Cuticular wax is a component of the plant cuticle, the lipid barrier which covers the surface of above ground primary plant organs and plays important protective roles. The isolation of wax deficient mutants from Arabidopsis and other plants resulted in identification and isolation of genes required for wax deposition, and broadened our knowledge of this process in plants. To identify additional components involved in cuticular wax production, I investigated the role of the XTC1 gene, defective in the xtcl (extra cotyledon 1) mutant. This mutant was reported to have reduced levels of cuticular wax on its inflorescence stems and accumulate a large number of oil bodies in the primordia of its extra cotyledons. Stem wax extraction and gas chromatography analysis showed that the total xtcl stem wax is decreased 3-fold in comparison to the wild type, and that all wax components were reduced to a similar extent. Compositional analyses of leaf and seed fatty acids demonstrated that saturated fatty acid content was decreased by around 55%, and unsaturated fatty acid content was approximately 20% lower in xtcl mutants. A detailed examination of xtcl seeds revealed seed deformities, altered seed coat permeability and defective seed mucilage extrusion. Positional cloning of the XTC1 gene resulted in the discovery that it is identical to FATB, an already characterized gene known to encode the fatty acid thioesterase B. The FATB enzyme releases saturated free fatty acids (C16:0and C18:0) from ACP in the plastid and allows their export across the plastid envelope. Analysis of FATB gene expression pattern showed that FATB is transcribed ubiquitously in all tissues and in different development stages. It is therefore not surprising that FATB disruption results in multiple lipid associated phenotypes, including decreased cuticular wax amounts and altered fatty acid compositions of leaves and seeds. Additional phenotypes caused by mutations in FATB that affect embryo and seed development and lead to appearance of extra cotyledons, altered permeability of the seed coat and defective seed mucilage extrusion are difficult to explain at present.

The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants.

The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants.

Cuticular wax is a component of the plant cuticle, the lipid barrier which covers the surface of above ground primary plant organs and plays important protective roles. The isolation of wax deficient mutants from Arabidopsis and other plants resulted in identification and isolation of genes required for wax deposition, and broadened our knowledge of this process in plants. To identify additional components involved in cuticular wax production, I investigated the role of the XTC1 gene, defective in the xtcl (extra cotyledon 1) mutant. This mutant was reported to have reduced levels of cuticular wax on its inflorescence stems and accumulate a large number of oil bodies in the primordia of its extra cotyledons. Stem wax extraction and gas chromatography analysis showed that the total xtcl stem wax is decreased 3-fold in comparison to the wild type, and that all wax components were reduced to a similar extent. Compositional analyses of leaf and seed fatty acids demonstrated that saturated fatty acid content was decreased by around 55%, and unsaturated fatty acid content was approximately 20% lower in xtcl mutants. A detailed examination of xtcl seeds revealed seed deformities, altered seed coat permeability and defective seed mucilage extrusion. Positional cloning of the XTC1 gene resulted in the discovery that it is identical to FATB, an already characterized gene known to encode the fatty acid thioesterase B. The FATB enzyme releases saturated free fatty acids (C16:0and C18:0) from ACP in the plastid and allows their export across the plastid envelope. Analysis of FATB gene expression pattern showed that FATB is transcribed ubiquitously in all tissues and in different development stages. It is therefore not surprising that FATB disruption results in multiple lipid associated phenotypes, including decreased cuticular wax amounts and altered fatty acid compositions of leaves and seeds. Additional phenotypes caused by mutations in FATB that affect embryo and seed development and lead to appearance of extra cotyledons, altered permeability of the seed coat and defective seed mucilage extrusion are difficult to explain at present.

