Copyright by Kyung-Jin Min 2003 · The Dissertation Committee for Kyung-Jin Min certifies that this is the approved version of the following dissertation: NEUROENDOCRINE REGUALTION

Copyright

by

Kyung-Jin Min

2003

The Dissertation Committee for Kyung-Jin Min

certifies that this is the approved version of the following dissertation:

NEUROENDOCRINE REGUALTION OF MIGRATION AND

REPRODUCTION IN THE GRASSHOPPER

MELANOPLUS SANGUINIPES FABRICIUS

Committee:

_________________________________Mary Ann Rankin, Supervisor

_________________________________Lawrence Gilbert

_________________________________Ulrich Mueller

_________________________________Beryl Simpson

_________________________________ Harold Zakon

NEUROENDOCRINE REGULATION OF MIGRATION

AND REPRODUCTION IN THE GRASSHOPPER


by

KYUNG-JIN MIN, B.S.; M.S.

DISSERTATION

Presented to the Faculty of the Graduate School of

the University of Texas at Austin

in Partial Fulfillment

of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

The University of Texas at Austin

December, 2003

This work is dedicated to my family, friends, and my Lord Jesus.Without them it would not have been possible.

v

ACKNOWLEDGMENTS

I am grateful to the members of my dissertation committee, Drs. Mary Ann Rankin,

Lawrence Gilbert, Harold Zakon, Ulrich Mueller and Beryl Simpson, for their advice,

encouragement, and helpful questions. I am indebted to my supervisor, Dr. Mary Ann

Rankin for her support and guidance. I really appreciate the time, energy and patience she

has devoted to me during last five and half years.

I acknowledge all members of the Rankin Lab. I thank Dr. Tina Taub-Montemayour and

Jack Kent for their encouragement and hospitality. Some of my work was not possible

without them. Special thanks for Tina. She was like a sister to me throughout my

graduate career. I was lucky to spend time with Cristina Jaramillo, Nathan Jones and

Terri Bartlett. They were all good friends and mentors. I thank Nathan for his valuable

work on the feeding experiment.

I want to thank Dr. David Borst at Illinois State University, who helped me establishing

the radioimmunoassay technique for JH titer determination. Without him, JH titer

determination, the most important part of my work, was not possible. He helped me with

many insightful discussions and critical review of several papers.

Dr. Klause Linse of the UT Protein Micro Facility helped using HPLC and Peptide

Analysis. Thanks for his time and efforts.

vi

I had the luck to have the help of exceptional undergraduate researchers: Leena Patel,

Alexa Lim, Jessica Weinkle, Santosh Dixit, Payal Patel and Harish Mangipudi. Some of

my work was helped or initiated by them. Maintaining the grasshopper colony would not

have been possible without the help of work-studies. Thanks to Murray Sanders, Marie

Nguyen, Jose Sanjorjo , JD Dong and Andria Rodriguez for their work.

I am grateful to the San Carlos Apache Reservation for permission to collect

grasshoppers on tribal land. Larry Lawrence at USDA helped me with the complete

nymphal survey of the grasshoppers, and Clark Richens at tribal land operations helped

locating grasshoppers. Grasshopper collection in Colorado was helped by many helpers.

Thanks to Chang Wook Lim, Kwang Hyun Kim, Hyun Sik Choi and Hyo Sik Choi.

This work was supported in part by NSF grant IBN-0235892, the Orthopterists’ Society

Fund and the Program of Zoology Fellowships: Zoology Fellowship, Blair Fellowship

and Hamilton Fellowship.

Finally, I give my love and thanks to my family and friends. My parents deserve much

credit for their constant support and encouragment. Many Korean friends in Austin,

including the choir members of the Korean Presbyterian Church, were an energy booster

in the tedious life in the US. Su Yeon, you have been the oak that has let this cypress

grow. Thank you for your support.

vii

NEUROENDOCRINE REGULATION OF MIGRATION

AND REPRODUCTION IN THE GRASSHOPPER


Publication No. ________

Kyung-Jin Min, Ph.D.

The University of Texas at Austin, 2003

Supervisor: Mary Ann Rankin

This study examines the endocrine regulation of migration and reproduction in the

migratory grasshopper, Melanoplus sanguinipes. Even though, in many insects, migration

is considered as energetically costly and seems to extract a cost in terms of reproduction,

this species has shown to exhibit enhanced reproduction after performance of even one

long, tethered flight to exhaustion. This study sought to understand the physiological

mechanisms underlying this enhanced reproduction after long flight.

The lipid content of eggs of long fliers flown to exhaustion was not different from

that of non fliers. In contrast, protein content of eggs was significantly lower in long

fliers flown to exhaustion compared to non fliers and 1 hr fliers.

Titer determinations of hemolymph AKH (Adipokinetic Hormone) were done at

rest and after various periods of flight. No significant differences in AKH levels in CC

viii

(Corpora Cardiaca) were detected between non-migrants, animals that had flown for one

hour to identify them as migrants, and animals that had flown to exhaustion. Adipokinetic

response to AKH I was greater in migrants than in non-migrants.

JH (Juvenile Hormone) levels increased on days 5 and 8 in animals flown to

exhaustion on day 4 but not in one-hour or non-flier controls. Treatment of grasshoppers

with JH III induced early reproduction as did performance of long duration flight. This

suggests that increased JH titer after performance of a long-duration flight is at least one

component of flight-enhanced reproduction. AKH treatment had no effect on JH titers.

There was no difference in the amount of peptide in the CC after long duration

flight. However, there were differences in the amount of peptides in hemolymph, and

partial sequence information of the peptides revealed that it is identical to that of serine

protease inhibitor in locusts.

No difference in mating frequency or duration after long flight was observed

between non fliers and long fliers. However, females after long flight consumed

significantly greater quantities of food and displayed increased digestive capacity for

lipid. These results suggest that nutritional compensation by changes in feeding behavior

and food utilization could partially account for increased reproduction after long flight in

M. sanguinipes.

ix

TABLE OF CONTENTS

List of Tables.……………………………………………………………………...xiv

List of Figures...…………………………………………………………………….xv

Chapter 1 General Introduction .……………………………………………………..1

Adaptive Significance of Flight...…………………………………………..……..1

Cost of Insect Flight……………...…………………………………………….….2

Counteractive Measures to Reproductive Cost of Flight...….……………….…....3

Physiology of Flight in M. sanguinipes………………..…………………….……4

Possible Linking Factors- Endocrine Factors…...……..……………………….....5

Possible Linking Factors- Nutritional Factors…..……..…………………….…....7

Summary of Possible Hypotheses…….………………..……………………..…...9

Significance of the Present Study…………..…...……..…………………….…....9

Chapter 2 General Methods; Confirmation of Flight Enhanced Reproduction…...11

General Methods……………………………………………………………..11

Field Collection of Grasshoppers…………………………………….11

Lab Culture………..…………………………………………………12

Hatching…………………………………………………………...…13

Tethered-flight Test…..……………………………………………...14

Comparison of Migrants and Non Migrants.………………………...15

Mating and Egg Pod Collection……………………………………...16

Statistical Test……………………………………………………………16

Confirmation of Flight Enhanced Reproduction……..……………………..16

x

Chapter 3 Lipid Transport into Ovary during Long Duration Flight………………23

Introduction………………………………………………………………….23

Materials and Methods……………………………………………………….25

Lipid, Carbohydrate and Protein Analyses.....……………………….25

Tethered Flight Test………………………...……………………….25

Results………………………………………………………………………..25

Discussion……………………………………………………………………26

Chapter 4 Relationship among Adipokinetic Hormone, Migratory Propensity and Reproduction……………………………………………………………….....34

Introduction…………………………………………………………………..34


Hemolymph Collection………………...…………………………….35

Hemolymph Extraction and Separation of AKH I by HPLC………..35

Radioimmunoassay...………………………………………………...36

Measurement of Hemolymph Volume……………………………….37

CC Dissection and Extraction……….……………………………….37

Quantification of CC AKH Content………………………………….38

Examination of AKH Response……..……………………………….40

Injection of AKH to Simulate Long Duration Flight and OvipositionCheck………..…………………………………………………………...40

Results………………………………………………………………………..41

xi

AKH titers...……………………………………………………………...41

Storage of AKH in CC.…………………………………………………..42

Adipokinetic Response to Synthetic Lom AKH I and Scg AKH II…43

Effect of AKH Injections on Reproduction………………………….43

Discussion……………………………………………………………………44

Chapter 5 Juvenile Hormone Titer after Long Duration Flight……………………55

Introduction…………………………………………………………………..55


Flight Assay and Hemolymph Collection...………………………….55

Radioimmunoassay for JH III……………...………………………...56

JHE Assay……………………………………………………………57

JH III Treatment and Oviposition Check….…………………………57

AKH I Injections..……………………………………………………58

Results………………………………………………………………………..58

JH titer after long flight………………………………………………59

Correlation between increased JH levels and early reproduction……59

Effect of JH on AKH titer.. ………………………………………….59

Existence of circadian rhythm of JH...……………………………….60

Discussion……………………………………………………………………61

Chapter 6 Search for Peptidic Molecular Makers in CC and Hemolymph after LongFlight………...…………………………………………………...71

Introduction…………………………………………………….………………...71

xii

Materials and Methods………………………………………………….………..72

Hemolymph collection………..………………………………………….72

Hemolymph extraction………………………………….………………..72

RP-HPLC………………………………….……………………………..72

MALDI-MS…………………………………………….………………..73

HPLC tendem ESI-MS...……………………………….………………..73

Results……………………………………………………………………………74

CC Profile Comparison………...……………………….………………..74

Hemolymph Profile Comparison……………………….………………..74

Discussion….….…………………………………………………………………75

Chapter 7 Nutritional Supplementation after Long Duration Flight by Mating andFeeding………………………………………………………………...……81

Introduction…………………………………………………………………..81


Observation of Mating…………………....………………………….82

Grasshopper Rearing in Feeding Experiment…...…………………...83

Adult Feeding Rate Determination…………………………..………83

Collection of Weight Data……………..….…………………………83

Frass Analysis…..……………………………………………………84

Calculation of Body Weight…………………………………………85

Preliminary Experiment…….………………………………….……85

xiii

Results………………………………………………………………………..86

Mating Frequency and Duration after Long Duration Flight...………86

Feeding Rate and Digestibility after Long Duration Flight…….……86

Discussion……………………………………………………………………87

Chapter 8 Conclusions….....……………...……………………………………………...95

Bibliography......…………………………………………………………………………99

Vita……………………………………………………………………………………...110

xiv

LIST OF TABLES

Table 5.1. Hemolymph JH titers (ng/ml) after topical application of 50µg JH III in

methanol on day 5. (N=5 each group) ………………………………………….............66

Table 5.2. Hemolymph JH titers (ng/ml) by time of collection (N=8 each group)...........66

Table. 7.1. Mating frequency and duration after long duration flight…………………...90

xv

LIST OF FIGURES

Figure 2.1. Age of first oviposition versus flight behavior……………………………....19

Figure 2.2. Number of eggs in the first egg pod versus flight behavior…………………20

Figure 2.3. Hatching rate versus flight behavior…………………………………………21

Figure 2.4. Percentage of females ovipositing……………………………………….…..22

Figure 3.1. Schematic diagram of lipid mobilization by lipophorin during flight in

locust……………………………………………………………………………………..30

Figure 3.2. Lipid content of eggs………………………………………………………...31

Figure 3.3. Carbohydrate content of eggs………………………………………………..32

Figure 3.4. Protein content of eggs………………………………………………………33

Figure 4.1. Standard curve for RIA assay of AKH……………………………………...49

Figure 4.2. Change in AKH titers and lipid concentrations during flight……………….50

Figure 4.3. Comparison of AKH in one pair of CC of flight-tested females……………51

Figure 4.4. Standard curves for synthetic AKH I and II used for calibration…………...52

Figure 4.5. Adipokinetic response to injected synthetic hormones……………………..53

Figure 4.6. Effect of repeated AKH injections on the age of first oviposition…….……54

Figure 5.1. Long duration flights cause an increase in JH levels………………….……67

Figure 5.2. Juvenile hormone esterase levels after long flight…………………………..68

Figure 5.3. Change in age at first oviposition after JH treatment……………………….69

Figure 5.4. JH titers after treatment with physiological doses of AKH I……………….70

Figure 6.1. Mass profile of CC extracts…………………………………………………77

xvi

Figure 6.2. HPLC profile of pooled hemolymph after long duration flight to

exhaustion………………………………………………………………………………..78

Figure 6.3. Amplified insert of fig. 6.2…..………………………………………………79

Figure 6.4. Mass data of fraction 1-5 in unflown control (UF) and long fliers flown to

exhaustion (LF-E)…………………………………………………………………….….79

Figure 7.1. Average feeding rate of non-fliers (NF) and long-fliers flown to exhaustion

(LF-E)……………………………………………………………………………………91

Figure 7.2. Lipid content in frass of non-fliers (NF) and long-fliers flown to exhaustion

(LF-E)……………………………………………………………………………………92

Figure 7.3. Carbohydrate content in frass of non-fliers (NF) and long-fliers flown to

exhaustion (LF-E)………………………………………………………………………..93

Figure 7.4. Protein content in frass of non-fliers (NF) and long-fliers flown to exhaustion

(LF-E)……………………………………………………………………………………94

1

CHAPTER 1

GENERAL INTRODUCTION

This study examines the relationship between migration and reproduction in a

migratory grasshopper, Melanoplus sanguinipes. In many insects, migration is considered

to be an energetically costly activity and seems to be correlated with a decrease or delay

in reproduction (Dingle, 1972; Roff, 1977; Denno and Dingle, 1981; Lidicker and

Caldwell, 1982). This species has been shown to exhibit enhanced reproduction after

performance of even one long, tethered flight to exhaustion (i.e. voluntary cessation of

the flight) (McAnelly and Rankin, 1986b; Rankin and Burchsted, 1992). Long fliers

displayed earlier onset to first oviposition, production of more egg pods and better

hatching success compared to migrants that had not made a long-duration flight. This

study seeks to understand the physiological mechanisms underlying this enhanced

reproduction after long flight. The results will provide important information about

physiological interactions between two key life history traits, migration and reproduction

in a migratory pest species, and will provide the basis for future work examining the

evolution of migration.

APAPTVE SIGNIFICANCE OF FLIGHT

Insects are the largest group of described species in the Animal Kingdom. This

success is due in part to their flying ability and the scope it offers for mobility, dispersal

and migration. Flight provides insects with many benefits such as escape from danger or

2

harsh environmental conditions, food or mate location, range extension and colonization

of new habitat patches (Johnson, 1969; Dingle, 1985).

COST OF INSECT FLIGHT

Even though there are many benefits associated with insect flight, there are also

potential costs as well. First, insect flight can increase the risk of predation. Flight

increases the visibility of an insect and thus may increase the possibility of being

captured by some predators. Second, insect flight entails energetic and developmental

costs associated with constructing the flight apparatus. Lastly, there are also metabolic

and reproductive costs associated with flight itself (Johnson, 1969; Dingle, 1985; Roff,

1977; Zera, 1984). In this study I have focused on the reproductive cost of migration.

