The oviposition and movement behaviour of bt resistant and bt susceptible helicoverpa armigera (hubner) (lepidoptera noctuidae) on bt cotton and non bt cotton plants

The oviposition and movement behaviour of
Bt-resistant and Bt-susceptible Helicoverpa
armigera (Hübner) (Lepidoptera: Noctuidae)
on Bt cotton and non-Bt cotton plants
Luong, Thi Anh Tuyet
Bsc. MPh.

A thesis submitted for the degree of Doctor of Philosophy at
The University of Queensland in 2016
The School of Biological Sciences
i


Abstract
Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) has caused poor yields to a range of
agricultural crops, particularly to cotton. Pesticides have been used to control this pest with serious
undesirable side effects, including the rapid development of high levels of resistance. Since 1996,
genetically modified cotton (Bt cotton) has been planted to control H. armigera in Australia.
However, it is reported that surviving larvae of all sizes can be found in fields from time to time in
all growing regions. Research has shown that the survival of these larvae on Bt cotton is not
necessarily due to physiological resistance, and behavioural resistance has been inferred. Extensive
work on various aspects of behaviour of H. armigera in Australian has been conducted; however, to
date those experiments were carried out using a Bt-susceptible H. armigera strain. Experiments in
this thesis were undertaken with both physiologically Bt-resistant and Bt-susceptible lines of H.
armigera. One might expect larvae that are susceptible to Bt to show differences in behaviour in
comparison to Bt-resistant larvae.
Oviposition choice experiements consistently showed that both Bt-resistant and Bt-susceptible
moths did not choose plants or plant parts that were less toxic in terms of Bt toxin on which to lay
eggs. There was one exception in that Bt-susceptible moths were more likely to lay eggs on squares
of Bt cotton plants than those of non-Bt cotton. As expected the mortality of Bt-susceptible H.
armigera larvae was significantly higher on structures of Bt cotton plants than on those structures of

conventional cotton, and survival was greater on flowers than on other structures of Bt cotton.
Bt-susceptible neonates of H. armigera, which were significantly heavier, could starve longer and
recover better than Bt-resistant neonates. Although H. armigera neonates did not shift their
behaviour towards Bt toxin on artificial diet before their first feeding event, Bt-susceptible neonates
showed a tendency to remain on non-Bt diet and move off Bt diet. These behaviours may have
allowed them to survive in a Bt environment, and led to a higher percentage of survival and
pupation in situations where a choice was offered.
The behaviour of Bt-susceptible larvae, which differed from Bt-resistant larvae, could help them
avoid Bt toxin by moving off Bt substrates (drop-off behaviour) or staying on Bt substrates but
eating their conspecific eggs and so survive the first instar stage (cannibalism behaviour). There
was a significant difference in the numbers of Bt-susceptible larvae dropped off the two lines of
cotton, more Bt-susceptible larvae dropped off Bt cotton than non-Bt cotton plants over time.
Significantly more Bt-susceptible larvae remained on squares and flowers, and this may offer an
opportunity for them to survive on Bt-cotton plants. The survival of Bt-susceptible H. armigera
larvae significantly improves on Bt cotton plant when they cannibalize eggs before feeding on the
ii


plant. Cannibalism may play a significant part in the survival of Bt-susceptible H. armigera larvae
on Bt cotton plants. Egg cannibalism could explain the relatively small, but surprising number of
Bt-susceptible larvae surviving in Bt cotton fields.
In conclusion, subtle differences in the behaviour of Bt-susceptible female moths and first instar H.
armigera larvae may allow the larvae to survive on Bt cotton plants. Some of Bt-susceptible larvae
laid on squares or flowers that contain less Bt toxin and are generally more nutrient rich have higher
chances to survive. In addition, egg cannibalism contributes to the higher survival of Bt-susceptible
larvae on other structures of Bt cotton plants. Larvae can move off plant parts with high Bt levels
and may encounter less toxic plant parts. Bt-susceptible larvae can survive without food for 48 h,
suggesting they have time to re-establish on less Bt toxic plant parts after moving away from higher
toxic areas.

iii


Declaration by author
This thesis is composed of my original work, and contains no material previously published or
written by another person except where due reference has been made in the text. I have clearly
stated the contribution by others to jointly-authored works that I have included in my thesis.
I have clearly stated the contribution of others to my thesis as a whole, including statistical
assistance, survey design, data analysis, significant technical procedures, professional editorial
advice, and any other original research work used or reported in my thesis. The content of my thesis
is the result of work I have carried out since the commencement of my research higher degree
candidature and does not include a substantial part of work that has been submitted to qualify for
the award of any other degree or diploma in any university or other tertiary institution. I have
clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.
I acknowledge that an electronic copy of my thesis must be lodged with the University Library and,
subject to the policy and procedures of The University of Queensland, the thesis be made available
for research and study in accordance with the Copyright Act 1968 unless a period of embargo has
been approved by the Dean of the Graduate School.
I acknowledge that copyright of all material contained in my thesis resides with the copyright
holder(s) of that material. Where appropriate I have obtained copyright permission from the
copyright holder to reproduce material in this thesis.

iv


Publications during candidature
T.T.A. Luong, M.P. Zalucki, B. Cribb, L.E. Perkins and S.J. Downes. Oviposition site selection by
adults and the survival of susceptible and resistant first instar larvae of Helicoverpa armigera
(Hübner) (Lepidoptera: Noctuidae) on genetically modified and conventional cotton. Bulletin of
Entomological Research (Accepted, 2016)

Publications included in this thesis
Chapter 2
T.T.A. Luong, M.P. Zalucki, B. Cribb, L.E. Perkins and S.J. Downes. Oviposition site selection by
adults and the survival of susceptible and resistant first instar larvae of Helicoverpa armigera
(Hübner) (Lepidoptera: Noctuidae) on genetically modified and conventional cotton. Bulletin of
Entomological Research (Accepted, 2016)
A modified and re-formatted version of this has been incorporated into Chapter 2.
Contributor

Statement of contribution

Tuyet T. A. Luong (Candidate)

Designed experiments (80%)
Wrote the paper (70%)

Myron P. Zalucki (Principal supervisor)

Designed experiments (20%)
Wrote and edited paper (15%)

Lynda E. Perkins (Co-supervisor)

Wrote and edited paper (5%)

Sharon J. Downes (Co-supervisor)

Wrote and edited paper (5%)

Bronwen Cribb (Co-supervisor)

Wrote and edited paper (5%)

v


Contributions by others to the thesis
Professor Myron P. Zalucki, Lynda E. Perkins, Sharon J. Downes and Bronwen Cribb made
significant contributions to the conception and design of the project, and provided advice and
guidance regarding analyses throughout the research.

