Effects on Attraction, Feeding and Mortality of tryoni (Froggatt) (Diptera:) and Beneficial Organisms with Protein Bait- Mixtures

Author Mahat, Kiran

Published 2009

Thesis Type Thesis (Masters)

School Griffith School of Environment

DOI https://doi.org/10.25904/1912/3805

Copyright Statement The author owns the copyright in this thesis, unless stated otherwise.

Downloaded from http://hdl.handle.net/10072/367286

Griffith Research Online https://research-repository.griffith.edu.au

Effects on attraction, feeding and mortality of Bactrocera tryoni (Froggatt)(Diptera:Tephritidae) and beneficial organisms with protein bait-insecticide mixtures

Kiran Mahat BSc (Horticulture) Dr Y.S. Parmar University

Griffith School of Environment Science, Environment, Engineering and Technology Griffith University

Submitted in fulfillment of the requirements of the degree of Master of Philosophy

June 2009

Abstract

ABSTRACT

This thesis examines the effects of malathion, chlorpyrifos, fipronil and spinosad mixed in protein bait on attraction, feeding and mortality of the Queensland fruit fly, Bactrocera tryoni (Froggatt). The effects of weathering of the protein bait- insecticide mixtures on the mortality of B. tryoni were also measured along with attraction, feeding response and toxicity of the protein bait sprays on important natural enemies particularly the red scale parasitic wasps, Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard), the green lace wing, Chrysoperla carnea (Stephens) and the mealy bug predator, Cryptolaemus montrouzieri (Mulsant).

In field cage experiments, protein-starved male B. tryoni showed the same level of attraction to protein baits mixed with malathion, chlorpyrifos, fipronil, spinosad and protein alone used as the control. However, protein-starved females elicited a difference in attraction with protein baits containing chlorpyrifos and spinosad. Traps with spinosad bait mixtures captured significantly more females compared to traps containing chlorpyrifos bait mixtures. Laboratory feeding experiments on protein-starved female demonstrated that baits containing malathion and chlorpyrifos deterred flies from feeding on them. In contrast, no such deterrence was detected with baits containing spinosad, fipronil and protein alone. These results demonstrated that the type of toxicant mixed with protein bait sprays can influence the attraction and feeding responses of B. tryoni. Therefore the process of screening toxicants for use in protein bait mixtures is important and should entail field and laboratory tests.

In terms of toxicity, protein baits mixed with malathion and chlorpyrifos caused significantly higher fly mortality and demonstrated a more rapid fly knock down than did spinosad, fipronil and protein alone as the control. Spinosad however was a slow acting toxicant, causing a gradual increase in fly mortality over time.

II Abstract

Fly mortality obtained with protein bait mixtures containing malathion, chlorpyrifos and fipronil, applied on citrus leaves, and weathered out doors for up to 6 days did not vary significantly from freshly applied baits. However, the residual effectiveness of bait mixed with spinosad waned significantly after 3 days of out door weathering. Fly mortality caused by 3 day aged spinosad bait mixture was significantly lower than fresh bait mixtures, suggesting a rapid break down of spinosad under field conditions.

The parasitism rates of the two most important parasitoids of red scales, A. lingnanensis and C. bifasciata, measured before and after commencement of fruit fly bait spraying in two commercial citrus orchards, did not show any significant negative trend. Aphytis were not attracted to Pinnacle protein, the most commonly used fruit fly protein lure in . A significantly higher number of Aphytis were attracted to honey solution and protein bait mixed with 20% sugar, compared to protein bait alone. A no-choice test further confirmed this result, demonstrating no difference in attraction between protein and water.

Protein bait containing malathion, chlorpyrifos, fipronil, and spinosad, fresh and weathered for up to 12 days, caused high mortality in Aphytis. In contrast protein bait mixed with spinosad caused a lower Aphytis mortality after 12 days out door weathering, compared to chlorpyrifos bait mixture. However, for other weathering periods, no such differences in mortality between the treatments were observed. In addition, the parasitizing capacity of Aphytis, after being exposed to these weathered residues, was reduced. Except for the control, aged bait mixtures significantly reduced the fecundity of Aphytis. Therefore, while integrating chemical based field control along with bio-control agents, appropriate measures should be in place to reduce the negative impacts of toxic residues.

Overall, the findings from this study indicate that spinosad is a suitable alternative to the older toxicants, for incorporation into fruit fly protein baits. Moreover, fruit fly protein baits in the field are less likely to disrupt the activites of important natural enemies. However, chemical-based control of pests in a cropping system

III Abstract should be designed carefully with the objective to prevent potential harm to susceptible biological control agents such as Aphytis.

IV Acknowledgements

ACKNOWLEDGEMENTS

Many people have, directly or indirectly, helped me complete my thesis and therefore deserve a special mention here.

Firstly, this thesis would not have been possible without the support of my supervisors, Professor Richard Drew and Dr Vijay Shanmugam. I remain ever greatful to my principal supervisor, Professor Richard Drew for his advice, guidance and encouragement. I thank him for devoting his precious time going through this thesis and all the constructive comments. Your positiveness always motivated me and has been one of the driving forces which helped me complete this course of study. I thank my associate supervisor, Dr Vijay Shanmugam for his advice, guidance and encouragement. I remain greatful for all the critical comments and the discussions we have had that contributed a lot in my learning process.

I would like to express my gratitude to all the staff in the International Center for the management of Pest Fruit Flies. Cameron Drew for the great company and assistance provided to carry out the field cage trials and accompanying me to Mundubbera. Peter Halcoop for helping me rear large number of flies and maintaining the laboratory culture of flies. Meredith Romig and Sarah Romig for their assistance in various aspects related to my work.

I would like to acknowledge the help of Janet Chaseling for her assistance and advice provided in my statistical analysis and Narit Taochan for introducing and initially helping me pick up the techniques involved in biostatistical analysis.

I greatfully acklowledge Dan Papacek for his generous help and support in the field work in Mundubbera and supplying me with the beneficial used in this study. Wes Allen has been prompt in packing and dispatching the insects required for the experiments, and I thank him for that. I also acklowledge Ed Carlton and Jenny Grigg for their help in assisting me to learn the technique involved in determining Aphytis

V Acknowledgements

parasitism in red scale on citrus fruit samples. I am greatful and thank Allen Jenkin and Craig Wallis for their support and corporation in allowing me collect large numbers of citrus from their orchards for the Aphytis sampling work. I would also like to thank Paul Thorn for allowing me to use his farm for the field cage trials.

Many thanks to Richard Bull for his support in a number of ways such as supplying fipronil, providing the ground sheets and helping me fix the field cages. I also thank Don Dennis and Bruce Mudway for their help in providing various materials for my experiments and also Lynita Howie for helping me with some materials needed for my experiments.

I am very greatful for the initial support from Professor Richard Drew in helping me secure the JohnAllwright fellowship from the Australian Centre for the International Agricultural Research (ACIAR). I owe my deepest gratitude to ACIAR for funding my fellowship. The Support and generosity from ACIAR is very much appreciated. I would also like to sincerely thank the Ministry of Agriculture, Bhutan for granting me approval for this study and assisting me in times of need.

Finally, I thank my Dad and Mom for always being on my side. Your unconditional love and guidance shaped my life and made me the person I am today. It is hard to express my gratitude to my wife, Bhawana, for accompanying me through every step of this journey and for her constant love, support and sacrifice.

VI Table of contents

TABLE OF CONTENTS

ABSTRACT ...... II ACKNOWLEDGEMENTS ...... V TABLE OF CONTENTS ...... VII LIST OF FIGURES ...... XI LIST OF TABLES ...... XIV STATEMENT OF ORIGINALITY ...... XVII

CHAPTER 1: GENERAL INTRODUCTION AND LITERATURE REVIEW ...... 1

1.1 GENERAL INTRODUCTION ...... 2 1.2 Biology of the Queensland fruit fly ...... 4 1.3 Fruit fly field management strategies ...... 5 1.3.1 Physical control ...... 6 1.3.2 Cultural control ...... 7 1.3.3 Biological control ...... 7 1.3.4 (SIT) ...... 9 1.3.5 Male annihilation technique (MAT) ...... 10 1.3.6 Chemical control ...... 12 1.3.7 Fruit fly control using cover sprays ...... 16 1.3.8 Attraction of fruit fly to protein ...... 17 1.3.9 Overview of fruit fly attractant bait development and use ...... 19 1.3.10 protein bait in fruit fly control ...... 22 1.4 Concerns associated with the use of malathion in protein bait spray and the development of alternative toxicants ...... 24 1.5 Fruit fly protein bait use and its effect on arthropod natural enemies ...... 26 1.6 Beneficial organism- The study species and its food source ...... 28 1.7 Thesis outline ...... 30

VII Table of contents

CHAPTER: 2 GENERAL METHODS AND MATERIALS ...... 33

2.1 GENERAL METHODS AND MATERIALS ...... 34 2.1.1 Fruit fly culture room ...... 34 2.2 Fruit fly rearing ...... 34 2.2.1 Laboratory stock colony of adult flies ...... 34 2.2.2 Fruit fly egg collection ...... 35 2.2.3 Larval rearing ...... 35 2.2.4 Adult flies for the experiments ...... 36 2.3 Experimental room ...... 36 2.4 Protein bait and ...... 36 2.5 Statistical analysis ...... 37

CHAPTER 3: Attraction and feeding responses of Bactrocera tryoni (Froggatt) to different combinations of protein bait and insecticides under laboratory and field conditions ...... 39

3.1 Introduction ...... 40 3.2 Methods and Materials ...... 44 3.2.1 Experiment 1: Field cage Attraction ...... 44 3.2.2 Experiment 2: Laboratory Feeding study ...... 46 3.2.3 Statistical analysis ...... 47 3.3 Results ...... 48 3.3.1 Field cage attraction ...... 48 3.3.2 Laboratory feeding experiment ...... 49 3.4 Discussion ...... 49

Chapter 4: Toxicity and impact of ageing of protein bait-insecticide mixtures on Bactrocera tryoni (Froggatt) ...... 55

4.1 Introduction ...... 56 4.2 Materials and methods ...... 59 4.2.1 Toxicology experiment ...... 59 4.2.2 Effects of aging of protein bait mixtures containing different insecticides on fly mortality ...... 60

VIII Table of contents

4.3 Statistical analysis ...... 62 4.4 Results ...... 62 4.4.1 Toxicology experiment ...... 62 4.4.2 Effects of weathering of protein bait mixtures containing different insecticides on fly mortality ...... 65 4.5 Discussion ...... 70

Chapter 5: Impact of protein bait sprays on parasitoids of red scale, Aonidiella aurantii (Maskell) (Homoptera:Diaspididae) and laboratory evaluation of attraction and feeding of beneficial insects on protein baits 75

5.1 Introduction ...... 76 5.2 Materials and Methods ...... 79 5.2.1 Experiment 1: Impact of protein bait sprays on A. lingnanensis and C. montrouzieri ...... 79 5.2.2 Experiment 2: Attraction of A. lingnanensis to protein bait, protein +sugar (20%), water and honey ...... 82 5.2.3 Experiment 3: Feeding propensity of green lace wings, C. carnea and the mealy bug predator, C. montrouzieri on protein bait, water and honey ...... 84 5.3 Statistical analysis ...... 85 5.3.1 Experiment 1: Impact of protein bait sprays on A. lingnanensis and C. bifasciata ...... 85 5.3.2 Experiment 2: Attraction of A. lingnanensis to protein bait ...... 86 5.3.3 Experiment 3: Feeding propensity of C. montrouzieri and C. carnea on protein bait ...... 86 5.4 Results ...... 86 5.4.1 Experiment 1: Impact of protein bait sprays on A. lingnanensis ...... 86 5.4.2 Experiment 2: Attraction of A. lingnanensis to protein bait, protein + sugar (20%), water and honey ...... 91 5.4.3 Experiment 3: Feeding propensity of C. montrouzieri and C. carnea on protein bait ...... 92 5.5 Discussion ...... 94

IX Table of contents

Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on the red scale parasite, Aphytis lingnanensis (Compere) (Hymenoptera:Aphelinidae) ...... 101

6.1 Introduction ...... 102 6.2 Materials and Methods ...... 104 6.2.1 Test Insects...... 104 6.2.2 Protein bait-insecticides application ...... 104 6.2.3 Residual toxicity of protein bait-insecticide mixtures to adult Aphytis lingnanensis ...... 105 6.2.4 Effect of protein bait-insecticide exposure on the fecundity of Aphytis lingnanensis ...... 107 6.3 Statistical analysis ...... 108 6.4 Results ...... 108 6.4.1 Residual toxicity of protein bait-insecticide mixtures to adult Aphytis lingnanensis ...... 108 6.4.2 Effect of protein bait-insecticide exposure on the fecundity of Aphytis lingnanensis ...... 110 6.5 Discussion ...... 112

CHAPTER 7: GENERAL DISCUSSION ...... 119

7.1 GENERAL DISCUSSION...... 119 7.2 Future research ...... 123

REFERENCES ...... 125

APPENDICES………………………………………………………………………………………….…158

X List of figures

LIST OF FIGURES

Figure 1.1 Schematic representation of the life cycle of Bactrocera tryoni (Froggatt) and potential intervention points for control procedures with minimum dependence on insecticides 5

Figure 3.1 Steiner trap fitted with paper cones containing protein bait- insecticide mixture in a beaker 42

Figure 3.2 Steiner trap suspended around the perimeter of the tree canopy with a flexible copper wire 43

Figure 4.1 Comparison of percent mortality (mean +s.e.) of Bactrocera tryoni (Froggatt) between leaves aged for 2-hr, 3-d and 6-d with protein bait containing chlorpyrifos, malathion, spinosad and fipronil after 72-hr exposure period 65

Figure 4.2 Maximum and minimum temperature recorded during the experiment days 66

Figure 4.3 Average relative humidity (%) recorded during the experiment days 66

Figure 5.1 Schematic design of the feeding apparatus. Treatment placed at one end on a sponge. Insects walk up the apparatus and feed. Once feeding commenced, actual feeding time was recorded 82

Figure.5.2 Percent parasitism of red scales, Aonidiella aurantii (Maskell) by Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) over four sampling period between January and April 2008 in Nova variety in the Auburnvale citrus orchard 85

Figure.5.3 Percent parasitism of red scales, Aonidiella aurantii (Maskell) by Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) over four sampling period between January and April 2008 in Nova variety in the Iron bark citrus orchard 85

XI List of figures

Figure 5.4 Number of red scales, Aonidiella aurantii (Maskell) parasitised by Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) and their emergence out of 200 scales sampled over four periods (between January to April 2008) in the Auburnvale citrus orchard 86

Figure 5.5 Number of red scales, Aonidiella aurantii (Maskell) parasitised by Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard et al.) and their emergence out of 200 scales sampled over four periods (between January to April 2008) in the Iron bark citrus orchard 86

Figure 5.6 Number of unparasitised red scales, Aonidiella aurantii (Maskell) including live unmated scales, mated scales and dead scales recorded over four sampling periods between January to April 2008 in the Nova variety in the Auburnvale citrus orchard 87

Figure 5.7 Number of unparasitised red scales, Aonidiella aurantii (Maskell) including live unmated scales, mated scales and dead scales recorded over four sampling periods in the Nova variety of the Iron bark citrus orchard 87

Figure 6.1 A schematic design of the exposure unit used to test the residual toxicity of protein bait-insecticide mixtures to adult Aphytis lingnanensis ( compere). The unit was made of a transparent plastic tube (2cm diameter: 4cm height) with four holes (0.5cm diameter) (two used for ventilation and two for Feeding honey + water). Treated leaves were placed on glass slides and exposed to Aphytis lingnanensis (compere)by placing on the top and bottom of the tube. (a) Components of the exposure units. (b) An assembled exposure unit 103

Figure 6.2 Maximum and minimum temperature recorded during the experiment days 108

Figure 6.3 Average relative humidity (%) recorded during the experiment days 108

XII List of tables

LIST OF TABLES

Table 1.1 Major class of insecticides and their discovery dates 13

Table 1.2 Overview of fruit fly bait use and its development 19

Table 2.1 Larval diet 35

Table 2.2 Insecticides used in protein bait mixtures 35

Table 3.1 Mean ± s.e. number of female Bactrocera tryoni (Froggatt) captured in Steiner type fruit fly traps with different protein bait- insecticide mixtures 46

Table 3.2 Mean ± s.e. number of male Bactrocera tryoni (Froggatt) captured in Steiner type fruit fly traps with different protein bait- insecticide mixtures 47

Table 3.3 Feeding time (mean ± s.e.) obtained with female Bactrocera tryon (Froggatt) on protein bait-insecticide mixtures 47

Table 4.1 Percent mortality (mean ± s.e.) of Bactrocera tryoni (Froggatt) induced by different protein bait-insecticide mixtures after 24- hr exposure to flies 60

Table 4.2 Percent mortality (mean ± s.e.) of Bactrocera tryoni (Froggatt) induced by different protein bait-insecticide mixtures after 48- hr exposure to flies 60

Table 4.3 Percent mortality (mean ± s.e.) of Bactrocera tryoni (Froggatt) induced by different protein bait-insecticide mixtures after 72- hr exposure to flies 61

XIV List of tables

Table 4.4 Percent knockdown of Bactrocera tryoni (Froggatt) induced by different protein bait- insecticide mixtures after 1-hr, 2-hr and 3-hr period exposure to flies 61

Table 4.5 Percent mortality (mean ± s.e) of Bactrocera tryoni (Froggatt) adults observed after 24 hours exposure to leaves with different protein bait-insecticide mixtures weathered for 2-hr, 3-d and 6-d 63

Table 4.6 Percent mortality (mean ± s.e) of Bactrocera tryoni (Froggatt) adults observed after 72 hours exposure to leaves with different protein bait-insecticide mixtures weathered for 2-hr, 3-d and 6-d 64

Table 5.1 Number of red scales, Aonidiella aurantii (Maskell) parasitised and unparasitised by both Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) over four sampling periods between January and April 2008 in the Nova citrus variety in the Auburnvale orchard 84

Table 5.2 Numbers of red scales, Aonidiella aurantii (Maskell) parasitised and unparasitised by both Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) over four sampling periods between January and April 2008 in the Nova citrus variety in the Iron bark orchard 84

Table 5.3 Mean ± s.e number of by Aphytis lingnanensis (Compere) attracted to honey, protein and water used as a control 88

Table 5.4 Mean ±s.e number of by Aphytis lingnanensis (Compere) attracted to honey, protein + sugar (20%) and water used as a control 89

Table 5.5 Mean±s.e number of by Aphytis lingnanensis (Compere) attracted to honey and water used as a Control 89

Table 5.6 Feeding times of adult mealy bug predator, Cryptolaemus montrouzieri (Mulsant) on honey, protein and water under laboratory conditions 90

XV List of tables

Table 5.7 Feeding times of adult green lace wing, Chrysoperla carnea (Stephen) on honey, protein and water under laboratory conditions 90

Table 6.1 Percent mortality (mean ± s.e) of Aphytis lingnanensis (Compere) adults observed after 24-h exposure to citrus leaves with different protein bait-insecticide mixtures weathered for 2-hr, 3-d and 7-d and 12-d 106

Table 6.2 Percent parasitism inflicted by surviving adult Aphytis lingnanensis (Compere) on Oleander scales, Aspidiotus nerii (Boucher) on butternut pumpkins after being exposed to protein bait-insecticide residues for 24-h 107

XVI Statement of originality

STATEMENT OF ORIGINALITY

This work has not been previously submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

………………… Kiran Mahat June 2009

XVII Chapter 1: General introduction and literature review

CHAPTER 1: GENERAL INTRODUCTION AND LITERATURE REVIEW

1 Chapter 1: General introduction and literature review

1.1 GENERAL INTRODUCTION

Fruit flies (Diptera: Tephritidae) are pests of global importance and are widely distributed through the tropical, subtropical and temperate regions (Christenson and Foote 1960). Among agricultural pests of economic importance in the world, fruit flies traditionally occupy an important position (Aluja and Liedo 1993). Dacine fruit flies, a major sub family under Tephritidae, are one of the most economically important groups within the order Diptera (Fletcher 1987).

From a total of approximately 4500 described species of Tephritidae, 70 are considered destructive agricultural pests causing serious damage through losses in fruit and vegetable crops in many parts of the world (Edwards 1961, Bateman 1972, Fletcher 1987, Hardy and Foote 1989, White and Elson-Harris 1992). In tropical and sub tropical regions, the most damaging fruit fly species belong to the genera Bactrocera, Anastrepha, Ceratitis, Dacus and Rhagoletis (White and Elson-Harris 1992).

Apart from causing direct yield losses, fruit flies cause major economic impacts especially through quarantine and regulatory programmes, costly survey and field control strategies, eradication programs, disinfestation treatments and the prevention of the development of desirable food crops (Christenson and Foote 1960, Bateman 1991, White and Elson-Harris 1992).

From about 80 native fruit fly species in Australian, the Horticultural Policy Council (HPC) has identified six fruit fly species as important horticultural pests (Drew 1989a, HPC 1991). They are Bactrocera tryoni (Froggatt) Bactrocera neohumeralis (Hardy), Bactrocera cucumis (French), Bactrocera musae (Tryon), Bactrocera jarvisi (Tryon) and Bactrocera aquilonis (May). However, the most important and destructive species is the Queensland fruit fly, B. tryoni (Bateman 1991). The Queensland fruit fly is distributed from Cape York in the north to Melbourne in the south and Darwin to the west (Osborne et al. 1997) and is prominent throughout Costal and sub-costal districts of Eastern Australia, where it is the most important fruit fly pest species of

2 Chapter 1: General introduction and literature review

fruit and vegetables (May 1958a, Drew 1989b). Apart from Australia, it has also been recorded in , the Society Group of Islands, Easter Island, the Austral Islands and Torres Strait Islands. A record from Papua New Guinea (Drew 1989a) was probably an introduction that has never become established.

B. tryoni is highly polyphagous and attacks a wide range of vegetable crops, most cultivated tropical, subtropical and temperate fruits, with the exception of Pineapples and some strawberry varieties (May 1958a, Edwards 1961, Fletcher 1987). It has also been recorded from around 60 wild hosts which help them build up large populations in forest areas and major urban environments that act as reservoirs from which cultivated crops are invaded (Botha et al. 2000). Management costs in Australia for B. tryoni are estimated to reach US$ 25.7–49.9 million annually, resulting in it being classified as one of the most costly horticultural pests (Sutherst et al. 2000).

Several strategies are employed to control fruit flies. One of the most commonly used techniques is the use of protein bait sprays. This method involves the use of a mixture of protein bait plus an insecticide which is applied as a spot of approximately 50ml per tree. This method reduces markedly the amount of insecticide sprayed into the environment compared to cover spray applications (Prokopy et al. 2003). Currently the need to replace the established insecticide, malathion, in fruit fly bait has resulted in a number of studies evaluating new compounds which have a relatively safe profile, suitable for controlling fruit flies in conjunction with other field control strategies such as biological control. For instance, several field trials have indicated promising results for compounds like spinosad and the phototoxic dye, phloxine B, that were evaluated for controlling various pest fruit flies (McQuate et al. 1999, Peck and McQuate 2000, Burns et al. 2001, Moreno et al. 2001, Vargas et al. 2001, McQuate et al. 2005b). However, such studies have been lacking for the Queensland fruit fly. Hence, this thesis presents research designed to evaluate compounds that may serve as potential alternatives to older insecticides in the baits and provide data to improve our understanding of

3 Chapter 1: General introduction and literature review

the suitability of protein bait sprays used in association with important biological control agents.

The following section reviews the general biology of the Queensland fruit fly and delves into topics related to fruit fly control.

1.2 Biology of the Queensland fruit fly

Almost all Dacinae, a major sub family of Tephritidae, have a similar life cycle. Under favourable conditions the development of adult B. tryoni from eggs usually takes about 3-4 weeks. Females pierce the skin of the ripening host fruit with the ovipositor and eggs are laid directly under the skin. Eggs are white, banana shaped and after 42 hours (at 25°C) larvae hatch and tunnel through the fruit pulp (Bateman 1967, Dominiak 2007). Fruit damage results from both larval feeding and secondary infections by microorganisms (Heather and Corcoran 1985).

After 7-10 days, depending on the surrounding temperature, mature larvae leave the fruit to pupate within the top 2-3-cm of the soil (Bateman 1972, Meats 1981, Fletcher 1987). Puparial development (at 25 °C) takes about 12 days, after which teneral adults emerge and become sexually mature after 8-10 days (Meats 1981). B. tryoni is an “r-selected” species with a short generation time and high fecundity, and females are able to lay as many as 100 eggs per week over several months (Bateman 1972, Bateman 1977). Adult females can live up to 7 months (Bateman 1977). B. tryoni is a multi-voltine species and depending on the surrounding temperature can have as many as 8 or more overlapping generations per year in warmer areas like North Queensland and around 4 in the Sydney region (Meats 1981, Fletcher and Bateman 1982).

Like all dacine flies, adult B. tryoni require water and carbohydrate for survival and females require a source of protein for egg maturation (Christenson and Foote 1960, Bateman 1972). Mating occurs at dusk and females usually mate once which is

4 Chapter 1: General introduction and literature review

sufficient to produce fertilized eggs for the first 7 weeks of their adult life (Barton- Browne 1957 , Tychsen and Fletcher 1971). It overwinters as adults and then starts to breed as temperatures begin to rise over spring (Bateman 1967, Bateman and Sonleitner 1967, Bateman 1968).

1.3 Fruit fly field management strategies

As Fruit flies are one of the major and most destructive pest insects in food crops world wide, the development of effective field control strategies has been given top priority.

