PHAON CRESCENT, Phyciodes Phaon : LIFE CYCLE, NUTRITIONAL ECOLOGY and REPRODUCTION

PHAON CRESCENT, Phyciodes phaon : LIFE CYCLE, NUTRITIONAL ECOLOGY AND REPRODUCTION

By HANIFE GENC

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

Hanife Gene To my wonderful husband, and my daughter ACKNOWLEDGMENTS

I express my gratitude and appreciation to all the people who supported my

graduate studies at the University of Florida. First, I express my sincere gratitude and appreciation to my major professor and advisor, Dr. James L. Nation. I thank him for his constant support, encouragement, advice and guidelines for my research during my entire

program. I also thank him for his help in preparation of this dissertation. I thank Drs. T.

Emmel, C. Guy, S. Halbert and G. Bowes for serving on my committee and for helpful

suggestions and comments during my research, and for reviewing my dissertation. I

thank Dr. Charles Guy for letting me use his laboratory facilities and equipment to prepare freeze-dried host plant leaves.

I extend my gratitude to Kathy Milne who made several computer drawings and

took care of my colony during my absence. I thank Dr. Kenneth Portier for help with

statistical analyses. I thank Dr. A1 Handler for his help, support and use of his laboratory

facilities at USDA in Gainesville to make digital pictures of my specimens. I offer

special thanks to the members of Dr. Handler’s lab, Grazyna Zimowska, Rob Harrell, and

Pat Whitmer for all their help and understanding. I thank Rob Harrell for help with the

photographs. I thank Nancy Lowman for help with diet preparation and some supplies. I

also want to thank my friends in Gainesville and around the world for their support and

friendship. My deepest thanks and appreciation go to my parents for the constant support

and unconditional love they have provided throughout my life. I appreciate the endless

IV V

love and support I receive from my brother Tanfer and my sister Hande. I also thank my in-laws for their support and encouragement.

I give my deepest thanks and love to my wonderful husband, Levent Gene, for his constant love, support, understanding, encouragement, and limitless help in preparation

of this dissertation. I extend my love to my special daughter, Destina E. Gene, for her

love, help and understanding. Finally, I thank the Turkish Ministry of Education for their

generous financial support during my graduate studies at the University of Florida. TABLE OF CONTENTS page

ACKNOWLEDGMENTS iv

LIST OF TABLES viii

LIST OF FIGURES x

ABSTRACT xii CHAPTER

1 NUTRITIONAL ECOLOGY AND GENERAL PRINCIPLES OF ARTIFICIAL DIETS FOR INSECTS 1

Introduction 1 Measurement of Nutritional Quality 4

2 THE LIFE HISTORY AND BIOLOGY OF Phyciodes phaon (EDWARDS) ON Phyla (Lippia) nodiflora 6

Introduction 6 Materials and Methods 8 Results 9 Description of Phyciodes phaon Immature and Adult Stages 9 Food Plant and Life History 24 Discussion and Conclusions 24

3 NUTRITIONAL ECOLOGY AND REQUIREMENTS OF AN ARTIFICIAL DIET FOR THE BUTTERFLY Phyciodes phaon 30

Introduction 30 Materials and Methods 31 Study Organism: Butterfly and Host Plant 31 Commercial and Published Diets Tested 31 Preparation of the Pinto Bean Diet from Components 32 Preparation of the Host Plant for Addition to Diets 33 Analyses of Diets and Diet Components 33 Food Consumption and Utilization 33 Extractions of the Host Plant and Addition of Extracts to the Pinto Bean Diet 39 Statistical Analyses 42 Results 45

vi vii

Commercial and Published Diets Available 45 Testing Extracted Host Plant with the Pinto Bean Diet 55 Measurements of Food Intake 57

Survival of the Phaon Crescent Larvae on White Clover ( Trifolium repens) 66 Survival of the Phaon Crescent Larvae on Other Verbenaceae Species 67 Conclusion and Discussions 67

4 DETERMINATION OF POLYUNSATURATED FATTY ACID COMPOSITION OF LARVAE AND ADULTS OF THE PHAON CRESCENT BUTTERFLY AS AFFECTED BY ARTIFICIAL DIET 75

Introduction 75 Composition of Polyunsaturated Fatty Acids in Insects 77 Materials and Methods 79 Study Organism 79 Artificial Diets 79 Extraction of Lipids and Fatty Acid Analyses 80 Gas Chromatography (GC) to Analyze Fatty Acids 81 Total Lipid Extraction to Isolate Plant Sterols from Phyla nodiflora 81 Gas Chromatography (GC) to Analyze Plant Sterols 81 Thin Layer Chromatography (TLC) 82 Identification of Sterols in Pinto Bean Diet (PBD) 82 Results 83 Rearing P. phaon Larvae on PBD with Added Oils 83 Fatty Acid Composition 83 Plant Sterols in Phyla nodiflora 86 Plant Sterols in Pinto Bean Diet 94 Comparison of Sterols Isolated from Phyla nodiflora and the PBD 94 Rearing P. phaon Larvae on PBD with Addition of Sterols 94 Conclusion and Discussions 105

5 INTERNAL REPRODUCTION SYSTEMS OF Phyciodes phaon 107

Introduction 107 Materials and Methods 109 Study Organism 109 Dissection 110

Results 1 10 Discussion and Conclusions 118

6 CONCLUSIONS 120

LIST OF REFERENCES 125

BIOGRAPHICAL SKETCH 132 2- LIST OF TABLES

3- Table page

2-1 The measurements of head capsule, weight and length Phyciodes phaon 16

2 Externally sexing P. phaon pupae 19

1 The list of several commercially available artificial diets and their ingredients 34

3-2 Components of the pinto bean diet 35

3-3 Typical analysis of ground wheat germ 36

3-4 Typical analysis of torula yeast 37

3-5 Typical analysis of Beck’s salt mixture 38

3-6 Typical analysis of vitamin mixture 38

3-7 Procedures for calculating the efficiency of food utilization by insects 40

3-8 Growth and survival of Phyciodes phaon on several artificial diets 47

3-9 Survival and development time of P. phaon larvae on Pinto Bean Diet, Pinto Bean Diet with 10% host plant and on Phyla 48

3-10 Survival on modified diet, on PBD with 20% host plant, on PBD with 5% host plant, on PBD with 1% host plant, PBD with 10% host plant and PBD only 51

3-11 Survival on PBD with 1% cellulose, 5% cellulose, on PBD with 10% host plant and on PBD 53

3-12 Survival on PBD with addition of 1% glucose, 5% glucose, 1% starch, 5% starch, PBD with 10% host plant and PBD only 54

3-13 Survival on PBD with addition of 1% Beck’s salt, 5% Beck’s salt, PBD with 10% host plant and on PBD only 56

3-14 Survival on modified diet and on PBD with 20% host plant 58

viii IX

3-15 Survival on PBD with chloroform: methanol extract, PBD with chloroformimethanol extracted residue and on PBD with 10% host plant 59

3-16 Survival on PBD with chloroform extract, PBD with chloroform extracted residue, PBD with 10% host plant and on PBD only 60

3-17 Survival on PBD with carotinoid extract, PBD with chlorophyll extract, PBD with 10% host plant and on PBD only 61

3-18 Survival on PBD with methanol extract, on PBD with methanol extraction of flowers, on PBD with 10% host plant and on PBD only 62

3-19 Survival on PBD with acetone extract, on PBD with acetone extracted residue,on PBD with 10% host plant and on PBD only 63

3-20 Survival on PBD with hot ethanol extract 64

3-3-21 Survival on PBD with hot water extract, on PBD with hot water extracted residue on PBD with 10% host plant and on PBD only 65 4- 3-22 Added components into PBD 67

3-23 The growth and utilization of food by the phaon crescent (fifth instars) on PBD with 10% host plant and PBD only 68

3-24 Separation of the means of food consumption calculated by t-test 69

3-25 Survival on PBD with addition of 5% and 10% clover 70

26 Survival on PBD with addition of 10% Lantana camara, on PBD with addition of 10% S.jama, on PBD with 10% host plant 71

1 Survival of phaon crescent on PBD with oil 84

4-2 The distribution of the methyl esters of long-chain fatty acids for lipids of P. phaon larvae and adults 93

4-3 The plant sterols standards: /3-sitosterol, stigmasterol, campesterol and their TMS and acetate derivatives 102

4-4 The isolated sterols from Phyla nodiflora and their acetate and TMS derivatives 102

4-5 Comparison of the amount of sterols isolated from P. nodiflora and Pinto Bean Diet 103

4-6 Survival on PBD with addition of sterols, PBD with 10 % host plant 104 0

LIST OF FIGURES

Figure page

2-1 Phaon crescent butterfly larval host plant of, Phyla nodiflora 1

2-2 Egg clusters of Phyciodes phaon 12

2-3 Micrographs of Phyciodes phaon eggs 13

2-4 Instars 14

2-5 The prepupa 17

2-6 Pupal color variations 18 2-

2-73- Micrographs of Phyciodes phaon pupae 20 3- 2-8 Micrographs of Phyciodes phaon pupae 21 4-

2-9 Sex determination of Phyciodes phaon pupae 22

2-10 The newly emerged adult’s pupal case 23

2-11 P. phaon female 26

2-12 Life cycle summary of Phyciodes phaon 27

13 Survival of laboratory reared Phyciodes phaon colony on Phyla nodiflora 28

1 The method for fractionation of chloroform:methanol extract ofR. nodiflora 43

2 The egg color variation in the egg clusters of P. phaon 49

1 Gas chromatography analysis of standard fatty acid methyl esters 87

4-2 Gas chromatography analysis of fatty acids on Phyla nodiflora 88

4-3 Gas chromatography analysis of fatty acids on PBD with host plant 89

4-4 Gas chromatography analysis of fatty acids on PBD 90

4-5 Gas chromatography analysis of fatty acids on PBD with addition of oil 91

x XI

4-6 Gas chromatography analysis of fatty acids of P. nodiflora and PBD 92

4-7 Gas chromatography analysis of /3-sitosterol 96

4-8 Gas chromatography analysis of stigmasterol 97

4-94- Gas chromatography analysis of campesteol 98

5- 4-10 Gas chromatography analysis of /3-sitosterol, stigmasterol and campesterol 99

4-11 Separation of P. nodiflora sterols by thin layer chromatography 100

12 The common plant sterols isolated from PBD 101

1 Internal reproduction structure of newly emerged female 1 12

5-2 Internal reproduction structure of female 113

5-3 Internal reproduction structure of 10-day-old female 1 14

5-4 Internal reproduction structure of male 115

5-5 Internal reproduction structure of Phyciodes phaon 116

5-6 Ovary dissected from a newly emerged female reared on pinto bean diet 117 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

PHAON CRESCENT, Phyciodes phaon : LIFE CYCLE, NUTRITIONAL ECOLOGY AND REPRODUCTION

Hanife Gene

December 2002

Chair: Dr. James L. Nation Department: Entomology and Nematology

The butterfly Phyciodes phaon can be reared to the adult stage on a pinto bean based diet (PBD) previously developed to rear moths. Larvae go through five instars. Adults reared on the PBD do not lay eggs. As many as 65-78% adults could be produced when this diet was improved by adding 5% glucose, 5% Beck’s salt mixture, and olive oil, linseed oil, or wheat germ oil. None of these additives promoted egg laying by the females. Addition of 10% by weight (of all dry components) of ground, freeze-dried leaves of the host plant Phyla nodiflora to PBD increased adult production up to 90% for newly hatched larvae started on the diet. Adult females laid eggs, indicating that some component or components in the host plant are necessary for reproduction. Extraction of the host plant leaves with polar and nonpolar solvents and adding them to PBD failed to improve the production of adults and failed to enable females to lay eggs. Gas chromatographic analysis of fatty acids in the body of larvae and adults reared on PBD showed low levels of linolenic acid (18:3). Insects reared on any one of the oils noted above had high levels of 18:3 fatty acids. Sterols in the host plant were tentatively identified by gas chromatography as campesterol, stigmasterol, and (3-sitosterol, and

these were added as synthetic compounds to the artificial diet in the approximate concentration found in the host plant. No improvement in adult production was found,

indicating that sterol is not a limiting factor in the artificial diet. The ovary and eggs in the ovary of females reared on PBD seem to be normal, but the male system appears to be abnormal. Adults reared on PBD do not appear to mate, and the reason may be that the

male reproductive system is not normal. CHAPTER 1 NUTRITIONAL ECOLOGY AND GENERAL PRINCIPLES OF ARTIFICIAL DIETS FOR INSECTS

Introduction

The principles underlying the major insect nutritional requirements for growth and reproduction have been established over the past 50 years through studies on representatives of the major insect groups (Dadd, 1973; Davis, 1968; House, 1965;

House, 1974; Nation, 2002; Simpson and Raubenheimer, 1995; Slansky and Scrieber

1985). Nevertheless, at most a few dozen insects have been studied critically to leam their nutritional requirements. Unusual nutritional requirements were found in a few insects from even these few studies, so surprises are likely. Nutrition concerns the

chemicals required by an organism for its growth, tissue maintenance, reproduction and energy (Chapman, 1998). Although insects generally have the same nutritional

requirements as large animals, the balance of nutrients is very important to most insects studied (House, 1965; Dadd, 1985). Balance may be important because of small body size and the stress placed on several physiological systems by having to deal with excesses while attempting to capture suboptimal levels of some nutrients.

Insects need a dietary source of arginine, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, trythophan, and valine, the same essential amino

acids needed by large animals. Carbohydrate is a major source of energy for most,

perhaps all, insects. Although some insects are known to have an absolute requirement

for carbohydrate in the diet, most do not have specific carbohydrate requirements. For all

1 2

insects, carbohydrate is an important fuel source. Some insect groups, such as

Lepidoptera, Orthoptera, and some Homoptera, use both carbohydrates and lipids to supply the energy for flight, but most insects can only utilize carbohydrate to support flight. A very few insects use carbohydrate and the amino acid proline to support flight.

A dietary source of polyunsaturated fatty acids and sterol are important to many insects. Lepidoptera and some other groups require a dietary source of the polyunsaturated fatty acids linoleic and linolenic acids (Fraenkel and Blewett, 1946).

Moths deficient in these fatty acids have defects in wing formation and their scales adhere to the pupal case on emergence (Vanderzant, 1974). All insects are believed to require a sterol in the diet because they cannot synthesize the sterol rings, or cannot synthesize enough to meet their physiological requirements. In addition to being a part of

all cellular membranes, a sterol is the precursor for synthesizing the 27-carbon ecdysteroid molting hormone of insects. Thus, deficiency of a sterol in the diet results in inability of the insects to molt and they typically die in an early instar. Most insects that have been studied can utilize cholesterol, the typical 27-carbon sterol of animal tissues.

Phytophagous insects typically convert the 28- and 29-carbon plant sterols into

cholesterol (Feldlaufer et al. 1995; Svoboda et al. 1975), but a few species synthesize a

28- or 29-carbon ecdysteroid from a dietary sterol acquired in their feeding. Even trace amounts of lipids or sterols in a diet may affect positively growth and development

(Vanderzant, 1974).

Typically, insects require the vitamins thiamine, riboflavin, nicotinic acid, pyridoxine, pantothenic acid, folic acid, and biotin. Some insects, notably Musca 3

domestica and Manduca sexta, require dietary vitamin A or P-carotene for normal morphology of the compound eyes and for most acute vision.

The mineral requirements of insects are poorly known. Given the known

composition of insects, it is reasonable to assume that sodium, potassium, calcium, magnesium, chloride and phosphate are essential minerals for the function of insects

(Nation, 2002). Trace amounts of other minerals may be necessary, but critical studies on mineral requirements are lacking.

Development of artificial or synthetic diets for insects was stimulated by the need to understand comparative nutritional requirements, as well as to enable rearing in the laboratory on foods more convenient than their natural food. Vanderzant (1974) defined

an artificial diet as one that is not the natural food of the insect (Vanderzant, 1974).

Artificial diets are useful in mass production of insect predators and parasites for

Integrated Pest Management (IPM) control programs, and for producing large numbers of insects for sterile release programs (Nation, 2002). Although basic nutritional needs of

insects are well understood, developing suitable diets for specific insects can still be quite

difficult.

Chemical attractants or phagostimulants and chemical deterrents play a role in the acceptance or rejection of potential foods by insects. Taste receptors on the mouthparts, and sometimes on the tarsi or other body parts, enable insects to taste and identify potential foods. Phagostimulants may be nutritional components or nonnutritional allelochemicals. Most food chemical components stimulate feeding in one insect or

another, but if a particular chemical cue that an insect needs is lacking, the insect may reject an otherwise nutritionally adequate food. Some insects feed almost continuously, 4

while others cease to feed when satiated, even in the presence of feeding stimuli. Hexose sugars and sucrose are primary nutrients and phagostimulants for many leaf-eating insects (Chapman, 1998). Glucosides, a combination of glucose with a non-sugar moiety, serve as phagostimulants for some insects. For example, Pieris larvae that feed on plants in the Brassicaceae family are stimulated to feed by mustard oil glucosides (David and

Gardiner, 1966; Dethier, 1980; Renwick and Huang, 1995). In many cases the phagostimulant is not a nutrient, and may even be a defensive chemical evolved by the plant to reduce general herbivory, such as cardenolides in milkweed plants (Brower,

1972; Brower et al. 1984), cucurbitacins in cucurbits, mulberin in mulberry leaves (Ito,

1960) and iridoid glycosides (Bowers, 1983; Bowers, 1984) in many plants.

