Contents

Abstract 3

Introduction The Queensland Fruit Fly Cost of Q-fly and the Fruit Fly Exclusion Zone (FFEZ) Current Control Climate, Chemicals and the Future of Q-fly Aims & Objectives of This Study Methods & Materials Preparation of Q-fly material for irradiation Irradiation of Q-fly material Rearing of Diachasmimorpha kraussii Exposure of Irradiated Q-fly to Diachasmimorpha kraussii Egg/larval mortality Q-fly longevity Q-fly reproductive sterility Rearing of Q-fly larvae for presentation to D. kraussii Parasitism success of grouped Diachasmimorpha kraussii Diachasmimorpha kraussii longevity Analysis Results Eggs to second instar Q-fly egg/larval mortality Eggs to third instar egg/larval mortality Eggs to second instar Q-fly emergence Eggs to third instar Q-fly emergence Eggs to second instar Q-fly longevity Eggs to third instar Q-fly longevity Eggs to second instar Q-fly sterility Eggs to third instar Q-fly sterility Eggs to second instar wasp emergence Eggs to third instar wasp emergence Eggs to second instar wasp survivorship Eggs to third instar wasp survivorship Eggs to second instar - successful parasitism by grouped D. kraussii Eggs to third instar - successful parasitism by grouped D. kraussii Discussion Bactrocera tryoni Diachasmimorpha kraussii Implications Conclusions

4 4 5 7 9 12 13 13 15 16 17 19 19 19 21 21 23 23 25 25 25 26 28 29 31 32 33 34 35 36 37 39 39 40 40 43 46 48

Acknowledgements

49

References

50

Appendix I

56

Appendix II

57

Appendix III

62

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 nonemergence. 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 79.9 Gy) 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 insect release. 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 of D. 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.

Introduction
The Queensland Fruit Fly 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 et al., 2000). Following oviposition, after 1-3 days (42 hours at 25oC, 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 of B. tryoni in Australia is throughout the 4

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 Qfly 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 Fruit Fly 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 to B. 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 MidMurray districts (see Figure 1) (Osborne et al., 1997; TriState Fruit Fly Strategy Steering Committee, 2002).

Figure 1 The 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 fly free 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). 6

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). Qfly are monitored using Lynfield traps charged with the parapheromone cue-lure (4-(pacetoxyphenyl)-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 are monitored 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 7

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 potentially spread 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 as South 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 et al., 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). 8

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 Future of Q-fly

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.

Control option

Current Sustainability/ use/ future effectiveness priority

Robustness under climate change Endemic **

Adaptability under climate change

Constraints

Cover spray

***/***

*/*

**

Bait spray SIT suppression

**/*** */**

***/*** **/*** FFEZ

*** *

*** *

Residues & contamination, public health, cost Public health, cost Fruit fly population size, cost Political, public health, cost Political, fruit fly population size, cost Political, cost Effectiveness/ politics

Bait spray SIT eradication Exclusion Strip fruit

***/*** **/*** ***/*** */*

*/*** **/*** **/*** */0

*** ** ** **

*** ** ** *

Table 1 Adaptability 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 the risks 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. Baker et 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 10

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 parasitoids are 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 establish permanently 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 11

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 over the 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 longevity of 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 its parasitoid, 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 of D. 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.

12

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 material for irradiation 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 caged separately 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 (1/8th ) of an organic 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.

13

Plate 1 Egging 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 for B. tryoni is 42hr at 25oC, 80%RH (Anderson, 1962)), the irradiation of which was not the aim of this experiment. 14

Irradiation of Q-fly material 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 irradiation or 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 an experiment 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 of B. 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.

15

Plate 2 Irradiation 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 Qfly 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 first flight cage were recorded. Male wasps generally emerged before females, but males were added to 16

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 Irradiated 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 larvae were 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 17

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 the D. kraussii (see Plate 3).

Plate 3 One 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, 18

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 medium plastic 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 sterility 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 (PerezStaples 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 19

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 Wales Department 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).

Plate 4 Small egging cup

Plate 5 Oviposition by Q-fly into small egging cup

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 20

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

Rearing of Q-fly larvae for presentation to D. kraussii Batches 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).

Parasitism success of grouped Diachasmimorpha kraussii 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 21

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 to D. 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 Qfly 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 5 Diachasmimorpha kraussii parasitism chamber 22

Diachasmimorpha kraussii 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 their exact 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 2 Summary 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 23

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-significant explanatory variables were removed. This was handled by R.

