PARASITOIDS Bactrocera Tryoni Irradiation of Eggs Pratt 2008

7/28/2019 PARASITOIDS Bactrocera Tryoni Irradiation of Eggs Pratt 2008

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Contents

Abstract 3

Introduction 4

The Queensland Fruit Fly 4Cost of Q-fly and the Fruit Fly Exclusion Zone (FFEZ) 5Current Control 7Climate, Chemicals and the Future of Q-fly 9Aims & Objectives of This Study 12

Methods & Materials 13

Preparation of Q-fly material for irradiation 13Irradiation of Q-fly material 15Rearing ofDiachasmimorpha kraussii 16Exposure of Irradiated Q-fly toDiachasmimorpha kraussii 17Egg/larval mortality 19Q-fly longevity 19Q-fly reproductive sterility 19Rearing of Q-fly larvae for presentation toD. kraussii 21Parasitism success of groupedDiachasmimorpha kraussii 21

Diachasmimorpha kraussii longevity 23Analysis 23

Results 25

Eggs to second instar Q-fly egg/larval mortality 25Eggs to third instar egg/larval mortality 25Eggs to second instar Q-fly emergence 26Eggs to third instar Q-fly emergence 28Eggs to second instar Q-fly longevity 29Eggs to third instar Q-fly longevity 31Eggs to second instar Q-fly sterility 32Eggs to third instar Q-fly sterility 33Eggs to second instar wasp emergence 34Eggs to third instar wasp emergence 35Eggs to second instar wasp survivorship 36

Eggs to third instar wasp survivorship 37Eggs to second instar - successful parasitism by groupedD. kraussii 39Eggs to third instar - successful parasitism by groupedD. kraussii 39

Discussion 40

Bactrocera tryoni 40Diachasmimorpha kraussii 43Implications 46

Conclusions48


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Acknowledgements 49

References 50

Appendix I 56

Appendix II 57

Appendix III 62


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Abstract

The Queensland fruit fly (Q-fly) Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) is a major

horticultural pest in Australia causing significant levels of crop destruction and economic loss.

Biological control of this pest would be a welcome technology. This thesis presents results from a

study that aims to assess the potential for mass rearing parasitoid wasps on host material exposed to

gamma irradiation at the egg stage. The practical advantages of such a rearing system are that (a)

the egg stage of the host is easy to handle and can be densely packed for transport without

competition between individuals and (b) irradiation of the host may result in fly sterility or non-

emergence. This would negate the requirement to separate flies and wasps prior to release, thus

reducing production costs and eliminating biological or political concerns related to the escape of

fertile Q-fly. Q-fly eggs were irradiated at a range of doses (0.0, 4.7, 9.1, 15.9, 27.6, 47.0 and 79.9Gy) and exposed to Diachasmimorpha kraussii as larvae. D. kraussii adults emerged from hosts

exposed to parasitoids as second instar larvae for the 0, 4.7 and 9.1 doses. All produced offspring.

Adult Q-fly of both sexes emerged from these irradiation treatments, though numbers declined with

increasing dose. None of the adult Q-fly from the 9.1 Gy treatment survived long enough to allow

mating and sterility testing, indeed the majority were disfigured, unable to fly, unable to walk or

otherwise deformed. This suggests that they are likely to be functionally (if not biologically) sterile.

Sterility in flies from doses 0 and 4.7 was not of a high enough level to qualify for sterile insectrelease. A similar trend was apparent for hosts exposed to parasitoids as third instar larvae.

Importantly, however, for the 15.9 Gy treatment, wasps emerged but no flies were produced. Wasps

at this dose were found to be fertile. Overall results suggest that there is potential to develop a

protocol involving a carefully calibrated irradiation dose to which host eggs would be exposed, that

would allow mass production ofD. kraussii, yet would result in Q-fly non-emergence from any

unparasitised hosts. Potential lines of research to follow on from this study are discussed.


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Introduction

The Queensland F ru it F ly

The Queensland fruit fly (Q-fly), Bactrocera (Dacus) tryoni (Froggatt), is one of the most

economically damaging horticultural pests in Australia (see Appendix I) (Fitt, 1990; Sutherst et al.,

2000; Yonow et al., 2004). The fly is a member of the family Tephritidae, which comprises the

most important group of quarantined pests of fresh produce (Hallman & Loaharanu, 2002) and

worldwide includes major pest species such as the Mediterranean fruit fly, Ceratitis capitata, the

Oriental fruit fly, Bactrocerra dorsalis and the melon fly, B. cucurbitae. Conversely, there are

several beneficial tephritid species that play important roles in the biological control of weeds; for

example the false peacock fly, Chaetorellia succinea , which, following unintentional introduction,now plays an important role in controlling the yellow starthistle (Centaurea solstitialis) in western

USA (Balciunas & Villegas, 2001). Bactrocera. tryoni is a highly polyphagous frugivore, attacking

a range of fruits (and some vegetables) including apples, oranges, pears, figs, plums, chillies and

olives (Botha et al, 2000; Sutherst et al., 2000). The damage caused by Q-fly occurs initially as a

sting to the fruit surface as the adult female uses her ovipositor to penetrate the skin and lay a

number of eggs under the surface. These stings can provide an access point for secondary invasion

by bacteria and fungi which in turn lead to necrosis of the sting site and rotting of the fruit (Botha etal., 2000). Following oviposition, after 1-3 days (42 hours at 25 oC, 80%Relative Humidity (RH) in

lab conditions (Anderson, 1962)), larvae hatch inside the fruit and feed on its tissues, causing direct

damage and making the product unmarketable where the fruit is a crop. Q-fly may cause damage

levels of up to 100% in unprotected fruit (Botha et al., 2000). The larvae complete first (36-40

hours at 25oC in carrot medium) and second (36-40 to 65-70 hours at 25oC in carrot medium)

instars within the fruit before emerging towards the end of the third instar (65-70 hours to 7 days

at 25oC in carrot medium) to pupate in the soil (see Appendix I) (Anderson, 1963a; CABI/EPPO, no

date). Adult flies emerge after a period of 1-2 weeks (longer at low temperatures) and their

probability of mating increases with age from 4-12 days as the flies mature (Perez-Staples, 2007).

In addition to its polyphagous feeding habit and a short generation time allowing a multivoltine life

history (with up to 16 generations per year possible in the North of its range (Yonow & Sutherst,

1998)), the pest status of Q-fly is enhanced by high fecundity levels, with females capable of

producing over 1000 eggs in a lifetime (Fitt, 1990). This combination of traits result in the Q-fly

having a high intrinsic rate of increase (Fitt, 1990), particularly in the more climatically favourable

regions to the North of its range. The current range ofB. tryoni in Australia is throughout the


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eastern half of Queensland, eastern New South Wales and the extreme east of Victoria as far south

as East Gippsland (CABI/EPPO, no date; Yonow & Sutherst, 1998), though there is some debate

over the presence of an overwintering population in Melbourne (OLoughlin et al., 1984; Yonow &

Sutherst, 1998). The abundance of Qfly is highest in Tropical to sub-Tropical Queensland,

decreasing southward (OLoughlin et al., 1984). In the southern half of its range only the adult Q-fly is able to overwinter, with this lifestage exhibiting some ability to withstand repeated frosts

(Meats & Fitt, 1987). In 1989-90 there was an outbreak of Q-fly in Perth, however this was

eradicated using male annihilation, chemical baiting and sterile insect technique (SIT) releases

(which are discussed further below) (Fisher, 1996). Western Australia actively quarantines against

Q-fly (Meats et al., 2003). Elsewhere in the world Q-fly may be found in French Polynesia, New

Caledonia, Pacific Islands and Vanatu (Botha et al., 2000), though it is indigenous only to Australia

(CABI/EPPO, no date).

Cost of Q-fly and the Fru it F ly Exclusion Zone (FFEZ)

In Australia, the three major horticultural earners after grapes and bananas (which are considered to

be marginal hosts of Q-fly) are apples, oranges and pears (Sutherst et al. 2000). In their economic

analysis, Sutherst et al. (2000) estimated the annual cost to these three industries of control

measures and lost production attributable toB. tryoni to be $A28.5 million per year (with a range of

$AU25.7-49.9 million) with 60% of this cost shouldered by commercial growers. The total annual

costs of Q-fly damage and control in Australia may, however, be in excess of $A125 million

(Horticultural Policy Council, 1991). This total includes the cost of maintaining Area Freedom

from fruit fly in the Tri-State Fruit Fly Exclusion Zone (FFEZ); a region approximately 1,000km x

800km in South-Eastern Australia comprised of territory in South Australia, New South Wales and

Victoria and including some of Australias most important fruit producing regions such as the

Murrumbidgee Irrigation Area, the Riverland area, the Goulburn Valley and the Sunraysia and Mid-

Murray districts (see Figure 1) (Osborne et al., 1997; TriState Fruit Fly Strategy Steering

Committee, 2002).