Measurement of Nutritional Quality

Nutritional quality of artificial diets has been determined by measuring growth, weight gains, time between molts, time to pupation and number of adults produced

(Dadd, 1985; House, 1965; House, 1974; Schoonhoven, 1972; Simpson and

Raubenheimer, 1995; Slansky, 1982, Slansky and Scriber, 1985; Reinecke, 1985;

Lazarevic et al. 2002; Vanderzant, 1974; Waldbauer and Fredman, 1991).

Currently, many moths, beetles, crickets, grasshoppers, and other insects can be reared on an artificial or semiartificial diet. However, only 2 or 3 butterfly species can be

reared on an artificial diet. The cabbageworm butterfly ( Pieris rapae ) (Webb and

Shelton, 1988) and the painted lady butterfly ( Vanessa carclui) have been reared on

artificial diets. The painted lady diet is commercially available (Carolina Biological

Supply Co., 2700 York Road, Burlington, NC; or Bioserv, Entomology Division, One 8th

St., Suite 1, Frenchtown, NJ 08825). Diets that contain host-plant material have been

published for rearing the Monarch butterfly, but success with Monarch rearing is difficult 5

and uncertain. Moths and butterflies are closely related and belong to the order

Lepidoptera. Thus, it might seem strange that so many moths can be reared and so few butterflies. One possible explanation may be that the moths that can be reared are often economic pests whose larvae feed on multiple plant species, whereas butterflies tend to be restricted to one or only a few host plants as larval food plants. This may mean that butterflies are more dependent on the balance of nutrients and/or presence of specific feeding cues in their host plants.

The main objective of this study is to develop an artificial diet for rearing the phaon

crescent, Phyciodes phaon. Since little has been published about the detailed life history and biology of the phaon crescent, secondary objectives aimed at providing more detail on the biology and developments of the butterfly are as follows:

• Describe number and appearance of the immature stages, and life history and biology of P. phaon.

• Test several commercially available diets for moths and butterflies to determine their suitability for rearing P. phaon.

• Determine the possibility of improving an existing diet formula by addition of relatively inexpensive nutritional components that may be particularly important to the phaon crescent.

• Measure and compute of standard indices of food consumption and its conversion to body components by phaon crescent larvae.

• Identify sterols in the host plant Phyla nodiflora, and determine the effect of adding them to the diet of the larvae.

• Describe female and male internal reproduction systems of adult P. phaon reared on the host plant and on artificial diets. CHAPTER 2 LIFE HISTORY AND BIOLOGY OF Phyciodes phaon (EDWARDS) ON Phyla (Lippia) nodiflora

Introduction

The butterfly genus Phyciodes Huebner is restricted to the Americas, and many species of this genus are tropical. There are 12 known species in the United States:

Phyciodes texana (Texas crescent), P. frisia (Cuban crescent), P. protlyca (Black crescent), P. vesta (Vesta crescent), P. phaon (Phaon crescent), P. tharos (Pearl crescent),

P. cocyta (Northern crescent), P. batesii (Tawny crescent), P. campestris (Field crescent),

P. pictus (Painted crescent), P. orseis (Orseis crescent), P. pallida (Pallid crescent), and

P. mylitta (Mylitta crescent). The wing shape and color pattern are diverse and cannot be used to characterize the genus (Howe, 1975). The species can be determined on the basis of the male genitalia (Howe, 1975). According to Scott (1994), there are three groups in this genus: the tharos species-group, the mylitta species-group, and the phaon species- group. There are two seasonal forms of most species: the marcia-spring form and morpheus-summer form (Scott, 1994).

Phyciodes phaon belongs to the phaon species group, a group characterized by strongly contrasting coloring of both surfaces of the forewing. Phyciodes pictus Edwards and Phyciodes campestris camillus Edwards also have similar contrasting coloring on the

underside of the forewing. Phyciodes phaon resembles the pearl crescent (P . tharos ) in the distribution of the wing coloring, but differs by having more checkered spots on the

wings (Emmel and Kenny, 1997). The phaon crescent is distributed from coastal North

6 7

Carolina throughout the southern parts of the Gulf States to southern Texas and westward

to Southern California. It emigrates north to Iowa and Nebraska. Emmel and Kenny,

(1997 p.30) described the origin of “phaon” from Greek mythology; “phaon was an old man who ferried the goddess of love, Aphrodite, to her destination. In return for his services, she gave him youth and beauty.”

The only known host plants of P. phaon. Phyla (Lippia ) nodiflora and P.

lanceolate, belong to the Verbenaceae (Riley, 1975). Little is known about P. phaon

biology and its evolution and why this particular species evolved in association with the

Verbenaceae family. The phaon crescent is the only species in the genus that utilizes the

Verbenaceae family as a larval host plant. All other species in the genus use larval food plants belonging to the families Acanthaceae and Asteraceae. Justicia ovata, which

belongs in the family Acanthaceae, is another larval host plant of P. phaon (Wahlberg,

2000). Riley (1975) associated Phyciodes phaon with Wedelia trilobata, creeping daisy, but did not specifically determine that the larvae could use this as a host.

The larval host plant. Phyla nodiflora, is widely distributed in the southern United

States. It is a perennial herb with long creeping stems and small whitish, yellowish or

reddish flowers (Figure 2-1). It roots readily at the nodes and gives rise to a new plant. It has slender purplish stems 20-45 cm long covered with hairs. Leaves are opposite, wedge shaped, thick, leather-like, and finely serrated along the edges but rounded at the

tip. It prefers moist areas, and characteristically grows near rivers, roadsides and other disturbed areas, showing a strong preference for wet, grassy areas. In the literature,

Verbena nodiflora L. and Lippia nodiflora (L.) Michx are two common synonyms for P.

nodiflora (Verdcourt, 1992). Phyla nodiflora is also known as fog fruit, matchweed, 8

capeweed, creeping charlie and match heads (Verdcourt, 1992). The common buckeye,

Junonia coenia, the white peacock, Anartia jatrophae, and the sphinx moth, Manduca rustica also use Phyla nodiflora as a larval host (Minno and Minno, 1999).

The aim of this chapter is to describe the life history, biology and immature stages of Phyciodes phaon and determine the external sex characters of P. phaon pupae.

Materials and Methods

During the summer of 1999, P. phaon adults (N^O) were collected alive in the vicinity of Gainesville, Florida. Eggs were obtained from these adults by placing them in a screen cage with a potted host plant. Phyla nodiflora. Eggs were removed daily, counted, and kept in a Petri dish on moist filter paper. Larvae were fed on freshly cut host-plant material collected from the University of Florida campus and vicinity. Larval food was changed every other day by transferring all larvae to new plants. Pupae were harvested daily, and transferred to a new cage with a potted host plant. Adults were

allowed to feed ad libitum from a 10% honey solution or Fruit Punch Gatorade . The colony was maintained under controlled laboratory conditions at 27°C, 16:8 (light:dark) hours photoperiod with multiple fluorescent lights. Small plastic Dixie cups having lids with screen were used to rear larvae individually to determine instars. Larval head capsule, weight and length were measured daily for an experimental group to determine the number of instars. Larval food in the cup was changed daily. Shed larval head capsules from each cup were collected and preserved in 70% alcohol.

Pupae were sexed on the basis of external sex characteristics on the ventrally located genital plates. In certain experiments, sexed pupae were held in individual cups. 9

and after they emerged, each adult was checked to confirm the sex designation. Eggs, all instars, pupae, and adults were photographed and preserved.

Results

Description of Phyciodes phaon Immature and Adult Stages

Eggs

The eggs are nearly spherical, about 0.63 ± 0.03 mm in length and 0.36 ± 0.01 mm in diameter (N=25 eggs) with a flattened base and slight depression at the micropyle

(Figure 2-2). They are adorned by 18-20 vertical ridges. Micrographs of Phyciodes phaon egg clusters, micropylar (dorsal view), egg surface under high magnification and the pores on the lateral surface of the egg are shown in Figure 2-3.

The eggs are light-green when laid, and this appearance persists for about 4 days if

they are fertile. About 5% of eggs were infertile in one count that I made. Females may lay an entire cluster of infertile (sterile) eggs if they are not mated. Although infertile eggs are similar to the fertile eggs in appearance when laid, within several days they shrink. Eggs typically are laid in a cluster on the undersides of the host plant leaves

(Figure 2-2). The number of eggs in a cluster varies, averaging 78 eggs, but rarely there are as few as 5 or as many as 187 per cluster. Usually, eggs are laid on top of each other in a cluster but sometimes they are laid in a line next to the main leaf vein. Development takes 5.1 ± 0.3 days at 27°C, and the color of the egg changes from light green to brownish black as development proceeds (Figure 2-2). Just before hatching, the mandibles and head become visible through the chorion. 10

(Verbanecaeae)

nodiflora

Phyla

plant.

host

larval

butterfly

crescent

Phaon

2-1.

Figure 11

Larvae

First instars. The first instar is olive green to olive brown with long setae over the whole body. (Scott, 1994). The head is cream colored with two large brown to black patches. The legs and prolegs are light brown and tarsal segments are black to brown.

The anal proleg is dark brown. The antennae are cream and the basal area is brown. The labrum is brown. The labial and maxillary palpi are light cream in color. The facial

suture margins are darkened. Ocelli are black. Head capsule setae are numerous and all oriented anteriorly. Brown and cream spots are randomly distributed on the integument

(Figure 2-4A-B).

The first instars eat their eggshells and stay together, spinning some silk web on the leaf. Generally larvae rest on top of the silk web, but sometimes larvae may rest and feed

beneath part of it. They eat small amounts of the undersides of the leaf and stick their

heads into these pits to feed on the internal leaf tissue. The first instar lasts for 3.6 ± 0.8 days (N=25).

Second instars. The second instar is light brown in color, having dark subdorsal

bands (Figure 2-4C). There is a row of short, branching small spines across each

segment of the body. The head is black with two long cream dorsal stripes extending posterior to the neck. The mouth parts are dark brown. The head capsule setae are more

numerous than in the first instar. The integument is textured with brown, dark brown and cream spots. The longitudinal, dorsal and subdorsal bands are more evident in the second instar than they were in first instar. The thoracic legs are light brown or cream in color with the tarsal claws darkened. The spiracles are brown. Second instars continue to feed on the underside of the leaves. The instar lasts 3.8 ± 0.8 days (N=25). 12 13

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Fifth

instar.

Fourth

instars.

Third

instars.

Second

instar.

First

instars.

first

emerged

Newly

Instars: instar.

2-4.

Figure .

Third instars. The third instar is similar in appearance to the second instar (Figure

2-4D). The cream stripes are more visible on the head. The head capsule is similar in appearance to that of the second instar, except that the cream patches are more evident.

Third instars generally feed solely on the upper sides of the leaves. They no longer aggregate, but distribute themselves over the whole plant and appear to feed continuously. They spend 4.1 ± 0.8 days in the third instar (N=25).

Fourth instars. The appearance of the fourth instar is similar to the third instar

(Figure 2-4E). Fourth instars consume a large quantity of host leaves. Larvae stay in the fourth instar 4.3 ± 0.8 days (N=25).

Fifth instars. The color and the appearance of the fifth instars is the same as that

of fourth instars (Figure 2-4F). Fifth instars eat the leaf by eating at the edge of the leaf.

The dorsal dark stripes are mottled; it is about 50% white and 50% brown. The duration of this instar is 3.9 ± 0.8 days (N=25). Larval head capsule measurements, weight and lengths are shown in Table 2-1

Prepupae. The mature larva attaches with the cremaster to a stem or leaf and

remains in a crescent shape about 8-10 hours. It then hangs straight down and changes within 2-3 minutes into the characteristic pupal appearance (Figure 2-5).

Pupae. Initially, the pupa is very soft and has the same color of mature larva; soon

it sclerotizes the cuticle and its color becomes light tan speckled with black and white

(Figure 2-6). It has dark and paler areas over the wings. It also has a brown “U-shaped” mark around the front of the head. 16

The pupa measures 12.2 ± 0.09 mm in length and 5.75 ± 0.06 mm in width

(dorsoventrally in the thoracic region) and weighs an average 82 ± 40 mg (N=25). The

pupal stage lasts for 4.6 ± 0.8 days.

Table 2-1. The measurements of head capsule, weight, and length of Phyciodes phaon in each instar (n=25)

Instars Head Capsule Weight (mg) Length (cm) Measurement (mm)

First 0.259 ± 0.02 6 ± 1 0.210 ±0.03

Second 0.578 ± 0.03 24 ±6 0.678 ± 0.02

Third 0.756 ±0.05 40 ± 1 1.25 ±0.06

Fourth 1.26 ±0.07 83 ±6 2.03 ± 0.02

Fifth 1.93 ±0.11 130 ± 3 2.97 ±0.08

The pupal abdomen consists of 10 segments. Segments 1-8 each have a pair of spiracles.

Segments 1-4 and 7-10 are immovably attached, but segments 4-7 are movable. The

genital aperture is located ventrally on segments 8 and 9. In the females, there are

separate apertures for the bursa copulatrix and the oviduct. In the males, there is a single

aperture on segment 9. In both females and males, the anal opening is located on segment 10. Segment 10 also contains the cremaster.

Pupae of the phaon crescent can be sexed by observation of the morphology around the genital plate. The genital plate covers the mid-ventral region of the entire eighth and the anterior margin of the ninth abdominal segments. 17

leaf.

the

cremaster

the

with

attaches

larvae

mature

The

prepupae.

The

2-5.

Figure 18

view.

Lateral

view.

Ventral

pupa.

view

Dorsal

A-D)

variations.

color

Pupa

2-6.

Figure 19

Female pupae (Figure 2-7, Figure 2-9A) have a longitudinally slotted mid-ventral pad between two “nipple-like” structures. The slot extends forward across the eight segment to the posterior margin of the seventh abdominal segment. This slotted pad seems to be homologous with the distal ends of the female genital ducts in the adult. Male pupae

(Figure 2-8 and Figure 2-9B) lack this vertically slotted pad, but have two small paired swellings located between the nipples at the ninth abdominal segment. External sexing of pupae was accomplished with 100% accuracy, as shown in Table 2-2.

Table 2-2. Externally sexing Phyciodes phaon pupae.

No of Pupae No of Adults Female Male Total Female Male Total Phyciodes phaon 32 26 58 32 26 58

Adults.

Adtilts of both sexes are similar in appearance, but females are a little larger than the males, and the female abdomen is noticeably wider than that of males (Figure 2-11). The wingspan of females is 30.7 ± 0.02 mm and 23.4 ± 0.01 mm in males (N=25). Both sexes show seasonal forms in their natural environment (Opler and Krizek, 1984). The spring (sometimes fall) form called “Marcia” has more dark lines and brown areas and more cream markings instead of white. The summer form “Morpheus” has less orange coloration on the upper hindwing and more orange on the upperside (Opler and Krizek,

1984). P. phaon is distinguished from other Phyciodes species by having a creamy

yellow band across the middle of the forewing. This band is clearly evident on both sides of the wings. The undersides of the hindwing have white with brown markings. 20 21

up.

closes

pupa

Male

pupa.

Male

pupae.

phaon

Phyciodes

Micrographs

2-8.

Figure 22

pupa.

Male

pupa.

Female

pupae.

phaon

Phyciodes

determination

Sex

2-9.

Figure 23

'• V > ‘

case.

pupal

adult’s

emerged

newly

The

2-10.

Figure 24

Food Plant and Life History

In the vicinity of Gainesville, FL, Phyciodes phaon uses Phyla nodiflora

(Verbeneceae) as a larval host. The summary of the life cycle of P. phaon is shown in

Figure 2-12. I have observed feral females ovipositing on these plants in nature, but I

have only found first instars on P. nodiflora in nature. Even though P. nodiflora is widely distributed in Gainesville, and in mid- to late summer the butterflies are common, mortality of P. phaon larvae appears to be high (95%) (Pyle, 1998). The majority are killed by parasitic flies, wasps, and other predators such as ants. Predatory mites feed on the eggs (Pyle, 1998). Figure 2-13 shows the survival of laboratory reared P. phaon.

The first instars suffer the highest mortality. They drop from the plant when disturbed,

and die if they do not find the host again. In the laboratory, survival of older larvae is good. Generally all fifth instars successfully pupate in the laboratory and emerge as an

adult. Mortality in the laboratory is low in the pupal stage. Newly emerged adult females have well developed ovaries with eggs that appear ready to be laid. Mating often occurs within a day after emergence and a mating pair may stay together 4-5 hours.

Males emerge before females in the laboratory. Mated females start laying eggs within 2 days following mating. A single female can lay from 200-250 eggs (N=25). Adults

survive in the laboratory about 2 weeks. The duration of one generation takes 23-3 1 days at 27°C, 16 h light and 8 h dark photoperiod in the laboratory. Adults of the phaon crescent typically occur all months of the year in the Gainesville area. Adults fly low to

the ground and moderately fast.

Discussion and Conclusions

Studies on the life history of Phyciodes phaon provide tools for identification of immature stages and for identification of the sex in the pupal stage. There are five instars 25

based on head capsule measurements. The external characteristics of each instar are described.

Little information is available regarding accurate methods for sexing pupae of

butterflies. It is well known that larger pupae typically develop into females, whereas the smaller ones generally turn out to be males. In addition to that, the majority of males usually eclose at the beginning of a brood, and females emerge later. However, such

methods are not very accurate. Sexing pupae is quite important, especially when virgin males and females are needed in a study. Therefore, accurate methods for sexing pupae are desirable.

