Bactrocera Invadens

DIVERSITY AND POPULATION DYNAMICS OF Bactrocera invadens AND OTHER TEPHRITID FRUIT FLY SPECIES INFESTING MANGO (Mangifera indica) IN ZIMBABWE AND RELATIVE EFFICACIES OF SELECTED INSECTICIDES INCORPORATED IN FOOD BAITS

VENGAI COLLEN MAFIRAKUREWA

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Tropical Entomology

University of Zimbabwe

Faculty of Science

Department of Biological Sciences

December 2014

DECLARATION

I hereby declare that this thesis is my own original work and has not been submitted for a degree in any other university.

Vengai Collen Mafirakurewa Date

I, as the supervisor, confirm that the work reported in this thesis was carried out by the student under my supervision. The thesis was examined and I approved it for final submission.

Dr. P. Chinwada Date

DEDICATION

This study is dedicated to parents, my wife, Joyce, our two children, Tawonga and Tadiwa and all the farmers who were gracious enough to let me into their homes and lives.

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ACKNOWLEDGEMENTS

I wish to extend my profound gratitude, appreciation and deepest respect to all the mango growers in Mutoko (Mrs Mudzonga, Mrs Mudzengerere and Mrs Machita) Murewa (Mr and Mrs Mukurazhizha, Mrs Magirivha and Mr Chiwanza), Domboshava (Mr and Mrs Magama, Mr and Mrs Parirewa and Mrs Chogugudza) and Seke (Mr and Mrs Chiruwa, Mr and Mrs Muronzi and Mrs Chimanikire) who allowed me — a total stranger — to carry out my study in their orchards and backyards. I will forever be indebted to my supervisor, Dr. P. Chinwada for his valuable advice and guidance during the course of this study and the many hours he spent correcting my work. Your way of doing things will always have a lasting impression in my scientific work. You taught me well and for that I will always be thankful.

I also want to thank the Director of the Research Services Division, Dr C. Mguni, and the Head of Plant Protection Research Institute (PPRI), Dr. G.P. Chikwenhere, for allowing me to use the laboratory and equipment at PPRI during my study. Their advice and comments during the course of the study were highly valuable. I am also very grateful to the following people from PPRI: Miss T. Makanza (Research Officer) for her valuable advice and support and Mrs Mary Nyamangodo (Research technician) for the long hours she had to be out with me in the field and laboratory. I also wish to acknowledge the assistance of Messrs Pawandiwa and Ngara who helped me with part of the data analysis. I also thank Mr O. Chipfunde (Research Officer, Gene Bank) for his efforts with drawing the map of the study areas.

Last, but by no means least, I wish to thank and appreciate my wife Joyce, for allowing me to finance a greater part of this work from our personal savings. Words fail me, many thanks for your support, sacrifice and understanding throughout my study period. May the gracious Lord richly bless you all.

ABSTRACT

Studies were conducted from November 2013 to October 2014 in Zimbabwe to determine the temporal population dynamics of the invasive fruit fly pest Bactrocera invadens (Diptera: Tephritidae), to determine the pest’s host range and incidence and to evaluate its control using a food bait consisting of protein hydrolysate and each of the insecticides dimethoate (Dimethoate 40® EC), malathion (Malathion 50® EC), trichlorforn (Trichlorfon 90® SP), deltamethrin (Deltamethrin 2.5%® SC) and lambda-cyhalothrin (Lambda-cyhalothrin 50® EC). Fruit fly temporal dynamics studies were conducted in Mutoko, Murewa, Domboshava and Seke using methyl eugenol-baited traps while host range and incidence were assessed through “fruit rearing”. At each of the four locations (intensive study sites), fruits sampled from mixed mango varieties every two weeks were held in the laboratory at room temperature to determine the associated fruit fly species spectrum. As further confirmatory tests on host fruit and fruit fly species associations, a once-off extensive fruit survey covering the areas in which traps were located as well as outside them was conducted. Overall, there was a significant site x month interaction, with the highest catches of (over 3,400 in February 2014) being recorded in Murewa between February and April 2014. The lowest catches (below 25) were recorded in Seke and Domboshava in October 2014. The highest fruit fly diversity was recorded in Murewa, Seke and Domboshava where, in addition to B. invadens, Ceratitis cosyra and Ceratitis rosa were also recorded infesting mangoes. In the extensive surveys, Harare and Mahusekwa had the highest fruit fly diversity, recording three fruit fly species (B. invadens, C. cosyra and C. rosa). The highest B. invadens fruit infestations were recorded from stringless mango fruits collected in Guruve, which had an infestation rate of 80.5%. No B. invadens infestations were recorded from fruits collected in Chegutu, Zvimba and Buhera. The mango seed weevil, Sternochaetus mangifera, which is a quarantine pest of mangoes worldwide, was also recorded from all the four trapping locations, with Mutoko having the highest infestation rate of 22.5%. In laboratory studies to assess B. invadens control using various insecticides incorporated in hydrolysed protein food baits, Trichlorfon 90® SP (a.i. trichloforn) gave the highest adult fruit fly mortality (87.5%) while Dimethoate 40® EC (a.i. dimethoate) was the least effective with mortality of 40%.

In conclusion, this study demonstrated that though B. invadens occurs throughout the year, populations are at their highest from January to April. Its abundance is greatly influenced by location. Trichlorfon 90® SP and Deltamethrin 2.5%® SC (a.i. deltamethrin) have the potential to be used as fruit fly killing agents in case of non- availability of malathion which is the traditional fruit fly insecticide that is incorporated in food baits in Zimbabwe. Further local studies on B. invadens biology, overwintering mechanisms and behaviour are recommended so as to generate information that, when combined with results from the present study, could be useful in coming up with ‘smart’ strategies for the management of the pest in specific localities.

TABLE OF CONTENTS

DECLARATION ...... ii DEDICATION ...... iii ACKNOWLEDGEMENTS ...... iv ABSTRACT ...... v TABLE OF CONTENTS ...... vii LIST OF TABLES ...... x LIST OF FIGURES ...... xi LIST OF PLATES ...... xii CHAPTER 1 ...... 1 INTRODUCTION ...... 1 1.1 Overview ...... 1 1.2 Justification ...... 3 1.3 Objectives ...... 5 1.4 Hypotheses ...... 5 CHAPTER 2 ...... 6 LITERATURE REVIEW ...... 6 2.1 Mango Production Overview ...... 6 2.2 General Overview of Mango Pests and Diseases ...... 6 2.3 Tephritid Fruit Flies ...... 7 2.3.1 General classification ...... 7 2.3.2 Biology and ecology ...... 7 2.3.3 Some economically important fruit fly pests of mango ...... 8 2.4 Economic Impact of Tephritid Fruit Flies ...... 10 2.5 Bactrocera invadens Spread in Africa and Zimbabwe ...... 12 2.6 Host Range of B. invadens ...... 13 2.7 Species Status of B. invadens Inferred from Morphometrics, Molecular, Cytogenetic and Behavioural and Chemoecological Data ...... 13 2.8 Inference of B. invadens status from work done so far worldwide ...... 16 2.9 Ecological Studies of B. invadens...... 16

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2.10 Interaction studies between native and invasive fruit flies ...... 17 2.11 Effect of altitude on seasonal activity and distribution of fruit flies ...... 18 2.12 Fruit Fly Monitoring ...... 20 2.13 Fruit Fly Management ...... 22 CHAPTER 3 ...... 28 MATERIALS AND METHODS ...... 28 3.1 Study Sites ...... 28 3.1 General Methods ...... 28 CHAPTER 4 ...... 33 OCCURRENCE OF B. INVADENS AND ITS TEMPORAL POPULATION DYNAMICS ...... 33 4.1 Introduction ...... 33 4.2 Materials and Methods...... 33 4.3 Results ...... 35 4.4 Discussion ...... 38 CHAPTER 5 ...... 43 DIVERSITY OF TEPHRITID FRUIT FLIES ASSOCIATED WITH MANGOES IN THE DIFFERENT STUDY LOCATIONS ...... 43 5.1 Introduction ...... 43 5.2 Materials and methods ...... 43 5.2.1 Intensive survey ...... 43 5.2.2 Extensive survey ...... 45 5.3 Results ...... 45 5.3.1 Intensive surveys...... 45 5.3.2 Extensive survey ...... 48 5.4 Discussion ...... 49 5.4.1 Intensive study ...... 49 5.4.2 Extensive survey ...... 52 CHAPTER 6 ...... 53 EVALUATION OF DIFFERENT INSECTICIDES FOR USE WITH BAITS ...... 53 6.1 Introduction ...... 53

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6.2 Materials and methods ...... 53 6.2.1 Test insects ...... 53 6.2.2 Insecticides...... 54 6.2.3 Bioassays ...... 54 6.3 Results ...... 55 6.4 Discussion ...... 56 CHAPTER 7 ...... 58 GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS ...... 58 7.1 General Discussion ...... 58 7.2 Conclusion ...... 61 7.3 Recommendations ...... 62 REFERENCES ...... 64

LIST OF TABLES

Table 1. GPS coordinates of the four selected sites in Mashonaland East ...... 30

Table 2. Seasonal Bactrocera invadens and Ceratitis cosyra host availability and maturation period of the most important fruits in Mutoko, Murewa, Seke and Domboshava ...... 30

Table 3. General characteristics of four trapping and fruit sampling sites of fruit flies in Mashonaland East province ...... 32

Table 4. Mean male B.invadens trap catches of the study sites for the period November 2013 to October 2014 ...... 35

Table 5. Overall species composition (%) of fruit flies that emerged from mango fruit in the intensive study sites from November 2013 to April ...... 46

Table 6. Non-tephritid insect pests and parasitoids recorded during the intensive ...... 46

Table 9. Percentage of fruit fly species abundance and infestation levels on mangoes sampled from different extensive survey locations across the country...... 49

Table 10. Cumulative percentage adult fruit fly mortality at 30 minutes post-exposure .. 55

LIST OF FIGURES

Figure 1. Map showing spatial distribution of the four locations in Mashonaland East Province ...... 29

Figure 2. Fluctuations in B. invadens trap catches across four sites from November 2013 to October 2014 ...... 37

LIST OF PLATES

Plate 1. Modified 500 ml empty plastic bottle for trapping male B. invadens (with parapheromone and DDVP insecticide block) ...... 34

Plate 2. Individual weighing of mangoes before placement in individual containers ...... 44

Plate 3. Rearing containers placed inside hessian bags showing fruit flies that had just emerged ...... 44

Plate 4. Special fruit fly trap for collecting B. invadens flies for use in laboratory bioassays ...... 54

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CHAPTER 1

INTRODUCTION

1.1 Overview

Fruit flies are a well-defined group of flies belonging to the family Tephritidae. The family is represented in all continents except Antarctica but the major genera have limited natural distributions (N’depo et al., 2013). Worldwide, there are an estimated 4,000 species of fruit flies of which around 350 are of economic importance (Plant Health Australia, 2011). Over 900 tephritid species are known to occur in the Afrotropical region with an estimated fifth of this population deemed to be present in Zimbabwe (Hancock, 1986). While non-frugivorous tephritids are rarely pestiferous (Boykin et al., 2012), the frugivorous tephritids contain many genera of major economic importance such as Bactrocera, Ceratitis, Rhagoletis and Anastrepha (White and Elson-Harris, 1992). Fruit flies are easily recognizable, being of moderate size and of almost cosmopolitan distribution. The larvae are generally phytophagous, usually living inside fruits of both cultivated and wild fruits, but some are found living on flower heads, inside stems and leaves. Although commonly named ‘fruit flies’, larval development can take place in other parts of host plants besides fruits, including flowers and stems. About 35% of fruit fly species attack soft fruits, including many commercially important ones (White and Elson-Harris, 1992).

Fruit flies are probably the most serious cause of pest losses to many fruit trees and most cucurbits (Dale and Nair, 1966). Mango (Mangifera indica L.) is one of the world`s most important cultivated tropical fruits. World production of mango was estimated at 28.5 million tonnes and was estimated to reach 31 million tonnes by 2010, accounting for nearly 50% of the world tropical fruit production (Ambele et al., 2012). The production of this fruit is, however threatened by a wide range of insect pests and diseases (Veerish, 1989). The Mango/Marula Fruit Fly, Ceratitis cosyra (Walker) has traditionally been one of the most damaging tephritid fruit fly pests of mango (Hill, 1987; Ekesi et al., 2009). Few other tephritid fruit flies, such as Ceratitis fasciventris, Ceratitis rosa, Ceratitis anonae and, to a limited extent, Ceratitis capitata, also coexist with C. cosyra in different

parts of Africa (Ekesi et al., 2009). Ceratitis cosyra has been documented as widely occurring in Zimbabwe (Hancock, 1986). More importantly, mango production is under threat from the African Invader Fly, Bactrocera invadens (Drew, Tsuruta and White) which has been described as one of the most devastating fruit fly pests in Africa (Ambele et al., 2012). Both ripe and unripe fruits are vulnerable to fruit fly attack although they show preference for oviposition on ripe and fully-ripe mangoes (Rattanapun et al., 2009).

Ceratitis cosyra is believed to be indigenous to the African continent where it is widespread whilst B. invadens is an invasive fruit fly species of Asian origin (Drew et al., 2005; Ekesi and Muchugu, 2007). Bactrocera invadens is believed to belong to the Bactrocera dorsalis (Hendel) complex of tropical fruit flies (Ekesi et al., 2006). The Bactrocera dorsalis complex contains 75 described species, largely endemic to South East Asia (Clarke et al., 2005). This complex comprises over 50 species that are found largely widespread in South East Asia and a further 16 species native to Australasia (CABI, 2007). Prominent members within this complex include B. dorsalis, B. papayae, B. philippinensis, and B. carambolae and B. invadens. Species falling within the B. dorsalis complex are regarded as pests of great economic importance where they occur, or as high-level quarantine threats in countries where they are not present but able to invade, establish, integrate and spread (Clarke et al., 2005). A number of the pest species, largely members of the B. dorsalis complex, have become widespread through accidental introduction associated with agricultural trade. Bactrocera invadens, for instance has spread across Africa in the last decade and become a major agricultural pest (San Jose et al., 2013). The B. dorsalis complex includes several morphologically and ecologically similar pests, making species designations uncertain (Leblanc et al., 2013).

