Natural variation and Biogeography of the melon fruit fly, Zeugodacus cucurbitae (Diptera : Tephritidae), in Southeast-Asia and the west-pacific

This thesis used multidisciplinary approaches which greatly enhance our understanding of population structure and can be particularly powerful tools for resolving variation of melon fly over geographic and temporal scales, and for determining invasive pathways. The results from this thesis reinforce the value of integrating multiple data sets to better understand and resolve natural variation within an important pest to determine whether there are cryptic species, discrete lineages or host races, and to identify dispersal pathways in an invasive pest. These results are instructive for regional biosecurity, trade and quarantine, and provide important background for future area-wide management programmes. The integrative methodology adopted in this thesis is applicable to a variety of other insect pests.

Natural Variation In Recovery From A Short Period Of Anoxia Due To A cGMP-Dependent Protein Kinase in Drosophila melanogaster

[EN] Protein Kinase G (PKG) or cGMP-dependent protein kinases (PKG) have been shown to play an important role in resistance to abiotic stressors such as high temperatures or oxygen deprivation in Drosophila melanogaster. In Drosophila, the foraging gene encodes a PKG; natural variants for this gene exist, which differ in the level of expression of PKG: rovers (forR allele) which express high PKG levels, and sitters (forS allele) which express lower PKG levels. This project explores the differences in recovery from short periods of anoxia between natural variants (focusing on forS2, flies with a sitter gene in a rover background), as well as mutants with insertions in the foraging gene and RNAi recombinants that show a reduced PKG expression. The parameters measured were time to recovery and level of activity after anoxia. The results showed lower activity after anoxia in sitters than in rovers, reflecting a worse recovery from the anoxic coma in flies with lower PKG levels.

Natural variation of rice strigolactone biosynthesis is associated with the deletion of two MAX1 orthologs

Rice (Oryza sativa) cultivar Azucena--belonging to the Japonica subspecies--exudes high strigolactone (SL) levels and induces high germination of the root parasitic plant Striga hermonthica. Consistent with the fact that SLs also inhibit shoot branching, Azucena is a low-tillering variety. In contrast, Bala, an Indica cultivar, is a low-SL producer, stimulates less Striga germination, and is highly tillered. Using a Bala × Azucena F6 population, a major quantitative trait loci--qSLB1.1--for the exudation of SL, tillering, and induction of Striga germination was detected on chromosome 1. Sequence analysis of the corresponding locus revealed a rearrangement of a 51- to 59-kbp stretch between 28.9 and 29 Mbp in the Bala genome, resulting in the deletion of two cytochrome P450 genes--SLB1 and SLB2--with high homology to the Arabidopsis SL biosynthesis gene, MAX1. Both rice genes rescue the Arabidopsis max1-1 highly branched mutant phenotype and increase the production of the SL, ent-2'-epi-5-deoxystrigol, when overexpressed in Bala. Furthermore, analysis of this region in 367 cultivars of the publicly available Rice Diversity Panel population shows that the rearrangement at this locus is a recurrent natural trait associated with the Indica/Japonica divide in rice.

Measurement of the natural variation of 13C:12C ratio in leaves at Reserva Ducke Forest, central Amazonia

Investigation of carbon isotope fractionation by plants was carried out at two sub-areas located in Reserva Ducke, central Amazonia: open reserve (virgin forest with low density of plant species); and closed reserve (virgin forest with high density of plant species). Preliminary results (δ‰ 13C: 12C values, PDB) of leaf analysis at different plant heights indicate the following: Eschweilera matamata Hub. (Lecythidaceae), common name 'matamata', -31.55±0.61; Protium heptaplyllum March. (Burseraceae), common name 'breu branco', -32.34±1.39; Calophyllum brasiliense Camb. (Guttiferae), common name 'jacareúba', -30.72±0.23; Scleronema micrantthum Ducke. (Bombacaceae), common name 'cardeiro'. -28.81±0.68; and Carapa guianensis Aubl. (Meliaceae), common name 'andiroba', -31.07±0.51. It is possible that the plant species analysed belong to the C3 photosynthetic cycle. In general, the species in the open reserve show differences of the order of 1.66±0.34‰ (greater in 13C) as compared with the same species in the closed reserve. The old leaves show differences in the relative isotopic enrichment (δ) of the order of 1‰, being smaller in new leaves in both reserves. The probable occurrence of an isotopic gradient from the lower (2-5 m) to the upper part (15-20 m) of the plant, of the order of 1.3‰, smaller in 13C, in species from the dense forest was noted. However, only two plants from each species were analysed during a two-year period, data obtained to far are still preliminary, and results should, therefore, be revised. Moreover, according to the literature, the natural carbon isotope fractionation by plants shows metabolic, physiological and environmental dependence. © 1991.

Natural Variation in Maize Aphid Resistance Is Associated with 2,4-Dihydroxy-7-Methoxy-1,4-Benzoxazin-3-One Glucoside Methyltransferase Activity

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.

Natural variation in expression of genes associated with carotenoid biosynthesis and accumlation in cassava (Manihot esculenta Crantz) storage root.

2016

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

ABSTRACT: BACKGROUND: Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. RESULTS: Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total ß-carotene, containing all-E-, 9-Z-, and 13-Z-ß-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no ?-carotene was observed, variable amounts of a ?-ring derived xanthophyll, lutein, was detected; with greater accumulation of ?-ring xanthophylls than of ß-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total ß-carotene accumulation. CONCLUSIONS: Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total ß-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.

