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WATER BALANCE, HOST UTILISATION, AND
MASS REARING IMPROVEMENTS OF THE
COCKROACH OOTHECAL PARASITOIDS,
Aprostocetus hagenowii (HYMENOPTERA:
EULOPHIDAE) AND Evania appendigaster
(HYMENOPTERA: EVANIIDAE)
TEE HUI SIANG
UNIVERSITI SAINS MALAYSIA
2016
WATER BALANCE, HOST UTILISATION, AND MASS
REARING IMPROVEMENTS OF THE COCKROACH
OOTHECAL PARASITOIDS, Aprostocetus hagenowii
(HYMENOPTERA: EULOPHIDAE) AND Evania appendigaster
(HYMENOPTERA: EVANIIDAE)
BY
TEE HUI SIANG
Thesis submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy
APRIL 2016
ii
ACKNOWLEDGEMENTS
I would like to thank my advisor Prof. Lee Chow Yang for his academic
guidance, support and expertise, which are fundamental to the completion of this
thesis, fulfilling the academic requirements for graduation. Nevertheless, his
mentoring, wisdom, enthusiasm, insightfulness, resourcefulness and kindness have
inspired, motivated and guided me while pursuing my doctorate studies at his Urban
Entomology Laboratory at USM. I am indebted to him for all these.
My sincere thanks to Dr. Yoshitaka Kamimura (Keio University, Japan) for
providing valuable discussions on my research and his time reviewing my manuscripts.
I am grateful to past and present Urban Entomology Labmates, Dr. Neoh Kok
Boon, Dr. Bong Lee Jin, Dr. Lee Ching Chen, Dr. Ang Ling Hui, Dr. Foo Foong Kuan,
Dr. Tay Jia Wei, Dr. Veera Singham, Nellie Wong Su Chee, Goh Xin Yu, Ikhsan
Guswenrivo, Nadiah Mohamed, Nurainie Ismail, Chris Kim Daeyun, Sophia Liew
Chiew Liang, Mai Duyen, Mark Ooi Chuan Sen, Jong Zheng Wei, Amy Low Kar Yin
and Kathleen Low for their friendship, assistance and support.
I would like to thank to Prof. Zairi Jaal, Dr. Hamdan Ahmad as well as the
staffs of Vector Control Research Unit, particularly Mr. Adanan Che Rus, Mr. Abdul
Malik Yahaya, Mr. Rohaizat Baba, Mr. Haslan Salim, Mr. Mohd Nasir Hassan, Mr.
Mohd Nasir Mohd Noor, Mr. Muhammad Noor Hisham Halim, Eugene Chan, Ms.
Ashiah Abu Seman and Ms. Nurul Mazni Yusuf for their assistance and technical
support (laboratory facilities, materials, etc.).
I am grateful to Universiti Sains Malaysia (USM Fellowship) and Agricultural
Crop Trust for their financial support of my PhD studies.
iii
My special thanks to my wife, Shin Yih, and my parents. This thesis would not
have been completed without their endless support.
iv
TABLE OF CONTENTS
Acknowledgements ii
Table of contents iv
List of Tables viii
List of Figures ix
List of Plates xiv
Abstrak xv
Abstract
xviii
CHAPTER ONE ‒ INTRODUCTION
1
CHAPTER TWO ‒ LITERATURE REVIEW
2.1 Periplaneta americana 5
2.2 Aprostocetus hagenowii 8
2.3 Evania appendigaster 11
2.4 Interspecific competition and coexistence of competing
species in parasitoid wasps
13
2.5 Water balance in insects 15
2.6 Factors influencing host selection and foraging in parasitoids 17
2.7 Rearing of A. hagenowii and E. appendigaster for biological
control of cockroaches
21
2.8
Biological control efficacy of A. hagenowii and E.
appendigaster
24
CHAPTER THREE ‒ WATER BALANCE PROFILES, HUMIDITY
PREFERENCE AND SURVIVAL OF TWO
SYMPATHRIC COCKROACH OOTHECA
PARASITOIDS Evania appendigaster and
Aprostocetus hagenowii
3.1 Introduction 25
v
3.2 Materials and methods 27
3.2.1 Rearing of E. appendigaster and A. hagenowii 27
3.2.2 Water balance profile 30
3.2.3 Survival of parasitoids at different RHs in the presence
and absence of a sugar solution
31
3.2.4 RH preference 32
3.2.5 Data analysis 34
3.3 Results 36
3.3.1 Water balance of E. appendigaster and A. hagenowii 36
3.3.2 Survival time of E. appendigaster reared at different
RHs
40
3.3.3 Survival time of A. hagenowii reared at different RHs 40
3.3.4 RH preference of E. appendigaster 43
3.3.5 RH preference of A. hagenowii 45
3.4
Discussion 47
CHAPTER FOUR – OOTHECA AGE AND TEMPERATURE
EFFECTS ON THE LIFE HISTORY OF
COCKROACH OOTHECA PARASITOID
Aprostocetus hagenowii
4.1 Introduction 55
4.2 Materials and methods 57
4.2.1 Insect rearing 57
4.2.2 Influence of age of oothecae incubated at different
temperatures on fitness traits of A. hagenowii
58
4.2.3 Influence of temperature on immature development of
A. hagenowii
59
4.2.4 Influence of temperature on survival, parasitism
activities and lifetime realized fecundity of A.
hagenowii
60
vi
4.2.5 Data analysis 62
4.3 Results 64
4.3.1 Influence of age of oothecae incubated at different
temperatures on fitness traits of A. hagenowii
64
4.3.2 Influence of temperature on immature development of
A. hagenowii
66
4.3.3 Influence of temperature on survival and parasitism
activities of A. hagenowii
70
4.4
Discussion 74
CHAPTER FIVE ‒ COCKROACH OOTHECA PARASITOID Evania
appendigaster EXHIBITS OVIPOSITION
PREFERENCE TOWARDS OOTHECA AGE
MOST VULNERABLE TO HOST
CANNIBALISM
5.1 Introduction 78
5.2 Materials and methods 81
5.2.1 Cockroach and parasitoid cultures 81
5.2.2 Cannibalism of healthy and parasitoid-parasitized
oothecae of different ages by P. americana
82
5.2.3 Ootheca age preference of E. appendigaster 83
5.2.4 Handling time and fitness traits of E. appendigaster on
different-aged oothecae
85
5.2.5 Data analysis 87
5.3 Results 88
5.3.1 Cannibalism of healthy and parasitoid-parasitized
oothecae of different ages by P. americana
88
5.3.2 Ootheca age preference by E. appendigaster 91
5.3.3 Handling time and fitness traits of E. appendigaster on
different-aged oothecae
91
5.4 Discussion 96
vii
CHAPTER SIX ‒ FEASIBILITY OF COLD-STORED Periplaneta
americana OOTHECAE FOR REARING THE
OOTHECA PARASITOIDS Aprostocetus hagenowii
and Evania appendigaster: EFFECT OF OOTHECA
AGE AND STORAGE DURATION
6.1 Introduction 101
6.2 Materials and methods 103
6.2.1 Rearing of P. americana 103
6.2.2 Rearing of A. hagenowii and E. appendigaster 104
6.2.3 Effect of heating on hatchability of oothecae 105
6.2.4 Rearing of A. hagenowii and E. appendigaster from
live, heat-killed, and cold-stored oothecae aged 1–4
weeks old
106
6.2.5 Data analysis 108
6.3 Results 110
6.3.1 Effect of heating on hatchability of oothecae 110
6.3.2 Biological parameters of A. hagenowii reared on live,
heat-killed, and cold-stored oothecae
110
6.3.3 Biological parameters of E. appendigaster reared on
live, heat-killed, and cold-stored oothecae
117
6.4
Discussion 121
SUMMARY AND CONCLUSION 128
REFERENCES 135
List of Publications and Presentations 155
viii
LIST OF TABLES
Page Table 3.1 Fresh mass, percentage of total body water, cuticular
permeability, and surface area to volume ratio (mean ±
SE) of males and females of E. appendigaster and A.
hagenowii.