In this study, sexes can be readily determined by observation of certain external characters of pupae. The egg-adult developmental time on the natural food plant. Phyla

nocliflora, is about 26 days under constant laboratory conditions. This is an easy butterfly to rear in the laboratory; the sexes mate readily in the laboratory in small containers or

cages, and it has relatively low mortality after the first instar. I have not seen any pathogen or disease associated with the phaon crescent during three years of continuous

rearing. The host plant is widely available, and requires minimal care to keep in small pots or in outdoor plots. These characteristics of butterfly and host plant make the phaon

crescent a valuable model butterfly for further research. It is also potentially useful as a teaching tool in schools and a convenient display butterfly for butterfly houses.

According to Riley (1975), P. phaon is associated with Wedelia trilobata (creeping daisy). In the laboratory, different instars were tested and none fed on W. trilobata. The butterfly may associate with the flowers of the W. trilobata, but this plant cannot be used as a larval host plant of Phyciodes phaon. 26

phaon.

P. of

side

Ventral

phaon.

P. of

side

Dorsal

female.

phaon

Phyciode

2-11.

Figure 27

2-3days

phaon.

Phyciodes

days of

5-6

summary

cycle

Life

2-12.

Figure 28

instars

first

250

with

started

experiment

The

nodiflora.

Phyla

colony

phaon

Phyciodes

reared oO o o replications. co eg

laboratory sjoAj/uns jo jeqiunN

three of of

Survival each

2-13.

Figure 29

An egg cluster of P. phaon was found on the underside of white clover leaves in the laboratory when the clover was growing in a pot of Phyla nodiflora. First instars from the egg cluster laid on the clover began feeding on the clover leaves. Freshly cut clover leaves were provided as needed and the larvae eventually pupated after a longer larval development time. Mature larvae and the pupae were smaller than those ones reared on

Phyla. The adults that emerged were smaller in size and survived in the laboratory only 3 days. CHAPTER 3 NUTRITIONAL ECOLOGY AND REQUIREMENTS OF AN ARTIFICIAL DIET FOR THE BUTTERFLY Phyciodes phaon

Introduction

The phaon crescent Phyciodes phaon is a small butterfly present over much of the

southeastern United States. Its larval food plants are two species of plants in the genus

Phyla, P. nodiflora and P. lanceolata in the family Verbenaceae (Minno, 1999; Emmel

and Kenny, 1997). Wahlberg (2000) also reported Justicia ovata in the family

Acanthaceae as a larval host plant. Phyla nodiflora is widely distributed and present year round in the Gainesville, FL area, and appears to be the host plant for feral populations.

The availability of the host plant, the lack of a diapause in the butterfly, and the ease with

which it can be cultured in the laboratory on the host plant make it a good candidate for

studies to develop an artificial diet on which it can be reared. An additional positive feature is that adults mate readily in the laboratory in small cages; some butterflies (for example, checkered white Pieris protodice adults) do not mate under laboratory conditions, but mate when placed in large screened cages in the natural environment.

The goals in this chapter are to describe (1) experimental tests of several of the commercial diets for moths and butterflies in order to determine their suitability for rearing Phyciodes phaon, the phaon crescent butterfly, (2) modifications of the best of the tested diets to try to improve growth and survivability, and (3) addition of solvent extracts of the host plant to an artificial diet in an attempt to determine what components are critical to the butterfly.

30 31

Materials and Methods

Study Organism: Butterfly and Host Plant

A colony of Phyciodes phaon was started from females collected on the Unij/ersity

of Florida campus and vicinity. The host plant Phyla nodiflora was either collected from

natural areas as needed or maintained in small plots or planted in containers. Leaves of

the host plant were frozen in liquid nitrogen, ground while frozen in a mortar, and freeze-

dried. The freeze-dried host plant material was stored at -20°C until needed; additional

freeze-dried host plant leaves were prepared as needed from time to time.

The butterfly was reared for several generations on potted or freshly cut host plants

in order to become more familiar with its biology and development time. In later

experiments, newly hatched first instars were placed on diets and monitored daily for

feeding, growth and development to the adult stage, or until they died if the diet was not

suitable. In all experiments, adults were kept in cages with a pot of the host plant and provided with 10% honey in water. Leaves of the host plant were checked daily for eggs.

Usually eggs were left on the host plant until larvae hatched because egg survival was found to be much better when left on the plant than when the leaf with eggs was removed

and kept in a petri dish. Nevertheless, eggs could be kept in petri dishes on moist filter paper if care was taken to prevent the paper from drying. A breeding colony of the butterfly was maintained continuously on potted host plants in the laboratory.

Commercial and Published Diets Tested

Rather than attempting to formulate a diet from individual components, it seemed wise to determine if any of the several available diets for Lepidoptera might work or be modified to work for rearing the phaon crescent. The commercial diets tested were the

following: A gypsy moth ( Lymantria dispar) diet, the painted lady butterfly ( Vanessia 32

cardui) diet, the monarch butterfly (Danaus plexippus) diet, the large cabbage white

butterfly ( Pieris brassicae ) diet, a “general purpose” diet, and a pinto bean diet. The pinto

bean diet was developed to rear the moths Trichoplusia ni and Spodoptera spp. (Guy et

al. 1985).

All diet mixes (and components for mixing the pinto bean diet) were obtained from

Carolina Biological Supply, NJ or Bioserv. Directions for preparing the diets also were

supplied by the manufacturer. All except the pinto bean diet are agar-based, and in

preparation a weighed quantity of agar was dissolved in boiling water and heated at

boiling for about 5 minutes. After brief cooling, but before the agar gelled, the dry diet

mixture was stirred in and thoroughly blended for 1 minute. The pinto bean diet uses

gelcarin as a thickening and gelling agent instead of agar and it is only necessary to heat

the mixture of dry components, gelcarin and water to 70 °C for 15-20 minutes. Heat

labile vitamins were added with mixing after the pinto bean diet mixture had cooled

briefly. Upon cooling, the gel formed in the pinto bean diet is not as hard as is typical of agar-based gels.

Preparation of the Pinto Bean Diet from Components

Since the pinto bean diet is the only diet tested that had to be mixed from individual components, the procedure followed is given in some detail. The weight or volume of components in Table 2.5 was sufficient to make 250 mL of diet, the typical amount of diet made at one time. Pinto bean meal, wheat germ, torula yeast, casein, gelcarin, methyl paraben, and sorbic acid were added to 182 mL cold water with stirring by a mechanical mixer. The mixture was heated slowly on a hot plate to 70°C with continuous stirring and then the formaldehyde was added and mixing without further heat was continued for about 3 minutes. Secondly, ascorbic acid was added and mixing continued 33

an additional 3 minutes. Thirdly, tetracycline, the vitamin mixture (400 g/1000 mL

water), and propionic acid were added with additional mixing for 2-3 minutes. When

10% by weight of freeze-dried plant material (actual weight of freeze-dried host plant

material was 5.1 g) was added, it was added at the last with the vitamin mix to avoid heat

damage to the host plant material. The diet was poured into 175 mL cups and kept in a

refrigerator until used.

Preparation of the Host Plant for Addition to Diets

Leaves of the host plant, Phyla nodiflora, were frozen in liquid nitrogen, ground in

a mortar, and freeze-dried. Ground, freeze-dried leaves of the host plant equal to 10% of

the dry diet mixture (actual weight = 5.1 g) were added with the dry diet mixture in the

gypsy moth, general purpose and pinto bean diets. All mixed diets were kept in a

refrigerator and small portions taken as needed for feeding larvae.

Analyses of Diets and Diet Components

Although some of these diets are proprietary formulations, limited data supplied by

BioServ on composition are given in Table 3-1 for the commercial diets and in Table 3-2

for the pinto bean diet. Nutrient analysis of some diet components such as ground wheat germ, torula yeast, Beck’s salt mixture and Vitamix premix results have been provided by

Bioserv Company (Table 3-2).

Food Consumption and Utilization

The consumption index measures the mean weight of an insect during the

feeding period as a function of the food eaten. The gain in weight of an insect is obtained by direct weighing at the beginning and at the end of the feeding period. 34

Table 3-1. The list of several commercially available artificial diets and their ingredients. All listed diets were used in this study. All diet mixtures are available from Bio-Serv Company.

Commercially available diets Ingredients*

Gypsy Moth (Lymantria clispar) Agar, wheat germ, cellulose, cholesterol, vitamin mix, inositol, choline chloride, dextrose, Wesson salt mix and casein

Painted Lady ( Vanessa cardui) Soy flour, wheat germ, brewer’s yeast, sucrose. Wesson salt mix, ascorbic acid, potassium sorbate, methyl paraben, aureomycin, choline chloride, potassium hydroxide, and acetic acid.

Monarch (Danaus plexippus) Whole wheat, soy flour, cholesterol, cellulose, Wesson salt mix, methyl paraben, sorbic acid, vitamin mix, aureomycin, choline chloride, safflower

oil, soybean oil, dried ground milkweed.

Imported Large Cabbage Butterfly Raw or stabilized wheat germ, high (Pieris brassicae)** nitrogen casein, Wesson salt mixture, vitamin premix, sorbic acid, agar, methyl paraben.

General Purpose Insect Diet Composition not available from the company

* Volume or amounts are not available from the Bio-Serv Company. ** Diet was published by Webb, 1988, and also available from Bio-Serv Company. 35

Table 3-2. Components of the pinto bean diet.

Ingredients Amount or Volume Raw pinto bean meal 19 g

Ground wheat germ 14 g Torula yeast 8g Casein 7g Gelcarin 3g

Methyl paraben 0.5 g

Sorbic acid 0.3 g

Distilled water 182 mL

Formaldehyde 1 mL

Ascorbic acid 0.9 g

Tetracycline capsules 0.01 g (250 mg/per capsule)

Vitamin mixture 2 mL

Propionic acid 0.3 mL

Small insects are usually weighed in groups and an average calculated. The average of

daily weights gives almost identical value when the growth curve is smooth (Waldbauer,

1964). The utilization of food for growth is measured by the efficiency of conversion of

ingested food to body substance (E.C.I.). The increase or decrease of E.C.I. value

depends upon the digestibility of a food (A.D) and the efficiency of conversion of

digested food to body substance (E.C.D.). The classical standard gravimetric method was used to measure the utilization of food (Waldbauer, 1964 and 1968).

The larval weight gain was calculated by subtracting its weight at the beginning

of a feeding period from its weight at the end of the feeding. The amount of ingested

food was determined by subtracting the weight of uneaten food from the weight of the 36

Table 3-3. Typical analysis of ground wheat germ (Bio-Serv Product #G1659).

Minerals and Vitamins Amino Acid Profile per kg % Calcium 0.50 gm Lysine 1.54 Copper 10.00 mg Tryptophane 0.3 Iron 51.00 mg Phenylalanine 0.94 Magnesium 2.50 gm Methionine 0.43 Manganese 134.0 mg Threonine 0.97 Phosphorus 9.2 gm Leucine 1.54 Potassium 9.7 gm Isoleucine 0.90 Sodium 0.30 gm Valine 1.17

Zinc 1 19.00 mg Arginine 1.87 Iodine n/a Histidine 0.65 Tyrosine 0.73 Pantothenic acid 20.10 mg Cysteine 0.48 Choline 3062 mg Serine 1.13 Folic acid 2.20 mg Glutamic acid n/a Inositol n/a Aspartic acid n/a Niacin 72.0 mg Glycine 1.47

Pyridoxine 1 1.40 mg Alanine n/a Thiamine 22.8 mg Proline n/a Riboflavin 6.00 mg Vitamin B12 n/a

* n/a: not available

Chemical Characteristics Protein (N x 6.25) 25% Fat 7% Moisture 13.7% Fiber 6% Mesh Size 30 US Standard Screen 37

Table 3-4. Typical analysis of torula yeast (Bio-Serv Product #G1720).

Minerals and Vitamins Amino Acid Profde per kg % Calcium 3.00 gm Lysine 8.70 Copper 2.90 pg Tryptophan 1.10 Iron 200.00 mg Phenylalanine 5.50 Magnesium 1.50 gm Methionine 0.60 Manganese 0.02 mg Threonine 5.40 Phosphorus 13 gm Leucine 8.70 Potassium 20 gm Isoleucine 5.60 Sodium 0.30 gm Valine 6.50 Zinc 120.00 mg Arginine 5.90 Iodine 1-00 pg Histidine 2.30 Tyrosine 3.50 Pantothenic acid 75.00 mg Cysteine 0.40 Choline 2.50 mg Serine 5.10 Folic Acid 2.00 mg Glutamic acid 14.80 Inositol 4.70 gm Aspartic acid 10.30 Niacin 0.45 gm Glycine 5.10 Pyridoxine 43.00 mg Alanine 6.60 Thiamine 0.15 gm Proline 3.90 Riboflavin 50.00 mg Vitamin B12 5.00 pg

Chemical Characteristics Protein (N x 6.38) 45 % Fat 4 % Moisture 7 % Carbohydrate 44.5% Mesh Size 30 US Standard Screen 38

Table 3-5. Typical analysis of Beck’s salt mixture (BIO-SERV Product #F8537).

Beck’s Salt Mixture Components Amount (g/kg)

Calcium 13.88 Chlorine 30.33 Chromium 0.00 Copper 0.995 Fluorine 0.00 Iodine 0.00 Iron 7.35 Magnesium 32.32 Manganese 1.82 Phosphorus 150.65 Potassium 274.81 Selenium 0.00 Sodium 19.67 Sulfur 48.38 Zinc 1.78

Table 3-6. Typical analysis of Vitamin Mixture (BIO-SERV Product #6265)

Ingredients Units/lb

Vitamin A 10,000,000 IU

Vitamin E 3,636 IU

Vitamin B12 0.91 mg

Riboflavin 227 mg

Niacinamide 460 mg

D-Pantothenic Acid 418 mg

Choline 19,727 mg

Folic Acid 1 14 mg

Pyridoxine 93 mg

Thiamine 101 mg

D-Biotin 9.1 mg

Inositol 9,091 mg 39

food provided. All of the fecal pellets were separated carefully from the uneaten food

and weighed. Newly molted fifth instars (the last instar) were used to determine food

utilization. Relative growth rate (R.G.R.), consumption index (C.I.), assimilation

efficiency (A.D.), gross growth efficiency or efficiency of conversion of ingested food

(E.C.I.) and net growth efficiency or efficiency of conversion of digested food (E.C.D.)

were calculated according to the formulas listed in Table 3-7 (Waldbauer 1968; Slansky

and Scriber 1985).

Extractions of the Host Plant and Addition of Extracts to the Pinto Bean Diet

Ground, freeze-dried host plant leaves were extracted with polar solvents (water,

methyl alcohol, ethyl alcohol, acetone, chloroform) and with the nonpolar solvents

pentane and hexane. Extracts were concentrated by evaporation with a gentle stream of

compressed nitrogen gas and mixed with dry casein, and then mixed with other dry

components. Sufficient extract was added to the diet to provide extracted substances

from at least 5.1 grams of freeze-dried leaves (5.1 g is equal to 10% of ground host plant

leaves in the diet). Growth, survival, larval development time, number pupating and

adults emerging were monitored daily. Adult males and females were held together and provided with a potted host plant for possible egg laying.

Pentane extraction

Pentane (125 mL) was used to extract 10 g freeze-dried Phyla nodiflora for 15 minutes to remove lipids, sterols, and nonpolar compound. The solution was filtered and the extract was concentrated to 20 mL by evaporating the solvent in a hood in a gentle stream of compressed nitrogen. The entire extract was then added to the dry components.

The host plant residue from the extraction was dried and tested separately by adding to the pinto bean diet. 40

Table 3-7. Procedures for calculating the efficiency of food utilization by insects (Waldbauer, 1964).

- F Consumption index : C.I. Tx A

° The relative growth rate: R.G.R.- TxA

The conversion of ingested food to body matter : E.C.I. = xlOO WFI (W7-W0 Approximate digestibility: oo WFI

WG The conversion of digested food to body matter: E.C.D.- x 100 (WFI - WF)

Where: F=fresh or dry weight of food eaten T=duration of feeding period (days) A=mean fresh or dry weight of insect during the feeding period G=fresh or dry weight gain of insect during feeding period WG=weight gained WF=weight of feces WFI= weight of food ingested.

Hexane extraction

Hexane (175 mL) was used to extract 40 g freeze-dried Phyla. The extract was

filtered, evaporated to 20 mL and added to the dry components of the pinto bean diet

ingredients. The residue was dried and tested also.

Chloroform: methanol (2:1) extraction

Chloroform: methanol (2:1) mixture (125 mL) was used to extract 50 g freeze-dried

Phyla. The solvent was removed and fresh solvent added several times over the period of

a week. All extracts were combined and filtered. A portion of the extract equal to 5.1 g

dried leaves was evaporated to 5 mL and added to the dry components. The residue was 41

dried and tested separately. The rest of the chloroform: methanol (2:1) extract was

fractionated by using 0.5 g silicic acid in a Pasteur pipet to separate carotinoids (yellow

extract) and chlorophylls (green extract) into different fractions (Figure 3-1). Each

fraction and the residue were tested separately in the diet.

Chloroform extraction

Chloroform (800 mL) was used to extract 100 g freeze-dried Phyla. The residue was re-extracted 10 more times with chloroform. The extracts were combined,

evaporated to 20 mL, and 5 mL (equivalent to 20 g dried host leaves) was added to the pinto bean diet. The residue was dried and tested separately by adding to the diet.

