MULTI-LEVEL ANALYSIS OF REPRODUCTIVE CHANGES IN HONEY BEE QUEENS (APIS ...

375 Pages·2012·5.63 MB·English

The Pennsylvania State University

The Graduate School

Department of Entomology
MULTI-LEVEL ANALYSIS OF REPRODUCTIVE CHANGES
IN HONEY BEE QUEENS (APIS MELLIFERA L.)
A Dissertation in

Entomology

by

Elina Lastro Niño
 2012 Elina Lastro Niño
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy

May 2012
ii
The dissertation of Elina Lastro Niño was reviewed and approved* by the following:

Christina M. Grozinger
Associate Professor of Entomology
Dissertation Advisor
Chair of Committee

James Tumlinson
Ralph O. Mumma Professor of Entomology

Diana Cox-Foster
Professor of Entomology

James Marden
Professor of Biology

Anita Collins
Adjunct Professor of Entomology

Gary W. Felton
Professor of Entomology
Head of the Department of Entomology

*Signatures are on file in the Graduate School
iii
ABSTRACT
Mating is an intricate process that causes many changes on behavioral, physiological, and
molecular level in females of sexually-reproducing species. Here, I study factors involved in
triggering and maintaining reproductive changes in honey bee queens. These changes can be
regulated by multiple factors including manipulation of the reproductive tract, insemination
volume, and seminal fluids. In honey bees, carbon dioxide, which is used as an anesthetic during
instrumental insemination, can also trigger post-mating changes. Previous studies on honey bees
suggest that insemination volume and seminal fluid components both play a role in triggering
queen post-mating changes. However, these studies only examined changes in newly
mated/inseminated queens and did not directly uncouple the effects of volume and substance.
The long-term effects on queens that have reached their final reproductive state – egg-laying –
had not been previously characterized.
By manipulating the mating process I examined the factors causing immediate behavioral
changes and longer-term physiological and molecular changes in queens that reached their final
reproductive state of high ovary activation. In chapter 1, I examined the behavioral (mating
flights and egg-laying), physiological (ovary activation), and molecular (gene expression)
changes in the queens treated with CO alone and queens treated with both CO and physical
2 2
manipulation. In chapter 2, I used the same parameters to characterize the queens inseminated
with low and high volumes of either semen or saline. My data indicate that CO , physical
2
manipulation of the genital tract, and insemination volume cause early behavioral changes, while
seminal fluid components play a role in maintaining long-term physiological and transcriptional
changes.
Mating also causes changes in production of queen pheromones which are important for
regulating the behavior and physiology of workers, as well as overall colony organization.
iv
Therefore, an important question is whether or not mating quality can have long-term effects on
pheromone production as well as on queen-worker interactions. Indeed, in chapter 3 I found that
the above mentioned factors have distinct effects on two different pheromone producing glands in
queens, and workers can detect these differences. Furthermore, in chapter 4 I demonstrated that
the queen insemination volume affected worker behavior and physiology in colonies, as well as
queen overwintering survival.
In conjunction with previous studies, my results show that multiple factors are involved in
regulating short- and long-term post-mating changes in honey bee queens. More importantly, in
social insects, this not only affects the reproductive female, but can alter social interactions in
entire colony by altering pheromone communication systems. The results of my dissertation will
lead to improved breeding techniques of honey bees and other agriculturally important insects,
and it will also provide fundamental insights into insect reproduction in general.
v
TABLE OF CONTENTS
LIST OF FIGURES ................................................................................................................. viii
LIST OF TABLES ................................................................................................................... ix
PREFACE ................................................................................................................................ x
ACKNOWLEDGMENTS ....................................................................................................... xii
Introduction: Honey Bee Queen (Apis mellifera L.) Biology .............................................. 1
Honey Bee Social Organization ....................................................................................... 2
Female Caste Differentiation in Honey Bees ................................................................... 2
Queen Development and Mating ..................................................................................... 5
Instrumental Insemination ................................................................................................ 8
Queen Reproductive Changes .......................................................................................... 9
References ........................................................................................................................ 15
Chapter 1. Genome-wide analysis of brain transcriptional changes in honey bee
queens (Apis mellifera L.) exposed to carbon dioxide and physical manipulation ... 25
Abstract ............................................................................................................................ 26
Introduction ...................................................................................................................... 27
Results .............................................................................................................................. 30
Behavioral Observations ............................................................................................. 30
Ovary Activation ......................................................................................................... 30
Vitellogenin Expression in Fat Bodies ........................................................................ 31
Brain Gene Expression ............................................................................................... 31
Comparative Analyses ................................................................................................ 32
Discussion ........................................................................................................................ 33
Experimental Procedure ................................................................................................... 39
General Bee Breeding ................................................................................................. 39
Behavioral Observation .............................................................................................. 40
Ovary Activation ......................................................................................................... 41
Vitellogenin Expression in Fat Bodies ........................................................................ 41
