothesis

that 0. depleta is an opportunistic feeder rather than an obligate honeydew feeder. It

would seem that an obligate honeydew feeder would produce fewer offspring from being

fed only a melezitose-free diet. This study showed no such reduction. Whether melezitose

might have an effect on the fecundity of 0. depleta when combined with other sugars

remains to be tested. This study does support the hypothesis that 0. depleta has the

necessary enzymes to hydrolyze melezitose.

Previous research showed that sucrose is quickly broken down in the crop of the

fly, but melezitose is hydrolyzed very slowly (Welch 2000). Burkett (1998) found similar

results in carbohydrate hydrolysis in mosquitoes. The likely location for melezitose

hydrolysis is the midgut. There may be some advantage to feeding on melezitose in that it

would be absorbed more slowly than other sugars, prolonging the time over which energy

could be derived from the food. This may make it less necessary to store energy in the

form of trehalose, fats or glycerol, which require energy to synthesize. Another possible

advantage of feeding on melezitose lies in its availability. The visual and/or olfactory

attractants associated with honeydew have not been investigated as they apply to O.

depleta. Whether these flies search out honeydew or come upon it randomly is unknown.









But once found, it is an easy food source to access. No specialized mouthparts are

required to reach the source, in contrast to many floral nectars, nor is it necessary to

pierce the skin of fruits or other plant tissues. The apparently non-specialized mouthparts

of 0. depleta could be the result of two possibilities. Either they are truly opportunistic

and feed on whatever carbohydrate sources they are able to locate, including honeydew,

and have never evolved any type of specialized feeding apparatus, or their mouthparts are

specialized and have evolved to feed primarily on honeydew. A good test for this would

be to determine whether 0. depleta have other behavioral or physiological traits which

show a specialization towards honeydew feeding. One such characteristic would be the

flies' ability to locate honeydew deposits. If 0. depleta has evolved the ability to locate

honeydew by olfactory or visual cues, then it is likely that it could be considered more of

a specialist feeder on honeydew than if it were to only find it by chance. The high

percentage of field-caught 0. depleta which had fed on honeydew, as indicated by the

presence of melezitose in the crop (Welch 2000), seems to support the hypothesis that

they are not merely encountering honeydew randomly. Future research into the attractant

properties of honeydew, honeydew constituents, and plant/aphid systems to 0. depleta

would be very useful both for determining the true specialization of this species as well

as for making banker plant recommendations for localized augmentation of fly

populations.













CHAPTER 3
INTRASPECIFIC COMPETITION FOR RESOURCES BY ORMIA DEPLETA
(DIPTERA: TACHINIDAE) LARVAE

Introduction

Ormia depleta can be a difficult organism to maintain in a lab colony. One of the

factors that makes them difficult lies in the variable and generally low proportion of

gravid females obtained under the current lab rearing protocol. A colony of 100

individuals may in one generation produce 20 gravid females and in the next only one or

two or even zero. Therefore, it is necessary to determine the best way to use the number

of planidia available in any one generation to produce the maximum number of healthy

pupae to start the next generation. This must also be balanced with the expense of rearing

the mole cricket hosts, which are very labor intensive to maintain. Current laboratory

protocol dictates that three planidia be placed under the posterior margin of the pronotum

of each Scapteriscus host when inoculating by hand (Personal communication with

Robert Hemenway, University of Florida). If there is competition among the larvae for

resources within the host, fewer planidia per host may increase the chances of survival

for those planidia and may produce larger pupae. This would, however, require more

hosts to produce enough pupae to maintain the colony. Inoculating hosts with more

planidia may increase the number of pupae and reduce the cost associated with host

rearing, but superparasitoidism should be avoided as well as the production of pupae and

adults with reduced fitness.








The effect of superparasitoidism on the developing larvae of many insect species

has been studied extensively. Superparasitoidism has been shown to increase larval

development time in Cotesia glomerata (L.) (Gu et al. 2003), Microplitis croceipes

(Cresson) (Eller et al. 1990), and Cotesiaflavipes Cameron (Potting et al. 1997).

However, Bai and Mackauer (1992) found no increase in the larval development time of

Aphidius ervi Haliday when subjected to superparasitoidism. In some species, it has been

shown to decrease survivorship (Vinson and Sroka 1978, Gu et al. 2003) but not in other

species (Harvey et al. 1993). The offspring of the ichneumonid wasp Venturia canescens

(Gravenhorst) were smaller under the condition of superparasitoidism (Harvey et al.

1993), but the wasp A. ervi actually increased in size (Bai and Mackauer 1992). The most

common effects of superparasitoidism apparently are an increase in the number of brood

produced, but with an overall reduction in the fitness of those offspring (van Dijken and

Waage 1987, Vet et al. 1994).

Previous research with 0. depleta showed that there was no relationship between

the number of planidia used to inoculate the host and the number of pupae produced

(Fowler 1988), but preliminary research done here showed that higher numbers of pupae

could be produced than previously recorded. Additionally, Fowler and Martini (1993)

found a weak correlation between host size used and the weights of the flies produced. In

the present experiment, this relationship was also examined to determine whether host

weight is an important factor in determining which hosts should be used. The goal of this

experiment was to determine if an increase in the number of pupae produced per host

could be achieved without sacrificing the survivability or vigor of the larvae. In addition

to varying the number of planidia applied to each host, the weights of the host mole








crickets were measured during inoculation to see whether larger hosts could provision

more parasitoids. These factors of host mole cricket weight and number of planidia used

to inoculate the host were examined to determine their effect on the number of pupae

produced, the mean weight of those pupae, and the survivability of the larvae to the pupal

stage.

Materials and Methods

During the maintenance of the laboratory colony of 0. depleta, S. abbreviatus

from the University of Florida mole cricket rearing lab were individually weighed and

inoculated with varying numbers of 0. depleta planidia. The weights of the hosts ranged

from 0.54-1.59 g and the weights of the hosts were not considered in determining the

number of planidia used to inoculate each individual. The number of planidia per host

ranged from 2-8, with most of the mole crickets being inoculated with either 3, 4, or 5

planidia. These numbers were favored because they are the numbers most frequently used

in the routine maintenance of the colony. The number of planidia placed on each host was

randomly determined. The numbers of mole crickets inoculated with 2, 3, 4, 5, 6, 7, and 8

planidia were 12, 108, 110, 52, 43, 32, and 11, respectively. Each mole cricket was then

returned to an individual 20 dram plastic vial filled with moist sand, and the larvae were

allowed to develop for 12 days at a room temperature of 260C. At that time, the pupae

were collected and weighed. Statistical analysis was performed using the general linear

model procedure (SAS Institute 2001). Regression analysis was used to determine how

differing numbers of planidia affected the number of pupae produced, the mean pupal,

and the survivability of the planidia. Additionally, the weights of the host mole crickets

were analyzed to determine their effect on the survivability of the planidia used. Where








applicable, the differences between the means were determined by Duncan's multiple

range test (SAS Institute 2001). Regression analyses were conducted to determine the

relationships between each of these factors (SAS Institute 2001).

To determine the effect that host mole cricket weight had on planidia survival, the

number of pupae produced and the mean weights of those pupae, mole cricket weights

were rounded up to the nearest 0.1 gram to place them into weight classes. Additionally,

weight classes which had only two or fewer samples were eliminated. In this case, the

smallest weight class, 0.70 grams (n = 2) and the two largest weight classes, 1.5 grams (n

= 2) and 1.6 grams (n = 2) were eliminated from the statistical analysis. The survival of

the planidia on hosts in the remaining weight classes were analyzed by ANOVA (SAS

Institute 2001).

Results

The mean number of the pupae produced relative to the number of planidia used

can be seen in Figure 3. There is an increase in the number of pupae produced as the

number of planidia increases (F = 15.77, P < 0.0001) and significant differences between

the means of the treatments. The regression analysis (Figure 4) supports this trend and

indicates an increase of 0.41 pupae for each increase in planidia (F= 83.77; P < 0.0001;

r2 = 0.19).

Figure 5 shows the survival of planidia grouped by the number of planidia placed

on each host. ANOVA is significant for the model (F=2.57, P < 0.02). Figure 6 is the

regression analysis of the same data set (F = 9.16; P < 0.002; r2 = 0.03), indicating an

approximate 3% reduction in survival for each increase in the level of planidia density.

The analysis of the number of planidia used as it affected the mean weight of the

pupae produced was found to be nearly significant by ANOVA (F=2.06, P = 0.057).









There was some significance among the means as indicated by the letters over the bars in

Figure 7. The regression analysis for the mean weights of pupae produced is in Figure 8

(F = 8.33; P < 0.004; r2 = 0.02) and indicate a reduction in the mean weight of the pupae

of 2.2 mg for each additional planidia.


6 e-

0d
Scd
4 be
C-

S3 a ab






0
2 3 4 5 6 7 8
Number of planidia inoculated per host


Figure 3: The effect of planidia density used to inoculate mole crickets on the number of
pupae produced (error bars indicate standard deviation, significantly different
means indicated by letters over bars as determined by Duncan's procedure, a
= 0.05)










8
S7 -

S6 y= 0.4105x+ 0.6515
5 r2 = 0.19, P < 0.0001

2



1o
0
1 2 3 4 5 6 7 8 9
Number ofplanidia inoculated per host



Figure 4: The effect of number of planidia used to inoculate mole crickets on the number
of pupae produced regression analysis with 95% confidence bands





a
0.8
ab ab ab
S0.6 b b

0.4

0.2


2 3 4 5 6 7 8
Planidia

Figure 5: The effect of number of planidia used to inoculate mole crickets on the survival
rate of the larvae to the pupal stage (error bars indicate standard deviation,
significantly different means indicated by letters over bars as determined by
Duncan's procedure, a = 0.05)













S1.0 y= -0.0317x+0.7129
2
.8 r = 0.03, P < 0.002
0.8

i 0.6

S0.4

S0.2

0.0
1 2 3 4 5 6 7 8 9
Number ofplanidia inoculated per host


Figure 6: The effect of number of planidia used to inoculate mole crickets on the survival
rate of the larvae to the pupal stage regression analysis with 95% confidence
bands



70 a
ab

60 T ab ab ab ab


S50 b

` 40

30
2 3 4 5 6 7 8
Planidia

Figure 7: The effect of number of planidia used to inoculate mole crickets on the mean
weight of the pupae produced (error bars indicate standard deviation,
significantly different means indicated by letters over bars as determined by
Duncan's procedure, a = 0.05)









120

,a 100 y = -2.2456x + 62.649
r2 = 0.02, P <0.004
Z 80

60 -

S 40-

20-

0
0 --- i ---- i ----- ---- i -

0 2 4 6 8

Number ofplanidia inoculated per host


Figure 8: The effect of number of planidia used to inoculate mole crickets on the mean
weight of the pupae produced regression analysis with 95% confidence
bands



The effect that host mole cricket weight had on the number of pupae produced

was not found to be significant when analyzed by ANOVA (F = 1.06, P = 0.39). The

effect that host mole cricket weight had on the survivability of the larvae was found to be

marginally significant (F = 2.12, P = 0.0505). The effect that host mole cricket weight

had on the mean weight of the pupae produced was highly significant (F = 3.49, P <

0.002) (Figure 9). The regression analysis can be seen in Figure 10 (F= 20.62; P <

0.0001; r2 = 0.05).










ab abc bc bc c
,R 80- ab
E a
60







0.8 0.9 1.0 1.1 1.2 1.3 1.4
Mole Cricket Weight Class (grams)
Figure 9: The effect of host cricket weight on mean pupal weight (error bars indicate
standard deviation, significantly different means indicated by letters over bars
as determined by Duncan's procedure, a = 0.05)


y =31.346x + 20.163
r = 0.05, P < 0.0001


7 0.9 1.1 1.3 1.

Host cricket weight class (g)


Figure 10: The effect of host cricket weight class on the mean weight of the pupae
produced regression analysis with 95% confidence bands

Discussion

The number of planidia used to inoculate host mole crickets as well as the weight

of those mole crickets are important factors to the rearing of 0. depleta in the laboratory.

Although these data do not clearly dictate a specific protocol that should be used, they do








provide a framework that would allow for anyone rearing 0. depleta to structure an

inoculation protocol specific to their needs. At times when large numbers of planidia are

available but few hosts are available, the data suggest that inoculating mole crickets with

more planidia would increase the production of pupae. Too many, however, would result

in reduced pupal size. At times when fewer planidia are available and maximum

survivability is required, inoculating two or three planidia per host would be more

effective. Alternatively, if larger pupae are desired, reducing the number of planidia per

host along with using larger hosts would achieve the desired goal. Therefore, the current

method of inoculating three planidia per host is less efficient than inoculating four or

five, since there is no significant reduction in pupal size, but there is a significant increase

in the number of pupae produced. The reduction in size that results from the use of eight

planidia, or possibly more, would likely be detrimental to the colony of flies. Some

preliminary data (Appendix A) has shown that pupae less than 40 mg have reduced

survivorship to the adult stage. This reduction is greater than the increase in number that

is gained when going from 6 or 7 planidia to 8 planidia. Furthermore, these data only

show a reduction in the emergence rate of the pupae, they do not indicate other negative

factors which may be associated with reduced size. Future research may be needed to

determine whether these smaller individuals show any reduction in longevity, ability to

mate, or in fecundity as well as how the reduction in size of a generation may affect the

size or fitness of future generations of flies.

According to the University of Florida's Integrated Pest Management Website

(http://ipm.ifas.ufl.edu/extension-resources/glossaries/glossary.htm), the definition of

superparasitoidism is








the situation in which more individuals of a parasitoid species develop in a host
than can obtain adequate resources to complete their development. Females of
some parasitoids may lay more than one egg in or on a host, resulting in
superparasitoidism, although the behavior of females tends to avoid this condition
by discriminating against already-parasitoidized hosts. In fact, five conditions can
be distinguished (a) only one parasitoid exists within the host; (b) there is more
than one parasitoid within the host, but all survive and produce adults of normal
size [this is not superparasitoidism]; (c) there is more than one parasitoid within the
host and they all survive but produce adults of subnormal size because of
competition for resources [this is viewed here as superparasitoidism]; (d) there is
more than one parasitoid within the host and some of them die due to competition
for resources (including attack by conspecifics) [this is superparasitoidism]; and (e)
there is more than one parasitoid within the host and all die because the resources
are too few [this is superparasitoidism].

By this definition, the situation of 0. depleta in this experiment qualifies as

superparasitoidism only under the highest number of planidia inoculated on the host.

Although some reduction in size is apparent at almost all levels above three planidia,

most of the reductions are not significant.

This raises an interesting question as to the natural behavior of the flies. Under

field conditions, the mean number of 0. depleta larvae found within trapped Scapteriscus

hosts is less than two (Amoroso 1990). It would seem likely that, due to the flies'

phonotactic search method for hosts and the solitary nature of adult mole crickets, it

would be to the flies' advantage to maximize the number of offspring that it would be

able to produce from the seemingly limited number of hosts. But apparently, this is not

the case. The closely related 0. ochracea Bigot, a parasitoid of Gryllus spp. crickets,

have an optimal laboratory clutch size of 4-5 larvae per host, but under field conditions

only deposit 1.7 + 1.0 S.D. larvae (Adamo et al. 1995). There must be some ecological

advantage to depositing fewer larvae than what appears to be the optimal number.

It may be that 0. depleta does not suffer from any shortage of hosts. Mole

crickets are certainly abundant and calling during certain times of the year, but at other








times seemingly unavailable. Ormia depleta may be able to find non-calling mole

crickets in other ways, or there may be alternative hosts (this is discussed more in

Chapter 4). Adamo et al. (1995) concluded that host availability was not a likely factor in

determining the number of larvae deposited on hosts by 0. ochracea. Another possibility

is that 0. depleta is responding to a factor in the field that is greatly reduced in the

laboratory, the mortality of the hosts. Under laboratory conditions, mole crickets suffer

little disease and no predation. It may be that in the field, the higher mortality of the hosts

would make it advantageous to partake in bet hedging and spread offspring out over

many hosts so that the loss of one host has a lesser effect on the total number of

offspring. This hypothesis is somewhat strengthened by the fact that 0. depleta does not

deposit eggs, but planidia larvae, so the female's investment in parasitoidizing a host is

already greater than that of an egg layer. Another laboratory factor that should be

considered is hand-inoculating. The mole crickets that are hand-inoculated are unable to

protect themselves in any way and have no opportunity to use whatever natural defenses

they may have available in the field. It may be that, although it would be advantageous

for 0. depleta to parasitoidize hosts with a greater number of planidia, the natural

behavioral defenses of Scapteriscus mole crickets prevent it, whether those defenses

involve brushing off planidia or simply retreating underground when the presence of O.

depleta is detected. This type of grooming has been observed in Gryllus spp. crickets

after an encounter with 0. ochracea (Adamo et al. 1995).

One possibility that has been suggested is that 0. depleta are parasitoidizing hosts

with higher numbers of planidia in the field, but we are unaware of it. The trapping

methods used to determine parasitoid levels of mole crickets in the field are usually








sound traps, which require the mole crickets to fly into the trap, or pitfall traps, which

require the mole crickets to be actively crawling on the soil surface to be captured. It may

be that those mole crickets with larger parasitoid loads are under greater physical stress

and are less able or likely to venture to the surface where they can be trapped. Zuk et al.

(1993) found that the calling of the field cricket Teleogryllus oceanus (Le Guillou) was

inhibited by infestation of 0. ochracea in Hawaii. Although Zuk did not report on these

crickets' ability to move, it does suggest that there is a physical liability to carrying

parasitoids. Collecting mole crickets by digging and sifting soil would probably produce

more accurate results than the current trapping methods, but the labor and expense

involved would not justify the change in methods.

The final reason for the low numbers of larvae found in field captured hosts may

be that there is a reduction in fitness caused by the high numbers of larvae used in this

experiment that were not investigated. Reduced size is the easiest type of fitness

reduction to observe, but many others may be at work. It may be that, due to competition,

certain key resources are not available in sufficient amounts for the flies reared under

superparasitoid conditions for the resulting adult flies to develop, mate, locate hosts, or

reproduce properly. Many physiological deficiencies may result from superparasitoidism,

and they may not be obvious either externally, or immediately (Waage and Ng 1984).

These possibilities still remain for future research.













CHAPTER 4
REARING ORMIA DEPLETA (DIPTERA: TACHINIDAE), A PARASITOID OF
MOLE CRICKETS (ORTHOPTERA: GRYLLOTALPIDAE), ON A FACTITIOUS
HOST, ACHETA DOMESTICUS (ORTHOPTERA: GRYLLIDAE)

Introduction

The maximum life span of adult 0. depleta in the lab is approximately 60 days

(Welch 2000), which would not allow them to survive between times of peak mole

cricket calling activity, which are from February to June (Walker and Moore 2000).

Therefore, some sort of diapause might be expected to allow the flies to carry over from

June to the Following February. Some preliminary research was made to determine

whether 0. deplela pupae could be stimulated to diapause by exposing them to cold

temperatures (0 5 C), but refrigeration attempts at these temperatures resulted in 100%

mortality. Additionally, work done by Cabrera (2000) found that attempts to rear larvae

at temperatures below 170 C resulted in the death of the larvae. Therefore, cold-induced

diapause does not seem to be a probable part of 0. depleta's life history. Other

experimentation (Appendix B) suggests that there is no photoperiod-induced diapause.

The question then remains: what happens to the population of 0. depleta during the

months between the period of peak calling activity of Scapteriscus spp. hosts?

It is possible that 0. depleta locate mole cricket hosts when the mole crickets are

not known to call in large numbers. The flies may be able to find enough to maintain their

population until the next calling season. Calling traps located at the Gulf Coast Research

and Education Center in Bradenton, Florida, catch most of their 0. depleta during the

seasons that follow peak adult Scapteriscus flight activity, which are from March through








May and from September through November, but some mole crickets are caught during

every month of the year (Walker et al. 1992a, b). Dr. Thomas Walker has also observed

individuals of S. borellii and S. vicinus calling in each month of the year, albeit in much

smaller numbers than in the peak seasons (Walker and Moore 2000). If only few flies

were able to locate Scapteriscus hosts during these times, it could maintain a seed

population until the next calling season.

Another possible solution is the use of alternate hosts. Ormia depleta is able to

survive through the winter in central Florida. Specimens are collected in Bradenton,

Florida, throughout the year, although the greatest numbers coincide with the peak mole

cricket flight seasons (Walker et al. 1992b). Bradenton does occasionally experience cold

weather and even frost. This may suggest that during these times when adult or pupal 0.

depleta would not be able to survive, that perhaps the larvae are able to survive within a

host. Being within a host would provide a great deal more protection from the

environment. If O depleta were unable to reliably locate Scapteriscus hosts during these

times when they are not calling, it could be possible that they were using an alternate

host. This has been suggested by Fowler and Mesa (1987) with the recovery of O. depleta

from a species of Anurogryllus. Additionally, Justi et al. (1988) reported that under

laboratory conditions, 0. depleta successfully developed within an unnamed Gryllus

species. Although there is no evidence to suggest that 0. depleta is phonotactic to any

taxa other than Scapteriscus (Fowler and Garcia 1987), it is plausible that it may

occasionally encounter enough hosts by other means to maintain a population between

the calling seasons of its preferred host.








Multiple hosts among tachinids are not unusual. Some tachinids have host ranges

that cover three orders of insects (Cantrell and Crosskey 1989). Ormia depleta is thought

to be and reported as an obligate parasitoid of Scapteriscus mole crickets (Frank et al.

1998), and under most conditions, it probably is. The mechanism for the specificity of O.

depleta lies in its phonotaxis. There are no reports of it being attracted to any calls other

than those of S. vicinus and S borellii (Walker et al. 1996). Under field conditions, this

greatly limits the fly's ability to select new hosts. Under laboratory conditions, especially

when hand-inoculating hosts, it may be possible to manipulate this characteristic. It

would be advantageous to be able to rear 0. depleta on an alternate host when

Scapteriscus are unavailable or simply to reduce the costs associated with rearing

Scapteriscus. The effect of rearing tachinid flies on factitious hosts has been examined

many times. Baronio et al. (2002) found there to be no decrease in the pupal weights of

Pseudogonia rufifrons Wiedemann when reared on the factitious host Ostrinia nubalis

(Hubner) rather than on Galleria mellonella L., its natural host. The tachinid fly Exorista

larvarum (L.) was found to be even more successful on the factitoius host G. mellonella

than on its natural host, Lymantria dispar (L.) (Dindo et al. 1999). With this in mind, a

factitious host for 0. depleta was examined.