Adult emergence

Dispersal Population flushing (SIT) Control using protein

Protein feeding bait sprays

Over wintering Maturation Male annihilation technique (MAT)

Mating Sterile Insect Technique (SIT) Host seeking Visual cues Dispersal Physical protection (eg.nets) Oviposition

Egg Biological control

Larva

Pupa

Figure 1.1 Schematic representation of the life cycle of Bactrocera tryoni (Froggatt) and potential intervention points for control procedures with minimum dependence on insecticides (Fletcher and Bateman 1982).

5 Chapter 1: General introduction and literature review

From 1960 and 1970 there was a major paradigm shift in insect pest control that focused more on integrated pest management control rather than relying solely on which can induce pest resistance, resurgence, and a rise of minor pests to major pest status(Hoyt and Burts 1974). By definition, IPM is a “decision support system for the selection and use of pest control tactics, singly or harmoniously coordinated into a management strategy, based on cost/benefit analyses that take into account the interests of and impacts on producers, society, and the environment” (Kogan 1998). IPM in general involves the use of a combination of techniques such as resistant plants varieties, cultural practices, the use of predators and parasites, microbial pesticides such as entomopathogenic bacteria, viruses and fungi, botanical insecticides, chemical insecticides, insect growth regulators and semiochemicals.

In fruit fly control, major emphasis has been placed on the development of management techniques that fit well with the concepts of Integrated Pest Management (IPM) (Allwood 1997). Fruit fly control can encompasses fruit bagging/wrapping, cultural control, use of natural enemies, sterile insect technique (SIT), chemical control employing insecticides, male annihilation technique (MAT) and protein bait sprays, or a combination of some of these to align with the principles of an IPM approach (Vijaysegaran 1994 , Allwood 1997). The strategies employed in fruit fly pest control are discussed below.

1.3.1 Physical control

The main method of physical control is fruit bagging or wrapping which prevents fruit flies from ovipositing into the fruit. Fruits are normally wrapped with paper at an early stage before becoming susceptible to fruit fly attack. This strategy is common in Asian countries, but is labour intensive (Vijaysegaran 1985b, 1994 ). It can be used effectively for fruits such as mango, carambola, guava and some gourds with stems around which the bags can be closed, but is not easily applied to fruits like papaya, citrus and sapodilla (Vijaysegran 1997). Fruit bagging has been reported

6 Chapter 1: General introduction and literature review

to be commonly used in carambola in Malaysia, mangoes in Philippines and Thailand and in melon in Taiwan (Hapitan and Castillo 1976, Vijaysegaran 1985b, Vijaysegaran 1989, Cheng and Lee 1991, Lin 2005). Another physical control technique includes netting (Fletcher and Bateman 1982). It is a system of enclosing individual trees or entire orchards with a fine mesh netting that excludes fruit flies. However, netting requires expensive infrastructure and is susceptible to the impacts of weather.

1.3.2 Cultural control

This method involves the collection and destruction of fallen, infested fruits which would otherwise provide the next generation of breeding flies. While it can contribute to a reduction in the fruit fly population, to be effective it needs to be implemented over a large geographic area and would need government and legal support (Allwood 1997). For instance, in China collection and destruction of 8 million host fruits of Bactrocera citri (now Bactrocera minax Enderlein) in Jangjen country, Sichauan province brought down the infestation levels from 25% to 0.5% in the next growing season (Yang 1991). Similarly, in another province destruction of 17 million infested fruits brought down the infestation levels from 80% to 5% within two years (Yang 1991). Collection and destruction of fallen fruits once every 10 days has also been recommend as one of the major control strategies in controlling the Chinese fruit fly, B. minax in citrus orchards in Bhutan (Dorji et al. 2006). Other cultural control strategies involve growing less susceptible fruit varieties, early harvesting and avoiding growing crops during seasons of peak fruit fly activity (Allwood 1997).

1.3.3 Biological control

Fruit flies are attacked by some predators and a variety of parasitoids under the order Hymenoptera. In fruit fly biological control programmes, more emphasis has been given to studies involving parasitoids with less attention to predators (Gingrich 1993).

7 Chapter 1: General introduction and literature review

Most introduced fruit fly parasitoids are solitary endoparasitoids belonging to the family Braconidae, subfamily Opiinae (Purcell 1998). Other parasitoids belong to the families Chalcididae , Pteromalidae and Eulophidae while predators include ants, spiders, carabid beetles, assassin bugs, staphylinid beetles and lygaeid bugs (Bateman 1972, Allwood 1997). These natural agents, to some extent, help reduce fruit fly populations. However, in terms of effectiveness, biological control alone is not known to provide the required level of economic control against fruit flies and tropical fruit flies in particular, with their extremely large populations, are not known to be good candidates for biological control (Bateman 1972, Waterhouse 1993, Vijaysegaran 1994).

The first fruit fly biological control programme was started by the Australian government in 1902 (Wharton 1989). However, the first large classical biological control program took place in Hawaii after the introduction of the Oriental fruit fly, Bactrocera dorsalis (Hendel) where a total of 32 natural enemies were released between 1947 and 1952 (Bess et al. 1950, Bess et al. 1961, Clausen 1965). Some of these natural enemies did establish successfully under the Hawaiian climatic conditions. There was spectacular success against the two introduced species, Mediterranean fruit fly, (Wiedemann) and the Oriental fruit fly, B. dorsalis (Hendel) but not against the introduced melon fly, Bactrocera cucurbitae (Coquillett) (Waterhouse 1993). Diachasmimorpha longicaudata (Ashmead) and Fopius arisanus (Sonan) were among the released parasitoids, but F. arisanus later became the dominant parasitoid species (van den Bosch and Haramoto 1953). F. arisanus is now one of the most important natural enemies of B. dorsalis and C. capitata in Hawaii (Haramoto and Bess 1970, Vargas et al. 2001). Among opiine tephritid biological control agents, D. longicaudata is one of the most widely used in biological control programmes (Ovruski et al. 2000).

As documented by Purcell (1998), biological control of fruit fly pests using parasitoids has been successful in Hawaii, Florida, Fiji and Southern Europe, but in Australia, Mexico and Costa Rica there has been relatively limited success.

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Another important strategy is the integration of biological control with environmentally safe chemical control and this can provide effective pest management with minimum disruption to natural enemies. For instance, compounds like spinosad and phloxine B have been suggested as being compatible with natural enemies like F. arisanus and Pysttalia fletcheri (Silvestri) for use in area wide management of C. capitata (Vargas et al. 2001, Stark et al. 2004b).

1.3.4 Sterile Insect Technique (SIT)

Knipling (1955) conceptualized the idea of suppressing and/or eradicating pest populations of insects through the mass release of sterile insects. The sterile insect technique (SIT) involves rearing and exposing large numbers of male flies to gamma rays at a level that induces sexual sterility without affecting viability when released into wild populations of the same species. The sterile males mate with field females which then produce infertile eggs (Knipling 1955, Klassen and Curtis 2005, Robinson 2005). With the females failing to reproduce, the wild populations over time are reduced to levels where an entire population in an area can be eradicated. The first success with SIT was in eradicating the screw worm fly, Cochliomyia hominivorax (Coquerel) from the south-eastern region of the United States in the late 1950s (Knipling 1960).

SIT is normally deployed as part of an area-wide integrated pest management strategy aimed at suppressing, eradicating, or containing pest fruit flies. This technique is most effective when used as a component of an area-wide integrated pest management (AW-IPM) program by “mopping up” sparse pest populations (Klassen 2005). The first large–scale program on fruit flies was in 1970 which prevented the incursion of the Mediterranean fruit fly, C. capitata into southern Mexico from Central America (Hendrichs et al. 1983). It has been used successfully in eradicating the melon fly and the oriental fly in Okinawa, Japan after which Japan was declared free from these pests (Kawasaki 1991, Kuba et al. 1996, Koyama et al. 2004). In Australia, this technique has been successful in eradicating a large

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infestation of B. tryoni in Perth and C. capitata in Carnarvon in Western Australia (Fisher et al. 1985, Fisher 1996). Currently, the Tri-State Fruit Fly Committee, comprising the states of , Victoria and South Australia maintains a “Fruit Fly Exclusion Zone” using SIT (Jessup et al. 2007). These states support a production facility that supplies sterile male B. tryoni which are released into target areas (Jessup et al. 2007). In Western Australia, trials on SIT in conjunction with protein bait sprays have been successful in regional towns to control C. capitata (Jessup et al. 2007). Successful suppression programmes are currently being undertaken in Israel, South Africa and Thailand, while countries like Brazil, Portugal, Spain, and Tunisia are organizing similar programs (Klassen and Curtis 2005). However, the successful application of SIT necessitates the capacity to rear, sterilize and release large numbers of insects and requires sterile males with a capacity to mate with wild females and compete for mates with wild males (Lance and McInnis 2005). Hence, such programs require specialized training, a high degree technical support and huge financial investments.

1.3.5 Male annihilation technique (MAT)

Male dacine fruit flies are highly attracted to various chemical lures often referred to as parapheromones, although this phenomena remains a complex mystery of tephritid biology (Chambers 1977, Cunningham 1989a). Some of these synthetic lures were discovered serendipitously and are referred to as male lures because of their capacity to be highly attractive to males of various fruit fly species (Drew 1974, Cunningham 1989a, Jang and Light 1996). Male lures are often used with suitable traps for fruit fly detection in the scheduling of spray control programs, population assessment in ecological studies, quarantine surveys, suppression and eradication programs (Drew 1974, Drew and Hooper 1981).

Methy eugenol is one powerful male lure, first discovered as strongly attracting male oriental fruit flies, B. dorsalis and many other species of fruit flies (Steiner 1951, 1952a, Drew and Hooper 1981). Cue-lure attracts both male melon fly and the

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Queensland fruit fly and tri-medlure attracts the males of the Mediterranean fruit fly (Chambers 1977).

Often referred to as the male annihilation technique (MAT), this involves the use of a male lure mixed with a toxicant which is then impregnated into an absorbent material such as fiberboard squares or cotton wicks. It may be applied as spot sprays in urban areas or sprayed from aircraft over large agricultural areas (Asquith and Burny 1998), although this is not recommended as the solution is phytotoxic and will corrode painted surfaces of cars etc. When males are attracted to the mixture and killed, the chances of females finding a mate are very low. Eventually the entire breeding population within an area may be eliminated (Cunningham 1989b).

A formulation based on a 1:1 mixture of Malathion and male lure is commonly used in MAT programs (Bateman 1982). MAT can be used in combination with protein bait in Area-wide suppression of localized populations (Bateman 1982, Cunningham 1989b). This technique in conjunction with other fruit fly control strategies has been successfully employed in several fruit fly suppression and eradication programs. For example, the successful eradication of the oriental fruit fly from the Pacific islands of Rota (Marianas Islands), Saipan and Tinian, California and Japan (Steiner et al. 1965, Steiner et al. 1970, Chambers et al. 1974, Koyama et al. 1984, Kawasaki 1991). In Australia cue-lure is widely employed in detecting the most important fruit fly pest species B. tryoni (Drew 1982). B. tryoni was successfully eradicated from Easter Island, a Chilean territory in the Pacific, employing both cue-lure and protein bait sprays (Bateman and Arretz 1973). Similarly, in conjunction with other control measures, the male annihilation technique (Caneite blocks treated with malathion plus male lures) has been employed in the eradication of B. tryoni from Western Australia and the Asian papaya fruit fly, Bactrocera papayae Drew and Hancock from Northern parts of Australia (Lloyd et al. 1998, Jessup et al. 2007).

The male annihilation technique is considered a very environmentally friendly technique with no possible impact on non-target insects (Chambers et al. 1974, Vayssieres et al. 2007). However, novel compounds as potential replacements for

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broad spectrum organophosphate insecticides, have been evaluated. Compounds such as Spinosad, considered safe to handle and environmentally friendly, and fipronil, have been proposed as substitutes for organophosphate insecticides in methyl eugenol and cue-lure dispensers for area-wide suppression programs in Hawaii against B. dorsalis and B. cucurbitae (Vargas et al. 2003, Vargas et al. 2005, Vargas et al. 2008). Neonicotinoid compounds, such as imidacloprid and acetamiprid, have also been suggested as alternatives to broad spectrum insecticides for use against B. dorsalis in fly traps (Chuang and Hou 2008).

1.3.6 Chemical control

A brief history

The historical development of pest management has been exclusively reviewed by Norris et al. (2003). The first chemicals used against insects were inorganic sulfur, arsenic, lead arsenate, cryolite and boric acid, followed by botanicals such as pyrethrum and nicotine which were limited in availability and very expensive (Tschirley 1979, Ecobichon 1994, Casida and Quistad 1998). The two economically important natural insecticides, Derris and pyrethrum, were introduced in the mid- 1800s (Ecobichon 1994, 2001).

The first synthetic insecticide, dichlorodiphenyltrichloroethane (DDT), was discovered by Paul Muller in 1939 (Cremlyn 1978). It demonstrated powerful insecticide properties and together with other highly poisonous chemicals such as dieldrin, aldrin, endrin, heptachlor and lindane, was widely used with great success in agricultural insect pest control (Madden 1971, Evans 1998).

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Table 1.0.1 Major classes of insecticides and their discovery dates (Casida and Quistad 1998).

Class Chemical example and entry year

Chlorinated hydrocarbons DDT-1939, lindane-1942, chlordane-1945, toxaphene-1947, aldrin/dieldrin-1949, endosulfan- 1956

Organophosphates TEPP-1938, Parathion-1946, malathion-1952, diazinon-1953, chlorpyrifos-1965, chlorethoxyphos- 1986 Methylcarbamate Carbaryl-1957, carbofuran-1965, aldicarb-1965, alancarb-1984 Pyrethroid Allethrin-1949, resmethrin-1967, permethrin- 1973, deltamethrin-1974, lambdacyhalothrin-1984, silafluofen-1990 Other chemicals Fipronil-1992, Imidacloprid-1982, spinosad-1995 Biological Bacteria or baculovirus-1986

Starting in the early 1950s, it was realized that a single or one system control approach would fail to provide a sustainable and permanent answer to various crop protection problems (Brader 1979). Extensive use of Chlorinated hydrocarbons led to a devastating effect on human health, wild life and the environment. This was portrayed in Rachel Carson’s book Silent Spring (1962) where she strongly opposed and challenged the indiscriminate use of pesticides, stirring public sentiment against use. Shortly after these events, many developed countries banned the use of persistent chlorinated hydrocarbon compounds like DDT realizing its adverse effect on human health, non-target organisms and the environment (Metcalf 1973, Brooks 1974, Evans 1998, Gillette 2008). Therefore, the period 1960 -1970 saw more emphasis being placed on integrated control of insect pests (Hoyt and Burts 1974).

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After chlorinated hydrocarbons, the organophosphates (OPs), methylcarbamates (MCs) and pyrethroids were introduced to the market (Casida and Quistad 1998). The OPs was first synthesized by Dr Gerhard Schrader and his team in Germany in 1937 and were used in chemical warfare as a nerve gas (Cremlyn 1978). The era of OP and MC helped recognize the following lessons (Casida and Quistad 1998). It was observed that OPs and MCs were selective, less persistence and could be detoxified more rapidly in mammals. Moreover, they had different modes of action in different insect species, minimum chronic effects on mammals, and some compounds with a systemic action could be easily synthesized. This led to wide spread use of these compounds in insect pest control.

The history, development and mode of action of various insecticides like the chlorinated hydrocarbons, the OPs and MCs have been reviewed extensively (Spencer and O'Brien 1957, Winteringham and Lewis 1959, Roan and Hopkins 1961, O'Brien 1966, Frazer 1967, Chambers and Levi 1992, Ecobichon and Joy 1994, Casida and Quistad 1998, Ecobichon 2001). The following section introduces and briefly reviews the insecticides used in this study.

The OPs are neurotoxic compounds containing carbon and are derivatives of an acid containing phosphorus (Ecobichon 1994 ). Insecticides in this group are commonly and widely used in agriculture and horticulture pest management (Gupta 2004, Bouchard et al. 2006, Aker et al. 2008). Their mode of action kills insects and some vertebrates by inhibiting acetylcholinesterase (AChE) (an enzyme responsible for stopping the activity of the neurotransmitter acetylchlorine) in the synaptic junctions, which consequently generates a continuous electrical firing of chemical signals along the nerve followed by repeated muscle contraction (hyperstimulation) and death by exhaustion (Ehrich et al. 1995, Gupta 2004, Guizzetti et al. 2005).

However, development of insect resistance to OPs and their environmental and human health concerns prompted physiology studies to investigate and understand insect neurobiology and novel biochemical targets that helped formulate new or “reduced risk” compounds with selective and novel modes of action (Casida and

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Quistad 1998, Ecobichon 2001). Some of the new compounds developed are nitromethylene heterocycles (developed from the cyclodienes and cyclohexanes), nitroimino derivatives (chloronicotinyl or neonicotinoids), the phenylpyrazoles and the bacterial product, spinosad, all promoted as being effective at low rates, not environmentally persistent and causing minimal mammalian toxicity (Casida and Quistad 1998, Ecobichon 2001).

These insecticides have different "targets” or "sites of action", usually a molecular receptor such as a specific enzyme site (Winteringham 1969). Unlike the OPs, these insecticides usually act on different target sites which can circumvent cross- resistance problems (Ecobichon 2001). For instance, imidacloprid, belonging to the class chloronicotinyls or neonicotinoids, and spinosad act on the insect nicotinic acetyl receptors (nAChR) (Nauen et al. 1999, Nauen and Bretschneider 2002).

Spinosad, an insecticide with a novel mode of action, was introduced by Dow AgroSciences for control of lepidopterous pests in cotton in 1997 (Salgado 1998). Spinosad is a bacterially derived insect control agent consisting of two active compounds, spinosyns A and D which are fermentation products produced by the actinomycete Saccharopolyspora spinosa, a bacterial organism isolated from soil (Crouse et al. 2001, Cleveland et al. 2002). Spinosad primarily acts as a stomach poison and exposure results in cessation of feeding followed by tremors, paralysis and death (Medina et al. 2003, Williams et al. 2003, Penagos et al. 2005b). Currently, spinosad-based products have been registered in more than 30 countries for control of pest Lepidoptera, Diptera, some Coleoptera, ants and thrips and this compound is classified by the United States Environmental Protection Agency as an environmentally and toxicologically reduced risk material (Thompson et al. 2000).

Another compound fipronil, a phenylpyrazole insecticide, is classified as a “new generation” insecticide effective against numerous insect pests at very low concentrations (Vargas et al. 2005). It is believed to be less damaging to ecosystems compared to the organophosphates insecticides, although there are widespread concerns about its persistence (Tomlin 1997, Stark and Vargas 2005, Gunasekara et

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al. 2007). Fipronil has a unique toxicity mechanism and acts by blocking the GABA- gated chloride channel in the nervous system, resulting in a disruption of neuron signalling and eventually death caused by hyper excitation and paralysis (Cole et al. 1993, Ecobichon 2001). As it acts on a different site it is effective against insects that are resistant to organophosphates, carbamates and pyrethroid insecticides (Bobe et al. 1998 ).

With the advent of these softer or “reduced risk” insecticides, their evaluation for use in fruit fly pest management has been proceeding.

1.3.7 Fruit fly control using cover sprays

In earlier years, fruit fly control relied on many inorganic insecticides such as lead arsenate and sodium fluorosilicate (Back and Pemberton 1918). However, soon after the discovery of synthetic chemical insecticides like DDT, these replaced the inorganic chemicals (Allwood 1997). In Australia, for the control of the Queensland fruit fly, DDT was recommended as a cover spray every two weeks (May 1958a, May 1958b). There was also a similar trend for the control of fruit flies in other parts of the world. However, quick acting OPs later replaced the chlorinated hydrocarbons and these have been used in fruit fly control programs for a considerable period of time (Roessler 1989, Moreno et al. 2001).

In Australia, organophosphorus materials including dipterex, dimethoate and fenthion are commonly recommended and used as cover sprays. Dimethoate and fenthion are popular because they are systemic insecticides capable of attacking eggs and larvae within the fruit (Edwards 1961, May 1961, Shedley 1961, Jessup et al. 2007). In Brazil, the use of malathion is common and currently eight organophosphate and two pyrethroids are registered for fruit fly pest management as either cover sprays or incorporation into toxic baits (Raga and Sato 2005). Cover sprays are also commonly employed in controlling temperate tephritid pest species. Temperate fruit fly species like Rhagoletis pomonella (Walsh), one of the most

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important pests in apple in apple growing states of North America, and Rhagoletis mendax (Curran) are commonly controlled using up to three cover sprays of organophosphates like phosmet, diazinon, malathion, or azinphosmethyl (Aliniazee 1984a, Prokopy et al. 1990, Wise et al. 2003, Yee 2007). Similarly, Rhagoletis indifferens (Curran) a pest in cherries in North America, is controlled using cover sprays of malathion, diazinon or dimethoate (Aliniazee 1984b). Many countries in Asia use cover sprays as an important tool in minimizing damage caused by pest fruit flies (FAO 1986). For instance in Pakistan, insecticide cover sprays against fruit flies are widespread and their use is reported to be increasing (Stonehouse et al. 1998). In India after widespread use of contact poisons for fruit fly control, alternatives such as neem are being investigated (Shivendra 2003). Cover sprays are also used in South East Asian countries like Vietnam, Indonesia and Malaysia, but currently more emphasis is being placed on protein bait spot sprays, using a locally produced product manafuctured from brewery yeast waste (ICMPFF 2005).

In general, the use of cover sprays in fruit fly control programs is losing popularity because of residues in fruit, human health concerns and their impact on beneficial and non-target organisms (Vijaysegaran 1994). Although cover sprays provide good control (Allwood 1997) increasing public awareness has led to the use of safer and effective alternatives such as protein bait sprays (Romoser and Ferro 1994).

1.3.8 Attraction of fruit fly to protein

Knowledge of feeding and associated searching for food in nature is essential for the development of lures and baits widely used to monitor, detect and control pest fruit flies (Landolt and Davis-Hernandez 1993). Tephritid fruit flies require a source of protein to complete egg maturation (Hagen and Finney 1950) and because of the importance of protein in the fruit fly diet, bait sprays based on such material have been successfully used for the control of these pests throughout the world (Roessler 1989). Olfactory attractants from the basis of all currently used tephritid detection and monitoring strategies and some control techniques (Jang and Light 1996).

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Protein baits are basically olfactory attractants (Jang and Light 1996) and when incorporated with an insecticide, attract and kill adult flies.

A number of studies have been conducted to define the attractive substances in protein baits, in order to standardize the specifications for these products and develop improved formulations (Rossler, 1989). Jarvis (1931) first revealed that baits containing ammonia were successful in attracting fruit flies. However, McPhail (1939) tested the attractiveness of gelatin that did not produce ammonia, and still found that it was attractive to Anastrepha striata (Schiner) and thus concluded that ammonia alone was not responsible for the attractiveness. Gow (1954) tested various protein hydrolysates and ammoniacal baits and recognized that the attractive nature of proteinaceous baits was due to some microbial action (bacteria) on the protein and not due to ammonia.

Morton and Bateman (1981) extensively studied the role of ammonia as an attractant to the Queensland fruit fly. They concluded that gaseous ammonia, at specified rates of release, was a strong attractant. They found that standard protein hydrolysate at pH 8.5 was more attractive to the fruit flies and they attributed this to the release of other unidentified volatile compounds. They also found that allowing micro-organisms to grow in the protein hydrolysate increased the attraction as well as ammonia production. Mazor, Gothilf and Galun (1987) in a similar study on the role of ammonia in the attraction of female med flies to protein hydrolysate agreed with the findings of Morton and Bateman (1981), noting that ammonia was very attractive to female med flies and that other unidentified volatiles also play an important role in the olfactory reaction of adult fruit flies to protein baits.

In summery these studies indicated that ammonia was the main olfactory factor in protein baits while amino acids provide the phagostimulatory factors that induced the flies to feed on them (Roessler 1989).

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However, Drew and Fay (1988) dismissed ammonia as the only factor in the attractive nature of the proteinaceous baits. They compared the attraction of Yeast autolysate protein bait, at two different pH levels 6.5 and 9.0, inoculated with the bacterium Providencia rettgeri. More male Queensland fruit flies were attracted to the bacteria inoculated bait at pH 6.5 which favoured the bacterial growth than at pH 9.0 which inhibited the micro-organism growth. Hence, it was concluded that the increase in attraction was due to the volatile metabolites produced by bacteria and not due to ammonia alone.

However, the identity of the attractive chemicals in protein baits remains unknown and there is little published information on the volatile compounds associated with them (Morton and Bateman 1981, Roessler 1989).