Brain Gene Expression ............................................................................................... 42
Comparative Analyses ................................................................................................ 44
Acknowledgments ............................................................................................................ 45
References ........................................................................................................................ 46
Chapter 2. Differential effects of insemination volume and substance on
reproductive changes in honey bee queens (Apis mellifera L.) ................................... 57
Abstract ............................................................................................................................ 58
Introduction ...................................................................................................................... 60
Results .............................................................................................................................. 63
vi
Behavioral Observation .............................................................................................. 63
Ovary Activation and Sperm Counts .......................................................................... 63
Gene Expression in Fat Bodies ................................................................................... 64
Comparative Analyses ................................................................................................ 66
qRT-PCR Confirmation of Expression of vitellogenin and transferrin Fat Bodies .... 67
Discussion ........................................................................................................................ 67
Materials and Methods ..................................................................................................... 72
General Bee Breeding ................................................................................................. 72
Behavioral Observation .............................................................................................. 73
Ovary Activation and Sperm Counts .......................................................................... 74
Gene Expression in Fat Bodies ................................................................................... 74
Comparative Analyses ................................................................................................ 76
qRT-PCR Confirmation of Expression of vitellogenin and transferrin Fat Bodies .... 77
Acknowledgments ............................................................................................................ 79
References ........................................................................................................................ 80
Chapter 3. Pheromone production in two glands is differentially regulated by
distinct mating factors in honey bee queens (Apis mellifera L.) ................................. 97
Abstract ............................................................................................................................ 98
Introduction ...................................................................................................................... 99
Results .............................................................................................................................. 102
Effects on the Dufour’s Gland Pheromone ................................................................. 102
Effects on the Mandibular Gland Pheromone ............................................................. 102
Retinue Choice Assay ................................................................................................. 103
Discussion ........................................................................................................................ 103
Materials and Methods ..................................................................................................... 109
General Bee Breeding ................................................................................................. 109
Gland Preparation ....................................................................................................... 110
Chemical Analysis of Gland Extracts ......................................................................... 110
Retinue Choice Assay ................................................................................................. 112
Acknowledgments ............................................................................................................ 114
References ........................................................................................................................ 115
Chapter 4. Effects of honey bee (Apis mellifera L.) queen insemination volume on
worker behavior and physiology................................................................................... 129
Abstract ............................................................................................................................ 130
Introduction ...................................................................................................................... 131
Materials and Methods ..................................................................................................... 134
General Beekeeping .................................................................................................... 134
Queen Longevity Measurements ................................................................................ 134
Brood Area Measurements ......................................................................................... 135
Worker Behavior ......................................................................................................... 136
Retinue Response ..................................................................................................... 136
Queen Cup and Cell Counts ..................................................................................... 137
Worker Physiology ..................................................................................................... 138
vii
Characterization of Juvenile Hormone and Methyl Farnesoate Hemolymph
Titers ........................................................................................................................ 138
Characterization of Worker Dufour’s Gland Extracts .............................................. 140
Results .............................................................................................................................. 141
Queen Longevity Measurements ................................................................................ 141
Brood Area Measurements ......................................................................................... 141
Worker Behavior ......................................................................................................... 142
Retinue Response ..................................................................................................... 142
Queen Cup and Cell Counts ..................................................................................... 142