37
Table 3.2 Statistics for the linear regression between cumulative
%TBW loss and desiccation time (h) for males and
females of E. appendigaster and A. hagenowii.
39
Table 4.1 Mean (± SE) percentage emergence, development time
(days), number of progeny/ootheca and percentage female
progeny of A. hagenowii emerged from P. americana
oothecae at 20‒35 °C.
68
Table 5.1 Results of three-way ANOVA of the effects of ootheca
age, cockroach life stage, and ootheca parasitism status on
percentage cannibalism of oothecae in P. americana.
89
Table 6.1 Two-way ANOVA of the % parasitism, % emergence and
log of the developmental time (days) for A. hagenowii and
E. appendigaster.
112
Table 6.2 The effect of progeny sex and cold storage conditions on
developmental time of E. appendigaster.
122
ix
LIST OF FIGURES
Page
Figure 3.1 Diagram of the linearly connected chambers. Four
different salt solutions (MgCl2, NaBr, NaCl, and KNO3)
were used to create a gradient of increasing RH among
the chambers.
33
Figure 3.2 Cumulative % total body water loss of males and females
of E. appendigaster and A. hagenowii with desiccation
time (0‒24 h) at 24.0 ± 0.1°C and 0‒2% RH.
38
Figure 3.3 Survival (ST50, d) of E. appendigaster males (A) and
females (B) when kept at 38%, 56%, 76%, and 87% RH
in the absence and presence of food (10% sugar solution).
Different letters and asterisks above bars denote
significant differences in survival of parasitoids between
different RHs in each food condition and between
different food conditions in each RH, respectively. The
vertical line on each bar represents the ± 95% fiducial
limit. Numbers inside bars indicate sample size.
41
Figure 3.4 Survival (ST50, d) of A. hagenowii males (A) and females
(B) when kept at 38%, 56%, 76%, and 87% RH in the
absence and presence of food (10% sugar solution).
Different letters and asterisks above bars denote
significant differences in survival of parasitoids between
different RHs in each food condition and between
different food conditions in each RH, respectively. The
vertical line on each bar represents the ± 95% fiducial
limit. Numbers inside bars indicate sample size.
43
Figure 3.5 Mean % distribution of E. appendigaster males (A) and
females (B) in linearly connected chambers with a
gradient of 44%, 56%, 76%, and 87% RH. Different
letters above each bar indicate significant differences in
the distribution of parasitoids among RH chambers at
each time interval (LSD test, P < 0.05). The vertical line
on each bar represents standard error of the mean (n = 8).
44
Figure 3.6 Mean % distribution of A. hagenowii males (A) and
females (B) in linearly connected chambers with a
gradient of 44%, 56%, 76%, and 87% RH. Different
letters above each bar indicate significant differences in
46
x
the distribution of parasitoids among RH chambers at
each time interval (LSD test, P < 0.05). The vertical line
on each bar represents standard error of the mean (n = 10).
Figure 4.1 (A) P. americana embryonic development time at 20, 25
and 30 °C. (B) Percentage emergence of A. hagenowii on
different-aged oothecae incubated at 20, 25 and 30 °C.
Asterisk *, ** and ** indicate no nymphs hatching from
oothecae aged 30 d at 30 °C, 40 d at 25 °C and 60 d at 20
°C, respectively. Vertical line on each bar indicates
standard error of the mean (n = 5).
65
Figure 4.2 Development time (A), number of progeny
produced/ootheca (B), percentage female
progeny/ootheca (C) and HTL (D) of A. hagenowii in
different-aged oothecae incubated at 20, 25 and 30 °C.
For each incubation temperature, bars with different
letters indicate significant difference between different-
aged oothecae (Tukey’s HSD test, P < 0.05). Sample size
for each ootheca age at each incubation temperature; 20
°C: 1, 10, 20, 30, 40 and 50 d = 22, 20, 25, 21, 22; 25 °C:
1, 10, 20 and 30 d = 24, 24, 24, and 22; 30 °C: 1, 10, 20
d = 23, 23 and 21, respectively. Vertical line on each bar
indicates standard error of the mean.
67
Figure 4.3 Linear (dotted line) and nonlinear model (solid line) fitted
to the observed developmental rate (days‒1) (white circle)
of A. hagenowii at 20‒32 °C. Sample size for 20, 25, 30
and 32 °C = 27, 29, 29 and 26, respectively.
69
Figure 4.4 Kaplan-Meier survival curves of A. hagenowii males and
females reared with water alone or a 10% sugar solution
at 20‒35 °C.
71
Figure 4.5 The number of oothecae parasitized (A) and lifetime
realized fecundity (B) of A. hagenowii females reared
with water alone or a 10% sugar solution at 20‒32 °C.
Asterisk (*) indicates significant difference between food
treatments at each temperature (Student’s t-test, P <
0.05). Vertical line on each bar indicates standard error of
the mean (n = 12).
72
xi
Figure 4.6 Parasitism activities (%) of A. hagenowii females at
different time intervals when reared with water alone (A)
or a 10% sugar solution (B) at 20‒35 °C.
73
Figure 5.1 Experimental set up for ootheca age preference of E.
appendigaster.
84
Figure 5.2 Mean (± SE) percentage cannibalism of different-aged
(A) healthy and (B) E. appendigaster-parasitized
oothecae by P. americana mid-nymphs, males, and
females. Letters without and with parentheses indicate
significant differences in percentage cannibalism among
different-aged oothecae by each cockroach life stage and
among cockroach life stages in each ootheca age,
respectively (Tamhane’s test, P < 0.05). The vertical line
on each bar represents standard error of the mean (n = 6).
90
Figure 5.3 Oviposition preference (%) of E. appendigaster females
(n = 60) when presented simultaneously with five
oothecae each at 1, 10, 20, 30, and 40 d old. Bars labeled
with different letters are significantly different (chi-
square test followed by Tukey-type multiple
comparisons, P < 0.05).
92
Figure 5.4 Relationship between ootheca age and various fitness
traits of E. appendigaster: (A) handling time (y = 1140.5
+ 50.75x – 0.5684x2, F2, 57 = 30.77, r2 = 0.0.435, n = 12
for each ootheca age, P < 0.001); (B) proportion
parasitism attempted; (C) proportion successful
parasitism; and (D) sex ratio. In (B), (C), and (D), binary
data were analyzed using logistic regressions (n = 120,
120, and 75, respectively) but are presented as a
proportion for each ootheca age. A significant
relationship was established only in (C), in which the
fitted logistic regression line shows the estimated
probability (P) of an ootheca successfully producing a
wasp (P = exp (2.013 ‒ 0.019x) / (1 + exp (2.013 ‒
0.019x)), x = ootheca age).