Hot ethanol extraction

Hot boiling ethanol (100 mL) was used to extract 12 g freeze-dried Phyla. The residue was re-extracted with 100 mL hot ethanol for another 20 minutes. Extracts were combined and filtered and evaporated to 10 mL and tested. The residue was dried and also tested.

Cold ethanol extraction

Cold ethanol (100 mL) was used to extract 20 g freeze-dried Phyla. The extract was filtered and evaporated to 10 mL and tested. After the cold ethanol residue was

dried, it was also tested.

Methanol extractions

Methanol (200 mL) was used to extract 50 g fresh Phyla by stirring overnight, filtered and evaporated to 5 mL for addition to the dry components of diet. The residue was dried and also tested. The same type of extraction was made with 15 g freeze-dried

Phyla flowers, and this extract was also used in a diet test. 42

Acetone extraction

Acetone (50 mL) was used to extract 20 g freeze-dried Phyla by stirring for an

hour. The extract was filtered, evaporated to 5 mL and then tested. The residue was dried

and tested separately by adding to the diet.

Hot water extract

Hot water (50 mL) was used to extract 20 g freeze-dried Phyla for ten minutes.

The extract was filtered and the entire volume was combined with enough additional water needed to make a diet. The residue was dried and tested separately by addition to

the diet.

Statistical Analyses

Analysis of probability of survival

A comparison of the relative difference in probability of survival was performed with binary logistic regression analysis. The logistic regression analysis models the natural log of the odds of survival as a linear function of treatments, in this case different diets. The number of adults surviving to 35 days on diet j is represented by nij, and the number not surviving to 35 days is represented by nqj. The log of odds is computed as log (nij/noj). Probability of survivors was calculated as P(S), for example; 12 adults

produced out of 50 individuals, P(S) = 12/50. The linear model used in the analysis is thus: 43

Figure 3-1 . The method for fractionation of chloroform: methanol (2:1) extract of Phyla nodiflora on silicic acid columns. A) Chlorophyll fraction. B) Carotinoid fraction. 44

( P(S) ' In = ju + aj+ Sj (1 -P(S) where the model parameters, the overall mean p and treatment effects a, are as defined in regular analysis of variance models. In this model, the means of adult production on each tested diets were transformed from non-linear function to the linear function. The residual terms are assumed to follow a normal distribution. Least square estimates are obtained. Unlike traditional regression, Chi Square tests are used to determine the

statistical significance of the model parameters and an overall Chi Square test is available to assess the hypothesis of no overall treatment (diet) difference. Given a significant overall Chi Square test, a result similar to multiple comparisons testing in ANOVA can be obtained by examining all sub-models that test the hypothesis that the alpha associated with two diets are the same. Finally, (least squares) estimates of the diet specific

probabilities, pj of survival are obtained by inverting the log odds model (Harrell, 2001;

Hosmer and Lemeshow, 2000).

exp (ju + cij) _[ 1 ^(l + exp(// + a).

Chi-square values, along with the multiple comparisons results are presented in Tables to follow.

Comparisons of the food consumption indices

An unpaired t-test was performed to compare the means of food consumption indices of larvae reared on PBD alone and larvae reared on PBD with addition of 10% host plant. The results are presented in Table 3-23. 45

Results

Commercial and Published Diets Available

The results from rearing tests of the phaon crescent on the gypsy moth diet with

10% added freeze-dried host plant material, painted lady diet, monarch butterfly diet,

cabbage butterfly diet, and the general purpose diet with 10% added freeze-dried host

plant material are shown in Table 3-8. One hundred newly emerged P. phaon larvae

were placed on each diet. Overall, growth and survival were low on all of the diets.

Previous data on gypsy moth and general purpose diets (not shown) indicated that

addition of some host plant material into the diets might increase survival. Ground,

freeze-dried host plant leaves equal to 10% by weight of PBD dry components were

added to the gypsy moth and general purpose diets. The monarch diet and the painted

lady diet contain dried plant material (but not the host plant for the phaon crescent).

Adult phaon crescents (2 adults from each diet) were only produced on the two diets containing host plant leaves; no adults were produced on the other diets. The adults were smaller than normal, lived only a few days, and laid no eggs. Development to the adult stage required about 30 days, whereas adults are produced in about 15-17 days when the larvae are reared on the living host plant. Larvae did not survive more than 20 days on the painted lady and monarch diets.

The results from feeding phaon crescent larvae with the pinto bean diet (PBD) developed by Guy et al. (1985) are shown in Table 3-9. Newly hatched larvae (25 larvae

on each of two replicates) were placed on PBD, PBD with 1 0% freeze-dried, ground host plant leaves added, and on a potted living host plant. Larval survival on the PBD diet was moderate, and the larvae completed their larval development in 20 days. A total of

25 adults (12 in one replicate and 13 in the other; 50% successful adults) were produced 46

on the pinto bean diet with no added host plant material. These adults survived in the

laboratory for 10-12 days but never laid eggs. The size and the appearance of the adults were the same as adults reared on the PBD with 10% added host plant material and those reared on a potted living host plant. A total of 41 adults were reared from the two replicates on PBD plus 10% host plant material (23 females and 18 males). The 23 females laid a total of 895 eggs that hatched and the larvae were continued on PBD with

10% host plant. Collection of eggs from adults reared on PBD with 10% host plant material was continued for 4 generations to determine that the results were repeatable.

The females reared on PBD without host plant material did not lay eggs. Nevertheless, that a substantial number of adults could be reared on the pinto bean diet was encouraging. Figure 3-2 shows the color difference between the egg clusters laid by adults reared on living host plants and on PBD with 10% ground, freeze-dried host plant material.

Subsequent approaches to improving the diet occurred along two lines of investigation: (1) addition of components to the pinto bean formula to increase production of adults on the diet, and (2) to try to determine what components are supplied by adding 10% freeze-dried host plant to the diet that enable adults to lay eggs. Although there was no difference in adult appearance, the adults reared on PBD with 10% Phyla laid pale green eggs and those reared on the living host plant laid darker green eggs. *

hatched

3 a. Q* newly 13

Initially,

diets.

° S3 CD I— 60 «

artificial P3 "3 no no oo (N

available

JS so c6 commercially O 3 cn 2 33 o >

3 several 00

on o 32 T3 (D phaon *— c '3 .2 Oh T3 0\ CN

diet. Phyciodes

CD each Q of on ^ s: 1 ^ survival placed 2 c/3

no a £ \o no (N 'O

Growth larvae TD O 0) c c3

on on are wise

and diets pair significantly produced

Phyla the by

not 10% adults

separated indicating are of with

letter are 0.001, number Diet

diets < same the Bean P of the

different df=4 Pinto with

Analysis

the

(PBD), on co estimates

Diet

replicates.

production Chi-Square=32.590, of CO

placed in 4— The CO phaon

were co

P. regression £ adults. i-t of (25) *c3 cx W) time Chi-Square

larvae logistic ’co producing

a >

Survival C— Phyla. highly the

3-9.

Table 49

* c o T3

3 g

V3 cS O

<£ ,t$ JJ £ Q jg® m g ? G u ° to "O 3

+-» »h -g •s §

ri mi H

At the suggestion of supervisory committee member Dr. Charles Guy, I conducted

an experiment with differing levels of host plant material in the diet. This experiment was

expected to reveal whether the need for host plant to enable egg laying could be met by

less than 10% by weight of host plant material, and if the mechanism by which host plant material worked could be saturated by adding more than 10%. The results for 1%, 5%,

10%, and 20% by weight of ground, freeze-dried host plant material incorporated into the pinto bean diet are shown in Table 3-10. Statistical analyses show that the means for number of adults produced by the diet with 1% and 5% host plant material are not different from the mean for PBD, but are different from mean numbers of adults produced by the diets containing 10% and 20% host plant material. The diets with 10%

and 20% host plant leaves are not different from each other, so it appears that the need for host plant is saturated by including about 10% freeze-dried host plant leaves into PBD.

The adults from PBD with 1% and 5% Phyla laid no eggs, but females from PBD with

10% host plant laid eggs.

PBD is a high protein diet and developed for pest moths. The diet may be imbalanced to rear P. phaon. This may result in longer development time because they may not get enough nutrients from an imbalanced diet so they may need to feed longer to accumulate enough nutrients and for to excrete excesses of some components.

Cellulose is a natural material in plants. Although most insects cannot digest cellulose, it has been shown to improve digestion in some insects (Nation, 2002). PBD was modified by reducing the amount of proteins such as casein and pinto bean meal in

half and adding cellulose instead, and this diet is called PBD modified (Table 3-10). 51

with were C

’ logistic co

producing G Chi-Square *3 PBD larvae >

on in binary 25 a

plant, by of i

significant

hatched diets a

-4-J host G comparison

20% different Newly highly T3

with - wise 4—* are the § only. 0 pair on PBD diets 1 PBD on the by produced o diet, and G

separated 1- Cd plant indicating

adults

modified

-4—* are host of

0.005,

with different the

phaon of df=5, PBD co the -4—* cd CO

Analysis on

host production

of Chi-Square=16.731, 1%

replications.

time

adult with

shows for PBD three c development 5

on of means o o Minitab £ M

Survival analysis

3-10.

Table 52

Larval survival and adult production were poor on the modified diet. Statistical analyses

show that the means for number of adults produced by the modified diet are not different

from the mean for PBD.

The results from addition of 1% and 5% cellulose to PBD are shown in Table 3-11.

Means for the number of adults produced by PBD with 1% and 5% of cellulose are not different than the mean for PBD. Addition of cellulose into PBD did not have a significant effect on adult production.

Results from addition of 1% glucose, 5% glucose, 1% starch and 5% starch to PBD

2 are shown in Table 3-12. Adult production was different on the six diets (X = 30.793, df

= 5, P < 0.001) and pair-wise comparison of binary logistic regression coefficient showed that addition of 5% glucose to PBD and PBD with 10% host plant are not significantly different.

On the other hand, the means for number of adults from diets containing 1% glucose, 1% starch or 5% starch and PBD alone are not different from each other. (Table

3-12). Addition of 5% glucose improved PBD, allowing 68% survival to the adult stage.

Larvae survived and grew better on this diet, along with good adult production, but the adults laid no eggs.

Insects need small amounts of many minerals because metal ions are required as enzyme co-factors. Phytophagous insects typically ingest more potassium than sodium, and they have a higher K/Na ratio in their hemolymph. A common salt mixture,

Wesson’s salt mixture, is designed for vertebrates and is high in Ca, Fe and Na (Osborne and Mendel, 1932). 53

and are letter adults 0.001,

plant diets of < same P host

number different the

10% df=3, with

the the

of on

cellulose, estimates CO 0J Analysis 15

production 5% of E CO Chi-Square=163.021,

replications. CO

for GO shows 1% 03

in GO each CD on

adults. regression

on analysis

larvae '3 placed Q-

producing phaon logistic W) g

Chi-Square "co were

in 'a>

binary > Phyciodes larvae a of significant 50 by of &

diets

comparison --* time C hatched o highly H

different are ”5 wise development Newly

diets pair c the rt wo the on only. by ’E and ho

PBD o produced separated indicating Survival c

3-1

Table 54

Chi- adult

starch,

coefficient. o replications. for QQ 1% shows G

means

glucose, three

Minitab

regression of *The 5% in each

adults.

logistic glucose, on analysis

placed 1%

producing binary of

were Chi-Square of in

addition

larvae a

25 significant comparison with by

diets

PBD hatched

wise highly on

different pair Newly are larvae

the diets by phaon only. on the

separated PBD

produced Phyciodes

and indicating

are

plant of

adults diets 0.001, time host of < 10% P different

number development with

df=5, the

the PBD on and of

starch,

production

Survival Analysis

Square=30.793, 5%

3-12.

Table 55

This salt mixture is not an appropriate salt mixture for most insects. For example, the Ca level in Wesson’s salt mix is toxic to the German cockroach (Gordon, 1959). A salt mixture relatively high in K and Mg and low in Na and Ca was developed for culturing

the European com borer, Ostrinia nubilalis (Beck et al. 1968), and this salt mixture, called Beck’s salt mix, is commercially available. Results from addition of 1% and 5%

Beck’s salt mix to PBD, PBD with 10% host plant leaves, and PBD only are shown in

2 Table 3-13. Means for survival on the diets were different (X = 16.98, df = 3, P < 0.001), and separation of means by pair-wise comparison of the binary logistic regression coefficient showed that the adult production on PBD with addition of 5% Beck’s salt and addition of 10% host plant are not significantly different. On the other hand, the means for survival on diets containing 1% Beck’s salt and PBD alone are not different.

Addition of 5% Beck’s salt supports betters survival and 64% adult production, but none of these adults laid eggs.

Testing Extracted Host Plant with the Pinto Bean Diet

The results from the bioassay of extracts of the host plant and the insoluble residue remaining after extraction are presented in Tables 3-14 through 3-21. In all tests, PBD with 10% host plant and PBD alone were tested concurrently with extracted components

and the insoluble residue left after extraction.

PBD with additions of extracts prepared with polar and non polar solvents were not different, with one exception, from PBD alone in promoting overall survival and adult production. The exception is methanol extraction of host plant flowers (Table 3-18), which promoted survival equal to that of PBD with 10% host plant leaves. 56

adult PBD Chi-

coefficient. replications. salt, for shows G

C/3

Beck’s

regression

5% *The of G in CL salt, each &J)

adults. .G logistic C/3 on analysis G Beck’s 13 > placed

PBD wise c hatched highly 0 on in different pair are 1 larvae Newly

the diets by o c phaon

produced o3

Phyciodes PBD

diets of adults and 0.001, time of

leaves <

different C/3 P (D

number §3 plant 1 development .£ 03 the df=3, 00 £ the C/3 host on

Square=16.98, Survival C/3 with O G CU «

3-13.

Table 57

Chi-square values and data for separation of means by pair wise binary logistic regression coefficient are given in each table. Moreover, the residues remaining after solvent extraction also failed to enhance survival, suggesting that solvent extraction had removed

or destroyed one or more critical components in the freeze-dried leaves. If such a critical

component was extracted into any of the solvents, it appears that it was not preserved long enough to have a beneficial effect on survival.

Pinto Bean Diet was modified for Phyciocles phaon larvae by addition of 5% glucose, 5% Beck’s Salt Mixture, linseed oil, and 10% Phyla (Table 3-22). Although adult production was 54% on pinto bean diet, adult production increased by 80% with these additions.

Measurements of Food Intake

The nutritional indices of fifth instars feeding on PBD with 10% host plant and

PBD only are listed in Table 3-23. Food utilization and all measurements were on a fresh weight basis from larvae in the last instar and during their last 3 days before pupation. A total of ten individuals on PBD diet and ten individuals on PBD with 10% host plant leaves were used in the experiment.

The mean larval relative growth rate (RGR) was 0.22 ± 0.1 mg/mg/day on PBD with 10% host plant. The mean larval relative growth rate (RGR) on PBD was 0.14 ±

0.06 mg/mg/day. Larvae on PBD with 10% host plant consumed more food, grew faster, and completed their development more rapidly than the larvae on PBD (Table 3-23). The mean of larval relative growth rate (RGR) was linear during the experiment. Larvae on

PBD with 10% host plant were also more efficient at converting ingested food into biomass (ECI). 58

O DO G placed hexane adults. ’55 analysis .3 OO 3 o _

were

residue, producing C —

Chi-Square 'S o

larvae o Is 50 in C 3 extracted O -- a w C by 'C B

significant hatched

diets £ O a^3 pentane

are only. the CO X> O on diets C pentane PBD 12 g 15 d on the produced rrt H a £ with and 8 u

d> indicating

adults *3 £ PBD o3 C/3 C/3

10% d Td phaon < >-< :> ,

u 'i time P c J- Oh extracted Chi-Square=160.170, 0_

replications. —h • (D

development 3 .2 o o

hexane shows £*§ S’ three o C/3 o C/3 and d (D and of cs a _o o d-d C/3 Minitab >-H c/3 • r- each c/3 ^> Survival 0)

extract, ob •— £ a3 on in r 2 cx

3-14.

Table »

with were

louistic 55

producing Chi-Square 13 PBD larvae % >

extract, by of i

significant -4— hatched diets c

comparison

wise 4—* are the c only. Oed pair chloroform: on diets X PBD :I the bv

produced on o with c and separated

indicating * -

PBD plant adults J-i CD -4—*

are on of host 0.001,

diets E % 03 larvae C/3 number < 10 P X

different phaon with the

df=3, of C/3

Analysis on s a Phyciodes on CO

-4—* — Chi-Square=148.262, 03

time replications. -5 cr 03 C/3 X) ^ extracted adult O c/3 »- o3 CU

shows for .

3-15.

Table » I I * ~

X m o <+hO chloroform E of o3 G c p cd IS 50 4b o chloroform P X o 'E c & M o C/J hatched c 03 with G X D, P M t-i c£

PBD Newly 33 cd

3-16.