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Figure 1The Fruit Fly Exclusion Zone (FFEZ) (Source: Tri-State Fruit Fly Program, no date, a)

The status of area freedom provides growers within the FFEZ with major marketing advantages,

allowing access to local and export markets that will trade only with formally recognised fruit flyfree regions (TriState Fruit Fly Strategy Steering Committee, 2002). The annual benefits of

maintaining the FFEZ have been estimated to be $A14.9million, with a $A6million annual cost of

maintenance, giving an estimated cost benefit ratio of 2.5:1 in 2002 (though recent costs are likely

to be closer to be $A8million) (PriceWaterhouseCoopers, 2001;TriState Fruit Fly Strategy Steering

Committee, 2002). The benefits accounted for are on farm savings from the absence of fruit fly

resulting in an absence of control costs and reduced eradication costs, benefits associated with

access to the international market and benefits of access to the domestic market

(PriceWaterhouseCoopers, 2001). Fruit from this region can be sent directly to market without the

requirement for costly post-harvest chemical, cold or irradiation treatment (TriState Fruit Fly

Strategy Steering Committee, 2002). Further benefits that are less easily quantified include regional

economic benefits, benefits from reduced chemical control usage and associated residues, and

supply chain industry benefits (PriceWaterhouseCoopers, 2001). Exporters of citrus that handle fruit

from the FFEZ, for example, benefit by $A6.4million per year in price premiums in the US market,

access to which relies wholly upon the region maintaining its status of area freedom

(PriceWaterhouseCoopers, 2001; TriState Fruit Fly Strategy Steering Committee, 2002).


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The FFEZ, established in 1995, is monitored using an extensive surveillance grid in local towns and

on horticultural sites in and around the zone, with almost 7000 fruit fly trapping sites in southern

NSW, Victoria and South Australia (TriState Fruit Fly Strategy Steering Committee, 2002;

Technical Review Team, 2001). At each site there are traps for Q-fly and Mediterranean fruit fly(Medfly), with some also having methyl eugenol (4-allyl-1,2-dimethoxybenzene-carboxylate)

baited traps to catch other fruit flies (Technical Review Team, 2001). Within the FFEZ itself there

are approximately 3000 sites with traps for Q-fly and Medfly (Technical Review Team, 2001). Q-

fly are monitored using Lynfield traps charged with the parapheromone cue-lure (4-(p-

acetoxyphenyl)-2-butanone) to attract mature adult male Q-flies, and maldison (= malathion) as the

killing agent. Traps are placed in a grid, with one trap every 400m in urban areas and one trap every

1km in horticultural regions within the FFEZ (Technical Review Team, 2001). The traps aremonitored on a weekly basis during the high risk period (November to May) and fortnightly for the

remainder of the year. If two or more flies are trapped within 1km in the space of two weeks then a

further 16 traps within a 200m radius of the outbreak location are deployed and fruit within this area

is examined for larvae (Technical Review Team, 2001). McPhail traps charged with a protein bait

solution are also deployed within this area, primarily to trap females, however these are largely

ineffective and are maintained only to satisfy USA quarantine requirements (Technical Review

Team, 2001). There is a standard Code of Practice for monitoring Q-fly within the trap network

within the FFEZ, which gives threshold numbers of Q-fly for a given area per unit of time, for

example five males trapped in two weeks in the 400m grid; a single gravid female; or the finding of

any larvae within fruit (Technical Review Team, 2001; TriState Fruit Fly Strategy Steering

Committee, 2002). If a threshold is reached or exceeded, an outbreak is declared and properties

within a 15, 30 or 80km radius of the outbreak epicentre (depending on the market or size of

outbreak) lose their fruit fly free status until the outbreak has been eradicated (TriState Fruit Fly

Strategy Steering Committee, 2002).

Current Control

For the FFEZ, the primary method employed to retain area freedom from fruit flies is to avoid

introductions to the region in the first instance. This is achieved by controlling imports into the area,

with fines of up to $A2500 for those found to be carrying fruit into the FFEZ illegally (Tri-State

Fruit Fly Program, no date, b). Education is also an important way of making the public aware of

the risks associated with fruit flies, and media campaigns and education kits along with road signs

indicating the penalties for fruit transportation into the FFEZ have proven effective in mitigating


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fruit fly incursions (Technical Review Team, 2001). Proactive control methods in towns bordering

the FFEZ are also important to help reduce incursions into the FFEZ and maintain area freedom.

The primary region requiring this action is known as the Risk Reduction Zone (RRZ) to the east of

the FFEZ, within which Q-fly outbreaks are fairly common. Baiting and male annihilation programs

are frequently carried out in towns in the RRZ to reduce Q-fly populations that could potentiallyspread into the Riverina region of the FFEZ, as occurred in 1999-2000 (Technical Review Team,

2001). Male annihilation involves the use of cue lure to attract males, combined with an insecticide

(typically maldison (=malathion)) to reduce numbers of male Q-flies and thus reduce Q-fly matings.

It is most effective when used in combination with other control methods (such as bait spraying)

that offer protection against inseminated females (Bateman et al., 1966).

In the event of an outbreak within the FFEZ (or in other regions that are free from Q-fly such asSouth Australia and Western Australia), eradication is carried out using chemical baiting or through

SIT releases (Sutherst et al., 2000; TriState Fruit Fly Strategy Steering Committee, 2002). Q-fly

(and other fruit fly species) require a source of protein for mating, egg maturation and for increased

longevity (Perez-Staples et al., 2007), with bacteria from leaf surfaces being the primary protein

source in nature (Drew et al., 1983). Chemical bait mixture contains a yeast hydrolysate or

autolysate protein source mixed with an insecticide (Hardy et al., 2007). Flies are attracted to the

protein component of the mixture, but upon feeding are killed by the insecticide, which is generally

maldison (=malathion) or chlorpyrifos (both of which are organophosphate compounds) (Hardy et

al., 2007). The mixture is applied to the lower foliage and skirts of trees and can be highly effective

in eradicating Q-fly following an outbreak (Hardy etal., 2007; Sutherst et al., 2000), particularly if

combined with good hygiene and the removal of any potentially infested fruit (Hardy et al., 2007).

Sterile insect technique (SIT) involves the release en mass of sterile males that flood the wild

population and mate with wild females, inducing reproductive sterility in them (Knipling, 1955).

Females may lay eggs, but they will not yield offspring, and the chances of mating with a fertile

male are reduced due to the huge numbers of sterile males. There is a Q-fly mass rearing facility

located at the Elizabeth Macarthur Agricultural Institute, Camden, NSW, from which Q-fly pupae

are taken for irradiation at the Australian Nuclear Science and Technology Organisation (ANSTO)

facility at Lucas Heights, NSW. Sterile flies resulting from the irradiation procedure may then be

used in strategic Q-fly management and research programs in SA, NSW and Victoria (Technical

Review Team, 2001; Dr. Olivia Kvedaras, personal communication). This technique has proven

effective for the eradication of Q-fly outbeaks from regions aiming to achieve area freedom and has

also been used in the RRZ as part of an integrated pest management (IPM) system, but the future

role of SIT in NSW is currently under review (Dr. Olivia Kvedaras, personal communication).


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Outside the FFEZ and other regions claiming area freedom, eradication of Q-fly is often not the aim

of control strategies due to its expense and the unsuitability of the regions biogeography and levels

of infestation to eradication techniques. In these regions the primary aim is to reduce Q-fly numbers

below an economic threshold. To do this SIT may be used to a limited extent, but bait spraying,cover spraying and male annihilation are the primary methods of control (Sutherst et al., 2000;

Dominiak, 2007). Cover sprays are applied to the tree and the fruit itself to kill fruit flies in the trees

and maggots in the fruit (Dominiak, 2007). The insecticide component of these sprays is usually

dimethoate, fenthion or trichlorfon, and is applied at a far higher rate than a bait spray, potentially

having a higher impact on non-target species.

Climate, Chemicals and the Fu ture of Q-f ly

Temperature and rainfall are the key limiting factors to the range of Q-fly, with annual fluctuations

in these parameters explaining much of the variation in Q-fly population dynamics year to year

(Yonow & Suherst, 1998; Technical Review Team, 2001). Whilst annual fluctuations in temperature

and rainfall are likely to be of major importance in the control of Q-fly and maintaining the FFEZ in

the short term, a longer term outlook allowing for climate change is also important. Sutherst et al.

(2000) describe the likely increase in suitability of much of the FFEZ and other currently marginal

regions to Q-fly with increasing global temperature, particularly in combination with irrigation that

can counter the range limiting effects of low rainfall to an extent. The team questions the likelihood

of being able to maintain area freedom within the FFEZ under these conditions, the risks to market

freedom and whether the associated increase in control costs would justify keeping the FFEZ.

Sutherst et al.(2000) also predict increases in Q-fly population sizes over much of its endemic

range, with associated increases in damage and control costs. Table 2, below provides a summary of

the major current control and eradication techniques used against the Queensland fruit fly, and their

adaptability to predicted climate change.