Acheta domesticus (L.) (the house cricket) can be reared in large groups

inexpensively and would be an economical substitute for Scapteriscus spp. as a host in

laboratory rearing, if able to support the development of 0. depleta. Any failure of O.

depleta to successfully develop in an A. domesticus host would lead to the question of

why they did not develop well. Was there failure to penetrate the host? Did A. domesticus

lack sufficient nutritional value? One of the major factors differentiating A. domesticus








and Scapteriscus spp. behaviorally is that Scapteriscus spp. are primarily subterranean

whereas A. domesticus is not. Although this would not seem to have a direct effect on

internally developing larvae, it would be likely to have an effect on the ability of the

larvae to pupate after leaving the host. There may also be differences in the cuticles of the

two crickets that could hinder the development or the emergence of the larvae. To study

this, the larval behavior of 0. depleta inside both S. abbreviatus and A. domesticus was

observed.

Materials and Methods

Larval Development of Ormia depleta in Acheta domesticus

Ten adult A. domesticus were obtained from the colony kept at the University of

Florida mole cricket rearing lab. Each cricket was inoculated with four planidia from a

freshly sacrificed gravid female 0. depleta from the Piracicaba strain laboratory colony.

Inoculated crickets were placed in a 30 x 23 x 10 cm clear, plastic container with a screen

ventilated top. The bottom of the container was filled with approximately 3 cm of moist,

autoclaved sand. A screen of 0.6 cm galvanized mesh was suspended approximately 3 cm

above the sand to keep the crickets off the sand but to allow newly emerged larvae to

drop through to the sand. Cardboard tubes 5 cm in diameter were placed on the

galvanized screen to provide hiding places for the crickets. Water was made available by

placing a Petri dish on the galvanized screen wicked with cotton. Food was also provided

(Nutrena Cricket and Earthworm Feed, Minneapolis, MN), also in a Petri dish on the

screen. Food and water were available ad lib. The crickets were observed daily and their

food and water changed as needed. Approximately 10 days later, when all crickets were

dead and emerged larvae had pupated, the pupae were removed and their number and

weight were recorded. This process was repeated for four generations of 0. depleta.









Larval Behavior within host

Acheta domesticus and S. abbrevialus were obtained from the mole cricket rearing

lab and inoculated with Os6rio strain 0. depleta planidia. Twenty-one crickets from each

species had four planidia placed under the posterior margin of the pronotum. Acheta

domesticus were then placed in a small container containing food (Nutrena Cricket and

Earthworm Feed, Minneapolis, MN) and water in a Petri dish wicked with cotton.

Scapteriscus spp. were returned to their vials of sand. The planidia were then allowed to

develop in their hosts. Three crickets were sacrificed at each of seven 24-hour intervals,

dissected, and the larvae searched for. When found, the size, number, location, and

apparent tissues being fed upon were all noted. A t-test was used to compare the mean

pupal weights of pupae reared on A. domesticus to those reared on S. abbreviatus (SAS

Institute 2001).

Results

Larval Development of Ormia depleta in Acheta domesticus

Acheta domesticus was able to act as a factitious host for 0. depleta in that some

of the larvae were able to survive to the pupal stage. However, the survivorship of the

planidia was only 29% versus 57% in S. abbreviatus (as determined in Chapter 3).

Additionally, the weight of the pupae produced was significantly less than those

produced in S. abbreviatus. The mean pupal weight when reared in A. domesticus was

39.85 mg + 1.53 (SE), which was significantly smaller than those in S. abbreviatus,

where the mean pupal weight was 54.60 mg 0.50 (SE) (P < 0.001; df= 950).









Larval Behavior within host

Twenty-one A. domesticus and twenty-one S. abbreviatus were examined in this

experiment. At least one planidium was found in each host species for each day of this

experiment. The mean number of planidia found and the mean length of those planidia

are shown in Table 6.

Table 6: Mean number and length of 0. depleta larvae found in S. abbreviatus and A.
domesticus hosts
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
S. abbreviatus
Mean number of larvae found 0.67 1.33 2.67 3.00 2.33 2.33
Meanlength(mm) 0.62 0.72 2.29 4.86 9.78 11.87


A. domesticus
Mean number of larvae found 0.33 1.00 1.00 1.67 2.33 1.67
Mean length (mm) 0.58 0.64 0.85 1.44 4.57 7.25

The following is a timeline of the activity of the larvae inside each host.

Scapteriscus abbreviatus-

Day 1 (24 hours after inoculation)- A few planidia were dead on the exterior of the host.

The larvae that succeeded in penetrating the host are very difficult to find only

two were found among the three hosts dissected. Those that were found were in

the fatty tissues just below the area where they had been placed, apparently

feeding on those tissues. (Figure 11).

Day 2- Still some dead planidia are visible on the exterior of the host. Larvae are easier to

find, though still small. The larvae are now distended and almost transparent.

Some of the larvae are still found in the fatty tissues under the pronotum while a

few have moved to a more posterior and lateral position. The farthermost

observed was located along the left side of the abdomen, just posterior to the

metacoxa (Figure 12).


Day 7

2.33
12.69


2.00
8.94








Day 3 All larvae found were in the abdomen of the host. They were found in clusters on

either side in the area near the gonads. They appeared to be feeding in fat tissue

and/or reproductive organs (Figure 13).

Day 4 Larvae are still in the gonadal region. The ovaries/testes are no longer visible in

large amounts. The larvae are now posteriorly attached to the host exoskeleton.

The alimentary canal of the host, though in reach of the larvae, does not appear to

be damaged (Figure 14).

Day 5 The attachment points of the larvae are now visible on the exterior of the host

and appear as small, dark brown spots (Figure 15). The fat bodies within the reach

of the attached larvae are now greatly reduced or gone. The alimentary canal still

appears to be undamaged (Figure 16).

Day 6 Hosts are starting to act very sluggish and showing signs of dying. The larvae

now occupy approximately half of the area within the hosts' abdomens (Figure

17). The heads of the larvae can now reach into the thorax and they are apparently

feeding there. The inside of the abdomen now looks very "dry" as there are no

remaining fat reserves, no gonads, and no hemolymph in any large amount. The

alimentary canal, though well within reach of the larvae, shows no signs of

damage. The attachment points of the larvae are now visible from the outside of

the host and appear as small brown patches, approximately twice the size of the

hosts' spiracles. On the inside, the points of attachment seem to open into small

scleritized cones that appear to be attached to the posterior ends of the larvae by

what appear to be the old exuviae (Figure 18).







Day 7 Larvae are much less active in the host. Some still appear to be feeding
on the remaining tissues in the thorax of the host, others appear to be at rest. The
alimentary canal of the host remains undamaged (Figure 19).


Figure 11: Larva of 0. depleta in S. abbreviatus host one day after inoculation


,- r0 a1


gt'~IQgI%


Figure 12: Larva of 0 depleta in S abbreviatus host two days after inoculation






















Figure 13: Larva of 0. depleta in S abbreviatus host three days after inoculation




a











Figure 14: Larva of 0. depleta in S ahhbreviatus host four days after inoculation


























Figure 15: Scapteriscus abbreviatus host five days after inoculation showing the external
evidence of the attachment point of O. depleta larva












Figure 16: Larvae of deplete in abreiaus host five days after inoculation





Figure 16: Larvae of O depleta in abbreviatus host five days after inoculation
















Figure 17: Larvae of 0. deplete in S. ahhreviatus host six days after inoculation


!N ,t A496*\MIWAMA


Figure 18: Two 0. depleta larvae in S abhhreviatus host six days after inoculation
showing detail of the larval attachment to the host


























Figure 19: Larva ofO depleta in S abbreviatus host seven days after inoculation



Acheta domesticus-

Day 1 (24 hours after inoculation)- Only one larva was found feeding on the fatty tissue

just below the area where placed. This larva does not appear to have grown much

although it has become slightly lighter in color, most likely due to feeding on fat

bodies (Figure 20).

Day 2 Larvae are easier to see, though still small. They are apparently feeding on fat

bodies in the thorax (Figure 21).

Day 3 Two larvae were feeding on fat tissues near the gonads of the host. One larva

was in the anterior region of abdomen (Figure 22).

Day 4 All larvae found were in the abdomen of the host. All but one were feeding on

fat and reproductive tissues. The remaining larva was in the anterior region of

abdomen feeding on fat tissue (Figure 23).








Day 5 Larvae are all now feeding in the gonadal region. Larvae are all now posteriorly

attached to the exoskeleton of host. The fat bodies of the host do not appear to be

depleted nearly as much as in S. abbrevialus hosts (Figure 24).

Day 6 Larvae continue to feed in the gonadal region. The size of the larvae appears to

be increasing, but at a reduced rate as compared to larvae in S. abbreviatus

(Figure 25).

Day 7 Larvae are still feeding in the gonadal region. The fat and reproductive tissues of

the host appear to be somewhat reduced. Some larvae appear to be showing signs

of slowing down or stopping feeding. The alimentary canal of the host is

undamaged (Figure 26).



















Figure 20: Larva ofO. depleta inA. domesticus host one day after inoculation
























Figure 21: Larva of 0. depleta in A. domesticus host two days after inoculation


















Figure 22: Larva of 0. deplete in A domesticus host three days after inoculation


























Figure 23: Larva of C0 depleta in A. domesticus host four days after inoculation

















Figure 24: Larvae ofO. depleta in A. domesticus host five days after inoculation








aa






g re 25 As


Figure 25: Larvae of O. depleta va A. domesticus host six days after inoculation


Figure 26: Larvae ofO. depleta in A. domesticus host seven days after inoculation


One larva was removed from each host species on day seven and photographed
next to each other to compare the difference between the S. abbreviatus-reared larva and
the A. domesticus-reared larva (Figure 27). The larval length for each day was measured
using an ocular micrometer and the mean plotted for each host species (Figure 28).






















J


Figure 27: Seven day old larvae of 0. depleta from S. abbreviatus (top) and A.
domesticus (bottom)


14


- S. abbrevitus
-- A domestiu


0 1 2 3 4 5 6 7
Days After Inoculation
Figure 28: The growth of 0. depleta larvae in alternate hosts: S. abbreviatus and A.
domesticus

Discussion

Although A. domesticus may be easier and less expensive to rear than

Scapteriscus spp., these results do not support its use as a factitious host for 0. depleta.

For many generations, there is a great abundance ofplanidia. Therefore, the survival of

the planidia is not a weighty factor in determining rearing procedures. Additionally, by








using large numbers of A. domesticus as hosts, the number of gravid flies available may

be able to be augmented and therefore produce even more planidia. In such a case, a drop

from 57% to 29% may not be too damaging to a colony's survival. Considering that this

represents approximately a doubling in mortality of planidia, from an economic

standpoint this would be acceptable because the cost of rearing a mole cricket host is so

much than the cost of rearing a house cricket. The main issue is that of the pupal size.

Previous experimentation has shown that for pupae weighing less than 40 mg, there is a

reduction in survival (Appendix A). Roughly half of the A. domesticus-reared 0. depleta

were in this low-emergence category. This is considering emergence only, no research

has been done to determine the effect that this small size may have on survivorship,

breeding ability, or fecundity, but it seems likely that any or all of these factors could be

affected negatively by significantly reduced weight. Among hymenopterans, reduced

female size has been demonstrated to cause a reduction in fecundity in Trichogramma

evanescens Westwood and Goniozus nephantidis (Muesebeck) (Waage and Ng 1984:

Hardy et al. 1992). Under laboratory rearing conditions, these problems may be further

exacerbated in that the offspring of these under-sized flies would become the parents of

the next generation of flies, potentially producing fewer or smaller individuals and

reducing the vigor of the colony as a whole.

It is possible that the poor results of A. domesticus as a factitious host were due to

the techniques used here. The number of planidia used was not altered within this

experiment. Perhaps a smaller number per host would have produced larger pupae,

although this is not supported by the physiological evidence. There may also be room for

improvement of the rearing cages. The 0.6 cm wire mesh used was too large to prevent








the crickets from getting through to the sand. House crickets do not survive well in very

moist conditions and this may have affected the larvae. Also, there were problems with

keeping the sand moist enough in the well-ventilated cages. Although this would not have

an effect on the size or number of pupae produced, in a larger-scale operation this may

effect the ability of the pupae produced to survive. Additionally, all of the pupae

recovered were found on the surface of the sand and several seemed unusually dry,

indicating the inability of the larvae to burrow into the sand effectively. Perhaps in the

future a different substrate, such as vermiculite, could be used to better retain moisture.

In considering the larval behavior in the hosts, no single factor stood out as being

the cause for the poor performance ofA. domesticus as a factitious host for 0. depleta.

Rather there appear to be multiple factors. The slowed growth and development of the

larvae in A. domesticus suggest that there may be a nutritional deficiency associated with

A. domesticus as a host for 0. depleta. This deficiency could possibly be a lack of

specific proteins or other components necessary or an overall shortage in the volume of

palatable or usable tissues. The fact that after seven days there was still a large amount of

fat tissue remaining in the host does not support the position that these tissues are simply

lacking in volume. It is more likely that they are nutritionally deficient or unpalatable.

Another explanation would be that there is a chemical or immunological incompatibility

between A. domesticus and 0. depleta. Further research in these areas is still needed.

Although smaller, the larvae of 0. depleta in A. domesticus do appear to survive

as well as those in S. abbreviatus, as evidenced by the similarity between the mean

number of larvae found in each over the last few days before emerging from the host.

There is, however, great reduction in the number of pupae produced by the different








hosts. There must be a cause of mortality between these two events. The most probable

cause for this mortality would be a problem with the ability of the larvae to emerge from

the host. Scapteriscus abbreviatus are subterranean and their bodies are well sclerotized,

however, their abdomens are quite soft and the cuticle thin. By contrast, A. domesticus

have a relatively hard and thick abdominal cuticle. This could very well be a barrier to 0.

depleta larvae being able to emerge successfully. This would especially be true for larvae

of reduced size and vigor. Emergence may also be affected by the factors present as the

larvae leave the host and are exposed to the laboratory environment. Ormia depleta

would probably be adapted to emerging from its host into a subterranean environment

where the relative humidity would normally be very high. Those larvae emerging from A.

domesticus would find themselves immediately exposed to much drier conditions. The

pupae of 0. depleta are well protected from desiccation, but before the formation of the

pupal sclerotia, the larvae could be extremely vulnerable, perhaps enough to keep it from

being able to fully emerge from the host.













CHAPTER 5
IDENTIFICATION OF TWO SOUTH AMERICAN GEOGRAPHICAL ISOLATES OF
ORMIA DEPLETA BY ANALYSIS OF CUTICULAR HYDROCARBONS

Introduction

With the release of the Os6rio strain of 0. depleta, it became desirable to find a

way to differentiate the two strains to make it possible to identify the origin of any flies

captured. Although no flies have yet been trapped where the Os6rio strain was released in

states north of Florida (personal communication with Dr. J. H. Frank, University of

Florida), those flies may perhaps be established in those areas, and in the areas between

the new release sites and the established range of the Piracicaba strain of flies. Gas

chromatographic (GC) analysis of cuticular hydrocarbons was chosen as a method to

differentiate the strains. GC has been used to identify different species, subspecies, and

races of insects (Sutton and Carlson 1993) and is a quick and simple testing method.

Materials and Methods

Gravid female 0. depleta of the Os6rio strain were obtained from the laboratory

colony maintained in the mole cricket rearing laboratory at the University of Florida.

Gravid female Piracicaba strain flies were collected from the sound traps maintained at

the Gulf Coast Research and Education Center in Bradenton, Florida. Planidia from each

strain were inoculated onto S. abbreviatus and kept under normal conditions as outlined

previously. Colonies of both Os6rio and Piracicaba strains of 0. depleta were maintained

until maturity (approximately four weeks after pupation). Males and non-gravid females

from both colonies were removed and frozen until time for cuticular hydrocarbon








extraction. Both individual and collective samples were taken. Individual samples were

collected by placing a single frozen fly in a vial containing 5 mL of hexane and agitating

for 30 seconds. The fly was then removed and discarded. The hexane solution was then

filtered through a glass pipette containing a 5 cm column of silicic acid. The pipette was

then flushed with an additional 5 mL of hexane. The sample was then evaporated down to

approximately 1 mL of solution. This process was repeated for eight flies for both males

and females from each of the two strains. Collective samples were taken by placing 10

frozen flies of the same sex and strain into vials containing 10 mL of hexane. These

samples were also filtered through glass pipettes containing a 5 cm column of silicic acid

and flushed with an additional 5 mL of hexane. Samples were evaporated down to

approximately 3 mL of solution. Both individual and collective samples were analyzed

by gas chromatography using a Hewlett Packard 6890 GC with a DB-5 fused silica

capillary column (30m x 0.25p1m, J & W Co., Folsum, CA using hydrogen carrier gas

@1.2 ml/min and a cold on-column injector system held at 600C). The temperature ramp

for the samples was as follows:



Initial temperature 600 C hold for 2 minutes

Increase at a rate of 16C/min to 170C

Increase at a rate of 80C/min to 234C

Increase at a rate of 6.50C/min to 2530C

Increase at a rate of 5.4C/min to 3200C

Hold at 3200C for 20 minutes








The purpose for this specific temperature ramp is that it separates straight-chained

alkanes into peaks with retention times approximately one minute apart (pers. comm.

with Dr. David Carlson, USDA-CMAVE, Gainesville, Florida). The resulting

chromatograms were analyzed by stepwise selection in multiple logistic regression to

determine discriminatory peaks between strains. Additionally, ajackknife analysis was

performed to assess the probability of the resulting model to correctly predict the strain of

an unknown sample (SAS Institute 2001).

Results

Ten predominant peaks were present in the cuticular hydrocarbon GCs of all

specimens tested. (Figures 29-32) These peaks were designated by the letters A through J

and their retention times listed in Table 7. The strongest variation between the two strains

was found to be between the peaks of the males, when analyzed by multivariate analysis,

with the Piracicaba strain having a higher percentage in both peaks. A stepwise logistic

procedure found complete separation of data points when comparing peaks F and I, with

the Piracicaba strain having a higher percentage of both peaks. Figure 33 shows a scatter

plot of the values for peaks F and I for both strains. The jackknife analysis showed that,

based on these data for peaks F and I, the probability of correctly identifying an unknown

specimen is dependent upon the strain of that specimen. Os6rio strain flies were correctly

identified with 100% accuracy. Piracicaba strain flies were only identified correctly 60%

of the time with the remaining 40% being falsely identified as Os6rio strain.









Table 7: Retention times of GC peaks for the cuticular hydrocarbons of 0. depleta
k Retention Time
Peak
(in minutes)
A 21.00
B 23.00
C 23.50
D 25.00
E 25.18
F 27.00
G 27.15
H 27.20
I 27.35
J 27.45


40
8


30.
a
0-
I- 20



0

is 8
o-
Retention Time (in minutes)



Figure 29: The mean percentage of total peak area for the gas chromatograms of the
cuticular hydrocarbons of female Piracicaba strain Ormia depleta















< 30



1 20


8
10




Retention Time (in minutes)



Figure 30: The mean percentage of total peak area for the gas chromatograms of the
cuticular hydrocarbons of female Os6rio strain Ormia depleta





50

8
e 40





0
30
.-

20 8
20 N
s.2 -


1 s I I


Retention Time (in minutes)



Figure 31: The mean percentage of total peak area for the gas chromatograms of the
cuticular hydrocarbons of male Piracicaba strain Ormia depleta


a














S40


30
30
S


20
a 20

I o


a 0

Retention Time (in minutes)



Figure 32: The mean percentage of total peak area for the gas chromatograms of the
cuticular hydrocarbons of male Os6rio strain Ormia depleta


9.0
-^-i
7- .0
A
5.0-


3 0


A Osorio Strain

o Piracicaba
Strain


0 0


A A


-1.0 1.0 3.0 5.0 7.0 9.0
Percentage of total peak area accounted for by peak F


Figure 33: Scatter plot representation of the values for peaks F and I for male 0. depleta
of the Piracicaba and Os6rio strains


K








Discussion

The complete separation of data points shows that, based on these data, males of

0. depleta can be identified to strain by GC cuticular hydrocarbon analysis with the use

of only two GC peaks. The difficulty in this lies in that males are rarely, if ever,

collected. However, because cuticular hydrocarbon makeup is influenced by a number of

factors including the age of the insect and its diet (Liang and Silverman 2000), specimens

prepared for GC analysis must be laboratory-reared under the protocol outlined above.

Therefore, gravid females collected would have to be used to inoculate mole crickets and

their offspring laboratory reared and tested. Because of this, it would be just as easy to

test males as females.

The lack of better predictability of identification of unknowns is somewhat

disappointing. This failure is most likely due to the rather small sample size with which

the jackknife analysis had to work as opposed to the actual differences in the cuticular

hydrocarbon makeup of the two strains. A more sophisticated model using more than two

peaks may be able to correct this even with the current data size. For this reason, the raw

data taken for both strains, as well as a hybrid of the two strains, has been presented in

Appendix C.













CHAPTER 6
SURVIVAL OF ORMIA DEPLETA WHEN CAGED WITH HONEYDEW-
PRODUCING APHISNERII

Introduction

The establishment of Ormia depleta, along with Larra bicolor and Steinernema

scapterisci, has been shown to have a strong negative effect on the overall population of

Scapteriscus mole crickets in Florida (Walker et al. 1992, Parkman et al. 1996). Although

0. depleta is apparently well established throughout most of central and southern Florida

(Frank et al. 1996), there has not yet been established any method to augment local

populations of the flies in areas where an increased level of mole cricket control would be

desired. Areas such as golf courses, which have a very low threshold for turf damage,

could benefit from an increased local population of natural enemies, which would reduce

the population of pest mole crickets.

Augmentation of biological control agents has been achieved in many ways.

Inundative releases of 0. depleta are not practical due to the difficulty in rearing them.

Banker plants have been used to introduce and disperse parasitoids into field situations

(Goolsby and Ciomperlik 1999). Banker plants may also be useful in providing food

sources for adult parasitoids and even acting as an attractant. Investigation into

determining a suitable banker plant for 0. depleta led to the discovery that much of their

diet consists ofhomopteran honeydew (Welch 2000). Therefore a plant nectar source

would not be likely to make a suitable banker plant. Alternatively, a plant which would

reliably maintain a population of honeydew producing homopterans could be established








in areas where a higher population density of parasitoids was needed. If the honeydew

produced was attractive to 0. depleta, and the homopteran population could be

maintained, it would very likely achieve the desired augmentation of the local population.