Table * »*

~ ~ C/3 o » . '— o g. a o o .2 cd U X 3 C/3 © - ."3 O t/3 O. O X33 Q. 2c r3 -C 3 X G - —* 4 H u 73 o CT lh c/3 C/3 cd £ CL *— L. 09 o cd Q £ jD CQ X G c/3 C/3 V Oh m O X) o X o 3 "3 bo .22 I c3 >3 Oh

5 . f!-! ^ >>^H -O T3 3 T3 2 c C/3 u O 3 3 Q s . 3 5; CQ o . ib 3 <+-, Oh 3 O o "O Oh tO c o ' 3 3 o hO bo E T3 3 S e T3 C/3 „ 3 G 1 " 3 ++ S o O 3 cd G CO ’’SCd cd o JS C-H G CL c > ^ (D X G L4hGii S-i— CO O 73 73

i CO XJU Hcd »'

O C/3 +J 07 c3 G c '*-* i? "ti G 07 £ ^ • G G o> — <4-» r- O tZ G | E £ 0 <17 07 S (U 0/} £ G c -c •£ • S o H O t/3 C/3 * s-S c (D ’(73 ~ ? o •-O cd ^ jg (U Q U G CQ « S -o 2 * an ,_ ap o - 2 u c3 03 C O §•! -S ao s=

, r->c c3 CL 07 cx M 1 C/3 Os — CO G ^ G 07 " II S G S . « O (D £ CO S-. T3 O c 2 O S •—° S- <4-4 C/3 •a cr ^ 07 £ c3 CO "g o § o 07 o .i Lh — c ~ -C D. ^ G G O a. rj G ’-G a> cr . 1-1 CO 3 X> > O T3 03 CA) C 2 (U % 03 X3 g is C/3 2 O >- 2 72 OQ X o 07 0) •S -S u— cl 00

J7 HG * , i< —*i >

d> CO • d) X -+— o ’£ o CO c 73 § d) o d) -4—* d) c O c _B ’co CO a3 o d> CO -5 T3 - H — o w *C7 00 Lh > O >, 3 ^ 03 E 03 t/3 CL s Q a. x 2 00 3 3 -*— * c « "2 CO Mh t- DJj ^ § * 3 M O ^ CT ^ 13 *s

o I 3 £ 3 ’ 1 2 & 3 « 2 o S X o d> ° s >> .£ ‘s S x> ^ * O "O -4— CO 5 C 3 tJ g —' cz o O X d) o 3 ^ 3 O ‘-3 c S "C - +3 X t3 CL , j-h co £ S O "O $2 ^ 0 Q £ M—I F-H >N no •- X £ X 00 d> X Cl, Z CO c 3 <3 IS~ ’> cZ X u > O ° >, #— l— X C g « 3 ° & •a £ t.l W .<3 C/3 J2 Q o S3 CQ o e •a u X O Cl, X 3 o *-* i— e •S §° CL 00 ad & Cl 1/3 3 s-. . Jo T3 3 3 g.^ ^ T3 3 S o L— O O 3 — a, o. X ^ X) o O £ o g 3 o o X,Jl "3 .52 CO a. c U3 d> O !> >3 — •4— c3 3 O r“ > Q 3 ^7 g o CQ on < oo CO ^ Oh (D T3 _ • II 3 OS 523 — 3 O 3 73 "3 o' *X 3“3- cr 3 O X) CO > 3 O C/3 3 o3 T3 '— > .3 -i T3 X) CO L- „ V)C/3 CL _3 O O a3 }-< 5- 3 (D d> on >- 2 U a- 0—i as m x Hc3 > * »* S.

C/3 o ^ cd _ c/3 o3 3 i? ° .3 3 x •° 3 s Si c C to •a O ^3 ? CQ 73 .+; CLh co ^ 3 Q 3 m r^ _>. £ co QQ O co 0) 3- 3 M-» ^ , <3 < 3 3 3 2 3 .S 3 ? 3 O cx52 1 I ^~ 11 CO "* £ > •2 3 § 3 73 36 c .3 ~ 2 £ 3 X> 3 X s "o 3 32 '3 3 ^O O 4= ^ 00 o 2 43 >* C — 3 k, 2 >~> 3 * CL 3 II 43 3 3 Q z ?S 00 3 "O ^ ,3 2 QQ . o 5C C Oh >, 3 3 Cm 73 a. _ 3 "S 3 2 § ° _>> 4) 3 > CD Q o 3 3 CD 3 3 CQ V, CU , — O 73 4—* X 3 30 00 73 73 O 73 •

d> w c d> J! ? o M G •- G co £ C o O < 0) &/) G -C - ^ D Cl O H Lh tn G * G3 CLh iS o °P c a, b .£ G »— o g ^ § g o a3 £ >? JZ O "O "w 8 c c >* c JG —CO >, o o d) X5 C s- on TD a> d> O TD G

~~G CO d) c T3 _cd a5 -G 0 G 0 # •»>*4 a. Cl_ 0 -a t—1~~

c/3 O o O i- V JO (X ox e O G CO Ji d> G C4H £ Xd> "O £ u-T CO Q c ° o CQ co 'O ° >, Oh § Q. •s 2 — cr _o g g " ~ II °5 o G d) 53 "w > d> G o 3 u G 3 -g cd •a < 3 "5 8 rs cr — >-M -o CO 00 u> CD d> CO 1 Gh JO c s- G •4—* cd ’.£ G "O O _d) d> 4—» U Id G G ’0 > O O C/3 "DG G C+-i d> 'E Oh O V-i CO G d) — ,0 O d> u. IZ) C/3 d— O

~~04 co JJ HG 66~~

~~Addition of 10% host plant material increased the growth efficiency on PBD by~~

~~improving its digestibility. Both gross (ECI) and net efficiency (ECD) were increased in~~

~~larvae feeding on PDB with 10% host plant. Much of the change in gross efficiency~~

~~(ECI) was due to the greater digestibility of PBD with 10% host plant. The food ingested~~

~~and the consumption index (Cl) (food intake per unit of body weight per day) for PBD~~

~~with 10% host plant were different from PBD alone (Table 3-24).~~

~~Survival of Phaon Crescent Larvae on White Clover ( Trifolium repens)~~

~~On 9-21-2001, in the laboratory, an egg cluster of P. phaon was found on leaves~~

~~of white clover ( Trifolium repens), which was accidentally potted with the host plant,~~

~~Phyla nodiflora. The egg cluster (46 eggs) hatched and 46 first instars started feeding on~~

~~clover leaves. Care was taken to ensure that they did not have further access to the~~

natural host plant. Twelve larvae were able to complete their development on clover in about 30 days. They were smaller in size than normal. Nine larvae pupated and 4 adults emerged. They were small and the wings were deformed, and they survived for only 4 days in the laboratory. This suggested an experiment in which ground freeze-dried clover leaves were added to PBD and newly emerged larvae were put on this diet. The results with PBD containing 5% and 10% dried clover leaves are presented in Table 3-25.

Surprisingly, PBD with 10% dried clover leaves was as effective as PBD with 10% freeze-dried host plant leaves, and adults from the clover diet laid eggs. When these eggs hatched, the larvae were again placed on PBD with 10% dried clover leaves and successfully developed to the adult stage. A total of 4 generations was reared and each time adults laid eggs. Addition of 5% clover did not improve PBD. 67

~~Table 3-22. Added components into pinto bean diet to modify it for P. phaon larvae.~~

~~Added Components into Adults Reared (%) Pinto Bean Diet~~

~~5% Glucose 70.8~~

~~5% Beck’s Salt 66.7~~

~~Linseed Oil 80.5~~

~~1 0% Phyla 87.5~~

~~Survival of Phaon Crescent Larvae on Other Verbenaceae Species~~

~~There are two plant species (Phyla nodiflora and Phyla (=Lippia) lanceolata)~~

~~which phaon crescent larvae use as a host plant and both belong to the Verbenaceae~~

~~family. Lantana camara and Stachytarpheta jamaicensis are also species of Verbenaceae~~

~~family, are readily available in nurseries, and were tested as a larval host. The larvae did~~

~~not feed on either species. Leaves of both plants were freeze-dried and tested in the PBD~~

~~diet. The survival rates on PBD with addition of 10% L. camara and S. jama are shown~~

~~in Table 3-26. P. phaon larvae survived poorly and none survived to the adult stage.~~

~~Conclusion and Discussions~~

~~Numerous studies have been performed to develop artificial diets for pest insects.~~

~~Many insects, mostly having some economic importance and feeding upon multiple host~~

~~plants, can be reared on artificial diet. Butterflies tend to be restricted to one or only a~~

~~few host plants as larval food plants, and a complete synthetic diet is available for only a~~

~~few butterfly species. The imported cabbageworm butterfly (Pieris rapae) (Webb and~~

~~Shelton, and the 1988) painted lady butterfly ( Vanessa cardui) have been reared on~~

~~artificial diets. Although there are many moth species that have been reared on several~~

~~artificial diets, there is no diet that has been used for rearing a large number of butterflies. d — 3 ^ 1 <~~

~~The ON 00 ON It CN b the -H 41 only. Q ^ CN xo CN © from U w sb- it CN PBD~~

~~taken cn 00 in and oi nb~~

~~were -H 41 plant hH oo in © ON PQ 5d d~~

~~host ro cn 0% calculations CO~~

~~PBD~~

~~used si Q o it OO on GO CN -H '5t> cb CN Data instar. e E -H 41 o3 instars) ~St) CN ON ~ E o On (last) s U & ON rb~~

~~utilization. c Si fifth +-» 03 NO T3 .— o '3a © d the O -H 41 crescent Dh food <2 50 CN it of ro CN H o id 3 d d days c phaon m cn by o~~

~~were of 13 (N r— during O © &~~

~~and cd CN CN of on O O & ID 03 X d d~~

~~growth _ S M 41 41 instars rrt NO NO (D • T3 individuals 1- t; C o o PG £~~

~~The fifth a -C *5* >> 3-23. o -c ’S £ a. O CQ no DQ Q © Q Table 0h — CP i <~~

~~the Oh_ by o o o o o o followed -s o o o o o o X) o o o o o o o s- d © d d d d CU V V V V V V~~

~~row~~

~~same~~

~~the in~~

~~Means~~

~~t-test. by~~

~~calculated~~

JO n J3 X X5 00 CN NO On fr> O O r~~ were t-test. d o o CN d o § -H -H -H -H -H -H S CN Q § m ON lO 00 CQ *2 NO >— 1 On i/S On Os SO unpaired CL S o o d -C r-H*

~~consumptions~~

~~by 5S £ a* food different NO ox of O Q 00 ro On u-i O NO 00 O O © — ON ON means o O — o o £ -H e -H -H -H -H -H significantly Q (N © (N the CQ .~~

~~not~~

~~are Separations ^ ^~~

~~letter~~

~~& g s 3-24. g, T& 13b ^ c E S £ £ £ c4 Q Table a U V Pl c4 U < W W — •i » «~~

<+H - Ui ° 33 43 O 2 i§ O o 73 sO « 3 o' 'w in -C o >, •*- ^H *— o g G u G oo ° II £ < £ C +-» C/D _o e 3 .2 O M "c/D (D a* o G 2 ^ .2 °? •§ in -y Cw CO # 3 2 r*> &• GO G W 15 'O '5b '-E o .g jUj oH U G- C/D "(3 b-> G o a3 C s- ^ ™‘O 2 &> '2 >. -*— •S ^ ^ T3 Jd G ^ ~s: -H coCO E £ g ’§ ^ E si ^ I op c" B G ’T"E i . ? > O G -4—* ,*_» CO G O 2 ° £ c - c3 G G r3 G -rd G< a> 0/5 " a> tH 2. .S uS (N mi jj jz HG 71

~~with~~

~~PBD~~

~~camara,~~

~~Lantana~~

~~10%~~

~~addition~~

~~with~~

~~only. PBD~~

~~on PBD~~

~~larvae~~

~~plant,~~

~~phaon~~

~~host %~~

~~Phyciodes 10~~

~~with of~~

~~PBD time~~

~~on ,~~

~~development S.jama~~

~~10% and of~~

~~Survival~~

~~addition~~

~~3-26.~~

~~Table 72~~

~~In this study, several artificial diets were tested for rearing Phyciodes phaon. Pinto~~

~~Bean Diet (PBD) was the best of several diets tested. Phaon crescent larvae successfully~~

completed five generations on this diet with a little longer development time compared to larvae developing on the host plant. Mature larvae were able to pupate and adults emerged after about 5 days. Adults survived in the laboratory for 10 days without laying any eggs.

~~PBD was synthetically modified for P. phaon by addition of 5% glucose and 5%~~

~~Beck’s salt. Larval survival and adult production were higher on this modified PBD than~~

~~PBD only but adults reared did not lay any eggs.~~

Freeze-dried host plant materials were added into PBD to modify it for phaon crescent larvae. Larvae completed their development in 15 days. Egg laying was successfully achieved. Addition of host plant material (10% dry weight of the diet components) is a requirement for egg laying by artificial diet reared P. phaon adults.

~~The host plant, Phyla nodiflora, was extracted with different solvents. Larval survival and adult production were increased only with addition of a methanol extract of~~

flowers added to PBD. However, it did not promote egg laying. Flowers are rich in sugars and the higher survival and adult production on PBD with the methanol extract of flowers may have been due to sugar, similar to the beneficial effect of adding 5% glucose to PBD as reported earlier. Other extracts and the extracted residues added to PBD did not improve the diet.

Clover is not known as a larval host plant for P. phaon. The larvae survive poorly on living clover leaves but addition of 10% freeze-dried clover into PBD works as well as addition of the host plant into PBD. This suggests that the beneficial effect of adding 73

~~10% by weight of Phyla nodiflora may not be due to a component or components specific to the host plant, but may be due to a component common to many plants. The~~

poor growth of larvae on living clover may be related to its well known cyanogenic properties (Jones, 1998). Freezing in liquid nitrogen, grinding in a mortar and then freeze-drying may destroy any cyanogenic compounds present so that larval feeding and survival was improved. Moreover, and more significantly, the adults reared on 10% freeze-dried clover in PBD mated and laid eggs, further suggesting that the beneficial

~~effect ofP. nodiflora in the diet is not absolutely host specific.~~

~~In the laboratory, P. phaon larvae were reared on PBD with 10% freeze-dried~~

~~clover for four generations without any problems. It is very significant that P. phaon can~~

~~be reared on this diet with addition of clover rather than the host plant, because clover is a more widely distributed plant that can be added to PBD.~~

~~The balance of nutrients is very important to most insects. It has been found that~~

imbalanced nutrients may reduce food intake, and optimum growth is also dependent upon proper ratio of amino acids to minerals. The excess nutrients may cause stress on insects and development may not be completed or they may not tolerate the imbalanced diet and die.

~~In general, low growth rate could be the result of a low rate of feeding and/or nutritional inadequacy of the diet (Waldbauer, 1964). Dethier (1954) and Fraenkel and~~

~~Blewett (1943) emphasized that host plant selection is caused by the presence or absence of non-nutritive secondary plant substances that act as feeding deterrents or stimulants.~~

~~Waldbauer (1964) described feeding behavior and utilization of food by a series of nutritional indices. Various imbalances in the nutrients in a diet can reduce food intake. 74~~

All measurements of food consumption and utilization show that growth, digestion and nutrient assimilation are greater on PBD with 10% host plant leaves than on PBD alone. CHAPTER 4 DETERMINATION OF POLYUNSATURATED FATTY ACID COMPOSITION OF LARVAE AND ADULTS OF THE PHAON CRESCENT BUTTERFLY AS AFFECTED BY ARTIFICIAL DIET

~~Introduction~~

~~The lipid requirements of vertebrates and insects differ with respect to sterols.~~

~~Insects cannot synthesize sterols, which they must obtain from their food and/or from~~

~~their symbiotic microorganisms (Dadd, 1985; Nation 2002). The sterol requirement as a~~

~~first growth factor was discovered by Hobson (1935) in blowfly larvae (Lucilia sericata)~~

~~and it is now well established that insects require a dietary sterol. Two dermestid species,~~

~~Dermestes vulpinus and Attagenus piceus only grow when cholesterol or 7-~~

~~dehydrocholesterol is supplied in the diet (Clark and Bloch, 1959). Sterol is required as a~~

~~part of all cell membranes and a precursor for synthesis of the molting hormone. Failure~~

~~to have an appropriate sterol in the diet results in inability to molt successfully.~~

~~Cholesterol usually is able to satisfy the sterol requirement for most insects (Gilbert,~~

~~1967), and it is typically added to artificial diets. Many insects are phytophagous and do~~

~~not obtain cholesterol from the diet, they must utilize a plant sterol such as P-sitosterol~~

and stigmasterol (Agarwal, 1970). The plant sterols typically are converted into cholesterol by removing one carbon (C28 plant sterols) or two carbons (C29 sterols) from the side chain of the plant sterol. The conversion of P-sitosterol to cholesterol has been

~~studied in several plant- feeding insects including Bombyx mori (Ikekawa et al. 1966) and the tobacco homworm, Manduca sextci (Svoboda and Robbins, 1967). The conversion~~

~~apparently proceeds via the 24-dehydrocholesterol intermediate, desmosterol (Svoboda et~~

~~75 76~~

~~al. 1967). A few insect species are known to have a specific requirement for a sterol~~

~~found in their food, such as Drosophila pachea which requires an unusual sterol, 7-~~

~~stigmasterol-3p-ol, found in its host plant, senita cactus (Kircher et al. 1967). A~~

~~deficiency in dietary sterol usually manifests itself during larval development but may be~~

~~expressed in a later stage. For example, newly hatched silkworm larvae on a sterol-~~

~~deficient diet failed to grow and eventually died after they had used up sterols from the~~

~~eggs (Ito, 1960 and Ito, 1961). Houseflies, Musca domestica, laid normal numbers of~~

~~eggs when maintained on a sterol-deficient diet, but egg hatch was reduced by 80%.~~