24

Results
Eggs to second instar Q-fly egg/larval mortality 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 3 Eggs to second instar - proportion of Q-fly eggs developing to pupae according to dose

Eggs to third instar egg/larval mortality 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 were similar 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) 25

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

Eggs to second instar Q-fly 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 5 Eggs to second instar Proportion of pupae resulting in adult Q-fly emergence according to sex and irradiation dose. Table 2 Eggs to second instar - mean proportions of pupae resulting in Q-fly emergence (1 s.e., n=6) Male Dose (Gy) 0.0 4.7 9.1 15.9 27.6 47.0 79.9 Exposed 0.40 0.03 0.26 0.04 0.11 0.04 0.00 0.00 0.00 0.00 0.00 0.00 Na Unexposed 0.45 0.03 0.30 0.05 0.20 0.07 0.00 0.00 0.00 0.00 0.00 0.00 Na Exposed 0.47 0.04 0.31 0.03 0.12 0.03 0.00 0.00 0.00 0.00 0.00 0.00 Na Female Unexposed 0.45 0.04 0.38 0.04 0.23 0.07 0.00 0.00 0.00 0.00 0.00 0.00 Na

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Figure 6 Eggs to second instar Proportion of pupae resulting in adult Q-fly emergence according to exposure and irradiation dose.

Eggs to third instar Q-fly 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 3 Eggs to third instar - mean proportions of pupae resulting in Q-fly emergence ( 1 s.e., n=12) Dose (Gy) 0.0 4.7 9.1 15.9 27.6 47.0 79.9 Exposed 0.20 0.02 0.16 0.03 0.06 0.01 0.00 0.00 0.00 0.00 0.00 0.00 Na Unexposed 0.38 0.01 0.21 0.02 0.03 0.01 0.00 0.00 0.00 0.00 0.00 0.00 Na

Figure 7 Eggs to third instar Proportion of pupae resulting in adult Q-fly emergence according to exposure and irradiation dose.

Eggs to second instar Q-fly longevity Dose was found to have a highly significant effect on the survivorship of emerged Q-fly adults (z = 29

-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 also found 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 4 Eggs 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) 0.0 4.7 9.1 Female 78.25 31.70 13.60 Male 100.96 40.90 17.55

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

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

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

Eggs to second instar Q-fly sterility 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 instar sterility trial data Experiment I I F F F I I F I I F F F I I F I I F F F I I F I I F F F I I F Dose (Gy) 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 0 0 0 0 0 0 0 0 Exposure Un Un Un Un Exp Exp Exp Exp Un Un Un Un Exp Exp Exp Exp Larvae 30 457 117 68 345 397 39 112 196 508 140 336 474 294 207 551 Eggs 97 323 208 41 74 350 217 253 210 425 797 258 247 340 579 282 Total 127 780 325 109 419 747 256 365 406 933 937 594 721 634 786 833 Fertility (%) 23.62 58.59 36.00 62.39 82.34 53.15 15.23 30.68 48.28 54.45 14.94 56.57 65.74 46.37 26.34 66.15

Eggs to third instar Q-fly sterility 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 6 Eggs to third instar sterility trial data Experiment I I F F F I I F I I F F F I I F I I F F F I I F I I F F F I I F Dose (Gy) 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 0 0 0 0 0 0 0 0 Exposure Un Un Un Un Exp Exp Exp Exp Un Un Un Un Exp Exp Exp Exp Larvae 77 515 11 736 16 644 227 171 136 682 401 788 509 506 340 435 Eggs 71 502 6 450 74 421 140 327 134 644 179 510 170 363 219 338 Total 148 1017 17 1186 90 1065 367 498 270 1326 580 1298 679 869 559 773 Fertility (%) 52.03 50.64 64.71 62.06 17.78 60.47 61.85 34.34 50.37 51.43 69.14 60.71 74.96 58.23 60.82 56.27

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Eggs to second instar 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 with the remaining doses, where no wasp emergence was observed. Males were found to emerge two or three days prior to females in general. Table 6 Eggs to second instar - Mean Proportion of Pupae Resulting in Wasps Dose (Gy) 0.0 4.7 9.1 15.9 27.6 47.0 79.9 Mean Proportion of Pupae Resulting in Wasps 0.01 0.01 (1 s.e., n=12) 0.03 0.01 (1 s.e., n=12) 0.02 0.01 (1 s.e., n=12) 0.00 0.00 (1 s.e., n=12) 0.00 0.00 (1 s.e., n=12) 0.00 0.00 (1 s.e., n=12) Na