Aphids feed on a number of ornamental plants that may be suitable as banker

plants for 0. depleta. Although almost any plant could be incorporated into various

landscapes to attract 0. depleta, there are considerations to be made when choosing a

banker plant. Honeydew production of the aphids associated with each plant is important,

as are the host range of those aphids and their pest status. Pittosporum spp., Pyracantha

spp., and Viburnum spp. are all infested by Aphis spiraecola Patch, but this species of

aphid also attacks Citrus spp. (Fasulo et al. 2003). Rosa spp. are host to the rose aphid,

Macrosiphum rosae (L.), which may be a suitable banker organism. Roses, however, are

not generally the type of plant used in golf course landscape, and encouraging a pest of

roses may not be as acceptable as encouraging pests of other ornamentals. Gardenia spp.

and Hibiscus spp. are host to Aphis gossypii Glover, but this species is also a pest of

Citrus spp., making it unsuitable for recommendation. Camellia spp. and Gardenia spp.

are hosts to the green peach aphid, Myzus persicae (Sulzer), but as this species has a wide

host range and produces a relatively small amount of honeydew, it is not a good

candidate (Capinera 2001). The crepe myrtle aphid, Sarucallis kahawaluokalani

(Kirkaldy), feeds on Lagerstroemia spp. and along with the podocarpus aphid,

Neophyllaphis podocarpi Takahashi, which feeds on Podocarpus spp., may prove to be

reliable banker organisms for 0. depleta.

The oleander aphid, Aphis nerii Boyer de Fonscolombe, is a minor pest of

oleander (Nerium oleander L.) and milkweeds (Asclepias spp.) (Johnson and Lyon 1991).








These aphids are able to use some of the toxins of their host plants for their own defense

(Malcolm 1986), but they are still susceptible to generalist predators and the braconid

wasp Lysiphlebus testaceipes Cresson (Hall and Ehler 1980). Whether these toxins would

be in the honeydew and whether they would act as a deterrent to feeding by 0. depleta

was unknown. Although A. nerii colonies can quickly grow to huge numbers on host

plants, they do not generally cause a decline in the health of the plant as damage is

generally restricted to unsightly sooty mold caused by the secretion of honeydew (Hall

and Ehler 1980). Due to this mild pest status, the narrow host range, and the observation

that colonies of A. nerii are present in Florida throughout much of the year, this species

was chosen for evaluation as a potential banker organism to provide honeydew for

populations ofO. depleta. The objective of this experiment was to determine whether

Aphis nerii honeydew was a suitable diet for 0. depleta.

Materials and Methods

Pupae of Piracicaba strain 0. depleta were obtained from the laboratory colony at

the University of Florida, which was reared from females collected from the Gulf Coast

Research and Education Center in Bradenton, Florida. Six cylindrical rearing cages (30

cm diameter x 60 cm tall) were placed in an outdoor heated greenhouse. Each cage

contained a 20 dram vial of moist sand with six 0. depleta pupae, a small (53 mm

diameter) Petri dish of water wicked with cotton, and an approximately 20 cm tall

milkweed (Asclepias curassavica L.) plant in a 10 cm diameter pot. Milkweed plants in

five of the six cages were inoculated with ten adult A. nerii each, which were field

collected from A. curassavica plants, and the colonies were allowed to grow undisturbed

for two weeks before exposure to the flies. By this time, each colony was well established








and covered the new growth of each plant. The milkweed in the sixth cage was not

inoculated with aphids to serve as the "starvation" control. None of the plants were in

bloom at the time of the experiment, so the only carbohydrate source available was from

the honeydew secreted by the aphids. At the time of the introduction of the pupae,

copious amounts of honeydew were visible on the plants as well as the sides of the

rearing cage. The adults emerged over a two-day period. The number of flies remaining

alive in the cages was recorded each day, and the experiment ran until all the flies were

dead. Each fly acted as a replicate to produce a mean lifespan for each treatment.

Results

The flies that had access to the honeydew of A. nerii lived an average of 21.7 days

(SD = 7.1, n = 21). The flies without honeydew lived an average of 15.7 days (SD = 4.1,

n = 4). A t-test showed this difference to be significant (P < 0.04). Neither set of flies

produced any gravid females.

Discussion

Although the results of this experiment did show significance, the level of

significance would have likely been much greater had it not been for several factors.

Running this experiment in a heated greenhouse resulted in environmental factors that

were not conducive to the health of the flies. The levels of temperature and humidity

fluctuated greatly. The mean life span of the honeydew-fed flies was far less than the

recorded life span of flies fed other diets in previous experiments. Under laboratory

conditions the mean life spans of flies fed hummingbird nectar and melezitose solution

were 32.7 days and 39.0 days respectively (Welch 2000). Honeydew-fed flies in this

experiment only averaged 21.7 days. Although this may seem to indicate that this

particular honeydew is not a suitable diet, the flies with no food only had mean life spans








of 15.7 days in this experiment whereas the mean life span under previous laboratory

conditions for starved flies was 22.2 days (Welch 2000). This indicates an overall life

span reduction caused by another factor. In this case, the most likely factor was humidity.

This experiment took place from December to January and because the greenhouses were

heated, the air remained quite dry. Additionally, the host plants for the aphids had been

kept very well watered prior to this experiment by placing them in shallow dishes of

standing water. This was done to reduce the stress of the aphid load on the plants. When

the plants were placed in the cages, they were kept watered, but the standing water dish

was removed to keep the flies from drowning. The resulting reduction in available water

was apparently the cause of the plants beginning to decline. By approximately day ten of

the experiment, all the plants were dead. The experiment was allowed to continue

because the amount of honeydew that had already been produced was significant.

Honeydew had been heavily deposited on the leaves of the plants, the wall of the cage,

and the plant pots. The amount of honeydew that flies were able to consume was enough

to produce significant results, but had the plants lived longer, the difference would have

likely been more dramatic.

The results do seem to confirm that the honeydew ofA. nerii is not toxic to O.

depleta and that it is a suitable food source. Because of the difficulties encountered in this

experiment, no real conclusions can be made as to the suitability of Aphis nerii honeydew

as a food source for the augmentation of 0. depleta in the field. This experiment was not

repeated because the laboratory colony of 0. depleta died out while it was being

conducted and a new colony was not obtained until it was too late to run the experiment






65


again. The ability ofA. nerii honeydew to act as an attractant in the field was not

investigated here.













APPENDIX A
THE EFFECT OF PUPAL SIZE ON EMERGENCE

To determine if the fitness of the pupae of smaller-than-avaerage 0. depleta was

reduced, a short experiment was conducted. Pupae were divided into 5 groups based on

their weight. Each group was then placed in a separate emergence box containing a 2 cm

layer of moist sand and covered with a 1 cm layer of moist sand. The boxes were kept at

room temperature (approximately 250 C) and when the adult flies had emerged, the

number of adults was recorded. The results are shown below in Table 8. There was a



Table 8: The percent emergence of 0. depleta pupae of varying sizes
Number of Number of
Pupal Size Pupae Adults % Emergence
<40 mg 28 20 71.4
40-50mg 31 29 93.6
50-60mg 50 45 90.0
60-70mg 50 46 92.0
>70mg 60 51 85.0

trend toward decline in survival of pupae less than 40 mg. Whether this is due to some

genetic or physiological problem or just a function of having less of a buffer from

desiccation is unknown.













APPENDIX B
ATTEMPTED INDUCTION OF DIAPAUSE BY SHORT DAY PHOTOPERIOD IN
OSORIO STRAIN ORMIA DEPLETA

An experiment was conducted to determine if diapause could be induced in O.

depleta by subjecting the pupae and adults to short day photoperiods. The Os6rio strain

pupae were obtained and placed in two large (30 cm diameter x 60 cm tall) rearing cages.

The pupae were shallowly buried in moist sand as per the normal rearing technique. One

cage was designated as the short-day cage and one as the long-day cage. Both cages were

placed near a window that received indirect sunlight. After sundown each day, the short-

day cage was covered with black plastic, consisting of two black plastic bags, one inside

the other, to block sunlight. In the morning, the long-day cage received sunlight but the

short-day cage remained covered until four hours after sunrise. After four hours, the

short-day cage had its black plastic removed and it received light until sundown. Both

cages were exposed to sundown, as twilight was considered an important mating

stimulant by Wineriter and Walker (1990). The pupae and resulting adults were kept

under these conditions. After gravid females had been produced, some from each

treatment were sacrificed and their planidia used to inoculate S. abbreviatus. These mole

crickets were then placed in their individual vials of moist sand and put back into either

the short-day cage or the long-day cage in accordance with the planidia with which they

had been inoculated. After 10 days, the vials which had contained the mole cricket hosts

were removed and the pupae harvested. These pupae were then shallowly buried in moist

sand as per the normal rearing technique. The resulting pupal duration was recorded for






68


each individual pupae. The results showed the mean pupal duration for the short-day

pupae to be 14.6 days (0.74 SD) and 13.8 days (0.73 SD) for the long-day pupae. A t-test

showed no difference between the two treatments.














APPENDIX C
GAS CHROMATOGRAPHIC ANALYSIS OF THE CUTICULAR HYDROCARBONS
OF ORMIA DEPLETA TO IDENTIFY STRAINS

Raw data showing the percentage each peak contributed to the total peak area.


U
cj J


Peak Retention Time

21.00 23.00 23.50 25.00 25.18 27.00 27.15 27.2 27.35 27.45


P C F 4.27 22.81 0.60 5.98 4.24 1.31 37.56 13.66 2.07 7.51
P C F 5.43 29.07 0.84 7.98 3.75 1.21 34.71 11.38 1.08 4.56
P S F 4.67 26.87 0.00 7.42 3.22 35.40 12.22 1.48 5.97 0.00
P S F 5.29 18.50 0.00 5.00 5.93 43.66 16.47 0.00 3.91 0.00
P S F 4.28 26.76 0.00 6.92 3.64 37.21 11.72 1.34 5.63 0.00
P S F 4.15 28.74 0.71 7.41 3.41 35.37 11.94 1.25 5.05 0.00
P S F 4.02 24.24 0.69 6.64 4.18 37.51 12.66 1.52 5.95 0.00
P S F 3.68 24.81 0.81 5.73 3.80 37.39 15.41 1.23 5.16 0.00
P S F 3.95 19.49 2.54 5.15 4.06 42.23 15.36 1.26 5.32 0.63
P S F 6.60 28.55 0.98 7.71 3.15 33.94 10.30 1.23 4.73 0.00
P C M 16.75 36.19 1.57 7.56 0.43 2.48 6.34 11.75 7.53 9.41
P C M 17.90 37.24 1.48 7.30 0.46 1.70 4.58 12.57 7.52 9.25
P S M 15.91 40.60 1.54 9.22 3.03 5.57 6.46 3.98 3.52 4.76
P S M 12.52 42.04 1.29 10.07 3.04 5.69 9.01 4.97 3.99 5.74
P S M 13.57 35.53 1.23 8.76 2.08 7.34 7.53 5.60 6.03 9.00
P S M 13.32 34.47 1.01 7.55 3.02 6.56 9.56 5.93 5.51 8.39
P S M 16.35 47.10 2.42 10.60 2.12 3.48 5.15 3.77 2.65 4.66
P S M 11.92 50.00 1.49 10.99 2.90 5.11 5.13 5.09 2.04 3.56
P S M 15.80 41.31 2.34 10.23 3.00 3.26 8.71 5.36 4.25 3.96
P S M 15.11 26.39 1.61 4.96 3.89 4.22 8.13 11.76 8.92 8.13


*Piracicaba, Os6rio, or hybrid (Fl generation from Piracicaba females and Os6rio males)

**Single or Composite










So Peak Retention Time

S 21.00 23.00 23.50 25.00 25.18 27.00 27.15 27.2 27.35 27.45
O C F 3.22 25.91 0.79 8.00 2.09 1.31 36.61 11.18 2.36 8.54
0 C F 3.18 27.25 1.12 11.19 1.88 0.98 32.79 11.80 1.89 7.92
O S F 0.00 23.89 3.58 12.40 12.40 24.58 9.77 1.16 7.05 2.33
O S F 3.66 52.78 6.90 7.08 2.54 31.42 12.74 1.42 8.46 0.00
O S F 1.75 22.90 10.36 10.83 1.44 26.88 10.28 2.01 11.15 2.40
O S F 5.18 16.86 0.72 3.82 2.59 35.57 14.03 2.76 10.69 1.59
O S F 1.36 26.02 3.84 9.67 1.43 33.78 13.02 0.00 1.53 7.25
0 S F 1.35 25.14 5.02 12.34 1.55 33.42 13.75 0.00 0.00 4.69
0 S F 3.66 23.77 2.62 5.96 2.72 36.00 13.20 0.00 2.12 9.94
0 C M 20.88 44.61 1.49 10.83 0.79 1.40 4.24 5.39 3.76 6.62
O C M 23.24 44.55 1.49 11.16 1.14 1.32 4.04 5.67 2.90 4.47
O S M 25.63 42.57 2.65 10.55 0.95 3.19 2.71 3.32 2.69 5.73
O S M 16.46 33.75 0.00 7.11 2.47 1.70 9.46 11.17 4.49 6.14
O S M 19.96 38.46 0.00 7.87 1.38 1.38 4.97 8.66 3.54 6.21
O S M 28.07 42.17 1.89 9.52 0.00 1.99 1.63 2.31 1.09 2.65
O S M 18.27 40.80 0.00 8.96 0.00 1.57 6.61 10.43 3.34 5.30
0 S M 17.16 46.53 3.11 15.75 0.00 0.00 0.00 1.44 0.00 3.16
0 S M 18.52 41.76 1.79 11.69 1.07 1.16 3.89 4.23 4.41 8.03
0 S M 16.27 44.64 0.00 10.09 0.00 0.00 8.02 8.14 3.21 4.37
H S F 3.80 12.04 0.60 3.61 5.76 47.91 14.85 1.10 3.14 0.00
H S F 3.25 18.88 0.51 5.61 4.66 42.55 14.78 1.17 4.41 0.00
H S F 2.82 24.89 0.00 6.86 4.13 43.40 6.98 1.72 6.02 0.00
H S F 3.37 22.13 0.62 6.50 2.98 42.76 6.19 2.44 8.14 0.00
H S F 4.00 14.89 0.00 4.25 6.31 43.69 18.73 1.11 4.06 0.00
H S F 2.01 20.28 0.53 6.54 3.49 40.77 7.64 2.51 11.10 1.14
H S F 3.05 22.34 0.80 6.92 2.98 42.24 5.94 2.43 8.33 0.00
H S F 3.82 21.91 0.00 5.51 3.52 42.53 10.24 2.04 7.20 0.00


*Piracicaba, Os6rio, or hybrid (F 1 generation from Piracicaba females and Os6rio males)


**Single or Composite














LIST OF REFERENCES

Adamo, S.A., D. Robert, J. Perez, and R.R. Hoy. 1995. The response of an insect
parasitoid, Ormia ochracea (Tachinidae), to the uncertainty of larval success
during infestation. Behav. Ecol. Sociobiol. 36: 111-118.

Amoroso, J. 1990. Ormia deplela parasitism of Manatee Co. trapped Scapteriscus
borellii. Annu. Report Mole Cricket Res. 12: 190-191.

Andersen, S. 1996. The Siphonini (Diptera: Tachinidae) of Europe. E.J. Brill; Leiden,
Netherlands. 148 pp.

Bai, B., and M. Mackauer. 1992. Influence of superparasitism on development rate and
adult size in a solitary parasitoid wasp, Aphidius ervi. Func. Ecol. 6: 302-307.

Baronio, P., M.L.Dindo, G. Campadelli, and L. Sighinolfi. 2002. Intraspecific weight
variability in Tachinid flies: response of Pseudogonia rufifrons to two host
species with different size and of Exorista larvarum to variations in vital space.
Bull. Insect 55: 55-61.

Buss, E.A, J. L. Capinera, and N. C. Leppla. 2002. Pest mole cricket management.
University of Florida Cooperative Extension Service document ENY-324.
Published world wide web at: http://edis.ifas.ufl.edu/LH039.

Burkett, D.A., D.A. Carlson, and D.L. Kline. 1998. Analysis of composition of sugar
meals of wild mosquitoes by gas chromatography. J. Amer. Mosq. Control Assoc.
14: 373-379.

Cantrell, B.K., and R.W. Crosskey. 1989. Family Tachinidae. pp. 733-784. In Evenhuis,
N.L. (ed). Catalog of the Diptera of the Australasian and Oceanian regions.
Bishop Museum Special Publication 86. Bishop Museum Press, Honolulu, and
E.J. Brill. 1155 pp.

Capinera, J.L. 2001.Green peach aphid in Featured Creatures (EENY-222). On world
wide web at: http://creatures.ifas.ufl.edu/veg/aphid/green_peach_aphid.htm.

Dindo, M.L., L. Sighinolf, G. Campadelli, and P.Baronio. 1999. Laboratory evaluation of
parasitism of wax moth and gypsy moth larvae by Exorista larvarum (L.) cultured
in vivo and in vitro. Boll. Inst. Entomol. 53: 109-119.








Eller, F.J., J.H. Tumlinson, and W.J.Lewis. 1990. Intraspecific competition in Microplitis
croceipes (Hymenoptera: Braconidae), a parasitoid of Heliothis species
(Lepidoptera: Noctuidae). Ann. Entomol. Soc. Am. 83: 504-508.

Fasulo, T.R., R.F.Mizell, and D.E. Short. 2003. WoodyPest. UF/IFAS. On world wide
web at: http://woodypest.ifas.ufl.edu/.

Fowler, H.G. 1987. Field confirmation of the phonotaxis of Euphasiopteryx depleta
(Diptera: Tachinidae) to calling males of Scapteriscus vicinus (Orthoptera:
Gryllotalpidae). Fla. Entomol. 70: 409-410.

Fowler, H.G. 1988a. Traps for collecting live Euphasiopteryx depleta_(Diptera:
Tachinidae) at a sound source. Fla. Entomol. 71: 654-656.

Fowler, HG. 1988b. Suitability of Scapteriscus mole crickets (Ort.: Gryllotalpidae) as
hosts of Euphasiopteryx depleta (Dip.: Tachinidae). Entomophaga 33: 397-401.

Fowler, H.G., and J.N. Kochalka. 1985. New record of Euphasiopteryx depleta (Diptera:
Tachinidae) from Paraguay: Attraction to broadcast calls of Scapteriscus acletus
(Orthoptera: Gryllotalpidae). Fla. Entomol. 68: 225-226.

Fowler, H.G., and A.V. Martini. 1993. Influencia do tamanho hospedeiro (Scapteriscus
borellii: Gryllotalpidae: Orthoptera) sobre a produgao experimental do parasit6ide
(Ormia depleta: Tachinidae: Diptera). Cientifica 21: 339-343.

Fowler H.G., and C.R. Garcia. 1987. Attraction to synthesized songs and experimental
and natural parasitism of Scapteriscus mole crickets (Orthoptera: Gryllotalpidae)
by Euphasiopteryx depleta (Diptera: Tachinidae). Rev. Bras. Biol. 47: 371-374.

Fowler, H.G., and A. Mesa. 1987. Alternate orthopteran hosts (Anurogryllus sp.) of
Euphasiopteryx depleta (Diptera: Tachinidae). Florida Entomol. 70: 408-409.

Frank, J.H. 2002. A parasitic fly that kills mole crickets. USGA Green Record. 40: 9-11.

Frank, J.H., E.A. Buss, and K. Barbara. 2002. Beneficial nematodes in turf: good for how
many years against pest mole crickets? Florida Turf Digest 19: 48-50.

Frank, J.H., T.R. Fasulo, and D.E. Short. 1998. MCRICKET. Published on world wide
web at: http://molecrickets.ifas.ufl.edu/.

Frank, J.H., C. Grissom, C. Williams, E. Jennings, C. Lippi, and R. Zerba. 1999. A
beneficial nematode is killing pest mole crickets in some Florida pastures and is
spreading. Fla. Cattleman Livestock J. 63: 31-32.









Frank, J.H., J.P. Parkman, and F.D. Bennett. 1995. Larra bicolor (Hymenoptera:
Sphecidae), a biological control agent of Scapteriscus mole crickets (Orthoptera:
Gryllotalpidae), established in northern Florida. Fla. Entomol. 78: 619-623.

Frank, J.H., T.J. Walker, and J.P. Parkman. 1996. The introduction, establishment and
spread of Ormia depleta in Florida. Biol. Control 6: 368-377.

Goolsby, J.A., and M.A. Ciomperlik. 1999. Development of parasitoid inoculated
seedling transplants for augmentative biological control of silverleaf whitefly
(Homoptera: Aleyrodidae). Fla. Entomol. 82: 532-545.

Hall, R.W., and L.E. Ehler.1980. Population ecology of Aphis nerii on oleander. Environ.
Entomol. 9: 338-344.
Hardy, I.C.W, N.T. Griffiths, and H.C.J. Godfray. 1992. Clutch size in a parasitoid wasp:
a manipulation experiment. J. Anim. Ecol. 61: 121-129.

Harvey, J.A., I.F. Harvey, and D.J. Thompson. 1993. The effect of superparasitism on
development of the solitary parasitoid wasp, Venturia canescens (Hymenoptera:
Ichneumonidae). Ecol. Entomol. 18: 203-208.

Hodge, S., and P. Mitchell. 1997. Inhibition of Drosophila melanogaster and D. hydei by
Aspergillus niger. Drosophila Information Service 80: 6-7.

Jacob, H.S., and E.W. Evans. 2004. Influence of different sugars on the longevity of
Bathyplectes curculionis (Hymenoptera: Ichneumonidae). J. Appl. Entomol. 128:
316.

Janzen, D.H. 1977. What are dandelions and aphids? Am. Nat. 111: 586-589.

Johnson, W.T., and H.H. Lyon. 1991. Insects that feed on trees and shrubs. Comstock
Pub. Associates. Ithaca, NY. 560 pp.

Justi J. Jr., J.R.P. Parra, and J.H. Frank. 1988. Alternate hosts for the production of
Euphasiopteryx depleta. Annu. Rep. Mole Cricket Res. 10: 183-184.

Leatemia, J. A., J.E. Laing, and J.E. Corrigan. 1995. Effects of adult nutrition of
longevity, fecundity, and offspring sex ratio of Trichogramma minutum Riley
(Hymenoptera: Trichogrammatidae). Can. Entomol. 127: 245-254.

Liang, D. and J. Silverman. 2000. "You are what you eat": Diet modifies cuticular
hydrocarbons and nestmate recognition in the Argentine ant, Linepithema humile.
Naturwissenschaften 87: 412-416.

Nickle, D.A., and J.L. Castner. 1984. Introduced species of mole crickets in the United
States, Puerto Rico and Virgin Islands (Orthoptera: Gryllotalpidae). Ann.
Entomol. Soc. Am. 77: 450-465.










Nguyen, K.B., and G.C. Smart, Jr. 1990. Steinernema scapterisci, new species,
(Rhabditida: Steinernematidae). J. Nematol. 22: 187-199.

Oesterbroek, P. 1998. The families of Diptera of the Malay archipelago. E.J. Brill;
Leiden, Netherlands. 227 pp.

Owen, D.F. 1978. Why do aphids synthesize melezitose? Oikos 31: 264-267.

Parkman, J.P., J.H. Frank, K.B. Nguyen, and G.C. Smart, Jr. 1994. Inoculative release of
Steinernema scapterisci (Rhabditida: Steinerematidae) to supress pest mole
crickets (Orthoptera: Gryllotalpidae) on golf courses. Environ. Entomol. 23:
1331-1337.

