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thesis entitled

The Endangered Karner Blue Butterfly
(Lepidoptera: Lycaenidae) in Michigan: Habitat
Suitability, Potential Impacts of Gypsy Moth
(Lepidoptera: Lymantriidae) Suppression, and

Laboratory Rearing
presented by

Catherine Papp Herms

has been accepted towards fulfillment
of the requirements for

Masters degree in Entomology

 

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THE ENDANGERED KARNER BLUE BUTTERFLY (LEPIDOPTERA:
LYCAENIDAE) IN MICHIGAN: HABITAT SUITABILITY, POTENTIAL IMPACTS
OF GYPSY MOTH (LEPIDOPTERA: LYMANTRIIDAE) SUPPRESSION,
AND LABORATORY REARIN G

By

Catherine Papp Herms

A THESIS

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

MASTER OF SCIENCE

Department of Entomology

1 996

ABSTRACT
THE ENDANGERED KARNER BLUE BUTTERFLY (LEPIDOPTERA:
LYCAENIDAE) IN MICHIGAN: HABITAT SUITABILITY, POTENTIAL IMPACTS
OF GYPSY MOTH (LEPIDOPTERA: LYMANTRIIDAE) SUPPRESSION,
AND LABORATORY REARING
By

Catherine Papp Herms

The Karner blue butterfly (Lycaeides melissa samuelis Nabokov) is an endangered
species found in oak savanna and pine barren habitats of the northeastern and central
United States. Populations have declined drastically or become extirpated as a result of
habitat destruction. In 1993 and I994, studies were conducted on the Kamer blue in
Michigan to investigate habitat suitability, potential impacts of gypsy moth (Lymantria
dispar) suppression and methods for laboratory rearing. Habitat studies revealed that
Karner blue abundance was highly associated with densities and frequencies of wild
lupine (Lupinus perennis), the sole larvae food source. Ant tending was Observed for
over 80 percent of Karner blue larvae that were found. Thirteen species of tending ants
were identified for Michigan. In field phenology surveys, Kamer blue larvae were found
to be phenologically susceptible to gypsy moth suppression activities using Bacillus
thuringiensis var. kurstaki (Btk). In a laboratory bioassay, mortality of Kamer blue larvae
was significant when larvae were fed foliage treated with two levels of Btk. Larvae were
highly physiologically susceptible to Btk. In 1994, spring generation female butterflies
were collected and housed in the laboratory to collect eggs. Larvae were successfully

reared through to adulthood, and released back into collection sites.

ACKNOWLEDGMENTS

I thank my major advisor, Deborah G. McCullough, for her excellent guidance,
unending enthusiasm, and friendship. Her outstanding scientific and creative abilities
contributed significantly to the development of my program at all levels, as well as to my
own development as a scientist. I also thank the other members of my committee, Robert
Haack, James Miller, Kurt Pregitzer, James Stevens and Mark Scriber, for their insightful
comments and guidance throughout my program. I am especially grateful to James
Stevens for coming on board midway, and to Mark Scriber for joining at the last minute.

I gratefully acknowledge Mary Rabe, Michigan Natural Features Inventory,
Joseph Kelly, Huron-Manistee National Forest, John Lerg, Allegan State Game Area, and
Nancy Sferra, The Michigan Nature Conservancy for their technical support, especially at
the initial stages of project development. I also thank Susan Walker, Michael DeCapita
and Kevin Stubbs, Fish & Wildlife Service, Ronald Priest, Michigan Department of
Agriculture, and Thomas Weise, Michigan Department of Natural Resources, for their
support and assistance.

I especially want to thank my research collaborators with the USDA Forest
Service, Robert Haack, Leah Bauer, Deborah Miller, North Central Forest Experiment
Station, and Normand Dubois, Northeastern Forest Experiment Station, for their many

contributions and assistance. I gratefully acknowledge John Peacock, Northeastern

iii

Forest Experiment Station, and Dale Schweitzer, The Nature Conservancy, for their
support of my research. I also thank Daniel Herms for his insightful scientific
perspective and advice, and Eileen VanTassell for her photographic assistance. I am
extremely grateful to Gordon Michaud, Katie Albers, and Kevin Ellwood for their
excellent help throughout the summer.

I thank the people with the USDA Forest Service, State & Private Forestry, for
making my experience in St. Paul as a cooperative education student very rewarding and
enjoyable. I especially want to acknowledge James Hansen for facilitating the
opportunity, Dennis Haugen for being an outstanding supervisor, and Barb Spears for her
technical support and friendship.

One of the most rewarding aspects of my program was the opportunity to interact
with graduate colleagues. I am grateful to Bryan Bishop, Joseph Chichester, Timothy
Work, Eileen Eliason, and Lyle Buss for providing input and the necessary distractions. I
am also grateful to Cynthia Lane, University of Minnesota, for her comments and advice.

I thank D. R. Smith with the USDA Agricultural Research Service, Beltsville,
Maryland, for his taxonomic assistance with ant specimens.

This project was funded through the USDA Forest Service, North Central Forest
Experiment Station, East Lansing, Michigan (cooperative agreement 23-92-61), and
USDA Forest Service, Northeastern Area State & Private Forestry in St. Paul, Minnesota.
Funding was also obtained through a USDA NAPIAP (National Agricultural Pesticide
Impact Assessment Program) grant (N C-26-93). I thank the Michigan Department of
Natural Resources for two years of support through the Nongame Wildlife Fund and

Living Resources Small Grants Program. Additional funding was provided by The

iv

Michigan Nature Conservancy, and Michigan State University, Department of
Entomology Hutson Grant program.

Federal permits for this study were obtained in 1993 (subpermit 93-23-1)
(Appendix 2) and 1994 (subpermit 94-23-R) (Appendix 3) from the US Fish & Wildlife
Service. Threatened / Endangered Species permits (Appendix 4) and Use permits
(Appendix 5) were also obtained in 1993 and 1994 from the Michigan Department of
Natural Resources and Allegan State Game Area, respectively.

Finally, I thank my parents for their support and love. And most of all, I thank
Daniel Herms for his unending patience, encouragement, and understanding through it

all.

TABLE OF CONTENTS

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

LIST OF FIGURES ....................................................................................................... xiii

INTRODUCTION ............................................................................................................ 1
CHAPTER 1

Laboratory Rearing of the Endangered Karner Blue Butterfly (Lepidoptera: Lycaenidae)

in Michigan ....................................................................................................................... 3

Abstract ................................................................................................................. 3

Introduction ........................................................................................................... 4

Methods & Materials ............................................................................................ 6

Lupine foliage ........................................................................................... 6

Field collection of Karner blue adults ....................................................... 6

Housing of butterflies ............................................................................... 8

Egg collection and care ............................................................................. 9

Larval rearing .......................................................................................... 10

Pupae ....................................................................................................... 11

Adult butterflies ...................................................................................... 11

Statistical analysis ................................................................................... 12

Results ................................................................................................................. 12

Collection and housing of female butterflies .......................................... 12

Egg collection and hatch ......................................................................... 12

Development of larvae, pupae, adults ..................................................... 13

Discussion ........................................................................................................... 15

Tables .................................................................................................................. 23
CHAPTER 2

Susceptibility of the Endangered Karner Blue Butterfly (Lepidoptera: Lycaenidae) to
Bacillus thuringiensis var. kurstaki Used for Gypsy Moth (Lepidoptera: Lymantriidae)

Suppression in Michigan ................................................................................................ 26
Abstract ............................................................................................................... 26
Introduction ......................................................................................................... 28
Methods & Materials ......................................................................... ; ................ 32

Phonology of Karner blue with respect to gypsy moth suppression ....... 32

vi

Btk susceptibility bioassays .................................................................... 34

Bioassay treatments .................................................................... 34
Experimental insects and foliage ................................................ 35
Btk application ............................................................................ 35
Initiation and monitoring of bioassays ....................................... 36
Statistical analysis ....................................................................... 38
Results ................................................................................................................. 38
Phenology of Karner blue with respect to gypsy moth suppression ....... 38
Predicted Btk application ............................................................ 38
Actual Btk application ................................................................. 39
Btk bioassays ........................................................................................... 40
Overall survival .......................................................................... 40
Karner blue survival .................................................................... 40
Gypsy moth survival ................................................................... 41
Karner blue vs. gypsy moth survival .......................................... 42
Sublethal effects on Karner blue ................................................. 42
Discussion ........................................................................................................... 43
Tables .................................................................................................................. 49
Figures ................................................................................................................ 52
CHAPTER 3
The Endangered Karner Blue Butterfly (Lepidoptera: Lycaenidae) in Michigan Oak
Savanna: Associations among Butterfly Abundance and Habitat Variables .................. 58
Abstract ............................................................................................................... 58
Introduction ......................................................................................................... 59
Methods & Materials .......................................................................................... 64
Study sites ............................................................................................... 64
Karner blue adult abundance estimates .................................................. 65
Indirect estimates of Karner blue larval abundance ................................ 67
Lupine density, frequency, flowering and quality .................................. 67
Spring and summer flower density, frequency, nectaring and
diversity ............................................................................................ 69
Canopy cover and shade ......................................................................... 70
Ant-tending of larvae .............................................................................. 71
Statistical analysis ................................................................................... 71
Results ................................................................................................................. 72
Karner blue adult adundance .................................................................. 72
Indirect estimates of summer Karner blue larval abundance .................. 74
Lupine density, frequency, flowering and quality .................................. 74
Flowering plants ..................................................................................... 76
Canopy cover .......................................................................................... 79
Karner blue larvae and shade .................................................................. 8O
Ant-tending of Karner blue larvae .......................................................... 80
Associations among Karner blue abundance and larval feeding
damage estimates ................ 2 ............................................................. 81

vii

Associations among Karner blue abundance and habitat variables ........ 81

Discussion ........................................................................................................... 82

Tables .................................................................................................................. 92

Figures .............................................................................................................. 120
APPENDICES .............................................................................................................. 139
APPENDIX 1 - Record Deposition of Voucher Specimens ......................................... 139
APPENDIX 1.1 — Voucher Specimen Data. ................................................................. 140
APPENDIX 2 - 1993 Federal Endangered and Threatened Species Subpermit ........... 142
APPENDIX 3 - 1994 Federal Endangered and Threatened Species Subpermit ........... 147
APPENDIX 4 - 1993 and 1994 State of Michigan Threatened / Endangered Species
Permits .......................................................................................................................... 154
APPENDIX 5 - 1993 and 1994 State of Michigan Use Permits .................................. 157

APPENDIX 6 - Tables of Flowering Plant Species Observed in Study Sites but Not
Occurring in Transect Surveys ..................................................................................... 160

APPENDIX 7 - Tables of Nectaring Observations of Summer Generation Karner Blue

Adults ............................................................................................................................ 164
APPENDIX 8 - Table of percentage canopy cover and frequency by tree species ...... 167
LIST OF REFERENCES .............................................................................................. 169

viii

LIST OF TABLES

CHAPTER 1

Table 1.1 - Adult flight periods of 1994 spring and summer Karner blue generations in
Allegan State Game Area (Allegan Co) and Huron-Manistee National Forest (Oceana
Co) in Michigan .............................................................................................................. 23

Table 1.2 - Total numbers of eggs obtained and hatched from caged female Karner blue
butterflies collected from Allegan State Game Area (Allegan Co) and Huron-Manistee

National Forest (Montcalm Co and Newaygo Co) in Michigan ..................................... 23
Table 1.3 - Mean duration (i SE) of Karner blue life stages captively reared at

24°C ................................................................................................................................. 24
Table 1.4 - Average body length (t SE) of captively reared Karner blue larvae at the
onset of each instar ......................................................................................................... 25
CHAPTER 2

Table 2.1 - Phenological development of first and second generation Karner blue and
gypsy moth in Allegan State Game Area (Allegan Co) and Huron-Manistee National
Forest (Oceana Co) in Michigan, 1993 - 1995. Life stages of Karner blue that were
observed at the time of hypothetical Btk application, predicted from gypsy moth
development, are in bold. Surveys for second generation eggs and larvae were

conducted only in 1995 ................................................................................................... 49
Table 2.2 - Actual timing of Btk applications for gypsy moth suppression in Michigan
counties near Karner blue study sites, 1993 - 1995 ........................................................ 51
CHAPTER 3

Table 3.1 - Size, origin, recent disturbance, average percentage canopy cover (1 SE), and
frequency of canopy cover (proportion of transects with canopy cover; all tree species
combined) of Karner blue study sites in Allegan State Game Area ............................... 92

ix

Table 3.2 - Dates and degree days of peak counts for spring and summer Karner blue
flight periods, and of spring and summer flower density surveys for study sites, 1993
and 1994 .......................................................................................................................... 93

Table 3.3 - Estimations of Karner blue abundance based upon peak counts of adults, and
mean values (:t SE) of lupine density and flowering plant density for Karner blue study
study sites, 1993 and 1994 .............................................................................................. 94

Table 3 .4 - Average number of lupine stems (per m2) (i SE), average percentage of
lupine stems (per m2) (1 SE) with larval feeding damage, and feeding damage frequency
(proportion of quadrats with feeding damage), from quadrat surveys of Karner blue
summer generation larval feeding damage in Allegan State Game Area study sites,

1994 ................................................................................................................................ 96

Table 3.5 - Frequencies (proportions of transects occupied) of lupine and of overall spring
and summer flowers from transect surveys in Allegan State Game Area study sites, 1993
and 1994 .......................................................................................................................... 97

Table 3.6 - Average percentage of lupine stems (per m2) with flower spikes and average
percentage of lupine flower spikes (per m2) at different stages of bloom (no buds open (0)
- flower spike in full bloom (1); seed pods present (Seed); flower spike bare (Bare)) from
weekly quadrat surveys of lupine flowering phenology in Allegan State Game Area study
sites, spring 1994 ............................................................................................................ 98

Table 3.7 - Densities of individual flower species from transect surveys in Allegan State
Game Area study sites, spring 1993 ............................................................................. 104

Table 3.8 - Densities of individual flower species from transect surveys in Allegan State
Game Area study sites, spring 1994 ............................................................................. 105

Table 3.9 - Frequencies of individual flower species (proportions of transects with
flowers) fi'om transect surveys in Allegan State Game Area study sites, spring 1993 and
1994 .............................................................................................................................. 106

Table 3.10 - Densities of individual flower species from transect surveys in Allegan State
Game Area study sites, summer 1993 .......................................................................... 108

Table 3.11 - Densities of individual flower species from transect surveys in Allegan State
Game Area study sites, summer 1994 .......................................................................... 109

Table 3.12 - Frequencies of individual flower species (proportions of transects with
flowers) from transect surveys in Allegan State Game Area study sites, summer 1993 and
1994 .............................................................................................................................. 111

Table 3.13 - Numbers of flower species encountered, and Shannon’s diversity index (H’)
and Simpson’s dominance index (expressed as reciprocal, l/D) of flowering plant

species, based on transect surveys in Karner blue study sites conducted during peak
spring and summer flight of Karner blue, 1993 and 1994 ............................................ 114

Table 3.14 - Spring and summer flowering plant species encountered (indicated by NI) in
transect Stu'veys conducted during the peak of the respective Karner blue flight periods in
study sites, and numbers of Karner blue adult nectaring events observed during butterfly
abundance surveys in study sites, 1993 and 1994 ........................................................ 115

Table 3.15 - Numbers of ant-tended and untended Karner blue larvae, by larval body
length (cm), observed in Allegan State Game Area study sites, summer 1993 and spring
and summer 1994 .......................................................................................................... 118

Table 3.16 - Thirteen species of ants (Hymenoptera: Fonnicidae) representing three
subfamilies observed tending Karner blue larvae. Ant specimens were collected in
Allegan County (Allegan State Game Area) and Oceana County (Huron-Manistee
National Forest), Michigan, during the 1993 and 1994 spring (Spr) and summer (Su)
Karner blue larval generations ...................................................................................... 119 ‘

APPENDIX 6

Table A6.1 - Plant species observed in flower in Allegan State Game Area study sites
during the Karner blue spring flight period, but not encountered in transect surveys, 1993
and 1994 ........................................................................................................................ 161

Table A6.2 - Plant species observed in flower in Allegan State Game Area study sites
during the Karner blue summer flight period, but not encountered in transect surveys,
1993 and 1994 ............................................................................................................... 162

APPENDIX 7

Table A7.1 - Total numbers of nectaring observations per survey week (# male, # female)
of summer generation Karner blue adults on individual flower species in Allegan State
Game Area study sites, 1993 ........................................................................................ 165

Table A7.2 - Total numbers of nectaring observations per survey week (# male, # female,
# unknown) of summer generation Karner blue adults on individual flower species in
Allegan State Game Area study sites, 1994 .................................................................. 166

xi

APPENDIX 8

Table A8 - Percentage canopy cover (P) (:t SE) by tree species and frequency (F) of tree
species from transect surveys in Allegan State Game Area study sites, 1994 .............. 168

xii

LIST OF FIGURES

CHAPTER 2

Figure 2.1 - Michigan counties where Karner blue butterfly study sites were located
(Allegan, Oceana), where Btk was applied at least once in 1993 - 1995 for gypsy moth
suppression (Muskegon, Newaygo, Oceana, Ottawa) and where the Btk laboratory
bioassay was conducted (Ingham) .................................................................................. 52

Figure 2.2 - Larval survival of (A) Karner blue butterfly and (B) gypsy moth over 13 days
on control (untreated) foliage, on foliage treated with Btk (Bacillus thuringiensis var.
kurstala') at a low dosage (30 - 37 BIU/ha), or on foliage treated at a high dosage (90
BTU/ha). On Day 7 (indicated by arrow), all surviving larvae were placed on untreated
foliage ............................................................................................................................. 54

Figure 2.3 - Survival over 13 days of early (lst, 2nd; E) and late (3rd, 4th; L) instar
Karner blue reared on lupine foliage treated with low (30 - 37 BIU/acre) or high (90
BTU/acre) dosages of Btk. On Day 7 (indicated by arrow), all surviving larvae were
placed on untreated lupine foliage. No further mortality occurred after day 13 ............ 56

Figure 2.4 - Mean pupal weight (mg) (+ 1 SE) 2 days afier pupation of surviving female
and male Karner blue larvae used in the Btk bioassay. There were 8, 4, and 1 female
survivors, and 7, 2, and 2 male survivors on control, low Btk (30 - 37 BIU/ha) and high
Btk (90 BIU/ha) treatments, respectively. For within-gender comparisons, bars with the
same letters were not significantly different by AN OVA at p < 0.05 (female pupal weight
for the high Btk treatment was not included in AN OVA) .............................................. 57

CHAPTER 3

Figure 3.1 - Map of Lower Peninsula of Michigan showing the location of Allegan State
Game Area study sites (Allegan Co) and Huron-Manistee National Forest (Oceana

Co) ................................................................................................................................ 120

Figure 3.2 - 1993 summer flight period of the Karner blue butterfly on six study sites in
Allegan State Game Area (Allegan Co), Michigan ...................................................... 122

xiii

Figure 3.3 - 1994 spring and summer flight periods of the Karner blue butterfly on seven
study sites in Allegan State Game Area (Allegan Co), Michigan ................................ 123

Figure 3.4 - 1993 and 1994 summer flight periods of the Karner blue butterfly on six
study sites in Allegan State Game Area (one site used only in 1994 not included) ..... 124

Figure 3.5 - Distribution of percentage canopy cover of individual transects (25-m) from
surveys in each study site ............................................................................................. 125

Figure 3.6 - Scatterplot of 1994 summer Karner blue abundance versus percentage (SE)
of lupine stems (per m2) with summer larval feeding damage from feeding damage
surveys in study sites .................................................................................................... 126

Figure 3.7 - Scatterplot of 1994 summer Karner blue abundance versus frequency of
summer larval feeding damage (proportion of quadrats with feeding damage) from
surveys in study sites .................................................................................................... 127

Figure 3.8 - Scatterplot of 1993 summer Karner blue abundance versus 1993 lupine
density estimates (SE) in study sites ............................................................................. 128

Figure 3.9 - Scatterplot of 1994 summer Karner blue abundance versus 1994 lupine
density estimates (SE) in study sites ............................................................................. 129

Figure 3.10 - Scatterplot of 1994 spring Karner blue abundance versus 1994 lupine
density estimates (SE) in study sites ............................................................................. 130

Figure 3.11 - Scatterplot of 1993 summer Karner blue abundance versus 1993 lupine
frequency (proportion of transects with lupine) from transect surveys in study sites.. 131

Figure 3.12 - Scatterplot of 1994 summer Karner blue abundance versus 1994 lupine
frequency (proportion of transects with lupine) fiom transect surveys in study sites.. 132

Figure 3.13 - Scatterplot of 1994 spring Karner blue abundance versus Shannon diversity
index (H’) for 1994 spring flowering plants in study sites ........................................... 133

Figure 3.14 - Scatterplot of 1994 summer Karner blue abundance versus Shannon
diversity index (H’) for 1994 spring flowering plants in study sites ............................ 134

Figure 3.15 - Scatterplot of percentage canopy cover (SE) versus 1993 summer flower
density estimates (SE) in study sites ............................................................................. 135

Figure 3.16 - Scatterplot of percentage canopy cover (SE) versus 1994 summer flower
density estimates (SE) in study sites ............................................................................. 136

xiv

Figure 3.17 - Scatterplot of percentage canopy cover (SE) versus the number of 1993
summer flowering plant species encountered in transect surveys in study sites .......... 137

Figure 3.18 - Scatterplot of 1994 transect estimates of percentage canopy cover versus

1994 transect estimates of spring flower density (stems / m2) in the ‘Jay’ study
site ................................................................................................................................. 138

XV

INTRODUCTION

Many species of invertebrates are declining as a result of habitat alteration and
destruction. The federally endangered Karner Blue butterfly (Lycaeides melissa samuelis
Nabokov; Lepidoptera: Lycaenidae), is a prime example. This butterfly species occupies the
declining oak savanna and pine barren habitats of the northeast and central United States.
These habitats support wild lupine (Lupinus perennis L.), the only known larval food plant of
the Karner blue. The butterfly was added to the United States’ federal endangered species list
in December 1992 as a result of drastic population declines within the last 20 years. The
species is currently extirpated in several states. The Karner blue is recognized as an indicator
species 03116 disappearing oak savanna and pine barre; communities. Current management
programs are focused on Conserving and restoring Karner blue populations based upon its
habitat requirements, for long-term maintenance of the Karner blue and of the savanna and I,
barrens communities as a whole. The potential for captive rearing is also being explored.
Concern has been raised regarding the—mgr; spread 'Of 7 gypsy moth (Lymantria dispar L.;
Lepidoptera: Lymantriidae), an introduced forest pest, into Karner blue habitat and the
potential threats from gypsy moth suppression using a bacterial insecticide, Bacillus
thuringiensis Berliner var. kurstaki (Btk).