Over the past century, many studies have tried to examine the ecological basis for the abundance, distribution and the various management options available for fruit flies. Various studies have indeed focused on the biology of the key pest species such as B. dorsalis, B. zonata and B. curcubitae. Often in the studies a high correlation of abundance with temperature has been observed allowing estimates to be made of relative abundance in various zones and seasons. According to Shukla and Prasad (1985), the key determinants of fruit fly abundance are host availability, median temperature and relative

humidity. Thus, the study of spatial and temporal distribution of fruit flies, which are important ecological parameters, is imperative as it forms the basis for coming up with practical management options for the farmer.

1.2 Justification

Africa is known to be the native home of a number of fruit fly species and the origin of several fruit fly introductions and establishments across the globe, the most notorious species to date being the Mediterranean Fruit Fly (C. capitata). The species is rather curiously named for it is actually an African insect first recognized as a pest in the Mediterranean region and one that has spread throughout the world. The continent has, however, also become highly exposed to introduction of alien invasive fruit fly species with the intensification of international fruit and vegetable trade. Of all the indigenous and alien fruit fly species recorded to date, B. invadens is considered to be responsible for causing extensive economic losses to horticultural crops throughout the African continent since its first report in Kenya in 2003. Zimbabwe appears to be no exception and the danger posed by the invasive species to the local fruit and vegetable industry cannot be overemphasized.

Mangoes are widely distributed throughout Zimbabwe and have nutritional and economic uses especially to the poorly resourced communal farmer. Most importantly, mangoes are known be hosts for a number of economically important fruit fly species, including the exotic B. invadens. According to previous work done by Ndlela (2008) in one of the Zimbabwean localities─ Domboshava ─ also covered in this particular study, B. invadens was not detected in Domboshava or elsewhere in the other areas he covered in the 2007/08 mango season hence the need to also revisit some of the areas following the recent declaration of the presence of B. invadens in the country in 2012 (EPPO, 2013).

In order to formulate an ecologically sound integrated pest management (IPM) programme for the management of B. invadens, it is important to monitor its occurrence and population dynamics in different parts of the country (for example, Murewa, Mutoko, Domboshava and Seke that were used in this study). This information will provide baseline data for further studies as well as to support the implementation of control

measures. This is especially important as our understanding of its off-season survival, altitudinal limits and factors that regulate its population abundance in the field under local conditions are still very sketchy and fragmented. There are indeed considerable gaps in knowledge with regard to B. invadens behaviour and dispersal ability under Zimbabwean climatic conditions. Thus, knowledge about fruit fly species and their respective seasonalities in relation to host plant phenology is crucial to understanding their population dynamics.

It is also important to determine the diversity of the local fruit fly fauna associated with mango production in Zimbabwe and determine parameters necessary for the formulation of a comprehensive integrated pest management programme. Work covered herewith thus seeks to report on the diversity of fruit flies in the four respective locations based on a one calendar year trapping and fruit rearing period.

Various control measures such as chemical, biological and cultural are being followed, though the latter has not proved vital for the management/control of the fruit flies. Chemical control has been the most important measure so far of fighting fruit flies. Insecticides for the control of fruit flies have been applied in two ways, mainly, baiting and cover spray and have proved successful in a number of cases. The equipment used for applying the bait is simple, so the technique is appropriate for control of fruit flies at either village or commercial levels (Allwood and Drew, 1997). In either case of application method, development of resistance is something that needs to be considered all the time (Haider and Khan, 2011). It is therefore important that other alternative insecticides that can also be used as killing agents should be evaluated in order to ease the pressure of resistance build up on the already existing pesticide list. Thus, the importance of inventorying the fruit fly species in the country as a prelude to formulating a sustainable management strategy of these pests cannot be overemphasized.

1.3 Objectives

1. To determine the diversity and abundance of tephritid fruit fly pests of mango in each of four selected areas of Zimbabwe. 2. To monitor the fluctuation of B. invadens population throughout one calendar year in each of four selected areas of Zimbabwe. 3. To evaluate the efficacy of various insecticides applied in food baits for the control of B. invadens.

1.4 Hypotheses

1.1 There is more than one tephritid fruit fly pest of mango in each the four study areas 1.2 The different fruit fly pest species of mango occur in equal proportions in each area. 2. Populations of B. invadens are maintained at a constant level throughout the year in each study site. 3. Bactrocera invadens is equally susceptible to all insecticides that are incorporated in food baits.

CHAPTER 2

LITERATURE REVIEW

2.1 Mango Production Overview

Mangoes are produced in over 90 countries worldwide. Asia accounts for approximately 77% of global mango production while the Americas and Africa account for approximately 13 and 9%, respectively (FAOSTAT, 2007). For the people of Asia, Central and South America and Africa, fruit production provides essential components to their diet/nourishment and also acts as a source of income. For several tropical fruits, the production is mainly by small landholders and a large proportion is intended for the rapidly growing local urban market (Mwatawala et al., 2006a). Mango is traded and consumed as fresh or in processed form. A number of products produced from mango like mango juice, mango pulp, mango flavour, mango kernel oil, mango pickles and powder, etc., have been well introduced and accepted in different market segments. In Zimbabwe, mango production is mainly undertaken by smallholder communal farmers who normally supply the domestic market during the mango fruiting season.

2.2 General Overview of Mango Pests and Diseases

Mango is attacked by a wide range of insect pests and diseases (Veerish, 1989). The fruit is under threat from such insect pests as the mango mealybug (Drosicha mangiferae), mango gall midges (Erosomyia indica, Dasineura amaramanjarae), mango shoot gall psylla (Apsylla cistellata, Pauropsylla brevicornis), fruit flies (Bactrocera, Ceratitis and Anastrepha spp.), fruit-sucking moths (Eudocima, Achaea), mango aphid (Toxoptera odinae) and the mango seed weevil (Sternochetus mangiferae). Some 25 fungus diseases affect mango, the most serious and widespread being anthracnose (Glomerella cingulata) which infects shoots, flowers and fruits. Powdery mildew (Oidium mangifera) is also an important fungal disease and can cause substantial crop losses as it affects flowers and fruitlets as well as the leaves. Infection symptoms include leaf spots and various storage rots of the fruit. Of all the insect pests attacking mangoes, fruit flies such as the Mango or Marula Fruit Fly (C. cosyra) and the African Invader Fly (B. invadens) in particular, have

been described as the most devastating pest of mangoes in Africa (Ekesi et al., 2009; Ambele et al., 2012).

2.3 Tephritid Fruit Flies

2.3.1 General classification

The Tephritidae (true fruit flies) is one of the most species-rich families within the order Diptera (Boykin et al., 2014). This is a major, cosmopolitan family containing about 4,000 species in 500 genera. Larvae of most species develop in fruits of wild and cultivated plants and the family is therefore commonly known as 'fruit flies' and are not to be confused with the Drosophilidae (vinegar flies) which share this common name (Skaife, 1979).

There is at present no generally accepted higher classification of the family Tephritidae, but three subfamilies are currently recognised: Dacinae, Trypetinae and Tephritinae, each of which is divided into a number of tribes and subtribes. The family is represented in all continents except Antarctica but the major genera have limited natural distributions. Thus, Anastrepha spp. occur in South and Central America and the Caribbean; Bactrocera spp. are native to tropical Asia, Australia and the South Pacific; Ceratitis and Dacus are native to tropical Africa, and Rhagoletis spp. are found in South and Central America and in temperate areas of North America and Europe. In a few cases, species have been accidentally introduced and have become established outside these natural ranges, mainly as a result of human activity.

2.3.2 Biology and ecology

Typically, fruit flies lay their eggs in semi-mature and ripe fruit. The female fruit fly has a retractable, sharp egg-laying appendage (the ovipositor) at the tip of the abdomen that is used to insert up to six eggs into a small chamber about 3 mm under the fruit skin. The eggs are white, banana shaped and nearly 1 mm long. Infested fruit may show ‘sting’ marks on the skin and may be stung more than once by several females. In two or three days, larvae (maggots) hatch from the eggs and burrow through the fruit. To the naked eye, the larvae resemble blowfly maggots. They are creamy white, legless, blunt-ended at

the rear and tapered towards the front where a pair of black mouth hooks are often visible. Female flies may have an association with bacteria resident in their gut, which they regurgitate onto the fruit before ovipositing. Most of the damage sustained by the fruit is actually caused by the bacteria and the maggots simply lap up the juice.

The mouth hooks allow the larvae to readily tear the fruit flesh. The larvae develop through three instars to become about 9 mm long and pale yellow when fully grown. Several larvae can develop in each fruit, and when fully developed, they leave the fruit, falling to the soil beneath the tree and burrowing down about 5 cm to form a hard, brown, barrel-like pupal case from its own skin where it completes its development. Many larvae leave the fruit when it is already on the ground. Most insects cannot pupate successfully in the presence of excess moisture and fruit flies have a prepupal stage when they can 'flick' themselves over some distance, presumably to distance themselves from the host fruit. The duration of pupal developmental is dependent on temperature with each stage taking from nine days to several weeks to complete. Adult flies emerge from their pupal cases in the soil and burrow towards the surface where they inflate their wings and fly away. Adults are able to mate within a week of emerging, living for many weeks with females continuing to lay eggs throughout their life cycle (Fletcher, 1987; Plant Health Australia, 2011).

Adult fruit flies feed on carbohydrates from sources such as fruit and honeydew secretions from aphids and scale insects, as well as natural protein sources, including bird droppings and bacteria. Fruit fly larvae can be attacked by parasitoids although they appear to have little impact on populations of most fruit flies, with 0-30% levels of parasitism typical (CABI, 2007). However, mortality due to vertebrate consumption of infested fruit can be very high, as can pupal mortality in the soil, either due to predation or environmental factors.

2.3.3 Some economically important fruit fly pests of mango

2.3.3.1 Bactrocera invadens, African Invader Fly/Asian Fruit Fly

The eggs of B. invadens are small, white and slender (0.8 mm x 0.2 mm). Larvae are white maggots. Adults have a pair of black spots on the face, one under each antenna.

Wing length is 4-7 mm with distinctive dark markings along the anterior edge. Two raised areas just below and behind the wing base are yellow. There is a lateral pair of broad yellow stripes on the side of the thorax. The abdomen has distinctive markings; a clear, black, mid-longitudinal line from tergite III-V; black marking extend on most of tergites III-IV with broad lateral markings almost rectangular in shape. A distinctive characteristic is that the males are attracted to the chemical lure of methyl eugenol (Ekesi and Muchugu, 2007; Allotey et al., 2010). Following a preliminary survey of fruit flies of Bangladesh, Leblanc et al. (2013) observed that the species in Bangladesh exhibited a broad range of scutum colour pattern variation which ranged from predominantly pale to predominantly dark. This was similar to the scutum colour pattern variation documented in B. invadens from Sri Lanka.

Bactrocera invadens is multivoltine in nature, and has all year host availability. Females lay eggs in the pulp of fruits. Larvae hatch from the eggs in 1-2 days, and feed on the fruit of the host plant. They leave the fruit after about 11 days and pupate in the soil at a depth of 2-6 mm below the ground. After a pupal period of about 10 to 21 days, the adult flies emerge from the pupae (Allotey et al., 2010). Pupariation can, however, last for up to 90 days under cool conditions as is the case with other members of the B. dorsalis complex (CABI, 2007).

Bactrocera invadens is a polyphagous species and has been recorded on over 50 host species belonging to 25 plant species belonging to 25 plant families, but especially on mango, guava, banana, and citrus. Other known hosts are papaya, avocado, tomato, cashew, pepper and several wild African fruits (Ekesi and Muchugu, 2007; Allotey et al., 2010).

2.3.3.2 Ceratitis cosyra (Walker), Mango or Marula Fruit Fly

The scutum of C. cosyra is predominantly yellow or pale brown with three black areas on the apical half of the scutellum. The male orbital setae are not expanded, and tibiae are not feathered. The fore femur is yellow on both sides. It is also recognized by the four black spots on either side of the thorax and the four black spots on the dorsal surface of the thorax. The female has a sharp and pointed aculeus and its post-pronotal spot

relatively large, with seta situated anteriorly. The costal band is continuous with no gaps. Ceratitis cosyra is attracted to terpinyl acetate, but not trimedlure or methyl eugenol. It has a post-pronotal lobe with a dark spot (Billah et al., 2007).

The female flies pierce the ripening fruit and insert the eggs into the puncture. The maggots feed on the pulp, making it worthless as a fruit. Pupation takes place either inside the fruit or underground. The adult is a small fly, which holds its wings partly at rest, and is about 4-5 mm long, and 10 mm wingspan. There are probably only two generations per year (Hill, 1987).

The mango fruit fly or marula fruit fly is a widespread species found throughout Africa and was reported to be the key pest of mango across the continent prior to 2003 (Lux et al., 2003; Ekesi et al., 2006). It is a polyphagous pest, with its host range including fruits such as mango, guava, sour orange, marula and wild apricot (Ekesi and Muchugu, 2007). It is considered the main fruit fly found in mangoes, accounting for the major losses in mango production (Mwatawala et al., 2006a).

2.4 Economic Impact of Tephritid Fruit Flies

Fruit flies cause direct damage by puncturing the fruit skin to lay eggs. During egg laying bacteria from the intestinal flora of the fly are introduced into the fruit. These bacteria cause rotting of the tissues surrounding the egg. When the eggs hatch, the maggots feed on the fruit flesh making galleries. These provide entry for pathogens and increase fruit decay and thereby making fruits unsuitable for human use (Ekesi and Muchugu, 2007). Direct damage caused by the fruit flies usually range from 20 to 80% (Ekesi et al., 2009). Larvae of the Mediterranean fruit fly (C. capitata) for instance, can cause extensive damage to fruit crops of up to 100% by tunneling into the fruit, making it unfit for human consumption. The larval tunnels also provide entry points for bacteria and fungi that cause the fruit to rot. These factors normally lead to reduced income, and increased costs of control. Production losses and costs of field control are the direct impacts of fruit fly attack, while indirect losses result from the implementation of regulatory controls and loss of export markets (Vijaysegaran, 1997; Boykin et al., 2014).

In Kenya, where about 90,000 tonnes of mangoes are produced annually, losses of between 20-80% due to fruit fly infestation have been reported (Lux et al., 1998). A similar situation prevails in the other mango-producing African countries such as Côte- d’Ivoire, Mali, Senegal and Burkina Faso, where the infestation has been reported to reach 70% in small-holder orchards. The production of mangoes is not only threatened by C. cosyra but especially of late by B. invadens, which has been described as the most devastating fruit fly pest in Africa (Allotey et al., 2010; Ambele et al., 2012). In Benin, losses of more than 60% on the main mango cultivars due to fruit flies have been recorded in the second half of the mango season, and phytosanitary pressure led to uprooting mango plantations in one area (Borgou) at one time (De Meyer et al., 2010). As a result of the threat posed by invasive fruit flies, the Mauritian and South African governments banned the importation of mango and avocadoes from Kenya (Ekesi et al., 2009).