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Control

option

Current

use/

effectiveness

Sustainability/

future

priority

Robustness

under climate

change

Adaptability

under climate

change

Constraints

Endemic

Cover spray ***/*** */* ** **

Residues &contamination,

public health,cost

Bait spray **/*** ***/*** *** ***Public health,

cost

SITsuppression

*/** **/*** * *Fruit fly

population size,cost

FFEZ

Bait spray ***/*** */*** *** ***Political, public

health, cost

SIT

eradication**/*** **/*** ** **

Political, fruitfly population

size, cost

Exclusion ***/*** **/*** ** ** Political, cost

Strip fruit */* */0 ** *Effectiveness/

politics

Table 1Adaptability of current control (endemic regions) and eradication (FFEZ) options against

Q-fly under climate change. After Sutherst et al. (2000)

Another important issue regarding the future of Q-fly control is that of continued chemical pesticide

usage. As indicated in Table , there is increasing concern from the public and growers alike over therisks associated with a heavy reliance on pesticides. Continued use of pesticides leads to the risk of

resistance developing in Q-fly, with a high selection pressure for flies able to withstand the effects

of the pesticide. With cover spraying in particular there is also a high degree of non-specificity in

pesticide application (Sutherst et al., 2000), meaning that non-target species such as Q-fly natural

enemies may be killed, potentially allowing the unchecked resurgence of a Q-fly population if

resistance to the pesticides were to develop or if the control measures were stopped. There are then

issues of residues and contamination having an effect on public health. Malathion for example,which is one of the major pesticides used in bait sprays, has been the cause of a number of

accidental poisonings as well as having an unfortunate association with suicides (e.g. Bakeret al.,

1978; Thompson et al., 1998). It is being withdrawn from use in a number of countries including

member states of the European Union (Kyprianou, 2007) and may be destined for a similar fate in

Australia. There is evidently a need for effective non-chemical controls for use against Q-fly.

SIT is a highly specific, non-chemical control method and can be very effective for suppression or

eradication of pests, however it can be expensive when used against large or widely dispersed


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populations (Sutherst et al., 2000; Parker & Mehta, 2007). There is potential to develop a

complementary, environmentally friendly, non-chemical control method that involves the use of

parasitoid wasps for biological control of Q-fly. Augmentative parasitoid release against fruit flies

can be used effectively as part of an integrated pest management (IPM) program alongside methods

such as SIT and male annihilation (Purcell, 1998; Montoya & Cancino, 2004).Braconid parasitoidsare the major natural enemies of tephritid fruit flies and have been used in various parts of the world

for biological control (Rungrojwanich & Walter, 2000a). Nowhere is that more apparent than in

Hawaii, where several opiine braconid fruit fly parasitoids have been released resulting in the

effective suppression of fruit fly pests including the melon fly (Bactrocera cucurbitae) and the

Oriental fruit fly (Bactrocera dorsalis), (Duan & Messing, 1997). One species of particular interest

is the opiine braconid,Diachasmimorpha kraussii (Hymenoptera: Braconidae: Opiinae), which was

introduced to Hawaii between 1947 and 1952 and again more recently (it did not establishpermanently from the earlier releases) to control the invasive Mediterranean fruit fly (Ceratitis

capitata) (Duan & Messing, 2000; Rungrojwanich & Walter, 2000a; Wang & Messing, 2002).

Diachasmimorpha kraussii (see Appendix 1) is a larval parasitoid native to Australia and is a

natural enemy of Q-fly, making it an ideal candidate for inundative release to provide control over

B. tryoni. It parasitizes second and third instar Q-fly larvae, eclosing from the pupal case and is

present along the east coast of Australia (Purcell, 1998). Inundative release generally requires the

rearing of parasitoids on their host at a large scale, followed by separation of eclosing parasitoids

and host adults. Separation is crucial so as not to release the host and further augment the pest

population, but is an expensive process and may limit the efficiency and practicability of a

biocontrol program. Another issue with rearing a parasitoid on a fertile host is that in some areas

such as the FFEZ this practice is not permitted due to the risk of pest escapes. One method that has

been attempted with good results uses the principles of SIT, but rather than irradiating the pupae of

the pest species, it involves irradiating earlier developmental stages (i.e. the eggs or larvae)

(Sivinski & Smittle, 1990; Cancino et al., In Press) and then exposing them at a suitable stage of

development (usually as eggs or larvae) to an appropriate parasitoid. It has been proposed that

irradiation of host material may actually improve parasitism rates, presumably by compromising the

immune response of the host (E. Burns, unpublished data, in Sivinski, 1996). Due to the advanced

nature of larvae compared with eggs, there is a higher degree of development in the gonads

(Anderson, 1962) potentially making this stage more prone to sterilisation, in which case eclosing

parasitoids and sterile hosts developing from non-parasitized larvae would not require separation

and could be released together, with sterile hosts contributing to local SIT programs. The less

developed egg stage may be less susceptible to sterilisation, but it has been demonstrated that

irradiation of fruit fly eggs can result in the non-emergence of the host as an adult, whilst allowing


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the successful development of its braconid parasitoids (Cancino et al., in press). Once more, the

expensive separation process would not be required with this outcome, as any unparasitized hosts

would result in non-emergence. Developing parasitoids (and potentially sterile hosts) could also be

transported and released whilst within the pupal case, which would be logistically easier and more

efficient than handling adult parasitoids.The use of eggs also has a number of practical benefits overthe use of larvae. Firstly, eggs are very small and could be irradiated in very high numbers. They are

also an immobile, non-feeding stage and could therefore be handled and transported more easily

than active larval stages.

Aims & Objectives of This Study

1.

To investigate the effects of gamma irradiation on the development, fecundity and longevityof the Queensland fruit fly, Bactrocera tryoni when irradiated at the egg stage.

2. To evaluate the suitability of irradiated host material (B. tryoni) for the development of itsparasitoid,Diachasmimorpha kraussii.

This study will investigate the suitability of Q-fly irradiated at the egg stage as a host for its

parasitoid,D. kraussii. It will look at the effects of irradiation on the Q-fly host, and the parasitism

success and longevity of any parasitoids developing from that host in order to establish the

suitability of this procedure for the mass production and release ofD. kraussii. This study will pave

the way for future investigation and potential commercialisation of the techniques used, with the

long term aim of environmentally sound, IPM-compatible, economically viable control of the

Queensland fruit fly,B. tryoni through the augmentative release of its parasitoid,D. kraussii.


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Methods and Materials

The following methods were carried out for both Q-fly eggs irradiated and reared out to second

instar before being exposed to D. kraussii and Q-fly eggs irradiated and reared out to third instar

prior to exposure.

Preparation of Q-fly materi al f or i rr adiation

The Q-fly eggs used in the irradiation procedure were obtained from a laboratory stock of Q-fly

held at Gosford Horticultural Institute of the New South Wales Department of Primary Industries.

Adult Q-fly were housed in large meshed cages inside a controlled temperature (CT) room

(262C, 655% relative humidity (RH), 12:1:10:1 Light:Dusk:Dark:Dawn) and were provided

with water, sugar and yeast hydrolysate enzymatic as a source of protein. The flies were cagedseparately by age cohort and at four weeks of age flies were disposed of. Eggs were obtained from

three to four week old adults and then reared on carrot medium (see Appendix II) through the larval

stages to pupation, also within the CT room. Pupae were placed into a large meshed cage and

emerging adults were classed as a new cohort. For the irradiation experiment, three week old Q-fly

adults that had not been egged previously were used in order to obtain a large volume of high

quality eggs (Dr. Katina Lindhout, personal communication; Fitt, 1990). These flies were presented

with a yellow egging cup (150m in height, 100mm diameter) containing a wedge (1/8

th

) of anorganic orange screwed into the lid, with orange juice applied to the outside of the cup, to act as cue

for the Q-fly and to stimulate oviposition by the females (which readily oviposit into oranges in the

wild). The cup had holes down its sides of a suitable size to allow penetration by the female Q-fly

ovipositor. The cup contained tap water to a level slightly below that of the lower holes in the cup,

into which eggs could fall preventing desiccation. Egging cups were left in the Q-fly enclosure for a

period of 10 hours (as opposed to the standard 24 hour egging period practiced at Gosford) in order

to obtain the required volume of eggs whilst maintaining a fairly even age-spread (see Plate 1).

After 10 hours, the egging cup was removed, eggs were poured out in a suspension of water into a

100ml glass beaker and left to settle. Excess water was poured off to a level approximately 1cm

above the level of the settled eggs. The eggs were then allowed to re-settle.


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Plate 1Egging cups with ovipositing Q-fly

30 Petri dishes (90mm diameter)for each experiment were filled with carrot medium (see Appendix

II) as a diet for hatching larvae, which was gently firmed in, filling the dishes to approximately

2mm below the rim. A Gilson 'Pipetman' micropipette was used to pipette five aliquots of 3l of

eggs into a 25ml glass beaker, to which 2.5ml of water was added. This was agitated to lift the eggs

into suspension and the contents were poured evenly across the surface of the carrot medium in the

Petri dish. A further 0.5ml of water was used to rinse out the beaker and poured onto the carrot

medium to ensure that all eggs had been added. This process was carried out for every Petri dish.

3l of eggs equated to 521.49 (1 s.e., n=15) eggs, so around 260 eggs were added to each Petri

dish. Lids were fitted to the Petri dishes to maintain humidity and secured using masking tape.

The Petri dishes were stored in the Gosford Horticultural Institute insectary CT room (262C,

655% RH, 12:1:10:1 Light:Dusk:Dark:Dawn) for the remainder of the day, and overnight until the

morning of the irradiation. At the time of irradiation the eggs were approximately 26-38 hours old.

