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.

Ο δάκος της ελιάς, Bactrocera oleae, αποτελεί το κυριότερο παράσιτο του καρπού της ελιάς. Λόγω της μεγάλης οικονομικής σημασίας της ελιάς, ιδιαίτερα για τις Μεσογειακές χώρες, ο αποτελεσματικότερος έλεγχος του εντόμου αυτού είναι απαραίτητος. Η εφαρμογή μεθόδων ολοκληρωμένης διαχείρισης του παρασίτου αυτού, μεθόδων δηλαδή φιλικών προς το περιβάλλον, επιβάλλει την καλύτερη γνώση της βιολογίας του και ιδιαίτερα της γενετικής και της γενετικής δομής των φυσικών πληθυσμών του. Στα πλαίσια αυτά, στόχος της παρούσας μελέτης ήταν η ανάπτυξη στο δάκο DNA δεικτών, των μικροδορυφόρων, οι οποίοι είναι άφθονοι στα γονιδιώματα και επιπλέον υψηλά πολυμορφικοί, για την ανάλυση φυσικών πληθυσμών του. Για την απομόνωση των μικροδορυφορικών δεικτών ακολουθήθηκαν τρεις διαφορετικές στρατηγικές: η κατασκευή και διαλογή γονιδιωματικών βιβλιοθηκών, η κατασκευή εμπλουτισμένων σε μικροδορυφόρους βιβλιοθηκών και ο έλεγχος ζευγών εκκινητών που είχαν σχεδιαστεί για την ενίσχυση μικροδορυφόρων στα συγγενικά είδη B. tyroni και C. capitata για το κατά πόσο ενισχύουν τις αντίστοιχες περιοχές και στο γονιδίωμα του εντόμου B. oleae (cross-species amplification). Από τις βιβλιοθήκες απομονώθηκαν συνολικά 69 κλώνοι που περιείχαν μικροδορυφόρους. Ακολούθησε ο σχεδιασμός ζευγών εκκινητών στις μοναδικές περιοχές που περιβάλλουν τους μικροδορυφόρους. Συνολικά σχεδιάστηκαν 42 ζεύγη εκκινητών. Οι εκκινητες αυτοί ελέγχθηκαν για το αν ενισχύουν το αναμενόμενο προϊόν. Ο έλεγχος έγινε με PCR και ηλεκτροφόρηση των προϊόντων σε πήκτωμα αγαρόζης. Παράλληλα ελέγχθηκαν είκοσι ζεύγη εκκινητών που είχαν σχεδιαστεί για μικροδορυφόρους του εντόμου B. tryoni και 42 ζεύγη εκκινητών που είχαν σχεδιαστεί για μικροδορυφόρους του εντόμου C. capitata. Οι τρεις διαδικασίες απομόνωσης έδωσαν συνολικά 67 λειτουργικά ζεύγη εκκινητών. Στη συνέχεια ελέγχθηκε μέσω PCR ο πολυμορφισμός των εκκινητών αυτών και το αν ενισχύουν διακριτά αλληλόμορφα, με μήτρα DNA γενετικό υλικό εννέα ατόμων. Τα προϊόντα της PCR αναλύθηκαν σε πήκτωμα ακρυλαμιδίου. Από τα 49 ζεύγη εκκινητών που ελέχθησαν, τα 28 έδωσαν πολύ καθαρό σήμα, ενώ τα 25 από αυτά βρέθηκαν πολυμορφικά. Μια πρώτη πληθυσμιακή ανάλυση έγινε με τη χρησιμοποίηση 24 από αυτούς τους δείκτες για την ανάλυση μικρού δείγματος ατόμων. Βασικός σκοπός της ανάλυσης αυτής ήταν να ελεγχθεί η ποιότητα των δεικτών και οδήγησε στον αποκλεισμό έξι δεικτών: ο ένας ήταν μονομορφικός, ένας δεύτερος έδειξε ασθενή ενίσχυση και μικρή επαναληψιμότητα και άλλοι τέσσερις εμφάνισαν απόκλιση από την ισορροπία κατά H-W, πιθανότητα λόγω της παρουσίας null αλληλομόρφων. Δώδεκα από τους υπόλοιπους δεκαοκτώ δείκτες χρησιμοποιήθηκαν για μια εκτεταμένη ανάλυση των πληθυσμών του δάκου στην Ευρωπαϊκή πλευρά της λεκάνης της Μεσογείου. Αναλύθηκαν δεκαεννέα δείγματα, μεγέθους εννέα ως πενήντα ατόμων, από έξι διαφορετικές χώρες (Ελλάδα, Κύπρος, Τουρκία, Ιταλία, Ισπανία και Πορτογαλία). Η ανάλυση αποκάλυψε σχετικά μικρές γενετικές αποστάσεις, που έδειχναν όμως μια στατιστικά σημαντική διαφοροποίηση σε τρεις υποπληθυσμούς. Ο πρώτος αποτελείτο από τα δείγματα της Κύπρου, ο δεύτερος από τα δείγματα Ελλάδας, Τουρκίας και Ισπανίας και ο τρίτος από τα δείγματα της Ιβηρικής χερσονήσου. Οι στατιστικές αναλύσεις που έγιναν έδειξαν τη σημαντική επίδραση της γεωγραφικής απόστασης στη δημιουργία αυτών των ομαδοποιήσεων. Οι τρεις αυτές ομάδες χαρακτηρίζονται από διαφορά και στο επίπεδο του πολυμορφισμού, εμφανίζοντας μια καθαρή μείωσή του από την Ανατολή προς τη Δύση. Η μείωση αυτή είναι στατιστικά σημαντική και με βάση την υπόθεση ότι η πορεία εποικισμού ενός είδους συνοδεύεται από μείωση του πολυμορφισμού, δίνει σημαντικές ενδείξεις για μία προς Δυσμάς πορεία εποίκισης του είδους στον Ευρωπαϊκό χώρο, με πρώτο κέντρο εξάπλωσης την Ανατολική