1.3.9 Overview of fruit fly attractant bait development and use

TABLE 1.2 Overview of fruit fly attractant bait development and use. Author/Year Major findings/developments

Tryon (1889) Recommended quassia (an insecticidal extract from a south-american tree,Quassia amara Linneaus), treacle, fruit essence and water to trap and control the Queensland fruit fly. Mally (1908) Mally’s mixture was sugar, lead arsenate and water recommended for controlling the Mediterranean fruit fly (Back and Pemberton 1918). Berlese (1908-1909) Berlese in Italy used baits against the olive fruit fly, Bactrocera oleae (Gmelin) that consisted of a stomach poison like arsenic combined with a food attractant such as molasses(Chambers 1977, Moreno et al. 2001). Lounsboury (1912) Berleses method was improved by Lounsboury in

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South Africa for controlling the Mediterranean fruit fly (Moreno et al. 2001). Newman (1913-1914) Newman, in Australia, also improved baits used by Berlese for the control of the Queensland fruit fly (Moreno et al. 2001). Maxwell-Lefroy (1916) Used 24-h old mixture of casein, brown sugar and water in equal parts. 1928-1929 The Florida State Agriculture Department carried out a massive campaign in eradicating the Mediterranean fruit fly from Florida and one technique involved the use of poisoned baits containing lead arsenate (Headrick and Goeden 1996, Selhime 2008). Jarvis (1931) He formulated a lure containing ammonia, water and vanilla essence which was successful in attracting the Queensland fruit fly, B. tryoni, the Mediterranean fruit fly, C. capitata, the Jarvis fruit fly, B. jarvisi, the small black fruit fly, Bactrocera nigra (Tryon), the Solanum fruit fly, Bactrocera cacuminata (Hendel), the boatman fruit fly, Rioxa musae (Froggatt). McPhail (1939) This was the first study that discovered the attraction of fruit flies to protein sources. McPhail found out that various sources of protein mixed with sodium hydroxide held in traps attracted wild Anastrepha striata (Schiner). Dean (1941) Found the apple maggot, R. pomonella to be highly attracted to various protein sources. Also, more female flies were attracted than male flies. Boyce and Bartlett (1941) Found glycine and sodium hydroxide attracted large numbers of walnut husk fly, Rhagoletis completa (Cresson), and this mixture was more attractive than Mc Phail’s baits. Hagen and Finney(1950) Hagen and Finney in their nutritional studies with the

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oriental fruit fly and melon flies found that enzymatic yeast hydrolysate in their diet shortened the pre oviposition period and increased fecundity. Steiner (1952b) Steiner first recorded and demonstrated that foliar sprays of protein hydrolysates plus the organophosphate, parathion, was effective in controlling the Mediterranean fruit fly and the Oriental fruit fly in Guava. This led to the development of a standard commercial bait formulation that was widely used (Roessler 1989). 1956-1957 Steiners bait formulation used in the Mediterranean fruit fly eradication program in Florida (Steiner et al. 1961). 1984-2001 Over this period Spinosad was developed and then commercialiazed in 1997 by Dow AgroSciences (Dow AgroSciences 2001). GF-120 fruit fly protein bait (Dow AgroSciences Indianapolis, IN) a mixture of the insecticide spinosad (0.02% active ingredient) and microbially hydrolysed protein, sugars, adjuvants and a series of conditioners was developed (Barry et al. 2003, Mangan et al. 2006). GF-120 was developed as an alternative to protein bait containing malathion (Wang et al. 2005) and was registered for use in as many as 50 countries for fruit fly control (Dow AgroSciences 2001).

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1.3.10 protein bait in fruit fly control

Since the early 1950’s protein based bait sprays have been used worldwide in fruit fly control programs. Because female fruit flies require protein to develop to sexually mature and produce eggs, (Hagen and Finney 1950), they are highly attracted to protein sources. Therefore, the technique of controlling fruit flies using protein bait sprays involves the use of a protein attractant mixed with a small amount of insecticide and applied as “spots” or “squirts” each about 50-100 ml per tree or 6-10L/ha every 7-10d, on foliage in the lower part of the tree, when fruit are susceptible to attack (Bateman 1982, Vickers 1996, Smith et al. 1997). Female flies in particular are highly attracted to the bait. Bait sprays applied as spot sprays greatly reduce the amount of insecticide sprayed into the environment. An application rate of 100 spots per hectare equates to approximately 100m² ground area or 1% of the ground surface (Anon 1996, Thomas and Meats 1999). This strategy has important environmental advantages over the conventional cover sprays in that there is considerable reduction in the proportion of crop and land area covered with spray droplets through chemical drift, a reduced impact on beneficial insects, lower residues in fruits and the general environment (Prokopy et al. 1992, Chueca et al. 2008). The disadvantage of using protein bait sprays is that it may not be very successful when the fruit fly population density is exceptionally high, it has to be applied every 7 days and reapplied after hot and rainy weather (Vickers 1996). However, the advantages of using bait sprays clearly outweigh its disadvantages (Allwood 1997).

In Australia, acid hydrolysates were the first baits used and these gave way to yeast autolysate preparations as the bait component and malathion as the insecticide (Fletcher and Bateman 1982, Allwood 1997). Yeast autolysate has been available in Australia since 1975 and is commercially sold as Mauri’s Pinnacle Protein Insect Lure (Mauri foods, Toowoomba, Queensland) (Smith and Nannan 1988). It consists of yeast cells killed by low heat and contains a very low salt concentration (Smith and Nannan 1988). Protein hydrolysates on the other hand are produced by hydrolysing plant protein with hydrochloric acid, a process which is stopped by adding sodium

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hydroxide which, in turn, results in a high salt concentration often causing phytotoxic damage to foliage and fruits (Allwood 1997). Other commercially available fruit fly protein baits are Mazoferm E802 (Corn products,Agro II,USA), NU- Lure Insect Bait(Miller Chemical and Fertilizer,Hanover,PA,USA), Provesta 621 autolysed yeast extract(Integrated Ingredients,Bartlesville,OK,USA) and GF-120 Fruit Fly Bait(Dow-Agro Sciences,Indianapolis,IN) (Vargas and Prokopy 2006b).

Protein bait sprays have been used in large scale fruit fly eradication programs as aerial applications. For instance, in the USA aerial application used in the Mediterranean fruit fly control includes three or four parts of Staley’s protein Bait 7 (PIB-7) and 91% to 95% malathion, prepared as an ultra low volume concentrate (ULVC) and sprayed from an aircraft from 10-m height treating alternative 50-m wide strips (Roessler 1989, Prokopy et al. 1992).

In Australia, Jones and Skepper (1965) and Bateman et al. (1966) used protein hydrolysate bait sprays for the first time in a large scale field suppression trial to control the Queensland fruit fly. Then Hargreaves et al. (1986) reported the use of spot sprays of hydrolysed protein bait plus Maldison to attract and kill the Queensland fruit fly as an alternative to cover sprays. In Australian commercial horticulture, protein bait sprays for fruit fly control are advocated and given more emphasis than cover sprays (Fletcher and Bateman 1982).

Bait mixtures have also been used in fruit fly control programs in several Mediterranean countries, based on formulations of hydrolysed protein and organophosphorous insecticides. These have been applied either by air or as spot sprays, particularly to control major fruit fly pests of Olives (Nadel 1966, Manousis and Moore 1987). The Mediterranean fruit fly, considered as one of the most economically damaging pests of citrus in Spain, is mainly controlled using aerial sprays or ground based spot sprays of protein bait and malathion (Magana et al. 2007). They have in California, also been used to control the walnut husk fly, R. completa and med fly in Chile, Argentina and Brazil (Steiner 1969).

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In Southeast Asia, protein bait sprays have reduced the damage levels caused by melon fly by 70-90% in hydroponic growing systems (Vijaysegaran 1985a), and in Carombola the damage levels have been reduced from 100% to 1.6% (Vijaysegaran 1989). Similarly protein bait spray use has been reported in Thailand (Meksongsee et al. 1991), Philippines (Rejesus et al. 1991), Taiwan (Huang et al. 2008), India (Fabre et al. 2003) and also in Vietnam where locally manufactured protein from brewery yeast waste is used (Vijaysegaran et al. 2005). However, the bait spray technique in Southeast Asia has not been widely adopted because locally manafuctured protein has not been available and the imported products are too expensive (Vijaysegran 1997).

1.4 Concerns associated with the use of malathion in protein bait spray and the development of alternative toxicants

One of the most widely used insecticides in protein bait sprays for fruit fly pest management has been Malathion. It was first used in baits against Mediterranean fruit fly in 1956 and still remains the most commonly used toxicant (Roessler 1989, Ecobichon 1994 ). Its popularity is due primarily to having a low mammalian toxicity (March et al. 1956, Dauterman 1971) and being inexpensive (Roessler 1989). It has also had widespread use in other areas of pest management (Weeks et al. 1977, Newhart 2006).

Inspite of the popularity of malathion its use in protein bait sprays has raised serious concerns mainly due to potential adverse effects on non target organisms and the environment. Several field and laboratory studies have documented the detrimental effects of malathion bait sprays on non target or beneficial organisms (Troetschler 1983, Ehler and Endicott 1984, Gary and Mussen 1984, Hoy and Dahlsten 1984, Cohen et al. 1988, Daane et al. 1990, Hoelmer and Dahlsten 1993, Messing et al. 1995).

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To address a need for softer toxicants in protein baits, the USA has developed GF- 120, a new bait combination consisting of an attractant, a feeding stimulant and spinosad insecticide (Mangan et al. 2006). In Hawaii, GF-120 has been used successfully in an area-wide management program in conjunction with natural enemies (Parasitoids), the sterile insect technique, male annihilation and field sanitation (Vargas et al. 2001, Vargas et al. 2002, Barry et al. 2003, Prokopy et al. 2003, Vargas et al. 2003, Stark et al. 2004b, Vargas et al. 2008).

In Australia spinosad as an insecticide has only been registered for use as a cover spray on cotton, brassica vegetables, grapes, pome fruit, tomatoes, , peppers, lettuce, spinach and sweet corn (West et al. 2000, ARDS 2003). There is now a need to test spinosad and other alternative toxicants in protein baits for field control of the Queensland fruit fly. In particular, it is imperative that such research measures the effect on the environment, human health and non-target organisms (Kinney 1993).

New compounds that have been investigated in fruit fly protein baits include spinosad, Phloxin B, fipronil and neonicotinoids (acetamiprid, clothianidin, or imidacloprid). However, more emphasis has been placed on spinosad due to the belief that it has the potential to be an effective alternative to the organophosphates.

Studies evaluating the influences of spinosad in bait sprays on attraction, feeding and toxicity have been carried out in the Caribbean fruit fly, Anastrepha suspensa (Leow) (King and Hennessey 1996), the blueberry maggot fly, R. mendax (Barry and Polavarapu 2005, Barry et al. 2005), the Western cherry fruit fly, R. indifferens (Curran) (Yee and Chapman 2005, Yee 2006, Yee and Alston 2006), the South American fruit fly, A. fraterculus (Wiedemann) (Raga and Sato 2005), the peach fruit fly, Bactrocera zonata (Saunders) ( El-Aw et al. 2008), C. capitata (Adan et al. 1996, Vargas et al. 2002, Barry et al. 2003, Stark et al. 2004b, McQuate et al. 2005b, Raga and Sato 2005), B. cucurbitae (Revis et al. 2004, Stark et al. 2004b, McQuate et al. 2005b, Barry et al. 2006, Vargas and Prokopy 2006a), B. dorsalis (Stark et al. 2004b,

25 Chapter 1: General introduction and literature review

McQuate et al. 2005b, Barry et al. 2006, Vargas and Prokopy 2006a) and R. pomonella (Yee 2007, Yee et al. 2007).

Spinosad bait sprays have been tested for their persistence in the field in the Walnut husk fly, R. completa (Cresson) (VanSteenwyk et al. 2003), Mexican fruit fly, A. ludens (Mangan et al. 2006) and in R. Pomonella (Yee et al. 2007). It has been evaluated as a border bait spray for the control of B. cucurbitae (Prokopy et al. 2003, Prokopy et al. 2004) and as an alternative insecticide in Methyl Eugenol and Cue- Lure bucket traps to attract and kill male Oriential fruit flies and Melon flies (Vargas et al. 2003, Vargas et al. 2008). Large-area field trials have also demonstrated its effectiveness in controlling C. capitata (Peck and McQuate 2000, Burns et al. 2001, McQuate et al. 2005a, Chueca et al. 2007) R. mendax (Barry et al. 2005, Pelz et al. 2005) R. Pomonella (Pelz et al. 2005) and important mango fruit fly pests like Ceratitis cosyra (Walker) and Bactrocera invadens (Drew, Tsuruta & White) (Vayssieres et al. 2009).

Inspite of this worldwide attention to spinosad, it has not been evaluated for use in bait sprays against the Queensland fruit fly and the ecosystem in which this fruit fly pest needs to be controlled.

1.5 Fruit fly protein bait use and its effect on arthropod natural enemies

The impacts of pesticides on the environment and non-target organisms became a major problem with the advancement of technologies used for widespread applications, particularly aerial spraying and large power sprays, together with the availability of relatively low cost synthetic pesticides (Buckley 1979). One of the first studies that demonstrated the adverse effects of chemical control treatments on natural enemy populations was that of DeBach and Bartlett (1951) in citrus. Apart from this, several reviews on the impact of pesticides on various arthropod natural enemies have been published (Ripper 1956, Bartlett 1964, Croft and Brown 1975, Croft 1990b, Croft 1990a, Hardin et al. 1995 , van Emden and D. B. Peakall 1996).

26 Chapter 1: General introduction and literature review

Research into the impact of pesticides on arthropod natural enemies, and the development and implementation of suitable techniques to mitigate adverse effects, has a relatively short history (Croft et al. 1998). However, in the past 20 years, this subject has generated more interest and the reason for this, as pointed out by Croft et al. (1998), is due to the availability of more pesticides, a clearer understanding on the roles natural enemies play in regulating insect pest populations, improved techniques for mass rearing of natural enemies for augmentative and inundative release and an increased resistance of important insect pests to pesticides.

The Working Group ‘Pesticides and beneficial organisms’ of the International Organisation for the Biological Control/West Palaearctic regional Section (IOBC/WPRS), laid down a set of guidelines for testing and evaluating the effects of insecticide toxicity and persistence on natural enemies, through laboratory testing, semi-field and field trials (Hassan 1988, Hassan 1989, Hassan 1998a, b). These standardized techniques were designed to assist researchers to screen suitable chemical compounds and thus assist growers in choosing the least disruptive insecticides for use in an integrated pest management program (Bellows and Fisher 1999). The reports of this group underpinned the importance of selecting and using safe insecticides in modern pest management programs to avoiding impacts on natural enemies that play a crucial role in regulating pest populations.

Killing beneficial insects and thus disrupting the biological control process within a cropping system, is a major problem associated with pesticide cover sprays or broadcast applications (Michaud and Grant 2003). Several publications have reported the negative effects of insecticide cover sprays on various beneficial and non-target organisms, based on large scale field trials (Basedow 1985, Longley et al. 1997, Jenser et al. 1999, Pekar 1999, Peveling et al. 1999, Epstein et al. 2000, Holland et al. 2000, Jansen 2000, Atanassov et al. 2003, Bel'skaya and Esyunin 2003, Langhof et al. 2003).

Unlike the wide spread effects of conventional cover sprays on beneficial organisms, it has been assumed that protein bait sprays have considerably smaller negative

27 Chapter 1: General introduction and literature review

impacts, especially as very low volumes are applied (Roessler 1989, Prokopy et al. 1992). However, it is possible that some non target come into random contact or are attracted to the bait mixtures (Asquith and Messing 1992, Messing and Seiler 1993). It has also been hypothesized that a considerable number of insect species including pollinators may be attracted to protein baits (Ichinohe and Hashimoto 1977). However, very few studies have investigated the attraction of non target organisms to the protein component of fruit fly baits (Smith and Nannan 1988, Asquith and Messing 1992) or their capture in traps baited with protein bait (Thomas 2003). Some studies have reported little or only moderate effects on some non target organisms. For instance, Thomas and Mangan (2005) found no difference in population levels of Aphytis sp before and after the spray of GF-120 fruit fly bait in Texas citrus.

Apart from reports of adverse effects of large scale applications of protein bait sprays, on non-target arthropods (Troetschler 1983, Ehler and Endicott 1984, Gary and Mussen 1984), no published work has documented the possible negative effects of malathion protein bait sprays, used as spot sprays, on the population dynamics of natural enemies in an orchard cropping system.

In this context, it can be hypothesized that certain beneficial organisms may also positively respond to the commonly used Pinnacle protein used in Australia for fruit fly control. It is probable that food scarcity in the field can result in some beneficial insects feeding on protein bait sprays. Consequently, it is important to research attraction, feeding and the impact of protein bait sprays on some important arthropod natural enemies used in Queensland citrus.

1.6 Beneficial organism- The study species and its food source

In Queensland, the red scale (RS) (or also called California red scale), Aonidiella aurantii (Maskell) (Homoptera: Diaspididae) is one of the most important pests in citrus (Smith 1978, Hassan and Summers 1997). This pest has been managed both by

28 Chapter 1: General introduction and literature review

chemical and biological control techniques and the latter involves mass releases of Aphytis lingnanensis (Compere) (Hymenoptera:Aphelinidae) and Comperiella bifasciata (Howard) (Hymenoptera: Encyrtidae), the only red scale parasites sold commercially in Australia (Hassan 1997, Smith et al. 1997). However, due to the pest having developed resistance to insecticides such as organophosphates and carbamates, biological control remains one of the most effective and reliable control strategies (Forster et al. 1995).

Various important aspects of the biology, behaviour, discovery and importation of Aphytis sp and the family Aphelinidae, into various countries, have been reviewed (Aanders 1953, Doutt 1959, DeBach and P. Sisojevic' 1960, DeBach et al. 1978, Gordh and DeBach 1978, Viggiani 1984, DeBach and D.Rosen 1991, Smith et al. 1997). Forster et al. (1995) and Smith et al.(1997) have also reviewed the life cycle of the red scale (RS) and important aspects such as the stages of scale most prone to attack by the parasites, signs of parasitism and the life stages of the two important parasitoids, Aphytis sp and C. bifasciata. Detailed practical field evaluation procedures for parasitism of red scale by these parasitoids have also been described by Forster et al. (1995) and Papacek (1999).

Apart from these parasitoids, some predators have also been mass released into Queensland citrus, including the green lace wing, Chrysoperla carnea (Stephenson and McClung) (Neuroptera:Chrysopidae) a very important predator of aphids and soft bodied insects in the larval stage (New 1975, Tauber et al. 2000, Medina et al. 2003), and Cryptolaemus montrouzieri (Mulsant) (Coleoptera: Coccinellidae) an important predator of a range of mealybug species and soft scales (Smith et al. 1997).

Adult female parasitoid species survive in the field through “host feeding” and feeding upon various sugar sources such as floral and extrafloral nectar and honeydew excreted by homopteran insects (Jervis and Kidd 1986, Evans and Watt 1993, Jervis et al. 1993, Heimpel and Collier 1996, Jervis and Kidd 1996, Wackers 2003, Rohrig et al. 2008).

29 Chapter 1: General introduction and literature review

Predators feed on liquid and solid plant substrates (eg. pollen) and some such as chrysopids and coccinellids have been reported to depend on honey dew excreted by homopeterous insects (Heidari and Copland 1993, Hogervorst et al. 2008).

Some research has been undertaken on the use of food sprays to lure populations of natural enemies, both parasitoids and predators, into fields more favourable for them. For example, higher numbers of natural enemies, both predators and parasitoids, were found in a sugar-treated maize field than in one treated with water alone (Canas and O’Neil 1998). Potato plants treated with artificial honey dew made from molasses, honey and tryptophane alone or combined with Feed-Wheast (a product produced by culturing yeast sp on cotton cheese), attracted and significantly increased the number of predatory insects such as coccinellids and chrysopids (Ben Saad and Bishop 1976). Sucrose dissolved in water, has also been used successfully to concentrate adult lady beetles and lacewings in crops (Ewert and Chiang 1966, Schiefelbein and Chiang 1966, Carlson and Chiang 1973) and lace wings and lady beetles have been observed responding positively to alfalfa crops treated with protein hydrolysate of brewer's yeast (Hagen et al. 1976, Evans and Swallow 1993).

Because protein bait sprays for fruit fly control are applied on a weekly basis, it is possible that various non target and beneficial arthropods can come in direct contact with them, and this requires further research.

1.7 Thesis outline

The purpose of my thesis is to evaluate important toxicants, both existing and novel, that can be incorporated into protein bait sprays for effective control of the Queensland fruit fly. It also reports research that investigated the impacts of fruit fly protein bait on some important arthropod natural enemies in citrus.

30 Chapter 1: General introduction and literature review

The first chapter reports studies on the attraction and feeding response patterns by Queensland fruit fly, to protein bait sprays mixed with different insecticides (chapter 3). The fruit fly protein bait-insecticide attraction study was carried out in large field cages and the feeding study was undertaken under laboratory conditions.

The next chapter, chapter 4, presents research that evaluated the toxicity of these protein bait-insecticide mixtures on B. tryoni and also their toxicity to this fruit fly species after periods of out door weathering on citrus foliage.

Chapter 5 documents data on the effects of protein bait sprays on A. lingnanensis in two commercial orchards and, through laboratory studies, whether they are attracted to fruit fly protein lure. Further, studies are reported on the feeding responses of two predators, on the protein component of the fruit fly bait sprays.

Because modern pest management requires the use of selective compounds that are less likely to disrupt beneficial organisms in cropping systems, chapter 6 presents results of research that evaluated the impact of exposure of fresh and aged protein bait-insecticide formulations on A. lingnanensis survival and fecundity.

Chapter 7 is a discussion of the findings of the research together with recommendations for new areas of investigation that can be undertaken to further improve techniques involved in fruit fly control using protein bait sprays.

31

Chapter 2: General methods and materials

CHAPTER: 2 GENERAL METHODS AND MATERIALS

33 Chapter 2: General methods and materials

2.1 GENERAL METHODS AND MATERIALS

This chapter describes some general methods and materials employed during the course of the study, in order to avoid repeating them in the following experimental chapters. However, specific methods and materials are described in each chapter.

2.1.1 Fruit fly culture room

Fruit fly colonies were held in the laboratory at 25 ± 2 °C, with a relative humidity of 65 ± 5%, and a photoperiod of 12:12(L:D). The lights in the room were automatically regulated to turn off and on at set times in order to present the flies with natural dawn and dusk conditions. Artificial illumination was provided with four fluorescent lights and natural light through glass windows.

2.2 Fruit fly rearing

2.2.1 Laboratory stock colony of adult flies

New generations of adult B. tryoni, to be used for the experiments, were produced from a laboratory colony maintained at Griffith University, Nathan campus for 20 generations. Field collected wild flies could not be used due to the large number required for each experiment. Adult flies in the stock colony were held in 60cm by 60cm by 60cm nylon screened (0.5mm by 0.25mm mesh) aluminium framed laboratory cages. They were supplied with sugar cubes (CSR Sugar Australia PL Epping, NSW) as a source of carbohydrate and Yeast Hydrolysate (Enzymatic) powder (MP Biomedicals Inc, Aurora, Ohio, USA) as a protein source and water ad libitum. Water was provided to the flies through a piece of sponge made from cellulose (Wettex®, Freudenberg household products, Victoria, Australia) extending through a cut in the top of a plastic container containing water. The rearing techniques were adopted from those described by Heather and Corcoran (1985).

34 Chapter 2: General methods and materials

2.2.2 Fruit fly egg collection

Eggs from the laboratory stock colony of adult flies were collected using apple cut into half and the flesh removed to leave a dome of apple skin. A large number of small holes were made in the apple dome using a needle, the dome then sealed onto a Petri dish with paraffin wax and placed inside a cage of mature adult flies. Eggs were deposited through the holes in the apple dome. After 4-h the dome was removed from the cage and the eggs washed from the internal surface, using a water sprayer, into a clean sterile Petri dish.

2.2.3 Larval rearing

Table 2.1 Larval diet. Ingredients Quantity Dehydrated carrot (grinded) 300g Torula yeast (Sanitarium,Castle Hill,NSW) 100g Nipagen(methyl p-hydroxy benzoate)(Sigma 10g Aldrich Pty Ltd. Castle Hill,NSW) Hydrochloric acid 21ml Water 2000ml

The larval diet was similar to that described by Heather and Corcoran (1985). The carrot, yeast and nipagin were mixed together and soaked overnight in 1000ml of water. The additional 1000 ml of water and hydrochloric acid were then added and the medium then mixed to a thick consistent paste. This paste was placed on plastic saucer (4cm by 30-cm diameter) and approximately 30-40 B. tryoni eggs were placed on top of the carrot diet using a pipette. The plastic saucer with the eggs was then placed inside a cardboard box, to exclude light and thus prevent yeast growth, containing sterilized sawdust (heated on 60 °C for 12h). The box was covered with plastic wrap. When the larvae pupated in the sawdust they were separated with a sieve.

35 Chapter 2: General methods and materials

2.2.4 Adult flies for the experiments

As per the requirement of each experiment, the pupae obtained were counted and either placed in aluminium framed laboratory nylon screened (0.5mm by 0.25mm mesh) cages (30cm by 30cm by 30cm) or in plastic containers (6cm by 12-cm diameter) containing a 1-cm layer of moist sterilized sawdust. The lids of each of the plastic containers were fitted with nylon mesh (0.2mm by 0.25-mm) to provide aeration, sugar and water. Sugar was placed on top of the nylon mesh and water provided through a cellulose sponge wick placed on the top of the nylon lids with one end inside a Petri dish containing water. For the flies maintained in the aluminium framed cages, sugar cubes and water were placed inside the cages.

Flies started to emerge approximately 6 to 7-days after development of the puparia. Flies tested in the experiments were all deprived of protein and were used when they were between 10 to 12- d after eclosion from the puparia.

2.3 Experimental room

All laboratory based experiments were conducted under controlled conditions of 24 ± 2°C temperature and a relative humidity of 75 ± 5%. The laboratory was illuminated with overhead fluorescent tube lights and day light through glass windows.

2.4 Protein bait and insecticides

Table 2.2 Insecticides used in protein bait mixtures. Active ingredient Trade name % active ingredient (a.i) and Application rate formulation Malathion Amgrow 500g/l EC 0.2% Chlorpyrifos Superway 50g/l EC 0.2% Fipronil Reagent 800WP 0.005% Spinosad Success 10g/l EC 0.02%

36 Chapter 2: General methods and materials

Each insecticide was mixed into Pinnacle protein solution that was diluted at a rate of 50 ml per 1-L water. The insecticides were added to this protein solution at a rate that achieved the dilution rates listed in Table 2.2. Protein alone without any insecticide was used as the control. The Protein used was Pinnacle Protein Insect Lure (420g/L protein) (Mauri Yeast Australia Pty Limited, Toowoomba, Australia). On each experiment day, these protein bait-insecticide mixtures were prepared in the morning outside the laboratory. These mixtures were held separately in 250ml capped plastic containers under refrigeration until required for use in the experiments.

