The maternal rearing environment (MRE) of an organism can be a key determinant of an organism's host choice decisions, its own fitness, or the fitness of its offspring. Here, it is investigated if the MRE of an organism can influence lower or higher trophic levels. A series of reciprocal cross-over experiments was conducted using pea aphids (Acyrthosiphon pisum), bean (Vicia faba) or pea (Pisum sativum) plants, and an aphid natural enemy, the parasitoid wasp Aphidius ervi, as model organisms. In each experiment, pea aphid offspring experienced either the same or an alternative plant host to that experienced by their mothers. This PhD showed that the MRE of pea aphids and parasitoid wasps was not a main contributory factor of host choice decisions or offspring fitness but influenced mother parasitoid wasp fecundity. Additionally, the MRE of pea aphids influenced the foliar nutrient concentration of pea plants when infested with the aphid's offspring. First, over shorter infestation periods, variation in foliar nitrogen and essential amino acid concentrations of pea leaves could be explained by pea aphid MRE. Over longer infestation periods, variation in foliar nitrogen and essential amino acid concentrations of pea leaves was explained by a combination of pea aphid MRE and aphid genotype. Second, the 13C concentration of pea leaf tissue, an indicator of stomatal aperture and leaf water stress, varied with pea aphid MREs over longer infestation periods. However, stomatal conductance and the expression of abscisic acid-responsive genes did not vary in a manner that was consistent with leaf water stress. Additional components of an organism's maternal rearing conditions are considered, including symbioses, as a more realistic MRE compared with that observed in nature. Taking account of MREs could provide a better understanding of the factors influencing the fitness of many organisms interacting in natural and managed ecosystems.

Field pea (Pisum sativum L.) is highly susceptible to ascochyta blight, primarily caused by the pathogen Mycosphaerella pinodes (Berk. & Blox.) Vestergr. Grasspea (Lathyrus sativus L.) has been reported to possess a moderate level of resistance to ascochyta blight caused by M. pinodes. The work reported in this thesis aimed to develop the various techniques that would be required to transfer ascochyta blight resistance from grasspea into field pea via somatic hybridisation. This thesis also assesses the feasibility of achieving this goal. Field pea shoot cultures were established on hormone-free MS medium, and a protoplast isolation protocol developed for both species. Grasspea shoot cultures were established on both RL and SSB8 medium. Friable grasspea callus was achieved on media supplemented with 2,4-D in the range 4.523 µM, whereas kinetin tested at any concentration did not appear to influence callus growth. A suspension culture of grasspea was developed for the first time, in B5 medium supplemented with 4.5 µM 2,4-D and 0.5 µM kinetin. Grasspea protoplasts were isolated from both in vitro seedlings and suspension cultures. Protocols for hybrid shoot culture on KM8p medium were developed via organogenesis and somatic embryogenesis.

Field pea (Pisum sativum L.) is highly susceptible to ascochyta blight, primarily caused by the pathogen Mycosphaerella pinodes (Berk. & Blox.) Vestergr. Grasspea (Lathyrus sativus L.) has been reported to possess a moderate level of resistance to ascochyta blight caused by M. pinodes. The work reported in this thesis aimed to develop the various techniques that would be required to transfer ascochyta blight resistance from grasspea into field pea via somatic hybridisation. This thesis also assesses the feasibility of achieving this goal. Field pea shoot cultures were established on hormone-free MS medium, and a protoplast isolation protocol developed for both species. Grasspea shoot cultures were established on both RL and SSB8 medium. Friable grasspea callus was achieved on media supplemented with 2,4-D in the range 4.523 µM, whereas kinetin tested at any concentration did not appear to influence callus growth. A suspension culture of grasspea was developed for the first time, in B5 medium supplemented with 4.5 µM 2,4-D and 0.5 µM kinetin. Grasspea protoplasts were isolated from both in vitro seedlings and suspension cultures. Protocols for hybrid shoot culture on KM8p medium were developed via organogenesis and somatic embryogenesis.

