Doctor of Philosophy / A number of types of molecular polymorphisms can be used for studying genetic relationship and evolutionary history. Microsatellites are hypervariable and can be very useful tools to determine population structure, distinguish sibling species, as well as verifying parental relationships and pedigrees. However, while microsatellite polymorphisms are useful for solving relationships between populations within a species, relations among species or genera will probably be obscured due to a high degree of homoplasy —identity arising from evolutionary convergence not by descent. For long range evolutionary history, such as phylogeny from old world monkey to human, mtDNA markers may be better candidates. The aim of this thesis is to assess molecular polymorphisms of different types and their optimal use in different situations. Two widely separated taxa were used for testing –the green monkey Chlorocebus sabaeus, and the sibling dipteran flies Bactrocera tryoni and B. neohumeralis, known collectively as the Queensland fruit fly. In the present study a complete 16,550 bp mtDNA sequence of the green monkey Chlorocebus sabaeus is reported for the fist time and has been annotated (Chapter 2). Knowledge of the mtDNA genome contributes not only to identification of large scale single nucleotide polymorphisms (SNPs) (Chapter 4) or other mtDNA polymorphisms development, but also to primate phylogenetic and evolutionary study (Chapter 3). Microsatellites used for the green monkey paternity and pedigree studies were developed by cross-amplification using human primers (Chapter 5). For studies of population structure and species discrimination in Queensland fruit fly (Chapter 7), microsatellites were isolated from a genomic library of Bactrocera tryoni (Chapter 6) The total length of 16550 bp of complete mtDNA of the green monkey C. sabaeus, which has been sequenced and annotated here, adds a new node to the primate phylogenetic tree, and creates great opportunity for SNP marker development. The heteroplasmic region was cloned and five different sequences from a single individual were obtained; the implication of this are discussed. The phylogenetic tree reconstructed using the complete mtDNA sequence of C. sabaeus and other primates was used to solve controversial taxonomic status of C. sabaeus. Phylogenies of primate evolution using different genes from mtDNA are discussed. Primate evolutionary trees using different substitution types are compared and the phylogenetic trees constructed using transversions for the complete mtDNA were found close to preconceived expectations than those with transversions + transitions. The sequence of C. sabaeus 12SrRNA reported here agrees with the one published by ven der Kuyl et al. (1996), but additional SNPs were identified. SNPs for other regions of mtDNA were explored using dHPLC. Twenty two PCR segments for 96 individuals were tested by dHPLC. Fifty five SNPs were found and 10 haplogroups were established. Microsatellite markers were used to construct a genealogy for a colony of green monkeys (C. sabaeus) in the UCLA Vervet Monkey Research Colony. Sixteen microsatellites cross-amplified from human primers were used to conduct paternity analysis and pedigree construction. Seventy-eight out of 417 offspring were assigned paternity successfully. The low success rate is attributed to a certain proportion of mismatches between mothers and offspring; the fact that not all candidate fathers were sampled, the limitations of microsatellite polymorphisms; and weakness of the exclusion method for paternity assessment. Due to the low success rate, the pedigree is split into a few small ones. In a complicated pedigree composed of 75 animals and up to four generations with multiple links a power male mated with 8 females and contributed 10 offspring to the pedigree. Close inbreeding was avoided. Population structure within two species of Queensland fruit fly Bactrocera tryoni and Bactrocera neohumeralis (Tephritidae: Diptera) is examined using microsatellite polymorphisms. Queensland fruit flies B. tryoni and B. neohumeralis are sympatric sibling species that have similar morphological and ecological features. They even share polymorphism at the molecular level. Mating time difference is the main mechanism by which they maintain separate species. In the present study, 22 polymorphic and scorable microsatellites were isolated from B. tryoni and tested in the two species sampled from sympatric distribution areas. Pairwise genetic distance analysis showed explicit differentiation in allele frequencies between the two species, but very weak differences between conspecific populations. Gene flow is higher within B. tryoni than within B. neohumeralis, and gene exchange between the two species exists. An averaging linkage clustering tree constructed by UPGMA showed two major clusters distinguishing the two species, and it appears that population structure is highly correlated with geographic distance. The relationship between molecular markers, evolution, and selection are discussed using comparative studies within two large taxa: primate and insect. The degree of conservation and polymorphism in microsatellites varies between taxa, over evolutionary time.