Worker Physiology ..................................................................................................... 142
Characterization of Juvenile Hormone and Methyl Farnesoate Hemolymph
Titers ........................................................................................................................ 142
Characterization of Worker Dufour’s Gland Extracts .............................................. 143
Discussion ........................................................................................................................ 143
Acknowledgments ............................................................................................................ 148
References ........................................................................................................................ 149
Conclusions and Future Research ........................................................................................ 158
Multiple insemination factors are involved in triggering and maintaining queen
reproductive changes ................................................................................................ 158
Multiple factors modulate long-term changes in queen pheromone profiles and
queen-worker interactions ........................................................................................ 160
Future Directions .............................................................................................................. 161
References ........................................................................................................................ 164
Appendix A. Supplementary Material for Chapter 1 ......................................................... 167
Appendix B. Supplementary Material for Chapter 2 ......................................................... 286
viii
LIST OF FIGURES
Figure 1: Worker and Queen Ovaries. ..................................................................................... 4
Figure 2: Uncapped Queen Cell. .............................................................................................. 6
Figure 3: Queen Mating Sign. .................................................................................................. 7
Figure 4: Instrumental Insemination ........................................................................................ 8
Figure 1-1: Queen Behavior and Ovary Activation ................................................................. 52
Figure 1-2: Vitellogenin Expression ........................................................................................ 53
Figure 1-3: Principal Components Analysis of Significant Brain Genes ................................ 54
Figure 2-1: Queen Reproductive Behavior and Ovary Activation ........................................... 85
Figure 2-2: qRT-PCR Confirmation of Expression Levels of vitellogenin and transferrin. ... 86
Figure 2-3: Fat Body Gene Expression Hierarchical Clustering and Principal Components
Analysis ............................................................................................................................ 87
Figure 2-4: Biological Processes and Pathways Regulated Dependent on the
Insemination Substance and Volume ............................................................................... 88
Figure 3-1: Queen Dufour’s Gland Pheromone Production .................................................... 121
Figure 3-2: Linear Discriminant Analysis of Queen Mandibular Gland Pheromone
Components ..................................................................................................................... 122
Figure 3-3: Linear Discriminant Analysis of Queen Mandibular Gland Pheromone
Components ..................................................................................................................... 123
Figure 3-4: Queen Retinue Choice Assay ................................................................................ 124
Figure 4-1: Queen Survival. ..................................................................................................... 155
Figure 4-2: Worker Behavior .................................................................................................. 156
Figure 4-3: Worker Physiology ............................................................................................... 157
ix
LIST OF TABLES
Table 1-1: Significant Gene Ontology terms for genes differing significantly among all
three treatment groups ...................................................................................................... 55
Table 1-2: List of significantly regulated transcripts overlapping between the present
study and those of Kocher et al. (2008) and (2010). Directionality of the gene
expression among the studies is provided in Table S18................................................... 56
Table 2-1: Top 20 significant GO biological processes and GO pathways that were
overrepresented for the significantly regulated genes among all five queen groups ....... 90
Table 2-2: Significant GO biological processes and GO pathways that were
overrepresented for genes regulated by both saline and semen, saline only, and
semen only regardless of the insemination volume ......................................................... 92
Table 2-3: Significant GO biological processes and GO pathways that were
overrepresented for genes regulated by both 1and 8µl, 1µl only, and 8µl only
regardless of the insemination substance ......................................................................... 94
x
PREFACE
This dissertation contains multiply authored chapters with Elina Lastro Niño being the first
author.
In chapter 1, ELN carried out the behavioral, physiological, and genetic studies,
performed the statistical analysis, and drafted the manuscript. DRT participated in conception
and design of the study, manipulated the queens, and reviewed the manuscript. CMG participated
in the conception and design of the study, helped to draft the manuscript and coordinated the
research.
In chapter 2, ELN carried out the behavioral, physiological, and genetic studies,
performed the statistical analysis, and drafted the manuscript. DRT participated in conception
and design of the study, inseminated the queens, and reviewed the manuscript. CMG participated
in the conception and design of the study, helped to draft the manuscript and coordinated the
research.
In chapter 3, ELN carried out the physiological observations, performed the mandibular
gland analysis, performed the statistical analysis, and drafted the manuscript. DRT
participated in conception and design of the study, manipulated and inseminated the queens, and
reviewed the manuscript. OM and AH performed Dufour’s gland analysis and reviewed the
manuscript. CMG participated in the conception and design of the study, helped to draft the
manuscript and coordinated the research.
In chapter 4, ELN carried out the behavioral observations and physiological
manipulations, performed the statistical analysis, and drafted the manuscript. OM and AH
performed Dufour’s gland analysis and reviewed the manuscript. PT performed the JH and MF