2.5 Statistical analysis

Data analyses were carried out using SPSS 14.0 (SPSS Inc 2005) statistical analysis software. Prior to analysis, normal distribution was tested using the Kolmogorov- Simirnov test and homogeneity of variances was tested using the Levene’s test. In order to normalize or stabilize variances, the data were either log transformed [log (x+1)], square root transformed√ [ (x+0.5)], or arcsine-square root transformed [Arsin√p1, where p1= %].

Data were either subjected to an analysis of variance, a t-test comparison or a chi- square test. Where the assumptions of the ANOVA were violated the non- parametric equivalent of ANOVA, Kruskal-Wallis test, was used instead.When a significant difference was detected by the ANOVA, treatment means were compared using either Tukey’s HSD test, Fisher’s LSD test or a Games-Howell test (for heteroscedastic data ) (Zar 1999). Differences in treatment means were considered statistically significant at the P = 0.05 level.

37

Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

CHAPTER 3: Attraction and feeding responses of Bactrocera tryoni (Froggatt) to different combinations of protein bait and insecticides under laboratory and field conditions

39 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

3.1 Introduction

Most species can differentiate between various odours and insects have sensitive chemosensory organs which assist them in detecting and discriminating a range of chemicals (Hildebrand and Shepherd 1997). In insects, olfactory and gustatory systems play important roles in reproductive success, location and response to food types, mates and ovipositing sites (Hallem et al. 2006). In fruit fly species, olfactory organs located on the head are used in the detection of odours through the olfactory sensory neurons (OSNs) present in the sensillae on the third segment of the antenna and the maxillary palps (Vosshall and Stocker 2007). Various olfactory and phagostimulatory semiochemicals are emitted by natural food sources that makes them attractive to fruit flies (Prokopy et al. 1992, Prokopy et al. 1993). Thus this mechanism of detecting and discriminating a distinct odour source by fruit flies can be favourably employed in controlling pest species, by using an attractive food like protein combined with a toxicant (Prokopy et al. 2004).

Fruit flies require protein in their diet for development to sexual maturity and food- based attractants based on protein have been successfully employed as olfactory attractants in controlling, monitoring and detecting a number of pest species (McPhail 1939, Jang and Light 1996). From the late 1950’s malathion, an organophosphate insecticide, has been the most commonly used toxicant in protein bait spray formulations deployed in fruit fly control and eradication programs.

Malathion is considered to have a low mammalian toxicity (Steiner 1952b, Roessler 1989), and consequently its use has been common in areas with large urban populations. However, because of its widespread use in many fruit fly control and eradication programs such as the Californian Mediterranean fruit fly eradication program, reports highlighting the adverse effect of large scale application of malathion on non-target beneficial organisms, has been recorded. In the Mediterranean fruit fly control programs in California and Morocco, malathion bait sprays killed numerous arthropods, compared to the unsprayed control sites (Harris et al. 1980, Troetschler 1983). Another example was an out break of scale insects in olive and citrus orchards in Northern California which was attributed to the destruction of natural enemies of these scale insects after 19 applications of protein

40 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures malathion bait sprays (Ehler and Endicott 1984). The Mediterranean fruit fly eradication program carried out in the 1980’s in northern California has been reported to have caused a significant mortality of adult bees, as a result of weekly applications of malathion bait sprays (Gary and Mussen 1984).

Simulated malathion bait spray residue was highly toxic to the ice plant scaleparasite, Encyrtus saliens (Prinsloo and Annecke), and degraded bait residue made them walk faster which increased their risk of coming in contact with the bait droplets (Hoy and Dahlsten 1984). White fly parasitoids, Euderomphale flavimedia (Howard) and Encarsia peltate (Cockerella), were found to be more susceptible to simulated malathion bait sprays compared to the White fly, Aleyrodes spiraeoides (Quaintance) (Hoelmer and Dahlsten 1993).The longevity of two aphid parasitoids, Aphidius liriodendrii (Liu) and Trioxys curvicaudus (MacKauer), after exposure to malathion bait sprays was found to decline rapidly (Daane et al. 1990).

In humans, malathion has the potential to cause cancer and various reproductive complications and its use is believed to have an adverse effect on the environment (Flessel et al. 1993, Newhart 2006). Use of malathion as cover sprays over large areas has also been attributed to the development of resistance in the Oriental fruit fly, Bactrocera dorsalis (Hendel) in Tawain (Hsu and Feng 2000). Similarly, in Spain field populations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), were found to be more resistant to malathion than the laboratory population, as a result of frequent and large scale application of malathion bait sprays in citrus and other fruit crops (Magana et al. 2007).

With concerns associated with the use of broad spectrum organophosphate insecticides, the Food Quality Protection Act (1996) in the United States, restricts the use of certain broad spectrum insecticides in field control programs for the Apple Maggot, R. pomonella , and the Blueberry maggot, R. mendax, and new “reduced- risk” compounds are being encouraged for registration (Reissig 2003, Barry et al. 2004). As a consequence, research is being conducted with new classes of insecticides which can replace the organophosphate insecticides (Vargas et al. 2002).

41 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

Concerns associated with the use of malathion in fruit fly control treatments have evoked numerous empirical studies evaluating various toxicants, with an objective to replace its use. Among the toxicants tested, spinosad and phloxine B, being environmentally safe, have commonly been considered as replacement candidates for malathion (Miller et al. 2004). Field tests comparing the efficacy of malathion, spinosad and phloxin B in controlling wild populations of C.capitata were carried out by Peck and McQuate (2000). Although malathion was found to be the most effective insecticide, spinosad and phloxine B also gave good levels of control. After field evaluation studies comparing spinosad and organophosphate insecticides (naled, dichlorvos (DDVP) and malathion), Vargas et al. (2003) suggested spinosad as a safe alternative for use in the male annihilation technique using methyl eugenol and cue-lure, in bucket traps for controlling the Oriental fruit fly, Bactrocera dorsalis (Hendel) and the Melon fly, Bactrocera cucurbitae (Coquillett) in Hawaii. Compared with fields treated with malathion, a rapid population build up of the braconid parasitoid, Fopius arisanus (Sonan), was reported in fields treated with spinosad and phloxin B, indicating that the use of these ‘softer’ insecticides would be preferred in an area wide management program for C. capitata (Vargas et al. 2001). In Hawaii, spraying adjacent coffee plantings with spinosad-protein bait sprays also suppressed C. capitata infestations in adjacent persimmon orchards as well as in the coffee cherries, without significantly affecting the level of parasitisation of C.capitata by F. arisanus in coffee cherries (McQuate et al. 2005a).

Different protein bait formulations, and toxicants used in protein baits, can induce varying degrees of responses in terms of attraction to and feeding by different fruit fly pest species. Fabre (2003) determined the relative attractiveness of six commercial protein baits (Buminal, Corn Steepwater, Hym-Lure,Pinnacle, Nulure, and SolBait) to B. cucurbitae, and found SolBait to attract a larger number of flies than the other bait mixtures. Attraction and feeding studies comparing different protein bait mixtures for the Blueberry maggot, R. mendax (Curran), suggested SolBait to be more attractive than Nu-lure but no such significant differences existed between AY50 % ( Mauri Yeast Australia), SolBait and Nu-lure. However, in terms of feeding times, adult flies fed significantly longer on Solbait as compared to AY50%

42 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures and Nu-lure (Barry and Polavarapu 2004). In protein-starved Mediterranean fruit flies, Prokopy (1992) reported that mixing 20% malathion ultra low volume concentrate (ULVC) in Staley’s Protein Insect Bait-7 (PIB-7) did not affect attraction, but did repel flies from feeding. In protein starved Mediterranean fruit fly, attraction and feeding were reduced significantly when malathion was added to Provesta, but not when spinosad and phloxine B were added to the bait (Vargas et al. 2002). B. cucurbitae in comparison to B. dorsalis had a better attractant response pattern to bait and was 4.8 times better in discriminating baits from water (Barry et al. 2006). Therefore, keeping in mind these variations in response patterns from different fruit fly pest species, attraction and feeding responses for each fruit fly pest species needs to be clearly understood.

In Australia, a number of pest tephritids including the Queensland fruit fly, B. tryoni, are controlled using protein bait sprays mixed with an insecticide (Barry and Polavarapu 2004). However, despite the use of such baits in the field, attraction and feeding responses of the Queensland fruit fly to protein bait combined with different insecticides has seldom been investigated.

A series of experiments were thus conducted both in field cages and in the laboratory to evaluate the Queensland fruit fly attraction and laboratory feeding responses on Pinnacle protein bait containing malathion, chlorpyrifos, spinosad and fipronil.

43 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

3.2 Methods and Materials

3.2.1 Experiment 1: Field cage Attraction

3.2.1.1 Field study site

The study was carried out in a Lychee (Litchi chinensis Sonn.) orchard in Shailer Park, a suburb of Logan city, located 27 km south of Brisbane city. The study was carried out between mid April to May, 2007.

3.2.1.2 Field-cages and traps

Four evenly spaced nylon screen field cages (4m by 4m by 2.5m) were erected. To present the flies with natural conditions, each field cage enclosed one non-fruiting Lychee tree of an average height of 2m. Cages were separated from each

Figure 3.1 Steiner trap fitted with paper cones at entrances and containing protein bait-insecticide mixture in a beaker.

44 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

Figure 3.2. Steiner trap suspended around the perimeter of the tree canopy with a flexible copper wire.

other with a tree in between them. 30ml of each insecticide bait mixture and the protein control were held in separate 100 ml glass beakers (Boeco, Germany) which were placed in separate Steiner type fruit fly traps. The beakers were covered with a nylon mesh to prevent the flies from entering and drowning in the test solutions. The beakers were secured inside the traps by fixing them with double-sided sticky tape (Scotch, USA) attached to the base. To prevent the escape of attracted flies, the openings of the traps were fitted with cones made from 110mm filter paper (Whatman, England) (Figure 3.1).

In each cage, five Steiner traps each with a different test solution, were randomly suspended in one of the five positions around the perimeter of the canopy with a flexible copper wire (Figure 3.2). The traps were suspended 0.65m above the ground, 1.3m from the cage sides and were 0.63m apart. When all traps were in place around the tree, approximately 3000 protein starved 10-14 days old adult B. tryoni were released into the field cage from two nylon screened aluminium framed laboratory cages containing 1500 flies each. Approximately equal numbers of male

45 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures and female flies were released as the sex ration in the B. tryoni colony is known to be approximately 1:1. The traps were rotated in a clockwise direction every 30 minutes to avoid any positional effect. The experiment was carried out between 0900 and 1130 hours. One experiment lasted for two and half hours and ended when each trap occupied all five positions around the tree. The flies were collected at the end of the experiment and the number of male and female flies attracted to each treatment recorded for each experiment day. In one experiment day four replicates were run for each treatment, and after four separate experiment days a total of sixteen replicates were carried out for each of the five treatments.

After the end of each experiment the remaining, non- responding flies inside the cages were removed by placing white ground sheets on ground below the trees and then fogging with pyrethrum. Pyrethrum was chosen because it has a very short residual activity, due to its quick degradation under sunlight, and a good knockdown ability (Katsuda 1999). However, after spraying, the cages were not used for at least five days. For the next experiment, new sets of Steiner traps, filter paper cones and beakers were used so as to avoid any contamination with insecticide residues and residual attractant that could influence the new data set.

3.2.2 Experiment 2: Laboratory Feeding study

All flies in this experiment were protein starved female flies and used when they were 10-12 days old. The experiments were carried out during September and October 2007, between 0900 and 1400 hours.

On the day the experiments were conducted, the cages of flies were relocated into the experimental area for few hours in order to acclimatize them to the actual experimental conditions (Barry and Polavarapu 2005).

A single fly, no choice feeding test was conducted in the laboratory under controlled conditions (24 ± 2°C) in single aluminium framed, nylon screened laboratory cages (30cm by 30cm by 30cm). The protocol followed was partly similar to that described in the Mediterranean fruit fly, C.capitata (Vargas et al. 2002), the Blueberry

46 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures maggot, R. mendax,(Barry and Polavarapu 2004, Barry and Polavarapu 2005), the Melon fly, B.cucurbitae and the Oriental fruit fly, B.dorsalis (Barry et al. 2006). In order to avoid mixing of chemicals cues, the feeding test for each treatment was conducted in a separate isolated cage.

From each pre-mixed treatments solution, 0.5 ml was placed on a sponge (Wettex®, Freudenberg household products, Victoria, Australia) cut in a rectangular shape (1.5cm by 0.5cm), using a micropipette. The sponge with the test solution was placed on a glass slide (7.5cm by 2.5 cm) and the glass slide then placed on the centre of the cage floor, which had one side open to observe the fly activity and to record the data. A single female fly was captured randomly using a transparent plastic cup (5cm high by 4 cm diameter) and gently placed within 2cm of the sponge containing the test material. Each fly was allowed 300 seconds to feed on the test solution. Feeding was defined as actual contact of the labellum with the test solution and when feeding commenced the total amount of time spent feeding was recorded with a stopwatch. Each fly was tested only once and discarded. Flies landing on the sponge and staying for < 5 seconds were considered to be in an agitated state (Vargas et al. 2002, Barry and Polavarapu 2004) and not counted.

A single replicate consisted of a single female B. tryoni tested on each of the treatments. In total, for each treatment, 30 replicates were conducted. The treatments were randomly selected and a feeding test for each treatment was conducted separately in its designated cage.

3.2.3 Statistical analysis

The data were log transformed [log (x+1)] to stabilize variances and to normalize the data. For the field cage attraction experiment, the total numbers of male and female of B. tryoni attracted and captured in the Steiner type fruit fly traps for each field experiment day were subjected to a repeated-measures analysis of variance. Sampling days were taken as repeated measures. The response data for the total number of male and female attracted to the treatment were analyzed separately.

47 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

The laboratory feeding experiment data were square root transformed [√ (x+0.5)]. Feeding response data obtained for each treatment were subjected to a one-way ANOVA.

3.3 Results

3.3.1 Field cage attraction

The numbers of protein-starved female fruit flies attracted to the different protein- insecticide bait mixtures were significantly different between the treatments (F 4,15 = 4.815, P = 0.011) (Table 3.1). Post-hoc comparison showed that the protein- spinosad treatment attracted significantly more female flies than the protein- chlorpyrifos mixture. For protein starved males, no significant difference occurred in terms of attraction to protein baits with the different insecticides (F4,15 = 1.059, P = 0.411) (Table 3. 2).

Table 3.1. Mean ± s.e. number of female Bactrocera tryoni (Froggatt) captured in Steiner type fruit fly traps with different protein bait-insecticide mixtures.

Treatment Mean ± s.e. Chlorpyrifos 25.88 ± 3.37 a Malathion 29.06 ± 3.36 ab Protein 33.06 ± 4.22 ab Fipronil 33.25 ± 3.01 ab Spinosad 37.63 ± 4.30 b

Means followed by the same letter do not differ significantly (Tukey’s HSD test on log [x+1] transformed data; P < 0.05).

48 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

Table 3.2. Mean ± s.e. number of male Bactrocera tryoni (Froggatt) captured in Steiner type fruit fly traps with different protein bait-insecticide mixtures.

Treatment Mean ± s.e. Chlorpyrifos 21.38 ± 2.37 a Malathion 28.75 ± 3.57 a Protein 24.44 ± 2.13 a Fipronil 23.94 ± 2.63 a Spinosad 27.75 ± 2.90 a

Means followed by the same letter do not differ significantly (Tukey’s HSD test on log [x+1] transformed data; P < 0.05).

3.3.2 Laboratory feeding experiment

Addition of insecticide to protein bait had a significant effect on the feeding duration of female B. tryoni flies (F 4,145 = 149.276, P = 0.001) (Table 3.3). Compared with the protein control, protein-spinosad and protein-fipronil, the duration of feeding was significantly less on the protein-malathion and protein-chlorpyrifos formulations. No significant differences were detected in feeding times between the control and bait containing spinosad and fipronil. The longest feeding times were observed on the control and protein-spinosad and protein-fipronil mixtures and shortest feeding times on protein-malathion and protein-chlorpyrifos mixtures.

Table 3.3. Feeding time (mean ± s.e.) obtained with female Bactrocera tryoni (Froggatt) on protein bait-insecticide mixtures.

Treatments Feeding time in seconds (mean ± s.e. ) Protein 98.67 ± 3.66 a Spinosad 94.13 ± 4.77 a Fipronil 92.33 ± 4.09 a Malathion 37.83 ± 1.65 b Chlorpyrifos 13.80 ± 3.12 c Means followed by the same letter do not differ significantly (Fisher’s LSD test on square-root transformed data; P < 0.05).

49 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

3.4 Discussion

The use of insect attractants in field control of pest species of insects is extremely important in Integrated Pest Management (IPM) programs. This is particularly the case in citrus pest management where the control of fruit fly pest species with attractants is essential to avoid high levels of mortality of beneficial species inflicted by cover sprays. For maximum efficacy using protein baits, it is not only important to have the most attractive protein bait but also the added insecticide that does not repel or have anti feedant properties. This study evaluated attraction and feeding responses of B. tryoni to food bait mixtures combined with different insecticides. Record of such studies does not exist and have not hitherto been documented for the Queensland fruit fly.

From the field cage attraction experiment, a significant difference in attraction between the protein-chlorpyrifos and protein-spinosad mixtures was recorded in the protein–deprived female flies. Except for this difference, no differences in attraction among treatments in both protein-deprived male and female flies were observed. Adding chlorpyrifos to protein bait reduced the attractiveness of the bait compared to adding spinosad. Previous findings by Prokopy et al. (1992) using protein-deprived C. capitata did not record a reduction in attraction when malathion was added to the protein bait. Contrary to this, Vargas et al. (2002) reported that protein-deprived C. capitata flies demonstrated a significant reduction in attraction when malathion was mixed with protein bait, but this difference was not observed in protein fed flies. In B. dorsalis and B. cucurbitae, Vargas and Prokopy (2006a) observed that malathion mixed with Provesta protein bait attracted significantly fewer flies than a mixture of Provesta and spinosad. The results of this attraction study in B. tryoni were in agreement with the findings of Prokopy et al. (1992), but contrary to those of Vargas et al. (2002), and Vargas and Prokopy (2006a) who recorded protein- deprived C. capitata, B. dorsalis and B. cucurbitae flies having a reduced attraction to protein bait containing malathion.

The feeding response data confirmed that B. tryoni had reduced feeding times on protein baits containing malathion and chlorpyrifos, compared to baits with fipronil,

50 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures spinosad and the control (protein alone). The shortest feeding duration was on baits containing chlorpyrifos. The feeding times on baits containing fipronil, spinosad and the control were much longer compared to baits with chlorpyrifos and malathion. These results indicated that protein bait mixtures containing malathion and chlorpyrifos did not elicit a strong phagostimulatory response from the flies. Prokopy et al. (1992) and Vargas et al. (2002) reported that adding malathion to bait mixtures deterred C. capitata flies from feeding on it. However, organophosphates such as malathion and chlorpyrifos which primarily act as contact toxicants do not require long feeding times to be effective, but reduced risk insecticides like spinosad, need to be consumed in larger amounts and therefore should be used with baits that induce longer feeding times to be effective in killing flies (Barry and Polavarapu 2004). Spinosad and fipronil did not induce any deterrence in feeding in B. tryoni. Both these insecticides, unlike malathion and chlorpyrifos, when mixed with protein bait induced feeding almost equal to the period of time observed with protein bait alone. Spinosad was developed as an alternative to malathion for use in protein baits and considered safe to non target insect species. Consequently, GF-120 containing this insecticide has been recommended as a potential replacement for baits containing broad spectrum organophosphate insecticides such as malathion to control many important fruit fly pest species (Peck and McQuate 2000, Barry et al. 2003, Revis et al. 2004). Spinosad in the USA is registered as suitable for organic producers and has been marked compatible for use in organic agricultural production (Organic Materials Review Institute 2002). Hence, as an alternative to broad spectrum insecticides, it can befit conventional as well as organic growers in reducing fruit fly populations with an associated minimum impact on non target organisms.

Fipronil, classified as a new generation insecticide because of it unique mode of action, ( acts by blocking the GABA-regulated chloride channels and interrupts the normal nerve transmission) is effective against insects that have developed resistance to organophosphates and carbamates (Gunasekara et al. 2007). It is effective at low dose rates (Bobe et al. 1997) and using insecticides with such low dose rates drastically reduces the total amount of active ingredient used, resulting in

51 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures a reduction in insecticide residue levels accumulating in the environment, compared to organophosphates and carbamates (Boiteau and Noronha 2007). In Vietnam, large scale field trials using protein manufactured from brewery yeast waste plus fipronil, applied as spot sprays, have demonstrated that this formulation has a high level of efficacy in controlling Bactrocera pyrifoliae (Drew & Hancock ) and B. dorsalis in peach, and Bactrocera correcta (Bezzi) and B . dorsalis in Barbados cherry (Malpighia glabra) (Vijaysegaran et al. 2005).

The feeding times observed in B. tryoni with baits containing spinosad and fipronil were long enough for the fly to ingest a lethal amount of insecticide. Mauri Yeast Autolysate proved to be an ideal bait to mix with spinosad and fipronil which, notably, induced longer feeding times without any significant changes in attracting B. tryoni. The attraction and feeding response data obtained in B. tryoni indicates that spinosad and fipronil can be potential alternatives for malathion for use in protein bait sprays.

From the attraction data, comparing the mean number of flies captured in the traps, more female than male flies responded to all the treatments. This trend aligned with the data obtained in attraction and feeding response studies in C. capitata by Vargas et al. (2002) where more females than males were attracted to protein baits. Malo (1992) similarly observed more females than males of five different Anastrepha fruit fly species collected in McPhail traps with proteninaceous baits. These data also support the findings that female Dacinae respond more strongly to protein than do males as they require protein for ovarian and sexual maturation (Hagen and Finney 1950).

Possible interactions between odour plumes, in a choice test, may occur in a field cage situation while no choice test employing multiple cages will avoid such interactions (Barry et al. 2006). To further support the findings here and to elucidate any differences in attraction in B. tryoni between protein-bait insecticide mixtures, such methodology can be employed, but would demand more inputs and resources than a choice test.

52 Chapter 3: Attraction and feeding responses of B. tryoni to protein bait-insecticide mixtures

The field cage attraction experiment was carried out on clear sunny days over a 2-h period when the average temperature did not fluctuate significantly. Though the methodology employed in the field cage experiment was consistent and aligned with what (Rousse et al. 2005) had suggested, some heterogeneity in the data was still noted. Although this heterogeneity was reduced after suitable data transformation, it still raises some concern over the type of methodological parameters researchers should adhere to in field cage studies. Variability in such parameters, to an extent, can influence the type of data obtained in a field cage study. Differences in some techniques may possibly skew the results obtained. This was demonstrated by Rousse et al. (2005) who also made recommendations for techniques that can be employed to improve consistency of data.

Finally, in Australia, bait sprays with malathion are commonly used to control tephrid fruit flies (Anon 1996). With a growing concern on the use of broad- spectrum insecticides like organophosphates, there is an ever increasing need to evaluate novel, reduced risk compounds, in order to employ some that can be used as toxicants that fit suitably into an Integrated Pest Management approach in controlling the Queensland fruit fly. This further necessitates the evaluation of some of these compounds to test their toxicity and the effects of weathering, on the mortality of B. tryoni. The next chapter assesses some of these important aspects on bait sprays use with the compounds tested here on B. tryoni.

53

Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

Chapter 4: Toxicity and impact of ageing of protein bait-insecticide mixtures on Bactrocera tryoni (Froggatt)

55 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

4.1 Introduction

An effective fruit fly protein bait should attract flies and then stimulate them to feed on it (Revis et al. 2004). The attractiveness of new applications of protein bait sprayed onto foliage, can decrease in dry weather conditions within a day (Prokopy et al. 1992, Vickers 1996), and insecticides in the baits, after application, also degrade rapidly as a result of weathering, metabolic degradation and other environmental factors (Gunther and Blinn 1956). Along with an attractive lure, protein bait sprays also require an effective toxicant (Nestel et al. 2004) which should demonstrate maximum toxicity to the flies. As bait sprays are now a standard recommendation for fruit fly control (Roessler 1989), use of an attractive bait combined with a suitable toxicant with an optimum residual action, would help to maximize their efficacy and ensure successful field pest management programs.

Many recent studies have focused on evaluating reduced risk insecticides that have advantages over broad spectrum ones, especially those known to be less toxic to humans and beneficial organisms, can be used at lower dose rates and have a lower risk of ground water contamination and development of insect resistance (Boiteau and Noronha 2007). A number of such compounds have been studied and compared with broad spectrum toxicants in terms of attraction, toxicity and residual effectiveness. One such compound that has been evaluated several times for many tephritid pest species is spinosad (Yee 2007) which has been considered a suitable replacement for malathion in protein bait sprays (DowElanco 1994).

Adan et al. (1996) found spinosad to be toxic to C. capitata and, when compared to the organophosphate insecticide fenthion, it significantly reduced the fecundity of the fly. Laboratory evaluation of spinosad in Provesta protein bait showed that it was highly toxic to C. capitata, B. cucurbitae and B. dorsalis (Stark et al. 2004). Nestle et al. (2004) also reported spinosad, mixed with 1% yeast hydrolysate containing 10% sucrose, to be highly toxic to C. capitata and the Ethopian fruit fly, Dacus

56 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni ciliatus (Loew). Adults of the Western cherry fruit fly, R. indifferens, had higher levels of mortality with spinosad than imidacloprid and thiacloprid (Yee 2006). In the Peach fruit fly, B. zonata laboratory feeding tests comparing the toxicity of different insecticides showed that the carbamate insecticide (methomyl) was more toxic than the neonicotinoid (thiamethoxam), the biorational insecticide (spinosad) and the organophosphate (malathion) (El-Aw et al. 2008). Moreover, malathion was less effective than the other compounds tested and female B. zonata were more tolerant to spinosad than male flies.