In many insects, the utilization of energy during flight seems to result in a cost in

terms of reproduction. After long duration flight some insects produce fewer eggs and/or

delay the age at first oviposition, in addition to the increased risk of mortality (Dingle,

1972; Roff, 1977; Denno and Dingle, 1981; Lidicker and Caldwell, 1982). This is

particularly true in species that display morphological differences between non migrants

and potential migrants where possession of wings is reflected in slower nymphal

development, increased time to first oviposition, decreased numbers of developed

ovarioles, decreased numbers of eggs per clutch, or decreased overall fecundity (Zera,

1984; Dixon, 1972; Roff, 1977; Walters and Dixon, 1983). The idea of reproductive cost

due to migration is also supported, in some insects, by interactions between the ontogeny

of flight and reproduction. Johnson (1969) proposed the “oogenesis-flight syndrome”

3

suggesting that migration is restricted to the post-teneral, pre-reproductive period in most

insects, and that migration and reproductive development are physiologically competing

processes.

COUNTERACTIVE MEASURES TO REPRODUCTIVE COST OF FLIGHT

Although conventional wisdom might predict that performance of migratory flight

would reduce or delay reproduction, an insect colonist might be expected to display a

suite of life history characters that involve both adaptations for long flight and rapid and

prolific reproduction (Lewontin, 1965; Mayr, 1965; Palmer, 1985; Rankin and Burchsted,

1992). Indeed, long duration flight has no observed deleterious effect on reproduction in

some insects and even stimulates reproduction in a few species (Johnson, 1958; Highnam

and Haskell, 1964; Rygg, 1966; Slansky, 1980). This is, however, very unusual, and the

underlying mechanisms by which long-distance flight stimulates reproduction are not

understood in any insect.

Melanoplus sanguinipes, a grasshopper that can be a highly mobile and successful

insect colonist, is a species in which long duration migratory flight appears not to elicit a

cost in terms of reproductive output. Indeed, in highly migratory populations, migration

appears to accelerate reproductive development and to enhance reproductive success over

the entire lifetime of the insect. After even one long duration flight to exhaustion (i.e.

voluntary cessation), it shows early onset of first oviposition, production of a larger

number of egg pods, increased hatching success and lifetime fecundity (McAnelly and

Rankin, 1986b; Rankin and Burchsted, 1992). This observation leads to questions

4

concerning mechanisms by which such flight-enhanced reproduction could be

accomplished. Understanding the underlying mechanisms of this effect is the subject of

this study.

PHYSIOLOGY OF FLIGHT IN M. sanguinipes

Lipid reserves in the thorax and eviscerated abdomen are significantly reduced

after flight to voluntary cessation in M. sanguinipes (Kent, et al., 1997; Kent & Rankin,

2001), indicating that lipid is an important flight fuel in this species as it is in locusts

(Goldsworthy, 1983). Also as in locusts, hemolymph carbohydrate is rapidly consumed

during the early stages of flight, while hemolymph lipid is elevated during the same

period. Glycogen stores are relatively larger in M. sanguinipes than in locusts and are

mobilized over a relatively longer period of time (Kent et al., 1997).

The adipokinetic hormones (AKHs—at least three have been isolated in locusts)

are members of a family of short peptides with similar amino acid sequences that have

various metabolic functions in different insects (Keeley et al., 1991). In locusts and in M.

sanguinipes, AKH I is responsible for lipid mobilization during flight. The AKHs are

produced in the glandular lobes of the CC, a neurohemal/endocrine gland that both stores

brain neurosecretions and produces neurohormones. We have identified only AKH I and

II in M. sanguinipes, with amino acid sequences identical to that of the corresponding

locust hormones, Lom-I and Scg-II, respectively (Taub-Montemayor et al., 1997, Taub-

Montemayor et al., 2002). Injection of exogenous AKH or extract of M. sanguinipes CC

5

elevates hemolymph lipid but does not affect hemolymph carbohydrate (Kent et al.,

1997).

POSSIBLE LINKING FACTORS – ENDOCRINE FACTORS

Previous work suggests that the neuroendocrine system may be the linking factor

between migration and reproduction. For example, both migration and reproduction are

influenced by juvenile hormone in some migratory insects (Rankin, 1974, 1978, 1980;

Coats et al., 1987). In locusts, increase in oocyte growth with flight is accompanied by

decrease in paraldehyde fuchsin staining material in the corpora cardiaca (CC) (Highnam

and Haskell, 1964). Cyclic changes in the volume of the corpora allata (CA) associated

with oocyte growth are also affected by flight activity. These results suggest that flight

may stimulate release of neuroendocrine factors necessary for oocyte growth and/or CA

activity. A similar phenomenon may be occurring in M. sanguinipes that perform long

duration flights.

I chose to study two hormones possibly involved in the interaction between

migration and reproduction. These are juvenile hormone (JH) and adipokinetic hormone

(AKH). In most insects, these two hormones play a significant role in flight and

reproduction with little known interaction between them.

Juvenile hormone

Juvenile hormone is a sesquiterpenoid that has various functions and is produced

by the corpora allata (CA). It regulates embryogenesis, larval and adult development,

6

metamorphosis and reproduction in insects (Riddiford, 1994). It is also involved in

control of diapause, migration, polyphenism, behavior and metabolism (Roe and

Venkatesh, 1990).

Control of reproductive development in M. sanguinipes seems to be similar to

locusts. As in locusts, a peak of JH synthesis occurs with the onset of previtellogenic

growth in each cycle of oocyte development in M. sanguinipes. A decline in JH synthesis

is correlated with onset of oviposition (McCaffery and McCaffery, 1983), suggesting that

JH may act as an on/off switch in the process of oocyte maturation and oviposition. In

locusts, as mentioned earlier, cyclical changes in the volume of CA (the JH producing

organ) are associated with oocyte growth and also affected by flight activity (Highnam

and Haskell, 1964). It means there could be possible regulation of CA activity or juvenile

hormone production by flight activity. The idea of possible regulation of CA activity or

JH by flight activity is also supported by the fact that both migration and reproduction are

regulated by juvenile hormone. Therefore, in order to elucidate the relationship between

flight and reproduction, it is necessary to examine possible JH changes in response to

long duration flight that may accelerate oocyte maturation and oviposition.

Adipokinetic hormone

Although many studies focus on its role in lipid mobilization during flight, there

is some evidence that AKH may have other roles in control of reproduction. Moshitzky

and Applebaum (1990) found that the AKH I titer in female locusts increases as

vitellogenesis proceeds, reaching the highest titer by the end of the reproductive cycle

7

(6mm oocyte diameter). This suggests that AKH may be involved in some aspect of the

final stage of oogenesis such as chorionization. In addition, AKH has been shown to

affect the rate of JH production in vitro in locusts (Applebaum et al., 1990). Given the

possibility of a direct effect of AKH on reproduction and the possible interaction of AKH

with JH in locusts, it was clearly important to examine the possible role of AKH in flight-

enhanced reproduction in M. sanguinipes.

Lipid transport

In addition to JH and AKH, another link between flight and reproduction may

occur via lipid transport during (and/or after) flight. Some evidence suggests that there

could be lipid transport from the fat body to the ovary that can be activated by flight.

Chino et al. (1977) first found that the same system that transports lipid from the fat body

to flight muscles also serves to carry lipid from the fat body to the ovary in the moth

Philosamia cynthia. Kawooya and Law (1988) showed that the protein complex that

carries lipid from the fat body to flight muscles also unloads its lipid at the ovary in

Manduca sexta. Thus, it is reasonable to deduce that when mobilization of lipid from the

fat body to flight muscle is stimulated by flight via AKH, lipid released into the

hemolymph during flight may also become available to the reproductive tract, and

contribute to the production of more and/or better-provisioned eggs, resulting in

enhanced reproduction after long flight.

POSSIBLE LINKING FACTORS – NUTRITIONAL FACTORS

8

Nutrient transfer during mating

In addition to endocrine factors, nutritional supplementation after flight could be

another linking factor between migration and enhanced reproduction. M. sanguinipes

females derive significant nutritional contributions to oogenesis from spermatophores

transferred during mating (Friedel & Gillott 1977) and there is evidence that mating

significantly enhances fecundity both because of nutrient transfer in the spermatophore

and because two proteins from the male accessory glands stimulate oviposition activity

(Friedel & Gillott 1976). If M. sanguinipes females change their mating behavior after

long flight and increase mating frequency and/or duration, the resultant increase in

nutrient transferred during copulation might result in increased reproductive success.

Feeding and Digestive Efficiency

Flight-enhanced reproduction could also occur via increased feeding behavior or

digestive efficiency after long flight that could compensate for the energetic cost of long

flight. In an interesting paper, Mole and Zera (1993) report that, while there is no

difference in amount of food ingested by adult short- and long-winged Gryllus rubens,

there is a significant difference in the conversion of assimilated nutrient into biomass

(mainly ovaries) between the two morphs. In this species it is the non-flying, short-

winged morph that is more fecund, but otherwise the parallel with the M. sanguinipes

system is striking. It is possible that performance of long duration flight changes food

utilization and these better assimilated nutrients can be available for reproductive

development, resulting in enhanced reproduction after long duration flight.

9

SUMMARY OF POSSIBLE HYPOTHESES

In summary,

some possible explanations for the enhanced reproduction after migration are:

Endocrinological hypotheses

n Hormone-stimulated lipid transport into eggs during flight enhances reproduction

after long flight

n AKH released during flight accelerates terminal oocyte development and

oviposition

n Long duration flight changes juvenile hormone release

Nutritional hypotheses (could be hormonally controlled)

n Supplemental feeding or improved digestive efficiency after long flight

compensates for the cost of flight

n Long flight increases mating behavior and thus increases nutritional

supplementation from males

The following chapters describe the experiments done to test these hypotheses.

SIGNIFICANCE OF THE PRESENT STUDY

10

This study examined physiological relationships between two key life history

characters, migration and reproduction, in an economically significant migratory insect.

A deeper analysis of the endocrine and other physiological mechanisms behind these

relationships will clarify the adaptations that have made this species a successful

colonizer. It is expected that the results of this work will be of interests to physiologists

interested in resource allocation and endocrine regulation of reproduction and migration,

ecologists studying life history theory and evolution, and entomologists interested in the

prediction and control of outbreak species.

11

CHAPTER 2

GENERAL METHODS;

CONFIRMATION OF FLIGHT ENHANCED REPRODUCTION

All grasshoppers used in this study were first generation, field-collected animals

from the San Carlos Apache Reservation near Globe, AZ. Methods of collection, rearing

and tethered-flight test were similar in most experiments in this study, and these common

methods are described in this chapter. Modifications of these general methods and

methods used for specific physiological experiments, are described in the relevant

chapter.

Even though flight-enhanced reproduction was reported by two other researchers

(McAnelly and Rankin, 1986b; Burchsted, 1990), it has been over a decade since it was

reported, and it is possible, particularly since these experiments were all done on first

generation field-derived animals, that this phenomenon might not be occurring anymore

due to changes in the environmental conditions and a relaxation of selection for

migration. Therefore, it was necessary to confirm this phenomenon before I started to

investigate the mechanism of flight-enhanced reproduction.

GENERAL METHODS

Field collection of grasshoppers

In mid and late June of all years of this study, dense populations of grasshoppers

were found on arid, elevated rangeland on Ash Creek Ranch, in the southern part of the

12

San Carlos Apache Reservation (average annual precipitatation in the range of 35-45cm;

approximate altitude = 1350-1450m above sea level). In most years, grasshoppers were

collected on the margins of Indian Highway 8, where vegetation is protected from

grazing and has a greater coverage of forbs compared to the adjacent fenced pasture.

Grasshoppers were collected with sweep-nets. In most years total grasshopper density at

the collection sites exceeded 10 per m2 except in 2002. M. sanguinipes was the

predominant species collected. Collected grasshoppers were sent to the University of

Texas at Austin by UPS overnight express.

Lab culture

Field-collected M. sanguinipes (N = 400-1000 adults/annual collection) were

maintained and allowed to mate in 30 cm cubical screen and Plexiglas cages

(N=100/cage) at the University of Texas at Austin. Cages were held in an environmental

chamber at 30 ºC in a LD 16:8h photocycle. Grasshoppers received ad libitum fresh

iceberg lettuce and a 1:1 w/w mixture of wheat germ and wheat bran, all changed daily.

In most cases, wheat germ/bran mixture was treated with sulfa drugs (0.6% w/w

sulfathiazole, 0.4% sulfapyridine, 0.3% sulfamethazine) to control Malameba locustae

infections (Henry and Oma, 1975).

To prevent infection of the grasshoppers by Malameba locustae, 1) individual

cages, sand and vermiculite were autoclaved before use and 2) Plexiglas cages and re-

used plastic ware such as bran dishes and some egg cups were sterilized overnight in

Clorox bleach solution.

13

M. sanguinipes females were allowed to mate repeatedly with males. It is possible

that females mated already in the field prior to collection, but this was not confirmed.

Two 8 oz. Styrofoam cups containing autoclaved sand moistened with 0.1% w/v

methyparaben (p-hydroxy benzoic acid methyl ester) to reduce growth of mold were

placed in each cage and replaced with new cups every three days. Females oviposited in

the sand within the Styrofoam cups. Pulled egg cups were covered with plastic lids and

held in the oviposition chamber for 10 days. Egg pods that did not hatch within that 10-

day period were removed and transferred to vermiculite moistened with 0.1%

methylparaben. Egg pods in vermiculite were stored in the refrigerator maintained at 10-

12 ºC until used. They are viable up to 12 months in this condition.

Hatching

All experiments in this study used the offspring of field-collected animals. After

at least 6 weeks storage in cold conditions, stored eggs from field-collected animals were

transferred from cold storage to an environmental chamber at 30 ºC in a LD 12:12h

photocycle. Under these conditions hatching typically occurred within 5-7 days. Nymphs

were reared in 30 cm cubical screen and Plexiglas cages with same diet as described

above, except that diet was supplemented with fresh alfalfa sprouts as a protein source.

Nymphal development proceeded through 5 instars, each lasting approximately one

week. At the final molt to adult, each grasshopper had wings and identifiable external

genitalia.

14

Eclosed adult grasshoppers were removed daily from the rearing cages between

12-2 PM and maintained individually in 46 cm3 metal and screen cages (‘The Bug

House,’ Tierney Specialties, Stockton CA) at 30 ºC in a LD 16:8h photocycle, and fed

with lettuce and wheat germ/bran/sulfa drug mixture as described above.

Tethered-flight test

Grasshoppers were evaluated for flight propensity using the approach of

McAnelly and Rankin (1986a,b). A small stick was attached to the pronotum of each

grasshopper with wax, and the insect was then suspended in front of a fan providing a

headwind of 8-11 km/h. Eight 150W incandescent lamps mounted above and behind the

grasshoppers provided illumination and, together with two electric heaters behind the fan,

maintained an ambient temperature of 31-34 ºC. The fan, an electric heater, and

incandescent lamps were used to simulate the conditions of wind speed, illumination, and

temperature associated with migratory flights in the field (Parker et al., 1955).

In this study, flight was defined as a period during which an individual had four

wings open and moving. If an individual did not fly or ceased flying, up to 3 attempts to

stimulate flight were made by the experimenter waiving a hand in front of the

grasshopper’s eyes. If all 3 attempts failed to elicit re-initiation of flight, time for flight

was recorded.

