1. INVESTIGATION INTO THE BIOLOGY OF
SEGESTIDEA DEFOLIARIA DEFOLIARIA
(UVAROV) AND ITS EGG PARASITISM BY
DOIRANIA LEEFMANSI WATERSTON
Tabitha Manjobie
A thesis submitted in fulfillment of the requirements for the degree of
Master of Philosophy in Agriculture
Department of Agriculture
Papua New Guinea University of Technology
November 2014

2. i
STATEMENT OF ORIGINALITY
I declare that this thesis is my own original work and to the best of my knowledge and
belief, it has not been previously published or submitted to meet the requirements for
either an undergraduate or a higher degree in any other higher education institution
except where due acknowledgement or reference is made in the thesis.
Tabitha Manjobie
Signature:
Date: 23rd
February 2015

3. ii
ABSTRACT
Oil palm, Elaeis guineensis Jacquin, 1763 is an important cash crop in Papua New
Guinea (PNG). With the ongoing expansion of new plantings by both the milling
companies and the smallholder growers, the trend for increased production and
revenue is expected to continue. Despite the importance of the crop, a variety of pests
cause considerable economic damage. Among them are the long-horned grasshoppers
collectively known as sexavae. There are four (4) pest species (Segestes decoratus,
Segestidea novaeguineae, S. defoliaria defoliaria, S. defoliaria gracilis) currently
known from the country. Two (2) of the species (S. decoratus and S. defoliaria) occur
in West New Britain Province (WNBP) and cause extensive damage, but their biology
as well as those of their biological control agents are poorly understood.
In an attempt to understand the basic biology of sexavae specifically S. defoliaria as
the study insect and its egg parasitoid, Doirania leefmansi, three (3) studies were
conducted. The first study investigated the reproductive potential and embryonic
development of S. defoliaria, the second study evaluated the rate of parasitism on S.
defoliaria by D. leefmansi in laboratory based experiments, whilst the final study
investigated the level of sexavae egg parasitism in the field.
In the first study, distinct developmental stages were noted where the adult stage
(males: 307 days, females: 288 days) took longer than the nymph (males: 113 days,
females: 112 days) and egg (79 days) stages. There was a distinct peak egg laying
period and the entire egg laying period may continue up to 16 weeks. The number of
eggs laid did not correlate positively with the number of times the females mated.
During egg development, there were distinct embryonic development stages with
corresponding weight increases. Six (6) nymphal instars lasting a mean of 112 days
for females and 112 days for males were identified. The life cycle was long,
completing a full cycle in more than a year (univoltine).
In the laboratory based parasitism evaluation study, sexavae eggs at different times
after deposition were presented to individual parasitoids to evaluate preference and
also over different periods to assess the efficacy of parasitism. Five (5) day old eggs
(stage 0) were preferred over 15 day (stages 18-20) and 28 day (stages 21-25) old

4. iii
eggs. Parasitism rate on host eggs by D. leefmansi over time was low with each
female parasitizing less than 5 eggs per hour.
Having understood the parasitism rate in the controlled environment in the laboratory,
I extended my final study to the field and investigated the rate of parasitism on
sexavae eggs by the parasitoids in the field. The level of parasitism in the field varied
from site to site. Apart from D. leefmansi, locally occurring parasitoid(s) and
predators were effectively controlling the eggs. Factors influencing the disparity in
the levels of egg parasitism across different sites can be multiple, but the absence of
floral resources as a food source for the adult parasitoid populations was noted as the
key factor.
Results from the different studies suggest that the pest can be effectively managed if
management options are implemented in close synergy with the timing of the different
life stages. Parasitoid releases need to be done during stage 0 of embryo
development, and beneficial plants should be planted in areas where biological control
agents will be released if the floral resources are low. Insecticide treatment, if
necessary, should be applied during the nymphal and adult pre-oviposition periods. If
overlapping populations exist, a follow up treatment is necessary.

5. iv
CONTENTS
Page
STATEMENT OF ORIGINALITY I
ABSTRACT II
CONTENTS IV
LIST OF TABLES VI
LIST OF FIGURES VII
LIST OF PLATES VIII
ACKNOWLEDGEMENTS IX
1 INTRODUCTION AND LITERATURE REVIEW 1
1.1 History, establishment and production of oil palm in PNG 3
1.2 Economic benefits of Oil Palm to PNG 4
1.3 Oil palm pests in PNG 5
1.4 Sexavae pests (taxonomy, biology and pest status) of oil palm 5
1.5 Management of sexavae pests 6
1.6 The study insects 8
1.6.1 Segestidea defoliaria (Uvarov, 1924) (Orthoptera: Tettigoniidae) 8
1.7 Doirania leefmansi Waterston (Hymenoptera: Trichogrammatidae) 12
1.7.1 Origin and Distribution 12
1.7.2 Taxonomy and Biology of D. leefmansi 13
1.7.3 Laboratory rearing techniques for field releases 15
1.8 Segestidea defoliaria and Doirania leefmansi as the study Insects 16
1.9 Structure of the thesis 17
2 GENERAL METHODOLOGY 19
2.1 Sampling site 19
2.2 Sampling 19
2.3 Study site 20
2.4 Experimental Conditions 21
2.5 Data analyses 23
3 INVESTIGATION INTO THE REPRODUCTIVE POTENTIAL AND
EMBRYONIC DEVELOPMENT OF SEGESTIDEA DEFOLIARIA (UVAROV)
(ORTHOPTERA: TETTIGONIIDAE) 24
3.1 Introduction 24
3.2 Materials and Methods 26
3.2.1 Investigation of the reproductive potential and the embryonic development of
S. defoliaria 26
3.3 Results 29
3.3.1 Investigation into the reproductive potential of S. defoliaria 29

6. v
3.3.2 Investigation into the embryonic developmental stages of S. defoliaria 32
3.4 Discussion 36
4 LABORATORY STUDY ON THE PARASITISM RATE OF S. DEFOLIARIA
DEFOLIARIA (UVAROV) (ORTHOPTERA: TETTIGONIIDAE) EGGS BY
DOIRANIA LEEFMANSI WATERSTON (HYMENOPTERA:
TRICHOGRAMMATIDAE) 39
4.1 Introduction 39
4.2 Materials and Methods 40
4.2.1 Experimental conditions 40
4.2.2 Host eggs and the parasitoid 40
4.2.3 Investigation into the rate of parasitism on different age eggs of S. defoliaria
by D. leefmansi 40
4.2.4 Investigation of the parasitism rate of S. defoliaria eggs by individual D.
leefmansi 41
4.3 Results 42
4.3.1 Investigation into the rate of parasitism on different age eggs of S. defoliaria
by D. leefmansi 42
4.3.2 Investigation of the parasitism rate of S. defoliaria eggs by individual D.
leefmansi 43
4.4 Discussion 45
5 FIELD PARASITISM OF SEXAVAE EGGS BY DOIRANIA LEEFMANSI
WATERSTON (HYMENOPTERA: TRICHOGRAMMATIDAE) 46
5.1 Introduction 46
5.2 Materials and Methods 47
5.2.1 Field egg sampling for parasitism assessment (baseline data) 47
5.2.2 Investigation of the parasitism rate on S. defoliaria eggs in the field by D.
leefmansi 47
5.3 Results 48
5.3.1 Field egg sampling for parasitism assessment 48
5.3.2 Investigation of the parasitism rate of S. defoliaria eggs in the field by
Doirania leefmansi 51
5.4 Discussion 51
6 GENERAL DISCUSSION 53
REFERENCES CITED 55

7. vi
LIST OF TABLES
Table 1. Frequency of mating and the number of eggs laid by females (n=10)....................................31
Table 2. Proportion (%) of S. defoliaria eggs from the different age stages that were parasitized,
unparasitized, unhatched (viable) and desiccated (dead). ...................................................................43
Table 3. Status of S. defoliaria eggs after exposure to D. leefmansi for different exposure periods......44
Table 4. Percentage (%) parasitism by Doirania leefmansi and an unknown locally occurring parasitoid
across the four (4) OPIC Division sites surveyed. ..............................................................................50

8. vii
LIST OF FIGURES
Figure 1. Oil palm project areas in PNG with the Head Quarter of NBPOL Main Operations based in
West New Britain Province (map from New Britain Palm Oil Ltd., 2011)............................................4
Figure 2. Schematic outline of the sexavae management IPM programme (Adapted from Caudwell,
2000)..................................................................................................................................................7
Figure 3. Map showing the distribution of sexavae on oil palm in WNBP for the years between 1993-
2011 with S. defoliaria occurring mostly on the north eastern part of the island. ................................10
Figure 4. Mean number of days (±SE) of the different life stages in female S. defoliaria (n = 30).......30
Figure 5. Relationship between the number of eggs laid by S. defoliaria and the frequency of matings.
........................................................................................................................................................31
Figure 6. Number of eggs laid by S. defoliaria during the egg laying period per female per week.......32
Figure 7. Mean weight (g) of S. defoliaria eggs across the different embryonic developmental stages.33
Figure 8. Number of days taken before moulting for each instar (n= 30) of immature male and female
S. defoliaria......................................................................................................................................35
Figure 9. The life cycle of S. defoliaria. ............................................................................................36
Figure 10. Mean (± SE) number of different aged S. defoliaria eggs parasitized by D. leefmansi........43
Figure 11. Mean (± SE) number of S. defoliaria eggs parasitized at different time intervals by D.
leefmansi. Different letters above the error bars denote significant differences. ..................................44
Figure 12. Status of sexavae eggs sampled from the field in 4 OPIC Divisions [the upper case letters
above the error bars indicate the significance levels among the different status of eggs within each site,
whilst the lower case letters indicate significant difference levels for each egg status across the sites].50
Figure 13. Percentage (%) S. defoliaria eggs parasitized by D. leefmansi at the three study sites. Site 1
(Banaule Village Oil Palm), Site 2 (Dami Oil Palm Research Plantation, close to the station) and Site 3
(Dami Oil Palm Research Plantation, close to the beach)...................................................................51

9. viii
LIST OF PLATES
Plate 1. Example of oil palms severely defoliated by sexavae (Photo: C F Dewhurst)...........................2
Plate 2. Lateral view of the green form of the adult female and male S. defoliaria (A), where the female
has a long ovipositor (B), whilst the male has a short genital plate (C). Scale bar = 5mm (Photos: W.W.
Page)..................................................................................................................................................9
Plate 3. Photographs of sexavae egg parasitized by D. leefmansi (A) adult emergent spot shown by blue
arrows, (B) dissected egg showing D. leefmansi larvae, (C) D. leefmansi adults emerging from the egg,
and (D) D. leefmansi adults. Scale bar = 0.5mm. ...............................................................................14
Plate 4. Washed white pollination bags used for transporting sexavae. The black circle shows the flap
cut through the clear plastic window for inserting the insects.............................................................19
Plate 5. Example of how the eggs were collected from palm base (A), and frond base (B). (Photo:
Margaret Gavuri)..............................................................................................................................20
Plate 6. Insect rearing cages set up used for rearing insects (A) large cage, (B) walk-in cage, (C)
cemented water drainage PVC pipe, and (D) oil palm seedlings in black polythene bags....................22
Plate 7. Summary of mating and egg laying (A) cage set up for feeding and mating, (B) mating, (C)
mated pair, (D) oviposition, (E) eggs laid, and (F) set up for embryonic development. .......................27
Plate 8. Embryonic stages of Decticus verrucivorus (Linnaeus 1758) (A), and four (4) clearly
distinguishable embryonic stages of S. defoliaria under the stereo-microscope (i = stage 14, ii = stage
20, iii = stage 24, iv = stage 25). Scale bar = 10mm...........................................................................34
Plate 9. Segestidea defoliaria egg set up in the field for parasitism (A) placement of eggs and filling of
moistened sand in mosquito wire, (B) positioning of mosquito wire cage, (C) release of parasitoid and
(D) all set up. Left side photos are of the 3 sites where trials were set up............................................48