However, Prokopy et al. (2000) reported reduced oviposition and high mortality in the Mexican fruit fly, Anstrepha ludens(Loew) with coloured spheres treated with dimethoate and imidacloprid, while spheres treated with spinosad did not have the same effect compared to untreated spheres. In a laboratory study comparing a range of new insecticides, Reissig (2003) found spinosad to be moderately toxic to the apple maggot, R. pomonella (Walsh), whilst in field trials, it did not provide satisfactory control whereas thiacloprid was as effective as organophosphate insecticides.

Studies comparing the effect of field ageing of protein bait-insecticide mixtures, on the mortality of flies, have also been undertaken in different fruit fly species. In the melon fly, B cucurbitae, attraction and toxicity of GF-120 fruit fly bait significantly decreased over time, rainfall greatly reduced the toxicity of the bait while those aged for 2 and 24 hours significantly lost their attractiveness in comparison to fresh baits (Revis et al. 2004). Yee (2007) reported a reduction in mortality of R. pomonella when spinetoram and spinosad were aged for 7 and 14 days, whilst the organophosphate insecticide azinphos-methyl, under the same conditions, induced a higher mortality. In laboratory studies, NuLure with malathion and spinosad gave similar levels of mortality in the Walnut husk fruit, R. completa, but the mortality with spinosad in field trials, after three days of application, declined by 50% (VanSteenwyk et al. 2003). In Hawaii, Prokopy et al.(2003) reported that GF-120 bait spray droplets containing spinosad lost 50% of its toxicity after 4 days of outdoor weathering, without being exposed to rainfall. However, exposure to 8mm of

57 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni simulated rainfall rendered it almost ineffective in killing protein deprived adult B. cucurbitae.

An important point to consider in evaluating insecticides in baits is that those tested toxic in laboratory tests may not be as effective under field conditions. For instance, the typical tropical climate with high temperatures, rainfall and relative humidity can greatly reduce the effectiveness of bait sprays as a result of weathering (Revis et al. 2004). Therefore, research to determine the residual effectiveness of baits after field weathering would be useful in establishing recommendations with regard to concentrations and application frequency, and in turn, assist in minimizing spray costs (Yee et al. 2007).

In Australia, the tropical and subtropical climatic pattern can have a rapid weathering effect on bait sprays and consequently reduce their effectiveness in field pest management programs. To date no study has been reported on experiments to evaluate the toxicity and effects of weathering of protein bait-insecticide formulations on mortality of the Queensland fruit fly, B. tryoni.

In this study, the toxicity and effects of weathering of different insecticides in protein baits, on mortality of the Queensland fruit fly, B. tryoni, under hot and dry weather conditions in South east Queensland has been evaluated.

58 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

4.2 Materials and methods

4.2.1 Toxicology experiment

Twenty sets of 80 puparia were placed in separate plastic containers (6 by 12-cm diameter) containing a 1-cm layer of moist sterilized sawdust. The lids of each container were fitted with nylon mesh (0.2mm by 0.25-mm) to provide aeration and access to a diet of sugar and water. The use of plastic containers instead of laboratory cages was necessary due to limited space in the rearing room. Flies started to emerge approximately 6 to 7-d after development of puparia. Flies tested in the experiment were all deprived of protein and were used when they were between 10 to 12- d old.

The treatments used were 1. protein + chlorpyrifos; 2. protein + malathion; 3. protein + spinosad; 4. protein + fipronil and 5. protein alone as control.

On the day of the experiment, flies were transferred from the plastic containers to nylon screened aluminium framed laboratory cages (30cm by 30cm by 30-cm). 20 such cages with 80 flies each were placed on a long wooden table 80-cm high. With a pipette, 0.5-ml of the test solution was placed on a sponge made from cellulose (Wettex®, Freudenberg household products, Victoria, Australia) cut in a rectangular shape (1.5cm by 0.5-cm). The sponge with the test solution was then placed on a clean glass microscope slide (7.5cm by 2.5-cm), at the centre of the cage floor. The treatments were removed from the cages after 72-hr, when the final fly mortality data were recorded. Throughout the experiment, flies were provided with unlimited access to sugar and water. After the introduction of the treatments, time periods were recorded using a stopwatch. The number of dead flies in each treatment was recorded after 24, 48 and 72-hr. All treatments were replicated 4 times and percent mortality estimated for each. Fly knockdown for all treatments was also recorded after 1-hr, 2-hr and 3-hr intervals after the treatments were introduced into the cages.

59 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

4.2.2 Effects of aging of protein bait mixtures containing different insecticides on fly mortality

The impact of ageing of protein bait-insecticide mixtures after field application was measured by assessing the mortality of adult B. tryoni after feeding on aged test solutions applied to citrus leaves. The study was conducted in the fruit fly research centre, Griffith University, from May 15th to May 27th, 2008.

Five 2-yr-old sweet orange, Citrus sinensis (Linnaeus), plants were used for the experiment. Plants were purchased from a nursery in Rochedale, Brisbane. The citrus plants were grown in black polythene containers with leaf mulch and top soil mixed with NPK fertilizers. Plants which had no pesticide treatment for the previous 3 months and were approximately 1.5-m tall, with numerous healthy leaves, were selected. The selected plants were watered and kept outdoors for approximately 3 weeks, before the beginning of the experiment.

Plants to be treated with each protein-insecticide mixture were clearly labelled and each treated with just a single treatment. Before treating the plants with the respective mixtures, the leaves were thoroughly rinsed with water in order to wash off dirt and other residues. The leaves were allowed to dry and then were treated with the bait-insecticide mixtures.

On each of the 5 plants, eight to nine healthy and uniform sized leaves were labeled and treated with the protein bait-insecticide mixtures. To apply the mixtures, the selected leaves were carefully dipped in the pre mixed protein bait-insecticide solutions contained in a 15-ml plastic container. Each of the treated leaves was clearly labelled with a yellow ribbon tag. Rather than simulating field spraying, the leaves were dipped in the insecticide bait mixtures to provide a uniform coverage. This was done in order to avoid any anomaly in the data as a result of variation in the amount of insecticide bait mixture present on each of the treated leaves. Moreover, this technique of pesticide application by leaf dipping is commonly

60 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni practiced and was used in similar studies with beneficial and pest arthropods (Rowland et al. 1991, Bellows and Morse 1993, Cahill et al. 1996, Martinson et al. 2001).

The leaves were then allowed to age for 2-hr, 3-days and 6- days. Fresh bait mixtures were prepared and applied on the leaves 2-hr, 3-days and 6-days before the actual mortality test commenced. The plants were left in the open to weather and were exposed to full sunlight with maximum temperatures during the experiment ranging between 19.8°C and 27.0°C and minimum ranging between 5.9°C to 11.8°C (Figure 4.2.). Most days were sunny and the average humidity ranged from of 37.5% to 77%. For the duration of the experiment, the plants were not exposed to rain (Figure 4.3.). The daily temperature was recorded with a maximum and minimum thermometer and the relative humidity was recorded at 9 am in the morning and 3 pm in the afternoon, during the period of the experiment. Following the periods of ageing, the leaves were carefully separated from the trees with a scissor and cut into uniform sizes of 5cm by 2-cm and placed on a labelled Petri dish for use in the mortality test.

Flies initially held in the plastic containers were transferred to aluminium framed, nylon screened laboratory cages (30cm by 30cm by 30-cm). Twenty such cages each with 80 flies (mixed sex) were placed on a long 80-cm high wooden table. Before the experiment commenced, flies were placed in the cages for several hours to assist them acclimatize to the laboratory conditions.

The flies, throughout the period of the experiment, were provided with an unlimited supply of water through a sponge made from cellulose and sugar cubes placed on top of the cages. The treated leaves from the Petri dish were placed on a clean glass microscope slide (7.5cm by 2.5-cm) and gently placed at the centre of the cage. After the introduction of the leaves into the cages, time was recorded with a stop watch. There were 4 replicates for each treatment and fly mortality data in all the treatments were recorded after 24 and 72-hr.

61 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

4.3 Statistical analysis

A one-way ANOVA was conducted, for the toxicity test, on the mortality data obtained after 24-hr, 48-hr and 72-hr. Fly knockdown data recorded after 1-h, 2-h and 3-d were also individually subjected to a one-way ANOVA. For the persistence test the mortality data obtained for the five treatments for each age period (2-hr, 3- d and 6-d) were subjected to a one-way analysis of variance. Also, mortality data obtained from bait mixtures containing chlorpyrifos, malathion, spinosad and fipronil were individually grouped for each of the three age periods and were subjected to a one-way analysis of variance. Prior to analyses, the percent mortality data were arcsine-square root transformed. Treatment means were separated by Fisher’s LSD test (P = 0.05).

4.4 Results

4.4.1 Toxicology experiment

There was a significant difference between some treatments in fly mortality, after 24-h, 48-h and 72-h. Malathion and chlorpyrifos were not significantly different in fly mortality after 24, 48 and 72-h but produced a higher mortality than spinosad, fipronil and the control (Table 4.1, 4.2 and 4.3). More than 90% fly mortality was recorded in baits containing malathion and chlorpyrifos after 24-h, 48- h and 72-h (Table 4.1, 4.2 and 4.3). Fipronil caused a significantly higher mortality than spinosad up to 48-h exposure but not after 72-hr, when both treatments recorded fly mortality up to 90 % (Table 4.1, 4.2 and 4.3). In terms of fly knockdown, spinosad and fipronil caused a significantly lower rate of knockdown, compared to malathion and chlorpyrifos, after 1-hr, 2-hr and 3-hr period exposure, with spinosad demonstrating the least knockdown rate among the three treatments (Table 4.4).

62 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

Table 4.1. Percent mortality (mean ± s.e.) of Bactrocera tryoni (Froggatt) induced by different protein bait-insecticide mixtures after 24-hr exposure to flies.

Treatments Percent mortality (mean ± s.e.) Malathion 94.69 ± 2.07a Chlorpyrifos 95.94 ± 0.94a Fipronil 78.44 ± 5.11b Spinosad 28.13 ± 2.99c Control 0.31 ± 0.31d

Means followed by the same letter do not differ significantly (Fisher’s LSD test on Arcsine square-root transformed data; P = 0.05) (F 4,15 = 140.334, P < 0.001).

Table 4.2. Percent mortality of Bactrocera tryoni (Froggatt) induced by different protein bait-insecticide mixtures after 48-hr exposure to flies.

Treatments Percent mortality (mean ± s.e.) Malathion 96.25 ± 1.61a Chlorpyrifos 99.38 ± 0.63a Fipronil 90.63 ± 1.49b Spinosad 60.63 ± 6.47c Control 0.94 ± 0.31d

Means followed by the same letter do not differ significantly (Fisher’s LSD test on Arcsine square-root transformed data; P = 0.05) (F 4,15 = 152.251, P < 0.001).

63 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

Table 4.3. Percent mortality of Bactrocera tryoni (Froggatt) induced by different protein bait-insecticide mixtures after 72-hr exposure to flies.

Treatments Percent mortality (mean ± s.e.) Malathion 98.43 ± 0.94a Chlorpyrifos 99.69 ± 0.31a Fipronil 92.50 ± 1.14b Spinosad 90.00 ± 2.22b Control 0.94 ± 0.31c

Means followed by the same letter do not differ significantly (Fisher’s LSD test on Arcsine square-root transformed data; P = 0.05) (F 4,15 = 277.687, P < 0.001) n =1600 flies.

Table 4.4. Percent knockdown of Bactrocera tryoni (Froggatt) induced by different protein bait- insecticide mixtures after 1-hr, 2-hr and 3-hr period exposure to flies.

Percent knockdown (mean ± s.e.) Treatments 1h 2h 3h Malathion 45.94 ± 6.70 a 68.13 ± 6.40 a 75.63 ± 5.56 a Chlorpyrifos 43.43 ± 5.36 a 66.56 ± 5.96 a 74.69 ± 6.56 a Fipronil 0.00 ± 0.00 b 7.19 ± 2.25 b 25.00 ± 5.71 b Spinosad 0.00 ± 0.00 b 0.31 ± 0.31 c 1.25 ± 0.51 c Control 0.00 ± 0.00 b 0.00 ± 0.00 c 0.00 ± 0.00 c

Means followed by the same letter in a column do not differ significantly (Fisher’s LSD test on Arcsine square-root transformed data; P = 0.05) n= 1600 flies.

64 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

4.4.2 Effects of weathering of protein bait mixtures containing different insecticides on fly mortality

There was a significant difference in mortality between some treatments (chlorpyrifos, malathion, spinosad, fipronil and the control) after exposure of flies for both 24 and 72-hr, at all three weathering time periods of the baits on leaves. At all three weathering times, chlorpyrifos and malathion produced significantly higher rates of mortality than spinosad, fipronil and the control, after 24 and 72-hr exposure to flies (Table 4.5 and 4.6). The mortality rates increased for chlorpyrifos, malathion, spinosad and fipronil after 72-hr exposure to flies, in all leaves aged for 2- hr, 3-d and 6-d (Table 4.6). However, weathering of the treated leaves from 2-hr to 6-days resulted in a decrease in mortality levels. Chlorpyrifos and malathion recorded a 4 % and 5% reduction in kill respectively with leaves aged for 6-d as compared to 2-hr, after 72-hr exposure to flies. With similar weathering, spinosad and fipronil recorded a much larger decrease in mortality of flies as compared to chlorpyrifos and malathion. Spinosad recorded a 25% reduction while fipronil an 11% reduction, in fly mortality after 6-days of bait weathering as compared to 2-hr, after 72-hr exposure to flies.

A separate one-way ANOVA conducted singly on the mortality data for each treatment (chlorpyrifos, malathion, spinosad and fipronil) over the three weathering periods of 2-hr, 3-d and 6-d, indicated no significant difference in mortality between chlorpyrifos, malathion and fipronil after 72-hr exposure to flies (ANOVA, F2,9 =

1.500, P = 0.274 ; F 2,9 = 1.693, P = 0.238 and F 2,9 = 3.765, P = 0.065 respectively) (Figure 4.1). But for spinosad a significant different in mortality was recorded between the three weathering periods (F 2,9 = 14.070, P = 0.002 ) (Figure 4.1). For spinosad, there was a decline in mortality rate from 2-hr weathering on leaves to 3- days and 6-days, when exposed to flies, particularly at the 72-hr-exposure period.

65 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

Table 4.5. Percent mortality (mean ± s.e) of Bactrocera tryoni (Froggatt) adults observed after 24-hr exposure to leaves with different protein bait-insecticide mixtures weathered for 2-hr, 3-d and 6-d.

Aged Leaf Treatment Percent mortality of B. tryoni after 24-hrs 2-hours Chlorpyrifos 95.62 ± 0.48 a Malathion 92.05 ± 1.60 a Spinosad 60.13 ± 3.33 b Fipronil 37.52 ± 4.00 c Control 0.00 ± 0.00 d

One-way ANOVA F 60.538 df= 4, 15 P <0.001

3-days Chlorpyrifos 95.65 ± 1.83 a Malathion 94.48 ± 2.11 a Spinosad 40.73 ± 3.31 b Fipronil 54.58 ± 5.14 c Control 1.33 ± 0.67 d

One-way ANOVA F 154.023 df= 4, 15 P <0.001

6-days Chlorpyrifos 87.56 ± 4.77 a Malathion 86.20 ± 5.00 a Spinosad 31.60 ± 2.11 c Fipronil 51.16 ± 3.70 c Control 2.08 ± 0.79 d

One-way ANOVA F 40.538 df= 4, 15 P <0.001

Means followed by the same letter are not significantly different (Fisher’s LSD test on Arcsine square-root transformed data: P =0.05) n=1600 flies.

66 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

Table 4.6. Percentage mortality (mean ± s.e) of Bactrocera tryoni (Froggatt) adults observed after 72-hr exposure to leaves with different protein bait-insecticide mixtures weathered for 2-hr, 3-d and 6-d.

Aged Leaf Treatment Percent mortality of B. tryoni after 72-hrs 2-hours Chlorpyrifos 98.53 ± 0.60 a Malathion 98.48 ± 1.07 a Spinosad 78.16 ± 0.20 b Fipronil 77.81 ± 0.45 b Control 1.54 ± 0.88 c

One-way ANOVA F 240.562 df= 4, 15 P <0.001

3-days Chlorpyrifos 97.69 ± 1.33 a Malathion 96.69 ± 0.92 a Spinosad 54.64 ± 1.74 b Fipronil 75.72 ± 3.55 c Control 1.33 ± 0.67 d

One-way ANOVA F 231.254 df= 4, 15 P <0.001

6-days Chlorpyrifos 94.78 ± 2.39 a Malathion 93.43 ± 2.22 a Spinosad 53.68 ± 6.24 c Fipronil 66.98 ± 3.60 c Control 2.91 ± 1.04 d

One-way ANOVA F 73.929 df= 4, 15 P <0.001

Means followed by the same letter are not significantly different (Fisher’s LSD test on Arcsine square-root transformed data: P =0.05) n=1600 flies.

67 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

Figure 4.1. Comparison of percent mortality (mean +s.e.) of Bactrocera tryoni (Froggatt) between leaves aged for 2-hr, 3-d and 6-d with protein bait containing chlorpyrifos, malathion, spinosad and fipronil after 72-hr exposure period.

a a a a a 100 a 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 Mean (+s.e) percent fly mortality fly percent Mean (+s.e) 0 mortality fly percent ) (+s.e Mean 0 Two hours Three days Six days Two hours Three days Six days

Aged bait (chlorpyrifos) Aged bait (malathion)

100 100 90 90 a 80 a a 80 a 70 b 70 60 b 60 50 50 40 40 30 30 20 20 10

Mean(+s.e) percent fly mortality fly percent Mean(+s.e) 10 Mean(+s.e) percent fly mortality fly percent Mean(+s.e) 0 0 Two hours Three days Six days Two hours Three days Six days

Aged bait (spinosad) Aged bait (fipronil)

Bars between age period with the same letter are not significantly different (Fisher’s LSD test on Arcsine square-root transformed data: P = 0.05) n=1600 flies.

68 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

Minimum Maximum

30 28 26 24 22 20 18 16 14 12

Temperature (°C) 10 8 6 4 2 0 15/05/2008 16/05/2008 17/05/2008 18/05/2008 19/05/2008 20/05/2008 21/05/2008 22/05/2008 23/05/2008 24/05/2008 25/05/2008 26/05/2008 27/05/2008

Experiment days

Figure 4.2. Maximum and minimum temperature recorded during the experiment days.

100

90

80

70

60

50

40

30

20 Average realtive humidity(%) humidity(%) realtive Average 10

0 15/05/2008 16/05/2008 17/05/2008 18/05/2008 19/05/2008 20/05/2008 21/05/2008 22/05/2008 23/05/2008 24/05/2008 25/05/2008 26/05/2008 27/05/2008

Experiment days

Figure 4.3. Average relative humidity (%) recorded during the experiment days.

69 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni

4.5 Discussion

Insecticides incorporated into protein bait sprays used in fruit fly field control programs should be toxic to the flies. Moreover, they should have residual effectiveness on leaves longer than the preoviposition period of a particular species of fruit fly (Roessler 1989). This study examined the toxicity and residual effectiveness of different protein insecticide bait mixtures on B. tryoni. To my knowledge no earlier published work documents such research on B. tryoni.

In the toxicity experiment, the percent mortality obtained with the organophosphate insecticides malathion and chlorpyrifos revealed that they were more toxic to adult B. tryoni than spinosad and fipronil. Malathion and chlorpyrifos also caused a faster knockdown of flies, with almost 95% being killed after 24-hr. In contrast, spinosad and fipronil recorded 28% and 78 % mortality respectively after 24-hr. Fipronil produced a steady increase in fly mortality with exposure, with 28% of the flies being killed in the first 24-hr and 90% after 48 hours. However, fipronil was significantly different and recorded a lower fly mortality after 24, 48 and 72-hr, compared to malathion and chlorpyrifos. Mortality with spinosad gradually increased from 28% in 24-hr of feeding exposure to 60% and 90% after 48 and 72-hr respectively. Spinosad caused an increase in fly mortality over time indicating it to be a slow acting toxicant. The percent knockdown data observed for each of these protein bait-insecticide combinations after 1, 2 and 3-hr exposure further substantiated this result. Spinosad caused the lowest fly knockdown within the first three hours of exposure, whereas malathion and chlorpyrifos recorded the highest, followed by fipronil. Spinosad acts as a stomach poison and has very little contact toxicity, hence needs to be to be ingested to be effective (Prokopy et al. 2003, Mangan et al. 2006). Conversely, malathion and chlorpyrifos are contact poisons (Matsumura 1975, NRA 2000) which results in a rapid kill. Because malathion and chlorpyrifos are contact poisons, random contact with these compounds when used in protein baits may prove lethal to any non-target, beneficial organisms. On the contrary, spinosad can have a minimum effect on non-target organisms even after a random contact with it. Hence such novel compounds are considered more

70 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni appropriate for environmental reasons and for use in conjunction with biological control systems.

In field control programs for fruit flies, particularly those based on protein baits, fast acting insecticides are considered more appropriate than slower ones as fruit flies are killed before they can oviposit (Roessler 1989). Consequently, malathion and chlorpyrifos may be more effective in protein bait based fruit fly control programs than spinosad and fipronil in baits. However, adult mortality is not the only factor to be taken into consideration when evaluating the effectiveness of an insecticide (Yee et al. 2007). For example, some insecticides can have a non-lethal effect on adult flies. Apart from death and reduced fecundity, a toxicant can induce sublethal effects such as shortened life span, mutations in offspring, weight loss, changes in fertility rates, pre-oviposition time, developmental rates and sex ratio (Stark et al. 2004a). However, research to determine non-lethal effects of insecticides is difficult and a time consuming process (Provost et al. 2003, Williams et al. 2003).

This study did not investigate non-lethal effects of the insecticides on B. tryoni adults, but such research could give more insight into their impacts. When the protein bait-insecticide mixtures were applied to citrus leaves, the mortality levels of adult B. tryoni remained high for the organophosphate insecticides over the test weathering period of 6 days. Malathion and chlorpyrifos induced a mortality rate of 93% and 94% respectively after 6 days of weathering on leaves after 72-hr exposure to flies, only a slight reduction in rate from the 2-hr field weathering time. Dry conditions can enhance the persistence of residues (Williams III et al. 2003) and this residual effectiveness recorded for the tested toxicants may have waned rapidly if it had been exposed to rainfall. Peck and McQuate (2000) recorded an increase in the population of C. capitata in coffee plantations treated with malathion, spinosad and phloxine B under high rainfall and this was attributed to bait sprays being washed from the foliage. These results, however, confirm that organophosphate insecticides like malathion in bait sprays remain very effective for, at least, a week after spraying under dry conditions. Similarly, fipronil which is a relatively new compound considered to be selective and having a reduced impact on the ecosystem, as

71 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni compared to organophosphate insecticides (Stark and Vargas 2005), demonstrated a similar trend. Fipronil had a longer residual effectiveness than spinosad and was comparable to chlorpyrifos and malathion. However, the mortality obtained with fipronil for each of the three weathering periods was slightly lower than malathion and chlorpyrifos.

The residual effectiveness of spinosad, as measured by induced mortality, rapidly diminished 3-days after application to the citrus leaves. In fact the mortality obtained with leaves 2-hr after application of bait was significantly different from those weathered for 3-days and 6-days. These results confirmed that spinosad breaks down very quickly under dry weather conditions, in comparison to the two organophosphate insecticides malathion and chlorpyrifos, and the phenylpyrazole insecticide fipronil. The findings here are consistent with several other studies that reported that spinosad loses its residual effectiveness within 3 to 7-d after application (Williams et al. 2003). Under Hawaiian weather conditions, Peck and McQuate (2000) reported 95% and 25% fly mortality after a 2 week period for malathion and spinosad respectively, in field cage tests and suggested a rapid weathering of spinosad compared to malathion. Though spinosad breaks down quickly under both natural and artificial light, it still can maintain a level of stability adequate to perform under field conditions (Berard and Graper 1996, Crouse et al. 2001). As observed here, and suggested by Peck and McQuate (2000), field level control with spinosad may not be comparable with organophosphate insecticides like malathion. However, addition of ingredients, such as photostabilizers or adjuvants to improve its photostability, and to extend its toxicity, has been suggested (Thompson et al. 2000, Yee et al. 2007).

In summary, malathion and chlorpyrifos were more toxic over time to B. tryoni than spinosad and fipronil. Nonetheless spinosad and fipronil gave adequate mortality over time. Although, spinosad had a shorter residual effectiveness than malathion, chlorpyrifos and fipronil, it is considered to have an advantage of being safe to non-

72 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni target organisms and the environment. In addition, short-lived formulations could still be effective and useful in integrated plant protection programs as they would have less impact on the natural enemies, compared to persistent toxicants which can be deleterious to beneficial insects (Mazomenos et al. 2002, Grutzmacher et al. 2004).

Application rates and timing of treatments can be modified if insecticides tend to break down rapidly under field conditions but the higher costs of this strategy, especially in large scale field control programs, can be prohibitive. Also, the reduced risk insecticides are more expensive than the organophosphates (Reissig 2003) and consequently a careful technical nad economic evaluation in selecting an appropriate toxicant would prove useful before embarking on large a scale field control program. Selection and use of an insecticide in a field situation would largely be influenced by its effectiveness to control a broad range of pest species, impact on non-target organisms, ease of application costy, safety profile and pre-harvest treatment intervals (Morse and Bellows 1986). A grower may choose to use reduced risk compounds because of residual concerns and the negative impact of broad spectrum insecticides on beneficial organisms. However, this ultimately would be determined by the grower’s choice based on the grower’s level of awareness of the effectiveness, advantages and disadvantages in using different toxicants in bait sprays and the farming practices he adheres to.

