Table Of ContentCHEMICAL DEFENCES IN WEB-
BUILDING SPIDERS
SHICHANG ZHANG
A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF BIOLOGICAL SCIENCES
NATIONAL UNIVERSITY OF SINGAPORE
2013
I
ACKNOWLEDGEMENTS
I thank my PhD supervisor, Associate Professor Daiqin Li for his strong support throughout the
four years of my candidature. I deeply appreciate his advice, concern and help during my
experiments, which have inspired my interest in exploring the largely unknown research field—
chemical defences in spiders. Moreover, I have made great progress in ability of scientific writing,
logic reasoning, and critical thinking, which are all crucial to my future scientific career.
I am deeply indebted to Associate Professor Yee Hing Lai and Assistant Professor Yu Zhao
from the Department of Chemistry, NUS, who have shown generous chemistry technological
support during my experiments. Without help from them, I could not have made any progress
easily in chemical analysis during the experiments and revision of papers.
My appreciation goes to our research collaborators, Dr. Matjaž Kuntner, Dr. Simona Kralj-
Fišer, and Dr. Matjaž Gregorič in Slovenian Academy of Sciences and Arts, and Dr. Mark
Elgar from University of Melbourne. I have learnt a lot through collaborating with them, who
have developed my ideas and extended my knowledge in arachnology.
My sincere thanks go to Poh Moi Goh from the Department of Biological Sciences, NUS, for
maintenance of fruit flies and house flies, Dr. Qiping Liu and Woon Yew Siau from the
Department of Chemistry, NUS, Dr. Joanne Yew from Tamasek Life Science Laboratory (TLL)
for their support and advice in usage of GCMS throughout the duration of my research, and
Jeremy Woon from Singapore National Park Board for research permits, and all lab members
from Behavioural Ecology and Sociobiology Lab for their help throughout these years,
especially seniors Diego Pitta de Araujo, Dr. Teck Hui Koh, Dr. Wee Khee Seah and Dr.
Laura Yen Ling Yap for the generous welcome, tutorial, support and encouragement when I
first joined the lab and Jun Hao Tang for the identification of ants.
I am also grateful to Dr. Yong-Chao Su from University of Kansas, Dr. André Walter from
University of Melbourne, Fengxiang Liu, Xin Xu and Chen Deng from Hubei University, Dr.
Hui Ai from Central China Normal University and Zhanqi Chen from NUS for either collected
silk samples, caught spiders, or accompanied me in the field.
My four years of research journey would not have been smooth and successful without the
unwavering support of my parents. It was only with their constant encouragement did I
continually obtain new and exciting results.
Finally, I would like to thank National University of Singapore for the provision of the research
scholarship, the President’s Graduate Fellowship (PGF) and the research grants R-154-000-435-
112 and R-154-000-262-112 from Singapore Ministry of Education Academic Research Fund
(AcRF) to Professor Li for this research.
I
TABLE OF CONTENTS
Acknowledgements I
Table of contents II
List of tables XI
List of figures XII
Summary 7
CHAPTER 1 OBJECTIVES AND OUTLINE 9
CHAPTER 2 ACTIVE CHEMICAL DEFENCES IN ANIMALS 13
2.1. Introduction 14
2.2. Chemical defences in protecting animals themselves 15
2.2.1. Application of externally derived chemicals 15
2.2.2. Application of self-produced chemicals 18
2.2.3. Chemical camouflage and chemical deception for defence 20
2.3. Active chemical defence in habitat/nest/den protection 21
2.4. Active chemical defence in mating right protection 23
2.4.1. Prevent female from re-mating with competitors 23
2.4.2. Winning dominance in sperm competition 25
2.4.3. Protecting females from their natural enemies 26
2.5. Active chemical defence in egg protection 27
2.5.1. Directly application of exogenous chemical sources 27
2.5.2. Application of endogenous/self-produced chemical sources 28
2.5.3. Defend against competition from conspecifics 30
2.6. Active chemical defence in food resources protection 31
II
2.7. Conclusions and future research 33
CHAPTER 3 A NOVEL PROPERTY OF SPIDER SILKS: CHEMICAL 51
DEFENCE AGAINST ANTS
3.1. Introduction 52
3.2. Materials and Methods 54
3.2.1. Study subjects 54
3.2.2. Chemical analysis 54
3.2.3. Behavioural bioassays 56
3.2.4. Spider body size and web silk diameter 58
3.3. Results 60
3.4. Discussion 66
CHAPTER 4 THE SOURCE OF ANT DETERRENT 2- 70
PYRROLIDINONE IN THE GOLDEN ORB-WEB SPIDER NEPHILA
ANTIPODIANA (ARANEAE: NEPHILIDAE)
4.1. Introduction 71
4.2. Materials and Methods 73
4.2.1. Study objects 73
4.2.2. Testing the eggs for the presence of 2-pyrrolidinone 73