Statement of parts of the thesis submitted to qualify for the award of another degree
None

vi


Acknowledgements
It has been a long, tough and challenging journey and I am so grateful that I have finally finished
my PhD. However, this journey wouldn’t have been possible without the help, support and
encouragement of so many people around me to whom I would like to take this opportunity to
express my gratitude and appreciation.
I am grateful and would like to express my sincere thanks to my principal supervisors, Professor
Myron P. Zalucki for his patience and supervision of the research. I greatly appreciate and value the
help he provided me in developing logical thinking and teaching me on how to propose good
questions and finding ways to get the answers.
I would like to thank my co-supervisors Dr. Bronwen Cribb for her general support and helpful
advice during my PhD. I am eternally gratefully for Dr. Lynda E. Perkins for her useful advice on
statistics and writing. Thank you also to Dr. Sharon J. Downes, who, with her own practical
knowledge, has given me some excellent suggestions on the design of some of my experiments, and

gave me a chance to have wonderful experience on the field in Narrabri, NSW.
I am grateful to Vietnamese Government Scholarship (MOET) in conjunction with The University
of Queensland for funding my research during my PhD studying. I also acknowledge CSIRO, and
The Cotton Research and Development Corporation for supplying cotton seeds and Helicoverpa
armigera source for my experiments.
My field-based experience was wonderful due to the generous support of a handful of people from
CSIRO Agriculture, Australian Cotton Research Institute, Narrabri, Australia who gave their
knowledge, time and friendship to support me. In particular I would like to thank Tracey Parker,
who in the initial stages of my PhD gave me valuable experience in H. armigera, and continuously
sending cotton seeds and H. armigera eggs for my experiments.
Thank you to my friends, both inside and outside of the university, who have made my time during
this research a fun one. In particular to Corinna L. Lange and Jason Callander for their support and
often giving me their critical comments on certain chapters of this thesis.
Finally I would like to thank my family for their love and support throughout my PhD. My parents,
Luong Minh Son and Nguyen Thi Trinh, gave me a childhood that inspired a love of nature and
instilled a drive to further my education. Thank you also to my brother, Luong Minh Tung, who
encouraged me to begin my PhD, and to finish it. I deeply appreciate the support of my parents-inlaw, Tran Van Muoi and Nguyen Thi Hong Phuong during my PhD.
Lastly, I would like to thank my husband, Tran Xuan Hiep, for his love, support and encouragement
over my PhD. Luckily for me Hiep has an excellent understanding of computers and has assisted
me with computer skills. He has also given up his down time to assist with editing most of the
chapters.
vii


Keywords
Helicoverpa armigera, Bt-resistant, Bt-susceptible, Bt cotton, Bt-resistant behaviour, oviposition
behaviour, time to starvation, recovery ability, Bt-detection ability, drop-off behaviour, egg
cannibalism

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060801, Animal behaviour, 70%
ANZSRC code: 070308, Crop and Pasture Protection, 20%
ANZSRC code: 060499, Genetics not elsewhere classified, 10%

Fields of Research (FoR) Classification
FoR code: 0608, Zoology, 70%
FoR code: 0703, Crop and Pasture Production, 20%
FoR code: 0699, Other Biological Sciences, 10%

viii


Table of Contents
Abstract ..............................................................................................................................................ii
Acknowledgements ..........................................................................................................................vii
Chapter 1.............................................................................................................................................1
Literature Review
1.1 General introduction ................................................................................................................2
1.2 Host-plant selection behaviour ................................................................................................3
1.3 Oviposition preference .............................................................................................................9
1.4 The movement behaviour of first instar H. armigera..........................................................12
1.5 Bacillus thuringiensis toxins ..................................................................................................14
1.6 Bt crops and Bt cotton............................................................................................................14
1.7 The expression of Bt genes on Bt cotton ...............................................................................16
1.8 Behavioural resistance ...........................................................................................................17
1.9 Cannibalism in natural population .......................................................................................18
1.10 Structure of the thesis...........................................................................................................21
Chapter 2...........................................................................................................................................24
Oviposition site selection and survival of Bt-resistant and Bt-susceptible larvae of Helicoverpa
armigera (Hübner) (Lepidoptera: Noctuidae) on Bt and non-Bt cotton

2.1 Introduction ............................................................................................................................26
2.2 Materials and methods...........................................................................................................27
2.2.1 Insects ................................................................................................................................27
2.2.2 Plants..................................................................................................................................28
2.2.3 Oviposition preference.......................................................................................................30
2.2.4 Survival of newly hatched larvae.......................................................................................31
2.2.5 Data analysis ......................................................................................................................31
2.3 Results......................................................................................................................................33
2.3.1 Oviposition preference.......................................................................................................33
2.3.2 Survival ..............................................................................................................................34
2.4 Discussion ................................................................................................................................36
Chapter 3...........................................................................................................................................39
Feeding and survival of Bt-resistant and Bt-susceptible larvae Helicoverpa armigera (Hübner)
(Lepidoptera: Noctuidae) when exposed to a diet with Bt-toxin
3.1 Introduction ............................................................................................................................41
3.2 Materials and Methods ..........................................................................................................43
3.2.1 Insects ................................................................................................................................43
3.2.2 How long can larvae survive starvation? ...........................................................................43
3.2.3 Can H. armigera larvae recover after a period of starvation? ...........................................44
3.2.4 Can H. armigera larvae detect Bt toxin on artificial diet?.................................................44
3.2.5 Bt detection assay: detailed observations ..........................................................................45
3.2.6 Data Analysis .....................................................................................................................46
3.3 Results......................................................................................................................................47
3.3.1 How long can H. armigera larvae survive starvation? ......................................................47
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3.3.2 Can H. armigera larvae recover after a period of starvation? ...........................................48
3.3.3 Can H. armigera larvae detect Bt toxin on artificial diet?.................................................49
3.3.4 Bt detection assay: detailed observations ..........................................................................50