93
Figure 5.5 Relationship between ootheca age and various fitness
traits of E. appendigaster progeny: (A) development time
of males (y = 0.102x + 45.10, F1, 29 = 30.77, r2 = 0.515, P
< 0.001) and females (y = 0.099x + 45.61, F1, 42 = 36.25,
r2 = 0.463, P < 0.001); (B) hind tibia length of males (y =
‒0.008x + 3.47, F1, 29 = 12.36, r2 = 0.299, P = 0.002) and
females (y = ‒0.009x + 3.42, F1, 42 = 20.22, r2 = 0.325, P
95
xii
< 0.001); (C) longevity of males (y = ‒0.254x + 30.88, F1,
29 = 12.95, r2 = 0.309, P = 0.001) and females (y = ‒
0.172x + 22.31, F1, 42 = 9.56, r2 = 0.185, P = 0.004); and
(D) lifetime realized fecundity (P > 0.05 for both linear
and nonlinear models). Sample size for oothecae aged 1,
10, 20, and 30 d old: males = 10, 8, 8, and 5; females =
16, 15, 8, and 5, respectively.
Figure 6.1 Mean percentage mortality of oothecae aged 1–4-week
old after exposure to heating at 48, 50 and 53°C for 15,
30, 45 and 60 min. Vertical line on each bar represents
standard error.
111
Figure 6.2 Mean percentage parasitism (A) and emergence (B) of A.
hagenowii in live, heat-killed and 0.5–4-month cold-
stored oothecae of P. americana aged 1–4-week old.
Letters above each bar are assigned for comparison of
ootheca age groups within each storage duration and
letters in parentheses are used for comparison of storage
durations within each ootheca age group. Bars labelled
with the same letter are not significantly different at α =
0.05 (Tukey’s HSD test). Vertical line on each bar
represents standard error.
113
Figure 6.3 Mean number of A. hagenowii progeny produced per
ootheca in live, heat-killed and 1–4-month cold-stored
oothecae of P. americana aged 1–4-week old. Bars
labelled with the same letter are not significantly different
at α = 0.05 (Dunn’s test). Vertical line on each bar
represents standard error. Numbers inside bars are sample
size.
116
Figure 6.4 Mean percentage female progeny of A. hagenowii
emerged from live, heat-killed and 1–4-month cold-
stored oothecae of P. americana aged 1–4-week old. Bars
labelled with the same letter are not significantly different
at α = 0.05 (Dunn’s test). Vertical line on each bar
represents standard error. Numbers inside bars are sample
size.
118
Figure 6.5 Mean developmental time (days) of A. hagenowii
emerged from live, heat-killed and 1–4-month cold-
stored oothecae of P. americana aged 1–4-week old. Bars
labelled with the same letter are not significantly different
at α = 0.05 (Dunn’s test). Vertical line on each bar
119
xiii
represents standard error. Numbers inside bars are sample
size.
Figure 6.6 Mean percentage parasitism (A) and emergence (B) of E.
appendigaster in live, heat-killed and 0.5–4-month cold-
stored oothecae of P. americana aged 1–4-week old.
Letters above each bar are assigned for comparison of
ootheca age groups within each storage duration and
letters in parentheses are used for comparison of storage
durations within each ootheca age group. Bars labelled
with the same letter are not significantly different at α =
0.05 (Tukey’s HSD test). Vertical line on each bar
represents standard error.
120
xiv
LIST OF PLATES
Page
Plate 2.1 Adult male of P. americana. 6
Plate 2.2 Aprostocetus hagenowii female attempts to drill and
insert its ovipositor into a P. americana ootheca.
9
Plate 2.3 Evania appendigaster female remains quiescence on an
ootheca with its tip of ovipositor touching the surface of
ootheca.
12
xv
KEIMBANGAN AIR, PENGGUNAAN PERUMAH, DAN PEMBAIKAN
KULTUR MASSA PARASITOID OOTEKA LIPAS, Aprostocetus hagenowii
(HYMENOPTERA: EULOPHIDAE) DAN Evania appendigaster
(HYMENOPTERA: EVANIIDAE)
ABSTRAK
Tesis ini menumpu kepada keseimbangan air, penggunaan perumah and
peningkatan kultur massa Aprostocetus hagenowii (Ratzeburg) (Hymenoptera:
Eulophidae) dan Evania appedigaster (Linnaeus) (Hymenoptera: Evaniidae), dua
parasitoid ooteka lipas Amerika Periplaneta americana (Linnaeus) (Blattidae:
Dictyoptera). Evania appendigaster mempunyai kebolehtelapan kutikal yang lebih
tinggi daripada A. hagenowii. Kesemua parasitoid, kecuali jantan A. hagenowii,
mempunyai kadar kehilangan jumlah kandungan air dalam badan yang sama.
Kelanjutan usia kesemua parasitoid, kecuali jantan E. appendigaster, berkurangan
dengan penurunan kelembapan relatif (RH) tetapi fenomena tersebut tidak berlaku
antara parasitoid-parasitoid yang diberi gula. Dalam kebuk yang mengandungi satu
gradien RH 44‒87%, kedua-dua jantina E. appendigaster menunjukkan
kecenderungan kepada kebuk yang paling lembap. Bagi A. hagenowii, betina
menunjukkan kecenderungan kepada kedua-dua kebuk yang paling kering dan yang
paling lembap dan jantan menunjukkan kecenderungan kepada kebuk yang paling
kering. Pengaruh umur ooteka dan suhu malar ke atas sejarah kehidupan A. hagenowii
telah dikaji. Aprostocetus hagenowii mampu berkembang dalam ooteka-ooteka
mencapai sehingga 72.9% daripada jumlah jangka waktu perkembangan embrio tanpa
sebarang kesan ke atas ciri-ciri kecergasan mereka. Ambang suhu bawah, atas dan
xvi
optimum suhu adalah 9.5, 34.2 and 31.1 °C, masing-masing. Suhu tidak memeberikan
ciri reproduksi betina tetapi menpengaruhi activti parasitisme melalui masa. Kesan
umur ooteka ke atas kanibalisme ooteka pada P. americana dan pengaruhannya ke atas
pemilihan umur ooteka dan kecergasan dalam E. appendigaster telah diperiksa.
Periplaneta americana lebih cenderung memakan ootheca berumur 1 hari berbanding
dengan ooteka berumur 10‒40 hari. Walaubagaimanapun, parasitoid betina lebih
cenderung memparasit ooteka berumur 1 hari daripada yang berumur 10‒40 hari.
Kadar kemunculan adalah lebih tinggi dan masa pengendalian lebih pendek pada
ooteka berumur 1 hari berbanding dengan yang lebih tua. Bagi progeni parasitoid,
masa perkembangan meningkat, dan saiz badan dan jangka hayat berkurangan dengan
umur ooteka. Keputusan-keputusan ini mecadangkan bahawa betina E. appendigaster
mendagangkan kemandirian progeni untuk keuntungan kecergasan bagi mereka
sendiri dan progeny mereka. Masa pemanasan minium yang diperlukan untuk
membunuh ooteka telah ditentukan dan kesesuaiannya sebagai perumah setelah
disimpan dalam berberapa keadaan yang berbeza (secara hidup, dibunuh dengan
pemanasan dan disimpan sejuk selama 0.5‒4 bulan pada 4 °C) untuk pembiakan
parasitoid A. hagenowii dan E. appendigaster telah dikaji. Pemanasan pada 48 °C
selama 45 min membunuh ooteka-ooteka yang pelbagai umur. Rawatan pemanasan
meningkatkan kesesuaian ooteka berumur 4 minggu sebagai perumah untuk
pembiakan parasitoid. Bagi A. hagenowii, kadar penetasan yang rendah telah
didokumenkan antara ooteka-ooteka berumur 3‒4 minggu yang telah disimpan sejuk.