Parkman, J.P., J.H. Frank, T.J. Walker, and D.J. Schuster. 1996. Classical biocontrol of
Scapteriscus spp. (Orthoptera: Gryllotalpidae) in Florida. Environ. Entomol. 25:
1415-1420.

Petelle, M. 1980. Aphids and melezitose: a test of Owen's 1978 hypothesis. Oikos 35:
127-128.

Potting, R.P.J., H.M. Snellen, and L.E.M. Vet. 1997. Fitness consequences of
superparasitism and mechanism of host discrimination in the stemborer parasitoid
Cotesiaflavipes. Entomol. Exp. Appl. 82: 341-348.

Reinert, J.A., and D.E. Short. 1980. Southern turfgrass insect pests with emphasis on
mole cricket biology and management. Proc. Fla. Turfgrass Manag. Conf. 28: 33-
43.

Reinert, J.R., and D.E. Short. 1981. Managing mole crickets. Grounds Maint. 16: 16-20.

SAS Institute Inc. 2001. SAS system for Windows Rel. 8.2. SAS Institute Inc. Cary,
North Carolina, USA.

Short, D.E., and J.A. Reinert. 1982. Ch. 21 Biology and control of mole crickets in
Florida. In Niemczyk, H.D., and B.G. Joyner (eds). Advances in turfgrass
entomology. Chemlawn; Columbus, OH. 150 pp.

Soper, R.S., G.E. Shewell, and D. Tyrrell. 1976. Colcondamyia auditrix nov. sp.
(Diptera: Sarcophagidae), a parasite which is attracted by the mating song of its
host, Okanagana rimosa (Homoptera: Cicadidae) [New taxa]. Can. Entomol. 108:
61-68.

Sutton, B.D. and D.A. Carlson. 1993. Interspecific variation in tephritid fruit fly larvae
surface hydrocarbons. Archives Insect Biochem. Physiol. 23: 53-65.









Tsitspis, J.A. 1980. Relative humidity effects at 20 Celcius on eggs of the olive fruit fly
Dacus oleae (Diptera: Tephritidae) reared on artificial diet. Entomol. Exp. Appl.
28: 92-99.

van Dijken, M.J., and J.K. Waage. 1987. Self and conspecific superparasitism by the egg
parasitoid Trichogramma evanescens. Entomol. Exp. Appl. 30: 77-82.

Vet, L.E.M., A. Datema, A. Janssen, and H.M. Snellen. 1994. Clutch size in a larval-
pupal endoparasitoid: consequences for fitness. J. Anim. Ecol. 63: 807-815.

Vinson, S.B., and P. Sroka. 1978. Effects of superparasitism by a solitary endoparasitoid
on the host, parasitoid and field samplings. Southwestern Entomol. 3: 299-301.

Waage, J.K., and S.M. Ng. 1984. The reproductive strategy of a parasitic wasp. I.
Optimal progeny allocation in Trichogramma evanescens. J. Anim. Ecol. 53: 401-
415.

Walker, T.J. (ed.). 1985. Mole crickets in Florida. Univ. Fla. Agric. Exp. Stn. Bull. 846:
i-iv; 1-54.

Walker, T.J. 1988. Acoustic traps for agriculturally important insects. Fla. Entomol. 71:
484-492.

Walker, T.J. 1989. A live trap for monitoring Euphasiopteryx and tests with E. ochracea
(Diptera: Tachinidae). Fla. Entomol. 72: 314-319.

Walker, T.J., J.J. Gaffney, A.W. Kidder, and A.B. Ziffer. 1993. Florida reach-ins:
environmental chambers for entomological research. Am. Entomol. 39: 177-182.

Walker, T.J., and T.E. Moore. 2000. Singing insects of North America. On the world
wide web at: http://buzz.ifas.ufl.edu.

Walker, T.J., and D.A. Nickle. 1981. Introduction and spread of pest mole crickets:
Scapteriscus vicinus and S. acletus reexamined. Ann. Entomol. Soc. Am. 74: 158-
163.

Walker, T.J., J.P. Parkman, and D.J. Schuster. 1992a. Seasonal distribution and
population trends of Ormia depleta in Florida. Annu. Rep. Mole Cricket Res. 14:
118-123.

Walker, T.J., J.P. Parkman, and D.J. Schuster. 1992b. Sound-trap assays of population
trends: Annual update. Annu. Rep. Mole Cricket Res. 14: 1-5.

Walker, T.J., J.P. Parkman, J.H. Frank, and D.J. Schuster. 1996. Seasonality of Ormia
depleta and limits to its spread. Biol. Control 6: 378-383.






76


Welch, C.H. 2000. Gas chromatographic analyses of crop sugars of Ormia depleta
established in Florida. Master's Thesis. University of Florida.

Wineriter, S.A., and T.J. Walker. 1990. Rearing phonotactic parasitoid flies (Diptera:
Tachinidae, Ormiini, Ormia spp.). Entomophaga 35: 621-632.

Wood, D.M. 1987. Chapter 110. Tachinidae. In McAlpine, J.F. et al. (eds.), Manual of
Nearctic Diptera. Volume 2. Agriculture Canada Monograph 28: 1193-1269

Zuk, M., L.W. Simmons, and L. Cupp. 1993. Calling characteristics of parasitized and
unparasitized populations of the field cricket Teleogryllus oceanus. Behav. Ecol.
Sociobiol. 33: 339-343.














BIOGRAPHICAL SKETCH

Craig Hinton Welch was born on February 15, 1971, in Knoxville, Tennessee, to

Sara H. and Robert N. Welch. He grew up in Dover, Delaware, where he attended school

and spent summers at a YMCA camp in North Carolina where his parents worked as

administrators and his interest in biology began. In 1986, his family moved to Salisbury,

North Carolina, where he graduated from high school in 1989. He then went on to earn

his Bachelor of Science degree in biology from the University of North Carolina at

Charlotte. He married his wife Celeste on May 13, 1995. In 2000, he earned his master's

degree from the University of Florida in entomology. He and his wife have four

daughters: Brooke, Courtney, Natalie, and Valerie.










I certify that 1 have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.


J. Howard Frank, Chair
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.


Eileen Buss
Assistant Professor of Entomology and
Nematology

1 certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosoph.


Rbert McSorley
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.


Bijan gan
Professor of Horticultural Science

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 q *ty, as a
dissertation for the degree of Doctor of Philosophy


Frank lansky
Professor of Entomolog} \id Nematology








This dissertation was submitted to the Graduate Faculty of the College of
Agricultural and Life Sciences and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

August 2004

Dean, College of Agricultural fe
Sciences G



Dean, Graduate School



























I) tUI


3 1262 08556 6268




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Ormia depleta:
LABORATORY MAINTENANCE, STRAIN IDENTIFICATION,
AND EVALUATION OF Aphis nerii AS A BANKER SPECIES
By
CRAIG H. WELCH
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2004

ACKNOWLEDGMENTS
I would like to thank Dr. J. Howard Frank for the help, support, and patience he
has offered me over the past years and my committee for the advice and guidance offered
in the development of this dissertation. I would like to thank Dr. Robert Hemenway for
his countless hours of work in maintaining fly and cricket colonies, without which none
of this research would have been possible. I would like to thank the many people at the
Gulf Coast Research and Education Center in Bradenton, Florida, for their help in
running the sound traps and collecting flies. I would like to thank the Green Section of
the US Golf Association for their financial support of this research. Their support is what
made it possible to import the Osorio strain of Ormia depleta, which was crucial to this
project. I would also like to thank Dr. Ken Portier for his help in statistical analysis.
Finally, I want to express my love and appreciation to my wife, Celeste, who for the past
seven years of my time in graduate school has shown me great love and patience, has
cooked dinners, done five million loads of laundry, managed our household, paid our
bills, and given birth to four daughters, who are the joy of our lives.

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT viii
CHAPTER
1 REVIEW OF LITERATURE 1
2 VARIATION OF PARAMETERS FOR REARING ORM1A DEPLETA (DIPTERA:
TACHINIDAE) AND INDUCING DIAPAUSE 7
Introduction 7
Materials and Methods 10
Environmental Chamber Rearing Parameters 10
Melezitose and Fecundity 12
Results 13
Environmental Chamber Rearing Parameters 13
Melezitose and Fecundity 15
Discussion 16
3 INTRASPECIFIC COMPETITION FOR RESOURCES BY ORM1A DEPLETA
(DIPTERA: TACHINIDAE) LARVAE 20
Introduction 20
Materials and Methods 22
Results 23
Discussion 28
4 REARING ORMIA DEPLETA (DIPTERA: TACHINIDAE), A PARASITOID OF
MOLE CRICKETS (ORTHOPTERA: GRYLLOTALPIDAE), ON A FACTITIOUS
HOST, ACHETA DOMESTIC US (ORTHOPTERA: GRYLLIDAE) 33
Introduction 33
iii

Materials and Methods 36
Larval Development of Ormia depletet in Acheta domesticus 36
Larval Behavior within host 37
Results 37
Larval Development of Ormia depleta in Acheta domesticus 37
Larval Behavior within host 38
Scapteriscus abbreviatus- 38
Acheta domesticus- 44
Discussion 49
5 IDENTIFICATION OF TWO SOUTH AMERICAN GEOGRAPHICAL ISOLATES
OF ORMIA DEPLETA BY ANALYSIS OF CUTICULAR HYDROCARBONS....53
Introduction 53
Materials and Methods 53
Results 55
Discussion 59
6 SURVIVAL OF ORMIA DEPLETA WHEN CAGED WITH HONEYDEW-
PRODUCING APHIS NERII 60
Introduction 60
Materials and Methods 62
Results 63
Discussion 63
APPENDIX
A THE EFFECT OF PUPAL SIZE ON EMERGENCE 66
B ATTEMPTED INDUCTION OF DIAPAUSE BY SHORT DAY PHOTOPERIOD IN
OSORIO STRAIN ORMIA DEPLETA 67
C GAS CHROMATOGRAPHIC ANALYSIS OF THE CUTICULAR
HYDROCARBONS OF ORMIA DEPLETA TO IDENTIFY STRAINS 69
LIST OF REFERENCES 71
BIOGRAPHICAL SKETCH 77
iv

LIST OF TABLES
Table page
1 Levels of parameters tested and treatment numbers assigned 12
2 Percentage of females gravid - arcsine square root transformation F-values and p-
values for the main factors and interactions 14
3 Percentage of females becoming gravid within each treatment combination 14
4 The effect if temperature, humidity, and photoperiod on the percentage of mortality of
O. depleta 15
5 Mortality F-values and p-values for the main factors and interactions 15
6 Mean number and length of O. depleta larvae found in S. abbreviates and A.
domesticus hosts 38
7 Retention times of GC peaks for the cuticular hydrocarbons of O. depleta 56
8 The percent emergence of O. depleta pupae of varying sizes 66
v

LIST OF FIGURES
Figure page
1 Old style rearing cages for O. depleta 11
2 Experimental style of rearing cage for O. depleta 11
3 The effect of planidia density used to inoculate mole crickets on the number of pupae
produced (error bars indicate standard deviation, significantly different means
indicated by letters over bars as determined by Duncan’s procedure, a = 0.05) 24
4 The effect of number of planidia used to inoculate mole crickets on the number of
pupae produced - regression analysis with 95% confidence bands 25
5 The effect of number of planidia used to inoculate mole crickets on the survival rate of
the larvae to the pupal stage (error bars indicate standard deviation, significantly
different means indicated by letters over bars as determined by Duncan’s
procedure, a = 0.05) 25
6 The effect of number of planidia used to inoculate mole crickets on the survival rate of
the larvae to the pupal stage - regression analysis with 95% confidence bands 26
7 The effect of number of planidia used to inoculate mole crickets on the mean weight of
the pupae produced (error bars indicate standard deviation, significantly different
means indicated by letters over bars as determined by Duncan’s procedure, a =
0.05) 26
8 The effect of number of planidia used to inoculate mole crickets on the mean weight of
the pupae produced - regression analysis with 95% confidence bands 27
9 The effect of host cricket weight on mean pupal weight (error bars indicate standard
deviation, significantly different means indicated by letters over bars as determined
by Duncan’s procedure, a = 0.05) 28
10 The effect of host cricket weight class on the mean weight of the pupae produced -
regression analysis with 95% confidence bands 28
11 Larva of O. depleta in S. abbreviates host one day after inoculation 40
12 Larva of O. depleta in S. abbreviates host two days after inoculation 40
vi

13 Larva of O. depleta in S. abbreviatus host three days after inoculation 41
14 Larva of O. depleta in S. abbreviatus host four days after inoculation 41
15 Scapteriscus abbreviatus host five days after inoculation showing the external
evidence of the attachment point of O. depleta larva 42
16 Larvae of O. depleta in S. abbreviatus host five days after inoculation 42
17 Larvae of O. depleta in S. abbreviatus host six days after inoculation 43
18 Two O. depleta larvae in S. abbreviatus host six days after inoculation showing detail
of the larval attachment to the host 43
19 Larva of O. depleta in S. abbreviatus host seven days after inoculation 44
20 Larva of O. depleta in A. domesticus host one day after inoculation 45
21 Larva of O. depleta in A. domesticus host two days after inoculation 46
22 Larva of O. depleta in A. domesticus host three days after inoculation 46
23 Larva of O. depleta in A. domesticus host four days after inoculation 47
24 Larvae of O. depleta in A domesticus host five days after inoculation 47
25 Larvae of O. depleta in A. domesticus host six days after inoculation 48
26 Larvae of O. depleta in A. domesticus host seven days after inoculation 48
27 Seven day old larvae of O. depleta from S. abbreviatus (top) and A. domesticus
(bottom) 49
28 The growth of O. depleta larvae in alternate hosts: S. abbreviatus and A. domesticus49
29 The mean percentage of total peak area for the gas chromatograms of the cuticular
hydrocarbons of female Piracicaba strain Ormia depleta 56
30 The mean percentage of total peak area for the gas chromatograms of the cuticular
hydrocarbons of female Osorio strain Ormia depleta 57
31 The mean percentage of total peak area for the gas chromatograms of the cuticular
hydrocarbons of male Piracicaba strain Ormia depleta 57
32 The mean percentage of total peak area for the gas chromatograms of the cuticular
hydrocarbons of male Osório strain Ormia depleta 58
33 Scatter plot representation of the values for peaks F and I for male O. depleta of the
Piracicaba and Osório strains 58
vii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
OR MIA DEPLETA-.
LABORATORY MAINTENANCE, STRAIN IDENTIFICATION,
AND EVALUATION OF APHIS NERII AS A BANKER SPECIES
By
Craig H. Welch
August, 2004
Chair: J. Howard Frank
Major Department: Entomology and Nematology
Ormia depleta (Wiedemann) is a tachinid fly used in the biological control of
Scapteriscus spp. mole crickets. Several changes to the current method of rearing O.
depleta in the laboratory were attempted. Manipulations of temperature, humidity, and
photoperiod during the pupal and adult stages showed that the percentage of gravid
females was higher at 23°C than at 27°C and at 70% relative humidity than at 85%, but
photoperiod did not have a significant effect. A diet of melezitose did not increase the
fecundity of flies compared to a melezitose-free diet. Increasing the number of planidia
used to inoculate host mole crickets increased the number of pupae produced, but reduced
the survivability of those planidia and the mean weights of the pupae produced. Larger
host mole crickets were also found to produce larger pupae. Acheta domesticus was tried
as a factitious host and found to be unsuitable. Although pupae were produced, they were
small and few in number. Dissections of hosts with developing larvae showed that the
viii

larvae in Acheta developed slowly and apparently had trouble emerging from the host.
Identification of two Brazilian strains of O. depleta was performed by gas
chromatographic analysis of cuticular hydrocarbons. The results indicated that, using the
model used in this experiment, the strains could be identified with 80% confidence. An
evaluation of the honeydew of Aphis nerii on its milkweed host indicated that it is an
adequate carbohydrate source for O. depleta, but further study is needed in this area
before any recommendations can be made.
IX

CHAPTER 1
REVIEW OF LITERATURE
Four species of mole crickets are currently established in Florida. Three of these
are immigrant pests of the genus Scapteriscus: S. vicinus Scudder (the tawny mole
cricket), S. abbreviates Scudder (the short-winged mole cricket), and S. borellii Giglio-
Tos (the southern mole cricket). These three species can be differentiated by several
features including dactyl spacing and the characteristics of the male song (Nickle and
Castner 1984). All three Scapteriscus spp. mole crickets are believed to have arrived
from South America around the year 1900 (Walker and Nickle 1981). S. vicinus and S.
borellii are thought to have initially arrived in the United States through Brunswick,
Georgia, in 1899 and 1904, respectively (Walker and Nickle 1981, Nickle and Castner
1984). Scapteriscus abbreviates apparently arrived several times between 1899 and 1923
into several ports in Florida and southernmost coastal Georgia (Walker and Nickle 1981).
Since then, Scapteriscus mole crickets have become major agricultural pests throughout
Florida and into Georgia, Mississippi, South Carolina, and other states (Frank et al.
1998). They are the most damaging pests of turf and pasture grasses in Florida and are
also pests of vegetable and field crops (Reinert and Short 1980). Bahiagrass, Paspalum
notatum Fluegge, is apparently the preferred, and therefore most damaged, plant species
(Reinert and Short 1980). Damage is typically caused by either the tunneling of the mole
crickets, which exposes the roots to desiccation, or by direct feeding on the roots (Reinert
and Short 1980). Traditionally, these pests have been controlled through the use of
chemical pesticides such as carbaryl, chlorpyrifos, acephate, bifenthrin, fipronil,
1

2
imidacloprid, and others (Buss et al. 2002). Pesticide use is only a short-term solution
because most of Florida’s over three million hectares of bahiagrass are in low-
maintenance areas such as pasture and roadside (Short and Reinert 1982), providing an
unlimited supply of mole crickets during flight periods in September-October and April-
May (Walker et al. 1992a). However, because multiple treatments are required to achieve
adequate levels of control (Parkman et al. 1996) and because of other concerns raised by
pesticide use, alternative methods of control have been sought.
At least four biocontrol agents have been considered for control of immigrant mole
crickets, three of which have already been released in the field. The entomopathogenic
nematode Steinernema scapterisci Nguyen and Smart has been effective against mole
cricket populations, with infected individuals being found up to eight years in pastures
after initial treatment and twelve years on golf courses after initial treatment (Frank et al.
1999, Frank et al. 2002). Steinernema scapterisci shows a much higher specificity toward
its mole cricket hosts than do other Steinernema species (Nguyen and Smart 1990) and
has had a measurable effect on mole cricket populations in treated areas (Parkman et al.
1994). The sphecid wasp Larra bicolor F. has been released and established in Florida,
and its population is spreading in northern Florida. However, little research has been
done on its effectiveness to date. The bombardier beetle Pheropsophus aequinoctialis L.,
a predator of mole cricket eggs, has not yet been released in Florida, but may be released
in the future (personal communication from Dr. J.H. Frank, University of Florida).
The tachinid fly Ormia depleta (Wiedemann) is an obligate parasitoid of
Scapteriscus mole crickets. Female flies are phonotactic to the call of the male
Scapteriscus spp. crickets (Fowler 1987, Fowler and Garcia 1987, Walker et al. 1996).

3
Phonotaxis is an uncommon host-locating method known in only one other dipteran,
Colcondamyia auditrix Shewed, a sarcophagid parasitoid of cicadas (Soper et al. 1976).
Ormia spp. are nocturnal and crepuscular, and collection can only be done reliably with
sound emitter traps that mimic the call of the host. Ormia depleta was originally brought
from Piracicaba, Brazil, to be used as a biocontrol agent against Scapteriscus spp. mole
crickets and was first released in 1988 (Frank et al. 1996). Since then, it has become
established in 38 counties in Florida, and in some it has been demonstrated to suppress
mole cricket populations (Parkman et al. 1996). This original strain seemed to have a
northern limitation to its establishment around 28° N latitude (Walker et al. 1996).
Because the strain was collected near Piracicaba, Estado de Sao Paulo, around 23° S
latitude (Piracicaba strain), it was speculated that flies collected from more temperate
latitudes would be more cold-hardy and able to establish farther north in the United
States. Dr. Howard Frank returned to Brazil in 1999, and collected a new strain of flies
near Osorio in Estado do Rio Grande do Sul, approximately 30° S latitude (Osorio strain)
(Frank 2002). The Osorio strain was laboratory-reared and in 2000-2001, several releases
were made in North Carolina, Georgia, Texas, and Louisiana. Adequate trapping and
monitoring of flies in these locations have not yet occurred to determine whether the
Osorio strain has established (Frank 2002).
Flies in the family Tachinidae are all parasitoids of other arthropods, almost
exclusively other insects (Cantrell and Crosskey 1989, Andersen 1996). In the more
primitive tachinid species, eggs are deposited directly on the host, whereas other species
deposit microtype eggs on their host’s food plant, inject eggs into the host’s body wall, or
drop newly hatched larvae on or near the host (Cantrell and Crosskey 1989). The most

4
common hosts are the larvae of Lepidoptera and Coleóptera, but Hymenoptera,
Hemiptera, Orthoptera, Dermaptera, other Diptera and, in some species, centipedes and
isopods are hosts (Cantrell and Crosskey 1989, Andersen 1996). Tachinids are among the
most important natural regulators of insect populations (Oesterbroek 1998). Few
tachinids are known to be species-specific in their choice of host, with some species
attacking hosts from three orders of insects (Cantrell and Crosskey 1989). Their success
as biocontrol agents has been mixed, with many attempts failing (Cantrell and Crosskey
1989). Adult tachinid flies, in general, are known to feed at flowers and do not require
any significant source of protein (Andersen 1996). They are very active and require a
daily source of sugar from plant nectar, homopteran honeydew, or sap from tree wounds
(Oesterbroek 1998).
Wineriter and Walker (1990) developed the original rearing protocol for O. depleta.
This is the protocol used throughout this dissertation unless specified otherwise. This
protocol requires that gravid female flies be sacrificed and the oviducts removed. The
oviducts are then placed on moist filter paper and gently tom open, releasing the planidia.
Three fully developed planidia are then placed behind the posterior margin of the
pronotum of the host. The hosts (usually S. abbreviates) are then placed in individual
vials of moist sand while the larvae develop. Both male and female mole crickets are
used. Twelve days after inoculation, the vials are emptied and the pupae retrieved from
the sand and placed in emergence boxes. Each box contains ~2 cm moist sand and 100
pupae are placed on the sand and then covered with another ~ 5 mm layer of moist sand.
Each emergence box is then placed in a large rearing cage consisting of a clear acetate
tube covered on both ends with bucket lids. These cages are placed near a window that

5
receives indirect sunlight and the flies are allowed to emerge into the cage. Food and
water are offered in Petri dishes wicked with cotton balls. After approximately four
weeks in the rearing cage, gravid females are removed and sacrificed to produce the next
generation. For the colonies kept at the University of Florida mole cricket rearing
laboratory, two rearing cages are maintained under normal circumstances. Each cage is
started with approximately 100 pupae. To obtain this number of pupae, approximately
200 S. abbreviates are inoculated with three planidia each, with the expectation of
obtaining two viable pupae per host (personal communication with Robert Hemenway,
University of Florida).
Although this rearing protocol has been fairly successful in maintaining laboratory
colonies of O. deplete, a new protocol was desired that would reduce the unpredictable
variability in the percentage of gravid flies produced in each generation as well as
allowing for smaller colonies to be maintained. These smaller colonies could be reared in
environmental chambers to control the factors of photoperiod, temperature, and humidity.
The number of planidia per host could be manipulated to determine the optimal use of
this limiting factor in colony growth.
Because the Osorio strain has been released but not yet recaptured, a method was
needed to differentiate between the two strains to identify the strain of individuals
recaptured in the future. This is especially important in identifying flies from areas that
lie between the established range of the Piracicaba strain and the new release sites of the
Osorio strain.
In addition, there is some demand for a method to locally augment populations of
O. deplete in areas where a greater level of mole cricket control is desired, such as golf

6
courses and bahiagrass pastures. The use of a plant/aphid banker system for the
production of honeydew and subsequent attraction of O. depleta was investigated to
determine whether the honeydew produced by this system is an adequate food source for
the flies.