10. ix
ACKNOWLEDGEMENTS
Before I proceed with my acknowledgements, I would like to first of all take this time
to thank the source of all knowledge, wisdom and understanding the Almighty God
for answering my prayers and making this dream of completing a Masters Degree
become a reality. I owe it to him for everything.
I am most grateful to PNG Oil Palm Research Association Inc. (PNGOPRA)
management for giving me this opportunity to continue my higher degree studies.
The former Director of PNGOPRA, Mr. Bill Page is thanked for the endorsement for
me to pursue this MPhil. study.
PNG University of Technology, particularly the Agriculture Department and the
School of Post Graduate Studies are thanked for accepting my application to enroll for
the study.
I am indebted to extend my sincere gratitude to my two supervisors, Dr. Mark Ero and
Dr. Lastus Kuniata who constantly provided the guidance along the journey of my
study. Dr. Mark Ero, Head of Entomology with PNGOPRA was a constant guide and
mentor throughout my studies and the thesis write-up. Dr. Lastus Kuniata, Head of
Research and Development with Ramu Agri Industries provided critical comments to
my thesis outline, study outlines and the thesis write-up. Both are also thanked for
attending my seminars and providing support.
Rachael Pipai, Takis Solulu and Solomon Sotman all provided literature that I was not
able to access, and also shared experiences and provided encouragement. Solomon
Sar helped develop all my maps.
Further words of thank you are extended to both current and former employees of
PNGOPRA. Dr. Luc Bonneau, the current Acting Director of PNGOPRA provided
critical comments at various stages of my thesis write-up and was also supportive of
my study. It would be remiss, if I did not acknowledge the encouragement and the
motivation that the former PNGOPRA Head of Entomology, Charles Dewhurst
provided to me as well as his push for financial support from my employer. He was
very supportive and passionate for me to pursue the study. Dr. Murom Barnabas,

11. x
PNGOPRA Head of Agronomy visited my office occasionally and encouraged me to
continue. Pole Crompton, Elizabeth Kibikibi and Joe Rusu were always prompt in
organizing my travels and accommodation for the registration and seminar travels to
the university.
The PNGOPRA Entomology staff Brian Kiely, Simon Makai, Seset Komda, Paul
Mana, Richard Dikrey and Sonia Yuan provided assistance with the experiments
when required.
And finally, a brain draining journey like this would not have been possible without
the help and support of my immediate family members. My dear parents, Dorothy
and Simon Manjobie were very supportive throughout the journey. A very special
thank you extends to my mother (a best and dearest friend), for the spiritual guidance
and also for sometimes staying up with me late in the night to keep me awake during
my thesis write up. My siblings Benjamin, Miriam and Samson, were always by my
side when I needed their support. Thank you all for the smiles and encouragement
when I needed them.

12. 1
CHAPTER 1
1 INTRODUCTION AND LITERATURE REVIEW
Oil Palm (E. guineensis Jacquin, 1763) is one of the most important agricultural crops in
Papua New Guinea (PNG). In 2013, Fresh Fruit Bunch (FFB) production volume was
1,496,146 tonnes for plantations and 589,524 tonnes for out-growers with total revenue of
US$558,652 million generated from both sectors for New Britain Palm Oil Limited (NBPOL)
(New Britain Palm Oil Ltd, 2013). With the ongoing expansion of new plantings in both
sectors, the trend for increased production and revenue is expected to continue. The company
(NBPOL) is also committed to producing sustainably certified palm oil that meets global
standards. It joined the Roundtable on Sustainable Palm Oil (RSPO) in 2002 and obtained its
certification in 2008 including that for its smallholder suppliers (New Britain Palm Oil Ltd,
2012).
A variety of insect pests have been recorded from oil palm in PNG that have severe impact on
palm oil production (PNG Oil Palm Research Association, 1992). The most serious pests are
the long-horned grasshoppers (Orthoptera: Tettigoniidae) commonly known as sexavae, with
two (2) species (S. decoratus, S. novaeguineae) and two (2) subspecies (S. defoliaria
defoliaria, S. defoliaria gracilis) known to cause economic damage to oil palm in PNG
(Dewhurst, 2012; Page & Dewhurst, 2010). All are nocturnal and both the nymphs and adults
can cause severe foliar damage by feeding on the leaflets (Plate 1).

13. 2
Plate 1. Example of oil palms severely defoliated by sexavae (Photo: C F Dewhurst).
Segestidea defoliaria,-hereafter referred to as S. defoliaria- is one of the two pest species of
sexavae (the other is Segestes decoratus) attacking oil palm in WNBP. Both species account
for about 80% of the pests reported on oil palm in the Province (PNG Oil Palm Research
Association, 2010).
Understanding the biology of a pest species is essential for developing an effective Integrated
Pest Management (IPM) strategy. Whilst an IPM strategy for sexavae as a group has been
developed using generalized biological information, species specific information for each pest
species as well as their biological control agents is still lacking. Availability of such
information will help to improve the management efforts against this pest. The most
important aspects of their biology in terms of pest management decisions are life stages of the
pest (egg laying, embryo development, egg duration period, sex ratio, fecundity, egg and
larval mortality, nymph and adult life span and survival). This information will improve
understanding of the life stage that cause most economical damage to palms, the adult stage at
which treatment can be targeted, the period during which most eggs are laid, and the
embryonic stage at which eggs become susceptible to parasitism.
This study will investigate these key aspects of S. defoliaria, as well as the effects of
parasitism on its eggs by one of its main egg parasitoids, D. leefmansi (Hymenoptera:

14. 3
Trichogrammatidae) that is mass reared in the laboratory and released in the field as part of
the general IPM program for the management of sexavae pests on oil palm.
This study was undertaken both in the laboratory and the field, and was carried out at Dami
Oil Palm Research Station, WNBP, PNG.
The following review develops a theoretical framework for the study presented in this thesis.
Rather than concentrating the review on the study insects, background information to the
importance of the crop (oil palm) that must be protected against the pest is also presented.
Sections 1.1, 1.2 and 1.3 provide information on the crop, whilst the next five (5) sections
(Sections 1.4 to 1.8) provide information on the study insects (S. defoliaria Uvarov 1924 and
D. leefmansi Waterstone 1928). The final section (Section 1.9) outlines the structure of the
thesis.
1.1 HISTORY, ESTABLISHMENT AND PRODUCTION OF OIL PALM IN PNG
Oil palm, Elaeis guineensis Jacquin, 1763 belongs to the family Arecaceae (Corley & Tinker,
2003). It is native to West Africa, and has spread to the other tropical parts of Africa and
other tropical regions of Asia and America through commercialisation for palm oil production
(Moll, 1987). Oil palm is grown mostly in countries that lie within 5-10 degrees north and
south of the equator. It needs a high but evenly spread rainfall of between 1800 to 5000mm
per year. It also requires over 2000 hours of sunshine per year, and grows better below 500m
above sea level (Page & Lord, 2006).
Oil palm was introduced to PNG in the 1920s, but was not commercially developed until
1968 when large plantations were established, initially in West New Britain Province
(WNBP) where it is the most important cash crop at present (Koczberski et al., 2001).
According to the same authors, the crop is now grown in five (5) project areas in PNG:
Hoskins and Bialla in WNBP, Northern, Milne Bay and New Ireland projects. More recently
it has been planted in Morobe and Madang Provinces (Nelson et al., 2010; New Britain Palm
Oil Ltd., 2011). However, WNBP is still the leading oil palm producing province in PNG
(Allen et al., 2009). In 2013, Fresh Fruit Bunch (FFB) production volume was 1,496,146
tonnes for plantations grown on an area of 79,884ha including the Guadalcanal oil palm
project in the Solomon Islands (New Britain Palm Oil Ltd., 2013) and 589,524 tonnes for out-
growers grown on an area of approximately 50,000ha (ZSL Living Conservation, 2012) with

15. 4
total revenue of US$558,652 million generated from both sectors for NBPOL (New Britain
Palm Oil Ltd., 2013). Figure 1 shows the locations of the project areas including its main
operation site in WNBP.
Figure 1. Oil palm project areas in PNG with the Head Quarter of NBPOL Main Operations
based in West New Britain Province (map from New Britain Palm Oil Ltd., 2011).
1.2 ECONOMIC BENEFITS OF OIL PALM TO PNG
Prior to 2000, coffee was the largest foreign currency earner for PNG, however after 2000
palm oil took over (Kumar, 2001) and it has become the most important export crop (Allen et
al., 2009). It was further reported that oil palm produced more edible vegetable oil per unit
area of land than any other commercial crop. Unlike other commercial crops, numerous
products can be derived from the fruits of oil palm, and they include crude palm oil (CPO)
and palm kernel oil (PKO) which are the most significant in terms of export volume, refined
palm oil and palm kernel expeller. The highest recorded production was in 2011 with
591,477 tonnes of oils (both crude palm oil and palm kernel oil) produced (New Britain Palm
Oil Ltd., 2012). Seeds produced by the seed production unit (SPU) of New Britain Palm Oil
Limited (NBPOL) at Dami are also sold to other oil palm growing areas around the world,
and within PNG. In 2012 oil palm seed sales from Dami Seeds had their strongest
performance with 14.7 million seeds sold within an estimated global market of 200 million

16. 5
seeds. This was a 25% increase from 2011, and generated USD$4.2 million profit before tax
(New Britain Palm Oil Ltd., 2012). An estimated 200,000 farmers in PNG that grow oil palm
depend on it as their principle source of income (Nelson et al., 2010). According to the same
authors, the benefit of this crop compared to other crops is that the profits are high and
income is earned regularly with harvesting done every two (2) weeks for more than 20 years
before replanting.
1.3 OIL PALM PESTS IN PNG
Dewhurst (2012) provides a comprehensive listing of the main invertebrate pests of oil palm
from PNG. Although the listing specifically covers pests occurring in WNB and New Ireland
Provinces, a few of the pests also occur on the mainland of PNG. The list identifies pests
from four (4) insect Orders and ten (10) Families, and they include two (2) Families
(Tettigoniidae and Phasmatidae) from the Order Orthoptera, two (2) Families (Scarabaeidae
and Curculionidae) from Coleoptera, five (5) Families (Hesperiidae, Lymantriidae, Noctuidae,
Peleopodidae and Psychidae) from Lepidoptera, and one (1) Family (Lophopidae) from
Hemiptera. The most damaging pests of oil palm in WNBP are from the family Tettigoniidae
(Caudwell & Orrell, 1997; Page 2005; Page & Dewhurst, 2010), with stick insects
(Orthoptera: Phasmatidae) also causing considerable localised damage particularly on palms
growing near the edges of natural forests (Dewhurst, 2012).
1.4 SEXAVAE PESTS (TAXONOMY, BIOLOGY AND PEST STATUS) OF OIL
PALM
Sexavae is a colloquial name used to refer to a group of Tettigoniidae commonly known as
bush crickets, katydids, long-horned grasshoppers or tree hoppers (Dewhurst, 2012). Whilst
the taxonomic status of many of the species of sexavae still remain to be confirmed, there are
four (4) species (which include two (2) sub-species) from two (2) different genera (Segestes
decoratus, Segestidea novaeguineae, Segestidea defoliaria defoliaria, Segestidea defoliaria
gracilis) both from the subfamily Mecopodinae (Orthoptera: Tettigoniidae) that are known to
cause economic damage to oil palm in PNG (Page, 2005). Of the four (4) economically
important species, two (2) (S. decoratus and S. defoliaria) occur in WNBP. Although there is
sympatric occurrence of these species in some areas, S. decoratus is more common in many
parts of the Province where oil palm is grown, and is facultatively parthenogenetic (Figure 3).