The following chapters discuss investigations into various aspects of conservation

of the Karner blue butterfly. The first chapter discusses methods used to rear Karner blue

2

in the laboratory from eggs to adulthood. Karner blue eggs were obtained from spring
generation female butterflies that were collected in the field and housed in the laboratory.
The goal of the second chapter was to determine the phenological and physiological .
susceptibility of Karner blue larvae to Btk as used for gypsy moth suppression in
Michigan. The last chapter presents results from an investigation of habitat suitability of
Karner blue in the oak savanna, focusing on larval and adult resources, and other aspects
of the butterfly’s environment. I hope that information from these studies will contribute

to the conservation of this species.

CHAPTER 1

Laboratory Rearing of the Endangered Karner Blue Butterfly
(Lepidoptera: Lycaenidae) in Michigan

Abstract

The Karner blue butterfly (Lycaeides melissa samuelis) is a federally listed
endangered species in the United States, occupying oak savanna and pine barren habitats
from eastern Minnesota to New Hampshire. In 1994, we successfully reared Karner blue
larvae under controlled laboratory conditions for experimental purposes, and report on
those rearing methods here. We collected 20 female Karner blue adults of the spring
generation from two areas in Michigan, and housed them in cages in an environmental
chamber at 24° - 26°C for 5 days. The female butterflies produced 154 eggs, of which 72
hatched in an average of 4.5 days, and 68 first instars survived. All larvae used as
controls for a related research project, plus those not used in the research, successfully
completed the 4 instars and survived to adulthood. Eggs, larvae and pupae were kept in a
growth chamber at 24°C. Developmental time from egg to adult averaged 26 days; the
average duration of each instar ranged from 3 to 4 days, and the average pupal duration
was 8 days. In total, 33 laboratory-reared Karner blue adults were released into the
maternal collection sites. Laboratory rearing may be a viable means of providing Karner
blue individuals for reintroduction into areas where the species has already gone extinct,

for supplementation of small populations, or for research with minimal risk to wild

3

4

populations. Ultimately, such methods may become an integral part in the recovery of

this and other rare invertebrate species.

“ac—4- u..‘-._.....- _,,_,-_.. .n.‘.¢-’4
,—

Introduction

Lycaeides melissa samuelis Nabokov (Lepidoptera: Lycaenidae), commonly
referred to as the Karner blue butterfly, is a federally endangered species, and occurs in
discontinuous populations along a narrow band from eastern Minnesota to New
Hampshire (Shapiro 1969; USFWS 1992; Haack 1993). This species occupies oak
savanna in the Midwest and pine barrens in eastern states, both of which are xeric,
sparsely wooded, prairie-like communities (Schweitzer 1989). The butterfly’s range
corresponds generally with the northern limits of its only known larval hostplant, wild
lupine (Lupinus perennis L.), which grows in the sandy soils of the savanna and barrens If
habitats (U SF WS 1992; Dirig 1994). The Karner blue overwinters in the egg stage and
has two generations per year. Larvae of both the spring and summer generations feed on
wild lupine, and adults utilize a variety of nectar sources (Schweitzer 1989; Haack 1993;
Dirig 1994; Swengel 1995).

The Karner blue was added to the United States federal endangered species list in
December 1992 in response to dramatic rangewide reductions in butterfly abundance and
distribution (U SFWS 1992). Karner blue numbers have. declined an estimated 99 percent
over the last 100 years, with. 90Mpercent of that decline occurring within the past decade
(Schweitzer 1989). Population declines are attributed to habitat loss and fragmentation
resulting fronLanthropogenic activities such as agriculture, residential and commercial

development, off-road vehicle use and fire suppression (Packer 1987; USFWS 1992;

5

Haack 1993; Dirig 1994). Currently, the species occurs in localized areas in Minnesota,
Wisconsin, Indiana, Michigan, New York and New Hampshire, and is extirpated in
Massachusetts, Pennsylvania, Ohio, Ontario and most likely Illinois (U SFWS 1992;
Haack 1993; Baker 1994; Grigore and Windus 1994; Packer 1994). Michigan, New York
and Wisconsin harbor the greatest numbers of Karner blue populations (Bleser 1992;
Haack 1993; Baker 1994).

Conservation of Karner blue is mandated by the Endangered Species Act of 1973,
which provides federal protection for the butterfly and its designated critical habitat, and
requires the development and implementation of management plans for species recovery
(U SFWS 1992). Specific recovery measures to-date include ongoing research to
elucidate Karner blue ecology and critical habitat needs, habitat restoration and
management, and investigation into the potential for Karner blue propagation and
reintroduction (U SFWS 1992; Baker 1994). Researchers have yet to define all the
components of critical habitat, limiting the abilities of managers to restore or improve
habitat (Andow et a1. 1994). The potential for propagation of Karner blue through
captive rearing is gaining increasing attention, especially in states such as Minnesota and
New Hampshire, where only a few, small Karner blue populations are known to occur
(Schweitzer 1994). These populations could become extirpated before necessary
information regarding Karner blue ecology is acquired, or before the habitat has time to
respond to management activities (Packer 1994).

Investigations into techniques for captive rearing have been conducted as part of
the conservation of other declining butterfly species in the family Lycaenidae (New

1993). Captiye rearing may provide a means to supplement low butterfly populations,

6

reestablish recently extirpated populations (New 1993), or provide individuals for
research, with minimal risk to existing butterfly populations (Lane and Welch 1994).
However, only recently have attempts been made to identify methods for collection and
captive rearing of the Karner blue (Savignano 1992; VanLuven 1993, 1994; Lane and
Welch 1994). We describe the methods and success of our efforts to rear Karner blue
from spring generation butterflies under controlled laboratory conditions in 1994. Larvae
acquired from this study were used in a related study to evaluate the susceptibility of
Karner blue to Bacillus thuringiensis Berliner var. kurstaki (Btk), a microbial insecticide
specific to Lepidoptera, commonly used for gypsy moth (Lymantria dispar L.;

Lepidoptera: Lymantriidae) suppression in Michigan (Chapter 2).

Methods & Materials

Lupingfoliage: Wild lupine foliage used for Karner blue rearing activities were
obtained from a small field in Ingham County, Michigan, which supports lupine and other
remnant prairie plant species but no Karner blue. The lupine was harvested by cutting
stems, placing them in a large, water-filled container, and then recutting the ends of the
stems under water. In the laboratory, the container with lupine was refrigerated at 5°C
until needed. A plastic bag was placed over the top of the foliage to reduce desiccation.
New lupine stems were harvested and the old stems discarded every 4 - 5 days. Leaves
with previous insect feeding or other damage were not used for rearing Karner blue
larvae.

Windus: We collected a total of 20 female Karner

blue adults during the spring flight in June 1994 fi'om five collection sites in the Lower

7

Peninsula of Michigan. Three sites were located in the Allegan State Game Area
(Allegan County) in the southwest, and two sites were in the Huron-Manistee National
Forest (Montcalm and Newaygo Counties) farther north. Sites were chosen in
cooperation with officials from the Michigan Natural Features Inventory, the Michigan
Field Office of The Nature Conservancy, the Allegan State Game Area, and the Huron-
Manistee National Forest, and were approved by the US Fish & Wildlife Service. Ten
females were collected from the Game Area and 10 from the National Forest. We
collected only in sites that had 1993 summer generation adult counts of more than 200
butterflies (Michigan Natural Features Inventory, unpublished data; Huron-Manistee
National Forest, unpublished data), with no more than five females collected from any
one site to minimize possible impacts on local populations.

We collected the Karner blue females 2 weeks after the first spring generation
adults were observed, approximately halfway into the spring flight period (Table 1).
Since butterflies began flying approximately 5 days sooner in the more southerly sites of
Allegan State Game Area than in the Huron-Manistee National Forest, Karner blue
females were collected on 1 June 1994 in the Game Area and on 9 June 1994 in the
National Forest. We attempted to select females with moderate wing wear, rather than
extremely fresh-looking females or those with worn wings, assuming that females with
moderate wear would have already mated but still retain much of their egg complement.
At the time of collection in the Game Area, the ratio of males to females in Karner blue
populations near the collection sites ranged from 2:1 to 3:1 (no butterfly surveys were

conducted in the collection sites) (Chapter 2).

8

Collections were initiated around 11 am and completed by 1 pm. On both days,
the weather was sunny, with temperatures around 22°C. We caught each Karner blue
female individually in a butterfly net, and transferred it to a glassine envelope by holding
the wings. Envelopes with butterflies were then placed in individual plastic containers to
prevent crushing, and kept in a slightly chilled cooler (approximately 20°C) in the shade
(Saul-Gershenz et a1. 1995). A layer of newspaper was used to prevent direct contact of
the containers with ice packs at the bottom of the cooler. Transportation time from each
collection site to our laboratory at Michigan State University was ca. 2 hours.

Wigs: In the laboratory, butterflies were transferred to aluminum
fiame cages (61 x 61 x 61 cm) with 32 mesh Lumite screen (BioQuip Products, Gardena,
CA). We opened each envelope inside the cage and allowed the female to walk out onto
lupine foliage (described below). Butterflies were caged together by site. Cages were
kept on fluorescent-lighted shelves in a walk-in environmental chamber maintained at 24
- 26°C, with an 18:6 hr lightzdark photoperiod, and relative humidity of 57 - 68 percent.

We provisioned each cage with a water source, partial shading, nectar source, and
ovipositional site. The water source was a wet sponge cut to tightly fit the bottom of a
petri dish (100 x 15 mm). One sponge was provided per cage, and was moistened daily.
Any standing water or condensation was wiped up immediately, to prevent butterflies
from becoming trapped or drowning (Lane and Welch 1994). We provided partial
shading by placing layers of paper towels over one corner of the top of the cage.

The nectar source was a 5 percent honeyz95 percent water solution presented as

per Lane and Welch (1994). The solution was placed in a sterile ISO-ml flask, and then

sealed with parafilm. Cotton dental wicking (Accu Bite Dental Supply Inc., East

9
Lansing, MI) was pushed partially into the flask through the parafihn, leaving 3 - 5 cm of

wicking protruding, to provide a suitable place for butterflies to perch and feed. We
provided two nectar flasks in each cage, and replaced them every 2 days.

The ovipositional site consisted of a wild lupine stem, 20 - 30 cm tall, with
flowers and leaves, in a water-filled 250-ml flask with a parafilm seal. We placed two
flasks with lupine in each cage, and replaced them every 2 days with fresh lupine.

We housed the females for 5 days in the cages, and then returned all survivors to
their original collection sites. Female butterflies were transported in a ca. 20°C cooler, in
glassine envelopes and plastic containers as above, to the appropriate site. At the sites,
we released each female by opening the envelope near a lupine plant, and allowing the
butterfly to walk onto a leaf.

MW: We removed the lupine stems from the cages and
inspected them for Karner blue eggs once per day. Eggs were carefully dislodged from
the plant using a small blade (Lane and Welch 1994), and placed individually into 30-ml
plastic cups (Jet Plastica Industries, Hatfield, PA). When lupine stems were replaced, the
old stems were kept with the flasks in the environmental chamber, and examined
periodically for any eggs or developing larvae that had been initially overlooked.

Plastic cups containing individual eggs were placed in large, lidded plastic boxes
(19 x 10 x 8 cm; Tri-State Plastics, Dixon, KY) lined with moist paper towels, and kept in
a fluorescent-lighted growth chamber maintained at 24°C, with an 18:6 hr light:dark
photoperiod and ambient relative humidity. Relative humidity inside each box with

moist paper towels was ca. 80 - 85 percent, as measured with a Bionaire instrument

10

(model BT-254F, accuracy :I: 5 percent; Bionaire Environmental Air Products, Blauvelt,
NY). We checked the eggs once per day for hatch. Two days alter the eggs were
collected, we added a small piece of lupine foliage to each cup in anticipation of hatch.
The paper towels in each box were rewetted once at most, but only if there was no
condensation on the sides of the box or in the cups. No additional moisture was added to
the boxes once the lupine foliage was added to the cups, and the box lids were propped
for short periods when necessary to allow excess moisture to dissipate.

Manning: We kept larvae in the same growth chamber as the eggs, and
checked them daily for molting, mortality, food supply and condition of container.
Molting was noted via presence of exuvia. Larval length was measured at the beginning
of each instar using a dissecting microscope fitted with an ocular micrometer.

First and second instars were reared individually in 30-ml plastic cups, which
were kept in the growth chamber in lidded plastic boxes as the eggs. Larvae were
transferred while on the lupine foliage to fresh cups every 2 days. If necessary, a #000
paintbrush was first used to place each larva on the lupine foliage. We supplied flesh
pieces of lupine every 2 days for first instars, and daily for second instars. Old foliage
was removed the following day after larvae had moved to the new leaves.

Third and fourth instars were reared individually in petri dishes (100 x 15 mm),
which were kept in the growth chamber on trays. We provided an entire lupine leaf to
each larva by placing the leaf stern in a water-filled 0.5 dram (2-ml) glass vial stoppered
with a cotton plug. In this way, the vials and leaves could be placed in the petri dishes

horizontally without water leakage, thus preventing larvae from drowning. Lupine leaves

11

were replaced when more than half of the leaf was eaten, usually every 1 - 2 days. Third
instars were transferred to new petri dishes every 2 days, and fourth instars were
transferred to new dishes daily. When replacing old lupine or transferring larvae to new
dishes, we cut the leaflets that had the larvae, and then moved the larvae while on the
leaflets.

After daily use, paintbrushes, forceps and scissors were sterilized by first soaking
in a bleach:water solution (1:4), then washing with soapy water and rinsing in distilled
water, and finally autoclaving. To avoid potential disease transmission between
individuals, we also cleaned utensils after use with each larva by dipping utensils in the
bleach solution, and then rinsing thoroughly with water.

him: We kept pupae in the same growth chamber as the eggs and larvae. Pupae
were placed individually in small, lidded plastic boxes (14 x 7 x 4 cm; Tri-State Plastics,
Dixon, KY) to allow room for adult emergence. When pupae were attached to a lupine
leaf, we cut away excess foliage from around the pupal case to avoid leaf molding. When
pupae were attached to the petri dish, we sterilized the dish surface around the pupa with
70 percent ethyl alcohol, and placed the open dish in the box.

Admumtterflies: After emergence, each Karner blue adult with its container was
removed from the growth chamber, and kept in a refrigerator at 5°C for 1 or 2 days prior
to field release. On the day of release, we transported adults in their boxes in a ca. 20°C
cooler to the maternal collection sites. The boxes were then removed from the cooler,

and opened in a shady area to allow each butterfly to acclimate and fly away.

12
Sjafisticalanalysis: Developmental times for male and female Karner blue were

compared by AN OVA using SYSTAT (Wilkinson 1990). All statistical analyses were

conducted at p < 0.05 level of significance.

Results

WW: All 20 Karner blue adult females
were collected and transported without mortality from the collection sites to Michigan
State University. The butterflies appeared to adjust quickly to the cages, and began using
the nectar and water sources within the first few hours. Females from Allegan State
Game Area and Huron-Manistee National Forest began laying eggs 2 and 3 days after
collection, respectively.

Ten of the 20 Karner blue females were still alive after 5 days (five each from
Allegan State Game Area and Huron-Manistee National Forest), and were returned to the
original collection sites. We observed male Karner blue butterflies of the spring
generation in the sites when the females were released, so presumably all females could
have mated. The ten females that did not survive died afier 4 - 5 days in captivity of
apparently natural causes. These specimens were donated to the Center for Insect
Diversity Studies, Department of Entomology, Michigan State University, East Lansing,
Michigan.

MW: We collected a total of 154 eggs from the caged
butterflies, of which, 61 percent were from Allegan State Game Area females, and 39/
percent were fiom Huron-Manistee National Forest females (Table 2). Once females

began laying eggs, we collected from O - 23 eggs per cage per day. Eggs were most often

13

found on the leaves, petioles and stems of the lupine, and occasionally on flowers. We
did not find eggs on the sides of the cages or flasks. Nine eggs laid by the Huron-
Manistee National Forest females were overlooked, and were later discovered as second
and third instars on the old lupine stems in the environmental chamber. Since females
were caged in groups, the exact number of eggs from each female could not be
distinguished. Based upon cage averages, the average overall number of eggs per female
ranged fiom 1 :16.

Overall egg hatch was 47 percent; however, egg hatch varied by region and site
(i.e. cage) (Tableg2). Forty-three percent of eggs from Allegan State Game Area, and 53
percent of eggs from the Huron-Manistee National Forest hatched (Table 2). Of the 72
first instars Obtained, two died (one was deformed so that it could not feed properly and
one became diseased), and two escaped (and presumably died), leaving 68 first instars.

A total of 82 Karner blue eggs (53 percent) did not hatch. Of these eggs, we
observed six cases where two eggs were stuck together (each was counted as 1 egg, not
2), two eggs which were oddly shaped as compared to the others, and an unidentified
species ofmite'on five of the unhatched eggs. Mold developed on 47 eggs, even though
no excessivemoisture was apparent. Twenty of those eggs became moldy 5 - 6 days after
they were collected, and the other 27 eggs developed mold in 8 - 11 days.

WWW: We used 59 of the 68 Karner blue larvae in
a related study (Chapter 2) to determine the susceptibility of Karner blue to Btk used for
gypsy moth suppression. The other nine Karner blue that were found as larvae on the old

lupine were not used in the Btk study, and were reared under normal conditions. Of the

larvae used in the Btk study, 15 were reared under normal conditions for controls, and the

14

other 44 larvae were placed at varying instars on Btk treatments. Information reported
here regarding larval and pupal development (Table 3, 4) was taken from the 15 control
larvae, and the 44 treatment larvae up to their placement on the treatments.

Total developmental time of Karner blue from egg collection to adulthood at 24°C
averagedi6 days overall; however, developmental time for females differed significantly
from males by 2 days on average (F = 11.47, df = 1; p < 0.005) (Table 3). Karner blue
eggs hatched on average 4 days after egg collection, with several eggs hatching after only
2 days (Table 3). One egg hatched after only 1 day; however, this egg was probably
overlooked during egg collection and left on the lupine foliage for a day. No eggs
hatched more than 6 days after collection. Total larval duration (first - fourth instar)
averaged 13 days overall; larval duration was ca. 1.5 days longer for females than males
on average, but was not significantly different (F = 4.41, df = 1; p < 0.056) (Table 3).

The duration of individual instars averaged 3 - 4 days (Table 3). At the prepupal stage,
which lasted ca. 1 day (Table 3), Karner blue larvae stopped feeding and became
stationary, attaching themselves to the petri dish or to a lupine leaf with a few silk
threads. The pupal stage averaged 8 days (Table 3) for both Karner blue males (11 = 7, SE
= 0.2) and females (11 = 8, SE = 0.2). Pupae darkened significantly 1 day before adult
emergence.

Larval body length was difficult to measure accurately because larvae were often
moving, appearing more elongate than when stationary. Based on the average initial
lengths for each instar, larvae grew 1 mm from first to second instar, 2.7 mm from second

to third, and 3.3 mm from third to fourth (Table 4).

15

The nine larvae not used in the Btk study and the 15 control larvae, plus nine of
the 44 treatment larvae that survived the Btk bioassay, developed successfully to
adulthood, producing 33 Karner blue adults for release. Nineteen adults (9 males, 10
females) were released into Allegan sites; 14 adults (6 males, 8 females) were released
into Huron-Manistee sites. We observed summer generation Karner blue adults from the

wild populations in the sites at the time of release (Table 1).

Discussion

Laboratory, or captive, rearing and subsequent reintroduction have been
successful components in the conservation of several butterfly species in the family
Lycaenidae, such as the atala hairstreak (Eumaeus atala Poey; New 1993) in Florida, and
the large blue (Maculinea arion L.; Clarke 197 7; New 1993) and large copper (Lycaena
dispar Obth.) in England (Duffey 1977; Pyle et a1. 1981). Our results confirm those of
recent Karner blue studies (Savignano 1992; VanLuven 1993, 1994; Lane and Welch
1994) that eggs can be collected from females in the laboratory, and can be reared
successfully from larva to adult. I

In the present study, we obtained 154 eggs, and subsequently 72 first instars, from
20 spring generation Karner blue females. Survival of larvae, pupae and adults reared
under normal conditions was high; only four first instars died. Developmental time from
egg to adult averaged 26 days at 24°C. The controlled environments of the walk-in
environmental chamber and growth chamber used to maintain butterflies and other
lifestages ensured that individuals would not experience detrimental temperature

extremes. Although most Karner blue larvae were used in related research (Chapter 2),

16

the 24 larvae reared under normal conditions, plus nine experimental larvae, survived to
adulthood (a total of 33), and were released into maternal collectiOn sites. We observed
summer generation Karner blue adults from wild populations at the time of release, a
fortuitous result. The rate at which Karner blue developed in the laboratory at 24°C was
similar enough to that of field individuals to allow for overlap. Ultimately, synchronous
development of lab and field populations would be a desired outcome for a reintroduction
program.

In Wisconsin, Lane and Welch (1994) reported the highest oviposition and
hatching rates of any rearing study to date. They obtained 876 eggs from 40 spring
generation Karner blue females after a 2-day housing period, and 88 percent of the eggs
hatched. Two hundred larvae were use in a laboratory experiment, and 149 survived to
adulthood. The remaining 570 larvae were placed out in the field, with 5 percent
survival. Lane and Welch (1994) concluded that captive rearing produced large numbers
of larvae with minimal or no impact to local populations, and that survival of larvae to
adulthood was higher in the laboratory than in the field.

Summer generation Karner blue females have been used successfully for captive
rearing activities in New Hampshire, although overwintering of the eggs and providing
lupine for newly hatched larvae in the spring posed some challenges (V anLuven 1993,
1994). In 1992, VanLuven (1993, 1994) obtained 117 eggs from 11 summer generation
females that were housed for 3 - 5 days. These eggs were placed outdoors in jars to
overwinter, and 110 hatched the following spring. Of those, 88 developed successfully to

adulthood.

