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The congeneric wasps, Encarsia formosa and E. pergandiella (Hymenoptera: Aphelinidae) are solitary endoparasitoids with an overlapping host range. Despite their similarities these wasps produce eggs that differ markedly in size. This dissertation details research conducted to determine differences in the egg provisioning strategies of these two wasp species and to understand how these differences correlate with differences in their early development. The use of the yolk protein, vitellogenin, was examined in the two wasps. A vitellogenin gene was isolated from E. formosa and the mature gene product observed in ovary extracts. Evidence for the use of vitellogenin was not found in an analysis of ovary extracts from E. pergandiella and a gene for vitellogenin was not detected in the genome of this wasp. Embryonic development times for the two parasitoids were studied in hosts of different ages. The embryonic development time of E. formosa is significantly shorter than that of E. pergandiella regardless of the host stage parasitized. This suggests that the rate of embryonic development of E. pergandiella is much more closely linked to that of the host than the development rate of E. formosa. The quantity and composition of amino acids in the eggs of the two wasps was followed over the course of embryonic development. The quantity of amino acid in the eggs of E. formosa does not increase during embryonic development while the eggs of E. pergandiella absorb more than 30 times the quantity of amino acids from the host hemolymph during embryonic development than is present in the eggs at the time oviposition. Only E. pergandiella appeared capable of absorbing and utilizing [¹⁴C]-labeled lysine in an in vitro system. The capacity of E. pergandiella eggs to absorb host nutrients is correlated with the development of a multinucleate extraembryonic membrane that grows to completely encompass the embryo at a very early stage of development. Evidence was found for pinocytosis of material by the embryo from the space bounded by the extraembryonic membrane. A similarly developed extraembryonic membrane was not observed in the eggs of E. formosa .

Nutrient storage is an important, but understudied life history trait. One approach to characterizing the role of nutrient storage in insect life histories begins with determining the rules that organisms use to allocate nutrients to storage in comparison to other life history traits. My dissertation research focuses on understanding rules of allocation to nutrient storage in two types of insects that differ in their life histories, grasshoppers and ants. First, I showed that the grasshopper, Schistocerca americana , differs from other known grasshoppers by having only a single dominant storage hexamerin that occurs in both larvae and adults. Second, I showed that allocation between somatic growth and lipid storage at the last larval-adult molt in females of the grasshopper, Schistocerca americana, was plastic in response to larval resource availability. On the best growth diet, individuals were larger and contained proportionally greater lipid stores than individuals on the lowest nutrient content diet. Prioritization of nutrient allocation to somatic growth over lipid storage on poor diets in Schistocerca americana females suggests that larvally derived lipid stores have some role in adult fitness, but that body size is more important. Third, I showed that two closely related members of the Camponotus festinatus species complex of desert carpenter ants differ in their life histories with respect to lipid storage. Dark-form workers and soldiers stored significantly more lipid per unit lean mass than light-form workers or soldiers, but light-form colonies involved a slightly larger proportion of soldiers in storing lipid. Fourth, we showed that there was a relationship between storage protein content of queens and founding strategies in five species of Harvester Ants in the genus Pogonomyrmex. Independent claustral-founding species contained the greatest protein stores, followed by independent facultative-foraging queens. Independent obligate-foraging queens, and queens of a dependent social parasite contained almost no protein stores. Last, we showed that arboreal and terrestrial ants differed in both seasonal foraging activity and nutritional preferences in a lowland neo-tropical forest. Differences in foraging and nutritional preferences likely reflect fundamental differences in nutrient availability in the two habitats.

Evolution of new adaptations in response to novel environmental selection pressures is often associated with negative pleiotropic costs. Fitness costs may be ameliorated by further evolutionary processes if selection pressure is maintained. Modifier mutations that ameliorate the fitness costs of resistance mutation may arise. Alternatively, resistance mutations that incur lower fitness costs at the same locus or other loci will replace the resistance mutations with high fitness costs. Taylor and Feyereisen (1996) proposed a hypothesis of genetic "succession" for such a mechanism in the evolution of resistance to toxins. Numerous disruptive mutations in the target of a toxin may confer resistance, but with the likelihood of high fitness costs at an early stage in the evolution of resistance. Rare specific mutation(s) with less fitness costs will replace the early resistance allele(s) at a later stage and form a genetic succession. The genetic succession hypothesis is examined in this dissertation. Theory and modeling predicts that genetic succession is likely a process involved in resistance evolution against stimulatory toxins. Thus, numerous disruptive mutations on the target site will confer resistance with high fitness costs initially, but later will be replaced by rare specific mutation(s) with lower fitness costs. Genetic succession is also investigated in the case of sodium channel mutations for pyrethroid resistance in Heliothis virescens. Three distinct resistance mutations in or near the H. virescens sodium channel gene hscp have been determined by sequence comparisons; Val 421 to Met (V421M), Leu1029 to His (L1029H), and the third factor linked to Hpy5 allele that involves neither of the mutations V421M or L1029H. Frequency changes in these mutations during the time of sampling (1990 to 1997) suggest a successional replacement of both V421M and an unknown Hpy5-linked mutation by L1029H in Louisiana H. virescens populations. Further information on relative fitness costs of the mutations is necessary before we can conclude with confidence that the apparent replacement of the V421M and Hpy5 linked resistance by L1029H H. virescens represents a genuine successional event.

