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thesis entitled

LATITUDINAL COMPARISONS OF DIAPAUSE
INDUCTION BETWEEN POPULATIONS OF
PAPILIO GLAUCUS AND PAPILIO TROILUS
(LEPIDOPTERA: PAPILIONIDAE)

presented by

Patricia A. Tidwell

has been accepted towards fulfillment
of the requirements for

 

MS degree in Entomology

// Major professor

 

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LATITUDINAL COMPARISONS OF DIAPAUSE INDUCTION BETWEEN
POPULATIONS OF PAPILIO GLAUCUS AND PAPILIO TROILUS
(LEPIDOPTERA: PAPILIONIDAE)

By

Patricia Ann Tidwell

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Entomology

1995

 

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ABSTRACT

LATITUDINAL COMPARISONS OF DIAPAUSE INDUCTION BETWEEN
POPULATIONS OF PAPILIO GLAUCUS AND PAPILIO TROILUS
(LEPIDOPTERA: PAPILIONIDAE)

By
Patricia Ann Tidwell

The multivoltine butterflies Papilio glaucus and P. troilus rely on seasonal
environmental cues to avert or induce diapause. The most reliable seasonal
cue in their range is thought to be photoperiod. Offspring of butterflies from
different latitudinal populations were reared in the laboratory under varying
photoperiods. Data were collected as to whether pupae did or did not
diapause. The purpose of this research was to determine critical photoperiods
for diapause and variation between these populations and to better
understand diapause induction in these two species. Statistical analysis
indicated that P. glaucus likely used a cue(s) in addition to photoperiod to
induce diapause, and that P. troilus used photoperiodic cues to induce
diapause. Understanding diapause dynamics can shed light on the evolution
of a species and on adaptations to the local environment. Diapause dynamics
can limit or allow wider distribution of a species by eliminating

nondiapausing individuals during adverse conditions.

To Penny Tidwell, my mother-in-law, who told me,
”Never let anyone tell you, ’You can’t do it.”’

iii

 

 

 

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ACKNOWLEDGMENTS

I would first like to thank my major professor, J. Mark Scriber, for the
opportunity to conduct this research with one of the most enthusiastic
scientists I know. He always got me excited about doing science and taught
me how to do it efficiently and to have fun. I really enjoyed the collecting
trips with him—watching a pro in action is a wonderful sight. I would like to
thank my committee members, Jim Miller, Deb McCullough, and Fred Dyer,
for their input and assistance. Thanks also to Bob Lederhouse for his
expertise and advice on experimental design and manuscripts, and for the
opportunities he gave me to collect butterflies with him and to pick his brain.
I would also like to thank Kelly Johnson and Janice Bossart for guidance in
the early stages of my project, and James Nitao for always being there to
answer questions about experimental design, tables and figures, and statistics.
He was a tremendous help. Thanks to Doozie Snider who helped me mature
as a graduate student and who provided me with a seemingly endless supply
of black cherry leaves. I would also like to thank Ahnya Redman for being
there to talk to when I needed to air my thoughts and for the stimulating
conversation that always followed. I am also thankful for her skill in writing
and grammar. Thanks to Wayne Wehling for advice and reading early
manuscripts, and also for collecting butterflies in addition to those caught by
Ted Herig and Jim Maudsley. Additional thanks go to Jari Kouki, John Gill
and Jan Eschbach. Thanks to Cheryl Frankfater for sharing pizza and keeping

me company on late-night larval feedings, and for listening to my complaints

iv

about 5
Meals
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Reneé
thouse

which

about stupid things. Thanks to my husband, Craig, for bringing me Happy
Meals from McDonalds when I had to stay late, and for helping me feed
butterflies when he wasn’t harrassing the larvae. And a special thanks to
Reneé Brenner for staying with me for three seasons through thousands and
thousands of eggs, larvae, pupae and adults. She was a blessing without

which I would not have been able to do this research on such a big scale.

”The significance of photoperiodic adaptations in the formation of
physiologically different local races has also been demonstrated,
enabling us to approach from a new angle the problems of
understanding the first stages of intraspecific differentiation and of
evaluating the possibilities of acclimatization and extending the
distribution range of insects.”

Danielevskii 1965

vi

 

list of I;
list of f:
lntroduc

Chapter

Chapl

 

TABLE OF CONTENTS

List of Tables ........................................................................................................ ix
List of Figures ...................................................................................................... xi
Introduction .......................................................................................................... 1
Chapter 1 Photoperiodic Induction of Diapause in Populations of

Chapter 2

Papilio glaucus Occurring at Different Latitudes ................ 10
Abstract ......................................................................................... 10
Introduction ................................................................................ 11
Materials and Methods ............................................................. 11
Results .......................................................................................... 16
Discussion .................................................................................... 32
Conclusion .................................................................................. 4O

Photoperiodic Induction of Diapause in Populations of

Papilio troilus Occurring at Different Latitudes .................. 41
Abstract ......................................................................................... 41
Introduction ................................................................................ 42
Materials and Methods ............................................................. 42
Results .......................................................................................... 46
Discussion .................................................................................... 58
Conclusion .................................................................................. 6O

vii

 

l

Chapter .-

Appendii

Appendi

 

Appendi

list of R

Chapter 3 Interspecific Comparison of Diapause Induction in the

Tiger Swallowtail and the Spicebush Swallowtail ............. 62
Appendix 1. Record of Deposition of Voucher Specimens ...................... 71
Appendix 1.1 Voucher Specirnan Data ........................................................... 72
Appendix 2 Information by mother for female butterflies ..................... 73
List of References ............................................................................................... 77

viii

 

 

 

Table 1.

Table I.

Table 3.

Table 4

 

Table 5

Table 6

Table '

Table

Table

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

LIST OF TABLES

Larval development periods of P. glaucus and P. troilus in
the wild, based on flight periods of adults. These dates are
approximate due to annual variation in the accumulation
of degree days .................................................................................... 13

Papilio glaucus, Florida Population, Spring 1993; number of
pupae in diapause per number of pupae formed, for each
family line ......................................................................................... 18

Papilio glaucus, Florida Population, Spring 1994; number of
pupae in diapause per number of pupae formed, for each
family line ......................................................................................... 18

Papilio glaucus, Florida Population, Spring 1995; number of
pupae in diapause per number of pupae formed, for each
family line ......................................................................................... 20

Papilio glaucus, Georgia Population, Late Summer 1993;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 22

Papilio glaucus, Georgia Population, Late Summer 1994;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 23

Papilio glaucus, Ohio Population, Late Summer 1993;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 26

Papilio glaucus, Ohio Population, Late Summer 1994;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 27

Papilio glaucus, Michigan Population, Late Summer 1993;

number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 29

ix

 

Table 10.

Table ll
Table l1
Table 13

Table 1»:

Table l5

 

Table 1.6

Table 1'

Table ]

Table

Tabk}

Table

Tabi.

Tab}

Table 10.

Table 11.

Table 12.

Table 13.

Table 14.

Table 15.

Table 16.

Table 17.

Table 18.

Table 19.

Table 20.

Table 21.

Table 22.

Table 23.

Papilio glaucus, Michigan Population, Late Summer 1994;
number of pupae in diapause per number of pupae formed,

for each family line .......................................................................... 30
Results determined by logistic regression analysis, by state... 32
Climatic information about collection sites ............................... 35
General climatic information about collection states .............. 35
Papilio troilus, Florida Population, Spring 1993; number of
pupae in diapause per number of pupae formed, for each
family line ......................................................................................... 47
Papilio troilus, Florida Population, Spring 1994; number of
pupae in diapause per number of pupae formed, for each
family line ......................................................................................... 47
Papilio troilus, Florida Population, Spring 1995; number of

pupae in diapause per number of pupae formed, for each
family line ......................................................................................... 48

Papilio troilus, Georgia Population, Late Summer 1994;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 50

Papilio troilus, Ohio Population, Late Summer 1993;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 52

Papilio troilus, Ohio Population, Late Summer 1994;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 53

Papilio troilus, Michigan Population, Late Summer 1993;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 56
Papilio troilus, Georgia Population, Late Summer 1994;
number of pupae in diapause per number of pupae formed,
for each family line .......................................................................... 56

Results determined by logistic regression analysis, by state... 54

Information by mother number about female P. glaucus ...... 73

 

Table 24.

 

Table 24. Information by mother number about female P. troilus ........ 75

xi

 

 

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Figure .,

  
 

figure 3.

figure «l.

 

Figure 3

Flgure t

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Figure 1.

Figure 2.
Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

LIST OF FIGURES

Difference in Hours of Daylight between the longest and
shortest days, vs. N° Latitude. Twilight is not included. ....... 5

Range of Papilio glaucus and Papilio troilus ............................. 7
Counties where butterflies were collected from the wild ....... 9

Papilio glaucus, Florida Population, Spring 1993. Percent
Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 7, range in
number of pupae: 4-12 ................................................................. 17

Papilio glaucus, Florida Population, Spring 1994. Percent
Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 7, range in
number of pupae: 4-10 ................................................................. 17

Papilio glaucus, Florida Population, Spring 1995. Percent
Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 12, range in
number of pupae: 8-16 ................................................................. 19

Papilio glaucus, Georgia Population, Late Summer 1993.
Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 4,

range in number of pupae: 1-7 ................................................... 21

Papilio glaucus, Georgia Population, Late Summer 1994.
Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 5,

range in number of pupae: 2-9 ................................................... 21

Papilio glaucus, Ohio Population, Late Summer 1993.

Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 17,

range in number of pupae: 11-22 ............................................... 25

xii

 

figure T.

Figure T

Figure I

Figure 1

Figure T

Figure ‘

 

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FlguI-e

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Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Figure 16.

Figllre 17.

Figure 18.

Papilio glaucus, Ohio Population, Late Summer 1994.

Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 4,

range in number of pupae: 1-10 ................................................. 25

Papilio glaucus, Michigan Population, Late Summer 1993.
Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 12,

range in number of pupae: 7-20 ................................................. 28

Papilio glaucus, Michigan Population, Late Summer 1994.
Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 5,

range in number of pupae: 1-9 ................................................... 28

P. glaucus Average Percent Diapause vs. Hours of Light larvae
were reared at. A comparison between populations from
Michigan, Ohio and Georgia. CP = Critical Photoperiod ...... 31

Papilio troilus, Florida Population, Spring 1995. Percent
Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 6, range in
number of pupae: 3-10 ................................................................. 49

Papilio troilus, Georgia Population, Late Summer 1994.
Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 7,

range in number of pupae: 3-11 ................................................. 49

Papilio troilus, Ohio Population, Late Summer 1993.

Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 37,

range in number of pupae= 39-45 ............................................... 51

Papilio troilus, Ohio Population, Late Summer 1994.

Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 47,

range in number of pupae: 36-59 ............................................... 51

Papilio troilus, Michigan Population, Late Summer 1993.
Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 10,

range in number of pupae: 6-17 ................................................. 55

xiii

 

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Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

Figure 24.