Doctor of Philosophy / A number of types of molecular polymorphisms can be used for studying genetic relationship and evolutionary history. Microsatellites are hypervariable and can be very useful tools to determine population structure, distinguish sibling species, as well as verifying parental relationships and pedigrees. However, while microsatellite polymorphisms are useful for solving relationships between populations within a species, relations among species or genera will probably be obscured due to a high degree of homoplasy —identity arising from evolutionary convergence not by descent. For long range evolutionary history, such as phylogeny from old world monkey to human, mtDNA markers may be better candidates. The aim of this thesis is to assess molecular polymorphisms of different types and their optimal use in different situations. Two widely separated taxa were used for testing –the green monkey Chlorocebus sabaeus, and the sibling dipteran flies Bactrocera tryoni and B. neohumeralis, known collectively as the Queensland fruit fly. In the present study a complete 16,550 bp mtDNA sequence of the green monkey Chlorocebus sabaeus is reported for the fist time and has been annotated (Chapter 2). Knowledge of the mtDNA genome contributes not only to identification of large scale single nucleotide polymorphisms (SNPs) (Chapter 4) or other mtDNA polymorphisms development, but also to primate phylogenetic and evolutionary study (Chapter 3). Microsatellites used for the green monkey paternity and pedigree studies were developed by cross-amplification using human primers (Chapter 5). For studies of population structure and species discrimination in Queensland fruit fly (Chapter 7), microsatellites were isolated from a genomic library of Bactrocera tryoni (Chapter 6) The total length of 16550 bp of complete mtDNA of the green monkey C. sabaeus, which has been sequenced and annotated here, adds a new node to the primate phylogenetic tree, and creates great opportunity for SNP marker development. The heteroplasmic region was cloned and five different sequences from a single individual were obtained; the implication of this are discussed. The phylogenetic tree reconstructed using the complete mtDNA sequence of C. sabaeus and other primates was used to solve controversial taxonomic status of C. sabaeus. Phylogenies of primate evolution using different genes from mtDNA are discussed. Primate evolutionary trees using different substitution types are compared and the phylogenetic trees constructed using transversions for the complete mtDNA were found close to preconceived expectations than those with transversions + transitions. The sequence of C. sabaeus 12SrRNA reported here agrees with the one published by ven der Kuyl et al. (1996), but additional SNPs were identified. SNPs for other regions of mtDNA were explored using dHPLC. Twenty two PCR segments for 96 individuals were tested by dHPLC. Fifty five SNPs were found and 10 haplogroups were established. Microsatellite markers were used to construct a genealogy for a colony of green monkeys (C. sabaeus) in the UCLA Vervet Monkey Research Colony. Sixteen microsatellites cross-amplified from human primers were used to conduct paternity analysis and pedigree construction. Seventy-eight out of 417 offspring were assigned paternity successfully. The low success rate is attributed to a certain proportion of mismatches between mothers and offspring; the fact that not all candidate fathers were sampled, the limitations of microsatellite polymorphisms; and weakness of the exclusion method for paternity assessment. Due to the low success rate, the pedigree is split into a few small ones. In a complicated pedigree composed of 75 animals and up to four generations with multiple links a power male mated with 8 females and contributed 10 offspring to the pedigree. Close inbreeding was avoided. Population structure within two species of Queensland fruit fly Bactrocera tryoni and Bactrocera neohumeralis (Tephritidae: Diptera) is examined using microsatellite polymorphisms. Queensland fruit flies B. tryoni and B. neohumeralis are sympatric sibling species that have similar morphological and ecological features. They even share polymorphism at the molecular level. Mating time difference is the main mechanism by which they maintain separate species. In the present study, 22 polymorphic and scorable microsatellites were isolated from B. tryoni and tested in the two species sampled from sympatric distribution areas. Pairwise genetic distance analysis showed explicit differentiation in allele frequencies between the two species, but very weak differences between conspecific populations. Gene flow is higher within B. tryoni than within B. neohumeralis, and gene exchange between the two species exists. An averaging linkage clustering tree constructed by UPGMA showed two major clusters distinguishing the two species, and it appears that population structure is highly correlated with geographic distance. The relationship between molecular markers, evolution, and selection are discussed using comparative studies within two large taxa: primate and insect. The degree of conservation and polymorphism in microsatellites varies between taxa, over evolutionary time.