17

In this study, we observed lower oviposition rates (Karner blue eggs per female)
and hatching success than in previous studies (Savignano 1992; VanLuven 1993; Lane
and Welch 1994). These results may have been due to random, uncontrollable variables,
such as field conditions experienced by the females prior to collection, that impacted egg
production and viability. Savignano (1992) reported year-to-year variability in egg hatch
among rearing experiments, ranging flom 6O - 90 percent hatch. Lederhouse and Scriber
(1987) obtained low oviposition rates and/or egg viability for 10 - 20 percent of field-
collected female tiger swallowtail butterflies (Papilio glaucus L.; Lepidoptera:
Papilionidae) in each of several trials; they attributed these results to random mating
failure. However, oviposition and hatching rates in this study may also have been
affected by experimental variables such as age (based on wing wear) of collected females,
handling of females (collection, transport), size and type of ovipositional cage, and
environmental conditions (temperature, relative humidity, light) used to maintain females
and eggs in the laboratory. Of these four variables, female age and environmental
laboratory conditions are the most probable ones to explain our results.

Like VanLuven (1993, 1994), we attempted to collect females with moderate
wing wear, assuming that these females would have mated (Friedrich 1986) but still
retain many eggs. In contrast, Lane and Welch (1994) captured flesh females, many of
which were observed ovipositing in the field and were presumed to be gravid. It is
possible that the moderately worn females collected in our study had already laid a large
proportion of their eggs in the field (Friedrich 1986), which would explain the low
numbers of eggs obtained. Age of the Karner blue females may also have impacted egg

viability. Lederhouse and Scriber (1987) reported significant declines over time in egg

18

viability of female tiger swallowtail butterflies. Although unlikely, some of the Karner
blue females we collected may not have been gravid, as proposed by VanLuven (1994) to
explain low egg numbers in his 1993 study; any eggs laid by these females would have
contributed to the low hatching success we recorded.

Our adult butterfly collection and transportation methods differed somewhat flom
other studies. After netting the Karner blue adults, we transferred individuals to glassine
envelopes to confine their movement, and kept them in a ca. 20°C cooler for transport
(Saul-Gershenz et a1. 1995). We handled the females only by the wings. In other studies,
butterflies were not directly handled, and had some fleedom of movement during
transport (V anLuven 1993, 1994; Lane and Welch 1994). Lane and Welch (1994) also
provisioned butterflies with water and nectar sources. Transport time flom the field to
the laboratory was considerably longer in our study than in the other studies. Keeping the
butterflies immobile and cool ensured that they would not experience temperature
extremes (Saul-Gershenz et al. 1995), reduced their need for resources during
transportation, and did not appear to stress or damage them.

Small butterflies, such as lycaenids, can be induced to oviposit in small containers
that restrict movement (Friedrich 1986). VanLuven (1993, 1994) used 240-ml glass jars
to house summer generation females for oviposition, with varying success. For this
study, we chose to use larger mesh cages, with access provided by a cloth sleeve, to
facilitate the provisioning of resources such as lupine stems for oviposition and
honeywater, and to minimize the risk of butterflies escaping. Lane and Welch (1994)
used a similar type of mesh cage to ours; however, their cage was half the size (30 x 30 x

30cm), which caused the lupine stems to touch the top of the cage. Females were ofien

19

observed walking on the cage top and coming into contact with the lupine (C. Lane,
University of Minnesota, pers. com.). A smaller cage may be more successful to induce
oviposition of Karner blue females by increasing the likelihood of contact between
butterflies and ovipositional sites.

Environmental laboratory conditions, such as temperature, relative humidity and
light, used to maintain females and eggs in this study may have affected oviposition rate
and egg hatch (Singh and Ashby 1985). Our rearing methods mimicked field conditions
less than other studies because of our use of an environmental walk-in chamber to house
caged butterflies and growth chambers to house the other butterfly lifestages.

Temperature is an important variable for determining insect activity and
development (Goodenough and Parnell 1985; Singh and Ashby 1985; Saul—Gershenz et
a1. 1995). We housed female butterflies at 24 - 26°C, temperatures slightly lower than
daytime temperatures in the field. VanLuven (1993) observed that female Karner blue
butterflies of the summer generation were relatively inactive when housed in the
laboratory at temperatures below 27°C. However, guidelines for butterfly rearing have
suggested 25°C as an acceptable temperature for oviposition (Friedrich 1986). Lane and
Welch (1994) kept caged females at ambient room temperature, which averaged 28°C,
but fluctuated widely flom 23° to 31°C during the day. Temperatures higher than what
were used in this study, or fluctuating temperatures, may be important to facilitate egg
production or stimulate oviposition with Karner blue. The same may be true for egg
development. We maintained eggs at 24°C, whereas Lane and Welch (1994) kept eggs in

ambient room temperature, which averaged 24°C, but ranged daily flom 20° - 28°C.

20

The appropriate level of relative humidity for insect deveIOpment varies with
different lifestages (Saul-Gershenz et a1. 1995). Relative humidity can impact egg
development (Goodenough and Parnell 1985) by either causing desiccation when
humidity is too low or molding when humidity is too high (Singh and Ashby 1985;
Friedrich 1986). In our study, molding appeared to have reduced egg hatch;
approximately half of the unhatched eggs developed mold, some within 6 days and others
within 11 days of collection. After collection, eggs were kept in plastic boxes in a growth
chamber with ambient relative humidity. We attempted to control the humidity in the
boxes in two ways: adding wetted paper towels (prior to the addition of foliage) to
increase humidity, or propping the lids of the boxes to reduce condensation. Lane and
Welch (1994) similarly reported molding as a significant factor in preliminary rearing
attempts with Karner blue. Surface disinfection of eggs would presumably reduce this
problem (Singh and Ashby 1985). The remaining unhatched eggs in our study neither
developed mold, nor appeared desiccated.

The quality of light, both wavelength and intensity, and photoperiod, can impact
insect physiology, biochemistry and behavior, including oviposition behavior (Singh and
Ashby 1985; Saul-Gershenz et a1. 1995). In our study, lighting experienced by caged
Karner blue females was provided entirely by fluorescent bulbs, with an 18:6 hr
light:dark photoperiod. In the studies by Lane and Welch (1994) and VanLuven (1993,
1994), caged butterflies experienced some indirect natural lighting. However, in the
study by Lane and Welch (1994), most lighting came flom fluorescent bulbs, with a 16:8
hr light:dark photoperiod. VanLuven (1993, 1994) supplemented the natural light with

an incandescent lamp during cloudy days.

21

We did not encounter any problems rearing larvae to adulthood in the laboratory.
Karner blue larvae developed successfirlly without the provision of tending ant species;
however, this may be a requirement for other ant-tended lycaenid species (New 1993).
Only one larva died flom an apparent disease. We emphasized sanitation throughout the
rearing process (Singh and Ashby 1985; Saul-Gershenz et a1. 1995), especially during
larval rearing. Protocols included housing larvae in individual containers which were
changed often, keeping larval containers flee of flass and moisture build-up, supplying
clean foliage regularly, and using sterilized tools.

Karner blue larvae appeared to do well on cut foliage flom wild lupine plants.
Our initial intention was to rear larvae on wild lupine grown flom seed in the greenhouse,
and a preliminary attempt in 1993 to produce greenhouse lupine was successful.
Unfortunately, in our 1994 study, the lupine seedlings became infested with western
flower thrips (Frankliniella occidentalis Pergande; Thysanoptera: Thripidae), a common
greenhouse pest, and no plants survived. Savignano (1992) successfully reared Karner
blue larvae flom eggs of spring generation butterflies on Russell Hybrid, a cultivated
lupine hybrid that grows more quickly in the greenhouse and produces larger leaves than
does wild lupine. Cultivation of lupine in the greenhouse may become a useful way of
providing foliage for Karner blue rearing projects, especially when overwintered eggs are
used and wild lupine may be difficult to obtain in the spring.

While we need more information on proper laboratory conditions for Karner blue
oviposition and development, captive rearing appears to be a viable means of producing
large numbers of Karner blue individuals with potentially little impact to source

populations. These individuals can be used to supplement or reestablish populations, or

22

used in research. In considering the use of reared Karner blue for reintroduction, some
questions still remain, such as which generation of Karner blue (spring or summer)
should be used for the egg source, and which life stage should be released in the field
(Lane and Welch 1994; Schweitzer 1994). Based upon previous recommendations for
captive rearing programs, reintroductions should occur only within the historic range of
the Karner blue, and reared individuals that are to be used for supplementation or re-
establishment should be genetically similar to native individuals in or near the release site
(Pyle 1976; New et a1. 1995). While captive rearing does not replace the need for
conservation of butterfly populations in the natural environment (New 1993; Robinson
1995), it appears to be a viable option in the overall conservation program of the Karner

blue.

23

Table 1.1. Adult flight periods of 1994 spring and summer Karner blue generations in
Allegan State Game Area (Allegan Co) and Huron-Manistee National Forest (Oceana Co)
in Michigan.

 

 

Area Flight period First adult seen Last adult seen
Allegan State Spring May 19 June 18
Game Area Summer June 27 August 12
Huron-Manistee Spring May 24 not recorded
National Forest Summer July 5 not recorded

 

Table 1.2. Total numbers of eggs obtained and hatched flom caged female Karner blue
butterflies collected flom Allegan State Game Area (Allegan Co) and Huron-Manistee
National Forest (Montcalm Co and Newaygo Co) in Michigan.

 

 

 

 

 

No. eggs
Karner blue No. Karner blue
collection area Cage no. adult females
Laid Hatched
Allegan State 1 4 64 29
Game Area 2 3 13 2
3 3 17 9
Subtotal 10 94 40
Huron-Manistee 4 5 54 28
National Forest 5 5 6 4
Subtotal 10 60 32

Total 20 154 72

 

24

Table 1.3. Mean duration (:I: SE) of Karner blue life stages captively reared at 24°C.

 

Duration of life stages (days)

 

Life stage Sample size1

 

Meand: SE Range
Egg 62 4.1 :1: 0.2 1 - 62
1stinstar 38 3.23:0.2 2-6
2nd instar 36 3.13: 0.1 1- 5
3rdinstar 31 3.4i0.1 2-5
4th instar 15 4.0 i 0.2 3 - 6
Prepupa 15 1.2 :t 0.1 1 - 2
Pupa 15 7.9 i 0.2 7 - 9
1st-4thinstar 15 13.1i0.4 11-16
Males 7 12.4 :I: 0.5 a 11 - 14
Females 8 13.8 :I: 0.5 a 12 -16
Egg - adult 15 26.0 :I: 0.4 24 - 29
Males 7 25.0 i 0.2 a 24 - 26
Females 8 26.9 i 0.5 b 25 - 29

 

NOTE: For gender comparisons, means followed by the same letter are not significantly
different by ANOVA at p < 0.05.

1 Some larvae reared in this study were used in related research (Chapter 2). Data
reported here represent development of ‘treatment’ Karner blue larvae before they were
assigned to treatments, and ‘control’ larvae in the related research.

2 Only one egg hatched 1 day after collection; however, it was probably overlooked
during egg collection and left on the lupine foliage for 1 day.

25

Table 1.4. Average body length (:1: SE) of captively reared Karner blue larvae at the onset
of each instar.

 

 

 

Body length (mm)
Instar Sample size1
Mean i SE Range
1st 25 1510.04 1.1 - 1.9
2nd 18 2.5 d: 0.12 1.9 - 3.5
3rd 31 5.23: 0.20 3.3 — 6.8
4th 28 8.5 :I: 0.25 6.2 - 12.5

 

1 Some larvae reared in this study were used in related research (Chapter 2). Data
reported here represent development of ‘treatment’ Karner blue larvae before they were
assigned to treatments, and ‘control’ larvae in the related research.

CHAPTER 2
Susceptibility of the Endangered Karner Blue Butterfly (Lepidoptera: Lycaenidae)

to Bacillus thuringiensis var. kurstaki Used for Gypsy Moth
(Lepidoptera: Lymantriidae) Suppression in Michigan

Abstract

Management conflicts have arisen in Michigan due to the recent spread of gypsy
moth (Lymantria dispar), an introduced forest pest, into oak savanna habitat occupied by
the endangered Karner blue butterfly (Lycaeides melissa samuelis). Microbial
insecticides formulated flom Bacillus thuringiensis var. kurstaki (Btk), a naturally
occurring soil bacterium, are commonly used for gypsy moth suppression; however,
widespread use has raised concern regarding the impacts of Btk on nontarget Lepidoptera.
In this study, we investigated the phenological and physiological susceptibility of Karner
blue to Btk as used for gypsy moth suppression in Michigan. In the spring of 1993 -
1995, we monitored phenology of spring generation Karner blue populations in two
regions of Lower Michigan to determine if larval stages overlapped temporally with the
Btk spray period for gypsy moth in nearby areas. In 1993, some late instar Karner blue of
the spring generation were found during Btk application in one region. In 1994 and 1995,
no spring generation larvae overlapped the Btk spray periods in either region; however,
spring generation Karner blue adults were observed up to 11 days prior to Btk application,

and in 1995, newly laid eggs were observed at the time of or a few days before Btk

26

27

application. Since Karner blue eggs hatch quickly, summer generation early instars were
most likely present during or shortly after Btk application in 1994 and 1995, and
assuming that Btk persists in the field for 4 - 6 days post-spray, some larvae would have
been at risk.

In a laboratory bioassay, captively-reared Karner blue larvae (first through fourth
instars) were fed foliage of the host plant, wild lupine (Lupinus perennis), which were
untreated or treated with the Btk formulation Foray 48B, at rates of ca. 30 - 37
BIU/hectare (12 - 15 BIU/acre) and 90 BIU/hectare (36 BIU/acre). A similar bioassay
with second instar gypsy moth larvae on white oak foliage (Quercus alba) was conducted
concurrently. Karner blue larval survival was 27 percent and 14 percent on low and high
Btk treatments, respectively, and was significantly lower for all instars on both Btk
treatments than for controls. Survival of gypsy moth larvae was 33 percent and 5 percent
on low and high Btk treatments, respectively. Overall survival of Karner blue did not
differ significantly flom that of gypsy moth; however, Karner blue mortality was
significantly higher than gypsy moth mortality in the first 3 - 6 days of the bioassay,
suggesting that Karner blue may be more sensitive to Btk than gypsy moth. We conclude
that Karner blue is highly susceptible physiologically to Btk, and is phenologically
susceptible to gypsy moth suppression activities, though the extent of phenological
overlap and the larval generation (spring vs. summer) at risk may vary flom year to year.
Information regarding the susceptibility of nontarget Lepidoptera to Btk, including
physiological susceptibility and temporal overlap of larval stages with Btk application and

the period of toxic persistence, must be considered in management plans for gypsy moth.

28

However, impacts of gypsy moth defoliation, in the absence of suppression, on nontargets

must also be considered.

Introduction

The Karner blue butterfly (Lycaeides melissa samuelis Nabokov; Lepidoptera:
Lycaenidae) is a federally endangered species occurring in localized areas of the
northeastern and midwestem United States. Recently, in Michigan, gypsy moth
(Lymantria dispar L.; Lepidoptera: Lymantriidae), an introduced defoliator of
hardwoods, has spread into oak savanna habitat, to which the Karner blue is restricted.
Bacillus thuringiensis Berliner var. kurstaki (Btk), a microbial insecticide, is widely
sprayed in Michigan to suppress gypsy moth populations. However, concern regarding
potential nontarget impacts of Btk on Karner blue has brought about management
conflicts in areas where gypsy moth and Karner blue co-occur.

The Karner blue was added to the United States’ federal endangered species list in
December 1992 due to dramatic population declines throughout its range (Schweitzer
1989; USFWS 1992). Historically, Karner blue populations occurred in a narrow band
flom Minnesota to New Hampshire. However, the species is currently extirpated in Ohio,
Pennsylvania, Massachusetts and Ontario (U SFWS 1992; Haack 1993). Habitat of the
Karner blue consists primarily of oak savannas in the Midwest and pine barrens in the
Northeast (Schweitzer 1989). These dry, sandy, sparsely wooded habitats support many
grasses and herbaceous plants including wild lupine (Lupinus perennis L.), the only
known host plant of Karner blue larvae (Schweitzer 1989). The butterfly completes two

generations per year; both larval generations feed on lupine, and spring and summer

29

adults require nectar sources (Schweitzer 1989; Dirig 1994). Rangewide decline of
Karner blue is attributed to loss of suitable habitat due largely to human activities, such as
agriculture, residential and commercial development and fire suppression (Packer 1987;
USFWS 1992; Haack 1993; Dirig 1994; Lane 1994). As with all federally listed species,
the Endangered Species Act of 1973 mandates that conservation measures be provided
for the Karner blue to ensure its survival (U SFWS 1992).

Gypsy moth was first recorded in eastern Michigan in 1954 (O’Dell 1955).
Despite control efforts, populations have continued to spread west throughout the state,
causing severe defoliation of oak-dominated woodlands (Gage et al. 1990; Witter and
Stoyenoff 1992). Current efforts to suppress gypsy moth populations in wooded
residential areas and high-value recreation sites in Michigan are administered jointly by
the Michigan Department of Agriculture and the United States Department of Agriculture
(USDA) Forest Service through the Michigan Voluntary Cooperative Gypsy Moth
Suppression Program (USDA 1994a). This is a large program, which recently has
involved aerial application of Btk to more than 91,200 hectares in Michigan in 1993,
56,720 hectares in 1994, and 42,800 hectares in 1995 (USDA 1994a, 1994b; USDA
1995)

Bacillus thuringiensis var. kurstala' is an entomopathogenic bacteria that occurs
naturally in the soil (DeLucca et a1. 1981; Dulmage and Aizawa 1982; Martin and
Travers 1989), and is selectively toxic to larvae of some lepidopteran species (Dubois and
Lewis 1981). The Bacillus thuringiensis group of bacteria produce proteinaceous
crystalline inclusions, or crystals, during spore formation (Cherwonogrodzky 1980;

Dubois and Lewis 1981; Gill et a1. 1992). The crystals of Btk are a matrix within which

30

glycoproteins, known as 6-endotoxins or insecticidal crystal proteins (ICP) (Gill et al.
1992; Bauer 1995), are contained. The insecticidal activity of Btk is largely attributed to
the solubilization of the crystal in the gut of the insect and activation of the S-endotoxins
(van Frankenhuyzen et a1. 1991). Gut perforations occur and the spores invade the
haemolymph and cause septicemia; death occurs flom ICP toxicity and is enhanced by
septicemia (Bauer 1995; Dubois and Dean 1995). Most, if not all, commercial
preparations of Btk contain both crystals and spores (Lilthy et al. 1982; Bauer 1995).

Bacillus thuringiensis var. kurstakr' is widely used as a microbial pesticide for
control of forest defoliating Lepidoptera in North America (van Frankenhuyzen 1990;
Beegle and Yamamoto 1992; Reardon et a1. 1994). Due to its selective toxicity, safety to
vertebrates, and apparently short field persistence of 4 - 6 days on foliage (Beegle et al.
1981; Reardon et a1. 1994; Wagner and Miller 1995), Btk is thought to present little risk
to nontarget organisms compared to alternative insecticides (Morris et al. 1975; Ltlthy et
a1. 1982; Dirnond and Morris 1984; Meadows 1993; Bauer 1995). However, as a result
of Btk’s extensive use, there is growing concern regarding the potential impacts on
nontarget Lepidoptera (Laird 1973; Brower 1986; Miller 1990), especially for declining
species such as the Karner blue. In addition, recent evidence suggests that Btk may
remain toxic to some lepidopteran species for much longer than generally thought
following field application (Johnson et a1. 1995).

Management conflicts have arisen in areas of Michigan where gypsy moth and
Karner blue populations overlap. Public pressure to treat gypsy moth-infested woodlands

is on the rise, especially in residential or recreational areas (USDA 1994a), and in

31

nurseries, Christmas tree plantations, and other plant industry production areas (D.
McCullough, Michigan State University, and R. Priest, Michigan Department of
Agriculture, pers. comm). However, according to US federal regulations, areas inhabited
by Karner blue cannot be treated with Btk (USDA 1994a), except through formal
consultation with the US Fish & Wildlife Service (U SF WS 1992), because of potential
negative impacts. In addition, a 0.8 km spray buffer must be maintained around known
Karner blue-occupied sites to protect them against drift (Borak 1994).

A limited number of field and laboratory studies to date have addressed the issue
of susceptibility of nontarget Lepidoptera to Btk. In field studies in Oregon and West
Virginia where only a single application of Btk was used for western spruce budworrn
(Choristoneura occidentalis Freeman; Lepidoptera: Tortricidae) and gypsy moth,
respectively, larval abundance and species richness of Lepidoptera were reduced for at
least two years after treatment (Miller 1992; Sample et a1. 1993). Decreases in species
richness and larval abundance of oak-feeding lepidopterans were also observed for up to
two years following repeated Btk applications over one season for gypsy moth eradication
in Oregon (Miller 1990). Btk toxicity has been determined for the cinnabar moth (Tyria

jacobaeae L.; Lepidoptera: Arctiidae) (James et al. 1993), a biocontrol agent of tansy
ragwort (Seneciojacobaea L.), and for two swallowtail butterfly species (Papilio glaucus
L. and P. canadensis Rothschild and Jordan; Lepidoptera: Papilionidae) and the
promethea moth (Callosamia promethea Drury; Lepidoptera: Saturniidae) (Johnson et al.
1995) in field trials. Laboratory bioassays have demonstrated Btk susceptibility for
several other native species of butterflies and moths (Peacock et a1. 1993; Wagner and

Miller 1995). Though negative effects of Btk have been demonstrated for a broad range

32

of nontarget lepidopteran species, Btk susceptibility cannot be generalized flom one
family or species to another (Wagner and Miller 1995), and must be considered on a
species-by-species basis (Peacock et a1. 1993). To date, no studies have examined the
susceptibility of Karner blue or other lycaenid species to Btk.

Surveys to locate all Michigan populations of Karner blue have not been
completed. Many new populations were discovered in 1993 - 1995, following listing of
the Karner blue as an endangered species (J. Kelly, Huron-Manistee National Forest,
pers. comm). As gypsy moth populations expand into new areas, it is possible that
unknown Karner blue populations will be inadvertently treated with Btk. Information on
phenological and physiological susceptibility of Karner blue to Btk is required to ensure
that populations are not negatively affected by gypsy moth management programs.

In this study, we investigated the susceptibility of the Karner blue butterfly to Btk,
as used for gypsy moth suppression activities in Michigan. _ Our first objective was to
monitor development of Karner blue in the field to determine if larval instars or other life
stages overlap temporally with the Btk spray period. Our second objective was to
evaluate the physiological susceptibility of Karner blue larvae to Btk in a laboratory

bioassay.

Methods & Materials
Phenology of Karner blue with respect to gypsy moth suppression

We monitored the phenological development of Karner blue and gypsy moth
populations in two regions of Lower Michigan in the springs of 1993 - 1995 to determine

if Karner blue larval stages would coincide temporally with the timing of aerial Btk

33

spraying for gypsy moth suppression. Btk application in the Michigan Voluntary
Cooperative Gypsy Moth Suppression Program is timed to occur when the majority of
gypsy moth larvae are late first instars and early second instars, and when oak foliage is
40 - 50 percent expanded (USDA 1985; Dubois 1991).