The use of stable nitrogen isotope data in ecological and physiological studies is based in the assumption that nitrogen fractionates predictably during metabolism, leading to a broadly conserved pattern whereby consumers are isotopically enriched with respect to their diets. The application of stable isotope data to such studies is limited is by our understanding of the factors in that cause variability in the Δ15N values of consumers. In particular, parasites and fluid-feeders have been shown to demonstrate isotopic depletion with respect to their food sources. One factor that has been suggested to influence the Δ15N values seen in fluid-feeding consumers is the presence of endosymbionts and their contribution to nitrogen metabolism. The experiments described in this thesis directly test the hypothesis that the endosymbiotic bacteria Buchnera aphidicola is influencing the Δ15N value of the pea aphid on host alfalfa plants. Here I find that although aphids cured of their bacterial symbionts are less isotopically depleted than untreated aphids, they are still not enriched with respect to their phloem sap diet, indicating that endosymbiont metabolism alone is not responsible for the isotopic depletion observed in pea aphids. Metabolism of nitrogen in the pea aphid-Buchnera symbiosis has been well described with decades of physiological studies and with the publication of the pea aphid and Buchnera genomes. The two key features of metabolism in the pea aphid-Buchnera symbiosis are the recycling of waste ammonia by the aphid and the upgrading of the nonessential amino acids found in phloem sap to essential amino acids through collaborative metabolism between the pea aphid and Buchnera. Consistent with the described role of Buchnera in nitrogen metabolism, amino acid analyses of symbiotic and aposymbiotic aphids demonstrates an accumulation of the nonessential amino acids glutamine and glutamate and lower amounts of essential amino acids in the aposymbiotic aphids. I tested the influence of dietary amino acid profile on the Δ15N value of pea aphids and found that aphids are only isotopically depleted when they feed on diets with unbalanced amino acid compositions and are isotopically enriched when fed on a diet with a balanced profile of amino acids. I used isotopically labeled fructose to determine whether the difference in Δ15N value of pea aphids on diets of varying amino acid profiles is correlated to the amount of de novo amino acid synthesis occurring in the aphid. I found that there was a significantly higher incorporation of the labeled carbon backbone in the protein of pea aphids feeding on the unbalanced diets, supporting the idea that increased de novo amino acid synthesis are responsible for the differences in Δ15N values among aphids feeding on the two diets. The findings of this study highlight the influence of endosymbionts on the Δ15N values for pea aphids, demonstrate that dietary amino acid composition can influence the Δ15N value of pea aphids through the demand for metabolic upgrading of amino acids, and provide a model for the study of Δ15N values in systems where metabolism has been well characterized by experimental and genomic data.

Constipation is a serious problem in the pediatric population and often requires medical management with laxatives and enemas. Participants (2-10 years of age, n=13) with a history of mild constipation were assigned randomly to a fibre treatment or placebo group. After three weeks, subjects were crossed over to the other treatment. Pea hull fibre (4.0-7.6 g/day = 3.6-6.8 g/day of dietary fibre) was added to snack foods and an inulin supplement (5.0 g/day = 4.5 g/day of dietary fibre) was given, whereas the placebos were non-fortified snacks and maltodextrin (5.0 g/day).<p> Subjects or their parents documented stool frequency, stool consistency, occurrence of abdominal pain and intake of snack foods and the supplement. Over the final two weeks, there was a trend towards an increase in the mean number of daily bowel movements in the fibre treatment group compared to the placebo group (n=11, 0.68 ± 0.18 vs. 0.59 ± 0.26, p=0.064). Exclusion of one subject with diarrhea-type stools led to a significant difference between groups (n=10, 0.54 ± 0.18 vs. 0.67 ± 0.22, p=0.002). Stool consistency, using the Bristol Stool Form Rating Scale, showed no significant differences in stool consistency between groups (p=0.379) nor was there a difference in the incidences of abdominal pain (p=0.129). Not all subjects experienced abdominal pain. The inulin supplement (91% compliance rate; 1 serving per day) was consumed more consistently than were the snack foods fortified with pea hull fibre (77% compliance rate; 2 servings per day). There were no significant differences in the intake of the snacks or supplement when the placebo and treatment groups were compared. Energy intake was significantly lower during the fibre treatment period compared to placebo (n=12, 1307 ± 296 kcal/day vs. 1441 ± 285 kcal/day, p=0.035). The addition of pea hull fibre to typical snack foods and an inulin supplement to beverages were well accepted by children and no adverse effects were reported. Fibre fortification of snack foods with pea hull fibre and fibre supplementation of beverages with inulin may provide an alternative means to treat pediatric constipation.