Figure 11 Eggs to second instar proportion of pupae resulting in adult D. kraussii emergence according to dose 34

Eggs to third 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, however the 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 Wasps Dose (Gy) 0.0 4.7 9.1 15.9 27.6 47.0 79.9 Mean Proportion of Pupae Resulting in Wasps 0.05 0.01 (1 s.e., n=12) 0.06 0.03 (1 s.e., n=12) 0.03 0.01 (1 s.e., n=12) 0.04 0.01 (1 s.e., n=12) 0.00 0.00 (1 s.e., n=12) 0.00 0.00 (1 s.e., n=12) Na

35

Figure 11 Eggs to third instar proportion of pupae resulting in adult D. kraussii emergence according to dose

Eggs to second instar wasp survivorship 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.

36

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

Eggs to third instar wasp survivorship As was observed in the eggs to second instar experiment, dose was found not to have a significant effect on the longevity of D. 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.

37

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

Figure 14 Eggs to third instar D. kraussii survivorship curve with regard to sex 38

Eggs to second instar - successful parasitism by grouped D. kraussii 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 third instar - successful parasitism by grouped D. kraussii As 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.

39

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 in larval 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 the proportion 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 40

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 of A. 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 properly functioning 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 harboured parasitoids, 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 41

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 of irradiation 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 in either 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 42

insect release program (e.g. <0.5% hatch for sterile males x fertile females) (FAO/IAEA/USDA, 2003). Therefore, the proposed target outcome of irradiation and exposure of Q-fly eggs is to produce D. kraussii with non-emergence of adult Qfly, rather than parasitoid production combined with sterility of emerging Q-fly adults. This proposal is similar to that put forth by Cancino et al., (In Press) who achieved similar results of non-emergence in a related fruit fly species exposed to opiine braconid wasps.

Diachasmimorpha kraussii There was no significant difference in the proportions of pupae resulting in wasp emergence in relation to sex for either the eggs to second or eggs to third instar experiments. In both experiments there was a trend for higher proportions of pupae resulting in wasp emergence at lower doses. This trend was found to be significant in the eggs to third instar, but not for the eggs to second instar, probably due to the very low total numbers of wasps emerging in this experiment giving reduced statistical power to the model. Wasps emerged from the control as well as doses 4.7 Gy and 9.1 Gy in both experiments, but also from dose 15.9 Gy in the eggs to third instar study.This is particularly noteworthy, as at this radiation dose there was no adult Q-fly emergence, potentially making this a selective dose, allowing the production of D. kraussii without the need to separate out emerging Qfly or risk escapes of this pest. No wasps emerged from dose treatments higher than 15.9 Gy in either experiment (or from 15.9 Gy in the eggs to second instar). The proportion of pupae resulting in wasps was low in both experiments even for the control treatments. There are a number of possible reasons for this, for example conditions in the CT room may have been suboptimal for parasitism by D. kraussii. Temperature in the exposure CT room was slightly lower and more variable than that used in a study of D. Kraussii life history by Rungrojwanich & Walter (2000a) (24 2C compared with 25 1C). Possibly more importantly, the humidity was very high (92 5% RH) in the exposure boxes which had very little ventilation. Whilst there does not appear to be any literature on the specific effects of humidity on parasitism rates for D. kraussii or related species, this relative humidity is very high compared with the majority of the natural Australian range of this parasitoid and is far higher than that generally used for rearing experiments involving D. kraussii (e.g. 605% RH - Rungrojwanich & Walter, 2000a). This high humidity may have resulted in reduced rates of parasitism by the wasps, which are capable of producing 15-20 offspring per day at peak levels (Rungrojwanich & Walter, 2000a). Rungrojwanich & Walter (2000a) found these peak levels of offspring production to occur at days 7-8 of adult life, which was the age of the wasps used in this experiment. Future studies should address the problem of ventilation to reduce the levels of humidity within the exposure boxes. Another possible improvement may be to increase the numbers 43

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/larval mortality 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 these hosts, 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 similar numbers of wasp offspring (without producing adult Qfly) when compared to the control . In terms of emergence, it would appear that third instar Q-fly larvae reared from irradiated eggs make better D. kraussii hosts than second instar larvae, though because the exposure process for each of these experiments was carried out independently on separate days with potentially slightly different conditions and with wasps differing by one day of age, absolute conclusions are not appropriate.