17. 6
Segestidea defoliaria is mainly localised in the north-eastern part of WNBP, and reproduces
through normal heterosexual reproduction (male-female mating). Segestidea novaeguineae
occurs on the mainland, whilst S. defoliaria gracilis occurs only on New Ireland.
Sexavae have long life cycles with relatively high fecundity and longevity (PNG Oil Palm
Research Association, 2011). According to Dewhurst (2012) some studies on the biology and
ecology of some of the species from PNG have previously been undertaken (Zelazny &
Hosang, 1987; 1991; Young, 2001; Page, 2005), but the information available is sparse and
more detailed studies for the rest of the species are still required. Generally, sexavae are well
suited to the warm wet climate of PNG. Eggs that are laid in the soil or palm frond bases
exhibit two (2) clearly defined egg diapause periods– the first is in the early stages of embryo
development and is obligatory; the second is at later stages in the development of the embryo
and is facultative, influenced by the environmental conditions such as prolonged dry weather
(Page, 2005). To further understand the biology and ecology of the pest species and make
informed management decisions, Papua New Guinea Oil Palm Research Association
(PNGOPRA) has initiated a series of studies into the biology and ecology of the pest species
and the current study forms part of that effort.
1.5 MANAGEMENT OF SEXAVAE PESTS
In many instances, pest management strategies around the world focus on individual
technologies such as chemical control, biological control, resistant plant varieties and cultural
control for the management of pests (Ooi et al., 1992; Kumar, 2001). However, in PNG an
Integrated Pest Management (IPM) approach has been promoted for the management of all oil
palm pests (Prior & Sar, 1992; Caudwell & Orrell, 1997; Caudwell, 2000) and there are six
(6) components (Figure 2). Knowledge of the biology and ecology of the pests and the use of
biological control agents are important elements of the management efforts.

18. 7
Figure 2. Schematic outline of the sexavae management IPM programme (Adapted from
Caudwell, 2000).
Each component of the IPM strategy plays an important role in the management of the pests.
Targeted Trunk Injection (TTI) is the standard pesticide application technique used by the oil
palm industry (both milling company plantations and smallholder growers) in PNG and is
carried out upon recommendation by PNGOPRA (Dewhurst, 2006). PNGOPRA Entomology
section recommends TTI for blocks with “moderate” to “severe” levels of infestation and for
monitoring for fields with “light” infestation. Recommendations are normally issued after
conducting extensive field surveys.
Populations of three (3) biological control agents of sexavae are still being reared at
PNGOPRA Entomology Laboratory on Dami Oil Palm Research Station and released in the
field for the management of the two (2) pest species in WNBP. Two (2) Hymenoptera egg
parasitoids, Doirania leefmansi Waterstone, 1928 (Hymenoptera: Trichogrammatidae) and
Leefmansia bicolour Waterstone, 1928 (Hymenoptera: Encyrtidae) are mass reared in the
laboratory and field released for the control of both S. decoratus and S. defoliaria eggs, whilst
the internal parasitoid Stichotrema dallatoreanum Hofeneder, 1910 (Strepsiptera:
Myrmecolacidae) (Solulu et al., 1998) is used only for the control of S. defoliaria in WNBP.
Infected male S. defoliaria carrying one or more mature female S. dallatoreanum are released
in the field (infection is confirmed by the protrusion of the parasitoid’s head on the host

19. 8
abdominal cuticle). Stichotrema dallatoreanum fails to successfully complete its life cycle in
S. decoratus from WNBP, although it does develop successfully in S. defoliaria (and S.
novaeguineae on the mainland). Leefmansia bicolor was introduced into PNG in 1933 by
Froggatt from materials collected from in (Indonesia) and was subsequently released in
Manus and New Hanover (Young, 1990). It was further released to the other parts of the
country including WNBP in the succeeding years. Doirania leefmansi is native to PNG and
was originally discovered from New Hanover (Froggatt, 1937). The original stock of D.
leefmansi to WNBP was introduced from there (New Hanover) by PNGOPRA in the early
1980s (Simon Makai pers. comm., 2014).
Whilst the overall IPM strategy has been developed as a guide for the management of oil
palm pests in PNG, specific information on the biology and ecology of individual pest species
and their biological control agents are still lacking. Furthermore, large numbers of egg
parasitoids (D. leefmansi and L. bicolor) are still being released on ad hoc basis, but there is
no empirical data to show if all eggs in the field are successfully parasitized. Hence, this
study is intended to generate information that will partially fill in this knowledge gap for S.
defoliaria.
1.6 THE STUDY INSECTS
1.6.1 Segestidea defoliaria (Uvarov, 1924) (Orthoptera: Tettigoniidae)
1.6.1.1 Origin and Distribution
Segestidea defoliaria (Uvarov, 1924) was reported to have originated from East New Britain
Province (ENBP) where it was defoliating coconut palms (Willemse, 1977; 1979). The initial
description was made using a male (holotype) and females (paratypes) specimens collected
from near Rabaul in 1923 (Willemse, 1979). Additional specimens used in the descriptions
were of the sub-species collected from Dami Oil Palm Research Station, Buvussi and Banaule
Village of WNBP. Thus, it can be argued that, the species spread from ENBP to WNBP,
feeding initially on coconut leaflets and then oil palm leaflets (Greve & Ismay, 1983; Young,
1987; Kumar, 2001; PNG Oil Palm Research Association, 2005). Segestidea leefmansi
(Willemse), a similar species not to be confused with S. defoliaria is also present on the Lihir
group of Islands and New Ireland (Young, 1987). Apart from its spread and distribution in

20. 9
parts of New Britain, S. defoliaria has not been recorded from the mainland of PNG. Plate 2
shows a female and male S. defoliaria.
According to Dewhurst (2012), the distribution of S. defoliaria in WNB is sympatric with S.
decoratus in some areas. The distribution map in Figure 3 shows the distribution of the two
(2) species in the oil palm growing areas of the Province. Segestidea defoliaria is mainly
concentrated towards the north eastern part of the Province particularly around Bialla, whilst
S. decoratus is mainly concentrated towards the south eastern part of the Province (Hoskins-
Talassea areas), with overlap of populations of both species in areas between those extremes.
Because of such overlapping distribution patterns, it is important that their biology is well
understood as this will help to differentiate the species responsible for the damage and further
develop effective species specific control measures.
Plate 2. Lateral view of the green form of the adult female and male S. defoliaria (A), where
the female has a long ovipositor (B), whilst the male has a short genital plate (C). Scale bar =
5mm (Photos: W.W. Page).

21. 10
Figure 3. Map showing the distribution of sexavae on oil palm in WNBP for the years
between 1993-2011 with S. defoliaria occurring mostly on the north eastern part of the
island1
.
1.6.1.2 Taxonomy and Biology of S. defoliaria
Segestidea defoliaria defoliaria (Uvarov, 1924) belongs to the subfamily Mecopodinae,
family Tettigoniidae and order Orthoptera. Uvarov in 1924 originally described it as Habetia
defoliaria, but in 1977, Willemse proposed the current name combination and relegated it to a
sub-specific level synonymising it with S. defoliaria simulatrix after studying the type
material (Willemse, 1977).
Little is known about the detailed biology of S. defoliaria. Page (2005) described the general
biology of sexavae indicating that they pass through 6-7 nymphal instars with adults having
both green and brown colour forms. He studied egg diapause in S. decoratus and identified
two (2) distinct diapause periods: an initial diapause at the early stages of embryo
development and late diapause in the late embryo development stages mainly influenced by
dry weather conditions. He speculated for S. defoliaria, S. gracilis and S. novaeguineae to
have similar diapause periods. Young (2001) stated that embryonic development of S.
1
Map courtesy of Solomon Sar, PNGOPRA

22. 11
defoliaria do not start until 15 days after oviposition. Females may lay 14-90 eggs in the soil
around the palm base or in frond bases and are very similar to those of S. decoratus (Page,
2005). The eggs hatch between 37-198 days. Young (1984) described the life cycle of S.
decoratus and assumed for the parameters studied to be common among the other species of
sexavae including S. defoliaria.
In 2011, PNGOPRA began a study on the fecundity of S. defoliaria and apart from the known
Pre-Oviposition Period (POP) a term often used by entomologists to refer to the period
between the final moult and the laying of the first lots of eggs (Lockwood & Lockwood,
2008), four (4) additional terms were developed for the current fecundity study. They
included Egg Laying Period (ELP), which is the period from the laying of the first egg to the
last egg lay; Post Egg Laying Period (PELP), is the period of time after the laying of last egg
to the time of death, and Nymph Duration Period (NDP), is the duration period for nymph
stages. All these terms are used in this Thesis.
Segestidea defoliaria is found mainly feeding on leaves of coconut and oil palm (Dewhurst,
2012). Other host plants include Pandanus leaves, banana leaves, Heliconia and kunai grass
(Kumar, 2001). The feeding patterns of S. defoliaria and S. decoratus are distinctly different.
Segestidea defoliaria feeds from the leaflet tip inwards towards the rachis, whilst S. decoratus
feeds from the leaflet base towards the tip and this feature can be used for rapid identification
of the species responsible for the damage in the field (Dewhurst, 2012). Both species can
cause serious damage to oil palm and result in large financial losses if left unchecked (Page &
Dewhurst, 2010). Leaf damage is caused by both the adults and nymphs, but adults are more
destructive (Ero et al., 2013). Outbreaks can lead to severe defoliation hence, potential
reduction of photosynthetic area and subsequent loss of yield (Page, 2005; Page & Dewhurst,
2010). In coconut palms, it can take up to two (2) years to recover from severe defoliation by
S. defoliaria (Froggatt & O'Connor, 1940; Young, 1987). Page & Dewhurst (2010) noted a
recovery time of at least four (4) years for oil palm.
1.6.1.3 Infestation levels
Segestidea defoliaria is one of the two (2) species of sexavae often causing severe damage to
oil palm in WNBP. Severe infestations by S. defoliaria on oil palm around Bialla area were
noted in the late 1970s (Young, 2001). PNG Oil Palm Research Association (1988) reported