15

Comparison of Migrants and Non-migrants

In tethered flight tests M. sanguinipes, like a number of other insect migrants, fly

for only a few moments or for several hours at a time. Thus, if an individual flies at least

60 min in a single flight test, it is very likely to fly much longer. One can, therefore,

classify 1-hr fliers as migrants (McAnelly, 1985) in terms of behavioral proclivity

without actually allowing the animals to perform flights to “exhaustion” (i.e., to

voluntary cessation). Three 60-min flight tests on succeeding days, can reliably

distinguish all migrants (long-fliers) from non-migrants (non-fliers) in a population (Kent

& Rankin, 2001), but these 1-hr flights do not elicit the accelerated or enhanced

reproduction seen after flights to exhaustion. By comparing animals that have made a 1-

hr flight (long-flier to 1 hr, LF-1) with those that have flown to exhaustion (long-fliers to

exhaustion, LF-E) and those that have refused to make a long flight (non-fliers, NF) we

can compare characteristics of migrants with non-migrants and, among the migrants we

can identify the effects of performance of flight from the characteristics shared by all

migrants. Thus, in the experiments described in this study, animals that had actually

performed flights to exhaustion (LF-E) were compared with animals that had made a one-

hour flight (LF-1), and therefore could be identified as migrants, as well as with those of

non-migrant controls (NF).

16

Mating and egg pod collection

Long-flier (LF-1 and LF-E) and non-flier (NF) females are mated with unflown

same aged males on day 8 (after all 3 trials of flight assay). Each pair was placed in an

individual cage and a sand cup was placed in each cage to collect egg pods. Egg cups

were examined daily to check for the first egg pod. This egg pod was dissected carefully

with fine forceps and the number of eggs in the first egg pod was examined. Counted

eggs were transferred into vermiculite and incubated at 30 º C until they hatched. The

number of hatchlings was counted daily.

Statistical Analysis of Data

One way ANOVA test was used for data analysis unless otherwise specified. In

each figure, single and double asterisks mean significant differences with one way

ANOVA P< 0.05 and P< 0.01 respectively.

CONFIRMATION OF FLIGHT ENHANCED REPRODUCTION

As Burchsted (1990) found, age at first oviposition was earlier in long-fliers

flown to exhaustion (LF-E) than in non-fliers (NF) and in long-fliers flown for 1 hr (LF-

1). The difference was smaller than in the previous study (Fig. 2.1); First oviposition

occurred on day 20 in both NF and LF-1 groups (19.8 ± 0.7 and 20.2 ± 1.3 respectively,

mean ± standard error), whereas first oviposition by LF-E grasshoppers occurred 17 days

after adult emergence (16.8 ± 1.1). The number of total eggs in the first egg pod was

17

higher in LF-E group than NF and LF-1 group (Fig. 2.2). The number of eggs in the first

egg pod was approximately 18 eggs in NF and LF-1 grasshoppers (17.6 ± 0.5 and 18.5 ±

1.6 respectively, mean ± standard error), whereas that of LF-E grasshoppers was 25 eggs

(25.3 ± 1.3). After the number of eggs in the first egg pod was counted, their hatching

rate was examined. The hatching rate was 42.3 ± 5.6, 53.9 ± 11.9, 70.1 ± 10.2 % (mean±

standard error) for NF, LF-1, LF-E grasshoppers, respectively (Fig. 2.3). The hatching

rate for LF-E grasshoppers was significantly different from that of NF grasshoppers.

Not all female grasshoppers that paired with males oviposited, and the number of

female grasshoppers oviposited was also higher in LF-E group than NF and LF-1 group

(Fig. 2.4). While only 50% of non-fliers oviposited, 80% of long-fliers flown to

exhaustion oviposited. This difference might be due to difference in mating behavior. It is

possible that long-duration flight stimulates mating tendency of females. If it is the case,

long-fliers flown to exhaustion are more enthusiastic in finding their mates and the

number of females oviposited could be higher than non-fliers. This possibility was partly

examined and discussed in chapter 6.

I have shown that flight enhanced reproduction is still occurring in the population

of San Carlos, AZ, even though the details of flight enhanced reproduction are a little bit

different from those of Burchsted (1990). Burchsted showed that females flown to

exhaustion began first oviposition about 10 days earlier than either non fliers or 1 hr

fliers, but the difference in day of first oviposition was not that substantial in this

experiment. It is possible that changes in environmental factors may have led to a

18

somewhat less extreme difference between long fliers to exhaustion and 1 hr fliers or non

fliers or led to relaxation in selection for migration.

The present study included smaller sample size than the previous study. It is

possible that increasing sample numbers could have diminished the difference between

the present and previous study.

19

Figure 2.1. Age of first oviposition versus flight behavior. First oviposition date wasearlier in long-fliers that flew to exhaustion (LF-E) than non-fliers (NF) and long-flierflown for one hour (LF-1). LF-E grasshopper laid their eggs significantly earlier than NFand LF-1 grasshoppers. One way ANOVA P < 0.05. Single asterisks means significantdifference with one way ANOVA P< 0.05.

0

5

10

15

20

25

NF (n=9) LF-1(n=6) LF-E (n=8)

Days

af

ter

emer

genc

e

*

20

Figure 2.2. Number of eggs in the first egg pod versus flight behavior. Number ofeggs in the first egg pod was higher in long-fliers that flew to exhaustion (LF-E) thannon-fliers (NF) and long-flier flown for one hour (LF-1). F (2.20) = 15.42, one wayANOVA P< 0.001. Double asterisks means significant difference with one way ANOVAP< 0.01.

0

5

10

15

20

25

30

NF (n=9) LF-1 (n=6) LF-E (n=8)

Num

ber

of e

ggs

**

21

Figure 2.3. Hatching rate versus flight behavior. Hatching rate was greater in long-fliers flown to exhaustion (LF-E) than non-fliers (NF). F (1.13) = 5.26, one way ANOVA P= 0.04. Single asterisks means significant difference with one way ANOVA P< 0.05.

0

10

20

30

40

50

60

70

80

90

NF (n=7) LF-1 (n=5) LF-E (n=8)

Pe

rce

nta

ge

*

22

Figure 2.4. Percentage of females ovipositing. Percentage of females ovipositing washigher in long-fliers that flew to exhaustion (LF-E) and long-flier flown for one hour (LF-1) than non-fliers (NF).

0

10

20

30

40

50

60

70

80

90

NF (n=18) LF-1 (n=8) LF-E (n=10)

23

CHAPTER 3

LIPID TRANSPORT INTO OVARY DURING LONG DURATION FLIGHT

INTRODUCTION

One link between flight and reproduction may occur via the lipid transport during

(and/or after) flight. The mobilization of fat stores in the fat body for energy utilization in

the flight muscle is done by macromolecular complexes, lipophorins. In the resting state,

lipophorin exists as a form of HDLp (high density lipophorin) that has two protein

subunits, Apo-Lp I and Apo-Lp II. When insects start to fly, adipokinetic hormones

(AKH) are released from the corpora cardiaca (CC) and convert stored triacylglycerol

into diacylglycerol in the fat body. Then, upon loading diacylglycerol from the fat body,

the HDLp particles are transformed into LDLp (low density lipophorin) and also

associate reversibly with another protein called Apo-Lp III (Wheeler and Goldsworthy,

1983). Diacylglycerol is delivered into flight muscles and hydrolyzed into fatty acids,

which are oxidized for energy production in flight muscle. After release of

diacylglycerol, both HDLp and Apo-Lp III are regenerated and can be re-used for LDLp

production at the fat body again forming the lipid shuttle system (see Fig. 3.1, reviewed

in Shapiro et al., 1988).

Lipophorin works also as the major vehicle for lipid transport to oocytes. As

mentioned in Chapter 1, Chino et al. (1977) first found that the same system that

transports lipid from the fat body to the flight muscle probably also serves to carry lipid

from the fat body to the ovaries in the moth Philosamia cynthia. In addition, Kawooya

24

and Law (1988) showed that LDLp unloads its lipids at the ovary and recycles proteins

back to the hemolymph in Manduca sexta. Thus, it is reasonable to deduce that when

mobilization of lipid from the fat body is stimulated by sustained flight via release of

AKH from the CC, then the system for the transfer of lipid from the fat body to the

ovaries is likewise stimulated and may carry lipid to the eggs during (and/or after) flight.

Lipid released into the hemolymph during flight may also become available to the

reproductive tract and contribute to the production of more and/or better-provisioned

eggs after flight, resulting in enhanced reproduction after long flight.

In addition to lipid transport to ovaries, change of partitioning of energy reserves

between the eggs after flight is also of interest. Fuels utilized during insect flight are quite

different in various groups of insects. In general, Lepidoptera and Orthoptera burn

carbohydrates together with lipids; (Beenakkers et al., 1981; Goldsworthy, 1983). Most

species of the orders Diptera and Hymenoptera exclusively oxidize carbohydrates during

flight (Wheeler, 1989). Tsetse flies and certain beetles utilize proline as a fuel for flight

(Bursell, 1981). Even though there are many studies of fuel utilization during flight in

various insects, few studies have examined nutrient changes in eggs after flight

(Blackmer and Byrne, 1995). As mentioned earlier, long-distant flight could be

energetically costly, and there could be a change in partitioning of energy reserves

between eggs after flight. To check this possibility, the lipid, carbohydrate and protein

contents of eggs laid after flight were examined.

25

METERIALS AND METHODS

Grasshopper Collection, Rearing methods are described in Chapter 2.

Tethered-flight test method is described in Chapter 2.

Comparison of Migrants and Non-migrants method is described in Chapter 2.

Mating and egg collection methods are described in Chapter 2.

Lipid, Carbohydrate and Protein analysis

In 10X 75 mm tube, each egg was homogenized in 500 ml distilled water. One ml

of chloroform was added, vortexed and centrifuged for 5 min. The aqueous (upper) layer

was removed and the extraction was repeated with an additional 500 ml of distilled water.

The combined aqueous extracts were analyzed for carbohydrates using the anthrone assay

(Van Handel, 1965) and for proteins using the Bradford assay (Bradford, 1976). The

organic (lower) layer was brought to 1 ml chloroform, and 100 ml were taken for the

vanillin assay to determine lipid concentration (Van Handel, 1985).

RESULTS

The lipid content per egg produced by grasshoppers that performed a long flight

to exhaustion (LF-E) was not different from that of non-fliers (NF). In contrast, 1 hr fliers

(LF-1) had higher lipid content per egg compared with the other two groups; 180 ± 5.5

mg, 203 ± 4.9 mg and 172 ± 5.5 mg (mean ± standard error) for NF, LF-1 and LF-E

respectively (F (2.102) = 8.29, one way ANOVA P< 0.001, Fig. 3.2). Carbohydrate content

per egg was not significantly different between groups (64 ± 4.4 mg, 57 ± 2.1 mg and 57 ±

4.9 mg for NF, LF-1 and LF-E respectively, Fig. 3.3). However, protein content per egg

26

was significantly lower in eggs laid after flight to exhaustion than those laid by non-fliers

or 1 hr fliers (F (2.106) = 9.85, one way ANOVA P< 0.001. Fig. 3.4). Long-fliers flown to

exhaustion contained 410 ± 14.9 mg protein per egg whereas non-fliers and 1 hr fliers

contained 470 ± 14.3 and 502 ± 13.6 mg protein per egg respectively. The major nutrient

in egg was found to be protein, followed by lipid, then carbohydrate in all groups.

DISCUSSION

Much attention has been paid to the regulation of protein synthesis and vitellogenin

uptake during insect oogenesis, and it is natural to suspect that protein or nitrogen

reserves would be the limiting resource for a phytophagous insect. However, eggs are

also very high in lipid in M. sanguinipes (Burchsted, 1990), and although much less

attention has been paid to the regulation of lipid production and uptake during oogenesis,

lipids are the predominant energy source during embryonic development of insects. In

Melanoplus differentialis eggs, 75% of the oxygen uptake during embryogenesis is due to

the oxidation of fat (Beenakkers et al., 1981). Thus, conceivably, lipid could also be a

critical limiting resource for egg production. Chino et al. (1977) has shown that the same

system that transports lipid from the fat body to flight muscles probably also serves to

carry lipid from the fat body to the ovaries in the moth Philosamia cynthia. So, it is

possible that this may be at least part of the explanation for the enhanced reproduction

after long duration flight we have observed in M. sanguinipes.

Enhanced reproduction after long flight occurs only in long fliers flown to

exhaustion. If enhanced reproduction after long flight is caused by lipid mobilization

27

during (and/or after) flight, long fliers flown to exhaustion should have a higher lipid

content in eggs than that of non fliers and 1 hr fliers. However, lipid content per egg was

not higher in grasshoppers that performed long flight to exhaustion than non-fliers and 1

hr fliers. Interestingly, lipid content per egg of 1 hr fliers was higher than that of the other

two groups (Fig. 3.2).

If, however, we consider total lipid content in an entire egg pod (= lipid content per

egg multiplied by the total number of laid eggs in first egg pod (data from chapter 2)), the

mean lipid content would be 3.13, 3.76, 4.35mg for NF, LF-1 and LF-E respectively.

Thus, lipid mobilization during (and/or after) flight may play a role, possibly in the

improved hatching success that is observed in long fliers, but because the eggs are

(smaller and) more numerous after long flight, lipid content per egg may be lower. A key

factor that was not examined in this experiment is egg weight/size. This experiment is

being repeated to include determination of individual egg weights as well as lipid content.

Comparing lipid content per gram of egg will give more information regarding how well

the eggs of each flight group are provisioned with lipid. These experiments are in

progress.

Higher lipid content per egg of 1 hr fliers may be due to a flight-induced increase in

lipid availability that results from even a one-hour flight. Since the number of eggs in the

first egg pod is not different in one-hour vs. non-fliers, increased ovarian lipid would

result in higher lipid content per egg. In long-fliers flown to exhaustion, however, the

number of eggs in the first egg pod is increased, and lipid content per egg is lower than in

1 hr fliers even though the total lipid content of the egg pod is greater. Carbohydrate

28

content of eggs was not different between groups. Since carbohydrate is used primarily at

the beginning of flight, and gives way to lipid as the primary fuel after 30 min

(Goldsworthy, 1983), it is not surprising that the effect of long-duration flight does not

differ from one-hour flights on carbohydrates per egg. In contrast, protein content per egg

is much smaller in grasshoppers that performed long flight than grasshoppers flown for 1

hour or than non fliers. Again, one explanation for this difference is that grasshoppers

flown to exhaustion produce eggs with lower protein content per egg because they

produce more eggs per pod. When protein source is limited and they produce more eggs,

they may have to put less protein in an egg. However, Kent (1999) found that the amount

of protein in the thorax declined with flight and suggested that there may be consumption

of muscle mass as a putative fuel for flight as occurs in avian flight (Pennycuik, 1998).

This means that less protein content in eggs of long duration fliers could be due to

depleted protein reserve after flight. This possibility needs to be checked in future

research.