This study provided a comparative evaluation of different compounds to B. tryoni in terms of their toxicity and residual effectiveness. Studies determining the lethal doses and sub-lethal effects of the toxicants tested here would further help understand their effectiveness in controlling B. tryoni. Field evaluation studies in controlling B. tryoni with novel compounds and insecticides like malathion and other organophosphates can provide a comparative effectiveness of the compounds when tested in a practical field situation. Additional studies should also be carried out to study the impact of the compounds tested here on beneficial organisms, under laboratory and field situations. The next chapter investigates attraction and feeding

73 Chapter 4: Toxicity and impact of ageing of protein bait-mixtures on B. tryoni responses of some beneficial insects to Pinnacle Autolysed Yeast and the impact of protein bait sprays on important parasitoids in citrus.

74 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

Chapter 5: Impact of protein bait sprays on parasitoids of red scale, Aonidiella aurantii (Maskell) (Homoptera:Diaspididae) and laboratory evaluation of attraction and feeding of beneficial insects on protein baits

A. lingnanensi C. bifasciata

75 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

5.1 Introduction

Ever increasing demands for food for human consumption, worldwide, has led to the widespread use of plant protection chemicals, especially cover sprays to prevent crop losses through insect pest infestation. This trend often has severe negative impacts on the non-target organisms, especially beneficial insects which play major roles in pollinating plants and suppressing pest insect populations. This predicament has been clearly recognized and discussed by biologists for many years (Ripper 1956, Newsom 1967, Johansen 1977, Ulmer et al. 2006). Beneficial insects encounter insecticides through direct spray contact, movement over residues and ingesting contaminated food and, if not killed, may receive sub lethal doses that reduce their ability to behave normally and perform their roles in the ecosystem, often resulting in increased pest populations (Hoy and Dahlsten 1984, Desneux et al. 2007).

Fruit fly control using attractive protein bait mixed with insecticide and applied as a spot spray, in contrast to cover sprays, is recognised as having fewer adverse effects on non-target species. However, protein bait-insecticide mixtures may also serve as a potential food source for, and pose risks to, non-target insect species (Wang and Messing 2006). It is essential that baits only attract and kill the target pest species and not attract and kill the beneficial species within the crop ecosystem (Asquith and Messing 1992, Wang and Messing 2006). Moreover, multiple applications of bait sprays and chances of repetitive exposure in a field situation, could compound the risk and impacts of bait sprays on beneficial organisms, if there is evidence of such negative impacts (Nadel et al. 2007).

Malathion, an organophosphate insecticide commonly used in fruit fly baits, has been shown to have a significant negative impact on beneficial insects when employed in large scale field control programs and also in laboratory studies ( also discussed in chapter3) (Hoy and Dahlsten 1984, Daane et al. 1990, Hoelmer and Dahlsten 1993, Messing et al. 1995). Such concerns have led to the development of novel fruit fly bait formulations such as the GF-120 spinosad–based bait commercialized in countries like the USA from 2002, as a replacement for

76 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies malathion-based baits (Wang and Messing 2006). Different studies have evaluated feeding, attraction and non-target impacts of protein bait-insecticide sprays on beneficial insects, with recent studies focusing on evaluating novel toxic compounds such as spinosad. For instance, Vargas et al.(2002) and Stark et al.(2004b) reported that two important braconid parasitoid species of tephritid fruit flies, Fopius arisanus (Sonan) and Pysttalia fletcheri (Silvestri), did not feed directly on the protein baits Mazoferm, Provesta, Nu-Lure and USB. Wang (2005) also observed that F. arisanus, P. fletcheri and another braconid parasitoid, Diachasmimorpha tryoni (Cameron), did not directly feed on GF120 fruit fly bait but were attracted to honey. However, (Michaud 2003) in laboratory based cage experiments found GF-120 fruit fly bait to be attractive and toxic to Aphytis melinus (De Bache) but not as toxic as the malathion-nulure mixture. Williams et al. (2003) in their review of studies evaluating the impact of spinosad on natural enemies, concluded that Hymenopteran parasitoids were more susceptible to spinosad than predatory insects. Approximately 80% of the field and laboratory studies reviewed indicated a moderately harmful or harmful result on parasitoid wasps. Wang and Messing (2006) observed no significant difference in feeding times on GF-120 and honey for C. capitata and Drosophila melanogaster (Meigen), however some beneficial tephritid species, Eutreta xanthochaeta (Zhang), Tetreuaresta obscuriventris (Loew), Ensina sonchi (Linnaeus), and an endemic Hawaiian tephritid Trupanea dubautiae (Ulmer) preferred to feed longer on honey than GF-120. Such non-target fly species can therefore be ‘behaviourally’ and ‘physiologically’ susceptible when GF-120 bait sprays are used on an area-wide fruit fly control program (Wang and Messing 2006). However, some studies did not indicate any negative impact on some parasitoid species. For instance, yellow sticky traps deployed in Texas citrus before and after the application of spinosad GF-120 bait sprays did not differ in the capture rates of Aphytis spp and C. bifasciata (Thomas and Mangan 2005).

To date, no studies have been conducted to determine the possible impacts of fruit fly bait sprays on some of the important parasitoids in Queensland citrus. Similarly, limited information exists on attraction and feeding responses, of some important

77 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies beneficial insects, to the most commonly used fruit fly bait in Australia.This study was therefore designed to address these queries and had three objectives.

• Firstly, determine the impact of protein bait sprays, applied in commercial orchards to control B. tryoni, on A. lingnanensis and C. bifasciata, by assessing their parasitism levels on the red scale (RS), A. aurantii, over one growing season.

• Second, to investigate whether A. lingnanensis is attracted to or is repelled by protein bait in the presence or absence of other food resources such as honey solution.

• As noted by Nadal et al.(2007) while assessing the non-target impact of insecticide-bait mixtures, equal importance should be given in evaluating the feeding propensity and feeding preference of the beneficial organism on the bait and other natural food sources. Therefore the final experiment examined whether general predators like the adult green lace wing, C. carnea and the mealybug predator, C. montrouzieri fed directly on protein and compared the total feeding time on protein, water and honey as a measure of comparative feeding preference.

78 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

5.2 Materials and Methods

5.2.1 Experiment 1: Impact of protein bait sprays on A. lingnanensis and C. montrouzieri

5.2.1.1 Field study site

The field assessment on the impact of protein bait sprays on the parasitoids of citrus red scale was carried out in Mundubbera, Queensland (25° 36’ S, 151° 18’ E) located 405 kilometres north-west of Brisbane city.

The field sampling in Mundubbera was carried out in two separate citrus orchards, Auburnvale and Ironbark. Both orchards grow different varieties of citrus covering a large area of land. However, the study was carried out only on an early ripening variety of citrus called Nova. One block each of this variety was chosen in both Auburnvale and Ironbark.

The block in Auburnvale consisted of 24 rows of mature Nova trees totalling 795 trees. The trees were healthy with new flushes of leaf growth. They were an average height of approximately 1.5-m with a canopy diameter of 3-m. Adjacent to this Nova block on one side, was a block of another variety of citrus (Ellendales) while the other side was vacant. The block chosen in Ironbark consisted of 6 rows of mature Nova trees totalling 1792 trees. The trees were an average height of approximately 2.5-m with a canopy diameter of 3-m.Bordering this block on two sides were blocks of Grape fruit and Nova varieties, respectively.

5.2.1.2 Protein bait spray and A. lingnanensis releases

Adult Aphytis wasps are released in the two orchards, Auburnvale and Ironbark, to control red scale infestations. Before release, 10,000 Aphytis were produced and held in cups containing green paper strips. The wasps within these cups climb on to

79 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies the paper strips which are placed on the trees. A standard release pattern is followed which varies according to the tree density per hectare, however approximately two and half cups are used over this area.

According to records obtained from the two orchards, Aphytis were released in the Auburnvale orchard on a frequent basis in the 2008 growing season but not at the Ironbark site. In the blocks that were sampled, 9 releases of Aphytis were carried out in Auburnvale compared to only 1 release in Ironbark.

Protein bait sprays for fruit fly control were applied as spot sprays in both orchards. Approximately 50 ml spots were applied on the leaves present on the lower part of the tree. In Auburnvale the first bait treatment commenced on 29th of January, 2008 and continued till harvest. In Ironbark the first spray was applied out on the 21st of January, 2008 and continued till harvest. The spraying was carried out at weekly intervals or more frequently in some instances when fruit fly infestations were observed to increase. Auburnvale used protein bait mixed with Abamectin as the insecticide, while Ironbark used Protein bait and malthion. The choice of insecticide used was the grower’s preference.

5.2.1.3 Sampling

In order to collect unbiased fruit samples from each of the experimental blocks, Auburnvale and Ironbark, each was divided into four equal sized plots. On each sampling day, 25 fruits were randomly sampled from each plot giving a total of 100 fruits from each block. In order to assess parasitism of red scales by the two parasitoids, fruits that had at least five or more scales were chosen. The fruits were initially collected into plastic bags and transferred to brown paper bags within 30 minutes and labelled with date and site information. The fruits were held under refrigeration and then two scales from each fruit were dissected under a microscope using a dissecting needle. In total 200 scales was dissected from each of the two experimental citrus blocks, at each sampling period.

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Four samples from each block, Auburnvale and Ironbark were taken between January 2008 and April 2008. Only one sample was taken before the protein bait spraying program commenced, at both the sites, due to an early increase in fruit fly infestation which required earlier than normal introduction of protein baits. The first sample was taken on the 16th January 2008, the second on the 12th February, 2008, the third sample on the 3rd April 2008 and the final one on the 29th April, 2008 when the fruits were being harvested. The first sample of fruits was dissected under the supervision of experienced staff at the Bugs for Bugs P/L laboratory in Mundubbera.

The next three sample fruits were dissected in the fruit fly laboratory, Griffith University, Brisbane.

5.2.1.4 Scale parasitism assessment

The scales on the fruits were dissected and assessed for parasitism by the two red scale parasitoids, A. lingnanensis and C. bifasciata. The sampling and dissection followed that described by Papacek (1999). From the total of 200 scales dissected in each sampling period, total number of parasitised and unparasitised scales by A. lingnanensis and C. bifasciata was recorded. For Aphytis, parasitism was assessed on each scale by recording the presence of eggs, larvae, pupae and the number of exit holes that indicated the number that had emerged. Similarly, for Comperiella the total number of larvae, pupae and the emerged adults were recorded. Comperiella eggs are very difficult to detect as they act as endoparasites where the adult females lay eggs inside the scale insect (Papacek 1999). Therefore, this information could not be recorded. The total number of scales parasitised in one sampling period was the combined number of scales parasitised by Aphytis and Comperiella. The total number of unparasitised scales was assessed as the total of dead scales, live unmated scales and live mated scales that did not contain parasites. The data were recorded on a standard field scale parasitism assessment sheet used in citrus orchards in Mundubbera by the Bugs for Bugs P/L monitoring staff.

81 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

The following formulae were also used to compute different levels of parasitism including the live scales.

Live scale = (l/t) ×100

Aphytis parasitism (%) = a/ (a+u) ×100

Comperiella parasitism (%) = c/(c+m) ×100

Total parasitism (%) = p/ (p+l) ×100

Where l = total live mated and live unmated scales, t = total scales sampled, a = total Aphytis parasitism, u = live unmated scales, c = total Comperiella parasitism, m = live mated scales, p = total parasitism by Aphytis and Comperiella.

The method used here to compute total parasitism (%) (rates of parasitism) for the four sampling periods is also similar to that described by Itioka et al. (1997). This technique has also been adopted to compute the total parasitism rates for other species of Aphytis like A. yanonensis DeBach et Rosen(Matsumoto et al. 2004).

5.2.2 Experiment 2: Attraction of A. lingnanensis to protein bait, protein +sugar (20%), water and honey

The insects used for this experiment were reared and supplied from the Bugs for Bugs P/L insect rearing facility in Mundubbera. As the experiments were conducted in Brisbane, adult Aphytis were transported from Mundubbera to the fruit fly centre, Griffith University, through an overnight courier service. For transportation, approximately 1000 Aphytis were placed in vials with a few drops of honey placed on the lids which contained small holes for aeration. The vials were packed in a 5 litre enclosed container with ice packs (Rmax/celluform) and filled with shredded paper packing. After the Aphytis were received at Brisbane, they were transferred to clean aerated vials without honey, in order to starve the insects for a period of 12 hours.

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The experiment was conducted under controlled conditions. Both choice and no choice experiments were conducted. The treatments for the choice test included protein (Pinnacle Protein Insect Lure) honey and water while protein and water as a control were used in the no choice test. 12.5ml of protein was diluted in 250ml of water and honey was diluted in water at a ratio 1:1. For the experiments, 6 clear plastic containers (18cm by 18-cm) were placed on a 80-cm high wooden table covered with clean white paper sheeting. The 6 containers were placed at an equal distance apart. Approximately 1000 Aphytis were released, from a single vile, into each container. Using a fine paint brush (Windsor & Newton LTD, Sablehair-07 brush), paper strips, 6cm by 1-cm, were treated with either protein, water or honey over an area of 1cm at one end of the strip.

For the choice test, 3 paper strips each treated with either protein, honey or water were placed randomly inside each container, equidistant from each other. In the no choice test, strips with protein and the water control were introduced. Upon the introduction of these treatments, time was recorded using a stop watch. The number of adult Aphytis feeding on the top and sides of the paper strips were recorded every 30 minutes, ten times. Aphytis demonstrated a quick random movement inside the containers (personal observation), as a result those moving over the treatments may not represent actual attraction. Consequently, counts were based on the insects alighting on the test strips. After each count, the strips were rotated clock wise in each container to avoid any possible positional effect. The choice and no choice tests were conducted on different occasions and each treatment was replicated 6 times in both the tests.

In addition, a choice test with protein, water and protein + 20% sugar was also conducted. The methodology was similar to the other attraction experiments, but this experiment had only four replicates.

83 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

5.2.3 Experiment 3: Feeding propensity of green lace wings, C. carnea and the mealy bug predator, C. montrouzieri on protein bait, water and honey

The insects for this experiment were supplied from the Bugs for Bugs P/L rearing facility in Mundubbera and the experiment was conducted in the fruit fly research laboratory, Griffith University, Brisbane.

The insects were transported from Mundubbera to Brisbane in cohorts of 10 individuals, each within a separate plastic container (15cm by 10-cm size) and placed in a 5 litre enclosed ice pack (Rmax/celluform) filled with shredded paper. During transportation, the insects were supplied with honey on strips of fabric. In Brisbane, the insects were transferred to clean containers without honey and starved for a period of 12-h before the actual experiment commenced. The experimental conditions and the test solutions (Protein, water and honey) were the same as those used and described for the Aphytis protein attraction experiment (Experiment 2).

Preliminary feeding tests indicated that C. carnea and C. montrouzieri did not feed readily on the treatments presented to them under laboratory conditions. Therefore, to overcome this, a simple technique was devised which allowed the insects to feed without any interruption. A 3.5ml plastic pipette (Sarstedt, Germany) was modified and used for the feeding test. The pipette was cut with a scissor to a length of 90-mm (Figure 5.1). The top was also cut open and a double folded sponge (2.5cm by 1.5-cm) made from cellulose (Wettex®, Freudenberg household products, Victoria, Australia) was inserted and then treated with 0.25-ml of one of the three treatments. Separate pipettes were used for each of these 3 treatments.

The tapered end of the pipette was also cut in order to attract the insects to the treatment. The device was placed on a support (35mm height) and when a single test insect was introduced at the lower end, the insect had the opportunity to exhibit positive phototaxis and moved towards the treatment.

84 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

35mm 10mm

55mm

Treatment placed on a sponge here

8mm 35cm

Support to raise the treatment at one end Insects placed here to walk up and feed

Figure 5.1 Schematic design of the feeding apparatus made out of a plastic pipette. Treatment placed at one end on a sponge. Insects walk up the apparatus and feed. Once feeding commenced, actual feeding time was recorded.

Insects were randomly selected and introduced to one of the three devices containing protein, water or honey. After feeding commenced, the actual feeding time was recorded using a stop watch. A single replicate consisted of testing a single insect on a treatment. For each of the three treatments, 30 replicates were conducted.

5.3 Statistical analysis

5.3.1 Experiment 1: Impact of protein bait sprays on A. lingnanensis and C. bifasciata

The parasitism data obtained for Aphytis lingnanensis and Comperiella bifasciata were summed for the individual sampling periods for each sampling site. A Chi- Square test was used to test whether any differences in parasitism existed over the four sampling period.

85 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

5.3.2 Experiment 2: Attraction of A. lingnanensis to protein bait

Prior to analysis the data obtained for the attraction of Aphytis to protein, honey and water were log transformed [log (x+1)] for the choice tests and square root transformed [√ (x+0.5)] for the no choice test. Data obtained from the choice test with protein, water and honey were subjected a one-way ANOVA and Fisher’s LSD test was conducted to separate differences in treatment means. Data with protein, protein + sugar and water was subjected to Kruskal-Wallis test and treatment means treatment means were separated using the Games-Howell test for heteroscedastic data and least significant difference (LSD) for homoscedastic data (Zar 1999). The data for the no choice test were subjected to a t-test comparison.

5.3.3 Experiment 3: Feeding propensity of C. montrouzieri and C. carnea on protein bait

In the feeding test comparing the feeding propensity of beneficial insects on protein, water and honey, the data were square root transformed [√ (x+0.5)] and a one-way ANOVA conducted. Treatment means were separated using Games-Howell tests.

5.4 Results

5.4.1 Experiment 1: Impact of protein bait sprays on A. lingnanensis

There was no significant difference in parasitism of red scales by Aphytis lingnanensis in the Auburnvale site (χ2 = 3.241, d.f. = 3, P = 0.356) (Table 5.1) over the four sampling periods. Similarly, for the Iron bark site, no significant difference in parasitism was detected over the four sampling periods (χ2 = 7.259, d.f. = 3, P = 0.064) (Table 5.2).

86 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

Table 5.1 Number of red scales, Aonidiella aurantii (Maskell) parasitised and unparasitised by both Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) over four sampling periods between January and April 2008 in the Nova citrus variety in the Auburnvale orchard.

Sampling period Total scales Total parasitized Total unparasitised sampled First 200 146 54 Second 200 95 105 Third 200 91 109 Fourth 200 106 94

Table 5.2 Numbers of red scales, Aonidiella aurantii (Maskell) parasitised and unparasitised by both Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) over four sampling periods between January and April 2008 in the Nova citrus variety in the Iron bark orchard.

Sampling period Total scales Total parasitized Total unparasitised sampled First 200 28 172 Second 200 36 165 Third 200 33 167 Fourth 200 50 150

In Auburnvale (Figure 5.2) and Ironbark (Figure 5.3), the percent parasitism by A. lingnanensis was higher than that by C. bifasciata. Between the two sampling sites, Auburnvale had a higher rate of parasitism than Ironbark. Total percent parasitism in Auburnvale during the first, second, third and the fourth sampling periods were 80%, 56%, 60% and 62.6% respectively while Ironbark recorded 16%, 22.6%, 13.5% and 33% for the first, second, third and the fourth sampling periods, respectively.

The total number of scales parasitised by Aphytis compared to Comperiella in both Auburnvale and Ironbark was higher in all four sampling periods (Figure 5.4 and 5.5). The total number of scales that attained the mated stage was lower in Auburnvale and this can be attributed to higher rates of parasitism which parasitised and killed them before they reached this stage (Figure 5.5). Conversely, Ironbark with lower

87 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies rates of parasitism had a higher number of mated scales over all the four sampling periods (Figure 5.7).

Aphytis Comperiella

100 90 80 70 60 50 40 30 % parasitism 20 10 0 First Second Third Fourth

Sampling period

Figure.5.2 Percent parasitism of red scales, Aonidiella aurantii (Maskell) by Aphytis lingnanensis (Compere) and Comperiella bifasciata Howard over four sampling periods between January and April 2008 in Nova variety in the Auburnvale citrus orchard.

Aphytis Comperiella

60

50

40

30

20 % parasitism 10

0 First Second Third Fourth

Sampling period

Figure.5.3 Percent parasitism of red scales, Aonidiella aurantii (Maskell) by Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) over four sampling periods between January and April 2008 in Nova variety in the Iron bark citrus orchard.

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Aphytis 120 Comperiella Emerged 100

80

60 Total parasitism Total 40

20

0 First Second Third Fourth Sampling period

Figure 5.4 Number of red scales, Aonidiella aurantii (Maskell) parasitised by Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard)and their emergence out of 200 scales sampled over four periods (between January to April 2008) in the Auburnvale citrus orchard.

Aphytis 70 Comperiella Emerged 60

50

40

30 Total parasitism Total 20

10

0 First Second Third Fourth Sampling period

Figure 5.5 Number of red scales, Aonidiella aurantii (Maskell) parasitised by Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) and their emergence out of 200 scales sampled over four periods (between January to April 2008) in the Iron bark citrus orchard.

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Live unmated scale 80 Mated scale 70 Dead scale

60

50

40

30 Total number Total

20

10

0 First Second Third Fourth

Sampling period

Figure 5.6 Number of unparasitised red scales, Aonidiella aurantii (Maskell) including live unmated scales, mated scales and dead scales recorded over four sampling periods between January to April 2008 in the Nova variety in the Auburnvale citrus orchard.

Live unmated scale 160 Mated scale 140 Dead scale

120

100

80

60 Total number Total

40

20

0 First Second Third Fourth Sampling period

Figure 5.7 Number of unparasitised red scales, Aonidiella aurantii (Maskell) including live unmated scales, mated scales and dead scales recorded over four sampling periods in the Nova variety in the Iron bark citrus orchard.

90 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

5.4.2 Experiment 2: Attraction of A. lingnanensis to protein bait, protein + sugar (20%), water and honey

In the choice test comparing the attraction of Aphytis to protein, honey and water a one-way ANOVA detected a significant difference in attraction between the

treatments (F 2,177 = 197.260, P = 0.001). Protein and water were not significantly different from each other and attracted an equal number of Aphytis. Honey was significantly different from protein and water and attracted significantly more Aphytis than did protein and water. In the choice test comparing the attraction of Aphytis to protein, Protein+ sugar (20%) and water a Kruskal-Wallis test showed a significant difference in attraction between the treatments (H = 261.65, d.f = 2, P = 0.001). Protein mixed with 20% sugar attracted more Aphytis than protein alone and water. In the no choice test, a t-test comparison showed no difference in attraction

between protein and the control (t59 = -0.875, P = 0.385).

Table 5.3 Mean ± s.e number of by Aphytis lingnanensis (Compere) attracted to honey, protein and water used as a control.

Treatments Mean ± s.e Honey 71.96 ± 3.20a Protein 22.75 ± 1.07b Water 18.00 ± 1.13b

Means followed by the same letter are not significantly different (Fisher’s LSD test on log [x+1] transformed data; P = 0.05).

91 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

Table 5.4 Mean ± s.e number of by Aphytis lingnanensis (Compere) attracted to protein, protein + sugar (20%) and water used as a control.

Treatments Mean ± s.e Protein 0.94 ± 0.09a Protein + sugar 13.44 ± 0.35b Water 0.65 ± 0.82a

Means followed by the same letter are not significantly different (Games-Howell test on log [x+1] transformed data: P = 0.05).

Table 5.5 Mean ± s.e number of by Aphytis lingnanensis (Compere) attracted to protein and water used as a control.

Treatments Mean ± s.e Protein 16.93±0.96a Water 18.10±1.15a

Means followed by the same letter are not significantly different (t-test on square root [√ (x+0.5)] transformed data; P = 0.05).

5.4.3 Experiment 3: Feeding propensity of C. montrouzieri and C. carnea on protein bait

The adult mealy bug predator, C. montrouzieri showed a significant difference in

feeding times for honey over protein and water (ANOVA, F 2,45 = 70.582, P = 0.001). It fed for an equal time period on protein and water but significantly longer on honey.

92 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

The adult green lace wing, C. carnea also showed a significant difference in feeding

times between protein, water and honey (ANOVA, F 2,78 = 43.952, P = 0.001). Post- hoc comparisons showed a significant difference between all the three treatments in terms of its feeding time with longer feeding times on honey than protein and water.

Table 5.6 Feeding times of adult mealy bug predator, Cryptolaemus montrouzieri (Mulsant) on honey, protein and water under laboratory conditions.

Treatments Feeding time in seconds (Mean ± s.e.) Honey 142.78 ± 21.81a Protein 0.16 ± 0.03b Water 0.12 ± 0.02b

Means followed by the same letter are not significantly different (Games-Howell tests on square root [√ (x+0.5)] transformed data; P = 0.05).

Table 5.7 Feeding times of adult green lace wing, Chrysoperla carnea (Stephen) on honey, protein and water under laboratory conditions.

Treatments Feeding time in seconds (Mean ± s.e.) Honey 213.73 ± 16.68a Protein 71.37 ± 16.04b Water 20.14 ± 7.85c

Means followed by the same letter are not significantly different (Games-Howell tests on square root[√ (x+0.5)] transformed data; P = 0.05).

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5.5 Discussion

Heavy and frequent use of insecticides has created serious problems in agricultural ecosystems, the most prominent being the impact on non target organisms such as parasites, predators and pollinators (Katsura 1977). One major challenge in controlling insect pests within Integrated Pest Management programs that use pesticides is to devise techniques that are selective and that target only the pest species, resulting in minimum disruption to the beneficial insects (Michaud and McKenzie 2004). All chemical based pest management techniques do pose some risk to non-target organisms, but selective compounds that specifically target the pest species and spot application techniques that reduce the volumes applied, can help minimise the negative impact on the non-target, beneficial insects (Michaud and McKenzie 2004).