La localisation des plantes-hôtes est un processus déterminant dans la stratégie adaptative des insectes phytophages et les stimuli olfactifs, qui sont des composés volatils émis par les plantes, jouent un rôle essentiel dans ce processus. Bactrocera cucurbitae (Diptera, Tephritidae) est responsable d'importants dégâts sur maintes cultures maraîchères à l'échelle mondiale et à La Réunion. Si la bio-écologie et la structuration génétique des populations de cette espèce sont relativement bien connues, des connaissances sur la médiation chimique se produisant lors de la localisation des fruits-hôtes restent nécessaires pour améliorer la gestion des populations de cette espèce. L'objectif de la thèse est de caractériser les stimuli olfactifs impliqués dans la localisation à distance par les femelles de B.cucurbitae, d'une large gamme de fruits-hôtes de Cucurbitaceae sauvages et cultivées de La Réunion. Une approche bidisciplinaire (comportementale et chimique) a été adoptée afin d'identifier, parmi 27 fruits-hôtes, les composés volatils responsables de l'attraction des femelles vers leurs sites de ponte. Les résultats soulignent, qu'au sein de la gamme de plantes-hôtes, la réponse des femelles aux effluves de ces fruits est très hétérogène (taux d’attraction de 1 % à 74 %), variant selon l'espèce végétale, la variété et le stade de maturité des fruits. Parallèlement, l'étude de la composition chimique des effluves de ces fruits révèle une chimiodiversité inter- et intraspécifique considérable : 280 composés volatils identifiés au total, avec des profils d'émission différents d'un fruit à l'autre. Ces résultats, complétés par de l’électroantennographie couplée à la chromatographie gazeuse, ont permis de déterminer 34 composés potentiellement impliqués dans l'attraction des femelles. De plus, un mélange à deux composés volatils de synthèse, aussi attractif qu'une odeur de fruits frais, a été identifié à partir des réponses des femelles à 10 des 34 composés, présentés individuellement ou en mélange. Les rôles des composés volatils de fruits-hôtes dans le processus de localisation des sites de ponte et dans la spécialisation écologique de cette espèce de ravageurs sont discutés. Par ailleurs, l'association des résultats de préférence des femelles et de performance larvaire représente une perspective de recherche prometteuse, qui contribuerait à acquérir une connaissance plus fine du phénomène de spécialisation d'hôte. Enfin, il est désormais possible d'envisager l'utilisation du mélange simple à deux composés volatils dans un système de piégeage des femelles, à intégrer dans un schéma de gestion agroécologique des populations de B. cucurbitae. / Locating host-plants is a key process in the adaptive strategy of phytophagous insects, and olfactory stimuli, which arevolatile compounds emitted by plants, play an essential role in this process. Bactrocera cucurbitae (Diptera,Tephritidae) is responsible for significant damage to many vegetable crops in Reunion Island and worldwide. Whilethe bioecology and genetic population structure of this species are relatively well known, understanding the chemicalmediation occurring during host-fruit location is needed to improve the management of this pest. The aim of this thesis is to characterize the olfactory stimuli involved in the location by females of B. cucurbitae, of awide range of wild and cultivated host fruits of Cucurbitaceae in Reunion Island. A multidisciplinary approach (behavioural and chemical) was adopted to identify volatile compounds responsible for the attraction of females to their oviposition sites in 27 host fruits. The results highlight that within the range of host plants the female response to fruit odours is very heterogeneous(attraction rates from 1% to 74%). These levels of female response vary according to the plant species and the varietyand the stage of fruit maturity. Meanwhile, the study of the chemical composition of fruit odours reveals aconsiderable inter- and intraspecific chemodiversity: 280 volatile compounds identified in total, with differentemission profiles from one fruit to another. These results, complemented by coupled gas chromatographyelectroantennographic detection, have identified 34 compounds potentially involved in female attraction. In addition, ablend of two synthetic compounds as attractive as fresh fruit odour was identified by measuring attractiveness of 10 ofthe 34 compounds presented individually or in combination. The roles of volatiles in oviposition sites location and ecological specialization of this pest are discussed. Moreover,the association of these results with data on female preference and larval performance represents a promising researchdirection, which would contribute to a better understanding of the phenomenon of host specialization. Finally, theintegration of a simple two-component blend in a trapping system for females into an agroecological management scheme for B. cucurbitae populations is proposed.

The objective of this dissertation is the isolation of DNA sequences that are differentially represented in males and females of the Oriental fruit fly Bactrocera dorsalis, specifically by initiating a molecular characterization of Y chromosome sequences in this species. Cytological observations have established the presence of a diminutive Y chromosome in B. dorsalis males. To isolate DNA sequences from the Y chromosome, a special method of genomic DNA isolation known as Representational Difference Analysis (RDA) was utilized to obtain DNA sequences unique to the B. dorsalis male genome. Genomic DNA from B. dorsalis males served as the "tester" DNA and female genomic DNA as the "driver" DNA. Six distinct RDA products were obtained following two complete rounds of DNA hybridization and difference enrichment via the Polymerase Chain Reaction (PCR). One ofthese products (RDA product 1) was used to isolate a genomic DNA clone (3.1a) from a B. dorsalis male genomic DNA minilibrary. This sequence shows similarity to the reverse transcriptase of R1 retrotransposable elements. The presence of R1 elements in the Tephritid insects has heretofore been undescribed, although these elements have been previously described in the genomes of other Dipteran species. Oligonucleotide primers for PCR were designed for the 3.1a clone. These primers consistently produce different amplification patterns in PCRs ofgenomic DNA from B. dorsalis males vs. females. Amplification using male genomic DNA produces 325 bp and 2.6 kb products while only a 2.6 kb product is obtained from female DNA. The amplification products obtained with these primers are also produced in PCRs of genomic DNA from B. dorsalis embryos and third instar larvae, suggesting the ability of this method to infer sex at pre-adult stages ofthe B. dorsalis life cycle. Similar amplification products have also been obtained in other Bactrocera species. Both the 325 bp male PCR product and the 2.6 kb products have regions of sequence similarity to R1 elements. The 2.6 kb product contains a putative 1.7 kb open reading frame (ORF) encoding 583 amino acids. Three amino acid motifs found in Drosophila R1 element reverse transcriptases are present in comparable locations within the hypothetical ORF product. Both of these sequences are also repetitively represented in the B. dorsalis male and female genomes. However, the 325 bp male product produces some bands that are male specific when used as a probe for Southern blots of B. dorsalis male and female genomic DNA. The amplification pattern produced by the 3.1a primers is consistent with what would be expected if the 2.6 kb and 325 bp PCR products originated from the B. dorsalis X and Y chromosomes, respectively. Thus, the cloned male-specific sequence recovered here is potentially useful both as a gateway into the relatively uncharacterized B. dorsalis Y chromosome and as a tool for the characterization of other aspects of the B. dorsalis genome.