Bilangan progeni yang dihasilkan daripada ooteka berumur 1‒2 dan 3-4 minggu
berkurangan setelah disimpan sejak 3‒4 dan 0.5‒4 bulan, masing-masing. Evania
appendigaster mempunyai kadar penetasan yang rendah dalam ooteka yang disimpan
sejuk.
xvii
WATER BALANCE, HOST UTILISATION, AND MASS REARING
IMPROVEMENTS OF THE COCKROACH OOTHECAL PARASITOIDS,
Aprostocetus hagenowii (HYMENOPTERA: EULOPHIDAE) AND Evania
appendigaster (HYMENOPTERA: EVANIIDAE)
ABSTRACT
This thesis focuses on the water balance, host utilization and mass rearing
improvements of Aprostocetus hagenowii (Ratzeburg) (Hymenoptera: Eulophidae)
and Evania appeindgaster (Linnaeus) (Hymenoptera: Evaniidae), two ootheca
parasitoids of the American cockroach, Periplaneta americana (Linnaeus) (Blattidae:
Dictyoptera). Evania appendigaster had higher cuticular permeability than A.
hagenowii. All of the parasitoids, except A. hagenowii males, had similar percentage
total body water loss rate. The survival times of all the parasitoids, except E.
appendigaster males, reduced with decreasing relative humidity (RH) but this
phenomenon did not occur among sugar-fed parasitoids. In chambers with a 44‒87%
RH gradient, both sexes of E. appendigaster preferred the wettest chamber. For A.
hagenowii, females preferred both the driest and the wettest chambers and males
preferred the driest ones. The effect of ootheca age and constant temperature on the
life history of A. hagenowii were studied. A. hagenowii could successfully develop in
oothecae attaining up to 72.9% of the total embryonic development time without any
effect on their fitness traits. The lower, upper and optimal temperature thresholds were
9.5, 34.2 and 31.1 °C, respectively. Temperature did not affect females’ reproductive
traits but did influence parasitism activities over time. The effect of ootheca age on
ootheca cannibalism in P. americana and its influence on ootheca age selection and
xviii
fitness in E. appendigaster was examined. Periplaneta americana differentially
cannibalized 1 d versus 10‒40 d old oothecae. However, parasitoid females preferred
to parasitize 1 d old than 10‒40 d old oothecae. The emergence rate was greater and
the handling time was shorter on 1 d old compared to older oothecae. For parasitoid
progeny, development time increased and body size and longevity decreased with
ootheca age. These results suggest that E. appendigaster females traded progeny
survival for fitness gains for themselves and their progeny. The minimum time of
heating required to kill oothecae was determined and their suitability as hosts after
different storage conditions (live, heat killed, and stored for 0.5‒4 months at 4 °C) for
rearing parasitoids A. hagenowii and E. appendigaster was investigated. Heating at 48
°C for 45 min killed oothecae of all ages. Heat treatment improved the suitability of 4-
week-old oothecae as hosts for parasitoid rearing. For A. hagenowii, reduced
emergence rates were documented among cold-stored 3‒4-week-old oothecae. The
number of progeny produced in 1–2-week-old and 3–4-week-old oothecae decreased
after cold-stored for 3–4 and 0.5–4 months, respectively. Evania appendigaster had
low emergence rates in cold-stored oothecae.
1
CHAPTER ONE
INTRODUCTION
The American cockroach, Periplaneta americana (Linnaeus), is one of the major
cosmopolitan pest cockroaches of economic and medical importance (Cornwell, 1968;
Lee, 1997, 2007; Rust, 2008). In the human living environment, this species lives in large
numbers in moist and warm habitats such as sewers, drainage systems, and rubbish chutes
(Lee and Ng, 2009; Rust et al., 1991; Tee et al., 2011a) and consequently provides
reservoir populations to fuel indoor and outdoor infestations (Robinson, 2002). Because
it inhabits areas associated with human waste and disease organisms, P. americana had
been reported to be a potential mechanical vector of pathogenic bacteria, viruses, fungi,
protozoans, and helminthes (Lee, 1997; Roth and Willis, 1957). Cockroach allergens also
contribute to the occurrence of asthma and allergy among sensitized individuals (Gore and
Schal, 2007).
Chemical control is widely used for cockroach management. However, awareness
of the adverse effects of insecticide exposure in the human environment has prompted
research of possible alternative approaches. Biological control of cockroaches using
oothecal parasitoid wasps is an alternative approach that warrants further study. Because
cockroach eggs are enclosed within oothecae and deposited in hidden locations (Rau, 1943;
Yeh, 1995), they often are not eliminated by insecticide treatment. Use of oothecal
parasitoid wasps is one way to control this immobile stage.
2
Evania appendigaster (Linnaeus) (Hymenoptera: Evaniidae) and Aprostocetus
hagenowii (Ratzeburg) (Hymenoptera: Eulophidae) are ootheca parasitoids of the
American cockroach. Evania appendigaster is a solitary species (only one wasp emerge
from an ootheca) whereas A. hagenowii is a gregarious species (numeruous wasps
produced per ootheca). Females lay their eggs inside oothecae in which 16 cockroach eggs
are enclosed in two rows. The immature stages of both parasitoid wasps consume all of
the cockroach eggs and take 32‒46 d (25‒30 °C) to develop before they chew an exit hole
on the surface of the ootheca to emerge as adult wasps (Bressan-Nascimento et al., 2010;
Cárcamo et al., 2013). Aprostocetus hagenowii is smaller in body size (1.3‒2.1 mm in
length) but a superior larval competitor compared to E. appendigaster (6.5‒8.0 mm)
(Narasimham, 1984; Kumarasinghe and Edirisinghe, 1987).
The use of ootheca parasitoids for cockroach control faces several constraints: (1)
limited information about how environmental factors (humidity and temperature)
influence the foraging and fitness of ootheca parasitoids. Several field release studies of
ootheca parasitoids had been conducted at different geographical regions (tropics to
subtropic) and microhabitats (e.g., treehole habitats, plumbing chases, sewer systems,
around and inside buildings). These studies reported mixed results (parasitism rates on
oothecae range 0‒100%). Knowledge of the environmental factors on parasitoids’
foraging and fitness is critical to improve their use as biological control agents in
cockroach control programs. (2) Host utilization pattern of each ootheca parasitoid is
poorly understood. Host age is one important criterion of host quality that affects
parasitoid fitness. It is important to assess the effect of ootheca age on parasitoid fitness
as this would help to improve the rearing of the parasitoids and their use for cockroach
3
control, such as estimation of the parasitism dynamics in the field, release schedule and
strategies (single or multiple species). (3) Methods for mass rearing of these ootheca
parasitoids are not adequately developed, and to date an artificial diet is not available.
Because P. americana has a slow ootheca production rate (females produce an average of
1–2 oothecae per week for their entire lifespan) (Gould and Deay, 1938; Roth and Willis,
1956), limited numbers of cockroach oothecae can be obtained from laboratory rearing of
cockroaches. Therefore, finding a way to stock pile oothecae may assist in the mass
production of ootheca parasitoids for use in biological control of cockroach populations.