Papilio troilus, Michigan Population, Late Summer 1994.
Percent Diapause vs. Hours of Light larvae were reared
under. Average number of pupae per treatment: 17,

range in number of pupae: 6-24 ................................................. 55

P. troilus Average Percent diapause vs. Hours of Light larvae
were reared at. A comparison between populations from
Michigan, Ohio and Georgia. CP = Critical Photoperiod.
*These two points wer estimated; all Georgia larvae reared
at 13.5h and 14h died ..................................................................... 57

An Interspecific Comparison. Average Percent Diapause

vs. Hours of Light larvae were reared at for Florida
Populations of P. glaucus and P. troilus. CP = Critical
Photoperiod ..................................................................................... 63

An Interspecific Comparison. Average Percent Diapause

vs. Hours of Light larvae were reared at for Georgia
Populations of P. glaucus and P. troilus. CP = Critical
Photoperiod *These points were estimated, all P. troilus
larvae died in these treatments ................................................... 64

An Interspecific Comparison. Average Percent Diapause

vs. Hours of Light larvae were reared at for Ohio
Populations of P. glaucus and P. troilus. CP = Critical
Photoperiod ..................................................................................... 65

An Interspecific Comparison. Average Percent Diapause

vs. Hours of Light larvae were reared at for Michigan
Populations of P. glaucus and P. troilus. CP = Critical
Photoperiod ..................................................................................... 66

xiv

 

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INTRODUCTION

Preface

Diapause in insects is important because it gives insects the ability to
withstand adverse conditions; it also synchronizes insect life cycles to the
changing seasons. I investigated photoperiodic induction of diapause in two
species of swallowtail butterflies, Papilio glaucus and P. troilus. Butterflies
were collected from different latitudinal populations in the United States and
their offspring were reared in the laboratory under varying photoperiods
until pupation, the stage at which they diapause. Data were taken as to
whether pupae diapaused or not, and used to determine the critical
photoperiod for diapause.

This paper is divided into three chapters with the general introduction at
the beginning. The first chapter describes diapause in P. glaucus, and the
second describes the experiments with P. troilus. The third chapter is a
discussion comparing and contrasting the two species of butterflies with

respect to diapause induction.

Background
Many insects diapause to avoid adverse conditions (Lees 1955). Tauber et
a1. (1986) define diapause as

a neurohormonally mediated, dynamic state of low metabolic activity.
Associated with this are reduced morphogenesis, increased resistance
to environmental extremes, and altered or reduced behavioral
activity. Diapause occurs during a genetically determined stage(s) of
metamorphosis, and its full expression develops in a species-specific
manner, usually in response to a number of environmental stimuli

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that precede unfavorable conditions.

Diapause should not be confused with ”quiescence,” a rapidly reversible state
of suppressed metabolism, allowing insects to withstand aseasonal periods of
stress. The environmental cues regulating diapause are called ”token
stimuli.” Token stimuli are not themselves favorable or unfavorable for
growth but indicate that a change in environmental conditions is forth-
coming. The most widely used token stimulus is photoperiod (Tauber et al.
1986, Danks 1987). The critical photoperiod for diapause, as defined by Tauber
et a1. (1986), is the photoperiod which induces 50% of a population to enter
diapause, given other constant conditions. The critical photoperiod may be
changed due to factors such as different temperatures, host plant quality or
moisture levels.

Diapause in insects can occur at various stages of development, but the
stage is usually species-specific (Danilevskii 1965, Danks 1987). Most
swallowtail butterflies (Lepidoptera: Papilionidae) in temperate regions
overwinter as diapausing pupae, but Parnassius swallowtails overwinter as
eggs (Tyler et a1. 1994). Diapause in Papilio can be ”obligate” and genetically
determined, or it can be ”facultative” and influenced by environmental
factors (Scriber 1994). Thus the number of generations per year, or voltinism
patterns, are genetically determined as well as environmentally cued (Slansky
1974, Scriber and Slansky 1981, Scriber and Lederhouse 1992). The genotype
can determine whether a population is univoltine or multivoltine, but
voltinism can be altered by environmental factors such as temperature,
moisture, food quality, or day length (Beck 1980, Danks 1987).

The onset of diapause in insects can occur during the sensitive
developmental stage when a critical environmental stimulus(i) is

experienced (Eizaguirre et a1. 1994). However, Tauber et a1. (1986) have shown

 

 

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that insects can detect changes in the environment long before the diapause

stage and can store this information. Some species can integrate different
types of stimuli to determine when to enter diapause. The Mediterranean
corn stalk borer, Sesamia nonagrioides (Noctuidae), which spends most of its
larval development inside plant tissue, integrates both photoperiod and
thermoperiod cues before diapausing (Eizaguirre et a1. 1994). The anise
swallowtail, Papilio zelicaon, can also use more than one cue. It has both
univoltine and multivoltine populations, which occur respectively in
ephemeral habitats or in more stable environments (Sims 1983). The
incidence of diapause in P. zelicaon can be modified by temperature and host

plant species (Sims 1980).

Diapause and Voltinism

At any given location, the maximum number of generations per season
depends on environmental conditions such as thermal unit accumulations
(season length) and host plant quality (Scriber and Lederhouse 1992, Scriber
1994). If the host plant quality declines after one brood and the growing
season is short, the butterflies in that area are probably univoltine and will
diapause after one generation. If food plant quality remains high for many
months and the thermal accumulations are sufficient, then the butterflies in
that area will be able to produce at least two generations before going into
diapause. This is the case for P. zelicaon in habitats with a more permanent

host species.

Diapause in other Papilio
P. demoleus:

Insects use different cues as indicators of season, depending on the biome
which they inhabit. Ishii (1987) showed that for tropical species such as
Papilio demoleus, seasonal changes in temperature trigger diapause. When
larvae reared at 14:10 (L:D cycle) were compared to larvae reared at 10:14 at the
same temperature, there were no diapause differences. When he investigated
different temperatures, all larvae reared at 25°C direct-developed, while 66%
of the larvae reared at 20°C went into diapause. This shows that P. demoleus
relies partially on temperature as opposed to a change in day length as a cue to
seasonal change.

In tropical regions, seasonal change in day length is nominal and may be
inadequate as an indicator of season (Figure 1) (Beck 1980). In temperate
regions however, day length is a reliable seasonal cue, and photoperiod plays
a major role in diapause induction in insects occurring there (Andrewartha
1952, Danilevskii 1961, Beck 1980, Saunders 1982). Temperature
(thermoperiod) may also play a role in season determination in temperate
environments, but it may not be an accurate indicator because of warm spells
in winter or cold spells in summer.

P, gangdgnsis:

Papilio canadensis R 8: J, the northern tiger swallowtail butterfly, occurs in
northern latitudes from 42° to 45° from Minnesota to New England and
northward. It is an ”obligate” diapauser having only one generation per
season. The growing season in these regions is very short and host plant
quality can usually only support one generation per season. P. canadensis is
univoltine for all photoperiods (Scriber 1982, Scriber and Lederhouse 1983,

Rockey et al. 1987a, Scriber and Lederhouse 1992). Even when larvae were

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reared under 24h of light in the laboratory, almost 100% of the pupae

diapaused (Rockey et al. 1987a, b). They also showed that in the P. glaucus
group, diapause behavior was a sex-linked gene and inherited from the male

(in butterflies, females are heterogametic).

P. glaucus and P. troilus

Papilio glaucus L., the eastern tiger swallowtail, and Papilio troilus L., the
spicebush swallowtail, are two multivoltine species of butterflies. P. glaucus
occurs in eastern North America from southern Ontario south to the Gulf
Coast, and west to eastern Colorado and central Texas (Figure 2). P. troilus
occurs in eastern North America from southern Ontario and southern New
England south to Florida and the Gulf Coast, and west to Oklahoma and
central Texas (Figure 2) (Opler 1992). Each has three or more generations
annually in the southern-most portions of their ranges and two generations
in the northern-most portion of their ranges (Scriber and Lederhouse 1992).
Photoperiod experienced during larval development cues pupae to develop
directly into adults or diapause until the next season (Tauber et a1. 1986).
Larvae reared under short day conditions generally go into diapause when
they pupate, whereas larvae reared under long day conditions direct-develop
into adults.

Much of the range and many of the flight periods of P. glaucus and P.
troilus overlap, which prompted my interest in comparing these two species.
I wanted to know how these two closely related species respond to the same
photoperiodic conditions, given that they are located in the same areas and fly
at the same time. Results from a preliminary experiment indicated that there
may be differences between these two species with respect to diapause.

Data were collected to determine the critical photoperiod for diapause in

 

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populations of both species occurring at different latitudes in the United

States, namely from Florida, Georgia, Ohio, and Michigan (Figure 3). It has
been shown that at a 16:8 photozscotophase, a decline in direct development
(ie. more diapause) occurs in populations of P. glaucus at higher latitudes
(Hagen and Lederhouse 1985, Rockey et al. 1987a), and this research allowed
for both 1) intraspecific comparisons between populations at different
latitudes and for 2) interspecific comparisons between species at the same
latitude.

The above locations were chosen because I wanted to investigate how
latitude affects diapause induction, and I needed to do a transect through the
range of P. glaucus and P. troilus . These areas were known to contain
populations of both species of butterflies and could also serve as a recurrent
source of butterflies. Populations from Florida were included to serve as a
comparison between populations from a sub-tropical environment with

those from a temperate environment.

 

 

 

 

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. = Papilio troilus

 

 

 

Figure 3. Counties where butterflies were collected from the wild.

 

 

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CHAPTER 1

Photoperiodic Induction of Diapause in Populations of Papilio glaucus

Occurring at Different Latitudes

Abstract

Facultative diapause in P. glaucus is induced by seasonal environmental
cues. The most reliable seasonal cue in temperate regions is day length.
Females were collected from Florida, Georgia, Ohio and Michigan and their
offspring were reared in controlled environments with constant
temperatures, at different photoperiods. The purpose of this research was to
determine the extent of differences among populations in the critical
photoperiod for diapause and to better understand diapause induction in this
species. The critical photoperiod for diapause for the Florida population
appeared to be between 12.0h and 12.5h of light. For the Georgia population,
the critical photoperiod appeared to be between 14.5h and 15h of light. The
critical photoperiod for the Ohio population was different for the two years
tested, but when averaged together the critical photoperiod was between 14.5h
and 15.0h of light. For the Michigan population, the critical photoperiod was
at least 18h of light, even though Michigan larvae never experience 18h days.
The R2 values obtained from the logistic regression suggest that P. glaucus
used a cue(s) in addition to photoperiod to induce diapause. These data help
explain the diapause dynamics of this species which can shed light on

adaptations to local environments and on the evolution of this species.

10

 

 

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11

Introduction

P. glaucus is a facultative diapauser, that relies on environmental cues to
determine the season (Scriber 1985, Lederhouse et a1. 1995). P. glaucus is a
multivoltine species, producing one to three generations annually depending
on the environmental conditions and host plant quality. Photoperiod is the
major seasonal indicator for larvae of temperate butterflies that overwinter as
pupae (Tauber et a1. 1986). The purpose of this research was to determine the
critical photoperiod that induced diapause in populations of P. glaucus
occurring in Florida, Georgia, Ohio and Michigan, and to determine if there

are any differences between these populations.