~~In addition to sterol in the diet, the majority of insects that have been studied~~

~~require the polyunsaturated fatty acids, linoleic and linolenic acids, in the diet. These and~~

~~other fatty acids are part of the complex lipids in all cell membranes. The saturated fatty~~

~~acids can be interconverted, but the 3 double bonds of linolenic acid cannot be inserted~~

~~into an 1 8-carbon chain by most animals, including insects.~~

~~The fatty acid composition of the oil or triacylglycerol (fat) in the diet of some~~

~~insects dictates the tissue fat composition of certain insects, while other insects may~~

~~rework the dietary fatty acids into their own characteristic tissue lipids (Nation and~~

~~Bowers, 1982). In classical studies on flour moths (Ephestia kuehniella, E. elutella, E.~~

~~cautella and Plodia interpunctella), Fraenkel and Blewett (1946) demonstrated that when~~

~~larvae were reared on an artificial diet deficient in polyunsaturated fatty acids they grew well as larvae, but suffered deformation of wings and/or wings without scales as adults.~~

Usually the need for polyunsaturated fatty acid can be met by linolenic acid (9,12,15- octadecatrienoic acid), and most of the oils that are added into synthetic diets have both linoleic and linolenic acids as a part of the triacylglycerols (Grau and Terriere, 1971). 77

~~David and Gardner (1966) demonstrated that the cabbage butterfly Pieris brassicae (L.)~~

~~could be reared on a wheat-germ diet with addition of dried cabbage leaves, or in the~~

~~absence of the cabbage leaves, with addition of linseed oil, which contains linoleic and~~

~~linolenic acids.~~

~~Not only Lepidoptera, but some other groups of insects as well, need these~~

~~polyunsaturated fatty acids for normal adult development. A polyunsaturated fatty acid~~

~~deficiency has been recognized in Hymenoptera, and the symptoms can be relieved by~~

~~linolenic acid (Yazgan, 1972). In Coleoptera, deficiency of fatty acids slows larval~~

~~development and decreases fecundity (Wardojo, 1969). Symbionts in the gut or tissues~~

~~of some insects can synthesize the polyunsaturated fatty acids; for example, aphids~~

~~(Order Homoptera) can be reared on a lipid free artificial diet for many generations.~~

~~Typically, oil from com, cottonseed, linseed, olive, peanut, sunflower, or wheat germ is~~

~~added to an artificial diet. Any of these supplies both linoleic acid and linolenic acid,~~

~~although in different proportion in the different oils.~~

~~Composition of Polyunsaturated Fatty Acids in Insects~~

~~Tissue and body triacylglycerol composition may change during development in~~

~~some insects. For example, the whole body of newly molted last instars of the silkworm,~~

~~Bombyx mori, contains 16% palmitic acid (Cl 6:0), 15% oleic acid (Cl 8:1), and 16%~~

~~linoleic acid (Cl 8:2) and these values change to 29%, 24%, and 7%, respectively, for last~~

~~instars that are 8 days old (Nakasone and Ito, 1967; Turunen, 1974a). Changes in body composition typically occur in pre- and postmetamorphic stages (Thompson and~~

~~Turunen, 1974a; Turunen, 1974b; Richeson et al. 1971). Kinsella (1966) and Thomas~~

~~(1974) demonstrated that the proportion of unsaturated fatty acids is much higher than saturated fatty acids in body lipids of Oncopeltus fasciatus and assumed that the natural 78~~

~~host plant may supply the unsaturated fatty acids. The fatty acid composition of~~

~~numerous insect species has been determined, including honeybees, Apis mellifera~~

~~(Robinson and Nation, 1970), milkweed bugs, Oncopeltus fciscicitus (Nation and Bowers,~~

~~1982), velvetbean caterpillar, Anticarsia gemmciatalis (Cookman et al. 1984), Caribbean~~

~~fruit flies, Anastrepha suspensa (Chow and Nation, 1975), bollworms, Heliothis zea~~

~~(Schaefer, 1968), Egyptian cotton leafworms, Prodenia litura (Levinson and Navon,~~

~~1969), southwestern com borers, Diatraea grandiosella (Chippendale and Reddy, 1972), silkworms, Bombyx mori (Ito and Nakasone, 1967), and flour moths, Ephestia spp.~~

~~(Fraenkel and Blewett,1946).~~

~~Larvae of the phaon crescent feed only on two species in the family Verbenaceae.~~

There is no study to elucidate the chemical basis of this specificity, but many studies have been done for other insects such as Manduca sexta by testing various host-plant extracts and fractions (Yamamoto and Fraenkel, 1960). Stadler and Hanson (1978) extracted nonpolar compounds from host leaves and showed them to be feeding stimulants for

Manduca sexta. De Boer and Hanson (1982) demonstrated that nonpolar feeding stimulants of the host plant, tomato, for tobacco homworm, M. sexta, are neutral lipids, many of which are found on the outer surface of the leaves. When an insect contacts the leaf surface, such lipids or other compounds may signal suitability of the plant as a feeding and/or oviposition host (Chapman, 1977; Woodhead and Chapman, 1986). p- sitosterol isolated from mulberry leaves is a specific feeding stimulant for silkworms

~~(Hawamuraet al. 1961).~~

~~The pinto bean diet appears to be a low-lipid diet; nevertheless, the pinto bean diet supports development of adult phaon crescents although those adults do not lay eggs. As 79~~

~~originally formulated, it does not have added oils or fats (Guy et al. 1985), although some~~

~~of the components (pinto bean meal, wheat germ, torula yeast) may contain various~~

~~lipids.~~

~~The objectives in this chapter are to report (1) the effect of adding various oils to~~

~~the pinto bean diet upon growth, development, and adult production of the phaon~~

~~crescent, (2) analysis of the body fat of larval and adult phaon crescents to determine the~~

~~fatty acid composition of those reared on the various diets with added oil, on PBD as~~

~~originally formulated and on the host plant. Phyla nodiflora, and (3) identify the sterols in~~

~~the host plant, Phyla nodiflora, and to determine what effect addition of synthetic sterols~~

~~or plant extracts containing the sterols might have upon the phaon crescent.~~

~~Materials and Methods~~

~~Study Organism~~

~~Phyciodes phaon colony was maintained in the laboratory at 27°C, 16 h L: 8 h D~~

~~cycle and fed on the host plant. Phyla nodiflora, collected from the University of Florida~~

~~campus and vicinity. The eggs were obtained from this laboratory colony and newly~~

~~hatched larvae were placed on diets to be tested.~~

~~Artificial Diets~~

~~The pinto bean diet (formula in Chapter 3) was enriched by adding 0.75 mL oil~~

~~/250 mL diet (a typical batch formulation to rear 25 or more larvae through to the adult~~

~~stage). Separate diets were formulated with wheat germ oil, linseed oil and olive oil.~~

Newly hatched larvae were reared on these diets, on PBD without oil, on PBD with 10% host plant and on living host plants. Survival and number of adults was recorded periodically. Adults were held with a host plant to see if they would lay eggs. 80

~~Extraction of Lipids and Fatty Acid Analyses~~

~~Mature, last instars were taken from each diet and starved 24 h before lipid~~

~~extraction to eliminate gut contents, and then were extracted or frozen until extraction.~~

* Adults were frozen within 24 h after eclosion for later extraction. Frozen insects were

~~held only a few days at -20°C before extraction. The total lipids were extracted by the~~

~~method of Folch et al. (1957), James (1960), and Luddy et al. (1960). Approximately 2 g~~

~~of fresh Phyla leaves and 0.5- lg wet weight of whole insects were extracted with a few~~

~~mL of chloroform: methanol (2:1, v/v) by grinding the tissue or leaves in a mortar. The~~

~~chloroform: methanol extract was filtered through Whatman #1 filter paper into a~~

~~separatory funnel, and an additional 2 mL of chloroform: methanol solvent was poured~~

~~over the residue and added to the filtrate. The chloroform: methanol extract was shaken~~

~~in a separatory funnel with an equal volume of 3 % aqueous sodium chloride solution to~~

~~remove methanol and small water soluble molecules. The bottom chloroform layer~~

~~containing lipids was saved and a small quantity of anhydrous sodium sulfate was added~~

~~to dry the extract. The extract was filtered to remove the sodium sulfate and evaporated~~

~~to dryness with a gentle stream of nitrogen. The triacylglycerols were saponified to~~

~~release fatty acids by addition of 1 mL of 0.5 N methanolic KOH (Luddy et al. 1960).~~

~~This mixture was allowed to sit overnight and then methyl esters were formed from the~~

~~potassium salts of the fatty acids by addition of 0.5 N HC1. The resulting fatty acid~~

~~methyl esters (FAMEs) were removed by gently shaking the aqueous layer with 5 mL of~~

~~pentane. The upper pentane layer containing the FAMEs was removed, dried with~~

~~sodium sulfate, and evaporated in a gentle stream of nitrogen to approximately 1 mL. An~~

~~aliquot of 1-2 pi was injected into a gas chromatograph for analysis. 81~~

~~Gas Chromatography (GC) to Analyze Fatty Acids~~

The fatty acid methyl esters were analyzed on a Varian model 3700 gas chromatograph equipped with a flame ionization detector. The methyl esters were separated by a 4 mm x 1.3 m column packed with 10% Silar 10C coated on 100/120 mesh Gas Chrom Q. The column was initially at 140 °C and held there for 30 minutes, and then programmed to 150 °C at 5 °C per minute. Injector and detector temperatures were 170 °C and 180 °C temperatures, respectively. Nitrogen was used as a carrier gas and the flow rate was 30 mL/min. Identification of the fatty acid methyl esters (FAMEs) was made by comparing retention times with fatty acid methyl ester standards (Applied

~~Science Laboratory Inc., State College, PA). FAME quantitative mixture K 108 (Applied~~

~~Science, 1 C14:0, C16:0, C18:0, C 8: 1 , C 1 8:2, C 1 8:3) showed the chromatographic technique to be ± 4% of the expected values (Nation and Bowers, 1982).~~

~~Total Lipid Extraction to Isolate Plant Sterols from Phyla nodiflora~~

~~About 40 g (wet weight) Phyla was extracted by grinding the leaves in a mortar with 6 mL chloroform: methanol (2:1) for 5 minutes. The extraction was filtered through~~

~~Whatman #1 filter paper into a vial, concentrated, dissolved in pentane and analyzed by gas chromatography.~~

~~Gas Chromatography (GC) to Analyze Plant Sterols~~

~~Plant sterols from Phyla nodiflora were analyzed on a Shimadzu GC 14A gas chromatograph equipped with a flame ionization detector. Injector and detector~~

temperatures were 250 °C and 320°C, respectively. Helium was used as a carrier gas at a flow rate of 27 cm/sec. The capillary column was 0.25 mm X 25 m coated with 0.2 pm thickness of RSL-150 non polar coating. The column was initially at 200°C, and upon sample injection, the temperature increased at 4 °C/min to 300 °C, with a 20 minute hold. 82

~~Identification of the plant sterols was made by comparing retention times with authentic~~

~~standards (jS-Sitosterol, campestrol and stigmasterol) (Applied Science Laboratory Inc.,~~

~~State College, PA). Acetate derivatives of the sterols were formed by adding a few drops~~

~~of acetyl chloride to a solution of the sterol, and holding for 1-2 hours before gas~~

~~chromatography. Trimethylchlorosilyl (TMCS) derivatives were made by adding a few~~

~~pi of the TMCS reagent to the sterols. Sterols from Phyla nodiflora were analyzed~~

~~quantitatively with cholesterol as an internal standard.~~

~~Thin Layer Chromatography (TLC)~~

~~Thin layer chromatography was used to separate compounds in the total lipid~~

~~extraction of P. nodiflora. The extract was washed with 3% (w/v) NaCl and the~~

~~chloroform layer containing lipids was applied in a series of spots in order to get~~

~~sufficient volume on glass plates coated with silicic acid. Cholesterol was run as a~~

~~standard to locate the sterol spots. The plate was placed upright in a vessel that contained~~

~~a shallow layer of chloroform, which ascends the layer of absorbent on the plate by~~

~~capillary action. Development of the plate in the solvent required about 45 minutes. The~~

~~plate was allowed to dry in the hood until free of chloroform. A second plate containing~~

~~sterol standard, cholesterol, and a few spots of the extract was developed in the same~~

~~chamber and sterol position on the plate was located with iodine vapor in an iodine~~

~~chamber. When the spots were evident, the plate was removed from the iodine chamber~~

~~and the spots were outlined with a pencil because they are not permanent.~~

~~Identification of Sterols in Pinto Bean Diet (PBD)~~

The dry components for the pinto bean diet were mixed and extracted with chloroform: methanol (2:1) in order to extract the sterols from the diet. The sterol extract was analyzed by gas chromatography in the same way as sterols were analyzed in Phyla. 83

~~Results~~

~~Rearing P. phaon Larvae on PBD with Added Oils~~

The survival and adult produced on PBD with addition of linseed oil, olive oil, wheat germ oil, PBD with addition of 10% Phyla and PBD alone were significantly different with Chi-Square=33.590, df=4, P <0.001 (Table 4-1). The means for numbers of adults produced on different diets were separated by pair wise comparison of the binary logistic regression coefficient. Adult production on PBD with addition of linseed oil, olive oil and wheat germ oil was not significantly different from numbers of adults produced on PBD with 10% host plant material, but all were different from the number of adults reared on PBD. Thus addition of any one of the oils improved the diet and allowed nearly 80% survivals to the adult stage. The adults, however, did not lay eggs when the

~~diet did not contain 1 0% host plant leaves.~~

~~Fatty Acid Composition~~

~~A chromatogram of the FAME standard (C14:0, C16:0, 1 6: C18:0, 8: C 1 , Cl 1,~~

C18:2, C 1 8:3) used in this study is shown in Figure 4-1. Six fatty acids were detected in extracts of whole body lipids from larvae and adults of the phaon crescent fed on Phyla nodiflora (Figure 4-2), PBD with 10% P. nodiflora (Figure 4-3), PBD only (Figure 4-4),

PBD with linseed oil (Figure 4-5), and the living host plant, Phyla nodiflora and pinto bean diet (Figure 4-6). The fatty acid composition of individuals reared on PBD with olive oil and wheat germ oil was very similar. Only PBD with linseed oil reared larvae and adults are presented. »* *

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~~Based on comparative retention times, the fatty acids in the larvae and adults were~~

~~identified as (palmitic), 1 6: 1 C16:0 C 1 (palmitoleic), C18:0 (strearic), C 8: 1 (oleic), C18:2~~

~~(linoleic) and C 1 8:3 (linolenic acid). In addition, a trace amount of myristic (C14:0) acid~~

~~was detected. The percent composition of fatty acids in the whole body of larvae and~~

~~adult stages on different diets is shown in Table 4-2. Trace amounts of certain fatty acids~~

~~were not considered in the percentage calculations. The predominant fatty acids were~~

~~1 1 C 6:0, C 8: 1 , C18:2 and C18:3, which together accounted for over 97% of the total in~~

~~larvae feeding on P. nodiflora, 77% in larvae on PBD, 83% in larvae on PBD with~~

~~addition of linseed oil, and 85% in larvae on PBD with addition of 10% Phyla (Table 4-~~

~~2). The percentages of these four fatty acids in adults were 92% on P. nodiflora, 81% on~~

~~PBD, 83% on PBD with addition of linseed oil and 98% on PBD with addition of 10%~~

~~Phyla (Table 4-2), respectively.~~

~~The most striking difference was that only traces of Cl 8:3 fatty acids were present~~

~~in total body lipids from larvae and adults fed PBD. Larvae are unable to selectively~~

~~accumulate Cl 8:3 from the PBD and adults have a very low Cl 8:3 content compared to~~

~~those on the other diets (Figure 4-4). When the diet was prepared with linseed oil, there~~

~~was a general trend for a 1 stronger dietary influence of C 8: 1 , C18:2 and C18:3 fatty acids~~

~~in the composition of total body lipids (Figure 4-5 and Table 4-2). Moreover, larval~~

~~survival and adult production were promoted on PBD with addition of linseed oil (Table~~

~~4-1). Larvae reared on PBD with addition of 10% Phyla had more C18:3, less C18:2 and~~

~~08:1, and more 06:0 compared to larvae read on PBD (Figure 4-3 and Figure 4-4).~~

~~The amount of polyunsaturated fatty acids in body lipids remained high (70-83%) in both larvae and adults fed on host plant (Figure 4-2), PBD with addition of linseed oil (Figure 86~~

~~4-5) and PBD with addition of 10% Phyla (Figure 4-3). When larvae were reared on P.~~

~~nodiflora, 44% of the total body fatty acids were Cl 8:3, and in adults 33% (Figure 4-2).~~

~~The composition of fatty acids in the living larval host plant. Phyla nodiflora and~~

~~pinto bean diet is shown in Figure 4-6. The amount of polyunsaturated fatty acids in~~

~~pinto bean diet was high (83%) and (79%) in P. nodiflora.~~

~~Plant Sterols in Phyla nodiflora~~

~~Based on comparative retention times of /3-sitosterol (RT= 27.41 min), stigmasterol~~

~~(RT= 26.42 min), and campesterol (RT= 25.72 min) (Table 4-3, Figure 4-7, Figure 4-8,~~

~~and Figure 4-9), peak retention times in the sample were approximately matched with the~~

~~common plant sterols. To effect partial purification, the chloroform solution of the plant~~

~~extract was chromatographed on glass plates coated with silica gel, and developed with~~