CHAPTER 2
VARIATION OF PARAMETERS FOR REARING ORMIA DEPLETA (DIPTERA:
TACHINIDAE) AND INDUCING DIAPAUSE
Introduction
The protocol for rearing O. depleta set by Wineriter and Walker (1990) has been
successful in keeping colonies at the University of Florida’s mole cricket rearing
laboratory almost continuously since 1988. The greatest limitation of this method is
primarily in the large number of flies required per cage in order to produce gravid
females. Many advantages would be gained if a method for rearing O. depleta in smaller
colonies were to be devised.
Although the Piracicaba strain of O. depleta has become well established
throughout southern Florida and somewhat established in central Florida, its range falls
far short of that of its host (Frank et al. 1996). The releases of the Osorio strain may
increase the fly’s range, but this has not yet been demonstrated (Frank 2002).
Augmentative releases may be a possible solution, but O. depleta is a very difficult
organism to reliably rear under the protocol set by Wineriter and Walker (1990). The
populations show a great deal of instability and reproductive success is unpredictable. In
an attempt to control some of the unpredictability of laboratory colonies, a revised
protocol was tested to rear flies under the controlled conditions of an environmental
chamber. The effects that had the greatest probability of affecting the outcome of the
colonies (humidity, temperature, and photoperiod) were tested at two levels each. Natural
sunlight, one of the factors long thought to be necessary for O. depleta to mate
7

8
successfully (Wineriter and Walker 1990), was not investigated. Dependence on natural
sunlight introduces another uncontrollable variable and other research has proved that
Ormia depleta can reproduce successfully in the absence of natural light (Welch 2000).
Perhaps the most compelling reason for a new rearing method is to maintain smaller
colonies. The current method requires at least 100 pupae to be used in each large (30 cm
diameter x 60 cm tall, approximately 35 liter) cage (Figure 1). Maintaining fewer flies in
smaller cages would facilitate experimentation and reduce the risk to the entire laboratory
colony, should one colony fail. This experiment was designed to determine the levels of
temperature, humidity, and photoperiod that would be most conducive to rearing O.
depleta in small colonies in environmental chambers.
Many homopterans, including aphids, feed on the liquids they extract from plants
by piercing the phloem tissues with their stylets (Owen 1978). The result is a diet very
high in sugars, and excess sugars are excreted as honeydew, which is used as a food
source for many organisms such as fungi, bacteria, bees, moths, ants and flies, including
O. depleta (Owen 1978, Welch 2000). Most of the sugars found in homopteran honeydew
are also occur in the saps of the plants on which they feed, such as fructose, glucose and
sucrose (Percival 1965). Melezitose (a-D-glucose-[l-*3]-f3-D-fructose-[2->l ]- a-D-
glucose) is a trisaccharide found in homopteran honeydew, but not in plants (Petelle
1980). The enzyme which makes melezitose has been isolated from the midgut of aphids
and shown to produce melezitose, fructose, and glucose from sucrose (Petelle 1980).
It is not fully understood why homopteran insects produce melezitose. Owen
(1978) hypothesized that aphids produce honeydew because it offers them an
evolutionary advantage. His theory has two requirements. The first requirement is that an

9
aphid and all her parthenogenically-produced offspring be considered a single
evolutionary individual (Janzen 1977) such that any benefit provided to offspring can be
considered benefit to self. The second, and most crucial to his argument, is that
melezitose must be a sugar source which is not as widely usable to other organisms as
other sugars, but highly usable to nitrogen-fixing bacteria. Aphids that produced
melezitose-heavy honeydew would be benefiting their host plants by providing a nitrate
source and, therefore, benefiting their offspring and, from the evolutionary individual
standpoint, themselves. This theory was refuted by Petelle (1980), who found that
fructose was nine times better than melezitose as a carbohydrate source for nitrogen¬
fixing bacteria. Instead, he postulated that, because the enzymatic action which produces
melezitose prevents sucrose from breaking down into fructose and glucose and in the
process ties up two molecules of glucose and one of fructose, the production of
melezitose reduces the concentration of monosaccharides in the midgut of the insect. This
reduction in concentration would be beneficial to the insect in that it would reduce the
uptake of these sugars. Too much sugar uptake would increase the osmotic pressure and
require the insect to ingest a large amount of water.
The question of how melezitose influences the longevity of O. depleta has been
investigated (Welch 2000). It was found to be a suitable diet, indicating that O. depleta
has the necessary enzymes to hydrolyze melezitose, but it did not perform better than a
diet of hummingbird feeder nectar containing no melezitose. Although this may provide
some insight into the importance of melezitose in the diet of O. depleta, for the purpose
of maintaining laboratory colonies, longevity is not as important as the ability to produce

10
offspring. An experiment was designed to determine the influence of melezitose on the
fecundity of mated female O. depleta.
Materials and Methods
Environmental Chamber Rearing Parameters
Scapteriscus abbreviates were obtained from the mole cricket rearing laboratory
at the University of Florida. This colony was originally collected from Broward County,
Florida, and was inoculated with planidia from Osorio strain O. depleta. Each mole
cricket was inoculated by placing four planidia behind the posterior margin of the
pronotum and then placing it in a vial of moist sand. After 12 days, pupae were collected
from the mole cricket vials. Four small cups (approximately 10 cm diameter, 4 cm deep)
were filled with approximately 2 cm damp sand, and 35 O. depleta pupae were placed in
each cup on top of the sand with the spiracles pointing upward. The pupae were then
covered with a thin layer (0.5-1.0 cm) of moist sand and each cup was placed in a small
plastic rearing box (10 x 15 x 20 cm) lined with paper towel and covered with a lid
(Figure 2). The lid was ventilated with a 10-cm-diameter hole covered with a fine screen
mesh. A nutrient solution containing a 1:1:1 solution of sucrose, fructose, and melezitose
(40g each /L) was kept in a small plastic Petri dish wicked with cotton and placed in each
rearing box. Each rearing box was also provided with water in a small plastic Petri dish
wicked with cotton. The nutrient solution was colored green for identification with
approximately 25pL/L of food coloring (McCormick, Hunt Valley, MD). Both the
nutrient solution and water were refilled as needed and replaced if mold appeared. Each
box with pupae was then placed in a separate Florida Reach-In environmental chamber
(Walker etal. 1993)

11
Figure 1: Old style rearing cages for O. depleta
Figure 2: Experimental style of rearing cage for O. depleta
set to a specific temperature, humidity and photoperiod. Each of these three variables had
two levels determined by preliminary experimentation (Table 1) and was set up according
to a balanced incomplete block design (a=8, b=14, k=4, r=7, k=3). This design was
necessary to accommodate all eight treatment combinations. A fully randomized model

12
would have been preferred, but this would require eight environmental chambers, which
were not available, and enough pupae to set up eight experimental units, which were a
greater number than our laboratory colony could reliably produce. The low humidity of
70% was used because, in preliminary trials, flies kept much below that range suffered
high mortality. The high humidity of 85% was chosen because the Florida Reach In units
do not do a good job of maintaining relative humidity higher than that amount. After 30
days in the chambers, all flies were removed and checked for gravidity. Mature planidia
are easily visible through the abdomen of gravid females. The numbers of dead males,
dead females, live males, live females, and gravid females were recorded. Gravid females
were then sacrificed and their planidia used to inoculate mole crickets for the next
generation. For statistical analysis, the percentage of gravid females was transformed
using the arcsine square root transformation (sin1 Vx). Both the arcsine square root of the
percentage of gravid females and the mortality of the flies were analyzed by ANOVA
(SAS Institute 2001).
Table 1: Levels of parameters tested and treatment numbers assigned
Treatment # 1
2
3
4
5
6
7
8
TT M- 70%
Humidity
85%
85%
70%
70%
85%
70%
85%
23 C
Temperature
23 C
27 C
27 C
23 C
23 C
27 C
27 C
n, . , 10 hrs
rhotopenod
14 hrs
10 hrs
10 hrs
14 hrs
10 hrs
14 hrs
14 hrs
Melezitose and Fecundity
Colonies of Osorio strain O. depleta were obtained from the mole cricket rearing
laboratory at the University of Florida. The colonies were reared on S. abbreviates and

13
maintained following the protocols outlined previously, with the exception of the food
provided. Colonies were provided a diet of either hummingbird nectar (Perky-Pet®
Denver, Colorado, Instant Nectar, containing: unspecified proportions of sucrose,
glucose, tartaric acid, sodium benzoate, artificial color and flavor), which had long been
used as a sugar source for laboratory rearing at the University of Florida mole cricket
rearing laboratory, or a solution of melezitose only (100 g/L). Both diets were provided
ad lib and replaced if signs of mold appeared. When gravid females appeared
(approximately 6 weeks after mole crickets were inoculated), females were sacrificed and
their oviducts removed by dissection of the abdomen. The planidia were removed and
used to inoculate mole crickets for the next generation. During inoculation, the number of
mature planidia was counted and recorded. Only dark, well-sclerotized larvae were
counted. Immature, light colored planidia and eggs were not counted. This procedure was
repeated over several generations of O. depleta during the course of maintaining the
laboratory colonies.
Results
Environmental Chamber Rearing Parameters
Determining the statistical significance of the variables temperature, humidity and
photoperiod was not possible using the variable “ratio gravid” (number of gravid females
/ number of live females). Only 18 out of the 56 trials produced gravid females. The large
number of zeros shifted the distribution so that parametric statistics were not meaningful.
By changing the unit of measurement from treatments to individual flies, it was possible
to produce a binomial distribution of female flies that survived the duration of the
treatment and indicate whether or not each one became gravid. Using the arcsine square
root transformation, the ANOVA results indicated that the lower temperature (23°C)

14
produced a higher percentage of gravid females (F = 9.40, P = 0.004) and that humidity
was marginally significant (F = 4.05, P = 0.052), with the lower humidity (70% RH)
producing more gravid females. Photoperiod was not significant (P > 0.05) nor were any
of the two-way or three-way interactions (Table 2). The analysis of these data remains
somewhat suspect, however, due to the fact that blocks 3, 10 and 12 failed to produce any
gravid females. Because of this, the statistical software was unable to converge on a
likelihood confidence interval, so some observations were eliminated from the analysis
(SAS Institute 2001). Both the lower temperature and the lower humidity produced a
larger percentage of gravid females (Table 3).
Table 2: Percentage of females gravid - arcsine square root transformation F-values and
p-values for the main factors and interactions
Block
0.54
0.884
Temperature
9.40
0.004
Humidity
4.05
0.052
Photoperiod
2.56
0.119
Temperature x Humidity
1.60
0.214
Temperature x Photoperiod
0.75
0.394
Humidity x Photoperiod
0.01
0.994
Temperature x Humidity x Photoperiod
1.01
0.323
Table 3: Percentage of females becoming gravid within each treatment combination.
23 C 27C
10 Hours 14 Hours 10 Hours 14 Hours
70% RH 15.98% 17.92% 3.90% 3.57%
85% RH 18.79%
1.10%
0.00%
0.00%

15
An ANOVA analysis of the mortality of the flies showed the treatment effect to be
highly significant (F = 3.67, P < 0.003). The percentage mortality for the eight treatments
ranged from 30.4% to 72.6% (Table 4). The higher temperature and longer photoperiod
produced higher mortality, whereas humidity and all two and three way interactions were
not significant (Table 5). The block effect was not significant (F= 1.51, P > 0.15).
Table 4: The effect if temperature, humidity, and photoperiod on the percentage of
mortality of O. depleta
23 C 27C
10 Hours 14 Hours 10 Hours 14 Hours
70 % RH 39.1% 41.3% 44.7% 67.3%
85% RH 30.4% 44.0% 55.6% 72.6%
Table 5: Mortality F-values and p-values for the main factors and interactions
Source
F -value
P Block
1.51
0.156
Temperature
13.87
0.0005
Humidity
0.65
0.424
Photoperiod
11.24
0.002
Temperature x Humidity
1.46
0.232
Temperature x Photoperiod
0.77
0.386
Humidity x Photoperiod
0.43
0.514
Temperature x Humidity x Photoperiod
2.85
0.098
Melezitose and Fecundity
The planidia of 11 melezitose-fed flies and 12 non-melezitose-fed flies were
counted. The mean number of planidia for melezitose-fed females was 248 + 73 (SD)
with a range of 121-392. For non-melezitose fed females, the mean was 232 ± 71 (SD)

16
with a range of 117-341. A t-test showed no difference between these two treatments (P =
0.61).
Discussion
The rearing of O. deplela inside environmental chambers was only somewhat
successful. For some generations, more flies became gravid in the four environmental
chambers than in the two large laboratory colonies. However, as previously stated, there
were several generations that did not produce any gravid females in the environmental
chambers. No tests were conducted to determine whether the colonies inside the
chambers would be more successful if maintained as large colonies versus the small
colonies used in this experiment.
Temperature was the factor of greatest significance. Although the flies can
develop normally at temperatures above 30° C (Cabrera 2000), mold appeared more often
in the chambers kept at 27° C versus those kept at 23° C. Food containers had to be
changed very frequently and fungi were visible growing on the paper towel lining the
bottom of the cage on several occasions. The increase in mortality from 23° C to 27° C
(38.7% vs. 60.1%) could have also been due in part to problems caused by fungal growth.
Fungi have been known to cause high mortality in laboratory colonies of Drosophila
hydei Sturtevant and D. melanogaster Meigen (Hodge and Mitchell 1997).
It was surprising that humidity was not a more significant factor in the successful
rearing of O. depleta than the data show (P = 0.052). It may have been discounted
somewhat in this experiment due to the fairly narrow range of humidities used. The lower
humidity level most likely was more successful again because of reduced fungal growth.
There was a greater incidence of fungi in the high humidity chambers over the low

17
humidity chambers. The mortality between the two levels of humidity was essentially the
same. Humidity is often a major source of mortality in rearing insects (Tsitsipis 1980,
Leatemia et al. 1995); this further supports the hypothesis that the range of humidity
tested here was too narrow to show the real significance of this factor.
Photoperiod was tested due to the traditional thinking that sunlight was in some
way necessary for O. depleta to mate successfully. The production of gravid females
within the environmental chambers showed that sunlight is not a necessity for O. depleta
to reproduce; however, it would be irresponsible to discount that the regular laboratory
colonies did, for some generations, produce more gravid females than those in the
environmental chambers. Until specific testing has been conducted, no changes would be
suggested to that particular practice. It was unexpected to find such a drastic difference
between the mortality of the flies kept on the two levels of photoperiod. The higher
mortality caused by the longer photoperiod may have been due to these flies having to go
longer periods without eating, as the flies do not often appear to feed actively during the
daylight hours.
Because some generations did not produce any gravid females in the small colonies
in the environmental chambers, it cannot be recommended that this method be adopted
for regular laboratory use. Future research in this area may investigate how larger
colonies of flies, such as those currently used in laboratory rearing of O. depleta, are able
to produce gravid females in environmental chambers. The most likely parameters to be
successful would be in the range of the lower temperature and lower humidity used in
this experiment.

18
Previous research has shown that melezitose does not increase or decrease the
survival of O. depleta compared to other carbohydrate diets (Welch 2000). In the
ichneumonid wasp Bathyplectes curculionis Thomson, longevity was significantly
reduced when fed a diet of melezitose versus other sugars (Jacob and Evans 2004). This
indicates that O. depleta does have some benefit from melezitose. There is, however, no
evidence that it is important for reproductive success. Comparable fecundities were
produced by both a diet of melezitose and a non-melezitose diet supports the hypothesis
that O. depleta is an opportunistic feeder rather than an obligate honeydew feeder. It
would seem that an obligate honeydew feeder would produce fewer offspring from being
fed only a melezitose-free diet. This study showed no such reduction. Whether melezitose
might have an effect on the fecundity of O. depleta when combined with other sugars
remains to be tested. This study does support the hypothesis that O. depleta has the
necessary enzymes to hydrolyze melezitose.
Previous research showed that sucrose is quickly broken down in the crop of the
fly, but melezitose is hydrolyzed very slowly (Welch 2000). Burkett (1998) found similar
results in carbohydrate hydrolysis in mosquitoes. The likely location for melezitose
hydrolysis is the midgut. There may be some advantage to feeding on melezitose in that it
would be absorbed more slowly than other sugars, prolonging the time over which energy
could be derived from the food. This may make it less necessary to store energy in the
form of trehalose, fats or glycerol, which require energy to synthesize. Another possible
advantage of feeding on melezitose lies in its availability. The visual and/or olfactory
attractants associated with honeydew have not been investigated as they apply to O.
depleta. Whether these flies search out honeydew or come upon it randomly is unknown.

19
But once found, it is an easy food source to access. No specialized mouthparts are
required to reach the source, in contrast to many floral nectars, nor is it necessary to
pierce the skin of fruits or other plant tissues. The apparently non-specialized mouthparts
of O. depleta could be the result of two possibilities. Either they are truly opportunistic
and feed on whatever carbohydrate sources they are able to locate, including honeydew,
and have never evolved any type of specialized feeding apparatus, or their mouthparts are
specialized and have evolved to feed primarily on honeydew. A good test for this would
be to determine whether O. depleta have other behavioral or physiological traits which
show a specialization towards honeydew feeding. One such characteristic would be the
flies’ ability to locate honeydew deposits. If O. depleta has evolved the ability to locate
honeydew by olfactory or visual cues, then it is likely that it could be considered more of
a specialist feeder on honeydew than if it were to only find it by chance. The high
percentage of field-caught O. depleta which had fed on honeydew, as indicated by the
presence of melezitose in the crop (Welch 2000), seems to support the hypothesis that
they are not merely encountering honeydew randomly. Future research into the attractant
properties of honeydew, honeydew constituents, and plant/aphid systems to O. depleta
would be very useful both for determining the true specialization of this species as well
as for making banker plant recommendations for localized augmentation of fly
populations.

CHAPTER 3
INTRASPECIFIC COMPETITION FOR RESOURCES BY ORMIA DEPLETA
(DIPTERA: TACHINIDAE) LARVAE
Introduction
Ormia depleta can be a difficult organism to maintain in a lab colony. One of the
factors that makes them difficult lies in the variable and generally low proportion of
gravid females obtained under the current lab rearing protocol. A colony of 100
individuals may in one generation produce 20 gravid females and in the next only one or
two or even zero. Therefore, it is necessary to determine the best way to use the number
of planidia available in any one generation to produce the maximum number of healthy
pupae to start the next generation. This must also be balanced with the expense of rearing
the mole cricket hosts, which are very labor intensive to maintain. Current laboratory
protocol dictates that three planidia be placed under the posterior margin of the pronotum
of each Scapteriscus host when inoculating by hand (Personal communication with
Robert Hemenway, University of Florida). If there is competition among the larvae for
resources within the host, fewer planidia per host may increase the chances of survival
for those planidia and may produce larger pupae. This would, however, require more
hosts to produce enough pupae to maintain the colony. Inoculating hosts with more
planidia may increase the number of pupae and reduce the cost associated with host
rearing, but superparasitoidism should be avoided as well as the production of pupae and
adults with reduced fitness.
20

21
The effect of superparasitoidism on the developing larvae of many insect species
has been studied extensively. Superparasitoidism has been shown to increase larval
development time in Colesia glomerata (L.) (Gu et al. 2003), Microplitis croceipes
(Cresson) (Eller et al. 1990), and Cotesia flavipes Cameron (Potting et al. 1997).
However, Bai and Mackauer (1992) found no increase in the larval development time of
Aphidius ervi Haliday when subjected to superparasitoidism. In some species, it has been
shown to decrease survivorship (Vinson and Sroka 1978, Gu et al. 2003) but not in other
species (Harvey et al. 1993). The offspring of the ichneumonid wasp Venturia canescens
(Gravenhorst) were smaller under the condition of superparasitoidism (Harvey et al.
1993), but the wasp A. ervi actually increased in size (Bai and Mackauer 1992). The most
common effects of superparasitoidism apparently are an increase in the number of brood
produced, but with an overall reduction in the fitness of those offspring (van Dijken and
Waage 1987, Vet et al. 1994).
Previous research with O. depleta showed that there was no relationship between
the number of planidia used to inoculate the host and the number of pupae produced
(Fowler 1988), but preliminary research done here showed that higher numbers of pupae
could be produced than previously recorded. Additionally, Fowler and Martini (1993)
found a weak correlation between host size used and the weights of the flies produced. In
the present experiment, this relationship was also examined to determine whether host
weight is an important factor in determining which hosts should be used. The goal of this
experiment was to determine if an increase in the number of pupae produced per host
could be achieved without sacrificing the survivability or vigor of the larvae. In addition
to varying the number of planidia applied to each host, the weights of the host mole

22
crickets were measured during inoculation to see whether larger hosts could provision
more parasitoids. These factors of host mole cricket weight and number of planidia used
to inoculate the host were examined to determine their effect on the number of pupae
produced, the mean weight of those pupae, and the survivability of the larvae to the pupal
stage.
Materials and Methods
During the maintenance of the laboratory colony of O. depleta, S. abbreviatus
from the University of Florida mole cricket rearing lab were individually weighed and
inoculated with varying numbers of O. depleta planidia. The weights of the hosts ranged
from 0.54-1.59 g and the weights of the hosts were not considered in determining the
number of planidia used to inoculate each individual. The number of planidia per host
ranged from 2-8, with most of the mole crickets being inoculated with either 3, 4, or 5
planidia. These numbers were favored because they are the numbers most frequently used
in the routine maintenance of the colony. The number of planidia placed on each host was
randomly determined. The numbers of mole crickets inoculated with 2, 3, 4, 5, 6, 7, and 8
planidia were 12, 108, 110, 52, 43, 32, and 11, respectively. Each mole cricket was then
returned to an individual 20 dram plastic vial filled with moist sand, and the larvae were
allowed to develop for 12 days at a room temperature of ~26°C. At that time, the pupae
were collected and weighed. Statistical analysis was performed using the general linear
model procedure (SAS Institute 2001). Regression analysis was used to determine how
differing numbers of planidia affected the number of pupae produced, the mean pupal,
and the survivability of the planidia. Additionally, the weights of the host mole crickets
were analyzed to determine their effect on the survivability of the planidia used. Where

23
applicable, the differences between the means were determined by Duncan’s multiple
range test (SAS Institute 2001). Regression analyses were conducted to determine the
relationships between each of these factors (SAS Institute 2001).
To determine the effect that host mole cricket weight had on planidia survival, the
number of pupae produced and the mean weights of those pupae, mole cricket weights
were rounded up to the nearest 0.1 gram to place them into weight classes. Additionally,
weight classes which had only two or fewer samples were eliminated. In this case, the
smallest weight class, 0.70 grams (n = 2) and the two largest weight classes, 1.5 grams (n
= 2) and 1.6 grams (n = 2) were eliminated from the statistical analysis. The survival of
the planidia on hosts in the remaining weight classes were analyzed by ANOVA (SAS
Institute 2001).
Results
The mean number of the pupae produced relative to the number of planidia used
can be seen in Figure 3. There is an increase in the number of pupae produced as the
number of planidia increases (F= 15.77, P < 0.0001) and significant differences between
the means of the treatments. The regression analysis (Figure 4) supports this trend and
indicates an increase of 0.41 pupae for each increase in planidia (f= 83.77; P < 0.0001;
r2 = 0.19).
Figure 5 shows the survival of planidia grouped by the number of planidia placed
on each host. ANOVA is significant for the model (F=2.57, P < 0.02). Figure 6 is the
regression analysis of the same data set (F= 9.16; P < 0.002; r2 = 0.03), indicating an
approximate 3% reduction in survival for each increase in the level of planidia density.
The analysis of the number of planidia used as it affected the mean weight of the
pupae produced was found to be nearly significant by ANOVA (F=2.06, P = 0.057).