Few insects therefore have a greater impact on international marketing and world trade in agricultural produce than tephritid fruit flies. With increasing international trade, fruit flies as major quarantine pests of fruits and vegetables have taken on added importance, triggering the implementation of transboundary control programmes. Due to globalization, trade in fresh fruits and vegetables is gradually being liberalized on a global scale (IAEA, 2003). As a result of the discontinuous distribution of some species of fruit fly, and their enormous potential as fruit pests, several species are subject to quarantine legislation in different countries, involving restriction of importation of fruit likely to contain the larvae. The main species involved are C. capitata, B. dorsalis, B. tyroni and B. invadens (Hill, 1987; CABI, 2007).

In April 2012, the South African National Plant Protection Organisation (NPPO) imposed a ban on the importation of fruits and vegetables from Zimbabwe owing to the “suspected” presence of B. invadens in the country. In addition, the South African NPPO instituted stringent measures on the movement of in-transit fruits from Zimbabwe across South Africa via its sea ports to other international destinations. Similarly in Asia, India, which is arguably the world largest mango producer, lost its European Union market following the detection of fruit flies in mangoes exported to the EU. Brussels has blocked

imports of Indian mangoes until December 2015 after inspectors found some consignments infested with fruit flies (Anand, 2014).

2.5 Bactrocera invadens Spread in Africa and Zimbabwe

The timing and exact pathway of invasion by B. invadens into Africa are not known (De Meyer et al., 2010). During a routine fruit survey activity at the Coast Province of Kenya, three specimens of unknown fruit fly (two from protein-baited trap and one from unidentified indigenous fruit, probably from the genus Strychnos were collected by the ICIPE-led African Fruit Fly Initiative (AFFI) in March 2003. Intensive surveys covering 75 sites in seven of Kenyan provinces which are the major fruit growing and trading areas were initiated two days later using methyl eugenol and Cue lure® covering (Lux et al., 2003). Large numbers of the same fruit fly species were attracted to methyl eugenol, while none was attracted to Cue lure®. This coupled with the numerous adults of the same fly reared from mango at the same time suggested beyond doubt that the pest was a new species in the country. Using basic morphological characteristics, Lux et al. (2003) concluded that the species was probably Bactrocera dorsalis which is native to Asia. Specimens were later shipped to Prof Dick Drew in Australia who later described it in 2005 as B. invadens (Drew et al., 2005). Although Drew et al. (2005) at the time distinguished B. invadens from its close relatives, for example B. dorsalis and B. kandiensis, they, however, indicated that some specimens of B. invadens were almost inseparable from B. dorsalis, as B. invadens can have a different array of colour patterns on the scutum as well as on the abdomen. The exact identity of B. invadens is therefore still controversial to date (Papadopoulos, 2010).

Bactrocera invadens has since been reported in many eastern, western and central African countries and in 2008, it was also reported in Mozambique, Zambia and Namibia. Following its detection in a methyl eugenol-baited surveillance trap in May 2010 in the northernmost part of the Limpopo Province of South Africa, in an area adjacent to the Zimbabwe border, a delimiting survey was carried out and subsequently a quarantine area of approximately 1,100 km2 surrounding the area was implemented by the South African NPPO to regulate the movement of hosts fruits (Manrakhan and Holtzhausen, 2011).

Consequently, in April 2012, the South African NPPO instituted stringent measures for the movement of in-transit fruit from Zimbabwe to international destinations across South Africa via its sea ports. Zimbabwe’s National Plant Protection Organisation declared the presence of B. invadens in the country in February 2013 following the detection of male specimens of the fruit fly in methyl eugenol traps in Bindura district of Mashonaland Central province (EPPO, 2013).

2.6 Host Range of B. invadens

Bactrocera invadens is a polyphagous pest of both cultivated and wild fruit species and has been recorded on over 50 plant species belonging to over 25 families (Allotey et al., 2010). Among the cultivated fruits that were found to be heavily infested are mango (Mangifera indica), guava (Psidium guajava) citrus (Citrus limon, Citrus reticulata and Citrus sinensis), avocado (Persea americana) and paw paw (Carica papaya). Among the wild fruits Marula (Sclerocarya birrea) and Terminalia catappa were the most preferred hosts (Rwomushana et al., 2008). The invader fly host range also stretches to vegetable crops such as solanaceous plants like tomato (Lycopersicon esculentum), pepper (Cucumis annum) and curcubits (Curcubita pepo) (Allotey et al., 2010; Manrakhan et al., 2010).

2.7 Species Status of B. invadens Inferred from Morphometrics, Molecular, Cytogenetic and Behavioural and Chemoecological Data

The concept that species are the basic units of evolution, each with its own unique genetic makeup, is widely accepted amongst evolutionary biologists (Drew, 2004). Defining species accurately is a critical need in disciplines such as ecology and evolutionary biology and in applied arenas such as pest management (Drew et al., 2008). The current disagreements about the theoretical concept of the species are closely tied to the issue of species delimitation, specifically how to determine the boundaries and numbers of species from empirical data (De Queiroz, 2007). Species delimitation directly impacts on global biosecurity. It is a critical element in the decisions made by national governments in regard to the flow of trade and to the biosecurity measures imposed to protect countries from the threat of invasive species (Boykin et al., 2012).

Of all the species in the genus Bactrocera, the B. dorsalis complex to which B. invadens belong, is regarded as the most economically important and arguably the most taxonomically challenging (Clarke et al., 2005). Members of this complex include B. dorsalis, B. papayae, B. philippinensis, B. carambolae and B. invadens. These species are widely acknowledged as either serious agricultural pests where they occur, or as high level quarantine threats in countries they are absent but capable of invasion and establishment (Clarke et al., 2005). Thus the biological species status of the taxa within the B. dorsalis complex, particularly of these pest species, has been a topic of considerable debate (Clarke et al., 2005; Drew et al., 2008).

Very significant research has been undertaken to date within an integrative taxonomic framework, to biologically delimit the target taxa. The tools used so far have included: i) morphological examination, including traditional morphology, morphometrics of genitalic characters, and fine-scale wing shape analysis using geometric morphometrics; ii) molecular-based approaches using both phylogenetic and population genetic analyses of nuclear DNA sequence and microsatellite data coupled with mitochondrial DNA sequence analysis and comparative transcriptome analysis (Schutze et al., 2012); and iii) studies of mating and post-zygotic sexual compatibility and analysis of rectal gland constituents. Hereunder are discussed some of the research and findings involving of the work done this far.

2.7.1 Morphometric studies

Morphometry of different populations of B. invadens distributed across the species range of tropical Africa and a sample from the pest`s assumed home of Sri Lanka was investigated. Morphometry using wing veins and tibia length was used to separate B. invadens populations from other closely related Bactrocera species. The Principal Component Analysis yielded 15 components which corresponded to the 15 morphometric measurements. The first two principal axes contributed to 90.7% of the total variance and showed partial separation of these populations. Canonical discriminant analysis indicated that only the first five canonical variates were statically significant. The first two canonical variates contributed a total of 80.9% of the total variance clustering B. invadens

with other members of the B. dorsalis complex while distinctly separating B. correcta, B. curcubitae, B. oleae and B. zonata. Evolutionary history inferred by Neighbor-Joining method clustered the Bactrocera species populations into four clusters. The first cluster consisted of the B. dorsalis complex (B. invadens, B. kandiensis and B. dorsalis sensu stricto) branching from the same node while the second group had paraphyletic clades of B. correcta and B. zonata. The last two were monophyletic clades, consisiting of B. cucurbiate and B. oleae, respectively (Khamis et al., 2012).

2.7.2 Molecular (DNA) studies

When one mitochondrial and two nuclear genes were sequenced from 73 specimens belonging to 19 species within the genus Bactrocera and several species of the B. dorsalis complex to address the placement of B. invadens, the results indicated that the B. dorsalis complex is polyphyletic (San Jose et al., 2013). Bactrocera invadens, B. dorsalis, B. papayae and B. philippinensis were also found to also found to be paraphyletic with respect to each other and probably represent a single genetically indistinguishable, phenotypically plastic, pest species that has spread throughout the world (San Jose et al., 2013). Phylogenetic analysis of COI and rDNA sequence data from B. philippinensis, B. dorsalis s.s., B. invadens and B. papayae (all members of the B. dorsalis species complex) also revealed a close relationship among the four taxa (Tan et al., 2013).

2.7.3 Cytogenetic, mating compatibility and behavioural studies

Compatibility tests and subsequent postzygotic analysis of hybrid viability, survival , fertility and sex ratios that were conducted under field-cage conditions showed that B.invadens from Kenya mated randomly with B.dorsalis from Pakistan and China, producing fully viable offspring to the hybrid F2 generation (Bo et al., 2014)

2.7.4 Chemoecological studies

The same rectal gland pheromone constituents (2-allyl-4, 5-dimethoxyphenol (DMP) and (E)-coniferyl alcohol (E-CF) that were produced by B.invadens in chemoecological studies were the same as those produced by B.dorsalis (Tan et al., 2013)

2.8 Inference of B. invadens status from work done so far worldwide

Evidence presented above based on chemical ecology, chemotaxonomy, and results from DNA analysis, together with other evidence from mating compatibility and hybrid viability and molecular analysis strongly indicate that B. invadens and B. dorsalis sensu stricto belong to the same biological species hence should be revised as junior taxonomic synonyms of that species. Though phenotypic and genotypic differentiation occurs across the species ranges, such variation is consistent with population rather than species level variation (Tan et al., 2011; Clarke et al., 2014). However, Clarke et al. (2014), emphasised that it is important to note that conclusions drawn from any single species delimitation tool used in isolation may be open to interpretation or criticism. Clarke et al. (2014) pointed out that greater confidence is obtained when several tools are independently applied, particularly when consistent results emerge from different approaches.

2.9 Ecological Studies of B. invadens

Dacine fruit flies emerge from puparia as sexually immature adults that need to forage for resources to facilitate survival and reproduction. Key resources include moisture for metabolism, sugars to fuel flight, protein to attain sexual maturity and, in conjunction with lipids, for egg production. Sugar sources include honeydew and other plant exudates. Protein is derived from sources such as phylloplane bacteria and bird droppings while moisture is derived from dew and rain. Adult flies forage for these resources in the environment. In addition, adults may also actively seek out certain plant-derived phenyl propanoids (for example, methyl eugenol) that are hypothesized to play a role in the mating behaviour of dacine species. With respect to frequency of mating, female flies are considered to monandrous (i.e. mate only once), while male flies are polygynous (i.e. mate several times) (Clarke et al., 2005).

Data with positive response observed in 184 sites covering virtually the complete range of climatic and ecological conditions prevailing in West and Central Africa clearly show that B. invadens is adapted to most ecological and climatic zones of the Afrotropics ranging from lowland rainforest to moist and dry savanna zones. In West Africa, the

observed ecological boundaries were found to fit the transition zone demarcating the dry savanna zone from semi-arid conditions. These limits, where the plant growing season is short and occupation by B. invadens can be only temporary, constitute an ecological condition that has not been observed in earlier studies in East Africa (Ekesi et al., 2006; Rwomushana et al., 2008; Goergen et al., 2011). Results from research work conducted elsewhere seem to suggests that B. invadens prefers hot and humid environments. Annual precipitation must be high, although it does not have to be continuous (De Meyer et al., 2010). Mwatawala et al. (2006b) trapped B. invadens in orchards in the Morogoro region of central Tanzania continuously for 61 weeks in 2004–2005. Morogoro is situated in the transition zone between bimodal and unimodal rainfall belts in Tanzania with a distinct dry season. Bactrocera invadens was found to be present year-round, although populations increased dramatically during the rainy season. Similar observations were also made in Benin, in areas also demonstrating fly activity during a clear dry season (Vayssières et al., 2005).

2.10 Interaction studies between native and invasive fruit flies

Invasive species are defined as those that have recently been introduced and have established viable populations far from their original distribution areas (Duyck et al., 2007). Understanding the strength and modes of interspecific interactions between introduced and resident species (native or previously introduced) is necessary to predict invasion success. The impacts of invasion of Bactrocera sp. on indigenous fruit fly species have not received much attention and interest (Clarke et al., 2005). In Kenya for instance, before the invasion of B. invadens, C. cosyra was the major pest of mango with damage due to it ranging from 60-70% in 1998-2001 at Nguruman (Lux et al., 1999). At the same location years later in 2006, it was noted that over 80% of mango fruits damaged were infested with both C. cosyra and B. invadens, with the latter, accounting for 91% of puparia collected from infested fruits. The results gathered during the study whereby there was a large difference in pupal harvest from mangoes seem to indicate some evidence of competitive displacement of C. cosyra by B. invadens (Ekesi et al., 2006). The genus Bactrocera is renowned for its strong competitive abilities and capacity to largely displace other fruit fly species (Duyck et al., 2006).

In larval and behavioural interference experiments measuring the ability of one female to displace another from a fruit, four species of polyphagous tephritid fruit flies from the island of La Réunion which included one endemic species, Ceratitis catoirii, and three exotic ones, C. capitata, C. rosa and Bactrocera zonata were studied. It was observed that there were asymmetric and hierarchical interactions among species in both larval and adult interference competition. In agreement with the hypothesis that invasion is competition-limited, the competitive hierarchy coincided with the temporal sequence of establishment on the island. Thus, each newly established species tended to be competitively dominant over previously established ones (Duyck et al., 2006). In Hawaii, the disappearance of C. capitata at low elevation was initially assumed to be as a result of the displacement by B. invadens. Native pest species, such as C. cosyra appear to be outcompeted by this invasive species, although pre-invasion data are largely lacking (De Meyer et al., 2010). Recent studies have, however, attributed the species displacement to differences in life history strategy between the two respective flies. Ceratitis capitata is considered an r-strategist (smaller and capable of rapid colonization) whilst B. invadens is considered a K-strategist (Vargas et al., 2000). According to Duyck et al. (2007), the whole genus Bactrocera in general, has a more K-orientated profile from Ceratitis, which could explain why the former has often displaced the latter during recent invasions, but not the reverse. Overall, it appears B. invadens possesses some characteristics of both K- strategy (very aggressive, adaptation to new environments, strong competitor) (Rwomushana et al., 2008) and some traits of r-strategy of being highly fecund (Ekesi et al., 2006). In addition to the significant threat to many economically important fruit crops, with considerable implications for loss of agricultural trade, B. invadens could also be an ecological sink of native parasitoid species due to its strong immune system. Exposure of B. invadens eggs to native Pystallia and Tetrastichus species using sentinel fruit domes in field and subsequent dissection of the host eggs showed heavy encapsulation of the eggs of native parasitoid species (Mohamed et al., 2006).