By irradiating eggs in this age range, the eggs had some chance to mature and develop, thereby

potentially increasing their ability to withstand the effects of gamma irradiation (Hallman, 2000;

Cancino et al., in press). Whilst sterilisation of eggs using gamma irradiation may not be possible,

the gonads complete their within-egg development by 28 hours of age (Anderson, 1962), so if

sterilisation of this developmental stage were possible, eggs would ideally be aged 28 hours or

older, as was the case for the majority of eggs in this experiment. Had the eggs been any older at the

time of irradiation, there would have been the potential for some of them to have hatched into first

instar larvae (egg development time from laying to hatching forB. tryoni is 42hr at 25

o

C, 80%RH(Anderson, 1962)), the irradiation of which was not the aim of this experiment.


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I rr adiation of Q-fly materi al

On the morning of irradiation, the Petri dishes were packed into a large polystyrene box with

ventilation holes cut into its sides. The material was then transported by car to the Australian

Nuclear Science and Technology Organisation (ANSTO) site at Lucas Heights, Sydney. The

conditions in the car were 192C and 505% RH. At Lucas Heights, material awaiting irradiationor post-irradiation was kept at 182C and 505%RH. For irradiation, three Petri dishes per

irradiation run were selected at random and taped flat, side by side onto the front surfaces of two

cardboard boxes (250mm x 500mm, see Plate 2). The boxes were taken to the Gamma Technology

Research Irradiator (GATRI) to be exposed to cobalt-60 gamma radiation (see Appendix II for

irradiation conditions). Seven doses of gamma radiation were selected based on a log1.75 scale

starting with a lowest dose of 5.0. Due to limitations on the number of visits possible to the

irradiation facility, the range of doses was selected to satisfy the irradiation requirements for anexperiment using second and third instar Q-fly larvae which are more developed and likely to

withstand higher levels of irradiation whilst still having a range of lower-end doses for this

experiment with eggs, which tend to be more sensitive to the effects of gamma irradiation (Balock

et al., 1963; Hallman, 2000). The target doses were 0.0 (control), 5.0, 8.8, 15.3, 26.8, 46.9 and 82.1

Gy. The upper dose was within the range 75-100 Gy, which is the range required to prevent adult

emergence ofB. tryoni from third instars in cherry, mango, orange and avocado (Hallman &

Loaharanu, 2002). For the first irradiation run, Fricke (ferrous ammonium sulphate) dosimeters

were situated throughout the boxes at the expected minimum and maximum dose zones. The boxes

were then positioned on a rig 1300mm from the radiation source screen (see Plate 2). The material

was then processed in the GATRI facility for a period of time expected to deliver the maximum,

82.1 Gy, dose. Results from this run were used to establish the extreme doses and this information

was used in subsequent runs. The average doses achieved were 4.7, 9.1, 15.9, 27.6, 47.0 and 79.9

Gy (for dose range see Appendix II). The Petri dishes for the control dose (0.0 Gy) were not

exposed to gamma radiation but were kept in the same room that irradiated material was stored in

prior to and following irradiation. Three spare unirradiated dishes were frozen for later confirmation

that all Q-fly were eggs at the time of irradiation and had not developed further.


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Plate 2Irradiation set up in the rig in the GATRI chamber

The irradiated material was then transported to Wagga Wagga Agricultural Institute (WWAI) of the

Department of Primary Industries New South Wales by car. The conditions were 192 oC and

505%RH and the journey time was around six hours. Upon arrival at WWAI, the irradiated (and

control) Q-fly material was placed into a CT room (27 3C, 65 15% RH, 12:12 L:D).

Rearing of Diachasmimorpha kraussii

Parasitoid wasps, Diachasmimorpha kraussii, were obtained from a laboratory stock held by the

Entomology Department of the Department of Primary Industries, Indooroopilly, Queensland. At

this establishment B.tryoni larvae were exposed to D. kraussii and then allowed to continue

development. They were received as pupae at WWAI and placed onto vermiculite (see Appendix II)

in Petri dishes (90mm diameter) inside flight cages (30x30x30cm) (see Appendix II), into which

were placed two water cups with cotton wicks (see Appendix II). The walls and ceiling of each

flight cage were streaked with honey (see Appendix II) using a fine camel hair brush to provide

nutrition for eclosing wasps. The flight cages were placed into the laboratory CT room at (27 3C,

65 15% RH, 12:12 L:D). The flight cages were checked daily for emergence. Q-fly tended to

emerge first. The dishes containing pupae were removed and placed into new flight cages with

honey and water and the flight cages containing emerged fruit flies were placed into the freezer,

dead Q-fly removed and the flight cages washed. When there was overlap of emergence between Q-

fly and D. kraussii, the flies were removed using an aspirator and disposed of. The Petri dishes

containing uneclosed pupae were removed and placed into new flight cages with honey and water,

and the approximate number, sex ratio and date of emergence of the wasps remaining in the firstflight cage were recorded. Male wasps generally emerged before females, but males were added to


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cages containing all-female cohorts at least one week prior to being presented with irradiated Q-fly

(host) material to allow ample time for mating (Rungrojwanich & Walter, 2000b) in a ratio of

approximately 1:1 males:females (Rungrojwanich & Walter, 2000b). Fresh water and honey were

provided as and when required.

Exposure of I r radiated Q-fly to Diachasmimorpha kraussii

Development of the Q-fly irradiated as eggs was closely monitored. Larvae in spare dishes that had

been kept under the same conditions as the irradiated material (i.e. spare 'control' dishes) were

analysed to determine the developmental stage of the Q-fly larvae that had hatched. The mouthparts

of the Q-fly were used to identify the larval instar, as described by Anderson (1963a). Upon

reaching the second instar for the irradiated eggs to second instar experiment, or the third instar for

the irradiated eggs to third instar experiment, 'exposure boxes' were set up within which the larvaewere to be exposed to D. kraussii females. Five female D. kraussii were carefully captured using

gelatine capsules (size 00, Healthy Life, Wagga Wagga, NSW) and released into a plastic box

(length 16.5cm, width 11cm, height 7cm, see Appendix II). The females used in the second instar

exposure were seven days old at the time of exposure and the females used in the third instar

exposure were eight days old at the time of exposure. Rungrojwanich & Walter (2000a) found that

D. kraussii offspring production was highest for 7-8 day old wasps. Each box contained a single

water cup and the sides were streaked with honey. The box was then covered with a fine mesh (25

filaments per cm) with apertures large enough to allow penetration by the ovipositor of the

parasitoids, but narrow enough to prevent escape of larvae or wasps. Two circular holes, each

45mm in diameter and positioned 30mm apart were cut into a lid, which was then fitted to the box.

The contents of each Petri dish containing the Q-fly larvae and carrot medium was quartered. Each

quarter was placed into a 55mm Petri dish, which was then filled to the rim with fresh carrot

medium and fitted with a lid which was secured using masking tape to prevent larval escape. This

resulted in 12 Petri dishes containing larvae and carrot medium for each irradiation dose for each of

the experiments. Of the 12 dishes per dose, half were to be exposed the parasitoids and half were to

be left unexposed, giving six replicates per treatment in total. Within each dose (and separately for

the two experiments), dishes were randomly assigned to replicates and to exposed or unexposed

treatments. Two dishes of the appropriate dose and replicate were inverted and positioned over the

circular holes in the lid of the box, the 'exposed' dish with its lid removed, and the 'unexposed' dish

with its lid still in place. The reason for having an unexposed treatment that was not subjected to

parasitoid exposure was to ensure that data on the effects of the irradiation on Q-fly could be

obtained; even under the circumstance where exposed treatments had high to total parasitism (i.e.

only data on emerging wasps would be obtainable). The dishes were then secured in place using Blu


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Tack (see Appendix II) around the rim, which also served to prevent the escape of larvae from the

dishes. This procedure was carried out for all of the replicates at each dose. The exposure boxes

were then placed into a CT room (24 2C, 60 15% RH, 12:12 L:D) grouped by replicate and

positioned randomly within that replicate. The direction of the exposed versus unexposed ends of

the boxes were also randomised. Data loggers were also placed into 'dummy' exposure boxes,identical in setup and positioned in the relevant sections of the CT room in order to assess the

within-box conditions during the experiment (24 2C, 92 5%RH). The boxes were then left for a

period of 24 hours to allow oviposition into the Q-fly larvae by theD. kraussii (see Plate 3).

Plate 3One replicate of exposure boxes in the CT room

After 24 hours, the dishes containing the Q-fly larvae and carrot medium were removed from the

lids of the exposure boxes. Lids were removed (from the unexposed dishes only) and the dishes

were placed individually into new boxes of the same dimensions, each of which contained a layer of

vermiculite approximately1.5cm in depth. Fresh carrot diet (1 teaspoon) was added to each dish,

then the box was covered using mesh (25 filaments per cm) held in place with elastic bands. The

boxes were then returned to their shelves in the CT room to allow the larvae to continue

development and pupate in the vermiculite. After 12 days (allowing time for all larvae to pupate

(Anderson, 1963a)), the vermiculite was sieved using a laboratory test sieve with 1.70mm aperture

(see Appendix II) in order to remove all pupae. Removed pupae were then counted and placed onto

fresh vermiculite in a 55mm Petri dish. The pupae were returned to their boxes, the walls of which

had been streaked with honey and to which had been added a water cup and a sugar cube (see

Appendix II). Boxes were returned to the same positions in the CT room (219C,45 25% RH,12:12 L:D) for the eggs to third instar experiment and to another CT room (22 7C, 60 15% RH,


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12:1:10:1 Light:Dusk:Dark:Dawn) for the eggs to second instar experiment (due to space

limitations) and observed every 24 hours for emergence. Q-fly were first to emerge and were

removed from the boxes using an aspirator. Those flies that emerged from replicates 1-4 were

separated by sex and placed into flight cages by dose and exposure for later use in sterility trials.