λεκάνη της Μεσογείου. Τα πειράματα δια-ειδικής ενίσχυσης έδειξαν στενές φυλογενετικές σχέσεις των ειδών που εξετάστηκαν και κυρίως μεταξύ B. tryoni - B. oleae και B.oleae - C. capitata. Τα πειράματα αυτά υποστηρίζουν την χρησιμότητα των δεικτών που απομονώθηκαν σε ενδεχόμενες φυλογενετικές μελέτες στα είδη αυτά, καθώς και σε άλλα συγγενικά είδη. Η εύρεση ενός μεγάλου ποσοστού συντηρημένων μικροδορυφόρων σε είδη που έχουν διαχωριστεί εδώ και πολλά εκατομμύρια χρόνια ενισχύει την υπόθεση ότι οι μικροδορυφόροι δεν είναι γενετικό υλικό χωρίς ρόλο (junk DNA), αλλά επιτελούν συγκεκριμένες λειτουργίες στο γονιδίωμα. Η ανάλυση πολυμορφισμού που έγινε στις ατομικές διασταυρώσεις ατόμων από εργαστηριακούς ήταν ιδιαίτερα ενθαρρυντική. Ο υψηλός πολυμορφισμός που βρέθηκε δείχνει την δυνατότητα χρησιμοποίησης τους και στη γενετική χαρτογράφηση του είδους. / The olive fruit fly, Bactrocera oleae, is the main pest of the olive fruit. Because of its great economic importance, especially for the Mediterranean countries, there is a need for a more effective control method. The application of an integrated, environmental friendly, management of this pest requires a better knowledge of its biology and of the genetic structure of its natural populations. The aim of the present study was the development of DNA microsatellite markers for the analysis of the natural populations of the olive fruit fly. These markers are abundant in the genome of any species studied so far and highly polymorphic. Three different strategies were used for the isolation of microsatellite markers. The first was the construction and screening of genomic libraries of the insect, the second was the construction of genomic libraries, enriched for microsatellites and the third was the use of primer pairs that were designed for the amplification of microsatellite markers in the closely related species Bactrocera oleae and Ceratitis capitata(cross-species amplification). A total of 69 microsatellite containing clones were isolated from libraries. The next step was the design of primer pairs in the microsatellite flanking sequences. A total of 42 primer pairs was designed and tested for their abillty to amplify the expected product. Test was performed through PCR and analysis of the PCR products through electrophoresis on agarose gel. Twenty primer pairs designed for the amplification of Bactrocera tryoni’ s microsatellites and 42 primer pairs designed for the amplification of Ceratitis capitata microsatellites were also tested. All three strategies gave 67 primer pairs that amplified the expected product. The degree of polymorphism of these primer pairs and their ability to amplify easily resolvable alleles was tested through PCR with DNA template of nine individuals. PCR products were analysed through polyacrylamide gel electrophoresis. Twenty eight out of the 49 primer pairs tested produced clear bands and twenty five of them were polymorphic. A small scale population analysis was then performed, using tventy four of the markers available. The main purpose of this analysis was to demonstate the quality of the markers and lead to the exclusion of six markers: one of them was monomorphic, another didn’t show reproducible results and four more showed deviations from H-W equilibrium, probably because of the presence of null alleles. Twelve of the remaining loci were used in a large scale analysis of B. oleae’s populations in the European part of the Mediterranean basin. Nineteen samples, varying from nine to fifty individuals, were analysed. These samples were collected from six different countries (Greece, Cyprus, Turkey, Italy, Spain and Portugal). The analysis revealed relatively low genetic distances, which, however, demonsrated a statistically important differentiation