24
There was some significance among the means as indicated by the letters over the bars in
Figure 7. The regression analysis for the mean weights of pupae produced is in Figure 8
(F = 8.33; P < 0.004; r2 = 0.02) and indicate a reduction in the mean weight of the pupae
of 2.2 mg for each additional planidia.
Number ofplanidia inoculated per host
Figure 3: The effect of planidia density used to inoculate mole crickets on the number of
pupae produced (error bars indicate standard deviation, significantly different
means indicated by letters over bars as determined by Duncan’s procedure, a
= 0.05)

25
Number of planidia inoculated per host
Figure 4: The effect of number of planidia used to inoculate mole crickets on the number
of pupae produced - regression analysis with 95% confidence bands
Planidia
Figure 5: The effect of number of planidia used to inoculate mole crickets on the survival
rate of the larvae to the pupal stage (error bars indicate standard deviation,
significantly different means indicated by letters over bars as determined by
Duncan’s procedure, a = 0.05)

26
5
¡5
'c
o.
1.0 -
0.8 -
0.6 -
0.4 -
0.2 -
0.0 -
1
y = -0.0317x+0.7129
r2 = 0.03, P < 0.002
-i 1 1 1 1 1 1 1
23456789
Number ofplanida inoculated per host
Figure 6: The effect of number of planidia used to inoculate mole crickets on the survival
rate of the larvae to the pupal stage - regression analysis with 95% confidence
bands
Planidia
Figure 7: The effect of number of planidia used to inoculate mole crickets on the mean
weight of the pupae produced (error bars indicate standard deviation,
significantly different means indicated by letters over bars as determined by
Duncan’s procedure, a = 0.05)

27
120 -i
'qo 100 -
a
£ 80 -
bp
‘5
- 60 -
cx
(2 40 -
§
2 20-
0 --
0
Figure 8: The effect of number of planidia used to inoculate mole crickets on the mean
weight of the pupae produced - regression analysis with 95% confidence
bands
The effect that host mole cricket weight had on the number of pupae produced
was not found to be significant when analyzed by ANOVA (F = 1.06, P = 0.39). The
effect that host mole cricket weight had on the survivability of the larvae was found to be
marginally significant (F = 2.12, P = 0.0505). The effect that host mole cricket weight
had on the mean weight of the pupae produced was highly significant (F= 3.49, P <
0.002) (Figure 9). The regression analysis can be seen in Figure 10 (F= 20.62; P <
0.0001; r2 = 0.05).
y = -2.2456x+62.649
r2 = 0.02, P < 0.004
2 4 6 8
Number of planidia inoculated per host

28
Figure 9: The effect of host cricket weight on mean pupal weight (error bars indicate
standard deviation, significantly different means indicated by letters over bars
as determined by Duncan’s procedure, a = 0.05)
120 -i
'S)
B
100 -
£
00
80 -
£
60 -
c3
£L
-
o-
40 -
a
-
2
20 -
0 -
0.7
y=31.346x + 20.163
r2 = 0.05, P <0.0001
—i 1 1—
0.9 1.1 1.3
Host cricket weight class (g)
1.5
Figure 10: The effect of host cricket weight class on the mean weight of the pupae
produced - regression analysis with 95% confidence bands
Discussion
The number of planidia used to inoculate host mole crickets as well as the weight
of those mole crickets are important factors to the rearing of O. depleta in the laboratory.
Although these data do not clearly dictate a specific protocol that should be used, they do

29
provide a framework that would allow for anyone rearing O. depleta to structure an
inoculation protocol specific to their needs. At times when large numbers of planidia are
available but few hosts are available, the data suggest that inoculating mole crickets with
more planidia would increase the production of pupae. Too many, however, would result
in reduced pupal size. At times when fewer planidia are available and maximum
survivability is required, inoculating two or three planidia per host would be more
effective. Alternatively, if larger pupae are desired, reducing the number of planidia per
host along with using larger hosts would achieve the desired goal. Therefore, the current
method of inoculating three planidia per host is less efficient than inoculating four or
five, since there is no significant reduction in pupal size, but there is a significant increase
in the number of pupae produced. The reduction in size that results from the use of eight
planidia, or possibly more, would likely be detrimental to the colony of flies. Some
preliminary data (Appendix A) has shown that pupae less than 40 mg have reduced
survivorship to the adult stage. This reduction is greater than the increase in number that
is gained when going from 6 or 7 planidia to 8 planidia. Furthermore, these data only
show a reduction in the emergence rate of the pupae, they do not indicate other negative
factors which may be associated with reduced size. Future research may be needed to
determine whether these smaller individuals show any reduction in longevity, ability to
mate, or in fecundity as well as how the reduction in size of a generation may affect the
size or fitness of future generations of flies.
According to the University of Florida’s Integrated Pest Management Website
(http://ipm.ifas.ufl.edu/extension-resources/glossaries/glossary.htm), the definition of
superparasitoidism is

30
the situation in which more individuals of a parasitoid species develop in a host
than can obtain adequate resources to complete their development. Females of
some parasitoids may lay more than one egg in or on a host, resulting in
superparasitoidism, although the behavior of females tends to avoid this condition
by discriminating against already-parasitoidized hosts. In fact, five conditions can
be distinguished (a) only one parasitoid exists within the host; (b) there is more
than one parasitoid within the host, but all survive and produce adults of normal
size [this is not superparasitoidism]; (c) there is more than one parasitoid within the
host and they all survive but produce adults of subnormal size because of
competition for resources [this is viewed here as superparasitoidism]; (d) there is
more than one parasitoid within the host and some of them die due to competition
for resources (including attack by conspecifics) [this is superparasitoidism]; and (e)
there is more than one parasitoid within the host and all die because the resources
are too few [this is superparasitoidism].
By this definition, the situation of O. depleta in this experiment qualifies as
superparasitoidism only under the highest number of planidia inoculated on the host.
Although some reduction in size is apparent at almost all levels above three planidia,
most of the reductions are not significant.
This raises an interesting question as to the natural behavior of the flies. Under
field conditions, the mean number of O. depleta larvae found within trapped Scapteriscus
hosts is less than two (Amoroso 1990). It would seem likely that, due to the flies’
phonotactic search method for hosts and the solitary nature of adult mole crickets, it
would be to the flies’ advantage to maximize the number of offspring that it would be
able to produce from the seemingly limited number of hosts. But apparently, this is not
the case. The closely related O. ochracea Bigot, a parasitoid of Gryllus spp. crickets,
have an optimal laboratory clutch size of 4-5 larvae per host, but under field conditions
only deposit 1.7 ± 1.0 S.D. larvae (Adamo et al. 1995). There must be some ecological
advantage to depositing fewer larvae than what appears to be the optimal number.
It may be that O. depleta does not suffer from any shortage of hosts. Mole
crickets are certainly abundant and calling during certain times of the year, but at other

31
times seemingly unavailable. Ormia depleta may be able to find non-calling mole
crickets in other ways, or there may be alternative hosts (this is discussed more in
Chapter 4). Adamo et al. (1995) concluded that host availability was not a likely factor in
determining the number of larvae deposited on hosts by O. ochracea. Another possibility
is that O. depleta is responding to a factor in the field that is greatly reduced in the
laboratory, the mortality of the hosts. Under laboratory conditions, mole crickets suffer
little disease and no predation. It may be that in the field, the higher mortality of the hosts
would make it advantageous to partake in bet hedging and spread offspring out over
many hosts so that the loss of one host has a lesser effect on the total number of
offspring. This hypothesis is somewhat strengthened by the fact that O. depleta does not
deposit eggs, but planidia larvae, so the female’s investment in parasitoidizing a host is
already greater than that of an egg layer. Another laboratory factor that should be
considered is hand-inoculating. The mole crickets that are hand-inoculated are unable to
protect themselves in any way and have no opportunity to use whatever natural defenses
they may have available in the field. It may be that, although it would be advantageous
for O. depleta to parasitoidize hosts with a greater number of planidia, the natural
behavioral defenses of Scapteriscus mole crickets prevent it, whether those defenses
involve brushing off planidia or simply retreating underground when the presence of O.
depleta is detected. This type of grooming has been observed in Gryllus spp. crickets
after an encounter with O. ochracea (Adamo et al. 1995).
One possibility that has been suggested is that O. depleta are parasitoidizing hosts
with higher numbers of planidia in the field, but we are unaware of it. The trapping
methods used to determine parasitoid levels of mole crickets in the field are usually

32
sound traps, which require the mole crickets to fly into the trap, or pitfall traps, which
require the mole crickets to be actively crawling on the soil surface to be captured. It may
be that those mole crickets with larger parasitoid loads are under greater physical stress
and are less able or likely to venture to the surface where they can be trapped. Zuk et al.
(1993) found that the calling of the field cricket Teleogryllus oceanus (Le Guillou) was
inhibited by infestation of O. ochracea in Hawaii. Although Zuk did not report on these
crickets’ ability to move, it does suggest that there is a physical liability to carrying
parasitoids. Collecting mole crickets by digging and sifting soil would probably produce
more accurate results than the current trapping methods, but the labor and expense
involved would not justify the change in methods.
The final reason for the low numbers of larvae found in field captured hosts may
be that there is a reduction in fitness caused by the high numbers of larvae used in this
experiment that were not investigated. Reduced size is the easiest type of fitness
reduction to observe, but many others may be at work. It may be that, due to competition,
certain key resources are not available in sufficient amounts for the flies reared under
superparasitoid conditions for the resulting adult flies to develop, mate, locate hosts, or
reproduce properly. Many physiological deficiencies may result from superparasitoidism,
and they may not be obvious either externally, or immediately (Waage and Ng 1984).
These possibilities still remain for future research.

CHAPTER 4
REARING ORMIA DEPLETA (DIPTERA: TACHINIDAE), A PARASITOID OF
MOLE CRICKETS (ORTHOPTERA: GRYLLOTALPIDAE), ON A FACTITIOUS
HOST, ACHETA DOMESTICUS (ORTHOPTERA: GRYLLIDAE)
Introduction
The maximum life span of adult O. depleta in the lab is approximately 60 days
(Welch 2000), which would not allow them to survive between times of peak mole
cricket calling activity, which are from February to June (Walker and Moore 2000).
Therefore, some sort of diapause might be expected to allow the flies to carry over from
June to the Following February. Some preliminary research was made to determine
whether O. depleta pupae could be stimulated to diapause by exposing them to cold
temperatures (0 - 5° C), but refrigeration attempts at these temperatures resulted in 100%
mortality. Additionally, work done by Cabrera (2000) found that attempts to rear larvae
at temperatures below 17° C resulted in the death of the larvae. Therefore, cold-induced
diapause does not seem to be a probable part of O. depleta’s life history. Other
experimentation (Appendix B) suggests that there is no photoperiod-induced diapause.
The question then remains: what happens to the population of O. depleta during the
months between the period of peak calling activity of Scapteriscus spp. hosts?
It is possible that O. depleta locate mole cricket hosts when the mole crickets are
not known to call in large numbers. The flies may be able to find enough to maintain their
population until the next calling season. Calling traps located at the Gulf Coast Research
and Education Center in Bradenton, Florida, catch most of their O. depleta during the
seasons that follow peak adult Scapteriscus flight activity, which are from March through
33

34
May and from September through November, but some mole crickets are caught during
every month of the year (Walker et al. 1992a, b). Dr. Thomas Walker has also observed
individuals of S. borellii and S. vicinus calling in each month of the year, albeit in much
smaller numbers than in the peak seasons (Walker and Moore 2000). If only few flies
were able to locate Scapleriscus hosts during these times, it could maintain a seed
population until the next calling season.
Another possible solution is the use of alternate hosts. Ormia depleta is able to
survive through the winter in central Florida. Specimens are collected in Bradenton,
Florida, throughout the year, although the greatest numbers coincide with the peak mole
cricket flight seasons (Walker et al. 1992b). Bradenton does occasionally experience cold
weather and even frost. This may suggest that during these times when adult or pupal O.
depleta would not be able to survive, that perhaps the larvae are able to survive within a
host. Being within a host would provide a great deal more protection from the
environment. If O. depleta were unable to reliably locate Scapteriscus hosts during these
times when they are not calling, it could be possible that they were using an alternate
host. This has been suggested by Fowler and Mesa (1987) with the recovery of O. depleta
from a species of Anurogryllus. Additionally, Justi et al. (1988) reported that under
laboratory conditions, O. depleta successfully developed within an unnamed Gtyllus
species. Although there is no evidence to suggest that O. depleta is phonotactic to any
taxa other than Scapteriscus (Fowler and Garcia 1987), it is plausible that it may
occasionally encounter enough hosts by other means to maintain a population between
the calling seasons of its preferred host.

35
Multiple hosts among tachinids are not unusual. Some tachinids have host ranges
that cover three orders of insects (Cantrell and Crosskey 1989). Ormia depleta is thought
to be and reported as an obligate parasitoid of Scapteriscus mole crickets (Frank et al.
1998), and under most conditions, it probably is. The mechanism for the specificity of O.
depleta lies in its phonotaxis. There are no reports of it being attracted to any calls other
than those of S. vicinus and S. borellii (Walker et al. 1996). Under field conditions, this
greatly limits the fly’s ability to select new hosts. Under laboratory conditions, especially
when hand-inoculating hosts, it may be possible to manipulate this characteristic. It
would be advantageous to be able to rear O. depleta on an alternate host when
Scapteriscus are unavailable or simply to reduce the costs associated with rearing
Scapteriscus. The effect of rearing tachinid flies on factitious hosts has been examined
many times. Baronio et al. (2002) found there to be no decrease in the pupal weights of
Pseudogonia ruflfrons Wiedemann when reared on the factitious host Ostrinia nubalis
(Hubner) rather than on Galleria mellonella L., its natural host. The tachinid fly Exorista
larvarum (L.) was found to be even more successful on the factitoius host G. mellonella
than on its natural host, Lymantria dispar (L.) (Dindo et al. 1999). With this in mind, a
factitious host for O. depleta was examined.
Acheta domesticas (L.) (the house cricket) can be reared in large groups
inexpensively and would be an economical substitute for Scapteriscus spp. as a host in
laboratory rearing, if able to support the development of O. depleta. Any failure of O.
depleta to successfully develop in an A. domesticus host would lead to the question of
why they did not develop well. Was there failure to penetrate the host? Did A. domesticus
lack sufficient nutritional value? One of the major factors differentiating yl. domesticus

36
and Scapteriscus spp. behaviorally is that Scapleriscus spp. are primarily subterranean
whereas A. domesticus is not. Although this would not seem to have a direct effect on
internally developing larvae, it would be likely to have an effect on the ability of the
larvae to pupate after leaving the host. There may also be differences in the cuticles of the
two crickets that could hinder the development or the emergence of the larvae. To study
this, the larval behavior of O. depleta inside both S. abbreviatus and A. domesticus was
observed.
Materials and Methods
Larval Development of Ormia depleta in Acheta domesticus
Ten adult A. domesticus were obtained from the colony kept at the University of
Florida mole cricket rearing lab. Each cricket was inoculated with four planidia from a
freshly sacrificed gravid female O. depleta from the Piracicaba strain laboratory colony.
Inoculated crickets were placed in a 30 x 23 x 10 cm clear, plastic container with a screen
ventilated top. The bottom of the container was filled with approximately 3 cm of moist,
autoclaved sand. A screen of 0.6 cm galvanized mesh was suspended approximately 3 cm
above the sand to keep the crickets off the sand but to allow newly emerged larvae to
drop through to the sand. Cardboard tubes 5 cm in diameter were placed on the
galvanized screen to provide hiding places for the crickets. Water was made available by
placing a Petri dish on the galvanized screen wicked with cotton. Food was also provided
(Nutrena Cricket and Earthworm Feed, Minneapolis, MN), also in a Petri dish on the
screen. Food and water were available ad lib. The crickets were observed daily and their
food and water changed as needed. Approximately 10 days later, when all crickets were
dead and emerged larvae had pupated, the pupae were removed and their number and
weight were recorded. This process was repeated for four generations of O. depleta.

37
Larval Behavior within host
Acheta domesticus and S. abbreviatus were obtained from the mole cricket rearing
lab and inoculated with Osorio strain O. depleta planidia. Twenty-one crickets from each
species had four planidia placed under the posterior margin of the pronotum. Acheta
domesticus were then placed in a small container containing food (Nutrena Cricket and
Earthworm Feed, Minneapolis, MN) and water in a Petri dish wicked with cotton.
Scapteriscus spp. were returned to their vials of sand. The planidia were then allowed to
develop in their hosts. Three crickets were sacrificed at each of seven 24-hour intervals,
dissected, and the larvae searched for. When found, the size, number, location, and
apparent tissues being fed upon were all noted. A t-test was used to compare the mean
pupal weights of pupae reared on A. domesticus to those reared on S', abbreviatus (SAS
Institute 2001).
Results
Larval Development of Ormia depleta in Acheta domesticus
Acheta domesticus was able to act as a factitious host for O. depleta in that some
of the larvae were able to survive to the pupal stage. However, the survivorship of the
planidia was only 29% versus 57% in S. abbreviatus (as determined in Chapter 3).
Additionally, the weight of the pupae produced was significantly less than those
produced in S. abbreviatus. The mean pupal weight when reared in A. domesticus was
39.85 mg ± 1.53 (SE), which was significantly smaller than those in S. abbreviatus ,
where the mean pupal weight was 54.60 mg ± 0.50 (SE) (P < 0.001; df = 950).

38
Larval Behavior within host
Twenty-one A. domesticus and twenty-one S. oiireviatus were examined in this
experiment. At least one planidium was found in each host species for each day of this
experiment. The mean number of planidia found and the mean length of those planidia
are shown in Table 6.
Table 6: Mean number and length of O. depleta larvae found in S. abbreviates and A.
domeslicus hosts
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
S. abbreviatus
Mean number of larvae found
0.67
1.33
2.67
3.00
2.33
2.33
2.33
Mean length (mm)
0.62
0.72
2.29
4.86
9.78
11.87
12.69
A. domesticus
Mean number of larvae found
0.33
1.00
1.00
1.67
2.33
1.67
2.00
Mean length (mm)
0.58
0.64
0.85
1.44
4.57
7.25
8.94
The following is a timeline of the activity of the larvae inside each host.
Scapteriscus abbreviatus-
Day 1 (24 hours after inoculation)- A few planidia were dead on the exterior of the host.
The larvae that succeeded in penetrating the host are very difficult to find - only
two were found among the three hosts dissected. Those that were found were in
the fatty tissues just below the area where they had been placed, apparently
feeding on those tissues. (Figure 11).
Day 2- Still some dead planidia are visible on the exterior of the host. Larvae are easier to
find, though still small. The larvae are now distended and almost transparent.
Some of the larvae are still found in the fatty tissues under the pronotum while a
few have moved to a more posterior and lateral position. The farthermost
observed was located along the left side of the abdomen, just posterior to the
metacoxa (Figure 12).

39
Day 3 - All larvae found were in the abdomen of the host. They were found in clusters on
either side in the area near the gonads. They appeared to be feeding in fat tissue
and/or reproductive organs (Figure 13).
Day 4 - Larvae are still in the gonadal region. The ovaries/testes are no longer visible in
large amounts. The larvae are now posteriorly attached to the host exoskeleton.
The alimentary canal of the host, though in reach of the larvae, does not appear to
be damaged (Figure 14).
Day 5 - The attachment points of the larvae are now visible on the exterior of the host
and appear as small, dark brown spots (Figure 15). The fat bodies within the reach
of the attached larvae are now greatly reduced or gone. The alimentary canal still
appears to be undamaged (Figure 16).
Day 6 - Hosts are starting to act very sluggish and showing signs of dying. The larvae
now occupy approximately half of the area within the hosts’ abdomens (Figure
17). The heads of the larvae can now reach into the thorax and they are apparently
feeding there. The inside of the abdomen now looks very “dry” as there are no
remaining fat reserves, no gonads, and no hemolymph in any large amount. The
alimentary canal, though well within reach of the larvae, shows no signs of
damage. The attachment points of the larvae are now visible from the outside of
the host and appear as small brown patches, approximately twice the size of the
hosts’ spiracles. On the inside, the points of attachment seem to open into small
scleritized cones that appear to be attached to the posterior ends of the larvae by
what appear to be the old exuviae (Figure 18).