Flies from replicates 5 and 6 were removed using an aspirator and placed individually into mediumplastic cups measuring 10cm in height, with a base diameter of 4.8cm and rim diameter of 7.5cm.

Each cup contained a sugar cube and water cup and was covered with a piece of mesh (25 filaments

per cm) secured by elastic bands. Emergence dates and fly sex were recorded and cups were

grouped by replicate in the CT room from which they came. Wasps, emerging after Q-fly and from

the exposed treatment only, were carefully removed from the flight cages and released into large

cups (height 110mm, top diameter 115mm, dase diameter 84mm , see Appendix II) according to

dose and replicate. Emergence date and sex of wasps were recorded.

Egg/larval mortality

The proportion of eggs developing to pupae was analysed as a measure of mortality of eggs and

larvae for each dose and for exposed and unexposed treatments.

Q-fly longevity

The cups containing individual flies from reps 5 and 6 were checked every 24 hours for fly

mortality. Fly death was determined by an absence of any movement in the fly, even when

disturbed. The date of death was recorded. Water cups within the longevity cups were refilled as and

when required, using a syringe. The syringe was used to penetrate the mesh covering the cup and

then to inject water into the water cup, thus minimising disturbance to the fly and preventing escape

that may otherwise have occurred with removal of the mesh.

Q-fly reproductive ster il ity

Flies from reps 1-4 were separated into flight cages by dose, exposure and sex following

emergence. Each flight cage contained two sugar cubes, three water cups and a teaspoon of yeast

hydrolysate enzymatic (see Appendix II) as a protein source for mating and egg production (Perez-

Staples et al., 2007). The flight cages were kept in the CT rooms from which they came. The flies

from the eggs to second instar experiment may have benefited from the lighting cycle in their CT

room, with dusk acting as a stimulus for sexual activity in this species (Bateman, 1972), though

matings were observed in both CT rooms soon after mixing of males and females. The sterility

testing methodology was adapted from the International Atomic Energy Association fruit fly

sterility quality control procedures (FAO/IAEA/USDA, (2003)) with additional methods based on


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those described by Collins et al. (2008). After one week, the following mating crosses were set up

for each dose and exposure: irradiated females x irradiated males; unirradiated females x irradiated

males; irradiated females x unirradiated males; unirradiated females x unirradiated males (control).

Each mating cross contained 10 male and 10 female Q-flies. Unirradiated Q-fly were obtained as

pupae from a laboratory stock held at the Elizabeth McArthur Institute of the New South WalesDepartment of Primary Industries which was established from the same mother colony as that for

the Gosford stock (as used in the irradiation procedure). Emergence dates for these flies coincided

with the emergence dates of the irradiated Q-fly, and upon emergence the unirradiated Q-fly were

separated by sex into flight cages containing yeast hydrolysate enzymatic, three water cups and 3

sugar cubes prior to being used in the described crosses. The unirradiated flies were kept in the

same CT room as the irradiated Q-fly throughout their development.

Following the mating crosses, flies were kept in the same CT rooms and provided with fresh diet

and water as and when required. At three weeks of age, small egging cups mounted upon inverted

medium cups (see Appendix II and Plates 4 and 5) were placed into each flight cage. Three weeks

of age was chosen as the egging age as Q-fly maintain high levels of mating between 12 and at least

30 days of age (Perez-Staples et al., 2007), and the stock from which the Q-fly were obtained is

egged at three weeks of age up to four weeks as this is the period in which the Q-fly produce the

highest quality eggs (Dr. Katina Lindhout, personal communication).

After 24 hours, the egging cups were removed from the flight cages. The contents of the egging

cups were poured into medium cups (see Appendix II) and were then rinsed thoroughly to ensure

that all eggs were transferred into the second cup. The eggs were allowed to settle, before all eggs

Plate 4Small egging cup Plate 5Oviposition by Q-fly into

small egging cup


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from the cup were pipetted onto a 90mm diameter charcoal filter paper (see Appendix II) placed

inside a 90mmPetri dish. The eggs were then gently streaked with a fine camel hair brush to give

an even distribution over the filter paper surface. Water was added to the point of saturation of the

filter paper to provide moisture for the developing eggs, with any excess water carefully removed

with a pipette. The Petri dishes were then placed into the laboratory CT room at (27 3C, 65 15% RH, 12:12 L:D) for four days, with 1ml of water being added to each dish after 2 days to

prevent desiccation of the eggs. The four day period was to allow fertile eggs to hatch (Collins et

al., 2008). After four days, the Petri dishes were observed under a microscope and the total numbers

of unhatched eggs and larvae were counted for each dish. Both the eggs and larvae were translucent

to white and hence stood out against the black charcoal filter paper background.

Reari ng of Q-f ly lar vae for presentati on to D. kraussiiBatches of Q-fly pupae were received weekly from the Elizabeth McArthur Institute of NSW DPI.

Pupae were placed onto vermiculite in 90mm diameter Petri dishes. The dishes were then placed

into flight cages inside each of which were three water cups, three teaspoons of yeast hydrolysate

enzymatic and four sugar cubes. These were replaced as and when required. Flight cages were kept

in the lab CT room (27 3C, 65 15% RH, 12:12 L:D). As flies eclosed from the pupae,

remaining uneclosed pupae were moved to new flight cages in order to keep the Q-fly density down

to avoid fighting and reduce competition. At three weeks of age, the Q-fly were presented with large

egging cups (as per Gosford, but transparent not yellow due to availability) for a period of 24 hours.

Following a day of egging, the flies were left for 24 hours before next being presented with an

egging cup. Following the egging period, the cups were removed and the contents were poured into

a glass beaker (200ml) and the eggs were allowed to settle. After settling, 3ml of eggs was pipetted

over the surface of carrot medium filled to a depth of 4cm within a plastic box (length 16.5cm,

width 11cm, height 7cm). A lid was pierced with several small holes to allow the inflow of fresh air

and fitted to the box. These boxes were set up daily and placed into the lab CT room (27 3C, 65

15% RH, 12:12 L:D ) to allow continued development and feeding of hatched larvae. Upon

reaching late second to early third instar, larvae were spooned into 55mm diameter Petri dishes

along with fresh diet. The number of larvae per dish was approximately 80-100 (in order to achieve

a ratio of larvae to female wasps of at least 15:1 in order to limit superparasitism (Lawrence et al.,

1978).

Parasit ism success of grouped Diachasmimorpha kraussi i

Wasps that emerged from the exposed irradiated material were placed into large cups by dose and

replicate as they emerged. Each cup was streaked with honey, contained two water cups and was


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covered with a section of mesh (25 filaments per cm) held in place with elastic bands.

Unfortunately in the experiment looking at irradiated Q-fly eggs that were exposed toD. kraussii as

second instar larvae, low numbers of wasps emerged, so replicates were paired for this study. Wasps

from the eggs to third instar experiment were then left in the CT room in which they emerged

(219C,45 25% RH, 12:12 L:D) for 48 hours to allow mating to occur (Rungrojwanich &Walter, 2000b), whilst the wasps emerging from the eggs to third instar experiment were moved to a

new CT room (227C, 65 15% RH, 12:12 L:D) due to space limitations. After 48 hours, the

wasps were presented with late second / early third instar Q-fly larvae. For this the lid was removed

from a 55mm Petri dish containing the larvae and carrot medium. The dish was then inverted and

placed onto the mesh on top of the cup containing the wasps, exposing the larvae in the carrot

medium to the parasitoids. The dish was secured in place using Blu Tack (see Appendix II), which

also formed a seal around the rim which served to prevent the escape of Q-fly larvae. See Plate 5.The dishes were removed after 24 hours and replaced with new dishes containing carrot medium

and larvae. This process was repeated daily for each cup until all wasps contained within that cup

had died. Removed dishes containing larvae that had been exposed to the wasps were placed into

plastic boxes (length 16.5cm, width 11cm, height 7cm ) containing vermiculite at a depth of

approximately 1.5cm. Upon the death of a female wasp, a new box was introduced, so that any D.

kraussii offspring that may later emerge could be attributed to a known number of females. This

process was carried out for the lifetime of the female wasps. The boxes containing the exposed Q-

fly larvae and carrot medium were kept in the same CT room and allowed to continue developing,

through pupation and on to emerge as either adult Q-fly or adult D. kraussii. When emergence

appeared to be completed, allowing at least a week after the last emerged insect had died, the

contents of the boxes were sifted, the Q-fly were removed and discarded and any wasps that had

emerged were sexed and counted.

Plate 5Diachasmimorpha kraussii parasitism chamber


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Diachasmimorpha kr aussii longevity

The sex and date of emergence of each wasp was recorded as it emerged and prior to mixing with

other wasps for the parasitism success study. Wasps were checked for mortality every 24 hours, and

the sex and date of death of any dead wasps were recorded. Death was determined by an absence of

any movement in the wasp, even when disturbed. Individual wasps could not be identified to theirexact emergence date having been mixed with other wasps, so the longevity calculations were based

on median time of emergence for all same sex wasps within each treatment to exact date of death.