Five Karner blue-occupied sites in Allegan State Game Area (Allegan County) in
southwestern Michigan, and one site located farther north on the Huron-Manistee
National Forest (Oceana County) (Figure 1) were chosen for monitoring activities. We
surveyed the sites for spring generation Karner blue larvae and adults once a week flom
late April through late May in 1993 and 1994, and flom early May through early June in
1995 (Table 1). In 1995, surveys for eggs and larvae of summer generation Karner blue
were also conducted.

For each larval survey, approximately 500 - 1000 randomly chosen wild lupine
stems were examined for window-feeding damage indicative of Karner blue larvae (Dirig
1994). Lupine stems with feeding damage were inspected for larvae. When Karner blue
larvae were found, larval length was recorded, and the plant’s location was flagged so
that plants could be relocated. Larval length was used to classify larvae as either early
(first and second) or late (third and fourth) instars. During subsequent surveys, we
rechecked all previous larval locations and searched new lupine stems for additional
larvae. Surveys for eggs in 1995 were conducted in a similar manner by visually
inspecting 500 - 1000 randomly chosen lupine plants. To survey for the presence of
Karner blue adults, we randomly walked through each site for ca. 30 - 60 minutes.

We monitored gypsy moth larval development in one population located

approximately 16 km east of the Karner blue study sites in Allegan State Game Area, and

34

in one population which occurred in our Karner blue study site in the Huron-Manistee
National forest. Foliage of 20 - 30 understory host trees with or near gypsy moth egg
masses were inspected for gypsy moth larvae once a week flom egg hatch through early
June. We recorded the larval stage of up to 100 larvae found.

We evaluated the potential overlap of Karner blue larval stages with gypsy moth
suppression activities in two ways. We used the information gathered on gypsy moth
larval development to predict the timing of a hypothetical Btk application in each of the
two Karner blue areas. We also compared Karner blue phenology with dates of actual
Btk application in spray areas near the Karner blue study sites in Allegan and the Huron-

Manistee (Ottawa County, and Muskegon, Newaygo and Oceana Counties, respectively)

(Figure 1).

Btk susceptibility bioassays

WW3 We measured the susceptibility of Karner blue larvae fed
wild lupine leaves treated with Foray 48B (Abbott Laboratories, North Chicago, IL), a
commercial Btk formulation commonly used in Michigan for gypsy moth suppression
(USDA 1994a, 1995). A concurrent bioassay with second instar gypsy moth larvae on
Btk-treated white oak (Quercus alba L.) leaves was conducted as a check for the Foray
48B dosages. Bioassays with each species consisted of three treatments: control
(untreated foliage), a low Btk dose equivalent to 30 - 37 Billion International Units
(Elm/hectare (l2 - 15 BIU/acre) field rate, and a high Btk dose equivalent to 90
BTU/hectare (36 BIU/acre) field rate. Typical rates of Btk application for gypsy moth

range flom 40 - 90 BIU/hectare (16 - 36 BIU/acre) (Dubois et al. 1993; Reardon et a1.

35

1994). Application rates used in the 1994 Michigan Voluntary Cooperative Gypsy Moth
Suppression Project ranged flom 40 - 60 BIU/hectare (16 - 24 BIU/acre) (USDA 1994a,
1995)

Exmrimemalinsectsandfqliage: Karner blue larvae were reared in the
laboratory flom eggs of spring generation female butterflies as described in Chapter 1.
Twenty female butterflies were collected flom two areas in Michigan during the first 2
weeks of June 1994, and housed in the laboratory for five days to obtain eggs. Collection
sites of the butterflies were located in Allegan State Game Area (Allegan Co.) and Huron-
Manistee National Forest (Montcalm and Newaygo Counties) (Chapter 1). Overall, 59
larvae were available for the bioassay.

Gypsy moth larvae were obtained flom USDA APHIS (Animal and Plant Health
Inspection Service) Methods Development Center insect rearing facilities, Otis Air
National Guard Base, Massachusetts. Larvae were shipped as first instars on artificial
diet several days prior to the bioassay, and were checked daily for second instars. All
second instars used for the bioassay were no more than 24 hours old.

Wild lupine foliage, obtained flom an isolated lupine population in a small field in
Ingham County, Michigan (Chapter 1), was used for general rearing and for the Btk
bioassay of the Karner blue larvae. White oak leaves used for the gypsy moth bioassay
were obtained flom a semi-residential site located in Ingham County, Michigan. Lupine
and oak foliage used in the bioassay were harvested 1 day prior to application of Btk
treatments.

W: Low and high Btk doses were applied to lupine and oak foliage

using a cylindrical spray tower, 2.5 m in diameter and ca. 4 m high (Hubbard and Lewis

36

1973), located at the USDA Northeastern Forest Experiment Station in Hamden,
Connecticut. The spray tower was designed to simulate aerial Btk application, and was
equipped with a Mini-Beecomist nozzle calibrated to generate Btk drops between 75 -
125 um volume median diameter (V MD) (Hubbard and Lewis 1973), the drop size range
generally used in gypsy moth suppression spray programs (Reardon et a1. 1994).

One day before foliage treatment, fleshly harvested wild lupine and white oak
leaves were placed as bouquets of five leaves in water picks. Excess lupine and oak
foliage was harvested for the control treatments and kept at 5°C in water-filled containers.
The bouquets of foliage were secured in a chilled cooler and flown that evening to
Hamden, Connecticut. The following morning, the oak and lupine bouquets were
brought to room temperature and sprayed at the doses described above. Kromekote spray
cards (Mead Corporation, Dayton, OH) were also placed next to the leaves and later
analyzed to confirm actual spray deposition rates. Btk treated foliage was returned to
Michigan by 6 pm the same day.

W: The bioassays were set up ca. 7 - 8 hours
after foliar application of Btk. Treatment leaves were labeled without reference to the
dosage to maintain a “blind” experiment. Due to differences in collection dates of female
butterflies, 22 of the 59 Karner blue larvae were early instars (all from Huron-Manistee
National Forest females), and 37 were late instars (36 flom Allegan State Game Area
females, and 1 flom a Huron-Manistee female). Fifieen late instar Karner blue larvae
were randomly chosen for controls. Twenty-two larvae (11 early and 11 late instars)
were randomly assigned to each Btk treatment. We felt it was necessary to use only late

instars as controls because of the limited number of larvae available for the test. Prior to

37

the bioassay, only two of 61 larvae died (one was deformed upon hatch, one died of
unknown causes). All available early instars were used in the bioassay to evaluate
possible stage-specific differences in Btk susceptibility. Each larva was placed in a clean
petri dish (100 x 15 mm) with one lupine leaf (untreated, or low or high Btk treatments),
which had been transferred to a water-filled 2-ml vial plugged with cotton.

For the gypsy moth bioassay, 40 l-day old second instars were randomly assigned
to each of the three treatments and placed in large, lidded plastic boxes (19 x 9 x 8 cm)
(Tri-State Plastics, Dixon, KY), 10 larvae per box, for a total of four replicates per
treatment. Each box contained a bouquet of five white oak leaves (untreated, or low or
high Btk treatments) in a water pick. Paper towels were used to line the bottom of the
box.

Karner blue and gypsy moth larvae were maintained on the same treated or
untreated leaves for up to 7 days, and were checked daily for molting and mortality. All
larvae were kept in a growth chamber at 24°C. Larvae were considered dead if they did
not respond to physical stimulus. Petri dishes and plastic boxes were kept flee of flass to
avoid buildup of secondary bacteria. Sanitation practices included daily removal of flass
flom the leaves, replacing the paper towel lining in the gypsy moth boxes every 2 days,
and replacing petri dishes for Karner blue every 1 - 2 days.

At the end of 7 days, surviving Karner blue and gypsy moth larvae were placed in
clean containers (petri dishes and plastic boxes, respectively) with flesh, untreated
foliage. Karner blue pupae were weighed several times prior to adult emergence to assess
potential sublethal effects of Btk on pupal weight. The gypsy moth bioassay was

terminated after 13 days (Figure 2). Surviving Karner blue were reared to adulthood

III.

by;

38

following protocol described in Chapter 1 and subsequently released into the parental
collection sites.

Sjafisfigaljnalysis: Percent survival of Karner blue and gypsy moth larvae on
control and Btk treatments were analyzed together as a two-dimensional contingency
table using SAS CATMOD, a nonparametric procedure for categorical data analysis
(SAS Institute Inc., 1987). Two separate analyses were conducted with this procedure,
the first to test for effects of Btk, species and Btk x species, and the second to test for
linear effects of the incremental doses of Btk (no, low and high Btk). The nonparametric
one-sided Smimov test (Conover 1980) was used to evaluate differences in larval
survival, for all paired combinations of insect species and treatments, at selected times
throughout the bioassay. Differences in survival between early and late instar Karner
blue were evaluated for each Btk dose as a nonparametric 2 x 2 contingency table using
the chi-square test of independence (Conover 1980). To assess sublethal effects, mean
pupal weights (measured 2 days after pupation) of female and male control Karner blue
were compared with those of female and male survivors, respectively, of the Btk
treatments by AN OVA using SYSTAT (Wilkinson 1990). All statistical analyses were

conducted at p < 0.05 level of significance.

Results

Phenology of Karner blue with respect to gypsy moth suppression

WW: Based upon the phenology of gypsy moth larvae, i.e.

when the majority of larvae were late first instars and early seconds, we predicted that

hypothetical Btk applications for gypsy moth management near Allegan State Game Area

39
would have occurred during the week of 18 May in 1993, 24 May in 1994 and 22 May in

1995 (Table 1). Spring generation Karner blue larvae were found during the predicted
Btk application only in 1993, when late instars were observed. In 1994 and 1995, spring
generation Karner blue adults were observed during the predicted spray times, and in
1994, adults had already been flying for approximately 5 days (Table 1).

For the Huron-Manistee National Forest, we predicted that hypothetical Btk
applications for gypsy moth management would have occurred during the week of 25
May in 1993, 30 May in 1994 and 29 May in 1995 (Table 1). During all of these periods,
we observed only spring generation Karner blue adults, and in 1994, the first adults were
seen 6 days prior to the predicted spray date (Table 1).

WW: Areas in Ottawa County, north of Allegan State Game
Area, were sprayed with Btk flom 1993 to 1995 for gypsy moth suppression (Table 2;
Figure 1). In 1993, we observed late instar spring generation Karner blue in Allegan
State Game Area during the Ottawa County spray period (Table 1). In 1994 and 1995, no
larvae were found during the spray periods; however, we first observed spring generation
Karner blue adults 4 days prior to the 1994 spray period, and 3 - 11 days prior to 1995
spray applications (Table 1). In 1995, Karner blue eggs were first seen 4 days into the 8-
day spray period, 1 week after adults were observed, and the first observation of a
summer generation early instar Karner blue larva was made 3 days after the end of the
Ottawa County spray period, 2 weeks after the first adults were seen (Table 1).

Areas in Muskegon, Newaygo and Oceana Counties, near our Karner blue site in
the Huron-Manistee National Forest, were also treated with Btk for gypsy moth

suppression. Btk applications occurred in Oceana and Newaygo Counties 1993 - 1995,

40

and in Muskegon County 1994 and 1995 (Table 2; Figure 1). For the years considered,
no spring generation larvae were observed during the spray periods in those counties. In
1993, the first spring generation Karner blue adults were observed 1 - 3 days prior to Btk
application in Oceana and Newaygo Counties (Table 1). In 1994, adults began flying in
the Huron-Manistee site 7 - 10 days before Btk treatments were completed in Newaygo
and Oceana Counties, and close to 3 weeks before the second Btk application in Newaygo
County (Table 1). In 1995, we first observed spring generation Karner blue adults 1 - 4
days prior to Btk application in Muskegon and Oceana Counties, and 7 days prior to
treatment in Newaygo County (Table 1). Karner blue eggs flom spring generation adults
were first seen on 5 June, the date of Btk application in Newaygo County, and 3 and 5

days after the Muskegon and Oceana County spray periods, respectively (Table 1).

Btk bioassays

mm: Categorical analysis indicated that overall survival of larvae on
leaves sprayed with Btk was significantly reduced (chi-square = 259.1, p < 0.001), but
there were no significant effects of insect species or Btk x species interactions (chi-square
= 2.2 and 3.9, respectively), suggesting that Karner blue and gypsy moth did not differ in
their overall response to Btk. Linear analysis showed a significant tendency for increased
mortality of each species with increased Btk dose (chi-square = 362.3 for both species
combined; chi-square = 459.1 and 111.4 for Karner blue and gypsy moth, respectively; p
< 0.001).

W: All Karner blue larvae (n=15) on untreated leaves survived

to adulthood (Figure 2A). With both Btk treatments, Karner blue larval mortality began

Sll

41

on Day 3, with a subsequent steep drop in survival (Figure 2A). By Day 7, 32 percent of
larvae on the low Btk, and 14 percent of larvae on the high Btk larvae had survived
(Figure 2A). After removing larvae flom the treatments to clean foliage, one additional
low Btk larva died (larva was unable to complete pupation), decreasing larval survival on
the low Btk dose to 27 percent (Figure 2A). The remaining six larvae exposed to low Btk
and three exposed to high Btk survived to adulthood. In total, 24 out of 59 Karner blue
larvae used in this study were released as adults (13 females, 11 males).

The Smimov test indicated significant differences in larval survival between the
control and each of the two Btk doses (p < 0.001) as suggested by categorical analysis.
However, mortality did not differ significantly between the low and high doses at any
time during the bioassay (p > 0.05).

On the low Btk dose, survival of early instar Karner blue was significantly higher
than survival of late instars on Day 3 (chi-square = 4.70; p < 0.05) and Days 7 - 12 (chi-
square = 5.24; p < 0.025) Of the bioassay (Figure 3); however, differences in overall
survival were not significant (chi-square = 3.67; p < 0.1). On the high Btk dose, survival
of early instar Karner blue was not significantly lower than survival of late instars at Day
13 (chi-square = 3.47; p < 0.1) or at any time during the bioassay (p > 0.05) (Figure 3).
Overall survival of early instars was significantly higher on the low versus high Btk
treatment (chi-square = 6.47; p < 0.025), but survival of late instars on the two treatments
did not differ significantly (chi-square = 1.22; p < 0.5).

W: All gypsy moth larvae on untreated control foliage
survived to Day 8. Some mortality occurred after Day 8, and 80 percent of the larvae

survived to Day 13 (Figure 23). For the two Btk treatments, some mortality occurred on

42

Day 3, but we did not observe a steep drop in survival until Day 6 (Figure 2B). By Day
13, 33 percent of low Btk and 5 percent of high Btk larvae had survived (Figure 2B).

As with the Karner blue, results flom the Smimov analysis indicated that survival
of gypsy moth larvae on each Btk treatment differed significantly flom the control (p <
0.001), but differences between the low and high Btk dose were not significant (p > 0.05).

WSW: Although overall survival of Karner blue
did not differ significantly flom survival of gypsy moth on any of the Btk treatments, the
steep decrease in survival observed for Karner blue on Day 3 suggests that Karner blue
larvae were affected more quickly by Btk than gypsy moth (Figure 2). Smimov analysis
indicated that gypsy moth larvae had significantly higher survival than Karner blue on
Days 4 - 6 (p < 0.01) for the low Btk treatments, and on Days 3 - 5 (p < 0.05) for the high
Btk treatments (Figure 2).

WW: There appeared to be a Btk concentration-
dependent decrease in mean pupal weight of female and male Karner blue on control and
Btk treatments (Figure 4). However, the only statistically significant difference occurred
between male pupal weights for the control versus high Btk treatment (F = 6.84; df = 1, p
< 0.05); all other within-gender comparisons of mean pupal weight were not significant
(p > 0.05), possibly due to the small sample sizes. Female pupal weight for the high Btk

treatment could not be included in an AN OVA because there was only a single sample

(Figure 4).

43

Discussion

Conflicts between management of forest pests such as gypsy moth, that involve
Btk and nontarget endangered Lepidoptera are likely to increase. Management problems
regarding the use of Btk similar to those in Michigan exist in Wisconsin, where Karner
blue have been found in jack pine (Pinus banksiana Lambert) stands infested with jack
pine budworrn (Choristoneura pinus Freeman; Lepidoptera: Tortricidae) (Baker 1994).
In general, susceptibility of nontarget Lepidoptera to Btk will depend on three conditions,
the presence of vulnerable larval stages around the time of Btk application, larval
consumption of foliage treated with Btk, and toxicity and/or viability of Btk to larvae
when ingested (Dubois and Lewis 1981; Venables 1990), and will be greatly influenced
by the length of time that toxic effects of Btk persist post-spray (Johnson et a1. 1995).

Btk application for gypsy moth suppression is timed to occur when most gypsy
moth larvae have hatched, and are predominantly highly susceptible first and second
instars, and when 50 percent canopy development has occurred (Dubois 1991).
Typically, there is a 2 week “window” for effective Btk application (Smitley and Davis
1993). However, timing varies considerably flom year to year due to factors such as
weather, and rates of canopy and larval development (Dubois 1991; Reardon et a1. 1994).

Our phenological data over a three-year period indicated that Btk application for
gypsy moth suppression in Michigan could impact Karner blue. For example, in 1993,
late instar Karner blue of the spring generation were actively feeding during both the
predicted and actual Btk spray periods in southwestern Michigan, and would likely have
been at risk. In 1994 and 1995, we observed spring generation Karner blue adults, rather

than larvae, during Btk application in nearby counties that had gypsy moth suppression

44

programs. However, early-instar larvae of the summer generation would likely have been
at risk.

In 1994 and 1995, Karner blue adults of the spring generation were present in
Allegan State Game Area 3 - 11 days prior to nearby Btk applications, and were present in
the Huron-Manistee National Forest as much as 7 - 10 days prior to nearby Btk
applications (ca. 3 weeks prior to a second Btk application in one county in 1994). Spring
generation Karner blue can begin laying eggs within one week of the first emerged adults,
as confirmed by our 1995 observations. Egg hatch is estimated to occur within 1 week in
the field (Schweitzer 1989; Dirig 1994), and in Chapter 1, I found that Karner blue eggs
laid in the laboratory took between 2 - 6 days to hatch at 24°C (average of 4 days). Based
on this information, we predict that summer generation larvae could begin hatching
approximately 9 - 10 days after the first spring adults emerge. Thus, assuming Btk
persistence of 4 - 6 days, Karner blue first instars could have begun to hatch during the
time of or a few days after Btk application in 1994 and 1995, and would have been at risk.
In 1995, we conducted searches for early summer generation Karner blue larvae in
Allegan State Game Area; first-instar Karner blue are small (ca. 1.5 mm), well-
camouflaged and difficult to locate when newly hatched (Chapter 3). We found the first
early instar 14 days afier spring generation adults were first observed, which was only 3
days after the end of the Btk spray period in a nearby area.

Persistence of Btk crystals and spores in the field is a necessary consideration for
evaluating the phenological susceptibility of Karner blue. Btk is generally thought to
breakdown within 4 - 6 days of field application due to environmental factors such as

sunlight, temperature, vapor pressure deficit, and rain (Ignoffo et a1. 1974; Pinan et al.

45

1974; Leong et al. 1980; Beegle et al. 1981; Reardon et al. 1994), and spore viability is
impacted much more than crystal activity by UV light (Lilthy et al. 1982). However,
recent studies have found Btk to remain toxic for longer periods of time in the field.
Beckwith and Stelzer (1987) reported significant Btk mortality for western spruce
budworm 10 days after application. Johnson et al. (1995) found that Btk was toxic to first
instars Of P. glaucus for at least 30 days in the field after application, potentially due to
low levels of viable spores remaining of the leaf surface for long periods of time (Leong
et al. 1980). Further research has revealed that increased sensitivity, several hundred- to
several thousand-fold, to Btk doses occurs in four Papilio spp. as compared to gypsy
moth sensitivity (Johnson et al. 1995). Thus, persistence may be determined, in part, by a
particular species’ sensitivity to Btk. In considering our Karner blue phenology data flom
1994 and 1995, the longer the toxic persistence of Btk, the greater the number of early
instars possibly impacted. Field bioassays would be the most conclusive way of
determining persistence of Btk toxicity for Karner blue (Leong et al. 1980).

Toxicity of Btk to Lepidoptera depends upon the physiological makeup of each
species. Afier ingestion by lepidopteran larvae, Btk crystals become toxic if conditions
within the larval gut solubilize crystals into specific 6-endotoxins, which then bind to
receptors on the gut wall (Reardon et al. 1994). The binding of 6-endotoxins causes gut
wall cells to swell and lyse, creating perforations in the gut lining, leading to mortality by
bacterial septicemia (Gill et al. 1992; Bauer 1995). Factors in the gut that determine
Btk’s insecticidal activity include the presence of Btk spores, appropriate gut pH,

digestive enzymes, receptors on the gut wall, and other factors that facilitate active pore

46

formation (Cherwonogrodzky 1980; van Frankenhuyzen et al. 1991; Bauer 1995).
Though the exact role of spores in the synergism of crystal toxicity is not known, their
presence in Btk formulations can have a significant influence on toxicity for some
lepidopteran species (Moar et al. 1990; Johnson et al. 1995). Other bacteria present as
opportunists could also significantly affect the observed mortality (Dubois and Dean
1995)

We found that Btk was toxic to Karner blue when larvae were fed treated lupine
foliage. Karner blue larvae did not differ flom gypsy moth larvae in their overall percent
survival. However, Karner blue mortality was significantly higher than gypsy moth
mortality in the first 3 - 6 days of the bioassay, suggesting that Karner blue may be more
sensitive to Btk than gypsy moth.

Early (first and second) and late (third and fourth) instar Karner blue were equally
susceptible. Generally for Lepidoptera, including gypsy moth, early instars are much
more susceptible than later instars to Btk (Peacock and Schweitzer 1992; Reardon et al.
1994; Wagner and Miller 1995). However, many exceptions have been reported (Wagner
and Miller 1995). Btk caused high mortality for late (fourth and fifth) instars of the
cinnabar moth while early instars appeared to be impervious (James et al. 1993). Peacock
and Schweitzer (1992) and Peacock et al. (1993) found substantial variation in early-
versus late-instar susceptibility to Btk for related species within the families Geometridae
and Noctuidae. As with Karner blue, early and late (fourth) instars of two species of
swallowtails and the promethea moth were reported to be susceptible to Btk (Johnson et
a1. 1995). Since all instars of Karner blue were negatively affected by the Btk treatments,

the late instar larvae observed in the field during the 1993 gypsy moth suppression

47

activities would have been at risk, along with the earlier instars which were most likely
present during or soon after Btk application in 1994 and 1995.