23. 12
S. defoliaria activity to have remained low during most of the year but was noted to be
gradually increasing during the last quarter of the year. According to Caudwell & Orrell
(1997), the record levels of damage occurred in 1994 and 1995. The damage in most
plantations and smallholder blocks were caused by a combination of S. defoliaria and S.
decoratus. In recent years the levels of damage by S. defoliaria in some parts of the province
have been increasing.
1.6.1.4 Control methods
Effective control method for S. defoliaria currently applied in WNBP is through IPM. The
IPM programme involves the use of biological control agents, cultural methods and
insecticide (methamidophos) application through Targeted Trunk Injection (TTI). The
insecticide is applied to the vascular tissue bundle by drilling at 45° angle at a 1.5cm diameter
hole 15cm deep at 45° angle 1m above the ground using a motorised STIHL® drill
(Dewhurst, 2006). Targeted trunk injection confines the insecticide to the palm and only
controls insects that are feeding on the foliage or the stem of the treated oil palm, thereby
preventing undesired impact on other non-target organisms. It also prevents run off into water
ways. Targeted trunk injection is only used when the infestation levels are “moderate” to
“severe” as defined by PNGOPRA. When infestation levels are “light”, the fields are
recommended for monitoring. Moderate to severe infestations can greatly affect production;
hence treatment needs to be conducted once treatment recommendations are issued.
There are three (3) biological control agents currently mass reared and released in the field for
the control of S. defoliaria in conjunction with the other control measures and they include
two (2) Hymenoptera egg parasitoids (L. bicolor and D. leefmansi) and one (1) internal
parasitoid (S. dallatorreanum) which attacks late instar nymphs and adults (Page, 2005;
Dewhurst, 2012).
1.7 DOIRANIA LEEFMANSI WATERSTON (HYMENOPTERA:
TRICHOGRAMMATIDAE)
1.7.1 Origin and Distribution
The genus Doirania has a widespread distribution around the world including North America
and other parts of the Palaearctic (Pinto, 2004). In early studies, there were only two species

24. 13
described; Doirania longiclavata Yashiro (1980) native to Japan and D. leefmansi Waterstone
(1928) native to PNG as reported by Pinto (2004). A later revision by Pinto (2006) has an
additional species, Doirania elegans described from South America. In PNG, D. leefmansi
was first found as a local parasitoid by Froggatt in 1930 on New Hanover where it was reared
from sexavae eggs in the area (Froggatt, 1935). It was also reported to be found in Amboina,
in Indonesia by Leefmans (Froggatt, 1935; Froggatt & O'Connor, 1940). Breeding and life
history studies were conducted in Manus Province where it was multiplied for distribution to
other parts of the country including Dami, WNBP where the breeding and release programme
is still ongoing.
1.7.2 Taxonomy and Biology of D. leefmansi
The wasps within the Hymenoptera Family Trichogrammatidae remain little studied due to
their very small size. Doutt& Viggiani (1968) were the first to review all the described genera
and species of Trichogrammatidae. The main diagnostic characteristic of Trichogrammatidae
is the size of the adult wasps where they are minute to small with the body length of 0.3 –
1.2mm (excluding the ovipositor); non metallic; funicle with no more than two (2) segments
and the tarsi with three (3) distinct segments (Pitkin, 2003).
All known species of the family have three-segmented tarsi (Hayat & Viggiani, 1984).
Doirania leefmansi has a relatively uniform morphology like all the other species of
Trichogrammatidae and this makes the taxonomic studies of the species more difficult,
resulting in many nomenclatorial problems (Nagarkatti & Nagaraja, 1977). Despite the
difficulties, Doutt & Viggiani (1968) through a comprehensive revision of the family,
developed keys for seventy (70) genera and subgenera, and synonymised many of the groups.
This work formed a basis for the subsequent studies of Trichogrammatidae (Hayat &
Viggiani, 1984). Froggatt & O'Connor (1940) briefly described the phenotype, life cycle,
feeding and general behavior of D. leefmansi and the other species. Viggiani (1984), after
doing a comparative morphological study on the external male genitalia of 44 species of
Trichogrammatidae belonging to 28 genera including Doirania, concluded that “the phallus
features appear to be constant among the species of the same genera and are of high
diagnostic value for generic and specific discrimination”. This was an important diagnostic
character used to confirm the taxonomy and phylogeny of Trichogrammatidae. Pinto (2004),

25. 14
in reviewing the genus Doirania Waterstone (Hymenoptera: Trichogrammatidae) developed
the keys to the species, especially traits pertaining to females using specimens from PNG and
Amboina, Indonesia. Pinto (2004), observed the females to be light brown in colour; antenna
with scape sub-equal in length to the club; maxillary palp regular at apex and not narrowing
asymmetrically. He found that the asymmetrically narrowing maxillary palp occurred in D.
longiclavata and D. elegans but not in D. leefmansi. The forewing of D. leefmansi has a
small “fumate cloud (patch) directly behind the stigma; the sensilla anterior to retinaculum on
the dorsal surface of disk is small and acuminate”. The ovipositor is elongate, and distinctly
longer than hind tibia extending beyond cerci. The male genitalia in D. leefmansi are simple,
reduced to a single tube with two short apodemes at the base (Pinto, 2004). Plate 3 shows
aspects of a parasitized sexavae egg.
Plate 3. Photographs of sexavae egg parasitized by D. leefmansi (A) adult emergent spot
shown by blue arrows, (B) dissected egg showing D. leefmansi larvae, (C) D. leefmansi adults
emerging from the egg, and (D) D. leefmansi adults. Scale bar = 0.5mm.

26. 15
1.7.3 Laboratory rearing techniques for field releases
Between 1977 and 1980, attempts were made at Dami Oil Palm Research Station, PNG to
laboratory rear L. bicolour using the technique described by Froggatt (1935); however the
cultures failed after three (3) to four (4) generations. The first attempt to mass-rear the two
(2) parasitoids were in October 1985 with material brought in from New Hanover. Froggatt
(1935) noted that emergence of D. leefmansi took 40 days from exposure with 280 wasps
emerging from one (1) egg. Page (2005) recorded 250 D. leefmansi adults emerging from a
parasitized egg. The parasitoids emerged from an egg over a range of 4-7 days (T Manjobie,
2014, unpublished data). The number of emerging parasitoids recorded by Froggatt (1935)
and Page (2005) differed by 30 (i.e. around 10% difference). According to PNG Oil Palm
Research Association (2009), there were differences in emerging numbers depending on the
host egg being used either larger (S. decoratus eggs ca 0.22g) or the smaller (S. defoliaria
eggs ca 0.02g). Another influencing factor is the environmental conditions such as moisture
and temperature under which the parasitoids were reared which affected the total number of
emerging parasitoids as observed by Froggatt & O'Connor (1940). Mass rearing is being
done successfully however the quest for improving the techniques continues at present as past
techniques and materials used by other authors are being modified and improved for the
production of greater numbers. Although drawbacks such as high humidity resulting in
fungal contamination of host eggs, with cumbersome and labour intensive rearing processes,
populations of D. leefmansi and the other two (2) parasitoids are successfully maintained and
multiplied in thousands at PNGOPRA Laboratory in Dami for field releases.
1.7.3.1 Parasitism rates in the field
O'Connor (1937) and Froggatt & O'Connor (1940) recorded the level of parasitism by L.
bicolor and D. leefmansi and compared the level of parasitism by the two (2) parasitoids on
sexavae eggs, but the information was incomplete. Young (1987) alluded to the fact that there
are very few data available on the levels of parasitism for one (1) of the sexavae species
studied, S. decoratus. The eggs that were laid on frond bases and oil palm crowns had higher
level of parasitism than the eggs laid in the soil. Apart from this information, much still
remain to be understood about the level of parasitism in the field.

27. 16
1.7.3.2 Field release programmes of parasitoids and their effectiveness
Caudwell & Orrell (1997) noted that several million egg parasitoids are released each year
without properly determining the efficacy of the egg parasitoids for the control of sexavae
populations. They further queried if the majority of sexavae eggs laid in the soil are being
parasitized by the parasitoids when released as biological control agents and proposed that
their field performances be rigorously evaluated and some of the existing mass release
programmes be carefully revised to ensure effective parasitism. To date none of these
suggestions have been thoroughly investigated.
1.7.3.3 Conservation
Conservation of D. leefmansi is an important part of biodiversity management. When they
survive longer in the field, they parasitize more eggs and the field parasitoid population is
sustained in nature. The parasitoids feed on the nectar of flowering plants. Some flowering
plants produce sweet nectar and provide shelter valuable for maintaining populations of
parasitoids. Planting of such beneficial weeds at the edges of plantations and smallholder
blocks should be encouraged. This is essential for the conservation of the parasitoid
populations within the oil palm cropping system (Page, 2005).
1.8 SEGESTIDEA DEFOLIARIA AND DOIRANIA LEEFMANSI AS THE STUDY
INSECTS
Segestidea defoliaria is one of the most important pests of oil palm in WNBP, and Doirania
leefmansi is an effective biological agent that is used to contribute towards the management of
S. defoliaria in the Province. Apart from generalized information on sexavae as a group and
taxonomic descriptions of Trichogrammatidae parasitoids, no specific information exists on
the biology and ecology of either of the species, particularly in terms of the life cycle and host
egg utilization by the parasitoid. The focus of this study will be to understand the life cycle
and the fecundity of S. defoliaria and its egg utilization by D. leefmansi. This information
will be critical to help improve biological control efforts as part of the overall IPM
programme against sexavae in PNG.