Bactrocera tryoni, the Queensland fruit fly, is established along the entire Australian east coast. It is a major pest of horticulture and arguably the worst horticultural insect pest in Australia. Adult flies lay eggs into fruit and resultant larvae feed on the flesh of the fruit. The population biology of B. tryoni has been well studied in temperate regions, where it has been established that climatic factors, particularly temperature and rainfall, limit population growth. In contrast, in subtropical and tropical regions, the population dynamics of the fly have been little studied. This thesis investigates the fly's phenology and abundance changes across subtropical and tropical Queensland and asks what factors govern the population cycles of B. tryoni in this state. Winter breeding and abundance of the fly, a component of the seasonal cycle which in south-east Queensland is fundamentally different from that observed in temperate Australia, is also investigated. A historical, extensive multi-year and multi-site trapping data set with from across Queensland was analysed to look at the effects of temperature, rainfall and relative humidity on B. tryoni trap catch. Trap data was further compared with the predicted phenology data generated by a DYMEX® based B. tryoni population phenology model. The phenology model used was based on a previously published model, but was also modified to more explicitly look at the effects of host plant availability and the presence or absence of non-reproductive over-wintering flies. Over-wintering field cage studies and a winter-spring field trapping study, both carried out in Brisbane, supplied additional data on B. tryoni's population abundance and capacity to breed during winter in the subtropics. Results show significant variation of monthly fly abundance for nine sites across Queensland. Abundance changed across sites in non-predictable ways. Annual population phenology within a site was, for some sites, highly consistent from year to year, but inconsistent for other sites. All sites in the subtropics showed some form of population depression during the cooler months, but breeding was continuous, albeit reduced at nearly all sites. Some tropical sites, where the climate is regarded as highly favourable for B. tryoni, still showed dramatic peaks and troughs in annual population abundance. There were relatively few significant correlations observed between weather factors and fly populations for any site. Output from the DYMEX population model suggested that fruit availability is a major driver of population dynamics in the tropical north of the state, while weather is more important in the subtropical south. The population dynamics of B. tryoni at sites along the central Queensland coast, where it is assumed that a mix of both weather and host fruit availability drive local populations, were poorly captured by the population model. Field cage results showed that B. tryoni successfully bred during winter in Brisbane, with pupal emergence starting in mid-winter (1st week of August), peaking in early spring (2nd week of September). Trap catch at orchards in Brisbane increased with increasing temperature and fruit availability, but diminished with decreasing temperature and fruit availability. The results suggest that B. tryoni has an optimal climate for population growth in the tropics, but fruit availability for offspring production limits population growth. In the subtropics however, both climate and fruit availability determine the population size. Winter temperatures are marginal for B. tryoni population growth in the subtropics.