Figure 2Summary of experimental sequence

Analysis

Statistical analysis was carried out using the statistical package R 2.7.2. Egg/larval mortality was

analysed using a regression with binomial errors (quasibinomial when overdispersed) on

proportions of eggs developing to pupate. Longevity studies were analysed using Kaplan-Meier

survivorship analysis, with exponential or Weibull errors depending on the fit of the model to the

data. Individuals outlasting the experimental timeframe or escaping were accounted for with

censoring incorporated into the model. Analysis of covariance with poisson (or quasipoisson when

overdispersed) error structure was used to analyse the data on parasitism success of grouped D.

kraussii. Analysis of covariance with binomial (or quasibinomial when overdispersed) error


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structures were used to analyse proportions of pupae resulting in of Q-fly and also proportions of

pupae resulting in wasps. Analysis of covariance with binomial (or quasibinomial when

overdispersed) errors was also used to analyse Q-fly sterility. In all analyses model simplification

was carried out, non-significant terms were removed, and the simplest model that explained the data

was kept, hence analysis of covariance was simplified to a regression when non-significantexplanatory variables were removed. This was handled by R.


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Results

Eggs to second instar Q-f ly egg/larval mortali ty

Exposure (Exposed or Unexposed to parasitoids) had no significant effect on the proportion of

pupae forming from the original eggs (t = 1.306, d.f. = 81, p > 0.05 n.s.). The irradiation dose (Gy),

however, had a highly significant effect on the proportion of eggs resulting in the formation of

pupae (t = -8.070, d.f. = 82, p < 0.001). The highest proportion of eggs developing to pupae was for

the control dose (0.0 Gy) with a mean proportion of 0.72 0.07 (1 s.e., n=12), though for the 4.7

Gy dose the mean was only slightly lower at 0.65 0.08 (1 s.e., n=12). The proportion of eggs

developing to pupae then dropped with dose (9.1Gy: 0.34 0.03(1 s.e., n=12), 15.9 Gy: 0.20

0.02 (1 s.e., n=12), 27.6 Gy: 0.13 0.03 (1 s.e., n=12), 47.0 Gy: 0.07 0.01 (1 s.e., n=12)). to

a mean of just 0.02 0.01 (1 s.e., n=12) at the highest dose, 79.9 Gy (see Figure 3).

Figure 3Eggs to second instar - proportion of Q-fly eggs developing to pupae according to dose

Eggs to thir d instar egg/larval mor tality

Exposure had no significant effect on the proportion of pupae forming from the original eggs (t = -

0.445, d.f. = 81, p > 0.05 n.s.). Again, irradiation dose (Gy) had a highly significant effect on the

proportion of eggs resulting in the formation of pupae (t = -9.776, d.f. = 82, p < 0.001). Means weresimilar for doses 0.0 Gy and 4.7 Gy (0.67 0.07 (1 s.e., n=12) and 0.64 0.08 (1 s.e., n=12)


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respectively), as with the eggs to second instar experiment. There was also a similar trend in the rest

of the data, with the mean proportion dropping with dose (9.1Gy: 0.40 0.04(1 s.e., n=12), 15.9

Gy: 0.32 0.03 (1 s.e., n=12), 27.6 Gy: 0.15 0.01 (1 s.e., n=12), 47.0 Gy: 0.10 0.01 (1 s.e.,

n=12)) to a minimum of 0.01 0.00 (1 s.e., n=12) at the highest dose, 79.9 Gy (see Figure 4).

Figure 4Eggs to third instar - proportion of Q-fly eggs developing to pupae according to dose

Eggs to second instar Q-f ly emergence

Dose was found to have a significant effect on the proportions of pupae emerging as adult Q-fly (t =

0.086, d.f. = 156, p < 0.001), with that proportion decreasing with increasing dose, and with zero fly

emergence from doses including and above 15.9 Gy (see Table 2 for mean values). There were also

significant differences in the proportion of pupae emerging as Q-fly with regard to sex (t = -2.742,

d.f. = 156, p = 0.007, with a higher proportion of females eclosing and exposure (t = 2.714, d.f. =

156, p = 0.007), with a higher proportion of flies eclosing from the unexposed treatment. There

were no significant interactions between explanatory variables.


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Figure 5Eggs to second instarProportion of pupae resulting in adult Q-fly emergence

according to sex and irradiation dose.

Table 2Eggs to second instar - mean proportions of pupae resulting in Q-fly emergence (1 s.e.,

n=6)

Male Female

Dose (Gy) Exposed Unexposed Exposed Unexposed

0.0 0.40 0.03 0.45 0.03 0.47 0.04 0.45 0.04

4.7 0.26 0.04 0.30 0.05 0.31 0.03 0.38 0.04

9.1 0.11 0.04 0.20 0.07 0.12 0.03 0.23 0.07

15.9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

27.6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

47.0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

79.9 Na Na Na Na


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Figure 6Eggs to second instarProportion of pupae resulting in adult Q-fly emergence

according to exposure and irradiation dose.

Eggs to thi rd instar Q-fl y emergence

As in the eggs to second instar experiment, dose had a significant effect on the proportions of flies

emerging (z = -12.209, d.f. = 154, p < 0.001) with a higher proportion of flies emerging from pupae

at lower doses, again decreasing to zero emergence at 15.9 Gy. A significant difference was also

apparent between the two levels of exposure (z = 7.102, d.f. = 154, p < 0.001), with a higher

proportion of flies emerging from pupae from the unexposed treatment than from the exposed

treatment. There was also a significant interaction between dose and exposure (z = -2.566, d.f. =

154, p = 0.0103), with a decreasing difference between the proportions of flies emerging from

exposed and unexposed treatments with an increase in the dose (see Figure 7 and Table 3). Unlike

the eggs to second instar experiment, no significant difference was found between the proportions

of male and female flies emerging from pupae (z = 1.502, d.f. = 153, p > 0.05 n.s.) in this

experiment.


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Table 3Eggs to third instar - mean proportions of pupae resulting in Q-fly emergence ( 1 s.e.,

n=12)

Dose (Gy) Exposed Unexposed

0.0 0.20 0.02 0.38 0.01

4.7 0.16 0.03 0.21 0.02

9.1 0.06 0.01 0.03 0.01

15.9 0.00 0.00 0.00 0.00

27.6 0.00 0.00 0.00 0.00

47.0 0.00 0.00 0.00 0.00

79.9 Na Na

Figure 7Eggs to third instarProportion of pupae resulting in adult Q-fly emergence according

to exposure and irradiation dose.

Eggs to second instar Q-fl y longevity

Dose was found to have a highly significant effect on the survivorship of emerged Q-fly adults (z =


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-7.99, d.f. = 1, p < 0.001), in that survival time was lower the higher the dose. The predicted mean

values for survivorship accounting for censored individuals were 88.70 days (0.0 Gy), 35.81 days

(4.7 Gy) and 15.32 days (9.1 Gy) ( standard error for the model, 0.0242 (1 s.e., n=161)) (see

Figure 9). There was no fly emergence at any of the higher doses. Exposure was found not to have

had a significant effect on the fly survivorship (z = 0.1313, d.f. = 3, p > 0.05 n.s.). Sex was alsofound not to have a significant effect on survivorship (and was removed from the final model),

though it was very close to being significant (z = 1.94, d.f. = 2, p = 0.0526) and there does appear to

be a (non-significant) trend, with longer predicted survival for males compared to females at all

doses of radiation (where flies emerged) (see Table 4 and Figure 8).

Table 4Eggs to second instar - Predicted mean survival times (days) for male and female Q-fly,

accounting for censored individuals (standard error for the model, 0.1315 (1 s.e., n=161)Dose (Gy) Female Male

0.0 78.25 100.96

4.7 31.70 40.90

9.1 13.60 17.55


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Figure 8Eggs to second instar Q-fly survivorship curve according to dose (Gy) & Sex

Figure 9Eggs to second instar Q-fly survivorship curve according to Dose (Gy) Final Model

Eggs to thir d instar Q-f ly longevity

As for the eggs to second instar longevity study, dose had a significant effect on Q-fly survival for

the eggs to third instar (z = -5.92, d.f. = 1, p < 0.001), with predicted mean longevity (accounting

for censored individuals) decreasing with increasing dose (0.0 Gy: 209.71 days; 4.7 Gy: 43.97 days;

9.1 Gy: 10.19 days, standard error of the model, 0.0562 (1 s.e., n=118)) (see Figure 10). There

was no significant difference in fly longevity related to sex (z = 0.3143, d.f. = 3, p > 0.05 n.s.) or

exposure (z = -0.0378, d.f. = 2, p > 0.05 n.s.).