40
Day 7 - Larvae are much less active in the host. Some still appear to be feeding
on the remaining tissues in the thorax of the host, others appear to be at rest. The
alimentary canal of the host remains undamaged (Figure 19).
Figure 11: Larva of O. deplete! in S', abbreviates host one day after inoculation
//{Mill!
Figure 12: Larva of O. deplela in S. abbreviates host two days after inoculation

41
Figure 13: Larva of O deplela in S. abbreviates host three days after inoculation
Figure 14: Larva of O. deplela in S. abbreviates host four days after inoculation

42
Figure 15: Scapteriscus abbreviatus host five days after inoculation showing the external
evidence of the attachment point of O. deplela larva
Figure 16: Larvae of O. deplela in S. abbreviatus host five days after inoculation

43
Figure 17: Larvae of O. depleta in S. ahbreviatus host six days after inoculation
Figure 18: Two O. depleta larvae in S. ahbreviatus host six days after inoculation
showing detail of the larval attachment to the host

44
Figure 19: Larva of O. depleta in S abbreviates host seven days after inoculation
Acheta domesticus-
Day 1 (24 hours after inoculation)- Only one larva was found feeding on the fatty tissue
just below the area where placed. This larva does not appear to have grown much
although it has become slightly lighter in color, most likely due to feeding on fat
bodies (Figure 20).
Day 2 - Larvae are easier to see, though still small. They are apparently feeding on fat
bodies in the thorax (Figure 21).
Day 3 - Two larvae were feeding on fat tissues near the gonads of the host. One larva
was in the anterior region of abdomen (Figure 22).
Day 4 - All larvae found were in the abdomen of the host. All but one were feeding on
fat and reproductive tissues. The remaining larva was in the anterior region of
abdomen feeding on fat tissue (Figure 23).

45
Day 5 - Larvae are all now feeding in the gonadal region. Larvae are all now posteriorly
attached to the exoskeleton of host. The fat bodies of the host do not appear to be
depleted nearly as much as in S. abbreviates hosts (Figure 24).
Day 6 - Larvae continue to feed in the gonadal region. The size of the larvae appears to
be increasing, but at a reduced rate as compared to larvae in S. abbrevialus
(Figure 25).
Day 7 - Larvae are still feeding in the gonadal region. The fat and reproductive tissues of
the host appear to be somewhat reduced. Some larvae appear to be showing signs
of slowing down or stopping feeding. The alimentary canal of the host is
undamaged (Figure 26).
Figure 20: Larva of O. deplete in A. domesticus host one day after inoculation

46
tíínilir.
* * • ** 2 ~
Figure 21: Larva of O. depleta in A. domesticus host two days after inoculation
Figure 22: Larva of O. depleta in A. domesticus host three days after inoculation

47
Figure 24: Larvae of O. depleta in A. domesticus host five days after inoculation

48
Figure 25: Larvae of O. deplela in A. domesticus host six days after inoculation
Figure 26: Larvae of O. deplela in A. domesticus host seven days after inoculation
One larva was removed from each host species on day seven and photographed
next to each other to compare the difference between the S. abbrevialus-reaied larva and
the A. domesticus-reared larva (Figure 27). The larval length for each day was measured
using an ocular micrometer and the mean plotted for each host species (Figure 28).

49
Figure 27: Seven day old larvae of O. depleta from S', abbreviatus (top) and A.
domesticus (bottom)
Figure 28: The growth of O. depleta larvae in alternate hosts: S. abbreviatus and A.
domesticus
Discussion
Although A domesticus may be easier and less expensive to rear than
Scapteriscus spp., these results do not support its use as a factitious host for O. depleta.
For many generations, there is a great abundance of planidia. Therefore, the survival of
the planidia is not a weighty factor in determining rearing procedures. Additionally, by

50
using large numbers of A. domesticus as hosts, the number of gravid flies available may
be able to be augmented and therefore produce even more planidia. In such a case, a drop
from 57% to 29% may not be too damaging to a colony’s survival. Considering that this
represents approximately a doubling in mortality of planidia, from an economic
standpoint this would be acceptable because the cost of rearing a mole cricket host is so
much than the cost of rearing a house cricket. The main issue is that of the pupal size.
Previous experimentation has shown that for pupae weighing less than 40 mg, there is a
reduction in survival (Appendix A). Roughly half of the A. domesticus-reared O. depleta
were in this low-emergence category. This is considering emergence only, no research
has been done to determine the effect that this small size may have on survivorship,
breeding ability, or fecundity, but it seems likely that any or all of these factors could be
affected negatively by significantly reduced weight. Among hymenopterans, reduced
female size has been demonstrated to cause a reduction in fecundity in Trichogramma
evanescens Westwood and Goniozus nephantidis (Muesebeck) (Waage and Ng 1984:
Hardy et al. 1992). Under laboratory rearing conditions, these problems may be further
exacerbated in that the offspring of these under-sized flies would become the parents of
the next generation of flies, potentially producing fewer or smaller individuals and
reducing the vigor of the colony as a whole.
It is possible that the poor results of A. domesticus as a factitious host were due to
the techniques used here. The number of planidia used was not altered within this
experiment. Perhaps a smaller number per host would have produced larger pupae,
although this is not supported by the physiological evidence. There may also be room for
improvement of the rearing cages. The 0.6 cm wire mesh used was too large to prevent

51
the crickets from getting through to the sand. House crickets do not survive well in very
moist conditions and this may have affected the larvae. Also, there were problems with
keeping the sand moist enough in the well-ventilated cages. Although this would not have
an effect on the size or number of pupae produced, in a larger-scale operation this may
effect the ability of the pupae produced to survive. Additionally, all of the pupae
recovered were found on the surface of the sand and several seemed unusually dry,
indicating the inability of the larvae to burrow into the sand effectively. Perhaps in the
future a different substrate, such as vermiculite, could be used to better retain moisture.
In considering the larval behavior in the hosts, no single factor stood out as being
the cause for the poor performance of A. domesticus as a factitious host for O. depleta.
Rather there appear to be multiple factors. The slowed growth and development of the
larvae in A. domesticus suggest that there may be a nutritional deficiency associated with
A. domesticus as a host for O. depleta. This deficiency could possibly be a lack of
specific proteins or other components necessary or an overall shortage in the volume of
palatable or usable tissues. The fact that after seven days there was still a large amount of
fat tissue remaining in the host does not support the position that these tissues are simply
lacking in volume. It is more likely that they are nutritionally deficient or unpalatable.
Another explanation would be that there is a chemical or immunological incompatibility
between ¿4. domesticus and O. depleta. Further research in these areas is still needed.
Although smaller, the larvae of O. depleta in A. domesticus do appear to survive
as well as those in S. abbreviatus, as evidenced by the similarity between the mean
number of larvae found in each over the last few days before emerging from the host.
There is, however, great reduction in the number of pupae produced by the different

52
hosts. There must be a cause of mortality between these two events. The most probable
cause for this mortality would be a problem with the ability of the larvae to emerge from
the host. Scapteriscus abbreviates are subterranean and their bodies are well sclerotized,
however, their abdomens are quite soft and the cuticle thin. By contrast, A. domesticus
have a relatively hard and thick abdominal cuticle. This could very well be a barrier to O.
depleta larvae being able to emerge successfully. This would especially be true for larvae
of reduced size and vigor. Emergence may also be affected by the factors present as the
larvae leave the host and are exposed to the laboratory environment. Ormia depleta
would probably be adapted to emerging from its host into a subterranean environment
where the relative humidity would normally be very high. Those larvae emerging from A.
domesticus would find themselves immediately exposed to much drier conditions. The
pupae of O. depleta are well protected from desiccation, but before the formation of the
pupal sclerotia, the larvae could be extremely vulnerable, perhaps enough to keep it from
being able to fully emerge from the host.

CHAPTER 5
IDENTIFICATION OF TWO SOUTH AMERICAN GEOGRAPHICAL ISOLATES OF
ORMIA DEPLETA BY ANALYSIS OF CUTICULAR HYDROCARBONS
Introduction
With the release of the Osorio strain of O. depleta, it became desirable to find a
way to differentiate the two strains to make it possible to identify the origin of any flies
captured. Although no flies have yet been trapped where the Osorio strain was released in
states north of Florida (personal communication with Dr. J. H. Frank, University of
Florida), those flies may perhaps be established in those areas, and in the areas between
the new release sites and the established range of the Piracicaba strain of flies. Gas
chromatographic (GC) analysis of cuticular hydrocarbons was chosen as a method to
differentiate the strains. GC has been used to identify different species, subspecies, and
races of insects (Sutton and Carlson 1993) and is a quick and simple testing method.
Materials and Methods
Gravid female O. depleta of the Osorio strain were obtained from the laboratory
colony maintained in the mole cricket rearing laboratory at the University of Florida.
Gravid female Piracicaba strain flies were collected from the sound traps maintained at
the Gulf Coast Research and Education Center in Bradenton, Florida. Planidia from each
strain were inoculated onto S. abbreviatus and kept under normal conditions as outlined
previously. Colonies of both Osorio and Piracicaba strains of O. depleta were maintained
until maturity (approximately four weeks after pupation). Males and non-gravid females
from both colonies were removed and frozen until time for cuticular hydrocarbon
53

54
extraction. Both individual and collective samples were taken. Individual samples were
collected by placing a single frozen fly in a vial containing 5 mL of hexane and agitating
for 30 seconds. The fly was then removed and discarded. The hexane solution was then
filtered through a glass pipette containing a 5 cm column of silicic acid. The pipette was
then flushed with an additional 5 mL of hexane. The sample was then evaporated down to
approximately 1 mL of solution. This process was repeated for eight flies for both males
and females from each of the two strains. Collective samples were taken by placing 10
frozen flies of the same sex and strain into vials containing 10 mL of hexane. These
samples were also filtered through glass pipettes containing a 5 cm column of silicic acid
and flushed with an additional 5 mL of hexane. Samples were evaporated down to
approximately 3 mL of solution. Both individual and collective samples were analyzed
by gas chromatography using a Hewlett Packard 6890 GC with a DB-5 fused silica
capillary column (30m x 0.25pm, J & W Co., Folsum, CA using hydrogen carrier gas
@1.2 ml/min and a cold on-column injector system held at 60°C). The temperature ramp
for the samples was as follows:
Initial temperature 60° C - hold for 2 minutes
Increase at a rate of 16°C/min to 170°C
Increase at a rate of 8°C/min to 234°C
Increase at a rate of 6.5°C/min to 253°C
Increase at a rate of 5.4°C/min to 320°C
Hold at 320°C for 20 minutes

55
The purpose for this specific temperature ramp is that it separates straight-chained
alkanes into peaks with retention times approximately one minute apart (pers. comm,
with Dr. David Carlson, USDA-CMAVE, Gainesville, Florida). The resulting
chromatograms were analyzed by stepwise selection in multiple logistic regression to
determine discriminatory peaks between strains. Additionally, a jackknife analysis was
performed to assess the probability of the resulting model to correctly predict the strain of
an unknown sample (SAS Institute 2001).
Results
Ten predominant peaks were present in the cuticular hydrocarbon GCs of all
specimens tested. (Figures 29-32) These peaks were designated by the letters A through J
and their retention times listed in Table 7. The strongest variation between the two strains
was found to be between the peaks of the males, when analyzed by multivariate analysis,
with the Piracicaba strain having a higher percentage in both peaks. A stepwise logistic
procedure found complete separation of data points when comparing peaks F and I, with
the Piracicaba strain having a higher percentage of both peaks. Figure 33 shows a scatter
plot of the values for peaks F and 1 for both strains. The jackknife analysis showed that,
based on these data for peaks F and I, the probability of correctly identifying an unknown
specimen is dependent upon the strain of that specimen. Osório strain flies were correctly
identified with 100% accuracy. Piracicaba strain flies were only identified correctly 60%
of the time with the remaining 40% being falsely identified as Osório strain.

56
Table 7: Retention times of GC peaks for the cuticular hydrocarbons of O. depleta
Peak
Retention Time
(in minutes)
A
21.00
B
23.00
C
23.50
D
25.00
E
25.18
F
27.00
G
27.15
H
27.20
I
27.35
J
27.45
Figure 29: The mean percentage of total peak area for the gas chromatograms of the
cuticular hydrocarbons of female Piracicaba strain Ormia depleta

Percentage of Total Peak Area 57
30: The mean percentage of total peak area for the gas chromatograms of the
cuticular hydrocarbons of female Osorio strain Ormia deplela
Figure 31: The mean percentage of total peak area for the gas chromatograms of the
cuticular hydrocarbons of male Piracicaba strain Ormia depleta

58
Figure 32: The mean percentage of total peak area for the gas chromatograms of the
cuticular hydrocarbons of male Osorio strain Ormia depleta
B
o o
A A
4
O
AO
A Osorio Strain
O Piracicaba
Strain
0°
-1.0 1.0 3.0 5.0 7.0 9.0
Percentage of total peak area accounted for by peak F
Figure 33: Scatter plot representation of the values for peaks F and I for male O. depleta
of the Piracicaba and Osorio strains

59
Discussion
The complete separation of data points shows that, based on these data, males of
O. depleta can be identified to strain by GC cuticular hydrocarbon analysis with the use
of only two GC peaks. The difficulty in this lies in that males are rarely, if ever,
collected. However, because cuticular hydrocarbon makeup is influenced by a number of
factors including the age of the insect and its diet (Liang and Silverman 2000), specimens
prepared for GC analysis must be laboratory-reared under the protocol outlined above.
Therefore, gravid females collected would have to be used to inoculate mole crickets and
their offspring laboratory reared and tested. Because of this, it would be just as easy to
test males as females.
The lack of better predictability of identification of unknowns is somewhat
disappointing. This failure is most likely due to the rather small sample size with which
the jackknife analysis had to work as opposed to the actual differences in the cuticular
hydrocarbon makeup of the two strains. A more sophisticated model using more than two
peaks may be able to correct this even with the current data size. For this reason, the raw
data taken for both strains, as well as a hybrid of the two strains, has been presented in
Appendix C.

CHAPTER 6
SURVIVAL OF ORMIA DEPLETA WHEN CAGED WITH HONEYDEW-
PRODUCING APHIS NER1I
Introduction
The establishment of Ormia depleta, along with Larra bicolor and Steinernema
scapterisci, has been shown to have a strong negative effect on the overall population of
Scapteriscus mole crickets in Florida (Walker et al. 1992, Parkman et al. 1996). Although
O. depleta is apparently well established throughout most of central and southern Florida
(Frank et al. 1996), there has not yet been established any method to augment local
populations of the flies in areas where an increased level of mole cricket control would be
desired. Areas such as golf courses, which have a very low threshold for turf damage,
could benefit from an increased local population of natural enemies, which would reduce
the population of pest mole crickets.
Augmentation of biological control agents has been achieved in many ways.
Inundative releases of O. depleta are not practical due to the difficulty in rearing them.
Banker plants have been used to introduce and disperse parasitoids into field situations
(Goolsby and Ciomperlik 1999). Banker plants may also be useful in providing food
sources for adult parasitoids and even acting as an attractant. Investigation into
determining a suitable banker plant for O. depleta led to the discovery that much of their
diet consists of homopteran honeydew (Welch 2000). Therefore a plant nectar source
would not be likely to make a suitable banker plant. Alternatively, a plant which would
reliably maintain a population of honeydew producing homopterans could be established
60

61
in areas where a higher population density of parasitoids was needed. If the honeydew
produced was attractive to O. depleta, and the homopteran population could be
maintained, it would very likely achieve the desired augmentation of the local population.
Aphids feed on a number of ornamental plants that may be suitable as banker
plants for O. depleta. Although almost any plant could be incorporated into various
landscapes to attract O. depleta, there are considerations to be made when choosing a
banker plant. Honeydew production of the aphids associated with each plant is important,
as are the host range of those aphids and their pest status. Pittosporum spp., Pyracantha
spp., and Viburnum spp. are all infested by Aphis spiraecola Patch, but this species of
aphid also attacks Citrus spp. (Fasulo et al. 2003). Rosa spp. are host to the rose aphid,
Macrosiphum rosae (L.), which may be a suitable banker organism. Roses, however, are
not generally the type of plant used in golf course landscape, and encouraging a pest of
roses may not be as acceptable as encouraging pests of other ornamentals. Gardenia spp.
and Hibiscus spp. are host to Aphis gossypii Glover, but this species is also a pest of
Citrus spp., making it unsuitable for recommendation. Camellia spp. and Gardenia spp.
are hosts to the green peach aphid, Myzus persicae (Sulzer), but as this species has a wide
host range and produces a relatively small amount of honeydew, it is not a good
candidate (Capinera 2001). The crepe myrtle aphid, Sarucallis kahawaluokalani
(Kirkaldy), feeds on Lagerstroemia spp. and along with the podocarpus aphid,
Neophyllaphis podocarpi Takahashi, which feeds on Podocarpus spp., may prove to be
reliable banker organisms for O. depleta.
The oleander aphid, Aphis nerii Boyer de Fonscolombe, is a minor pest of
oleander (Nerium oleander L.) and milkweeds (Asclepias spp.) (Johnson and Lyon 1991).

62
These aphids are able to use some of the toxins of their host plants for their own defense
(Malcolm 1986), but they are still susceptible to generalist predators and the braconid
wasp Lysiphlebus leslaceipes Cresson (Hall and Ehler 1980). Whether these toxins would
be in the honeydew and whether they would act as a deterrent to feeding by O. depleta
was unknown. Although A. nerii colonies can quickly grow to huge numbers on host
plants, they do not generally cause a decline in the health of the plant as damage is
generally restricted to unsightly sooty mold caused by the secretion of honeydew (Hall
and Ehler 1980). Due to this mild pest status, the narrow host range, and the observation
that colonies of A. nerii are present in Florida throughout much of the year, this species
was chosen for evaluation as a potential banker organism to provide honeydew for
populations of O. depleta. The objective of this experiment was to determine whether
Aphis nerii honeydew was a suitable diet for O. depleta.
Materials and Methods
Pupae of Piracicaba strain O. depleta were obtained from the laboratory colony at
the University of Florida, which was reared from females collected from the Gulf Coast
Research and Education Center in Bradenton, Florida. Six cylindrical rearing cages (30
cm diameter x 60 cm tall) were placed in an outdoor heated greenhouse. Each cage
contained a 20 dram vial of moist sand with six O. depleta pupae, a small (53 mm
diameter) Petri dish of water wicked with cotton, and an approximately 20 cm tall
milkweed (Asclepias curassavica L.) plant in a 10 cm diameter pot. Milkweed plants in
five of the six cages were inoculated with ten adult A. nerii each, which were field
collected from A. curassavica plants, and the colonies were allowed to grow undisturbed
for two weeks before exposure to the flies. By this time, each colony was well established

63
and covered the new growth of each plant. The milkweed in the sixth cage was not
inoculated with aphids to serve as the “starvation” control. None of the plants were in
bloom at the time of the experiment, so the only carbohydrate source available was from
the honeydew secreted by the aphids. At the time of the introduction of the pupae,
copious amounts of honeydew were visible on the plants as well as the sides of the
rearing cage. The adults emerged over a two-day period. The number of flies remaining
alive in the cages was recorded each day, and the experiment ran until all the flies were
dead. Each fly acted as a replicate to produce a mean lifespan for each treatment.
Results
The flies that had access to the honeydew of A. nerii lived an average of 21.7 days
(SD = 7.1, n = 21). The flies without honeydew lived an average of 15.7 days (SD = 4.1,
n = 4). A t-test showed this difference to be significant (P < 0.04). Neither set of flies
produced any gravid females.
Discussion
Although the results of this experiment did show significance, the level of
significance would have likely been much greater had it not been for several factors.
Running this experiment in a heated greenhouse resulted in environmental factors that
were not conducive to the health of the flies. The levels of temperature and humidity
fluctuated greatly. The mean life span of the honeydew-fed flies was far less than the
recorded life span of flies fed other diets in previous experiments. Under laboratory
conditions the mean life spans of flies fed hummingbird nectar and melezitose solution
were 32.7 days and 39.0 days respectively (Welch 2000). Honeydew-fed flies in this
experiment only averaged 21.7 days. Although this may seem to indicate that this
particular honeydew is not a suitable diet, the flies with no food only had mean life spans

64
of 15.7 days in this experiment whereas the mean life span under previous laboratory
conditions for starved flies was 22.2 days (Welch 2000). This indicates an overall life
span reduction caused by another factor. In this case, the most likely factor was humidity.
This experiment took place from December to January and because the greenhouses were
heated, the air remained quite dry. Additionally, the host plants for the aphids had been
kept very well watered prior to this experiment by placing them in shallow dishes of
standing water. This was done to reduce the stress of the aphid load on the plants. When
the plants were placed in the cages, they were kept watered, but the standing water dish
was removed to keep the flies from drowning. The resulting reduction in available water
was apparently the cause of the plants beginning to decline. By approximately day ten of
the experiment, all the plants were dead. The experiment was allowed to continue
because the amount of honeydew that had already been produced was significant.
Honeydew had been heavily deposited on the leaves of the plants, the wall of the cage,
and the plant pots. The amount of honeydew that flies were able to consume was enough
to produce significant results, but had the plants lived longer, the difference would have
likely been more dramatic.
The results do seem to confirm that the honeydew of A. nerii is not toxic to O.
depleta and that it is a suitable food source. Because of the difficulties encountered in this
experiment, no real conclusions can be made as to the suitability of Aphis nerii honeydew
as a food source for the augmentation of O. depleta in the field. This experiment was not
repeated because the laboratory colony of O. depleta died out while it was being
conducted and a new colony was not obtained until it was too late to run the experiment

65
again. The ability of A. nerii honeydew to act as an attractant in the field was not
investigated here.

APPENDIX A
THE EFFECT OF PUPAL SIZE ON EMERGENCE
To determine if the fitness of the pupae of smaller-than-avaerage O. depleta was
reduced, a short experiment was conducted. Pupae were divided into 5 groups based on
their weight. Each group was then placed in a separate emergence box containing a 2 cm
layer of moist sand and covered with a 1 cm layer of moist sand. The boxes were kept at
room temperature (approximately 25° C) and when the adult flies had emerged, the
number of adults was recorded. The results are shown below in Table 8. There was a
Table 8: The percent emergence of O. depleta pupae of varying sizes
Pupal Size
Number of
Pupae
Number of
Adults
% Emergence
<40 mg
28
20
71.4
40-50mg
31
29
93.6
50-60mg
50
45
90.0
60-70mg
50
46
92.0
>70mg
60
51
85.0
trend toward decline in survival of pupae less than 40 mg. Whether this is due to some
genetic or physiological problem or just a function of having less of a buffer from
desiccation is unknown.
66

APPENDIX B
ATTEMPTED INDUCTION OF DIAPAUSE BY SHORT DAY PHOTOPERIOD IN
OSORIO STRAIN ORMIA DEPLETA
An experiment was conducted to determine if diapause could be induced in O.
deplela by subjecting the pupae and adults to short day photoperiods. The Osorio strain
pupae were obtained and placed in two large (30 cm diameter x 60 cm tall) rearing cages.
The pupae were shallowly buried in moist sand as per the normal rearing technique. One
cage was designated as the short-day cage and one as the long-day cage. Both cages were
placed near a window that received indirect sunlight. After sundown each day, the short-
day cage was covered with black plastic, consisting of two black plastic bags, one inside
the other, to block sunlight. In the morning, the long-day cage received sunlight but the
short-day cage remained covered until four hours after sunrise. After four hours, the
short-day cage had its black plastic removed and it received light until sundown. Both
cages were exposed to sundown, as twilight was considered an important mating
stimulant by Wineriter and Walker (1990). The pupae and resulting adults were kept
under these conditions. After gravid females had been produced, some from each
treatment were sacrificed and their planidia used to inoculate S. abbreviatus. These mole
crickets were then placed in their individual vials of moist sand and put back into either
the short-day cage or the long-day cage in accordance with the planidia with which they
had been inoculated. After 10 days, the vials which had contained the mole cricket hosts
were removed and the pupae harvested. These pupae were then shallowly buried in moist
sand as per the normal rearing technique. The resulting pupal duration was recorded for
67

68
each individual pupae. The results showed the mean pupal duration for the short-day
pupae to be 14.6 days (0.74 SD) and 13.8 days (0.73 SD) for the long-day pupae. A r-test
showed no difference between the two treatments.