Black scavenger flies are characterized by sexual behaviours that are very unusual in insects. I have studied two of the most remarkable elements of their mating systems: the timing of copulations immediately after an oviposition bout (post-oviposition matings) and the males' escorting of ovipositing females. In a study of the patterns of sperm precedence in one sepsid species, I found that the sepsids' peculiar timing of matings is not associated with unusual patterns of sperm precedence: sepsid males displace rival sperm and achieve a large last male advantage, which is the most common outcome of sperm competition in insects. I discuss the potential significance of sperm transfer mechanisms for the sepsids' timing of matings, and I consider factors that may favour the maintenance of post-oviposition matings in sepsid populations. In a survey of sepsid mating patterns, I found that post-oviposition matings are typical of many black scavenger flies and that mating systems characterized by the absence of copulations with gravid females may have arisen early in the family's evolutionary history. In several black scavenger flies, ovipositing females are commonly accompanied by an escorting male, and in all but one of the species I have studied, escorting is pre-copulatory. In several species, I found pronounced geographic variation in the expression of this trait. I argue that sepsids share certain characteristics which may have facilitated multiple independent origins of escorting behaviour. In order to investigate the adaptive significance of escorting, I have conducted a comparative study of patterns of sexual size dimorphism and sex ratios at oviposition sites in conspecific populations that show great divergence in the expression of this trait. The results of this research support the pre-copulatory mate guarding hypothesis for the adaptive significance of escorting behaviour, and they suggest that conspecific populations vary significantly in the degree or nature of sexual selection acting both on morphology and behaviour of males. Furthermore, in a study of the genetic architecture of escorting behaviour, I found that the observed behavioural variation has a genetic basis: the expression of escorting behaviour is a quantitative trait with a significant sex-related component of inheritance.

Three resistance monitoring methods, leaf disk, sticky trap, and vial, were tested to evaluate their relative reliability, discriminating ability, convenience, and practicality for monitoring insecticide resistance in Arizona whiteflies. Each method was evaluated against two field populations divergent in susceptibility using a mixture of fenpropathrin + acephate and two single chemicals, endosulfan and fenpropathrin. Correlations of field efficacy and leaf disk bioassays resistance estimates were conducted with the Yuma population and a comparatively resistant Maricopa population. At each location egg, immature, and adult whitefly densities were monitored before and after fenpropathrin + acephate treatments. The three methods had advantages and disadvantages. The leaf disk method had the greatest discriminating ability, the vial method was the most practical, and the sticky trap method was good at discriminating populations that had large differences in susceptibility. The field efficacy trials indicated good concordance between the leaf disk assays results and field performance.