Apart from the traditional lethal dose estimates, effects of pesticides on target and non-target species can also be measured through biological assessed such as studying the life histories and population fitness through the use of demography and population growth rates (Stark and Banks 2003). In this study the impact of protein bait-insecticide sprays on the two parasitoids, A. lingnanensis and C. bifasciata, were assessed by studying their parasitism levels on red scale, A. aurantii, over one fruit cropping season.

Most parasitic Hymenoptera are dispersive species (Longley et al. 1997) and when observed on plants and fruits move rapidly in search of their host (personal observation). This random movement also increases their chances of coming in contact with protein bait applications in the field (Hoy and Dahlsten 1984, Daane et al. 1990). In addition, residual effects of insecticide bait sprays can also have an adverse effect on these parasitoids. For instance, in Florida, Rehman et al. (1999) showed field residues of insecticides several days post treatment to be still lethal to Aphytis holoxanthus (Debach), a major biological control agent used in controlling the Florida red scale, Chrysomphalus aonidum (Linneaus). They observed that

94 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies

Carbaryl was more persistent and killed the parasitic wasps over a much longer period of time than did Dicofol residues.

However, the results obtained in this study from the field scale parasitism assessment showed a constant rate of parasitism over the fruiting season, suggesting no significant negative impact on these parasitoids as a result of protein bait spray applications. The parasitism levels recorded over the four sampling periods did not show a significant reduction over time by both the parasitoids. However, A. lingnanensis demonstrated higher rates of parasitism compared to C. bifasciata at both study sites. The most obvious reason for this is that A. lingnanensis adults were released into the orchards where as C. bifascuta were not. Moreover, C. bifasciata, being endoparasites, are difficult to detect inside the scales (Forster et al. 1995), especially the egg stage, consequently the egg count data could not be taken into account. The overall parasitism rates were higher at the Auburnvale orchard and this can be attributed to the multiple releases of Aphytis carried out in this orchard. Regardless of the frequency and number of releases, no significant changes in parasitism rates were observed in both study sites over the four sampling periods. Use of two different insecticides as the toxicants in the baits, in the two orchards, also did not result in any site specific difference in parasitism. Auburnvale used Abamectin which is a novel natural compound derived from the soil actinomycete, Streptomyces avermitilis, and is considered safe environmentally (Lasota and Dybas 1991, Clark et al. 1995). It has been recommended as an alternative for malathion in protein bait spray programs against the Caribbean fruit fly, A. suspensa, in Florida (Hennessey and King 1996). In contrast, Iron Bark used the organophosphate insecticide malathion, which has been more widely used in protein bait sprays over a long time period.

Attraction of parasitoids to protein bait sprays may be influenced by several factors (Hoy and Dahlsten 1984). For instance, under field conditions, hunger combined with a shortage of food may result in the parasitoids being attracted to protein bait. Hence, attraction levels may vary with the insect’s physiological state and conditions

95 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies such as hunger may make them more vulnerable to bait sprays. The second experiment which evaluated attraction of Aphytis to protein baits, in a choice test, showed that honey was significantly more attractive compared to protein and water. The non attractiveness of protein bait was confirmed through the no-choice test which demonstrated no significant difference in attraction between protein and water as the control. Adding sugar to the protein bait increased its attraction to Aphytis. This suggests that adding sugar to enhance attraction of fruit flies to protein baits, as proposed by some fruit fly control recommendations, can have an impact on non-target insects.

Aphytis in the presence of other natural food sources, such as honey dew, would avoid feeding on the available formulation of protein bait sprays even when hungry, resulting in minimal impact on its populations. However, attraction should generally be only taken as conservative criterion for predicting an effect (Asquith and Messing 1992). Nonetheless results from the field analyses in this study, combined with the laboratory attraction study, indicate that the observed non significant impact of protein bait sprays on A. lingnanensis and C. bifasciata is largely due to their unattractiveness to these parasitoids. One would expect higher mortality rates and a consequent reduction in parasitism, if protein baits were attractive to these insects.

These results are consistent with the findings of Purcell (1994), where D. longicaudata an important larval-parasitoid of B. dorsalis was reported to be at a lower risk to malathion in Staley’s protein bait because of their lack of attraction to the bait. In a previous study, Smith and Nannan (1988) reported the unattractiveness of yeast autolysate to A. lingnanensis in a choice test, but they did not examine the attraction of this insect to protein in a no choice situation and on the adults at different physiological states. Moreover, their study lacked proper statistical design as repeated counts of adult A. lingnanensis taken over time, were considered as replicates.

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It should also be noted that, in the past, in the few Hymenoptera that have been studied for protein attractancy, no strong positive response has been found. Hoy (1984) found three species of hymenoptera, Encyrtus saliens (Prinsloo and Annecke), a parasite of ice plant scale , Trioxys pallidus (Haliday), a parasite of walnut aphid and Euderomphale flavimedia (Howard), a parasite of iris white fly, showed either neutral or negative responses to protein bait. Daane (1990), similarly observed that the two aphid parasitoids, Aphidius liriodendrii (Liu) and Trioxys curvicaudus (MacKauer) were neither attracted nor repelled by protein bait containing malathion. However, in Hawaii, Asquith (1992) reported a diverse group of litter- inhabiting invertebrates including nondiptera, Diptera and Drosophilidae to be significantly attracted to pitfall traps baited with protein bait. Attraction may therefore vary between closely related species as it is a trait that may be specific to a particular species rather that at the genus or family level (Asquith and Messing 1992).

The third experiment assessing the feeding propensity of hungry adult C. carnea and C. montrouzieri, on Pinnacle Autolysed Yeast showed that they preferred feeding on honey rather than protein or water. The feeding time of adult C. carnea on Pinnacle Autolysed Yeast was significantly longer than on water but significantly shorter than on honey. As adult C. carnea feed on nectar, honeydew and pollen in the field (Principi and Canard 1984), this result suggest that Pinnacle protein would be the least preferred food compared to sugary food like honey dew in the field. These results are in agreement with the findings of Nadel et al. (2007) where they reported that the feeding time of adult C. carnea on GF-120 fruit fly bait in the presence of honey was significantly shorter and suggested that it would demonstrate a similar response pattern in the presence of honey dew or nectar in the field. They suggested that their preference to feed on sugary food like honey can lower their chances of coming in contact with protein bait sprays in the field. However, a remote chance does exist that hungry adult C. carnea could feed on bait sprays if natural food sources are in short supply, because in this study the feeding time obtained on Pinnacle protein was significantly longer than on water. Conversely,

97 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies hungry adult C. montrouzieri did not exhibit such feeding behaviour, as no significant difference in feeding time was observed between Pinnacle protein and water.

It could be hypothesised, therefore, that this insect is highly unlikely to feed and come in contact with protein bait sprays in the field other than through accidental contact.

Hagen (1986 ) found that applying artificial honey dew to crops helped in attracting and retaining various predatory insects that normally fed on honey dew as a food source. Similarly, several reports exist in which artificial honey dew with sucrose as the main ingredient, has been employed in attracting and retaining major predators in field crops (Ewert and Chiang 1966, Schiefelbein and Chiang 1966, Hagen et al. 1971, Ben Saad and Bishop 1976, Nichols and Neel 1977, Evans and Swallow 1993, Evans and Richards 1997). Field and laboratory studies have shown that the fitness of some Aphytis species such as A. melinus is directly related to the availability of carbohydrate sources like honey and flower-nectar (Jeing-Wei 1987). Moreover, its searching capacity was found to be greatly dependent on the availability of a carbohydrate source. Hence, providing such food as alternative sources may be useful in conserving and improving the effectiveness of predators, parasitoids and natural enemies, whilst minimizing the risk of them coming in contact with bait spray applications.

In summary, this study confirmed no possible impact of protein bait sprays on A. lingnanensis and C. bifasciata. Hungry A. lingnanensis are most unlikely to be attracted to bait sprays even in the absence of other natural food sources. Important predators like C. carnea and C montrouzieri preferred feeding on sugary food like honey rather than the Pinnacle Autolysed Yeast. Future studies with experiments carried out over a large geographic area, over a longer period of time, would give more insight into the impact of pesticides on the non target species (Burn 1988). Also, studies should focus on testing the impact of residual effects, behavioural responses and sub-lethal effects of some of the insecticides tested here in this study, on some of the important parasitoids and natural enemies commonly

98 Chapter 5: Attraction, feeding and impact of protein bait sprays on arthropod natural enemies occurring in Queensland citrus. Such studies would help gauge the advantages of using novel toxicants in place of older compounds like malathion. A precise understanding of the biological impacts of new toxicants on beneficial organisms can prove useful in incorporating improved strategies and techniques into existing fruit fly pest management programs using protein bait sprays, which in turn can help minimise the impact on beneficial and non-target organisms.

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Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on the red scale parasite, Aphytis lingnanensis (Compere) (Hymenoptera:Aphelinidae)

101 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

6.1 Introduction

Apart from target pest species, pesticide applications often harm beneficial insects that play an important role in biological control of pest insect populations (Ulmer et al. 2006). A clear understanding of the impact of specific pesticides on the survival and effectiveness of different natural enemies is crucial in an integrated pest management (IPM) program (Desneux et al. 2006). Further a modern pest management program requires the use of selective insecticides that are effective in controlling target pest species while having minimal impact on important natural enemies (Hassan 1989).

Chapter 3 presented data on the toxicity of different protein bait-insecticide mixtures on B. tryoni. Apart from evaluating the toxicological effect of an insecticide on the target pest, its negative impact on beneficial organisms should also be assessed (Wang et al. 2008). Therefore this chapter presents research aimed at determining the acute contact toxicity of protein bait-insecticide formulations on A. lingnanensis.

Previous research has shown that two natural enemies of the red scale, Aphytis melinus Debach and C. bifasciata, were susceptible to malathion and consequently its integration with biological control of red scales was considered unsuitable (Abdelrahman 1973). Residues of 0.15% malathion were highly lethal to the parasite, A. melinus, released to control the California red scale, A. aurantii Maskell(Campbell 1975). Hence, it was recommended that the release of this parasite be delayed by 30 to 50-d post treatment with malathion, to minimize the insecticide impact on these parasitoids. A 48-hr residual toxicity test indicated that A. melinus was more susceptible to major pesticides used for control of citrus thrips, Scirtothrips citri (Moulton) and California red scale, A. aurantii, compared to C. montrouzieri a large and robust predator of various mealybug species (Morse and Bellows 1986). Aphytis sp. exposed to citrus leaves treated with chlorpyrifos at the rates of 0.1 and 0.5%, enclosed in a Petri dish, were highly susceptible to the pesticide when first applied

102 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

and then 48-h after spraying (Hassan 1997). Laboratory feeding tests with fruit fly baits containing spinosad (GF-120) and Nu-lure containing malathion, demonstrated high mortality to two parasitoid wasps, A. melinus and Lysiphlebus testaceipes (Cresson) within a 24-h period (Michaud 2003). However, the percent mortality obtained with Nu-lure containing a high concentration of malathion (195,000ppm) was higher than that obtained with GF-120. Rill et al. (2008) observed a high level of mortality of emerging adults of A. melinus, after 48-h exposure of insects to residues of the neonicotinoid insecticide acetamiprid, but indicated no significant effect on the development of the pupal stage to adults. However, two insect growth regulators, buprofezin and pyriproxyfen, had no significant effect on the fecundity of A. melinus and its development of immature stages to adults.

Assessing the effects of pesticides on minute Hymenoptera is difficult because of their very small size and delicate external structure (Williams III and Price 2004). To overcome this difficulty, Williams III and Price (2004) developed and recommended a novel bioassay technique specifically designed to assess the impact of foliar resides of different insecticide on acute mortality of minute Hymenoptera. Using this technique, they tested spinosad, thiamethoxam and oxamyl and found these compounds to be highly toxic, each with a varying degree of toxicity on the two egg parasitoids, Anaphes iole (Girault) and Trichogramma pretiosum (Riley). In this study, they concluded that pesticide effects on parasitoid species, and closely related insect species, can vary greatly and therefore should not be generalized.

No previous studies have tested the residual toxicity and other negative effects of protein bait-insecticide mixtures on the fecundity of A. lingnanensis. This study was designed to evaluate the toxicity of fresh and aged protein bait-insecticide mixtures, applied on citrus leaves, to A. lingnanensis and to determine their effect on its fecundity.

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6.2 Materials and Methods

6.2.1 Test Insects

The test insects for this experiment were supplied from the Bugs for Bugs P/L insect rearing facility in Mundubbera. A. lingnanensis was reared on Butter nut pumpkins (Cucurbita moschata) infested with Oleander scale, Aspidiotus nerii (Boucher). They were transported to the International Centre for the Management of Pest Fruit Flies (ICMPFF) laboratory, Griffith University, Brisbane, in a similar manner as described in chapter 5. On arrival, the insects were placed in plastic jars (18 cm height: 16 cm width) with their end removed and replaced with fine nylon fabric mesh for aeration. They were fed with honey and water solution (1:1) by applying to the nylon fabric with a paint brush. Water was sprayed twice daily on the nylon fabric by an atomizer. The experiments were conducted in the ICMPFF laboratory.

The protein bait-insecticide mixtures tested were the same as those used in chapter 3 and 4.

6.2.2 Protein bait-insecticides application

Treatments were applied to leaves of five sweet orange trees, Citrus sinensis (Linnaeus) and aged for 2-h, 3-d, 7-d and 12-d. Citrus leaves were used because exposure of natural enemies to leaves treated with pesticides is considered the most appropriate method of determining the toxicity of post-application residues on insects that are not sprayed directly with the pesticide, in an orchard situation (Williams III et al. 2003).

The method of applying the protein bait-insecticide mixtures to the citrus plants was similar to that described for the protein bait-insecticide ageing test conducted with Queensland fruit fly, in chapter 4. The daily temperature was recorded with a

104 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

maximum and minimum thermometer and the relative humidity was recorded at 9 am in the morning and 3 pm in the afternoon, during the period of the experiment.

6.2.3 Residual toxicity of protein bait-insecticide mixtures to adult Aphytis lingnanensis

The method and equipment used to expose A. lingnanensis to the protein bait- insecticide mixtures was adopted, with slight modification, from that described by Williams III and Price (2004) which were designed for specifically assessing foliar contact residues of pesticides to minute Hymenoptera.The The exposure units consisted of plastic cylinders (2cm diameter: 4cm height) with their ends uniformly cut and removed. Four holes (0.5cm diameter) were cut equidistant from each other, in the centre of the tube. Two opposite holes were sealed with fine nylon mesh for ventilation. The other two were plugged with cotton wool soaked with few drops of water or a 1:1 honey and water mixture for the insects to feed.

105 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

Citrus leaf Glass slide

Feeding and 4cm ventilation Cotton with honey

2cm (b)

(a)

Figure 6.1. A schematic design of the exposure unit used to test the residual toxicity of protein bait-insecticide mixtures to adult Aphytis lingnanensis (Compere). The unit was made of a transparent plastic tube (2cm diameter: 4cm height) with four holes (0.5cm diameter) (two used for ventilation and two for feeding honey + water). Treated leaves were placed on glass slides and exposed to Aphytis lingnanensis (Compere) by placing on the top and bottom of the tube. (a) Components of the exposure units. (b) An assembled exposure unit.

For the experiment 30-35 A. lingnanensis were transferred to holding glass vials (1.5 cm diameter: 4 cm height) through a flexible plastic tube connected to an electric pooter. Citrus leaves with the various treatments were removed from the plants and attached to microscope slides (7cm by 2 cm) with the aid of double sided tape (Scotch, USA). Two slides with the leaves attached were placed on the tube in such a

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manner that both the upper and lower surfaces of the leaves were exposed. This arrangement simulates field conditions by exposing the upper and lower side of the leaves to the parasitoids. After the parasitoids were transferred to the tubes from the holding glass vials, the slides with the leaves were held in place by two rubber bands. There were five replicates for each treatment and the control. The exposure units were held under controlled conditions of 22-23 °C and relative humidity of 65 ± 10%, and a photoperiod of 12:12 (L:D).The units were placed under a continuous cycle of fresh air, pumped by an air conditioner, to avoid any possible mortality through a localised fumigation effect. The laboratory was illuminated with a combination of fluorescent lights and natural light through glass windows. After 24- h, the dead parasitoids were counted and recorded.

6.2.4 Effect of protein bait-insecticide exposure on the fecundity of Aphytis lingnanensis

The ability of adult A. lingnanensis to parasitise scale insects, after a 24-hr period of exposure to the five different protein bait-insecticide mixtures, was recorded by introducing them to Butter nut pumpkins that were infested with unparasitized Oleander scale. As the pumpkins were fully covered with scales, some of the scales were scrapped from the pumpkins using a scalpel blade in order to expose a similar surface area of scales to parasitoids from the different treatments. The scales were approximately 6-w old third instars, the preferred stage of Aphytis spp. for oviposition (Forster et al. 1995). For each treatment three butternut pumpkins were used. The scrapped part of each pumpkin was cleaned with a tissue paper and placed separately in a plastic container (18 cm wide: 11 cm height) with its top covered and sealed with a fine nylon mesh. Live parasitoids from the exposure units were gently placed on the pumpkins. The inside of the container was streaked on one side with honey-water mixture, with a fine paint brush, for the parasitoids to feed. Every consecutive day, water was provided by a single spray using an atomizer. The pumpkins were held under controlled conditions of 22-23°C and relative humidity of 65 ± 10%, and a photoperiod of 12:12 (L:D). They were placed under a

107 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

continuous cycle of fresh air circulated through an air conditioner. After 14 days of exposure to A. lingnanensis, 60 scales from each of the three pumpkins were randomly selected and dissected for parasitisation. Presence of Aphytis eggs, pupae and emerged parasitoids (scales with exit holes), if present, were recorded.

6.3 Statistical analysis

The percent mortality data was corrected for control mortality using Abbott’s fromula [(% treatment mortality − % control mortality)/ (100 − % control mortality)] x 100 (Abbott 1925). In spite of standard transformations the data failed to meet the assumptions of the ANOVA. Therefore, the non-parametric equivalent, Kruskal- Wallis test was used.

6.4 Results

6.4.1 Residual toxicity of protein bait-insecticide mixtures to adult Aphytis lingnanensis

A Kruskal-Wallis test showed a significant difference in parasitoid mortality between the treatments with 2-hr ( H = 23.641, d.f = 4, P= 0.001), 3-d ( H = 23.641, d.f = 4, P = 0.001 ) , 7-d ( H = 15.694, d.f = 4, P = 0.003) and 12-d ( H = 16.476, d.f = 4, P = 0.002) aged leaves. For 2-hr, 3-d and 7-d aged leaves, post-hoc comparisons revealed a significant difference between the protein control and the four protein treatments (spinosad, chlorpyrifos, fipronil and malathion) (Table 6.1). Leaves aged for 12-d showed a significant difference in mortality between the control and the four treatments (spinosad, chlorpyrifos, fipronil and malathion) and between spinosad and chlorpyrifos) (Table 6.1). Parasitoid mortality obtained for the 12-d aged leaves was significantly lower for spinosad than chlorpyrifos (Table 6.1).

108 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

Table 6.1. Percent mortality (mean ± s.e) of Aphytis lingnanensis (Compere) adults observed after 24-h exposure to citrus leaves with different protein bait- insecticide mixtures weathered for 2-hr, 3-d and 7-d and 12-d.

Aged Leaf after 24-h Treatment % mortality of A. lingnanensis 2-hours Chlorpyrifos 100.0 ± 0 a Malathion 100.0 ± 0 a Spinosad 100.0 ± 0 a Fipronil 100.0 ± 0 a Control 2.2 ± 0.66 b

3-days Chlorpyrifos 100.0 ± 0 a Malathion 100.0 ± 0 a Spinosad 100.0 ± 0 a Fipronil 100.0 ± 0 a Control 5.5 ± 0.78 b

7-days Chlorpyrifos 100.0 ± 0 a Malathion 94.9 ± 5.41 a Spinosad 95.8 ± 2.53 a Fipronil 95.0 ± 3.05 a Control 2.0 ± 0.83 b

12-days Chlorpyrifos 100.0 ± 0 a Malathion 98.5 ± 0.59 ab Spinosad 89.9 ± 4.93 b Fipronil 95.0 ± 3.05 ab Control 1.6 ± 0.67 c

Means followed by the same letter are not significantly different (Games-Howell test (for 2-h, 3-d,7-d and 12 d data) on Arcsine square-root transformed data: P = 0.05).

109 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

6.4.2 Effect of protein bait-insecticide exposure on the fecundity of Aphytis lingnanensis

Due to an insufficient number of live adult A. lingnanensis, parasitism levels for two exposure periods (2-h and 3-d aged residues for all treatments) and one treatment (chlorpyrifos for all weathering periods) could not be assessed. A Kruskal-Wallis test showed a significant difference in percent parasitism for 7-d (H = 57.83, d.f = 3, P = 0.001) and 12-d (H = 70.99, d.f = 3, P = 0.001) residues. Post-hoc test revealed a significant difference only between control and protein bait-insecticide containing spinosad, malathion and fipronil. The percent parasitism for the control for 7-d and 12-d was 81.03% and 77.03% respectively (Table 6.2). The percent parasitism of A. lingnanensis exposed to 7-d and 12-d aged protein bait-insecticide containing spinosad, malathion and fipronil was < 2.7% and < 5.1% respectively (Table 6.2).

Table 6.2. Percent parasitism inflicted by surviving adult Aphytis lingnanensis (Compere) on Oleander scales, Aspidiotus nerii (Boucher) on butternut pumpkins after being exposed to aged residues of protein bait-insecticide mixtures for 24-h.

Treatment % parasitism by A. lingnanensis 7-day aged residue 12-day aged residue Malathion 1.5 a 1.8 a Spinosad 2.7 a 5.1 a Fipronil 2.4 a 2.0 a Control 81.0 b 77.0 b

Means followed by the same letter are not significantly different (Games-Howell test on Arcsine square-root transformed data: P = 0.05).

110 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

Minimum Maximum

35

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15 Temperature(ºC) 10

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0 19/01/09 20/01/09 21/01/09 22/01/09 23/01/09 24/01/09 25/01/09 26/01/09 27/01/09 28/01/09 29/01/09 30/01/09

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Figure 6.2. Maximum and minimum temperature recorded during the experiment days.

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30 Average relative humidity(%) 20

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Figure 6.3. Average relative humidity (%) recorded during the experiment days.

111 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

6.5 Discussion

Chemical treatments applied to insect pests can, as a side effect, induce a variety of negative impacts on natural enemies. Insecticides cause lethal and sub lethal effects on natural enemies, where lethal effects are often expressed as acute or chronic mortality resulting from contact with or ingestion of insecticides, while sub lethal effect are not always apparent (Haseeb and Amano 2002). The sub lethal effects of pesticide use on natural enemies of pests are poorly known, and without researching this selectivity, risks and hazards associated with pesticides can not be understood (Jones et al. 1998, Amano and Haseeb 2001). Indeed, knowledge of the effect of the impacts of pesticides on natural enemies would be crucial when combining chemical and biological control strategies in an integrated pest management program (Greathead 1995).

By nature many minute adult parasitoids spend a significant amount of time on foliage where they feed, mate, rest and search for hosts. Therefore experiments designed to assess the toxic effects of direct contact between parasitoids and insecticide residues, using leaf contact, provides a closer representation of field conditions (Williams III and Price 2004). This study was conducted to determine the contact toxicity of fresh and aged residues of protein bait containing spinosad, malathion, chlorpyrifos and fipronil on citrus leaves, to adult A. Lingnanensis, and their effect on its ability to parasitize scale insects after a 24-h period of exposure to these compounds. There are two aspects involved in determining the contact toxicity of insecticide residues (Williams III et al. 2003). Firstly, the initial mortality resulting from a fresh application and secondly the changes in mortality as the residues age and/or dissipate.

The results obtained from this study strongly indicated that all compounds tested were highly toxic to the parasitoids through direct contact. Freshly applied baits and 3-d aged baits were equally toxic, resulting in 100% mortality for all the treatments except the control. A slight weathering effect was recorded in baits aged for 7-d and

112 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

12-d where the mortality for spinosad, malathion and fipronil declined below 100 %. Spinosad aged for 12-d inflicted a significant lower mortality than 12-d aged chlorpyrifos, indicating spinosad to be less toxic than chlorpyrifos after that period of weathering. Though no significant differences between chlorpyrifos, malathion and fipronil were observed, chlorpyrifos inflicted 100% mortality, after all periods of weathering. In other research, chlorpyrifos had the highest acute contact toxicity to Anagrus nilaparvatae (Pang et Wang), a major natural enemy of the rice planthopper Nilaparvata lugens (Stal) (Wang et al. 2008). Similarly chlorpyrifos residue was persistent in nature in its toxicity to two predatory bugs, Dicyphus tamaninii (Wagner) and Macrolophus caliginosus (Wagner), for up to 30 days (Figuls et al. 1999). Moreover, the results here demonstrated a significant difference in parasitism between A. lingnanensis exposed to protein bait-insecticide residues and the control, indicating that if parasitoids come in direct contact with aged residues of spinosad, malathion, chlorpyrifos and fipronil, their capacity to parasite their hosts would be markedly reduced.

The results here also indicate that the reduced-risk compound spinosad is also toxic to adult A. lingnanensis and these align with results from other research where spinosad has been reported to be toxic to many hymenopteran parasitoids (Mason et al. 2002, Williams III et al. 2003, Haseeb et al. 2004, Schneider et al. 2004, Williams III and Price 2004, Jones et al. 2005, Penagos et al. 2005b). A review of published studies on spinosad and its effect on predators and parasitoids pointed out that spinosad is one of the most judicious insecticides available for use in pest management systems that involve predator populations (Williams et al., 2003). However, hymenopteran parasitoids were found to be significantly more susceptible to spinosad than predatory insects.

