WATER BALANCE, HOST UTILISATION, AND MASS REARING ...

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


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


4.2.1 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


4.3 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


5.2.1 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


5.3 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


6.2.1 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


6.3 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


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


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




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



119

xiii


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


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.

WATER BALANCE, HOST UTILISATION, AND MASS REARING ...

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