3.4 Discussion ................................................................................................................................55
Chapter 4...........................................................................................................................................58
The drop-off behaviour of Bt-resistant and Bt-susceptible Helicoverpa armigera (Hübner)
(Lepidoptera: Noctuidae) larvae on Bt-cotton and non-Bt cotton plants
4.1 Introduction ............................................................................................................................60
4.2 Material and Methods ............................................................................................................61
4.2.1 Plants..................................................................................................................................61
4.2.2 Insects ................................................................................................................................61
4.2.3 Drop-off behaviour of Bt-resistant and -susceptible H. armigera neonates on artificial diet
with and without Bt toxin ...........................................................................................................62
4.2.4 Drop-off behaviour of Bt-resistant and -susceptible H. armigera neonates on leaves of Bt
cotton and non-Bt cotton.............................................................................................................63
4.2.5 Drop-off behaviours of Bt-resistant and -susceptible neonates of H. armigera on different
structures of Bt and non-Bt cotton plants ...................................................................................64
4.3 Results......................................................................................................................................65
4.3.1 Drop-off behaviour of Bt-resistant and Bt-susceptible H. armigera larvae on artificial diet
with and without Bt toxin ...........................................................................................................65
4.3.2 Drop-off behaviour of Bt-resistant and -susceptible H. armigera larvae on leaves of Bt
and non-Bt cotton plants .............................................................................................................67
4.3.3 Drop-off behaviour of Bt-resistant and -susceptible H. armigera neonates on different
structures of Bt and non-Bt cotton plant .....................................................................................68
4.4 Discussion ................................................................................................................................72
Chapter 5...........................................................................................................................................76
Egg cannibalism in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae:
overcoming the plant establishment hurdle
Abstract .........................................................................................................................................77
5.1 Introduction ............................................................................................................................78
5.2 Materials and Methods ..........................................................................................................80
5.2.1 Egg cannibalism by Bt-susceptible H. armigera neonates on different eggs in a no choice
experiment...................................................................................................................................80

5.2.2 Comparison of egg cannibalism by Bt-resistant and -susceptible H. amigera neonates in a
no choice experiment ..................................................................................................................81
5.2.3 Egg cannibalism occurring on leaf discs ...........................................................................81
5.2.4 Does the first meal matter? ................................................................................................81
5.2.5 Analysis..............................................................................................................................83
5.3 Results......................................................................................................................................84
5.3.1 Egg cannibalism by Bt-susceptible H. armigera neonates on different eggs in a no choice
experiment...................................................................................................................................84
5.3.3 Egg cannibalism occurring on leaf discs ...........................................................................85
5.3.4 Does the first meal matter? ................................................................................................86
5.4 Discussion ................................................................................................................................91
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Chapter 6...........................................................................................................................................93
General Discussions
6.1 Oviposition behaviour of female moths and the survival of first instar H. armigera
larvae..............................................................................................................................................99
6.2 Feeding behaviour and the survival of first instar H. armigera larvae on Bt and non-Bt
diet................................................................................................................................................100
6.3 Staying or moving?...............................................................................................................101
6.4 Significance of this study......................................................................................................102
6.5 Future research.....................................................................................................................103
References .......................................................................................................................................104

xi


List of Figures
Figure 2.1 Materials used for oviposition experiments in glasshouse. (A) large cage; (B) cotton

plants arranged in large cage; (C) Bt cotton plants; (D) non-Bt cotton plants...................................29
Figure 2.2 Helicoverpa armigera eggs were laid on different cotton plant structures. (A) young
leaf; (B) mature leaf; (C) stem; (D) square; (E) flower; (F) boll .......................................................32
Figure 2.3 Mean percentage (± SE) of eggs that Bt-resistant (left) and Bt-susceptible (right) H.
armigera female moths laid on different plant structures on Bt cotton (white bars) and non-Bt
cotton (black bars) plants ...................................................................................................................33
Figure 2.4 Mean percentages (± SE) of Bt-resistant (left) and -susceptible (right) H. armigera
larvae that survived on plant parts (young leaf, mature leaf, square, and flower) of Bt cotton (white)
and non-Bt cotton (grey) after 2 d. Asterisks identified the significant difference in survival of Btsusceptible neonates on flowers between Bt cotton and non-Bt cotton. ............................................34
Figure 2.5 Mean percentages (± SE) of Bt-resistant (left) and -susceptible (right) H. armigera
larvae that survived on artificial diet after 6 d; 2 d on plant parts (young leaf, mature leaf, square,
and flower) of Bt cotton (white) and non-Bt cotton (grey) followed by 4 d on artificial diet.
Asterisks identified the significant difference in survival of Bt-susceptible larvae on flowers
between Bt cotton and non-Bt cotton.................................................................................................35
Figure 3.1 Diagram of a petri dish (6 cm diameter) with one H. armigera neonate (Bt-susceptible or
Bt-resistant neonate) between two cubes of artificial diet (Bt and non-Bt diet, 3 cm apart) .............45
Figure 3.2 Regression of time to death from starvation (y, hours) against the initial weight (x, µg)
of Bt-resistant (n = 80) (left) and –susceptible (n = 87) (right) H. armigera neonates......................47
Figure 3.3 The probability of survival (y) of Bt-resistant (n = 80) (dotted line) and Bt-susceptible (n
= 87) (black line) H. armigera larvae at a period of time to starvation (x, hours).............................48
Figure 3.4 Growth rate (µg weight per larva per day) of Bt-resistant (n = 122) (left) and Btsusceptible (n = 153) (right) H. armigera larvae after periods of starvation (x, hours) followed by
five days of growth on artificial diet (Growth rate = (ln (final weight) – ln (initial weight))/ 5 d) ...49
Figure 3.5 The mean percentage (± SE) of survival during 120 h of Bt-resistant (above) and Btsusceptible (below) H. armigera larvae released on different diets within test arenas: initial
release on diet + Bt toxin side (n = 5);  initial release on diet + water-side (n = 5);  control
treatments with Bt toxin on both sides (n = 3); Ο control treatments with water on both sides (n =
3).........................................................................................................................................................50
Figure 3.6 The mean percentage (± SE) (y, %) of Bt-resistant (n = 367) (above) and Bt-susceptible
(n = 325) (below) H. armigera neonates on Bt diet (dashed and dotted line), non-Bt diet (solid line)
and not on diet (dotted line) at different points of observation (x, min or hour). Observation was
conducted every 15 min within first 2 h and then at 12 h. .................................................................51