High natural gene expression variation in the reef-building coral Acropora millepora: potential for acclimative and adaptive plasticity

Background: Ecosystems worldwide are suffering the consequences of anthropogenic impact. The diverse ecosystem of coral reefs, for example, are globally threatened by increases in sea surface temperatures due to global warming. Studies to date have focused on determining genetic diversity, the sequence variability of genes in a species, as a proxy to estimate and predict the potential adaptive response of coral populations to environmental changes linked to climate changes. However, the examination of natural gene expression variation has received less attention. This variation has been implicated as an important factor in evolutionary processes, upon which natural selection can act. Results: We acclimatized coral nubbins from six colonies of the reef-building coral Acropora millepora to a common garden in Heron Island (Great Barrier Reef, GBR) for a period of four weeks to remove any site-specific environmental effects on the physiology of the coral nubbins. By using a cDNA microarray platform, we detected a high level of gene expression variation, with 17% (488) of the unigenes differentially expressed across coral nubbins of the six colonies (jsFDR-corrected, p < 0.01). Among the main categories of biological processes found differentially expressed were transport, translation, response to stimulus, oxidation-reduction processes, and apoptosis. We found that the transcriptional profiles did not correspond to the genotype of the colony characterized using either an intron of the carbonic anhydrase gene or microsatellite loci markers. Conclusion: Our results provide evidence of the high inter-colony variation in A. millepora at the transcriptomic level grown under a common garden and without a correspondence with genotypic identity. This finding brings to our attention the importance of taking into account natural variation between reef corals when assessing experimental gene expression differences. The high transcriptional variation detected in this study is interpreted and discussed within the context of adaptive potential and phenotypic plasticity of reef corals. Whether this variation will allow coral reefs to survive to current challenges remains unknown.

A reliable externally fixated murine femoral fracture model that accounts for variation in movement between animals.

Fifty-two CFLP mice had an open femoral diaphyseal osteotomy held in compression by a four-pin external fixator. The movement of 34 of the mice in their cages was quantified before and after operation, until sacrifice at 4, 8, 16 or 24 days. Thirty-three specimens underwent histomorphometric analysis and 19 specimens underwent torsional stiffness measurement. The expected combination of intramembranous and endochondral bone formation was observed, and the model was shown to be reliable in that variation in the histological parameters of healing was small between animals at the same time point, compared to the variation between time-points. There was surprisingly large individual variation in the amount of animal movement about the cage, which correlated with both histomorphometric and mechanical measures of healing. Animals that moved more had larger external calluses containing more cartilage and demonstrated lower torsional stiffness at the same time point. Assuming that movement of the whole animal predicts, at least to some extent, movement at the fracture site, this correlation is what would be expected in a model that involves similar processes to those in human fracture healing. Models such as this, employed to determine the effect of experimental interventions, will yield more information if the natural variation in animal motion is measured and included in the analysis.

Warming and acidification experiment on early life-stage F. vesiculosus under natural fluctuation and seasonal variations (spring 2013 - 2014)

Genetic diversity of baltic F. vesiculosus is low compared to other populations which might jeopardize their potential for adaptation to climate change. Especially the early life-stage F. vesiculosus may be threaten by ocean warming and acidification. To test this, we exposed F. vesiculosus germlings to warming and acidification in the near-natural scenario in the "Kiel Outdoor Benthocosms" maintaining the natural variation of the Kiel Fjord, Germany (54°27 'N, 10°11 'W) in all seasons (spring 2013 - 2014). Warming was simulated by using a delta treatment adding 5 °C and by increasing pCO2 at 1000 µatm. Warming positively affected germlings' growth in spring and in summer but decreased non-photochemical quenching in spring and survival in summer. Acidified conditions showed much weaker effects than warming. The high genotypic variation in stress sensitivity as well as the enhanced survival at high diversity levels indicate higher potential for adaptation for genetically diverse populations. We conclude that the combination of stressors and season determines the sensitivity to environmental stress and that genetic variation is crucial for the adaptation to climate change stress.

HKT2;2/1, a K+-permeable transporter identified in a salt tolerant rice cultivar through surveys of natural genetic polymorphism

We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5¢ region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3¢ region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na+ concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na+ and K+, even at high external Na+ concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K+ uptake under saline conditions.

Impact of changes in natural ultraviolet radiation on pigment composition, physiological and morphological characteristics of the Antarctic moss, Grimmia antarctici

The impact of ambient ultraviolet (UV)-B radiation on the endemic bryophyte, Grimmia antarctici, was studied over 14 months in East Antarctica. Over recent decades, Antarctic plants have been exposed to the largest relative increase in UV-B exposure as a result of ozone depletion. We investigated the effect of reduced UV and visible radiation on the pigment concentrations, surface reflectance and physiological and morphological parameters of this moss. Plexiglass screens were used to provide both reduced UV levels (77%) and a 50% decrease in total radiation. The screen combinations were used to separate UV photoprotective from visible photoprotective strategies, because these bryophytes are growing in relatively high light environments compared with many mosses. G. antarctici was affected negatively by ambient levels of UV radiation. Chlorophyll content was significantly lower in plants grown under near-ambient UV, while the relative proportions of photoprotective carotenoids, especially beta-carotene and zeaxanthin, increased. However, no evidence for the accumulation of UV-B-absorbing pigments in response to UV radiation was observed. Although photosynthetic rates were not affected, there was evidence of UV effects on morphology. Plants that were shaded showed fewer treatment responses and these were similar to the natural variation observed between moss growing on exposed microtopographical ridges and in more sheltered valleys within the turf. Given that other Antarctic bryophytes possess UV-B-absorbing pigments which should offer better protection under ambient UV-B radiation, these findings suggest that G. antarctici may be disadvantaged in some settings under a climate with continuing high levels of springtime UV-B radiation.