Longevity of emerged D. kraussii was not affected significantly by dose in either experiment, which suggests that fitness of wasps reared on irradiated hosts may be similar to that of wasps reared on 44

unirradiated Q-fly hosts, which is a promising sign for potential use in biological control. Longevity was found to be significantly affected by sex in the eggs to second instar experiment, with longer survivorship in males than females. In the eggs to third instar, a similar trend is apparent for sex (see Figure X), though it is not significant, probably due to the associated high levels of variance. Mean survival times and predicted mean survival times (accounting for censored individuals) were quite large between the eggs to second and eggs to third instar experiments respectively, however conditions for the eggs to third instar were far from ideal, with large temperature and humidity fluctuations over the course of experiments, particularly overnight drops in temperature. Wasp survival may have been affected by these fluctuating conditions and so the means for the two separate experiments cannot fairly be compared. Potential impacts of the fluctuating conditions in this CT room are discussed further below. Due to non-emergence of male wasps for certain replicates from the original exposure of the irradiated Q-fly material, some females in the longevity trial (and parasitism success study) were unmated. Unmated females were low in number, but it has been reported that longevity in unmated females tends to be longer than that for mated females (Rungrojwanich & Walter, 2000a). Ideally all females would have been mated, or mated and unmated females treated as separate treatments. The study into the successful parasitism by grouped D. kraussii was named as such as only emerging offspring were counted (successful cases of parasitism) and inhabitants of pupae that failed to eclose were not identified as Q-fly or wasp. Similar studies focusing on individual female wasps are often referred to as reproductive capacity studies, however it is important to appreciate the potential effects of grouping female parasitoids; namely the possibility of superparasitism and increased mortality of larvae through ovipositor probing; direct wasp competition, with females blocking or otherwise obstructing the exposure site; reduced longevity (and hence reduced offspring production) of females through competition. All of these factors could potentially affect the parasitism success of the females, and so it would not be fair to analyse the mean number of offspring produced per female as her true reproductive capacity. Ideally the parasitism of individual females would have been studied, but various constraints along with varying ratios of males to females across reps meant that grouping of wasps was necessary. No significant difference was found in the number of wasps produced per wasp day with dose, or with the start number of grouped females (or an interaction between these variables) for either the eggs to second or eggs to third instar experiments. The lack of significant difference between successful wasp parasitism at each dose implies that wasps emerging from irradiated host material have a comparable ability to wasps emerging from non-irradiated host material in terms of successfully parasitising Q-fly larvae, which is a very good sign for a potential production method of biological control agents. It must be noted, however, that the number of replicates for the eggs to second instar experiment was very low 45

due to the low numbers of wasps emerging from the initial exposure of irradiated Q-fly material and also that numbers of emerging wasps in the eggs to third instar successful parasitism experiment were very low. This low emergence may not be a fair representation of the reproductive ability of these, however, as the CT room in which they were kept functioned extremely poorly for the duration of the experiment, dropping as low as 12oC overnight, despite having a target temperature of 24oC. The room was slow to warm up, though it did occasionally increase above the target temperature to a maximum of 27.8oC. Relative humidity also fluctuated greatly, with a mean well below the target of 60%. Possibly as a direct result of this variation, particularly the low temperatures, little oviposition activity was observed and very low numbers of wasps emerged. A second factor that may have contributed to a reduction in the number of eclosing parasitoids in this experiment is the ability of certain parasitoid species to enter larval dormancy, which can last up to several months in opiine braconid species if the temperature is below 13 oC (Wong & Ramadan, 1992). Extended emergence periods have been seen for D. kraussii even at 25oC (Rungrojwanich & Walter, 2000a), so it is possible that dormant wasp larvae may have been present but unaccounted for in a proportion of the unemerged pupae resulting from this experiment.