~~chloroform (Figure 4-1 1). The sterol region detected by iodine vapor was scraped from~~

~~the plate having plant extract and the sterols were dissolved in pentane and analyzed by~~

~~GC. Results are shown in Figures 4-10. They were tentatively identified as /3-sitosterol~~

~~(RT= 27.38 min), stigmasterol (RT= 26.24 min) and campestrol (RT= 25.66 min) (Table~~

4-4 and Figure 4-10) based on comparation of retention times of the standards. In order to further confirm the identity of Phyla nodiflora sterols, TMS and acetate derivatives were made along with standards and analyzed by GC (Table 4-3 and Table 4-4). The retention times of TMS derivatives of ^-sitosterol (RT= 28.57 min) (Figure 4-7), stigmasterol (RT= 27.55 min) (Figure 4-8) and campestrol (RT- 26.80 min) (Figure 4-9),

~~peak retention times in Phyla sample were approximately matched with /3- sitosterol silyl~~

~~ether (RT= 28.5 min), stigmasterol silyl ether (RT= 27.29 min) and campesterol silyl 87~~

4-1 Figure . Gas chromatography analysis of the standard fatty acid methyl ester mixture (FAME) containing equal amounts ofC14:0, C16:0, C18:0, C18:l, C18:2, Cl 8:3. The retention times of the standard were compared with the samples. 88

~~Figure 4-2. Gas chromatography analysis of fatty acids of the phaon crescents reared on Phyla nodiflora; A) Larvae, B) Adults. 89~~

~~Figure 4-3. Gas chromatography analysis of fatty acids of the phaon crescents reared on PBD with addition of 10% host plant; A) Larvae, B) Adults. 90~~

~~Figure 4-4. Gas chromatography analysis of fatty acids of the phaon crescents reared on PBD only; A) Larvae, B) Adults. 91~~

~~Figure 4-5. Gas chromatography analysis of fatty acids of the phaon crescents reared on PBD with addition of linseed oil; A) Larvae, B) Adults. •~~

~~CR> -nf* r-> — u1< r--»~~

~~r-*> •v~~

~~Figure 4-6. Gas chromatography analysis of fatty acids of Phyla nodiflora and pinto bean diet; A) Phyla nodiflora, B) Pinto bean diet 93~~

~~(N r- ON a with 00 r-~~

~~expressed CM on *32~~

~~< -c ~ PBD, bonds. also. .ti o adult, VO oo 00 c OO n 5 "D NO o NO OO r- on 00 o H u no NO ON Urn Q d (N © CO m (N and g double~~

~~analyzed CU CM Q nodiflora, M < QJ of larvae +* CM~~

~~were w (N o cn NO o Q i/D o ON number i r rn phaon CO NO Phyla d d d On CL, (N NO " diets aJ m~~

~~on the ofP. the is 3) reared ON NO ON (N lipids from £ 00 NO O O II i~~

~~(t=trace). o and Newly~~

~~CM long-chain *32 atoms, areas plant. .p o < O r- VO NO VO - £ -o ITi 00 'I CN II 4—* r- oo of u peak U Q Z d d d d host C3 Oh 13 esters 06 10% < of~~

~~percentage~~

~~linseed number The 'Z "o CZ5 4-2. *3 H Q ® •£ © o -- ii ^ first c ^ • • na, • • H S cs ° 4r 00 0^ 00 00 00 U Oh 5 2 •*-* 2 < o. Urn "< E n a CM The Table u U U U Su 5u * 94~~

~~ether (RT= 26.7 min) (Figure 4-10 and Table 4-4). The acetate derivative of 0-~~

~~sitosterol acetate (RT= 30.1 min), stigmasterol acetate (RT= 28.8 min) and~~

~~campesterol acetate (RT= 27.6 min) from Phyla (Figure 4-10) were identified based~~

~~on comparative retention times of acetate derivatives of the standard 0-sitosterol~~

~~(RT= 30.18 min) (Figure 4-8), stigmasterol (RT= 28.88 min) (Figure 4-9) and~~

~~campestrol (RT= 28.15 min) (Figure 4-7).~~

~~Plant Sterols in Pinto Bean Diet~~

~~The peak retention times in the pinto bean diet were approximately matched with the common plant sterols 0-sitosterol (RT= 27.41 min), stigmasterol (RT= 26.42~~

~~min), and campesterol (RT= 25.72 min) (Table 4-3) and based on comparative retention times, they were identified in PBD as 0-sitosterol (RT= 27.37 min),~~

~~stigmasterol (RT= 26.29 min), and campesterol (RT= 25.72 min) (Figure 4-12).~~

~~Comparison of Sterols Isolated from Phyla nodiflora and the Pinto Bean Diet~~

The sterols in Phyla and PBD were quantified by using cholesterol as an internal standard and the results are presented in Table 4-5. The pinto bean diet has a trace amount of 0-sitosterol (3.10 pg/g), stigmasterol (1.04 pg/g) and campesterol

(1.03 pg/g). In contrast, Phyla nodiflora leaves contain 0-sitosterol (120.02 pg/g), stigmasterol (52.28 pg/g) and campesterol (17.74 pg/g). The reason for the low survival and adult production of phaon crescent on PBD may be that PBD does not supply sufficient amount of sterols for the growth and development of the phaon

~~crescent larvae. The host plant of the butterfly is relatively rich in these sterols.~~

~~Rearing P. phaon Larvae on PBD with Addition of Sterols~~

~~The amounts of sterols in Phyla nodifora and in PBD are shown in Table 4-5.~~

~~PBD was enriched by adding enough 0-sitosterol, stigmasterol and campestrol to 95~~

approximate the amount in the host plant. Newly hatched larvae were reared on this diet, on PBD only, and PBD with 10% host plant. Survival and number of adults were recorded. Adults were held with a host plant to see if they would lay eggs.

~~The survival and adults produced on PBD with addition of sterols, PBD with addition of 10% host plant and PBD only were significantly different with Chi-~~

Square=16.555, df=5, P <0.005 (Table 4-6). The means for numbers of adults produced on different diets were separated by pair wise comparison of the binary logistic regression coefficient. Adult production on PBD with addition of sterols was not significantly different from numbers of adults produced on PBD but was significantly less than the number of adults reared on PBD with 10% host plant

~~material. Thus addition of the sterols did not improve the artificial diet. 96~~

~~Figure 4-7. Gas chromatography analysis of /3-sitosterol (A) and derivatives; /3 sitosterol silyl ether (B) and /3-sitosterol acetate (C). 97~~

~~Figure 4-8. Gas chromatography analysis of stigmasterol (A) and derivatives; stigmasterol silyl ether (B) and stigmasterol acetate (C). 98~~

~~Campesterol acetate~~

~~i i n r~~

~~Figure 4-9. Gas chromatography analysis of campesteol (A) and derivatives; campesterol silyl ether (B) and campesterol acetate (C). 99~~

~~Figure 4-10. Gas chromatography analysis of /3-sitosterol, stigmasterol and campesterol from Phyla nodiflora (A), TMS derivatives (B) and acetate derivatives (C). 100~~

Figure 4-11. Separation of P. nodiflora sterols by thin layer chromatography. The sterols run along with the gray band as shown above. A region just above and including the gray band was scraped off the plate and the sterols were eluted with pentane. 101

campesterol, B) stigmasterol, and C) (3-sitosterol. » * * *

~~102~~

~~cd 2 -c 00 5! *- u 2 <0 derivatives. H id CN ,S Q Q ?£> *5 O~~

~~oo 27.60 IS o oo "O CN c w ro CN t"; CD acetate G -*— a3 «r> (D CO CN~~

~~and~~

c/3 00 > C/5 their o oo G \0 m 7.74 *c 'C and oo cu o £ §! •w •*— Cft « oo fa T3 in C campesterol c n- -C 7Z a3 stigmasterol, -*— id Oi CO CN o e cd (D OO 55, -C O w j(3-sitosterol, in o £ Lh 0,

~~4-3. o C/5 .2f u 02. jl> H on Table C/5 HG » v~~

~~103~~

~~o om 5 =1 CO~~

~~Q CD CQ SO cd CL. •*— ON ON oo G x Q> o ON On 41 U O u l/") CM 5~~

~~diet o e (D £ £ bean H G ON~~

~~and~~

~~nodiflora CM S w> O 00 CM t 'Sb o ; I =r CM cm" C P. 5~~

~~from~~

~~0 s- isolated 4> bO +-»~~

~~amount £ H ITT f-H co r- the Jg vd uS C (N~~

~~Comparison co o o o •- 2- 0> 4-5. a s H CO M E 1 *G co 'Q. CZ) u~~

~~Table •~~

~~104~~

~~.*3 Chi- 3 PBD T3 73 3 jo3 on 0 shows 44 o~~

~~and M CL, s 3~~

~~f—»~~

~~PBD T3 to Chi-Square 2 '5b Cl O~~

~~sterols, . s 2 • C3 a 1 C c • of by |i£ S he o “ 3 - diets bn 5 »— addition • H O o co co~~

~~V-.~~

~~C! Oh 03 with different ab £ S ° 3 o s S| PBD the 1~~

~~.>= on on~~

~~phaon 2 ^ § ’5-0 0 produced~~

~~•Sg-slS M <* 3 3 Phyciodes~~

~~of 1 0 c/3 L; O 13 ® C• .24 53 time o c number v c~~

~~Analysis 'C ° CO and OS 3 T 2 ^ II 53 p l- L_ 50 a. o~~

~~4-6.~~

~~Table 105~~

~~Conclusion and Discussions~~

~~In this chapter I have shown that PBD was improved by addition of small amounts~~

~~of linseed oil, olive oil and wheat germ oil. The original PBD formula does not contain~~

~~any additional oil supply. The modified PBD contains factors, which promote growth~~

~~and development of the phaon crescent. The modified diet enhances adult production and~~

~~makes adult production equal to PBD with 10% host plant. This represents a significant~~

~~and inexpensive improvement in the PBD formula. Even though the modified diet~~

~~produced more adults, they did not lay eggs.~~

~~The results of fatty acid composition of larvae reared on different diets are in~~

~~concurrence with reports from other species, including, the bollworms, Heliothis zea~~

~~(Schaefer, 1967), the Egyptian cotton leafworm, Prodenia litura (Levinson and Navon,~~

~~1969), the southwestern com borer, Diatraea grandiosella (Chippendale and Reddy,~~

~~1972), the silkworm, Bombyx mori (Ito and Nakasone, 1967), and the flour moths,~~

~~Ephestia spp. (Fraenkel and Blewett, 1946). Like many lepidopteran species (Thompson,~~

~~1973; Cookman et al. 1984), the predominant fatty acids of the phaon crescent (larvae~~

~~and adults) are C16:0, Cl 8: 08:2 and 1 , 08:3.~~

~~Dietary fatty acids enhance the growth rate of some lepidopteran larvae~~

~~(Chippendale and Reddy, 1972). The differences in fatty acid composition between~~

~~insects reared on the foliage (host plant, Phyla nodiflora) and those reared on the~~

~~artificial diet seem to be related to corresponding differences in larval food. Green leaves~~

~~of plants are generally high in 08:3, whereas PBD is high in 08:1 and 08:2 but low in~~

08:3. Thus, the low percentage of 08:1 and 08:2 and high percentages in 08:3 in P. nodiflora reared insects are associated with low levels of 08:1 and 08:2 and high level of 08:3 in P. nodiflora compared to the PBD. 106

~~Among foliage-reared insects, the fatty acid composition of larvae was similar to~~

~~that of their food, suggesting less synthesis in contrast to the artificial diet reared larvae~~

~~(Bridges and Phillips, 1972; Cookman et al. 1984 and Turunen, 1983). In contrast to the~~

~~phaon crescent larvae fed on PBD, Heliothis virescens (F.) larvae accumulate Cl 8:3~~

~~when fed on a pinto bean-based artificial diet (Dikeman et al., 1981). The larvae of~~

~~Tricoplusia ni (Hubner) did not accumulate Cl 8:3 when reared on artificial diets low in~~

~~C18:3 fatty acid (Grau and Terriere, 1971). Some lepidopteran species respond to low~~

~~level of Cl 8:3 in the diet by synthesizing more Cl 6:1 and Cl 8:1 (Turunen, 1974b).~~

~~In this study, fatty acid analyses show remarkable differences in the fatty acid~~

~~composition of body fat in phaon crescents (larvae and adults) reared on the different~~

~~diets, especially PBD versus PBD with added oils or 10% host plant. It is likely (but not~~

~~proven) that the beneficial effect of added oils on adult production is largely due to the~~

~~increase in available polyunsaturated fatty acids in the diet when one of the three oils is~~

~~added.~~

~~The sterols in the host plant Phyla nodiflora have been tentatively identified by GC~~

~~as /3-sitosterol, stigmasterol and campestrol. These are common sterols in many plants.~~

Phytophagous insects such as lepidopteran larvae ingest those major plant sterols and usually convert them to cholesterol. Unfortunately, addition of these three sterols together into the diet gave very poor adult production. The few adults produced did not lay eggs. CHAPTER 5 INTERNAL REPRODUCTION SYSTEMS of Phyciodes phaon

~~Introduction~~

~~The phaon crescent butterfly, Phyciodes phaon, can be reared to the adult stage on~~

~~a pinto bean diet, an artificial diet without addition of any host plant material. The~~

~~females, however, do not lay eggs when reared on this artificial diet. In order to maintain~~

~~a colony, it is necessary to rear some phaon crescents on the pinto bean diet with ground,~~

~~freeze-dried host plant leaves (10% in the diet) or on living host plants, Phyla nodiflora.~~

~~Adults from either of the latter two diets lay eggs that hatch. There may be several~~

~~reasons why females reared on the artificial diet fail to lay eggs, including (1) failure of~~

~~the ovaries to develop, (2) failure of vitellogenin production and growth of eggs, and/or~~

~~(3) failure to mate.~~

~~The reproductive system in female Lepidoptera consists of a pair of ovaries, lateral~~

~~oviducts, a medial oviduct and a gonopore that is covered in an inflection of the body~~

~~wall, forming a cavity, the genital chamber. The genital chamber is also called the bursa~~

copulatrix, and it initially receives the spermatophore containing the sperm. A duct from the bursa copulatrix leads to the sac-like spermatheca that stores the sperm. Paired lateral accessory glands are each connected to the median oviduct by a tubular duct. Each ovary is composed of a few to many ovarioles; each ovariole contains a series of egg chambers or follicles in which eggs accumulate yolk and mature. Although there may be many follicles in each ovariole, only a few eggs can be matured at one time because of the small size of the abdomen. Oocytes in progressively earlier developmental stages occur

~~107 108~~

~~in secondary follicles leading to the germarial region at the distal end of the ovariole. The~~

~~youngest oocytes occur near the apical germarium and the most mature oocyte occurs~~

~~near the pedicel leading to the lateral oviduct (Gullan and Cranston, 2000; Chapman,~~

~~1998). The distal end of each ovariole has a germarium in which mitosis gives rise to~~

~~primary oocytes. Each ovariole contains different sizes and stages of developing oocytes.~~

~~Ovarioles are anchored to the dorsal body wall by a terminal filament. Eggs ready to be~~

~~laid pass into the medial oviduct, receive sperm as they pass by the spermathecal duct,~~

~~and are deposited by the female. The accessory glands produce a glue-like material that~~

~~glues eggs to the substrate.~~

~~Vitellogenesis, the deposition of yolk in eggs, is under hormonal control in insects~~

~~and is variable in Lepidoptera with respect to temporal sequence and specific hormones~~

~~involved. Some Lepidoptera begin vitellogenesis in the late larval stages (the cecropia~~

~~moth, Hyalophora cecropia), or early pupal stage (the silkmoth, Bombyx mori), and the~~

~~hormone 20-hydroxyecdysone seems to be the main hormone. Some lepidopterans~~

~~mature eggs in the late pupal stage and have mature eggs when the adult emerges. Both~~

~~JH and 20-hydroxyecdysterone are thought to be important to vitellogenesis in these~~

~~lepidopterans. Some moths and butterflies have a relatively immature ovary at adult~~

~~emergence (the noctuid moth, Heliothis virescens and the monarch butterfly, Danaus~~

~~plexippus ) and only JH is believed to be important for vitellogenesis that follows adult~~

~~emergence.~~

~~The reproduction structures in male Lepidoptera consist of paired testes, the vas~~

~~deferens, paired accessory glands, the ejaculatory organ, and the aedeagus. In each 109~~

~~testis, there are a number of follicles in which spermatozoa are matured (Nation, 2002;~~

~~Davey, 1985).~~

~~In Lepidoptera, the internal organs of the male reproduction system begin to~~

~~develop in the larval stage and can be observed in mature larvae (Eaton, 1988). The~~

~~testes are fused to form a single median structure and attached dorsally to the fourth~~

~~abdominal segment. The vas deferens extends from the fused testes. The enlargement in~~

~~the vas deferens is called the seminal vesicle. The vas deferens is folded in the abdomen~~

~~and is about 25 mm long (Eaton, 1988). The accessory glands are about 35 mm long.~~

~~The ejaculatory duct transports the sperm package (spermatophore) to the gonopore.~~

~~Males have a single pair of accessory glands in Lepidoptera (Eaton, 1988; Chapman,~~

~~1998).~~

~~No information is available in the literature about the structure and physiology of~~

~~the reproductive system of female and male phaon crescent butterflies. The objectives of~~

~~this chapter are (1) to describe the female and the male internal reproduction system for~~

~~Phyciodes phaon and (2) to determine the developmental condition of the ovary of~~

~~female phaon crescents reared on the pinto bean diet.~~

~~Materials and Methods~~

~~Study Organism~~

~~The colony of Phyciodes phaon was established from females collected on the~~

~~University of Florida campus and vicinity. The host plant Phyla nodiflora was either~~

collected from natural areas as needed or maintained in small plots and planted in containers. The colony was maintained on potted or freshly cut host plants, and newly hatched larvae were transferred from the host plant to the pinto bean diet (PBD) for rearing. Newly emerged and 10-day-old adults from the host plant and pinto bean diet 110

~~were dissected and the internal reproductive organs were removed in a physiological saline solution and photographed.~~

~~Dissection~~

The abdomen of adults was brushed with a camel’s hair brush dipped in 70% ethyl alcohol to remove scales from the abdomen. The abdomen was opened ventrally from the first to the terminal abdominal segment. A physiological saline solution was used during dissection.