Coexistence of competing parasitoids parasitizing the same host species can be
facilitated by niche partitioning. Niche partition (temporal or spatial) can be mediated
when differences in the intrinsic biological attributes between competing species results
in differences in their abilities to exploit host of varying distribution, such as differences
in competitive and dispersal abilities (Lei and Hanski, 1998), ovipositor length (Hanks et
al., 2001), host detection behavior (van Dijken and van Alphen, 1998) and responses to
extrinsic environmental factors (humidity, temperature, nutrients, etc.) (Amarasekare,
2003; Sorribas et al., 2010). Both parasitoid species are widespread and commonly found
coexisting in areas where P. americana occurs (Roth and Willis, 1960; Piper et al., 1978;
Narasimham and Sankaran, 1979; Kumarasinghe and Edirisinghe, 1991). This
coexistence of ootheca parasitoids provide an excellent multiple-parasitoids-host model
to study the biological differences between competing species and, thus, allows the key
features that are unique to the ecological success of each parasitoid species to be identified.
This would also benefit the use of these parasitoid for cockroach control.
4
This study focuses on the host utilization pattern of A. hagenowii and E.
appendigaster in relation to ootheca age and extrinsic environmental factors (humidity,
temperature and sugar food resources), and their mass rearing improvements through the
following objectives:
1. To study the water balance profiles, humidity preference and survival of both
parasitoids in order to understand the impact of desiccation on forming their
ecological niche.
2. To investigate the effect of ootheca age on the effect of ootheca age on
cannibalism in the P. americana and its effect on host age selection and fitness
of E. appendigaster.
3. To examine the influence of ootheca age and temperature on the immature
development and adult fitness of A. hagenowii.
4. To assess the feasibility of using cold-stored P. americana oothecae for rearing
A. hagenowii and E. appendigaster.
5
CHAPTER TWO
LITERATURE REVIEW
2.1 Periplaneta americana
Periplaneta americana is one of the major pest cockroach species of medical and
economic importance in tropical and subtropical regions (Cornwell, 1968). It has been
found associated with pathogenic bacteria, viruses, fungi, protozoans and helminthes
(Roth and Willis, 1957, 1960; Lee, 1997; Lee and Ng, 2007). Seven allergens have been
identified in P. americana and these could potentially lead to household asthma and
allergy (Gore and Schal, 2007).
Adult cockroaches are 28‒44 mm in length and shinning dark-brown in color with
a pale-yellow strip around the edge of the pronotum (Plate 2.1) (Cornwell, 1968). Adult
males can live for 125‒362 days whereas adult females have a longer lifespan of 125‒706
days (Rust, 2008). Females produce 1‒2 oothecae during their peak reproductive period
and a total of 10‒84 oothecae during their entire lifespan (Gould and Deay, 1938; Roth
and Willis, 1956). Ootheca is an egg case in which 12‒16 cockroach eggs are enclosed
(ootheca generally refers to both the egg case and enclosed eggs) (Roth, 1968; Bell and
Adiyodi, 1981). Oothecal shell protects developing embryos from desiccation, physical
damage, and disease infections (Roth and Willis, 1955; Provine, 1981). Ootheca is soft
and reddish-brown in color upon newly laid, and is hardened and darkened within the next
few days after deposition. Incubation time of P. americana eggs range from 40 to 69 days
at 20‒30°C
6
Plate 2.1 Adult male of P. americana.
7
(Bressan-Nascimento et al., 2008). Nymphs undergo 6‒14 molts in 150‒450 days
(temperature-dependent) before maturing into the adult stage (Cornwell, 1968).
American cockroaches frequently shelter in warm and moist microhabitats inside
and around buildings such as tree holes, sewers, crack and crevices, bin-chute and
drainage systems (Appel, 1986; Suiter et al., 1998; Lee and Ng, 2009; Tee et al., 2011a).
This is partly due to their vulnerability to desiccation as Appel et al. (1983) have shown
that this species has a hygric-typed water loss rate (1.82% h‒1). Cockroaches aggregate in
shelters in groups, which is facilitated by attraction to aggregation pheromone presented
in their feces (Roth and Cohen, 1973; Bell, 1981). This group living enhances
development, sexual maturation, survival, sexual maturation and foraging efficiency for
food and shelter (reviewed by Bell et al., 2007 and Lihoreau et al., 2012). However, group
living also incur cannibalism among group members. There is little information about why
cockroaches exhibit cannibalistic behaviors but hypotheses have been proposed that (1) it
occurs as a by-product of their opportunistic and omnivorous feeding habit, where
cannibals take the advantage of consuming vulnerable conspecifics (oothecae, young and
individuals that are newly molted, injured and weak) and cannibalism frequency varies
depending on the cockroach density and food availability (quality and quantity); and (2)
it serves to recover and recycle essential nitrogenous resources back into cockroach
aggregation (Ross and Mullins, 1995; Nalepa and Bell, 1997; Bell et al., 2007).
Cannibalism of oothecae in blattid pest cockroaches is commonly observed (Roth and
Willis, 1960; Gordon et al., 1994). However, studies of the benefits of ootheca
cannibalism in cockroaches are limited. Nalepa and Mullins (1992) estimated that adult
female wood cockroach, Cryptocercus punctulatus Scudder, could recover up to 58.7%
8
of the nitrogen she invested into her brood inside a single ootheca by eating the oothecal
shell after hatching. The oothecal shell of P. americana consists of 87% protein (Kramer
et al., 1991) but little is known about the nutritional benefits of eating ootheca in this
species.
2.2 Aprostocetus hagenowii
Aprostocetus hagenowii is a gregarious parasitoid wasp of cockroach oothecae. Its
cockroach hosts include P. americana, Periplaneta australasiae (Fabricius), Periplaneta
fuliginosa (Serville), Blatta orientalis (Linnaeus), Parcoblatta spp., Eurycotis floridana
(Walker) and Eurycotis biolleyi Rehn (Roth and Willis, 1960; Lebeck, 1991).
Adult wasps are black in color and 1.8‒2.0 mm in length (Plate 2.2) (Narasimham,
1984). The lifespan of males and females reared without foods are 1.5‒1.8 and 3.8‒5.9
days, respectively. However, males and females can live for an extended time if fed with
carbohydrate-based foods (Narasimham, 1984). In the laboratory, A. hagenowii given
mucilage of Hibiscus rosa-sinensis L. and nectar of Leucas aspera (Wild.) had longer
lifespan than those reared without foods (Narasimham, 1984). This suggests that, similar
to many other parasitoid wasps, they may feed on floral food resources in natural
environments (Jervis et al., 1993). Females have a pair of ovaries each consists of 8‒12
ovarioles. Females have been documented to possess 45 mature eggs in their ovaries upon
emergence and continue to mature more eggs in the following days (Zhang et al., 2010).
9
Plate 2.2 Aprostocetus hagenowii female attempts to drill and insert its ovipositor into a
P. americana ootheca.
10
Females can parasitize 1‒5 oothecae in their entire lifespan, producing an average
of 103 progeny (range 50‒139) (Roth and Willis, 1954). Females lay a large portion of
their lifetime total number of mature eggs into the first ootheca they encountered and
smaller numbers of eggs into other oothecae they found subsequently (Roth and Willis,
1954). An ootheca that has been parasitized by an oviposition-inexperienced (virgin)
female can produce 45‒96 progeny with a female-biased sex ratio of 0.07‒0.09
(proportion of males) (Suiter et al., 1998; Tee et al., 2010). Developmental time of A.
hagenowii varies between 22 and 90 days (Roth and Willis, 1960). An ootheca can
produce a greater number of progeny (up to 261 wasps) if it is superparasitized by many
females (Lebeck, 1991). Aprostocetus hagenowii is a facultative hyperparasitoid of E.
appendigaster. It can successfully develop in an ootheca containing a developing E.
appendigaster of up to 32-days-old (Kumarasinghe and Edirisinghe, 1987). Piper et al.