28. 17
Segestidea defoliaria and Doirania leefmansi form an ideal system for this study as D.
leefmansi is successfully reared at Dami Entomology Laboratory; whilst large numbers of S.
defoliaria adults and nymphs can be easily collected from the field to maintain in the
laboratory for egg collection and rearing. Thus, they can be reared in large numbers for such
studies to understand the basic biology of the host insect and its utilization by the egg
parasitoid. An extensive literature search on both species shows a lack or little biological
information available for either of the species.
Finally, and more logically both species make ideal study organisms as they are typically
present in large numbers in the field both in smallholder oil palm blocks and plantations and
are dominant in many parts of WNBP where oil palm is grown. The eggs of S. defoliaria are
large enough for embryonic development to be observed by dissection and the number of
nymphal stages can be differentiated by the number of moults by constant observation for
evidence of shed skin. Although feeding and mating occurs at night for all sexavae species,
the number of times that a female has mated can be identified by the presence of an uneaten
spermatophylax in the genital opening of the female. Doirania leefmansi also parasitizes the
eggs of S. decoratus apart from S. defoliaria and can be mass reared on eggs from both
species to be used for the laboratory based trials. It is parthenogenetic (Page, 2005) and
therefore does not need a male to mate. Emergence holes of D. leefmansi are large enough to
be seen on eggs collected from the field with the naked eye, and this makes field observations
easy, an aspect which is also important for post- field release surveys.
1.9 STRUCTURE OF THE THESIS
In this thesis, the biology of S. defoliaria and the parasitism rate on its eggs by the parasitoid,
D. leefmansi were investigated. As outlined in the literature review section, the biology of all
pest sexavae species and their biological control agents is poorly understood. The thesis
chapters, while structurally independent have a common aim of expanding understanding of
the biology of pest sexavae species and their biological control agents so that informed
decisions can be made on their management.
In the first part of this study (Chapter 3), the reproductive potential of S. defoliaria was
investigated as part of understanding the biology of the pest. The study showed that the

29. 18
females of this species have distinct Nymph Duration Period, Pre-oviposition Period, Egg
Laying Period and Post egg laying Period, and that the embryonic stages are distinct and show
corresponding weight (g) increases. The number of eggs laid showed discrete peaks but was
not directly related to the number of times a female mated. These results imply that effective
management can be developed with the appropriate timing of the implementation of control
options.
Having information available on the biology of the pest, it was necessary that the parasitism
rate on host eggs by the parasitoids was understood. This was addressed in study 2 (Chapter
4) and the results showed that the level of parasitism by individual D. leefmansi was low and
that they preferred “stage 0” eggs for parasitism rather than the older stages. With this
information, parasitoid release programmes can be improved by releasing them at appropriate
times and improving the mass rearing programmes so that large numbers can be regularly
released for effective control.
In the last study chapter (Chapter 5), I went a step further from the laboratory work and
investigated the level of sexavae egg parasitism by the biological control agents in the field, to
help understand if the use of biological control agents for the control of sexavae had been
effective. The results showed that the level of parasitism varied from site to site. Apart from
D. leefmansi, other natural enemies (locally occurring parasitoids and predators) were found
to be effectively in killing the pest eggs. The disparity in the level of parasitism among the
sites was attributed to the lack of adequate supplies of floral resources for the adult parasitoid
populations.
In the final general discussion chapter (Chapter 6), the key findings from all three (3) studies
were drawn and implications for the effective management of the pest were outlined.
Recommendations were then provided on how the IPM programme for sexavae management
can be improved in relation to the information derived from the current studies.

30. 19
CHAPTER 2
2 GENERAL METHODOLOGY
2.1 SAMPLING SITE
Stocks of S. defoliaria were collected from NBPOL Malilimi Plantation, WNBP when
infestation levels were “moderate” to “severe” with the insects occurring in large numbers.
Doirania leefmansi used in these laboratory trials were collected from Lolokoru Plantation
(NBPOL) also in WNBP.
2.2 SAMPLING
More than 200 apparently healthy male and female S. defoliaria (adults and nymphs) were
hand collected from infested oil palm frond bases and around the palm bases in the field and
put into used (and washed) white pollination bags provided by SPU with some oil palm
leaflets as substrate and food source while in transit. Those insects without S. dallatoreanum
heads protruding from the insects’ abdomen were regarded as healthy. A flap was cut in the
clear plastic window through which to add insects to improve survival when transporting
them from the field to the study site (Plate 4).
Plate 4. Washed white pollination bags used for transporting sexavae. The black circle shows
the flap cut through the clear plastic window for inserting the insects.

31. 20
As many as 1000 to 2000 sexavae eggs were collected using soft point entomological forceps.
The eggs were carefully dug up from about 2cm beneath the soil surface around the base of
the palm and also collected from the palm trunk (Plate 5). They were placed in medical urine
sample pots that had a 1cm hole cut at the centre of the lids sealed with mesh cloth for
ventilation. The pots were filled with a small amount of soil to keep the eggs moist until they
were transferred back to the laboratory (Parasitoid Rearing Room) where they were washed
with tank (rain) water, dried on paper towel, sorted and kept for parasitoid emergence. The
collection bags and pots were labelled with the collection date, and location (with geo-
coordinates).
Plate 5. Example of how the eggs were collected from palm base (A), and frond base (B).
(Photo: Margaret Gavuri).
2.3 STUDY SITE
Studies were undertaken both in the laboratory and outdoor walk-in cages at Dami Oil Palm
Research Station (150°20’06”E, 5°31’53”S) in WNBP, PNG. Two (2) separate field
parasitism studies were done. The first study was done at four (4) separate sites in four (4)
OPIC Divisions (Buvussi, Kavui, Nahavio, Siki), and the second study was conducted in three

32. 21
(3) randomly selected sites at Dami (2 sites) and Banaule (1 site). Prior to conducting the
experiments, the fields were surveyed to find out if there were any beneficial plants growing
within each selected site, as these plants produce nectar which the adult parasitoids feed on to
survive in the wild. The first site was at Banaule Village Oil Palm (VOP) (150°20’22”E,
5°31’58”S); second site at Dami Oil Palm Research plantation close to the station
(150°19’51’E, 5°31’50”S) and the third site was also on Dami Oil Palm Research plantation,
but with the field backing close to Dami Recreational Beach (150°19’13”E, 5°31’15”S) about
5 minutes drive away from the first Dami site.
2.4 EXPERIMENTAL CONDITIONS
The outdoor walk-in cages were made from aluminum frames with four (4) wheels to allow
easy movement. They were covered with 50% green shade cloth and the floors of the cages
were of aluminum wire, covered with Pfeiffer® mosquito wire. The insects were maintained
in these cages under ambient environmental conditions where they were exposed to direct
rain, sunlight and wind. Two (2) palm seedlings grown in a black polythene bag each were
placed in the cages as food source. The seedlings were sprayed with water using a rubber
hose every morning and afternoon, and were replaced once the leaves were eaten. This
process continued until the required population of test insects were taken from the cage and
used for the studies. These cages were housed under a bigger green screen cage constructed
of similar materials as the small cages except that it had concrete floor (Plate 6). It also had
80% black shade cloth on the outer roof and 50% green shade cloth 2m beneath it. A small
PVC pipe with an outlet was cemented in the middle of the floor partially exposed. The
exposed pipe was cut randomly along the length of the pipe for draining water out during
watering of the feed palms and the cage wash up (Plate 6C).

33. 22
Plate 6. Insect rearing cages set up used for rearing insects (A) large cage, (B) walk-in cage,
(C) cemented water drainage PVC pipe, and (D) oil palm seedlings in black polythene bags.
The Quarantine facility where the trials were conducted comprised of three (3) separate rooms
separated by sealed doors. The facility is housed within the main PNGOPRA Entomology
Laboratory at Dami Oil Palm Research Station. The first is the “low security” quarantine
room not air conditioned and therefore with fluctuating room temperatures of 26-28°C and
relative humidity of 78- 90% and bench top work space. One (1) of the aluminium framed
movable cage was housed in this room to prevent unintentional S. dallatorreanum infection of
S. defoliaria. The second is a small transit room with well sealed doors on both sides. The
third room is the “high security” quarantine room and is air conditioned. It had work bench
tops, a sink and a freezer for freezing quarantine specimens before disposal. This room was
where most of the studies were conducted and was maintained at a constant temperature of

34. 23
260
C and 58% RH with 12:12 hour photo period. The feeding and rearing were done in
BugDorm® insect rearing cages set up on bench tops (Plate 3-1).
2.5 DATA ANALYSES
The data was analyzed using 17th
Edition of GenStat.

35. 24
CHAPTER 3
3 INVESTIGATION INTO THE REPRODUCTIVE POTENTIAL
AND EMBRYONIC DEVELOPMENT OF SEGESTIDEA
DEFOLIARIA (UVAROV) (ORTHOPTERA:
TETTIGONIIDAE)
3.1 INTRODUCTION
Reproduction can be through either sexual or asexual means. Sexual reproduction is where a
new individual is formed through the union of male and female gametes, thereby inheriting
genes from both parents, whilst in asexual reproduction an offspring arises from a single
female parent, thus inheriting the gene only from one parent (Johnson, 1989). Insects
reproduce either through sexual, asexual and/or both means of reproduction (Chapman, 1998).
Apart from some insect groups that go into diapause or hibernation at the egg or nymph/pupal
stages, which may extend their life cycle, almost all have short life cycles, but this is
compensated for by the utilization of diverse assemblage of reproductive potential that allow
them to produce prodigious numbers of offspring. The main reproductive strategies that
insects utilize to help compensate for their short life cycle include parthenogenesis,
paedogenesis, polyembryony, hermaphroditism, viviparity, oviparity and heterosexual
reproduction (Chapman, 1998; Klowden, 2013).
Whilst different reproductive strategies are employed by insects to maximize their
reproductive success, most natural environments do not present unlimited oviposition
opportunities, and actual fecundity is usually limited by the amount of time that the females
remain active and able to oviposit (Jervis et al., 2005). The key factors that influence
reproductive success of insects include environmental conditions, size and physiology of the
insects, quality of food sources, their genetic composition and the forms of reproduction
employed (Cherrill & Begon, 1989; Cordero, 1995; Ponsonby & Copland, 1998; Awmack &
Leather, 2002; Whitman, 2008). In environments with favourable conditions and abundant
suitable hosts, the amount of resources that a female converts to eggs should directly
determine her reproductive potential. Studies have also shown that larger females tend to be
more fecund than smaller females (Berger et al., 2008).

36. 25
Results from controlled studies have shown that the rate of increase in embryonic weight in
insects is directly proportional to temperature increase, when all other factors are kept
constant (Bodine, 2005). Egg maturation is strongly temperature-dependent, hence the
physiological process of converting resources to eggs may also be limiting under natural
conditions (Papaj, 2000).
Tettigoniidae are oviparous and embryonic development occurs in the egg after they are laid
utilizing the yolk as the food source. Ingrisch (1984), when studying morphological changes
during embryonic development of Decticus verrucivorus (Orthoptera: Tettigoniidae), detailed
26 embryonic stages. Prolonged embryonic development had also been noted in eggs of other
tettigoniids, and this has been attributed to either a dormancy sequence during embryogenesis
or prolonged diapauses (Ingrisch, 1984). In most genera, two (2) periods of diapause have
been noted during the course of embryogenesis (Hartley & Warne, 1972; Dean & Hartley,
1977). One appears to be natural and occurs during the early stages of development whilst the
second is triggered by dry weather and occurs at any time during the process of development.
According to Young (1990), embryonic development in S. defoliaria eggs did not start at least
until 15 days after oviposition. Hodek (2003), later found that moisture plays a key role in
breaking the diapause. Page (2005), studied diapause in S. decoratus and noted initial
(embryo development related) and late diapause (induced by dry weather) and speculated that
it could be the same for all species of sexavae.
Whilst there is a wealth of information available on the reproductive potential and the
embryonic developmental stages of Tettigoniidae as a family, no such information specific for
sexavae exists. Hence, this component of the study is intended to fill in this knowledge gap
by generating information on the reproductive potential and embryonic developmental stages
of S. defoliaria. Such information will form the basis for making informed management
decisions of this particular species of sexavae.