Figure 3.7 The percentages of Bt-susceptible H. armigera larvae (n = 325 larvae) at different
positions (Bt diet, n-Bt diet and off diet) at 120 min, 12 h, survival at 7 d, and pupation at 26 d of
xii


experiment. All numbers are shown as percentages of the initial % feeding. Note at 12 h all larvae
were placed on diet without Bt...........................................................................................................52
Figure 3.8 The percentages of Bt-resistant H. armigera larvae (n = 367 larvae) at different positions
(Bt diet, n-Bt diet and off diet) after 120 min, 12 h, survival after 7 d, and pupation at 21 d of
experiment. All numbers are shown as a percentage of the initial percentage of feeding. Note at 12 h
all larvae were placed on diet without Bt. ..........................................................................................53
Figure 3.9 (A), (B) small pot to contain H. armigera neonates in starvation assays. Bt detection
assay set up. (C) Bt-susceptible larvae on treatment without Bt toxin; (D) Bt-susceptible larvae on
treatment with and without Bt toxin; (E) Bt-susceptible larvae on treatment with Bt toxin; (F) Btresistant larvae on treatment with Bt toxin; (G) Bt-resistant larvae on treatment with and without Bt
toxin; (H) Bt-resistant larvae on treatment without Bt toxin .............................................................54
Figure 4.1 Diagram of an apparatus of 2 petri dishes connected by a 10 mm-high column (left) and
experiment with 20 complexes (right). Helicoverpa armigera larvae were introduced to the top Petri
dish and their drop off behaviour either observed or inferred by the presence of larvae on the lower
Petri dish.............................................................................................................................................62
Figure 4.2 Diagram drop-off experiment on leaves of Bt cotton (n = 22 plants) (row A) and non-Bt
cotton (n = 22 plants) (row B) plants arranged in laboratory. Five H. armigera Bt-resistant (n = 880
larvae) and Bt-susceptible (n = 880 larvae) larvae were introduced on different leaves of cotton
plants: (C) young leaf and (D) mature leaf. .......................................................................................64
Figure 4.3 Mean percentages (± SE) of Bt-resistant (n = 700) (above) and Bt-susceptible (n = 900)
(below) H. armigera neonates that dropped off Bt diet (dash dot line) and non-Bt diet (solid line).
Asterisks identified the significant difference in the percentages of Bt-resistant and –susceptible
larvae that dropped off Bt cotton and non-Bt diet..............................................................................65
Figure 4.4 The percentages of Bt-susceptible H. armigera larvae that dropped off or maintained on
Bt diet and non-Bt diet at 12 h and survived after 3 d .......................................................................66
Figure 4.5 Mean percentage (± SE) of Bt-resistant (n = 880) (left) and Bt-susceptible (n = 880)

(right) H. armigera larvae that dropped off Bt cotton (dash dot line) and non-Bt cotton plants (solid
line) at different time intervals. Asterisks identified the significant difference in the percentages of
Bt-susceptible larvae dropped off Bt cotton and non-Bt diet.............................................................67
Figure 4.6 Mean percentage (± SE) of Bt-resistant H. armigera larvae (n = 515) that dropped off
young leaves (solid line), mature leaves (dash line), squares (dot line), and flowers (dash dot line)
on Bt cotton (left) and non-Bt cotton plants (right) at different time intervals ..................................68
Figure 4.7 Mean percentage (± SE) of Bt-susceptible H. armigera larvae (n = 510) that dropped off
young leaves (solid line), mature leaves (dash line), squares (dot line), and flowers (dash dot line)
on Bt cotton (left) and non-Bt cotton plants (right) at different time intervals ..................................69
Figure 4.8 Mean percentage (± SE) of Bt-resistant (solid line) and Bt-susceptible (dot line) H.
armigera larvae that dropped off Bt (left) and non-Bt (right) young cotton leaves at different time
intervals ..............................................................................................................................................70
xiii


Figure 4.9 Mean percentage (± SE) of Bt-resistant (solid line) and Bt-susceptible (dot line) H.
armigera larvae that dropped off Bt (left) and non-Bt (right) mature cotton leaves at different time
intervals ..............................................................................................................................................70
Figure 4.10 Mean percentage (± SE) of Bt-resistant (solid line) and Bt-susceptible (dot line) H.
armigera larvae that dropped off Bt (left) and non-Bt (right) cotton squares at different time
intervals ..............................................................................................................................................71
Figure 4.11 Mean percentage (± SE) of Bt-resistant (solid line) and Bt-susceptible (dot line) H.
armigera larvae that dropped off Bt (left) and non-Bt (right) cotton flowers at different time
intervals ..............................................................................................................................................71
Figure 4. 12 Helicoverpa armigera neonates establish on different structures of cotton plant in
drop-off experiments. (A) young leaf; (B) small square; (C) mature leaf; (D) leaflet; (E) flower; (F)
H. armigera larvae moving on stem to find a better “food source”...................................................72
Figure 5.1 Experimental protocols were designed to assess the effects of the order in which food
was eaten: (5.1 a) cotton (Bt or non-Bt) or (5.1 b) conspecific eggs. Helicoverpa armigera larvae
were either introduced onto leaf discs for 24 h before being offered eggs for 48h, and their survival