Although there was a trend for reduced pupal weight, and possibly lower
fecundity (Honek 1993), when Karner blue were reared on Btk-treated foliage, mean
pupal weights differed significantly only between control and high Btk treatments for
male Karner blue. Since very few females and males survived the Btk treatments to
provide comparison, these data should be interpreted cautiously. However, potentially
sublethal effects of Btk have only been previously considered for beneficial insect
predators and parasitoids (Croft 1990). Possible sublethal or multi-generational impacts
of Btk on nontarget Lepidoptera need further investigation.

Data on the individual roles of each Btk 5-endotoxin and Btk spores in Karner blue
mortality could be usefirl in the future production of a Btk formulation which would
impact gypsy moth, but have no effect on Karner blue. Van Frankenhuyzen et al. (1991)
found that, of the three CryIA toxins in Btk, CryIA(c) toxin caused little gypsy moth
mortality compared to CryIA(a) and CryIA(b). Dubois and Dean (1995) also showed that
CryIA(a) was more toxic to gypsy moth than CryIA(c).

We conclude that Karner blue is highly physiologically susceptible to Btk, and is
phenologically susceptible to the timing of Btk application for gypsy moth suppression,
although the extent of phenological overlap and the larval generation (spring vs. summer)
at risk may vary flom year to year. The actual amount of risk posed by gypsy moth
suppression to the survival of a particular Karner blue population must take into

consideration the length of time that Btk remains toxic and/or viable to Karner blue larvae

48

after field application, as well as the size and level of isolation of each population. Small
or isolated Karner blue populations would face more of a risk than populations which
have large numbers of individuals or are in close proximity to other Karner blue areas to
allow for recolonization (Schweitzer 1994).

Information regarding the susceptibility of nontarget Lepidoptera to Btk, including
physiological susceptibility and the temporal overlap of larval stages with the application
of Btk or the period of its toxic persistence (which appears to be species-specific; Johnson
et al. 1995), must be considered in management plans for gypsy moth. However,
nontarget impacts of gypsy moth defoliation, in the absence of suppression, such as a
potential increase in parasitoids and predators, altered microclirnate or a decrease in the
availability or quality of host plants (Liebhold and Elkinton 1989; Sample et al. 1993;
Johnson et al. 1995; Wagner and Miller 1995) must also be considered. The potential for
development of modified Btk products that have higher specificity for gypsy moth, so as
to reduce the physiological impact on select nontarget lepidopteran species, should be

explored.

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51

Table 2.2. Actual timing of Btk applications for gypsy moth suppression in Michigan
counties near Karner blue study sites, 1993 - 1995.

 

 

 

Btk Applicationl
County Year Date Degree days
(base 50° F)3
Muskegon 1994 May 27 250
1995 May 30 - June 2 280 - 312
Newaygo 1993 May 28 300
1994 June 2 - 3 340 - 360
June 152 525
1995 June 5 358
Oceana 1993 May 26 284
1994 May 31 - June 2 320 - 340
1995 May 30 - 3l 275 - 282
Ottawa 1993 May 17 280
1994 May 23 320
1995 May 25 - June 2 272 - 370

 

1 Aerial application of Btk as conducted in the Michigan Voluntary Cooperative Gypsy
Moth Suppression Program which is administered by the Michigan Department of

Agriculture.
2 Date of second Btk application.

3 Degree days (base 50° F) based upon degree day accumulation since March 1st,
published in the Michigan State University Landscape Crop Advisory Team Alert
Newsletter. Degree days calculated using the Baskerville-Emin method (Baskerville and

Emin 1969).

52

 

 

-., Figure 2.1. Michigan counties where Karner blue butterfly study sites were located
(Allegan, Oceana), where Btk was applied at least once in 1993 - 1995 for gypsy moth
suppression (Muskegon, Newaygo, Oceana, Ottawa) and where the Btk laboratory
bioassay was conducted (Ingham).

 

53

 

 

 

 

 

Phenological monitoring activities
of Karner blue butterfly and gypsy
moth populations

Nearby counties where areas
were treated with Btk for
gypsy moth suppression

Location of Btk bioassay

 

 

 

 

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Oceana

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Ottawa

 

 

 

 

 

 

 

 

 

 

 

 

 

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54

Figure 2.2. Larval survival of (A) Karner blue butterfly and (B) gypsy moth over 13 days
on control (untreated) foliage, on foliage treated with Btk (Bacillus thuringiensis var.
kurstaki) at a low dosage (3O - 37 BIU/ha), or on foliage treated at a high dosage (9O
BIU/ha). On Day 7 (indicated by arrow), all surviving larvae were placed on untreated
foliage.

 

55

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Figure 2.3. Survival over 13 days of early (lst, 2nd; E) and late (3rd, 4th; L) instar
Karner blue reared on lupine foliage treated with low (30 - 37 BIU/acre) or high (90
BTU/acre) dosages of Btk. On Day 7 (indicated by arrow), all surviving larvae were
placed on untreated lupine foliage. No further mortality occurred after day 13.

 

 

 

57

 

 

I Female
Cl Male

 

 

 

 

Mean pupal weight (mg)
.k
C

 

 

 

 

 

 

 

 

 

 

Control Low Btk High Btk

Bioassay treatments

Figure 2.4. Mean pupal weight (mg) (+ 1 SE) 2 days afier pupation of surviving
female and male Karner blue larvae used in the Btk bioassay. There were 8, 4, and 1
female survivors, and 7, 2, and 2 male survivors on control, low Btk (30 - 37 BIU/ha)
and high Btk (9O BIU/ha) treatments, respectively. For within-gender comparisons,
bars with the same letters were not significantly different by AN OVA at p < 0.05
(female pupal weight for the high Btk treatment was not included in AN OVA).

 

 

 

CHAPTER 3

The Endangered Karner Blue Butterfly (Lepidoptera: Lycaenidae) in Michigan
Oak Savanna: Associations among Butterfly Abundance and Habitat Variables

Abstract

The Karner blue butterfly (Lycaeides melissa samuelis Nabokov) is an endangered
species occupying oak savanna and pine barren habitats. Local habitat requirements of
the butterfly appear to be adequate seasonal supply of wild lupine (Lupinus perennis L.),
the sole larval foodplant, and adult nectar sources, microclirnatic variation provided by
shading of woody plants, and ant-tending of Karner blue larvae. An integrated study was
conducted in oak savanna sites in southern Michigan to investigate habitat suitability for
the butterfly with respect to those habitat requirements. In 1993 and 1994, six and seven
Karner blue-occupied sites in Allegan State Game Area (Allegan Co), respectively, were
surveyed during the spring and summer Karner blue flight periods to assess relative
population sizes. Nectaring was also recorded. Indirect estimates of summer larval
abundance were made through feeding damage surveys. Select habitat variables, e.g.,
wild lupine density and frequency, density and frequency of flowers during spring and
summer Karner blue flight periods, and percentage canopy cover and frequency, were
quantified for each site. Larval surveys were conducted to assess the quality of lupine
used by larvae for feeding, to observe ant-tending, and to indirectly estimate female

oviposition on lupine in different shade conditions. The relationships among Karner blue

58

 

 

 

59

abundance and several of the habitat variables were analyzed. There were significant ~~

positive associations between butterfly abundance and lupine density (r > 0.8) and

.4. “51.-..” J1“ . .lvN a/u..s._,
- ‘Wmfi

frequency (r > 0.7) in both years, suggesting that lupine plays a significant role in Karner
blue population dynamics. Karner blue abundance was not significantly correlated with
flower density and diversity measures, or percentage canopy cover and frequency.
Summer Karner blue abundance was highly correlated with percentages and frequencies
of larval feeding damage (r > 0.9), suggesting that feeding damage may be used to
estimate adult population size. Some summer flower species that were favored one year
for nectaring were not available the other year, while some flower species that were used
less for nectaring were available consistently in both years. It may be important to have a
diversity of nectar sources in the Karner blue landscape due to these random phenological
differences. Summer Karner blue larvae fed on lupine leaves that appeared to be less
senesced than the overall clump. Karner blue larvae were found in both partial shade and
in open areas, which suggests that females use both shade conditions equally for
oviposition. Ant-tending was observed for almost 100 percent of the larvae found in
1993, and for 82 - 89 percent of the larvae in 1994. Thirteen species of tending ants from
three subfamilies were identified. The dominant tending species was Formica

obscuripes.

Introduction
The endangered Karner blue butterfly (Lycaeides melissa samuelis Nabokov;
Lepidoptera: Lycaenidae) is restricted to early successional, xeric oak savanna and pine

barren habitats of the central and northeastern United States (Ewert and Ballard 1990).

 

 

 

60

Addition of the Karner blue to the federal endangered species list in December 1992 was
the result of rangewide population declines (U SF WS 1992). Like other invertebrate
species in the United States and elsewhere, loss of habitat associated with human
settlement has been the major cause of the butterfly’s decline (New 1993). The primary
means of preserving this species is habitat conservation (Pyle 1976; Pyle et al. 1981), to
maintain remaining savannas and barrens as well as restore degraded areas. The Karner
blue has become a symbol for conservation of the threatened savanna and barren
landscapes and the other unique species they support (Ewert and Ballard 1990), as well as
for invertebrate conservation. Like all endangered species, conservation of the Karner
blue is mandated by the Endangered Species Act of 1973, which requires designation and
conservation of critical habitat (Pyle et al. 1981; USFWS 1992). Understanding of
Karner blue ecology and the critical habitat factors required by the butterfly is needed to
form sound management plans for effective species and habitat conservation.

The Karner blue has declined an estimated 99 percent over its historical range
from eastern Minnesota to New Hampshire in the past 100 years, with most of the decline
occurring in the last 10 to 20 years (Schweitzer 1989; USFWS 1992). The species is
extirpated in Massachusetts, Ohio, Ontario, and Pennsylvania (and most likely Illinois),
and occurs as a few small localized populations in Indiana, Minnesota and New
Hampshire (U SFWS 1992; Haack 1993; Baker 1994). Michigan, Wisconsin and New
York have the largest populations, and the best opportunities for species conservation

(Baker 1994).

 

61

Karner blue populations occur on sandy post-glacial lake and outwash plains
which support wild lupine (Lupinus perennis L.), the sole foodplant of larvae, along with
other xerophytic, fire-successional savanna or barren vegetation (Bleser 1992). The
butterfly’s range approximates the northern limits of its larval foodplant (U SFWS 1992).
The savanna and barren landscapes are characterized by open canopy with an understory
of grasses and other herbaceous plant species, historically maintained by fire (N uzzo
1985; Givnish et al. 1988). In eastern states, the Karner blue is closely associated with
grassy openings of fire-climax pine/oak barrens (Dirig 1994). In the Midwest, the
butterfly’s habitat represents the transition between native western prairies and eastern
deciduous forests, taking the form of oak savanna and oak/pine barren communities
(Shuey 1994).

The vast, historic savanna and barren landscapes have been drastically reduced
and fragmented since European settlement, from activities such as agriculture,
commercial and residential development, off-road vehicle use, timber production, and fire
suppression (USFWS 1992; Haack 1993; Shuey 1994). Of the 11 - 13 million hectares of
oak savanna that once covered the Midwest, only two percent remains (Nuzzo 1985).
The Albany Pine Bush in New York, at one time famous for its Karner blue population
numbering 100,000, was reduced from 25,000 acres to 2,500 acres by the mid-1980’s
(Givnish et al. 1988). Currently, most populations of Karner blue in New York number
fewer than one hundred butterflies (Sommers and Nye 1994). Disturbances, such as fire,
historically perpetuated lupine by preventing encroachment of the overstory and woody
vegetation (Givnish et al. 1988; Shuey 1994). Current fire-suppression practices in

remnant savanna and barren habitats often result in the exclusion of lupine and other

 

62

herbaceous plants necessary to the Karner blue, by allowing fire-intolerant species to
shade in the openings (Lane 1994a; Wilsmann 1994).

Destruction, modification and fiagmentation of Karner blue habitat as a result of
development and fire suppression has impacted butterfly populations at both the
landscape and local, or patch, level. At the broader scale, the Karner blue was thought to
exist as metapopulations, or dynamic clusters of populations (Givnish et al. 198 8).
Individuals of these populations could disperse and shift among a patchy landscape, to
colonize new areas created by fire, recolonize areas where populations had gone extinct,
and thus maintain gene flow (Givnish et al. 1988). Currently, the majority of extant
Karner blue populations are small and separated by unsuitable intervening habitat or by
distances which prevent successful dispersal, disrupting the metapopulation regime
(Shuey 1994). On a local scale, extreme disturbance and fire suppression have reduced
the suitability of habitat patches for survival and reproduction of butterfly populations
(Givnish et al. 1988; Lane 1994a; Shuey 1994).

The Karner blue overwinters in the egg stage and has two generations per year
(Schweitzer 1989). Larvae of both spring and summer generations feed solely on wild
lupine, and are tended by various species of ants, which feed on the sugary, protein-rich
fluid emitted by specialized larval glands, and provide protection for the larvae in return
(Dirig 1994; Schweitzer 1989; Savignano 1990b). Spring generation larvae hatch in late
April and feed for approximately three weeks (Bleser 1992; Lane 1992). The spring adult
flight period is from late May to early June, and adults live for 5 to 7 days (Schweitzer
1989). Eggs are laid on or near lupine plants (Dirig 1994; Schweitzer 1989). Summer

generation adult flight occurs mid-July to mid-August, and butterfly numbers are usually

 

63
higher than in spring flight (Bleser 1992; Lane 1992). Eggs that will overwinter are laid

on vegetation near senescing or senesced lupine. Adults of both generations require
nectar, and utilize a variety of native and exotic flowering plants (Packer 1987; Lawrence
and Cook 1989; Schweitzer 1989; Haack 1993). Moderate levels of interspersed canopy
cover in the habitat appear to provide butterfly adults and larvae with shelter from
daytime temperatures, as well as providing microclirnate heterogeneity (Leach 1992;
Lane 1994). Like other Lycaenidae, the Karner blue has low vagility, and butterflies
rarely disperse more than 1 km (Fried 1987; Lawrence and Cook 1989; Cushman and
Murphy 1993; Bidwell 1994).

Karner blue management has concentrated on improving habitat quality to stabilize
local populations, with the eventual goal of restoring metapopulation dynamics in the
landscape (Lane 1994b; Shuey 1994). Successful conservation of individual Karner blue
populations requires that key, local habitat needs are met (Packer 1987; Bleser 1992).
Past studies have suggested that the availability of lupine, nectar sources, microclirnate
heterogeneity provided by minimal shading and tending ants are critical components in
the Karner blue habitat (Packer 1987; Savignano 1987, 1990a,b; Lawrence and Cook
1989; Bleser 1992; Lane 1992, 1994a; Leach 1992). However, the associations, relative
importance and interactions of Karner blue with these aspects of its habitat are not fully
understood, and require further examination in an integrated autecological study. Our
primary objective was to investigate associations among Karner blue abundance and
several components of the butterfly’s habitat, primarily lupine density and frequency,
flowering plant density, and percentage canopy cover and frequency, in an integrated

study. We also investigated the extent of ant-tending of Karner blue larvae, the influence

64

of shading on female oviposition and use of lupine foliage by summer larvae. Our goal
was to firrther elucidate aspects of the butterfly’s ecology and habitat suitability to guide

future research and management.

Methods & Materials

W2 This study was conducted during the spring and summer of 1993 and
1994 in Allegan State Game Area (Allegan County) in southwest Michigan (Figure 3.1).
The Game Area is located on sandy deposits, from the Pleistocene glaciers, comprising
outwash plains, lake plains and moraines (USDA 1987). Presettlement vegetation
consisted of eastern white pine (Pinus strobus L.) forests, oak savannas and prairies
which were maintained by fire (W ilsmann 1994). With pioneer settlement, the Game
Area was altered by logging, fire suppression, and a brief period of cultivation practices
(Lawerence and Cook 1989). Currently, ca. 7 percent of Allegan State Game Area is oak
savanna and interspersed oak openings (Wilsmann 1994).

Six Karner blue-occupied sites were studied in 1993, and seven sites were studied
in 1994 (the six sites fi'om 1993 plus one additional site, the ‘Park’). F our sites
represented remnant oak savanna habitat (Table 3.1), and were most likely farmed for a
brief period in the early 1900’s (John Lerg, Allegan State Game Area, pers. comm).
These sites were located within a 2.6-km2 area in a northern region of the Game Area.
Sites were separated fiom one another by ca. 0.6 - 1.8 km of interspersed woodland and
dirt roads. The other three sites were narrow openings created within the last 20 t0 25
years for game management (Table 3.1), and were located within a 1.3-km2 area, ca. 3.6

km south of the remnant oak savanna sites. The ‘Jay’ and ‘Pipe’ sites were separated by

 

65
less than 0.2 km, and were both ca. 0.6 km from the ‘Horseshoe’. The three created sites

and the ‘Park’ site were surrounded completely by forested habitat, while the ‘48N89’,
‘Marsh’, and ‘Square’ sites were bordered by forest on three sides and a road on one side.
All Karner blue study sites were located on well-drained, fine sand soils of the Oakville
association, with 0 - 6 % slope (USDA 1987).

We selected sites wrth a‘range” of ' butterfly population sizes, based upon

..~w»—.—.—_—P-v (- I

A, preliminary surveys by the Michigan Natural Features Inventory. We intended to include
unoccupied sites in the study; however, all‘sites selected to represent unoccupied habitat
were later found to be occupied by Karner blue.

Two Karner blue sites in the Huron-Manistee National Forest in southcentral

Oceana County (Figure 3.1) were used for collection of tending ant species of Karner

blue larvae (described below) in addition to the Game Area sites.

WW5: Karner blue adult abundance in each

study site was estimated from timed-area transect counts of adults that were conducted
weekly during the 1993 summer flight period, and the 1994 spring and summer flight
periods. Methods used to estimate population sizes were analogous to the those

developed by Pollard (1977) and Thomas (1983). However, sampling Egon (e. g. the

~ “a“.h“ .W -~4-——r'—“-‘

amount of time spent per survey per site) was standardized based on the area of each site.

m-\-v

In each site, we established a transect route whichtraversed tlrefientiremsite. The
three created openings were narrow, no more than ca. 30 meters wide in any one spot;
therefore, the transect route for each created site followed a direct line from one end of

the site to the other. In each of the four remnant oak savanna sites, we partitioned the

entire site into ca. 30-meter wide strips, and then established a transect route which went

66

through the strips, alternating direction from one strip to the next. From preliminary
surveys, we determined that it took ca. 20 minutes to walk, at a moderate pace, a transect
route which traversed a 1 hectare opening. Based upon this and the size of the study
sites, the amount of time spent for each survey was 60 minutes in the ‘Marsh’, 50 minutes
in ‘48N89’ and ‘Park’, 30 minutes in the ‘Square’, and 20 minutes in the ‘Horseshoe’,
‘Jay’ and ‘Pipe’.

Each transect survey was conducted by two people, walking at the same pace
within adjacent halves of the 30 m wide strips. Ten-meter buffers were maintained

between surveyors. Karner blue adults seen within 3 to 4 meters on either side of the

.m-’

‘ ,HMW
M’fl’ _
. -.._..~

transect were recorded. Data on male/female, nectaring and wing wear were also

--~r_,.

 

recorded. Surveys were conducted between 10 am to 1 pm and 2 to 6 pm, and were not
conducted if the temperature was below 20°C or if it was raining.
Numbers of adults counted during each survey were standardized across sites by

con"...

Vconverting SPHEumbiriQf adults counted per” person hour. The highest standardized count
obtained in each site was used as the estimate of Karner blue abundance for that site I
during that specific flight period.

During the 1993 summer flight period, sites were surveyed twice each week when

weather permitted, with surveys .2, _d_ays_apan. During the 1994 spring flight period, sites

were surveyed once every 4 to 7 days. For both flight periods, the order ighchsites

. "WMMME‘rH‘fl‘ -ywh _'

' ' ,

Mere survengglqctfid randomly each survey date. During the 1994 summer flight
period, sites were surveyed twice every 6 to 7 days, with both surveys in each site
- occurring on the same day, once in the morning and once in the aftemoon. For each 1994

summer survey date, the order in which sites were surveyed in the morning was selected

67

randomly, and then reversed for the afiemoon surveys. The highest of the two daily
counts was used to determine the adult abundance estimate for each survey date in each
site.

In 1994, we documented the beginning and end of the spring and summer flight
periods in each site by initiating butterfly surveys 1 to 2 weeks prior to estimated adult
Vetfiérgence to get {319. counts, and continuing surveys through the flight period until zero

counts were again owed.

.—-—c — WWMM".

WWW: From 28 to 30 June 1994,
abundances of summer generation Karner blue larvae were indirectly estimated through
quadrat (1 -m2) surveys for feeding damage on lupine. In each study site, 20 lupine
clumps were chosen by randomly selecting points, and walking a randomly chosen
direction until the first lupine plants were encountered. A l-m2 quadrat was then placed
over the lupine clumps, and the numbers of lupine stems in the quadrat and the numbers
of stems with window feeding damage, made by summer generation Karner blue larvae,
were counted. The average percentage of lupine stems (per m2) with feeding damage and
feeding damage frequency (proportion of quadrats with damage) were calculated for each
site.

WW: From 2 to 4 June 1993 and 3 to
8 June 1994, density of lupine stems was estimated in each study. site using a transect -
quadrat method (Bonham 1989) (surveys done in conjunction with spring flowering plant
and percentage canopy cover surveys, below). The number of transects per site was
based upon site area. We randomly located 25-m transects throughout each site, at a

density of one transect per 1000 m2 in 1993 and one transect per 850 m2 in 1994. Lupine

68

stems were counted in six l-m2 quadrats placed at regular intervals along each transect
(Bonham 1989). For each site, the lupine density estimate was calculated as the average
number of lupine stems per m2 per transect, and lupine frequency was calculated as the
proportion of transects with lupine stems.

From 13 May to 10 June 1994, flowering phenology of lupine was monitored on 6
different days through quadrat(1-m2) surveys. In each study site, six lupine clumps were
randomly chosen using the same method as for larval feeding damage surveys (above).
Only lupine clumps that occupied 1/4 or more of the quadrat were sampled. The numbers
of lupine stems and flower spikes in each quadrat were counted. The stage of flowering

was recorded for each flower spike using the following scale:

0 = no flowers on spike open
< 1/4 = flowers beginning to expand and show color
1/4 = 1/4 of flowers on spike open
1/2 = 1/2 of flowers on spike open

3/4 = 3/4 of flowers on spike open
1 = all flowers on spike open
Seed = all flowers done, seed pods present
Bare = bare flower spike, no flowers or seed pods present

Average percentages of lupine stems (per m2) with flower spikes and flower
spikes at each stage of bloom were calculated.

