Cotyledon orbiculata, Hermannia depressa and Nicotiana glauca are widely used plants in traditional medicine for treatment of various infections and diseases. C. orbiculata is mostly used in treatment of epilepsy, earache and the removal of warts while H. depressa is used to relieve toothache, nerves and diarrhoea. Lastly, N. glauca has been mostly used as pain killer in relieving earache, toothache and seldom applied on body in treatment of soreness and inflammation. Traditional usages of these plants by indigenous people and local traditional healers have not yet been scientifically reported, that is why the current study was aimed at determining the anthelmintic and anticancer activity of these plants. The anthelmintic activity of acetone and water extracts from the shoots of Cotyledon orbiculata, Hermannia depressa and Nicotiana glauca were investigated using the egg hatch, larval development and larval mortality assays. In all extracts tested, C. orbiculata water extract at 7.5 mg/ml prevented nematode eggs from hatching with 82.63% success rate. Other extracts exhibited egg hatch inhibition in a degree of less than 50%. Similarly, C. orbiculata water extracts suppressed nematode larval development with 85.32% at the concentration of 2.5 mg/ml followed by 66.69% of H. depressa extract at 7.5 mg/ml concentration. However, N. glauca water and all acetone extracts induced the 100% larval development inhibition. The in vitro larval mortality rate revealed that the water extracts from all the plants were able to kill all larvae at 2.5 mg/ml within 48 to 96 h. The results from this study have shown that the extracts from the three plants have the potential to prevent and ameliorate diseases associated with gastrointestinal nematodes. The anticancer activity of the above mentioned plants was tested on two cancer cell lines, the MCF-7 and HeLa cells. All plants possessed anticancer activity at different durations and concentrations. The N. glauca demonstrated an activity against both cell lines, however, the plant acetone extracts were much effective on the MCF-7 line at 48 hours with inhibition percentages > 80% at all concentrations. The N. glauca acetone extracts were effective at 24 hours. The H. depressa acetone extracts also possessed much activity than water extracts at 24 hours, whereas C. orbiculata showed no activity at all on this cell line. The C. orbiculata acetone extracts followed by the water extracts were the most effective on the HeLa line ranging from 12 to 84% and 0 to 77% inhibition. H. depressa activity was observed at 48 hours of experimentation at all concentrations in both extracts. N. glauca exhibited significant inhibition percentages at 24 hours of water extracts and 48 hours of acetone extracts. This study has showed that the three plants are potential candidates for cancer treatment. Brine shrimp lethality test was conducted on the nauplii stage of the shrimps. Furthermore, two assays, MTT and LDH cytotoxicity assays were conducted on the MDBK cells. None of the plants was toxic on the brine shrimps as most of them survived through 24 and 48 hours. C. orbiculata and H. depressa brine shrimp larval mortality was observed at 96, 72, 72, 96 and 96 h at 0.2, 0.4, 0.6, 0.8, and 1.0 mg/ml of water extracts respectively and 72 h at all concentrations of acetone extracts while N. glauca larval mortality was observed at 120 and 96 hours at all concentration of both extracts. There was a cell survival decrease of 50% at 0.65 mg/ml and 50-60% in C. orbiculata water and acetone extracts treated wells, respectively. No significant cell decrease was observed from both H. depressa and N. glauca water and acetone extracts except for N. glauca acetone extract at 2.50 mg/ml. None of the plants induced a significant toxicity on both the brine shrimps and the bovine cells. Alkaloids, saponins, tannins and phenols were the constituents tested for in order to identify constituents responsible for observed activity of the whole study. No alkaloids were detected from all plants for both extracts, only saponins, tannins and phenols were present even though N. glauca acetone extracts possessed none of the tested constituents. Tested plants are therefore good candidates in treating parasitic gastrointestinal nematodes and cancer disease. Potassium permanganate (KMnOâ) is a widely used chemical substance in the rural communities as prophylaxis or in treatment of various infections and diseases; however, there is no scientific validation of its usage. Anthelmintic activity of potassium permanganate was tested against parasitic gastrointestinal nematodes in comparison to anthelmintic commercial drugs, Tramisol®, Noromectin® and Valbazen®. There was no egg hatch inhibition induced (<10%) by potassium permanganate at all concentrations as compared to >80% inhibition of commercial drugs, however, 85.24, 98.10 and 90.91% of larval development was induced at 0.5, 1.0 and 1.5 mg/ml, respectively. Larval mortality was observed at 216, 144 and 144 hours at 0.5, 1.0 and 1.5 mg/ml, respectively. The cytotoxicity of this chemical was tested on the brine shrimps and MBDK cells as well. Within 24 hours all the brine shrimps larvae treated with KMnOâ died. A significant decrease in MDBK cell survival was observed at 0.65 mg/ml at 24, 48 and 72 hours. This study has therefore showed that potassium permanganate have anthelmintic activity and concentration dependent cytotoxicity on the mammalian cell lines. In conclusion Cotyledon orbiculata, Hermannia depressa, Nicotiana glauca and potassium permanganate have the potential to supress the aggressiveness of parasitic gastrointestinal nematode and cancer diseases. These substances are therefore potential candidates in treatment of tested pathogens.