Burchsted (1992) found that the major component in eggs was lipid (mean = 188.8

mg/egg, 9.3% fresh weight), followed by protein (mean = 67.0 mg/egg, 3.3% fresh

weight) and carbohydrate (mean = 50.1 mg/egg, 2.5% fresh weight). This study found

similar concentrations for lipids and carbohydrates, but protein concentration was quite

different. The most abundant nutrient in M. sanguinipes eggs was found to be protein,

rather than lipid, and this finding is much more reasonable because the major component

of egg is vitellin (Hagedorn and Kunkel, 1979). Yolk vitellin represents the major

component of the insect egg and may account for up to 30% of the egg volume or up to

29

90% of the total protein content (Sander K., Gutzeit, H.O. and Jackle, H, 1985). The fact

the main component of eggs is protein, not lipid, suggests that there may be less

competition for fuel reserves between the ovaries and the flight muscle than has

previously been supposed. Unfortunately, the role of lipid and its importance compared to

protein in insect reproduction is poorly understood.

This study showed that lipid mobilization during (and/or after) flight may play a

role in flight enhanced reproduction but that role is modified (or obscured) by the number

of eggs produced. It also showed that there was no significant decrease in lipid and

carbohydrate content in eggs after long flight even though they are major fuels during

flight. This suggests that lipid and carbohydrate reserves are quite large in this

grasshopper and are not affected by long flight or decreased lipid and carbohydrate levels

are backed up to original levels by supplemental feeding after long flight. The second

possibility was checked in Chapter 7.

30

Figure 3.1. Schematic diagram of lipid mobilization by lipophorin during flight inlocust. Figure from Beenakkers (1991) . TG: Triacylglycerol, DG: Diacylglycerol, FFA:Free fatty acid, HDLp: High Density Lipophorin, LDLp: Low Density Lipophorin

31

Figure 3.2. Lipid content of eggs. Lipid content of an egg in non-fliers (NF),grasshoppers flown to one hour (LF-1) and long-fliers flown to exhaustion (LF-E). Eggslaid by 1 hr flier had higher lipid content than that of non fliers and long fliers flown toexhaustion. F (2.102) = 8.29, one way ANOVA P< 0.001. Double asterisks meanssignificant difference with one way ANOVA P< 0.01.

0255075

100125150175200225

NF (n=40) LF-1 (n=29) LF-E (n=40)

Con

cent

rati

on

(mg/

ml) **

32

Figure 3.3. Carbohydrate content of eggs. Carbohydrate content of one egg in non-fliers (NF), grasshoppers flown to one hour (LF-1) and long-fliers flown to exhaustion(LF-E). All three groups were not significantly different.

01020304050607080

NF (n=40) LF-1 (n=29) LF-E (n=40)Co

ncen

trat

ion

(mg

/ml)

33

Figure 3.4. Protein content of eggs. Protein content of one egg in non-fliers (NF),grasshoppers flown to one hour (LF-1) and long-fliers flown to exhaustion (LF-E). Eggsof long fliers flown to exhaustion had significantly lower protein than that of non fliersand 1 hr fliers. F (2.106) = 9.85, one way ANOVA P< 0.001. Double asterisks meanssignificant difference with one way ANOVA P< 0.01.

0

100

200

300

400

500

600

NF (n=40) LF-1 (n=29) LF-E (n=40)

Con

cent

rati

on

(mg/

ml)

**

34

CHAPTER 4

RELATIONSHIP AMONG ADIPOKINETIC HORMONE,MIGRATORY PROPENSITY AND REPRODUCTION

INTRODUCTION

In this chapter AKH action and titers in M. sanguinipes migrants versus non-

migrants were compared with those reported in locusts. In the first report to follow AKH

titer changes during flight using a radioimmunoassay (RIA) anti-Tyr1-Lom-AKH I serum

was employed to examine AKH I titer changes in animals that had performed flights of

various durations from 30 min to 6 hours. Investigating whether there be any difference

between migrants and non-migrants in amount of AKHs stored in the CC at rest and

whether there be differences in the adipokinetic response to AKH I or II in migrants vs.

non-migrants after hormone injection was also focused.

In chapter 1, the possibility was raised that AKH might have an action on final

stage of oogenesis, thereby hastening first oviposition. AKH I titer in female locusts

increases as vitellogenesis proceeds, reaching highest titer by the end of the reproductive

cycle (Moshitzky and Applebaum, 1990) and more numerous eggs laid earlier by long

fliers might be the result of such an action by AKH. Thus if we can know the amount of

AKH released during long duration flight, we can simulate its effect by injection of AKH

into unflown grasshoppers. Therefore in this chapter the effect of injection of

physiological dose of AKH, mimicking those that would be experienced during flight to

exhaustion, on reproduction of unflown grasshoppers was examined.

35

MATERIALS AND METHODS

Grasshopper Collection, Rearing methods are described in Chapter 2.

Tethered-Flight test method is described in Chapter 2.


Hemolymph collection

Hemolymph was collected before and 30, 60, 90, 180 or 360 min after flight from

4-6 day old adult males by puncturing the dorsal neck membrane between the head and

pronotum. On the assumption that multiple bleeding could affect subsequent AKH titer

and flight behavior, each hopper was bled at just one time point and then removed from

the flight apparatus. Hemolymph that bled freely from the puncture was collected in a

graduated glass micropipette, the amount collected being limited to 5 ml in flown animals.

In resting animals, 25 ml hemolymph was pooled from several animals.

Hemolymph Extraction and Separation of M. sanguinipes AKH I by HPLC

The extraction technique was modified from the method of Gäde et al. (1984).

Each hemolymph sample was dissolved in 400 µl 50 % methanol and centrifuged at 5000

¥g for 15 min. The supernatant was filtered through a Millipore Ultrafree-MC 0.1 µm

pore size centrifugal filter at 5000 ¥g for 15 min. The pooled filtrates from each

hemolymph sample were frozen in siliconized Eppendorf tubes, dried completely under

vacuum in a centrifugal concentrator, and stored at –80° C until analyzed.

M. sanguinipes AKH I was separated from potentially interfering substances by

performing narrow bore RP-HPLC on all hemolymph samples. Dried samples were

36

dissolved in 50 µl 12 % acetic acid prior to injection. Narrow bore RP-HPLC was

performed according to a modified method of Gäde et al. (1984) and Taub-Montemayor

et al. (2002) on a 1.0 ¥ 250 mm 300 micron C18 column (Separation Methods

Technologies). Absorbance at 210 nm and 280 nm was recorded with an Amersham-

Pharmacia SMART System UV-M, and absorbance peak areas were integrated with the

system software. Fractions that matched with Lom-AKH I fractions were collected and

stored at –80° C until analyzed by RIA for titer determination.

Radioimmunoassay

Hemolymph fractions that contained M. sanguinipes AKH I after HPLC were

dried and reconstituted with 100 ml RIA buffer (sodium phosphate buffer, pH 7.4, 0.05 M

NaCl, 0.1 % BSA, 0.1 % Triton X-100 and 0.01 % NaN3). Synthetic Lom-AKH I

standards (Peninsula Laboratories, Inc.) were diluted in 100 ml RIA buffer with a

concentration range of 1 pg to 1280 pg. Standards and samples were incubated with 100

ml of anti-Tyr1-Lom-AKH I antiserum (Peninsula Lab.) at 4° C for 16-24 hrs. Following

incubation (day 2), [125I] AKH I (Peninsula Lab. 10,000 - 15,000 dpm) in 100 ml RIA

buffer was added, mixed well and again incubated for 16-24 hrs at 4° C. On day 3,

pretitrated 100 ml goat anti-rabbit IgG serum and 100 ml normal rabbit serum (Peninsula

Lab.) were added, vortexed and incubated at room temperature for 90 min. 500 ml of RIA

buffer were added to these tubes and centrifuged at 3,000 rpm for 20 min at 4° C. The

supernatant was carefully aspirated, pellets were taken up in 4 ml liquid scintillation

cocktail, and levels of 125I were counted using a Beckman LS 400 scintillation counter.

37

Measurement of hemolymph volume

4-6 day old males were injected ventrally between the second and third abdominal

segments with [3H] methoxyinulin (15,000dpm in 1 ml insect saline) using a graduated

glass micropipette with a fine tip (Levenbrook, 1958). The abdomen was stretched and

relaxed three times to disperse the injected material in the hemocoel. Preliminary tests

indicated that 30 min were sufficient to distribute the radiolabel. Hemolymph was

collected after 30 min by puncturing the dorsal neck membrane between the head and

pronotum. One microliter of hemolymph that bled freely from the puncture was collected

in a graduated glass micropipette and used for measurement of radioactivity. The dilution

factor obtained in this way provided a measurement of the total hemolymph volume of

each animal. The mean hemolymph volume in these experiments was 62.4 ± 8.2 ml (n =

8).

CC dissection and extraction

Heads were severed from the prothorax with a razor blade, placed posterior

surface down on a glass slide, and sectioned with two downward cuts: one through the

centerline of the eyes and one immediately above the labrum. The center section was

fixed upright on a dissecting dish. Under a dissection microscope at 8¥ magnification,

blocks of tissue were lifted out, exposing the esophagus and the CC-CA gland complex

dorsal to it. At 15¥, the preparation was moistened with a drop of insect saline (0.13 M

NaCl, 0.005 M KCl; Stone and Mordue, 1980); the glands and esophagus were extended

38

by pulling gently on the esophagus and circumesophageal nerves. The nerves connecting

the gland complex to the brain were cut with a jeweler’s scissors or lancet (B&D

Ultrafine II); the esophagus and glands were removed to a dish of insect saline. At 25¥,

the gland complex and attached tissue were teased free from the esophagus and removed

to fresh saline for final dissection.

The corpora cardiaca extraction technique was modified from methods of Gäde et

al. (1984). Each CC complex was disrupted in 400 µl 50 % methanol for 3 min with a

Fisher 60 sonic dismembrator. The crude extract was centrifuged at 5000 ¥g for 15 min.

The supernatant was filtered through a Millipore Ultrafree-MC 0.1 µm pore size

centrifugal filter at 5000 ¥g for 15 min. The pellet from the first centrifugation was

resuspended in 400 µl 50 % MeOH and centrifuged and filtered as above. The pooled

filtrates from each CC complex were frozen in siliconized Eppendorf tubes, dried

completely under vacuum in a centrifugal concentrator, and stored at -80° C until

analyzed.

Quantification of CC AKH content

AKH I and II were separated and analyzed by on-line micro-capillary-LC-ESI-

MS (Aglient LC 1100 series; on-line with a Bruker Esquire LC-MS) performed on CC

extracts or synthetic hormone aliquots (Peninsula Labs) according to the methods of

Taub-Montemayor et al. (2002). Chromatograms containing the total-ion-current (TIC)

and base-peak-current were integrated for peak areas using the Esquire-LC DATA

analysis software package version 3.0 (Bruker Instruments). The TIC peaks

39

corresponding to the individual peptide hormones were identified by their precursor ion

mass peaks as determined via analysis of the synthetic peptide standards. The peaks were

detected by their base peak ion current, their areas integrated and compared with a series

of areas obtained for known amounts (ranging from 1.5 to 50 pmol) of synthetic AKH I

and II. To create the calibration curves, multiple injections were performed for each

selected amount of the synthetic AKHs. Peak areas were then plotted against the amount

of peptide to develop the standard curves for each AKH. Linear regression analysis was

then used to predict amounts from peak areas determined for each AKH. Experiments

performed to determine the linearity of response using both analytical systems, narrow

bore RP-HPLC and micro-capillary-LC-ESI-MS (Taub-Montemayor et al., 2002) showed

that narrow bore RP-HPLC maintained linearity in the range from 5 to 100 pmol, and the

latter system showed linearity in the range 0.5 to 50 pmol. Since our previous

experiments indicated that the peptides investigated were present in the glands around

and below 26 picomoles, we can assume that our experimental protocol is within the

linear range of absorbance. The operational mode of our analytical instruments was

verified prior to each set of analyses by running multiple injections of standard peptides.

The efficiency of our extraction method and its ability to quantitatively recover the

peptides from the glands was verified via spiking experiments. For this purpose we

selected individuals from the same physiological stage. Multiple sets of gland pairs (a

total of 4 per set) were extracted then divided into two halves and one half was spiked

with the synthetic Lom-AKH I peptide. Both samples were injected separately and

quantified.

40

Examination of AKH response

Either synthetic Lom-AKH I or Scg-AKH II (Peninsula Labs) was dissolved in

distilled water and diluted to make 2 pmol/ml and 20 pmol/ml concentrations. Injection

needles were made from graduated glass micropipettes drawn over a flame. 4-6 day old

males received 1 ml of AKH solution delivered as follows: a glass needle was introduced

into a puncture made with a sterile pin in the membrane between the third and fourth

abdominal sternites and the dose was delivered slowly under mouth pressure through a

blow-tube. The abdomen was stretched and relaxed three times to disperse the injected

material in the hemocoel.

Hemolymph was collected after 60 min by puncturing the dorsal neck membrane

between the head and pronotum. One microliter of hemolymph that bled freely from the

puncture was collected in a graduated glass micropipette. Hemolymph lipid

concentration was measured colorimetrically using a vanillin-reagent assay (Stone &

Mordue, 1980), with cholesterol (1 mg/ml in methanol) as standard.

Injections of AKH to simulate long duration flight and oviposition check

The amount of injection was determined first using total hemolymph volume and

titer of AKH after 30 min flight (see results section). Four day old unflown female

grasshoppers were treated with repeated injections of 2 and then 1 pmol AKH or insect

saline for 6 hrs with an1 hr interval to mimic the AKH levels in animals during a long

flight. Two similar aged males and sand cups were placed as the method mentioned in

Chapter 2 and sand cups were examined daily for first oviposition.

41

RESULTS

AKH titers

The success of radioimmunoassay is dependent on the development of a good

antibody and separation of the desired peptide from other interfering substances that can

affect the peptide-antibody binding. To separate M. sanguinipes AKH I from potentially

interfering substances, narrow bore RP-HPLC was performed on all hemolymph samples.

The fraction that had the same retention time as Lom-AKH I was further analyzed by

RIA for titer determination. At the time these experiments were done we had only

identified M. sanguinipes AKH I in the relevant fraction. Subsequent work has revealed

the additional presence of M. sanguinipes AKH II in the fraction with the same retention

time as Lom-AKH I in this study (Taub-Montemayor et al., 2002). Thus, the hemolymph

HPLC fraction that we used in this work probably contained both M. sanguinipes AKH I

and II. The results show that the method was sensitive to detecting as little as 8 pg Lom-

AKH I and Scg-AKH II had no affect on the binding between [125I] Lom-AKH I and

antiserum (Fig. 4.1). Since M. sanguinipes AKH II has the same amino acid sequence as

Scg-AKH II, the RIA results likely reflect only M. sanguinipes AKH I titer, rather than a

combination of AKH I and II. Thus, we believe the RIA actually identified only M.

sanguinipes AKH I in this study.

The concentration of Lom-AKH I required for half-maximal inhibition of binding

of [125I] Lom-AKH I analog was found to be approximately 153 pg (0.13 pmole). The

assay we used can detect as little as 8 pg of Lom-AKH I (Fig. 4.1). It was sufficiently

42

sensitive to measure AKH in 25 ml sample of hemolymph from unflown controls and in 5

ml samples of hemolymph from flown animals.