To examine the quality of lupine used by summer generation larvae, we surveyed
lupine clumps in late June 1994, 1 week afier the first senescent lupine stems were
observed. Twenty lupine clumps were chosen in each site using the same quadrat method
as for larval feeding damage surveys (above). The l-m2 quadrat was then placed over the

lupine plants, and an overall estimate of senescence for all lupine foliage in the quadrat

was made. A visual senescence scale of 1 to 5 was used to rate foliage, with 1

 

 

69

representing foliage with no apparent signs of senescence, 2 to 4 representing foliage with
increasing amounts of discolored and necrotic areas, and 5 representing complete
senescence. All larvae observed in the quadrat were measured and a senescence rating
was made for the leaves occupied by larvae.

: In 1993

 

and 1994, flowering plant density was surveyed during peak spring and summer Karner
blue flight periods (Table 3.2), using the same transect - quadrat design used for lupine
surveys (Bonham 1989). Since butterfly surveys were not conducted during the 1993
spring flight period, the peak flight period was estimated from casual observations of
butterfly numbers.

In each quadrat, we counted the numbers of stems of different plant species in
flower at the time of the survey. Stems that were done flowering or had only unopened
buds were not counted. As with lupine density, the overall mean number of flowering
stems per m2 per transect was calculated for each site, along with overall flower
frequency (proportion of transects with flowers; all species combined). In addition,
averages of each flower species were calculated and used to calculate diversity and
dominance indices (below) for each site. Nectaring by Karner blue adults was recorded
during the butterfly surveys.

Shannon’s diversity index (H’ ), and Simpson’s dominance index (expressed as the

 

reciprocal, 1/D) (Margurran 1988) were calculated for spring and summer flowering
plants 1n each site in 1993 and 1994. To calculate Simpson’s index, flower density
estimates were converted to number of stems per 100 m2 to avoid negative values. One

MW

of the assumptions of the Shannon diversity index, that all species from a community

70

were included in the sample (Margurran 1988), was not met; some flower species were
not encountered in the transect surveys.

WW9: Average percentage of canopy cover in each site was

estimated in late June 1994 after leaves were fully expanded using a transect-intercept
method (Bonham 1989). Transects were located randomly, at a density of one 25:m
transect per 850 m2. The number of meters along each transect with direct canopy cover
was recorded, along with the species of each tree intersecting the transect. Only trees 1.5
m in height or taller were included. The amount of cover along each transect was
expressed as a percentage and the overall mean percentage of canopy cover per site was
determined. Overall canopy cover frequency (proportion of transects with canopy cover;
all species combined) was calculated.

To indirectly investigate oviposition by Karner blue females on lupine plants
growing in different shade conditions, surveys for Karner blue larvae were conducted
weekly in each of the sites prior to the summer adult flight period in 1993, and the spring
and summer flight periods in 1994. Larval searches were done from 10 am to 6 pm, and
varied in duration fi'om 1 to 3 hours per site, based upon lupine density. For each survey,
randomly chosen lupine clumps were examined for evidence of larval feeding. When
feeding damage was found, the lupine foliage was searched thoroughly for larvae.
Growing conditions of lupine plants occupied by larvae were estimated as either open
(i.e., never shaded) or partially shaded (i.e., shaded for some part of the day by tree

trunks, foliage, etc.), and plants were flagged for relocation. During subsequent larval

searches, new plants were searched for additional larvae.

 

71

Antfiendinggflamae: In summer 1993 and spring and summer 1994, data on ant-
tending of Karner blue larvae were collected while conducting larval surveys (described
above) in Allegan State Game Area. For each larva found, presence or absence of
tending ants and larval length were recorded, and ant specimens were collected for
identification. Some ants were also collected during preliminary surveys in spring 1993
in Allegan State Game Area. In addition, tending ant specimens were collected in two
Karner blue sites in the Huron-Manistee National Forest in spring 1993 and spring and
summer 1994.

MW: All analyses were conducted with SYSTAT, Version 5.0

M “. ...

(Wilkinson 1990), at the p < 0.05 level of significance. In each study year, differences
among sites in lupine density, spring and summer flower densities, percentage larval
feeding damage, percentage flower spikes, percentage of spikes at each stage of bloom,

and percentage canopy cover were evaluated usng one-way analysis of variance

(AN OVA) and Tukey’s HSD multiple comparison. Weekly estimates of the percentage

5....

 

lupine stems with flowerspikes were also compared among sites by repeated measures.

Estimates of lupine density, flowering plant density and percentage canopy cover
were log-transformed, and percentage feeding damage estimates were arcsine-
transformed, before analysis (Little and Hills 1978). After transformation, the normality
assumption of homogeneous variances (Bartlett test of homogeneity of variances) was not
met for lupine density estimates (both years) and marginally for percentage canopy cover.
These data were analyzed with the nonparametric Kruskal-Wallis test in addition to

AN OVA.

72

Associations among Karner blue abundance, lupine density and frequency, spring
and summer flowering plant densities, percentage canopy cover, and diversity indices
(H’, l/D) were evaluated using Pearson’s correlation analysis. Only summer Karner blue
abundance estimates were available for 1993 correlations. Separate 1994 correlation
analyses were conducted using spring and summer Karner blue abundance estimates.
Also, associations between 1994 summer adult abundance and percentage feeding
damage and feeding damage frequency were analyzed. The critical value of significance
(for a one-tailed test) of correlation coefficients (r) was 0.729 (v = 4, p < 0.05) for 1993
comparisons among sites, and was 0.669 (v = 5, p < 0.05) for 1994 site comparisons (Zar
1974)

For each site in 1994, analyses were conducted to investigate the associations
between transect estimates of percentage canopy cover and corresponding transect

estimates of lupine density and spring flower density.

Results

WWW: In 1993, the Karner blue spring flight period
occurred from 25 May to 27 June, and the summer flight occurred from 8 July to 10
August, based upon first and last observations of adult butterflies in study sites and other
Karner blue-occupied areas in the Allegan State Game Area. In 1994, the spring flight
period occurred from 19 May to 18 June, and the summer flight period occurred from 27

June to 12 August.

73

Butterfly surveys were conducted in the study sites from 7 July to 3 August
(Julian Date (JD) 188 - 215) in summer 1993 (Figure 3.2), from 16 May to 22 June (JD
136 - 173) in spring 1994 (Figure 3.3), and fi'om 24 June to 17 August (JD 175 - 229) in
summer 1994 (Figure 3.3). For all flight periods, the first butterfly counts were low and
dominated initially by male butterflies. Afier counts peaked (Figure 3.2, 3.3), late counts
were dominated by female butterflies.

During the 1993 summer flight, Karner blue numbers on most sites peaked at
approximately the same time, except for the ‘Square’, which peaked ca. 5 days earlier
(Figure 3.2). In spring 1994, Karner blue abundance peaked at the same time in late May
for the ‘48N89’, ‘Marsh’, ‘Park’, and ‘Square’ sites, and 1 week later for the ‘Horseshoe’,
‘Jay’, and ‘Pipe’ sites (Figure 3.2). In summer 1994, the ‘Horseshoe’, ‘Jay’, ‘Marsh’,and
‘Square’ sites peaked at the same time mid-July, and the ‘48N89’, ‘Park’, ‘Pipe’ sites
peaked 6 days later (Figure 3.2). Overall peak summer counts were obtained within a
similar range of calendar dates and degree days in 1993 as in 1994 (Table 3.2); however,
calendar dates of peak counts in individual sites varied from one year to the next (Figure
3.4).

The ‘Jay’ site consistently had the greatest adult Karner blue abundance (adults
per hour), followed by the ‘Pipe’, ‘Square’ and ‘Horseshoe’ sites (Table 3.3). The
‘Marsh’ site had the lowest abundance in 1993, and the ‘Park’ site (only used in 1994)
had the lowest abundance in 1994 (Table 3.3). Summer abundance estimates in each site

were higher in 1994 than in 1993. Counts for summer flight were consistently higher

 

74

than counts for spring flight in all sites in 1994 (Figure 3.3). Peak summer abundance
was approximately two to three times greater than peak spring abundance (Table 3.3).

Indireetestimatemfsummenlhmenhluflarxalahundancez Percentages of lupine
stems with summer larval feeding damage differed significantly among sites (F = 6.487;
df = 6, p < 0.001) (Table 3.4). The ‘Pipe’ and ‘Jay’ sites had the highest percentages of
feeding damage, as well as the highest feeding damage frequencies (Table 3.4). The
‘Horseshoe’ site had the third highest percentage and frequency of feeding damage,
followed by the ‘Square’ (Table 3.4).

W The ‘JaY’, ‘Pipe’ and ‘Square’
sites consistently had the highest lupine densities, and the ‘Horseshoe’ had the lowest
densities in both years (Table 3.3). Lupine density estimates in the ‘48N89’,
‘Horseshoe’, ‘Marsh’ and ‘Pipe’ sites were similar from year to year, but varied
somewhat in the ‘Jay’ and ‘Square’ (Table 3.3). Lupine density differed significantly
among sites in 1993 (F = 17.698; df= 5, p < 0.001) and 1994 (F = 18.606; df= 6, p <
0.001). Based upon multiple comparison tests, sites could be grouped into one of two
statistically differing lupine density levels, high lupine density (‘Jay’, ‘Pipe’ and
‘Square’) or low lupine density (‘48N89’, ‘Horseshoe’, ‘Marsh’, and ‘Park’) (Table 3.3).
Results of the non-parametric Kruskal-Wallis test were consistent with AN OVA results.
Lupine density estimates differed significantly in 1993 (test statistic = 46.4; df = 5, p <

0.001) and 1994 (test statistic = 60.2; df = 6, p < 0.001).

75

As with lupine density, lupine frequencies were highest in the ‘Jay’, ‘Pipe’ and
‘Square’ sites, and lowest in the ‘Horseshoe’ in both years (Table 3.5). For each site,
frequency estimates were similar in both years (Table 3.5).

In general, sites did not differ widely in lupine flowering phenology (Table 3.6).
On 13 May 1994, the majority of lupine flower spikes in study sites had not begun to
open; 4 days later, all sites had a small percentage of flower spikes that were showing
some color (Table 3.6). On 20 May, sites did not differ significantly in percentage bloom
for any stage (Table 3.6). By 27 May, all sites had a percentage of flower spikes at each
stage of flowering from 0 to 1, full bloom (Table 3.6). Peak lupine bloom (the greatest
percentage of spikes with all flowers open) occurred on 1 June; however, several sites had
high percentages of spikes that had not begun to bloom (Table 3.6). The ‘Horseshoe’ site
had consistently high percentages of unopened flower spikes from 27 May to 1 June,
while percentages of unopened flower spikes rose during that period for the ‘Square’ and
‘Marsh’ sites (Table 3.6). By 10 June, the majority of lupine spikes were done flowering
and had seed pods or were bare (Table 3.6). Repeated measures analysis of weekly
percentages of lupine stems with flower spikes revealed that the ‘48N89’ and ‘Park’ sites
had significantly greater percentages of flowering lupine stems per area than all other
sites (F > 15.67; df = 1, p < 0.003), but the other sites did not differ significantly from
each other (F < 3.10; df= 1, p < 0.5).

On 28 June 1994, 46 summer generation Karner blue larvae were found in study
sites during quadrat surveys of lupine senescence. Seven larvae were 0.5 cm or smaller,
20 larvae were 0.6 to 1 cm long, and 19 larvae were 1 to 1.6 cm long. Of the 46 quadrats

with larvae, the numbers of quadrats with each overall-senescence rating were: rating 1 =

76

3 quadrats; rating 2 = 24 quadrats; rating 3 = 15 quadrats; and rating 4 = 4 quadrats, with
7 quadrats also containing some completely senesced lupine stems. Of the 46 leaves
occupied by larvae, rating 1 = 27 larvae; rating 2 = 16 larvae; and rating 3 = 3 larvae.
Larvae tended to occupy leaves appearing less senesced than overall lupine in the clumps.

W: In spring and summer 1993 and 1994, some plant species were
observed flowering in the sites but were not represented in the transect surveys due to
extremely low densities (T able A61, A62). Transect surveys to determine spring and
summer flowering plant densities were conducted within similar ranges of degree days
from 1993 to 1994 (T able 3.2).

Overall densities of spring flowers ranged more widely among sites in 1994 than
in 1993 (Table 3.3). Spring flower densities differed significantly among sites in 1993 (F
= 3.819; df= 5, p < 0.004) and 1994 (F = 14.846; df= 6, p < 0.001). The dominant
spring flower species in 1993 and 1994 surveys in all sites were wild lupine (Lupinus
perennis), mouse-ear hawkweed (Hieracium pillosella), and sheep sorrel (Rumex
acetosella), in addition to dewberry (Rubus sp.) in 1994 (Table 3.7, 3.8). These flower
species also had consistently high frequencies among sites in the above years, especially
for mouse-ear hawkweed (Table 3.9). In both years, ‘48N89’ and ‘Square’ sites had the
highest overall spring flower densities (Table 3.3), mostly because of high densities of
this hawkweed species (Table 3.7, 3.8). In addition, increased flower density fi'om 1993
to 1994 in the ‘48N89’, ‘Marsh’ and ‘Square’ sites, and decreased density in the ‘Jay’ site

were largely the result of changes in the abundance of mouse-ear hawkweed (Table 3.7,

77

3.8). Overall frequencies of spring flowers were high in both years, but frequencies were
generally lower in 1994 (Table 3.5).

The ranges of summer flower densities were similar in both years (Table 3.3);
densities differed significantly among sites in 1993 (F = 3.980; df = 5, p < 0.003) and
1994 (F = 6.3; df = 6, p < 0.001). The dominant summer flowers encountered in the 1993
and 1994 surveys across sites were flowering spurge (Euphorbia corollata) and St.

J ohnswort (Hwericum perforatum); horsemint (Monarda punctata) in 1993, and mouse-
ear hawkweed in 1994 (Table 3.10, 3.11). Of these, only flowering spurge and mouse-ear
hawkweed had consistently high frequencies among sites for the years considered (Table
3.12). The ‘Horseshoe’ site had the highest overall densities in both years, primarily as a
result of large abundances of hoary alyssum (Berteroa incana) and spotted knapweed
(Centaurea maculosa), which were rare or nonexistent in other sites (Table 3.10, 3.11,
3.12). As with spring flower densities, changes in mouse-ear hawkweed abundance
(Table 3.10, 3.11) explained the increase in summer flower densities from 1993 to 1994
for ‘48N89’ and ‘Square’ (Table 3.3). Overall frequencies of summer flowers were
generally higher in 1994 than 1993 (Table 3.5), and differences between 1993 overall
spring and summer flower frequencies for some sites were most likely explained by a
change in mouse-ear hawkweed frequency, as above (Table 3.9, 3.12).

Changes in numbers of flower species encountered in transect surveys per site
were not consistent from 1993 to 1994; in some sites, the number of species increased,
while in others, the number decreased (Table 3.15). When survey results from all sites
were combined, numbers of spring and summer flower species were higher in 1994 than

in 1993 (Table 3.16).

78

In 1993, the ‘Horseshoe’ site had the highest spring and summer Shannon
diversity (H’) and spring Simpson’s dominance (1/D) values, as well as the highest
number of flowering plant species (Table 3.15). The ‘Marsh’ had the next highest
number of summer flower species and the highest summer l/D (Table 3.15). The
‘Square’ site had the fewest species and values of H’ and ND in spring, and the ‘Jay’ had
the lowest values for those categories in the summer (Table 3.15).

In 1994, the ‘Pipe’ site had the highest H’ and ND values in both seasons (Table
3.15). The ‘48N89’ site had the lowest H’ in the spring and summer, and the lowest 1/D
value in the spring (Table 3.15). The ‘Park’ site, with the fewest summer flower species,
also had the lowest l/D value in the summer (Table 3.15). In contrast to 1993, the lowest
1994 values of diversity and dominance were not consistently associated with the lowest
numbers of species (Table 3.15). The highest diversity and dominance values were
associated with the highest number of species in spring 1993 and summer 1994 (Table
3.1 5).

In spring 1994, Karner blue adults were observed nectaring on eight flower
species. Nectaring was observed most frequently on cinquefoil (Potentilla spp.),
dewberry, mouse-ear hawkweed and wild lupine (Table 3.16). The latter three were also
dominant species in spring transect surveys (Table 3.7, 3.8, 3.9).

In the summers of 1993 and 1994, Karner blue adults were observed nectaring on
19 and 21 flower species, respectively, with nectaring most fi'equently observed on
butterfly weed (Asclepias tuberosa), flowering spurge, horsemint and spotted knapweed
(Table 3.16, A7 .1, A72). Nectaring was observed ca. 80 times on goat’s rue (Tephrosia

virginiana) and lance-leaved coreopsis (Coreopsis Ianceolata) in 1994, but almost no

116C

5116

fl0‘

obs

of‘

516'

Fr:

3111

C01

001'

6311

79

nectaring was observed on these species in 1993 (Table 3.16). It appeared that these two
species were past peak bloom in 1993 when summer Karner blue began flying, so no
flowers of goat’s rue and few coreopsis blooms were available. In support of this
observation, combined density estimates (estimates from all sites added together) for each
of these species were slightly higher in 1994 than in 1993 (for coreopsis, 0.31 vs. 0.01
stems per m2, respectively; for goat’s rue, 0.21 vs. 0 stems per m2, respectively).
Frequencies of these species were also higher in 1994 than in 1993 (Table 3.12).

Woodland sunflower (Helianthus divaricatus) and yellow hawkweed species
(Hieracium spp.) were used for summer nectaring to lesser extents in 1993 and 1994,
respectively (Table 3.16). All of the summer nectar sources mentioned above, with the
exception of goat’s rue and woodland sunflower, were encountered in both 1993 and
1994 transect surveys (Table 3.16). However, only flowering spurge in both years, and
horsemint in 1993 had consistently high density estimates among sites. Spotted
knapweed had a high density and frequency estimate only in the ‘Horseshoe’ site (where
most of nectaring observations were made) (Table 3.10, 3.11, 3.12). Butterfly weed was
consistently rare among the sites.

W: The dominant tree species in the sites were black oak (Quercus
velutina Lamarck), white oak (Quercus alba L.), black cherry (Prunus serotina Ehrhart)
and sassafi'as (Sassafias albidum (N uttall) Nees) (Table A8). Overall percentage canopy
cover and frequency estimates in the ‘Horseshoe’ site were extremely low (Table 3.1).
All other sites had a percentage canopy cover estimate of at least 20 percent and canopy
cover fiequency of at least 0.70 (Table 3.1). The ‘Jay’ site had the greatest cover

estimate, but the ‘Marsh’ had the greatest frequency estimate (Table 3.1). Percentage

80

canOpy cover was significantly different among the sites (F = 5.33; df = 6, p < 0.001),
primarily due to the low ‘Horseshoe’ cover estimate. The six other sites differed
significantly from the ‘Horseshoe’, but were not significantly different from each other
(Table 3.1). The among-site difference in percentage canopy cover was also significant
when tested with the non-parametric Kruskal-Wallis test (test statistic = 23.01; df = 6, p <
0.001).

At least 30 percent of transects in each site (and all of the transects in ‘Horseshoe’
site) had a percentage canopy cover of less than 10 percent (Figure 3.5). Most of the
remaining transects in each site had percentage canopy cover of 11 to 70 percent;
however, a few transects had cover greater than 70 percent (Figure 3.5).

MW: Of 46 larvae observed in summer 1993, 65 percent
were found on lupine in the open, and the other 35 percent were found in partially shaded
conditions. Of 69 larvae observed in spring 1994, 39 percent were found on lupine
growing in open conditions, and 61 percent were on lupine in partial shade. Of 198
summer larvae found in 1994, 62 percent were in the open, and 38 percent were in
partially shaded conditions.

W: In summer 1993, all but one Karner blue larva
was ant-tended at the time of observation (Table 3.15). In spring 1994, 83 percent of
larvae were tended, and 17 percent were untended (Table 3.15). In summer 1994, 89
percent of larvae were tended, and 11 percent were untended (Table 3.15). Presence or
absence of ants was not related to larval length. Ant-tending was observed for larvae of
all lengths, from 0.2 to 1.9 cm; untended larvae also ranged in length fiom 0.2 to 1.9 cm

(Table 3.15). Thirteen species of tending ants from three subfamilies were identified

81

from the collected specimens (Table 3.16). One of the predominant tending ant species
was Formica obscuripes F orel (Table 3.16).

; 11.. 11 snow ..u- y - -,- u... -:_u, .11. 61‘01°I,eooe°“.11-,‘:
In 1994, summer Karner blue adult abundance was highly correlated with the percentage
of lupine stems with summer larval feeding damage (r = 0.97) (Figure 3.6). Adult

abundance was also highly correlated with the frequency of larval feeding damage (r =

0.96) (Figure 3.7).

 

1993 and spring and summer 1994, there was a significant positive correlation of r > 0.8
between Karner blue abundance and lupine density (Figure 3.8, 3.9 and 3.10). Lupine
frequency was also significantly correlated with summer adult abundance in 1993 (r =
0.78) (Figure 3.11) and 1994 (r = 0.75) (Figure 3.12).

Summer Karner blue abundance was not significantly associated with summer
flower densities in either year (1993, r = - 0.14; 1994, r = - 0.06), nor was 1994 summer
abundance correlated with 1994 spring flower density (r = - 0.30). Spring butterfly
abundance in 1994 was not significantly correlated with 1994 spring flower densities (r =
- 0.33), or with 1993 summer flower densities (r = - 0.16).

Karner blue abundance was not significantly correlated with numbers of flower
species, flower diversity (H’), or flower dominance (l/D) for spring and summer of either
year. However, in 1994, correlations of spring and summer Karner blue abundance with
spring H’ were only marginally insignificant (Figure 3.13, 3.14, respectively).

There was no significant correlation between Karner blue abundance for summer

1993, spring 1994 and summer 1994 and percentage canopy cover (r = 0.52, 0.20, 0.23,

82

respectively), or canopy cover frequency (r = 0.10, - 0.01, 0.12, respectively). Percentage
canopy cover was not significantly associated with lupine density in either 1993 (r =
0.64) or 1994 (r = 0.35). The decrease in ‘r’ from 1993 to 1994 was due to the addition
of the ‘Park’ site.