on artificial diet was then assessed at 4 d (a) or larvae were offered eggs for 48 h before being
introduced onto leaf discs for 24 h, and their survival on artificial diet was assessed at 4 d (b).
83
Figure 5.2 The mean percentage (± 95% LSD interval) that Bt-susceptible H. armigera larvae
cannibalized different types of conspecific eggs: young eggs (n = 135), frozen eggs (n = 315) and
aged eggs (n = 360) over 48 h. ...........................................................................................................84
Figure 5.3 The percentages of Bt-resistant (grey bars) and Bt-susceptible (white bars) first instar H.
armigera larvae that cannibalized aged eggs or hatched neonates from the same strain in wells
without alternative food over 48 h. None is the percentage of larvae that did not eat eggs or
neonates..............................................................................................................................................85
Figure 5.4 The percentages of Bt-susceptible H. armigera larvae (n = 212 larvae) feeding (or not
feeding) on leaf discs (Bt and non-Bt leaf) for 24 h, cannibalizing conspecific eggs in wells for next
48 h, and their survival at 4 d on artificial diet. All numbers were shown in percentages relative to
106 larvae. ..........................................................................................................................................87
Figure 5.5 The percentages of Bt-susceptible H. armigera larvae (n = 200 larvae) cannibalizing or
not cannibalizing conspecific eggs in wells for 48 h, follow by feeding (or not feeding) on leaf discs
(Bt and non-Bt leaf) for next 24 h, and their survival at 4 d on artificial diet. All numbers were
shown in percentages relative to 100 larvae.......................................................................................88
Figure 5.6 Egg cannibalism of H. armigera larvae in wells without food after 48 h. (A), (B), (C)
larvae cannibalizing eggs; (D), (E), (F), (G), (H) show eggs damaged by larvae .............................89
Figure 5.7 Egg cannibalism by H. armigera larvae when on leaf-disc. (A), (B), (C), (D), (E), (F)
larvae cannibalizing eggs; (G), (H) larvae feeding leaf disc..............................................................90

xiv


List of Tables
Table 1.1 Comparison of plant defensive responses to at least one specialist and one generalist
insect herbivore from the same feeding guild (Ali & Agrawal 2012). Ordinal number in the result
column reflects consistency with the hypothesis: (1) no consistent pattern; (2) indicates that the

level of specialization was not predictive of plant responses and (3) indicates consistent, but only
two species are compared.....................................................................................................................5
Table 2.1 The mean (± SE) of eggs/ plant laid by Bt-resistant or -susceptible H. armigera female
moths on Bt cotton and non-Bt cotton plants in each cage (n =8) from December 2012 to April
2014. Means within a column followed by same letter are not significantly different (α = 0.05,
Tukey’s Multiple Range Test). The replicate with plants at different growth stages is highlighted .30
Table 3.1 The recovery ability (mean final weight (± SE) (µg) and mean mortality percentage (%
Dead)) of Bt-susceptible and -resistant H. armigera larvae after periods of starvation. Initial weight
was the mean weight (± SE) (µg) of larvae before artificial diet was introduced..............................48
Table 5.1 The percentage of Bt-resistant (n = 75) and Bt-susceptible (n = 60) larvae of H. armigera
that cannibalized old conspecific eggs on Bt and non-Bt cotton leaf discs after 24 h and 48 h
86
Table 6.1 The key findings of experimental chapters presented in this thesis ..................................97

xv


List of Abbrevations used in the thesis

Bt

Bacillus thuringiensis subsp. kurstaki

H.a / H. armigera

Helicoverpa armigera

vs.

Versus

SP15

Bt-resistant larvae

GR

Bt-susceptible larvae

LD 95

Lethal dose for 95% of mortality

Cry1Ac/ Cry 2Ab

Bt genes

ELISA

enzyme-linked immune-sorbent assay

RH

Relative humidity

L: D

Light: Dark

Non-Bt cotton

Conventional cotton (Sicot 71 RRF)

Bt cotton

Bollgard II® cotton (Sicot 71 BRR)

UC soil mix

a mixture of sand, bark and peat moss

min/ h/ d

minute(s)/ hour(s)/ day(s)

ANOVA

analysis of variance

df

Degree of freedom

SE

Standard Error

Fig.

Figure

xvi


Chapter 1
Literature Review

1


1.1 General introduction
Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is an economically important
polyphagous pest that causes major damage and poor yields to a range of agricultural crops (Zalucki
et al. 1986; 1994), particularly to cotton (Fitt 1994; Men et al. 2005), maize (Jallow & Zalucki
1998; Wu et al. 2004), various grain legumes (Sharma & Pampapathy 2004) and tobacco (Pang et
al. 2012; Zalucki et al. 2012). Pesticides have been used to control this pest with serious undesirable
side effects, including the rapid development of high levels of resistance. Pesticide resistance
management is now a necessity (Forrester et al. 1994; Tabashnik et al. 2009; Tabashnik & Carriere
2010; Zhang et al. 2011). Since 1996 cotton engineered to express insecticidal toxins from Bacillus
thuringiensis (Bt) has been planted to control Helicoverpa spp. in Australia (Fitt 2003), as in other
parts of the world (see below). It is now widely adopted, comprising nearly 90% of all cotton crops
in Australia (Zalucki et al. 2009, Jame 2015); subsequently there has been a significant reduction in
the use of insecticides against this pest (Fitt 2003; Wilson et al. 2013).
Although Bt cotton is effective in Australia in controlling Helicoverpa spp., farmers, scouts and
researchers occasionally report surviving larvae of all sizes for short periods in all growing regions
(Whitburn & Downes 2009). Specifically, a survey conducted from 2005-2008 estimated that on
average 15% of the area planted to Bt cotton carried larvae at or above the threshold levels
recommended for applying a control spray. Research has shown that the survival of larvae on Bt
cotton is not necessarily due to physiological resistance (Lu et al. 2011). A number of potential
alternative mechanisms may be responsible for the higher than expected survival, including poor

gene expression in genetically modified plants (Lu et al. 2011), pest load or pressure due to climate
(Zalucki & Furlong 2005), and/or behavioural mechanisms (Yang et al. 2008) including
behavioural resistance (Liu et al. 2010). Behavioural resistance could occur for instance, if larvae
survive on plants due to where females placed eggs or by moving to find ‘safe havens’, thereby
avoiding induced defenses and minimizing exposure to constitutive defenses (Perkins et al. 2013),
including Bt toxin (Downes & Mahon 2012). Such behaviours would require a genetic basis that
can be subject to selection in a Bt dominated environment.
The research reported here was designed to address a simple question: how do Bt-susceptible larvae
survive on Bt cotton plants? The focus of this research is the behaviour of H. armigera, especially
as it may relate to aspects of ‘behavioural resistance’ to Bt cotton. In particular, I address two major
areas of research to tease apart the potential role of egg deposition versus larval movement in
enabling Bt-susceptible larvae to survive on Bt cotton plants. Specifically I address a number of
smaller questions within these two areas:

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1. Do female moths choose oviposition sites within a crop randomly or do they place eggs on
plants and/ or locations within plants that express relatively low levels of Bt toxin? If the
latter, then I expect to find a relationship between oviposition site and the survival of first
instar larvae.
2. Do first instar larvae move the same way or differently on cotton with and without Bt toxin?
Can larvae detect Bt toxin levels in cotton plants and move to sites with relatively low
expression levels or do they move independently of toxin levels? Do larger neonates (from
bigger eggs) have greater reserves, which afford them a higher chance of finding sites that
express relatively low levels of Bt toxin or finding un-hatched eggs as a food source and so
survive the critical first instar stage?
In a first when addressing these questions I included a H. armigera strain that is physiologically
resistant to Bt toxin (Cry2Ab resistance, see below). The comparison of a Bt-susceptible and resistant strain enabled me to ascertain if there was any link between behaviour and physiological
resistance in this strain. Here firstly, I review the mechanisms of host-plant selection behaviour, and

the relationship between oviposition site, neonate movement and survival in general. The current
situation in Australia with regard to commercial production of Bt cotton, including managing for
resistance in Helicoverpa spp., is also covered. In particular, this review focuses on oviposition
behaviour of female moths, the movement and cannibalism behaviour of the first instars on both Bt
and non-Bt substrates (artificial diet and cotton plants).
1.2 Host-plant selection behaviour
Phytophagous insects (herbivores) have been conventionally categorized by their degree of dietary
specialization: from specialist to generalists. Insect herbivores, which feed on only one plant
species, are designated as monophagous (or specialists). Oligophagous species are insects that feed
on several plant species, usually within one botanical family. Finally, insects feeding on a wide
range of plants in many different families are polyphagous (or generalists) species (Bernays 1988;
Bernays & Chapman 1994; Bernays & Minkenberg 1997).
Finding suitable host plants is a challenging task. Insects use olfactory and visual cues to “choose”
or at least orient to their host from a distance. This process is integrated by numerous sensory
inputs, including olfactory or gustatory cues, and physical information, such as colour, shape and
texture from the plant (Visser 1988). Volatile chemical cues play a significant role enabling the
insect to recognize host plants from a distance (Dethier 1982; Bernays & Chapman 1994; Visser
1998). Plant volatiles are often complex mixtures of several hundred compounds (Visser 1986;
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Fraser et al. 2003). The blend of volatiles produced by host plants changes over time because of
physiological changes in the plant (Johnson et al. 2004). Helicoverpa armigera, for example, which
is a highly polyphagous herbivore with a host range of more than 100 species prefers the flowering
growth stage (Cunningham & Zalucki 2014). Moths respond to volatiles from flowering hosts
(Cunningham et al. 2004), and can locate host plants by learning the associated volatile blends
(Cunningham 2012).
In the conceptualization of the impacts of plant defensive compounds on herbivores, specialists,
which are considered as having physiological adaptations to cope with the plant defenses, are
thought to have greater tolerance of toxins than most generalist (Cornell & Hawkins 2003). Ali &

Agrawal (2012) confirmed that specialist insects did show a better tolerance to low level of toxins;
however, few insects were resistant to the various effects of plant toxins at higher levels. Generalist
herbivores can feed on a broad range of species by using mechanisms to suppress or reduce the
effects of plant defenses, more so than specialists (Dussourd & Denno 1994; Eichenseer et al.
1999). Polyphagous holometabolous insects in the larval feeding stages have extraordinarily high
and generalized tolerance to contact insecticides (Georghiou & Saito 1983). That could be the result
of selection for endurance to prolonged and varied biochemical stresses associated with the
diversity of their natural food plant (Gordon 1961). With respect to plant toxins, generalists are
more suppressive of plant defense (Musser et al. 2002; Zarate et al. 2007; Erb et al. 2012) probably
leading to a better survival or they can change their behaviour (behavioural resistance) in response
to plant toxins. Caterpillars of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) for example,
were able to secrete salivary glucose oxidase (GOX) (Eichenseer et al. 1999; Erb et al. 2012), which
is an effector responsible for the suppression of defense. A recent study of GOX levels in 85 species
(23 families of Lepidoptera) showed that there were higher levels of GOX in polyphagous species
compared with specialized species (Erb et al. 2012). On the other hand, specialists can be somewhat
tolerant of defenses (thus, not needing to be manipulative) or maximize their fitness in nonobvious
ways; e.g. time their feeding to less defended phenological stages or by location of less toxic
feeding sites (Ali & Agrawal 2012). Many studies and the predictions of the specialist–generalist
paradigm suggest that there could be consistency in plant recognition and herbivore elicitation
among different types of herbivore insects (Dussourd & Eisner 1987; Karban & Agrawal 2002; Ali
& Agrawal 2012). Cornelius & Bernays (1995) predicted three strategies of herbivores and their
expected relationships with plant toxins. Sequestering specialists benefited from the toxins at
intermediate levels, non-sequestering specialists were tolerant of toxin at low level, and generalist
benefited from suppressing induction. In the two first cases toxins finally impose a cost, while some
generalists specially benefited from feeding on toxic plants, even if they do not sequester the toxins.
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Ali & Agrawal (2012) found 20 studies to interpret this prediction by comparing the responses of a
plant to both specialist and generalist herbivores using one feeding guild (Table 1.1).