Materials and Methods
1 i P l i n 1
In March, female butterflies were collected from Highlands and Levy
Counties, Florida (Figure 3) and shipped via Federal Express or brought back
to Michigan in a cooler. Females were allowed to oviposit in the laboratory
and were fed a 1:4 honey:water solution. See Scriber, 1993 for oviposition
methods. Eggs were collected daily, put in a petri dish and placed in an 18°C
chamber, with a photoperiod of 18:6 (L:D). Eggs hatched in five to seven days.
Larvae were reared in controlled environmental Percival® chambers with
four 40 watt fluorescent bulbs at a constant 25°C. The neonates were set up in
150 mm wide x 25 mm deep petri dishes, one per dish, with foliage from the
field. The nine photoperiod treatments were as follows: 12:12 (L:D), 13:11,
13.5:10.5, 14:10, 14.5:9.5, 15:9, 15.5:8.5, 16:8, and 18:6. Twelve family lines were
set up, for a total of 490 larvae, which were set up between 3 - 7 April. Family

lines were followed throughout the experiment.

12
P. glaucus larvae were reared on sweet bay (Magnolia virginiana) collected

in Florida and shipped to Michigan via Federal Express or brought back to
Michigan in a cooler. Leaves were placed in aquapics (rubber-capped, water-
filled vials) to maintain turgor. Fresh leaves were provided 1-2 times per
week for early instars and 2-3 times per week for later instars, until pupation.
Pupae were weighed one day after pupation and then placed in individual
petri dishes with a 175 mm high x 125 mm wide screen cylinder, returned to
the same treatment chamber as they were reared in and checked daily for
emergence. Pupae that develop directly take about 14 days to eclose. After six
weeks live pupae were assumed to be in diapause and dead pupae were
discarded. Pupae were considered to be alive if the abdomen was still flexible.
All butterflies, eggs, larvae and pupae in subsequent experiments were treated

similarly unless otherwise stated.

Florida Population. 1294

Females were collected from Highlands and Levy counties, Florida in
March. The first instars were set up across six treatments with five siblings
per dish instead of one per dish to increase efficiency of space utilization.
Each female’s offspring were randomly assigned as done in the previous
experiment to one of six treatments, and reared at 25°C. One to two dishes
were set up per female. After 10d, larvae were placed into individual dishes
to complete their development. The six photoperiod treatments tested were
as follows: 8:16 (L:D), 9:15, 10:14, 11:13, 12:12, and 13:11. These photoperiods
were selected because in Florida, the longest day is about 14.5h (Beck, 1980), so
all of the treatments used in 1993 were ”long” days to the larvae except the
12:12 and 13:11 treatments. The modified photoperiodic treatments more

closely match and bracket what a Florida larva would experience (Table 1).

13

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14
Ten different family lines were set up between 5 - 11 April for a total of 479

larvae.

Florida Population, 1225

Butterflies were collected from the same counties as previous years and
allowed to oviposit in the laboratory. Larvae were set up in petri dishes with
three to five siblings per dish and one to four dishes per female per treatment.
Larvae were place into individual dishes at 10d to complete development. A
total of 682 larvae in fifteen family lines were set up between 4 - 12 April.
Results from the previous two experiments (in 1993 and 1994) suggested that
the critical photoperiod for diapause was likely to be between 12h and 13h of
light; therefore a 12.5:11.5 treatment was added and the treatments were
modified to the following five: 10:14(L:D), 11:13, 12:12, 12.5:11.5, and 13:11.
Each female’s offspring were randomly assigned as done in previous
experiments. Those pupae that did not eclose within six weeks were

considered to be in diapause.

ia h' M' hi a P l i 1

Butterflies were collected from Clarke County, Georgia; Gallia and
Lawrence Counties, Ohio; and St. Joseph and Clinton Counties, Michigan in
July 1993 (Figure 3). The offspring from twenty-three different females were
set up for a total of 973 larvae. First instar siblings were set up two to five per
dish, one to three dishes per female per treatment, and at 10d larvae were
subsequently placed into individual dishes. Each dish was randomly assigned
to one of the nine treatments so that each female line was represented in each
treatment. The treatments were: 12:12 (L:D), 13:11, 13.5:11.5, 14:10, 14595,

15:9, 15.5:8.5, 16:8 and 18:6. Larvae were reared to pupation on black cherry

15
(Prunus serotina), collected in Michigan. Black cherry was used because sweet

bay is not available to northern populations of P. glaucus. Georgia offspring
were set up between 19 — 22 August, Ohio offspring set up between 11 - 13

August, and Michigan larvae were set up between 10 - 19 August.

r i ' n ' i a P l i 1

Butterflies were collected from Clarke County, Georgia; from Gallia and
Lawrence Counties, Ohio; and from St. Joseph, Eaton, Calhoun, and Ingham
Counties, Michigan in July. There was also one female butterfly used from
each of Columbus, Franklin, Harris, and Union Counties, Georgia. Offspring
were randomly assigned to treatments as described above. The treatments
were as follows: 12:12 (L:D), 13:11, 14:10, 14.5:9.5, 15:9, 15.5:8.5, 16:8, and 18:6.
Larvae were reared to pupation on black cherry. Twenty-eight different
family lines were set up across the eight photoperiod treatments for a total of
1,948 larvae. Georgia offspring were set up between 9 - 12 August and 16 - 22
August. Ohio neonates were set up between 7 - 10 August and 17 - 20 August.
Michigan larvae were set up between 31 July and 8 August. Larvae and pupae

were treated as in other experiments.

Statistical Analysis

Data were analyzed using logistic regression analysis. Analysis was done
by testing percent diapause versus photoperiod for each state (with all years
lumped together). R2 values were used to determine how fit the model was
for predicting diapause induction, where 1 is a perfect fit and 0 means there is

no gain by using the model (JMP 1995).

16
Results

The following results should be interpreted with caution. High mortality

rates and possible interactions with pathogens may have biased the results.

Fl ' a P l ' 1 4 an 19

In 1993, 50% of the pupae diapaused when reared at 12h of light; only 11%
of the pupae reared at 13h diapaused. At 13.5h of light and longer all pupae
direct developed into adults except for 20% (n: 1 / 5) at 15h of light (Figure 4,
Table 2).

In 1994, when larvae were reared at shorter day lengths, 25% of the pupae
in the 13h treatment diapaused and 60% diapaused in the 12h treatment
(Figure 5, Table 3). Percent diapause increased from 60% to 80% with a
decrease in day length from 11h to 8h of light, respectively. In no treatment
was there 100% diapause for Florida butterflies for either year. Therefore, the
critical photoperiod for diapause for these experiments appeared to be less
than 13h of light.

In 1995, the critical photoperiod was more closely approximated and
appeared to be between 12.0h and 12.5h of light. Percent diapause decreased
from 70% at 12.0h to 0% at 12.5h of light (Figure 6, Table 4).

r ia ' and ' ' P l ' 1 1

In the 1993 Georgia population experiment, at 16h of light, 50% (n: 1/ 2) of
the pupae diapaused. No pupae diapaused at 15.5h and only 14% diapaused at
15h of light (Figure 7, Table 5). At 14.5h of light, percent diapause went up to
67%; the critical photoperiod appears to be between 15h and 14.5h of light. In
1994, the Georgia population responded with a low incidence of diapause at

18h, 16h and 15h, although 75% diapaused (n: 3 / 4) at 15.5h. Diapause

17

 

100-

Percent Diapause

 

 

 

// //W
12.0 13.0 13.5 14.0 14.5 15.0 15.5 16.0 18.0
Hours of Light
Figure 4. Papilio glaucus, Florida Population, Spring 1993.

Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 7, range in number of pupae: 4-12.

 

100-

75-

50-

Percent Diapause

 

 

 

8.0 9.0 10.0 11.0 12. 0 13. 0
Hours of Light

Figure 5. Papilio glaucus, Florida Population, Spring 1994.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 7, range in number of pupae: 4-10.

 

Tub:
pupae

ll

.blglh
ii I‘lEi
9?.

h
971
972
974
97?
To

°iDh

 

Tabb:
Pupae

18
Table 2. Papilio glaucus, Florida Population, Spring, 1993; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

Hr'tPhoh

Mother # 12.9 13.0 13.5 14.9 14.5 15.9 15.5 16.0 18.9

# neonates 40 40 38 38 41 39 39 39 39

9701 -- -- 0/1 0/2 0/1
9703 0/1 -- -- --
9718 2/4 0/1 0/3 0/4 0/3 1/3 0/3 0/2 0/3
9719 0/ 2 0/ 2 --- 0/ 2 0/1 0/1 0/3 0/ 3 0/1
9720 2/ 2 1/ 2 --- 0/ 2 0/ 2 --- 0/1 0/ 2 0/1
9744 0/2 0/2 0/1
9751 0/1 0/1 0/1
Total 4/8 1/9 014 0/12 0/9 1/5 0/7 0/7 0/5

%Diapause 50 89 0 0 0 20 0 0 0

 

Table 3. Papilio glaucus, Florida Population, Spring, 1994; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

Hmsinfluuihetnphase
Mother # 8.0 9.0 10.0 11.0 12.0 13.0

# neonates 35 37 39 38 39 43

10318 1/1 2/2 3/3 1/1 1/1
10323 0/1
10327 4/4 3/3 2/3 2/3 2/4
10329 0/1

10335 3/5 2/5 1/3 0/1 1/1 0/1
Total 8/10 7/10 6/9 3/5 3/5 1/4

%Diapause 80 70 67 60 60 75

19

 

100-

50-

25-

Percent Diapause

 

 

 

10.0 11.0 ‘ 12.0 12.5 13.0
Hours of Light

Figure 6. Papilio glaucus, Florida Population, Spring 1995.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 12, range in number of pupae: 8-16.

Table l. |
in diapa-

ll

 

 

20
Table 4. Papilio glaucus, Florida Population, Spring, 1995; number of pupae
in diapause per number of pupae formed, for each family line.

 

 

thus in the PthQphasg
Mother # 10.0 11.0 12.0 12.5 13.0

#neonates 129 122 111 118 126

11004 --- 1/ 3 --- 0/ 3 1/ 3
1 1046 --- 4/ 4 --- --- --
1 1048 1 /1 --- --- -- 0/ 4
11053 5/ 5 1/1 --- 0/1 0/ 3
11064 2/ 2 --- 3/ 6 0/ 3 0/1
11065 1/1 --- 1/1 -- ---
1 1066 --- --- --- 0/1 1 / 3
11070 --- 1/1 1/1 --- ---
1 1097 --- 3/ 3 1/1 --- ---
1 1099 --- 1 / 1 --- --- ---
11104 1/2 --- 1/1 --- ---

11106 2/2 1/1
Total 12/13 12/14 7/10 0/8 2/14

%Diapause 92 86 70 0 14

 

21

 

1.1
O
‘P

\1
9"

Percent Diapause
N U1
S" ‘P

  
  

 

 

E :
12.0 13.5 14.0 14.5 15.0 15.5 16.0 18.0 24.0
Hours of Light

 

‘P

Figure 7. Papilio glaucus, Georgia Population, Late Summer 1993.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 4, range in number of pupae: 1-7.