Implications This study has been very useful in establishing a suitable dose range for future studies into the irradiation of Q-fly eggs upon which parasitoids may be reared. Ideally experiments progressing from this study will use smaller increments between doses (e.g. increments of 2.5 Gy, as used by Cancino et al., (In press)) towards the lower end of the dose range that had to be used in this study, with particular focus on the potentially selective doses at and around 15.9 Gy. The use of a lower dose range with narrower increments would result in emergence at a greater number of doses and would also enable a more accurate analysis of the effects of irradiation on Q-fly and its parasitoid, D. kraussii, with the aim of establishing the optimum irradiation conditions to produce healthy, reproductive and competitive wasps in combination with non-emergence of adult Q-fly. Further suggestions to follow on this investigation would also include the use of third instar larvae as the most suitable stage for exposure to D. kraussii, as used for reproductive capacity studies on this parasitoid by Rungrojwanich & Walter (2000a). It may also be useful to use higher numbers of wasps in exposure treatments to increase the numbers of offspring produced, along with an increased number of eggs at the irradiation stage to maintain the ratio of host larvae to wasps and avoid superparasitism (Walters et al., 1978). It would certainly be worth investigating the use of other parasitoid species in this procedure, to compare their abilities to develop on irradiated Q-fly material. Potential species of interest include Diachasmimorpha longicaudata, D. tryoni and Fopius arisanus, all of which are present in 46

Australia and are natural enermies of B. tryoni (Purcell, 1998). All three of these species have been introduced to Hawaii in biological control programs and have been shown to be effective in contributing to the control of populations of Mediterranean fruit fly and oriental fruit fly (Funasaki et al., 1988). The ease of rearing D longicaudata has meant that it has been considered for biological control programs using augmentative release techniques in several studies (Purcell, 1998). Field trials may be carried out to determine the relative effectiveness of each of these parasitoids in the augmentative control of Q-fly. Future studies must ensure that consistent and reliable equipment is used, as the inconsistencies in the CT rooms in this experiment may have significantly affected certain outcomes and reduced analytical confidence. Depending on the success of future studies into the irradiation of eggs for the rearing of parasitoids, commercialisation and increased scales of production may also be considered, along with a financial analysis of the potential costs of this manner of parasitoid production in relation to the likely benefits resulting from increased control of B. tryoni (and potentially a number of other fruit fly species).

47

Conclusions
Diachasmimorpha kraussii is able to develop successfully on Q-fly irradiated at the egg stage. A selective dose of radiation that allows the development of D. kraussii in the irradiated host, but results in non-emergence of adult Q-flies has also been determined - an ideal scenario for biological control of Q-fly through augmentative D. kraussii release, negating the requirement for separation of emerging adult wasps and flies. Sterility was not detected in Q-fly that emerged from lower doses. Wasps eclosing from irradiated hosts had similar survival times and parasitism ability to unirradiated controls. Emergence of wasps was lower than expected, but this may have been due to fluctuating or unsuitable conditions in one of the CT rooms. Further study is required to determine an optimum dose of radiation to select for wasp development to adulthood whilst preventing Q-fly emergence and this study has revealed a suitable range of doses on which to focus. Upon establishing an optimum radiation dose, work must be carried out to determine the practicability and expense of commercial production of parasitoid wasps using this technique. A number of other parasitoid species may also be considered for further study. This study has yielded promising results, with the long term potential of increased levels of control over the Queensland fruit fly using this parasitoid augmentation approach, along with a decreased reliance on chemical pesticides.

48

Acknowledgements
Many thanks to Geoff Gurr and Olivia Kvedaras for their supervision and assistance throughout the project and to Andrew Jessup, for his contributions to the original project proposal and for his helpful and extensive advice. Thanks also to Catherine Gitau for her help rearing the parasitoid wasps and fruit flies. Many thanks to Katina Lindhout for her input at a number of stages in the project and for saving the day on several occasions.

Thanks to Connie Banos for her guidance though the intricacies of the irradiation procedure and to YongSeok Choi and Jennifer Spinner for their help with data collection. Thanks also to Roger Mandel and Michael Stout for their assistance at various stages and also to Julie, Adam, Jo and Rachael from the Entomology lab at WWAI for making us welcome!

Many thanks to Tricia Reader and Simon Leather for their support and proof reading skill. Thanks also to Skye Wassens, Tom Ezard, Brian Pickett and Mick Crawley for their help with data analysis.

Finally, very many thanks to Anna Harris for everything! I would not have been able to complete this project without her.