A Pest Risk Analysis was conducted on commodities imported as hand luggage from Cameroon, India and Kenya at O.R. Tambo International Airport. The initiation phase indicated that fruit, and to a lesser extent vegetables, are often imported concealed in hand luggage and not declared for inspection. Such undeclared commodities pose a risk to the South African agricultural industry and environment as it may serve as a pathway for quarantine pests. The qualitative pest risk assessment conducted on the commodities intercepted indicated that the risk is high, with the commodities serving as pathways for quarantine pests including insects, bacteria, mites, fungi, viruses and weeds. Bactrocera fruit flies were identified as a specific high risk and were further subjected to a quantitative pest risk assessment. Mitigation treatments for fruit flies were evaluated for efficacy and effect on the quality of the fruit. Hot water immersion treatment was deemed to be an effective mitigation treatment for fruit flies. This treatment on fruit flies was found effective at pulp temp of 46°C and 47°C where the pulp temp is held for 10 and 12 minutes respectively. / Dissertation (MInstAgrar)--University of Pretoria, 2009. / Plant Production and Soil Science / unrestricted

The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), a major pest of horticulture in eastern Australia, is a relatively poor coloniser of new habitat. This thesis examines behavioural properties that might limit the ability of B. tryoni to establish new populations. As the potential for B. tryoni to establish an outbreak population may be most directly limited by mechanisms associated with dispersal and mating behaviour, these two factors were the focus of this research project. The relevance of dispersal and mating behaviour for control of outbreak populations was assessed. Dispersal (i) Dispersal patterns of males and females are not different. Dispersal of post-teneral male B. tryoni from a point within an orchard near Richmond, New South Wales, was monitored following temporally replicated releases. Application of sterile insect technique (SIT) requires knowledge of dispersal from a release point so that effective release rates can be determined. In addition, dispersal following introduction to new habitat can lead to low or negative population growth and an Allee effect. In Spring and Autumn, 2001 � 2003, three different strains of B. tryoni were released: (1) wild flies reared from infested fruit collected in the Sydney Basin; (2) a laboratory-reared strain with a colour mutation (white marks); and (3) sterile flies obtained by gamma-irradiation of a mass-reared strain. Dispersal was monitored using a grid of traps baited with the male attractant, cuelure. During the majority of releases, flies were massmarked using a self-marking technique and fluorescent pigment powder to enable identification of recaptured flies. A preliminary study found that fluorescent pigment marks had no effect on adult survival and marks did not fade significantly in the laboratory over a period of five weeks after eclosion. As cuelure repels inseminated sexually mature female B. tryoni, unbaited, coloured flat sticky traps, and black and yellow sticky sphere traps baited with a food lure (protein autolysate solution) were used to supplement traps baited with cuelure. The effectiveness of these two sticky trap types was assessed, and recaptures used to compare patterns of dispersal from a release point by male and female B. tryoni. Fluorescent yellow (chartreuse), green, and clear unbaited flat sticky traps were relatively ineffective for monitoring dispersal of sterile male and female B. tryoni, recapturing only 0.1% of released sterile flies. Monitoring dispersal with sticky ball traps baited with protein autolysate solution was more successful, with yellow spheres and black spheres recapturing 1.7% and 1.5%, respectively. Trap colour had no effect on recaptures on flat sticky traps or sticky spheres. Equal recapture rates on yellow and black sticky sphere traps suggests that the odour of yeast autolysate solution was more important than colour for attraction of post-teneral flies to traps. Using the results of recaptures on odoriferous black and yellow sticky sphere traps within one week of release, regression equations of male and female recaptures per trap were found to be similar (Figure 4-3). This is the first study to clearly indicate that post-teneral dispersal patterns of male and female B. tryoni released from a point do not differ, enabling the use of existing models to predict density of both sexes of B. tryoni following post-teneral dispersal. (ii) Males disperse further in Spring than in Autumn, but this is not temperature-related. Analysis of replicated recaptures in traps baited with cuelure revealed that dispersal of male B. tryoni in an orchard near Richmond, New South Wales, was higher in Spring than in Autumn (Figure 5-6). As the maximum daily temperature was significantly higher in Spring than in Autumn this result was unexpected, since earlier studies have found that B. tryoni disperse at the onset of cool weather in search of sheltered over-wintering sites. Dispersal of post-teneral B. tryoni may have been affected by habitat suitability; it was found that seasonal trends in dispersal could have been influenced by local habitat variables. Low mean dispersal distances in Autumn may be explained by the presence of fruiting hosts in the orchard, or the availability of resources required by over-wintering flies. There was no significant correlation between temperature and mean dispersal distance, suggesting that higher rates of dispersal cannot be explained by temperature-related increases in activity. Recapture rate per trap was significantly negatively correlated with increasing daily maximum and average temperature. This may have consequences for detection of B. tryoni outbreaks in quarantine areas due to reduced cuelure trap efficiency. (iii) Maturity and source variation affect dispersal and response to cuelure. This research indicated that most male and female B. tryoni do not disperse far from a release point, suggesting that an invading propagule would not spread far in the first generation. However, there is considerable variation in flight capability among individuals. Comparison of wild, laboratory-reared white marks, and gamma-irradiated sterile male B. tryoni indicated that mean dispersal distance and redistribution patterns were not significantly affected by fly origin. Despite no difference in dispersal distance from the release point, recaptures of wild and sterile males per Lynfield trap baited with cuelure were highest within one week after release, whereas recaptures