 

100-

\1
S"

Percent Diapause
N U1
91 c

 

 

 

0: // //
12.0 13.0 14.5 15.0 15.5 16.0 18.0
Hours of Light
Figure 8. Papilio glaucus, Georgia Population, Late Summer 1994.

Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 5, range in number of pupae: 2-9.

22

Table 5. Papilio glaucus, Gerogia Population, Late Summer, 1993; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

Mother L 13.5

# neonates
10021
10025
10026
10027
10029

Total

22
1/1
1/1
0/1

2/3

%Diapause 67

Hr' P

ha

14.0 14.5 15.0 15.5 16.9 24.9

15 19 25
3/3 1/1 0/3
-- -- 0/1
0/1 0/1 0/2
-.. 1/1 1/1

3/4 2/3 1/7

75 67 14

26
1/1

1/3
0/1
2/5

40

 

23

Table 6. Papilio glaucus, Georgia Population, Late Summer, 1994; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

W
Mother # 12.0 13.0 14.5 15.0 15.5 16.0 18.0

# neonates 100 101 100 99 99 99 101

10727 -- 0/1
10728 1/1 3/3 0/2 0/2 0/1
10730 1/2 {
10731 1/1 1/1 0/1 -- 0/3
10737 1/1
10757 1/1 1/1 1/1 1/2 0/2
10760 -- 0/1 1/1
10779 1/1
10787 1/1
10790 1/1 --
10799 1/1 1/1 1/1

Total 4/4 4/4 2/2 2/6 3/4 1/5 3/9

%Diapause 100 100 100 33 75 20 33

 

24
increased from 33% at 15h to 100% at 14.5h of light (Figure 8, Table 6). There

appears to be no difference in the critical photoperiod between 1993 and 1994
for the Georgia population.

In the 1993 Ohio population, the the critical photoperiod was between 15h
and 14.5h of light, same as for the Georgia population; diapause at the longer
photoperiods ranged from 14% to 36% and increased up to 100% at 13.5h of
light (Figure 9, Table 7). In the 1994 Ohio population the long days 16h and
18h induced 60% and 50% diapause respectively; although only 17%
diapaused under the 15.5h photoperiod (Figure 10, Table 8). Many of these
larvae died of disease, with only about 5% survival.

For the Michigan population, diapause was induced in at least 55% of the
larvae reared at photoperiods as long as 18h in both years (Figures 11 and 12,
Tables 9 and 10). Only in the 24h photoperiod was there no diapause. (The
13:11 treatment was actually a 24:00 treatment due to a malfunction with the
lighting dial, which was not discovered until most of the pupae direct
developed where diapause was expected.) In 1993, 55% of the pupae
diapaused under 18h, while 80% diapaused in 1994. In addition, percent
diapause increased from 60% at 15.5h to 94% at 15h of light in 1993. 100%
diapause occurred under photophases shorter than 14.5h of light in 1993.
Percent diapause was at 100% from the 16h photophase down to the 12h
photophase, in 1994.

There appears to be an absolute photoperiod, for the populations north of
Florida, where all pupae will diapause, which was at 13h days or shorter

(Figure 13).

 

100-

75-

50-

25-

Percent Diapause

 

 

 

7.7

12.0 13.5 14.0 14.5 15.0 15.5 16.0 18.0 24.0
Hours of Light

9’

Figure 9. Papilio glaucus, Ohio Population, Late Summer 1993.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 17, range in number of pupae: 11-22.

 

100-
O)
‘3
a 75.
Du
.53
D 50-
E
Q)
a 25-
Q)
D-t

 

 

 

0.
12.0 13.0 14.5 15.0 15.5 16.0 18.0
Hours of Light

Figure 10. Papilio glaucus, Ohio Population, Late Summer 1994.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 4, range in number of pupae: 1-10.

26
Table 7. Papilio glaucus, Ohio Population, Late Summer, 1993; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

figurs in the PthQphsse
Mother # 12.0 13.5 14.0 14.5 15.0 15.5 16.0 18.0 24.0

 

# neonates 45 42 48 48 44 48 44 43 44

9953 2/ 2 2/ 2 1/1 0/1 0/1 1/ 2 O/ 3 0/1 ---
9960 -- --- 1/1 2/ 2 --- 1 / 3 -- --- ---
9965 2/2 1/1 2/2 2/2 1/1 2/3 3/3 2/3 3/3
9967 2/2 -- 1/3 1/1 1/3 0/1 0/2 -- 0/2
9968 1/1 3/3 2/3 1/1 0/3 0/3 0/1 0/1 0/5
9972 3/ 3 6/ 6 3/ 3 O/ 2 --- 0/ 3 0/ 4 0/ 2 0/ 4
9974 4/ 4 1/1 -- 2/ 2 1/ 4 --- --- 0/1 0/ 3
9975 -- --- 1 / 2 0/ 2 0/1 -- --- --- 0/1
9976 2/ 2 4/ 4 4/ 4 2/ 3 0/ 4 0/ 6 0/5 2/ 3 0/ 4
Total 16/16 17/17 15l19 10/16 3/17 4/21 3/18 4/11 3/22

%Diapause 100 100 79 63 18 20 17 36 14

 

27

Table 8. Papilio glaucus, Ohio Population, Late Summer, 1994; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

Mother# 12.0 13.0 14.5 15.0 15.5 16.0 18.0

# neonates 87 95 86 90 87 90 85

10705 --- --- --- m 0/1 --- ---
10708 --- --- --- 1/1 -- --- ---
10710 -- -- --- -- 1/1 2/ 3 --
10711 2/2 --- --- 1/2 0/1 --- 1/1
10713 4/4 1/1 -—- --- 0/1 1/2 0/1
10717 1/1 1/1 1/1 1/1 --- --- ---
10747 1/1 --- --- m 0/1 --- ---
10748 1/1 --- --- --- 0/1 --- --_
10750 --- 1/1 m m m --- ---
10758 1/1 -- m m --- --- _--
Total 10/10 3/3 1/1 3/4 1/6 3/5 1/2

%Diapause 100 100 100 75 17 60 50

 

28

 

1.1
C
j

\1
L

N
9‘

  

Percent Diapause
U1
O

 

 

 

/ . - //m'
12.0 13.5 14.0 14.5 15.0 15.5 16.0 18.0 24.0

Hours of Light

9

Figure 11. Papilio glaucus, Michigan Population, Late Summer 1993.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatement= 12, range in number of pupae: 7—20.

 

100-

\1
91

Percent Diapause
N U1
91 o

 

 

 

// //
12.0 13.0 14.5 15.0 15.5 16.0 18.0
Hours of Light

Figure 12. Papilio glaucus, Michigan Population, Late Summer 1994.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatement= 5, range in number of pupae: 1-9.

29
Table 9. Papilio glaucus, Michigan Population, Late Summer, 1993; number
of pupae in diapause per number of pupae formed, for each family line.

 

 

P t a

M er # 12. 1 . 14. 14. 15 15. 16.0 18.0 24.0

# neonates 43 47 39 36 39 44 39 39 40

9992 3/ 3 4/ 4 5/5 3/ 3 3/ 4 0/ 4 1 / 2 2/ 4 O/ 3
9994 --- -- --- -- --- 2/ 3 0/1 1 / 1 ---
9995 2/ 2 1 /1 --- 2/ 2 1/1 2/ 2 --- 0/ 2 ---
9996 1/1 --- -- 2/ 2 2/ 2 -- --- 1/ 3 0/ 2
10002 -- --- 1/1 --- 3/ 3 1/ 2 0/1 2/ 3 0/1
10005 2/ 2 1 /1 --- -- 3/ 3 2/2 -- 3/5 0/1
10008 -- -- --- --- -- 0/1 --- --- ---
10015 3/3 1/1 1/1 1/1 2/2 2/3 1/1 2/2 0/1
10017 2/ 2 --- 1/1 --- 1/1 3/ 3 2/ 2 --- ---
Total 13/13 7/7 8/8 8/8 15/16 12/20 4/7 11/20 0/8

%Diapause 100 100 100 100 94 60 57 55 0

 

30
Table 10. Papilio glaucus, Michigan Population, Late Summer, 1994; number
of pupae in diapause per number of pupae formed, for each family line.

 

 

r ' h a
Mother # 12.0 13.0 14.5 15.0 15.5 16.0 18.0

# neonates 55 56 55 53 55 55 53

10657 -- 1/1 m m m --- _..
10658 5/5 2/2 1/1 1/1 --- 2/ 2 0/1
10660 --- -- --- 1/1 --- --- 2 /2
10662 1/1 2/2 m m m -..- 2 /2
10663 --- 1/1 m m m --- ---
10674 --- 1/1 --- --- --- --- ---
10675 --- 2/ 2 2/ 2 3/ 3 1/1 1/1 ---

Total 6/6 9/9 3/3 5/5 1/1 3/3 4/5

%Diapause 100 100 100 100 100 100 80

31

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' a1 a1 i

The results of the analysis are contained in Table 11. All R2 values were

less than 0.5.

 

111. R 1 wt: in'od lvi ' re -osinanal i - ta.

State 112 iegiakemgd Climate 1L__u_e-va1

 

Florida .3672 24.98 49.96 0.00

Georgia .0544 2.22 4.44 0.03

Ohio .0252 26.72 53.44 0.00

Michigan 0.3427 24.84 49.69 0.00
Discussion

Disease

Disease may play a role in diapause induction. Hanski (1988) noted that
insects can go into diapause to avoid adverse conditions other than weather,
such as high disease conditions. The diseases that caused a high mortality
rate amongst the larvae may have altered the experimental conditions. One
pathogen was determined to be a bacteria, Seratia marsescens. I believe other
pathogens were also present such as viruses and other bacteria due to the

differences in the ways the larvae and prepupae died.

II'li 11101.}: It
The critical photoperiod for the Ohio population was lower in 1994 than

in 1993, and more pupae diapaused from the Michigan population in 1994

than in 1993. The difference between 1993 and 1994 may be attributed to small

sample size, but a high rate of disease in 1994 could have altered the critical

33
photoperiod, inducing diapause on longer days in the presence of disease.

Tauber et a1. (1986) stated that the critical photoperiod determined by
experiments are for specific conditions. Here the presence of disease altered

the specific conditions set up for the experiment.

Geergia Pepulan'en
The critical photoperiod for both 1993 and 1994 was between 14.5h and

15.0h of light. The photoperiods that larvae in the wild would likely be
experiencing at the time of year the tests were done are between 13.7h and
11.3h of light (Table 1). The critical photoperiod determined by these
experiments seems high for this latitude. Perhaps there is some token
stimulus in addition to photoperiod responsible for this result. See further

discussion.