2.11 Effect of altitude on seasonal activity and distribution of fruit flies

Altitude has long been known to have direct effects on both insect and plant phenology (Hopkins, 1919). Hopkins’ bioclimatic law states that seasonal activities (phenology

events), such as bloom time or adult emergence, are usually delayed for four days for each increase of 122 m in altitude. According to the bioclimatic law, the season is thus shortened not only by a day at the beginning but also by a day at the end for each 30 m increase in altitude. Insects are not actually affected by altitude per se but by the climatic changes that occur along the altitudinal gradient. Altitudinal gradients can serve as spatial analogues for climate change. Ecological gradients such as host plants and predators, as well as physical gradients like temperature, rainfall and humidity encountered along an altitudinal transect can have an impact on the density, diversity, and life history of insects and demands for phenotypic flexibility and genotypic adaptability of many species (Geurts et al., 2012).

Of all the climatic and environmental factors such as temperature, rainfall and host plants, it appears temperature is the most important as it decreases about 0.5-0.6oC with every 100 m increase in altitude (Ekesi et al., 2006; Kovanci and Kovanci, 2006). Thus temperature has the dominant role in determining development rates and is responsible for the timing of the population processes and their synchronization with changes in the environment. In temperate climates for example, fruit flies are seasonal in abundance and multivoltine species such as C. capitata increase their numbers up to a peak in later summer and early autumn and then decline rapidly (Radonjic et al., 2013). A species with a wide altitudinal range appears later in the season at higher altitudes, and at these locations it must be adapted to a shorter season. For instance, the emergence of Rhagoletis cerasi flies must coincide with the ripening period of sweet cherries in order to be able to lay eggs. However, adult emergence dates may vary from one location to another because, even though locations might be geographically close together, they may be subject to widely differing climatic regimes (Randall, 1982). Studies conducted by Ekesi et al. (2006) on field infestation rates of B. invadens at different locations in Kenya revealed that there was a significant inverse relationship between numbers of flies per kilogramme of fruit and elevation at which fruit was collected. From this work, it was quite evident that high level of infestation recorded at low elevations is an indication that B. invadens may well be adapted to a hot climate and thus represents a real threat to mango crops grown in the warmer low elevation regions of Kenya. Similarly, Mwatawala

et al. (2006) observed that in the orchards in different agro-ecological zones of the Morogoro region in Tanzania, the most abundant species at low and mid-elevation sites was B. invadens. On the other hand, C. cosyra appears to dominate at high elevations. Ceratitis rosa is able to develop in a wide range of temperatures and is therefore found from sea level up to an altitude of 1,500 m (Mwatawala et al., 2006; Duyck et al., 2010). Pupae of this species survive better in humid compared to dry conditions (Duyck et al., 2006). As for other fruit fly species, their development and reproductive ability are linked to temperature, with lower temperature causing decreased ovarian maturation rates and therefore decreased fecundity.

2.12 Fruit Fly Monitoring

Fruit fly monitoring helps to identify fruit fly pests in an area, determine distribution of pest species, identify local hot spots with high populations of the pest, track changes in population levels, determine efficacy of control measures and facilitate early detection of new fruit fly pests in a particular area. Tools used in fruit fly monitoring consist of attractant-based traps and host fruit surveys (Manrakhan, 2007).

2.12.1 Attractants

The three main types of attractants used in fruit fly monitoring include male and female lures, and food baits.

2.12.1.1 Male specific lures

The most widely used attractants are pheromones or parapheromones that are male- specific. Parapheromones are chemicals that are not naturally used in intraspecific communication but which do elicit responses similar to true pheromones. Parapheromones are available in both liquid form and polymeric plugs in the form of a controlled-release formulation. The parapheromone methyl eugenol (ME) captures a large number of species of the genus Bactrocera (including B. dorsalis, B. zonata, B. carambolae, B. invadens, B. philippinensis and B. musae). The pheromone Spiroketal® captures B. oleae. The parapheromone trimedlure (TML) captures species of the genus Ceratitis (including C. capitata and C. rosa). The parapheromone cuelure (CUE) captures

a large number of other Bactrocera species, including B. cucurbitae and B. tryoni. Parapheromones are generally highly volatile, and can be used with a variety of traps. Controlled-release formulations exist for TML, CUE and ME, providing a longer-lasting attractant for field use. It is important to be aware that some inherent environmental conditions may affect the longevity of pheromone and parapheromone attractants. Para- pheromones may also be mixed with a sticky material and applied to the surface of the panels. Killing agents used in panels, delta-traps and in bucket traps when used dry are usually a form of a volatile toxicant such as DDVP (2,2- Dichlorovinyl dimethyl phosphate), naled and malathion, although some of these are repellent at higher doses (Manrakhan, 2007; IAEA, 2013).

2.12.1.2 Female-biased lures

Female-biased attractants (natural, synthetic, liquid or dry) that are commonly used are based on food or host odours. Historically, liquid protein attractants have been used to capture a wide range of different fruit fly species. Liquid protein attractants capture both females and males. These liquid attractants are generally less sensitive than the parapheromones. In addition, the use of liquid attractants captures a high number of non- target insects. Several food-based synthetic attractants have been developed using ammonia and its derivatives. This may reduce the number of non-target insects captured. For example, for capturing C. capitata, a synthetic food attractant consisting of three components—ammonium acetate, putrescine and trimethylamine—is used. For capture of Anastrepha species, the trimethylamine component may be removed. A synthetic attractant will last approximately 4–10 weeks depending on climatic conditions, captures few non-target insects and captures significantly fewer male fruit flies, making this attractant suited for use in sterile fruit fly release programs. New synthetic food attractant technologies are available for use, including the long-lasting three-component and two- component mixtures contained in the same patch, as well as the three components incorporated in a single cone-shaped plug. In addition, because food-foraging female and male fruit flies respond to synthetic food attractants at the sexually immature adult stage, these attractant types are capable of detecting female fruit flies earlier and at low population levels than liquid protein attractants (IAEA, 2013).

2.12.1.3 Food baits

Food baits attract both male and female fruit flies. They are species-specific and are known to have a lower efficiency compared to male lures. Food baits can also attract a number of non-target insects, including beneficial ones. Food baits are available in both liquid and synthetic forms. Ammonia is the principal attractant emanating from food baits. There are a variety of food baits available commercially. These include liquid protein hydrolysates (Nu-Lure®, Buminal®, Corn Steepwater®, Hym-Lure®, Loklure® and Mazoferm®), yeast products, ammonium salts and the three-component lure (consisting of putrescine, ammonium acetate and trimethylamine). Field longevity of liquid protein hydrolysates, yeast product and ammonium salts is usually between 1-2 weeks while the three-component lure and Questlure capsule can last between 4-6 weeks. Minimum distance interval between food-baited traps should range from 10-30 m ((IAEA, 2013).

2.13 Fruit Fly Management

2.13.1 Sanitation

All damaged and rotten fruit on the ground should be removed and burned, as these promote pest population build up (Hill, 1987; CABI, 2007). The fruit can be buried, cooked, and fed to poultry or swine. Cultivating the soil beneath the trees to expose larva and pupa to ants, poultry, lizards, and song birds is also a worthwhile idea.

2.13.2 Fruit picking

Picking all the fruit from a tree has been used primarily in eradication programs. This was used a great deal in California. All fruit from the tree is picked to remove any ovipositional sites that would be available for the continued development of the fruit fly population (Sharp et al., 1989; Jacobi et al., 2001).

2.13.3 Wild host destruction

Elimination of non-economic or non-cultivated hosts that fruit fly populations need to survive is effective, especially in eradication programs (Messing, 1999; Smith, 2001).

Fruit of these wild hosts provide a source for survival when the cultivated hosts are absent or not fruiting.

2.13.4 Bagging of fruit

Bagging of fruit to prevent fruit fly oviposition has been used by many backyard growers and small farmers in Hawaii. The bag is removed 24-28 hours prior to harvest to allow natural colour of the fruit to develop. Small holes must be made in the paper bag in order for transpiration to take place. Plastic bags should not be used. Although labour- intensive, mechanical fruit protection is an effective method for high value fruit produced for export or fruits produced in backyard gardens for family use (Ekesi et al., 2007).

2.13.5 Biological control

Biological control is the use of fruit fly parasitoids, predators and pathogens to reduce the damage caused by a pest or related species of a pest (Elzinga, 2004; Ekesi et al., 2007). One of the most outstanding successes of classical biological control against fruit flies is attributed to the use of the egg parasitoid, Fopius arisanus against B. dorsalis, a close relative of B. invadens. This parasitoid is presently being released in Africa. Several species of parasitoids and predators abound in fruits and vegetable agro-ecosystems which can contribute to the suppression of fruit flies. Native parasitoid species may include Tetrastichus giffaardi, Psyttalia cosyrae, P. concolor, Fopius caudatus, Dirhinus giffardi and Spalangia spp.

Predators may include spiders, ants, carabid beetles and staphylinid beetles. The presence and foraging activity of the African weaver ant, Oecophylla longinoda, hinders the fruit flies from laying eggs (Van Mele et al., 2007; Vayssières et al., 2013). The predatory weaver ant can be utilized to protect mango and citrus fruits from damage by fruit flies provided that the tree hosts provides food resources (such as homopteran honeydew or plant nectar) to the ant.

A potent fungal pathogen isolate, Metarhizium anisopliae, is also effective against both developmental stages of the major fruit fly species such as B. invadens, B. cucurbitae, C. cosyra, C. fasciventris, C. rosa, C. capitata and C. anonae (Ekesi et al., 2007). The

efficacy of biological agents such as parasitoids and predators are, however, not considered as successful due to low fecundity of parasitoids as compared to fruit flies and poor searching ability of parasitoids to larval and pupal populations of fruit flies (Nadeem et al., 2014).

2.13.6 Male annihilation

Among the various alternate strategies available for the management of fruit flies, the use of methyl eugenol traps stands as the most outstanding alternative. Methyl eugenol has both olfactory as well as phagostimulatory action and is known to attract fruit flies from a distance of 800 m. Methyl eugenol, when used together with an insecticide impregnated into a suitable substrate, forms the basis of male annihilation technique. This technique has been successfully used for the eradication and control of several Bactrocera species (Ravikumar and Viraktamath , 2007). Male annihilation basically involves mass trapping using male lures such as Cuelure, methyl eugenol and Trimedlure with an approved killing agent. The tactic is useful if it used with an area-wide suppression strategy. The concept of male annihilation grew out of the use of lures for monitoring. Male lure traps are placed out on a given area in numbers sufficient to catch the majority of males in the population. The few remaining males fertilize fewer females, and the population gradually declines because of shortage of males. Lowering the number of males in a population minimizes the chances of successful reproduction and regeneration. The goal is to annihilate and totally remove the male fruit fly population from the area.

2.13.7 Sterile Insect Release Method (SIRM)

The sterile insect release method or sterile insect technique (SIT) is used to contain and exclude populations of fruit flies. The goal of SIT is to release a large amount of sterile males to mate with any introduced wild female, resulting in the production of infertile eggs. The potential of SIT for controlling pests has been around since the 1960s. Compared to insecticidal control methods, SIT has some advantages including increased specificity and can be targeted to affected regions (Knipling, 1959). SIT programs in the past have failed due to continual immigration into the areas being targeted.

The most common method used to make fruit flies sterile for SIT programs is to irradiate them. For irradiation to be most effective, it must be conducted when pupation is approximately 70% complete (Gilchrist and Crisafulli, 2006). The most effective irradiation dose rate for SIT programs should be at a level which makes an individual sterile without reducing its reproductive competitiveness. Studies have found that dose rate does not affect sterility induction, but higher dose rates can cause stress mortality. The “lowest practical” dose rate should be used when irradiating male insects for SIT control programs. Irradiated males are not reproductively disadvantaged against normal males in terms of females re-mating (Harmer et al., 2006).

Although irradiation of male flies causes changes in the temporal patterns of calling and courtship sounds which could potentially affect mating competitiveness, there is no difference between the proportions of irradiated and untreated males which copulate successfully (Mankin et al., 2008). SIT has been used against C. capitata in Costa Rica, Italy, Mexico, Nicaragua, Peru, Spain, Tunisia and the USA (California and Hawaii) (CABI, 2007). Success was also achieved with Bactrocera species; for instance B. dorsalis was eradicated from Guam in 1963, and B. cucurbitae from Rota in 1962-63 (Hill, 1987).

2.13.8 Chemical control

2.13.8.1 Baits

This method of fruit fly control involves the spot spraying of a combination of a dilute protein mixture and an insecticide. The protein (hydrolyzed proteins or their ammonium mimics) serves as an attractant, and when the fruit fly feeds on the protein mixture, the insecticide component causes death. This method targets both the male and female fruit flies. For example, a density of 100 spot sprays per hectare (around 6 to 8 per residential property) is used in the Fruit Fly Exclusion Zone of Eastern Australia (Gilchrist and Crisafulli, 2006). This amount of spraying is thought to be effective due to a bait spot being within the “daily wandering range of each fly within the treatment area. The effectiveness of this control method can be reduced by rain washing off bait spots and the pesticide degrading over. Malathion is used as an insecticide in bait spraying as it has a

short residual life and low mammalian toxicity. Bait spraying alone will not be enough to control high populations of fruit flies hence it should be used in combination with other control techniques. Bait sprays generally use much smaller quantities of chemical than cover sprays. Bait sprays are generally applied to foliage and not to the fruit (Dominiak, 2007).

The equipment used for applying the bait is simple, so the technique is appropriate for control of fruit flies at either village or commercial level (Allwood and Drew, 1997). Effectiveness of GF-120 spinosad fruit fly bait in suppressing B. invadens and other mango-infesting fruit flies was assessed by comparing treated orchards with untreated mango orchards in Benin. The larval infestation by B. invadens and other native fruit fly species was significantly lower in plots treated with GF-120 than in untreated control plots (Vayssières et al., 2009).