of white marks males per trap increased in the second week. This result may offer evidence to support the hypothesis that sterile male B. tryoni respond to cuelure at an earlier age. Rearing conditions used to produce large quantities of males for sterilisation by gamma-irradiation may select for earlier sexual maturity. Mating Behaviour (i) Density and sex ratio do not affect mating, except at low densities. Demographic stochasticity in the form of sex ratio fluctuations at low population density can lead to an observed Allee effect. The effect of local group density and sex ratio on mating behaviour and male mating success of a laboratory-adapted strain of B. tryoni was examined in laboratory cages. In the laboratory-adapted strain of B. tryoni used in this study, a group of one female and one male was sufficient for a good chance of mating success. The proportion of females mated and male mating success was not significantly affected by density or sex ratio, although variability in male mating success was higher at low density. This could indicate that mating success of B. tryoni can be reduced when local group density is low owing to decreased frequency in encounters between males and females. (ii) Mass-reared males exhibit aberrant mating behaviour, but this does not reduce mating success. Strong artificial selection in mass-rearing facilities may lead to decreased competitiveness of sterile males released in SIT programs as a result of alteration or loss of ecological and behavioural traits required in the field. The effects of domestication and irradiation on the mating behaviour of males of B. tryoni were investigated by caging wild, mass-reared and sterile (mass-reared and gammairradiated) males with wild females. Mating behaviour of mass-reared males was different from that of wild males, but behaviour of wild and sterile males was similar. Mass-reared males were found to engage in mounting of other males much more frequently than wild and sterile males, and began calling significantly earlier before darkness. Male calling did not appear to be associated with female choice of mating partners, although this does not exclude the possibility that calling is a cue used by females to discriminate between mating partners. Conditions used to domesticate and rear large quantities of B. tryoni for SIT may select for an alternative male mating strategy, with mass-reared males calling earlier and exercising less discrimination between potential mating partners. Despite differences in behaviour of wild, mass-reared and sterile males, frequency of successful copulations and mating success were similar. (iii) Pheromone-calling by males was increased in larger aggregations but this did not result in significantly more female visits. Finally, large laboratory cages with artificial leks were used to investigate the importance in B. tryoni of male group size for female visitation at lek sites and initiation of male pheromone-calling. Calling propensity of male B. tryoni was increased by the presence of conspecific males. Females visited the largest lek more frequently than single males, but there was no correlation between lek size and female visitation. Female B. tryoni had a limited capacity to perceive a difference between the number of calling males; female visitation at leks was only weakly associated with male calling, suggesting that lek size and the number of pheromone-calling males may not be the only factor important in locating mates in B. tryoni. The weak, but positive correlation between male calling and female visitation may indicate that passive attraction maintains lek-mating in B. tryoni. Further studies are essential on mating behaviour of B. tryoni, including identification of male mating aggregations in the field, measurement of habitat variables associated with male aggregations, the influence of density on wild B. tryoni mating success, and the role of pheromone-calling, in order to optimise use of SIT for control of this pest.

The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), a major pest of horticulture in eastern Australia, is a relatively poor coloniser of new habitat. This thesis examines behavioural properties that might limit the ability of B. tryoni to establish new populations. As the potential for B. tryoni to establish an outbreak population may be most directly limited by mechanisms associated with dispersal and mating behaviour, these two factors were the focus of this research project. The relevance of dispersal and mating behaviour for control of outbreak populations was assessed. Dispersal (i) Dispersal patterns of males and females are not different. Dispersal of post-teneral male B. tryoni from a point within an orchard near Richmond, New South Wales, was monitored following temporally replicated releases. Application of sterile insect technique (SIT) requires knowledge of dispersal from a release point so that effective release rates can be determined. In addition, dispersal following introduction to new habitat can lead to low or negative population growth and an Allee effect. In Spring and Autumn, 2001 � 2003, three different strains of B. tryoni were released: (1) wild flies reared from infested fruit collected in the Sydney Basin; (2) a laboratory-reared strain with a colour mutation (white marks); and (3) sterile flies obtained by gamma-irradiation of a mass-reared strain. Dispersal was monitored using a grid of traps baited with the male attractant, cuelure. During the majority of releases, flies were massmarked using a self-marking technique and fluorescent pigment powder to enable identification of recaptured flies. A preliminary study found that fluorescent pigment marks had no effect on adult survival and marks did not fade significantly in the laboratory over a period of five weeks after eclosion. As cuelure repels inseminated sexually mature female B. tryoni, unbaited, coloured flat sticky traps, and black and yellow sticky sphere traps baited with a food lure (protein autolysate solution) were used to supplement traps baited with cuelure. The effectiveness of these two sticky trap types was assessed, and recaptures used to compare patterns of dispersal from a release point by male and female B. tryoni. Fluorescent yellow (chartreuse), green, and clear unbaited flat sticky traps were relatively ineffective for monitoring dispersal of sterile male and female B. tryoni, recapturing only 0.1% of released sterile flies. Monitoring dispersal with sticky ball traps baited with protein autolysate solution was more successful, with yellow spheres and black spheres recapturing 1.7% and 1.5%, respectively. Trap colour had no effect on recaptures on flat sticky traps or sticky spheres. Equal