Elerida Pepglatien

The Florida population seems to be different from its northern
counterparts. The critical photoperiod determined by these experiments for
Florida populations was between 12.0h and 12.5h of light, and all northern
populations tested diapaused under these conditions. This may be due to
being reared on a different host, sweet bay, than the other populations. A
more likely explanation is that Florida is a subtropical environment and
experiences less change from season to season, including smaller changes in
day length. Cockrell et a1. (1994) found that monarch butterfly (Danaus
plexxipus) populations in Gainesville, FL avoid the very hot weather, while
other populations north and south of the area continue to reproduce without
interruption by diapause or quiescence. It was suggested that maybe P.

glaucus also avoids the hottest days of summer between June and September.

34
However the data do not support this hypothesis. Butterflies are flying in

Florida in March when days are about 12.1h long. The subsequent offspring
would experience days longer than 12.5h-above the critical photoperiod—and
direct develop into adults. Butterflies would be flying again by mid-May
when temperatures are frequently in the 90°s (Climates of the States 1985).
These butterflies may be using some cue other than photoperiod, otherwise
the butterflies and their offspring would be out in the midst of the Florida
heat.

In addition, not all Florida pupae direct develop on long days or all
diapause on short days. These butterflies may be ”bet-hedging” by spreading a
few offspring over the season. Normally pupae emerge in about two weeks,
but I have observed Florida pupae reared in the laboratory emerge 45 to 50
days after pupation. Florida butterflies may have a slightly different genetic
make up that allows for an extended pupal stage that other populations do
not have, thus increasing the butterfly’s chances for survival over the longer,

sub-tropical season.

Statistical Analysis

According to the R2 values obtained from the logistic regression,
photoperiod alone cannot explain diapause induction, at least for these
experiments. All values were less than 0.5, indicating that P. glaucus may be
using a token stimulus in addition to photoperiod to induce diapause. It
seems that photoperiod would override any other cue, especially in Michigan,
since the climate is not as predictable or stable as it is in the other locations
tested (Table 12 and 13).

Probit and Logit were not used to analyze data because I did not have

enough ’non-zero’ data points. Also, contingency table analysis or Chi-square

35

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36
analysis were mathematically impossible because I had expected values of

zero.

Twilight was not included in any of the given photoperiods. The
importance of twilight varies among species depending on their individual
light intensity thresholds (Beck 1980), and the chambers in which the larvae
were reared did not take into account twilight. The lights in the chambers
came on and went off at the prescribed times. The effect of twilight was not

investigated in this experiment.

A LATITUDINAL COMPARISON

The longest days that Michigan larvae would experience is 15.6h and by 1
October the days are about 11.7 hours long (Nautical Almanac). The critical
photoperiod determined by these experiments was 18h, even though
Michigan larvae never experience a day this long. Photoperiod may not be
the only cue Michigan larvae or the Ohio or Georgia populations use to
determine season, and thus to trigger diapause.

It is not known what the sensitive stage is for the tiger swallowtail.
Although we know it is probably the larval stage, we do not know if it is
limited to one or more instars. We also do not know how larvae interpret
the photoperiodic cue, e.g., whether they can detect decreasing day lengths or
take an average of the total light hours experienced. Some insects can only
detect change in day length instead of absolute day length, so the proper cue of
decreasing light hours would not have been available to the larvae reared at a
constant photoperiod (Tauber et a1. 1986). Larvae take about one month to

reach the pupal stage and the day length can change by almost one and a half

37
hours from 1 August to 1 September in Michigan, which is when wild larvae

would be growing outside.
Alternative hypotheses follow in the discussion as to why the critical
photoperiod in Michigan is greater than 18.0h of light and why there were

differences and similarities between the populations tested.

za 1

One explanation why the critical photoperiod for the Michigan population
seems high is that there may be hybridization and introgression between P.
glaucus and P. canadensis. Their ranges overlap in mid-Michigan at a ”hybrid
zone” and there may be gene flow between these two species, which would
account for the higher incidence of diapause at longer photoperiods. P.
canadensis is an obligate diapauser, but even a few individuals direct-
deve10ped when reared at 24h of light (Rockey 1987b). This gene flow may
only go in one direction. If a female P. glaucus hybridizes with a male P.
canadensis most females and some males diapause because they will all
inherit the obligate diapause gene from their father (Rockey et al. 1987a,b). If a
female P. canadensis hybridizes with a male P. glaucus some of the males will
diapause, since they are heterozygous for the X chromosome, and all of the
females will be facultative diapausers, inheriting the diapause gene on the X
chromosome of their P. glaucus father. It is not known to what extent
hybridization and introgression takes place between P. glaucus and P.
canadensis, although Hagen et a1. (1991) believe it is not very common. But
some P. canadensis genes may be increasing the genetic variability and thus
influencing this high critical photoperiod. Scriber (1990) has noted a ”Spring
form” of P. glaucus which resembles P. canadensis in color and wing pattern.

He proposes that this similarity is due to P. glaucus containing P. canadensz's

38
genes obtained through introgression. So it is possible that there is influence

from P. canadensis, at least in the Michigan population.

Gene Flow

This experiment could not detect a difference between the Georgia and
Ohio populations. Both populations appear to have a critical photoperiod of
14.5h of light. The fact that the critical photoperiod is the same may be due to
small sample size, or that there really is no difference between these
populations. This homogeneity may be maintained by gene flow between
Georgia and Ohio populations. Larger sample sizes, with less disease in
laboratory rearing could help differentiate the relative causes and the critical

photoperiod for diapause.

HostflanLQuaJiu
As stated earlier, P. glaucus is probably not primarily using day length as a

token stimulus. Host plant quality may be influencing diapause induction as
well. If larvae are reared on a ”good” host, they will grow fast and pupate
while the days are still long. If a larvae that hatched at the same time was
reared on a lower quality host species, they will grow slower and pupate while
when the days are getting shorter. The larvae on good hosts will
subsequently become adults earlier in the season and produce another
generation. The larvae on the lower quality host will pupate and probably go
into diapause because it is too late in the season to reproduce another
generation. Host plant quality and host plant species may be more important
in diapause induction than photoperiod, especially in areas where three
generations could be possible on high quality hosts, such as Ohio and Georgia.

There could be another explanation. P. glaucus larvae may incorporate

39
more than one cue to determine change of season. Host plant quality may be

influencing diapause induction (Hagen and Lederhouse 1985). They reported
a greater than 50% diapause induction in all late season populations of P.
glaucus north of about 38° latitude when reared at 16:8. This would coincide
with the results from the Michigan population.

The pink bollworm, Pectinophora gossypiella, was affected by diet. When
fed a diet of 1% lipid content versus 5% lipid content, the higher lipid content
diet favored induction of diapause (Ankersmit and Adkisson 1967). In
addition to the bollworm, P. zelicaon can take into account the quality of the
host plant and enter diapause on an ephemeral host or direct develop on a
more enduring host species. The P. glaucus larvae may have detected a
change in the host plant (black cherry) quality which would override any

photoperiodic stimulus (Sims 1983).

Maternal Effeet

Another hypothesis as to why there was an overall high percentage of
diapause in the Michigan population is that there may have been a maternal
effect. I refer to maternal effect in the sense that ”offspring of diapausing
females direct develop into adults, and offspring of direct developing females
go into diapause.” The Michigan butterflies collected from the field for these
experiments were second generation adults, offspring of diapausing mothers.
This second generation came from direct developing females. If there is a
maternal effect on diapause induction, all of the offspring should have
diapaused and most of them did from the Michigan population. Only under
24h of light was there 100% emergence of pupae. A maternal effect would
also ensure that there would be pupae diapausing late in summer for the next

generation in spring. A maternal effect is probably not apparent in the Ohio

40
or Georgia population where the climate is milder than in Michigan (Table

13). Further experiments are needed to confirm this hypothesis.

Conclusion
P. glaucus is likely using a cue(s) other than photoperiod or in addition to
photoperiod to determine the season and to induce or avert diapause. Other
cues that P. glaucus may be sensitive to are changes in thermoperiod or host
plant quality. Further investigation is needed to determine what the extent of
gene flow is, how host plant quality or host plant species can affect diapause

induction and the role the mother plays in diapause induction.

CHAPTER 2

Photoperiodic Induction of Diapause in Populations of Papilio troilus

Occurring at Different Latitudes

Abstract

For P. troilus, facultative diapause is induced by seasonal changes in the
environment. The most reliable seasonal cue in its range is day length.
Females were collected from Florida, Georgia, Ohio and Michigan and their
offspring were reared at different photoperiods. The purpose of this research
was to determine the extent of population differences with respect to the
critical photoperiod for diapause and to better understand diapause induction
in this species. The critical photoperiod for diapause for the Florida
population appeared to be between 12.0h and 12.5h of light. The critical
photoperiod for the Georgia population could not be determined but it was
longer than 13.0h and shorter than 14.5h of light. The critical photoperiod for
the Ohio population appeared to be between 13.0h and 14.5h of light. For the
Michigan population it was between 14.5h and 15.0h of light. There were
differences between populations. The more northern populations entered
diapause on longer days than more southern, and the R2 values obtained
from the logistic regression suggest that P. troilus is using photoperiod as a
cue to avert / induce diapause. These data support the idea that diapause
induction by photoperiod is synchronizing the life cycle of P. troilus with the

seasonal changes in the environment.

41

42
Introduction

P. troilus is multivoltine and a facultative diapauser which relies on
environmental cues. Photoperiod is thought to be the most reliable indicator
of season throughout its range. The purpose of this research was to
determine the critical photoperiod for diapause for populations of P. troilus
occurring in Florida, Georgia, Ohio and Michigan, and to determine if there

were any differences between these populations.

Materials and Methods
1 i P la i 19

Butterflies were collected from Highlands and Levy counties, Florida in
March (Figure 3) and shipped via Federal Express or brought back to Michigan
in a cooler. Females were allowed to oviposit in the laboratory and were fed a
1:4 honey:water solution (see Scriber 1993). Eggs were collected daily, put in a
petri dish and placed in an 18°C chamber, with a photoperiod of 18:6 (L:D).
Hatching occurred in five to seven days. The neonates were set up one per
dish in petri dishes with foliage from the field. Each female’s offspring were
equally distributed amongst the treatments, but assigned to a treatment in
random order. This was done to avoid any bias from early versus older
offspring. The larvae were reared at 25°C, and the nine photoperiod
treatments were as follow: 12:12 (L:D), 13:11, 13.5:10.5, 14:10, 14.5:9.5, 15:9,
15.5:8.5, 16:8, and 18:6. After nine larvae from a female were set up across the
treatments, the next nine larvae were randomly assigned in a similar
manner, until all larvae were evenly distributed. This was done to eliminate
bias between younger vs. older larvae. Offspring from four different females
were set up for a total of 456 larvae, which were set up between 3 - 10 April.

Family lines were followed throughout the experiment.