2.13.8.2 Ground spraying

Ground spraying is applied under host trees which are known to be infested with fruit flies. A spray of an appropriate insecticide, for example chlorpyrifosis, is applied to the ground under infested trees. The ground area which is sprayed includes the area from the trunk to the outer perimeter of the foliage. All compost heaps in the vicinity are also sprayed. No more than two ground sprays are usually necessary under one tree. This method targets larvae and emerging adults in the soil (Dominiak, 2007). In experiments conducted at ICIPE, it was found that application of a combination of Nulure/spinosad bait spray with soil inoculation of M. anisopliae reduced B. invadens population by 79% relative to control. Mean mango fruit infestation was 10% in the combined treatment of bait and fungus and was 73% in untreated control plots. Similarly, in field trials conducted during the 2006-2007 mango season, combined application of M. anisopliae and GF-120 spinosad bait spray achieved the highest reduction of B. invadens (92.1%) relative to the control of the last date of sampling (Papadopoulos, 2010).

2.13.8.3 Postharvest (Regulatory Control)

Many countries, such as mainland USA, forbid the importation of susceptible fruit without strict post-harvest treatment having been applied by the exporter (CABI, 2007).

Commodity treatments are needed in order to be able to transport host fruits from areas infested with fruit flies through quarantine barriers into areas that are free of the pest. These include use of fumigants and lethal temperatures.

Fumigants

Fumigants are chemicals which produce a gas or vapour that is toxic to insects, bacteria, or rodents. Methyl bromide and ethylene dibromide (EDB) have been used in the past but have since been withdrawn.

Lethal temperatures

The use of lethal temperatures is based upon the thermal tolerance of the insect and commodity. Mortality is a function of temperature and time. There are a number of treatments concerned with lethal temperatures. These include vapour heat treatment which involves heating air which is saturated with water vapour. The heated water vapour holds the commodity to a specific temperature for a prescribed period of time. This has proved effective for papayas and has also been tried and is utilized in other countries for fruit flies in mangoes. Another method involves the use of heat and cold treatments which involves hot and cold baths. This has been used with papayas which are immersed in hot water (49oC) for 20 minutes for disease control and then held in cold storage at 5- 6oC for 10 days. Hot-water treatment whereby mangoes for example are held in a hot water bath at 45.9-47.1oC for 67.5 minutes kills fly eggs and larvae. To kill eggs and larvae of C. capitata, the United States Department of Agriculture regulations require cold treatment for 10 days at 0oC or below, 11 days at 0.5oC or below, 12 days at 1.11oC or below, 14 days at 1.66oC or below, or 16 days at 2.22oC or below (Fletcher, 1987). Quick freezing is also an effective disinfestation treatment for fruits that can be used after freezing. Gamma irradiation can also be used to kill all stages of the fruit flies. The dosages that kill fruit flies are in the range 150-500 Gray. However, there are still some questions about consumer acceptance of irradiated food (Sharp et al., 1989; Jacobi et al., 2001).

CHAPTER 3

MATERIALS AND METHODS

3.1 Study Sites

The seasonal population dynamics of B. invadens and diversity of mango fruit fly pest species was studied from November 2013 to October 2014 at four locations in Mashonaland East province of Zimbabwe, namely, Mutoko, Murewa, Domboshava and Seke (Fig. 1). The climate of these areas is varied. Mutoko falls under Natural Farming Region IV which is characterised by mean minimum temperature of 11-20ºC, mean maximum temperature of 19-26ºC, mean annual temperature range of 18-24ºC and receives an average rainfall of 600-450 mm year (Mungandani et al., 2012). Murewa, Domboshava and Seke all lie in Natural Farming Region II which is characterised by mean maximum temperature of 19-23ºC, mean minimum temperature of 10-13ºC, mean annual temperature of 16-19ºC and annual rainfall of 700-1000 mm. These areas are composed mainly of mixed orchards and backyards which are all dominated by mango trees of mixed. All the mango varieties in the four locations were string mangoes. All the four locations are also within a communal land set up with all the farmers engaged in subsistence farming. At all the orchards where the study was conducted, no insect pest control measures were applied during the study. The fruit spectrum (wild and cultivated) at each respective site is given in Table 2. Information on maturation and availability of the different cultivated fruits across the four localities is also summarized in Table 2.

3.1 General Methods

Population fluctuations of B. invadens were determined through weekly recording of trapped male at each study site. Trapping was done using methyl eugenol which was dispensed in improvised 500 ml empty mineral water bottle traps with three windows made on the upper half of the bottle. A strip of dichlorovos was placed at the bottom to kill insects that entered the trap. The number of fruit flies caught per trap per week was recorded and flies identified and then preserved in 70% ethanol.

Location of sites

Figure 1. Map showing spatial distribution of the four locations in Mashonaland East Province

Table 1. GPS coordinates of the four selected sites in Mashonaland East Location Site Latitude Longitude Elevation (m) Mutoko 1 '643 S 032 20'20 E 1042 2 '954 S 032 '63 E 1072 3 '954 S 032 20'263 E 1039

Murewa 1 40'066 S 03 38'532 E 1241 2 3 ' 35 S 03 38' 2 E 1223 3 38' S 03 38'8 E 1177

Domboshava 1 34'235 S 03 ' 25 E 1519 2 34'2 0 S 03 0'8 4 E 1552 3 34' 5 S 03 0'8 2 E 1562

Seke 1 8 0 '6 6 S 03 5' 25 E 1538 2 8 0 '520 S 03 5'6 6 E 1532 3 8 0 '253 S 03 5'28 E 1531

Table 2. Seasonal Bactrocera invadens and Ceratitis cosyra host availability and maturation period of the most important fruits in Mutoko, Murewa, Seke and Domboshava Fruit host Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Mangifera indica Psidium guajava Citrus sinensis Citrus reticulata Citrus lemon Casimiroa endulis Prunus persica Persea americana Uapaca kirkiana

To determine the fruit fly species associated with mangoes of mixed varieties, two approaches were used. The first involved intensive fruit sampling every fortnight in the same four areas were trapping was conducted. The other approach involved an extensive

survey whereby mango fruits from other mango growing areas within the country were sampled in a once-off exercise. Mango sampling was done during the main mango fruiting and ripening season. The collected fruits were then transported back to the laboratory at the Plant Protection Research Institute (PPRI) were they were incubated. Sampled fruits were placed on a thin layer of sand in individual holding containers to allow exiting larvae to pupate. The emerging fruit fly species from each respective area were then identified before being preserved in 70% ethanol.

Laboratory bioassays were also conducted at PPRI to evaluate the efficacy of various insecticides as killing agents when incorporated in Loklure—a food bait—(for the control of B. invadens. The trials were conducted to measure mortalities of B. invadens males caused by five insecticides, four of them organophosphates and one pyrethroid. The fruit flies used in the experiment were from a wild population of unknown ages trapped at Plant Protection Research Institute, Harare, using methyl eugenol-baited traps (without the insecticide, dichlorovos).

Table 3. General characteristics of four trapping and fruit sampling sites of fruit flies in Mashonaland East province Elevation, agroclimatic region and its main Fruit and vegetable crops cultivated within the Wild fruits close to the homesteads characteristics study locations Mutoko Altitude 1,051 m; Natural Farming Region IV; Mango (average of 40 trees/homestead); avocado; Uapaca kirkiana [Wild loquat/ ‘mazhanje’ (S*)]; average annual rainfall of 600-450 mm; mean orange (Citrus spp.); lemon; White sapote Ziziphus abyssinica [masawu (S)]; Ximenia minimum temperature of 11-20ºC; mean maximum (Casimiroa edulis); mulberry; banana; different americana [‘nhengeni’ (S); Parinari curatellifolia temperature of 19-26ºC; mean annual temperature vegetable crops (mostly tomatoes, brassicas and [mobola plum/ ‘hacha’ (S)]; Vitex payos [‘tsubvu’ 18-24ºC curcubits) (S)]; Ficus capensis [cape fig/maonde (S)]; Strychnos spinosa [monkey orange/ ‘matamba’ (S)]; Strychnos innocua [‘hakwa’ (S)]; Azanza garkeana [ snot-apple/ ‘matohwe’ (S)]; Psidium guajava (feral guava); Opuntia vulgaris [‘zvinanazi’ (S)]

Murewa Altitude 1,214 m; Natural Farming Region II; Mango (average of 25 trees/homestead); guava; Wild loquat/ ‘mazhanje’ (S)]; masawu (S); Syzygium annual rainfall of 700-1,000 mm; mean maximum orange; naartjie; lemon; avocado; banana; White spp [waterberry/ ‘hute’ (S)]; Ficus sycomorus temperature 19-23ºC, mean minimum temperature sapote; mulberry; different vegetable crops [sycamore fig/ ‘muonde’ (S)]; mobola plum/ 10-13ºC; mean annual temperature 16-19ºC (mostly tomatoes, brassicas and curcubits) ‘hacha’ (S)]; monkey orange/ ‘matamba’ (S); ‘zvinanazi’ (S)

Domboshava Altitude 1,544 m; Natural Farming Region II; Mango (average of 6 trees/homestead); guava; Wild loquat/ ‘mazhanje’ (S*); sycamore fig/ annual rainfall 700-1000mm; mean maximum lemon; peach; mulberry; White sapote; different ‘muonde’ (S); mobola plum/ ‘hacha’ (S); Syzygium temperature 19-23ºC, mean minimum temperature vegetable crops (mostly tomatoes, brassicas and spp [waterberry/ ‘hute’ (S)]; snot-apple/ ‘matohwe’ 10-13ºC; mean annual temperature 16-19ºC curcubits) (S); ‘zvinanazi’ (S); feral guava

Seke Materera Altitude 1,534 m; Natural Farming Region II; Mango (average of 10 trees/homestead); guava; Wild loquat/ ‘mazhanje’ (S*); ‘hacha’ (S)]; annual rainfall 700-1,000 mm; mean maximum lemon; avocado; mulberry; White sapote; Syzygium spp [waterberry/ ‘hute’ (S)]; sycamore fig/ temperature 19-23ºC, mean minimum temperature different vegetable crops including tomatoes; ‘muonde’ (S); snot-apple/ ‘matohwe’ (S), monkey 10-13ºC; mean annual temperature range of 16- brassicas and curcubits orange/ ‘matamba’ (S); ‘zvinanazi’ (S); feral guava 19ºC * S – indicates Shona language

CHAPTER 4

OCCURRENCE OF B. INVADENS AND ITS TEMPORAL POPULATION DYNAMICS

4.1 Introduction

Mango (Mangifera indica L.) is an important tropical fruit for sub–Saharan African economies since it represents a fundamental source of nutrition for rural populations, provides basic production in reducing poverty and is a potential export product (Vayssières et al., 2009). In Zimbabwe, the three economically important species of fruit fly associated with mangoes are C. cosyra, C. rosa and B. invadens. Of all the native and exotic fruit fly species known to be present in Africa, B. invadens is thought to be responsible for causing extensive economic losses to horticultural crops on the continent since its first report in 2003 (Papadopoulos, 2010). Bactrocera invadens needs to be studied because it is a new species and is presently considered the most important tephritid fruit fly pest of small-scale and commercially grown fruits.

The aim of this study was to determine the occurrence and temporal population dynamics of B. invadens in a part of Zimbabwe where mangoes are important in the livelihood of smallholder farmers. These data would be invaluable in the implementation of sustainable fruit fly management strategies.

4.2 Materials and Methods

For this study, trapping guidelines for area-wide fruit fly programs (IAEA, 2003) were followed. Fruit fly traps were made from 500 ml empty plastic water bottles (Plate 1). Three small rectangular lateral holes (approximately 3 x 1.5 cm) were cut out near the top to facilitate entry of the flies. The traps were each baited with methyl eugenol and a strip of dichlorovos (DDVP) was placed at the bottom so as to kill trapped flies. The traps were set up in selected locations in Mutoko, Murewa, Domboshava and Seke in Mashonaland East province.

Plate 1. Modified 500 ml empty plastic bottle for trapping male B. invadens (with parapheromone and DDVP insecticide block)

At each site, traps were set up within a distance of 1.5 km from each other. The traps were hung in fruiting mango trees at a height of 1.5 to 1.8 m above the ground and on the southeastern side of the tree. The central coil of wire holding the trap was coated with thick grease in order to prevent predation of trapped dead adult flies by ants and spiders. A total of 12 traps, three in each respective location were used for this study. Re-baiting of the male attractant was done every six weeks according to guidelines given by International Atomic Energy Agency and FAO (IAEA, 2003). The traps were maintained at each of the four locations between November 2013 and October 2014. The traps were inspected and emptied every week. All insect specimens were placed in labeled vials containing 70% alcohol and transported back to the laboratory for species identification.

4.2.1 Data collection and specimen preservation

Catches were recorded as an index of fruit flies per trap per day (FTD). The flies per trap per day is a population index that estimates the average number of flies captured in one trap in one day that the trap is exposed in the field (IAEA, 2003). This population index provides a relative measure of the size of the adult pest population in a given space and time. It is used to compare the size of the population before, during and after the application of a fruit fly control programme. Its value is the result of dividing the total

number of captured flies by the product obtained from multiplying the total number of serviced traps by the average number of days the traps were exposed. That is,

Population index = F/T x D (IAEA, 2003)

where F = Total number of flies T= Number of serviced traps D = Trapping duration in weeks.

Analysis of variance (ANOVA) was performed after transforming the original count data by log (x+1) to determine the statistical differences in B. invadens catches between the sites, months and to determine if there was any significant interaction between site and month on the invader fly population. Where the F-ratio was significant, means were separated using Tukey-Kramer’s HSD test.

4.3 Results

4.3.1 Bactrocera invadens population dynamics

4.3.1.1 Effect of site

Murewa recorded the highest Flies per Trap per Day (FTD) of 50.1, followed by Mutoko with 23.8, Domboshava with 4.7 whilst Seke recorded the least FTD of 3.4 (Table 4 ). There were highly significant differences (P < 0.001) in the B. invadens trap catches between November 2013 and October 2014 among the four sites studied.

Table 4. Mean male B.invadens trap catches of the study sites for the period November 2013 to October 2014 Site Mean Flies per Trap per Day (FTD) Seke 3.4 Domboshava 4.7 Mutoko 23.8 Murewa 50.1

4.3.1.2 Effect of month

There were highly significant (P < 0.001) differences in mean monthly B. invadens trap catches within the different trapping sites.