49

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Meats, A.W., Clift, A.D. & Robson, M.K. (2003) Incipient founder populations of Mediterranean and Queensland fruit flies in Australia: the relation of trap catch to infestation radius and models for quarantine radius. Australian Journal of Experimental Agriculture, 43: 397-406.

Montoya, P. & Cancino, J. (2004) Augmentative biological control in fruit flies (Diptera: Tephritidae). Folia Entomologica Mexicana, 43(3): 257-270. OLoughlin, G.T., East, R.A. & Meats, A. (1984) Survival, Development Rates and Generation Times of the Queensland Fruit Fly, Dacus Tryoni, in a Marginally Favourable Climate: Experiments in Victoria. Australian Journal of Zoology, 32(3): 353-361.

Osborne, R., Meats, A., Frommer, M., Sved, J.A., Drew, R.A.I. & Robson, M.K. (1997) Australian Distribution of 17 Species of Fruit Flies (Diptera: Tephritidae) Caught in Cue Lure Traps in February 1994. Australian Journal of Entomology, 36: 45-50.

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Rungrojwanich, K. & Walter, G.H. (2000b) The Australian fruit fly parasitoid Diachasmimorpha 53

kraussii (Fullaway): Mating behavior, modes of sexual communication and crossing tests with D. longicaudata (Ashmead) (Hymenoptera: Braconidae: Opiinae). Pan-Pacific Entomologist, 76(1): 12-23.

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Wang, X.G. & Messing, R.H. (2002) Newly imported larval parasitoids pose minimal competitive risk to extant egglarval parasitoid of tephritid fruit flies in Hawaii. Bulletin of Entomological Research, 92: 423-429.

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Appendix I

Plate 6 Female Queensland Fruit Fly Bactrocera tryoni.

Plate 7 Male Queensland Fruit Fly Bactrocera tryoni.

Plate 8 Second instar larva - Queensland Fruit Fly Bactrocera tryoni.

Plate 9 Third instar larva - Queensland Fruit Fly Bactrocera tryoni.

Plate 10 Female Diachasmimorpha kraussii

Plate 11 Male Diachasmimorpha kraussii

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Appendix II

Materials
Petri Dishes: Size 90mm diameter Size 55mm diameter Colour: Natural LabServ, Biolab. 5 Caribbean Drive, Scoresby, Victoria 3179, Australia.

Carrot medium: Dried carrot Dried carrot, 3mm. H.J. Langdon & Co. PO Box 596, Sunshine, Vic 3020, Australia. Sodium Benzoate Sodium Bezoate Laboratory Reagent, Chem-Supply PTY. LTD. Gillman, South Australia. Citric Acid Cargil. Imported by Langdon Ingredients. H.J. Langdon & Co. PO Box 596, Sunshine, Vic 3020, Australia. Torula Yeast Yeast Torula Food Grade. H.J. Langdon & Co. PO Box 596, Sunshine, Vic 3020, Australia.

Method: 1. 7.5 litres Hot Water 2. 2.7 litres Dried Carrot 3. 20 grams Sodium Benzoate 4. 72 grams Citric Acid 5. 480 Torula Yeast

Place hot water into stainless steel container, add sodium benzoate and stir to dissolve. Add citric acid and stir to dissolve. Add yeast and mix thoroughly. Add carrot and stir to thoroughly mix. Lid on and leave in preparation room.

Stir medium throughout the day to make sure all the ingredients are mixed and the carrot has absorbed all the water. Carrot medium can be covered and stored in refrigerator.

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Irradiation Conditions: Irradiation Facility Gamma Technology Research Irradiator (GATRI) Radiation Type Gamma radiation (cobalt-60) Irradiation Date 14 May 2008 Facility Dose Rate 4.0 Gy.min-1 Required Doses 5.0, 8.8, 15.3, 26.8, 46.9, 82.1 Gy Irradiation Temperature Approximately 20oC Dosimeter Type Fricke Dosimeter Batch - F195

Irradiation dose range:

Target Dose 82.1 46.9 26.8 15.3 8.8 5.0

Measured dose range (Gy) 75.0-84.8 44.2-49.9 26.0-29.3 15.1-16.6 8.9-9.3 4.4-4.9

Average dose (Gy) 79.9 47.0 27.6 15.9 9.1 4.7

Sugar Cubes: Cane Sugar. 1 cube aprox. 4.5g. CSR Sugar Australia PTY. LTD., Epping, NSW 2121, Australia.