37. 26
3.2 MATERIALS AND METHODS
3.2.1 Investigation of the reproductive potential and the embryonic development of S.
defoliaria
3.2.1.1 Investigation of the reproductive potential of S. defoliaria
Male and female S. defoliaria of all stages were collected and kept outdoors in a walk in cage
which was housed under a large green screen insect rearing cage (L = 22m, W = 17m and H =
10m) at Dami Oil Palm Research Station (Plate 6).
From the large walk in cage, male-female pairs were removed two (2) days after they fledged
and set up in small feeding cages (60cm x 24cm rectangular Bug Dorm® cage) for mating
and egg-laying. Mating was confirmed by the presence of a spermatophylax (a white
gelatinous protein rich substance containing the spermatophore) plugging the female opening
(gonopore). A small round clear plastic pot (11cm in diameter and 8cm tall) was filled to the
top with sterilized moist river sand and placed inside the cage for oviposition. The sand
surface in the pot was gently compressed so as to make egg laying holes more visible (when
the female lays eggs, the ovipositor normally creates a visible hole on the sand surface). A
50ml plastic tube (9cm tall and 2.5cm in diameter) filled with water (without lid) was pushed
into the sand (base first) into the pot (Plate 7). The tube held two (2) fresh oil palm leaflets
which were changed at two (2) days intervals between 1400-1600hrs. The leaflets served as a
food source for the pair of sexavae. Two (2) leaflets were sufficient to provide food source
for a male-female pair of S. defoliaria for two (2) nights. Each cage was labelled according to
the replicate number.

38. 27
Plate 7. Summary of mating and egg laying (A) cage set up for feeding and mating, (B)
mating, (C) mated pair, (D) oviposition, (E) eggs laid, and (F) set up for embryonic
development.
The feeding cages were set up on benches at the laboratory in the “low security” quarantine
room, at fluctuating room temperature of 26-28°C and relative humidity of 78-90% monitored
daily. Once the females began laying eggs, the sand was checked and the number of eggs laid
was counted and recorded according to the replicate number and date laid. The retrieving of
eggs continued daily until the female died. From a batch of eggs laid on the same day, ten
(10) eggs were removed and placed in clear plastic pots with sterilized sand: the eggs were
covered with sand to a depth of 2cm, and the top of the pots covered with mesh cloth,
fastened with a rubber band. These were maintained in the large outdoor walk-in sexavae
rearing cage under ambient environmental conditions. The pots were watered with distilled

39. 28
water whenever the soil began to dry and the eggs monitored for nymph emergence until 150
days. Segestidea defoliaria eggs have a peak hatching period of between 60 to 80 days, and
may go into diapause thereafter (Tabitha Manjobie, unpublished). Eggs that did not hatch
after 150 days were dissected to see if they were still viable or had failed to develop (died).
For eggs that hatched, the dates of nymph emergence and sex were recorded. The nymphs
were fed with fresh clean young palm leaflets (washed with distilled water) and monitored in
the BugDorm® cages, held in another air conditioned room (high security quarantine room)
with temperature maintained constant at 260
C. For those that died, the date of death and the
sex was recorded. For those nymphs that fledged to become adults, the number of moults and
the date of fledging were recorded, and the nymphs transferred to larger cages and fed with
mature palm leaflets until they died, where the date of death was recorded. The parameters
measured were:
Nymph Duration Period (NDP): period from when the nymph hatched to the time of fledging
Pre-oviposition Period (POP): period from fledging to first egg laying
Egg Laying Period (ELP): period between first egg laying and the last egg laying
Total Eggs Laid (TEL): is the sum of all eggs laid by a single female during its life time
Post Egg Laying Period (PELP): survival period of the female after its last egg was laid
The nymph and adult life spans as well as the number of times the male-female pairs mated
were also recorded.
The trial was replicated 10 times with separate male-female pairs.
3.2.1.2 Investigation into the embryonic developmental stages of S. defoliaria
For embryonic development assessment, the same protocol as above was followed for mating
and egg laying. Five (5) female and two (2) male adults each of S. defoliaria were set up
together in small Bug Dorm® insect cages (32.5cm x32.5cm x 32.5cm). Once the females
started laying eggs, 50 eggs from the same cohort (all laid during the same day) were removed
from each cage and set up in separate holding pots containing moistened sterilized sand as
replicates (10 replicates). One egg from each pot (11 eggs) (replicated) was removed at days
7, 14, 21, 28, 42, 56, 70, 84, 98, 112, 126 intervals, and were weighed, dissected and the
embryonic stages of the eggs determined. The remaining eggs were left to hatch.

40. 29
A Stereo Microscope (Leica MZ75) was set up to obtain a critical image using the technique
described by Sumner & Sumner (1969). A Petri dish half filled with clean tap water was
placed at the centre stage of the microscope. The water in the Petri dish kept the egg tissues
moist and made observation easier during dissection. An unhatched egg was washed, dried
and placed in the Petri dish with water and dissected under the microscope. The egg was held
lightly (with the hatching line of the egg facing up) using fine forceps (with curved tips) and
dissected with a small ophthalmic scalpel. This was done by running the blade very lightly
along the length of the egg ensuring not to cut too deep as this may destroy the embryo
making it difficult to distinguish the stages. To get clear exposure of the embryo, the egg
shell was carefully peeled off with forceps. For the embryo that was visible, observation and
recording of the embryonic stage was done in reference to Ingrisch (1984) (Plate 8).
This experiment was replicated 10 times with the number of eggs at daily intervals for
dissection.
3.3 RESULTS
3.3.1 Investigation into the reproductive potential of S. defoliaria
According to a One Way ANOVA analysis, there was a significant difference in the number
of days taken by the different life stages of the female (F27,3 = 20.62, P < 0.001). The Scheffe
post-hoc pair-wise comparison test showed that the number of days taken for the Nymph
Duration Period (NDP) and Egg Laying Period (ELP) did not differ significantly from each
other but were both significantly different to Pre-Oviposition Period (POP) and Post Egg
Laying (PELP) which were also not significantly different from each other (Figure 4). The
sum of all four life stages put together gave the total longevity of females which lived for an
average of 286 days (n = 30, Range = 33 - 111) (Figure 9).
The frequency of mating between each male- female pair varied. The maximum number of
times a pair mated was 42 while the lowest number of times it mated was one (1) with the
female infected with S. dallatoreanum and four (4) times by two (2) other healthy pairs. The
total number of eggs laid also varied. The maximum number of eggs that a healthy female
laid was 287 with a mean of 176 (Table 1).
When a Pearson Correlation test was run between the number of times the pairs mated and the
number of eggs the female laid, there was a weak correlation (R2
= 0.167) (Figure 5). The

41. 30
egg laying period lasted over a period of 16 weeks with a peak number of eggs laid during
week 5. Most of the eggs were laid between weeks 2 and 10 and declined gradually until
week 16 where only around three (3) eggs were laid by three (3) different females (Figure 6).
Figure 4. Mean number of days (±SE) of the different life stages in female S. defoliaria (n =
30).

42. 31
Table 1. Frequency of mating and the number of eggs laid by females (n=10).
Replicate Frequency Mating Total number of eggs laid
1 8 252
2 17 122
3 5 135
4 15 287
5 * 1 49
6 42 247
7 * 7 50
8 4 247
9 4 175
10 19 198
*Stichotrema dallatoreanum infected females
Figure 5. Relationship between the number of eggs laid by S. defoliaria and the frequency of
matings.

43. 32
Figure 6. Number of eggs laid by S. defoliaria during the egg laying period per female per
week.
3.3.2 Investigation into the embryonic developmental stages of S. defoliaria
There was a strong correlation between the mean egg weight (g) and changing embryonic
stages with 60% (R2
= 0.60) of the egg weight influenced by embryonic stages (Figure 5).
The embryonic weight increased from one stage to another, being more pronounced
particularly from stages 10 to 25. Only four (4) embryo stages (stages 14, 20, 24 and 25)
were clearly distinguishable under the binocular microscope (Plate 8). Following Ingrisch
(1984), the key distinguishing characters in these four (4) stages were:
(i): Stage 14: the embryo was found almost at one end of the egg. This is not shown in the
photograph below because the embryo was removed from the egg shell for photographing.
Eye pigment was reddish, and the antennae reached the first pair of embryonic feet when
observed from ventral surface (Plate 8 A [15 -17]).
(ii): Stage 20: yolk was visible and protruded behind the caput (pronotal region), but not as
pronounced as in stages 21 and 22. Embryo was white and translucent.
(iii): Stage 24: pigmentation of the embryo was completed and was nearly dark green in
colour; antennae reached the last (4th
) abdominal segment when observed in ventral aspect.
(iv): Stage 25: embryo was fully developed; eye pigment was dark, the whole embryo was
dark green in colour, eye pigment was dark, the mandibles were dark and integument around

44. 33
the thorax was heavily swollen, the antennae reached the full length of the abdomen when
seen from ventral aspect.
The data for each instar after hatching were combined and a chi-square test was run to test if
there was a significant difference in the number of days taken by the male and female
immature stages to moult. There was a significant difference in the number of days taken by
the different nymphal instars of the male and female before moulting (χ² = 4.12, d.f = 5, P =
0.53). The developmental period for each male nymphal stage did not vary significantly
among the different instars, whilst for female nymphs the developmental period for the
second and sixth instar took longer than all but not the third instar (Figure 8).
Segestidea defoliaria has a long life cycle of more than 400 days, with the adults persisting
longer than for all nymphal stages combined. The egg stage lasted a mean of 79 days before
hatching (n= 30, range = 68 – 107 days). The mean number of days (combined across all
instars) taken by female nymphs (112 days, n =30, range = 107 – 120 days) was slightly
shorter than the number of days taken by the male nymphs (113 days, n = 30, range = 107 -
121). Thus, males had a slightly longer NDP than the females. In the adult stages, the males
also lived slightly longer than the females (Figure 9).
Figure 7. Mean weight (g) of S. defoliaria eggs across the different embryonic developmental
stages.

45. 34
Plate 8. Embryonic stages of Decticus verrucivorus (Linnaeus 1758) (A)2
, and four (4) clearly
distinguishable embryonic stages of S. defoliaria under the stereo-microscope (i = stage 14, ii
= stage 20, iii = stage 24, iv = stage 25). Scale bar = 10mm.
2
Adopted from Sigfrid Ingrisch (1984).

46. 35
Figure 8. Number of days taken before moulting for each instar (n= 30) of immature male
and female S. defoliaria.

47. 36
Figure 9. The life cycle of S. defoliaria.
3.4 DISCUSSION
The Nymph Duration Period (NDP) and the Egg Laying Period (ELP) were significantly
longer than the Pre-Oviposition Period (POP) and Post Egg Laying Period (PELP).
Segestidea defoliaria has a total of six (6) nymphal instars. The long nymphal stage period
can be attributed to the body size of the insect, as the insects are big. According to Callier &
Nijhout (2011), body size has a profound effect on many aspects of animal biology including
metamorphosis. The long egg laying period is likely influenced by the large body size and
physiology of the insect, and availability of food sources, as these factors are known to impact
on the reproductive potential of insects (Awmack & Leather, 2002; Berger et al., 2008;
Whitman, 2008).
Mating was observed during the late afternoon to early evening hours (1500-1800hrs) and
sometimes during the night as a spermatophore containing a spermatophylax was usually seen
on the female in the mornings of the next day. Copulation behaviour of S. defoliaria was
witnessed several times in the same manner as described by O'Connor (1937) for S.