In both years, there was a significant negative correlation between percentage
canopy cover and summer flower densities (Figure 3.15, 3.16). However, when the
‘Horseshoe’ site was removed from comparison, the 1993 correlation became positive
and not significant (r = 0.49), and the 1994 association remained negative but was also no
longer significant (r = - 0.56). A similar association occurred between percentage canopy
cover and 1993 numbers of summer flower species (Figure 3.17), which disappeared
when the ‘Horseshoe’ was removed (r = - 0.02).

For all sites, there was no significant correlation between transect estimates of
percentage canopy cover and lupine density. For comparisons between transect estimates
of percentage canopy cover and spring flower density, there was a significant negative
correlation for the ‘Jay’ site (df= 12, r = - 0.57; critical r = 0.53) (Figure 3.18), but

associations for all other sites were not significant.

Discussion

Habitat destruction and alteration have been the overwhelming causes of
invertebrate species declines (Hafernik 1992; New 1993; New et al. 1995). Like other
Lycaenidae, the Karner blue may be particularly susceptible to environmental changes,
and thus endangerment, because of its limited dispersal ability, dependence on one larval

hostplant found only in early successional habitats, and association with ant species

83

which may have patchy distributions and be impacted, as well, by altered habitat
(Cushman and Murphy 1993). Habitat conservation has emerged as the primary means of
preserving the Karner blue (Givnish et al. 1988; New et al. 1995), and autecology studies
are only just beginning to reveal aspects of the butterfly’s habitat requirements. As with
other Lepidoptera, larval and adult resources are presumed to be the basic prerequisites
(W iklund et al. 1977) of the Karner blue (Schweitzer 1989). However, overall habitat
suitability is most likely determined by a complex suite of components, interacting in
both time and space (Singer 1972). Microclimate heterogeneity provided by canopy
cover and ant-tending appear to be two additional components determining habitat
suitability for the Karner blue (Packer 1987; Leach 1992; Savignano 1994).

Karner blue larvae depend solely on wild lupine; therefore, it must be present in
some amount to support butterfly populations. In both years of this study, we found a
strong, positive correlation between abundance of Karner blue and lupine density, as has
been found by other researchers (Givnish et al. 1988; Lawrence and Cook 1989; Grundel
1994; Savignano 1994), as well as a strong correlation with lupine frequency. These
associations suggest that the amount and spatial distribution of lupine play a key role in
Karner blue population dynamics. However, studies done by Bleser (1992) in Wisconsin
and Lane (1992, 1994) in Minnesota did not show a consistently positive correlation
between density of lupine and Karner blue, and those researchers concluded that some
other variable was a limiting factor.

Savignano (1990a) suggested that Karner blue abundance and distribution may be
impacted by asynchronous timing of egg hatch and lupine development in the spring, and

early senescence of lupine in the summer. Swengel (1995) concluded that significant

hatching
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obscn'at
select in
clump.

Ital 1011,

and lup‘

limiting

84

hatching of Karner blue eggs prior to emergence of adequate lupine was unlikely.
However, larval starvation caused by early hostplant senescence has been documented for
some Lepidoptera other than the Karner blue (Ehrlich et al. 1980; Weiss et al. 1988). Our
observations of summer generation Karner blue larvae suggest that larvae may be able to
select individual lupine leaves of higher quality than the average quality of the overall
clump. Leaf quality may be affected by secondary plant compounds, nitrogen content,
leaf toughness and age, and can impact larval performance (F eeny 1970; Rausher 1981).
Mechanisms governing the positive association between Karner blue abundance
and lupine density are not known. The absolute amount of lupine does not appear to be
limiting, since the majority of lupine plants are not occupied by larvae (Lawrence and
Cook 1989), supporting the contention that herbivorous insects are rarely food limited
(Dethier 1959b; Hairston et al. 1960). However, lupine density and distribution may
function in the ability of larvae to find suitable food (Dethier 1959b). Hostplant location
is especially critical for larvae emerging in the spring. Newly emerged spring larvae have
only a short time after hatching to find lupine leaves (Lane and Welch 1994; Swengel
1995). Larvae are more likely to encounter lupine stems when the plants are more
abundant and randomly distributed. The same would be true for spring or summer
generation larvae, which often rest for part of the day in the litter and must relocate lupine
stems (Grundel 1994). Denser patches of lupine may also diffuse density-dependent
mortality of larvae, including parasitism and predation, and disease. Abundance of
hostplants would help to counteract any mistakes made in hostplant choice by ovipositing

females (Dethier 1959a). In addition, lupine density may play a role in Karner blue

85

female ovipositional behavior. Females may prefer to oviposit in areas with concentrated
plant resources (Root 1973).

Availability of nectar sources is an important requirement for the survival of both
spring and summer Karner blue adults. In some areas and in some years, nectar plants,
rather than lupine, may be the limiting factor for butterfly populations (Clench 1967;
Murphy 1983). Scarcity of nectar plants, especially during the summer flight period,
have been attributed with lower Karner blue numbers than would have been expected for
a particular site (Schweitzer 1989; Bleser 1992). Some qualitative studies reported that
absence of suitable nectar sources as a result of drought prevented establishment of
Karner blue populations in areas where adequate lupine was present (Packer 1987;
Schweitzer 1989). A previous study in Allegan State Game Area found a positive
correlation between abundance of nectar plants and Karner blue (Lawrence and Cook
1989). However, in our study, we did not observe an association between butterfly
abundance and flower density, suggesting that the minimal requirement for nectar was
met in all sites and nectar was not a limiting factor during the years of study.

Lepidoptera vary in their dependence on adult resources. Many moth species do
not feed, while many female butterfly species require nectar sources for egg maturation
and oviposition (Murphy et al. 1983). Female butterflies in the genus Euphydryas can
Pmduce many eggs without feeding (Murphy et al. 1983). However, Murphy et al.
(1933) found that nectar consumption increased female lifespan and fecundity, allowing
females to lay more eggs later into the season. Larvae hatching from these late eggs were

unlil(ely to survive in most years due to hostplant senescence; however, Murphy et al.

86

(1983) proposed that survival of late larvae in rainy years increased butterfly numbers,
providing a significant buffer against extinction in dry years. The dependence of Karner
blue females on nectar sources for egg production has not been investigated, but would
aid in further understanding Karner blue population dynamics.

The distributions of nectar sources in relation to lupine may impact the
oviposition behavior of Karner blue females. Murphy et al. 1984 and Grossmueller and
Lederhouse (1987) found that female butterflies preferred oviposition hostplants that
were in areas with high densities of preferred nectar plants.

We observed Karner blue adults utilizing a variety of nectar sources. As others
have reported (Packer 1987; Lawrence and Cook 1989; Bleser 1992; Haack 1993; Lane
1994), the two most widely used nectar sources were butterfly weed, which was
consistently rare in all the sites, and horsemint. Two other flower species, goat’s rue and
coreopsis, were used heavily in one year, but were not phenologically available to
butterflies in the other year. Flower species such as flowering spurge and mouse-ear
’ hawkweed, while not used as extensively as butterfly weed for nectaring, were the most
abundant flowers in the sites in both years. These less-preferred but abundant flower
Species may be especially important if they are predictably in flower during the Karner
blue flight periods. The ability of Karner blue to utilize a variety of nectar sources would
help to buffer the butterfly from temporal dissociations of flowering time of particular
nectar sources with the adult flight period (Carey 1994).

Microclimatic conditions have been shown to be important in determining habitat
Suitability for butterflies (Ehrlich et al. 1980; Dobkin et al. 1987; Weiss et al. 1988).

Shade in limited amounts could provide microclirnatic variation important for Karner

87

blue adults and larvae, as well as lupine (Givnish et al. 1988; Bleser 1992; Leach 1992;
Lane 1994). Tree-canopy shade reduces understory temperatures (Belsky et al. 1993).
Karner blue adults and larvae, like other butterflies, may require shady microhabitats to
escape hot mid-day temperatures (Lawrence and Cook 1989; Bleser 1992). Some studies
found Karner blue to be more abundant in sites with interspersed sun and shade versus
large xeric openings (Lawrence and Cook 1989; Leach 1992). We did not observe an
association between Karner blue abundance and percentage canopy cover in our study.
Study sites with low and high butterfly abundance had similar percentages of canOpy
cover. However, this suggests that canopy cover of 20 to 30 percent is not a limiting
factor for Karner blue, and that in the sites with low butterfly abundance, some other
factor was limiting.

Tree-canopy shade reduces soil- and foliage- moisture loss (Belsky et al. 1993).
Thus, shading may increase the amount of time which lupine is available to summer
generation larvae, providing a buffer to population losses in dry years (Carey 1994).
Lawrence and Cook (1989) observed lupine to desiccate prematurely in dry, sunny
Openings. In certain years, significant mortality of summer generation Karner blue larvae
could result if lupine senesces before larvae finish development, as reported by Ehrlich et
al. (1980) for the checkerspot butterfly. Lupine has been found to persist longer under
seEli-closed canopies than in open areas (Hess 1983; Leach 1992), which would provide
hoS’tplants for larvae for a longer period of time into the summer. And higher lupine
densities and frequencies may translate into a wider variety of microclirnates occupied by

1“Pine, and a greater likelihood of some plants being shaded.

88

In our study, we found Karner blue larvae on lupine in both partially shaded and
open areas, as did Lawrence and Cook (1989), suggesting that female Karner blue adults
unpreferentially use lupine plants in different shade conditions for oviposition. However,
conflicting data from other studies suggest oviposition preference for lupine plants in
partial shade (Packer 1987) as well as in open habitats (Savignano 1990a; Bleser 1992).
These data say nothing of survival of larvae in the different shade conditions. Results
from a laboratory study (Grundel 1994) suggest that summer generation Karner blue
larvae develop more quickly on lupine leaves from plants growing in partial shade versus
the open, perhaps due to decreased leaf quality of the sun-exposed lupine plants (Rausher
1981; Dudt and Shure 1994). However, some of this difference may be mediated by the
fact that larvae in the sunnier microenvironments would develop faster than in shadier
microclirnates (Weiss et al. 1988).

Myrrnecophilous associations of lycaenid larvae have been well documented
(New 1993). Though associations can range from commensalism to larval predationon
ant broods, the relationship is more ofien a facultative mutualism (Atsatt 1982; Pierce
1985), such as with the Karner blue (Savignano 1990b). Savignano (1987, 1990a,b) and
Packer (1987) found that larval survival was greater for Karner blue larvae that were ant-
tended than those that were not, suggesting that ants reduce the levels of larval mortality
due to parasitism and predation (Savignano 1990b).

We identified thirteen species of tending ants, many of which have been reported
tending Karner blue larvae in New York (Savignano 1994) and Ontario (Packer 1987).
Formica obscuripes Forel was a common and aggressive tending species. In our study,

ant-tending was observed for 82 percent or more of the Karner blue larvae in Michigan,

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similar to ant-tending percentages reported by others (Packer 1987; Savignano 1989).
More than 50 percent of larvae less than 0.5 cm were tended, which was surprising since
Savignano (1990b) reported that first and second instar Karner blue lack fully developed
ant-associated organs. Ant-tending appears to be a significant aspect of Karner blue
ecology in Michigan. Although Karner blue larvae can develop successfully without
tending ants (Savignano 1990b; Chapter 2), benefits of ant-tending may be important in

, years when parasitoid and predator populations are high, or when other factors make .
Karner blue populations more vulnerable to extinction. Past extirpations of other
lycaenids have been correlated with the disappearance of protective ant species due to
unfavorable habitat conditions or management practices (Packer 1987; New 1993). Many
of the species of tending ants we identified build nests above ground in logs and stumps
(Wheeler et al. 1994), and may be more prone to disturbance. Impacts of management on
ant species should be considered.

In this study, we estimated Karner blue abundance fiom weekly surveys
throughout the entire flight period. This methodology allowed us to identify peak flight.
Some sites peaked at different calendar dates from one year to the next, emphasizing the
necessity of conducting surveys throughout the entire flight period each year. We also
indirectly estimated larval abundance through larval feeding damage surveys, and found
that the results from these surveys were highly positively correlated with adult estimates
0f abundance. These results are consistent with Swengel’s (1995) results of positive
assOCiation between larval and adult abundance.

Our results suggest that lupine density is a significant factor in determining the

Poplilation dynamics of the Karner blue. Nectar sources are also important; however, the

90

low densities of flowers present in our study sites over the two years appeared to meet
some minimum requirement, and were not a limiting factor. Since some flower species
differed in their availability to Karner blue from year to year, a diversity of nectar sources
in the Karner blue habitat would help to buffer this phenomenon. The exact role of
canopy cover could not be determined fiom our study; sites with low and high
abundances of butterflies and lupine had similar canopy cover. However, this finding
suggests that 20 to 30 percent canopy cover does not limit Karner blue populations or
lupine, and may provide a benefit of microclirnatic variation in various ways including
prolonging the availability of lupine to the butterfly. Karner blue larvae in Michigan are
predominantly ant-tended, and ant-tending also appears to be a significant factor in the
butterfly’s survival, and thus in habitat suitability.

Current management activities for the Karner blue in different states are focused
on improving and maintaining habitat suitability for local butterfly populations (Baker
1994). The primary goals of habitat management are to increase amounts of lupine and
nectar plants by decreasing woody vegetation through hand-cutting, mowing and
Prescribed fire (Baker 1994; Shuey 1994). Management activities also include restoration
0f Karner blue-unoccupied oak savanna and pine barren habitats, which are ofien adjacent
to existing butterfly populations in the hopes of expanding the butterfly’s range (Baker
1994). In Ohio and Ontario where the Karner blue is now extirpated, habitat once
ocCupied by the species is being restored for future Karner blue reintroductions (Baker
1994; Packer 1994). Some states are involved in Karner blue propagation through
caPtive rearing (Lane and Welch 1994), and lupine and nectar plant propagation and

Planting (Baker 1994). Our results support management activities which increase lupine

 

 

91

densities and fiequencies, maintain a diversity of nectar sources, and maintain habitat
heterogeneity created by low levels of canopy cover.

Many questions regarding Karner blue ecology still need to be answered in
understanding the gradient between habitat suitability and unsuitability for this butterfly
species (Haack 1993). Future research activities need to address topics such as the ability
of butterflies to disperse through different types of intervening habitats, and the role of
lupine and nectar source density and distribution in Karner blue population dynamics.
On the local scale, the Karner blue requires some minimum level of lupine and nectar
sources to survive. Our results suggest that more lupine is beneficial for Karner blue
populations; however, the same may not be true for nectar sources once the minimum
requirements are met. Impacts of management activities on tending-ant species also need
to be explored.

Habitat suitability of an invertebrate can be difficult to identify through short-term
investigations, which do not reveal complex interactions or effects of sporadic climatic
events. This is especially true in dynamic habitats such as the savannas and barrens that
were historically maintained by natural processes (Shuey 1994). Long-term studies, like
those on the checkerspot butterfly, provide extremely useful information regarding a
SPeCies’ ecology and habitat suitability (Ehrlich and Murphy 1987), and should be
Persued for Karner blue. Ultimately, long-term viability of Karner blue populations will
depend upon restoration of metapopulation dynamics in the threatened oak savanna and
pine barren landscapes, allowing for local extinctions and recolonizations (Givnish et a1
1983; Shuey 1994). The Karner blue serves as a symbol for savanna lbarren conservation

and management at both the species and ecosystem levels.

92

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119

Table 3.16. Thirteen species of ants (Hymenoptera: Formicidae) representing three
subfamilies observed tending Karner blue larvae. Ant specimens were collected in
Allegan County (Allegan State Game Area) and Oceana County (Huron-Manistee
National Forest), Michigan, during the 1993 and 1994 spring (Spr) and summer (Su)
Karner blue larval generations.

 

Karner blue larval

 

generation tended
Tending ant species1 at time of collection County
Subfamily Myrmicinae
Crematogaster lineolata (Say) Spr, Su Allegan, Oceana (Su only)
Monomorium pharaonis (L.) Spr Oceana
Myrmica americana Weber Su Allegan
Myrmicafiacticornis Emery Spr, Su Allegan
. Subfamily Dolichoderinae
Dolichoderus mariae Forel Spr Oceana
Dolichoderus pustulatus Mayr Su Allegan
T apinoma sessile (Say) Spr, Su Allegan, Oceana
Subfamily Formicinae
Formica neogatates Emery Su Allegan, Oceana
Formica obscuripes F orel Spr, Su Allegan, Oceana
Formica obscuriventris Mayr Su Allegan, Oceana
Formica schaufizssi Mayr Su Allegan
Formica subsericea Say Spr Allegan, Oceana
Lasius neoniger Emery Su Allegan

 

1 Ant species were determined on 14 September, 1995 by D. R. Smith, Research
Entomologist, USDA Agricultural Research Service, Systematic Entomology Laboratory,
Communications & Taxonomic Services Unit, Beltsville Agricultural Research Center-
West, Beltsville, Maryland 20705-2350.

120

Figure 3.1. Map of Lower Peninsula of Michigan showing the location of Allegan State
Game Area study sites (Allegan Co) and Huron-Manistee National Forest (Oceana Co).

121

 

 

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Figure 3.6. Scatterplot of 1994 summer Karner blue abundance versus percentage

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damage surveys in study sites.

127

 

 

 

 

 

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130

 

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131

 

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Figure 3.11. Scatterplot of 1993 summer Karner blue abundance versus 1993 lupine
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132

 

 

 

 

 

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133

 

 

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0 0.2 0.4 0.6 0.8 l 1.2 1.4 1.6

Shannon Diversity Index (H') for Spring Flowers

Figure 3.13. Scatterplot of 1994 spring Karner blue abundance versus Shannon
diversity index (H') for 1994 spring flowering plants in study sites.

134

 

200 -- I

150 " r= 0.63

100 --

50 --

No. Summer Karner Blue Adults / hr

 

 

 

 

O l l 1 l I 1 l
I I I I r I I

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Shannon Diversity Index (H') for Spring Flowers

Figure 3.14. Scatterplot of 1994 summer Karner blue abundance versus Shannon
diversity index (H) for 1994 spring flowering plants in study sites.

135

 

Percentage Canopy Cover (SE)

 

 

 

 

O 1 2 3 4 5

No. Summer Flower Stems / m2 (SE)

Figure 3.15. Scatterplot of percentage canopy cover (SE) versus 1993 summer flower
density estimates (SE) in study sites.

 

136

 

4o .-

30 --

20 ac

Percentage Canopy Cover (SE)

10 «-

 

 

 

O : i. 1+

 

 

O l 2 3 4

No. Summer Flower Stems / m2 (SE)

Figure 3.16. Scatterplot of percentage canopy cover (SE) versus 1994 summer flower
density estimates (SE) in study sites.

137

 

A
O
1
I

 

= - 0.74

U.)
C
I
I

Percentage Canopy Cover (SE)
8
l—H

p—s

O
I
I

 

 

 

0 . : : J

0 4 8 12 16

No. Summer Flowering Plant Species

Figure 3.17. Scatterplot of percentage canopy cover (SE) versus the number of 1993
smnmer flowering plant species encountered in transect surveys in study sites.

138

 

 

 

 

 

100
I

E 80 --
O
0
>5
8‘
5 60 q.
o
0
.82?
§
3'5 401
D-u
E 20-

o.____+l_l : : : : . l—I—J

0 2 4 6 8 10 12 14

Transect Spring Flower Density (stems / m2)

Figure 3.18. Scatterplot of 1994 transect estimates of percentage canopy cover versus
1994 transect estimates of spring flower density (stems / m2) in the 'Jay' study site.

APPENDICES

APPENDIX ll

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.: 1996-3

 

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

The Endangered Karner Blue Butterfly (Lepidoptera: Lycaenidae) in
Michigan: Habitat Suitability, Potential Impacts of Gypsy Moth
(LepidOptera: Lymantriidae) Suppression, and Laboratory Rearing.

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

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigator's Name (3) (typed)
Catherine Papp Herms

 

 

 

Date' April 25, 1996

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in
Nerth 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.

139

APPENDIX 1.1

APPENDIX 1.1

Vbudher Specimen Data

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140

141

APPENDIX 1.1

Vbucher Specimen Data

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APPENDIX 2

APPENDIX 2

1993 Federal Endangered and Threatened Species Subpermit

l42

143

United States Department of the Interior 1.8:..-
=

 

IUSPIAIHDVVHJDLUHESERNHCEZ olllll ll
Bishop Henry Whipple Federal Building - -
manmflunmnm lfiakmdtkhe
Fort Snelling, MN 55111-4056
FWS/AES-TE

 

AUTHORIZATION TO USE REGION 3 ENDANGERED AND THREATENED SPECIES PERMIT
TO CARRY OUT THE FOLLOWING ACTIVITIES WITHIN THE STATE(S) OF
Michigan

 

SUBPERMIT #93-23-I ISSUED June 2, 1993
INDIVIDUALS COVERED BY THIS SUBPERMIT:

Catherine M. Papp, Deborah G. McCullough, Thomas Ellis, and two student
employees; all under the supervision of Catherine Papp.

SPECIES COVERED BY THIS SUBPERMIT:
Karner blue butterfly (Lycaeides melissa samuelis)

In accordance with Federal Endangered Species Permit PRT-697830, you are
authorized to conduct the following take activities on the above species for
scientific research, enhancement of propagation, or enhancement of survival
through September 30, 1993. Any activity related to Federally listed
threatened or endangered species that is not specifically permitted in this
document is prohibited.

The activities allowed under this subpermit, and the conditions under which
those activities must be conducted, are as follows:

1. Authorized activities to be conducted at Allegan State Game Area,
Allegan County, MI, and Huron-Manistee National Forest, Manistee Unit,
Newaygo and Oceana Counties, MI.

2. No specimens may be collected or removed from the wild for
laboratory studies.

3. Conduct census' to determine Karner blue butterfly habitat and the
population density and diversity of attending ants.

4. Census' are to be conducted at the same time of the day for all days
that a census is conducted.

5. Injuries and/or mortalities may not exceed five specimens. In the
event that this number is met, all permitted activities must cease. You
must contact the U.S. Fish and Wildlife Service (Service) within 48
hours explaining the circumstances in writing to the following: U.S.
Fish and Wildlife Service, 1 Federal Drive, Fort Snelling, MN 55111
(Attn: Carlita Shumate), and U.S. Fish & Wildlife Service, East Lansing

144

Field Office, 302 Manly Miles Bldg., 1405 South Harrison Road, East
Lansing, MI 48823 (Attn: Susan Walker); telefax (517) 337-6899.

6. Any specimens that are killed are to be preserved according to
standard museum practices, properly labeled (date, complete scientific
and common name, and location where obtained), and submitted to Ms.
Carlita Shumate of this office at the address above.