Mutoko

Mean monthly B. invadens trap catches for the months November 2013, December 2013, September 2014 and October 2014 were not significantly different from each other (Fig. 2). Similarly, the catches for the period January-April 2014 were also not statistically different and witnessed the highest trap catches of 2,131 fruit flies in March 2014. Catches for the period May-August 2014 followed almost the same trend though there was a rise in August 2014 (Fig 2).

Murewa

Trap catches in the months November 2013, December 2013 and October 2014 were not significantly different and had the lowest B. invadens catches during the entire trapping period in this locality. Catches for the months January-September 2014 were not significantly different from each other, though March 2014 had the highest numeric catch of 3,408 (Fig 2).

Domboshava

Bactrocera invadens trap catches for December 2013 and May-October 2014 were not significantly different from each other. The lowest trap catches were recorded during the months of November and December 2013, July 2014 and October 2014. Though not significantly different from each other, the period January to April 2014 had the highest catches (Fig. 2).

Seke

The highest trap catches were recorded in March 2014 (mean of 339 fruit flies) whilst the lowest occurred in November 2013 (mean of 5.0 fruit flies) (Fig. 2).

4.3.1.3 Interaction of site and month on B. invadens population dynamics

The period November 2013 had low B. invadens catches across all the sites (Fig. 2). There was a huge jump in catches from December 2013 to around March 2014, with Murewa and Mutoko recording the steepest increase. The population increases in Domboshava and Seke were less pronounced during the same period. From April 2014 up to around end of July 2014, there was a sharp decline in catches in Mutoko, Domboshava and Seke. However, in Murewa, catches fell slightly at the end of March 2014 but maintained high populations even during the months of June and July 2014. The month of August saw a second surge in catches across all the four sites, though the magnitude was more pronounced in Murewa. Thereafter, catches across all the four sites maintained a downward trend.

Figure 2. Fluctuations in B. invadens trap catches across four sites from November 2013 to October 2014

4.4 Discussion

This study demonstrated that B. invadens occurs throughout the year but its abundance varies significantly with location and month. It appears that the fluctuations in B. invadens populations in Mutoko and Murewa follow a similar pattern while those in Domboshava and Seke also follow a certain distinct pattern. Significantly higher catches of B. invadens were recorded in Murewa and Mutoko with the highest catches occurring in Murewa in March 2014. Interestingly, Mutoko also recorded its highest catches in the same month. Though with comparatively lower catches, Domboshava and Seke also had peak catches during the same month of March. Over the entire year, Murewa had high trap catches compared to the other sites. Across all four locations, populations (as indicated by trap catches) seem to be at their lowest levels during the months October and November.

So one is left to wonder what factors could be responsible for these B. invadens population fluctuations at the four sites. Thus even though some sites are close geographically (for example Mutoko and Murewa are only separated by about 60 km), it appears that their populations at some point in time behave quite different. One such factor could be altitude. As pointed out by Hopkins (1919), altitude has long been known to have direct effects on both insect and plant phenology. According to the Hopkins` bioclimatic law, seasonal activities (phenological events), such as bloom time or adult emergence, are usually delayed for four days for each increase of 122 m in altitude. Altitudinal gradients can serve as spatial analogues for climate change. Geurts et al. (2012) do concur, highlighting that ecological gradients such as host plants and predators, as well as physical gradients like temperature, rainfall and humidity encountered along an altitudinal transect can have an impact on the density, diversity, and life history of many insect species. Similar sentiments were also echoed by Ekesi et al. (2006) who stated that B. invadens is predominantly a lowland species. The results of the current study seem not to concur with those by Vargas et al. (1983) who reported that fruit infestation by B. dorsalis — a very close relative of B. invadens — in native and exotic forests on Kauai Island was moderate at middle (579–800 m) elevations and low at high (> 800 m) elevations. On the contrary, the findings in this study seem to suggest that B. invadens

actually occurs at high population densities at an altitude of around 1,200 ml as was observed in Murewa. This was even higher compared to Mutoko which is at a lower elevation (approximately 1,051 m). Data from more seasons would, however, be important in validating these findings.

Though abiotic factors such as temperature and relative humidity were not recorded during this study, it appears that these contribute significantly toward increasing or decreasing the number of fruit flies trapped with the help of methyl eugenol. From the trap data collected in this study, it was observed that the highest number of B. invadens was captured during the summer season when the rainfall season was at its peak. Temperature was also generally high during this period. Relative humidity, particularly for weekly data, also appeared to greatly influence the fruit fly populations caught as the numbers caught during periods of high humidity were generally higher compared to periods when relative humidity was generally low and temperatures were high. This was confirmed by farmers at the locations where traps were set especially in Mutoko and Murewa.

The impact of abiotic factors on the population dynamics was also noted by Mahomood et al. (2002) who indicated that mean maximum and minimum temperatures have a positive and highly significant correlation with the number of fruit fly caught per trap. Shukla and Prasad (1985) found a significant correlation between relative humidity and number of fruit flies caught per trap. Similar findings were also highlighted by Ganie et al. (2013) when studying the population dynamics and distribution of fruit flies on cucurbits in Kashmir Valley, India. The team observed that the populations of fruit flies were significantly correlated with the minimum and maximum temperatures.

Rainfall was reported to have a significant positive effect in fruit fly trap catches. Similar sentiments were also echoed by Alim et al. (2012) in studies they conducted on the seasonal variations of melon fly, Bactrocera cucurbitae in different agricultural habitats of Bangladesh. The team noted that the seasonal rise of melon fly population coincided with a rise in the air temperature, availability and fruiting period of the host plants. However, Su (1984) noted a negative correlation (r = -0.269) between number of male

fruit flies per trap and relative humidity. It was also noted during the current study that there was a gradual increase in B. invadens trap catches as the rainy season progressed and this was characterised by the ripening of most of the mango cultivars in all the areas covered. In the Northern Guinean savannah, Vayssières and Kalabane (2000) noted that there was a consistent increase in B. invadens population in the early rainy season leading to considerable damage to mid-season and late season mango cultivars and the seasonal increase coincided with the fruiting period of the main cultivars mango cultivars. This trend was also observed by Vayssières et al. (2009) in central and northern Benin who reported a significant increase in the population of B. invadens at the beginning of the rain season in the mango producing areas and also a positive correlation between the rate of losses of mangoes and abundance of the invasive fruit fly.

Another important factor which appears to be of importance in explaining the B. invadens population dynamics is fruit abundance. It appears that fruit abundance and duration of availability to some extent seem to influence B. invadens population dynamics as observed in the current study. For example, the highest catches were recorded in Murewa followed by Mutoko. Even though Murewa has slightly lower mango fruit tree densities compared to the latter, mangoes in Murewa were observed to be fruiting for longer periods compared to those in Mutoko which ripen early. These observations are similar to those obtained from studies by Vargas et al. (1983) on the population dynamics of C. capitata. In their study, it emerged that the main factor affecting population build up in the tropics is fruit abundance and availability. In support, Ye (2001) reported that there appears to be a relationship between the Oriental fruit fly population size and the area planted with fruit trees, the fruit production yields, and the fruiting period. Whilst the observation by Ye (2001) might be true, in this particular study it was, however, noted that the fruit fly population in Murewa was consistently higher compared to Mutoko even though the latter has a higher density of mango trees per household.

Vayssières et al. (2009) noted that generally availability and abundance of cultivated and wild host fruits are important factors which determine population fluctuations of Bactrocera species. There appears to be a seasonal fly population flux between the neighbouring native vegetation of the dry savannahs and the mango orchards. So another

important aspect of these population fluctuations is the presence of wild hosts near to the orchard. For example, wild hosts and cashew trees have been observed to be immensely important in the re-infestation of orchards by Ceratitis during the flowering and fruit set stages of mango.

The difference in B. invadens populations could also be explained by the fact that the mango marketing period in Mutoko is relatively shorter as traders flock to the area in search of mangoes for reselling in the urban areas. Mutoko is one of the few districts in the country to have mangoes ready for sale earlier to traders before the fruit becomes available to most areas within the country. Most mangoes are actually harvested as soon as they become physiologically mature but whilst they are still green. The net effect is that very few mangoes are left lying around on the ground as virtually all the fruits are harvested from the trees and traded. This seems to be a classic example of indirect “orchard sanitation”. As was noted by Hill (1987) and Ekesi et al. (2007), infested fruits left unattended in the field may serve as reservoirs for continuous presence of the fly. In contrast, by the time mangoes are ready for consumption and sale in Murewa, most of the other mango producing areas countrywide will also be offering mangoes for sale hence the market will be flooded. The consequence is that there is a lot of unattended fallen fruit on the ground. Together with altitudinal positioning of Murewa relative to Mutoko, this could therefore possibly explain the high numbers of B. invadens males caught during and soon after the mango fruiting season. Thus the duration the mango fruits are in the field and the total number of fruits available for oviposition to B. invadens could very well be playing important roles in the population dynamics of the invader fruit fly in any given area.

Another question to ponder is what triggers the sudden surge in the B. invadens populations witnessed across all the four sites during the month of August after a somewhat downward trend from the month of April. One would wonder if environmental factors alone are the ones responsible for this trend or there are other factors such as the insect physiology linked to the sudden rise in the fruit fly populations. Richards and Davies (1977) noted that ecological diapause permits survival under adverse environmental circumstances and allows synchronized resumption of development when

conditions improve. It was observed that just prior to the sudden upswing in trap catches, there was a sudden increase in temperatures in the same month which probably signaled the end of winter and the beginning of summer. Though the population surge observed during this period might appear insignificant, it could actually act as the “reservoir population” responsible for the population explosions observed later in the season when the mango fruits started to ripen. As highlighted by Ekesi et al. (2006), the life expectancy of B. invadens at pupal eclosion was 75.1 days in females and 86.4 days in males. The average net fecundity and net fertility were 794.6 and 608.1 eggs, respectively, while average daily oviposition was 18.2 eggs. Ekesi et al. (2006) further noted that the daily population increase was 11% and the mean generation time was 31 days. Based on such information, it seems highly probable that the emerging August population could well be responsible and likely to affect the level of infestation in the preceding mango fruiting season.

This information on the sudden surge in population around the month of August, if proved to be repetitive over several seasons of study, could be exploited in the management of the invasive fruit fly. In order to come up with strategies that suppress or eradicate populations of B. invadens during the winter period or when mangoes are off season, it is important to come in with control strategies when its populations are at their lowest, before the observed increase in August. It is imperative that the physiological mechanisms and environmental factors regulating diapause or seasonal carryover in B. invadens are fully elucidated.

Overall, it appears as was observed in this study that B. invadens is a lowland pest as noted by Ekesi et al. (2006). Its distribution and seasonal dynamics appear to be greatly influenced by biotic and abiotic factors. The role played by wild fruits in bridging the season when mangoes are off-season needs further study. Also, the role played by guavas (including feral guavas) and other fruits such as oranges and naartjies which occur in the absence of mango during the winter period need to be fully comprehended as these fruits could be responsible for sustaining a certain fruit fly population during the time when the preferred host, mango, would be out of season.

CHAPTER 5

DIVERSITY OF TEPHRITID FRUIT FLIES ASSOCIATED WITH MANGOES IN THE DIFFERENT STUDY LOCATIONS

5.1 Introduction

The mango fruit with 36% of the world’s production of tropical fruits represents the most universally produced fruit (N’depo et al., 2013). In Zimbabwe, mangoes are grown by both commercial and communal farmers, with the latter accounting for the bulk of production and supply to the market. The production of this crop is, however, threatened by the attack of many insect pests, including fruit flies, and especially B. invadens which has been described as the most devastating fruit fly pest in Africa (Ekesi and Billah, 2007; Ambele et al., 2012). This fruit fly attacks more than 50 fruit trees including citrus and berries. In the Afrotropical region, 140 fruit fly genera, including 65 Ceratitis, 14 Bactrocera and 170 Dacus spp. are known. Fruit flies are responsible for significant damage in mango orchards. For example, in Mali, losses due to the fruit flies on national production were estimated at 50% and in Benin, these losses vary between 10 and 60% (Vayssières et al., 2004, 2005). According to N’depo et al. (2013), mango losses in Côte d’lvoire due to fruit fly infestation range from 17% at the beginning of the mango season to 80% at the end of the season. As noted by Mwatawala et al. (2006), two of the main prerequisites for formulating an effective integrated pest management program are to establish the diversity of the local fruit fauna and to determine parameters necessary for its formulation, including the population ecology of those involved. The objective of this study was to establish an inventory of fruit flies associated with mango in different mango-producing areas of Zimbabwe. This study was divided into intensive and extensive fruit surveys.

5.2 Materials and methods

5.2.1 Intensive survey

This survey was conducted during the main mango fruiting season which normally stretches from end of November to beginning of April. The objective was to study the

occurrence and diversity of fruit flies associated with mangoes in the four selected areas of Mashonaland East province where fruit fly traps were set up. Every two weeks at ripening time, at least 30 mango fruits of mixed varieties were randomly sampled at each location. Ripe and fully ripe fruits were collected from trees and on the ground. The fruits were put in 20 litre buckets and taken to the Plant Protection Research Institute (PPRI) laboratory. Mangoes sampled from the different site were held separately. Fruits from Mutoko and Murewa were collected on the same day while those from Domboshava and Seke sites were also collected during the same week but on a different day. In the laboratory, fruits were washed, individually weighed and placed in 13 x 12.5 cm cylindrical plastic containers (Plate 2).

Plate 3. Rearing containers placed inside hessian Plate 2. Individual weighing of mangoes before placement in bags showing fruit flies that had just emerged

individual containers

The lids of the containers were removed and Plate.replaced Rearing with finecontainers hessian placed bag mesh inside (22 hessian x 34 cm). The plastic containers were placed inside the hessian bags. These bags were tied Plate. Individual weighing of mangoes bags showing fruit flies that had just emerged. at the end to prevent the emerging fruit flies from escaping (Plate 3). Each fruit sample before placement in individual was reared in a separate container. The bottom of the container contained a thin layer containers (about 5 cm) of moistened sandy soil to hold exudates dripping from the rotting fruit as

well as to serve as a pupariation medium for larvae exiting fruits. Sand for the pupariating medium was sourced from local sand suppliers and sieved to remove larger particles using a 1.60 mm aperture-sized sieve. The fruit samples were held for 6-8 weeks and checked weekly for the emergence of any adult fruit flies and other insects. Emerged adult fruit flies were provided with water-soaked cotton wool and honey on the roof of the rearing cages for feeding. Emerged flies were left in the cages until they died in order to allow for growth and full development of morphological features. All dead flies and other insects were then removed with an aspirator or a fine camel hairbrush and then placed in labeled vials containing 70% ethanol for further identification and preservation. Chi-square test (χ2) test of association was performed to determine whether there was an association between site (location) and the fruit fly species that emerged.