(1978), in their survey of the ootheca parasitoids around residential properties in Texas
and Louisiana, documented that 96.5% of the 113 parasitized oothecae produced A.
hagenowii and the remaining oothecae had E. appendigaster emerged.
Saliva used by P. americana females to glue ootheca to a substrate had been found
to serve as a host recognition cue in A. hagenowii (Vinson and Piper, 1986). In addition,
one short-range volatile hydrocarbon, identified as 6,9-heptacosadiene, had been detected
from P. americana frass, oothecae and females, which serves as a kairomone that attracts
A. hagenowii (Suiter et al., 1996). This hydrocarbon consists of a large portion of cuticular
hydrocarbons of P. americana and is absent in P. australasiae, Periplaneta brunnea
Burmeister, and P. fuliginosa (Saïd et al., 2005). This likely suggests the role of 6,9-
11
heptacosadiene in mediating the preference of A. hagenowii for P. americana over other
blattid cockroach species (Roth and Willis, 1954).
2.3 Evania appendigaster
Evania appendigaster is a solitary ootheca parasitoid of blattid pest cockroaches
which include P. americana, P. australasiae and B. orientalis. It is found in tropical and
subtropical regions (Roth and Willis, 1960). Adult wasp is black in color and 6.5‒8.0 mm
in body length (Plate 2.3). Wasps in the family Evaniidae can be easily recognized as they
have a laterally-flattened abdomen, which is attached to the top of the propodeum by a
petiole. This unique abdominal structure resembles a flag and, thus evaniids are
collectively known as ensign wasps (Stange, 2010).
Adult wasps can live for 1‒2 weeks without foods and can extend their lifespan to
3‒6 weeks when fed with carbohydrate-based foods (Yeh and Mu, 1994a; Fox and
Bressan-Nascimento, 2006). Females have a pair of ovaries each bearing an average of
six ovarioles. Females have a total of 12 mature eggs with each ovariole containing one
mature egg upon emergence (Yeh and Mu, 1994a). Females can produce up to 23 progeny
during their entire lifespan (Fox and Bressan-Nascimento, 2006).
Oviposition by a female on an ootheca takes an average of 1342 seconds, which
involves a sequence of behaviors; (1) host contact and probing using antennae, (2)
unsheathe ovipositor to probe and find a suitable drilling site, (3) female remains
quiescence with its tip of ovipositor touching the surface of ootheca, (4) drilling and
insertion of ovipositor, (5) oviposition, (6) withdrawal of ovipositor and leaving the
12
Plate 2.3 Evania appendigaster female remains quiescence on an ootheca with the tip of
its ovipositor touching the surface of the ootheca.
13
ootheca (Yeh and Mu, 1994b). Of these oviposition behaviors, the quiescent stage takes
up 70% (ca. 1000 s) of the time budget for oviposition. Yeh and Mu (1994b) proposed
that females might be releasing protein-denaturing enzymes during the quiescent stage to
facilitate the insertion of the ovipositor into ootheca.
Evania appendigaster females normally lay one egg into an ootheca (≤ 6% self-
superparasitism) and only one wasp is produced from an ootheca in the case of
superparasitism (Bressan-Nascimento et al., 2010; Fox et al., 2012). Developmental time
from egg to adult takes 36‒45 days at 25‒30°C (Bressan-Nascimento et al., 2010). After
hatching, the larva undergoes two molts and consumes all the cockroach eggs before
reaching the pupal stage. Upon emergence, parasitoid chews an exit hole through the
ootheca shell to emerge as an adult wasp (Fox et al., 2012). Mating and oviposition can
occur immediately after emergence. Females are monandrous (mating once with just one
male) whereas males are polygynous (mating with many female mates) (Yeh and Mu,
1994a).
2.4 Interspecific competition and coexistence of competing species in parasitoid
wasps
Parasitoid wasps often compete with other heterospecific parasitoids for the same
host resources in the same habitat. This interspecific competition can occur among
parasitoid larvae that compete for limited resource from a single host (intrinsic
competition) and between female foragers that search for and parasitize the same host
resources (extrinsic competition) (Cusumano et al., 2012). In natural ecosystems, it is
14
common that more than one competing parasitoid species can coexist. Typically only one
parasitoid species can survive from an intrinsic competition for a single host resource
(Cusumano et al., 2012; Mills, 2006). The outcome of such competition is affected by
several mechanisms such as physiological suppression, physical attack, sequence of and
time interval between ovipositions (Vinson and Hegazi, 1998; Mills, 2006; Uka et al.,
2006; Bai et al., 201l; Cusumano et al., 2012). Thus, whether competing parasitoids can
co-exist cannot be determined based solely on the outcome of intrinsic competition.
Several studies demonstrated mechanisms that shape the coexistence of parasitoid
wasps, such as responsiveness to host productivity gradient (Amarasekare, 2000), spatial
and temporal niche partitioning (Hanks et al., 2001; Sorribas et al., 2010). Among these
mechanisms, spatial niche partitioning is commonly found to facilitate coexistence of
parasitoids (Mills, 2006). Spatial niche partitioning can be mediated when parasitoids
differ in their biological attributes to exploit spatially separated hosts. For example,
difference in ovipositor length between Syngaster lepidus Brullé and Callibracon
limbatus (Brullé) had resulted in spatial partitioning of the larvae of eucalyptus
longhorned borer Phoracantha semipunctata Fabricius concealing inside tree bark of
varying depth; S. lepidus with a longer ovipositor (10.1 mm) than C. limbatus (8.6 mm)
was able to parasitize larvae concealed in thicker tree bark (Hanks et al. 2001). In another
study, Lei and Hanski (1998) demonstrated spatial partitioning in parasitoids is mediated
by trade-off between dispersal and competition ability. In their study, parasitoid species
that was inferior larval competitor but a good disperser efficiently utilized isolated host
patches, which might not have been colonized by another competing species that was a
superior larval competitor but a poor disperser.
15
2.5 Water balance in insects
Insects face the challenge of maintaining a balance body water in relation to the
desiccation stress of their habitats. Insects living in a drier area have lower water loss rates
than those found in more humid environments; they can be classified based on the water
loss rates (% body water loss h-1) introduced by Hadley (1994) as xeric (< 0.8% h-1), mesic
(0.8‒2.0% h-1) and hygric species (> 2.0% h-1). Insects lose their body water through
evaporation at the cuticle surface, respiration, defecation and secretion. Among these
water loss routes, cuticular water loss is a major water loss route that accounts for > 80%
of the total body water loss in most insects (Hadley, 1994; Gibbs and Rajpurohit, 2010).
This is because (1) cuticular water loss occurs as a function of the body surface area and
(2) insects that are small in size have a large body surface area relative to body volume
for body water to be lost through the cuticle. Thus, cuticular permeability and body size
are two interacting factors that influence water loss rates of insects.
Cuticular lipids at the surface of the cuticle have been recognized as the most
effective barrier in preventing cuticular water loss in most insects (Gibbs and Rajpurohit,
2010). Cuticular lipids mainly consists of hydrocarbons. The quantity and composition
(e.g., chain length, saturation and numbers of methyl-branching) of cuticular hydrocarbon
account for differences in water loss rates of insects at the intra- and interspecific levels
(Gibbs 1998; Parkash et al., 2008a; Gibbs and Rajpurohit, 2010). For example, under an
extended desiccation selection period, Drosophila melanogaster Meigen populations have
been found to develop a lower water loss trait, partly due to their longer-chained cuticular
hydrocarbons compared to those of control populations (Gibbs et al. 1997). Besides
16
cuticular lipids, melanization of cuticle also affects cuticular water loss (Parkash et al.