This study investigates insect-fungal-plant interactions on tree-nut crops (pistachio, walnut and pecan) in South Africa. The main aim was to determine whether insects occurring in tree-nut crop orchards are assocaited with fungal phytopathogens and whether they have the capability to harbour and dissiminate them. Isolations from Atelocera raptoria (powdery stink bug) on pistachio showed that the largest proportion of fungi associated with these insects are pathogenic, some serious pathogens of the crop. Isolations from Coenomorpha nervosa (grey-brown stink bug) and Empoasca citricola (green citrus leafhopper) on walnut showed that the insects were associated with fungi but the presence of pathogens was minimal. The isolation of fungi associated with insects occurring in pecan orchards showed that the most abundant fungal species from Empoasca sp. (leafhoppers) and the Sciobius cf. granosus (citrus snouted weevil) were pathogenic. Pathogens were also isolated from Panafrolepta dahlmani, however, a large proportion of the fungi isolated from these specimens were non-pathogenic. Fungi from the grey-brown stink bug, collected through a pecan nut production season and comparing it to other ways of dispersal (air, soil, leaf & nut samples), showed that the greatest diversity of fungi was found to be associated with the stink bug specimens. This indicates that stink bugs, most likely insects in general, play an important role in the dynamics of fungal populations within pecan orchards. As a result the isolation of fungi from insects in tree-nut crop orchards may give an indication of the mycoflora in the environment. Overall, the two most abundant fungal species isolated from stink bugs and other niches were Alternaria tenuissima and Cladosporium cladosporioides. The incidence of these two species on stink bugs followed a similar trend to the incidence of these two species in the air and soil. However, the incidence of these two species on the stink bugs differed from their incidence in the leaves and nuts. This indicates that the fungi occurring on the stink bugs are from their environment and that the presence of fungal species such as A. tenuissima and C. cladosporioides in the leaves and nuts cannot necessarily be attributed to vectoring by stink bugs. Neofusicoccum parvum was only isolated from the nuts and from the stink bugs. It was completely absent from all the other samples taken throughout the whole season. This suggests an association between the stink bugs and N. parvum, where the stink bugs act as disseminators spreading this fungus between developing nuts as they feed. Pathogenicity trials indicated that A. tenuissima and N. parvum are pathogenic on pecan nuts and leaves, while C. cladosporioides cannot be considered a pathogen of this crop. It was also found that wounding of pecan nuts increases disease incidence on nuts inoculated with A. tenuissima and is a necessity for both A. tenuissima and N. parvum to be able to infect, colonise and cause symptoms on pecan leaves. Wounding does not increase the rate of disease development, but it reduces the time required by N. parvum to infect and colonise pecan nuts. In contrast, wounding does not provide the same benefit to A. tenuissima. Overall, the study has shown that insects occurring in tree-nut orchards have the capability to harbour and disseminate fungal phytopathogens. These insects feed on various parts of the trees causing damage and, in addition, provide suitable entry points into the trees through which pathogens gain entry to infect, colonise and cause disease.