43
P. troilus larvae were reared to pupation on redbay, Persea borbonia,

collected in Florida and shipped to Michigan via Federal Express or brought
back to Michigan in a cooler. Leaf turgor was maintained using aquapics
(rubber-capped, water-filled vials). Fresh leaves were provided 1-2 times per
week for early instars and 2-3 times per week for later instars, until pupation.
Pupae were weighed one day after pupation, placed in individual petri dishes
with a 175 mm high x 125 mm wide screen cylinder, returned to the same
treatment chamber and checked daily for emergence. After six weeks live
pupae were assumed to be in diapause and dead pupae were discarded. Pupae
were considered to be alive if the abdomen was still flexible. All butterflies,
eggs, larvae and pupae in subsequent experiments were treated similarly A
otherwise stated. All larvae were reared at the same time, in the same

chambers with the P. glaucus larvae.

l r' l ' n

Females were collected from Highlands and Levy counties, Florida in
March. Butterfly adults were treated in a similar manner as in the previous
year. The first instars were set up five per dish instead of one per dish to
increase efficiency of space utilization. At 10d, larvae were put into
individual dishes to complete their larval development. Neonates were
randomly assigned to one of six photoperiod treatments, and reared at 25°C.
The six photoperiod treatments were as follows: 8:16, 9:15, 10:14, 11:13, 12:12,
and 13:11. These photoperiods were used because in Florida, the longest day
is about 14.5h (Beck, 1980), so all of the treatments used in 1993 were ”long”
days to the larvae except the 12:12 and 13:11 treatments. The modified
photoperiod treatments more closely match what a Florida larva would

experience. After six dishes of larvae from each female were set up across the

44
treatments, another six were randomly assigned to a treatment, and so on

until three dishes per treatment per female were set up. Larvae were reared
to pupation and treated in the same manner as in 1993. Three family lines
were set up across the six treatments for a total of 61 larvae, which were set up

between 7 — 10 April.

1 r' a '

Butterflies were collected from the same counties as in previous years.
Larvae were originally set up five per dish and then placed into individual
dishes at 10d. Due to the results in the previous two experiments, it was
hypothesized that the critical photoperiod for diapause may be between 12
and 13h of light, therefore the 12.5:11.5 treatment was added and modified to
the following five treatments: 10:14, 11:13, 12:12, 12.5:11.5, and 13:11.
Offspring from five different females were set up; one to three dishes per
female per treatment were randomly assigned as done in previous
experiments. A total of 232 larvae was set up between 4 - 16 April. Larvae

were reared to pupation on redbay and pupae were treated as before.

1 1 n u 1

Butterflies were collected from Gallia and Lawrence counties, Ohio and
from St. Joseph county, Michigan during July. No butterflies were collected
from Georgia in 1993. A total of 547 larvae was set up from twelve different
female lines. Larvae were set up two to three per dish, two to three dishes per
female per treatment. After 10d larvae were placed into individual dishes to
complete their development. Larvae were reared to pupation on sassafras
(Sassafras albidum), which was collected from the field, because redbay is not

available to northern populations of P. troilus. Leaf turgor was maintained

45
using aquapics. Each dish was randomly assigned as done before to one of the

following photoperiods (L:D): 12:12, 13:11, 13.5:10.5, 14:10, 14.5:9.5, 15:9, 15.5:8.5,
16:8, and 18:6. Larvae were reared to pupation and pupae were treated in a
manner similar to the Florida population. Ohio larvae were set up between
10 - 16 August, and Michigan neonates were set up between 13 - 14 August
and 21 - 22 August.

r i hi an Mi i an 1 ' 1

Butterflies were collected from Clarke and Echols counties, Georgia; Gallia
and Lawrence counties, Ohio; and from Ingham county, Michigan during
July. Fourteen family lines were set up, for a total of 768 larvae. Larvae were
reared on sassafras collected from the field. Five neonates per dish and one to
three dishes per female per treatment were set up. Larvae were placed into
individual dishes at 10d to complete development. Each female’s offspring
were randomly assigned to one of the following L:D treatments: 12:12, 13:11,
14:10, 14.5:9.5, 15:9, 15.5:8.5, 16:8, 18:6. Georgia larvae were set up between 18 -
25 August, Ohio neonates were set up between 7 - 12 August and Michigan
offspring were set up between 31 July and 8 August. Pupae were treated as

before.

Data were analyzed using logistic regression analysis. Analysis was done
by testing percent diapause versus photoperiod for each state (with all years
lumped together). R2 values were used to determine how fit the model was
for predicting diapause induction, where 1 is a perfect fit and 0 means there is

no gain by using the model (JMP 1995).

46

Results
The following results should be interpreted with caution. Small sample

size or possible interactions with pathogens may have biased the results.

Fl ' a P l ' 1 4 1

All larvae that survived to pupation in the 1993 experiment directly
developed into adults (Table 14). All larvae that survived to pupation in the
1994 experiment diapaused (Table 15). The critical photoperiod for diapause
appeared to be between 12h and 13h of light; The 1995 experiment more
closely approximated the critical photoperiod to be between 12h and 12.5h of
light (Figure 14, Table 16). Percent diapause dropped from 100% at 12h to 0%
at 12.5h of light.

r ia hi an Mi ' 1 1 9

For the Georgia population, only two family lines were represented and
only in 1994 (Figure 15, Table 17). All larvae reared in the 14h treatment died,
which was likely close to the critical photoperiod for diapause. At 14.5h of
light all pupae emerged and at 13h of light all pupae diapaused. The critical
photoperiod for diapause was either between 14.5h and 14h or between 14h
and 13h of light.

In the 1993 Ohio experiment, percent diapause increased from 3% at 15h to
64% at 14.5h of light (Figure 16, Table 18). The critical photoperiod for
diapause appears to be between 15h and 14.5h of light. In 1994, percent
diapause increased from 3% at 15h, to 28% at 14.5h and to 100% at 13h of light
(Figure 17, Table 19). All the larvae in the 14h treatment died. There were

insufficient data to determine whether the critical photoperiod for diapause

47
Table 14. Papilio troilus, Florida Population, Spring, 1993; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

Hoursinflefihmgplm
Mother # 12.0 13.0 13.5 14.0 14.5 15.0 15.5 16.0 18.0

# neonates 51 52 51 51 50 51 50 49 51

9711 --- 0/1 —-- --- 0/2 --- --- m m
9713 --- --- --- 0/ 2 0/ 2 --- 0 / 2 --- ---
9714 --- 0/1 --- --- O/ 2 --- 0/1 0/ 2 0/1
Total --- 0/2 --- 0/2 0/6 -- 0/3 0/2 0/1
%Diapause --- 0 --- 0 0 --- 0 0 O

 

Table 15. Papilio troilus, Florida Population, Spring, 1994; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

H in P t
Mother # 8.0 9.0 10.0 11.0 12.0

# neonates 29 32 34 31 32

10315 1/1 2/2 1/1 5/5 4/4
10316 1/1 2/2 1/1 --- 1/1
10332 2/2 1/1 1/1 1/1 1/1
Total 4/4 5/5 3/3 6/6 6/6

%Diapause 100 100 100 100 100

48

Table 16. Papilio troilus, Florida Population, Spring, 1995; number of pupae
in diapause per number of pupae formed, for each family line.

 

 

H

i

P

ha

Mether # 19,9 11.0 12.9 12.5 13.0

# neonates 37

11008 1/1
11071 1/1
11090 ---
11114 ---
11131 1/1
11135 ---
Total 3/3

%Diapause 100

40
3/ 3
2/ 2

4/ 4
1/1
10l10

100

43

42
0/ 3
0/1
0/1

40
1/6

49

 

     

 

Percent Diapause
U1
‘P

 

// ‘ I V
10.0 11.0 12.0 12.5 13.0
Hours of Light

Figure 14. Papilio troilus, Florida Population, Spring 1995.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatement= 6, range in number of pupae: 3—10.

 

100W
75+
50-

25-

Percent Diapause

 

 

 

12.0 13.0 14.5 15.0 15.5 16.0 18.0
Hours of Light

Figure 15. Papilio troilus, Georgia Population, Late Summer 1994.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatement= 7, range in number of pupae: 3-11.

50

Table 17. Papilio troilus, Georgia Population, Late Summer, 1994; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

Heme in the Phetephase
Mother # 12.0 13.0 14.5 15.0 15.5 16.0 18.0

 

# neonates 7 10 11 11 11 5 5
10800 2/2 2/ 2 0/3 1/ 3 0/3 0/ 2 0/2
10859 1/1 4/ 4 0/ 8 0/ 7 0/ 8 0/1 0/ 2
Total 3/3 6/6 0/11 1/10 0/11 0/3 0/4

%Diapause 100 100 0 10 0 0 0

 

51

 

 

 

 

100-
Q)
8
a 75
9..
.2
Q 50-
a
Q)
2 25-
a)
9-4
0 //t//¢7'
12.0 13.5 14.0 14.5 15.0 15.5 16.0 18.0 24.0

Hours of Light

Figure 16. Papilio troilus, Ohio Population, Late Summer 1993.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 37, range in number of pupae: 39-45.

 

100-

75

50‘

25-

Percent Diapause

 

  

 

 

//

12.0 13.0 14.5 15.0 15.5 16.0/18.0
Hours of Light

Figure 17. Papilio troilus, Ohio Population, Late Summer 1994.

Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatment: 47, range in number of pupae: 36-59.

52
Table 18. Papilio troilus, Ohio Population, Late Summer, 1993; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

W
Mother # 12.0 13.5 14.0 14 15.0 15.5 16. 18. 24.

# neonates 45 50 46 46 49 43 45 44 48

 

9978 5/5 2/2 1/4 6/6 0/6 1/6 0/6 0/5 0/6
9980 5/5 6/6 5/5 3/4 0/6 0/6 1/6 0/6 0/5
9981 6/6 5/5 4/5 2/5 0/4 0/6 0/5 0/6 0/5
9983 3/3 4/4 5/6 5/6 0/5 0/6 0/5 0/6 0/6
9984 6/6 3/3 6/6 2/3 0/1 0/4 0/4 0/4 0/6
9985 3/3 1/1 1/2 1/3 1/4 0/6 0/2 -- 0/4
9989 4/4 2/2 4/4 4/5 0/5 0/5 0/4 0/5 0/4
9990 4/4 6/6 3/5 0/4 0/4 0/6 0/6 0/5 0/4
Total 36/36 29/29 29/37 23/36 1/35 1/45 1/38 0/37 0/40

%Diapause 100 100 78 64 3 2 3 0 0

 

53
Table 19. Papilio troilus, Ohio Population, Late Summer, 1994; number of
pupae in diapause per number of pupae formed, for each family line.

 

 

r ' ha
Mother # 12.0 13.0 14.5 15.0 15.5 16.0 18.0

# neonates 57 65 58 55 67 66 66
10684 4/4 8/8 1/3 1/10 0/8 1/9 3/8
10687 5/5 6/ 6 1/ 7 0/ 4 0/5 0/ 8 0/ 5
10690 7/ 7 8/ 8 1/ 8 0/ 8 0/ 8 0/ 10 O/ 7
10691 6/6 5/5 2/4 0/2 0/6 0/4 1/4
10692 4/ 4 5/5 0/1 0/ 3 0/ 4 3/ 9 2/ 7
10696 4/4 7/ 7 0/2 0/4 0/4 0/ 2 1 / 3

10698 --- 3/ 3 0/ 2 --- 0/ 4 0/ 4 0/5
10699 --- -- -- ~-- -- -- 0/2
10702 9/ 9 8/ 8 3/ 6 0/ 7 0/ 8 2/ 10 0/ 7
10742 --- 4/ 4 2/ 3 O/ 3 0/2 --- ---
10744 --- --- --- --- O/ 1 0/ 3 ---

Total 39/39 54/54 10/36 1/41 0/50 6/59 7/49

%Diapause 100 100 28 3 0 10 14

 

54
was between 14.5h and 14h of light, or between 14h and 13h of light.