A copy of FRI-697830 is attached and the conditions of that permit must be
adhered to. This subpermit and PRT-697830 must be in your possession while
conducting authorized activities. You are reminded that necessary state
and/or local permits, if applicable, must also be acquired and adhered to;
this subpermit is invalid without such permits.

All specimens obtained under this subpermit remain the property of the United
States Government and must be clearly identified as such.

Reporting Requirements

A full report of activities conducted under the authority of this subpermit,
as well as copies of all data obtained from those activities, are due in this
office by close of business 01/31/94, and to Ms. Susan Walker of the Service's
East Lansing Field Office. In addition, copies of all reports and
publications resulting from those data must be submitted to this office as
they become available. The report required for this permit must include the
following:

1. A complete discussion of field procedures, data collection methods,
results, and conclusions.

2. The dates data are collected, a description of weather conditions
for each day of collection, and the location (state, county, section,
township, and range) of collection sites.

3. For each date data are collected, the report must specifically
provide the time of day, the location and size of each sampling quadrat
or plot; the number of Karner blue butterfly eggs, larvae and adults
observed in each quadrat or plot; a description of any larval and adult
behavior that is observed; the number, location, scientific, and common
name of vascular vegetation within each plot; a description of
distribution, percentage cover, and phenology of nectar and lupine
plants; the percentage cover of canopy, litter, and soil layers (e.g.,
percentage sandy substrate) in each quadrat or plot where Karner blue
butterfly eggs, larvae and adults are observed; the number of larvae
observed on each lupine plant; and the scientific name of ants tending
Karner blue butterfly larvae.

4. A complete description of injuries and/or mortalities to Karner blue
butterflies, the dates they occurred, any circumstances surrounding the
incidents, and describe steps that will be taken to reduce the
likelihood of such situations from occurring in the future.

I45

Failure to furnish any reports that are required by this permit is cause for
permit revocation and/or denial of future permit applications.

All correspondence related to this subpermit should reference subpermit 93-23-
I. Any questions you may have regarding this subpermit should be directed to
the Region 3 Chief, Division of Endangered Species, at (612) 725-3276.

  
 

Di ision of Endangered Species

Attachment

cc: FWS Ft. Snelling, MN (LE)
ES Field Office TE Coordinator for E. Lansing, MI
DNR/DOC Endangered Species Coordinator for Michigan
FWS RD-S, ES Regional Office TE, Chief

I46

' —_-_I'
United States Department of the Interior fl=

 

_
FISH AND WILDLIFE SERVICE _.-I=-
Bishop Henry Whipple Federal Building - .
murmur. 1 Federal Drive
Fort Snelling, MN 55111-4056
FWS/AES-TE

June 2, 1993

Miss Catherine Papp
Department of Entomology
Michigan State University
East Lansing, Michigan 48824

Dear Miss Papp:

Enclosed is your subpermit, 93-23-1, which authorizes research activities that
will be carried out on Karner blue butterflies (Lycaeides melissa samuelis).
The permit authorizes you and the individuals you included in your application
to conduct most of the activities requested in your April 9, 1993, permit
application. Please provide my office with the names, statement of
qualifications, and resumes of the two student employees you plan to hire for
this proposal within ten working days of their hire.

I have not granted authorization for you to collect and lethally take adult
butterflies from the wild for the Bacillus thuringiensis (Bt) susceptibility
studies for several reasons. First, my office will not authorize lethal take
of this endangered species for research purposes while alternatives exist. My
staff and I are being particularly cautious this year because of the large
number of individuals who want to work with Karner blue butterflies, the fact
that the studies are likely to affect the Karner blue butterfly throughout
their range in the midwestern states, and the potential for mortality
associated with some of those studies (particularly prescribed burns to
restore habitat).

For the moment, I recommend conducting your study using closely-related,
Lycaenidae that are not endangered species (for example, the melissa blue,
Plebjus melissa, or tailed blue, Everes comyntas, butterflies). Depending on
the results of your research with one of these species, you could consider
resubmitting an application for a permit to conduct Bt susceptibility studies
on the Karner blue butterfly.

Any questions regarding your application or this subpermit may be directed to
me at (612) 725-3276 or to Ms. Carlita Shumate of my staff at the same
telephone number.

 

 

Enclosure

cc: ES Field Office TE Coordinator for E. Lansing, MI
DNR/DOC Endangered Species Coordinator for Michigan
FWS RD-S, ES Regional Office TE, Chief

APPENDIX 3

APPENDIX 3

1994 Federal Endangered and Threatened Species Subpermit

147

148

United States Department of the Interior

IHSPIAmfl)VVDJDLIFEEHHUVKJE
Bishop Henry Whipple Federal Building
lFakdekne
Fort Snelling, MN 55111-4056

 

IN REPLYREFERTO:

IVS/ABS -TE

 

AUTHORIZATION TO USE REGION 3 ENDANGERED AND THREATENED SPECIES PERMIT
TO CARRY OUT THE FOLLOWING ACTIVITIES WITHIN THE STATE OF
Michigan

SUBPERMIT #94-23-R ISSUED May 23, 1994
EXPIRES December 31, 1994

INDIVIDUALS COVERED BY THIS SUBPERMIT:

Catherine Papp, Deborah McCullough, Robert Haack, Leah Bauer, and one
student employee; all under the supervision of Catherine Papp.

SPECIES COVERED BY THIS SUBPERMIT:
Karner blue butterfly (Lycaeides melissa samuelis)

In accordance with Federal Endangered Species Permit PRT-697830, you are
authorized to conduct the following take activities on the above species for
scientific research, enhancement of propagation, or enhancement of survival
through December 31, 1994. ‘Any activity related to federally listed
threatened or endangered species that is not specifically permitted in this
document is prohibited.

The activities allowed under this subpermit, and the conditions under which
those activities must be conducted, are as follows:

1. Collect no more than 20 female Karner blue butterflies during the
Spring generation (late May through early June) to study the effects of
Bacillus thuringiensis var. kurstaki (Btk) on this species. Collection
is limited by the following conditions:

A. Collect no more than ten butterflies from the following
locations within the Allegan State Game Area (ASGA), Allegan
County, with between three and five individuals collected per
site: 1. T2NR15W, Section 14 NE4NE4; 2. T2NR15W Section 2 NW4SE4;
3. T2NR14W Section 28 E2NE4; or, 4. T2NR15W Section 13 NW4NW4.

B. Collect no more than ten butterflies from the following
locations within the Manistee National Forest (MNF), Montcalm,
Newaygo, and Oceana Counties, with between three and five
individuals collected per site: 1. Tl3NR16W Section 26 NW4; 2.
T12NR12W Section 35 SE4; 3. T15NR12W Section 6 SW4SW4; 4. T12NR10W
Section 21 NW4; or, 5. T13NR10W Section 32 NE4SE4.

149

C. Collect adult female butterflies using a butterfly net and
transfer to small screen cages, then place in coolers and
immediately transport to Michigan State University laboratory
facilities at the Pesticide Research Center. Butterflies will be
kept there in an environmental chamber which regulates
temperature, light and humidity.

D. House captive adult butterflies in the laboratory for a period
of two to five days to permit ovipositioning on cultivated wild
lupine (Lupine perennis) and then immediately return butterflies
to the place of capture.

E. House eggs and larvae in an environmental chamber which
regulates day/night temperatures, dark/light cycles and humidity
and rear larvae according to the protocol developed by C.P. Lane
(copy on file).

2. Conduct Btk bioassay of Karner Blue butterfly larvae according to
the study protocol presented in the permit application (on file), as
outlined below:

A. Not more than 180 larvae will subject to the bioassay study.
Two larval stages of Karner blue butterfly (second and fourth
instars) will be subjected to three treatments. Thirty larvae of
each stage will be subjected to each of the following treatments:
1) control consisting of untreated lupine which has been subjected
to the same handling procedures as the treated lupine,

2) lupine which has been sprayed with the either the carrier of
Foray 48B. or autoclaved Foray 488., and

3) lupine treated with Foray 488..

B. Keep larvae on treated or untreated lupine foliage in petri
dishes for five to seven days, and check daily for lethal effects.

C. Transfer larvae surviving the treatments to fresh petri dishes
with fresh lupine and monitor daily through pupation and adult
emergence.

D. Return all Karner blue butterfly adults which survive the Btk
bioassay to the sites of parental capture within two days of
emergence.

E. Rear additional larvae produced but not used in the bioassay
according to the protocol developed by C. N. Lane and release the
adults within two days of emergence. Progeny must be released at
the sites of original parental capture.

3. Conduct walk-through survey and monitoring activities to assess
habitat characteristics of the oak savannah habitat that includes
determination of Karner blue butterfly larvae presence, measuring of
larvae, observation of attending ants, determination of wing wear and
sex ratio of adult butterflies, and wild lupine (Lupinus perennis)

150

phenology as described under section J, Objective 1 (A)-(C), through
Objective 3 of the March 14, 1994, permit application. The survey and
monitoring activities shall be conducted in a manner to minimize
disturbance to the Karner blue butterfly and wild lupine. Handling of
adults and larvae shall be kept to a minimum.

4. Census' are to be conducted at the same time of day for all days
that a census is conducted.

5. Injuries and/or mortalities may not exceed five specimens. In the
event that this number is met, all permitted activities must cease. You
must contact the U. 8. Fish and Wildlife Service (Service) within 48
hours explaining the circumstances in writing to the following: U. 8.
Fish and Wildlife Service, 1 Federal Drive, Fort Snelling, MN 55111
(Attn: Carlita Shumate), U.S. Fish and Wildlife Service, Ecological
Services Field Office, 302 Manly Miles Building, 1405 South Harrison
Road, East Lansing, MI 48823 (Attn: Charles Wealey, Field Supervisor);
telefax: 517/337-6899.

6. Any observed intact specimens of the Karner blue butterfly
accidentally killed or freshly dead shall be preserved according to
standard museum practices, and properly identified and indexed [include
date, complete scientific and common names“ and location (include
township, range, and section)]. The specimens shall be sent a public
scientific museum in the State of Michigan. All specimens obtained
under this subpermit remain the property of the United States Government
and must clearly be identified as such. A list of specimens collected
(if any) and pertinent location data shall be provided to the Service's
Regional Office, Division of Endangered Species and to the East Lansing,
Michigan, Field Office by December 31, 1994.

A copy of Federal Endangered Species Permit PRT-697830 is attached; you are
required to adhere to the conditions of that permit. This subpermit, Permit
FRI-697830, and your permit application (signed March 14, 1994) must be in
your possession while conducting authorized activities. Be advised that
necessary state and/or local permits must also be acquired and adhered to;
this subpermit is invalid without such permits.

Reporting Requirements

A full report of activities conducted under the authority of this subpermit,
as well as copies of all data obtained from those activities, and year-end
report are due in this office by the close of business on December 31, 1994,
and to the Service's East Lansing Field Office (Attn: Field Supervisor). In
addition, copies of all reports and publications resulting from those data
must be submitted to these offices as they become available. Failure to
furnish any reports that are required by this permit is cause for permit
revocation and/or denial of future permit applications. The 1994 year-end
report must include the following:

1. A complete discussion of field procedures, data collection methods,
results, and conclusions.

151

2. Legible photocopies of all field data sheets or complete summaries
of all field data sheets as described under the conditions of this
subpermit as well as the following analyses of studies done:
a) An assessment of the relationship between Karner blue
butterfly density and lupine density.

b) An assessment of the relationship between Karner blue
butterfly density and nectar source density.

c) An assessment of the relationships between Karner blue
butterfly and lupine density with percentage of canopy cover.

d) An analysis of the extent and importance of ant-tending to
Karner blue butterfly larvae.

e) Estimates of the population size of Karner blue butterflies at
each study site. ‘

f) A determination of whether the period when spring generation
Karner Blue butterfly larvae are present coincides with the period
of aerial application of Btk for gypsy moth suppression in
Michigan. .

g) An assessment of the susceptibility of Karner Blue butterfly
larvae to Btk, at the rate and of the formulation used in gypsy
moth suppression, in a controlled laboratory setting.

3. A complete description of injuries and/or mortalities to Karner blue
butterflies, the dates they occurred, and any circumstances surrounding
the incidents. In addition, steps should be identified to reduce the
likelihood of such injuries and/or mortalities occurring in the future.

4. The ultimate disposition of injured or dead butterflies (i.e.,
retained, returned to location of encounter, forwarded to a state or
Federal agency or educational institution, etc.). If a specimen was
retained, your report must identify the location where it is being
stored and the reason it is being retained. A list of specimens
collected (if any) and pertinent location data shall be provided also.

-All correspondence related to this subpermit should reference subpermit
94-23-R. Any questions you may have regarding this subpermit should be
directed to the Region 3 Chief, Division of Endangered Species, at
612/725-3276.

Is! John A. Blankenship
0

Job A. Blankenship g)
Ass :tant Regional Dire tor

cological Services

  
 
 
  

Attachment

152

RESBBVQE
SEND TO: ‘U.S.Fish and Wildlife Service
Division of Endangered Species .

1 Federal Drive 15 ' " ‘
Ft. Snelling, MN 55111 H 8- ILDLIFE SERIICE
c411: 512/725-3275 _ TE

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studies of the endangered Karner blue

butterfl (Lycaeides melissa
amounts... “mm-dflme-A-dm .
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Catherine M. Papp 2. Co lection of adult female Karner
Department of Entomology blue for rearing and Bacillus

' Michigan State University thuringiensis var. kurstaki suscepti-
East Lansing, MI 48824 bility studies.

(517) 355- 4552 p . ,

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153

United States Department of the Interior

FISH AND WILDLH’E SERVICE
Bishop Henry Whipple Federal Building
1 Federal Drive
Fort Snelling, MN 55111-4056

 

IN REPLY REFER TO:

FWS/AES-TE

May 24, 1994

Miss Catherine M. Papp
Department of Entomology
Michigan State University

East Lansing, Michigan 48824

Dear Miss Papp:

Enclosed is your subpermit, 94-23-R, which authorizes research activities that
will be carried out on Karner blue butterflies (Lycaeides melissa samuelis).
Please provide my office with the name, statement of qualifications, and
resume of the student employee you plan to hire for this proposal within 10
working days of hiring.

Any questions regarding your application or this subpermit may be directed to
Mr. Robert Adair, Chief, Division of Endangered Species, or Ms. Carlita
Shumate of his staff, at 612/725-3276.

Sincerely,

 

Enclosure

cc (w/enclosure):
FWS ES Field Office TE Coordinator, East Lansing, MI
Endangered Species Coordinator, Michigan DNR
Chief, ES/TE, FWS Region 5

APPENDIX 4

APPENDIX 4

1993 and 1994 State of Michigan Threatened / Endangered Species Permits

154

156

 

STATE OF MICHIGAN .
DEPARTMENT OF NATURAL RESOURCES PGIIIIII Number. 1446
WILDLIFE DIVISION ‘
PO. BOX 3002B ‘3 Date Issued: May 20, 1994
LANSING, MICHIGAN 48909 m

 

 

THREATENED/ENDANGERED SPECIES PERMIT

BY THE AUTHORITY OF 1974 PA 203 AND THE RULES AND REGULATIONS
ESTABLISHED THEREUNDER, PERMISSION IS HEREBY GRANTED TO:

 

Ms. Catherine M. Papp
Department of Entomology
243 Natural Science

Michipgn State University
East nsing, Michigan 48824

 

 

 

To conduct the scientific activities listed under special conditions on the threatened/endangered species listed below.
All activities are subiect to the standard permit conditiogs on the back of this permit.

This Permit shall be valid only on the following lands/locations:

Allegan State Game Area, Huron-Manistee National Forest.

SPECIES: Lycaeides melissa samuelis, Karner blue butterfly

 

 

SPECIAL CONDITIONS:

Permitted is the entry into known karner blue sites to conduct research into habitat features of oak savanna
affecting butterfly distribution and population size.

Permitted is field research including the collection of up to 20 adult females and the establishment of a
laboratory colony for research into their succeptibility to St in relation to the effcts of the Michigan MDA gypsy
moth control program on non-target species.

Collect a maximum of 3-5 butterflies from each location to reduce the impact to the local population. If

possible, release surplus eggs or larvae from the laboratory colony back into the areas where they were
collected.

Provide the Endangered Species Coordinator a copy of the results of the research including the techniques
needed to raise larvae under laboratory conditions.

UNLESS REVOKED SOCNER, THIS PERMIT EXPIRES ON ‘2’ 31494

7" W
THE DIRECTOR OF NATURAL RESOURCES BY: «47714.4- V7/ m

ENDANGERED SPECIES COORDINATOR

 

 

 

Revrsed 4792

 

APPENDIX 5

APPENDIX 5

1993 and 1994 State of Michigan Use Permits

157

159

 

 

 

 

 

STATE OF MICHIGAN am NUMBER m l ORY NUMBER
DEPARTMENT or NATURAL RESOURCES TI 20" -9 L{ ' NT”
USE PERMIT Issumo Locmon (Form. «an. NBA. Em
mm under some or Act I7. PA. 1921. as amended. “\me STEVE» 6%“?! “cm
WTOTMWOINLNWTNWWWWIBWOIMTOW WTOUBOWWWEGTNWW.

 

 

SSW... WW! ”SWEET”

beginning ”IR .19_ from I’IGVIO ICICI‘I .19_tobeg_ 3, .1923]

DESCRIPTION 0' STATE-OWNED LAND

Leonid-«.5 W AIIean.‘ $I‘CL‘I'Q, GM Afédx. as CIQL‘éLI/fd O‘YL-tflb
SCIKSJQ “\ch

 

 

 

 

 

 

AUTHORIZED LAND USE

 

SPECIAL CONUTIONS AND/OR PENALTIES NOT CITED BELOW.

1324M“, MONSLLIQ 'Ib V“W+“M\ I‘uIM—LLL “’5‘ éwa’... EHJMISO’LQc‘ .gm
Q‘MS (L3 mpr‘W-LLL’

 

ADDRESS OF AUTHORIZED DEPARTMENT REPRESENTATIVES TO CONTACT RELATIVE TO OPERATIONS UNDER THIS PERMIT. PAY ANY INSTALLMENTS HERE.
LOCATION NAME Bud STREET CITY NONE NUMBER

MI H‘fimo (pas-25130

 

 

 

THIS PERMIT IS SUBJECT TO THE FOLLOWING CONDITIONS AND REOUIREM
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I HAVE READ THE CONDITIONS GOVERNIM THIS PERMIT AND AGREE TO ASIDE DY THEM IN THE CDNDLCT OF MY DPERATDNS UNDER THIS PERMIT.
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APPENDIX 6

APPENDIX 6

Tables of Flowering Plant Species Observed in Study Sites but Not Occuring
Transect Surveys

160

161

Table A6. 1. Plant species observed in flower in Allegan State Game Area study sites
during the Karner blue spring flight period, but not encountered in transect surveys, 1993
and 1994.

 

 

 

Year observed

Flowering plant species1 1993 1994
Achillea millefolium Yarrow ‘I
Antennaria plantagim'folia Pussytoes ‘I ‘I
Apocynum androsaemifolium Dogbane ‘/
Arabis sp. Rock Cress ‘I
Claytom'a virginica Spring Beauty ‘I
Convolvulus spithameus Erect Bindweed ‘I
Coreopsis Ianceolata Lance-leaved Coreopsis ‘1 ‘I
Elaeagnus umbellata Autumn Olive ‘/
Pedicularis canadensis Wood Betony ‘/
Penstemon hirsutus Hairy Beardtongue NI

Saponaria ofi‘icinalis Bouncing Bet NI
Senecia aureus Golden Ragwort \I
Tradescantia ohiensz's Spiderwort ‘1 ‘I
Tragopogon dubius Yellow Goatsbeard ‘/

Vaccinium angustifolium Blueberry \/
Viola pedata Birdfoot Violet ‘I
Viola spp. other Violet spp. \I
Vicia cracca Cow Vetch NI

 

1 Some plant species listed here were not observed in every site.

162

Table A6.2. Plant species observed in flower in Allegan State Game Area study sites
during the Karner blue summer flight period, but not encountered in transect surveys,
1993 and 1994.

 

 

 

Year observed
Flowering plant species1 1993 1994
Achillea millefolium Yarrow ‘1
Anemone virginiana Thimbleweed
Apocynum androsaemifolium Dogbane
Aureolaria pedicularia False Foxglove
Campanula rotundifolia Harebell

Ceanothus americanus
Daucus carota
Desmodium rotundifolium
Desmodium spp.
Dianthus armeria
Gnaphalium obtusifolium
HeIianthemum canadense
Helianthus divaricatus
HeIianthus occidentalis
Hieracium aurantiacum
Lespedeza hirta
Lespedeza intermedia
Lespedeza violacea
Lithospermum canescens
Lotus corniculatus
Medicago sativa
Oenothera biennis
Opuntia humifilsa
Penstemon hirsutus
Potentilla recta

Rosa carolina

Rudbeckia hirta

Solanum dulcamara
Solidago j uncea
Solidago nemoralis

New Jersey Tea
Queen Anne’s Lace
Prostrate Tick-trefoil
Tick-trefoils

Deptford Pink

Sweet Everlasting

F rostweed

Woodland Sunflower
Western Sunflower
Orange Hawkweed
Hairy Bush-clover
Wandlike Bush-clover
Bush-clover

Hoary Puccoon
Birdsfoot Trefoil
Alfalfa

Evening Primrose
Prickily Pear

Hairy Beardtongue
Rough-fi'uited Cinquefoil
Pasture Rose
Black-eyed Susan
Bittersweet Nightshade
Early Goldenrod

Gray Goldenrod

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163

Table A6.2 (cont’d)

Solidago speciosa Showy Goldenrod
Specularia perfoliata Venus Looking-glass
T ephrosia virginiana Goat’s Rue

T radescantia ohiensis Spiderwort

T ragopogon dubius Yellow Goatsbeard
Verbascum thaspus Common Mullein
Vicia cracca Cow Vetch

«é

 

' Some plant species listed here were not observed in every site.

 

 

 

APPENDIX 7

 

 

APPENDIX 7

Tables of Nectaring Observations of Summer Generation Karner Blue Adults

164

165

 

 

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APPENDIX 8

 

APPENDIX 8

Table of Percentage Canopy Cover and Frequency by Tree Species

167

168

 

 

 

 

 

 

 

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LIST OF REFERENCES

LIST OF REFERENCES

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Baker, R. J. 1994. The Karner blue butterfly: 1993 and beyond. pp. 163-169. In: D. A.
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169

 

 

170

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DeLucca II, A. J ., J. G. Sirnonson and A. D. Larson. 1981. Bacillus thuringiensis
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171

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173

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