of the samples in three subpopulations. The first consisted of the samples from Cyprus, the second of the samples from Greece, Turkey and Italy and the third of the samples from the Iberian Peninsula. The statistical analyses performed showed the statistically important contribution of geographic distance to the generation of genetic distance. These three groups of samples also demonstrate a clear loss of polymorphism towards West. This loss is statistically important and, if we take into account the hypothesis that the colonization process of a species is followed by a loss in polymorphism, it suggests a colonization process of the olive fruit fly towards West in the European part of the Mediterranean basin, with a first expansion area in the Eastern part of the Mediterranean basin. Cross-species amplification experiments indicate close phylogenetic relationships among the species studied, mainly between B. tryoni-B. oleae and B. oleae-C. capitata. These results support the usefulness of the markers isolated in phylogenetic studies in these species, as well as in other, closely related species. The identification of a high percentage if conserved microsatellites in species that have been well separated for millions of years is in agreement with the hypothesis that microsatellites are not useless genomic regions (junj DNA) but they perform specific functions in the genome. Polymorphism analysis in the crosses of individuals from laboratory strains was very encouraging. The high degree of polymorpism showes that they can be used in genetic mapping of the species

Fruit flies (Tephritidae) can enter and establish in new territories due to the movement of fruit from one area to another through trade or tourism, which can negatively impact on fruit production and market access. An invader fruit fly species (Bactrocera invadens) has established on the African continent and has spread throughout sub-Saharan Africa. This newly described polyphagous fruit fly species is a successful invader species which continues to distribute and establish in new habitats. The introduction and establishment of B. invadens in South Africa may have serious market access consequences with regard to fruit exports due to its absence in the territories of many trading partners. The Musina area was considered as the study area as it is the first entry point from Zimbabwe. The national highway (N1) which runs through the area is a major route from several B. invadens infested countries in the Southern Africa region. A species initiated pest risk assessment was conveyed to determine the risk potential of this pest. The pest risk assessment (PRA) identified several pathways with a high risk to the Musina area, that B. invadens can follow. A detection survey was carried out to determine the status of B. invadens in the Musina area as support to the PRA. The detection survey continued over three years and by the second year B. invadens was detected for the first time in the study area. The detection survey was followed by a delimiting survey and the pest was eradicated in the area. After several months of no detection, it was however detected again in the area. Risk management options were suggested for regulatory control as an outcome of the pest risk assessment. These measures can be utilised by the National Plant Protection Organisation of South Africa for the commercial importation of host material of B. invadens, control of fruit imported by travellers, informal traders and national control in the event of pest incursions in the area. Corrective actions as well as quarantine actions should be implemented in an integrated approach in the affected areas. / Thesis (Master of Environmental Sciences)--North-West University, Potchefstroom Campus, 2013.