M. sanguinipes AKH I titer was examined at rest and after 30, 60, 90, 180 and

360 min of flight (Fig. 4.2). The titer before flight was relatively low (0.8 ± 0.2 ng/ml). It

increased up to 5.7 ng/ml (5.7 ± 1.4 ng/ml, mean ± S.E) after 30 min of flight, then had

dropped at 60 and 90 min to 4.2 – 4.3 ng/ml (4.2 ± 0.3 and 4.3 ± 0.3 at 60 and 90 min

respectively). After 3 and 6 hr of flight, it had dropped to 2.7 ng/ml (2.7 ± 0.2 ng/ml) and

2.2 ng/ml (2.2 ± 0.3 ng/ml) respectively.

Storage of AKH in the CC

In an early experiment, we identified migrants and non-migrants by the one-hour

assay. After a resting period of 7 days, we measured total AKH content of the CC (by

HPLC) in resting non-migrants, resting migrants, and migrants flight-tested to voluntary

cessation. No significant difference in one pair of CC content was observed between

resting migrants and non-migrants (effect of migratory propensity) nor between migrants

flown to voluntary cessation and resting migrants (effect of flight performance) (N = 35

females, 28 males; males were all 15 day old and females were 8, 12, 16, 20 day old).

Once we had developed methods to resolve M. sanguinipes AKH I and II by HPLC, we

repeated these experiments. No significant differences were found between 1-hr fliers

(migrants) and non-migrants in AKH content of CC for either AKH I or II (Fig. 4.3). The

mean amount of AKH I per individual gland pair for both long-flier and non-flier

combined was 26.5 ± 3.16 pmol (mean ± S.E , n = 15) and of AKH II 6.7± 1.43 pmol

43

(mean ± S.E , n = 15). These data are obtained from RP-HPLC fractions of extracts of CC

dissected from adults of both sexes following flight testing. They reflect LC peaks

corresponding in retention time to synthetic AKH I and II standards. The amount of

AKHs were estimated from 210 nm absorbance peaks relative to a standard curve

prepared using known amounts of synthetic AKH I & II (Fig. 4.4).

Adipokinetic response to synthetic Lom AKH I and Scg AKH II

Long-fliers (migrants) exhibited a consistently higher elevation of hemolymph

lipid levels in response to AKH I injection than non-fliers (non-migrants) as determined

by measuring hemolymph lipid increase after injection of synthetic Lom AKH I (Fig.

4.5). In contrast, injection of 20 pmol Scg AKH II showed negligible lipid concentration

increase compared to Lom-AKH I injection, and there was no significant difference in

response between non-fliers and long-fliers (Fig. 4.5). Injection of 2 pmol and 20 pmol of

synthetic locust AKH I caused hemolymph lipid increase, the higher dose approximately

doubling the response.

Effect of AKH injections on reproduction

Using the total hemolymph volume and hemolymph AKH titer after 30 min flight,

the total AKH amount in hemolymph was calculated. The average concentration of AKH

I in M. sanguinipes hemolymph during flight is approximately 2 pmol. The half-life of

AKH I in resting locusts has been calculated to be 53 min (Oudejans et al., 1996).

Assuming that the half life of AKH I in M. sanguinipes is similar to that of the locust, the

44

amount of AKH I that remains one hour after the first injection (2 pmol) will be about 1

pmol. Thus, to mimic the AKH experience during flight, I attempted to maintain a

physiological level of AKH I for 6 hours by hourly injections of AKH I in saline thus

maintaining the concentration at 2 pmol during that time. When similar amount of AKHs

were injected with 1 hr interval for 6 hrs to simulate long duration flight, there was no

difference in age of first oviposition between injected and saline injected group (Fig. 4.6).

DISCUSSION

The rise in hemolymph AKH during flight of Locusta migratoria was

demonstrated by Cheeseman et al. (1976) and Cheeseman and Goldsworthy (1979) by

means of a bioassay. L. migratoria is a large insect and has a correspondingly large

hemolymph volume (~250 µl) so that in monitoring AKH titer changes during flight, 50-

100 µl of hemolymph could be obtained from each individual locust after flight. The

hemolymph was then fractionated via Sephadex column (Cheeseman et al., 1976), and

the fraction containing AKH was assayed by monitoring the amount of lipid mobilization

it elicited when injected into a non-flying animal. This assay, while fairly sensitive, is

indirect and inherently subject to an additional source of variation (the assay animal)

relative to a purely biochemical assay. Furthermore, M. sanguinipes is a smaller insect

than L. migratoria, and the volume of hemolymph obtainable from a single bleeding is

correspondingly less (5-10 µl), thus we explored an alternative to bioassay. M.

sanguinipes AKH I has the same amino acid sequence as that of L. migratoria AKH I

(Taub-Montemayor et al., 1997), making it possible to use the commercially available

45

Lom-AKH I antiserum for the development of an RIA. Since AKH II does not affect

hemolymph lipid in M. sanguinipes, the fact that the RIA we used did not recognize M.

sanguinipes AKH II (which has the same sequence as Scg AKH II (Taub-Montemayor et

al., 2002)), was not a concern in examining the relationship between AKH I titers and

increasing hemolymph lipid levels after flight. Even though partitioning each

hemolymph sample by RP-HPLC requires time and labor, it improves the assay

sensitivity substantially by separating AKH from interfering substances (Moshitzky &

Applebaum, 1990). The M. sanguinipes AKH I titer without the RP-HPLC separation

step was significantly lower than the titer after separation, indicating that there are indeed

interfering substances in the hemolymph (data not shown).

AKH titer during flight increased sharply during the first 30 min of flight and

subsequently declined after extended flight. This initial increase is consistent with and

seems to slightly precede the increase in hemolymph lipid observed by Kent et al. (1997)

during the first 30 min of flight in M. sanguinipes (see Fig. 4.3). This pattern is also

similar to the hemolymph lipid concentration changes observed in locusts that reach a

steady state after about an hour and then slowly decline during extended periods of flight

(reviewed in Goldsworthy, 1983). Cheeseman & Goldsworthy (1979) found that the

maximal amount of AKH recovered from L. migratoria hemolymph as estimated by the

bioassay was about 5.0 ng/ml, as compared to 5.7 ng/ml after 30 min flight in M.

sanguinipes; this then decreased to about 2.13 ng/ml by 90 min in L. migratoria (as

compared to 4.3 ng/ml in M. sanguinipes). Recently, Candy (2002) showed that AKH I

titer after 60 min flight in S. gregaria was around 3.4 ng/ml (2.9 pmol/ml) using RIA.

46

If we assume that the half-life of M. sanguinipes AKH I during flight is similar to

that of Lom-AKH I (35 min; Oudejans et al., 1996), we can roughly estimate the amount

of AKH released during flight in M. sanguinipes. During the first 30 min after the

initiation of flight, the AKH titer is increased by 4.9 ng/ml. This total increase (DI, 4.9

ng/ml) should be total release (DR) minus the amount of degradation (DD) of AKH, i.e.

DI = DR - DD. For ease of calculation we assume that the AKH concentration decreases

to half of its original concentration in 30 min. At 0 min of flight, the original

concentration of AKH is 0.8 ng/ml. If there were no AKH release, titer would be

decreased to 0.4 ng/ml after 30 min of flight (DD = 0.4 ng/ml). The actual release (DR)

during 30 min of flight is the sum of the total increase (DI, 4.9 ng/ml) plus the amount

degraded (DD, 0.4 ng/ml), or about 5.3 ng/ml AKH I. Using the same method for the

calculation of released AKH I during flight until 90 min, we assume that about 8.9 ng/ml

AKH I is released during 90 min of flight. If the total hemolymph volume of M.

sanguinipes is around 62 ml, then, the total AKH release from the corpora cardiaca is

about 552 pg AKH I (0.48 pmol) during the 90 min of flight. Cheesman et al. (1976)

estimated that 920-1500 pg of hormone are released in L. migratoria during a short

period of flight.

When the storage of AKH in CC of M. sanguinipes was compared in non-

migrants and migrants, there was no significant difference in the stored amount of either

AKH I or II in different flight groups (Fig. 4.3). In addition, in initial experiments to

examine total AKH content, we found no significant decrease in total AKH content of the

CC as an effect of flight to voluntary cessation, even though AKHs are released during

47

flight. This apparent paradox may have been observed simply because the variances

among individuals were too large to permit statistical resolution, and/or because the

amount of AKH released during flight was small relative to the total stores in the CC (as

shown by our RIA titer determinations). It is also likely that AKH synthesis occurs

during flight. For example, in L. migratoria, flight activity increased levels of AKH

mRNAs in CC (Bogerd et al., 1995), and in a recent report Harthoon et al. (2002)

demonstrated that a continuous biosynthesis of AKHs is necessary for maintaining a

readily releasable pool of AKH-containing secretory granules during flight.

The AKH content of CC in M. sanguinipes was much smaller than that of locusts.

Siegert and Mordue (1986) quantified the AKH I and II content of CC in S. gregaria and

L. migratoria. They found that AKH I content in adult male and female S. gregaria is

about 300-1200 pmol/gland and in L. migratoria 100-750 pmol/gland. Amounts for

AKH II were determined to be 70-200 pmol/gland and 30-130 pmol/gland respectively.

There was a lot of variability between glands but amounts of AKHs were consistently

higher in females than males in both species. In contrast, M. sanguinipes has an average

of only 26 pmol AKH I and 7 pmol AKH II per gland, and no differences were observed

between the sexes. M. sanguinipes body size is 3 to 10 times smaller than that of locusts,

but even so the amount of stored AKH seems to be relatively low in this species.

It is interesting that, although long-fliers and non-fliers do not differ significantly

in body size, resting lipid reserves, or resting levels of AKH I or II, they do differ

significantly in their adipokinetic response to injections of exogenous AKH I. Injection

of 2 and 20 pmol of AKH I induced consistently greater increase of hemolymph lipid in

48

long-fliers than in non-fliers (Fig. 4.5). Since lipid is the major fuel for sustained flight, a

more intense adipokinetic response in long- fliers of M. sanguinipes could be

advantageous. Similar results have been observed in other acridids. For example,

gregarious L. migratoria showed greater increase of hemolymph lipid than solitary

locusts (Ayali and Pener, 1992) and young gregarious S. gregaria showed a higher flight-

induced hyperlipemic response than isolated young adults, while the difference was

insignificant in older insects (Schneider and Dorn, 1994). In contrast to AKH I, lipid

increase by AKH II of this species was almost negligible. It seems that AKH II does not

play a major role in lipid metabolism in this species. The mechanism for the difference in

the strength of the adipokinetic reaction might be a difference in density of receptors for

AKH in the fat body or a difference in the capacity to form low density lipophorin

(LDLp) as in L. migratoria (Chino et al., 1992).

The fact that repeated injections of AKH did not accelerate the age of first

oviposition means that released AKH during long flight does not have an effect on at

least final stage of oogenensis. This result is concurrent with the findings of Schaefer

(1998). Even though she did not know the amount of AKH I released during flight, she

injected animals with various amount of AKH I. Schaefer found that serial injections of

2.5 pmol AKH I which is physiological dose of AKH did not change first oviposition

date or average egg size.

49

Figure 4.1. Standard curve for RIA assay of AKH. Displacement of [125 I] AKHanalog by Lom-AKH I and Scg-AKH II (n=10 for AKH I and AKH II). Error bars showstandard error. The non-radioactive AKH I and II (1 pg-1280 pg) were pre-incubated withthe antiserum for 24 hrs at 4°C and [125I] AKH was added.

0

20

40

60

80

100

0 0.5 1 1.5 2 2.5 3

log [pg]

% b

ound

AKH I AKH II

50

0

2

4

6

8

10

12

14

0

2

4

6

8

10

0 60 120 180 240 300 360

Flight duration (min)

lipid concentration

AKH titer

Figure 4.2. Change in AKH titers and lipid concentrations during flight. Titer forAKH I (square) increases during the first 30 min of flight and then subsequently declines.The increase in lipid (circle) observed in the hemolymph correlates with the increase inAKH I titer but with a slight delay. AKH titer was examined before and 30, 60, 90 min,3 hrs and 6 hrs after flight. Hemolymph was collected once for each animal. N= 5 foreach time point and error bars show standard error. Hemolymph lipid increase data weretaken from Kent et al. (1997).

51

AKH IAKH II

LF

NF

0

5

10

15

20

25

30

35

Amount [pm]

Flight Status

Figure 4.3. Comparison of AKH in one pair of CC of flight-tested females. Nosignificant differences were observed for either AKH I or II when comparing migrantsand non migrants (ANOVA: F(1, 13) = 0.1918, P = 0.6685 and ANOVA: F(1, 13) = 0.6979,P= 0.4185, respectively). Error bars are standard error.

52

Figure 4.4. Standard curves for synthetic AKH I and II used for calibration. Theamount of AKH was calculated by using the base peak current observed during LC-MSruns as described in more detail in Material and Methods. Linear regression for bothcurves was calculated as R2 = 0.99.

53

Figure 4.5. Adipokinetic response to injected synthetic hormones. The response oflipid increase in the hemolymph was determined 60 min after injection of synthetic LomAKH I or Scg AKH II. Long-fliers exhibited a higher elevation of hemolymph lipids afterinjections of Lom AKH I. No significant difference in response to the injection of ScgAKH II was observed when comparing long-fliers (LF) and non-fliers (NF) (ANOVA:F(1,13) = 1.24, P = 0.28). Overall injections of 2 and 20pmol of AKH I caused an increaseof lipid in the hemolymph, the higher dose approximately doubling the response. 9-11day old males and females were used. N=10 for AKH I injections and 7 for AKH IIinjection. Error bars show standard error.

0

5

10

15

20

25

NF LF

lipid

in

crea

se

(mg/

ml)

2pmol Lom AKH I 20pmol Lom AKH I 20pmol Scg AKH II

54

0

5

10

15

20

25

30

Saine injected (n=5) AKH injected (n=7)

1st

ovip

osi

tion

days

Figure 4.6. Effect of repeated AKH injections on the age of first oviposition.Physiological dose of AKH or insect saline were injected to simulate AKH release duringflight for 6 hrs. Age of first oviposition was not significantly different between saline andAKH injected grasshoppers.

55

CHAPTER 5

JUVENILE HORMONE TITER AFTER LONG DURATION FLIGHT

INTRODUCTION

Another possible link between migration and reproduction may be juvenile

hormone (JH), an important control factor in insect reproduction (Engelmann, 1970;

Doane, 1973) and migration (Rankin, 1980). It was hypothesized in Chapter 1 that the

enhanced reproduction observed in M. sanguinipes after migration could be caused by a

flight-induced change in JH titer. Titer determinations were done using a well-

established, chiral-selective radioimmunoassay (RIA) (Huang et al., 1994; Hunnicutt et

al., 1989).


Grasshopper collection and rearing methods are described in Chapter 2.

Flight assay and hemolymph collection

Flight tests were done as described in Chapter 2. On day 4 after emergence,

grasshoppers were either flown for one hour and stopped or flown to voluntary cessation,

here termed exhaustion. After flight, the grasshoppers were divided into 3 groups. Each

group was bled every 3 days from day 5 to day 15. Thus, grasshoppers in group 1 were

bled on days 5, 8, 11 and 14; those in group 2 were bled on days 6, 9, 12 and 15; and

those in group 3 were bled on days 7, 10, and 13. All hemolymph samples were taken

between noon and 2 PM to control for possible circadian changes in JH titer (Zera and

Cisper, 2001; Ramaswamy et al., 2000). Hemolymph was collected by puncturing the

56

dorsal neck membrane between the head and pronotum. Hemolymph (5 ml) bleeding

freely from the wound was collected with a graduated glass micropipette.