Cuticular wax is a component of the plant cuticle, the lipid barrier which covers the surface of above ground primary plant organs and plays important protective roles. The isolation of wax deficient mutants from Arabidopsis and other plants resulted in identification and isolation of genes required for wax deposition, and broadened our knowledge of this process in plants. To identify additional components involved in cuticular wax production, I investigated the role of the XTC1 gene, defective in the xtcl (extra cotyledon 1) mutant. This mutant was reported to have reduced levels of cuticular wax on its inflorescence stems and accumulate a large number of oil bodies in the primordia of its extra cotyledons. Stem wax extraction and gas chromatography analysis showed that the total xtcl stem wax is decreased 3-fold in comparison to the wild type, and that all wax components were reduced to a similar extent. Compositional analyses of leaf and seed fatty acids demonstrated that saturated fatty acid content was decreased by around 55%, and unsaturated fatty acid content was approximately 20% lower in xtcl mutants. A detailed examination of xtcl seeds revealed seed deformities, altered seed coat permeability and defective seed mucilage extrusion. Positional cloning of the XTC1 gene resulted in the discovery that it is identical to FATB, an already characterized gene known to encode the fatty acid thioesterase B. The FATB enzyme releases saturated free fatty acids (C16:0and C18:0) from ACP in the plastid and allows their export across the plastid envelope. Analysis of FATB gene expression pattern showed that FATB is transcribed ubiquitously in all tissues and in different development stages. It is therefore not surprising that FATB disruption results in multiple lipid associated phenotypes, including decreased cuticular wax amounts and altered fatty acid compositions of leaves and seeds. Additional phenotypes caused by mutations in FATB that affect embryo and seed development and lead to appearance of extra cotyledons, altered permeability of the seed coat and defective seed mucilage extrusion are difficult to explain at present. / Science, Faculty of / Botany, Department of / Graduate

The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants. / Science, Faculty of / Chemistry, Department of / Graduate

The Common bed bug, Cimex lectularius L., suddenly reappeared in developed countries in the past 15 years. The factor contributing to the sudden resurgence of the bed bugs is insecticide resistance. In this study, we investigated the mechanisms of reduced cuticular penetration type insecticide resistance in bed bugs. First, we determined and compared the lethal dosage (LD50) of a pyrethroid insecticide using topical and injection application. The resistant strain not only had significantly greater resistance ratios, but also demonstrated significantly greater penetration resistance ratios. This provided the evidence of the reduced cuticular penetration in bed bugs. Second, we determined the levels of gene transcription (CPR-type cuticle protein genes) using real-time quantitative polymerase chain reaction (qRT-PCR). We identified 62 putative bed bug cuticle protein-encoding contigs based on the presence of the Chitin-binding 4 (CB4) domain. Based on the qRT-PCR analysis of the mRNAs, we found many of the genes were up-regulated in the resistant strain suggesting thickening of the cuticle or increasing the cuticular proteins might be involved in the reduced cuticular penetration. Third, we identified and described the cuticular proteins using the matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometry (MALDI-TOF/TOF). The total of 265 peptides were identified, among which 206 belonged to one of 50 confidently identified proteins. We identified the CPRL, CPF, CPFL, TWDL, and CPAP1 family proteins. The profile of the cuticular proteins between the resistant and the susceptible strains bed bugs were almost identical. Fourth, we determined and compared the cuticular thickness using Scanning Electron Microscopy (SEM). We found statistical differences of the cuticular thickness among different strains (populations), however, correlation between the levels of insecticide resistance and cuticular thickness were not found. Finally, we identified and described bed bug cuticular hydrocarbon profiles using Gas-Chromatography and Mass-Spectrometry (GC-MS). The total of 87 compounds in addition to n-alkanes were extracted and identified. There were no correlation found with the concentration and the levels of insecticide resistance. However, several additional compounds exhibited the correlation between the concentration of the compounds and the levels of insecticide resistance. Overall, we found three lines of evidence to support reduced cuticular penetration as a mechanism of insecticide resistance in some bed bug populations. This study provides additional evidence of the reduced cuticular penetration type resistance in bed bugs. / Ph. D.