In the 1993 Michigan experiment, percent diapause increased from 33% to
100% between the 15h and the 14.5h treatment (Figure 18, Table 20); in 1994,
percent diapause increased from 0% at 15.5h, to 50% at 15h and to 60% at 14.5h
(Figure 19, Table 21). There is a discrepancy between these two years of data as
was also the case for the Ohio population. The 1993 data implies that the
critical photoperiod for diapause is between 15h and 14.5h of light and the
1994 data implies that the critical photoperiod for diapause is between 15h and
15.5h of light.

In Figure 20, average percent diapause vs. hours of light is graphed for P.
troilus populations from Georgia, Ohio and Michigan. The critical
photoperiod seems to change by about one half hour as you move from one
population to the next: 15.0h for Michigan, 14.5h for Ohio and between 14.5h
and 13.0h for Georgia.

Statistieal Analysis

The results of the analysis are contained in Table 22. All R2 values were

greater than 0.5.

 

Al: __. " - 0‘ '1911‘0. 0. 014-! ‘1 ‘ lle'_ i - te.

State 1:2 WWW

Florida .9845 26.92 53.84 0.00
Georgia .7715 18.95 37.90 0.00
Ohio .5607 n / a n/ a 0.00

Michigan .6361 100.94 201.88 0.00

 

55

 

100-

\1
E”

N
9‘

  
     

Percent Diapause
01
O

 

 

//l//m
12.0 13.5 14.0 14.5 15.0 15.5 16.0 18.0 24.0

Hours of Light
Figure 18. Papilio troilus, Michigan Population, Late Summer 1993.

Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatement= 10, range in number of pupae: 6-17.

 

r—l
O
9’

Percent Diapause
U1
.9

 

 

.. ‘ f I. l I
12.0 13.0 14.0 14.5 15.0 15.5 16.0 18.0
Hours of Light

 

     

Figure 19. Papilio troilus, Michigan Population, Late Summer 1994.
Percent Diapause vs. Hours of Light larvae were reared under.
Average number of pupae per treatement= 17, range in number of pupae: 6-24.

56
Table 20. Papilio troilus, Michigan Population, Late Summer, 1993; number
of pupae in diapause per number of pupae formed, for each family line.

 

 

Hr'thP ha

Mamet # 12.0 13.5 14.0 14.5 15:9 15.5 16.0 18.0 24.0

# neonates 16 16 11 12 17 16 11 21 11

9997 5/ 5 3/ 3 5/ 5 5/5 1/ 3 0/ 6 0/ 2 O/ 6 0/ 4
10040 5/5 3/3 5/5 4/4 1/3 0/1 0/4 0/1 0/2
10041 --- 3/ 3 --- 1/1 --- 1 /2 --- 0/ 5 ---
10042 3/ 3 --- --- --- --- 4/ 5 --- 0/ 5 ---

Total 13/ 13 9/9 10/10 10/10 2/6 5/ 14 0/6 0/ 17 0/ 6

%Diapause 100 100 100 100 33 36 0 0 0

 

Table 21. Papilio troilus, Michigan Population, Late Summer, 1994; number
of pupae in diapause per number of pupae formed, for each family line.

 

 

MW
Mother # 12.0 13.0 14.0 14.5 15.0 15.5 16.0 18.0

# neonates 25 25 25 25 25 24 22 26

10659 12/ 12 11/11 6/ 6 9/ 12 7/ 12 0/ 10 1/ 15 O/ 12
10680 5/5 7/ 7 --- 5/ 8 3/ 8 0/ 5 2/ 9 0/ 6
Total 17/17 18/18 6/6 14/20 10/20 0/15 3/ 24 0/ 18

%Diapause 100 100 100 60 50 0 12 0

 

57

 

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58
Discussion

' h' hi 1a ° n

The difference in the Michigan population between the two years may be
due to sampling error. Only two family lines were represented in 1994 and
four family lines in 1993, although twice as many pupae survived in 1994 as
in 1993. Perhaps the second experiment better represents individual family
lines better than the population as a whole; although four family lines does
not represent a population very well; but those were the only females that
produced eggs that hatched.

For the Ohio population, the difference in the critical photoperiod
between 1993 and 1994 is not likely based on sample size; eight family lines
were represented in 1993 and eleven family lines in 1994. In addition to the
number of families represented, the number of pupae per treatment ranged
from 29 to 45 in 1993 and from 36 to 50 in 1994. One reason why there was a
difference between the two trials was that the P. troilus larvae were reared in
the same chambers as the P. glaucus larvae. P. troilus seemed to be unaffected
in terms of survival by being in the same chamber in which P. glaucus were
dying from disease. Perhaps the presence of a high level disease may have

somehow altered the P. troilus diapause response.

Giorgia—Wan

Only one year of data were available for the Georgia population. Another
experiment in which the larvae survive to pupation in all treatments would
enable me to determine the critical photoperiod for diapause. These results
fall in nicely with what has been determined for the Michigan and Ohio
populations, indicating that the critical photoperiod is probably less than for

the more northern populations.

59

Eldridafipulation

The Florida population seems to be different from its northern
counterparts. The critical photoperiod determined by these experiments for
Florida populations was between 12.0h and 12.5h of light, and all northern
populations tested diapaused under these conditions. This may be due to
being reared on a different host, redbay, than the other populations. A more
likely explanation is that Florida is a subtropical environment and
experiences less change from season to season, including smaller changes in
day length. The critical photoperiod corresponds with what larvae in the
wild would be experiencing in the wild (Table 1). Only in the Florida
population did percent diapause drop directly from 100% to 0% and in no
other populations tested. This indicates that Florida butterflies probably

integrate the token stimulus differently than their northern counterparts.

Staflsflgal Analysis

According to the R2 values obtained from the logistic regression analysis
(Table 22) P. troilus reared in this experiment primarily used photoperiod as
a cue to induce or avert diapause, since all R2 values were greater than 0.5.
Results also indicated that the proportion of variation in diapause percent is
mainly explained by the model.

Probit and Logit were not used to analyxe data because I did not have
enough ’non-zero’ data points. A contingency table analysis or Chi-square test

were mathematically impossible because I had expected values of zero.

60
A LATITUDINAL COMPARISON

Pupae generally diapaused when larvae were reared under short days and
direct developed when reared under long days. I could not pinpoint the
critical photoperiod for the Georgia population because no larvae survived to
pupation in the 13.5h and 14.0h treatments, but the critical photoperiod is
between 13.0h and 14.5 hours of light because percent diapause decreases from
100% at 13.0h to 0% at 14.5h of light. The critical photoperiod would also be
less than the critical photoperiod for both Michigan and Ohio populations.

It appears from these data that the Michigan population begins to enter
diapause at longer days than the Ohio population, which enters diapause at
longer days than the Georgia population. This timing of diapause ensures
that the populations are kept in phase with the changing seasons without
limiting the potential for multiple generations by inducing diapause in the

first generation when the growing season can still support another brood.

Conclusion

There appeared to be differences in the populations of P. troilus occung
at different latitudes with the northern populations going in to diapause at
longer days. This serves to synchronize those populations with the earlier
change of season while a shorter threshold for the southern populations
ensures that they maximize their output within the longer growing season.

This also occurs in other species with populations occurring at different
latitudes. Tauber and Tauber (1972) found that in the green lacewing,
Chrysopa carnea, the critical photoperiod for diapause for a New York
population at 42 N° was between 13.5 and 14.0 hours of light, and for an
Arizona population at 33 N° the critical photoperiod was between 12.5 and

13.0 hours of light. This is an hour difference in critical photoperiod between

61
these two populations reared under the same conditions.

Mousseau and Roff (1989) discovered a similar pattern in the striped
ground cricket, Allonemobious fasciatus. They reared crickets, originating
from different latitudes, at 14:10 (L:D), 30°C and found the percentage of
crickets in diapause increased as the latitude of origin increased. And Bell et
a1. (1979) investigated 23 different populations of Indian meal moth, Plodia
interpunctella ranging from 52 N° latitude to 35 5° latitude. They also found
that there was a tendency for the critical photoperiod to increase in length as
the latitude of origin increased. The longer critical photoperiods allow the
northern populations of the green lacewing, the ground cricket, the Indian
meal moth and the Spicebush swallowtail to enter diapause earlier in the
season which adapts these species to the early occurring winter conditions at
higher latitudes (Tauber and Tauber 1972). This ensures that these
population are in synch' with the unique seasonal changes of their

environment.

CHAPTER 3

Interspecific Comparison of Diapause Induction in the Tiger Swallowtail

and the Spicebush Swallowtail

As seen in the previous chapters P. glaucus and P. troilus did not
respond similarly to varied photoperiods. This research has shown that there
are differences between populations of the same species. The purpose of this
chapter is to compare and contrast these two species at each location. Now I
will look at and discuss the differences between species living in the same
area, which are active at the same time of the year.

In Figure 21 the Florida populations of P. glaucus and P. troilus are
graphed as Percent Diapause vs. Hours of Light, with all years lumped
together. Both species responded with the same critical photoperiod, between
12.0h and 12.5h. The diapause threshold for P. troilus dropped off sharply
between 12h and 12.5h of light, while for P. glaucus there was a more gradual
slope down toward no diapause. In addition to the slope of the lines, P.
glaucus never responded with 100% diapause, so some pupae always direct-
developed. Florida populations of both species appear to have the same
critical photoperiod, but P. troilus showed more of an ”all or none” response
while P. glaucus was more variable.

At the other three locations, there was a similar pattern (Figures 22, 23
and 24). Some P. glaucus diapaused in every treatment. Even when reared at

24h of light some pupae from Ohio and Georgia still went into diapause.

62

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67
Opler and Krizek (1984) stated that some offspring from every generation

overwinter, so maybe P. glaucus is just following what it does in the wild
despite the controlled conditions of a laboratory.

It seems clear from these results that P. troilus used photoperiod as a
major indicator of season, and P. glaucus did not. P. glaucus may be using
cues other than photoperiod to indicate what time of year it is, such as the
quality of the host plant. This high level of variation may be attributed to the
absence of natural selection for a specific diapause-inducing photoperiodic
response (Harvey 1957). There may simply be some token stimulus not
provided in laboratory rearing that would reduce the variation between P.
glaucus and P. troilus.

Another explanation for the variation between the two species is that
there may be more gene flow among populations of P. glaucus than among
populations of P. troilus. Diapause is a genetic component and if there are
genes coming in from other populations, this can influence what photo-
period is the critical photoperiod for each individual.