An analysis of the genetic structure of northern Nearctic wolf spiders from the genus Pardosa (Lycosidae) was conducted. Wolf spiders are common and abundant across the Nearctic, but despite their ecological importance, little is known about their phylogeography and taxonomically uncertain species exist. Wolf spiders were collected from sites across northern Canada to examine the phylogeographic history of Pardosa glacialis (Thorell) and taxonomic status of the Nearctic members of the Pardosa lapponica species-group. Results show that the high Arctic species P. glacialis occupied two major glacial refugia during the Wisconsin glaciation: Beringia and a lesser-known high Arctic refugium. Additionally, analyses support a P. glacialis population expansion in Beringia during the Wisconsin glaciation, likely facilitated by an increase in habitat due to suitable climate in the region. Post-glacial dispersal from the Beringian and high Arctic refugia produced a secondary contact zone in central high Arctic Canada. With regards to taxonomy, morphometric analyses of P. lapponica (Thorell) and P. concinna (Thorell), the only two members of the P. lapponica species-group in the Nearctic, revealed no species-specific variation beyond a single male morphological character. Genetic analyses showed there was more genetic variation between Palearctic and Nearctic P. lapponica specimens than between Nearctic P. lapponica and P. concinna specimens. The P. lapponica species-group is in need of taxonomic revision to resolve the species boundaries among its members. / Une analyse de la structure génétique des Lycoses néarctiques du Grand Nord appartenant au genre Pardosa (Lycosidae) a été effectuée. Les Lycoses sont des espèces communes et abondantes dans le néarctique. Malgré leur importance écologique, peu d'information est disponible sur leur phylogéographie et l'identité taxonomique de certaines espèces n'est pas clairement établie. Dans le contexte de cette étude, nous avons recueilli des Lycoses dans le nord du Canada afin d'étudier l'histoire phylogéographique de Pardosa glacialis (Thorell) et d'établir le statut taxonomique des membres néarctiques du groupe d'espèces Pardosa lapponica. D'après nos résultats, l'espèce arctique P. glacialis aurait autrefois occupé deux importants refuges lors de la glaciation du Wisconsin : la Béringie et un refuge situé dans le Haut-Arctique. Les analyses appuient une expansion de la population de P. glacialis en Béringie pendant la glaciation wisconsinienne, une situation vraisemblablement favorisée par une augmentation de l'habitat disponible grâce à un climat adéquat. La dispersion postglaciaire des espèces à partir de ces refuges a créé une zone de contact secondaire, située dans le centre du Haut-Arctique canadien. Par rapport au statut taxonomique, une étude morphométrique de P. lapponica (Thorell) et P. concinna (Thorell), les deux seules espèces membres du groupe P. lapponica dans le Néarctique, n'a dévoilé aucune variation discriminante, mis à part un caractère morphologique chez les mâles. L'analyse des caractères génétiques a démontré qu'il n'y a pas plus de variation entre les spécimens paléarctiques et néarctiques de P. lapponica qu'entre les spécimens de P. lapponica et P. concinna provenant exclusivement du Néarctique. Une révision taxonomique du groupe d'espèces P. lapponica qui réexaminerait les limites interspécifiques entre les membres de ce groupe est nécessaire.

Anaplasma marginale is a virulent intra-erythrocytic pathogen that causes bovine anaplasmosis, its closely related species, Anaplasma centrale causes mild sickness. The pathogen is transmitted biologically by tick vectors and mechanically through blood contaminated fomites. It has a worldwide distribution extending from tropical to subtropical regions in correlation with the vector distribution. Bovine anaplasmosis is often characterised by progressive anaemia, jaundice, decreased milk production, abortion and a sudden death. The commonly used method for the diagnosis of A. marginale of infected cattle in South Africa is microscopic examination of Giemsa stained blood smears and detection of antibodies from serum using cELISA. However the diagnostic methods have limitations in cases of low parasitemia and in carrier cattle (microscopy) and they fail to differentiate closely related Anaplasma spp due to antigenic similarity (serology), the detection limitations of the diagnostic methods influenced the aim of this study which is to develop molecular species -specific assays for the detection of A. marginale strains in South Africa, specifically Including conventional polymerase chains reaction, real-time polymerase chain reaction and loop-mediated isothermal amplification assay. Chapter one of this study discusses bovine anaplasmosis and its causative agent A. marginale, the diversity of the strains transmission, distribution, clinical signs, treatment and economic importance of the disease. The first objective of this study was to develop a species specific conventional PCR for detection of A. marginale in cattle in South African regions based on msp1b gene. The conventional PCR primers were designed through visual inspection and were named F3 and B3 primers. In the specificity test, the primers were specific whereby the amplified only A. marginale DNA and did not amplify control DNAâs: A. centrale, Babesia bovis, B. bigemina and Ehrlihia rumunantium. The sensitivity of the conventional PCR primers was examined using a 10 ng/ul DNA and the detection limit of the assay was 0.01 ng/ul, The assay was validated on field samples to confirm the infection of the cattle with A. marginale, out of 144 samples, (60%) infection rate was obtained with the newly developed conventional PCR, the homogeneity of the sequences were confirmed with the GenBank, the maximum similarity varied from 94 - 100%. The second objective of this study was to develop a species-specific real-time PCR for detection of A. marginale in cattle in South African regions based on msp1b gene. The real-time PCR primers and probes were designed using Genescript program, one set of primer (Prf 2, PrR2, and PrB2) was chosen to carry out the study as it showed high sensitivity with the detection limit of 0.001 ng/ul .The specific and sensitive TaqMan based real-time PCR was successfully developed for the of A. marginale infections in South Africa. Validation of the assay on field samples showed that the rate of infection was 74% in different sampled provinces of South Africa. The third objective of this study was to develop loop-mediated isothermal amplification for the detection of A. marginale in South African regions based on msp1b gene. The LAMP primers were designed using primer Explorer version 4, the LAMP primers were named LAF3, LA-B3, LA-FIP, LA-BIP,LA-LF and LA-LB. The LAMP assay showed positive results with specific amplification, but as far as the validation of the assay false positive results were obtained, troubleshooting involved the addition of additives, changing of primer purification and manufacturers, however the results were not consistent, false positive results were obtained, speculations were that it could be possible contamination of the laboratory resulting in the amplification of control DNA and distilled water. The first three objectives of this study were achieved. The newly developed assays were further compared for specificity, sensitivity and detection performance on field derived samples. The developed assays are specific and sensitive; they form a good tool of diagnosis of bovine anaplasmosis, with each assay having its own unique characteristic over the other, they are sensitive giving a correct determination of the infection status, aiding in compiling of epidemiological information. These assays will aid in understanding the major constraint to develop control measures due to the genetic diversity of A. marginale, and will also help in constructing of phylogenetic tree between strains from South Africa and other countries.