Fruit flies (Tephritidae) can enter and establish in new territories due to the movement of fruit from one area to another through trade or tourism, which can negatively impact on fruit production and market access. An invader fruit fly species (Bactrocera invadens) has established on the African continent and has spread throughout sub-Saharan Africa. This newly described polyphagous fruit fly species is a successful invader species which continues to distribute and establish in new habitats. The introduction and establishment of B. invadens in South Africa may have serious market access consequences with regard to fruit exports due to its absence in the territories of many trading partners. The Musina area was considered as the study area as it is the first entry point from Zimbabwe. The national highway (N1) which runs through the area is a major route from several B. invadens infested countries in the Southern Africa region. A species initiated pest risk assessment was conveyed to determine the risk potential of this pest. The pest risk assessment (PRA) identified several pathways with a high risk to the Musina area, that B. invadens can follow. A detection survey was carried out to determine the status of B. invadens in the Musina area as support to the PRA. The detection survey continued over three years and by the second year B. invadens was detected for the first time in the study area. The detection survey was followed by a delimiting survey and the pest was eradicated in the area. After several months of no detection, it was however detected again in the area. Risk management options were suggested for regulatory control as an outcome of the pest risk assessment. These measures can be utilised by the National Plant Protection Organisation of South Africa for the commercial importation of host material of B. invadens, control of fruit imported by travellers, informal traders and national control in the event of pest incursions in the area. Corrective actions as well as quarantine actions should be implemented in an integrated approach in the affected areas. / Thesis (Master of Environmental Sciences)--North-West University, Potchefstroom Campus, 2013.

Frugivorous insects, specialised herbivores that consume fruit and seeds, are considered detrimental to host plant fitness. Their direct link to genetic fitness via consumption of plant reproductive tissue, and their negative socioeconomic association with agriculture exacerbates their harmful status. However, empirical testing of insect frugivore effects on host plants, and ecological research on the contribution of insect frugivores to multitrophic frugivory systems, is lacking. In the current study, direct effects of a non-mutualistic, insect frugivore/host plant system were tested and results showed variable effects. Beneficial, detrimental, but predominantly neutral effects on germination and seed production were observed between the Queensland fruit fly (Bactrocera tryoni) and tomato and capsicum plants. Significant effects on seed production were unexpected because infestation occurs after seed set. It was also found that eggplant, although a recorded host of B. tryoni, is inconsistent in its ability to sustain B. tryoni larvae through to its final instar. These results confirmed a simplification and presumption associated with insect frugivore (specifically fruit fly)/host plant interactions. Larval movement, infestation-induced fruit decay, pulp removal and germination were then investigated. For all hosts (tomato, apple and paw paw), treatments infested by B. tryoni decayed significantly quicker and to a greater extent than uninfested treatments, with obvious but variable changes to the texture and appearance. The movement of B. tryoni larvae, pattern of infestation-induced decay and pulp removal was unique and host dependent for all hosts. Only seeds from infested tomato were shown to germinate during the experiment. This indicated that host fruit characteristics are responsible, in part, for variable direct effects on host plant fitness by insect frugivores. Variable direct effects between insect frugivores and host plants, combined with the more rapid decay of infested fruits is likely to have implications for seed dispersal and seed predation by a third trophic level. The characteristics of fruit that are changed by infestation by an insect frugivore were then tested for their effect on a vertebrate frugivore, to illustrate the importance of recognising multitrophic interactions and indirect effects in frugivory. Specifically, seed predating rodents were incorporated into the study and their response to infested and uninfested fruits were recorded, as well as their reaction to the changes in fruit caused by insect frugivores (i.e. texture, smell, larvae presence and sound). Apple and pear infested with B. tryoni larvae were found to attract rodents, while infested tomato and paw paw had a neutral effect on the native rats. This differed from the predominant finding in the literature, which was a deterrent effect on avian seed dispersers. Vertebrate response to fruit infested with insect frugivores therefore, is variable. Assessing the indirect effect of insect frugivores on host plant fitness by attracting or deterring another trophic level requires knowledge of the direct effect between the introduced trophic level and the host plant. For example, the attraction of a seed predator may be as detrimental to host plant fitness as the deterrence of a seed disperser. This illustrates the complexity associated with assessing insect frugivore effects on host plant fitness. Results also indicated that differences in pulp texture, caused by infestation, have a significant effect on rodent preference for infested or uninfested treatments. Pulp texture is likely to effect rodent foraging efficiency, whereas the presence of B. tryoni larvae was observed to be inconsequential to rodent response to fruits. For rodents, and indeed any trophic level motivated by foraging efficiency, this finding raises the issue that for long lived fruiting plants, outside factors such as food abundance and competition for food, may cause a variable response to fruits infested by insect frugivores. From these investigations it has become apparent that insect frugivores are not consistently harmful to host plant fitness, as suggested by their negative stigma, but are likely to contribute variable effects, directly and indirectly, on multiple components of plant fitness and multitrophic frugivory systems.