Radioimmunoassay (RIA) for JH III

Hemolymph samples were immediately added to 0.5 ml acetonitrile. 1.0 ml of

0.9% NaCl was added, and the sample was extracted twice with 1.0 ml hexane. The

pooled hexane fractions were stored at 80°C until analyzed by radioimmunoassay

(Hunnicutt et al., 1989; Huang et al., 1994). At the time of analysis, we dried each

hexane extract and re-suspended it in 20 ml methanol. About 4,000 – 5,000 dpm [3H] JH-

III was added to each RIA tube in 100 ml gel-PBST (137 mM NaCl; 2.7 mM KCl; 4.3

mM Na2HPO4; 1.4 mM KH2PO4; 0.1% gelatin; 0.01% Triton X-100; pH 7.2). Then, 4 ml

of the methanol re-suspension of an unknown sample was added to duplicate assay tubes.

Next, 100 ml of a rabbit anti-10R-JH III serum (1:16,000) diluted in gel-PBST was added

to each assay tube. The assay tubes were vortexed and then incubated for 2 h at room

temperature. After the incubation period, the samples were chilled on ice for 5 min and

then 0.5 ml of cold, stirred dextran-coated charcoal (5 mg dextran; 5.6 mg EDTA; 0.2 mg

sodium azide; 1.0 ml PBS; 100 mg charcoal; 23 ml dH2O) was added to each tube. The

samples were incubated for 5 min and then centrifuged at 2,000 g at 4°C for 5 min. The

supernatant (containing [3H] JH III bound to antibody but no free [3H] JH) was poured

into a scintillation vial and counted in a liquid scintillation counter. The radioactivity in

each unknown sample was compared to a standard curve to determine the amount of JH

III in the sample.

57

Methanol dilutions of racemic JH III (Sigma Chemical Co., St Louis, MO, USA)

ranging from 0 (=total bound) to 3,000 pg were used to generate a standard curve;

radioactivity from each sample was expressed as a percent of total bound (= % TB). The

standard curve was plotted as log JH versus % TB and fit to a quartic equation used to

estimate the JH levels in each experimental sample. Because this assay is chiral selective,

results are expressed as ng 10R-JH III/ml hemolymph; detection limit was approx. 5 ng

10R-JH III/ml hemolymph.

JHE assay method

Juvenile hormone esterase assay was performed by the partition method of

Hammock and Sparks (1977) as modified by Lefevere (1989). The substrate [H3]-JHIII

was dissolved in ethanol and stored at –20o C prior to assay. 0.5 µl hemolymph samples

were collected on sequential days post flight and diluted in phosphate buffer pH 7.4. JH

III was added to a final concentration of 5 x 10-6 M and the solution incubated at 30o C

for 15 minutes.

JH III treatment and oviposition check

JH III (NEN) was dissolved in MeOH to make final concentration of 50 mg/ml.

1ml of methanol with or without JH III was applied to the abdominal sternites of female

grasshoppers on day 5 and 8. After the second topical application of JH III on day 8, each

female was mated with 2 8-day-old males. Each trio was placed in an individual cage

with a sand cup for oviposition that was examined daily for egg pods.

58

AKH I injections

On day 4 after eclosion females received 2 ml of synthetic Lom-AKH I (Peninsula

Labs) at a concentration of 1 pmol/ml in distilled water at time zero (T = 0) and then 1 ml

of the AKH solutions at 1-hr intervals for the next 6 hrs to simulate AKH release during

flight (Chapter 4). Hemolymph samples were collected on day 5 and day 8 after eclosion

and analyzed by RIA for JH titer.

RESULTS

JH titer after long flight

JH titer of unflown grasshoppers was quite stable, and was in the range of 10-20

ng/ml from day 5 to day 15 after emergence (Fig. 5.1). Similarly, animals that had been

identified as migrants via a one-hour flight test, but had not made a flight to exhaustion

showed no significant difference in JH titer when compared with unflown controls. In

contrast, animals that had made a flight to exhaustion on day 4 after eclosion had elevated

JH titers on day 5 (30.9 ± 6.5 ng/ml; F(2.20)=4.40, One way ANOVA p=0.026) above

those values observed in unflown controls (18.8 ± 4.1 ng/ml) and one-hour flier controls

(14.7 ± 0.9 ng/ml) and again on day 8 (36 ± 6.9 ng/ml; F(2.13)=7.89, One way ANOVA

p=0.006) above those observed in controls (16.8 ± 4.63 ng/ml) and one-hour flier controls

(13.8 ± 0.7 ng/ml) .

JH esterase (JHE) levels (Fig. 5.2) were elevated on day 7 in animals that had flown

to exhaustion on day 4, remained higher than control levels throughout the remainder of

59

the experiment, but were significantly different from control levels only on day 7

(F(1,14)=6.51, One way ANOVA p=0.023) (Note that JHE and JH titer determinations

were done in different groups of animals.)

Correlation between increased JH III levels and early oviposition

To determine whether the rise in JH titer after flight actually causes or is merely

correlated with flight-enhanced reproduction, 50 mg JH III was topically applied in 1 µl

methanol on days 5 and 8, to mimic the increase in JH that follows long-duration flight.

JH III was used in preference to a JH mimic such as methoprene because we wanted to

induce transient increases in JH similar to those observed after flight. When hemolymph

levels of JH III were checked after topical applications, it was clear that application of

50mg JH III increased JH titer, though not to the extent observed after a long-duration

flight (Table 1). Nevertheless, oviposition occurred earlier in the treated than in the

control group (F(1.10) = 11.78, one way ANOVA p<0.01) and indeed the timing was

similar to that observed in animals that had performed a flight to exhaustion (Fig. 5.3).

These observations support the hypothesis that increased JH titers are part of the

system by which long-duration flight results in accelerated oviposition. Whether or not

the elevation of JH is also responsible for more long-term effects of migratory flight on

reproduction cannot be determined from these results.

Effect of AKH on JH titer

60

It is tempting to suggest that the AKHs might be a link between flight and

enhanced reproduction possibly even by stimulation of JH III synthesis. However,

Moshitzky & Applebaum (1990) and Applebaum et al., (1990) have shown that in L.

migratoria AKH can inhibit vitellogenesis and, at high doses, even CA activity.

Radioimmunoassay for AKH in female L. migratoria demonstrated a dramatic increase in

AKH titer near the end of the first ovarian cycle, and such levels were shown to depress

yolk deposition (Moshitzky & Applebaum, 1990).

Thus, the hypothesis that the elevation of AKH during flight might affect

subsequent JH production by the CA was tested. Unflown grasshoppers were treated with

repeated injections of 2 and then 1 pmol AKH, a physiological dose as determined by

AKH radioimmunoassay (Chapter 4) for 6 hours on day 4 to mimic the AKH levels in

animals during a long flight. JH titers were measured on days 5 and 8 in saline-injected

animals, AKH-injected animals, and animals flown to exhaustion. AKH treatment did

not induce the JH titer increase seen after flight to exhaustion (F(1,10) = 0.76, one way

ANOVA p=0.4) (Fig. 5.4) nor did it significantly affect the age at oviposition (Data in

Chapter 4).

Existence of circadian rhythm of JH

Time of day for sample collection was controlled carefully, but to check for the

possibility that JH titer changes after long duration flight might be due to the disruption

of a diurnal pattern of JH secretion, hemolymph samples are collected on day 5 after

eclosion in the morning (9-10AM, 2 hours after light on), late afternoon (5-6PM) and late

61

night (1-2AM, 2 hours after light off) to check any possible circadian rhythm of JH.

There was no significant difference in JH titer by time of hemolymph collection (

F(2,23)=1.45, One way ANOVA p=0.25, Table 2).

DISCUSSION

JH is produced by CA and one of the ways to measure JH is to measure the

amount of JH synthesized by CA and released into hemolymph over a defined period of

time (Radiochemical assay, RCA). This method, however, shows high variability of JH

production in Orthoptera generally and in M. sanguinipes in particular (McCaffery and

McCaffery, 1983). An alternative method for RCA is the radioimmunoassay (RIA).

Although it does not measure rates of synthesis directly, it does assay circulating titers of

JH, which in our experiments is really the more meaningful measurement. RIA also has

the advantages of not requiring sacrifice of the experimental animal and the possibility of

sequential determinations from one animal. Thus, for several reasons, RIA was the best

choice for these experiments. The RIA used in this study has successfully been employed

to measure JH levels from bees (Huang et al., 1994), beetles (Trumbo et al., 1995),

crickets (Cisper et al., 2000) and lubber grasshoppers (Hatle et al., 2000), and has been

validated against two other JH-RIAs (Goodman et al., 1990; Strambi et al., 1981) that

have, in turn, been validated against gas-chromatographic/mass spectrophotometric

(GC/MS) JH titer determinations (Huang et al., 1994). In M. sanguinipes it delivered

consistent results in both control and treated animals. The JH titer of unflown animals

was in the range of 10-20 pg/ml from day 5 to day 15. After flight to voluntary cessation

62

JH titers reached 30 or 36 pg/ml on day 5 and day 8, and were significantly higher than

those observed in unflown controls or one-hour fliers. The JH titer of M. sanguinipes at

rest or after a 1 hour flight is similar to that reported for L. migratoria (4-30 pg /ml) (Dale

and Tobe, 1986). Long-duration flight, on the other hand, results in two transient

increases in JH titers. This is the first report that JH titer is altered after long-duration of

flight. Furthermore, when unflown grasshoppers were treated with topical applications of

JH III in methanol, we observed a decrease in age at first reproduction that was similar to

that observed after a long flight. Together these observations indicate that the increased

JH titer that occurs after long-duration flight is at least one component of flight-induced

enhancement of reproduction in this species.

Whether or not the JHE increase is induced by the rise in JH (Venkataram et al.

1994) cannot be determined from the experiments reported here (SHOULD HAVE

DONE JHE ON JH III-TREATED FEMALES) nor can we tell whether or not the JHE is

having any real effect on JH levels in vitro—perhaps ensuring that any rise in JH is brief.

The prolonged elevation of JHE after a long duration flight suggests the possibility that

JH synthesis by the CA may be greater than the titer determinations indicate, perhaps for

some time. However recent evidence suggests that there are only small amount of JH

available for degradation due to the binding to JH binding proteins (JHBPs) and JHE has

little effect on controlling JH titer in vivo in the cockroach (Engelman and Mala, 2000),

so the fact that JHE remains elevated after long-duration flight may not have major

physiological import. There is also accumulating evidence which indicates that JH

degradation occurs mainly in tissues and that hemolymph JHE may only be important

63

during critical developmental stages in certain insect orders (de Kort and Granger, 1996).

However, there is also evidence that although virtually all JH exists as the JH binding

protein-JH complex in hemolymph, JHE nonetheless efficiently hydrolyze JH in JH

binding protein-JH complex in Manduca sexta (Touhara et al., 1996). A more detailed

information about regulation of JH titers is necessary.

AKH levels are elevated during long flight in M. sanguinipes (Chapter 4) and

might conceivably be the stimulus for increased JH production after flight. To mimic the

AKH experience during flight, I attempted to maintain a physiological level of AKH I for

6 hours by hourly injections of AKH I in saline thus maintaining the concentration at 2

pmol during that time. These treatments had no effect on JH titer or age of first

oviposition. Thus, it is seems that flight-induced increased JH levels are not caused by

the release of AKH during flight.

Applebaum et al. (1990) found that AKH I inhibited in vitro synthesis of JH by

the corpora allata, but the amount of AKH I required was relatively high (10-3 M) and was

probably not a physiological dose. Our results indicate that physiological levels of AKH

have no effect, positive or negative, on the in vivo titers (and presumably production) of

JH in M. sanguinipes.

In M. sanguinipes, as in locusts, JH, produced by the CA, is necessary for

reproductive development. Either cautery of the MNSC or removal of the CA prevents

vitellogenesis. It appears that the MNSC are necessary for general protein synthesis, that

both the CA and these cells are necessary for synthesis of vitellogenin, and that the CA

are necessary for the oocyte-follicle cell complex to become competent to sequester yolk

64

(Gillott & Elliott 1976). The specific effect of JH on the M. sanguinipes ovary has not

been examined as yet, but in L. migratoria JH is required for patency (withdrawal of the

follicle cells from the ovarian surface that allows uptake of Vg and other hemolymph

proteins to proceed) (Davey et al. 1993). As in locusts, a peak of JH synthesis occurs in

M. sanguinipes with the onset of previtellogenic growth in each cycle of oocyte

development. A decline in JH synthesis is correlated with onset of oviposition

(McCaffery & McCaffery 1983). (Note that this work was done with a non-diapausing

lab strain of M. sanguinipes in which onset of reproduction was much earlier than in our

field-derived animals. Increasing JH levels at key times in the ovarian cycle could have a

major impact on timing of oogenesis, and thus we believe that the increase in JH titers

after performance of long flight are a critical part of the phenomenon of flight-enhanced

reproduction. At the same time, it is unlikely to be the whole story. Little is known about

control of JH production in M. sanguinipes or the occurrence of neurohormones affecting

the ovary directly. However, in L. migratoria (Girardie et al. 1991) and in S, gregaria

(Girardie et al. 1998) a protein produced by the pars intercerebralis called ovary

maturating parsin (Lom/Scg-OMP) appears to act directly on the ovarian follicle cells to

induce production of 20-hydroxyecdysone (20HE) which in turn stimulates fat body

production of Vg even in the absence of JH (Girardie et al. 1991, 1998; Girardie &

Girardie 1996). Furthermore, in L. migratoria increase in size of the CA occurs after

flight on a roundabout (Highnam and Haskell, 1964) and there is some evidence that

neurosecretory cells (BNCS) in the locust brain release material during flight. The CA

are known to be under control of the brain or brain neurohormones in most insects, thus a

65

key link between flight and reproduction may involve control of one or more

neuroendocrine factors from the BNSC and possibly a reprogramming of the brain-CA

system.

McNeil and Tobe (2001) suggest another mechanism by which flight might affect

hormone levels. They have shown that body temperature increases significantly

following the initiation of flight in P. unipuncta, and there is very rapid and significant

increase in JH biosynthesis following the transfer of females from low to high ambient

temperature conditions (Cusson et al., 1990). It is possible that the elevated body

temperature associated with long-duration flight could similarly result in an overall

increase in the rate of JH biosynthesis. These and other possibilities are under

investigation at this time.

66

Table 5.1. Hemolymph JH titers (ng/ml) after topical application of 50µg JH III inmethanol on day 5. (N=5 each group)

Treatments T = 0 T = 12 hr T = 24hrMeOH 7.0 ± 1.0 8.2 ± 2.4 8.7 ± 1.7

50 mg JH III 7.1 ± 1.1 19.4 ± 2.6 12.7 ± 1.8

Table 5.2. Hemolymph JH titers (ng/ml) by time of collection (N=8 each group)

Collection time 9-10AM 5-6PM 1-2AMJH titer 10.1 ± 1.8 14.6 ± 3.1 9.6 ± 1.7

67

0

10

20

30

40

50

5 6 7 8 9 10 11 12 13 14 15

Days after emergence

JH ti

ter (

ng/m

l)

flight to exhaustion resting 1hr flight

***

Figure 5.1. Long duration flights cause an increase in JH levels. JH titerafter long flight. Animals were flown to exhaustion on day 4 and bled from days 5 to 15after eclosion.The animals were divided into three groups. Individuals from each groupwere bled once every three days to reduce the effect of repeated bleeding. Controls areunflown animals. N=6 each group. Single and double asterisks means significantdifference with one way ANOVA P<0.05 and P< 0.01 respectively.