5.2.2 Extensive survey

An extensive survey was also conducted at the same time as the intensive survey during the period end of November 2013 to end of May 2014. The objective of this survey was to study the diversity of fruit flies associated with mangoes in other mango-growing areas of Zimbabwe not covered by the intensive survey. Similarly, mangoes collected from the extensive survey were of mixed varieties. However, unlike in the intensive survey where only string mangoes were available in the orchards, both stringless and string varieties were available. The sampled mangoes were handled in the same manner as those collected during the intensive survey.

5.2.3 Insects identification

All emerged adult fruit flies were identified using taxonomic keys described by Billah et al. (2007). Voucher specimens were deposited at the Plant Protection Research Institute museum in Harare, Zimbabwe.

5.3 Results

5.3.1 Intensive surveys

A total of 1,058 mango fruits were collected during the main mango fruiting period from the four study locations. Three species of fruit flies were recorded infesting mangoes: B.

invadens, C. cosyra and C. rosa (Table 5). There was a significant association (χ2 = 1334.04, P < 0.001) between fruit fly species and location. In Murewa and Mutoko, B. invadens constituted 85.7 and 78.3%, respectively, of the recovered fruit flies. The highest numbers of C. cosyra were recorded at Domboshava (88.8%) and Seke (79.5%). Except for Seke, very low numbers of C. rosa were recorded in all study sites.

Table 5. Overall species composition (%) of fruit flies that emerged from mango fruit in the intensive study sites from November 2013 to April Site N B. invadens C. cosyra C. rosa Mutoko 187 78.3 21.7 0 Murewa 416 85.7 12.8 1.5 Domboshava 112 9.2 88.8 2.1 Seke 343 7.2 79.5 13.4

Other non-tephritid insect pests recovered from mango fruits included the Mango seed weevil, Sternochaetus mangifera (a pest of quarantine importance) and the False codling moth, Cryptophlebia leucotreta (Table 6). The Mango seed weevil was recorded at all four sites. At Seke, two fruit fly parasitoids were recovered: Tetrastichus giffardii and Psyttalia cosyrae.

Table 6. Non-tephritid insect pests and parasitoids recorded during the intensive Site Species recovered Category Mutoko Sternochaetus mangifera (Mango mango pest seed weevil) Murewa S. mangifera mango pest Cryptophlebia leucotreta (False mango pest colding moth ) Domboshava S. mangifera mango pest Seke S. mangifera mango pest C. leucotreta mango pest Tetrastichus giffardii parasitoid Psyttalia cosyrae parasitoid

On a month by month basis, adult fruit flies emerging from Murewa and Mutoko were largely dominated by B. invadens (Table 7). In Mutoko, for the period November 2013 to March 2014, B. invadens emerging from mango fruit rearing constituted 74.3-100% of all recovered fruit flies whilst in Murewa it constituted 76.8 to 93.2%. Ceratitis cosyra appeared to be more abundant in Murewa during the month of November 2013 (22.2%) though its numbers never picked up thereafter. Ceratitis rosa was only recovered from Murewa in March but only accounting for 7.1% of the total fruit flies recovered (Table 7).

Table 7. Percentage composition of fruit fly species that emerged from sampled mango fruits in Mutoko and Murewa from November 2013 to March 2014 and the associated B. invadens infestation levels Month Site N B. invadens C. cosyra C. rosa % B. invadens % Total fruit fruit infestation infestation Nov 2013 Mutoko 20 100 0 0.0 10.0 10.0 Murewa 34 77.7 22.2 0.0 14.7 20.6 Dec 2013 Mutoko 84 74.3 25.7 0.0 11.9 13.1 Murewa 78 93.2 6.8 0.0 24.4 25.6 Jan 2014 Mutoko 62 75.9 24.1 0.0 24.2 30.6 Murewa 110 80.8 19.2 0.0 31.8 32.7 Feb 2014 Mutoko 21 93.3 6.7 0.0 19.0 19.0 Murewa 137 92.3 7.7 0.0 51.1 53.3 Mar 2014 Murewa 57 76.8 16.2 7.1 52.6 54.4

The mango fruiting season for Domboshava only lasted for two months (January and February 2014) and all fruit rearings were dominated by C. cosyra (Table 8). In January 2014, 95.9% of the total fruit fly population recovered were C. cosyra whilst during the same period, only 2.8% were B. invadens. Very low incidences of C. rosa were recorded in Domboshava in January and February 2014 (1.9 and 2.9%, respectively). In Seke, the fruiting season lasted almost four months and C. rosa was the dominant species recovered in January 2014 (40.9%) and February 2014 (19.6%). However, in March and April 2014, C. cosyra appeared to be the dominating species accounting for 64.6 and

66.2%, respectively, of all the fruit flies that were reared from fruit during that period. Overall, B. invadens appeared to be less prevalent in Domboshava and Seke (Table 8).

Table 8. Percentage composition of fruit fly species that emerged from sampled mango fruit in Domboshava and Seke from January to April 2014 and the associated B. invadens infestation levels Month Site N B. invadens C. cosyra C. rosa % B. invadens fruit % Total infestation infestation Jan 2014 Domboshava 53 2.8 95.9 1.9 3.8 39.6

Jan 2014 Seke 41 3.4 9.1 40.9 4.9 36.6

Feb 2014 Domboshava 59 46.4 50.7 2.9 11.9 22.0

Feb 2014 Seke 102 2.7 8.8 19.6 2.9 12.7

Mar 2014 Seke 188 4.7 64.6 1.8 10.1 30.9

Apr 2014 Seke 12 8.8 66.2 7.4 1.7 41.7

5.3.2 Extensive survey

A combined total of 314 mango fruits were collected during the period November 2013 to May 2014 from twelve locations scattered across Zimbabwe. Bactrocera invadens was the dominant fruit fly species infesting mangoes with its abundance ranging from 47.6 to 100% across the various locations (Table 9). Ceratitis cosyra was the second most abundant species (up to 99.3%) which emerged from sampled fruit. Ceratitis rosa occurred in low numbers and was only recovered from Buhera, Harare and Mahusekwa with per cent abundances of 0.7, 10.8 and 42.9, respectively. The highest fruit infestations of 80.5% were recorded in Guruve whilst mango fruits sampled in Chegutu and Zvimba were free from fruit fly infestation.

Table 9. Percentage of fruit fly species abundance and infestation levels on mangoes sampled from different extensive survey locations across the country.

Location N Weight (g) % Species abundance (%) Fruit B. invadens C. cosyra C. rosa infestation Bindura 27 4.330 86.5 13.5 0.0 18.5

Buhera 26 3.208 0.0 99.3 0.7 76.9

Chegutu‡ 6 3.393 0.0 0.0 0.0 0.0

Chinhoyi 40 4.262 100 0.0 0.0 10.0

Chiredzi 24 2.690 100 0.0 0.0 4.2

Goromonzi 31 3.853 60.0 40.0 0.0 12.9

Guruve‡ 41 11.079 74.4 25.6 0.0 80.5

Harare 26 3.208 66.5 22.7 10.8 76.9

Mt Darwin 32 3.737 82.4 17.6 0.0 25.0

Mahusekwa 14 1.896 47.6 9.5 42.9 21.4

Nyazura 33 5.588 100 0.0 0.0 12.1

Zvimba 14 1.920 0.0 0.0 0.0 0.0 ‡ stringless mangoes

5.4 Discussion

5.4.1 Intensive study

Three fruit fly species were found to be associated with mangoes in the intensive survey sites. The species that were recovered during the study consisted of two native species, C. cosyra and C. rosa, and an invasive species, B. invadens. All the three species were recorded in Murewa, Domboshava and Seke whilst in Mutoko, no C. rosa was recovered from the fruit rearings. It is interesting to note that in three areas where all the three fruit flies were recorded, their dominance was distinctly different. For instance, while B.

invadens was the dominant species emerging from mangoes sourced from Murewa and Mutoko, C. cosyra appeared to be dominant in Domboshava and Seke.

This study confirms that the invasive fruit fly seems to be well established or establishing itself in the study areas where it occurs at relatively higher densities such as Murewa and Mutoko. Bactrocera invadens thus appears to becoming the most abundant and dominant fruit fly species infesting mango fruits. Though infestation levels in Domboshava and Seke still appear to be generally low, there is cause for concern as it appears that the African invader fly is now slowly creeping and establishing in those areas. Only recently in 2008, Ndlela (2008) conducted a similar study in Domboshava — which was again covered in this particular study — but recovered only C. cosyra and C. rosa. However, data from this present study from both trap and fruit rearing confirm the presence of the invasive fruit fly in this particular locality. Previously, Hancock (1986) had highlighted that C. rosa, C. capitata and C. cosyra were the most prevalent native afrotropical fruit flies species in Zimbabwe. These observations seem to be in line with Ekesi et al. (2009) who documented that C. cosyra was gradually being displaced by B. invadens on mango in an eight year study carried out in Kenya.

As was noted by Lux et al. (2003), C.cosyra has long been recognized as the most dominant and damaging tephritid fruit fly pest of mango in Africa. This dominance, however, appears to be waning away as evidenced by fruit rearing results from Murewa and Mutoko. One factor which can explain this shift could be the issue of inter-specific competition between native fruit fly species and B. invadens. Two possible mechanisms could be responsible for this displacement, namely; resource competition by larvae within mango fruits and aggression behaviour between adult fruit flies. Aggressive behaviour in the form of lunging, head butting and chasing off of other indigenous species from the mango dome were observed taking place during this particular study in mango orchards in Murewa and Mutoko. Interference competition through aggressive behaviour consequently will result in fewer of the local fruit flies being able to oviposit on the mangoes and hence resulting in fewer progeny emerging. These observations were also recorded by Ekesi et al. (2009) who observed that the number of times C. cosyra was

seen ovipositing was significantly lower under competitive interaction compared to the controls.

The new invasive species, B. invadens also appears to be stretching its altitudinal limits as it now seems to be capable of surviving even at mid to high altitude. According to Ekesi et al. (2006), B. invadens has predominantly been a pest of low-lying areas. However, in this study, the pest was recorded from 1,000 to 1,500 m.a.s.l. One would wonder what factors could be responsible for the ‘sudden’ appearance and establishment of this new exotic species across all the other non-traditional areas. According to Duyck et al. (2006), the genus Bactrocera is renowned for its strong competitive abilities and capacity to displace other native species. As was noted by N’depo et al. (2013), the strong pullulating, possessive character and aggressive behaviour of the adults of B. invadens could have induced the displacement or suppression of indigenous species towards other ecological niches. The laws of interspecific competition state that for any two competing species to share an ecological niche, equilibrium has to be established first. This balance can be achieved either by the elimination of one of the species or the establishment of a new stable equilibrium in which the two species coexist but at different population levels (Duyck et al., 2004). Another result from the current study was the conspicuous absence of C. capitata from all the mango samples collected from all the locations. This could well be as a result of it being eliminated from the mango fruit niche. Interestingly, from side experiments of other fruit rearings conducted during the same period (though not reported in this work), C. capitata was found emerging from some of the relatively smaller fruits such as peaches and Uapaca kirkiana [wild loquat/‘mazhanje’ (Shona)].

Abiotic environmental factors such as temperature and relative humidity also seem to have an effect on the occurrence and dominance of some of these fruit fly species. It appears there is a direct relationship between the arrival of the rains and the dominance and proliferation of certain fruit fly species. From the data presented herewith in this study, it can be noted that the numbers of B. invadens in the fruit rearings increased as the mango fruit season progressed as noted in Murewa and Mutoko.

5.4.2 Extensive survey

Out of the twelve extensive survey sites, ten had one or more fruit fly species being recorded infesting mango. As was also observed during the intensive survey, B. invadens, C. cosyra and C. rosa appear to be dominant fruit fly pests in the mango agroecosystems. Bactrocera invadens and C. cosyra appeared to share the same niche as evidenced by their coexistence in more than four locations. However, in Chiredzi, Chinhoyi and Nyazura, only B. invadens was recovered. Whilst it is difficult to ascertain that no other fruit fly species could be sharing the same niche with the invader fly in some areas where only B. invadens was recorded as sampling was only undertaken once, it appears that the invasive fruit fly is becoming dominant in most of the areas sampled. This could be a typical case of competitive exclusion taking place as was highlighted by Duyck et al. (2004). In Buhera, C. cosyra appears to be fully dominating that area. It will be necessary to monitor that locality to see if the status quo will still be obtaining in the foreseeable future.

The zero emergence of fruit flies from fruits collected from Chegutu and Zvimba is of interest particularly given the fact that some fruit flies were recorded in the area of Chinhoyi which is not so far away from those two locations. The small sample size used in this study from these respective areas could well have influenced the results. Thus long term sampling of fruits from multiple locations within and around these localities could give a better picture of abundance of the pests.

CHAPTER 6

EVALUATION OF DIFFERENT INSECTICIDES FOR USE WITH BAITS

6.1 Introduction

The African invasive fruit fly, B. invadens, which belongs to the B. dorsalis complex of tropical fruit flies, was detected in Africa in 2003 and has since spread to more than 30 African countries (Mwatawala et al., 2004; Ekesi and Billah, 2007). This insect is highly polyphagous and causes extensive damage to a wide variety of fruits and vegetables (Goergen et al., 2011). Various control measures such as chemical, biological and cultural are used to manage this pest; though the latter has not proved vital for the management or control of the fruit flies. Chemical control has been the most important approach used in the management of tephritid fruit fly pests. Insecticides for the control of fruit flies have mainly been applied in two ways: baiting and cover sprays (Haider et al., 2011; Abdullah et al., 2002). One challenge encountered with the use of chemicals is the behaviour of the fruit flies which lay their eggs beneath the exocarp of the fruit and the larvae develop inside the fruit. This implies that most of the insecticides have little chance of affecting internally-feeding larvae (Hill, 1987).

Several insecticides are registered for the control of fruit flies in Zimbabwe (Zimbabwe Crop Chemical Handbook, 2006). However, there is no local data available on the susceptibility of B. invadens to these insecticides as they were registered for pest control before the advent of the invader fly. Thus the objective of this study was to evaluate the efficacy of insecticides most commonly used by farmers (especially communal) in Zimbabwe to control this invasive pest.