APPENDIX C
GAS CHROMATOGRAPHIC ANALYSIS OF THE CUTICULAR HYDROCARBONS
OF ORMIA DEPLETA TO IDENTIFY STRAINS
Raw data showing the percentage each peak contributed to the total peak area.
Peak Retention Time
â– g
Zn
E
n
Sex
21.00
23.00
23.50
25.00
25.18
27.00
27.15
27.2
27.35
27.45
p
c
F
4.27
22.81
0.60
5.98
4.24
1.31
37.56
13.66
2.07
7.51
p
c
F
5.43
29.07
0.84
7.98
3.75
1.21
34.71
11.38
1.08
4.56
p
s
F
4.67
26.87
0.00
7.42
3.22
35.40
12.22
1.48
5.97
0.00
p
s
F
5.29
18.50
0.00
5.00
5.93
43.66
16.47
0.00
3.91
0.00
p
s
F
4.28
26.76
0.00
6.92
3.64
37.21
11.72
1.34
5.63
0.00
p
s
F
4.15
28.74
0.71
7.41
3.41
35.37
11.94
1.25
5.05
0.00
p
s
F
4.02
24.24
0.69
6.64
4.18
37.51
12.66
1.52
5.95
0.00
p
s
F
3.68
24.81
0.81
5.73
3.80
37.39
15.41
1.23
5.16
0.00
p
s
F
3.95
19.49
2.54
5.15
4.06
42.23
15.36
1.26
5.32
0.63
p
s
F
6.60
28.55
0.98
7.71
3.15
33.94
10.30
1.23
4.73
0.00
p
c
M
16.75
36.19
1.57
7.56
0.43
2.48
6.34
11.75
7.53
9.41
p
c
M
17.90
37.24
1.48
7.30
0.46
1.70
4.58
12.57
7.52
9.25
p
s
M
15.91
40.60
1.54
9.22
3.03
5.57
6.46
3.98
3.52
4.76
p
s
M
12.52
42.04
1.29
10.07
3.04
5.69
9.01
4.97
3.99
5.74
p
s
M
13.57
35.53
1.23
8.76
2.08
7.34
7.53
5.60
6.03
9.00
p
s
M
13.32
34.47
1.01
7.55
3.02
6.56
9.56
5.93
5.51
8.39
p
s
M
16.35
47.10
2.42
10.60
2.12
3.48
5.15
3.77
2.65
4.66
p
s
M
11.92
50.00
1.49
10.99
2.90
5.11
5.13
5.09
2.04
3.56
p
s
M
15.80
41.31
2.34
10.23
3.00
3.26
8.71
5.36
4.25
3.96
p
s
M
15.11
26.39
1.61
4.96
3.89
4.22
8.13
11.76
8.92
8.13
*Piracicaba, Osório, or hybrid (FI generation from Piracicaba females and Osorio males)
** Single or Composite
69

70
*
* u Peak Retention Time
.S "S.
3
H
CZ>
£
es
C/3
X

C/3
21.00
23.00
23.50
25.00
25.18
27.00
27.15
27.2
27.35
27.45
o
c
F
3.22
25.91
0.79
8.00
2.09
1.31
36.61
11.18
2.36
8.54
0
c
F
3.18
27.25
1.12
11.19
1.88
0.98
32.79
11.80
1.89
7.92
o
s
F
0.00
23.89
3.58
12.40
12.40
24.58
9.77
1.16
7.05
2.33
0
s
F
3.66
52.78
6.90
7.08
2.54
31.42
12.74
1.42
8.46
0.00
0
s
F
1.75
22.90
10.36
10.83
1.44
26.88
10.28
2.01
11.15
2.40
0
s
F
5.18
16.86
0.72
3.82
2.59
35.57
14.03
2.76
10.69
1.59
0
s
F
1.36
26.02
3.84
9.67
1.43
33.78
13.02
0.00
1.53
7.25
0
s
F
1.35
25.14
5.02
12.34
1.55
33.42
13.75
0.00
0.00
4.69
0
s
F
3.66
23.77
2.62
5.96
2.72
36.00
13.20
0.00
2.12
9.94
0
c
M
20.88
44.61
1.49
10.83
0.79
1.40
4.24
5.39
3.76
6.62
0
c
M
23.24
44.55
1.49
11.16
1.14
1.32
4.04
5.67
2.90
4.47
0
s
M
25.63
42.57
2.65
10.55
0.95
3.19
2.71
3.32
2.69
5.73
o
s
M
16.46
33.75
0.00
7.11
2.47
1.70
9.46
11.17
4.49
6.14
()
s
M
19.96
38.46
0.00
7.87
1.38
1.38
4.97
8.66
3.54
6.21
0
s
M
28.07
42.17
1.89
9.52
0.00
1.99
1.63
2.31
1.09
2.65
o
s
M
18.27
40.80
0.00
8.96
0.00
1.57
6.61
10.43
3.34
5.30
0
s
M
17.16
46.53
3.11
15.75
0.00
0.00
0.00
1.44
0.00
3.16
0
s
M
18.52
41.76
1.79
11.69
1.07
1.16
3.89
4.23
4.41
8.03
o
s
M
16.27
44.64
0.00
10.09
0.00
0.00
8.02
8.14
3.21
4.37
11
s
F
3.80
12.04
0.60
3.61
5.76
47.91
14.85
1.10
3.14
0.00
H
s
F
3.25
18.88
0.51
5.61
4.66
42.55
14.78
1.17
4.41
0.00
H
s
F
2.82
24.89
0.00
6.86
4.13
43.40
6.98
1.72
6.02
0.00
H
s
F
3.37
22.13
0.62
6.50
2.98
42.76
6.19
2.44
8.14
0.00
H
s
F
4.00
14.89
0.00
4.25
6.31
43.69
18.73
1.11
4.06
0.00
H
s
F
2.01
20.28
0.53
6.54
3.49
40.77
7.64
2.51
11.10
1.14
H
s
F
3.05
22.34
0.80
6.92
2.98
42.24
5.94
2.43
8.33
0.00
H
s
F
3.82
21.91
0.00
5.51
3.52
42.53
10.24
2.04
7.20
0.00
*Piracicaba, Osorio, or hybrid (FI generation from Piracicaba females and Osorio males)
**Single or Composite

LIST OF REFERENCES
Adamo, S.A., D. Robert, J. Perez, and R.R. Hoy. 1995. The response of an insect
parasitoid, Ormia ochracea (Tachinidae), to the uncertainty of larval success
during infestation. Behav. Ecol. Sociobiol. 36: 111-118.
Amoroso, J. 1990. Ormia depleta parasitism of Manatee Co. trapped Scapteriscus
borellii. Annu. Report Mole Cricket Res. 12: 190-191.
Andersen, S. 1996. The Siphonini (Díptera: Tachinidae) of Europe. E.J. Brill; Leiden,
Netherlands. 148 pp.
Bai, B., and M. Mackauer. 1992. Influence of superparasitism on development rate and
adult size in a solitary parasitoid wasp, Aphidius ervi. Func. Ecol. 6: 302-307.
Baronio, P., M.L.Dindo, G. Campadelli, and L. Sighinolfi. 2002. Intraspecific weight
variability in Tachinid flies: response of Pseudogonia rufifrons to two host
species with different size and of Exorista larvarum to variations in vital space.
Bull. Insect 55: 55-61.
Buss, E.A, J. L. Capinera, and N. C. Leppla. 2002. Pest mole cricket management.
University of Florida Cooperative Extension Service document ENY-324.
Published world wide web at: http://edis.ifas.ufl.edu/LH039.
Burkett, D.A., D.A. Carlson, and D.L. Kline. 1998. Analysis of compostion of sugar
meals of wild mosquitoes by gas chromatography. J. Amer. Mosq. Control Assoc.
14: 373-379.
Cantrell, B.K., and R.W. Crosskey. 1989. Family Tachinidae. pp. 733-784. In Evenhuis,
N.L. (ed). Catalog of the Diptera of the Australasian and Oceanian regions.
Bishop Museum Special Publication 86. Bishop Museum Press, Honolulu, and
E.J. Brill. 1155 pp.
Capinera, J.L. 2001.Green peach aphid in Featured Creatures (EENY-222). On world
wide web at: http://creatures.ifas.ufl.edu/veg/aphid/green_peach_aphid.htm.
Dindo, M.L., L. Sighinolf, G. Campadelli, and P.Baronio. 1999. Laboratory evaluation of
parasitism of wax moth and gypsy moth larvae by Exorista larvarum (L.) cultured
in vivo and in vitro. Boll. Inst. Entomol. 53: 109-119.
71

72
Eller, F.J., J.H. Tumlinson, and W.J.Lewis. 1990. Intraspecific competition in Microplitis
croceipes (Hymenoptera: Braconidae), a parasitoid of Heliothis species
(Lepidoptera: Noctuidae). Ann. Entomol. Soc. Am. 83: 504-508.
Fasulo, T.R., R.F.Mizell, and D.E. Short. 2003. WoodyPest. UF/IFAS. On world wide
web at: http://woodypest.ifas.ufl.edu/.
Fowler, H.G. 1987. Field confirmation of the phonotaxis of Euphasiopteryx deplela
(Díptera: Tachinidae) to calling males of Scapteriscus vicinus (Orthoptera:
Gryllotalpidae). Fla. Entomol. 70: 409-410.
Fowler, FI.G. 1988a. Traps for collecting live Euphasiopteryx depleta_(Diptera:
Tachinidae) at a sound source. Fia. Entomol. 71: 654-656.
Fowler, HG. 1988b. Suitability of Scapteriscus mole crickets (Ort.: Gryllotalpidae) as
hosts of Euphasiopteryx depleta (Dip.: Tachinidae). Entomophaga 33: 397-401.
Fowler, H.G., and J.N. Kochalka. 1985. New record of Euphasiopteryx depleta (Diptera:
Tachinidae) from Paraguay: Attraction to broadcast calls of Scapteriscus acletus
(Orthoptera: Gryllotalpidae). Fla. Entomol. 68: 225-226.
Fowler, H.G., and A.V. Martini. 1993. Influencia do tamanho hospedeiro (Scapteriscus
borellii: Gryllotalpidae: Orthoptera) sobre a produjo experimental do parasitóide
(Ormia depleta'. Tachinidae: Diptera). Científica 21: 339-343.
Fowler H.G., and C.R. Garcia. 1987. Attraction to synthesized songs and experimental
and natural parasitism of Scapteriscus mole crickets (Orthoptera: Gryllotalpidae)
by Euphasiopteryx depleta (Diptera: Tachinidae). Rev. Bras. Biol. 47: 371-374.
Fowler, FI.G., and A. Mesa. 1987. Alternate orthopteran hosts {Anurogryllus sp.) of
Euphasiopteryx depleta (Diptera: Tachinidae). Florida Entomol. 70: 408-409.
Frank, J.H. 2002. A parasitic fly that kills mole crickets. USGA Green Record. 40: 9-11.
Frank, J.H., E.A. Buss, and K. Barbara. 2002. Beneficial nematodes in turf: good for how
many years against pest mole crickets? Florida Turf Digest 19: 48-50.
Frank, J.H., T.R. Fasulo, and D.E. Short. 1998. MCRICKET. Published on world wide
web at: http://molecrickets.ifas.ufl.edu/.
Frank, J.H., C. Grissom, C. Williams, E. Jennings, C. Lippi, and R. Zerba. 1999. A
beneficial nematode is killing pest mole crickets in some Florida pastures and is
spreading. Fla. Cattleman Livestock J. 63: 31-32.

73
Frank, J.H., J.P. Parkman, and F.D. Bennett. 1995. Larra bicolor (Hymenoptera:
Sphecidae), a biological control agent of Scapteriscus mole crickets (Orthoptera:
Gryllotalpidae), established in northern Florida. Fla. Entomol. 78: 619-623.
Frank, J.H., T.J. Walker, and J.P. Parkman. 1996. The introduction, establishment and
spread of Ormia depleta in Florida. Biol. Control 6: 368-377.
Goolsby, J.A., and M.A. Ciomperlik. 1999. Development of parasitoid inoculated
seedling transplants for augmentative biological control of silverleaf whitefly
(Homoptera: Aleyrodidae). Fla. Entomol. 82: 532-545.
Flail, R.W., and L.E. Ehler.1980. Population ecology oí Aphis nerii on oleander. Environ.
Entomol. 9: 338-344.
Hardy, I.C.W, N.T. Griffiths, and H.C.J. Godfray. 1992. Clutch size in a parasitoid wasp:
a manipulation experiment. J. Anim. Ecol. 61: 121-129.
Harvey, J.A., I.F. Harvey, and D.J. Thompson. 1993. The effect of superparasitism on
development of the solitary parasitoid wasp, Venturia canescens (Hymenoptera:
Ichneumonidae). Ecol. Entomol. 18: 203-208.
Hodge, S., and P. Mitchell. 1997. Inhibition of Drosophila melanogaster and D. hydei by
Aspergillus niger. Drosophila Information Service 80: 6-7.
Jacob, H.S., and E.W. Evans. 2004. Influence of different sugars on the longevity of
Bathyplectes curculionis (Hymenoptera: Ichneumonidae). J. Appl. Entomol. 128:
316.
Janzen, D.H. 1977. What are dandelions and aphids? Am. Nat. Ill: 586-589.
Johnson, W.T., and H.H. Lyon. 1991. Insects that feed on trees and shrubs. Comstock
Pub. Associates. Ithaca, NY. 560 pp.
Justi J. Jr., J.R.P. Parra, and J.H. Frank. 1988. Alternate hosts for the production of
Euphasiopteryx depleta. Annu. Rep. Mole Cricket Res. 10: 183-184.
Leatemia, J. A., J.E. Laing, and J.E. Corrigan. 1995. Effects of adult nutrition of
longevity, fecundity, and offspring sex ratio of Trichogramma minutum Riley
(Hymenoptera: Trichogrammatidae). Can. Entomol. 127: 245-254.
Liang, D. and J. Silverman. 2000. “You are what you eat”: Diet modifies cuticular
hydrocarbons and nestmate recognition in the Argentine ant, Linepithema humile.
Naturwissenschaften 87: 412-416.
Nickle, D.A., and J.L. Castner. 1984. Introduced species of mole crickets in the United
States, Puerto Rico and Virgin Islands (Orthoptera: Gryllotalpidae). Ann.
Entomol. Soc. Am. 77: 450-465.

74
Nguyen, K.B., and G.C. Smart, Jr. 1990. Steinernema scapterisci, new species,
(Rhabditida: Steinemematidae). J. Nematol. 22: 187-199.
Oesterbroek, P. 1998. The families of Díptera of the Malay archipelago. E.J. Brill;
Leiden, Netherlands. 227 pp.
Owen, D.F. 1978. Why do aphids synthesize melezitose? Oikos 31: 264-267.
Parkman, J.P., J.H. Frank, K.B. Nguyen, and G.C. Smart, Jr. 1994. Inoculative release of
Steinernema scapterisci (Rhabditida: Steinemematidae) to supress pest mole
crickets (Orthoptera: Gryllotalpidae) on golf courses. Environ. Entomol. 23:
1331-1337.
Parkman, J.P., J.H. Frank, T.J. Walker, and D.J. Schuster. 1996. Classical biocontrol of
Scapteriscus spp. (Orthoptera: Gryllotalpidae) in Florida. Environ. Entomol. 25:
1415-1420.
Petelle, M. 1980. Aphids and melezitose: a test of Owen’s 1978 hypothesis. Oikos 35:
127-128.
Potting, R.P.J., H.M. Snellen, and L.E.M. Vet. 1997. Fitness consequences of
superparasitism and mechanism of host discrimination in the stemborer parasitoid
Cotesiaflavipes. Entomol. Exp. Appl. 82: 341-348.
Reinert, J.A., and D.E. Short. 1980. Southern turfgrass insect pests with emphasis on
mole cricket biology and management. Proc. Fla. Turfgrass Manag. Conf. 28: 33-
43.
Reinert, J.R., and D.E. Short. 1981. Managing mole crickets. Grounds Maint. 16: 16-20.
SAS Institute Inc. 2001. SAS system for Windows Rel. 8.2. SAS Institute Inc. Cary,
North Carolina, USA.
Short, D.E., and J.A. Reinert. 1982. Ch. 21 - Biology and control of mole crickets in
Florida. In Niemczyk, H.D., and B.G. Joyner (eds). Advances in turfgrass
entomology. Chemlawn; Columbus, OH. 150 pp.
Soper, R.S., G.E. Shewell, and D. Tyrrell. 1976. Colcondamyia auditrix nov. sp.
(Díptera: Sarcophagidae), a parasite which is attracted by the mating song of its
host, Okanagana rimosa (Homoptera: Cicadidae) [New taxa]. Can. Entomol. 108:
61-68.
Sutton, B.D. and D.A. Carlson. 1993. Interspecific variation in tephritid fruit fly larvae
surface hydrocarbons. Archives Insect Biochem. Physiol. 23: 53-65.

75
Tsitspis, J.A. 1980. Relative humidity effects at 20 Celcius on eggs of the olive fruit fly
Dacus oleae (Díptera: Tephritidae) reared on artificial diet. Entomol. Exp. Appl.
28: 92-99.
van Dijken, M.J., and J.K. Waage. 1987. Self and conspecific superparasitism by the egg
parasitoid Trichogramma evanescens. Entomol. Exp. Appl. 30: 77-82.
Vet, L.E.M., A. Datema, A. Janssen, and H.M. Snellen. 1994. Clutch size in a larval-
pupal endoparasitoid: consequences for fitness. J. Anim. Ecol. 63: 807-815.
Vinson, S.B., and P. Sroka. 1978. Effects of superparasitism by a solitary endoparasitoid
on the host, parasitoid and field samplings. Southwestern Entomol. 3: 299-301.
Waage, J.K., and S.M. Ng. 1984. The reproductive strategy of a parasitic wasp. I.
Optimal progeny allocation in Trichogramma evanescens. J. Anim. Ecol. 53: 401-
415.
Walker, T.J. (ed.). 1985. Mole crickets in Florida. Univ. Fla. Agrie. Exp. Stn. Bull. 846:
i-iv; 1-54.
Walker, T.J. 1988. Acoustic traps for agriculturally important insects. Fla. Entomol. 71:
484-492.
Walker, T.J. 1989. A live trap for monitoring Euphasiopteryx and tests with E. ochracea
(Díptera: Tachinidae). Fla. Entomol. 72: 314-319.
Walker, T.J., J.J. Gaffney, A.W. Kidder, and A.B. Ziffer. 1993. Florida reach-ins:
environmental chambers for entomological research. Am. Entomol. 39: 177-182.
Walker, T.J., and T.E. Moore. 2000. Singing insects of North America. On the world
wide web at: http://buzz.ifas.ufl.edu.
Walker, T.J., and D.A. Nickle. 1981. Introduction and spread of pest mole crickets:
Scapteriscus vicinus and S. acletus reexamined. Ann. Entomol. Soc. Am. 74: 158-
163.
Walker, T.J., J.P. Parkman, and D.J. Schuster. 1992a. Seasonal distribution and
population trends of Ormia depleta in Florida. Annu. Rep. Mole Cricket Res. 14:
118-123.
Walker, T.J., J.P. Parkman, and D.J. Schuster. 1992b. Sound-trap assays of population
trends: Annual update. Annu. Rep. Mole Cricket Res. 14: 1-5.
Walker, T.J., J.P. Parkman, J.H. Frank, and D.J. Schuster. 1996. Seasonality of Ormia
depleta and limits to its spread. Biol. Control 6: 378-383.

76
Welch, C.H. 2000. Gas chromatographic analyses of crop sugars of Ormia depleta
established in Florida. Master’s Thesis. University of Florida.
Wineriter, S.A., and T.J. Walker. 1990. Rearing phonotactic parasitoid flies (Diptera:
Tachinidae, Ormiini, Ormia spp.). Entomophaga 35: 621-632.
Wood, D.M. 1987. Chapter 110. Tachinidae. In McAlpine, J.F. et al. (eds.), Manual of
Nearctic Diptera. Volume 2. Agriculture Canada Monograph 28: 1193-1269
Zuk, M., L.W. Simmons, and L. Cupp. 1993. Calling characteristics of parasitized and
unparasitized populations of the field cricket Teleogryllus oceanus. Behav. Ecol.
Sociobiol. 33: 339-343.

BIOGRAPHICAL SKETCH
Craig Hinton Welch was bom on February 15, 1971, in Knoxville, Tennessee, to
Sara H. and Robert N. Welch. He grew up in Dover, Delaware, where he attended school
and spent summers at a YMCA camp in North Carolina where his parents worked as
administrators and his interest in biology began. In 1986, his family moved to Salisbury,
North Carolina, where he graduated from high school in 1989. He then went on to earn
his Bachelor of Science degree in biology from the University of North Carolina at
Charlotte. He married his wife Celeste on May 13, 1995. In 2000, he earned his master’s
degree from the University of Florida in entomology. He and his wife have four
daughters: Brooke, Courtney, Natalie, and Valerie.
77

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.
J. Howard Frank, Chair
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.
Ojlith. ¿t. f%tU4.
Eileen Buss
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
Robert McSorley/
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.
Bijan Dargan
Professor of Horticultural Science
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 qudl|ity, as a
dissertation for the degree of Doctor of Philosophy^
FrankVlansky
Professor of Entomologjl
id Nematology

This dissertation was submitted to the Graduate Faculty of the College of
Agricultural and Life Sciences and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
August 2004
s.
Dean, College of Agricultural
Sciences
^and'btfe
Dean, Graduate School

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UNIVERSITY OF FLORIDA
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