Water cups: Cups Plastic Souffles, P100, 1oz, 29.6ml. Dimensions: Height 30mm, Top Diameter 40mm, Base Diameter 32mm. Solo Cup Company, Highland Park, IL 60035-379, USA. Lids Clear Plastic Souffle Lids, PLI. Dimensions: Diameter 32mm. Solo Cup Company, Highland Park, IL 60035-379, USA. Wicks Roeko, Luna Cotton Rolls. Non-Chlorine Bleached. Size 2. Halas Dental Limited, 44 ODea Avenue, Waterloo, NSW 2017, Australia.

Method: Perpendicular cuts were made in each lid to form a cross, through which a wick was inserted with exposed on the underside of the lid, exposed above the topside of the lid. The cup was filled to slightly below the rim with water, and then the lid with wick was fitted (see Plate 12). 58

Plate 12 A Water Cup

Flight Cages: BugDorm 4030 Insect Rearing Cage (30cm x 30cm x 30cm). Made of plastic: research material. MegaView Science, Education Services Co., LTD. Taichung, Taiwan.

Honey: Woolworths Home Brand Australian Honey, Bella Vista, NSW, 2153, Australia. Per 100g: Energy Protein Fat 1416kJ 0.3g 0.0g

Carbohydrate 83.1g - Sugars 77.4g

Dietary Fibre 0.0g Sodium 15mg

Vermiculite: Exfoliators Vermiculite, Exfoliators (Aust.) PTY. LTD., Dandenong, Victoria, Australia.

Plastic Boxes: Plastic box 1000ml. Dimensions: Length 165mm, Width 110cm, Height 70mm. Genfac Plastics PTY. LTD., Melbourne, Australia.

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Yeast hydrolysate enzymatic: MP Biomedicals, LLC, Solon, Ohio, 44139, USA.

Test Sieve: Laboratory Test Sieve: Brass, Mesh - Stainless Steel, Aperture 1.70mm. Endecolts Ltd., London, England.

Small Egging Cups: Small Cups Plastic Souffles, P100, 1oz, 29.6ml. Dimensions: Height 30mm, Top Diameter 40mm, Base Diameter 32mm. Solo Cup Company, Highland Park, IL 60035-379, USA. Lids Clear Plastic Souffle Lids, PLI. Dimensions: Diameter 32mm. Solo Cup Company, Highland Park, IL 60035-379, USA. Medium Cups - Dimensions: Height 100mm, Top Diameter 75mm, Base Diameter 48mm. Genfac Plastics PTY. LTD., Melbourne, Australia. Staples Rexel Bambi, Number 25. Dimensions: Length

Method: A mounted needle was used to create seven evenly spaced columns of four evenly spaced holes (of a similar size to those in the large egging cups) around the upper of a small cup. The cup was filled to just below the lower holes with water, into which Q-fly eggs could fall preventing desiccation. A piece of organic orange (including peel) approximately 10mm x 10mm x 5mm was hung inside the cup from the lid using a staple. This was to act as an oviposition cue to the Q-fly, that in the wild readily oviposit into oranges. The small cup was then mounted on an inverted medium cup (secured with double-sided tape) following preliminary trials, as it was found that Qfly could locate the oviposition cup more readily when it was in their flight path than when on the floor. This setup was also more stable standing on the flexible floor of the flight cages.

Charcoal filter paper: FILTER-LAB filter paper with active carbon. Mixture of noble cellulose and active carbon. Filtration: Slow. Weight in grams g/m2: 155. Diameter: 90mm. Filtros Anoia, S.A., Cami de Baix, s/n, 08776 Sant Pere de Riudebitlles (Barcelona), Spain.

Blu Tack: Blu Tack, Bostik Australia Pty.Ltd. 52-72 High Street, Thomastown, Victoria 3074, Australia. 60

Medium Cups: Medium cup Dimensions: Height 100mm, Top Diameter 75mm, Base Diameter 48mm. Genfac Plastics PTY. LTD., Melbourne, Australia.

Large Cups: Large cup Dimensions: Height 110mm, Top Diameter 115mm, Base Diameter 84mm. Bonson Industrial Co. LTD. Auckland, New Zealand.