Ticks and tick-borne diseases are a burden in the livestock industry, decreasing productivity and compromising food security, leading to high socioeconomic impacts on agro-exporting nations. Apart from being agricultural pests they can transmit pathogens of zoonotic significance. The aim of the study was to therefore detect and determine with PCR the prevalence of tick-borne zoonotic pathogens i.e. Coxiella burnetii, Ehrlichia spp., Rickettsia spp., Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato from ticks collected from livestock. The sampling areas included both commercial and communal farms as well as domestic animals from KwaZulu-Natal, Free State, Eastern Cape, North West and Mpumalanga Provinces. As a result a total of 1947 tick samples were collected which were then identified and further processed for PCR amplification. Tick species collected included Rhipicephalus spp. (n = 570), R. sanguineus (n = 275), R. evertsi evertsi (n = 650), R. decoloratus (n = 228), R. appendiculatus (n = 10), Amblyomma hebraeum (n = 171), Hyalomma marginatum rufipes (n = 4), and Haemaphysalis elliptica (n = 38). The overall prevalence of infection with B. burgdorferi and A. phagocytophilum was 8±1.4% and 9±1.2% respectively, this was an unexpected finding since only one positive PCR confirmation of A. phagocytophilum has been reported in the country, since then no other studies have been successful in detecting this pathogen. There have been anecdotal cases of B. burgdorferi but the pathogen has, to the best my knowledge, not been detected and characterized by molecular methods. Both pathogens have not been isolated from ticks in South Africa previously. The tick vectors for these pathogens are not known to occur in the country, however this study managed to isolate A. phagocytophilum from R. sanguineus, R. appendiculatus, R. evertsi evertsi and H. elliptica and isolated B. burgdorferi from Rhipicephalus spp., R. evertsi evertsi, R. decoloratus and A. hebraeum which may act as main vectors and reservoir for livestock infections. I am however uncertain about their transmissibility neither to human hosts nor of their vectorial capacity, nevertheless tick species of Amblyomma readily bite humans and may be able to transmit both pathogens and could therefore pose a serious threat to the public. C. burnetii incidence was 16±1.6% amongst the ticks, this was also a first detection from ticks in the country but the findings seem to be consistent with previous serological studies, also all the tick species in the collection were found harboring the pathogen. The prevalence of E. ruminantium, E. canis and Ehrlichia/Anaplasma was determined to be 29±2.2%, 20±3.6% and 18±3.8% respectively. No significant Ehrlichia/Anaplasma species were characterized except for A. phagocytophilum as reported above. Rickettsia species that were isolated and characterized in the current study were R. africae and R. conorii as expected and the prevalence was 26±1.7%. All in all the target pathogens were successfully isolated, characterized and validated through sequencing reactions, however there still remains a task of determining the vectorial capacity of the ticks and evaluation of factors that could lead to their transmission to the public. In conclusion, these pathogens should be considered as part of routine screening in patients presenting with fevers of unknown origin especially amongst tourists where pathogens like Rickettsia seem to have become problematic in South Africa.