recapture rates on yellow and black sticky sphere traps suggests that the odour of yeast autolysate solution was more important than colour for attraction of post-teneral flies to traps. Using the results of recaptures on odoriferous black and yellow sticky sphere traps within one week of release, regression equations of male and female recaptures per trap were found to be similar (Figure 4-3). This is the first study to clearly indicate that post-teneral dispersal patterns of male and female B. tryoni released from a point do not differ, enabling the use of existing models to predict density of both sexes of B. tryoni following post-teneral dispersal. (ii) Males disperse further in Spring than in Autumn, but this is not temperature-related. Analysis of replicated recaptures in traps baited with cuelure revealed that dispersal of male B. tryoni in an orchard near Richmond, New South Wales, was higher in Spring than in Autumn (Figure 5-6). As the maximum daily temperature was significantly higher in Spring than in Autumn this result was unexpected, since earlier studies have found that B. tryoni disperse at the onset of cool weather in search of sheltered over-wintering sites. Dispersal of post-teneral B. tryoni may have been affected by habitat suitability; it was found that seasonal trends in dispersal could have been influenced by local habitat variables. Low mean dispersal distances in Autumn may be explained by the presence of fruiting hosts in the orchard, or the availability of resources required by over-wintering flies. There was no significant correlation between temperature and mean dispersal distance, suggesting that higher rates of dispersal cannot be explained by temperature-related increases in activity. Recapture rate per trap was significantly negatively correlated with increasing daily maximum and average temperature. This may have consequences for detection of B. tryoni outbreaks in quarantine areas due to reduced cuelure trap efficiency. (iii) Maturity and source variation affect dispersal and response to cuelure. This research indicated that most male and female B. tryoni do not disperse far from a release point, suggesting that an invading propagule would not spread far in the first generation. However, there is considerable variation in flight capability among individuals. Comparison of wild, laboratory-reared white marks, and gamma-irradiated sterile male B. tryoni indicated that mean dispersal distance and redistribution patterns were not significantly affected by fly origin. Despite no difference in dispersal distance from the release point, recaptures of wild and sterile males per Lynfield trap baited with cuelure were highest within one week after release, whereas recaptures of white marks males per trap increased in the second week. This result may offer evidence to support the hypothesis that sterile male B. tryoni respond to cuelure at an earlier age. Rearing conditions used to produce large quantities of males for sterilisation by gamma-irradiation may select for earlier sexual maturity. Mating Behaviour (i) Density and sex ratio do not affect mating, except at low densities. Demographic stochasticity in the form of sex ratio fluctuations at low population density can lead to an observed Allee effect. The effect of local group density and sex ratio on mating behaviour and male mating success of a laboratory-adapted strain of B. tryoni was examined in laboratory cages. In the laboratory-adapted strain of B. tryoni used in this study, a group of one female and one male was sufficient for a good chance of mating success. The proportion of females mated and male mating success was not significantly affected by density or sex ratio, although variability in male mating success was higher at low density. This could indicate that mating success of B. tryoni can be reduced when local group density is low owing to decreased frequency in encounters between males and females. (ii) Mass-reared males exhibit aberrant mating behaviour, but this does not reduce mating success. Strong artificial selection in mass-rearing facilities may lead to decreased competitiveness of sterile males released in SIT programs as a result of alteration or loss of ecological and behavioural traits required in the field. The effects of domestication and irradiation on the mating behaviour of males of B. tryoni were investigated by caging wild, mass-reared and sterile (mass-reared and gammairradiated) males with wild females. Mating behaviour of mass-reared males was different from that of wild males, but behaviour of wild and sterile males was similar. Mass-reared males were found to engage in mounting of other males much more frequently than wild and sterile males, and began calling significantly earlier before darkness. Male calling did not appear to be associated with female choice of mating partners, although this does not exclude the possibility that calling is a cue used by females to discriminate between mating partners. Conditions used to domesticate and rear large quantities of B. tryoni for SIT may select for an alternative male mating strategy, with mass-reared males calling earlier and exercising less discrimination between potential mating partners. Despite differences in behaviour of wild, mass-reared and sterile males, frequency of successful copulations and mating success were similar. (iii) Pheromone-calling by males was increased in larger aggregations but this did not result in significantly more female visits. Finally, large laboratory cages with artificial leks were used to investigate the importance in B. tryoni of male group size for female visitation at lek sites and initiation of male pheromone-calling. Calling propensity of male B. tryoni was increased by the presence of conspecific males. Females visited the largest lek more frequently than single males, but there was no correlation between lek size and female visitation. Female B. tryoni had a limited capacity to perceive a difference between the number of calling males; female visitation at leks was only weakly associated with male calling, suggesting that lek size and the number of pheromone-calling males may not be the only factor important in locating mates in B. tryoni. The weak, but positive correlation between male calling and female visitation may indicate that passive attraction maintains lek-mating in B. tryoni. Further studies are essential on mating behaviour of B. tryoni, including identification of male mating aggregations in the field, measurement of habitat variables associated with male aggregations, the influence of density on wild B. tryoni mating success, and the role of pheromone-calling, in order to optimise use of SIT for control of this pest.