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Figure 10Eggs to third instar Q-fly survivorship curve according to Dose (Gy)Final Model

Eggs to second instar Q-fl y ster il ity

Neither dose (t = 0.058, d.f. = 10, p > 0.05 n.s.) nor exposure (t = -0.503, d.f. = 11, p > 0.05 n.s.)

were found to have a significant effect on the proportions of eggs that hatched into larvae (see Table

5 for data). Significance was detected between the different types of mating cross used in the

experiment (t = -3.846 d.f. = 12, p = 0.002)) with mean hatching proportions of: 0.53 0.03 (1 s.e,

n=4) (fertile male x fertile female); 0.23 0.05 (1 s.e, n=4) (fertile male x irradiated female); 0.54

0.08(1 s.e, n=4) (irradiated male x fertile female); 0.55 0.13 (1 s.e, n=4) (irradiated male x

irradiated female). All flies from dose 9.1 Gy died prior to the onset of the mating and oviposition

experiment.


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Table 5 Eggs to second instarsterility trial data

Experiment Dose (Gy) Exposure Larvae Eggs Total Fertility (%)

I I 4.7 Un 30 97 127 23.62F F 4.7 Un 457 323 780 58.59F I 4.7 Un 117 208 325 36.00I F 4.7 Un 68 41 109 62.39I I 4.7 Exp 345 74 419 82.34F F 4.7 Exp 397 350 747 53.15F I 4.7 Exp 39 217 256 15.23I F 4.7 Exp 112 253 365 30.68II 0 Un 196 210 406 48.28F F 0 Un 508 425 933 54.45F I 0 Un 140 797 937 14.94I F 0 Un 336 258 594 56.57I I 0 Exp 474 247 721 65.74F F 0 Exp 294 340 634 46.37

F I 0 Exp 207 579 786 26.34I F 0 Exp 551 282 833 66.15

Eggs to thir d instar Q-fly steri li ty

No significant difference in proportions of eggs hatching was detected in this experiment in relation

to dose (t = -0.754, d.f. = 11, p > 0.05 n.s.), exposure (t = 0.023, d.f. = 10, p > 0.05 n.s.) or mating

cross (see Table 6 for data). Again, all flies from dose 9.1 Gy died prior to the onset of the mating

and oviposition experiment.

Table 6Eggs to third instarsterility trial data

Experiment Dose (Gy) Exposure Larvae Eggs Total Fertility (%)

I I 4.7 Un 77 71 148 52.03F F 4.7 Un 515 502 1017 50.64F I 4.7 Un 11 6 17 64.71I F 4.7 Un 736 450 1186 62.06I I 4.7 Exp 16 74 90 17.78F F 4.7 Exp 644 421 1065 60.47F I 4.7 Exp 227 140 367 61.85I F 4.7 Exp 171 327 498 34.34I I 0 Un 136 134 270 50.37F F 0 Un 682 644 1326 51.43F I 0 Un 401 179 580 69.14I F 0 Un 788 510 1298 60.71I I 0 Exp 509 170 679 74.96F F 0 Exp 506 363 869 58.23F I 0 Exp 340 219 559 60.82

I F 0 Exp 435 338 773 56.27


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Eggs to second i nstar wasp emergence

There was no significant difference for sex (z = 6.28e-16, d.f. = 77, p > 0.05 n.s.) or dose (z = -

1.294, d.f. = 78, p > 0.05 n.s.) in relation to the proportions of wasps emerging from pupae in the

eggs to second instar experiment, though a non-significant trend can be observed (see Table 6,

Figure 11) with higher proportions of wasps emerging from the three lowest doses compared withthe remaining doses, where no wasp emergence was observed. Males were found to emerge two or

three days prior to females in general.

Table 6Eggs to second instar - Mean Proportion of Pupae Resulting in Wasps

Dose (Gy) Mean Proportion of Pupae Resulting in Wasps

0.0 0.01 0.01 (1 s.e., n=12)

4.7 0.03 0.01 (1 s.e., n=12)9.1 0.02 0.01 (1 s.e., n=12)

15.9 0.00 0.00 (1 s.e., n=12)

27.6 0.00 0.00 (1 s.e., n=12)

47.0 0.00 0.00 (1 s.e., n=12)

79.9 Na

Figure 11

Eggs to second instar

proportion of pupae resulting in adult D. kraussii emergence

according to dose


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Eggs to thi rd instar wasp emergence

As for the eggs to second instar study, no significant difference in proportions of wasps emerging

was detected in relation to sex in this experiment (t = 1.284, d.f. = 73, p > 0.05 n.s.). There was a

similar trend in the previous study in the proportion of wasps emerging in relation to dose, howeverthe difference was found to be statistically significant in this case (t = -2.284, d.f. = 74, p = 0.0252)

(see Table 7 and Figure 12). Emergence was also observed at dose 15.9 Gy, whereas there was no

emergence at this dose in the eggs to second instar experiment. It is worth noting that overall wasp

emergence was higher in this experiment. Males were found to emerge two or three days prior to

females in general.

Table 7

Eggs to third instar - Mean Proportion of Pupae Resulting in WaspsDose (Gy) Mean Proportion of Pupae Resulting in Wasps

0.0 0.05 0.01 (1 s.e., n=12)

4.7 0.06 0.03 (1 s.e., n=12)

9.1 0.03 0.01 (1 s.e., n=12)

15.9 0.04 0.01 (1 s.e., n=12)

27.6 0.00 0.00 (1 s.e., n=12)

47.0 0.00 0.00 (1 s.e., n=12)79.9 Na


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Figure 11

Eggs to third instar

proportion of pupae resulting in adult D. kraussii emergenceaccording to dose

Eggs to second instar wasp sur vivorshi p

When wasps did emerge (doses 0.0 Gy, 4.7 Gy and 9.1 Gy), dose was not found to have a

significant effect on their longevity (z = -1.16, d.f. = 2, p > 0.05 n.s.). Sex, however, did play a

significant role (z = 7.05, d.f. = 1, p < 0.001), with mean survival for male wasps (27.67 days 2.77

(1 s.e, n=12)) far higher than for female wasps (17.42 days 0.39 (1 s.e, n=12)) (see Figure 12).

No individuals were censored in this study as all died within the experimental timeframe.


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Figure 12 Eggs to second instarD. kraussii survivorship curve with regard to sex - final model

Eggs to thi rd instar wasp sur vivorship

As was observed in the eggs to second instar experiment, dose was found not to have a significant

effect on the longevity ofD. kraussii (z = 0.826, d.f. = 2, p > 0.05 n.s.) (see Figure 13). Unlike the

eggs to second instar experiment, sex was found not to have a significant effect on the longevity of

the wasps, though it was close to being significant (z = 1.83, d.f. = 1, p = 0.0674) and there does

appear to be a trend in the data, with longer survival times for males (see Figure 14). The predicted

mean age at death accounting for censored individuals is 10.36 days for females and 17.00 days for

males (standard error of the model 0.271 (1 s.e., n=62)), though the variance is high (Female:

56.47, Male: 30.11) possibly explaining the lack of significance.


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Figure 13Eggs to third instarD. kraussii survivorship curve with regard to sex and dose

Figure 14Eggs to third instarD. kraussii survivorship curve with regard to sex


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Eggs to second instar - successful parasitism by grouped D. kr aussi i

Neither dose (z = 0.303, d.f. = 3, p > 0.05 n.s.) nor the total number of females per rep at each dose

(z = 1.579, d.f. = 2, p > 0.05 n.s.) were found to have a significant effect on the number of offspring

produced per wasp-day. There was also no significant effect of interaction between these variables(z = -0.183, d.f. = 1, p > 0.05 n.s.). Offspring were produced by females at every dose (where wasp

emergence had occurred from the initial irradiated material exposure experiment). Mean wasps per

wasp day were similar for doses 0.0 and 4.7 Gy and highest for dose 9.1 Gy (2.769, 2.237 and 3.177

wasps per wasp day respectively), though the number of replicates was low and this difference was

found not to be statistically significant.

Eggs to thir d instar - successfu l parasitism by grouped D. kraussiiAs for the eggs to second instar experiment, neither dose (z = -0.333, d.f. = 11, p > 0.05 n.s.) nor the

total number of females per rep at each dose (z = 0.484, d.f. = 10, p > 0.05 n.s.) were found to have

a significant effect on the number of offspring produced per wasp-day. There was also no significant

effect of interaction between these variables (z = -0.171, d.f. = 9, p > 0.05 n.s.). Offspring were

produced by females at doses 0.0, 9.1 and 15.9 Gy (not at 4.7 Gy) and generally the numbers of

wasp offspring emerging was very low (0.0 Gy: 0.076, 4.7 Gy: 0.000, 9.1 Gy: 0.059, 15.9 Gy: 0.019

mean wasps per wasp-day).

See Appendix III for data summary tables.