Helminths belonging to the genera Haemonchus and Trichostrongylus as well as those of the genus Fasciola are the causative agents of various helminthiases in animals and sometimes in humans. In both host types infections are often acquired through the ingestion of infected food as well as drinking contaminated water. In animals they disrupt the efficient conversion of food material and absorption of nutrients resulting in weakness and death. Infected humans often suffer from intestinal obstruction, insomnia, vomiting, weakness and stomach pains and sometimes temporary asthma. Infections are associated with huge economic losses globally. Diagnosed is achieved through the observation of clinical manifestations. In low infections alternative diagnosis is based on the recovery and identification of faecal eggs and cultivation of L3 by microscopy. More accurate diagnosis has recently been achieved through the use of molecular diagnostic tools such as PCR and LAMP. The aim of this study was to develop rapid, sensitive, specific and accurate molecular diagnostic assays. In particular the study focused on LAMP and PCR for the detection of H. contortus, Fasciola and Trichostrongylus spp. infections in four provinces of South Africa. The first study emphasized on the development of LAMP and PCR assays for detection of H. contortus infections in livestock. LAMP primers that specifically amplify the ITS2 gene of H. contortus were designed from this target gene. This set of primers was used to develop a PCR assay for species-specific gene amplification. Both assays were tested at various reaction conditions to optimize for primer annealing temperature. Sensitivity reactions were conducted using 10 fold serial dilutions of target DNA while primer specificity was determined using DNA extracted from closely related species. For the LAMP assay, the optimum annealing temperature was found to be 60°C and 55°C for the PCR assay. When tested for specificity both assays only amplified target DNA thereby proving to be specific. The sensitivity reactions for both the LAMP and PCR assay yielded a detection limit of 0.42 ng and 10-3 ng respectively as the lowest amount of the target DNA that can be detected by the assays. Screening of field samples by PCR yielded negative results on several occasions while the positive control amplified the target gene as expected. Failure to validate the assay using field samples was attributed to poor quality or lack of DNA. Validation of these assays is central to determining their efficacy and potential importance in diagnosing natural infections with high sensitivity and rapidity. Therefore we recommend close evaluation of DNA extraction from faecal material as well as ways of reducing or eliminating PCR inhibitors. The second study was aimed at developing a LAMP and PCR assay for the detection of Trichostrongylus spp. infections in livestock. In this study, the target gene used for LAMP primer design for genus-specific amplification was the ITS2 gene. Two primer sets were designed and from these primers, two PCR assays were developed. All these assays were subjected to various LAMP and PCR conditions respectively in order to determine suitable annealing temperatures for each assay. Various methods of DNA extraction were evaluated for troubleshooting together with the use of already published PCR primers. Both LAMP and PCR assays did not amplify the target gene at different ranges of annealing temperatures tested. Furthermore, no amplification was achieved from control DNA samples extracted using different methods. Negative results were obtained when the PCR was troubleshot using already published primers. The results achieved with the designed assays may be a direct consequence of improperly designed primers; however, the inability of all primer sets to function for LAMP and PCR as well as for already published primers suggests problems with the extraction of DNA. This can be attributed to ineffective disruption of worms or the presence of DNases that may degrade DNA and inhibit its amplification. The results of this study suggest the need to evaluate pre-treatment of worms prior to DNA extraction. Evaluation of the methods used to reduce the effects of PCR inhibitors during extraction and amplification of DNA may also be considered. The third study was aimed at developing a LAMP and PCR assay for detection of Fasciola spp. infections in livestock. Two sets of primers for species-specific amplification by LAMP were designed by targeting the ITS2 gene of both F. hepatica and F. gigantica. Species-specific PCR assays were developed from the latter primers and gene of the parasites and the assays were then tested at various reaction conditions. A genus-specific PCR assay was subsequently developed and tested. No amplification of DNA was observed with the F.hepatica-specific LAMP assay whereas the assay developed for specific detection of F. gigantica produced false positive results. All PCR assays yielded negative results following many attempts to optimize for primer annealing temperature. Reagent contamination was eliminated as the source of non-specific amplification with LAMP suggesting that improper primer design was a possible cause of this problem. On the other hand failed attempts to optimize all the other assays suggest that there is need to evaluate pre-treatment of worms prior to DNA extraction as well as the methods reducing the effects of PCR inhibitors during amplification. Overall, this study successfully achieved the development and optimization of a LAMP and PCR assay for detection of H. contortus DNA. However, subsequent validation of the assays using field derived samples was not possible. The overall results of this study mostly point to faulty primer design and the presence of PCR inhibitors in extracted DNA samples, extracted from either tissue or faecal samples. According to previous studies the presence of inhibitory substances may interfere with the lysis step, inactivate the thermostable DNA polymerase and even interfere with nucleic acids. Therefore evaluation of more accurate methods of mechanical disruption of the worms, DNA extraction, primer design and the use of amplification facilitators may yield desired results. Therefore, these factors should be taken into account for successful development and validation of the molecular diagnostic tool for detection of helminth infections.