The first part of this study examines vaccine candidates for African pathogenic trypanosomes. Nonvariant antigens (NVAs) limited to bloodstream form stages of Trypanosoma congolense, have been identified using immunodiffusion, immunoelectrophoresis, and Western blotting techniques. Results from experiments employing surface biotinylation, iodination, and immunogold labeling suggest that these NVAs are exposed on the parasite surface and thus, if appropriately presented might afford the mammalian host a protective immunity. Trypanosoma brucei brucei development in Glossina provides another opportunity to block parasite development via passive immunization against procyclic forms. Results obtained from studies designed to evaluate this possibility suggest that ingested anti-procyclic antibody had little influence on parasite development within Glossina. The apparent resistance of antibody to enzymatic degradation for up to 120 h postingestion, as determined by Western blotting, suggests that antibody inactivation, rather than destruction is responsible for these results and provides a rationale for future investigations. The second part of this study investigates virulence mechanisms and identifies possible diagnostic antigens of the human urogenital protozoan parasite Trichomonas vaginalis. Plasma membrane isolated from T. vaginalis strains differing in virulence contain qualitative but not quantitative differences in their protein composition as determined with the aid of Western blotting. Two proteins in the 150 kDa range appeared more abundant in mild strains, while proteins of 43 and 25 kDa appeared more abundant in the membranes of virulent strains. These latter proteins are apparently heavily glycosylated as indicated by their ability to bind ConA and SBA lectins. Possibly, these glycoproteins may contribute to virulence mechanisms of the parasite. In attempts to develop a reliable diagnostic assay, soluble antigens shed by T. vaginalis were identified using Western blotting techniques. Five soluble antigens were initially identified in supernatant fluids obtained from parasites grown on TYM medium supplemented with 1% normal human plasma (NHP). Four of these proteins immunologically cross-reacted with NHP and are thus of limited diagnostic potential. A 100 kDa antigen, lacking in NHP cross-reactivity, however, could be isolated from all parasite strains examined and maybe of some use in developing a diagnostic assay.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2005. / Title from PDF t.p. (viewed Dec. 2, 2008). Source: Dissertation Abstracts International, Volume: 66-01, Section: B, page: 0152. Chair: Peter Kloosterman.

The genus Ctenopseustis Meyrick (Lepidoptera: Tortricidae) is reviewed and the type species C. obliquana (Walker) is redefined. The species C. servana Walker and C. fraterna Philpott are reinstated and the synonomy and characteristics of each species are described and illustrated. Populations of the three leafroller species Epiphyas postvittana (Walker), C. obliquana and Planotortrix excessana (Walker) together with Stathmopoda “skelloni” (Butler) were monitored intensively from 1976 to 1979 in an insecticide free block of mature apple, cv ‘Dougherty’ trees in West Auckland. S "skelloni" is recorded as a pest of apple for the first time. The general biology, distribution and host range of these four species is described. A key to the larvae of the four species, as found on ‘Dougherty’, using head capsule measurements together with other characters, and keys to the leafroller eggs and pupae are provided. A technique to successfully mate C. obliquana and P. excessana in the laboratory was formulated by predicting the emergence time of female pupae and enclosing one such pupa with several males. A colony of C. obliquana was initiated from the progeny of a single mated female but collapsed in the fourth generation and possible reasons for this are discussed. The life history of all four species on ‘Dougherty’ apple in Auckland is described. E. postvittana completed four generations, P. excessana three and S. “skelloni” two but the number of C. obliquana generations per year was undetermined. The seasonal and daily flight periods were determined using two suction traps within the apple block. Female flight in all species occurred shortly after sunset, generally for two to three hours, whereas male flight extended for up to eight hours after sunset. Results of the suction traps were also used to show the lower temperature thresholds of flight for E. postvittana as 7-8°C, 8-9°C, and 10-11°C for the second summer, spring and first summer generations respectively and for S. “skelloni” as 9-10°C and 11-12°C for the autumn – winter and spring –summer generations respectively. Loss through pre-establishment failure of first instar larvae accounted for the greatest amount of mortality in the life history of all leafroller species. No eggs or pupae of S. “skelloni” were found on the trees throughout the study. Egg mortality in the leafrollers was low and larval parasitism was up to 33.6% in E. postvittana and 19.6% in P. excessana. The parasite species found associated with each leafroller host are listed, the most common being Apanteles tasmanica in E. postvittana and Goniozus sp. in P. excessana. Host preferences were shown by several parasite species. Parasitism of E. postvittana by Apanteles spp. reduced the larval growth rate and head capsule size of this leafroller. Larvae of C. obliquana did not complete development on the 'Dougherty’ trees due to its resistance to this leafroller species. Laboratory rearing experiments comfirmed this and showed cv 'Red Delicious’ was susceptible. A third culivar, 'Granny Smith’, showed some indication of resistence to the larvae of C. obliquana and P. excessana but E. postvittana showed high survival rates on all three cultivars. The distribution of each species within both the study site and the trees was examined. S. “skelloni” showed a significant preference for the calyx of fruit whereas all leafroller species preferred feeding sites on the foliage. Analysis of instar – feeding site comparisons showed that leafroller larvae moved to new feeding sites several times in their lives. S. “skelloni” probably remained in their initial sites until leaving the trees to pupate.