Constipation is a serious problem in the pediatric population and often requires medical management with laxatives and enemas. Participants (2-10 years of age, n=13) with a history of mild constipation were assigned randomly to a fibre treatment or placebo group. After three weeks, subjects were crossed over to the other treatment. Pea hull fibre (4.0-7.6 g/day = 3.6-6.8 g/day of dietary fibre) was added to snack foods and an inulin supplement (5.0 g/day = 4.5 g/day of dietary fibre) was given, whereas the placebos were non-fortified snacks and maltodextrin (5.0 g/day).<p> Subjects or their parents documented stool frequency, stool consistency, occurrence of abdominal pain and intake of snack foods and the supplement. Over the final two weeks, there was a trend towards an increase in the mean number of daily bowel movements in the fibre treatment group compared to the placebo group (n=11, 0.68 ± 0.18 vs. 0.59 ± 0.26, p=0.064). Exclusion of one subject with diarrhea-type stools led to a significant difference between groups (n=10, 0.54 ± 0.18 vs. 0.67 ± 0.22, p=0.002). Stool consistency, using the Bristol Stool Form Rating Scale, showed no significant differences in stool consistency between groups (p=0.379) nor was there a difference in the incidences of abdominal pain (p=0.129). Not all subjects experienced abdominal pain. The inulin supplement (91% compliance rate; 1 serving per day) was consumed more consistently than were the snack foods fortified with pea hull fibre (77% compliance rate; 2 servings per day). There were no significant differences in the intake of the snacks or supplement when the placebo and treatment groups were compared. Energy intake was significantly lower during the fibre treatment period compared to placebo (n=12, 1307 ± 296 kcal/day vs. 1441 ± 285 kcal/day, p=0.035). The addition of pea hull fibre to typical snack foods and an inulin supplement to beverages were well accepted by children and no adverse effects were reported. Fibre fortification of snack foods with pea hull fibre and fibre supplementation of beverages with inulin may provide an alternative means to treat pediatric constipation.

Open Access Article

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Pterocarpan phytoalexins are antimicrobial compounds produced by legumes when challenged by biotic stresses. Most legumes produce pterocarpan phytoalexins with (-)-stereochemistry but pea (Pisum sativum L.) produces as its major phytoalexin (+)-pisatin. Pea also occasionally produces a minor amount of (-)-maackiain as a pterocarpan phytoalexin, and studies on the biosyntheses of (+)-pisatin and (-)-maackiain have shown that up to (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] and 7,2'-dihydroxy-4',5'-methyl-enedioxyisoflavanol [(-)-DMDI]they have common intermediates with (-)-DMDI being where the two pathways diverge. The final step in (+)-pisatin biosynthesis is the methylation of (+)-6a-hydroxymaackiain [(+)-6a-HMK] by 6a-hydroxymaackiain methyltransferase (HMM2) but the steps from (-)-DMDI to (+)-6a-HMK are unknown.The shifting of the stereochemistry from (-)-DMDI to (+)-6a-HMK has been proposed to involve the achiral isoflavene, 7, 2'-dihydroxy-4', 5'-methylene-dioxyisoflav-3-ene (DMDIF). In this dissertation, I have shown that cis-(-)-DMDI is the enzymatic product of (-)-sophorol, and is the precursor of DMDIF which is produced by the dehydration activity of "isoflavene synthase" (IFVS). IFVS activity was not observed in elicited tissues of alfalfa, chickpea, beans, pepper, and broccoli, plants that do not produce (+) pterocarpans. Partial purification of IFVS demonstrated that it is either large in size or tightly complexed with other proteins. The SDS-PAGE of the 29-fold purified product revealed 12 major bands that aggregated into 3 bands in the non-denaturing PAGE. IFVS activity was in band 3 which co-migrated with marker proteins of>100 kDa in size. Proteins identified from LC-MS/MS peptide sequences of the proteins in band 3, when compared to three protein databases, did not identify any proteins with an enzymatic activity expected for IFVS. A disease resistance-response protein (a dirigent-like protein) and two protein-binding proteins were the most abundantly detected proteins in the pea transcriptome-translated database. Also, four of the known enzymes (isoflavone reductase, HMM1, HMM2, and sophorol reductase) involved in (+)-pisatin biosynthesis were among the proteins identified. It may be that IFVS is associated with these other proteins as a complex in vitro and in vivo. The lack of detection of IFVS in the databases could be because it has not yet been sequenced as it functions in a rare biosynthetic pathway.

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