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Discussion

Bactrocera tryoni

Though the results of the eggs to second and eggs to third instar experiments cannot be statistically

compared directly as the experiments were carried out independently due to various constraints,

broad comparisons are permissible as the methods used for each were the same, although

experimental conditions did vary to an extent. Dose was found to have a significant effect on the

proportion of eggs developing to form pupae in both the eggs to second and third instar

experiments, suggesting increase in direct egg mortality and/or mortality in larvae prior to pupation

with increasing radiation dose. Cancino et al. (in press) found that 24 and 48 hour old eggs of the

Mexican fruit fly (Anastrepha ludens) suffered decreased hatchability with increasing radiation

dose. Mutations in hatching larvae caused by gamma irradiation of the eggs may cause a decrease inlarval fitness or otherwise prevent larvae from developing to the pupal stage (Grosch, 1962). The

trend of increased mortality prior to pupation with increased dose may have appeared less marked

than it may otherwise have been, as the likely higher numbers of larvae surviving at the lower

irradiation doses may have suffered higher mortality through competition than those at lower

densities in the higher dose treatments. Means were very similar between the control (0.0 Gy) and

the 4.7 Gy treatments in both experiments suggesting that there may be a threshold dose of

radiation, below which very little mortality is caused. Exposure had no significant effect on theproportion of eggs developing to pupae in either the eggs to second instar or eggs to third instar

experiments, which may suggest that larval mortality was not significantly increased by the effects

of lid removal and exposure to parasitoids. Potential effects of exposure may have included

dehydration of the larval diet due to lid removal (though relative humidity in the exposure boxes

was high see Methods), as well as mortality of larvae due to probing by (potentially multiple)

ovipositing females, though Rungrojwanich & Walter (2000a) concluded that mortality through

ovipositor probing of Q-fly larvae by individual D. kraussii was probably not responsible for

additional mortality when compared with an unexposed control. The relatively high numbers of

eggs used for the irradiation may have helped to limit mortality through superparasitism and

multiple probing events by female parasitoids, as eggs surviving the irradiation procedure and

developing into larvae may have been in a high enough ratio to the five female parasitoids in each

exposure box to limit the number of interactions each larva had with the parasitoids. Mortality

through parasitoid probing and superparasitism has been reported in fruit fly larvae (Sime, et al.,

2006).

Radiation dose also had a significant effect on the proportions of pupae from which adult Q-flies

eclosed, with higher proportions emerging the lower the dose for both the eggs to second instar and


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eggs to third instar experiments, with no adult Q-fly emerging at or above 15.9 Gy. Cancino et al.

(in press) found a similar effect when they irradiated the eggs ofA. ludens, with adult emergence

decreasing with increasing dose to zero above a threshold dose, despite the formation of pupae. It

would appear that above a threshold dose, flies are unable to complete metamorphosis, with the

major transformation from larva into adult fly (Denlinger & Zdarek, 1994) reliant on properlyfunctioning genes that may be deleteriously and irreparably altered by irradiation (mutations are

generally deleterious (Grosch, 1962)). For the eggs to second instar experiment, a significantly

higher proportion of females emerged than males which may suggest that female flies are more able

to withstand the effects that gamma irradiation of eggs has on the pupation process, though this was

not apparent in the eggs to third instar experiment and would require further specific investigation.

Exposure was found to have a significant effect on the proportions of pupae emerging in both

experiments, which is logical as some of the pupae from the exposed treatment harbouredparasitoids, therefore decreasing the proportion of Q-fly that could emerge, whereas the unexposed

treatment did not result in parasitoid emergence. A second factor that may have resulted in a

reduction in the proportion of pupae resulting in Q-fly adults for the exposed treatments may have

been caused by the potential increase in mortality brought about by wasp probing, as mentioned

previously, however in this case resulting in delayed mortality, acting at the pupal rather than larval

stage. There was also a significant interaction effect between dose and exposure on the proportions

of pupae emerging as flies in the eggs to third instar experiment, with decreasing difference

between exposed and unexposed treatments with increasing dose. This is probably because at lower

doses, a proportion of the Q-fly that were exposed would develop into (or be killed by) parasitoids,

explaining the large gap between the two exposure treatments at these lower doses. At higher doses,

however, the decrease in Q-fly emergence appeared to be primarily due to the effects of irradiation

(i.e. smaller differences between exposed and unexposed treatments, yet decreasing proportions of

Q-fly emerging), and where no Q-fly emergence occurred at all due to these effects, there was

clearly no difference between exposed and unexposed treatments. A similar trend can be observed

in the eggs to second instar results, however the interaction effect appears less pronounced and was

not significant.

Proportions of Q-fly pupae developing to become adult Q-fly/D. kraussii were used in the statistical

analysis rather than raw emergence numbers, as the emergence of adult Q-flies and wasps was

confounded by the numbers of pupae that preceded them, with no more than 100% of the number of

pupae able to eclose. Even though similar numbers of eggs were irradiated for all replicates at all

doses, numbers of pupae varied greatly, so proportions of pupae resulting as flies/wasps were used

to make the data comparable.

Fly longevity was significantly affected by dose in both the eggs to second and eggs to third instar


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experiments, with significantly higher survivorship at lower doses (where fly emergence occurred)

in both cases. There was no significant difference in longevity related to exposure in either

experiment. It seems that whilst some irradiated flies were able to develop to adulthood, deleterious

effects of irradiation on fitness were still apparent in this stage when compared with un-irradiated

controls, with fitness costs in terms of survival time increasing with dose. The highest dose ofirradiation from which adult Q-fly developed was 14.9 Gy. The low numbers of flies emerging at

this dose generally died quickly and the majority emerging were malformed, often with shrivelled

wings or tiny bodies, most were unable to work, and only a small minority exhibited any flight

ability. Flies emerging from the 4.7 Gy radiation treatment generally survived much longer, were

able to walk and fly and displayed far fewer deformities than those at 14.9 Gy, though they did

display significantly lower predicted mean survival times than the control (accounting for censored

individuals). There was no significant difference in survival time between males and females ineither experiment, though there was an apparent trend for higher survivorship in males for the eggs

to second instar experiment at each dose (which was close to being significant). This was not the

case however, in the eggs to third instar experiment, and is contrary to the findings of Perez-Staples

et al. (2007) who observed higher longevity in female Q-flies.Non-emergence of adults from the

highest dose (79.9 Gy) is expected, as this is within the dose range (75-100 Gy) required to achieve

quarantine security against B. tryoni eggs and larvae in fruit (Hallman & Loaharanu, 2002). It is

worth mentioning that the majority of literature available regarding the irradiation of fruit fly eggs

is concerned with quarantine-type fruit treatments with the aim of egg and/or larval mortality (e.g.

Hallman, 1998; Hallman & Loaharanu, 2002). Few studies have investigated the potential of

irradiating fruit fly eggs with the aim of rearing parasitoids upon the irradiated host material, and

this study will prove useful to future investigations into this rearing approach.

Dose was not found to have a significant effect on the fertility of eggs produced by emerging flies

in either experiment, but this study only compared flies from the control and dose 4.7 Gy, as at the

only other dose from which adult flies emerged (9.1 Gy), all died prior to the sterility study. Their

short survivorship combined with the tendency for deformed and non-flying Q-fly to emerge at this

dose would suggest functional sterility of flies may be achieved at 9.1 Gy, if not biological sterility.

There was found to be a significant difference in the proportions of eggs hatching in relation to

mating cross for the eggs to second instar experiment, with the lowest proportion of eggs hatching

in the fertile male x irradiated female cross, which may be surprising, with most studies finding the

irradiated male x irradiated female cross to give highest sterility (e.g. Collins et al., 2008). The

analysis of these data, however, must be treated very carefully as it is based on only one replicate

and may not give a true representation of the situation. These data are very useful, however, in that

they demonstrate that levels of sterility observed in all reps are far below those required in a sterile


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insect release program (e.g.


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of parasitoids in each exposure box in order to increase the number of offspring produced, though

this would go against the recommendations of Lawrence et al., (1978) to use a ratio of at least15:1

hosts to female wasps. This may be rectified by increasing the number of eggs (and therefore

larvae) in each replicate, though competition for food and space between larvae may become an

issue. Numbers of pupae decreased with increasing dose as can be seen in the Q-fly egg/larvalmortality analysis, yet proportions of these pupae developing into wasps was fairly similar across

all doses where emergence occurred. It may be expected that due to the lower numbers of pupae at

the higher doses, the proportions developing to wasps (where any wasps emerged) would be higher

for these doses, if levels of parasitism were similar across all emerging doses. The fact that this does

not appear to be the case could be for a number of reasons. Firstly, it is difficult to detect the exact

nature of trends due to low numbers of emerging wasps, particularly for the eggs to second instar

experiment. Lower numbers of parasitoids at higher doses may also indicate that parasitism of thesehosts, or development within these hosts may be less successful due to irradiation effects compared

with unirradiated hosts, making them less suitable for parasitoid rearing. Also, larval development

may have been delayed due to the effects of irradiation, potentially resulting in some larvae that

were not suitably developed for parasitisation by D. kraussii, particularly in the eggs to second

instar experiment (where very low numbers of parasitoids eclosed). Rungrojwanich & Walter

(2000a) achieved much higher levels of parasitism in their study, which involved the presentation of

third instar Q-fly larvae to D. kraussii. If this were the case, larval development times prior to

exposure to the wasps may need to be increased to allow higher rates of parasitism, though further

study would be required to determine the necessity of this action. With the likely lower numbers of

larvae at the higher doses due to radiation-induced mortality, parasitism levels may have been lower

due to the decreased density of hosts compared with the lowest and control doses. Also with lower

numbers of available hosts may come an increased risk of superparasitism and larval mortality

through multiple probing events as the ratio of hosts to parasitoids would be lower. It may be that

higher numbers of eggs must be irradiated, for example, at the 15.9 Gy selective dose in the eggs to

third instar experiment to produce simila

Documents

PARASITOIDS Bactrocera Tryoni Irradiation of Eggs Pratt 2008