The aim of this study is to test for photoperiodic control of larval and pupal development in the New Zealand moth Planotortrix octo Dugdale. The photoperiodic response curves for larval and pupal development and especially for instar number at 17°C and 21°C indicate that a photoperiodic mechanism is involved. Superimposed on this response is the suggestion that daylength affects development rate directly, with larvae and pupae developing faster under longer photophases. This effect is especially strong for pupal development (Chapter 3). The effects of thermophotoperiods (Chapter 4), night interruption and resonance experiments (Chapter 6) provide further evidence for photoperiodic involvement. The response to resonance experiments suggests the involvement of an hourglass rather than a circadian mechanism. Larvae reared under short days accumulate significantly more lipids in the 5th and 6th instars than larvae reared under long days (Chapter 4). This finding, combined with the suppressed development rate and higher instar number under short days, suggests that a weak form of diapause may be present in this insect. This is significant in being the first recorded incidence of a photoperiodically induced diapause in a phyllophagous New Zealand insect for which a year round food supply is available (Chapter 1). By transferring insects from long to short days I found that long days have more influence than short days on larval development (Chapter 7). An attempt was made to measure juvenile hormone titres under long and short days using a Galleria bioassay. The test used was not sensitive enough however to measure any significant amounts of juvenile hormone (Chapter 8). Simulations of the experimental results were performed using a damped circadian oscillator model (Chapter 9). This model was considered the most appropriate to use, based on the experimental results and on a review of the literature (Chapter 2). Simulations showed good similarities with experimental results in most cases, but could not account for resonance responses.

In insects, olfaction is mediated by a large family of integral membrane proteins, called olfactory receptors (ORs), that mediate the transduction of odorant binding into a neuronal signal. A functional assay for insect ORs was developed utilising calcium imaging in Sf9 cells. The Drosophila melanogaster OR, Or22a, was expressed using transient transfection, and its activity measured by monitoring increased intracellular calcium levels using a calcium–sensitive dye. The interaction of the odorants ethyl butyrate, pentyl acetate and ethyl acetate with Or22a were both dose–dependent and sensitive, with EC50 values of 1.53 x 10−11 M, 5.61 x 10−10 M and 3.72 x 10−9 M, respectively. Furthermore, Or22a expressed in Sf9 cells has a similar response profile to a range of odorants previously tested in vivo. This assay system will provide a useful tool for the investigation of insect olfactory receptor structure and function. A consensus of eleven transmembrane (TM) domain prediction algorithms suggested a model for Or22a that contains seven TM domains, reminiscent of GPCRs. To test this model empirically, the membrane topology of Or22a was determined using epitope–tagging of predicted loops followed by immunochemistry. These experiments revealed that Or22a has seven TM domains but that its orientation in the membrane is opposite to that of GPCRs, having a cytoplasmic N–terminus. This orientation was also observed for Epiphyas postvittana Or1, which suggests that this inverted topology may be common to all insect ORs. To test whether Or22a forms higher order structures, fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins inserted into the intracellular loops of Or22a was employed. The third intracellular loop interacts strongly with itself in homo–multimers, with interactions between the first and first loops and first and third loops also observed. These experiments show that ligand binding ORs can form multimeric structures in heterologous cells. The co–transfection of Or83b into S2 cells had no impact on these interactions, however Or83b is likely expressed in this cell line. Finally, models of how a ligand binding OR interacts physically with the ion channel Or83b are presented, and approaches that could be used to distinguish between these models are discussed.

Invasive ant species represent a major threat to biodiversity through their detrimental impacts on native ecosystems. An important step to managing invasive ant species is determining the factors responsible for their current and potential distribution. This thesis examines the spatial distribution of invasive ant species at a range of scales. The focus is on the Pacific region, specifically Fiji and New Zealand, which have a relatively high number of invasive ant species. Taxonomic checklists of the ant fauna of Fiji and New Zealand are presented. Climate variables were used to model the distribution of, and predict suitable areas for, particular invasive ant species. In general, models had high evaluation success, especially for models that were built and tested within a region. However, the utility of models was far less when transferred and tested in new regions. A better understanding of how climate variables directly and indirectly affect a species is needed to improve the utility of species distribution modeling. On a finer spatial scale, habitat partitioning was evident at Colo-i-Suva Park in Fiji, where distinct ant communities were sampled from litter and canopy habitats. Results suggested that body size, habitat and resource utilisation interact to create opportunities for invasion, and influence the susceptibility of different habitats. Habitat also shaped the assembly of ant communities and mediated inter-specific competition in the Yasawa Islands, Fiji. Similarly, habitat was important in partitioning ant communities in New Zealand; endemic ant species were in forest, and invasive species in scrub and urban habitats. However, there was no evidence that inter-specific competition influenced community composition or the distribution of invasive ant species at regional or local scales in New Zealand. The extent and diversity of exotic ant species arriving at the New Zealand border was also investigated. A high proportion (>64%) of intercepted ants originated from the Pacific. The effectiveness of detecting exotic ant species at the New Zealand border ranged from 48-78% for different pathways, indicating a number of species remain undetected. Future work on invasive ants should focus on species-specific tolerances, and how the physical environment and small scale abiotic conditions influence distribution.