Table 1.1 Comparison of plant defensive responses to at least one specialist and one generalist
insect herbivore from the same feeding guild (Ali & Agrawal 2012). Ordinal number in the result
column reflects consistency with the hypothesis: (1) no consistent pattern; (2) indicates that the
level of specialization was not predictive of plant responses and (3) indicates consistent, but only
two species of insects are compared
Plant

Generalist Specialist

Brassicaceae
Arabidopsis
thaliana

(1) The generalist caused slightly more
changes in gene expression than did
the specialist (sequesterer). General
stress-responsive genes and
octadecanoid and indole GS synthesis
genes were similarly induced by
generalist and specialist (Mewis et al.
2006; Heidel & Baldwin 2004). The
specialist induced a lower GS response
than did the generalist (Bidart-Bouzat
& Kliebenstein 2011).
Aphididae Aphididae Transcriptional (1) Induction pattern by the two
M. persicaeB. brassicae responses, (GS) species depended on water status of the
plant (Winz & Baldwin 2001).
Noctuidae Pieridae
Transcriptional (1) Expression of GS genes was
Spodoptera Pieris rapae response, GS similar for generalist and specialist, but

exigua
GS levels only showed an increase in
response to S. exigua. Mean aliphatic
GS levels were equal. Pieris rapae
caused a higher increase in indolyl GS
content (Mewis et al. 2006).
Noctuidae Pieridae
Transcriptional (2) Transcription profiles were
S. littoralis P. rapae
response
indistinguishable (Reymond et al.
2004).
Noctuidae Pieridae
Parasitoid
(1) Parasitoid attracted to damaged
S. exigua P. rapae
specificity for plants over controls for both
herbivore
generalists and specialists. Parasitoids
Plutellidae induced plant only discriminate between induction
P. xylostella volatiles
by insects in different guilds (van
(HIPVs)
Poecke et al. 2003)

Brassicaceae
Brassica
oleraceae
Brassicaceae
A. thaliana

Brassicaceae
A. thaliana
Brassicaceae
A. thaliana

Brassicaceae
A. thaliana

Measure of
plant response
Aphididae Aphididae Transcriptional
Brevicoryne responses,
Myzus
persicae
brassicae glucosinolates
(GS)

Results

Noctuidae Pieridae
Transcriptional (2) Transcriptional responses and GS
Trichoplusia P. rapae,
responses, GS were not consistently influenced by
ni,
Plutellidae
degree of insect specialization (BidartS. exigua P. xylostella
Bouzat & Kliebenstein 2011).

5

Brassicaceae
Brassica nigra

Noctuidae Pieridae
 GS
Mamestra P. rapae,
brassicae Plutellidae
P. xylostella
Noctuidae (Pieridae) Foliar
T. ni
P. rapae
trichomes,
sinigrin, foliar
nitrogen

(2) Indole GS was significantly higher
after feeding by
P. rapae and M.
brassicae than after P. xylostella
feeding (Loon van et al. 2008).
Brassicaceae
(1) Differential induction by specialist
B. nigra
versus generalist led to increased
trichomes, but the effect reversed on
different leaf positions (Traw &
Dawson 2002).
Brassicaceae
Noctuidae Plutellidae Transcriptional (3) Specialist induced SA- and
Boechera

T. ni
P. xylostella response
ethylene-associated genes, whereas
generalist induced JA and ET genes
divaricarpa
(Vogel et al. 2007). The specialist
might be well adapted, but the plant
defends against the generalist.
Brassicaceae
Noctuidae Pieridae
Induced
(2) Variation in induction was found,
Raphanus
T. ni,�
P. rapae,
resistance,
but it was not associated with insect
sativus
S. exigua Plutellidae herbivore
specialization. P. xylostella and S.
P. xylostella performance exigua induced resistance to all,
whereas P. rapae only induced
resistance to P. rapae and S.
exigua.
T. ni did not induce resistance
(Agrawal 2000).
Brassicaceae
Noctuidae Tenthredinida GS,
(3) Specialist (sequesterer) and
S. frugiperdae
Sinapis alba

myrosinase
mechanical wounding induced GS and
Athalia
(MYR)
MYR threefold, whereas generalist
rosae
induced only GS (twofold) (TraversMartin & Mueller 2008) – generalist
might be adaptively suppressing
defense.
Lauraceae
Noctuidae Geometridae Peroxidase
(3) POD activity was more strongly
Lindera benzoin S. exigua Epimecis
activity (POD), induced by generalist than specialist
hortaria
C/N ratio,
(no difference in bioassay) (Mooney et
protein content, al. 2009) – plant might be adaptively
insect
defending against generalist.
bioassays
Plantaginaceae Nymphalidae Erebidae
Iridoid GS
(3) Higher IrGS induced by specialist
Junonia
Plantago
Spilosoma (IrGS), protein, (sequesterer) compared with generalist
ceonia
lanceolata
congra

foliar nitrogen (Stamp & Bowers 1994)– plant might
be adaptively defending against
generalist.
Poaceae
Chrysomel Chrysomeli Parasitoid
(3) Natural enemies preferred roots
Zea mays
idae
dae
specificity for attacked by specialist over roots
Diabrotica Diabrotica herbivore
damaged by generalist. The specialist
balteata
virgifera
induced plant induced significantly more (E)-bvolatiles
caryophyllene than the generalist.
Solanaceae
Noctuidae Sphingidae Phytohormones (3) Specialist induced JA/ET burst,
Nicotiana
S. exigua Manduca
generalist induced SA (Diezel et al.
attenuata
sexta
2009) – might be adaptive for
generalists to suppress resistance by
activating SA.
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Solanaceae

N. attenuata

Solanaceae
N. attenuata

Solanaceae
N. tabacum

Noctuidae Sphingidae Transcriptional
Heliothis M. sexta
response
virescens,
S. exigua