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Cuticular hydrocarbon (CHC) profiles of insects play roles in behavioural interactions within and between species, encompassing species-, colony- and mate-recognition. CHCs are largely genetically determined and are thus unique to each species, making them useful in chemotaxonomy. However, species exhibit intra-species variation in their CHC profile which can be the result of both intra-species genetic variation as well as environmental influences such as habitat effects, colony effects, diet, host switching, as well as adsorption of CHCs from other insects. Studies have found that the CHC profiles of a specific insect species will often exhibit variations between regions as well as the species of host the insect is associated with. Therefore, an ideal system to investigate the effects of genetic population structure and environment on the CHC profiles of insects is within the fig – fig wasp mutualism. Fig species occur in a wide variety of habitats and host a diverse complement of fig wasp species. We were therefore offered the opportunity to investigate a wide range of potential influences on fig wasp CHC profiles ranging from environmental to genetic effects. Firstly, through GC-MS we found that the CHC profiles of the fig wasps investigated are both species-specific and species-group-specific, with the species Elisabethiella glumosae, Elisabethiella stuckenbergi and Ceratosolen capensis, and two Otitesella species-groups (the Uluzi and Sesqui species-groups) separating out significantly. Consensus phylogenies (based on COI, Cytb and EF-1α) showed that within the galling fig wasp genus Otitesella there were multiple genetic lineages within a species-group which corresponds to species-level genetic variation, and that each genetic lineage was confined to a single host fig species. The CHC profiles reflected the genetic relationships between the two species-groups, and the CHC profiles within a species group could be differentiated by genetic lineage/host species. This indicated that although genetic lineage was mostly responsible for the observed variation in CHC profiles, factors associated with different host species also had an effect. Strong regional variation overriding both the influence of genetic lineage and factors associated with host species were observed in the CHC profiles of the fig wasps within a species-group. This regional variation in CHC profiles was also observed within two pollinating fig wasp species, Elisabethiella stuckenbergi and Ceratosolen capensis, which was not supported by population genetic data (COI and Cytb). In fact, very little genetic population structure was found within the pollinating species, even though the pollinators were collected across South Africa. The lack of genetic structure in pollinating fig wasps can be the result of high gene flow caused by the large dispersal capability of pollinating fig wasps. Our results indicated that fig wasp CHC profiles have the potential to be used in chemotaxonomy and are possibly used as species and mate-recognition cues by the fig wasps. Furthermore, we found both a regional and associated host species effect on the CHC profile. We suggest that the observed regional effect in this study could be attributed to habitat differences and differences in fig wasp community between regions. Moreover, the effect host species had on the CHC profiles may be as a result of dietary differences between galls in different host species. A possible consequence of the observed regional/host speciesassociated effect on fig wasp CHC profiles is that it could lead to pre-mating isolation within fig wasp species, which could ultimately result in speciation. In addition, our results indicated that the interpretation of the variation in the fig wasp CHC profile was dependent on the scale of the analysis: on a broad, inter-species-level scale, fig wasp CHC profiles were species-specific; on a finer intra-species scale, variation in CHC profiles occurred between fig wasps collected from different regions; and on a within-region scale, variation in CHC profiles within species-groups occurred between genetic lineages/host species. Future studies should look at the application of CHCs in chemotaxonomic studies on the fig wasp phylogeny, as well as the effect of fig wasp community composition on fig wasp CHCs. / AFRIKAANSE OPSOMMING: Kutikulêre