2008a, 2008b). Cuticle is hardened and darkened during melanization process, which
results in a layer of more compact and water-proofed cuticle (Gibbs and Rajpurohit, 2010).
For example, Parkash et al. (2008b) demonstrated that darker Drosophila immigrans had
lower water loss rates than those of lighter flies, even though both fly populations showed
no difference in the quantity of cuticular lipids.
Body size has been found responsible for the intra- and interspecific variation in
desiccation resistance in insects (Hood and Tschinkel, 1990; Addo-bediako et al., 2001;
Kaersgaard et al., 2004; Schilman et al., 2007; Yoder et al., 2010). It is expected that
insects of smaller sizes are vulnerable to water loss than those of bigger sizes because
smaller body size means larger body surface area/body volume for cuticular water loss.
Thus, increase in body size is one way for insects to be less susceptible to desiccation
(Danks, 2000; Benoit and Denlinger, 2010). For example, desiccation resistance was
positively correlated with body size in arboreal and terrestrial ant species (Hood and
Tschinkel, 1990). In a study of the foraging activity of ants on the forest floor, Kaspari
(1993) documented that small ant species forage primarily in moist environment in closed
canopy whereas large ant species could expand their foraging territory into drier habitats
in open canopy. In addition, two sympatric congeneric subterranean termites with similar
cuticular permeability were documented to adopt distinct foraging strategies due in part
to difference in their body size; bigger-sized Macrotermes carbonarius (Hagen) was
documented to be able to forage above ground in open-air condition during wet seasons
while smaller-sized M. gilvus (Hagen) foraged mainly in concealed conditions
(underground tunnels and mud tubes) and rarely in open-air environment (Hu et al., 2012).
17
Most insects possess body water content of 65‒75% of their fresh weight (Hadley,
1994; Danks, 2000). Total body water content is one physiological feature commonly
examined in the studies of desiccation resistance in insects. Conserving high body water
content is one strategy for insects to reduce desiccation risk (Gibbs et al., 1997; Danks,
2000; Schilman et al., 2007). In a study of physiological adaptations of insects towards
desiccation stress, Gibbs et al. (1997) showed high body water content (approximately 30%
more) was a trait selected for D. melanogaster subjected to desiccation selection, which
render these flies less susceptible to desiccation than that of control flies. Yoder et al.
(1998) also documented that the parasitoid Ageniaspis citricola (Logvinovskaya) had a
high body water content of 75%, which was at the upper range of the average value for
most insects.
2.6 Factors influencing host selection and foraging of parasitoids
Host quality is one important determinant of parasitoid fitness as parasitoid larvae
depend solely on a single host for acquisition of nutrient. Besides host species and size
variation, host quality is influenced by host age as host undergoes biochemical,
physiological and morphological changes during the course of its development. These
changes affect both the fitness of ovipositing females and their progeny developing
on/inside the host. Depending on host types (eggs, larvae, pupae or nymphs) and
parasitoids’ development modes (idiobiont: hosts are sessile and do not grow in size after
parasitism; koinobiont: hosts are mobile and continue to grow after parasitism),
parasitoids differ in their host-age related fitness trade-offs during host-parasitoid
interaction.
18
For egg and pupal parasitoid wasps, host quality generally decreases with age
because host resources do not increase in size and are converted into more complex host
tissues (e.g, well-developed embryos and sclerotized pupae), which become less
accessible and suitable for parasitoid development. Thus, egg and pupal parasitoids may
take longer time to develop and experience reduced body size, longevity and fecundity
when they develop in older hosts. Vinson (1998) has proposed three oviposition strategies
female egg and pupal parasitoids commonly used to maximize their fitness in relation to
host-aged issues (Vinson, 1998); (1) parasitoids should oviposit in only young hosts,
which can be facilitated by acceptance of only young hosts for oviposition through
assessment of host’s chemical and physical cues, presence before host eggs are laid
(phoretic behavior, attraction to host sex or aggregation pheromones), and attraction
towards female oviposition cues, such as oviposition secretions. Studies of host preference
in egg and pupal parasitoids have shown that females exhibited oviposition preference for
hosts of young or/and intermediate age compared to old hosts, via either innate preference
or learning of host cues (Kouamé and Mackauer, 1991; Ueno, 1997; Chow and Mackauer,
1999; Godin and Boivin, 2000). For example, Godin and Bivin (2000) found that, in a
multiple-choice condition, 70% of the 42 species and strains of trichogrammatid egg
parasitoids prefer to parasitize young and intermediate-aged over old host eggs. Vinson
(1998) postulated that the amount of host’s volatile compounds may change over time.
Female wasps may make use of these changes in volatility in their search for newly-laid
hosts; (2) parasitoid larvae should increase their development rates when they are being
laid in hosts of older ages. However, little is known about whether parasitoid larvae adjust
their development rate according to host age. Studies of egg and pupal parasitoids
commonly demonstrate that development time of parasitoid wasps increases with host age
19
due to a longer time needed to consume and assimilate well-developed host tissues (Otto
and Mackauer, 1998; Hirose et al., 2003); and (3) parasitoids can arrest or delay host
development and breakdown host tissue, rendering old host suitable for parasitoid larval
development. It has been demonstrated that newly-hatched larva of Trichogramma
pretiosum Riley consumed host embryos and yolk, which had been extraorally digested
by factors female wasp injected during oviposition (Strand, 1986; Pennacchio and Strand,
2006; Strand, 2014). In another study, Strand et al. (1986) discovered that female of the
scelionid egg parasitoid Telenomus heliothidis Ashmead release factors that arrest host
embryogenesis while teratocytes released from hatching egg of parasitoid facilitate
extraoral digestion of host embryo. Zhou et al. (2014) further shown that Telenomus podisi
(Say) could even successfully develop in hosts 24 h prior to its hatching (incubation time
takes six days) without incurring detrimental effects on its fitness traits (survival, body
size, fecundity and sex ratio, except developmental time).
For idiobiont larval parasitoids, host larvae increase in size with age. Thus,
parasitoid larvae developing in older and larger hosts attain greater body size (and
associated fitness correlates) than those utilizing young and small hosts (Liu et al., 2011).
However, female parasitoids need to invest more energy, time and venom to parasitize an
older and larger host, which costs them a greater handling time and risk of injury/death
and reduced parasitism success rate and fecundity (Wei et al., 2014). Consequently, there
are trade-offs between fitness traits in relation to host age (Liu et al., 2011; Wei et al.,
2014). By contrast, koinobiont parasitoid larvae could delay development when they are
deposited in smaller hosts in order to benefit a greater nutrient pool accumulated in later
20
host stages of larger sizes (Harvey and Strand, 2002; Colinet et al., 2005; Jenner and
Kuhlmann, 2006).