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Appendix III

Data Summary Tables Table 7 Eggs to second instar Q-fly emergence Dose (Gy) 0.0 4.7 9.1 15.9 27.6 47.0 79.9 Total Female Flies Male Flies Total Flies Pupae Exp Unexp Total Exp Unexp Total Exp Unexp Total 569 134 132 266 116 126 242 250 258 508 552 69 127 196 53 99 152 122 226 348 267 16 30 46 17 26 43 33 56 89 159 0 0 0 0 0 0 0 0 0 102 0 0 0 0 0 0 0 0 0 58 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 1719 219 289 508 186 251 437 405 540 945

Table 8 Eggs to third instar Q-fly emergence Dose (Gy) Pupae 0.0 547 4.7 509 9.1 312 15.9 253 27.6 118 47.0 78 79.9 10 Total 1827 Female Flies Male Flies Total Flies Exp Unexp Total Expo Unexp Total Expos Unexp Total 57 93 150 61 107 168 118 200 318 27 68 95 33 69 102 60 137 197 8 4 12 13 4 17 21 8 29 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 92 165 257 107 180 287 199 345 544

Table 9 Eggs to second instar mean Q-fly longevity Dose (Gy) 0.0 4.7 9.1 Total Females Survivorship (days) Males Survivorship (days) Exposed Unexposed Total Exposed Unexposed Total Grand Total 37.07 45.55 41.91 44.12 46.60 45.46 43.74 27.73 32.86 30.72 36.33 38.71 37.72 33.85 10.20 7.89 8.71 8.00 13.88 12.70 10.38 29.23 33.44 31.71 38.77 37.80 38.20 34.77

Table 10 Eggs to third instar mean Q-fly longevity Dose (Gy) 0.0 4.7 9.1 Total Females Survivorship (days) Males Survivorship (days) Exposed Unexposed Total Exposed Unexposed Total Grand Total 40.93 42.50 41.81 43.81 45.67 44.86 43.45 33.75 32.37 32.61 34.71 36.92 36.11 34.19 11.00 1.50 6.25 1.50 3.00 2.00 4.43 36.50 35.46 35.81 37.88 41.32 39.86 37.84 62

Table 11 - Eggs to second instar wasp emergence Dose (Gy) 0.0 4.7 9.1 15.9 27.6 47.0 79.9 Total Pupae 286 213 141 70 42 27 4 783 Females 2 7 3 0 0 0 0 12 Males 3 6 3 0 0 0 0 12 Total 5 13 6 0 0 0 0 24

Table 12 - Eggs to third instar wasp emergence Dose (Gy) 0.0 4.7 9.1 15.9 27.6 47.0 79.9 Total Pupae 287 205 181 146 58 44 2 923 Females 12 2 6 5 0 0 0 25 Males 15 13 3 6 0 0 0 37 Total 27 15 9 11 0 0 0 62

Table 13 - Eggs to second instar mean wasp survivorship Dose (Gy) 0.0 4.7 9.1 Total Female Survivorship (days) Male Survivorship (days) Total Survivorship (days) 18.00 30.33 25.40 18.00 29.17 23.15 15.67 22.00 18.83 17.42 27.67 22.54

Table 14 - Eggs to third instar mean wasp survivorship Dose (Gy) 0.0 4.7 9.1 15.9 Total Female Survivorship (days) Male Survivorship (days) Total Survivorship (days) 10.00 10.53 10.30 6.50 16.77 15.40 10.00 17.33 12.44 13.20 13.67 13.45 10.36 13.78 12.40

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Table 15 Eggs to second instar - successful parasitism by grouped D. kraussii Dose (Gy) 0.0 4.7 9.1 Total Mean female Mean Male Mean total Total wasp- Mean offspring per offspring offspring offspring days wasp-day 35.00 37.00 72.00 26.00 2.77 51.00 60.50 111.50 98.00 2.24 33.50 27.50 61.00 40.00 3.18 40.80 42.60 83.40 164.00 2.72

Table 16 Eggs to third instar - successful parasitism by grouped D. kraussii Dose (Gy) 0.0 4.7 9.1 15.9 Total Mean female Mean Male Mean total Total wasp- Mean offspring per offspring offspring offspring days wasp-day 0.80 1.20 2.00 69.00 0.08 0.00 0.00 0.00 4.00 0.00 1.00 0.00 1.00 33.00 0.06 0.25 0.00 0.25 42.00 0.02 0.62 0.46 1.08 148.00 0.05

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