(1) Despite large overlap, the plant
response to the generalists was more
similar than the response to the
specialist. This was correlated to
FACs/oral secretions. Both generalists
were noctuids and down regulated a
large number of similar genes
(Voelckel & Baldwin 2004)
Noctuidae Sphingidae Phytohormones (1) M. sexta induced a JA and SA
T. ni,�
M. sexta
response, whereas
S. littoralis and T.
ni induced stronger SA responses
S. littoralis
(Heidel & Baldwin 2004).
Noctuidae Noctuidae Lipoxygenase (1) Both herbivores induced a similar

HelicoverpaHelicoverpa (LOX),
defensive response, but response
armigera assulta
proteinase
intensity of plants was different:
inhibitors (PIs), specialist induced a lower PPO
nicotine,
response and more intensive nicotine
peroxidase
and POD response than generalist (JA,
(POD),
LOX and PIs were not different) (Zong
polyphenol
& Wang 2007).
oxidase (PPO)

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The success of larvae depends on their ability to establish on a host plant (Perovíc et al. 2008).
Dethier (1982) indicated that lepidopteran larvae have limited sensory abilities; they need to be
physically in contact with a host plant to pick up sensory cues. Finding another host should a
caterpillar be dislodged or leave the plant on which it hatched posses some difficulties (Perovíc et
al. 2008). These difficulties may not be serious for highly mobile late stage caterpillars, and/or
when hosts are at high density. Even trial and error would work, although the perceptive distance of
caterpillars is very small (Bierzychudek et al. 2009). For walking insects, however, especially at
low host densities, finding hosts by chance seems likely to pose high risks of exposure to abiotic
mortality sources or encountering predators, and seriously increases the possibility of death by
starvation (Rausher 1979; Cain et al. 1985; Perovíc et al. 2008).
After hatching, caterpillars will seek a place to feed. During movement they use various senses to

contact and evaluate the plant. These caterpillars will respond to chemical and physical signals
offered by the plant. Volatile plant compounds occurring at relatively high concentrations in the leaf
boundary layer can affect insect behaviour (Baur et al. 1993). Many species make a major initial
behavioural decision, which is either to continue to assess the plant and locate a feeding site, or
reject the plant on the first contact with the physical and/or surface chemical characteristics
(Elaysed 2011). The insect may damage the plant in the next stage of the evaluation sequence by
test biting. Sensory information is gathered during contact evaluation and during initial feeding and
adjudged by the central nervous system. If it is positive, the insect will make the final decision in
the host-plant selection process, namely food intake is started (Eigenbrode & Espelie 1995),
otherwise the insect may continue to move and repeat the sampling process (Perkins et al. 2010).
Some components of the plant surface are common to many plants. They are considered as the
deterrents of establishment or larvae feeding (Kantiki & Ampofo 1989; Yang et al. 1993). Leaf
waxes, for instance, affect the survival and establishment of neonates. Shelomi et al. (2010)
suggested that larvae of H. armigera respond differently to different plant surfaces. Larvae spend
more time feeding on the thicker leaves of canola, with longer first meals on waxy canola than on a
wax-less variety because they need longer processing time to remove leaf waxes on the surface.
Moreover, leaf waxes can contain specific chemicals unique to each plant including primary and
secondary metabolites. These chemicals can have a positive or negative influence on the
establishment of feeding or oviposition behaviour of insects on a plant (Chapman & Bernays 1989).
Other mechanical problems that the larvae have to overcome to locate leaf tissue are toughness (due
to cell walls) and hardness (due to localized amorphous silica) (Lucas 2000; Cribb et al. 2010).
These factors vary within and among plants and can affect the wear and tear on mandibles (Bernays
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& Chapman 1994). Additionally, exposure to the leaf micro-flora; bacteria, fungi, yeasts and other
microbes growing on the leaf surface is potentially dangerous when neonates are attempting to
establish on the surface of leaves (Zalucki et al. 2002). Microorganisms may have definite effects
on host selection of insects by producing their own volatiles or influencing the mixture of host-plant
volatiles. Neonates of some species are able to distinguish between diets containing toxins, although

the mechanism is not clear. Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) neonates, for
example, have the ability to discriminate between diets which contain Bt toxins Cry1Ac and
Cry1Ab (Harris et al. 1997). The mean proportion of Spodoptera exigua (Hübner) (Lepidoptera:
Noctuidae) larvae on non-Bt diet was higher than on Bt diet (Stapel et al. 1998). In the choice tests
with Bt and non-Bt cotton leaves, significantly more H. armigera neonates were found away from
the Bt leaf discs, and lower consumption occurred with Bt cotton than in the choice tests containing
non-Bt cotton leaves (Zhang et al. 2004) (see Chapter 3).
In the context of finding surviving larvae on Bt crops, some obvious questions arise. Specifically,
do larvae detect Bt toxin (or some other correlated factors) and as a consequence have reduced
exposure to Bt? Or do some larvae just move more before feeding and incidentally locate better
quality or less toxic diets. Thus, it is necessary to examine whether there are any changes in hostfinding and feeding behaviour leading to better survival on Bt toxin substrates. Answers to such
questions will help us to understand behavioural resistance and contribute to current resistance
management.
1.3 Oviposition preference
Oviposition behaviour is a part of host finding and involves searching, orientating, encountering,
landing and acceptance behaviours (Morris & Kareiva 1991). These stages in the process depend on
a variety of sensory cues (Renwick & Chew 1994). Most studies on the initial stages of host finding
have focused on odours and visual factors such as plant size, shape and colour (Rausher 1979;
Stanton 1984; Singer 1993). When a female moth lands on a plant, she identifies the suitability of
the host plant by using a combination of physical and chemical signals from leaves or other plant
surfaces (Jallow et al. 1999). Helicoverpa spp. moths prefer to lay eggs on rough or hairy surfaces
(Zalucki et al. 1986), flowering plants (Jallow et al. 1999) and fruiting bodies (Matthews 1999).
Recent studies (Cunningham et al. 2004) concentrating on nectar feeding behaviour of H. armigera
confirmed the preference of moths to flower odours; for a review see Cunningham and Zalucki
(2014).
In addition, learning or experience can affect an insect’s preferences (Stanton 1984; Papaj 1986;
9