6.2 Materials and methods

6.2.1 Test insects

The study used wild-caught insects that were trapped from mango trees. Adult B. invadens adult males were captured using special fruit fly collecting containers (Plate 4) hung on mango trees at the Department of Research and Specialist Services complex . However, in order to obtain live flies, only methyl eugenol without the killing agent was

used in the traps. Traps were checked 1-2 hours after set up. The captured flies were then taken to the laboratory for use in the bioassay.

Plate 4. Special fruit fly trap for collecting B. invadens flies for use in laboratory bioassays

6.2.2 Insecticides

Commercial formulations of the test insecticides were obtained from reputable agrochemical suppliers. The insecticides evaluated were deltamethrin (Cislin® SC), dimethoate (Dimethoate 40® EC), lamba-cyhalothrin (Lambda-cyhalothrin 50® EC), malathion (Malathion 50® EC) and trichlorfon (Trichlorfon 90® SP). The insecticides were mixed together with 4 ml of protein hydrolysate (425 g/litre) (Lok-Lure®, Arysta LifeScience) at the following rates per litre of water: 12 ml of Cislin® SC, 0.75 ml of Dimethoate 40® EC, 0.1 ml of Lambda-cyhalothrin 50® EC, 2 ml of Malathion 50® EC and 0.5 g Trichlorfon 90® SP. This bait-pesticide mixture was later used to impregnate the cotton plugs.

6.2.3 Bioassays

The bioassays were conducted at room temperature conditions in the laboratory. Ten field-collected B. invadens adult males of unknown age were transferred into cylindrical plastic containers containing cotton wool plugs that had been impregnated with the

Loklure/insecticide solution 24 hours previously. The plastic containers were then placed inside fine mesh hessian bags as previously described in the fruit rearing procedure. The number of insects that had died was recorded at 10, 20 and 30 minutes after exposure to the bait/insecticide solution was recorded. Cumulative percentage mortality was then calculated. Irreversible knockdown followed by death of the adult fruit flies was the criterion used to determine mortality. Moribund adults that could no longer stand on their own were considered dead. The experiment was laid down as a completely randomised design of six treatments (five insecticides and untreated control) each replicated four times. Data were analysed using the analysis of variance (ANOVA) after performing arcsine transformation. Mortality was not corrected using Abbot’s (1925) formula since there was zero mortality in the control.

6.3 Results

There were significant differences (P < 0.05) among the different insecticides. Although the highest fruit fly mortalities were given by Malathion 50® EC, Cislin® SC and Trichlorfon 90® SP, mean separation revealed that mortalities due to three treatments were not significantly different from each other (Table 10). Dimethoate 40® EC and Lambda-cyhalothrin gave the lowest mortalities (40.0 and 42.5%, respectively) and were not also significantly different from each other.

Table 10. Cumulative percentage adult fruit fly mortality at 30 minutes post-exposure Treatment N % Mortality (mean ± SE)

Dimethoate 40® EC 10 40.0 ± 8.2 c

Lambda cyhalothrin 50® EC 10 42.5 ± 8.5 bc

Malathion 50® EC 10 75.0 ± 2.9 ab

Cislin® SC 10 82.5 ± 4.8 a

Trichlorfon 90® SP 10 87.5 ± 4.8 a

Means within a column followed by the same letter are not significantly different (P = 0.05).

6.4 Discussion

In this study, the effectiveness of various insecticides belonging mainly to organophosphate group (Dimethoate 40® EC, Malathion 50® EC and Trichlorfon 90® SP) and two pyrethroids (Cislin® SC and, Lambda-cyhalothrin 50® EC) on male B.invadens was investigated. The adult male susceptibility bioassay of the field population of B. invadens demonstrated varying susceptibility to the five insecticides that were under evaluation in this particular study. The African invader fly was comparatively more susceptible to Trichlorfon 90® SP, Deltamethrin 2.5® EC and Malathion 40® EC than to Dimethoate 40® EC and Lambda-cyhalothrin 50® EC.

The results of this study seem to be in agreement with those of Ullah et al. (2012) who investigated the use of various insecticides as toxicant lure for fruit fly management in guava orchards in Pakistan. His team observed that Trichlorfon 90® SP was the most effective of the tested insecticides followed by malathion and Decis® (deltamethrin), respectively. Raga and Sato (2005) also reported that deltamethrin gave high mortality of C. capitata and Anastrepha fraterculus. The results of this study also appear to be in agreement with those of Mahmoudvand et al. (2011) who also noted that deltamethrin gave good control of Dacus ciliatus under laboratory conditions. However, their findings which showed that dimethoate gave equal good control of the same pest seem to be in contrast with findings of this study which found the same insecticide to be less effective against B. invadens. The pyrethroid, lambda cyhalothrin just like dimethoate, exhibited very low toxicity. This could be as a result of the some repellent effect associated with the insecticides. Thus, this could have forced the fruit flies to flee away from the protein source instead of attracting them.

It must, however, be noted that the results obtained in this study could well have been influenced by the environment in which the experiment took place. Thus, given that the bioassay was conducted in confined containers where the Loklure and insecticide mixtures were supplied using improvised cotton plugs, this could have meant that the fruit flies did not have much choice but to get close to the insecticide-impregnated plugs and in the process coming into contact with the insecticides. It was also observed during

the experiment that most of the fruit flies took more time (more than 10 min particularly in the case of the more pungent smelling insecticides such as dimethoate and lambda- cyhalothrin) to land onto the cotton plugs. Even on the not so strong smelling insecticides such as deltamethrin and malathion, the insects appeared not to be immediately attracted to the treated plugs. It can therefore be argued that given a choice, the fruit flies would have in some cases chosen the option to escape and forage elsewhere than to feed on the insecticide-treated protein bait. It would there be imperative to take the most promising insecticides in this study and evaluate them under field conditions where the fruit flies are not confined, hence have an option to feed or not. The use of Loklure as an attractant for B. invadens also needs also to be investigated further as it may be a poor attractant.

CHAPTER 7

GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS

7.1 General Discussion

The present study demonstrates that B. invadens occurs throughout the year but its abundance is generally higher from January to April and is greatly influenced by geographic location. The results from this study indicated fluctuations in the Flies per Trap per Day (FTD) depending on month and locality, with notable differences in adult population densities. The population of the invader fruit fly started to increase with the onset of the rains and generally coincided with the main mango fruiting period in all the locations investigated during this study. It may be stated that the presence of mango hosts is crucial in B. invadens population build up. Both biotic and abiotic factors such as temperature and relative humidity appear to have a complex influence on B. invadens population dynamics.

Altitude seems to play a pivotal role in influencing the activities of the invader fly. Research done elsewhere has indicated that there seems to be a positive linear relationship between altitude and the population of fruit flies up to around 1,200 m.a.s.l. However, elevation by itself does not determine fruit fly distribution but associated factors such as temperature, rainfall and host plants at such elevations seem to play a significant role. It was also observed that B. invadens is generally prevalent at low-mid altitude and its population is significantly higher at low-mid elevation compared to high altitude. Thus B. invadens populations were significantly higher in Murewa and Mutoko than Domboshava and Seke. Data from fruit rearings seem also to portray a similar picture. This is possibly an indication that the invader fruit fly is slowly dispersing into new areas. Partial results from the mango agroecosystems studied may suggest that significant competitive interactions between the invasive pest and the indigenous (Ceratitis spp.) fruit fly species are at play. Thus two possible mechanisms could be responsible for the displacement of primarily the native fruit flies, namely, resource competition by larvae within the mango fruits and aggression between adult fruit flies. These would require further investigation under local conditions.

Bactrocera invadens populations seemed to increase just after the onset of the rainy season from December reaching a peak in March. Incidentally, this period coincides with the main mango fruiting and ripening period across most parts of Zimbabwe. In addition, during the same period, guavas which are also another preferred host of the invader fly are also abundant in the orchards. The population seemed to decline during the dry season particularly in Mutoko, Domboshava and Seke. Although the population seems to decline after the last rains and the main mango fruiting season, B. invadens trap catches in Murewa continued to be quite high even during the coldest months of the year.

The role played by wild fruits in bridging the season when mangoes are off-season needs to be studied. Also the role played by guavas (including feral guavas) and other cultivated fruits such as oranges and naartjies which occur in the absence of mango during the winter period needs to be fully comprehended as these fruits could be responsible for sustaining a significant fruit fly population. This scenario appears to be playing out in Murewa where despite the absence of mango in winter, relatively high populations of B. invadens are still maintained. Fruit rearing of oranges and naartjies that were carried out during the citrus fruiting period showed that these are also hosts of B. invadens though infestation rates appeared to be quite low. It must be noted that Murewa has a strong presence of citrus fruits such as naartjies and oranges than any of the other sites studied in the intensive study. It can therefore be speculated that these could be responsible for maintaining the population at such high levels even in the absence of mango, which is the most preferred host.

From the fruit rearing studies, two prominent native tephritid fruit fly species (C. cosyra and C. rosa) and a newly detected exotic fruit fly pest, B. invadens were recorded to be associated with mangoes during this present study. The relative abundance of these indigenous fruit fly species appears to be affected by the recently detected B. invadens. It therefore seems quite probable that this new invasive fruit fly species is out-competing and displacing the indigenous fruit fly species particularly at low to mid altitude.

Among the species recorded to be infesting mangoes, the infestation levels of B. invadens were observed to increase soon after the first rains. This observation was also noted in the

trapping exercise B. invadens trap catches seemed to increase during the rainy season from December and reaching a peak in March. A similar trend was observed in all the intensive study sites. It must, however, be emphasised that though this new invasive species is able to quickly dominate the indigenous species, complete exclusion of the latter has not yet been recorded. Whether B. invadens will eventually become the dominant species in these areas and eventually out-compete the native fruit fly species remains to be seen. Nonetheless its negative impact in the fruit and vegetable sectors in Zimbabwe cannot be underestimated. Thus the issue of interspecific competition between the indigenous and the invasive fruit fly species need to be explored further.

The baseline information generated in this study could be useful in coming up with ‘smart’ strategies for the management of fruit flies in specific localities. For example, in areas such as Murewa and Mutoko where B. invadens far overshadows indigenous fruit fly species, it could be more appropriate to have attractants that are specific to B. invadens such as methyl eugenol (for B. invadens males) and Biolure 3C (for B. invadens females) though the latter attractant can also be used to attract even other fruit fly species within the genera Bactrocera and Ceratitis. At relatively higher altitude, such as Domboshava and Seke, it will be important to use more lures which are specific to C. cosyra and C. rosa (such as Ceratitislure, Capilure and Terpinyl acetate) than those specific to B. invadens (methyl eugenol) as they are the dominant species in these particular localities. This information could be practically useful in cases where the male annihilation technique is the most preferred option.

Fruit fly management has largely been dependent on the use of insecticides for their control. This has of late included the use of food baits which are normally mixed with a compatible insecticide and then applied to fruit trees. Traditionally, malathion has been the insecticide of choice with food baits. Application of broad spectrum insecticides has of late been discouraged and the continuous use of the same insecticide for prolonged periods of times has led to the development of resistance. Thus, results obtained in this study could well be used in the management of B. invadens and malathion, deltamethrin and trichlorfon could be useful in this regard.

7.2 Conclusion

Results from the current study show that B. invadens now occurs widely across Zimbabwe. While B. invadens was the dominant species in the mid altitudes of Mutoko and Murewa, C. cosyra and C. rosa dominated at relatively higher altitude in locations such as Domboshava and Seke. In general, although the seasonal occurrence of B. invadens was found to follow a similar pattern at all study sites, its population densities varied significantly across sites. It was apparently clear from the trap catches made that the African invader fly exhibits a broad range of colour pattern variation particularly on the scutum and abdomen — as has been reported in other localities in and beyond the African continent — And this clearly justifies the need to incorporate molecular techniques in its identification.

Some of the insecticides used in this study such as trichlorfon, deltamethrin and malathion remarkably affected males of B. invadens under laboratory conditions. These chemicals therefore show promise to be used in rotation. The suspected native fruit fly parasitoids (Psyttalia cosyrae and Tetrastichus giffardi) which were recovered during this study could also aid in further understanding the natural fruit fly dynamics which are obtaining in the local mango agro-ecosystems.

The ongoing taxonomic debate on the species status of B. invadens has clearly showed the inadequacies associated with solely depending on morphological characteristics for its identification. Since B. invadens belong to a complex, it is hoped that ongoing efforts in various spheres seeking to find its proper placement within the B. dorsalis complex will help ease quarantine restrictions and potentially contribute to its better management particularly if the sterile insect technique or eradication from particular locality or region is an option. In the meantime, it can be argued that phytosanitary treatments in place for other species within the B. dorsalis complex can be applied to B. invadens.

In conclusion, it can be stated that the preliminary results of this present study which largely focused on four selected sites in Mashonaland East province, together with data from other related past studies obtained within Zimbabwe and beyond show the need for further studies on B. invadens biology, overwintering mechanisms and behaviour. The

interaction between abiotic and biotic factors on fruit fly diversity and B. invadens in particular need to be explored further.

7.3 Recommendations

From the results of the study, several recommendations can be made going forward.

 To better understand the population dynamics of B. invadens in Zimbabwe, there is need to undertake this study over several years, locations and fruit hosts (both wild and cultivated).

 The population dynamics and impact of native parasitoids associated with B. invadens need to be studied.

 Competitive interactions between B. invadens and among mango-infesting indigenous fruit fly species in Zimbabwe need to be studied.

 There is need for studies to track the movement of B. invadens adult females so as to understand the role played by non-mango hosts in the seasonal carryover of the pest.

 The losses which are associated with fruit fly infestations, particularly under the smallholder need to be quantified in order to raise awareness of the magnitude caused by fruit fly infestations. Very little knowledge is available to smallholder farmers on the actual threat posed by fruit flies. In fact fruit fly damage is often misdiagnosed as rotting due to disease, thus very few farmers make any effort to control them.

 The potential of using guavas (another good host of B. invadens) as trap crops in mango orchards need to be explored as these two differ significantly in terms of value hence the guava could be ‘sacrificed’ in order to minimise fruit infestation levels of mangoes.

 To conduct studies aimed at investigating the effect of the B. invadens across all the local mango varieties (both string and stringless) grown in this country. Information from such studies could useful in breeding mangoes which can tolerate attack from the invasive fruit fly.

 The potential use of botanicals in the management of fruit flies as an alternative to synthetic insecticides needs to be investigated, since these pose less harm to the environment.

 Considering that the area under feral guavas is increasing in many areas, their importance in the sustenance of B. invadens needs to be investigated.

REFERENCES

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