Female parasitoids also assess the risk of predation and hyperparasitism during host
foraging and selection. Avoidance of high-risk host patches through detection of
predators/hyperparasitoids or their traces and switch to attack enemy-free or low-risk
patches are one way for parasitoid wasps to overcome predation or hyperparasitism risk
(Höller et al., 1993; Taylor et al., 1998). However, because majority of parasitoid wasps
are time-limited and have limited reproductive resources (due to lack of lipogenic ability,
and lipids, as an essential nutrient for egg production, are accumulated during parasitoid
larval development), females are expected to exhibit adaptive host foraging strategies in
order to maximize their reproductive fitness. In the context of host-parasitoid-
predation/hyperparasitoid interaction, adaptive host foraging in parasitoid wasps often
involves trade-off between survival and fitness of parasitoid. For example, females of the
aphid parasitoid Pauesia silvestris Stary were found to optimize host utilization by shifting
to attack hosts of lower quality when hosts of higher quality were distributed in
microhabitats with a high hyperparasitism risk (Völkl and Kroupa, 1997). This optimal
host foraging had resulted in a greater overall fitness gain for P. silvestris. In another study,
Ayal and Green (1993) reported that female aphid parasitoid Diaeretiella rapae (M’intosh)
optimized the number of aphids to be parasitized per aphid colony based on aphid density
and the frequency of hyperparasitoid visits. This optimal ovipsotition strategy maximizes
the number of parasitoids emerged per aphid colony because hyperparasitoids spent less
foraging time for aphid colonies with low density of D. rapae-infected aphids.
21
Besides adult female wasps, parasitoid larvae also exhibit adaptations to reduce
mortality risk due to predation and hyperparasitism. For example, Harvey and Strand
(2002) reported that a distinct difference in developmental trait among koinobiont
parasitoids in the families Icheneumonidae and Braconidae, which attack hosts of different
feeding ecology. They found that koinobiont parasitoid larvae would prolong their
development time in small host larvae that feed in concealed environment so that they
could obtain more nutrients along host development. By contrast, koinobiont parasitoid
larvae that develop in host larvae feeding at exposed environments would trade off
potential gain in body size for rapid development, which minimize their time window of
vulnerability to predation and hyperparasitism.
2.7 Rearing of A. hagenowii and E. appendigaster for biological control of
cockroaches
Yeh et al. (1995) described an efficient way to collect oothecae from P. americana
cultures for use in the maintenance of cockroach cultures and parasitoid rearing. They
demonstrated that oothecae can be collected from a piece of styrofoam board, which was
placed into a rearing container of P. americana females. This method takes the advantage
of the oviposition concealment behavior of P. americana female, where it tends to chew
out a cavity on a soft and thick substrate and cover its ootheca inside the cavity with debris
held in place with its saliva (Rau, 1943; Bell and Adiyodi, 1981; Yeh, 1995).
In biological control program, parasitoid rearing methods that produce female-
biased clutches are desirable as females are responsible to suppress insect pest populations.
22
Hagenbuch et al. (1988) developed a rearing method to produce A. hagenowii. In their
study, a parasitoid production unit was constructed using a chamber, which consisted of a
dark and a light side; the dark side was used to house parasitized oothecae so that, upon
emergence, parasitoids would be attracted to the light side (due to its positive phototactic
response), where 30‒40 healthy oothecae were exposed for parasitism. Although this
method provides a convenient way to prepared A. hagenowii-parasitized oothecae, the
resultant sex ratio (number of males/number of parasitoid produced) of parasitoids per
ootheca was less female-biased (0.47). Several studies have shown that sex ratio of A.
hagenowii produced from an ootheca could be greatly improved (ranged 0.07 to 0.12)
when oothecae were exposed individually to a female wasp for oviposition (Suiter et al.,
1998; Tee et al., 2010). This individually exposure method is time-consuming but it
produces A. hagenowii females with a larger body size and a longer lifespan than those
produced from superparasitized ootheca (Narasimham, 1984).
In biological control programs, use of killed hosts for parasitoid rearing is one
important step because it (1) eliminates the needs to handle individuals hatching or
emerging from unparasitized hosts into rearing cages, (2) prevents accidental releases of
insect pests from unparasitized hosts into the field, and (3) retains hosts at stages optimum
for parasitoid development (Suiter et al., 1998; Geden and Kaufman, 2007; Tee et al.,
2010). Techniques used to kill host insects without compromising their suitability as hosts
for parasitoid rearing include exposure of hosts to irradiation (e.g., ultraviolet (UV),
gamma-ray, X-ray) and high/ low temperatures (Suiter et al., 1998; Moreno et al., 2009;
Cancino et al., 2012).
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Studies of the rearing improvement showed that gamma-irradiation could terminate
embryonic development of P. americana and these-irradiated oothecae were as suitable
as live oothecae for rearing A hagenowii and E. appendigaster (Suiter et al., 1998: Hwang
and Chen, 2004). Similarly, heating of oothecae at 48‒50 °C provides an economical way
to produce killed oothecae for rearing A. hagenowii and E. appendigaster but long heating
time would reduce the quality of oothecae for parasitoid rearing (Hwang and Chen, 2004;
Tee et al., 2010). Tee et al. (2010) showed that a minimal heating time of 30 min at 48 °C
was sufficient to terminate embryonic development of 1‒2-days-old oothecae. By contrast,
exposure of oothecae to freezing temperatures (‒16 and ‒20 °C) can kill P. americana
eggs but such killed-oothecae are not suitable as hosts for A. hagenowii and E.
appendigaster as due to low emergence rates (Suiter et al., 1998: Hwang and Chen, 2004).
Alternatively, Bressan-Nascimento et al. (2008) demonstrated that oothecae subjected to
a chilling temperature of 0‒5 °C terminated P. americana embryogenesis without
compromising their quality for rearing both A. hagenowii and E. appendigaster. However,
methods to increase the shelf-life of these killed oothecae are not adequately developed.
Studies on other parasitoids have shown that storage at low temperatures can be a viable
way to stock-pile hosts for mass-rearing and field releases of parasitoids (Chen and
Leopold, 2007; Geden and Kaufman, 2007).
2.8 Biological control efficacy of A. hagenowii and E. appendigaster
Aprostocetus hagenowii and E. appendigaster are prevalent among natural
cockroach populations. Surveys of field collected oothecae showed that parasitism of A.
hagenowii rated 16‒48% in P. americana, 22‒84% in P. fuliginosa and 12.5% in B.
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orientalis (Cameron, 1955; Fleet and Frankie, 1975; Kanayama et al., 1976; Piper et al.,
1978; Narasimham and Sankaran, 1979) whereas parasitism of E. appendigaster was 29%
in P. americana (Cameron, 1957). However, placement of sentinel oothecae inside sewer
manholes, one of the primary breeding sites of P. americana, failed to detect the presence
of A. hagenowii (Reierson et al., 2005; Tee et al., 2011b).
Several studies have been conducted to evaluate the biological control efficacy of
A. hagenowii in indoor room condition and at natural cockroach habitats. These studies
reported varying parasitism rates. Releases made in room conditions generally produced
the most promising results. For example, Roth and Willis (1954) documented that a
release of 1000 female wasps into a laboratory room could achieve up to 83% parasitism
rate. Similarly, Hagenbuch et al. (1989) reported high parasitism rates (95‒98%) for an
evaluation, where 300 female wasps were released weekly into a simulated room.
In field evaluations, weekly releases of A. hagenowii at plumbing chases, crevices
around buildings and sewer systems resulted in 18.2‒30.6%, 4.2‒37.5% and 5.6‒24.1%
parasitism, respectively (Pawson and Gold, 1993). By contrast, Suiter et al. (1998)
documented promising results, where they reported parasitism rates that ranged from 50
to 100% for releases made around treeholes infested with blattid pest cockroaches.