4.2.3. Testing for the presence of 2-pyrrolidinone in fruit flies Drosophila 74
melanogaster
4.2.4. Testing for the presence of 2-pyrrolidinone in the web silk of 74
juveniles and adults
4.2.5. Testing for the presence of 2-pyrrolidinone in different kinds of web 75
silk
4.2.6 Testing for the presence of 2-pyrrolidone in the silk that was directly 76
pulled from spinneret spigots
VII
4.3. Results 77
4.4. Discussion 81
CHAPTER 5 CHEMICAL EGG DEFENCE IN SPIDERS 84
5.1. Introduction 85
5.2. Materials and Methods 87
5.2.1. Study species and maintenance 87
5.2.2. Chemical analysis 88
5.2.3. Bioassays 89
5.3. Results 89
5.3.1. Chemical analysis 89
5.3.2. Bioassays 93
5.4. Discussion 93
CHAPTER 6 EVOLUTION OF ANT DETERRENT IN WEB BULIDING 97
SPIDERS
6.1. Introduction 98
6.2. Materials and Methods 100
6.2.1.Taxon sampling 100
6.2.2. Silk sampling 101
6.2.3. Identification of ant deterrent 101
6.2.4. Silk diameter test 102
6.2.5. Evolutionary analysis 103
6.3 Results 104
6.4. Discussion 109
VIII
CHAPTER 7 MATE BINDING: MALE ADAPTATION TO SEXUAL 125
CONFLICT IN THE GOLDEN ORB-WEB SPIDER (NEPHILIDAE:
NEPHILA PIPIPES)
7.1. Introduction 126
7.2. Materials and Methods 128
7.2.1. Spiders and maintenance 128
7.2.2. Behavioural definitions 129
7.2.3. Function of mate binding 130
7.2.4. Mechanisms of mate binding 131
7.2.5. Data analysis 133
7.3. Results 134
7.3.1 Behaviour of males and females during mate binding 135
7.3.2. Function of mate binding 137
7.3.3. Mechanisms of male mate binding 137
7.4. Discussion 141
CHAPTER 8 GENERAL DISCUSSION 145
8.1. Active chemical defences in animals 145
8.2. Ant deterrence behaviour 145
8.3. Source of ant deterrent 2-pyrrolidinone 146
8.4. Chemical egg defence in spiders 147
8.5. Evolution of ant deterrence behaviour in web-building spiders 148
8.6. Chemical defences used by males to decrease female cannibalism during 149
mating
8.7. Problems and limitations of the study 149
8.8. Future studies 150
IX
REFERENCES 152
Appendix A
Appendix B
X
LIST OF TABLES
Table No. Title Page
2-1 List of some reported cases of typical active chemical defences in 36
animals
3-1 The influence of the presence and absence of 2-pyrrolidinone on 61
the number of Monomorium pharaonis workers that cross
different kinds of silk bridges
3-2 The effects of silk age on the number of trials in which at least 62
one Monomorium pharaonis worker crossed the bridges
constructed with multiple silk threads produced by adult Nephila
antipodiana females and subject to different treatments (see text)
6-1 Spider species that have been surveyed for presence and absence 113
of ant deterrent 2-pyrrlidinone
7-1 Binary logistic regression for testing the effects of female and 140
male treatments on female aggressiveness (calmed: reference; not
calmed: predicted) during male mate binding
XI
LIST OF FIGURES
Figure No. Legend Page
3-1 The bioassay chamber and ants crossing the silk bridge. (a) The 59
bioassay chamber. Workers had access to and from the food
source via different silk bridges that were constructed from the
scaffold threads of webs of Nephila antipodiana. (b) A photo
showing two Pheidole angulicollis workers crossing the bridge
(Deterrent removed; top) constructed with the silk threads
produced by adult N. antipodiana and one ant retreating upon
contacting the bridge constructed with natural silk (Control;
bottom) produced by adult N. antipodiana
3-2 Total ion count of the GC-MS spectrum for the methanol extracts 60
of web silk of adult N. antipodiana, and (inset) the mass spectrum
and the structure of 2-pyrrolidinone (peak 1)
3-3 The influence of the presence of 2-pyrrolidinone on whether 63
pharaoh ants traversed bridges of silk produced by adult or large
juveniles and comprising either (a) multiple silk threads; and (b) a
single silk thread. Fisher’s exact tests for all paired comparisons,
p < 0.01; different lower case letters indicate the significance
differences
3-4 Passage of spiders across bridges constructed with scaffold silk 64
(Deterrent removed) and the diameter of silk produced by spiders
of different sizes. (a) Mean (± s.e.m.) body lengths of adult (n =
8), small (n = 4) and large (n = 6) juvenile N. antipodiana used
for the measurements of silk diameters (ANOVA: F = 77.753,
2,15
p < 0.001); (b) Effect of the silk thickness (i.e. diameter) on the
number of trials (Fisher’s exact test: p < 0.0001) in which at least
one Monomorium pharaonis worker crossed the single-thread silk
bridges constructed by adult (n = 5), large juvenile (n = 5) and
small juvenile (n = 5) spiders; (c) Mean (± s.e.m.) diameter (µm)
of the multiple-thread scaffold silk from the webs of N.