koolwaterstof (KK) profiele van insekte speel rolle in die gedragsinteraksies binne sowel as tussen spesies, en behels die herkenning van spesieof kolonielidmaatskap asook potensiële maats. Kutikulêre koolwaterstowwe word meestal deur gene bepaal en is dus uniek vir elke spesie, wat dit handig maak vir chemotaksonomie. Spesies vertoon egter soms intraspesie variasie in hul KK profiele wat die gevolg kan wees van beide intraspesie genetiese variasie sowel as omgewingsinvloede soos habitat effekte, kolonie effekte, dieet, tussen-gasheer skuiwings, asook die adsorpsie van ander insekte se kutikulêre koolwaterstowwe. Studies het gevind dat die kutikulêre koolwaterstof profiele van ʼn spesifieke insek spesie op ʼn gereelde basis verskille vertoon tussen streke asook tussen die verskillende gasheer spesies waarmee die insek geassosieer is. Om hierdie redes is die vy – vy-wesp mutualisme ʼn ideale sisteem om die uitwerking van genetiese populasie struktuur en omgewing op die KK profiele van insekte te ondersoek. Vy spesies kom in ʼn wye verskeidenheid van habitatte voor en ondersteun ʼn diverse groep vy-wesp spesies. Dit het ons die geleentheid gebied om ʼn wye reeks moontlike invloede van vy-wesp KK profiele te ondersoek, van omgewings- tot genetiese invloede. Eerstens, deur die gebruik van GC-MS het ons gevind dat die KK profiele van die vy-wespe wat ondersoek was beide spesie-spesifiek en spesie-groep-spesifiek is, met die spesies Elisabethiella glumosae, Elisabethiella stuckenbergi en Ceratosolen capensis, asook twee Otitesella spesie-groepe (die Uluzi en Sesqui spesie-groepe) wat betekenisvol onderskei kon word. Konsensus filogenieë (gegrond op COI, Cytb en EF1-1α) het getoon dat daar in die gal-induserende vy-wesp genus Otitesella veelvuldige genetiese lyne binne die spesie-groepe voorgekom het ooreenstemmend met tussen-spesie genetiese variasie, en dat elke genetiese lyn beperk was tot ʼn enkele gasheer vy spesie. Die KK profiele het die genetiese verhoudings tussen die twee spesie-groepe weerspieël, en die KK profiele binne ʼn spesie-groep kon onderskei word op grond van hul genetiese lyn/gasheer spesie. Hierdie het getoon dat, alhoewel genetiese lyn meestal verantwoordelik was vir die waargeneemde variasie in KK profiele, faktore wat met verskille in gasheer spesies gepaard gaan ook ʼn effek gehad het. Sterk streeks-verbonde variasie wat beide die invloed van genetiese lyn, én faktore wat met verskille in gasheer spesie gepaard gaan, oortref het, was waargeneem in die KK profiele van die vy-wespe binne ʼn spesie-groep. Hierdie streeks-verbonde variasie in KK profiele was ook waargeneem in twee bestuiwende vy-wespe, Elisabethiella stuckenbergi en Ceratosolen capensis, ʼn resultaat wat nie ondersteun was deur die genetiese bevolkingsdata nie (COI en Cytb). In werklikheid was baie min genetiese bevolkings-struktuur opgespoor binne die bestuiwer spesies, selfs as was die bestuiwer spesies regoor Suid-Afrika ingesamel. Die tekort aan genetiese struktuur in die vy-wesp bestuiwers kan die gevolg wees van hoë geenvloei wat veroorsaak word deur die hoë verspreidingskapasiteit van bestuiwende vy-wespe. Die resultate toon aan dat vy-wesp KK profiele die potensiaal besit om in chemotaksonomie gebruik te word, en word moontlik deur vy-wespe gebruik as kenmerke vir die herkenning van spesie en potensiële maats. Verder was daar gevind dat daar beide ʼn streekseffek en ʼn effek geassosieer met gasheer spesie op KK profiele was. Ons stel voor dat die waargeneemde streekseffek in hierdie studie toegeskryf kan word aan verskille tussen habitatte asook streeksverbonde verskille tussen vy-wesp gemeenskappe. Boonop kan die effek wat gasheer spesie op die KK profiele gehad het ʼn gevolg wees van dieetverskille tussen die galle in verskillende gasheer spesies. ʼn Moontlike gevolg van die waargeneemde streeks/gasheer-spesie-geassosieerde effek op vy-wesp KK profiele is dat dit moontlik kon lei tot voor-paring-isolasie binne vy-wesp spesies, wat uiteindelik spesiasie kon veroorsaak het. Daarbenewens wys ons resultate dat die interpretasie van die variasie in die vy-wesp KK profiel was afhanklik van die skaal van die analise: op ʼn breë interspesie vlak was die vy-wesp KK profiele spesiespesifiek; op ʼn fyner intra-spesie vlak het variasie in KK profiele voorgekom tussen vy-wespe wat in verskillende streke ingesamel was; en op streeksvlak het variasie in die KK profiele binne spesie-groepe voorgekom tussen genetiese lyne/gasheer spesies. Toekomstige studies behoort te kyk na die toepassing van kutikulêre koolwaterstowwe in chemotaksonomiese studies van die vy-wesp filogenie, asook die effek wat vy-wesp gemeenskap samestelling het op vy-wesp kutikulêre koolwaterstowwe.