In addition, spinosad has also been reported to be harmful to natural enemies of fruit flies. Three major parasitoid species of tephritid fruit flies, Fopius arisanus (Sonan), Pysttalia fletcheri (Silvestri) and Diachasmimorpha tryoni (Cameron) were observed to be susceptible to GF-120 (Wang et al. 2005). Stark et al (2004b) also

113 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

reported spinosad, at high concentrations, to be highly toxic to F. arisanus (Sonan) and P. fletcheri (Silvestri), two important braconid parasitoid species of tephritid fruit flies. Ruiz et al.(2008), in laboratory studies, also reported GF-120 bait containing spinosad as toxic to another species of tephritid fruit fly parasitoid, D. longicaudata (Ashmead).

Contrary to results from laboratory studies, large scale field studies in Hawaii in coffee plantations treated with a protein bait malathion formulation indicated a slower recolonization and lower resulting population numbers of F. arisanus (Sonan) compared to fields treated with protein bait and spinosad (Vargas et al. 2001). Similarly, populations of specific indicator species of parasitoids such as Aphytis spp and C. bifasciata and other beneficial organisms did not fluctuate significantly when citrus groves were treated with spinosad bait sprays to control the Mexican fruit fly, Anastrepha ludens (Loew) in the Rio Grande valley of Texas (Thomas and Mangan 2005). These results suggest that spinosad in the field can be less toxic to parasitoids although this does require further evaluation.

Many studies have reported that spinosad residues degrade rapidly in the field. Published studies reviewed by Williams (2003) point out that it degrades quickly with little residual toxicity at 3-7 days post application and McLeod (2002) and Thompson et al. (2002) reported a post application toxicity for approximately 7-10 days. However, dry weather conditions have been reported to enhance the persistence of spinosad residues. For instance, Williams III (2003) showed that field aged residues (0, 2, 4, 8, 16, 23, and 30 days residue) of cyfluthrin, spinosad, and fipronil had a higher residual toxicity, compared to acephate, λ -cyhalothrin, imidacloprid, thiamethoxam and oxamyl, to Anaphes iole (Giraultan), an egg parasitoid of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). In addition, 50% parasitoid survival was observed when cyfluthrin, spinosad, and fipronil were weathered in the field for≥ 30 days which was significantly greater than that for other insecticides tested.

114 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

The results here also demonstrate a longer residual action for spinosad, where baits aged up to 12-d caused 89% parasitoid mortality. One explanation for this could be that the citrus plants treated with the protein bait-insecticide formulations were only exposed to sunlight and not rainfall. In field conditions, degradation and persistence of foliar residues of pesticides is a process dependent on various environmental factors (Willis and McDowell 1987, Bentson 1990). Rainfall is one important factor which can degrade insecticidal residues instantaneously. Heavy rain has been observed to wash away protein bait sprays from treated foliage rapidly, resulting in an increase in the Mediterranean fruit fly, C. capitata populations in plantations treated with different protein bait formulations mixed with either spinosad, phloxine B or malathion, in the Hawaiian island of Kauai (Peck and McQuate 2000).

Nonetheless, without exposure to rainfall, the degradation due to ultraviolet light in the field will normally be expected to be more than that on plants grown under cover or within glass houses, without exposure to direct sunlight. For instance, in Hawaii, Prokopy et al. (2003) reported that GF-120 bait spray droplets containing spinosad lost 50% of their toxicity after 4 days of outdoor weathering, without being exposed to rainfall, but exposure to 8mm of simulated rainfall rendered the bait almost ineffective in killing protein deprived adult B. cucurbitae. This indicated that ultraviolet exposure alone can also cause a rapid degradation of insecticide residues, without exposure to rainfall. Therefore, as pointed out by Williams and Price (2004) the level of toxicity of novel compounds purported to be safer on beneficial insects can vary greatly between insect species and natural enemies and the results obtained here support this as A. lingnanensis compared to other beneficial organisms or insect species, appeared very susceptible to protein bait-insecticide residues.

Spinosad acts primarily as a stomach poison and to a lesser degree by contact (Penagos et al. 2005b). Consequently, the parasitoid mortality resulting from ingestion of toxicants in bait would have been minimum, in the exposure units in

115 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

this study, as it was demonstrated in chapter 5 that adult A. lingnanensis are not attracted to protein bait sprays. Hence, compared to organophosphates such as malathion, that induce high mortality rates by contact, spinosad has been considered safer to beneficial organisms. However, the results obtained here indicated that A. lingnanensis adults to be highly susceptible to spinosad through contact as well. Reports from othe studies have also indicated parasitoids to be susceptible to this compound through ingestion as well as contact (Haseeb et al. 2004, Williams III and Price 2004). Consoli (2001) observed high adult mortality in Trichogramma galloi (Silva) while coming in contact with host eggs that were surface treated with spinosad. Similarly, Ruiz et al.(2008) in a laboratory study found that spinosad GF-120 bait proved highly toxic through both contact and ingestion and also severely affected the reproductive capacity of D. longicaudata (Ashmead), a braconid parasitoid of tephritid fruit flies.

The Working Group “Pesticides and Beneficial Organisms” of the “International Organization for Biological Control and Integrated Control of Noxious and Plants/West Palaearctic Regional Section (IOBC/WPRS)” classifies the effect of toxicants using specific IOBC toxicity ratings. The scale has four levels, 1= harmless (<30% mortality), 2= slightly harmful (30-79%), 3=moderately harmful (80-90%) and 4=harmful (>99%) (Hassan 1989). The results from the laboratory tests discussed herein indicated that all insecticides tested (spinosad, chlorpyrifos, malathion, fipronil) were harmful to A. lingnanensis and probably are in the 3 to 4 category. Pesticides found harmful to beneficial organisms under laboratory conditions should be tested further through semi-field and field tests as recommended by IOBC/WPRS (Hassan 1989).

In conclusion, careful consideration should be given when releasing A .lingnanensis after a spray program as these results indicate that they can be highly susceptibility to insecticide residues. Cover sprays will have a more adverse impact on parasitoids than selective spot spraying as the probability for parasitoids coming in direct contact with pesticide residue increases with the plant surface area treated. If possible, the impacts of insecticides on important predator and parasitoid 116 Chapter 6: Toxicity and effect of different protein bait-insecticide mixtures on A. lingnanensis

populations should be documented for every formulation used in insect pest control (Rill et al. 2008). Indeed, future research should concentrate more on evaluating novel compounds with a selective mode of action on these parasitoids. In particular, studies should be focused on measuring the residual toxicity of different insecticides in the field, and also comparing the impacts of cover and spot sprays on A. lingnanensis. Results from such field research would assist in making correct decisions on the timing of parasitoid releases after spray programs and thus enhance the effectiveness of these parasitoids, particularly where the integration of biological and chemical control strategies are of prime importance.

117

Chapter 7: General discussion

CHAPTER 7: GENERAL DISCUSSION

7.1 GENERAL DISCUSSION

A land mark development in fruit fly control was the discovery and effective use of protein based baits in Hawaii by Steiner (1952b). Ever since that discovery, protein bait sprays have been and will remain one of the most important and effective techniques available for field pest management of tephritid fruit fly pest species (Fletcher and Bateman 1982). As this technique has had a proven effectiveness in fruit fly control programs (Steiner et al. 1961, Bateman and Arretz 1973, Fletcher and Bateman 1982, Vijaysegaran 1989), there has never been a long-term ongoing effort to find new formulations with improved efficacy. However, the use of the organophosphate insecticide, malathion, in protein baits has always been a matter of contention and this toxicant has always come under scrutiny. Its use has raised concerns because of its adverse effects on human health (Flessel et al. 1993), the environment (Newhart 2006) and beneficial organisms (Daane et al. 1990, Hoelmer and Dahlsten 1993). Moreover, with increasing awareness of the roles biological control agents or natural enemies play in pest regulation (Croft et al. 1998), incorporation and use of selective or new generation of insecticides in integrated pest management program remains unequivocal.

In this thesis, I investigated the potential for incorporating new toxicants as alternatives to the old broad spectrum organophosphate insecticides in protein bait sprays for controlling B.tryoni. Moreover, I also examined fruit fly attraction and feeding responses and effects of different fruit fly protein bait-insecticide formulations on important arthropod natural enemies in citrus.

The power of protein bait sprays to attract fruit flies and stimulate feeding on them to a great extent determines the effectiveness of this strategy in field pest management. In order to determine the differences in attraction and feeding responses of B. tryoni to protein baits incorporated with various insecticides

119 Chapter 7: General discussion (chlorpyrifos, malathion, fipronil and spinosad), a comparative field cage attraction test and laboratory feeding test were conducted (Chapter 3). The field cage attraction study revealed that protein baits incorporated with spinosad, fipronil, malathion and protein alone attracted equal numbers of both male and female flies. The only difference detected in attraction was with female flies where significantly more were captured in traps baited with protein-spinosad than those containing protein-chlorpyrifos. This result indicates that the type of insecticide mixed with protein bait has the potential to reduce the attractiveness of the bait component. Consequently, the choice of the insecticide component in the protein bait mixture should also be given a careful consideration. Such differences in attraction and feeding responses of the fly to protein bait and toxicant mixtures can influence the outcome of a bait spray control programs (McQuate et al. 2005a).

The laboratory feeding test conducted with adult female B.tryoni demonstrated that protein baits containing malathion and chlorpyrifos deterred flies from feeding on them, while flies were not repelled from feeding on protein baits mixed with spinosad and fipronil. The feeding times recorded on the spinosad and fipronil mixtures did not vary significantly from the control which was protein alone. Similar feeding inhibition with with malathion protein bait mixture has also been reported in the Mediterranean fruit fly (Prokopy et al. 1992, Vargas et al. 2002).

The evaluation of toxicity and effects of ageing of protein bait-insecticide mixtures exposed on citrus leaves, on the mortality of adult B. tryoni (Chapter 4), showed the organophosphate insecticides (malathion and chlorpyrifos) to be more toxic than fipronil and spinosad. Protein baits containing malathion and chlorpyrifos applied on citrus leaves and weathered outdoors demonstrated longer residual effectiveness in killing flies than did those containing spinosad and fipronil. Spinosad degraded more rapidly than the other insecticides that were tested. Several other studies have also reported a short residual activity for spinosad (Williams et al. 2003). In summary, toxicants such as malathion, chlorpyrifos and fipronil in the field will be more persistent than spinosad.

120 Chapter 7: General discussion The mode of action of malathion and chlorpyrifos is through contact contact ingestion and to some extent by inhalation and affects the nervous system by inhibiting the activity of acetyl cholinesterase (Matsumura 1975, NRA 2000). Such contact insecticides can, therefore, kill non target organisms that come in random contact with them. As malathion and chlorpyrifos are highly toxic to fruit flies, they will most likely have a similar impact on non-target organisms. Spinosad, on the other hand, has to be ingested to be effective and is regarded as having minimum contact toxicity (Prokopy et al. 2003, Penagos et al. 2005a, Mangan et al. 2006). Therefore using spinosad can potentially reduce the chances of non-target organisms being killed by random contact. Apart from this, modern pest management requires pesticides that do not persist for a longer period than necessary to achieve the purpose for which they are applied (Frazer 1967). Also, because spinosad broke down after three days of out door weathering and, at the same time, demonstrated a high toxicity during that period, killing up to 90% of the flies in the toxicity test, it fits the concept of toxicant that can be used in modern pest management programs. In summery, this study has demonstrated that spinosad can be a suitable alternative to broad spectrum insecticides in baits sprays for controlling B. tryoni.

Our knowledge of the impact of protein bait formulations on arthropod natural enemies is scarce and often based on general field observations. In order to obtain a clearer understanding of such impacts, I investigated the influences of protein bait sprays specifically on A. lingnanensis by evaluating their parasitism rates of red scales over one growing season in two citrus orchards (Chapter 5). Moreover, I tested the attraction of Aphytis to Pinnacle protein bait through a choice and no- choice test and the feeding behaviour of the lace wing, C. carnea and mealy bug predator, C. montrouzieri on Pinnacle protein (Chapter 5). No significant differences in parasitisation rates were detected over the four sampling periods. Therefore unlike the potential side effects of cover sprays on beneficial organisms (Discussed in Chapter 1), the results obtained suggest that protein bait sprays applied as spot sprays per se will have a minimum negative impact on the population of these

121 Chapter 7: General discussion natural enemy in an orchard system. Moreover, food deprived Aphytis were not attracted to Pinnacle protein in both choice and a no-choice situations in the laboratory tests. The non attractiveness of protein bait was confirmed when adding 20% sugar solution to Pinnacle protein made it attractive to Aphytis. Hence, the current formulation of Pinnacle protein will just attract fruit flies while being unlikely to attract Aphytis, in the field. The feeding behaviour of the mealy bug predator did not indicate that it will feed on protein bait in the field, but lace wings are likely to feed on it if hungry.

Very little information is available on the specific influences of new compounds on important natural enemies, particularly their lethal and sub lethal effects (Kim et al. 2006). In the last chapter, I tested the toxicity of fresh and aged residues of protein bait-insecticides mixtures containing either chlorpyrifos, malathion, fipronil or spinosad, on citrus leaves, to Aphytis and its subsequent effect on its fecundity (Chapter 6). All the compounds aged for 2-h, 3-d, 7-d and 12-d caused high mortality and severely disrupted the fecundity of Aphytis. However, spinosad after 12-d of out door weathering, caused a lower mortality than chlorpyrifos. Other studies have reported spinosad to be toxic to hymenoptera parasitoids (Mason et al. 2002, Williams III et al. 2003, Haseeb et al. 2004, Schneider et al. 2004, Williams III and Price 2004, Jones et al. 2005, Penagos et al. 2005a). Predators are known to be more tolerant to insecticides than parasitoids (Croft and Brown 1975). Similarly spinosad is know to be less harmful to predators than to parasitoids, and therefore has been recommended as one of the most judicious insecticides for use where conservation of predators are of prime importance (Williams et al. 2003). Though spinosad may be classified as an environmentally and toxicologically reduced risk material (Williams et al. 2003), the data obtained with the toxicity test indicated that Aphytis was highly susceptible to all the insecticides tested. Hence, their release in the field should be timed appropriately in order to minimize the possible negative impact of any insecticidal based treatment. This result obtained here is the first of its type to demonstrate the toxicity of spinosad to A. lingnanensis and its adverse effect on the fecundity of the parasitoid.

122 Chapter 7: General discussion

7.2 Future research

1) The behavioral responses of B. tryoni especially attraction to and feeding on other commercially available fruit fly protein lure formulations needs to be evaluated. Such comparative studies will provide important insights that can help incorporate changes to improve the existing fruit fly protein bait used in controlling this and other pest species. In addition, the efficacy of various novel and selective toxicants as potential replacements for malathion in fruit fly protein bait, needs to be tested and validated through large scale field trials.

2) Cayol (2000) reviewed changes in behaviour and life history strategies that can occur due to selection in laboratory reared flies and highlighted the fact that wild flies can significantly differ in many aspects of behaviour compared to laboratory reared ones. Prokopy (1992) reported an equal degree of attraction with treatments containing bird feces and PIB-7 in wild and laboratory cultures of C. capitata, but a greater degree of discrimination between the treatments was elicited by wild flies compared to laboratory-cultured flies. Keeping in mind the paucity of behavioural response information in tephritid pest species to protein bait sprays (Prokopy et al. 1992), behavioural response patterns of wild and laboratory cultured B. tryoni flies, in different physiological states, requires further understanding that can be addressed through laboratory and field based studies.

3) Studies on the process of protein bait degradation in the field and means to improve its longevity and effectiveness need to be investigated. Novel compounds, which are relatively safe to on non target arthropods, possessing a longer residual effectiveness on pest organisms need to be screened through field testing. Furthermore, a study on the sub-lethal effects of spinosad and other novel toxicants needs to be carried out, as these may give clear insights into how these novel compounds act on pest species. This has not been clearly understood in the majority of important fruit fly pest species.

123 Chapter 7: General discussion

4) As spinosad is known to act as a stomach poison, and minimum of contact action (Penagos et al. 2005a), it did have a strong a contact action on Aphytis (Chapter 7). The mode of action of spinosad needs to be investigated and understood clearly for important arthropod natural enemies.

5) Our understanding of the effectiveness and use of other selective and safer alternatives for fruit fly control, especially entomopathogens, insect growth regulators (IGRs) and mineral oils requires upgrading. The larval stage of the eastern cherry fruit fly, R. cingulata (Loew) has been shown to be susceptible to various species of etomopathogenic nematodes like Steinernema carpocapsae (weiser) (Kostarides 2002). Moreover, insect growth regulators hold promise for use against Diptera (Greathead 1989), as do the horticultural and agricultural mineral oils against fruit flies (Liem 2004). The mineral oils have been reported to control a wide range of pests and diseases in different crops (Beattie et al. 2002). All these merit further investigation and study, particularly for a wide range of fruit fly pest species over a variety of ecosystems and climatic ranges.

124 References

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161 Appendices

APPENDICES

Appendix 1. Yeast Hydrolysate Enzymatic (Catalog number 103304)

Description: The product is an autolyzed yeast extract produced from 100% brewers yeast. It is a concentrated source of soluble protein components, carbohydrates, and B Complex vitamins natural to yeast.

Typical Analysis:

Protein (N × 6.25) 60.0% Total Nitrogen 8.8% Alpha Amino Nitrogen 4.2% Ratio of Amino Nitrogen 48.0% of Total Nitrogen Vegetable Oil 0.5% Salmonella Negative Standard Plate Count Max. 50000/g

Typical Vitamin Profile (ug/g):

Thiamine (B1) 45 Riboflavin (B2) 60 Niacin 350 Pyridoxine (B6) 30 Pantothenic Acid 170 Folic Acid 11 Biotin 3 Choline 2000 Inositol 1400

Typical Mineral Profile:

Sodium 0.13% Chloride 0.85% Potassium 3.24% Calcium 0.07% Magnesium 0.28% Copper 1.6 ppm Manganese 10.3 ppm Iron 48.0 ppm Zinc 43.0 ppm

Typical Amino Acid Profile (As % of Protein):

Alanine 7.3 Arginine 6.3 Aspartic Acid 10.0 Glutamic Acid 13.7 Glycine 5.0 Histidine 2.5 Isoleucine 4.5 Leucine 6.8 Lysine 7.5 Methionine 1.7 Phenylalanine 4.2 Proline 4.8 Serine 4.8 Threonine 4.5 Tryptophane 1.0 Tyrosine 3.5 Valine 5.5

MP Biomedicals Inc, Aurora, Ohio, USA Source : http://www.mpbio.com/product_info.php?products_id=103304

162 Appendices

Appendix 2. Sample of the data sheet used for red scale, Aonidiella aurantii (Maskell) parasitism assessment.

163 Appendices

Appendix 3. Statistical tables

Table A 1.1 Summary of results of repeated-measures ANOVA for the number of adult female and male Bactrocera tryoni (Froggatt) attracted to different protein bait-insecticide mixtures in field cage attraction study (Chapter 3: for Table 3.1 & 3.2).

Female flies

Source df Mean Square F Probability Female B. 4 .072 4.815 .011 tryoni Error 15 .015

Male flies

Source df Mean Square F Probability Male B. tryoni 4 .031 1.059 .411 Error 15 .030

Table A 1.2 Summary of results of a one-way ANOVA for the laboratory feeding test with adult female Bactrocera tryoni (Froggatt) on different protein bait-insecticide mixtures (Chapter 3: for Table 3.3).

Source df Mean Square F Probability Female B. 4 236.597 149.276 .001 tryoni Error 145 1.585

Table A 1.3 Summary of results of a one-way ANOVA for the toxicity test on Bactrocera tryoni (Froggatt) with different protein bait-insecticide mixtures (chapter 4: for Table 4.1, 4.2 and 4.3).

After 24-h exposure

Source df Mean Square F Probability Treatment 4 1.361 140.334 0.001 Error 15 .010

164 Appendices

After 48-h exposure

Source df Mean Square F Probability Treatment 4 1.359 152.259 0.001

Error 15 .009

After 72-h exposure

Source df Mean Square F Probability Treatment 4 1.432 277.687 .001

Error 15 .005

Table A 1.4 Summary of results of a one-way ANOVA for the fly knock down after 1-h, 2-h and 3-h exposure with different protein bait-insecticide mixtures (Chapter 4: for Table 4.4).

1-h exposure Source df Mean Square F Probability Treatment 4 .643 106.122 .001

Error 15 .006

2-h exposure Source df Mean Square F Probability Treatment 4 .955 95.827 .001

Error 15 .010

3-h exposure Source df Mean Square F Probability Treatment 4 1.029 72.886 .001

Error 15 .014

Table A 1.4 Summary of results of a one-way ANOVA for the effects on mortality of adult Bactrocera tryoni (Froggatt) after 24-h and 72-h exposure to leaves with different protein bait-insecticides mixtures weathered for 2-h,3-d and 6-d (Chapter 4: for Table 4.5 and 4.6).

2-h aged protein bait-insecticide mixtures

24-h exposure

Source df Mean Square F Probability Treatment 4 1.214 60.538 .001

Error 15 .003

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72-h exposure

Source df Mean Square F Probability Treatment 4 1.291 240.562 .001

Error 15 .005

3-d aged protein bait-insecticide mixtures

24-h exposure

Source df Mean Square F Probability Treatment 4 .987 154.023 .001

Error 15 .006

72-h exposure

Source df Mean Square F Probability Treatment 4 1.055 231.254 .001

Error 15 .005

6-d aged protein bait-insecticide mixtures

24-h exposure

Source df Mean Square F Probability Treatment 4 .839 40.538 .001

Error 15 .021

72-h exposure

Source df Mean Square F Probability Treatment 4 .975 73.929 .001

Error 15 .013

166 Appendices

Table A 1.5 Summary of results of a one-way ANOVA for mortality of Bactrocera tryoni (Froggatt) between leaves aged for 2-hr, 3-d and 6-d with protein bait containing chlorpyrifos, malathion, spinosad and fipronil after 72-hr exposure period(Chapter 4: for Figure 4.1).

chlorpyrifos Source df Mean Square F Probability Treatment 2 .093 1.500 .274

Error 9 .001

malathion Source df Mean Square F Probability Treatment 2 .020 1.693 1.693

Error 9 .012

spinosad Source df Mean Square F Probability Treatment 2 .081 14.070 .002

Error 9 .006

fipronil Source df Mean Square F Probability Treatment 2 .016 3.765 .065

Error 9 .004

Table A 1.6 Summary of results of a Chi-Square test for difference in parasitism of red scales, Aonidiella aurantii (Maskell) by Aphytis lingnanensis (Compere) and Comperiella bifasciata (Howard) over four sampling periods in two citrus orchards (Chapter 5: for Table 5.1 and 5.2).

Auburnvale orchard

Sampling periods Chi-Square 3.241 df 3 Asymp. Sig. .356

Iron bark orchard

Sampling periods Chi-Square 7.259 df 3 Asymp. Sig. .064

167 Appendices

Table A 1.7 Summary of results of a one-way ANOVA for attraction of Aphytis lingnanensis (Compere) to protein bait, water and honey (Chapter 5: for Table 5.3).

Source df Mean Square F Probability Treatment 2 6.133 197.260 .001

Error 177 .031

Table A 1.8 Summary of results of Kruskal-Wallis test for attraction of Aphytis lingnanensis (Compere) to protein bait, protein + 20 % sugar and water (Chapter 5: for Table 5.4).

Total number attracted Chi-Square 261.65 df 2 Asymp. Sig. .001

Table A 1.9 Summary of results of a t-test for attraction of Aphytis lingnanensis (Compere) to protein and water (Chapter 5: for Table 5.5).

Std. Std. Error 95% Confidence Interval Mean Deviation Mean of the Difference Sig. (2- Lower Upper t df tailed) Pair 1 protein - -1.16667 10.33042 1.33365 -3.83530 1.50196 -.875 59 .385 control

Table A 1.10 Summary of results of the feeding times of adult mealy bug predator, Cryptolaemus montrouzieri (Mulsant) and adult green lace wing, Chrysoperla carnea (Stephens) on honey, protein and water under laboratory conditions (Chapter 5: for Table 5.6 and 5.7).

Cryptolaemus montrouzieri Source df Mean Square F Probability Treatment 2 555.841 70.582 .001 Error 45 7.875

Chrysoperla carnea Source df Mean Square F Probability Treatment 2 905.712 43.592 .001 Error 78 20.607

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Table A 1.11 Summary of results of Kruskal-Wallis test for the mortality of Aphytis lingnanensis (Compere) adults after 24-h exposure to citrus leaves with different protein bait-insecticide mixtures weathered for 2-hr, 3-d and 7-d and 12-d (Chapter 6: for Table 6.1).

2-hr treatments Chi-Square 23.641 df 4 Asymp. Sig. .001

3-d treatments Chi-Square 23.641 df 4 Asymp. Sig. .001

7-d treatments Chi-Square 15.694 df 4 Asymp. Sig. .003

12-d treatments Chi-Square 16.476 df 4 Asymp. Sig. .002

Table A 1.12 Summary of results of Kruskal-Wallis test for the effect of weathered protein bait-insecticide exposure on the fecundity of Aphytis lingnanensis (Compere) (Chapter 5: for Table 6.2).

7-d residue treatments Chi-Square 57.83 df 3 Asymp. Sig. .001

12-d residue treatments Chi-Square 70.993 df 3 Asymp. Sig. .001

169 Appendices

Appendix 4: Photographs used

All photographs used in the thesis are by the author except for two photographs on page 88 by Dan Papacek.

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