~~Results~~

~~The structure of the female internal reproductive organs is illustrated in Figures 5-~~

1, 5-2, and 5-3. Females reared on the host plant have a large amount of bright yellow fat body surrounding the ovaries upon emergence (Figure 5-1). There are 4 ovarioles in each ovary (Figure 5-2). Newly emerged adult females appear to have mature or nearly mature eggs in the most proximal follicles of each ovariole. The eggs are pale green

(Figure 5-5A), which is the same color of eggs that are laid by females reared on PBD with 10% host plant leaves. In 10-day-old females the fat body has largely been used up in metabolism and in providing yolk for maturing eggs. The ovaries in females reared on

~~the pinto bean diet with 10% host plant added are surrounded by fat body tissue that is somewhat lighter yellow in color than those reared on the living host plant (Figure 5-5 A).~~

~~Females reared on either living host plant or on pinto bean diet with added host plant~~

~~(10%) mate and lay eggs that hatch within a few days after emergence.~~

~~Females have the potential to lay large numbers of eggs. An ovariole has~~

~~approximately 49 follicles that can be seen at emergence. I did not determine whether additional follicles may be elaborated from the germarial region as the female ages. Ill~~

~~However, 49 follicles in each ovariole give a female the potential to mature at least about~~

~~400 eggs in the two ovaries.~~

~~Females reared on the pinto bean diet appear to have a mature ovary and to have~~

~~mature eggs in the proximal follicles (Figure 5-6A). They also have a large amount of fat~~

~~body tissue surrounding the ovaries, but the fat body is cream colored, apparently lacking~~

~~in the yellow pigment in the fat body of females reared on the host plant. The mature-~~

~~looking eggs are pale green. These females, however, do not lay the eggs.~~

~~The general structure of the male internal reproduction organs is shown in Figure 5-~~

~~4. The male reproduction system consists of fused testes forming one testicular body that~~

~~is round and dark reddish brown in color. The vas deferentia are swollen near the~~

~~midlength where seminal vesicles are located. The long proximal ends open into the round, milky white duplex shown in Figure 5-4A. This duplex also receives the ducts~~

~~from the paired accessory glands. The duplex also is fused with a tube called the primary simplex, which is shown in Figure 5-4A on the left side. The primary simplex terminates~~

~~in the cuticular simplex, which is dark brown cuticular structure and is shown in Figure~~

~~5-4. The structure of the testes in males reared on the pinto bean diet with 10% host plant~~

~~(Figure 5-5B) looks like the testes reared on the living host plant (Figure 5-4A).~~

The male reared on the pinto bean diet appeared to have a little different testes structure (Figure 5-6B) than males reared on the living host plant (Figure 5-4) or PBD with 10% host plant (Figure 5-5B). The testes are light red in color and not round (Figure

~~5-6B). 112 113~~

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~~Discussion and Conclusions~~

Female phaon crescents have four ovarioles in each of 2 ovaries; each ovariole contains approximately 49 egg follicles, but additional follicles may be budded from the germarium as female ages. Newly emerged females reared on the host plant, pinto bean diet with added host plant leaves, and those reared on host-free pinto bean diet appeared to have mature ovaries containing some mature eggs ready to be laid. Only females reared on the host plant or artificial diet containing 10% by weight of host plant, however, lay eggs. Females reared on the pinto bean diet without host plant material do not lay their eggs. Females lay their eggs in clusters on the underside of host leaves. Up to 187 eggs have been observed in a single cluster, and a female can lay several clusters.

~~Thefiighest number of eggs observed to be laid by a female is 434.~~

Male phaon crescents also have a mature reproductive system at emergence. The male reproduction organs include fused testes, vas deferentia, paired accessory glands, an ejaculatory structure and duct. Enlarged regions of the vas deferentia serve as a sperm reservoir and seminal vesicle.

~~The failure of females reared on the host-free pinto bean diet to lay eggs cannot yet~~

~~be explained. The ovaries appear to be mature and contain mature eggs ready to be laid.~~

~~The only difference that has been observed in the appearance of ovaries from females~~

~~reared on artificial diet and those reared on the host plant is in the quantity of fat body~~

(more in those reared on the host plant) and in the color of the fat body (bright yellow in females from the host plant; cream colored in females from the artificial diet). The failure of the diet reared females to lay eggs may be related to failure to mate. The only difference between males reared on artificial diet and those reared on the host plant has been observed in the appearance of testes. The testes from artificial diet reared males are 119

~~bigger (swollen) and light red in color. The failure of the artificial diet reared males to mate may be related to defects in testes structure.~~

Mating has not been observed among males and females reared on host-plant free artificial diet. Mating is a stimulus for oviposition and sometimes oogenesis in some insects. For example, oviposition in the Australian field cricket, Teleogryllus sp., and the onion fly, Delia sp., is enhanced as a result of mating. Although unmated female laid only a few eggs, mated females laid many eggs (Chapman, 1998). In addition, males of some lepidopterans transfer prostaglandins or prostaglandins-synthesizing chemicals to the female during mating and these stimulate oviposition (Stanley-Samuelson, 1994).

Failure to mate might be caused by a failure of sex pheromone production in the butterflies. A pheromone has not been experimentally established in this species, but such a pheromone seems likely. If this should prove to be the principal reason for oviposition

failure, it may mean that some chemical substance present in the host plant is needed as a precursor for pheromone synthesis. Similar host plant precursors are known for some other insect pheromones. CHAPTER 6 CONCLUSIONS

~~This is an easy butterfly to rear in the laboratory; the adults mate readily in the~~

~~laboratory in small cages, and laboratory cultures have relatively low mortality after the~~

~~first instar. I have not seen any pathogen or disease associated with the phaon crescent~~

~~during three years of continuous rearing. The host plant is widely available, and requires~~

~~minimal care to keep in small pots or in outdoor plots. These characteristics of butterfly~~

~~and host plant make the phaon crescent a valuable model butterfly for further research. It~~

~~is also potentially useful as a teaching tool in schools and a convenient display butterfly~~

~~for butterfly houses.~~

~~Based on head capsule measurements, P. phaon has five instars. The external~~

~~characteristics of each instar are described. The sex of individuals can be determined~~

~~readily by observation of certain external characters of pupae. The egg-adult~~

~~developmental time on the natural food plant, Phyla nodiflora, is about 26 days under~~

~~laboratory conditions at 23-25°C.~~

~~Several artificial diets were tested for rearing Phyciodes phaon in the laboratory. A pinto bean diet (PBD) was found to be the best diet of several diets tested. The phaon~~

crescent larvae successfully completed larval development on this diet with a little longer development time comparing to the larvae developing on the host plant. Mature larvae were able to pupate and adults successfully emerged. Adults reared on PBD survived in the laboratory for 10 days without laying any eggs.

~~120 121~~

~~Freeze-dried host plant materials were added into PBD to modify it for the phaon~~

~~crescent larvae. Survival and adult production were improved and the adults produced~~

~~laid eggs.~~

~~PBD was improved for P. phaon by addition of 5% glucose and 5% Beck’s salt.~~

~~Larval survival and the adult production were higher on this modified PBD than PBD~~

~~alone but adults did not lay eggs.~~

~~The host plant, Phyla nodiflora, was extracted with polar and nonpolar solvents and~~

~~the extracts were added to PBD. Survival and adult production was increased only with~~

~~addition of methanol extraction of host plant flowers. However, this extract did not~~

~~promote egg laying. The addition of the other extracts and the extracted residues did not~~

~~improve the diet and had no effect on adult production.~~

~~PBD was modified for P. phaon larvae by adding freeze-dried clover. Although~~

~~the larvae survive poorly on living clover leaves, addition of 10% freeze-dried clover to~~

~~PBD works as well as addition of the host plant to PBD. Four generations were reared~~

on PBD with freeze-dried clover leaves without problems. One possible explanation might be that when the clover leaves are grounded in liquid nitrogen and freeze-dried some plant chemicals which may act as feeding deterrents are destroyed.

~~The original PBD formula does not contain any additional oil to supply lipids.~~

~~PBD was modified by addition of small amounts of linseed oil, olive oil and wheat germ oil. The PBD with any one of these oils enhances growth and survival of the phaon~~

~~crescent better than PBD alone. Moreover, it enhances adult production and makes adult production equal to PBD with 10% host plant. This represents a significant and 122~~

~~inexpensive improvement in the PBD formula. Even though the modified diet produced more adults, they did not lay eggs.~~

~~The fatty acids in the body of larvae and adults were determined by gas chromatography. The predominant fatty acids of the phaon crescent are Cl 6:0, Cl 8:1,~~

~~C18:2 and C18:3. The differences in fatty acid composition between insects reared on the host plant and those reared on the artificial diet seem to be related to corresponding~~

~~differences in larval food. The artificial diet (PBD) is high in Cl 8: 1 and C18:2 but low in~~

Cl 8:3 in the fatty acids. Insects reared on PBD alone were especially low in Cl 8:3 polyunsaturated fatty acid known to be important to Lepidoptera. Insects reared on the host plant had a high percentage of Cl 8:3 in their lipids.

The sterols in the host plant Phyla nodiflora have been tentatively identified by GC as /3-sitosterol, stigmasterol and campestrol. The phaon crescent larvae ingest those plant sterols and probably convert them to cholesterol, the sterol usually used by insects as a precursor to the molting hormone, ecdysone. Surprisingly, however, addition of these three sterols together into the diet gave very poor adult production, and the few adults produced did not lay eggs.

Female phaon crescents have four ovarioles in each of 2 ovaries; each ovariole contains approximately 49 egg follicles, but additional follicles may be budded from the germarium as female ages. Newly emerged females reared on the host plant, pinto bean diet with added host plant leaves, and those reared on host-free pinto bean diet appear to have mature ovaries containing some mature eggs ready to be laid. Only females reared on the host plant or PBD containing 10% host plant leaves, however, lay eggs. Females reared on the pinto bean diet do not lay their eggs. The failure of females reared on the 123

~~host-free pinto bean diet to lay eggs cannot yet be explained. The ovaries appear to be~~

~~mature and contain mature eggs ready to be laid. The only difference that has been~~

~~observed in the appearance of ovaries from females reared on artificial diet and those~~

~~reared on the host plant is in the quantity of fat body (more in those reared on the host~~

~~plant) and in the color of the fat body (bright yellow in females from the host plant;~~

~~cream colored in females from the artificial diet). The failure of the diet-reared females~~

~~to lay eggs may be related to failure to mate~~

~~Male phaon crescents reared on the PBD appeared to have some abnormal~~

~~differences in the structure of the internal reproductive organs. The testes on artificial~~

~~diet reared males are swollen and light red in color. The failure of the artificial diet-~~

~~reared males to mate may be caused by a failure in testes structure.~~

~~Another possible reason might be a failure of sex pheromone production in the butterflies. A pheromone has not been experimentally established in this species, but~~

~~such a pheromone seems likely. If this should prove to be the principal reason for failure~~

~~to mate, it may mean that some chemical substance present in the host plant is needed as a precursor for pheromone synthesis. Host plant precursors are known for some insect pheromones.~~

~~A few larvae were discovered feeding on white clover plants growing in a pot of~~

~~Phyla tiodiflora. These larvae were transferred to white clover leaves and were not allowed to feed upon the host plant in order to see if they could survive on the clover.~~

~~Survival was poor but a few adults were produced which only lived 3 days. This suggested, however, an experiment to add ground freeze-dried clover leaves to PBD.~~

~~Surprisingly, PBD with 10% freeze-dried clover produced results equal to PBD with 10% 124~~

~~host plant leaves, and adults reared on PBD with 10% freeze-dried clover laid eggs. This~~

~~suggests that plants other than the natural host plant may contain all the components~~

~~necessary for increased survival and normal reproduction. This also means that the~~

~~phaon crescent could be reared in locations where its natural host plant does not occur by~~

~~adding freeze-dried white clover leaves into PBD.~~

~~I have shown in this work that the phaon crescent butterfly can be reared on an~~

~~artificial diet with no host plant added. I have shown that artificial diet can be improved~~

~~in nutritive quality by addition of 5% glucose, 5% Beck’s salt mixture, and a small~~

~~quantity of one of several oils. All of these additions to PBD are readily available and~~

~~inexpensive and give adult production equal to adding host plant material to PBD. One~~

~~problem remains and that is those adults reared on the completely artificial diet do not~~

~~mate and lay eggs. This problem needs to be solved in future research.~~

~~The ease with which the phaon crescent butterfly can be reared in the laboratory on~~

~~an artificial diet and the wide availability of the host plant and/or white clover suggest~~

~~that this may be a good butterfly for rearing in classrooms to demonstrate butterfly life cycles, butterfly conservation and basic insect biology. LIST OF REFERENCES~~

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Bridges, J.R., and J.R. Phillips. 1972. Fatty acids of the imago of Heliothis zea (Lepidoptera:Noctuidae): an approach to determining larval hosts. In Distribution, Abundance and Control of Heliothis species in Cotton and Other Host Plants. Soth. Coop. Ser. Bull. 169:72-79.

Brower, L.P., P. B. McEvoy., K.L. Williamson., and M.A. Flannery. 1972. Variation in cardiac glycoside content of monarch butterflies from natural populations in eastern North America. Science 177:426-429.

Brower, L.P., J.N Seiber., C.J. Nelson., S.P. Lynch., M.P. Hoggard., and J.A. Cohen. 1984. Plant-determined variation in cardenolide content and thin layer chromatography profdes of monarch butterflies, Danaus plexippus reared on

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~~Chow, M.L., and J.L. Nation. 1975. Effect of age upon fatty acid composition of adult Caribbean fruitflies, Anasterpha suspensa. Short communication. Ext. Exp & Appl. 18:515-517.~~

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~~Cookman, J.E., M. J. Angelo, F. Slansky and J. L. Nation. 1984. Lipid content and fatty acid composition of larvae and adults of the velvetbean caterpillar, Anticarsia~~

~~gemmatalis, as affected by larval diet. J. Insect Physiol. 30:523-527.~~

~~Dadd, R.H. 1973. Insect nutrition: Current developments and metabolic implications. Annu. Rev. Entomol. 18: 381-420.~~

Dadd, R.H. 1985. Nutrition: Organisms, pp. 313-390. In G A. Kerkut and L.I. Gilbert [Eds.], Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 4. Pergamon, Oxford. National Academy Press, Washington, DC.

~~Davis, G.R.F. 1968. Phagostimulation and consideration of its role in artificial diets. Bull. Entomol. Soc. Am. 14: 27-29.~~

~~David, W.A.L., and B.O.C. Gardiner. 1966. Mustard oil glucosides as feeding stimulant for Pieris brassicae larvae in a semi-synthetic diet. Ent. Exp & Appl. 9:247-255.~~

~~Davey, 1985. K.G. The male reproduction tract, pp 1-13, in G. A. Kerkurt and L. I. Gilbert (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol.l, Pergamon Press, Oxford.~~

~~De Boer, G., and F.E. Hanson. 1982. Chemical isolation of feeding stimulants and~~

deterrents from tomato for the tobacco homworm, pp. 371-372, in J. A. Visser and A. K. Minks (eds.). Proceedings of the Fifth International Symposium on Insect-Plant Relationships. Pudoc, Wageningen, the Netherlands.

~~Dethier, V.G. 1954. Evolution of feeding preferences in phytophagous insects. Evolution 8:33-54.~~

~~Dethier, V.G. 1980. Evolution of receptor sensitivity to secondary plant substances with~~

~~special reference to deterrents. Am. Nat. 1 15:45-66.~~

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Hanife Gene was bom in 1971 in Bursa, Turkey. She earned a Bachelor of Science degree in plant protection at University of Uludag in June 1994 in Bursa, Turkey. After her graduation from Uludag University, she started her Master of Science degree in entomology at the same university. In 1995, she was awarded a scholarship by the

~~Ministry of Education of Turkey to pursue the Master of Science and Doctor of~~

~~Philosophy degrees in the USA. She received her Master of Science degree from the~~

Plant Pathology Department at the University of Florida in December 1998. She then continued working towards a Ph.D. degree in entomology with Dr. James L. Nation. She expects to receive the Ph.D. in December 2002.

~~132 —~~

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. z ones L. Nation, 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~~

~~Thomas C. Emmel Professor of Zoology~~

~~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.~~

~~Charles L. Guy (/ Professor of Environmental Horticulture~~

~~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.~~

~~— Susan Halbert Courtesy Assistant Professor, Division of Plant Industry~~

~~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.~~

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. December, 2002 Dean, College of Agricult’ Life Sciences

~~Dean, Graduate School~~