In wheat, cooler canopies have been associated with yield under high temperature stress. The objectives of this study were, i) to understand the role of leaf cuticular waxes as physiological adaptive mechanisms during reproductive stage high temperature stress ii) define quantitative trait loci (QTL) regulating leaf cuticular waxes and determine its link with the QTL for reproductive stage heat tolerance iii) define stable QTL associated with leaf cuticular waxes and yield stability across environments. For the first objective, thirteen wheat cultivars were subjected to a 2-day heat treatment at 38 degrees C at 10 days after pollination (DAP). Leaf cuticular waxes, canopy temperature depression and stomatal conductance were estimated during high temperature stress. At maturity the percent reduction in yield components in each cultivar was calculated. The wheat cultivars 'Kauz' and 'Halberd' had significantly high leaf cuticular wax content of 2.91mg/dm^-2 and 2.36mg/dm^-2 respectively and cooler canopies. Leaf cuticular waxes were significantly correlated with leaf temperature depression and reduction in yield components. A set of 121 recombinant inbred lines (RIL) population derived from the cross of heat tolerant wheat cultivar 'Halberd' and heat susceptible wheat cultivar 'Karl 92' was utilized for QTL mapping. The RIL population received a 2-day short-term high temperature stress at 38°C at 10DAP in 2008 and a long-term high temperature stress at 38 degrees C from 10DAP until maturity in 2009 in the greenhouse. The RIL population was also planted in College Station, Texas in 2009 and 2010 and in Uvalde, Texas in 2010. Leaf cuticular wax was estimated at 10DAP and leaf/spike temperatures were recorded during grain filling. Yield components were estimated after harvest. Heat susceptibility indexes for main spike yield components were estimated in the greenhouse. Overall ten significant QTL were identified for leaf cuticular waxes each explaining 8-19 percent of the variation respectively. Stable QTL for leaf cuticular waxes were located on chromosome 5A and 1B and co-localized with QTL for leaf/spike temperature depression and HSI for kernel weight and single kernel weight of main spike. Another QTL on chromosome 1B contributed by Karl92 was found in the greenhouse and field environments and co-localized with a previously identified QTL on 1B for spike non-glaucousness. The results suggest that leaf cuticular waxes may reduce leaf temperatures and improve adaptation during high temperature stress.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Subbaratnam Muthukrishnan / The exoskeleton of insects, the cuticle, functions as a support structure and a physical barrier that protects insects from mechanical damage and dehydration. The exoskeleton is mainly made of chitin and proteins, some cross-linked to one another into certain patterns to form the rigid and resistant cuticle. In previous studies from our laboratory, cuticular proteins analogous to peritrophins (CPAPs) and Knickkopf (Knk) were identified and characterized mainly at the pharate adult stage during insect development. However, the dynamic assembly of both CPAP and Knk into the cuticle and the functions of the CPAPs are still not fully understood. Our study is to investigate how these cuticular proteins are assembled into the cuticle during different developmental stages and carry out their functional characterizations in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) experiments that resulted in down-regulation of transcripts for CPAP 1-C, CPAP1-H, CPAP 1-J, CPAP 3-C and Knk genes resulted in molting defects. Confocal and transmission electron microscopic analysis examined protein expression at twelve stages of development, as well as the span from young larva through adult day 3 stages. The results suggested that the CPAP 3-C protein is present in the lower part of endocuticle in the so-called assembly zone and it was not distributed thoughout the procuticle with chitin. Down-regulation of CPAP 3-C transcripts revealed a disorganized assembly zone; however, no loss of chitin content or the laminar architecture of the procuticle was found. Knk protein was present throughout the procuticle and some of the protein was found inside of the epithelial cells.