In insects, olfaction is mediated by a large family of integral membrane proteins, called olfactory receptors (ORs), that mediate the transduction of odorant binding into a neuronal signal. A functional assay for insect ORs was developed utilising calcium imaging in Sf9 cells. The Drosophila melanogaster OR, Or22a, was expressed using transient transfection, and its activity measured by monitoring increased intracellular calcium levels using a calcium–sensitive dye. The interaction of the odorants ethyl butyrate, pentyl acetate and ethyl acetate with Or22a were both dose–dependent and sensitive, with EC50 values of 1.53 x 10−11 M, 5.61 x 10−10 M and 3.72 x 10−9 M, respectively. Furthermore, Or22a expressed in Sf9 cells has a similar response profile to a range of odorants previously tested in vivo. This assay system will provide a useful tool for the investigation of insect olfactory receptor structure and function. A consensus of eleven transmembrane (TM) domain prediction algorithms suggested a model for Or22a that contains seven TM domains, reminiscent of GPCRs. To test this model empirically, the membrane topology of Or22a was determined using epitope–tagging of predicted loops followed by immunochemistry. These experiments revealed that Or22a has seven TM domains but that its orientation in the membrane is opposite to that of GPCRs, having a cytoplasmic N–terminus. This orientation was also observed for Epiphyas postvittana Or1, which suggests that this inverted topology may be common to all insect ORs. To test whether Or22a forms higher order structures, fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins inserted into the intracellular loops of Or22a was employed. The third intracellular loop interacts strongly with itself in homo–multimers, with interactions between the first and first loops and first and third loops also observed. These experiments show that ligand binding ORs can form multimeric structures in heterologous cells. The co–transfection of Or83b into S2 cells had no impact on these interactions, however Or83b is likely expressed in this cell line. Finally, models of how a ligand binding OR interacts physically with the ion channel Or83b are presented, and approaches that could be used to distinguish between these models are discussed.

The Australian sheep blow fly Lucilia cuprina is an economically important dipteran pest whose circadian behavioural rhythms have been the subject of considerable scrutiny. The underlying biochemical nature of these rhythms however, has remained a mystery. The primary objective of this thesis was therefore to investigate the molecular control of circadian rhythms in L. cuprina using an integrative approach. To these ends, a dynamic molecular simulation model for L. cuprina was formulated using existing biochemical data on insect circadian clocks. The validity of this simulation model was subsequently tested at both molecular and behavioural levels. The basic molecular assumptions of the simulation model were tested by cloning a full length L. cuprina per cDNA and analysing its mRNA and protein expression levels. Isolation of the 4 Kb L. cuprina per cDNA revealed the conservation of three functional domains known to be important for circadian clock function; namely the PAS dimerisation motif (with 92% identity to D. melanogaster at the amino acid level), and the cytoplasmic and nuclear localisation domains (with 85% and 80% identity respectively). A fourth domain, the threonine-glycine (TG) repeat region, was also found to be conserved, but severely truncated in L. cuprina. No length variation was found in the TG repeat of flies collected from several different latitudinal zones, and no correlation was detected between sequences flanking the repeat and latitude of collection of flies. Thus, the contention that the TG repeat region plays a role in temperature compensation of the circadian clock is cast in doubt. Expression analyses (using quantitative RT-PCR) showed per mRNA levels to undergo diel oscillations with a period (24 h) and peak phase (Zt 12) consistent with the Drosophila data. PER-immunoreactive protein oscillations were also demonstrated, with peak immunoreactivity lagging approximately 3 h behind peak mRNA levels. The behavioural predictions of the model were tested by recording adult locomotor activity under different light regimes. The simulation model successfully predicted free-run, entrainment, the effect of short light pulses, and the effects of constant lighting on behavioural rhythms. Disparities between the simulated and real phase response cnrves for L. cuprina are hypothesised to be indicative of an ealier nuclear entry time of the PER-TIM dimer in L. cuprina compared with D. melanogaster. The three different approaches of simulation modellingo molecular analysis and behavioural investigation are integrated in the discussion in order to help provide a comprehensive explanation of circadian function in L. cuprina. The benefits of an integrated approach to the analysis of circadian function are discussed, as is the relevance of the present findings to the development of a clock-based control strategy for this economically important pest species.