The oriental fruit fly, Bactrocera dorsalis Hendel, is an economic tephritid that invaded Senegal in 2004 (Vayssieres et al. 2011). This work determined 1) Impact of B. dorsalis on mango crop production and value, and marketing practices, 2) Effect of neem and kaolin on its behavior and development (laboratory and field), and 3) Effect of treated soil with neem seed cake (NSC) and neem seed powder (NSP) on the larval-pupal survival and development (laboratory and field). Results revealed crop value (price/kg) was associated with infestation levels. P<0.0001, crop value decreased in highly infested orchards. Southern Orchards were heavily infested than northern orchards. Early harvest, orchard sanitation, decreasing the purchase quantity, sorting infested mangoes and lowering prices were strategies used by growers and traders. Laboratory bioassays showed females landed and spent time on fruit identically on control and neem. On kaolin there were fewer landings and less time spent. P<0.0001, mean pupae was higher in control than in treated mangoes. In the field, percent of infested mangoes was higher in neem than in control and kaolin. However, the number of emerged flies was higher in the control than in either the neem or kaolin-treated fruit. Treated soil with NSC and with NSP did not have effect on pupation; all larvae pupated in the lab. However, treated soil decreased significantly the number of emerged flies. In the field, there were no significant differences between untreated and treated soil in number of emerged flies. / Master of Science in Life Sciences

This thesis investigated the autecology of the dacine species, Bactrocera cacuminata (Hering) (Diptera: Tephritidae: Dacinae). I specifically focused on the adult phase of the life cycle and resources believed to be significant to this life stage. The prevailing paradigm in dacine ecology predicts that the larval host plant serves as the centre of dacine activity, a state mediated by mutualistic associations with fruit fly-type bacteria. Contrary to predictions, an explicit test of this hypothesis found that the host plant of B. cacuminata, Solanum mauritianum Scopoli, acted almost exclusively as a site for oviposition and larval development. Other key adult behaviours, most notably feeding and mating, were rare at the host plant. Even in disturbed habitats, the paucity of key adult behaviours such as mating was striking. Adult flies of this species were therefore hypothesized to be utilizing other components of their habitat, i.e. resources vital to their life history requirements. Some of the resources that B. cacuminata are known to respond to include sugar, protein, methyl eugenol and the host plant. The latter three resources are believed to be critical in the reproductive success of dacine flies in general. I assessed the physiological status of flies arriving at these resources to determine if flies of different status foraged for resources differently. In dacines, the internal reproductive structures of the male and female flies have been used as predictors of physiological status. I quantified expansion of the male ejaculatory apodeme in B. cacuminata with age of fly and found that there is a threshold apodeme size that is strongly correlated Abstract with sexual maturity. Maturity of female flies could be accurately predicted by ovarian development. Using these methods to assess the physiological and nutritional status of flies arriving at resources (larval host plant, protein and methyl eugenol) in the field, I discovered that only sexually mature and mated females were responding to the host plant, while the males at the host plant were sexually immature. This confirmed the hypothesis that the host plant primarily served as an oviposition site. Additionally, this study revealed that sexually mature males with high nutritional reserves were most commonly collected at methyl eugenol (a plant-derived chemical that elicits a strong response in males of many dacine species) at dusk, the time of peak sexual activity in this species. This indicated that methyl eugenol was perhaps a significant resource in the context of the reproductive behaviour of this species. Methyl eugenol (ME) is one of group of phenyl propanoids to which males of certain species of Dacinae respond. The current hypothesis of the role of these phenyl propanoids is that they function as pheromone precursor chemicals. Response to these chemicals is hypothesized to be a trait under sexual selection. In Bactrocera dorsalis (Hendel), this effect is so strong that a single feeding on ME results in a strong mating advantage up to a month after males feed on the chemical. Bactrocera cacuminata fed on multiple occasions on ME in a laboratory bioassay. After a single 24-hour exposure to ME, investigations of mating competitiveness did not reveal any obvious advantage for ME-fed males over unfed males. However, ME-fed males did enjoy a higher mating success 16 and 32 days after exposure to the chemical, suggesting that some physiological benefits unrelated to the pheromone synthesis was driving this delayed advantage. Investigation of the physiological consequences of feeding on ME revealed no enhancement of nutritional or energetic reserves, suggesting that the delayed mating advantage observed was more likely a chance event. An alternate hypothesis about the proximate function of ME, proposed by Robert Metcalf, is that it serves as a mate rendezvous site. As mating behaviour was notably absent at the host plant, I tested Metcalf’s hypothesis. A field-cage experiment, spatially separating adult resources (host plant, methyl eugenol, sugar and protein) clearly demonstrated that methyl eugenol was functioning as a mate rendezvous stimulus for B. cacuminata. This is the first direct support for Metcalf’s hypothesis. A synthesis of the literature revealed that significantly greater ecological and evolutionary information was required to understand the basis of dacine response to phenyl propanoids. Different dacine species may be utilizing these chemicals differently, even if their evolutionary origin may have been as a plant based kairomone. My studies show that generalizations on the ecology and behaviour of Dacinae, often extrapolated from research on a few pest species, do not hold up in the case of B. cacuminata. This suggests that a more autecological, species-specific approach is required in dacine research, before any predictive generalizations can be made.