antipodiana adults (n = 8), large juveniles (n = 6), and small
juveniles (n = 4) collected in nature (ANOVA: F =14.988, p <
2,15
0.001); (d) Mean (± s.e.m.) diameter (µm) of the single-thread
scaffold silk from the webs of N. antipodiana adults (n = 8), large
juveniles (n = 6), and small juveniles (n = 4) collected in nature
(ANOVA: F =54.67, p < 0.001); (e) the number of trials in
2,15
which at least one P. angulicollis worker traversed the bridges
constructed with Deterrent removed and Control (i.e. natural) silk
threads obtained from adult N. antipodiana (n = 5); (f) the
number of trials in which at least one Monomorium sp. worker
crossed the bridges constructed with Deterrent removed and
Control silk threads produced by adult N. antipodiana (n = 5).
Post hoc Tukey HSD tests for all paired comparisons if ANOVA
revealed a significant overall effect, and post hoc Fisher’s exact
XII
test for all paired comparisons if Fisher’s exact test showed a
significant overall effect, p < 0.01; different low letters indicate
the significance differences
4-1 Total ion count of the GC-MS spectrum for the methanol extracts 78
of (a) eggs of Nephila antipodiana (n = 5); (b) web silk of small
N. antipodiana juveniles (n = 3); (c) fruit fly Drosophila
melanogaster (n = 3); (d) web silk of adult N. antipodiana
females (n = 3). Peak of 2-pyrrodilidone was pointed out by
arrow
4-2 Total ion count of the GC-MS spectrum for the methanol extracts 79
of different types of web silk produced by Nephila antipodiana.
(a) scaffold silk (n = 20); (b) frame silk (n = 6); (c) spiral silk (n =
6); and (d) radial silk (n = 15). Peak of 2-pyrrodilidone was
pointed out by arrow
4-3 Total ion count of the GC-MS spectrum for the methanol extracts 80
of (a) silks that were directly pulled out from spinnerets of
Nephila antipodiana (n = 3); and (b) whole silk glands of N.
antipodiana (n = 3)
5-1 Total ion count of the GC-MS spectrum for the methanol extracts 92
of (a) Nephila pilipes eggs; (b) the silk that covered the eggs of N.
pilipes; (c) Argiope versicolor eggs; (d) the silk that wrapped the
eggs of A. versicolor; (e) Nephilengys malabarensis eggs; (f) the
silk that covered the eggs of N. malabarensis; (g) Scytodes
pallida eggsac; (h) Pardosa pseudoannulata eggsacs. All tests
were replicated for 3 times
5-2 Eggsacs of wolf spider Pardosa pseudoannulata were under 93
consuming of ant workers (a) Oecophylla smaragdina; (b)
Monomorium pharaonis
6-1 Evolutionary route of ant deterrent in web-building spiders. 106
Branches in red refer to the taxa in which 2-pyrrolidinone was
present in the web silk, while branches in white refer to the taxa
in which 2-pyrrolidinone was absent in the web silk
6-2 Corelation between the evolution of ant-deterrent 2-pyrrolidione 107
and web architectures in web-building spiders
6-3 Corelation between the evolution of ant-deterrent 2-pyrrolidione 108
and spiral silk diameter in web-building spiders
7-1 The orb-web spider Nephila pilipes male and female showing 135
mate binding and treatments. (a) Mating showing the male
inserting its right palp into the female’s epigynum; (b) dorsal
view of female male showing a layer of fine silk on female’s
dorsum laid by male (resting on dorsal abdomen) after mate
XIII
Description:with liquid super glue (S–); (g) male tapping on female palps . been scrutinized further into active chemical defence and passive defence, . peptides, which take effect inside of animals, are beyond the scope of this review. the skin of Bufo toads, stimulating toads to release their toxin and an