48. 37
novaeguineae and Gwynne (2001) for other Tettigoniidae. The male showed sexual
excitement and aligned itself alongside the female laterally with both facing the same
direction. The hooks of the epiphallus grasped the female sub-genital plate, the penis was
then introduced where the sperm was eventually released into the female genital tract
(Uvarov, 1966). After mating, the male deposited a substance known as the spermatophore
around the female sub-genital plate. The spermatophore was surrounded by the
spermatophylax, which is a large gelatinous protein rich sperm-free portion (Gullan &
Cranston, 1994). The spermatophylax was subsequently eaten by the female as a form of
parental investment in which nutrients obtained help to increase the number and size of eggs
laid (Gullan & Cranston, 1994). The presence of spermatophore was used to confirm mating,
and may remain attached to the female genitalia plate for 12 to 24 hours if the spermatophylax
is not eaten soon enough. Apart from mating, most of the night is used for feeding and egg
laying (females).
Males were able to mate multiple times with the females over their lifetime. Unparasitised
males mated 14 times on average. A mean of 208 (n = 8, range: 122 -287 eggs) eggs were
laid by each healthy female (excluding those laid by the parasitized females) implying that
each female is capable of laying a large number of eggs after mating. There was weak
correlation between the number of times mating had occurred and the number of eggs laid by
the females showing that the number of times mated did not influence the number of eggs
laid. For instance, two (2) females laid 247 eggs each but one mated 42 times whilst the other
mated only four (4) times. One (1) female infected with S. dallatorreanum mated only once
but laid 49 eggs. This showed that there was multiple-fertilization from a single mating.
Oviposition on average began 33 days after a female fledged and this is the same as reported
for S. novaeguineae by O'Connor (1937). The egg laying period was 16 weeks (an average of
101 days, range 3-186) with a unimodal peak of laying period at week five (5). There was a
rapid increase in egg laying activity during weeks one (1) to five (5) with a gradual decline
from week 6 to week 16 where only 1 egg each were laid by 3 different females. Whilst the
number of eggs laid is usually influenced by the egg laying capability of the female, the result
shows that there is generally a period where peak numbers of eggs are normally laid by the
females.

49. 38
There were 25 embryonic stages recognized in this species as described by Ingrisch (1984) for
Decticus verrucivorus. However, in the current study, the developing embryos only became
visible under the stereo-microscope (Leica MZ8) at 10x magnification from stage 9 to stage
25, where most of the features were fully developed ready for hatching. Stages 1 to 8 were
not clearly visible, as the stereo-microscope used for viewing and the camera (Dino–Lite®,
FC 150829) used for photographing were not powerful enough to pick up the finer details of
the earlier stages. Stage 14 was also the stage at which the weight increased. The mean egg
developmental period was 79 days, and this period may be attributed to the large number of
developmental stages (25 stages) that the eggs pass through prior to hatching. The eggs
developed progressively during the embryonic period and this correlated significantly with
weight increase. Around 60% of the weight was caused by the changes in the embryonic
stages, showing that the weight increase was directly related to changes during embryonic
development at each stage.
The instars were differentiated by the moulting process (shedding the cuticle from one stage
to another and thus changing instar). The difference in the developmental period for male
nymphs to that of females was only two (2) days but the reason is not clear. It was difficult to
differentiate males from females at first and second instars but became obvious from the third
instar onwards when the genitalia were clearly visible under the microscope. The newly
emerged nymphs were dark green in colour in both males and females and generally
resembled the adults but without wings. They developed wing buds at the 4th
instar (average
of 77 days). As they moulted to second instar, they turned light green. Some of the nymphs
were observed to change colour to brown at third instar stage. The vermiform stage is not
counted as the first instar because it still contains the embryonic cuticle which is shed before
moulting into first instar (Brown, 1990).
The long life cycle of S. defoliaria can be attributed to the long egg stage, 6 nymph instars
and the large size of the adult and availability of food sources (Awmack & Leather, 2002;
Berger et al., 2008). Whilst there maybe overlapping generations in the field, the results
indicate that each generation is univoltine completing a full life cycle well over one calendar
year.

50. 39
CHAPTER 4
4 LABORATORY STUDY ON THE PARASITISM RATE OF S.
DEFOLIARIA DEFOLIARIA (UVAROV) (ORTHOPTERA:
TETTIGONIIDAE) EGGS BY DOIRANIA LEEFMANSI
WATERSTON (HYMENOPTERA: TRICHOGRAMMATIDAE)
4.1 INTRODUCTION
Doirania leefmansi is an egg parasitoid of sexavae, and it is native to PNG. The species has
been mass reared and field released on an ad hoc basis as part of the sexavae IPM programme
in WNBP. It is one of the three (3) species (D. leefmansi, L. bicolor, and an unidentified
Mymaridae) of egg parasitoids that have been identified to be of economic importance as
biological control agents of sexavae (Froggatt, 1937; Young, 1990). Caudwell & Orrell
(1997) observed a higher number of sexavae eggs on the palm frond bases and trunks to be
successfully parasitized than those laid in the soil.
Doirania leefmansi is parthenogenetic and only females are found. Adults are approximately
0.5mm in length. Seen with the naked eye, they are the size of a small pin head, but when
magnified they have light coloured thorax and dark abdomen (Froggatt & O'Connor, 1940).
The species completes its life cycle in 38-41 days with average of 39.5 days (Froggatt, 1935),
but the adults survive 2-3 days without food and when they are fed (with honey) tend to
survive a bit longer for 4-6 days (PNG Oil Palm Research Association, 2008; 2009). Page
(2005), reported that around 250 D. leefmansi adults can emerge from a single S. decoratus
egg. The adult parasitoids emerge from a single oviposition, but it is not clear if large
numbers of eggs are deposited or that multiple numbers of larvae develop from a single egg.
The larvae pupate within the host egg and adults emerge by biting a hole in the chorion of the
eggs (Pitkin, 2003).
Adult parasitoids are known to utilise nectar resources from flowers of beneficial weeds as
food source (Young, 2001). According to Gurr et al., (2003), there may be a need to integrate
annual and perennial non-crop vegetation with crops to increase biodiversity at landscape
level, and this may include beneficial plants that support the adult biological control agents.

51. 40
Whilst this is more common for most parasitoid species, it is still not clear which species of
beneficial plants are able to adequately support the sexavae parasitoid populations in oil palm
systems. The laboratory parasitoid cultures are normally fed with processed honey purchased
from the local supermarkets.
Apart from generalized information on the biology, specific biological information for this
particular species such as parasitism rate and preferred host egg stage are still lacking. The
thrust by Caudwell & Orrell (1997) for rigorous investigation of the field performances of any
parasitoids that are released as part of oil palm pest IPM programmes have not been
undertaken.
This component of the study investigated the parasitism rate and the preferred stage of host
egg for parasitism by D. leefmansi.
4.2 MATERIALS AND METHODS
4.2.1 Experimental conditions
The study was conducted in the High Security Quarantine air conditioned room in the
Entomology Laboratory, at a constant temperature of 260
C and 12:12 hour photo-period, as
described earlier.
4.2.2 Host eggs and the parasitoid
Non-parasitized S. defoliaria eggs were obtained from eggs laid by laboratory reared females
(the same protocols as for study 1 were used for egg laying). The parasitoids were mass
reared in the laboratory in the Low Security quarantine room before their use for the studies in
High Security quarantine room.
4.2.3 Investigation into the rate of parasitism on different age eggs of S. defoliaria by
D. leefmansi
Fifteen (15) S. defoliaria eggs each of five (5) days (Stage 0), 15 days (Stage 18 - 20) and 28
days (Stage 21 - 25) old were exposed to five (5) mature females of Doirania leefmansi for 48
hours. D. leefmansi start ovipositing within 48 hours of hatching (Simon Makai, pers. comm.,
2014). After 48 hours, the parasitoids were removed and the eggs were monitored for
parasitoid emergence. A cotton bud was dipped in processed honey and provided as food

52. 41
source together, with water for the adult parasitoids. The parasitoids that emerged were
counted and the dates of emergence recorded. This process continued until no more
parasitoids emerged. Unhatched eggs were dissected after parasitoids stopped emerging (50-
121 days) to see if S. defoliaria embryos were still developing, or if they were dead.
This trial was replicated 15 times for each embryonic stage.
4.2.4 Investigation of the parasitism rate of S. defoliaria eggs by individual D. leefmansi
A sterilized (60ml) medical urine sample pot was used for this trial (Plate 7). A small hole
(1cm in diameter) was cut out at the centre of the lid and glued over the inside of the lid
(using super glue®) with muslin netting to allow for ventilation. A piece of Oasis® (flower
arranging foam) was cut to size and pushed to the bottom of the container. Ten (10) freshly
laid eggs (5 days old) were set up in the container and 1ml distilled water was added to the
Oasis® using a disposable plastic Pasteur pipette (3.5ml) to provide moisture for the eggs.
Emerged parasitoids were transferred from the main culture onto a plain white A4 paper using
a small soft paint brush as the insects were very small (0.5mm). The paper with the
parasitoids was held vertically above the opened container with the eggs, and a single
parasitoid was carefully guided into the container and the lid was screwed on tightly. The
parasitoids used were one (1) day old as females of this species normally start ovipositing
within 24-hours after emerging (Simon Makai, pers. comm., 2014). There were four (4)
different exposure periods for each parasitoid of 1, 12, 24 and 48 hours.
The trial was replicated ten (10) times for each exposure period, and each comprised of 10 S.
defoliaria eggs each.
After each of the assigned exposure periods lapsed, the parasitoids were removed from the
container containing the ten (10) eggs. Assuming that all ten (10) eggs were parasitized by
the single parasitoid, these eggs were then removed from the medical urine sample pots and
set up individually in smaller glass livestock tubes (50mm x 25mm). The lid of each tube had
a circular air hole covered in very fine bronze gauze. A piece of Oasis® was cut to fit the
base of the tube and an egg was placed on it repeating the procedure as above. The whole
process was repeated for the ten (10) replicates for all four (4) treatments and each tube was
labelled according to replicate, treatment and egg number. Parasitoid emergence was
monitored for 40-50 days (these were the range of days in which parasitoids complete

53. 42
emerging from an egg as observed in study 4.2.3). The number of eggs parasitized and the
number of parasitoids emerging were counted and recorded. Eggs that remained unhatched
were dissected to see if the S. defoliaria embryos were still developing or if they contained
parasitoid larvae. The same protocols as in study 1.2 were used for the dissections. As per
Young’s protocol for confirming parasitism of eggs (Young, 1987), those containing
immature stages of parasitoids, were recorded as parasitized; those eggs with S. defoliaria
embryo developing were recorded as hatched. The eggs that failed to develop were recorded
as not viable (dead).
The trial was replicated ten (10) times for each S. defoliaria age group.
4.3 RESULTS
4.3.1 Investigation into the rate of parasitism on different age eggs of S. defoliaria by
D. leefmansi
There was a significant difference (P < 0.001, n = 45) in the level of parasitism among the
different age eggs of S. defoliaria. The number of five (5) day old eggs parasitized was
significantly higher than the 15 and 28 day old eggs. The 15 day old eggs were least
parasitized (Figure 10).
The proportion of eggs that desiccated (died) from each age group was low compared to those
that were either parasitized, hatched or did not hatch but were still viable (Table 2).