An alternative hypothesis is that diapause in P. glaucus is influenced
by host species. P. glaucus larvae feed on host plants from at least nine
different families while P. troilus only larvae feed on hosts only from one,
the Lauraceae family (Nitao et al. 1,991, Scriber 1994). P. glaucus were reared
on only one host in these experiments, either sweet bay or black cherry, when
in nature they are probably spread out amongst the many hosts available to it.
Eggs would probably be distributed amongst high and low quality hosts. Since
swallowtail larvae generally remain on the host and tree in which they were
put, the offspring would be spread amongst various hosts. Each individual
larvae will grow at a different rate thus exposing the larva to different

amounts of light. By rearing larvae on only one host there may be some sort

68
of artificial selection for an increase in the propensity to diapause not usually

seen in nature when larvae are spread amongst many host plants.

As mentioned in the previous chapters, many P. glaucus larvae died of
disease. Both the P. glaucus and P. troilus larvae were reared in the same
chambers at all times, but almost all of the P. troilus larvae escaped death by
disease. Even the adults that emerged produced fertile offspring. While over
90% of the P. glaucus larvae succumbed to disease, most of the P. troilus
larvae survived. Most of the mortality of P. troilus was due to larvae not
feeding or larvae crawling into the aquapic and drowning. P. troilus may
simply escape disease by reaching the pupal stage faster than P. glaucus. In
addition, P. troilus generally take about 20 days to pupate from date of hatch
while P. glaucus usually take around 30 days. If P. glaucus do not pupate
within a few days of their 30 day larval life, they usually don’t survive to
pupation, the pupae die, or if they happen to make it to adulthood, they’re
usually deformed. Differential disease interactions with diapause physiology
may explain some of the differences between these two Papilio species.

[Currently the disease topic is under investigation, with initial steps
taken to completely sterilize and repaint the rearing room and to sterilize
foliage in a bleach solution. Other measures considered are creating an
artificial diet, looking at the gut physiology of P. troilus to determine if it has
any ”special” disease-fighting enzymes, and rearing P. glaucus on sassafras to
determine if sassafras is responsible for the low mortality rate in P. troilus.]

Understanding diapause dynamics and mechanisms determining
voltinism patterns sheds light on adaptations of species to local
environments and on the evolution of species. This also helps in the design
of pest management systems, especially where multivoltinism is likely.

Knowing the voltinism patterns of a pest species can help in the prescribing

69
of pest management strategies. If the pest has more than one generation per

season of a variable number, then special steps may have to be taken to
predict and control the pest when it returns. The European Corn Borer,
Ostrim'a nubilalis, causes damaged to many crop species such as corn and
potatoes. There are populations of both univoltine and bivoltine in some
areas. Being able to predict potential insect damage to crops is important to
insect management programs and the ability to predict this damage starts
with diapause and voltinism patterns (Scriber and Lederhouse 1992).

Pest management tactics can also affect non-target species such as P.
glaucus and P. troilus. Preliminary results of bioassays testing Bacillus
thuringiensis on swallowtail larvae versus gypsy moth larvae (Lymantria
dispar) revealed that the high doses of Bt needed to kill gypsy moth larvae are
persistent for weeks and highly lethal to swallowtail larvae (Johnson et al.
1995). Even Bt in very small doses was lethal. If, in the areas where gypsy
moths occur, Bt is being applied and non-target species are being affected, Bt
could wipe out a species in that area. However, understanding the diapause
dynamics and seasonal phenology of both the pest species and the non-target
species could help in planning pest management strategies. By knowing
when each species will be active in the areas targeted for management,
strategies can be used that will maximize kill on the pest and minimize
damage to the non-target species. I

Diapause dynamics of an insect can limit or allow wider distribution of
a species. The capacity of the Indian meal moth and the pink bollworm to
diapause increases their chances that they will survive when moved to a new
environment and has also facilitated in the spread of these pests (Ankersmit
and Adkisson 1967, Bell et a1. 1979). Diapause dynamics can also limit

further distribution as it does for P. glaucus. P. glaucus has not extended its

70
range farther north than the hybrid zone where it encounters P. canadensis.

This limit is likely due to the inability of P. glaucus to adapt to a univoltine
life cycle, which is necessary in regions north of the hybrid zone (Hagen et al.
1991)

Diapause is not just a way to avoid adverse conditions, but is also of
prime adaptive significance in adjusting the insect’s entire life cycle to
seasonal change; it serves as the primary means whereby insect life cycles are
kept in phase with the changing seasons (Tauber et al. 1986).

Understanding photoperiodic adjustments in animals and the
interactions of biotic and abiotic environmental factors influencing animal
behavior is also of importance in many other ways such as for determining

hibernation or migration cycles.

APPENDICES

 

APPENDIX 1

Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in
the named museum(s) as samples of those species or other taxa which were
used in this research. Voucher recognition labels bearing the Voucher
No. have been attached or included in fluid-preserved specimens.

Voucher No.: 1995-5

 

Title of thesis or dissertation (or other research projects):

LATITUDINAL COMPARISONS OF DIAPAUSE INDUCTION BETWEEN
POPULATIONS OF PAPILIO GLAUCUS AND PAPILIO TROILUS
(LEPIDOPTERA: PAPILIONIDAE)

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State university (MSU)

Other Museums:

Investigator's Name (3) (typed)
Patricia A. Tidwell

 

 

 

Date August 3, 1995

 

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in
North America. Bull. Entomol. Soc. Amer. 24:141-42.

Deposit as follows:

Original: Include as Appendix 1 in ribbon copy of thesis or
dissertation.

Copies: Included as Appendix 1 in copies of thesis or dissertation.
Museum(s) files.
Research project files.

This form is available from and the Voucher No. is assigned by the Curator,
Michigan State University Entomology Museum.

71

 

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APPENDIX 1.1
Voucher Specimen Data

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72

Table 23. Information by mother number about female P. glaucus.

harsherji
9701
9703
9718
9719
9720
9744
9751

10318
10323
10327
10329
10335

11004
11046
11048
11053
11064
11065
11066
11070
11097
11099
11104
11106

10021
10025
10026
10027
10021

C fen

n/ a

n / a
Highlands
Highlands
Levy
Highlands
Levy

Levy
Levy
Highlands
Highlands
Highlands

Highlands
Highlands
Highlands
Levy
Levy
Levy
Levy
Levy
Levy
Levy
Levy
Levy

Clarke
Clarke
Clarke
Clarke
Clarke

Wear Classl

Np—H—H—tr—IQON

2/ 3
3/4

N
\
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\
OJ

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APPENDIX 2

73

Lkufljlgflegkzi

3/22
3/ 23
3/27
3/27
3/ 31
4/ 6

4/1

3/26
3/26
3/27
3/27
3/27

3/ 24
3/ 26
3/26
3/ 26
3/26
3/26
3/26
3/26
3/ 28
3/ 28
3/ 28
3/ 28

8/11
8/ 11
8/11
8/11
8/ 11

Color2
yel
yel
yel
yel
dk
yel
dk

dk
yel

yel
dk

yel

yel
yel
yel
dk
dk
dk
dk
dk
yel
dk
dk
dk

dk
dk
dk
dk
dk

 

10727
10728
10730
10731
10737
10757
10760
10779
10787
10790
10799

9953
9960
9965
9967
9968
9972
9974
9975
9976

10705
10708
10710
10711
10713
10717
10747
10748
10750
10758

9992
9994
9995
9996
10002
10005
10008
10015
10017

Clarke
Clarke
Clarke
Clarke

Columbus

Union
Clarke
Clarke
Clarke
Clarke
Franklin

Gallia

Lawrence
Lawrence
Lawrence
Lawrence
Lawrence
Lawrence
Lawrence
Lawrence

Lawrence
Gallia
Gallia
Gallia
Gallia
Gallia
Gallia
Gallia
Gallia
Gallia

St. Joseph
St. Joseph
St. Joseph
St. Joseph
Clinton
Clinton
St. Joseph
Clinton
Clinton

F-‘Nt-‘NNNNOJNN ND-‘l-‘i-‘Ni-‘NV-‘N Hi—‘NHHND—‘HNNN

NNNOJNNNNN

74

8/ 3
8/ 3
8/ 3
8/ 3
8/ 6
8/ 7
8/ 10
8/ 10
8/10
8/ 10
8/ 10

8/4
8/4
8/4
8/4
8/4
8/4
8/4
8/4
8/4

7/ 30
7/ 30
7/ 30
7/ 30
7/ 30
7/ 30
8/ 9

8/ 9

8/ 9

8/ 10

7/ 30
8/ 6
8/ 6
8/ 6
8/ 7
8/ 7
8/ 8
8/11
8/ 11

dk
dk
yel
dk
dk
dk
yel
dk
dk
dk
dk

dk
dk
dk
dk
dk
dk
yel
yel
yel

dk
dk
dk
dk
dk
dk
dk
dk
dk
dk

dk
yel
dk
dk
yel
yel
yel
yel
yel

 

10657
10658
10660
10662
10663
10674
10675

Ingham
Ingham
Ingham
Calhoun
Calhoun
Eaton
Eaton

HNNHHHN

7/ 25
7/ 25
7/ 25
7/ 25
7/ 25
7/28
7/29

yel
yel
yel
yel
yel
yel
dk

 

 

Table 24. Information by mother number about female P. troilus.

t
9711
9713
9714

10315
10316
10332

11008
11071
11090
11114
11131
11135

10800
10859

9978
9980
9981
9983
9984
9985
9989
9990

10684
10687
10690
10691

C fQ . .
n/a
n/a
n/a

Levy
Levy
Highlands

Highlands
Highlands
Highlands
Levy

Highlands
Highlands

Clarke
Clarke

Gallia
Gallia
Gallia
Gallia
Gallia
Gallia
Lawrence
Lawrence

Gallia
Gallia
Lawrence
Lawrence

HNHHHHHH NN HUDNNt—‘H lop-AN

HHNH

75

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3/22
3/22
3/22

3/26
3/ 26
3/26

3/24
3/26
3/ 27
3/ 30
3/ 30
3/ 30

8/ 10
8/ 15

8/4
8/4
8/4
8/4
8/4
8/4
8/4
8/4

7/30
7/30
7/ 30
7/ 30

 

10692
10696
10698
10699
10702
10742
10744

9997

10040
10041
10042

10659
10680

Lawrence
Lawrence
Lawrence
Lawrence
Lawrence
Gallia

Gallia

St. Joseph
St. Joseph
St. Joseph
St. Joseph

Ingham
Ingham

NNNNHNH

WNr-Iv—I

H03

7/ 30
7/ 30
7/ 30
7/ 30
7/ 30
8/ 9

8/9

8/ 6

8/ 14
8/ 14
8/ 14

7/ 25
7/ 29

 

1 The approximate age of a butterfly can be determined by the wear of wings and abdomen.
1 is a very fresh female and a 4 is a very old female, see Lederhouse and Scriber 1987.

2 P. glaucus females are dimorphic, either yellow or dark (black).

76

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