68

10

15

20

25

30

5 6 7 8 9 10

Days after eclosion

nmol

s/m

in/m

l

Unflown LF-1 LF-E

*

Figure 5.2. Juvenile hormone esterase levels after long flight. JH esterasedetermination (as nM of JH III converted to JH acid/min/ml) after long flight performedon day 4. Controls were animals that had not been flight tested and those that made only aone hour flight. Same legend as A. N=6 for flown to exhaustion, 9 for one hour controls,12 for unflown controls. Single asterisks means significant difference with one wayANOVA P<0.05.

69

10

15

20

25

30

Unflown methanoltreatment

Flown toexhaustion

JH IIItreatment

1s

t o

vip

os

itio

n d

ay

** **

Figure 5.3. Change in age at first oviposition after JH treatment. Topical applicationof 50mg JH III induced early oviposition as did flight to exhaustion. N=10 for unflownand flown to exhaustion groups and 6 for each treatment group. JH III was used ratherthan the JH mimic, methoprene, because I was hoping to achieve a transient JH peak asthat occurs after flight, using the natural hormone that could be degraded by JHE. Doubleasterisks means significant difference with one way ANOVA P< 0.01.

70

0

10

20

30

40

50

Day5 Day8

JH t

iter

(ng

/ml)

Saline injected AKH injected Flight to exhaustion

Figure 5.4. JH titers after treatment with physiological doses of AKH I. AKH I wasinjected for 6 hrs at 1-hr intervals to simulate the rise in AKH observed during a long-duration flight on day 4 after eclosion. Sample size: 9 for saline injected group and 12 forAKH I injected group.

71

CHAPTER 6

SEARCH FOR PEPTIDIC MOLECULAR MARKERS IN CORPORA

CARDIACA AND HEMOLYMPH AFTER LONG FLIGHT

INTRODUCTION

Chapter 5 showed that JH titers are changed after long duration flight and these

changes are at least one component of flight-enhanced reproduction in M. sanguinipes.

However, JH changes are unlikely to be the full explanation of this phenomenon.

Apparent release of hormones from brain neurosecretory cells has been implicated in

endocrine changes after flight in Locusta migratoria (Highnam and Haskell, 1964). These

may in turn cause changes in the JH production, but may have other effects as well.

Furthermore, it is still not clear how the long term effects of flight on reproduction are

accomplished. Thus in a collaborative effort, the analysis of CC and hemolymph peptides

have been examined in long fliers compared to non-fliers.

The insect corpus cardiacum produces a number of bioactive peptides. This

chapter reports studies to determine whether there are significant changes in CC peptides

with long duration flight, and if so, whether these might be involved in flight-enhanced

reproduction. Furthermore, because release of neuroendocrine peptides might be a very

dynamic process during long flight, involving release and synthesis sites other than CC, I

also report studies in which we examined changes in hemolymph peptides after long

duration flight.

72



Tethered-Flight test method is described in Chapter 2. Long fliers were flown on tether

for 8 hours while unflown animals were not tethered.

Hemolymph collection

Hemolymph was collected from 6 day unflown adults or immediately following

tethered flight to exhaustion on 6 day by puncturing the dorsal neck membrane between

the head and pronotum. Hemolymph that bled freely from the puncture was collected in a

graduated glass micropipette in the amount of 15 ml for each individual.

Hemolymph extraction

Extraction for HPLC analysis followed the method of Clynen, et al (2002).

Briefly, an aliquot of 25 ml hemolymph was combined with 250 ml methanol/water/acetic

acid (90:9:1). After sonication and centrifugation, the pellet was discarded and 400 ml

0.01% aqueous TFA was added to the supernatant. The methanol was evaporated and the

remaining aqueous residue was re-extracted with 500 ml ethyl acetate and 500 ml n-

hexane to remove lipids. The aqueous phase was filtered through a Millipore Ultrafree-

MC 0.1 mm pore centrifugal filter prior to the HPLC analysis.

RP-HPLC.

Hemolymph extracts were injected directly following filtration. The following

73

conditions were used for HPLC on a Beckman, Analytical System Gold: diphenyl

column (1X250mm), solvent A: 0.8% TFA in water; solvent B: 80% acetonitrile in

0.75% aqueous TFA with flow rate of 40 ml/min. Chromatograms were integrated to

determine peak areas on an Amersham-Pharmacia SMART System UV-M. The

absorbance was monitored at 214 and 280mm. Fractions were automatically collected by

drops (8 drops/tube).

MALDI-MS

MALDI-MS analysis was performed on a PerSeptive Biosystems Voyager-STR

mass spectrometer with delayed extraction. Samples were irradiated with a nitrogen laser

(Laser Science Inc.) operated at 337 nm, and attenuated and focused on the sample target

using the built-in software. Ions were accelerated with a deflection voltage of 30kV. Ions

were differentiated according to their m/z using a time-of-flight mass analyzer or the

time-of-flight reflectron mass analyzer. Aliquots of samples were loaded on a multi-

sample target using sinapinic acid as a matrix.

HPLC tandem ESI-MS.

Experiments were performed on a liquid chromatography system modified to

allow for stable flow rates between 1-200 ml/min (System Gold, Beckman Instruments)

linked to an Esquire ion trap mass spectrometer (Bruker Instruments). Electrospray

samples were introduced into the mass analyzer at a rate of 8.0 ml/min for online

infusions and 1.0 ml/min when using direct infusions with an adjustable syringe pump

74

(Hamilton Instruments). In the standard operating mode, the positive ions, generated by

charged droplet evaporation, entered the analyzer through the orthogonal spraying

interface of the ion trap. An online capillary LC (Agilent 1100 series) was used for

analysis of hemolymph extracts.

RESULTS

CC Profile Comparison

Peptidomic analysis of CC extract from unflown animals and long fliers flown to

exhaustion were compared to determine whether there were any changes in the amount of

peptides in CC after long duration flight. Major peaks in CC extract were found to be

adipokinetic hormones (Fig. 6.1). AKH I and II have masses of 934 and 1159,

respectively (Taub-Montemayor et al., 1997, 2002). Major peaks in CC extracts were

observed as their sodium (+23 Da) or potassium adduct (+39 Da). Unflown animals and

long fliers flown to exhaustion did not show any clear differences in peptide amounts in

CC.

Hemolymph Profile Comparison

HPLC analysis of hemolymph extracts was undertaken to uncover differences

between the two flight groups to see the difference in peptide profile in hemolymph after

long duration flight. RP-HPLC data of pooled hemolymph samples showed that there are

differences in peak expression and size after long duration flight especially in retention

time between 15 min and 60 min (Fig. 6.2. and 6.3). Samples were collected with a

75

fraction collector and analyzed for mass with MALDI-MS. It was found that the amount

of peptides with masses of 3656, 3684 and 3702 Da increase after long duration flight

(Fig. 6.4). Edman sequence of the 3702 Da peptide allowed partial sequence

determination of TPGQTKKEDCN. Blast search of the partial sequence revealed

homology with the locust serine protease inhibitor I (Hamdaoui et al., 1998).

DISCUSSIONIn this collaborative project, it was found that there is differential display of

peptide levels in hemolymph between non fliers and long fliers flown to exhaustion after

long duration flight. At least three peptides are changed in amount after long duration

flight and one of them is tentatively identified as serine protease inhibitor.

Proteases are enzymes that catalyze the hydrolysis of covalent peptide bonds. In

the case of serine proteases, they attack peptide bonds between serine and other amino

acids. Serine proteases have been studied intensively and perform many important

functions, especially in digestion, blood clotting, embryogenesis and immune response

(Simonet et al., 2002). Many of these processes involve a proteolytic cascade; activation

and inactivation of this enzyme cascade must be precisely controlled by a set of protease

inhibitors, indeed, serine protease inhibitors.

In locusts, several serine protease inhibitors have been described. Two are found

in L. migratoria (LMCI-1-2: Locusta migratoria Chymotrypsin Inhibitor 1 and 2.

Boigegrain et al., 1992) and 5 serine protease inhibitors in S. gregaria (SGPI-1-5:

Schistocerca gregaria protease inhibitors 1-5. Hamdaoui et al.,1998). One of the

76

peptides that showed differential display after long duration flight in M. sanguinipes has a

sequence homology with that of LMCI-1 and SGPI-1.

Several biological functions of serine protease inhibitors have been suggested.

Initially, a role in the innate immune defense system was suggested (Boigegrain et al.,

1992). However, the confirmation of the presence of LMCI-1 and LMCI-2 in the pars

intercerebralis – CC system of the migratory locust (Harding et al., 1995) and in other

tissues [the fat body and the gonads (Kromer et al., 1994)], clearly suggests that these

locust inhibitors might be involved in other physiological processes such as

neurosecretion, molt, reproduction, or ovarian development (Vanden Broeck et al., 1998;

Simonet et al., 2002).

A possible role in reproduction or ovarian development of serine protease

inhibitors suggests that they might be relevant in flight enhanced reproduction. We

observed that long duration flight changed the amount of serine protease inhibitors in

hemolymph (Fig. 6.4). If these activated serine protease inhibitors can stimulate

reproduction or ovarian development, flight enhanced reproduction might be the result.

To test this possibility, it will be necessary to get the full amino acid sequences of the

peptides. Once these are obtained, the effect of the peptides can be simulated with

injection of synthesized peptides into unflown animals. The exact role of serine protease

inhibitors in reproduction or ovarian development should be clarified to obtain a clear

picture of relation between activated serine protease inhibitors and flight enhanced

reproduction.

77

Figure 6.1. Mass profile of CC extracts. Peptides with mass of 956.62 and 972.35 are

sodium and potassium adduct of AKH II (mass of 934) each. Peptides with mass of

1182.01 and 1198.35 are also sodium and potassium adduct of AKH II (mass of 1159)

respectively. Methanolic extracts of CC from unflown and long fliers flown to exhaustion

did not show any difference in peptide profiles.

78

Figure 6.2. HPLC profile of pooled hemolymph after long duration flight to

exhaustion. Pooled hemolymph (25ml collected from 5 animals) extracts are run on

HPLC system. There are several changes in pattern and amount of peptides especially in

retention time between 15 and 60 min. UF: unflown controls, LF-E: long fliers flown to

exhaustion.

0.0

0.0

0.1

0.1

0.2

0.2

0.3

0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120

Min

Ab

so

rb

an

ce

UF LF-E

79

Figure 6.3. Amplified insert of fig. 6.2. HPLC profile with retention time between 15

and 60 min. F1-F5 are fraction collected.

80

Figure 6.4. Mass data of fraction 1-5 in unflown control (UF) and long fliers flown toexhaustion (LF-E). Several peptides with mass of 3656, 3683 and 3702 Da showedchanges in amount after long duration flight (see fractions number 3 and 4).

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

LF5: 3892.9

4: 3656,3683, 3702

3: 3702

2

1

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens. UF

5: 3892.86

4: (1) 3656,3684

3: Traces of 3702

2

1

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

0 5 10 15 20 25 30 35 40 Time [min]0

1

2

3

5x10Intens.

377399

199217240

158779

158779

158779

158779

199217240

377321

199217240

655442611

199217240

199217240

199217240

199217240

158779

199217240

81

CHAPTER 7

NUTRITIONAL SUPPLEMENTATION AFTER LONG DURATION FLIGHT;SUPPLEMENTATION BY MATING AND FEEDING

INTRODUCTION

Even though a single insemination can provide enough sperm to fertilize the

entire lifetime production of eggs, many female insects mate repeatedly (Parker, 1970).

One of the possible explanations for this behavior is that females obtain beneficial

nutrients from males during mating that increase fecundity (Boggs and Gilbert, 1979;

Friedel and Gillot, 1977; Markow and Ankney, 1984). As mentioned in Chapter 1, M.

sanguinipes females derive significant nutritional contributions to oogenesis from

spermatophores transferred during mating (Friedel & Gillott 1977). In some cases

several spermatophores may be transferred in a single copulatory event, and the longer

the mating, the more spermatophores are transferred (Pickford and Gillot, 1971). There is

evidence that mating significantly enhances fecundity both because of nutrient transfer in

the spermatophore and because two proteins from the male accessory glands stimulate

oviposition activity (Friedel & Gillott 1976).

This chapter reports work that examined whether long duration flight can change

mating behavior of females after flight. Females are larger than males and can repel

unwanted mounting attempts of males. If long duration flight changes a female’s mating

behavior and results in an increase in frequency or duration of copulation, this could

82

result in a significant increase in nutrient available for oogenesis and might therefore be a

partial explanation of enhanced reproduction after migration.

As also mentioned in chapter 1, the flight-enhanced reproduction could be partly

achieved by increased nutritional supplementation after long duration flight. Changes in

feeding behavior (e.g., eating more) or food utilization (e.g. more complete digestion or

absorption) after long duration flight could compensate for the cost of long duration

flight. Unflown female and female flown to exhaustion grasshoppers were fed with

artificial diet, and any uneaten food and frass samples were collected at two day intervals.

Frass samples were analyzed for residual carbohydrate, lipid and nitrogen to examine

food utilization.



Tethered-Flight test method is described in Chapter 2.


Observation of mating

After identification of non-fliers, 1hr fliers and long-fliers flown to exhaustion in

females, two similar-aged males were paired with a female in plastic cage with lid. The

plastic cage was placed in an environmental chamber at 30 ºC in a LF 16:8h photocycle.

Their mating behavior was watched for a week with a time-lapse videocassette recorder

83

(Panasonic AG-TL950P) and flex camera (checked from Biological Science Store

Room). Red light was used to provide light during the dark phase. Bailey and Harris

(1991) showed that M. sanguinipes cannot detect light in that part of the spectrum.

Grasshopper rearing in the feeding experiment

Nymphs were provided artificial diets ad libitum. The diet formula was modified

from Henry and Oma’s (1975) diet used for the delivery of sulpha drugs. Sulpha drugs

were added to all diets to prevent infection with the parasite Malamoeba locusta.

Artificial diets were placed in circular Petri dishes 4 cm in diameter. The Petri dishes

contained three cotton dental wicks to keep the diet moist, with the diet in the middle.

The diet was changed as needed to prevent drying, and the wicks were moistened with

0.1% solution of methylparaben daily.

Adult feeding rate determination

A cohort of grasshoppers was fed the control diet until adult eclosion. Newly

eclosed adult females were separated into individual cages and provided the high protein

diet (see preliminary experiment later). For the determination of feeding rate, it was

necessary to know the starting weight of the diet pellet. Use of a standard mold to create

the pellets provided a predictable dry weight that could be used in the calculation of a rate

of consumption. This was very successful in that pellets produced with this mold were

extremely uniform: mean pellet wet weight= 0.652g (standard error= 0.0038) and mean

84

dry weight= 0.128g (standard error= 0.0006). A mean dry weight was determined for

each successive batch of diet pellets.