54. 43
Figure 10. Mean (± SE) number of different aged S. defoliaria eggs parasitized by D.
leefmansi.
Table 2. Proportion (%) of S. defoliaria eggs from the different age stages that were
parasitized, unparasitized, unhatched (viable) and desiccated (dead).
5 Days 15 Days 28 Days
Parasitized eggs 50.0 13.3 30.7
Unparasitized eggs (hatched) 16.7 42.0 20.7
Unhatched viable eggs 32.7 39.3 46.6
Desiccated eggs 0.6 5.4 2.0
4.3.2 Investigation of the parasitism rate of S. defoliaria eggs by individual D. leefmansi
A One-Way ANOVA test showed a significant difference in the number of eggs parasitized at
four (4) different time intervals (p = 0.003, n = 10). According to the Scheffe pair-wise test,
the number of eggs parasitized in 1, 12 and 24 hours did not differ significantly but all
differed significantly from those parasitized in 48 hours (Figure 11).

55. 44
The proportion of unparasitised eggs was highest for all four (4) exposure time periods
compared to the parasitized and desiccated eggs (Shutts, 1949). The proportion of unhatched
viable eggs was low for those exposed for 1 and 12 hours, but was high for those exposed for
24 and 48 hours (Table 3).
Figure 11. Mean (± SE) number of S. defoliaria eggs parasitized at different time intervals by
D. leefmansi. Different letters above the error bars denote significant differences.
Table 3. Status of S. defoliaria eggs after exposure to D. leefmansi for different exposure
periods.
1 Hour 12 Hours 24 Hours 48 Hours
Parasitized eggs 5.0 8.0 9.0 23.0
Unparasitized eggs (hatched) 93.0 86.0 45.0 36.0
Unhatched viable eggs 0.0 2.0 46.0 41.0
Desiccated eggs 2.0 4.0 0.0 0.0
a

56. 45
4.4 DISCUSSION
Doirania leefmansi preferred S. defoliaria eggs that were five (5) days old rather than those
that were either 15 or 28 day old eggs for oviposition. The adults of D. leefmansi are about
0.5mm in length and appear to have parasitized the host eggs when the chorion (outer
membrane of the egg shell) of the host eggs was soft. According to Shutts (1949),
grasshopper eggs that are freshly laid have a soft chorion that is about 20µ thick, so this may
have made it easier for ovipositor penetration. When five (5) day old eggs were dissected and
observed under the stereo-microscope, the egg contents (yolk) were clear, indicating that most
of the oviposition by the parasitoid occurred prior to the development of the embryo so that
the developing parasitoid larvae can feed on the embryonic fluid of the host for their
development.
When five (5) day old eggs were exposed to individual D. leefmansi at different time intervals
(1 hour, 12 hours, 24 hours and 48 hours) to assess the rate of parasitism, the percentage of
eggs parasitized for first three (3) time intervals (1, 12 and 24 hours) was below 10% albeit
more eggs were parasitized from those exposed for 48 hours. The actual number of eggs
being parasitized per hour was less than 5 (data not presented) which gave rise to the low
percentages. There are two (2) possible reasons for this low level of parasitism. Firstly, it
could be attributed to the number of times an individual female is able to oviposit eggs in its
life time. Because of the small size of the parasitoid, and the short life span (4-5 days), it
appears that and individual female is only able to parasitize less than five (5) eggs in its life
time, but this assertion requires further investigation. The second reason could be attributed
to the lack of natural food resources in the laboratory setting. In the field, adult parasitoids
forage and derive their food resources from the nectar, honeydew, pollen and sugar from
flowering plants that increase their chances of survival, fecundity and parsitism (Kidd &
Jervis, 1989; Leatemia, et al., 1995; Rusch, et al., 2010), however the insects used in the trial
were only feed processed honey. This diet restriction may have reduced ovarial development
thus limiting their egg laying capability. Futher studies need to be conducted to investigate
this possibility.

57. 46
CHAPTER 5
5 FIELD PARASITISM OF SEXAVAE EGGS BY DOIRANIA
LEEFMANSI WATERSTON (HYMENOPTERA:
TRICHOGRAMMATIDAE)
5.1 INTRODUCTION
Parasitoids are insects that live and feed on their host tissues, and in the process kill their
hosts (Vincent, 1976). Because of this ability to kill their hosts during feeding, they are most
effectively used as biological agents of insect pests. Parasitoids are mainly from the families
Hymenoptera, Strepsiptera and Diptera (Godfray, 1994).
In most cases adults are solitary and live most of their lives away from the hosts after having
laid their eggs. Most adults of parasitoids of Hymenoptera and Diptera are pollen and nectar
feeders (Schmidt et al., 2004) and tend to seek floral resources which may be distance away
from the hosts to feed and survive (Young, 2001). For parasitoids that are used as biological
control agents, it is important to ensure that adequate floral resources are available in areas
where biological control agents are released. Availability of both the hosts and the floral
resources within close proximity will enhance survival of parasitoids which will encourage
effective control of their hosts.
The potential for parasitic species to regulate host populations is dependent on many different
factors such as the reproductive potential, sex ratios, host finding ability of parasitoids,
temporal and spatial synchronism and environmental parameters (Salatic, 1963). When
selecting biological control agents to use in biological control programmes, it is critical to
adequately understand the influence attributed by each of these factors on the parasitoids.
This component of the study investigated the parasitism level of sexavae eggs in the field by
D. leefmansi. Whilst large populations of parasitoids have been field released on ad hoc basis
as part of the sexavae IPM programme in the Province, it is not fully understood if the
parasitoids are establishing successfully and exerting effective control on the pest populations.

58. 47
Hence, the information derived from this study is deemed to provide background information
to the efficiency of field parasitism.
5.2 MATERIALS AND METHODS
5.2.1 Field egg sampling for parasitism assessment (baseline data)
Four separate oil palm blocks in four (4) OPIC Divisions with a known history of sexavae
infestations and releases of biological control agents were selected and sexavae eggs were
sampled to assess the levels of parasitism. Once the blocks were selected, ten (10) palms
were chosen (each palm as a replicate) and the eggs were collected from palm bases using
small metal quadrats (25cm x 25cm) as the unit for sampling. The sampling was repeated
three (3) times on fortnightly basis in each block on ten (10) different palms selected
randomly. Eggs were collected and brought back to the laboratory and processed to
determine the levels of parasitism.
5.2.2 Investigation of the parasitism rate on S. defoliaria eggs in the field by D.
leefmansi
Two hundred (200) unparasitized S. defoliaria eggs (5 day old) were obtained from the
laboratory culture and a batch of 100 eggs each were placed into moistened sand placed in
small mesh size fly wire cages (Plate 9A). Five hundred (500) mature D. leefmansi (48 hours
after emergence) were collected from the laboratory culture using an aspirator and released
simultaneously with the host eggs in the field at selected sites and the point of release marked.
Each cage was randomly pinned down to the soil using U-shaped nails under a frond pile
(Plate 9B). This was done to prevent small rodents from carrying the egg cages away from
the points of release. The trial was left for one (1) week and checked daily for signs of
predation. After one (1) week, the eggs were retrieved and individual eggs were set up in
small gauze lid plastic bottles (same protocol as in study 4.2.4), and the number of parasitoids
that emerged from each egg were counted and recorded. For the eggs that did not hatch after
50 days, they were dissected to see if they were still viable or parasitized. For egg dissections
and determination of egg viability (whether host embryo or parasitoid embryo), the same
protocol as in study 1.2 and 4.2.3 was followed.

59. 48
This trial was replicated across three (3) sites. Two (2) were within Dami NBPOL Plantation
(150°19’51.4794’E, 5°31’50”S and 150°19’13”E, 5°31’15”S) and one at nearby Banaule
Village Oil Palm (VOP) block (150°20’22”E, 5°31’58”S).
Plate 9. Segestidea defoliaria egg set up in the field for parasitism (A) placement of eggs and
filling of moistened sand in mosquito wire, (B) positioning of mosquito wire cage, (C) release
of parasitoid and (D) all set up. Left side photos are of the 3 sites where trials were set up.
5.3 RESULTS
5.3.1 Field egg sampling for parasitism assessment
A Two-Way ANOVA was run to test if there was any interaction among the sites and the
different status of sexavae eggs sampled, as the field upkeep levels were not uniform among
the four (4) sampling sites. There was a significant interaction among sites (four divisional
sites) and the status of eggs sampled (F9,10.56 = 20.30, P < 0.001) with significant differences

60. 49
both among the different status of the eggs (F3,66.18 = 127.30, P < 0.001) and across the four
sampling sites (F3,2.29 = 0.01, P < 0.001). When the Scheffe pair-wise comparison test was
run, there were significant differences among all different status of eggs sampled from within
each division except for the number of unhatched and predated eggs sampled from Buvussi
Division where there was no significant difference in the number of unhatched and predated
eggs sampled. There was no consistent pattern in the number of eggs sampled for each egg
status across the sites. For the number of eggs hatched, the number that hatched from Buvussi
Division was significantly higher than those from the other three (3) divisions. The number
of unhatched eggs from Buvussi and Siki Divisions did not differ significantly but differed
from the number of unhatched eggs sampled from Nahavio and Kavui Divisions where the
number sampled from these divisions were also significantly different. The number of
parasitized eggs sampled from all four (4) divisions differed significantly. For the number of
eggs predated, those sampled from Nahavio and Siki Divisions did not differ significantly
from each other, but were significantly different to the number of eggs sampled from Buvussi
and Kavui Divisions which were significantly different from each other (Figure 12).
When the eggs were processed to assess the level of parasitism, they were found to be
parasitized by D. leefmansi as well as by other locally occurring parasitoid(s). The proportion
(%) of parasitism between the two (2) groups of parasitoids varied noticeably among the four
(4) sites. For Buvussi and Kavui, highest percentage parasitism was by the native
parasitoid(s) whilst for Nahavio and Siki Divisions, a high percentage of eggs were
parasitized by D. leefmansi (Table 4).

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Figure 12. Status of sexavae eggs sampled from the field in 4 OPIC Divisions [the upper case
letters above the error bars indicate the significance levels among the different status of eggs
within each site, whilst the lower case letters indicate significant difference levels for each
egg status across the sites].
Table 4. Percentage (%) parasitism by Doirania leefmansi and an unknown locally occurring
parasitoid across the four (4) OPIC Division sites surveyed.
Percentage (%) parasitism of sexavae eggs
D. leefmansi Locally occurring parasitoid
Buvussi 0.00 100.0
Kavui 23.5 76.5
Nahavio 100.0 0.0
Siki 90.0 10.0
Aa
Ca
Ba Ba
Ab
Bb
Cb
Db
Ab
Bc
Cc Dc
Ab
Ba
Cd
Dc