Single pellets were placed in small petri dishes, nestled between two dental wicks

moistened with 1% methyl paraben to prevent drying and unpalatability in the dry heat of

the environmental chamber.

Collection of weight data

Each time a pellet was replaced, the previous pellet was collected, labeled, and

placed in a drying oven over night. The dry weight was determined using an analytical

balance. When replacing the pellet, the concurrent accumulated frass was collected and

labeled in the same manner as the pellet. Labeled micro tubes were tared on the

analytical balance, and each frass sample was placed inside the tube. The samples were

dried in an oven over night. Dry weight of each frass sample was determined.

Frass Analysis

A compositional analysis of the frass samples was performed. Frass samples of

known weight were separated via a modification of the methods of Handel (1965). Frass

was softened in 1ml distilled H2O and sonicated for one minute to create slurry. 1 ml

chloroform was added and the solution vortexed for 1 minute. The solution was

centrifuged at 1000 g for 5 minutes. The resulting three phases of liquid were sampled.

The chloroform phase was analyzed by Vanillin assay for lipid using cholesterol as

standard (Burchsted, 1990). Carbohydrate content was determined in the aqueous phase

85

using the colorimetric Anthrone assay using glucose as standard. Protein content was

measured in the aqueous phase using the Bradford assay (Sigma) with bovine serum

albumin as standard. Results are expressed in grams of analyte per gram of dry weight

frass.

Calculations of body weight

A correlation exists between hind leg femur length and body weight in M.

sanguinipes (Pfadt 1949). Femur length of each grasshopper used was determined with a

caliper (Manostat Corp.).

Preliminary experiments

In the first preliminary experiment not described herein, a diet was adapted from

Henry and Oma (1964). No significant differences were observed between flight groups

using this diet. The preliminary diet, however, was later found to be nutritionally

insufficient for the grasshoppers by virtue of its inability to provide adequate nutritional

satisfaction for a high percentage of emerging nymphs to eclose as adults. The most

optimal diet was later identified and named the high protein diet. This high protein diet

was used in the present study.

The present study was repeated including a 1-hr flier control, but the results were

not included in this report because the grasshoppers were badly infected with parasites;

viability was poor and the experiment was judged invalid.

86

RESULTS

Mating frequency and duration after long duration flight

Pre-copulatory behavior is displayed by both sexes (e.g., leg waggle prior to mating),

and mating is observed throughout the photo- and scotophase in M. sanguinipes. Mating

occurred evenly both in photo- and scotophase. The percentage of mating that occurred

in the photophase was 69%, and the light and dark cycle was 16:8h. Most females

started their mating within 1 or 2 days after they were paired with males, and there was

no difference in starting day between groups. When mating frequency and duration were

compared among non-fliers, 1 hr fliers and long-fliers flown to exhaustion, there was no

difference among groups (Table 1).

Feeding rate and digestibility after long duration flight

I observed a significant difference between the amount of food consumed by non-

fliers vs. long-fliers flown to exhaustion (Fig. 7.1) (F (1,17)=10.28, one way ANOVA p =

0.005 at interval 2; F (1,17)= 7.51, one way ANOVA P = 0.013 at interval 3). The amount

of artificial diet consumed was corrected for grasshopper size (femur length, which is

highly correlated with body weight; Burchsted, 1990). The difference occurred during the

intervals concurrent with and immediately following flight-testing. The digestibility was

calculated by examination of lipid, carbohydrate and protein remaining in frass. No

significant difference in digestibility of protein and carbohydrate between the two groups

was found (Fig 7.3 and 7.4). However, lipid content of frass from long-fliers flown to

87

exhaustion was significantly lower than that of non-fliers during the sample interval

immediately after flight (Fig. 7.2) (F(1,16) = 6.51, one way ANOVA P=0.023 at interval

3).

DISCUSSION

Because juvenile hormone has previously been reported to stimulate sexual

behavior (Amerashinge, 1978; Pener, 1967; Ignell et al., 2001), and juvenile hormone

titer change was detected after long duration flight (Chapter 5), it is reasonable to

hypothesize that JH titer change after long duration flight might change female mating

behavior and result in enhanced reproduction. However, we did not demonstrate

differences in mating frequency or duration that might account for the enhanced

reproduction of females These data are in agreement with the result of Burchsted (1990)

who did not find any difference in transfer of soluble protein from males to females

during copulation between migrants and non-migrants. Male accessory glands produce

soluble proteins that are transferred to females during copulation (Friedel and Gillot,

1976). The fact that there was no difference in transfer of soluble protein between

migrants and non-migrants suggests that copulation frequency or duration may not

change after long duration flight.

Locusta migratoria has been used as a model organism to examine many aspects

of the regulation of feeding. The initiation of a given meal can be influenced by many

factors such as time since previous meal, hemolymph osmolarity, circadian rhythms, the

presence of food in the digestive tract, and various light, visual and chemical stimuli

88

(Simpson, 1982; Simpson and Ludlow, 1986; Simpson and Abisgold, 1985). The

termination of a meal is regulated by a less complicated suite of factors. A meal is

typically terminated by the feedback of both nerve on the interior of the crop and by the

nutritional content of the meal (Simpson, 1995).

It is known that many migrants are able to replenish expended reserves by feeding

during (Brower, 1985) or after flight (Slansky, 1980; Gunn et al., 1989), and the

magnitude of energy uptake during migration can be greater than pre-migratory lipid

loading (Brower, 1985). Whether or not feeding compensates for nutritional loss during

long duration flight differs among species. In Drosophila subobscura, enforced tethered

flight for 1 h may result in a permanent reduction in fecundity even when females are fed

after flight (Inglesfield and Begon, 1983). However, Gunn et al.(1989) have shown that

ad libitum access to a source of carbohydrate (10% sucrose solution) after flight restored

the fecundity of flown moths to levels independent of flight duration. If M. sanguinipes

can overcompensate the nutritional loss during long duration flight with increased

feeding rate or improved digestive efficiency, reproductive enhancement after long

duration flight might be the result.

Comparisons of feeding and digestion of nutrients in long-fliers vs non-fliers

showed that there was a difference in consumption of nutrients in M. sanguinipes females

after long duration flight that might compensate for the cost of flight. Long fliers ate

more food than non-flier controls. However, the increase was transient, and it is not sure

if it is equal to or exceeds the whole resources expended during long duration flight. It is

therefore difficult to say that this is the sole source of the reproductive success of females

89

after long duration flight. It could be surmised from these results that reproductive

success of females after long duration flight is a combined result of endocrine changes

and nutritional supplementation.

Lower lipid in the frass of grasshoppers flown to exhaustion indicates that lipid

may be better absorbed in fliers vs. non-fliers. As mentioned several times, lipid is a

predominant energy reserve used during long duration flight in M. sanguinipes and better

assimilation of lipid after long duration flight might compensate for lipid loss during long

duration flight.

In this study, 1hr fliers were not included for comparison. Since both long fliers

and non fliers have same body parameters (Kent, 2001), it is assumed that both non fliers

and long fliers consume same amount of food before flight. Thus, it is likely that feeding

compensation occurred in long-fliers flown to exhaustion is the actual result of long

flight, not one of the characteristics of long-fliers. Nevertheless, this experiment should

be repeated including 1 hr flier controls.

The existence of actual compensation of feeding in the field is a little bit

problematic. Food may sometimes be unavailable or scare in the filed. Even though

McAnelly (1986b) found migratory propensity is not altered with changes in food

quality, it is not clear whether reproductive enhancement after long duration flight still

occurs in nutritionally stressed animals. Further investigation is necessary to solve this

question.

90

Table. 7.1. Mating frequency and duration after long duration flight. Neitherfrequency or duration was significantly changed after long duration flight.

Number of mating Duration (min)NF (n=9) 6.6±1.57 745±210.5

LF-1 (n=12) 9.5±1.22 781±158.0LF-E (n=9) 8.3±0.95 632±92.9

91

Figure 7.1. Average feeding rate of non-fliers (NF) and long-fliers flown toexhaustion (LF-E). LF-E grasshoppers consumed significantly more artificial diet justafter long duration flight for a while. Single and double asterisks means significantdifference with one way ANOVA P< 0.05 and P<0.01, respectively.

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

1 2(fly) 3 4 5

Interval

g/

day/

fem

ur

mm

LF-E NF

** *

92

0

5

10

15

20

25

30

35

1 2(fly) 3 4 5 6 7

Sample Interval

g lip

id/g

fra

ss

LF-E NF

*

Figure 7.2. Lipid content in frass of non-fliers (NF) and long-fliers flown toexhaustion (LF-E). Lipid content in frass of LF-E grasshoppers was lower than that ofNF grasshoppers just after long flight. Single asterisks means significant difference withone way ANOVA P< 0.05.

93

0

20

40

60

80

100

1 2(fly) 3 4 5 6 7

Sample interval

g c

arb

/g

fra

ss

LF-E NF

Figure 7.3. Carbohydrate content in frass of non-fliers (NF) and long-fliers flown toexhaustion (LF-E). There was no difference in carbohydrate content in frass betweengroups.

94

0

5

10

15

20

1 2(fly) 3 4 5 6 7

Sample Interval

g p

rote

in/g f

rass

LF-E NF

Figure 7.4. Protein content in frass of non-fliers (NF) and long-fliers flown toexhaustion (LF-E). There was no difference in carbohydrate content in frass betweengroups.

95

CHAPTER 8

CONCLUSIONS

This study examined the neuroendocrine regulation of migration and reproduction

in the grasshopper, Melanoplus sanguinipes. The conclusions of this study are

summarized below.

The phenomenon of flight-enhanced reproduction is repeatable. Grasshoppers

flown to exhaustion laid their eggs earlier than non fliers or long fliers flown to 1 hr. The

number of eggs in the first egg pod was greater in long fliers flown to exhaustion than

non fliers or long fliers flown to 1 hr. Hatching rate and percentage of ovipositing

grasshoppers were also greater in long fliers flown to exhaustion than non fliers. These

observations illustrate that flight enhanced reproduction continues to occur in the

population of San Carlos, AZ.

Lipid transport during flight may not the reason of flight enhanced

reproduction. The possibility of lipid transport during flight from fat body to ovary that

could explain flight-enhanced reproduction was tested. Lipid content per egg after long

duration flight was not significantly different from that of non fliers but total lipid per

pod was higher. Thus it is as yet unclear whether accelerated reproduction is related to

lipid mobilization.

Adipokinetic hormone is released during flight. The titer of hemolymph

adipokinetic hormone I (AKH) during long-duration tethered flight was examined using

radioimmunoassay (RIA). The fraction that contained AKH I after RP-HPLC was

96

assayed using commercially available anti-Tyr1-AKH I serum. The AKH I titer during

flight was examined at rest and after 30, 60, 90, 180 and 360 min of flight. AKH I titer

before flight was quite low (about 0.8 ng/ml), and it increased up to about 5.7 ng/ml after

30 min flight. By 60 min, it had dropped to approximately 4.2-4.3 ng/ml where it

remained at 90 min. After 3 hr and 6 hr flight, it had dropped to 2.7 ng/ml and 2.2 ng/ml

respectively.

Content of adipokinetic hormones in CC is not different between non fliers and

long fliers. When resting levels of AKH I and II in corpora cardiaca of non fliers and

long fliers were examined with HPLC, no significant differences in AKH levels were

detected between non fliers, animals that had flown for one hour, and animals that had

flown to exhaustion. CC levels of both AKH I and II were less in this species than in

locusts.

However, response to AKH is different between non fliers and long fliers. When

the lipid mobilization in response to AKH I and II was compared in long fliers and non

fliers, the adipokinetic response to AKH I was greater in long fliers than in non fliers,

possibly indicating differences in level of sensitivity or number of receptors in the target

tissues. AKH II had little effect on hemoplymph lipid levels in either flight group and

may not play a significant role in lipid mobilization in this species.

Release of AKH during long duration flight does not accelerate first day of

oviposition. Repeated injections of a physiological dose of AKH did not accelerate the

first day of oviposition that means that the release of AKH during long duration flight

does not have an effect on final stage of oogenesis like chorionization.

97

Juvenile hormone levels are altered after long duration flight and may be at

least one component of flight-enhanced reproduction. Since juvenile hormone (JH) is

involved in the control of reproduction in most species, JH titer was examined after long

flight using a chiral selective radioimmunoassay. JH levels increased on days 5 and 8 in

animals flown to exhaustion on day 4 but not in one-hour or non flier controls. Treatment

of grasshoppers with JH III induced early reproduction as did performance of long

duration flight. This suggests that increased JH titer after performance of long-duration

flight is at least one component of flight-enhanced reproduction.

JH titer change is not caused by the release of AKH during flight. To test the

possibility that post-flight JH titer increases are caused by adipokinetic hormone released

during long flights, 2 or 1 pmole of Lom-AKH I were injected for 6 hours at 1 hour

intervals to simulate AKH release during long flight. This treatment had no effect on JH

titers. Therefore it appears that the increased JH titers observed after long-duration flights

are not caused by the release of AKH.

There is no change in the peptide amount in CC after long duration flight.

Peptidomic analysis of CC extracts showed that major peaks in CC extracts are

adipokinetic hormones. There was no difference in the peptide pattern or amount in CC

extracts after long duration flight. Thus it seems that long duration flight does not change

the amount of peptides in CC.

There are changes in the peptide pattern in hemolymph extracts after long

duration flight. Peptidomic analysis of hemolymph extracts showed that there were

several changes in peptide expression after long duration flight. Major changes were

98

increases in amount of peptides with mass of 3702, 3683, 3656 Da after long duration

flight. Partial edman sequencing of a peptide with mass of 3702 Da showed the sequence

of TPGQTKKEDCN, which is the same sequence as a serine protease inhibitor in locust.

There is no difference in mating frequency or duration after long duration

flight, but females after long duration flight consume greater amount of food and

digest lipid better. Flight enhanced reproduction might occur due to nutritional

supplementation through nutrient transfer via increased mating and spermatophore

transfer, feeding behavior changes or changes in food utilization after long flight. No

difference in mating frequency or duration after long flight was observed. However,

females after long flight consumed significantly greater quantities of food and displayed

increased digestive capacity for lipid. These results suggest that nutritional compensation

by changes in feeding behavior and food utilization could partially account for increased

reproduction after long flight in M. sanguinipes.

99

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Vita

Kyung-Jin Min (KJ) was born on January 21, 1970, in Seoul, Korea. He is the

son of Bong Hee Min and Jung Ok Cho. After graduating from Osung High School,

Daegu, in 1988, he studied biology at Korea University, Seoul, Korea, from which he

received the degrees of Bachelor of Arts (1995) and Master of Science (1997). He served

as a medic in the Korean Army for 28 months from 1990 to 1992. He entered The

University of Texas at Austin in 1998. His graduate studies were supported by a

University Fellowship, teaching assistantships, and research assistantships.

Permanent Address: 12370 Alameda Trace Circle #1425. Austin, TX 78727

This dissertation was typed by the author.

Documents

Copyright by Kyung-Jin Min 2003 · The Dissertation Committee for Kyung-Jin Min certifies that this is the approved version of the following dissertation: NEUROENDOCRINE REGUALTION